key: cord-004477-qu2o2iu1 authors: vlasova, anastasia n.; butler, john e. title: editorial: porcine anti-viral immunity date: 2020-03-06 journal: front immunol doi: 10.3389/fimmu.2020.00399 sha: doc_id: 4477 cord_uid: qu2o2iu1 nan the swine industry is an important part of agriculture in many countries, generating over $11 billion annually in the us alone. viral diseases in pigs pose a serious risk for swine health and significantly impact the economy of the global swine industry. pigs also serve as zoonotic reservoirs for viruses transmittable to humans and other species, including influenza a virus, nipah virus, and hepatitis e virus (1) . these problems are made worse by globalization and industrial livestock rearing (2, 3) . unlike bacterial pathogens, antibiotics do not protect against viral infections and cannot be used to control viral outbreaks or epidemics. since viruses can alter their genome >10 6 times faster than their mammalian hosts (4), the latter would have succumbed to microbial infection were it not for their adaptive immune system that uses somatic gene rearrangement and mutation to counter the rapid diversification of microbes. immediately following viral infection, neonatal survival depends on innate immunity and passive protection by lactogenic immune factors such as pathogen-specific antibodies, until an adaptive immune response can develop. thus, intervention against these moving targets, depends on availability of effective maternal and neonatal vaccines and strict biosecurity measures. wide-spread porcine reproductive and respiratory syndrome virus (prrsv) and swine influenza virus (siv) represent major health challenges in the large us swine production systems and possibly worldwide. in the us alone, economic annual losses due to prrsv are estimated to be 664 million u.s. dollar (usd) (5) . while the exact costs associated with siv and other endemic viruses, including rotavirus, are hard to evaluate, they are economically important due to their ubiquitous nature, which can reduce growth and increased morbidity (6) . emerging and re-emerging coronaviruses in pigs are quite often associated with diarrheal disease and high morbidity and mortality (7) . the emergence of seneca valley virus (svv) and increased incidence of svv-associated vesicular disease alarmed the swine industry in several countries, including the us, brazil, china, thailand, and colombia (8) . finally, a deadly swine disease, caused by african swine fever virus (asfv), that has been plaguing africa for decades, has now spread to south eastern europe and asia, and has severely impacted the world's largest swine industries in china (9) . in this special volume of frontiers in immunology, comparative immunology section, lager and buckley provide an overview of the importance of swine in the world food supply and a review of the major viral infection that threaten this species (lager and buckley). in introducing the topic of anti-viral immunity, we emphasize the genetic diversity of viruses, the virus life cycle and the pathology that viral infection can cause. a total of 12 articles are contained in this volume to which 86 researches have contributed. despite the advantage of internal offspring development and the provision for passive immune protection, fetuses and neonates of higher vertebrates still remain vulnerable to environmental pathogens. piglets in utero are protected from eukaryotic parasites and bacteria, but are vulnerable to viruses (including parvovirus, prrsv, and porcine circovirus 2) since they are able to cross the placental barrier (10) (11) (12) . these cause pathological injury and often fetal abortion. in a number of mammals, viruses infect the thymus and can also cause hypergammaglobulinemia. butler et al. discuss the implication of thymic atrophy and apoptosis of thymocytes and hypergammaglobulinemia with regard to the persistence of prrsv. however, apoptosis is also a host mechanisms used to eliminating virus-infected cells (13) , but can also promote spread of the virus (14) . fernandes et al. demonstrated that svv developed a 3c protease-dependent mechanism for late apoptosis that facilitates virus release from infected cells. thus, the role and importance of virus-induced apoptosis awaits further research. since the adaptive immune system of fetal piglets is underdeveloped, the innate immune system is very important. schäfer et al., emphasize its importance with the specific focus on porcine invariant natural killer t cells (inkt) and their role in the pathogenesis of asf and si. they discuss inkt agedependence, levels and distribution in relationship to various porcine viral infections. one of the first host responses to viral infection is the production of interferons, which are needed to drive other elements of innate immunity and adaptive immunity. likai et al. show how a porcine deltacoronavirus escapes from the immune system by suppressing ifn-α production, while shi et al. describe a novel immune evasive mechanism that depends on prrsv non-structural protein 1a which antagonizes tbk1-irf3-ifn signaling. while the initial antibody response depends on broadly specific natural igm antibodies, effective anti-viral immunity depends on an adaptive immune response that delivers igg and iga antibodies. since adaptive immune responses depend on stimulation through the innate immune system, viruses that impair or interfere with the innate immune response, also impair adaptive immunity. nedumpun et al. discuss how prrsv-induced il-1ra downregulates innate immune responses, t lymphocyte differentiation and proliferation. piglets, unlike humans, but like the offspring of other artiodactyls and perissodactyls (horses), are especially dependent on lactogenic immunity since there is no transplacental transfer of passive antibodies in this group of mammals. thus, providing ways to deliver anti-viral antibodies via colostrum/ milk is important. this topic is the focus of studies by langel et al.. antibodies can intercept and neutralize a virus before it infects additional cells and can prevent further infection by blocking the viral receptor; these are called virus neutralizing (vn) antibodies. naturally, a critical step in viral vaccine design is the identification of viral epitopes targeted by effective vn antibodies. this become increasingly important in the case of prrsv in which effective vn antibodies are slow to appear and current vaccines are of questionable efficacy. this is also an ongoing challenge in the case of asfv which is rapidly spreading and for which no vaccine exists. goldeck et al. describe a novel b cell cloning procedure to identify these epitopes on several strains of prrsv. while vn antibodies can block or delay infection, the ultimate elimination of a virus is to kill the cells in which the virus replicates which is the job of cytotoxic t cells. portions of the virus are displayed on infected cells. these are called a t cell epitope, i.e., a molecular structure that binds to the t cell receptor (tcr). in this volume, pan et al. describe their efforts to identify such epitopes for their potential use in stimulating expansion of the cytotoxic t cells that recognize them. viral vaccines take various forms, the simplest being the use of killed virus. a more tedious procedure is to use only parts of the virus as the vaccine (subunit vaccines) that target the immune response to those viral epitopes that elicit vn antibodies. killed viruses and subunit vaccines typically generate immediate responses that wane faster, making it imperative for long-lived mammals like humans, to receive booster vaccinations. a second approach to vaccine development is use of live attenuated virus that has been genetically modified or cell culture adapted and cannot produce a disease in the host but can still replicate. these are often more likely to stimulate virus-specific cytotoxic t cells and to induce longer term immunity. sharma et al. developed a recombinant svv strain using reverse genetics and tested its immunogenicity and protective efficacy in pigs. toman et al. provide comparative data on four vaccines for prrsv, two killed and two modified live. we believe that veterinary immunovirology should place more emphasis on how each viral pathogen effects and/or avoids the host immune system and on the identification of viral epitopes that are effective targets of vn antibodies and t cell epitopes that can promote the action of cytotoxic t cells. jb wrote the first draft and then both co-authors contributed equally to the final draft. reservoir host immune responses to emerging zoonotic viruses current drivers and future directions of global livestock disease dynamics the future of pork production in the world: towards sustainable, welfare-positive systems what contemporary viruses tell us about evolution: a personal view assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers impact of health challenges on pig growth performance, carcass characteristics, and net returns under commercial conditions emerging and reemerging coronaviruses in pigs emergence of a novel recombinant seneca valley virus in central china african swine fever virus in asia: its rapid spread and potential threat to unaffected countries apoptosis and necroptosis as host defense strategies to prevent viral infection viruses and apoptosis fetal infections and antibody profiles in pigs naturally infected with porcine circovirus type 2 (pcv2) effect of wean-tofinish management on pig performance transplacental infection and embryonic death following maternal exposure to porcine parvovirus near the time of conception the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 vlasova and butler. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-280924-g6062fwk authors: hachim, mahmood yaseen; al heialy, saba; hachim, ibrahim yaseen; halwani, rabih; senok, abiola c.; maghazachi, azzam a.; hamid, qutayba title: interferon-induced transmembrane protein (ifitm3) is upregulated explicitly in sars-cov-2 infected lung epithelial cells date: 2020-06-10 journal: front immunol doi: 10.3389/fimmu.2020.01372 sha: doc_id: 280924 cord_uid: g6062fwk current guidelines for covid-19 management recommend the utilization of various repurposed drugs. despite ongoing research toward the development of a vaccine against sars-cov-2, such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. therefore, there is an urgent need to develop a framework for the rapid identification of novel targets for diagnostic and therapeutic interventions. we analyzed publicly available transcriptomic datasets of sars-cov infected humans and mammals to identify consistent differentially expressed genes then validated in sars-cov-2 infected epithelial cells transcriptomic datasets. comprehensive toxicogenomic analysis of the identified genes to identify possible interactions with clinically proven drugs was carried out. we identified ifitm3 as an early upregulated gene, and valproic acid was found to enhance its mrna expression as well as induce its antiviral action. these findings indicate that analysis of publicly available transcriptomic and toxicogenomic data represents a rapid approach for the identification of novel targets and molecules that can modify the action of such targets during the early phases of emerging infections like covid-19. coronaviruses are a large family of viruses that were first described over 50 years ago (1) . since the turn of the millennium, there have been two major global outbreaks caused by coronaviruses, namely sars-cov in 2003 and mers-cov in 2012 (2) . the ongoing covid-19 pandemic caused by sars-cov-2 represents the third and most devastating of these outbreaks. these outbreaks, notably the covid-19 pandemic, are harsh reminders of the challenges posed by emerging infectious diseases. the global impact of the covid-19 pandemic has brought to the forefront the need to rapidly develop and deploy an effective vaccine. however, despite ongoing concerted research efforts, it is accepted that such a vaccine will not be available in time to contribute to the containment of the ongoing pandemic. current management guidelines include the use of repurposed drugs such as chloroquine and its analog hydroxychloroquine as well as antiviral agents (3) . however, the need for well-designed clinical trials to validate their efficacy continues to be highlighted. to effectively address the ongoing covid-19 pandemic, there is a recognized need for a framework for rapid identification of novel targets for diagnostic and therapeutic interventions as well as determine clinically approved drugs with high potential for repurposed use against sars-cov-2. publicly available transcriptomic datasets generated from sars-cov infected humans, and mammalian cells represent a wealth of data that could be used to identify consistent differentially expressed genes, which could then be validated against sars-cov-2 infected epithelial cells transcriptomic datasets. a comprehensive toxicogenomic analysis of the identified genes could potentially identify possible interactions with clinically proven drugs. this simple approach can be used for the rapid identification of novel targets and drugs for further validation. in this study, we have applied this approach, and our findings have identified ifitm3 as an early upregulated gene and indicate that valproic acid enhances ifitm3 mrna expression and antiviral action. publicly available transcriptomic datasets were retrieved from gene expression omnibus (geo) mouse lung tissue transcriptome response to a mouse-adapted strain of sars-cov in wild type c57bl6/nj mice and tlr3-/-mice c57bl6/nj ma15 gse68820 (11) (https://www.ncbi.nlm.nih.gov/geo/). only microarray gene expression datasets with the word "sars-cov, " virus, or modified strain infected vs. mock-infected and no more than 48 h after the infection. twelve datasets fulfilled the criteria, as detailed in table 1 . we used geoquery and limma r packages through the geo2r tool for each dataset (12) . after sorting the genes according to the false discovery rate (fdr), the top 2,000 differentially expressed probes with fdr <0.05 were selected from each dataset. the annotated genes of the 5,000 probes in each dataset were intersected with differentially expressed genes (degs) from all other datasets. the degs that were common in at least 9 out of the 12 (75%) datasets were identified as shared genes that are consistently deg in the first 48 h of sars-cov infection. enriched ontology clustering for the identified genes was performed to explore using the metascape (http://metascape.org/gp/index.html#/main/step1). the shortlisted genes expression was then explored in another dataset (gse147507), where rna-sequencing of primary human lung epithelium (nhbe) mock-treated or infected with sars-cov-2 was done to examine whether there is a difference in the response of sars-cov-2 from other strains in terms of degs (13) . in total, 9,692 genes were differentially expressed genes (degs) between mock-infected and virally infected models in the 12 studies. thirty-eight genes that were degs in at least 9 out of 12 studies (75%) were considered common degs due to sars-cov infection of the lungs in the first 48 h post-infection. these genes are listed in table 2 . in order to identify deg in sars-cov infected lung tissuespecific to each of the models used and those which are shared, we intersected the degs from datasets that use the same model. human epithelial cells datasets (gse17400, gse33267, gse37827, gse47960, and gse48142), mice datasets (gse33266, gse50000, gse52920, gse64660, and gse68820), ferret (gse11704) and cynomolgus macaques (gse23955) were all intersected with the covid-19 infected epithelial cells dataset as shown in figure 1 . the number of deg intersected between different species is listed in the table 3 . epithelial cells infected with sars-cov-2 shared 9 degs (mx1, oas3, xaf1, ifi44, mx2, irf7, stat1, ifit3, and ifit1) with human lung cells, mice, and cynomolgus maca. the identified genes are involved in the immune response against rna viruses as expected, the top genes identified are involved in innate immune responses against rna viruses. these include the cytosolic dna-sensing pathway, toll-like receptor signaling pathway, and negative regulation of binding. interferon (ifn) response to viral infections such as type i interferon signaling pathway, defense response to the virus, the antiviral mechanism by ifn-stimulated genes, regulation of type i interferon production, response to interferon-alpha, and regulation of defense response to virus and influenza a, were also upregulated. genes that play significant roles in activating immune systems such as regulation of response to cytokine stimulus, negative regulation of immune response, myeloid cell homeostasis, and positive regulation of the multi-organism process are also upregulated (figure 2 and table 4 ). the identified genes expression levels were higher in human bronchial epithelium infected with sars-cov-2 compared to those mock-infected (figure 3) . however, only ifitm3 showed a significant difference (p < 0.05), while two other genes oas2 and mx1 showed a trend of enhancement, although it was not statistically significant (p = 0.06 using the two-stage linear stepup procedure of benjamini, krieger, and yekutieli). ifitm3 mrna levels were one of the highly expressed genes compared to the other identified genes at baseline in mock-infected hbe and were further induced by the virus, which results in overall high mrna levels. valproic acid can upregulate the ifitm3 mrna expression next, we searched the comparative toxicogenomics database (http://ctdbase.org/) to identify drugs/chemicals that might affect the mrna expression of ifitm3 in at least two reference studies (14) . interestingly valproic acid, carbon nanotubes, nickel, and tert-butylhydroperoxide were shown to upregulate ifitm3 expression while pirinixic acid, acetaminophen, and ethinyl estradiol decreased such an expression ( table 5) . in order to examine the effect of valproic acid therapy on the ifitm3 mrna expression in immune cells of the blood, a publicly available transcriptomics dataset (gse143272) was extracted and reanalyzed. healthy controls were compared to responders and non-responders patients on valproic acid therapy. we found upregulation of the mrna expression of ifitm3 in patients, and the difference was significant in the responder group only (p < 0.05) compared to healthy controls (figure 4) . in response to viral rnas, like in the case of sars-cov-2, the innate immune system will unleash interferon (ifn), to activate antiviral mechanisms and effector cells like natural killers (15) . in mice infected with sars-cov, a delayed and prolonged type i interferon (ifn-i) signaling leads to lung immunopathology as it promotes the accumulation of pathogenic inflammatory cells with increased lung cytokine/chemokine levels and vascular leakage (16) . this prolonged ifn-i and virally induced il-10 set the scene for secondary bacterial infection, which can add a strong il1β and tnfα-mediated inflammatory response to magnify lung damage (17) . understanding how sars-cov-2 can manipulate ifn is vital in deciphering the battle of the body against the viral spread and consequence. our reanalysis of transcriptomic data showed that although the ifn pathway is upregulated consistently in sars-cov related infection, sars-cov-2 showed specific upregulation of the gene for a unique interferon-induced protein, namely ifitm3. ifitm3 is a 15-kda protein that localizes to endosomes and lysosomes and is possibly acquired by mammalian ancestral cells via horizontal gene transfer (18) . interferon-induced transmembrane proteins (ifitms 1, 2, and 3) are innate immune responders to virus infections as they regulate the fusion of invading virus and endocytic vesicles and direct it to the lysosomes (19, 20) . ifitm3 can further alter membrane rigidity and curvature to inhibit virus membrane fusion (21) . such action is important to prevent the release of viral particles into the cytoplasm, which controls viral spread (22) . during influenza a infection of human airway epithelial cells, ifitm3 was shown to clusters on viruscontaining endosomes and lysosomes within few hours postinfection, indicating its role in the early phase of viral entry (23) . even platelets and megakaryocytes were shown to remarkably upregulate ifitm3 to prevent viral progression during influenza infection (24) . bst2, ifi35, ifit2, ifit1, ifit3, irf7, isg20, mx1, mx2, oas2, oas3, sp100, stat1, isg15, ifitm3, usp18, xaf1, zbp1, rsad2, trim21, ddx58, il6, cxcl10, cxcl11 the epithelial cell and resident leukocytes in lung upper and lower airways that constitutively express ifitm3 can withstand viral infections, and this is vital to decide viral tropism as viruses favor cells with low ifitm3 expression (25) . ifitm3 enhances the accumulation of cd8+ t cells in airways to promote mucosal immune cell persistence (26) . lung and circulating immune cells were reported to express less ifitm3 than other tissues, and this was a suggestive reason for covid-19 severity and cytokine release syndrome (27) . interestingly, ifitm3-rs12252-c/c snp prevalence in the chinese population is 26.5%, and recent research confirmed that snps in ifitm3 could change the severity of influenza infection, as was shown in one case with covid-19 (28) . ifitm3 polymorphisms have been linked with hospitalization and mortality during influenza virus infection (29) . expressing the gene is not the only prerequisite to the antiviral action of ifitm3, as it was found recently that within the protein, an amphipathic helix is critical for its blocking effect of viral fusion of similar pathogenic viruses like influenza a virus and zika virus (30). another factor that regulates the ifitm3 trafficking specificity to such viruses is that it requires s-palmitoylation (19, 20) . s-palmitoylation (s-palm) is the reversible process of linking a fatty acid chain to cysteine residues of the substrate protein (31) . multiple zinc finger dhhc domain-containing palmitoyltransferases (zdhhcs) can palmitoylate ifitm3 to make it a fully functional antiviral protein (32) . it seems that bats (order chiroptera), which act as natural hosts for many viral infections, use ifitm3 as an antiviral mechanism if there is s-palmitoylation of the protein; however, if this modification is disturbed, the bat can develop viral infection (33) . based on that, we can suggest that severe covid-19 cases might be due to either non-functional ifitm3 by snp, failure of lung cells to upregulate ifitm3 in response to interferon, a mutation in amphipathic helix sequence or modification in s-palmitoylation. further examination and screening for the ifitm3 dynamics in covid-19 might explain the possible therapeutic and diagnostic options. our toxicogenomic analysis showed that valproic acid increased the mrna expression of ifitm3, supporting a new report that the sars-cov-2-human protein-protein interaction map showed that valproic acid might be a potential repurposing drug for covid-19 (34) . virtual screening, docking, binding energy calculation, and simulation show that valproic acid forms stable interaction with nsp12 of cov and can inhibit its function (14) . valproic acid is currently used for the treatment of epilepsy and known to target histone deacetylases (hdacs) that modify the gene expression epigenetically (35) . valproic acid was shown to inhibit mature and fully infectious enveloped viruses release as it alters cellular membrane composition (36) . the modest and broad antiviral activity of valproic acid made the drug an attractive possibility due to its availability, and limited side effects for a short term of use during acute viral disease (37) . the reported side effects like hepatotoxicity and teratogenicity are mainly associated with the parental compound valproate and can be avoided by the use of its derivatives like valpromide (vpd) and valnoctamide (vcd) . a recent open-label proof-of-concept trial of 10 days intravenous valproic acid for severe covid-19 showed a 50% reduction in the case fatality rate and length of stay (38) . more studies are needed to explore the promising potential of valproic acid in the treatment of covid-19. one limitation of the study is that it is based on the publicly available transcriptome dataset, which is limited in number, partly because this is a novel disease, but also because ongoing lockdowns have made it challenging for scientists to carry out the extensive laboratory work required. our evaluation showed that the analysis of publicly available transcriptomic data could be a reasonable approach to identify the novel target and suggest drugs that can modify the action of such targets during the early phases of emerging infections like covid-19 until a complete understanding of the disease become clear. this can justify the experimental use of clinically approved drugs and guide the clinicians in their limited options against such lethal disease. all datasets presented in this study are included in the article/supplementary material. coronaviruses in animals and humans three emerging coronaviruses in two decades covid-19 -navigating the uncharted lack of innate interferon responses during sars coronavirus infection in a vaccination and reinfection ferret model dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection comparative pathogenesis of three human and zoonotic sars-cov strains in cynomolgus macaques complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis release of severe acute respiratory syndrome coronavirus nuclear import block enhances host transcription in human lung cells a network integration approach to predict conserved regulators related to pathogenicity of influenza and sars-cov respiratory viruses the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection ncbi geo: archive for functional genomics data sets-update sars-cov-2 launches a unique transcriptional signature from in vitro, ex vivo, and in vivo systems. biorxiv the comparative toxicogenomics database: update 2019 viral innate immune evasion and the pathogenesis of emerging rna virus infections dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice a model of superinfection of virus-infected zebrafish larvae: increased susceptibility to bacteria associated with neutrophil death more than meets the i: the diverse antiviral and cellular functions of interferon-induced transmembrane proteins ifitm3 directly engages and shuttles incoming virus particles to lysosomes interferon-induced transmembrane protein 3 blocks fusion of sensitive but not resistant viruses by partitioning into virus-carrying endosomes ifitm proteins restrict viral membrane hemifusion antiviral protection by ifitm3 in vivo ifitm3 clusters on virus containing endosomes and lysosomes early in the influenza a infection of human airway epithelial cells human megakaryocytes possess intrinsic antiviral immunity through regulated induction of ifitm3 ifitm3: how genetics influence influenza infection demographically enhanced survival of lung tissue-resident memory cd8(+) t cells during infection with influenza virus due to selective expression of ifitm3 two potential novel sars-cov-2 entries, tmprss2 and ifitm3, in healthy individuals and cancer patients ifitm3 restricts the morbidity and mortality associated with influenza snp-mediated disruption of ctcf binding at the ifitm3 promoter is associated with risk of severe influenza in humans insights into protein s-palmitoylation in synaptic plasticity and neurological disorders: potential and limitations of methods for detection and analysis the palmitoyltransferase zdhhc20 enhances interferoninduced transmembrane protein 3 (ifitm3) palmitoylation and antiviral activity bat ifitm3 restriction depends on s-palmitoylation and a polymorphic site within the cd225 domain a sars-cov-2-human protein-protein interaction map reveals drug targets and potential drug-repurposing. biorxiv exploring the inhibitory activity of valproic acid against the hdac family using an mmgbsa approach new pharmacological strategies to fight enveloped viruses trends in antiviral strategies methods of an openlabel proof-of-concept trial of intravenous valproic acid for severe covid-19. medrxiv all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. key: cord-030803-6i0e2zkd authors: hu, wan-chung title: a framework of all discovered immunological pathways and their roles for four specific types of pathogens and hypersensitivities date: 2020-08-07 journal: front immunol doi: 10.3389/fimmu.2020.01992 sha: doc_id: 30803 cord_uid: 6i0e2zkd nan follicular helper t cells (tfh) are considered as the key helper cells for b-cell germinal centers in lymph nodes and are characterized as il-21-producing t cells (8) . follicular dendritic cells (cd14+) are antigen presenting cells (9) , whereas lymphoid tissue inducer cells (lti) are the innate lymphoid cells for tfh (10) . bcl6 is a key transcription factor in tfh development (11) . tgf-β induced by a stat5 signal can constrain the differentiation of il-21-producing helper t cells (12) . il-21 production is related to stat1and stat3 activation. il-21 production is also related to stat5 activation because immunosuppressive prolactin can cause stat5a to suppress bcl6 expression (13) . in contrast, stat5b can upregulate bcl6 (14) . stat5a and stat5b have distinct target genes in immune responses (15) , and stat5b is the transcription factor that induces tfh. tfh can induce b-cells to produce igm antibodies and il-21 produced by tfh facilitates b cell isotype switching to igg (16, 17) . besides the protein antigen recognized by b cells and t cells, natural killer t (nkt) cells also recognize lipid antigens. the subtype inktfh plays a role in tfh responses (18) . thus, t lymphocytes are the first to initiate adaptive host immunity (19) (20) (21) , wherein different stat proteins regulate different immunological pathways. if the infection tends to be eradicable, then the host immunological pathways mentioned in the following sections are generated along with other cytokines. th1 immune responses are driven by il-12 and are induced against intracellular bacteria or protozoa (22) . type 2 myeloid dendritic cells (cd141+ mdc2) are the antigen presenting cells eradicable immune responses include th1, th2, th17, and thαβ. tolerable immune responses include th1-like (th1l), th9, th22, in the th1 response (9) . the main th1 effector cells are stimulatory macrophages (m1), ifn-γ-secreting cytotoxic cd8 t cells (cd28+ tc1), ifn-γ-secreting cd4 t cells, inkt1 cells, and igg3 -producing b-cells (23, 24) . initiation of eradicable immunity also requires innate lymphoid cells to produce the initial cytokines that drive different immunological pathways. for th1 immune responses, the key innate lymphoid cells are ilc1 (25) . stat4 is the key transcription factor for th1 immunity but t-bet also plays a vital role. th1 responses against self-antigens present as type 4 delayed-type hypersensitivity, such as type 1 diabetes mellitus or crohn's disease (26) . th2 immune responses are driven by il-4 and is induced against extracellular parasites (helminths) (27) . the antigen presenting cells in th2 immune responses are langerhans cells (cd1a+) (9, 28) . the main th2 effector cells are eosinophils (ieos), basophils/pro-inflammatory mast cells (mct, mast cell tryptase), il-4-/il-5-secreting cd4 t cells, inkt2 cells, ilc2, and igg4/ige-producing b-cells (29) . igg4 activates eosinophils, and ige activates mast cells, as in acute anaphylaxis (30) . igg4eosinophils function to activate eosinophil-mediated cellular immunity against parasites or insects, whereas ige-mast cells act to expel helminths or insects through a physiological mechanism. mast cells activated by ige can release histamine, which causes bronchoconstriction, vomiting/nausea, rhinorrhea, skin itching, gastric acidification, increased local vascular permeability, or increased bowel movement. these actions can all help to physiologically expel helminths or insects. the key transcription factor in th2 response is stat6 and gata3 also plays a vital role in the th2 immunological pathway. th2 responses against selfantigens present as type 1 immediate allergy, such as food/drug allergy, anaphylaxis, or urticarial (31) . thαβ is distinct from traditional th1 immune responses (2) . thαβ cells are induced against viruses and were previously called as tr1 cells (4, 32) . thαβ immune responses are driven by ifnα/β or il-10. the antigen presenting cells for thαβ responses are plasmacytoid dendritic cells (pdc) (9) . the main effector cells of thαβ immune responses are il-10-producing stimulatory nk cells (cd56-cd16 + nk1 cells), il-10/il-27-secreting cd4 t cells, il-10-secreting cytotoxic cd8 t cells (cd28+ tc2), inkt10 cells, ilc10, and igg1-producing bcells (23, (33) (34) (35) . the cd27 molecule is important for antiviral immunity. the key transcription factors for thαβ response are stat1 and stat2 (36). thαβ immune responses against self-antigens present as type 2 antibody-dependent cytotoxic hypersensitivity, such as the acute stage of myasthenia gravis or graves' disease (37) . il-10 is not merely an immunosuppressive cytokine; it can also have potent stimulatory effects on nk cells, cytotoxic t lymphocytes (ctls), and b-cells. th22 responses are part of the host innate immunity against extracellular bacteria and fungi (38) . th22 response is driven by il-6 or tnfα (39) . the antigen presenting cells in th22 immune responses are type 1 myeloid dendritic cells (cd1c+ mdc1) (9) . the main th22 effector cells are neutrophils (n1), il-22-secreting cd4 t cells, inkt17 cells, ilc3(ncr+), and igg2-producing b-cells (6, 40) . the key th22 transcription factor is stat3; ap1 and cebp are also important transcription factors. tgf-β can suppress il-22 to skew the th22 immune response toward th17 (41) . th22 responses against selfantigens present as type 3 immune-complex and complementmediated hypersensitivity, such as the arthus reaction or rheumatoid arthritis (42) . the host immunological pathways induced are mainly decided by the extracellular or intracellular location of protozoa or fungi. four igg subtypes fit the four types of acute immunological pathways. murine igg antibodies also have four subclasses and are correlated with human igg subtypes as follows: human igg1<->murine igg2a; human igg2<->murine igg3; human igg3<->murine igg2b; and human igg4<->murine igg1 (43) . higg1/migg2a function against viral antigens; higg2/migg3 function against bacterial antigen, especially polysaccharides; higg3/migg2b act against intracellular bacteria; and higg4/migg1 act against parasite antigens (44) (45) (46) . notably, the immune response against fungi or protozoa is mainly based on their intracellular or extracellular location. extracellular fungi such as candida spp or aspergillus spp usually trigger th22 immune responses, whereas intracellular fungi such as histoplasma spp. trigger th1 responses. tregs are the host immune inhibitory cells (47) driven by il-2 and tgf-β. regulatory dendritic cells (dcreg) are the antigen presenting cells for tregs (48) . regulatory innate lymphoid cells (ilcreg) are the initial helpers for treg production (49) . the main effector cells for tregs are the tgf-β-producing cd4 t cells, foxp3 regulatory inkt cells, and iga-producing b-cells (50) . stat5, especially stat5a is the key transcription factor for the treg pathway. however, both stat5a and stat5b play non-redundant roles in treg generation (51) . they may first act sequentially with stat5b activation in tfh signaling. combined signaling stat5b and stat5a induces treg generation. the combination of tregs and the aforementioned four immunological pathways are important to shift adaptive immunity to tolerable immunity. during initial infection, acutestage fierce cytokines can rapidly kill pathogens and infected cells or tissues. however, if the pathogen infects numerous cells in a tissue such as the liver, killing the infected cells will completely destroy the organ (52) . thus, a regulatory t cell stat5 signal combined with th1/th2/th22/thαβ will allow the generation of cd4 t cells with less fierce inflammatory cytokines (51) . th1like/th9/th17/th3 immunological pathways are generated during chronic infection. iga1 and iga2 are the two types of iga antibodies, with iga1 being dominant in the serum, whereas iga2 is dominant in the mucosa. tgf-β can induce iga1 or iga2 depending on the lymphoid follicle location (53) . in the gutassociated lymphoid tissues (galts) or the peyer's patches, iga2 is the dominant iga antibody produced in the gastrointestinal mucosa. in the lymph nodes at other body locations, iga1 is the dominant iga antibody produced. however, iga1 is specifically related to viral protein antigens, whereas iga2 is especially related to bacterial antigens such as lps. the heavy-chain locus sequence of b-cell antibodies on the human chromosome 14 is igm, igd, igg3, igg1, iga1, igg2, igg4, ige, and iga2. b-cells co-express igm and igd. igg3, igg1, and iga1 comprise the first group for cellular immunity, whereas igg2, igg4, ige, and iga2 can be considered as the second group for humoral immunity. the gene sequence order is important as it affects the time sequence of the isotype switch. th1-like cells (non-classic th1) are initiated by tgf-β (stat5 signaling) and ifn-γ (stat4 signaling). th1-like cells with foxp3+ regulatory characteristics have been identified (7) . th1 helper cells and th1-like cells are closely related (54) . th1-like cells are induced in chronic th1 immune responses. thus, these cells may be related to chronic inflammation such as long-term tuberculosis or leishmania infection (55) . the effector cells of th1-like immune responses include suppressive macrophages (m2), ilc1, suppressive cd8 t cells (cd28-cd8+treg), iga1producing b-cells, inkt1 cells, and ifn-γ-/tgf-β-producing cd4 t cells (24, 40, 56) . the th1-like response induces type 4 delayed-type hypersensitivity, such as crohn's disease (26) . th9 cells are driven by il-4 (stat6 signaling) combined with tgf-β (stat5 signaling) (57) (58) (59) . thus, th9 cells are closely related to the th2 immunological pathway in parasite immunity (60) . the cells are characterized as il-9-secreting cd4 t cell. th9 cells are important under a chronic allergic condition such as asthma. thus, th9 helper cells are chronic t helper cells related to th2 immune response. the effector cells of th9 immunity include regulatory eosinophils, basophils/profibrotic mast cells (mcct, mast cell chymase, and tryptase), ilc2, il-9-producing cd4 t cells, inkt2 cells, and iga2-producing b-cells (40, 61) . th9 immune responses present as type 1 allergy, including asthma (29) . th17 cells are driven by il-6/il-1 combined with tgf-β (5). thus, th17 cells are closely related to the th22 immunological pathway against extracellular bacteria and fungi (62) . th17 cells are characterized as il-17-secreting cd4 t cells. th17 cells are important in chronic immune-complex-mediated diseases such as rheumatic arthritis. the th17 helper cell is the chronic t helper cell related to th22 immunity. tgf-β with stat5 can suppress the acute il-22-producing cells and enhance the chronic il-17-producing cells (41) . owing to the role of tgfβ in th17 immunity, regulatory il-17-producing cells have been noted. the effector cells of th17 immunity include regulatory neutrophils (n2), ilc3(ncr-), il-17-producing cd4 t cells, inkt17 cells, and iga2-producing b-cells (40, 63) . th17 immunity presents as type 3 immune-complex hypersensitivity, including ulcerative colitis or rheumatoid arthritis (42) . th3 cells are driven by il-10 and tgf-β (64, 65) . thus, th3 cells are closely related to the thαβ immunological pathway against viruses (66) . these cells also produce il-10 and tgf-β. thus, th3 helper cells are important for chronic antibody-dependent cellular cytotoxic hypersensitivity. th3 cells are the chronic helper t cells corresponding to thαβ helper cells. the th3 immune effector cells include il-13-producing regulatory nk cells (cd56 + cd16-nk2 cells), ilc10, il-10-and tgf-β-secreting cd4 t cells, suppressive cd8 t cells (cd28-cd8+ treg), inkt10 cells, and iga1-producing b-cells (34, 35, 56, 67, 68) . iga1 of th3 immune responses is produced in the serum and acts against viral protein antigens. th3 immune responses induce type 2 antibody-dependent cytotoxic hypersensitivity, including the chronic stage of systemic lupus erythematosus (sle) (69) . the summary diagram includes complete picture of the 4 × 2 + 2 immunological pathways ( table 1). the th1, th2, th22, and thαβ eradicable immune responses correspond with the th1-like, th9, th17, and th3 tolerable the author is thankful for the instructions provided by professors alan scott, louis august bourgeois, and pien-chien huang during his ph.d. study in the department of international health, johns hopkins university, bloomberg school of public health. the author is also grateful for the guidance from professors chi-huey wong and alice lin-tsing yu in his postdoctoral research at the genomics research center of academia sinica, taiwan. this manuscript has been released as a pre-print at biorxiv (70) . two types of murine helper t cell clone. i. definition according to profiles of lymphokine activities and secreted proteins human immune responses to plasmodium falciparum infection: molecular evidence for a suboptimal thalphabeta and th17 bias over ideal and effective traditional th1 immune response th3 immune responses in the progression of leprosy via molecular cross-talks of tgf-beta, ctla-4 and cbl-b ifn-alpha and il-10 induce the differentiation of human type 1 t regulatory cells interleukin 17-producing cd4+ effector t cells develop via a lineage distinct from the t helper type 1 and 2 lineages th22 cells represent a distinct human t cell subset involved in epidermal immunity and remodeling identification of t helper type 1-like, foxp3+ regulatory t cells in human autoimmune disease the development and fate of follicular helper t cells defined by an il-21 reporter mouse human dendritic cell subsets lymphoid tissue inducer cells: bridges between the ancient innate and the modern adaptive immune systems bcl6 mediates the development of t follicular helper cells tgf-beta prevents t follicular helper cell accumulation and b cell autoreactivity prolactin inhibits bcl6 expression in breast cancer through a stat5a-dependent mechanism stat5 regulates the self-renewal capacity and differentiation of human memory b cells and controls bcl-6 expression a sequence of the cis gene promoter interacts preferentially with two associated stat5a dimers: a distinct biochemical difference between stat5a and stat5b il-21 induces differentiation of human naive and memory b cells into antibody-secreting plasma cells cutting edge: il-21 is a switch factor for the production of igg1 and igg3 by human b cells development of invariant natural killer t cells cutting edge: stat1 is required for il-6-mediated bcl6 induction for early follicular helper cell differentiation cutting edge: induction of follicular homing precedes effector th cell development early th1 cell differentiation is marked by a tfh cell-like transition active tuberculosis is characterized by highly differentiated effector memory th1 cells type 2 cytotoxic t lymphocytes modulate the activity of dendritic cells toward type 2 immune responses tgfbeta signaling plays a critical role in promoting alternative macrophage activation innate lymphoid cells. innate lymphoid cells: a new paradigm in immunology th1-type responses mediate spontaneous ileitis in a novel murine model of crohn's disease host protective roles of type 2 immunity: parasite killing and tissue repair, flip sides of the same coin functional specializations of human epidermal langerhans cells and cd14+ dermal dendritic cells lungresident eosinophils represent a distinct regulatory eosinophil subset interleukin-4 promotes the development of tryptase and chymase doublepositive human mast cells accompanied by cell maturation lps promotes th2 dependent sensitisation leading to anaphylaxis in a pru p 3 mouse model. sci rep il-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice cd3/cd28-costimulated t1 and t2 subsets: differential in vivo allosensitization generates distinct gvt and gvhd effects differentiation of human nk cells into nk1 and nk2 subsets a novel il-10-producing innate lymphoid cells (ilc10) in a contact hypersensitivity mouse model interferon alpha/beta-mediated inhibition and promotion of interferon gamma: stat1 resolves a paradox interleukin 10 deficiency attenuates induction of anti-tsh receptor antibodies and hyperthyroidism in a mouse graves' model th22 cells are an important source of il-22 for host protection against enteropathogenic bacteria identification of a human helper t cell population that has abundant production of interleukin 22 and is distinct from t(h)-17, t(h)1 and t(h)2 cells innate lymphoid cells-how did we miss them? transcription factor c-maf mediates the tgf-beta-dependent suppression of il-22 production in t(h)17 cells elevated th22 cells correlated with th17 cells in patients with rheumatoid arthritis selective increases in antibody isotypes and immunoglobulin g subclass responses to secreted antigens in tuberculosis patients and healthy household contacts of the patients virally induced modulation of murine igg antibody subclasses correlation between serum igg-2 concentrations and the antibody response to bacterial polysaccharide antigens allergen-specific ige and igg4 are markers of resistance and susceptibility in a human intestinal nematode infection control of regulatory t cell development by the transcription factor foxp3 regulatory dendritic cells: there is more than just immune activation regulatory innate lymphoid cells control innate intestinal inflammation identification of regulatory foxp3+ invariant nkt cells induced by tgf-beta nonredundant roles for stat5a/b in directly regulating foxp3 cd4+ cd25+ foxp3+ regulatory t cells protect against t cell-mediated fulminant hepatitis in a tgf-beta-dependent manner in mice cd40 engagement triggers switching to iga1 and iga2 in human b cells through induction of endogenous tgf-beta: evidence for tgf-beta but not il-10-dependent direct s mu->s alpha and sequential s mu->s gamma, s gamma->s alpha dna recombination il-12 inhibits the tgf-betadependent t cell developmental programs and skews the tgf-beta-induced differentiation into a th1-like direction mononuclear cells from patients recovered from cutaneous leishmaniasis respond to leishmania major amastigote class i nuclease with a predominant th1-like response four functionally distinct populations of human effector-memory cd8+ t lymphocytes th9 cells that express the transcription factor pu.1 drive t cell-mediated colitis via il-9 receptor signaling in intestinal epithelial cells il-4 inhibits tgf-beta-induced foxp3+ t cells and, together with tgfbeta, generates il-9+ il-10+ foxp3(-) effector t cells stat6-dependent regulation of th9 development il-9 and th9 in parasite immunity human mast cell subsets-distinct functions in inflammation? lung-infiltrating t helper 17 cells as the major source of interleukin-17a production during pulmonary cryptococcus neoformans infection polarization of tumor-associated neutrophil phenotype by tgf-beta: "n1" versus "n2 il-10 and tgf-beta induce alloreactive cd4+cd25-t cells to acquire regulatory cell function antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by t(h)3/t(r)1-type cytokines il-10 and transforming growth factor-beta but not by a t(h)1 to t(h)2 shift airway memory cd4(+) t cells mediate protective immunity against emerging respiratory coronaviruses cd8+cd28-, suppressive t cells in systemic lupus erythematosus tgfbeta promotes conversion of cd16+ peripheral blood nk cells into cd16-nk cells with similarities to decidual nk cells continuous administration of anti-interleukin 10 antibodies delays onset of autoimmunity in nzb/w f1 mice a framework of all discovered immunological pathways and their roles for four specific types of pathogens and hypersensitivities the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 hu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-279105-e2zjxjox authors: lee, cheryl yi-pin; lin, raymond t. p.; renia, laurent; ng, lisa f. p. title: serological approaches for covid-19: epidemiologic perspective on surveillance and control date: 2020-04-24 journal: front immunol doi: 10.3389/fimmu.2020.00879 sha: doc_id: 279105 cord_uid: e2zjxjox since december 2019, the novel coronavirus, sars-cov-2, has garnered global attention due to its rapid transmission, which has infected more than two million people worldwide. early detection of sars-cov-2 is one of the crucial interventions to control virus spread and dissemination. molecular assays have been the gold standard to directly detect for the presence of viral genetic material in infected individuals. however, insufficient viral rna at the point of detection may lead to false negative results. as such, it is important to also employ immune-based assays to determine one's exposure to sars-cov-2, as well as to assist in the surveillance of individuals with prior exposure to sars-cov-2. within a span of 4 months, extensive studies have been done to develop serological systems to characterize the antibody profiles, as well as to identify and generate potentially neutralizing antibodies during sars-cov-2 infection. the vast diversity of novel findings has added value to coronavirus research, and a strategic consolidation is crucial to encompass the latest advances and developments. this review aims to provide a concise yet extensive collation of current immunoassays for sars-cov-2, while discussing the strengths, limitations and applications of antibody detection in sars-cov-2 research and control. the ongoing pandemic, which originates from a newly emerged coronavirus, sars-cov-2, was discovered in the city of wuhan in china's hubei province in december 2019 (1) . to date, due to rapid transmission globally, there are more than two million laboratory-confirmed human infection cases, with a few hundred thousand deaths across 210 countries and territories (https://www.who.int/emergencies/diseases/novel-coronavirus-2019/ situation-reports/). this unprecedented crisis led to a worldwide effort to rapidly characterize the immunobiology of sars-cov-2, while mitigating further spread of this deadly pathogen. sars-cov-2 is a single stranded, positive sense rna virus that belongs to the coronaviridae family of the betacoronavirus genus (2) . it has a genome size of ∼30 kilobases that encodes for multiple structural proteins comprising the spike (s), the envelope (e), the membrane (m), and the nucleocapsid (n), as well as non-structural proteins (3) (figure 1) . infection by sars-cov-2 causes an acute respiratory disease termed the coronavirus disease 2019 . the clinical manifestations of covid-19 form a spectrum, from being asymptomatic to fever with mild respiratory illness, to acute respiratory distress syndrome, and death from respiratory failure or associated complications (3) (4) (5) . as the reported incubation period varies among different patient cohorts, it is often difficult to ascertain the actual day of onset, and infected subjects who are asymptomatic or pre-symptomatic may go undetected (5) (6) (7) . early detection of sars-cov-2 infection is one of the crucial interventions to control virus transmission. with the discovery of the genome organization of sars-cov-2 viral rna, which is adapted from genbank accession number: mn908947, is characterized by sequence alignment against two representative members of the betacoronavirus genus. the entire genome sequence is ∼30 kilobases (kb) long. the virus, numerous diagnostic assays using quantitative reverse transcriptase pcr (qrt-pcr) were developed (3) . qrt-pcr is the reference standard for diagnosing infections with high sensitivity and accuracy in the acute phase of illness. sars-cov-2 viral rna has been detected in both throat and nasal swabs of infected individuals by qrt-pcr, which becomes almost undetectable by 14 days post-illness onset (pio) (or symptom onset) (8, 9) (figure 2) . apart from being costly and time consuming to perform, false negative results may arise due to improper handling of nucleic acid samples, inadequate and variable sampling resulting in insufficient viral genetic material at the point of detection (after 14 days pio), or biological variation on when viral rna is detectable by qrt-pcr (10, 15) . with the limitations of qrt-pcr, immunoassays may offer another figure 2 | schematic illustration on the window period of detection for either viral rna or antibodies in sars-cov-2-infected individuals. presence of sars-cov-2 viral rna (boxed in pink) in throat or nasal swab of patients are typically undetectable by 14 day post illness onset (pio) (8, 9) . sars-cov-2-specific antibodies (boxed in blue): igm is detectable as early as 3 days pio, and peaks between 2 and 3 weeks pio (10, 11) . igm response was still detectable after more than 1 month pio (12) . both iga and igg are present as early as 4 days pio, and peaks after 2 weeks pio in serum samples (10, 11, 13, 14) . there are currently no reports on the presence of these sars-cov-2-specific antibodies in the later phase pio, as indicated by dotted lines. this depicts the importance of serological studies to identify individuals with current or prior exposure to sars-cov-2 that went undetected, by testing for either igm, igg, or iga antibodies against sars-cov-2. illustration was created using biorender. avenue to reduce undiagnosed cases, with the advantage that rapid test formats may deliver results in a relatively shorter time and lower cost (10). immunoassays are another diagnostic approach that can provide information on both active viral infections and past exposures (figure 2 ). to date, many commercial companies and research institutes have developed serological assays to detect sars-cov-2 antibodies from patient serum or plasma samples (16, 17) . closely related to another pathogen, severe acute respiratory syndrome coronavirus (sars-cov), these assays mainly target immunogenic coronavirus proteins: s protein, which is the most exposed viral protein, and n protein, which is abundantly expressed during infection (3, 14, 18) . in addition, the receptor-binding domain (rbd), which is located along the s protein, is also a target of interest to detect the presence of sars-cov-2-specific antibodies (19, 20) . in recent pre-prints deposited in medxriv and bioxriv, it was shown that both anti-sars-cov-2-igm and igg levels increase gradually along with infection phases, with igm being detected as early as 3 days pio, which peaks between two to three weeks pio (10, 11) . one study has reported that sars-cov-2-specific igm is still present in the serum after 1 month pio (12) . sars-cov-2-specific igg antibodies, on the other hand, can be present as early as 4 days pio, and peak after 17 days pio (10, 11) (figure 2) . these observations are similar to what was previously reported during a sars-cov infection (21) . however, interestingly, one study demonstrated that longitudinal profiling of both antibodies in a population of 63 covid-19 patients showed no specific chronological order in terms of igm and igg seroconversion (10) , which was also observed in patients infected with sars-cov and another human coronavirus, middle east respiratory syndrome coronavirus (mers-cov) (22, 23) . in addition, there seems to be no correlation between seroconversion rates with age, gender or time of hospitalization (10) . these findings on sars-cov-2-specific antibodies seroconversion against the s viral protein suggest the importance to test for both igm and igg antibodies to confirm a positive infection. expectedly, similar to what was reported for sars-cov and mers-cov, both igm and igg levels seems to be correlated with disease severity, with a higher level of both antibodies present in patients with more severe sars-cov-2 infection (10, 11, 14, (24) (25) (26) . in contrast to other flu-like infections such as influenza, instead of igg1, igg3 appears to be the dominant igg subtype during sars-cov-2 infection (13, 27, 28) . as a majority of the human population has prior exposure to endemic human coronavirus infections including alphacoronaviruses (229e and nl63), and other betacoronaviruses (oc42 and hku1) (29) , it is crucial to validate the specificity and sensitivity of current immunoassays against sars-cov-2 to avoid false positive outcomes. within the s protein antigen, cross-reactivity was observed when samples were tested against sars-cov s and s1 subunit proteins, and to a smaller extent, with mers-cov s protein ( table 1) . interestingly, there was no cross-reactivity with the s1 subunit of mers-cov (14) . the high level of crossreactivity between sars-cov and sars-cov-2 can be attributed to the high degree of genetic homology (3, 14, 19) . furthermore, detailed analysis revealed a highly conserved s2 subunit domain across coronaviruses, which may explain for the cross-reactivity observed with only the s protein of mers-cov, and not with the s1 subunit (14, 19) . these data suggest that using an s1 subunit-based immunoassay may be more specific than the entire s antigen for diagnosing sars-cov-2 infections. another immunogenic target, the rbd, which lies along the s protein is usually the target of many neutralizing antibodies against sars-cov (30) . a substantial level of cross-reactivity by sars-cov rbd-induced antibodies to sars-cov-2 rbd was described ( table 1 ) (20) . of clinical relevance, these antibodies were also able to cross-neutralize sars-cov-2 pseudovirus infection, signifying the potential of an immunotherapy-based treatment (20) . while one non-peer reviewed study has shown that rbd-based serological assays are more sensitive than s1 subunit-based assays in identifying antibodies in mild covid-19 patients (14), other non-peer-reviewed studies have described a lower degree of antibody response to the rbd as compared to full-length s protein, plausibly reflecting the larger number of epitopes present on the larger s antigen (13, 19 ). due to a high level of similarity of 90% between sars-cov and sars-cov-2 n proteins, the n antigen of sars-cov was also used for serological detection of sars-cov-2-specific antibodies ( table 1 ) (14) . these n-based assays were reported to be more sensitive than s1 subunit-based tests (14) . the use of sars-cov antigens to diagnose sars-cov-2 infections may be reliable, given that sars-cov has not circulated in the human population since 2004 (3). in addition, an earlier report has demonstrated waning of sars-cov-specific antibodies, therefore being undetectable in 91% of patient serum samples after 6 years (31) . since respiratory diseases are the hallmark of coronavirus infections, which activate mucosal immunity, several studies have exploited the detection of iga to diagnose sars-cov-2 infection in patients (table 1 ) (13, 14) . although a strong iga response was also detected in covid-19 patients where peak seroconversion was achieved by two weeks pio (figure 2) , igabased immunoassay has been hypothesized to be less specific than igg-based elisa due to cross-reactivity with serum samples from patients infected by other coronaviruses (14) . with the availability of immunoassays utilizing various coronavirus structural proteins, the use of more than one different antigen-based serological approach may be essential to establish a true positive sars-cov-2 infection. in addition, frontiers in immunology | www.frontiersin.org the use of saliva samples and other bodily fluid swabs as a less invasive alternative, which have been done for other viral infections including hiv and measles, should also be explored for serological testing of sars-cov-2 infections (32, 33). apart from using immunoassays for the early detection of sars-cov-2 infected individuals, it is also critical to determine the regions where sars-cov-2-specific antibodies bind to help guide vaccine designs. using sars-cov-derived b-cell epitopes that have been experimentally identified from positive b-cell assays (34), 49 out of 298 linear b-cell epitopes have an identical match with sars-cov-2 protein sequences without any mutations (3) . notably, majority of these matches were located at both the s and n viral antigens, with only 4 from the m protein, and none in the e protein (3). on the other hand, 6 conformational b-cell epitopes identified from the same database were located on the s antigen. however, unlike the linear epitopes, none of these mapped identically to the sars-cov-2 protein (3). further mapping the residues of linear b-cell epitopes onto available sars-cov s protein structure revealed several regions on the s2 subunit that may allow cross-neutralization of both sars-cov and sars-cov-2 (3, 35) . in contrast, conformational b-cell epitopes mapped onto the s1 subunit, resulting in very few identical residues within sars-cov and sars-cov-2 (3). these findings indicate that sars-cov-specific antibodies targeting these discontinuous regions may not be able to cross-react with sars-cov-2 (3, 36) . as these regions are computationally predicted, serological studies using patient samples are necessary to validate the importance of these regions for serology and in controlling sars-cov-2 infection. it also remains imperative to identify other sars-cov antibodies that may recognize the conformational epitopes of sars-cov-2 s protein, which can greatly reduce the amount of time needed to develop novel neutralizing antibodies. the findings derived from serological assays can provide valuable information that would help to support the diagnosis, treatment, and prevention of sars-cov-2 infections. characterization of antibody profiles suggested that any suspected individuals with undetectable antibody levels against sars-cov-2 after 20 days pio may be a true negative case, since both anti-sars-cov-2 igm or igg seroconversion should have already occurred (10, 11) . however, these findings may be limited to the relatively small sample size (<300 patients) and may require further validation with a larger cohort. in order to reinforce diagnosis, it would be advisable to perform multiple assays against different viral antigen. in addition, the information of antibody seroconversion is crucial in determining the optimal timepoints to collect serum or plasma samples for immunoassay screening, as well as obtaining peripheral blood b cells for the generation of therapeutic monoclonal antibodies (37) . currently, in order to rapidly generate neutralizing monoclonal antibodies against sars-cov-2, repurposing of existing sars-cov-specific antibodies was demonstrated. to date, two human sars-cov-specific antibodies, cr3022 and 47d11, have been shown to recognize sars-cov-2 (38, 39) . cr3022 recognizes an epitope along the rbd of sars-cov-2, which differs largely at the c-terminus residues to the rbd of sars-cov (38) . unfortunately, this variation in sequence impacted the ability of cr3022 to crossneutralize sars-cov-2. monoclonal antibody 47d11, on the other hand, targets the rbd along the s1 subunit of both sars-cov and sars-cov-2 with similar affinities, thereby enabling cross-neutralization against sars-cov-2 infection (39) . while combinatory therapy has exhibited a stronger neutralization capability against sars-cov infection (40), a cocktail antibody approach for sars-cov-2 could be explored. surprisingly, reports on antibodies against the coronavirus e protein are scarce, possibly due to it being the smallest protein. however, the e antigen is involved in viral assembly, release of virions, as well as virus pathogenesis (41) . it was demonstrated that recombinant coronaviruses lacking the e protein displayed significantly reduced viral titers, impaired viral maturation and produced avirulent virus progenies, suggesting a similar importance of e protein during sars-cov-2 infection (42, 43) . thus, it would be worthwhile to identify or generate neutralizing antibodies that are specific against the viral e protein. during the course of an epidemic, one of the main challenges is the identification of asymptomatic infection. since these individuals do not present any distinguishable symptoms, they could be the major source of transmission (10) . immunoassays may be able to detect mildly infected cases (14) , which is important to ascertain the extent of community spread. while it is fast, robust and easy to perform, there are several limitations to serological assays. one of the major setbacks of immunoassays is the inability to detect the presence of infection during the early stage of disease, as antibodies take several days to be generated after exposure to foreign material (44) . as such, a recent infection may provide false negative results during serological testing. thus, the use of rt-pcr may be more suitable to diagnose an early acute sars-cov-2 infection. furthermore, due to the unique genetic makeup of each individual, there would be an inherent variability of the antibody response (45) . this could possibly explain the difference in antibody profiles elicited among individuals infected with sars-cov-2 (10). cross-reactivity could potentially be a limitation of immunoassays as it severely impacts the specificity and sensitivity of the test. although the phylogenetically closest coronavirus, sars-cov, has not been reported to be circulating in the human population since 2004 (3), other endemic human coronaviruses may still pose a problem to accurately diagnose patients with true sars-cov-2 infection. while a recent study has demonstrated negligible cross-reactivity from human coronavirus, nl63, to sars-cov-2 (13), validation with other human coronaviruses remains to be investigated. in addition, prior findings on the s protein sequence and neutralization antigenicity of other coronaviruses suggest that antibodies neutralizing clinical human coronavirus isolates may not have the same degree of cross-reactivity with laboratory strains of human coronaviruses, thereby affecting the sensitivity of immunoassays (46) (47) (48) . given the rapid increase in the number of confirmed covid-19 cases coupled with the shortage in test kits to meet rising demands, decentralized point-of-care tests (poct) may be another alternative to facilitate sars-cov-2 diagnosis. such tests include lateral flow assay (lfa), which is a paper-based platform for the detection and quantification of analytes in complex mixtures (49) . to design lfa for sars-cov-2 detection, an antibody specific to the viral antigen, or a viral antigen that is detectable by patient serum or plasma samples can be immobilized on a nitrocellulose membrane. detection of binding between the analyte and capture antibody by a detector antibody will give rise to a colored line, closely resembling home pregnancy kits (50) . poct is advantageous as it is usually designed to be rapid, sensitive, highly accessible, and easily performed, requiring only a small amount of sample (50) . meanwhile, several hundreds of candidate pocts are being evaluated for their applicability toward identifying sars-cov-2-infected individuals (50) . however, pocts can't replace rt-pcr and it is crucial that these developing tests are rigorously assessed prior to use. it is important to note that wrong use and interpretation could lead to disastrous public health consequences (51) . rapid development of diagnostic tools and immune-based assays are important early interventions against the ongoing sars-cov-2 pandemic. the availability of serological assays that target a diverse range of viral antigen has no doubt assisted in the accurate diagnosis of covid-19 patients. essentially, data generated through serological studies can greatly aid in supplementing the results from qrt-pcr, as well as contribute to seroepidemiology, which has been shown to help in the design of virus elimination programs (52) . moving forward, this extensive collation of the current immunoassays against sars-cov-2 will provide insights toward monoclonal antibodies discovery and characterization for the development of a sars-cov-2 vaccine. ln and lr conceived the presented idea. cl wrote the manuscript and prepared the figures. ln, lr, and rl revised the manuscript. all authors approved this manuscript for publication. this work was supported by core research grants provided to singapore immunology network by the biomedical research council (bmrc), and by the a * ccelerate gap-funded project (accl/19-gap064-r20h-h). new sars-like virus in china triggers alarm novel coronavirus 2019-ncov: prevalence, biological and clinical characteristics comparison with sars-cov and mers-cov preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical characteristics of coronavirus disease 2019 in china early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia viral load of sars-cov-2 in clinical samples sars-cov-2 viral load in upper respiratory specimens of infected patients antibody responses to sars-cov-2 in covid-19 patients: the perspective application of serological tests in clinical practice. medrxiv immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19. medrxiv profile of specific antibodies to sars-cov-2: the first report a serological assay to detect sars-cov-2 seroconversion in humans. medrxiv sars-cov-2 specific antibody responses in covid-19 patients chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing sars-cov-2 serology testing ramping up to address next stages of pandemic covid-19) update: fda expedites review of diagnostic tests to combat covid-19 identification of an epitope of sars-coronavirus nucleocapsid protein crossreactive antibody response between sars-cov-2 and sars-cov infections. biorxiv characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine immunofluorescence assay for serologic diagnosis of sars chronological evolution of igm, iga, igg and neutralisation antibodies after infection with sars-associated coronavirus viral shedding and antibody response in 37 patients with middle east respiratory syndrome coronavirus infection anti-sars-cov igg response in relation to disease severity of severe acute respiratory syndrome antibody response and disease severity in healthcare worker mers survivors mers-cov antibody responses 1 year after symptom onset, south korea age dependence and isotype specificity of influenza virus hemagglutinin stalk-reactive antibodies in humans analysis of anti-influenza virus neuraminidase antibodies in children, adults, and the elderly by elisa and enzyme inhibition: evidence for original antigenic sin sars-cov-2 vaccines: status report neutralizing epitopes of the sars-cov s-protein cluster independent of repertoire, antigen structure or mab technology lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study oral fluid for the serological and molecular diagnosis of measles detection of human immunodeficiency virus using oral mucosal transudate by rapid test vipr: an open bioinformatics database and analysis resource for virology research structure, function, and antigenicity of the sars-cov-2 spike glycoprotein cryo-em structure of the 2019-ncov spike in the prefusion conformation cov-2: virus dynamics and host response potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody. biorxiv a human monoclonal antibody blocking sars-cov-2 infection. biorxiv human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing coronavirus envelope protein: current knowledge a severe acute respiratory syndrome coronavirus that lacks the e gene is attenuated in vitro and in vivo absence of e protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway the immune system basic problems of serological laboratory diagnosis examination of seroprevalence of coronavirus hku1 infection with s protein-based elisa and neutralization assay against viral spike pseudotyped virus differences in neutralizing antigenicity between laboratory and clinical isolates of hcov-229e isolated in japan in 2004-2008 depend on the s1 region sequence of the spike protein antibodies to coronaviruses are higher in older compared with younger adults and binding antibodies are more sensitive than neutralizing antibodies in identifying coronavirus-associated illnesses lateral flow assays portable tests come online to curb coronavirus pandemic statement on point-of-care serology testing for sars-cov-2 (the virus that causes covid-19). (2020) seroepidemiology: an underused tool for designing and monitoring vaccination programmes in low-and middle-income countries diagrams are created with biorender. the authors wish to thank drs. siew-wai fong and yi-hao chan for critical comments of this manuscript. key: cord-260452-js4nr4d8 authors: yu, junyang; wu, yuzhang; wang, jingxue title: activation and role of nacht, lrr, and pyd domains-containing protein 3 inflammasome in rna viral infection date: 2017-10-31 journal: front immunol doi: 10.3389/fimmu.2017.01420 sha: doc_id: 260452 cord_uid: js4nr4d8 nacht, lrr, and pyd domains-containing protein 3 (nlrp3) inflammasome activation and effects during ribonucleic acid (rna) viral infection are the focus of a wide range of research currently. both the pathogen-associated molecule pattern derived from virions and intracellular stress molecules involved in the process of viral infection lead to activation of the nlrp3 inflammasome, which in turn triggers inflammatory responses for antiviral defense and tissue healing. however, aberrant activation of the nlrp3 inflammasome can instead support viral pathogenesis and promote disease progression. here, we summarize and expound upon the recent literature describing the molecular mechanisms underlying the activation and effects of the nlrp3 inflammasome in rna viral infection to highlight how it provides protection against rna viral infection. the ribonucleic acid (rna) virus has single-stranded (ss)rna or double-stranded (ds)rna as its genetic material. infections with rna viruses are responsible for a variety of diseases are significant threats to public health, including influenza, hepatitis, viral encephalitis, and autoimmune deficiency syndrome, to name a few of the most prominent. host antiviral defense mechanisms are critical for clearing rna viral infections and controlling the manifested diseases. the initiation of antiviral defense in response to rna viruses involves interferon (ifn) response and inflammasome activation. the rna viral infection usually leads to activation of the nacht, lrr, and pyd domainscontaining protein 3 (nlrp3) inflammasome, which is the intracellular multiprotein complex formed upon sensing pathogen-associated molecule patterns and/or damage-associated molecule patterns (1) . the inflammasome response is critical for the innate immune response's ability to initiate innate and adaptive immunity pathways during rna viral infections (2) . the nlrp3 inflammasome has been extensively investigated. its major components include pro-caspase-1 and apoptosis-associated speck-like protein containing a card (asc), in addition to the nlrp3 molecules. upon sensing activating signals, the nlrp3 oligomerize to generate a caspase-1-activating scaffold, resulting in autocleavage, and activation of pro-caspase-1. the activated caspase-1 then shears pro-interleukin (il)-1β and pro-il-18 into il-1β and il-18, leading to production of their functional forms and release from cells to mediate protective (or sometimes detrimental) inflammatory responses. the activated caspase-1, however, can also promote caspase-1-dependent pyroptosis, a type of inflammatory programmed cell death; although, the consequence of pyroptosis induced by the nlrp3 inflammasome remains to be fully elucidated in rna viral infection (3) . table 1 . this review will focus on recent advances in our knowledge of the activation mechanisms and functions of the nlrp3 inflammasome in response to such rna viral infections, discussing the related protective, and/or detrimental mechanisms that may represent manipulable targets for prevention and/or treatment. priming mechanisms of the nlrp3 inflammasome in rna viral infections nacht, lrr, and pyd domains-containing protein 3 inflammasome activation requires two types of signals. signal 1 primes the nlrp3 inflammasome and signal 2 initiates the sequence of its assembly, activation of the pro-caspase-1, and cleavage of pro-il-1β and pro-il-18. during rna viral infections, signal 1 is usually derived from the signals evoked upon pathogen recognition by toll-like receptors (tlrs) (6) and retinoic acid-inducible gene-i (rig-i)-like receptors (41) . recent research has shown that signal 1 triggering of the nlrp3 inflammasome involves transcriptiondependent and post-translation-dependent priming. the transcription-dependent priming is executed by activation of nuclear factor (nf)-κb and ifn-regulatory factor transcription factors, and subsequent up-regulation of transcription of genes encoding the major inflammasome components through the classical tlr signaling pathway: the myd88-mediated and trif-mediated pathway. the specific role of tlr7 was shown by the reduced pro-il-1β transcription in bone marrow-derived macrophages from tlr7 −/− mice in response to 24 h of iav infection (6) . another in vivo study of the iav infection model provided evidence that commensal bacteria lipopolysaccharide can provide priming signal 1 (42) . investigations of vsv infection showed that rig-i promotes the synthesis of pro-il-1β through engagement by the viral 5′-triphosphate rna (3prna). moreover, bone marrow-derived cells lacking rig-i, as well as the downstream molecules mavs and card9, showed reduced synthesis of pro-il-1β but normal caspase-1 p10 subunits when stimulated with vsv and 3prna (41) , suggesting that the rig-i/mavs/card9 signaling pathway provides the signal 1 in vsv infection. post-translation-dependent priming occurs upon the simultaneous engagement of tlrs and nod-like receptors with their ligands, even causing rapid activation of the nlrp3 inflammasome (within 15 min) (43, 44) . viral rna (vrna) moleculeinduced nlrp3 inflammasome activation also comes about by post-translational priming, but involving the rip1/caspase 8/rip3 signaling pathway (29, 45) , which may ultimately result in the deubiquitination of nlrp3 protein by brcc3 (46, 47) . rip3-deficient mice showed reduced caspase-1 activation and il-1β production following infection with vsv, sendai virus, or iav (29) . bone marrow-derived macrophages and mice lacking rip1 both displayed substantial reduction in caspase-1 cleavage and levels of il-1β and il-18 following challenge with iav (8, 29) . in various rna viral infection studies, the rip1/ rip3-mediated signaling pathway has been demonstrated to provide the endogenous signal 2 for the nlrp3 inflammasome, being derived from reactive oxygen species (ros) production by mitochondrial fission initiated by the rip1/rip3/drp pathway (29, 45, 48) . signal 2 is responsive to a wide range of stimuli [i.e., nlrp3 agonists, such as adenosine triphosphate (atp), silica, alum, and ultraviolet radiation]. however, the spectrum of different structures represented by these varied and distinctive agonists indicates that nlrp3 does not simply bind directly to each but instead relies on a common intracellular signal transmission pathway, triggered by the agonists themselves, for activation of the nlrp3 inflammasome. to date, five major signal mechanisms have been proposed as those responsible for or contributing to the nlrp3 inflammasome activation (1, 49) . in rna viral infections, in particular, viroporins and some viral functional proteins act as the stimuli for signal 2 (as shown in table 1) . viroporins are a group of virus-encoded proteins that enhance permeability of host cell membrane (through interactions with the lipid bilayer) to promote release of viral particles from cells (50) , making them a critical component of virus replication and virion release in the host system. the viroporin effect on permeability also modifies the cell's ability to regulate ion passage, disrupting ion homeostasis (51) . several viroporins, such as the iav m2 protein (6), rsv sh protein (15) , hcv p7 protein (21), sars-cov e protein (25), emcv 2b protein (27) , and rhinovirus 2b protein (32) , have been reported to activate the nlrp3 inflammasome by disturbing intracellular ionic concentrations, particularly through potassium efflux, calcium flux, and ph alteration. in the viroporin-induced nlrp3 activation itself, the host-encoded nek7 protein may contribute to the nlrp3 inflammasome assembly and activation via formation of the nlrp3-nek7 complex (52) . among the other viral proteins involved in nlrp3 inflammasome activation are iav pb1-f2 (a small protein encoded by an alternate + 1 open reading frame in the viral pb1 gene) (7) and ev71 3d protein (an rna-dependent rna polymerase) (34) . pb1-f2 activates the nlrp3 inflammasome through mitochondrial ros production, which could be a major mechanism of pathogenic inflammation induced by highly pathogenic iav strains (53) . the zbp1 protein can interact with rip3 upon sensing the viral protein pb1, and contributes to nlrp3 inflammasome activation in iav but not in vsv infection through post-translational regulation (8) . in contrast, the ev71 3d protein can directly bind to nlrp3 and asc, and promotes nlrp3 inflammasome assembly and activation (34) . plasma vrna, another common agonist for nlrp3, may bind with nlrp3 and act to subsequently activate it. although nlrp3 contains a nucleotide-binding domain (54), rna-sensing molecules have been reported to participate in the process by which vrna activates the nlrp3 inflammasome; these include the dexd/h-box helicase (dhx) family members (14, 41, 55, 56) and the 2′,5′-oligoadenylate synthetase (oas, or 2-5a)/rnase l system (30) . however, some debate exists as to the exact role of dhx33, in particular, in vsv-induced nlrp3 inflammasome activation (29) . the dhx family members contain some domains required for atp binding, hydrolysis, and nucleic acid binding and unwinding, which usually acts as a cytosolic rna sensor and triggers type i ifn response. dhx33, ddx19a, and ddx58 have been reported to regulate nlrp3 inflammasome activation. dhx33 was shown to combine directly with vrna to bind to nlrp3 through the dead domain of dhx33 and the nacht domain of nlrp3, forming a dhx33/nlrp3/asc inflammasome complex to promote nlrp3 inflammasome activation (14) . in that same study, down-regulation of dhx33 expression led to a substantial reduction in cleavage of caspase-1 and secretion of il-18 and il-1β from the human monocyte cell line, thp-1, upon stimulation with viral dsrna purified from reovirus, and produced by rsv during the replication process (14) . dhx33 has also been demonstrated to contribute to nlrp3 inflammasome activation and il-1β secretion during vsv and iav infections (30) , the process of which was found to be dependent upon the oas/rnase l system induced by ifn. oass recognize cytosolic viral dsrna and subsequently trigger synthesis of 2-5a in the presence of three atp molecules. then, rnase l is activated by 2-5a and cleaves intracellular vrna. these rnase l-mediated rna cleavage products will directly interact with dhx33 and induce a dhx33/mavs/nlrp3 complex to trigger assembly of the nlrp3 inflammasome. another study demonstrated that ddx19a binds porcine reproductive and respiration syndrome virus (prrsv) genomic rna through its atp-binding domain and c-terminal domain and directly interacts with the nlrp3 inflammasome in highly pathogenic-prrsv-infected primary porcine alveolar macrophages, dependent on the ddx19a atp-binding domain, and figure 1 | activation and effects of nacht, lrr, and pyd domainscontaining protein 3 (nlrp3) inflammasome in response to ribonucleic acid (rna) virus infections. viroporin, viral nucleic acids, and some virus-encoded functional proteins are able to activate the nlrp3 inflammasome. viroporin-mediated ion concentration change, virus replication-produced reactive oxygen species, and even viral protein direct binding promote assembly of the nlrp3 inflammasome. the cytokines interleukin (il)-1 and il-18 contain extensive downstream effector molecules and effector cells, which ultimately orchestrate innate and adaptive immunity, leading to antiviral defense, and/or inflammatory injury during rna virus infections. the nlrp3 nacht and lrr domains. knockdown of ddx19a expression led to reductions in both il-1β secretion and virus titers in the prrsv-infected porcine alveolar macrophages (56) . thus, ddx19a appears to be required for both antiviral defense and nlrp3 inflammasome activation. rig-i (ddx58) is a key intracellular sensor for cytosol ssrna and provides the transcriptional priming signal for nlrp3 inflammasome during infections with vsv (41) and iav (55) , upon recognition of the vrna. however, in primary respiratory epithelial cells, iav infection promotes rig-i binding with asc and caspase-1, implying that rig-i contributes to inflammasome assembly (55) . during the process of rna viral infection, nlrp3 inflammasome activation contributes to either enhancing antiviral defense and tissue healing, or inflammatory pathological injury, which depends mainly on the time point and extent of nlrp3 activation. the appropriate and early phase activation of the nlrp3 inflammasome in some rna viral infection systems, such those of iav, encephalomyocarditis virus, and west nile virus (wnv) (shown in table 1 ), usually provide protection, thereby decreasing mortality and viral load, as shown in virus-infected mouse models (4, 9, 16, 57) . in rhesus monkeys, neither nlrp3 inflammasome activation nor ifn response can be established successfully within 24 h following challenge with the simian immunodeficiency virus, which may be the key reason underlying the observation of viral dissemination occurring rapidly from the inoculation site to a distal site (within the first day of infection) (58) . reciprocally, when using hiv-1-specific broadly neutralizing antibodies, pgt121 is capable of inducing innate immune responses such as nlrp3 inflammasome activation in vrna-positive tissues within 24 h; in addition, viral replication is reduced in distal tissues significantly (59) . these findings imply the early nlrp3 inflammasome activation as well as ifn response play critical roles in establishment of effective antiviral defense. similarly, the administration of mc9550 (a specific nlrp3 inhibitor) on days 1, 3, and 5 but not days 3-5 following iav challenge also resulted in accelerated weight loss and mortality in iav-infected mice (9) . at 2 weeks after challenge with iav, the mortality rates among nlrp3 −/− , caspase-1 −/− (4, 5), and il-1ri −/− (57) mice are significantly higher (by 60-80%) than for the wild-type counterparts. thus, proper nlrp3 inflammasome activation in the early phase of rna viral infection protects the infected host. in contrast, if activated at a later phase or over-activated, the nlrp3 inflammasome response to rna virus results in inflammatory injury. production of il-1β and il-18, and pyroptosis are the critical and fundamental reactions directly induced by activated nlrp3 inflammasome (figure 1) . il-1β and il-18 serve to activate myriad downstream cell responses, and orchestrate innate and adaptive immunity through myd88/irak4/traf6-mediated nf-κb signaling and the jnk/p38 mitogen-activated protein kinase pathways (60-63), which may represent key events for the nlrp3 inflammasome-dependent antiviral defense. pyroptosis is a pro-inflammatory caspase-dependent programed cell death (64). nlrp3 inflammasome-dependent pyroptosis has been described in hcv-infected hepatoma cells (65) , dengue virus (dv)-infected monocytes (66) , and hiv-infected podocytes (10) . the data to date suggest that pyroptosis in viral infection leads to host injury and pathogenesis. in mice, iav-induced nlrp3 inflammasome activation initiates protective inflammation by recruitment of monocytes and neutrophils to the lung (as noted in the bronchoalveolar lavage fluid), which occurs within 3-7 days of the iav infection, and the subsequent promotion of secretion of various cytokines, such as tumor necrosis factor (tnf)-α, ifn-α, and mip-2α (4, 5) . furthermore, the impaired il-1 response by il-1ri deficiency results in impaired production of immunoglobulin m, reduced recruitment of cd4 + t cells in the bronchoalveolar lavage fluid (57) , and impaired priming of cd8 + t cells in the lung and draining mesenteric lymph nodes through the intervention of cd11b lo cd103 + dendritic cell migration and maturation (67) . in a recent study, il-18 was confirmed to be required for ifn-γ production in cd161 + vα7.2 + mucosal-associated invariant t cells, which has been observed clinically to be increased in recovered but not succumbed patients hospitalized with avian h7n9 influenza pneumonia and which implies a protective role in influenza virus infection (68) . in wnv infected mice, the il-1β produced by cd45 + cd11b + infiltrating t cells promotes the wnv-specific cd8 + t cell entry to the central neuronal system nlrp3 inflammasome induced by rna virus frontiers in immunology | www.frontiersin.org october 2017 | volume 8 | article 1420 via regulation of cxcl12-mediated t cell adhesion, eventually controlling the viral infection (16, 17) . in bv-2 mouse microglia cells infected by japanese encephalitis virus, the nlrp3 inflammasome induces production of il-1β and il-18 rapidly (within 3 h of exposure) and of tnf-α, ccl2, and il-6 later (within 6 h after exposure) (40) ; the findings suggest that the nlrp3dependent protective inflammatory response is a very early phase innate immune response against rna viral infection. in addition to initiating protective inflammation to regulate and orchestrate innate and adaptive immunity, the nlrp3 inflammasome also promotes tissue healing and reduces respiratory epithelial necrosis, which contributes to enhancing host disease tolerance (5, 69) . thus, the nlrp3 inflammasome provides protective roles through enhancing host tolerance capacity and orchestrating innate immunity and adaptive immunity during rna viral infections. aberrant nlrp3 inflammasome activation usually results in progression of viral pathogenesis and the manifested disease in host, such as the robust nlrp3 activation induced by pb1-f2 during the iav infection (7). the major pathogenic mechanism associated with pb1-f2-induced aberrant nlrp3 inflammasome activation involves excessive neutrophil influx (7, 53) . the nlrp3 activation induced by hcv promotes chronic intrahepatic inflammation in macrophages (18) and interrupts cellular lipid metabolism (20) in hcv-infected hepatoma cells, both of which contribute to chronic liver injury and liver disease progression. in hiv-infected microglia, nlrp3 inflammasome activation is involved in the occurrence of chronic neuroinflammation (12) . in addition, nlrp3-dependent pyroptosis during rna viral infection also supports the pathogenesis of virus infection diseases. hiv-induced podocyte pyroptosis could be associated with hiv-associated nephropathy (10) . nlrp3 inflammasome-mediated pyroptosis is also observed in dv-infected monocytes (66) and in hcv-infected and bystander hepatoma cells (65) , and could be associated with viral pathogenesis ( table 1) . unlike rna viruses, the dna viruses, which have a large dna genome, can encode viral homologs of some complex intracellular inflammasome-regulating molecules to regulate inflammasome activity; an example of this is the pyrin-only proteins and bcl-2 encoded by poxvirus (70, 71) . the rna viruses, in contrast, have evolved some simple yet effective strategies to inhibit activation of the nlrp3 inflammasome and evade host immunity. specifically, the proteases 3c and 2a of ev71 are able to cleave nlrp3 at the g493-l494 or q225-g226 junction (34) . furthermore, the protease 3c of ev71 is capable of degrading gasdermin d, a protein critical to the induction of pyroptosis (72) , and host failure to inhibit ev71 replication (73) . the ns1 protein of influenza virus can bind with nlrp3 directly, thereby inhibiting assembly of the nlrp3-asc-caspase-1 complex and secretion of il-1β; this inhibition is dependent on rna and the trim-25 domain of the ns1 protein (74) . the measles virus v protein also targets nlrp3, probably serving to interfere with the nlrp3 inflammasome assembly and ultimately facilitating escape from the host immune response (75) . finally, the non-structural protein 11 encoded by prrsv also antagonizes the nlrp3 inflammasome effect in the macrophages, through decreasing expression of pro-il-1β (76). nacht, lrr, and pyd domains-containing protein 3 inflammasome activation in response to rna virus is a fundamental innate immune response as well as an ifn response, which usually contributes to antivirus defense, although aberrant and inappropriate nlrp3 inflammasome response still results in the immunopathology and exaggeration of infectious disease, especially in some persistent virus infections. the activation itself is induced by different signals and probably occurs in different phases of the viral infection, triggering inflammatory response (appropriate/protective or inappropriate/detrimental), and affecting outcome of the viral infection substantially. thus, the proper regulation of nlrp3 inflammasome activation with specific nlrp3 inhibitors may have therapeutic benefit in controlling virus-related diseases and in clearance of the infecting virus. jy collecting literatures and drafting manuscript. jw design of the work, collecting literatures, drafting, and revising manuscript. yw discussing literatures, design of the work, and revising manuscript. this work was supported by grants from the national natural science foundation of china (grant no. 31670899 and 31070796) and the natural science foundation of chongqing (grant no. cstc, 2011bb5029). the inflammasomes inflammasome control of viral infection viruses and the diversity of cell death the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the intracellular sensor nlrp3 mediates key innate and healing responses to influenza a virus via the regulation of caspase-1 frontiers in immunology | www.frontiersin.org october influenza virus activates inflammasomes via its intracellular m2 ion channel activation of the nlrp3 inflammasome by iav virulence protein pb1-f2 contributes to severe pathophysiology and disease zbp1/dai is an innate sensor of influenza virus triggering the nlrp3 inflammasome and programmed cell death pathways reassessing the role of the nlrp3 inflammasome during pathogenic influenza a virus infection via temporal inhibition hiv promotes nlrp3 inflammasome complex activation in murine hiv-associated nephropathy hiv-1 tat primes and activates microglial nlrp3 inflammasome-mediated neuroinflammation hiv-1 viral protein r activates nlrp3 inflammasome in microglia: implications for hiv-1 associated neuroinflammation interleukin 18 coexpression during respiratory syncytial virus infection results in enhanced disease mediated by natural killer cells the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation il-1beta signaling promotes cns-intrinsic immune control of west nile virus infection il-1r1 signaling regulates cxcl12-mediated t cell localization and fate within the central nervous system during west nile virus encephalitis il-1beta production through the nlrp3 inflammasome by hepatic macrophages links hepatitis c virus infection with liver inflammation and disease hcv genomic rna activates the nlrp3 inflammasome in human myeloid cells the hepatitis c virus-induced nlrp3 inflammasome activates the sterol regulatory element-binding protein (srebp) and regulates lipid metabolism the p7 viroporin of the hepatitis c virus contributes to liver inflammation by stimulating production of interleukin-1beta platelets mediate increased endothelium permeability in dengue through nlrp3-inflammasome activation resistance of interleukin-1beta-deficient mice to fatal sindbis virus encephalitis the inflammatory cytokine, interleukin-1 beta, mediates loss of astroglial glutamate transport and drives excitotoxic motor neuron injury in the spinal cord during acute viral encephalomyelitis effect of interleukin-18 on viral myocarditis: enhancement of interferon-gamma and natural killer cell activity encephalomyocarditis virus viroporin 2b activates nlrp3 inflammasome the nlrp3 inflammasome detects encephalomyocarditis virus and vesicular stomatitis virus infection rna viruses promote activation of the nlrp3 inflammasome through a rip1-rip3-drp1 signaling pathway rnase l activates the nlrp3 inflammasome during viral infections rhinovirus infection of primary cultures of human tracheal epithelium: role of icam-1 and il-1beta rhinovirus-induced calcium flux triggers nlrp3 and nlrc5 activation in bronchial cells reciprocal regulation between enterovirus 71 and the nlrp3 inflammasome ev71 3d protein binds with nlrp3 and enhances the assembly of inflammasome complex stimulation of host-defense mechanism with synthetic adjuvants and recombinant cytokines against viral infection in mice critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna zika virus induces inflammasome activation in the glial cell line u87-mg involvement of nlrp3 inflammasome in cvb3-induced viral myocarditis japanese encephalitis virus differentially modulates the induction of multiple pro-inflammatory mediators in human astrocytoma and astroglioma cell-lines nlrp3 inflammasome: key mediator of neuroinflammation in murine japanese encephalitis recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production microbiota regulates immune defense against respiratory tract influenza a virus infection cutting edge: tlr signaling licenses irak1 for rapid activation of the nlrp3 inflammasome irak-1 bypasses priming and directly links tlrs to rapid nlrp3 inflammasome activation caspase-8 scaffolding function and mlkl regulate nlrp3 inflammasome activation downstream of tlr3 non-transcriptional priming and deubiquitination regulate nlrp3 inflammasome activation deubiquitination of nlrp3 by brcc3 critically regulates inflammasome activity the rip1-rip3 complex initiates mitochondrial fission to fuel nlrp3 regulation of inflammasome activation viroporins: structure and biological functions nek7 is an essential mediator of nlrp3 activation downstream of potassium efflux pb1-f2 derived from avian influenza a virus h7n9 induces inflammation via activation of the nlrp3 inflammasome cryopyrin/nalp3 binds atp/datp, is an atpase, and requires atp binding to mediate inflammatory signaling type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells ddx19a senses viral rna and mediates nlrp3-dependent inflammasome activation interleukin-1 is responsible for acute lung immunopathology but increases survival of respiratory influenza virus infection rapid inflammasome activation following mucosal siv infection of rhesus monkeys antibodymediated protection against shiv challenge includes systemic clearance of distal virus the role of il-18 in innate immunity the il-1 family: regulators of immunity interleukin-1 (il-1) pathway molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases hepatitis c virus infection of cultured human hepatoma cells causes apoptosis and pyroptosis in both infected and bystander cells dengue virus-infected human monocytes trigger late activation of caspase-1, which mediates pro-inflammatory il-1beta secretion and pyroptosis il-1r signaling in dendritic cells replaces pattern-recognition receptors in promoting cd8(+) t cell responses to influenza a virus human mucosal-associated invariant t cells contribute to antiviral influenza immunity via il-18-dependent activation mx1 reveals innate pathways to antiviral resistance and lethal influenza disease pyrin-and card-only proteins as regulators of nlr functions inflammasomes and its importance in viral infections cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death enterovirus 71 inhibits pyroptosis through cleavage of gasdermin d the rna-and trim25-binding domains of influenza virus ns1 protein are essential for suppression of nlrp3 inflammasome-mediated interleukin-1beta secretion measles virus v protein inhibits nlrp3 inflammasome-mediated interleukin-1beta secretion the endoribonuclease activity essential for the nonstructural protein 11 of porcine reproductive and respiratory syndrome virus to inhibit nlrp3 inflammasome-mediated il-1beta induction key: cord-003598-m2fsrwvw authors: elbahesh, husni; gerlach, thomas; saletti, giulietta; rimmelzwaan, guus f. title: response modifiers: tweaking the immune response against influenza a virus date: 2019-04-12 journal: front immunol doi: 10.3389/fimmu.2019.00809 sha: doc_id: 3598 cord_uid: m2fsrwvw despite causing pandemics and yearly epidemics that result in significant morbidity and mortality, our arsenal of options to treat influenza a virus (iav) infections remains limited and is challenged by the virus itself. while vaccination is the preferred intervention strategy against influenza, its efficacy is reduced in the elderly and infants who are most susceptible to severe and/or fatal infections. in addition, antigenic variation of iav complicates the production of efficacious vaccines. similarly, effectiveness of currently used antiviral drugs is jeopardized by the development of resistance to these drugs. like many viruses, iav is reliant on host factors and signaling-pathways for its replication, which could potentially offer alternative options to treat infections. while host-factors have long been recognized as attractive therapeutic candidates against other viruses, only recently they have been targeted for development as iav antivirals. future strategies to combat iav infections will most likely include approaches that alter host-virus interactions on the one hand or dampen harmful host immune responses on the other, with the use of biological response modifiers (brms). in principle, brms are biologically active agents including antibodies, small peptides, and/or other (small) molecules that can influence the immune response. brms are already being used in the clinic to treat malignancies and autoimmune diseases. repurposing such agents would allow for accelerated use against severe and potentially fatal iav infections. in this review, we will address the potential therapeutic use of different brm classes to modulate the immune response induced after iav infections. influenza viruses (ivs) are responsible for significant morbidity and mortality in the human population with ∼500,000 annual deaths worldwide. ivs can cause severe acute respiratory disease especially in high-risk populations like children, the elderly and the immunocompromised. while both influenza a and b viruses (iav and ibv, respectively) cause annual epidemics, the majority of severe human infections are caused by iav. ivs have segmented negative-sense single-stranded rna genomes. the lack of proof-reading activity of the viral rna-dependent rna polymerase (rdrp) and successive replication can lead to the accumulation of nucleotide mutations which drive antigenic drift. in addition, the segmented nature of their genome allows genetic reassortment between iv's to take place, which can produce novel strains that have acquired alternative antigenically distinct hemagglutinin, also known as antigenic shift. both antigenic drift and antigenic shift contribute to the iv's ability to evade pre-existing host immunity induced by previous infections. early recognition and responses to iv infection are largely mediated by innate immune sensors expressed by its primary target, the alveolar epithelial cells (1, 2). recognition of ivs is mediated by pattern recognition receptors (prrs) that include toll like receptors (tlrs), retinoinc acid inducible gene-i (rig-i), and nucleotide oligomerization domain (nod)-like receptor family pyrin domain containing 3 (nlrp3); all of which can recognize viral rnas during various stages of the infection cycle (3-5). activation of these sensors triggers signaling cascades that lead to the production of interferons as well as pro-inflammatory cytokines and chemokines ultimately resulting in an antiviral state within the surrounding cells/tissue (6). accordingly, ivs have multiple mechanisms to evade these responses mediated by the viral nonstructural 1 protein (ns1), polymerase basic 1 protein (pb1), polymerase basic 2 protein (pb2), polymerase acidic (pa) and nucleoprotein (np) [ in otherwise healthy individuals, iav infections are mild and the ensuing pro-and anti-inflammatory responses are balanced. in contrast, a "cytokine storm" is typically associated with severe infections including those caused by highly pathogenic iv strains. during a cytokine storm, chemokine and cytokine responses are dysregulated in both intensity and kinetics resulting in excessive damage to the host due to infiltration of inflammatory immune cells. acute lung injury (ali) caused by this inflammatory response is typically characterized by significant damage or destruction of the respiratory epithelium leading to acute respiratory distress syndrome (ards) (7, 8). clinical treatment options for severe influenza virus infections remain limited and relying heavily on the administration of antiviral neuraminidase inhibitors (nais) and supportive critical care (9). however, nais have not been effective in patients with severe h7n9 or h5n1 infections and there is evidence that fatal outcomes are associated with development of antiviral resistance in patients (10-12). while virus-targeted therapies remain the standard approach, iv's mutability and adaptation to current antivirals has highlighted the need for new therapeutic options that target host factors that regulate iv infections and resulting immune responses. in either approach, the focus is to prevent or limit damage to the lung epithelium due to exaggerated or dysregulated immune cell responses. biological response modifiers (brms) can alter the immune response thereby offering an additional therapeutic approach to treating severe infections. in this review, we highlight several studies that have shown the viability of brms as potential treatment options. for clarity, brms are categorized based on the type of biological agent (table 1) . therapeutic antibodies iav infections and some vaccines elicit broadly-neutralizing antibodies (abs) that target the viral ha-stem. however, their abundance and immune-subdominance is overshadowed by abs targeting the ha-head domain. the effectiveness of these hastem abs against a broad range of iav subtypes, makes them an attractive target not only for vaccine development but also as antivirals. indeed, several ha-stem specific human monoclonal abs are now being evaluated in clinical trials [reviewed in davidson (34) ]. mhaa4549a, medi8852, and vis410 are human monoclonal abs that have been shown to control viral replication and improve symptoms of human patients in phase 2 clinical trials (13-15). while virus-specific abs aim to reduce antigenic load, abs to host targets aim at limiting the secondary wave of cytokines and reduce prolonged damaging cellular infiltration during severe infections. host-target directed antibodies have been utilized to target key regulators of this inflammatory wave and could potentially be used to dampen these overt responses. angiopoietin-like 4 (angptl4) is a soluble angiogenicregulating protein. following proteolytic cleavage, the cterminal portion (cangptl4) is involved in integrin-dependent wound repair and can regulate vascular permeability (35, 36) . angptl4 was significantly elevated in lung biopsies from iav-induced pneumonia patients (16). in mouse studies, neutralizing anti-angptl4 abs reduced pulmonary tissue leakiness significantly accelerating lung recovery and improved lung tissue integrity (16). neutrophil infiltration into the alveolar space occurs within 1 day following iav infections (37) . neutrophil extracellular traps (nets) released during iav-induced pneumonia into the alveolar space caused alveolar damage (38) . the complement protein c5a was shown to induce nets release and administration of anti-c5a abs (ifx-1) reduced h7n9-induced ali due to reduced infiltration of lung macrophages and neutrophils as well as reduction of viral load in african green monkeys (17, 39). tumor necrosis factor alpha (tnfα) is a key cytokine for controlling severe iav infections. it regulates two main antiviral functions: the induction of (i) the nfkb pathway, which ultimately controls expression of several inflammatory cytokines and (ii) apoptosis through multiple signaling cascades (40, 41) . tnf upregulation during iav infections correlates with infection severity, especially following highly pathogenic iav-infections (42) (43) (44) . mice treated with anti-tnf abs showed reduced disease burden; however, the authors of that study reported no effect on viral replication (20). tnf-related apoptosis inducing ligand (trail) can trigger apoptosis in iav-infected cells. iav-infected human epithelial cells are sensitized to trail-mediated apoptosis while peripheral blood mononuclear cells upregulate trail expression. moreover, administration of monoclonal abs against trail increases survival rate following iav infections in mouse studies (18, 19). antimicrobial peptides (amps) are host proteins that have direct antibacterial and antiviral activities and can modulate immune responses to infections. while the literature is largely focused on the antibacterial aspects of amps, several studies have highlighted the antiviral potential of amps against several viruses including ivs [reviewed in hsieh and hartshorn (45) anti-angptl4 -reduced pulmonary tissue leakiness, significantly accelerated lung recovery and improved lung tissue integrity in mice. -mouse-adapted laboratory iav (h1n1) c5a ifx-1 antibody -reduced viral load and virus-induced ali due to reduced infiltration of lung macrophages and neutrophils in iav-infected african green monkeys. -highly-pathogenic avian iav (h7n9) trail anti-trail -increased survival rate following iav infections in mouse studies. -improved symptoms and increased survival of iav infected mice. ed survival after h1n1 or h5n1 mouse infections. -1957 pandemic iav (h2n2); mouse-adapted laboratory iav (h1n1); 2009 pandemic iav (h1n1) (31) (32) (33) and albericio and kruger (46)]. ll-37 is a human cathelicidin derived amp that is found predominantly in neutrophils and its expression can also be induced in epithelial cells and macrophages (47) . aerosol administration of either human ll-37 or its mouse counterpart mcramp led to reduced morbidity and mortality to similar levels as the neuraminidase inhibitor zanamivir that is used for the treatment of human influenza patients (21). both cellular and viral fadd-like il-1β-converting enzymeinhibitory protein (cflip and vflip, respectively) protect cells from death receptor mediated apoptosis. kα2 is a vflip-derived peptide that consists of 10 amino acids from the α2 helix of the kaposi's sarcoma herpes virus (kshv) death effector domain 1 protein. a synthetic version of this peptide, tat-kα2, was generated by fusing kα2 to a portion of the hiv tat protein (22, 48) . in mouse challenge studies, intranasal administration of tat-kα2 at the time of infection with highly pathogenic avian h5n1 virus resulted in protection of the treated mice. no replicating virus was detected in the lungs at either 3 or 5 days after infection suggesting complete protection from infection (22). it should be noted that this effect is largely due to direct destabilization of the virions by the tat-kα2 peptide and it is likely that infection in treated mice was not established; the efficacy of this amp has not been determined during an established infection and warrants further investigation. host kinases regulate not only iav entry and replication but also initiate antiviral signaling cascades that regulate expression of pro-inflammatory chemokines and cytokines during infections and present viable targets for intervention (24, (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) . iav infection has been shown to upregulate c-jun n-terminal kinases 1 and 2 (jnk1/jnk2). these kinases directly regulate the induction of pro-inflammatory responses. iav-induced jnk1/jnk2 activation mediates production of chemokines and cytokines including tnf-α, interferon β (ifn-β), and interleukin 6 (il-6) (24) . in vivo inhibition of jnk1/jnk2 resulted in reduced levels of pro-inflammatory cytokines and reduced viral titers (23, 24). the mitogen activated protein kinase (mapk), p38, regulates viral entry and replication (55, 59) . furthermore, p38 regulates ifn stimulated gene (isg) gene expression and ultimately cytokine production via stat1 phosphorylation (25) . using either of two specific p38 inhibitors (sb 202190 or sb 203580), mice were protected from lethal h5n1 infection exhibiting reduced mortality and pro-inflammatory responses (25) . activation of another mapk, mek, is required for efficient iav replication and its inhibition results in viral ribonucleoprotein (vrnp) retention and reduced titers of progeny virus (26, 60, 61) . importantly, treatment of mice with the clinically approved mek inhibitor (ci-1040) showed reduced lung viral load and mortality of mice following infection with a lethal dose of pandemic h1n1 iav; interestingly, this inhibitor significantly out-performed the clinically recommended oseltamivir in these studies (26) . another central regulator of immune responses at the epithelium as well as immune cells is the nf-κb signaling pathway. accordingly, iav has evolved several mechanisms to modulate this pathway to counteract antiviral responses including directly targeting the ikb kinase (ikk) (62, 63) . sc75741 is a potent nfkb inhibitor that functions by reducing the ability of the p65 subunit of the nfkb complex to bind dna; thereby limiting its transcription-regulating functions (64, 65) . in vivo administration of sc75741 at 4 days after lethal infection with either h5n1 or h7n7 avian viruses resulted in significant protection with most mice surviving and showing little to no clinical symptoms; similar results were obtained by prophylactic administration (27) . g-protein coupled receptor kinase 2 (grk2) is best known for its phosphorylation of gpcrs in cardiac tissue resulting in recruitment of β-arrestin to facilitate rapid receptor internalization and lysozomal degradation (66) . recent phosphoproteomic studies identified grk2 as a potentially proviral host protein for iav that plays a major role in virion uncoating (28) . although in vivo inhibition of grk2 using paroxetine led to a significant reduction in upper respiratory tract viral load and to a modest reduction in lower respiratory tract titers at 4 days post infection, this inhibition was not protective from lethal infections (28) . however, it is possible that the route of administration (intraperitoneal vs. intranasal) and dosing regimen influenced the results. sphingosin kinases (sphk) are lipid kinases that mediate conversion of sphingosine to bioactive lipid sphingosine 1phosphate (s1p) (67), a known modulator of central apoptotic pathways (68) . iav infections leads to increased expression and activation of sphk1 and sphk2 (29) and in vitro inhibition of sphk1 was shown to decrease iav rna synthesis via suppression of nfkb activation (69) . treatment of mice with specific inhibitors to either sphk1 or sphk2 or a pan-sphk inhibitor led to prolonged survival of mice following lethal iav infection (29) . peroxisome proliferator-activated receptors (pparα, pparβ, and pparγ) regulate metabolic homeostasis and are important mediators of the inflammatory response. several ppar agonists have been investigated for efficacy during iav infections with varying results. gemfibrozil (pparα agonist) not only improved symptoms when administered 4 days after infections with an h2n2 virus, but also increased survival of iav infected mice (31) . prophylactic treatment of h1n1-infected mice with pioglitazone (pparγ agonist) resulted in increased survival (32) . combined activation of pparγ and its downstream target ampk improved survival of mice infected with pandemic iav strains (33) . protease activated receptor (pars) link protease activity to inflammatory cellular responses (70) . par1 expression is upregulated in the mouse airways following iav infections (71) . intranasal administration of a par1 antagonist (sch79797) at the time of infection with various iav strains including highly pathogenic avian h5n1 and pandemic h1n1 viruses led to increased survival and a decrease in inflammatory responses. moreover, this effect was also observed when sch79797 was administered 48-72 h after infection (30) . the use of statins, angiotensin ii receptor blockers (arbs) and angiotensin converting enzyme inhibitors (acei) has been proposed to regulate the iav-induced cytokine storm in severe infections (72, 73) . retrospective studies conducted separately in mexico, netherlands, uk and usa reported an association of reduced iav-related pneumonia and lower case fatality due to lower respiratory tract iav infections with statin treatment (74) (75) (76) (77) . however, this association was contested in two additional studies that found no benefit of statin treatment on iavinduced disease burden (78, 79) . this uncertainty regarding the iav therapeutic potential of these widely used compounds warrants further investigations at the basic science level and in clinical trials. the continuous accumulation of adaptive mutations and the introduction of novel viruses in the human population continue to pose a threat to public health, especially to individuals at high risk to influenza. the emergence of strains resistant to existing classes of antiviral drugs and reduced vaccine effectiveness highlights the need for the development of alternative intervention strategies. therefore, therapeutic approaches that can diminish the potential for drug-resistance while being effective against multiple iav subtypes/strains are highly desirable. targeting host cell factors meets these criteria and is more likely to avoid overtly robust immune responses thereby reducing disease severity and improve patient outcome (figure 1) . a large effort has been made in recent years to identify host proteins to serve as intervention targets against iv infections. several genetic and proteomic screens have identified several promising hits with potential roles in the iv replication cycle (80) (81) (82) (83) (84) (85) (86) (87) (88) (89) (90) . in addition to these genome-wide screens, viral and host protein interactions can be mapped into networks that can also be used to identify host factors critical for iv replication (91, 92) . interestingly, meta-analysis of some these studies shows limited overlap in the genes/proteins identified as required host factors (87, (93) (94) (95) . this is likely due to study-specific variations in iv types/strains and cell-lines used, inclusion/exclusion criteria, limited hit-validations and methods used to "knock-down/out" these genes. local microenvironment within a given tissue can dictate the quality and intensity of an immune response. inhibition or activation of critical signaling pathways expressed in both respiratory tract epithelial and immune cells by brms can have opposite and unintended consequences. as discussed above, trail regulates immune cell-mediated apoptosis of infected cells and several studies have shown that blocking trail signaling by genomic deletion or depletion by monoclonal antibody administration can improve infection outcome in iavinfected mice. indeed inhibition of trail signaling in alveolar macrophages and other monocytes limits their ability to induce apoptosis in alveolar cells, prevents lung tissue damage and promotes survival (19, 96, 97) . however, cd8+ t cells from trail−/− mice are less able to protect mice from severe infections, consistent with impaired trail-mediated effector functions of cd8+ t cells (18). similarly, opposing beneficial and detrimental outcomes have also been observed in studies using bcl-2 inhibitors to treat iav infections (98, 99) . brm delivery should be guided by immune system "compartmentalization" to ensure they elicit balanced immune responses. ideally, mucosal delivery deposits brms that reduce viral titers at the site of iav replication; however, systemic delivery of certain brms might be required to dampen dysregulated responses. this not only depends on the brms used but also on the timing of their administration. moreover, the duration of treatment with brms must be considered because sustained inhibition of certain inflammatory responses can result in an immune status that increases susceptibility to secondary opportunistic infections. repurposing of clinically approved drugs could potentially be used as brms for the treatment of severe iav infectious and should be explored (86, 89, 90) . considering that susceptibility to severe iav infections is influenced by host genetics and hostspecific immune responses, selection of therapeutic brms should be carried out using in vivo model systems that are representative of the immune status spectrum and underlying conditions of high-risk influenza patients (young, immunocompromised, nonnaive, obese, pregnant, or aged). using these model systems will increase the likelihood of identifying brms with clinically relevant antiviral and immunomodulatory potentials. he, tg, gs, and gr conceptualized and composed the manuscript. gr and he oversaw all aspects of the manuscript preparation. this work was funded by the alexander von humboldt foundation in the framework of the alexander von humboldt professorship endowed by the german federal ministry of education and research. we apologize to any investigators whose relevant work was not included due to space limitations. regulatory roles of c-jun in h5n1 influenza virus replication and host inflammation inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal h5n1 infection the mek-inhibitor ci-1040 displays a broad anti-influenza virus activity in vitro and provides a prolonged treatment window compared to standard of care in vivo the nf-kappab inhibitor sc75741 protects mice against highly pathogenic avian influenza a virus phosphoproteomic-based kinase profiling early in influenza virus infection identifies grk2 as antiviral drug target transient inhibition of sphingosine kinases confers protection to influenza a virus infected mice par1 contributes to influenza a virus pathogenicity in mice increased survival after gemfibrozil treatment of severe mouse influenza tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection peroxisome proliferator-activated receptor and amp-activated protein kinase agonists protect against lethal influenza virus challenge in mice treating influenza infection, from now and into the future angiopoietinlike 4 interacts with matrix proteins to modulate wound healing role of angptl4 in vascular permeability and inflammation h5n1 and 1918 pandemic influenza virus infection results in early and excessive infiltration of macrophages and neutrophils in the lungs of mice excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis viable neutrophils release mitochondrial dna to form neutrophil extracellular traps molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression relevance of signaling molecules for apoptosis induction on influenza a virus replication role of host cytokine responses in the pathogenesis of avian h5n1 influenza viruses in mice innate immune responses to influenza a h5n1: friend or foe? new fronts emerge in the influenza cytokine storm the role of antimicrobial peptides in influenza virus infection and their potential as antiviral and immunomodulatory therapy therapeutic peptides cathelicidins, multifunctional peptides of the innate immunity flip-mediated autophagy regulation in cell death control the tyrosine kinase inhibitor tyrphostin blocks the cellular actions of nerve growth factor role of protein kinase c betaii in influenza virus entry via late endosomes from virus entry to release: the diverse functions of pi3k during rna virus infections influenza a virus-induced early activation of erk and pi3k mediates v-atpase-dependent intracellular ph change required for fusion development of cellular signaling pathway inhibitors as new antivirals against influenza novel roles of focal adhesion kinase in cytoplasmic entry and replication of influenza a viruses a novel p38 mitogen activated protein kinase (mapk) specific inhibitor suppresses respiratory syncytial virus and influenza a virus replication by inhibiting virus-induced p38 mapk activation focal adhesion kinase (fak) regulates polymerase activity of multiple influenza a virus subtypes role of c-jun terminal kinase (jnk) activation in influenza a virus-induced autophagy and replication influenza virus infections and cellular kinases toll-like receptor 4-mediated activation of p38 mitogen-activated protein kinase is a determinant of respiratory virus entry and tropism antiviral activity of the mekinhibitor u0126 against pandemic h1n1v and highly pathogenic avian influenza virus in vitro and in vivo combination of mek inhibitors and oseltamivir leads to synergistic antiviral effects after influenza a virus infection in vitro jnk2 and ikkbeta are required for activating the innate response to viral infection influenza a virus-encoded ns1 virulence factor protein inhibits innate immune response by targeting ikk a novel class of potent nf-kappab signaling inhibitors the nf-kappab inhibitor sc75741 efficiently blocks influenza virus propagation and confers a high barrier for development of viral resistance grk2 in the heart: a gpcr kinase and beyond regulation of sphingosine kinase and sphingolipid signaling sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases sphingosine kinase 1 serves as a pro-viral factor by regulating viral rna synthesis and nuclear export of viral ribonucleoprotein complex upon influenza virus infection participation in inflammation altered expression and in vivo lung function of protease-activated receptors during influenza a virus infection in mice pandemic influenza: a potential role for statins in treatment and prophylaxis treating influenza with statins and other immunomodulatory agents old drugs losing effectiveness against flu; could statins fill gap? influenza and copd mortality protection as pleiotropic, dose-dependent effects of statins experience in the management of the severe form of human influenza a h1n1 association between use of statins and mortality among patients hospitalized with laboratory-confirmed influenza virus infections: a multistate study influenza morbidity and mortality in elderly patients receiving statins: a cohort study an assessment of the effect of statin use on the incidence of acute respiratory infections in england during winters drosophila rnai screen identifies host genes important for influenza virus replication the ifitm proteins mediate cellular resistance to influenza a h1n1 virus, west nile virus, and dengue virus a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection the use of random homozygous gene perturbation to identify novel host-oriented targets for influenza genome-wide rnai screen identifies human host factors crucial for influenza virus replication human host factors required for influenza virus replication antiviral effects of inhibiting host gene expression influenza virus-host interactomes as a basis for antiviral drug development genomewide crispr/cas9 screen identifies host factors essential for influenza virus replication repurposing host-based therapeutics to control coronavirus and influenza virus repurposing of drugs as novel influenza inhibitors from clinical gene expression infection signatures comparative influenza protein interactomes identify the role of plakophilin 2 in virus restriction network-guided discovery of influenza virus replication host factors cellular networks involved in the influenza virus life cycle genetic screens for the control of influenza virus replication: from meta-analysis to drug discovery meta-and orthogonal integration of influenza "omics" data defines a role for ubr4 in virus budding the magnitude of the t cell response to a clinically significant dose of influenza virus is regulated by trail pathogenic potential of interferon alphabeta in acute influenza infection anticancer compound abt-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice antiviral properties of chemical inhibitors of cellular anti-apoptotic bcl-2 proteins key: cord-003724-705h5l06 authors: di lullo, giulia; calabresi, valentina; mariotti, feliciana; zambruno, giovanna; lanzavecchia, antonio; di zenzo, giovanni title: identification of a novel non-desmoglein autoantigen in pemphigus vulgaris date: 2019-06-19 journal: front immunol doi: 10.3389/fimmu.2019.01391 sha: doc_id: 3724 cord_uid: 705h5l06 pemphigus vulgaris (pv) is an autoimmune bullous disease of the skin and mucous membranes characterized by the presence of circulating and tissue-bound autoantibodies against keratinocyte cell surface antigens, specifically desmoglein (dsg) 1 and 3. the pathogenic role of anti-dsg antibodies is well-established, while the mechanism of blister formation is only partly defined. we have applied a previously developed method for the efficient immortalization of igg+ memory b cells to identify novel target antigens in pv. a human monoclonal antibody reactive with a hitherto unreported non-dsg antigen was isolated. immunoprecipitation and immunoblotting studies with keratinocyte extracts indicated α-catenin as the putative antigen, then confirmed by immunoblotting on the recombinant protein. four of ten pv sera reacted with recombinant α-catenin. although the isolated human monoclonal antibody was per se unable to dissociate keratinocyte monolayers and also to synergize with a pathogenic antibody in vitro, further studies are warranted to assess its possible in vivo contribution in the multifactorial pathogenesis and heterogeneous manifestations of pv disease. pemphigus vulgaris (pv) is a rare but highly disabling and, if untreated, almost always fatal immunobullous disease of the skin and mucous membranes. pv is characterized histologically by loss of cell-cell adhesion between suprabasal keratinocytes, leading to acantholysis, and immunopathologically by the presence of circulating and tissue-bound autoantibodies (autoabs) against keratinocyte cell surface antigens, specifically desmoglein (dsg) 1 and 3. pv is considered as a paradigmatic organ-specific autoimmune disease in view of (i) present knowledge of disease autoantigens and pathogenesis and (ii) reproducibility of major clinical and pathogenic features in animal models (1) . the existence of both pathogenic and non-pathogenic anti-dsg autoabs has recently been underscored by isolation of human monoclonal antibodies (hmabs) from pemphigus patients. anti-dsg hmabs characterization has shown that their pathogenic potential primarily depends on the targeted epitopes (1) . we have been interested in characterizing the repertoire of naturally occurring autoreactive epithelium-specific memory b cells in pemphigus vulgaris patients. in a first work, we focused on autoantibodies targeting dsg3 (2) . however, (i) the lack of tight correlation between anti-dsg autoab titers and disease activity in some patients and (ii) the significant degree of disease heterogeneity point at the importance of non-dsg autoabs, that have been progressively, even though not exhaustively, described (3, 4) . in fact, besides dsg3 and dsg1, other non-desmoglein autoabs, either pathogenic or nonpathogenic, have been identified in pemphigus patients. autoabs endowed with an acantholytic potential target desmocollin 3, α-acetylcholine receptor, pemphaxin, and keratinocyte mitochondria (5) (6) (7) (8) . on the other hand, the pathogenic role of autoabs recognizing other autoantigens, such as atp2c1, desmocollin 1, bp230, periplakin, e-cadherin, desmoglein 4, desmoplakin 1, and desmoplakin 2, remains to be demonstrated (9) . in line with this interest, our current work aimed to identify autoabs targeting non-dsg membrane-bound or membrane-associated intracellular antigens. in the present study, we report on the characterization of a hmab isolated from a pv patient and directed to a novel non-dsg antigen. the hmab reacts with α-catenin that is recognized by almost half of pv sera analyzed. peripheral blood was obtained from 2 patients (pvc and pvf) suffering from active mucocutaneous pv. the patients showed typical clinical, histological, and immunopathological features and had high-titer anti-dsg circulating autoantibodies (pvc: dsg3, 127 u/ml, dsg1, 90 u/ml; pvf: dsg3, 191 u/ml, dsg1, 170 u/ml), as assessed by elisa kits based on ectodomain of dsg1 and dsg3 (mbl, nagoya, japan). hmabs were isolated by a highly-efficient protocol for the immortalization of igg+ memory b cell with epstein barr virus (ebv) in the presence of a toll-like receptor agonist, as previously described (2) . in detail, igg+ memory b cells were isolated from cryopreserved peripheral blood mononuclear cells using anti-cd22 microbeads (miltenyi biotec, bo, italy) followed by depletion of cells carrying igm, igd, and iga by cell sorting. multiple replicate microcultures of 10-30 igg+ memory b cells/well (for a total of 2 to 8 × 10 4 purified cells) were infected with ebv and cpg as previously described (10) . culture supernatants were tested for binding to dsg1-and dsg3-coated elisa plates and for binding to hacat keratinocyte cell line monolayers (both on live cells and on fixed and permeabilized cells) by immunofluorescence (if) assay using an automated fluorescence microscope (pathway 855, bd). the specificity of positive polyclonal cultures was further assessed by if on primary human keratinocytes. positive reactivities were confirmed by the propagation of oligoclonal cultures. positive cultures were cloned by limiting dilution and expanded; antibodies were purified using protein g columns. serum samples were collected from 10 pv and 16 bullous pemphigoid (bp) patients and 10 healthy donors. this study was carried out in conformity with the helsinki guidelines and with approval of the idi-irccs ethics committee. all the biological samples were obtained after patient's informed consent. if studies were performed according to the procedure described in di zenzo et al. with minor modifications (2) . briefly, supernatants from immortalized human memory b cells were screened on monolayers of live and fixed/permeabilized hacat cells. after washing with phosphate-buffered saline, cells were stained with alexa fluor 488-conjugated goat anti-human igg (invitrogen, carlsbad, ca, usa). the isolated monoclonal antibodies were further tested on permeabilized hacat cells and on primary keratinocytes. non-keratinocyte cell lines, i.e., mrc9, hela, and skmel cells, were used as controls for if analyses on cell monolayers. human antibodies of irrelevant specificity were used as negative controls. serial images of stained keratinocyte monolayers were acquired by the bd pathway 855 automated fluorescence microscope. staining was also performed on cryo sections of normal human skin, guinea pig and monkey esophagus and revealed with fluorescein isothiocyanateconjugated anti-human igg antibody (agilent dako, santa clara, ca, usa). immunoprecipitation (ip) of 35 s-labeled keratinocyte extracts by hmab pvf144 was carried out as previously described (11) . the precipitated proteins, separated on sodium dodecyl sulfatepolyacrylamide gel electrophoresis on 6% gels under reducing conditions, were detected by autoradiography. immunoblotting (ib) experiments on keratinocyte extracts were performed as previously reported using both horseradish peroxidase (hrp)conjugated and alkaline phosphatase-conjugated secondary antibodies (12) . bands were quantified using imagej software (national institutes of health, bethesda, md, usa) and their intensities were normalized respect to the positive control signal obtained by anti-α-catenin antibody. the cutoff value was set as the medium value +2 standard deviations obtained measuring signals obtained with healthy donors. commercial primary antibodies were purchased from progen biotechnik gmbh (heidelberg, germany) (anti-desmocollin 2), bd biosciences (san jose, ca, usa; anti-α-catenin), and santa cruz biotechnology, inc (dallas, tx, usa; anti-γ-catenin, antiβ-catenin, anti-p120, and anti-e-cadherin). recombinant tagged human α1-catenin was purchased from abcam (cambridge, uk). to rule out that pv sera were mainly reacting against the fused glutathione s-transferase (gst) moiety of the α-catenin recombinant protein, ib experiments by using commercial tagged protein and equimolar gst were performed (data not shown). briefly, recombinant dsg1 and dsg3 ectodomains were produced in baculovirus and used for coating of elisa plates. the plates were, then, blocked with 1% bovine serum albumin and incubated with antibodies followed by hrp-conjugated antihuman igg (jackson immunoresearch, baltimore, pa, usa) (2). the assay was performed as previously reported (13) . briefly, primary human keratinocyte cells were seeded onto 12-well plates and 24 h post-confluence treated with monoclonal antibodies. after adding exfoliative toxin a to cleave dsg1 protein, the cell monolayers were detached with dispase i (merck, kgaa, darmstadt, germany) and subjected to mechanical stress by pipetting. the monolayer fragments, fixed by adding a 3% formaldehyde solution, were subsequently stained using crystal violet. to investigate a possible synergistic effect pvf144 was applied to the monolayer together with a pathogenic antibody (pvb28) (2) at optimal (4 µg/ml) and suboptimal concentrations (1 µg/ml and 0.25 µg/ml; figure 4 ) and a non-pathogenic antibody (2, pvb28 in a germline version that is not able to dissociate the keratinocyte monolayer; data not shown). in order to identify hmabs targeting non-dsg membranebound or membrane-associated intracellular antigens, we took advantage of the same strategy by which we had previously isolated and finely characterized several dsg-reactive pv patient-derived monoclonal autoantibodies (2) . in detail, peripheral blood samples were collected from 2 patients with mucocutaneous pv: one with long-lasting steroid-resistant disease (pvc) and the other prior to treatment initiation (pvf). igg+ memory b cells were isolated by magnetic and fluorescence-activated cell sorting, seeded in 96-well microplates, and immortalized with ebv in the presence of irradiated mononuclear cells and oligodeoxynucleotides containing cpg motifs, as previously described (10) . the reactivity of antibodies secreted in the supernatants of growing polyclonal cultures was screened by if staining both on live and on fixed and permeabilized cells from the human keratinocyte hacat cell line. the polyclonal antibodies produced by the vast majority of isolated cultures bound to surface antigens on the keratinocyte membrane and were reactive with dsg1 and/or dsg3 ectodomains by elisa, as previously reported (2, and data not shown). besides such prevalent pattern of reactivity, supernatants of rare cultures from both patients showed a distinctive membrane-associated fishnet reactivity, detected only on permeabilized keratinocytes, and were negative in elisa on dsg1 and dsg3 ectodomains. the human mabs pvf144 (igg1-isotype), pvc6 (igg1-isotype), and pvc33 (isotype igg3isotype) were cloned by limiting dilution and showed the same if pattern as the original polyclonal cultures also on permeabilized hacat keratinocytes (figure 1a) , suggesting their specificity for a membrane-associated intracellular (i.e., non-exposed) autoantigen. in addition, they showed an intercellular staining pattern by if on human skin, guinea pig, and monkey esophagus (figures 1b-d) , very similar to if staining pattern of anti-dsg3 antibodies (2, and data not shown). supernatants from the selected clones were tested on non-keratinocyte cell lines, i.e., mrc-9 (fibroblast), skmel (melanoma), and hela (epithelial) cells: they failed to stain both live and permeabilized cells, apart from a reactivity on permeabilized hela cells (data not shown), hinting at a specificity for membrane-associated intracellular antigens expressed on keratinocytes and other epithelial cells. ip studies with the 3 selected clones on radiolabelled keratinocyte extracts to identify the target antigen showed reactivity to a 100 kda antigen for pvf144 (figure 2a) and to 190-210 kda antigens for pvc6 and pvc33, respectively (data not shown). as the ip results from the latter two clones pointed at members of the plakin-family as candidate target antigens and autoreactivity of pv sera to plakin family members has been already documented (14, 15) , we chose to further characterize pvf144 with the aim to identify a possible novel membrane-associated pv autoantigen. subsequent ib experiments using commercial antibodies reacting with keratinocyte membrane-associated proteins with a molecular weight of ∼100 kda (desmocollin 2-115 kda; α-catenin-102 kda; e-cadherin-120 kda; β-catenin-92 kda; γ-catenin-83 kda; p120-catenin 80-100 kda) indicated α-catenin as the putative antigen ( figure 2b) . sequential ip and ib experiments further supported α-catenin as the target antigen of pvf144. specifically, a protein immunoprecipitated from keratinocyte extracts with pvf144 was recognized by a commercial anti-α-catenin antibody, and an anti-α-catenin antibody was in turn able to immunoprecipitate a protein recognized by pvf144 in ib ( figure 2c) . finally, ib experiments with the recombinant tagged protein unequivocally confirmed the specific binding of pvf144 to α-catenin ( figure 2d ). to determine whether autoabs of the same specificity as pvf144 were present in the sera of pv patients, we performed ib experiments with 10 pv sera on recombinant tagged α-catenin figure 2 | pvf144 binds a membrane-associated epithelial antigen: α-catenin. pvf144 immunoprecipitates (ip) an unknown antigen of 100 kda from radiolabeled normal human keratinocyte extracts (a). immunoblotting (ib) experiments on keratinocyte extracts by using pvf144 and commercial antibodies suggest that α-catenin could be the putative antigen of 100 kda: pvf144 and a monoclonal murine anti-α-catenin antibody (ab) react with a keratinocyte antigen of similar molecular weight (100 kda) (b). anti-α-catenin commercial antibody reacts to α-catenin from keratinocyte extracts by ib (lane 1); pvf144 immunoprecipitates α-catenin recognized by the commercial anti-α-catenin antibody by ib (lane 2) and, viceversa, anti-α-catenin antibody immunoprecipitates α-catenin recognized by pvf144 by ib (lane 3). the faint reactivity observed in lane 3 could be related to the epitope recognized (c). ib performed with a recombinant gst-tagged human α-catenin (120 kda) confirms that the target of pvf144 is α-catenin. the lower bands are likely degradation products (d). (figure 3) . four out of 10 pv patients (40%) showed the same reactivity of pvf144 to α-catenin, whereas the other pv sera and 10 normal human control sera showed only a faint signal, indicating that this autoreactivity was well-represented in pv patients, even though not shared by all patients. in order to evaluate whether this reactivity is disease-specific, 16 bp patient sera were analyzed on recombinant α-catenin by ib. only one of 16 bp (7 representative bp sera shown and other 9 not shown) reacted to α-catenin (supplementary figure 1) underlining the specificity of this autoantigen. the weak signals found in the other remaining pv, pb, and healthy donor sera might be ascribed to reactivity to the tag protein (gst). the presence of anti-α-catenin autoabs in several pv sera raised the question as to their pathogenic potential. previous studies showed the ability of intact autoabs to enter the cytosol or nucleus of living cells (16, 17) . more recently, marchenko et al. reported that pv autoabs could penetrate keratinocytes and react with intracellular mitochondrial proteins (8) . these findings suggested that a hmab to an intracellular antigen could exert its pathogenic ability on live keratinocytes. thus, an in vitro dissociation assay was used to evaluate the pathogenic activity of the hmab pvf144. this approach allows to measure the ability of a specific antibody or a mixture of antibodies, such as a serum, to fragment a monolayer of primary human keratinocytes seeded to confluence. the keratinocyte monolayers, incubated for 24 h with pvf144, remained intact, similarly to monolayers incubated with an unrelated hmab used as negative control (figure 4) . as expected, human (pvb28) and murine (ak23) pathogenic anti-dsg3 antibodies were able to dissociate the monolayers (2, 18) (figure 4) . in addition, pvf144 failed to synergize with a pathogenic antibody (figure 4) . these findings exclude a primary pathogenic function of anti-α-catenin autoabs in pv, nevertheless a potential secondary role in the immunobiology of the disease cannot be excluded and warrants future studies. in the present work we went forward in the characterization of the diverse targets of epithelium-specific autoreactive b cells from pv patients. to this purpose, we took advantage of the figure 3 | almost half of pv sera specifically react with recombinant α-catenin. immunoblotting with sera obtained from 10 pemphigus vulgaris (pv) patients shows that 4 of 10 sera (4, 8, 9, 10) react with the novel epithelial antigen, while 3 healthy donor sera (11, 12, 13) , representative of 10 sera analyzed, show only a background signal. the positive control (c+) is the commercial anti-α-catenin antibody. the background signal, could be also due to reactivity to the tag protein (gst). quantification and normalization of bands using imagej analysis (data not shown) have confirmed the reported results (see materials and methods section). high-efficiency immortalization protocol of igg+ memory b cells we had previously developed (10) and applied to isolate and molecularly characterize a number of anti-dsg3 hmabs (2). our present focus was to detect plasma-membrane-associated antigens, either expressed on the cell surface or intracellularly, therefore we chose to screen the isolated polyclonal cultures on both live and permeabilized keratinocytes. our choice was based on the hypothesis that antigens associated to the cell membrane, even those with intracellular localization, could have a higher chance to contribute to pemphigus pathogenesis, which is due to the loss of cell-cell adhesion. as expected, the vast majority of cultures resulted specific for dsg1 and/or dsg3, further confirming the major role of these antigens in pemphigus pathogenesis. among polyclonal cultures that reacted only with permeabilized keratinocytes, we selected and cloned those giving a membrane-associated fishnet-like staining pattern on stratified epithelia. among the cloned cultures, we further characterized clone pvf144 and demonstrated by if, ip and ib studies that α-catenin is its target. α-catenin is a component of adherens junctions (ajs), i.e., cellcell anchoring structures that, together with desmosomes, allow keratinocytes to adhere to one another and maintain epithelial integrity. α-catenin binds to β-catenin in ajs and is required for their formation and maintenance (19, 20) . in addition, α-catenin was reported to be necessary for the organization of desmosomes in epithelial cells (21) . a previous study reported a reactivity of pemphigus sera to another aj component, i.e., e-cadherin (22) . likewise, we showed that anti-α-catenin autoreactivity was: (i) wellrepresented in pv patient sera, as α-catenin was recognized by almost half of the pv sera tested; (ii) more frequently associated with pemphigus than with other autoimmune bullous diseases, as α-catenin was very rarely recognized by the bp sera analyzed. then, considering that previous studies demonstrated the ability of intact autoabs to enter living cells (8, 16, 17) , we addressed the potential pathogenicity of α-catenin-specific mab pvf144 by evaluating its acantholytic activity in an in vitro keratinocyte dissociation assay. in this regard, several observations suggest that ajs, and in particular e-cadherin, may be involved in pemphigus pathogenesis (23, 24) . of note, with the exception of anti-desmocollin 3 autoabs, all known non-dsg-reactive autoabs with reported pathogenicity do not possess acantholytic potential on their own but may act synergistically with anti-dsg antibodies (1, 25, 26) . accordingly, marchenko et al. described a pathogenic role for intracellular anti-mitochondrial autoabs, even though not on their own (8) . in our hands, the anti-α-catenin mab pvf144 was not able to dissociate the keratinocyte monolayer either alone or in combination with a suboptimal dose of a pathogenic anti-dsg3 antibody. nevertheless, we cannot exclude that regions of α-catenin different from that recognized by pvf144 could be recognized by pv sera and contribute to acantholysis. in addition, a possible role of anti-α-catenin hmabs as cofactors in disease initiation or maintenance could not be exhaustively addressed by our approach. in previous studies, several antibodies against intracellular antigens have been considered as triggers for autoimmunity. mabs specific for the cytoplasmic precursor form of dsg1 (predsg1) have been cloned from pemphigus patients and from healthy individuals (25, 26) . yamagami et al. postulated that the presence of anti-predsg1 b cells is involved in the initiation of the autoimmune response in pemphigus patients. in particular, in the context of tissue damage they could present peptides which are part of mature dsg1 (i.e., the extracellular autoantigen recognized by most pathogenetic autoabs) derived from the processing of predsg1. this intramolecular epitope spreading phenomenon could lead to the production of pathogenic autoabs targeting mature dsg1 and to the initiation of disease pathogenesis (27, 28). moreover, natural autoabs (naas), i.e., antibodies to intracellular autoantigens that naturally occur in the healthy population and are per se unable to cause immune phatology, have been theorized as cofactors in the onset of autoimmunity, possibly by participating in the mechanisms of chronic tissue injury at the basis of intermolecular epitope spreading (29) (30) (31) . in conclusion, while our previous study (2) demonstrated that pathogenic pv antibodies primarily target the dsg-3 cisinterface, thus leading to desmosome disruption, our current results reveal novel antibodies targeting intracellular keratinocyte proteins. we are tempted to speculate that the cellular damage figure 4 | pvf144 is not able to dissociate a keratinocyte monolayer even in the presence of suboptimal concentrations of pvb28. a representative keratinocyte dissociation experiment. primary human keratinocytes, seeded to confluence, were incubated with pvf144, an unrelated human mab (negative control) and, as positive controls, the human pathogenic mab pvb28 (7) and the murine pathogenic mab ak23 (14) , both dsg3-specific (a). to investigate synergistic potential of cloned antibody we have employed pvf144 (10 µg/ml) together with optimal (4 µg/ml) and suboptimal concentrations (1 µg/ml and 0.25 µg/ml) of the pathogenic anti-dsg3 antibody pvb28 without obtaining any difference in ability of pvb28, with or without pvf144, to dissociate the keratinocyte monolayer (b). induced by pathogenic anti-dsg antibodies may trigger an intermolecular epitope-spreading phenomenon resulting in an antibody response against intracellular antigens, among which α-catenin. further studies are needed: (i) to evaluate whether pvf144 may act synergistically with anti-dsg antibodies using more informative approaches, such as in vitro organ culture or in vivo models; (ii) to assess the possible role of anti-α-catenin autoabs in pemphigus initiation and evolution in vivo; and (iii) to characterize this novel antigen as a possible target of epitope spreading phenomena in pv. the datasets generated for this study are available on request to the corresponding author. this study was carried out in accordance with the recommendations of helsinki guidelines, idi-irccs ethics committee. the protocol was approved by the idi-irccs ethics committee. all subjects gave written informed consent in accordance with the declaration of helsinki. gdz and gdl have written the manuscript. gdz, gz, and al have designed the study. gdz, gdl, vc, and fm performed the experiments. all the authors have revised the manuscript and given the final approval for submission. the present work has been supported by the italian ministry of health (ricerca corrente 2017-2018 to gdz). immune response in pemphigus and beyond: progresses and emerging concepts pemphigus autoantibodies generated through somatic mutations target the desmoglein-3 cis-interface non-desmoglein antibodies in patients with pemphigus vulgaris the evolving story of autoantibodies in pemphigus vulgaris: development of the "super compensation hypothesis" igg autoantidodies against desmocollin 3 in pemphigus sera induce loss of keratinocyte adhesion novel human alpha9 acetylcholine receptor regulating keratinocyte adhesion is targeted by pemphigus vulgaris autoimmunity pemphigus vulgaris antibody identifies pemphaxin. a novel keratinocyte annexinlike molecule binding acetylcholine antimitochondrial autoantidodies in pemphigus vulgaris: a missing link in disease pathophysiology pemphigus vulgaris autoantibody profiling by proteomic technique an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus a truncating mutation in the laminin-332α chain highlights the role of the lg45 proteolytic domain in regulating keratinocyte adhesion and migration autoantibody profile of a cohort of 78 italian patients with mucous membrane pemphigoid: correlation between reactivity profile and clinical involvement in vitro keratinocyte dissociation assay for evaluation of the pathogenicity of antidesmoglein 3 igg autoantidodies in pemphigus vulgaris envoplakin and periplakin are components of the paraneoplastic pemphigus antigen complex mucosal dominant pemphigus vulgaris with antidesmoplakin autoantibodies the penetrating potential of autoantibodies into live cells in vitro coincides with the in vivo staining of epidermal nuclei proceedings of the second international conference on the penetration of autoantibodies into living cells induction of pemphigus phenotype by a mouse monoclonal antibody against the aminoterminal adhesive interface of desmoglein 3 identification of a neural alpha-catenin as a key regulator of cadherin function and multicellular organization alpha-catenin: at the junction of intercellular adhesion and actin dynamics identification of regions of alpha-catenin required for desmosome organization in epithelial cells e-cadherin is an additional immunological target for pemphigus autoantibodies cadherins in cutaneous biology a pathogenic autoantibody, pemphigus vulgaris-igg, induces phosphorylation of desmoglein 3, and its dissociation from plakoglobin in cultured keratinocytes critical role of the neonatal fc receptor (fcrn) in the pathogenic action of antimitochondrial autoantibodies synergizing with anti-desmoglein autoantibodies in pemphigus vulgaris synergy among non-desmoglein antibodies contributes to the immunopathology of desmoglein antibody-negative pemphigus vulgaris isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantidodies antibodies to the desmoglein 1 precursor proprotein but not to the mature cell surface protein cloned from individuals without pemphigus humoral epitope spreading in autoimmune bullous diseases cellular and genetic mechanisms of self tolerance and autoimmunity predominant autoantibody production by early human b cell precursors conflict of interest statement: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest we thank naomi de luca for the excellent technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01391/full#supplementary-material key: cord-015254-xtox2rxs authors: li, hao-ling; huang, yan; zhou, ya-lan; teng, run-hua; zhou, shu-zhuan; lin, jia-piao; yang, yan; zhu, sheng-mei; xu, hua; yao, yong-xing title: c-x-c motif chemokine 10 contributes to the development of neuropathic pain by increasing the permeability of the blood–spinal cord barrier date: 2020-03-20 journal: front immunol doi: 10.3389/fimmu.2020.00477 sha: doc_id: 15254 cord_uid: xtox2rxs neuropathic pain is among the most debilitating forms of chronic pain. studies have suggested that chronic pain pathogenesis involves neuroimmune interactions and blood–spinal cord barrier (bscb) disruption. however, the underlying mechanisms are poorly understood. we modeled neuropathic pain in rats by inducing chronic constriction injury (cci) of the sciatic nerve and analyzed the effects on c-x-c motif chemokine 10 (cxcl10)/cxcr3 activation, bscb permeability, and immune cell migration from the circulation into the spinal cord. we detected cxcr3 expression in spinal neurons and observed that cci induced cxcl10/cxcr3 activation, bscb disruption, and mechanical hyperalgesia. cci-induced bscb disruption enabled circulating t cells to migrate into the spinal parenchyma. intrathecal administration of an anti-cxcl10 antibody not only attenuated cci-induced hyperalgesia, but also reduced bscb permeability, suggesting that cxcl10 acts as a key regulator of bscb integrity. moreover, t cell migration may play a critical role in the neuroimmune interactions involved in the pathogenesis of cci-induced neuropathic pain. our results highlight cxcl10 as a new potential drug target for the treatment of nerve injury–induced neuropathic pain. neuropathic pain is caused by primary lesions or dysfunction in the nervous system, and it is among the most debilitating forms of chronic pain (1) . its etiology is poorly understood, and this hinders the development of therapeutic and preventative strategies (2) (3) (4) . one thing that is clear is that peripheral nerve injury leads to neuropathic pain by triggering radical changes that affect multiple components of the pain signaling pathway (5, 6) . over the past decade, inflammatory responses after nerve injury have become an important topic in neuropathic pain research, and recent evidence suggests that neuroimmune interactions are involved in the pathogenesis of chronic pain states (7) (8) (9) . accumulating evidence indicates that multiple proinflammatory mediators are released from injured nerve fibers and adjacent immune cells after nerve injury and that these mediators in turn promote central sensitization and behavioral hyperalgesia (5, 10) . furthermore, animal studies have shown that peripheral nerve injury induces circulating immune cells to enter the spinal cord parenchyma, a phenomenon that may contribute to pain-related behaviors during the development of neuropathic pain (11) (12) (13) . because the blood-spinal cord barrier (bscb) is the main structure regulating interactions between the immune system and the central nervous system (cns), it is reasonable to speculate that bscb dysfunction may play a critical role in the migration of circulating immune cells into the spinal cord (14) . however, little is known about the functional states of the bscb in the context of peripheral nerve injury-induced neuropathic pain. researchers do not know how, or even whether, a remote injury can affect bscb integrity. the potential consequences of compromised bscb integrity in terms of spinal cord homeostasis and the development of pathological pain are also unclear. this matter may be illuminated through research into chemokines, a family of small cytokines (i.e., signaling proteins) that are upregulated by primary proinflammatory mediators and tumor necrosis factors (15, 16) . in the cns, chemokines regulate myriad functions including neuronal development, synaptic transmission, and neuroinflammation (17) (18) (19) (20) . recent studies have shown that the c-x-c motif chemokine receptor 3 (cxcr3) and its ligand c-x-c motif chemokine 10 (cxcl10) are involved in the pathophysiology of allergic itches and neuropathic pain (21) (22) (23) . however, the mechanism by which cxcl10/cxcr3 signaling mediates neuropathic pain remains poorly understood. past studies have reported that cxcl10 promotes the entry of peripheral immune cells into the spinal cord (24, 25) . on the other hand, some studies and our recent report have shown that t cell infiltration of the dorsal horn may contribute to the onset of neuropathic and inflammatory hyperalgesia (11, 12, 26, 27) . however, it is unknown whether these processes contribute to hyperalgesia following peripheral nerve injury. in this study, we examined the integrity of the bscb and the migration of circulating immune cells into the spinal cord after chronic constriction injury (cci) of the sciatic nerve, which induces neuropathic pain. we also examined the activation of the cxcl10/cxcr3 signaling pathway after cci. we aimed to elucidate the pathophysiology underlying nerve injury-induced neuropathic pain and to identify potential drug targets for the treatment of neuropathic pain. all animal experiments were conducted in accordance with the arrive guidelines (28) and all relevant chinese laws. the experimental protocol was approved by the research ethics committee of the first affiliated hospital at zhejiang university. all measures were taken to minimize the animals' suffering and to reduce the number of animals used. adult male sprague-dawley rats (87 rats in total, 8 weeks at arrival) weighing 200-300 g were obtained from the animal center of the chinese academy of sciences. they were housed in groups (4 rats/cage) in a temperature-controlled room (22 ± 2 • c) with a 12-/12-h light/dark cycle and ad libitum access to food and water. the rats were randomly divided into the sham surgery and cci groups (n = 5-6 per group for the behavioral test; n = 3-4 for the others). after the baseline was determined, the rats underwent the corresponding procedures on experimental day 0. cci was surgically induced as described in our previous publication (29) and another study (30) . in brief, the rats were anesthetized with intraperitoneal pentobarbital injections (60 mg/kg), and the left sciatic nerve was exposed and isolated. three ligations were placed around the nerve with 4-0 chromic gut sutures (pudong jinghuan co. ltd., shanghai, china). a hindpaw twitch indicated successful nerve constriction. the muscles and skin overlying the sciatic nerve were then closed with sutures. the sham surgery was identical except for the omission of sciatic nerve ligation. all animals received hypodermic penicillin injections (0.5 ml/rat; 96 mg/ml) to reduce the risk of infection. to reduce variability, all surgeries were performed by a single proficient investigator. the rats were anesthetized with an intraperitoneal injection of pentobarbital (60 mg/kg) and perfused with normal saline (ns), followed by 4% ice-cold paraformaldehyde in phosphate buffer. the lumbar 4-5 segments were removed, post-fixed, and dehydrated in 30% sucrose at 4 • c. next, 30-µm freefloating transverse cutting was performed using a freezing microtome at −20 • c. after blocking with 10% goat serum for 2 h at room temperature to reduce non-specific binding, the sections were incubated for 48-72 h with the following primary antibodies: mouse anti-cxcr3 (1:100 dilution, santa cruz biotechnology, dallas, tx, usa), rabbit anti-iba1 (1:400; abcam, cambridge, uk), rabbit anti-gfap (1:500; proteintech, rosemont, il, usa), and rabbit anti-neun (1:400, cell signaling technology, danvers, ma, usa). subsequently, the sections were incubated with an appropriate secondary antibody (fitcconjugated goat anti-rabbit or cy3-conjugated goat anti-mouse, both 1:200 dilution; beyotime, shanghai, china) for 2 h at room temperature in the dark. fluorescence signals were observed using a fluorescence microscope with appropriate filters. after the intraperitoneal injection of an overdose of pentobarbital, the spinal dorsal horn segments (lumbar 4-5) were dissected rapidly and stored in liquid nitrogen. frozen samples were homogenized in lysis buffer containing pmsf (beyotime). after centrifugation at 10,600 rpm and 4 • c for 15 min, the supernatants were collected as protein samples. sample aliquots containing equal amounts of protein were separated via sds-page and transferred onto polyvinylidene difluoride membranes. the membranes were blocked in 5% nonfat milk for 1 h at room temperature, and incubated overnight at 4 • c with rabbit anti-cxcr3 (1:500 dilution; abcam), rabbit anti-cxcl10 (1:1,000; genetex, irvine, ca, usa), or mouse anti-gapdh (1:10,000; proteintech). the membranes were then washed with tbst buffer and incubated with an appropriate secondary antibody (horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit, 1:2,000; beyotime) for 2 h at room temperature. after extensive washing, the densities of labeled protein bands on the blots were detected using an enhanced chemiluminescence reagent (thermo fisher, waltham, ma, usa) and captured using a chemidoc mp system (bio-rad, hercules, ca, usa). to investigate the role of cxcl10/cxcr3 signaling in cciinduced neuropathic pain, rats were randomly divided into the sham surgery (n = 5-6) and cci groups. subgroups of rats that underwent cci were selected to receive intrathecal antibody or saline injections (n = 5). intrathecal administration was performed by lumbar puncture, as described in a previous study (31) . the rats were anesthetized with isoflurane (0.3 ml/rat, baxter international inc.; shanghai, china) in a home-made anesthesia box and placed on a plexiglass tube to broaden the intervertebral spaces. a 20-µl volume of normal saline or a solution containing an anti-cxcl10 antibody (200 ng/rat, proteintech) was injected into the subarachnoid space between the l5 and l6 vertebrae with a 30-gauge needle. an instantaneous and rapid tail-flick indicated a successful puncture. to determine the effect of cxcl10/cxcr3 signaling on the development of hyperalgesia, the first injection of anti-cxcl10 antibody was administered on experimental day 1 after cci. daily follow-up injections were performed until day 14 (for the behavioral experiment), unless the rats were sacrificed earlier for a bscb permeability evaluation or flow cytometry assay. to determine the effect on established hyperalgesia, the injections were administered on experimental days 5-7 after cci. rats that received normal saline injections are herein referred to as the cci + ns group, while those that received anti-cxcl10 antibody injections are herein referred to as the cci + anti-cxcl10 antibody group. hyperalgesia was assessed based on paw withdrawal responses to a calibrated series of von frey filaments (stoelting; wood dale, il, usa) as described in one of our earlier publications (32) . in brief, the rats were individually placed in a chamber (20 cm × 10 cm × 20 cm) in which the floor was a customized platform consisting of a grid of iron wires with 10-mm spacings between wires. the rats were allowed to acclimate to the chamber for ≥30 min before the experiment began. a series of von frey filaments with ascending buckling forces were applied to the midplantar surface of the hindpaw ipsilateral to the site of the cci or sham surgery (herein referred to as the ipsilateral hindpaw) and the hindpaw contralateral to the surgical site (herein referred to as the contralateral hindpaw). each von frey filament was held for 2 s, and the interval between filament applications was 15 s. a brisk withdrawal or flinching of the hindpaw upon filament application was regarded as a positive response, and the filament applications continued until a filament produced positive responses in at least three out of five consecutive applications. the paw withdrawal threshold (pwt) was defined as the buckling force (in grams) of that particular filament. pwt testing was performed by an investigator who was blinded to the rats' group assignments. daily pwt testing began on experimental day 0 (baseline) and continued until day 14 after cci or sham surgery. bscb permeability was assessed with the micromolecular tracer dye sodium fluorescein (naflu; molecular weight, 376 g/mol; sigma-aldrich; st. louis, mo, usa) according to a modified version of a published procedure (33) . in brief, subgroups of rats that underwent sham or cci surgeries were selected to receive intravenous injections of a 10% naflu solution (2 µl per gram of bodyweight) on experimental day 3. after an intraperitoneal injection of pentobarbital (60 mg/kg), naflu was allowed to circulate for 30 min, and the rats' bodies were then intracardially perfused with cold saline to remove intravascular naflu. the l4 and l5 spinal cord segments were removed and used for subsequent analyses aimed at quantifying the amount of naflu extravasated from circulation. after being weighed, the spinal cord samples were homogenized in 1 ml of phosphate-buffered saline (pbs), and a volume of 60% trichloroacetic acid equal to that of the resulting solution was added to precipitate proteins. after being vortexed for 2 min, the samples were cooled for 30 min and centrifuged at 14,000 × g for 10 min. the naflu concentration in the supernatant was measured with a spectrophotofluorometer (excitation wavelength, 440 nm; emission wavelength, 525 nm). a calibration curve was created by assaying solutions with controlled naflu concentrations under identical assay conditions. all experimental measurements were within the detection range established with the calibration curve, which had an r 2 -value of 0.85-0.90. naflu levels were calculated as micrograms per gram of spinal cord tissue. to assess t cell entry into the spinal cord after cci, flow cytometry was used to measure cd3-positivity levels in mononuclear cells extracted from the dorsal horn, as cd3 is a well-known t cell marker (34) . on day 3 of cci, after an overdose intraperitoneal injection of urethane (2 g/kg), the rats' lumbar spinal cord segments were harvested. the dorsal horn tissues ipsilateral to the site of cci or sham surgery were isolated, placed in 0.01-m pbs, and homogenized into single-cell suspensions with a cell strainer. homogenates were washed with 0.01-m pbs, suspended in a 30%/70% discontinuous-gradient percoll solution (sigma-aldrich), and centrifuged at 390 × g for 30 min. mononuclear cells were collected, washed with 0.01-m pbs, and resuspended in a fluorescence-activated cell sorting buffer solution for 30 min at 4 • c. the cells were labeled with fluorescein isothiocyanate-conjugated mouse anti-cd3 antibodies (1:100 dilution; ebioscience, san diego, ca, usa) for 20 min at room temperature, and ≥10,000-cell samples were analyzed with the facscalibur platform running with cellquest software (becton dickinson; franklin lakes, nj, usa) to determine the percentage of mononuclear cells that were cd3-positive. statistical analyses were performed with prism 5.0 software (graphpad software; la jolla, ca, usa). data were expressed as means ± standard errors. behavioral data were analyzed with two-way repeated-measures analysis of variance (anova) followed by bonferroni post-hoc testing. bscb permeability evaluation and flow cytometry data were analyzed with the independent t-test. mann-whitney u-test was used if equal-variance assumptions were not made. statistical significance was defined as p < 0.05. we initially performed immunohistochemistry for specific cell markers to determine the profile of cxcr3 expression in the spinal cord. the results revealed that cxcr3 is expressed abundantly in the spinal cord, where it colocalized with neun (neuron marker), but not with gfap (astrocyte marker) or iba1 (microglia marker; figure 1 ). relative to the sham surgery group rats, the cci group rats had decreased ipsilateral hindpaw pwts on experimental days 3, 5, 7, 10, and 14 (p < 0.001; n = 6, anova, figure 2a ), indicating cci-induced mechanical hyperalgesia. no significant between-group differences were observed for contralateral hindpaw pwts (figure 2a) . the cci group rats also had dramatically elevated lumbar spinal cord naflu concentrations on experimental day 3 (p = 0.029; n = 6, mann-whitney u-test, figure 2b ), which suggests that cci increased bscb permeability. compared to the sham surgery group, we observed increased cxcl10 expression in the ipsilateral spinal cord after cci injury (p = 0.003; n = 3, independent t-test, figure 3a ). the immunohistochemical results revealed that cci induced cxcr3 activation, as shown in figure 3b . this was further confirmed by western blotting, which demonstrated that the cxcr3 protein level was increased from 3 to 7 days after cci (p < 0.01; n = 3, anova, figure 3c ). relative to the rats treated with cci + saline group, rats in the cci + anti-cxcl10 antibody group exhibited a marked increase in pwts from experimental day 1 to day 14 or from day 5 to 7 (p < 0.05, n = 5, anova, figures 4a,b) . this indicates that the anti-cxcl10 antibody attenuated cciinduced mechanical hyperalgesia in both the developmental and established stages. on the other hand, anti-cxcl10 antibody dramatically reduced naflu concentrations in the lumbar spinal cord on experimental day 3 (p = 0.0049; n = 6, independent t-test, figure 4c) . these results suggest that the cxcl10/cxcr3 signaling pathway is involved in the pathophysiology of cci-induced hypernociception and increased bscb permeability. the percentage of ipsilateral dorsal horn mononuclear cells that were cd3-positive was more than two times higher in the cci group rats than in the sham surgery group rats (p < 0.0014; n = 3-4, independent t-test, figures 5a,b) and was lower in the cci + anti-cxcl10 antibodies group rats than in the cci + ns group rats (p < 0.0264; n = 3, independent t-test, figures 5b,c) . these results suggest that cci promotes t cell entry into the spinal cord, and that blocking the cxcl10/cxcr3 signaling pathway counteracts this effect, which provides further evidence for the cxcl10/cxcr3 signaling pathway being involved in the pathophysiology of cci-induced bscb disruption ( figure 5d ). in this study, we investigated the putative link between cxcl10/cxcr3 signaling-mediated bscb disruption and neuropathic pain. as in our previous studies, cci group rats exhibited robust post-operative behavioral hypersensitivity to mechanical stimuli, and this hypersensitivity persisted throughout the experimental period (29, 32) . we also determined that cci induced cxcl10/cxcr3 signaling, increased bscb permeability, and promoted t cell migration into the spinal dorsal horn. moreover, intrathecal administration of anti-cxcl10 antibodies attenuated the rats' behavioral hyperalgesia and reduced the cci-induced increases in bscb permeability and t cell infiltration into the dorsal horn. to the best of our knowledge, it is the first study to report that blocking cxcl10/cxcr3 signaling attenuates the increases in bscb permeability and t cell infiltration of the spinal cord induced by peripheral nerve injury. researchers have attempted to unravel the mechanisms underlying cci-induced inflammatory reactions in the spinal cord, and have made considerable progress (35, 36) . myriad inflammatory mediators in the spinal cord may contribute to the development of neuropathic pain, with interleukin-6, tumor necrosis factor alpha, and c-x-c motif chemokines being possible examples (5, 37, 38) . among the chemokines, cxcl10 has been identified as a potentially important trigger (39, 40) , and our results provide further evidence for its importance. previous studies have indicated that increased bscb permeability is a prerequisite for immune cell infiltration of the spinal cord during the development of neuropathic pain (41) , and we found that blocking cxcl10/cxcr3 signaling with anti-cxcl10 antibodies reduced the bscb's permeability to naflu, which suggests that cxcl10/cxcr3 signaling plays a critical role in cci-induced bscb dysfunction. the chemokines cxcl9, cxcl10, and cxcl11 compose a subfamily of chemokines that bind to cxcr3 and have various roles in nociceptive signaling. past investigations have suggested that cxcl10/cxcr3 signaling contributes to the pathophysiology of neuropathic pain, although spinal cxcl9 and cxcl11 levels do not seem to have important roles in the development of chronic pain (42, 43) . other reports have shown that t cell infiltration of the dorsal horn may contribute to the onset of neuropathic and inflammatory hyperalgesia (10, 12, 39) . our present findings further this line of research by elucidating the spinal cxcr3 protein level increased during days 3-7 after cci as shown by western blotting (**p < 0.01; ***p < 0.001; anova, n = 3). the data are presented as means ± standard errors. gapdh was used as a loading control. anova, analysis of variance; cci, chronic constriction injury; cxcl10, c-x-c motif chemokine 10; contra, contralateral to cci. figure 4 | neutralizing cxcl10 alleviated hyperalgesia and reduced cci-induced bscb disruption. (a,b) hindpaw pwts in cci + anti-cxcl10 antibodies group and cci + saline group rats on various experimental days (*p < 0.05, **p < 0.01, ***p < 0.001; anova, n = 5-6). (c) lumbar spinal cord naflu concentrations in the cci + anti-cxcl10 antibodies group, cci + saline group, and sham surgery group rats on experimental day 3 (*p < 0.01, cci + ns group vs. sham surgery group; mann-whitney u-test, n = 4-6; ## p < 0.01, cci + anti-cxcl10 antibodies group vs. cci + ns group; independent t-test, n = 6). the data are shown as means ± standard errors. anova, analysis of variance; bscb, blood-spinal cord barrier; cci, chronic constriction injury; cxcl10, c-x-c motif chemokine 10; naflu, sodium fluorescein; pwt, paw withdrawal threshold; ns, normal saline. the potential mechanistic role of cxcl10/cxcr3 signaling in the development of neuropathic pain following peripheral nerve injury. our observation that blocking cxcl10/cxcr3 signaling reduced cci-induced t cell migration into the spinal cord is consistent with past reports suggesting that cxcl10/cxcr3 signaling plays a role in the migration of t cells from the periphery into the cns (44) . within the spinal cord, neurons, and glia can secrete cxcl10, which in turn promotes the entry of circulating immune cells into the spinal cord (45) (46) (47) (48) . studies have shown that type 1 t helper cells secrete interferon gamma, and elevated spinal cord interferon gamma levels can induce cxcl10 secretion. cxcl10 in turn increases bscb permeability and promotes the migration of t cells into the spinal cord (44, 49) . this creates a positive feedback system that favors ever-increasing migration of activated t cells into the spinal cord. this implies that blocking the contribution of cxcl10/cxcr3 signaling to increased bscb permeability, as we did by administering anti-cxcl10 antibodies to the cci group rats, may disrupt this positive feedback loop. in conclusion, our study suggests that cxcl10/cxcr3 signaling triggers a positive feedback loop involving bscb permeabilization and t lymphocyte infiltration of the spinal cord. intrathecal administration of anti-cxcl10 antibodies prevents the development of cci-induced neuropathic pain. our findings highlight the cxcl10/cxcr3 signaling pathway as a new potential target for drugs designed to treat chronic pain. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by research ethics committee of the first affiliated hospital at zhejiang university. a new definition of neuropathic pain neuropathic pain. nat rev dis primers dna methyltransferase dnmt3a contributes to neuropathic pain by repressing kcna2 in primary afferent neurons a decade of research on tlr2 discovering its pivotal role in glial activation and neuroinflammation in neurodegenerative diseases the neuropathic pain triad: neurons, immune cells and glia microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential peripheral immune contributions to the maintenance of central glial activation underlying neuropathic pain delayed activation of spinal microglia contributes to the maintenance of bone cancer pain in female wistar rats via p2x7 receptor and il-18 neuroinflammation, bone marrow stem cells, and chronic pain. front immunol γδ t cells modulate myeloid cell recruitment but not pain during peripheral inflammation expression of ccr2 in both resident and bone marrow-derived microglia plays a critical role in neuropathic pain t-cell infiltration and signaling in the adult dorsal spinal cord is a major contributor to neuropathic pain-like hypersensitivity cns-infiltrating cd4+ t lymphocytes contribute to murine spinal nerve transection-induced neuropathic pain the blood-spinal cord barrier: morphology and clinical implications plasma ifn-gammainducible chemokines cxcl9 and cxcl10 correlate with survival and chemotherapeutic efficacy in advanced pancreatic ductal adenocarcinoma ccl26 and ccr3 are associated with the acute inflammatory response in the cns in experimental autoimmune encephalomyelitis chemokine cxcl10 and coronavirus-induced neurologic disease functions of the chemokine receptor cxcr4 in the central nervous system and its regulation by mu-opioid receptors role of chemokines in cns health and pathology: a focus on the ccl2/ccr2 and cxcl8/cxcr2 networks cl-channel is required for cxcl10-induced neuronal activation and itch response in a murine model of allergic contact dermatitis the involvement of iron responsive element (-) divalent metal transporter 1-mediated the spinal iron overload via cxcl10/cxcr3 pathway in neuropathic pain in rats high levels of cerebrospinal fluid chemokines point to the presence of neuroinflammation in peripheral neuropathic pain: a cross-sectional study of 2 cohorts of patients compared with healthy controls up-regulation of chemokine gene transcripts and t-cell infiltration into the central nervous system and dorsal root ganglia are characteristics of experimental european bat lyssavirus type 2 infection of mice blood coagulation protein fibrinogen promotes autoimmunity and demyelination via chemokine release and antigen presentation cd4+ αβ t cell infiltration into the leptomeninges of lumbar dorsal roots contributes to the transition from acute to chronic mechanical allodynia after adult rat tibial nerve injuries bidirectional modulation between infiltrating cd3 + t-lymphocytes and astrocytes in the spinal cord drives the development of allodynia in monoarthritic rats animal research: reporting in vivo experiments: the arrive guidelines n-methyl daspartate receptor subtype 2b antagonist, ro 25-6981, attenuates neuropathic pain by inhibiting postsynaptic density 95 expression a peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man disruption of glial function enhances electroacupuncture analgesia in arthritic rats dexmedetomidine attenuates neuropathic pain by inhibiting p2x7r expression and erk phosphorylation in rats morphological and functional changes of blood-brain barrier in kindled rats with cortical dysplasia cutaneous localization of plasmablastic multiple myeloma with heterotopic expression of cd3 and cd4: skin involvement revealing systemic disease neuroinflammation and the generation of neuropathic pain an inflammation-centric view of neurological disease: beyond the neuron cytokine mechanisms of central sensitization: distinct and overlapping role of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in regulating synaptic and neuronal activity in the superficial spinal cord chemokines, neuronal-glial interactions, and central processing of neuropathic pain chemokine cxcl10/cxcr3 signaling contributes to neuropathic pain in spinal cord and dorsal root ganglia after chronic constriction injury in rats delayed functional expression of neuronal chemokine receptors following focal nerve demyelination in the rat: a mechanism for the development of chronic sensitization of peripheral nociceptors quantifying blood-spinal cord barrier permeability after peripheral nerve injury in the living mouse promoted interaction of c/ebpalpha with demethylated cxcr3 gene promoter contributes to neuropathic pain in mice spinal cxcl9 and cxcl11 are not involved in neuropathic pain despite an upregulation in the spinal cord following spinal nerve injury expression of neuronal cxcl10 induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines/cytokines and enhancement of blood-brain barrier permeability cytokine regulation of cc and cxc chemokine expression by human astrocytes innate stat1-dependent genomic response of neurons to the antiviral cytokine alpha interferon induction of the genes for cxcl9 and cxcl10 is dependent on ifn-gamma but shows differential cellular expression in experimental autoimmune encephalomyelitis and by astrocytes and microglia in vitro spinal ifn-gamma-induced protein-10 (cxcl10) mediates metastatic breast cancer-induced bone pain by activation of microglia in rat models c motif) ligand (cxcl)10 in autoimmune diseases the authors would like to thank editage (www.editage.com) for english language editing. key: cord-269170-9f460xbq authors: kaneko, kazunari; akagawa, shohei; akagawa, yuko; kimata, takahisa; tsuji, shoji title: our evolving understanding of kawasaki disease pathogenesis: role of the gut microbiota date: 2020-07-24 journal: front immunol doi: 10.3389/fimmu.2020.01616 sha: doc_id: 269170 cord_uid: 9f460xbq kawasaki disease (kd) was first described by dr. tomisaku kawasaki in 1967. the etiology of kd has been studied comprehensively but remains largely unknown. the disease seems to result from the interplay of genetic and environmental susceptibility factors with infectious triggers, followed by a subsequent abnormal immune response characterized by increased levels of inflammatory cytokines and chemokines during the acute phase. evidence has mounted to suggest that an imbalance between t helper 17 cells (th17s) and regulatory t cells (tregs) is associated with aberrant immune responses in kd. recent advances in culture-independent techniques for detection and identification of intestinal commensal bacteria enabled the discovery that th17 and treg differentiation are regulated by short chain fatty acids (scfas), in particular butyrate, produced by the gut microbiota. this finding provided a mechanistic link between dysbiosis, defined as changes in the composition of the gut microbiota, and various inflammatory diseases. on this basis, we propose that dysbiosis, with reduced production of scfas leading to imbalances of th17s/tregs, could be involved in the etiology of kd. a pilot study supported this hypothesis, as only fecal concentrations of butyrate were significantly reduced in kd patients among scfas. this evolving perspective prompted us to undertake metagenomic analyses of bacterial dna from the feces of kd patients who were antibiotic-naïve at diagnosis. simultaneous measurements of th17s/tregs in peripheral blood and scfa concentrations in feces would provide valuable information regarding the association between dysbiosis and dysregulated immune responses in kd. kawasaki disease (kd), named after dr. tomisaku kawasaki deceased june 5th, 2020, mainly affects young children between the ages of 6 months and 4 years (1). kd is characterized by persistent fever, bilateral conjunctival congestion, changes of the lips and oral cavity, polymorphous exanthema, changes of peripheral extremities, and acute non-purulent cervical lymphadenopathy (2, 3) . although kd was originally reported to be self-limiting and benign (4) , it is now recognized as a systemic vasculitis with a specific predilection for forming coronary artery lesions. these develop in up to 25% of children with kd who are not treated with intravenous immunoglobulin (5) . coronary artery lesions associated with kd are the most common causes of pediatric heart disease in developed countries. the incidence of kd in the japanese population continues to increase and reached 330 cases per 100,000 children aged ≤4 years per year in 2015 (6) . despite extensive ongoing research into the etiology of kd, the underlying mechanisms of this enigmatic vasculitis are not fully understood (3, 7) . the current paradigm of kd pathogenesis is that the disease results from a pathologically amplified immune response against infectious agent(s) in a genetically and environmentally susceptible child (3) . this paradigm is based on the following observations. first, there is clinical overlap between kd and infectious diseases such as adenovirus and streptococcosis. second, seasonal clustering of kd in the winter and spring mimics that of several viral diseases (8) . third, temporal clusters of epidemics have been reported in japan, the us, canada, and finland (9) . moreover, an outbreak in japan began in tokyo and spread throughout the country over a period of 6 months (10). finally, low incidence in the first 3 months of life suggests at least partial protection from trans-placental antibodies (11) . the low incidence of kd in schoolchildren indicates a potential role of common antigen(s) that most children encounter uneventfully in early childhood and against which they mount an appropriate and protective immune response (12) . however, efforts to find a single unifying microbiological cause of kd have been, to date, unsuccessful. standard microbiological techniques, molecular methods and serological investigations have all failed to identify an etiological agent. it has long been held that infection by one or more widely distributed microorganism(s) might elicit dysregulated immune responses in genetically susceptible children resulting in kd. candidate pathogens include epstein-barr virus (13, 14) , human herpes virus (15), human immunodeficiency virus (16) , human adenovirus (17) (29) , and candida spp. (30) . candida albicans (c. albicans) has recently drawn attention, as administration of caws (water-soluble extracellular polysaccharide from culture supernatants of c. albicans) induces coronary arteritis similar to kd in mice (31) . several reports suggested that c. albicans plays an important role as an infectious trigger of kd (30, 32, 33) . considering the recent pandemic of coronavirus disease 2019 (covid-19), potential links between kd and covid-19 are also deserving of mention (34) (35) (36) (37) . clusters of children presenting with kd-like symptoms have been documented in the uk, us, france, and italy, and some of them were confirmed to have covid-19. it appears that hyperinflammation associated with covid-19 could act as primer for kd development in individuals having genetically or environmentally determined predisposition. however, the specific mechanism is not yet defined (34) . the higher incidence of kd in asian countries suggests a genetic predisposition for acquiring the disease (9) . increased risk in family members of kd patients in japanese populations (38) also hint at genetically determined susceptibility. recent advances have been made in identifying disease-susceptibility genes from genome-wide association studies. candidate genes contributing to kd susceptibility include b-lymphoid kinase (blk) (39) epidemiologic studies have extensively searched for environmental factors that may explain variation and seasonality in kd incidence. surveys from the us and japan have demonstrated that higher precipitation and lower temperatures were associated with higher incidence of kd (45, 46) . an investigation of the role of the early social environment in kd susceptibility in a japanese population found that higher household income, smaller family size, and urbanization were associated with increased kd incidence (47) . this study, however, did not find a significant association between absence of infectious exposures during early life and kd. the prominent role played by the immune system in kd has been confirmed by many studies demonstrating activation of neutrophils and other immune cells as well as overproduction of inflammatory cytokines and chemokines such as tumor necrosis factor-α, interleukin (il)-1, il-2, il-6, and il-8, and monocyte chemotactic protein-1 (3). levels of inflammatory cytokines and chemokines are reported to be elevated during the acute phase of kd. however, the mechanisms responsible for abnormal immune responses and overexpression of inflammatory cytokines remain unclear. several lines of evidence have revealed decreased numbers of regulatory t cells (tregs) and imbalances between t helper 17 cells (th17s) and tregs in acute kd (48) (49) (50) . jia et al. elegantly demonstrated that th17 proportions and cytokine (il-17, il-6, and il-23) levels were significantly increased, while treg proportions and expression of treg transcription factors (e.g., foxp3) were significantly decreased in patients with acute kd (49) . they concluded that th17 expansion and treg depletion were characteristic of acute kd. furthermore, recent studies suggested that treatment efficacy was associated with decreased th17 proportions and increased treg proportions, with both returning closer to a normal range (48, 51) . thus, th17/treg imbalances may contribute to exaggerated immune responses in kd patients. growing evidence suggests that disturbances within intestinal communities of commensal bacteria may lead to illness through aberrant immune system development. while the study of gut microbiology related to human health has a centennial history, recent technological advances have enabled us to explore this field in a more sophisticated manner. current approaches rely primarily on culture-independent methods such as amplification of conserved regions of the 16s rrna gene present in all bacteria (52) . studies using these techniques have demonstrated that an adult humans harbor 100 trillion gut bacteria comprising more than 1,000 different species and approximately 160 species per person per fecal sample (53) . development of the gut microbiota, defined as its colonization by microorganisms, might begin not at birth but in utero. however, the existence of viable bacteria in the womb was recently questioned (54) (55) (56) . the maternal microbiota provides the first microbial inoculum, and from birth, microbial diversity increases and converges toward an adultlike microbiome within the first 3-5 years of life (53) . the composition of the microbiota in childhood depends on various factors including sanitation, mode of delivery, maturity at birth, infant diet, antibiotic use during infancy, immunizations, and environmental factors such as geography or diet (52, 53, 57, 58) . ethnic and genetic factors may also give rise to dysbiosis (59, 60) . the factors that can alter the microbiome are being studied as potential drivers of changing trends in non-communicable diseases. dysbiosis, defined as changes in the composition of the gut microbiota, may be associated with several clinical conditions including obesity and metabolic diseases (61), cancer (62), autoimmune diseases (63), allergy (64), chronic inflammatory bowel diseases (65, 66) , chronic kidney diseases (67) , and autisticspectrum disorders (68) . we also found that dysbiosis was present in children with idiopathic nephrotic syndrome (69) . although research on the mechanism(s) through which dysbiosis impairs human health has just begun, regulation of the gut immune system by microbiota is believed to be involved. experiments with germ-free animals (deficient in commensal microbiota including gut microbiota) have demonstrated that microbial colonization promotes anatomical development of the intestinal epithelium, increases epithelial cell turnover rates, and initiates the maturation of gut-associated lymphoid tissue (70) . both tregs and th17s have received much attention in terms of their role in the gut immune system and its regulation by microbiota (71) . tregs were originally identified as cd4-positive, cd25-positive and foxp3-positive t cells that exerted inhibitory control over immune responses (72) , maintained tolerance to self-antigens, and prevented autoimmune disease (73) . there are several immunosuppressive mechanisms mediated by tregs, including secretion of immunosuppressive cytokines such as il-10 and tgf-β, functional modification or killing of antigenpresenting cells, and cell contact-dependent suppression via cytotoxic t-lymphocyte-associated protein 4 (72) . tregs mainly arise from naïve t cell precursors following stimulation by short chain fatty acids (scfas) such as acetate, propionate or butyrate produced by the gut microbiota (74, 75) . previous studies demonstrated that members of the genus clostridium were potent inducers of treg differentiation through butyrate production (76, 77) and that reduced luminal concentrations of scfas resulted in impaired development of intestinal tregs in germ-free mice (74, 76) . in these mice, reconstitution with commensal bacteria or administration of scfas, especially butyrate, restored treg frequency (76) , supporting the role of bacterial metabolites in treg development. therefore, it has been proposed that a decrease in the relative abundance of butyrateproducing microbes may disrupt mucosal immune homeostasis (78) . the gut microbiota also plays a crucial role in the induction of effector t cell responses in the intestine. th17s are a subtype of cd4-positive t cells specialized for mounting immune responses against fungi and some extracellular bacteria. in addition to il-17a, th17s produce il-17f, il-21, il-22, and il-26 (79) . because il-17 is a potent proinflammatory cytokine that amplifies ongoing inflammation, aberrant regulation of th17s contributes to development of inflammatory and autoimmune disorders. in germ-free mice, the number of th17s was markedly decreased. however, the th17 compartment was restored by reconstitution with conventional microbiota (80), thus indicating a crucial role for gut microbes in th17 development. among commensal bacteria, segmented filamentous bacteria (sfb) are one of the most potent inducers of th17s. colonization of mice by sfb causes abundant accumulation of th17s in the small intestine via enhanced production of serum amyloid a (81, 82) . the existence of human commensal bacteria equivalent to sfb in rodents is probable because mixtures of bacterial strains isolated from fecal samples ulcerative colitis patients could induce th17 development (81) . the gut, the largest interface between microbial factors and the host, contains the largest proportion of bacteria and the largest amount of lymphoid tissue in the body. thus, it was hypothesized that the intestinal environment might be reshaped in patients with kd. indeed, kd patients frequently exhibit gastrointestinal symptoms and complications (83) . the contribution of the gut microbiota to kd has been evaluated in limited numbers of small cohorts using culture-based methods. several studies have been performed to identify the causative microbial agent(s) of kd at disease onset. takeshita et al. showed that the gut microbiomes of kd patients were distinguished by a lack of lactobacilli during the acute phase (84), while nagata et al. isolated both hsp60-producing gram-negative bacteria and gram-positive cocci capable of inducing vβ2 t cell expansion from kd patients (85) . these results indicated that distinctive gut microbes might be involved in the pathophysiology of kd. however, because these studies of the microbiomes of kd patients were carried out using culture-based methods, microorganisms that cannot be cultured, which constitute more than half of the human gut microbiome, would have been overlooked. unlike culture methods, metagenomic analyses can reveal the composition of the intestinal microbiota irrespective of the ability to culture microbes. kinumaki of a metagenomic analysis of feces using culture-independent methods (86) . they collected 28 paired fecal samples from children with kd during the acute and non-acute phases and demonstrated that streptococcus spp., including s. pneumoniae, s. pseudopneumoniae, s. mitis, s. oralis, s. gordonii, and s. sanguinis, were more abundant during the acute phase while the genera ruminococcus, roseburia and faecalibacterium were less abundant. unfortunately, more than half of subjects were treated empirically with antibiotics during the early phase of kd when the fecal samples were collected because clinical and laboratory findings often do not fulfill the diagnostic criteria of kd (2), but are instead suggestive of bacterial infections (87) . as antibiotic administration rapidly perturbs the gut microbiota (88), these results may reflect the effects of antibiotic therapy on the gut microbiota and not dysbiosis associated with kd. here, we would like to focus on a novel viewpoint on the role of the gut microbiota in kd: dysbiosis, defined as changes in the composition of the gut microbiota and caused by various prenatal and postnatal factors that are not necessarily infectious agent(s), might contribute to genetically and environmentally determined predilection for kd. this perspective is illustrated in figure 1 and can be explained as follows: [1] various factors during the in utero and postnatal period drive dysbiosis in young children; [2] dysbiosis results in reduced intestinal production of scfas including butyrate; [3] reduced levels of scfas in the gut cause an imbalance of th17s/tregs; and [4] individuals with th17/treg imbalances develop hypercytokinemia triggered by ubiquitous infectious agents(s), followed by kd (figure 1) . recent observations revealed that viral respiratory infections may alter microbial growth in the gut leading to dysbiosis (89) . in addition, the gut microbiota has been shown to play an important role in regulating the generation of virus-specific cd4-positive and cd8-positive t cells and antibody responses following influenza virus infection (90) . therefore, we hypothesize that young children with dysbiosis are prone to a vicious cycle of hypercytokinemia following infection by viruses. this paradigm for the pathogenesis of kd contrasts with previous hypotheses, which focused on specific microorganisms, toxins, or pathogen-associated molecular patterns (91) (92) (93) . as butyrate has been reported to limit th17 differentiation and promote treg development (94) (95) (96) , we hypothesize that kd-associated dysbiosis might be characterized by lower abundance of butyrate-producing bacteria. interestingly, the genera roseburia and faecalibacterium, which were reported to be less abundant in patients with acute kd by kinumaki et al. (86) , are butyrate-producing bacteria (97, 98) . furthermore, the strong association between kd and allergic diseases (99) (100) (101) in which dysbiosis plays an important role (102) also supports this perspective. recent observations of a potential association between previous antibiotic therapy and development of kd also supports our hypothesis (103) . in this study, the median interval between the final dose of antibiotics and the onset of kd was 2.5 months, which was insufficient time for restoration of the gut microbiota and complete resolution of dysbiosis caused by antibiotic use (104) . to confirm our hypothesis, further investigations involving metagenomic analysis of bacterial dna from the feces of a larger number of antibiotic-naïve patients with kd is clearly needed. it would be worthwhile to compare the proportions of specific microbial species such as butyrate-producing bacteria. simultaneous analysis of th17/treg ratios in peripheral blood and measurement of fecal butyrate concentrations, reflecting the intestinal production of scfas, would also be helpful. we have just begun a project to test our hypothesis with approval from our institutional ethics committee (approval no. 2015127) and parental informed consent. fecal samples will be collected not only from kd patients and healthy children but also from controls with viral infections. this will allow us to specifically characterize dysbiosis in kd because recent observations suggested that viral infection itself may cause dysbiosis (89) . the results of our pilot study of four acute kd patients (median age 1.1 years, range 0.8-2.1 years; 3 boys and 1 girl) and four healthy children (median age 2.1 years, range 1.2-3.7 years, 3 boys and 1 girl) supports our perspective as fecal butyrate concentrations were significantly lower in kd patients (p < 0.05, mann-whitney u test). in contrast, fecal concentrations of acetate, lactate, and propionate did not differ between kd patients and healthy children (figure 2) . the kd patients studied had a median body mass index of 14.7 (range: 13.0-18.1), median maximal body temperatures of 39.0 • c (range: 38.0-39.7 • c), and median maximal c-reactive protein levels of 41 mg/l (range: 20-57 mg/l). all kd patients presented with typical clinical features and fulfilled the diagnostic criteria (2). none reported diarrhea or constipation and none received antibiotics. fecal samples were provided before intravenous immunoglobulin administration. no cases were complicated by coronary artery lesions. what factors perturb the gut microbiota and cause dysbiosis in young children? as shown in figure 1 , both prenatal and postnatal conditions affect the establishment of the intestinal microbiota in infancy. factors that influence the initial colonization of the gut by microbes include maternal factors such as the maternal gut microbiota, vaginal infection or periodontitis as well as postnatal factors such as cesarean delivery, formula feeding, excessive antibiotic use, host genetics, and the environment (105). interestingly, formula feeding (106) and social environment factors such as higher household income, smaller family size, and urbanization were associated with both increased risk of dysbiosis and increased kd incidence (47) . in addition, the peak age of kd onset ranging from 6 months to 4 years corresponds to the critical period for establishment of the gut microbiota during the first 1,000 days of life (107) . we believe that dysbiosis underlies kd and could contribute to genetically and environmentally determined predilections for kd. therefore, kd might be included in the growing list of dysbiosis-associated conditions. if our perspective is confirmed, it would be valuable to investigate whether supplying probiotics starting at birth could reduce the risk of kd in infancy. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by ethics committee of kansai medical university. written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. diagnosis, treatment, and long-term management of kawasaki disease: a statement for health professionals from the committee on rheumatic fever, endocarditis, and kawasaki disease, council on cardiovascular disease in the young diagnosis of kawasaki disease kawasaki disease: an evolving paradigm acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children kawasaki syndrome nationwide epidemiologic survey of kawasaki disease in japan a framework for understanding kawasaki disease pathogenesis seasonality of kawasaki disease: a global perspective epidemiology of kawasaki disease in asia, europe, and the united states nationwide epidemic of kawasaki disease in japan during winter of 1985-86 maternal antibody against toxic shock syndrome toxin-1 may protect infants younger than 6 months of age from developing kawasaki syndrome kawasaki disease: what is the epidemiology telling us about the etiology? epstein-barr virus genome-positive tubulointerstitial nephritis associated with kawasaki disease-like coronary aneurysms could a herpesvirus be the cause of kawasaki disease? pediatric kawasaki disease and adult human immunodeficiency virus kawasaki-like syndrome are likely the same malady adenovirus infection in patients with kawasaki disease association between a novel human coronavirus and kawasaki disease the etiology of kawasaki disease: retrovirus? active or recent parvovirus b19 infection in children with kawasaki disease kawasaki disease and human bocavirus-potential association? toxic shock syndrome toxin-secreting staphylococcus aureus in kawasaki syndrome polyclonal expansion of tcrbv2-and tcrbv6-bearing t cells in patients with kawasaki disease cytokine polymorphisms have a synergistic effect on severity of the acute sickness response to infection kawasaki disease-specific molecules in the sera are linked to microbeassociated molecular patterns in the biofilms mycoplasma pneumoniae: a possible trigger of kawasaki disease or a mere coincidental association? report of the first four italian cases presence in kawasaki disease of antibodies to mycobacterial heat-shock protein hsp65 and autoantibodies to epitopes of human hsp65 cognate antigen ehrlichia chaffeensis and rochalimaea antibodies in kawasaki disease kawasaki disease in european adult associated with serological response to coxiella burneti tropospheric winds from northeastern china carry the etiologic agent of kawasaki disease from its source to japan murine model of kawasaki disease induced by mannoprotein-betaglucan complex, caws, obtained from candida albicans neutrophil activation and arteritis induced by c. albicans water-soluble mannoprotein-beta-glucan complex (caws) caws administration increases the expression of interferon γ and complement factors that lead to severe vasculitis in dba/2 mice covid-19 and kawasaki disease in children kawasaki-like disease: emerging complication during the covid-19 pandemic an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study kawasaki disease linked to covid-19 in children parents with a history of kawasaki disease whose child also had the same disease a genome-wide association study identifies three new risk loci for kawasaki disease common variants in casp3 confer susceptibility to kawasaki disease genome-wide association study identifies fcgr2a as a susceptibility locus for kawasaki disease itpkc functional polymorphism associated with kawasaki disease susceptibility and formation of coronary artery aneurysms transforming growth factor-beta signaling pathway in patients with kawasaki disease variations in orai1 gene associated with kawasaki disease relationship of climate, ethnicity and socioeconomic status to kawasaki disease in san diego county increased kawasaki disease incidence associated with higher precipitation and lower temperatures association of early social environment with the onset of pediatric kawasaki disease th17-and treg-related cytokine and mrna expression are associated with acute and resolving kawasaki disease the t helper type 17/regulatory t cell imbalance in patients with acute kawasaki disease cd25+cd4+ regulatory t cells in patients with kawasaki disease infliximab regulates monocytes and regulatory t cells in kawasaki disease the intestinal microbiome in early life: health and disease the composition of the gut microbiota throughout life, with an emphasis on early life viable bacterial colonization is highly limited in the human intestine in utero lack of detection of a human placenta microbiome in samples from preterm and term deliveries a critical assessment of the "sterile womb" and "in utero colonization" hypotheses: implications for research on the pioneer infant microbiome linking long-term dietary patterns with gut microbial enterotypes impact of diet in shaping gut microbiota revealed by a comparative study in children from europe and rural africa ethnic and diet-related differences in the healthy infant microbiome mhc variation sculpts individualized microbial communities that control susceptibility to enteric infection an obesity-associated gut microbiome with increased capacity for energy harvest gut dysbiosis: a potential link between increased cancer risk in ageing and inflammaging commensal microbiota influence systemic autoimmune responses the human microbiota and its relationship with allergies celiac disease and the microbiome the treatment-naive microbiome in new-onset crohn's disease the gut microbiota and the brain-gut-kidney axis in hypertension and chronic kidney disease microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders gut microbiota dysbiosis in children with relapsing idiopathic nephrotic syndrome lymphoid tissue genesis induced by commensals through nod1 regulates intestinal homeostasis the microbiota and epigenetic regulation of t helper 17/regulatory t cells: in search of a balanced immune system dynamics of peripheral tolerance and immune regulation mediated by treg regulation of the immune system by the resident intestinal bacteria the microbial metabolites, short-chain fatty acids, regulate colonic treg cell homeostasis metabolites from intestinal microbes shape treg commensal microbe-derived butyrate induces the differentiation of colonic regulatory t cells induction of colonic regulatory t cells by indigenous clostridium species decreased abundance of faecalibacterium prausnitzii in the gut microbiota of crohn's disease transcriptional regulators of t helper 17 cell differentiation in health and autoimmune diseases specific microbiota direct the differentiation of il-17-producing t-helper cells in the mucosa of the small intestine th17 cell induction by adhesion of microbes to intestinal epithelial cells induction of intestinal th17 cells by segmented filamentous bacteria kawasaki disease and the pediatric gastroenterologist: a diagnostic challenge characteristic profile of intestinal microflora in kawasaki disease heat shock proteins and superantigenic properties of bacteria from the gastrointestinal tract of patients with kawasaki disease characterization of the gut microbiota of kawasaki disease patients by metagenomic analysis antibiotic use in children with kawasaki disease short-term effect of antibiotics on human gut microbiota respiratory viral infectioninduced microbiome alterations and secondary bacterial pneumonia microbiota regulates immune defense against respiratory tract influenza a virus infection editorial: infection-related immunemediated diseases and microbiota. front pediatrics a presumed etiology of kawasaki disease based on epidemiological comparison with infectious or immune-mediated diseases the gut microbiota-host partnership as a potential driver of kawasaki syndrome metabolites produced by commensal bacteria promote peripheral regulatory t-cell generation short-chain fatty acids regulate cytokines and th17/treg cells in human peripheral blood mononuclear cells in vitro butyrateproducing gut bacteria and viral infections in kidney transplant recipients: a pilot study impact of gut colonization with butyrate-producing microbiota on respiratory viral infection following allo-hct diversity, metabolism and microbial ecology of butyrateproducing bacteria from the human large intestine atopic predilection among kawasaki disease patients: a crosssectional study of 1,187,757 teenagers increased risk of atopic dermatitis in preschool children with kawasaki disease: a population-based study in taiwan atopic diathesis in patients with kawasaki disease the role of gut microbiota in atopic asthma and allergy, implications in the understanding of disease pathogenesis previous antibiotic use and the development of kawasaki disease: a matched-pair casecontrol study the pervasive effects of an antibiotic on the human gut microbiota, as revealed by deep 16s rrna sequencing the microbiome in early life: implications for health outcomes breastfeeding and risk of kawasaki disease: a nationwide longitudinal survey in japan shaping microbiota during the first 1000 days of life we thank edanz group (https://en-author-services.edanzgroup. com/) for editing a draft of this manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 kaneko, akagawa, akagawa, kimata and tsuji. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003914-ss8vdpox authors: wang, shenghua; geng, na; zhou, dong; qu, yi; shi, mengke; xu, yuliang; liu, kangping; liu, yongxia; liu, jianzhu title: oral immunization of chickens with recombinant lactobacillus plantarum vaccine against early alv-j infection date: 2019-10-02 journal: front immunol doi: 10.3389/fimmu.2019.02299 sha: doc_id: 3914 cord_uid: ss8vdpox in this study, a novel oral vaccine of recombinant lactobacillus plantarum (l. plantarum) containing the gp85 protein was explored, and the effects of this vaccine on the prevention of subgroup j avian leukosis virus (alv-j) infection were assessed. in the current study, the gp85 protein of alv-j was expressed on the surface of l. plantarum with the surface-display motif, pgsa, by constructing a shuttle vector pmg36e:pgsa:gp85. surface localization of the fusion protein was verified by western blotting and flow cytometry. subsequently, specific pathogen free hy-line brown layer chickens were orally vaccinated with the recombinant l. plantarum and presented with high levels of serum immunoglobulin g (igg) and secretory immunoglobulin a (siga) titers in bile and duodenal-mucosal fluid. after challenged with alv-j of a 3 × 10(3) 50% tissue culture infective dose (tcid50), serum samples of the chickens were collected and viremia was analyzed. results showed that, compared to the l. plantarum and pbs control group, the recombinant l. plantarum group showed a significant rise in antibody levels after inoculation, and provide improved protection against alv-j according to viremia detection. these results indicate that oral immunization with the recombinant l. plantarum provided an effective means for eliciting protective immune response against early alv-j infection. the j subgroup avian leukosis virus (alv-j) is a subgroup of retrovirus that was first reported and isolated from broiler chickens in the united kingdom in the early 1990s (1) . however, numerous cases of alv-j infection and tumors in commercial layer chickens and broiler breeders have emerged in recent years (2) (3) (4) (5) . this resulted in serious economic losses to breeding farms, including growth retardation, high mortality, tumor production and cost for eradication. therefore, here is an urgent need to take measures to protect chickens against alv-j infection. as we know, alv-j spreads via vertical and horizontal infections, with horizontal transmission accounting for most alv-j infections during the brood period (6) . vertical transmission from broiler breeders to progeny is frequent with alv-j (7); this is due to the current lack of an effective vaccine against alv. consequently, researchers have been trying to explore a vaccine that will increase the proportion of positive antibodies against alv through incubating breeder flocks, thereby reducing the chance of infection from infected chickens and improving the resistance of chickens by increasing alv maternal antibodies to later infection. however, the inactivated vaccine has no significant effect, because the antigenicity of the inactivated virus is commonly destroyed (8) . furthermore, considering the complexity of alv, the existing vaccine did not work well (9, 10) . the glycoprotein (gp) with mr of 85,000 (gp85) is encoded by the envelope (env) gene of alv, and that the gp85 protein forms globular structures on the surface of the virus, and is closely associated with viral neutralization which determines the antigenicity of alv-j (11) . previous researches have shown that the protein could be used as a vaccine to help alv-j infect susceptible cells and induce the production of specific antibodies among infected chickens (12, 13) . therefore, this study set out to search for a live vehicle that could carry the surface protein gene gp85 to prevent the alv-j infection. the mucosal immune barrier is the first line of defense of the immune system, and mucosal inoculation is a practical, noninvasive and efficacious method for the simultaneous induction of systemic and mucosal immune responses (14, 15) . oral vaccination is more favored and convenient than other routes of mucosal vaccination (16) (17) (18) . the edible lactic acid bacteria, lactobacillus plantarum, is well-known as a safe candidate live vector vaccine to deliver antigen (17). many recent investigations have analyzed and validated the potentiality of recombinant lactic acid bacteria for the delivery of heterologous antigens to the mucosal immune system (15, 16, (18) (19) (20) . poly-γ-glutamate synthase a (pgsa) is a constituent protein of bacillus subtilis polyglutamate synthetase (pga), which can be used as a bacterial surface display element to immobilize the enzyme system on the surface of the cell membrane. in view of the characteristics of pgsa protein, it has been applied to various prokaryotic proteins on the surface display, in particular the lactic acid bacteria and other gram-positive receptor strains (21) . these findings provided a theoretical basis for studying the process of immobilizing exogenous proteins on the cell wall of l. plantarum. in the current study, the recombinant l. plantarum harboring pmg36e-pgsa-gp85 was constructed using genetic engineering technology, and then expressed on the surface of l. plantarum. the live recombinant l. plantarum was then used to orally vaccinate chickens. igg and iga antibodies against alv-j, as well as viremia were detected to assess the effect of the recombinant l. plantarum. the alv-j-nx0101 strain, pmd18t-env recombinant vector (containing the gp85 gene), gp85-specific mouse monoclonal anti-body (mab je9), alv-j antibody test kit, and alv p27 antigen enzyme-linked immunosorbent assay (elisa) test kits (idexx usa inc., beijing, china) were donated by prof. zhizhong cui. the alv iga antibody test kit was purchased from lanpai biotechnology company (shanghai, china). the pgsa gene and purified gp85 protein were stored in our laboratory. the pmg36e expression vector was obtained commercially (invitrogen, shanghai, china). l. plantarum hq542228 was purchased from china center of industrial culture collection (cicc) and grown anaerobically at 37 • c, without agitation in mrs broth medium (250 g, hopebiol, qingdao, china). the e. coli strains were cultured at 37 • c with continuous shaking in luria-bertani (lb) medium (250 g, hopebiol). hy-line brown layer chickens (1-day-old) were purchased from the hylan breeder company (shandong province, china) and housed in a specific-pathogen-free environment at the laboratory animal and resources facility, shandong agricultural university. the animals had free access to water and commercial standard pellet diet from liuhe jingwei farming and animal husbandry co., ltd. (taian, china). each chicken was confirmed negative for the alv-j antibody and virus by alv-j-antibody elisa initiation of the experiment. the experimental procedure was approved by the animal care and use committee of the shandong agricultural university and performed in accordance with animal welfare and ethics guidelines (sdaua-2016-037). the primers were designed according to the bacillus subtilis pgsa genes, complete cds sequence published in genebank (number ab016245.1) by primer 5.0 and synthesized in sangon biotech (shanghai, co. ltd). the forward and the reverse primer of the pgsa gene was 5 ′ -cgagctcgcgaactgagctttcatgaaaag-3 ′ and 5 ′ -ct agtctagactatgatcaatatcaaacgtca-3 ′ , containing the saci and xbai sites (underlined) respectively, with the plasmid t7-pgsbca as the template. pcr amplification was performed as follows: 95 • c for 5 min, 30 cycles of 94 • c for 30 s, 58 • c for 30 s and 72 • c for 72 s, and 72 • c for 5 min of final extension. the pcr product was confirmed by dna sequencing. the gp85 gene was amplified using the forward primer (5 ′ -tcatctagagggagttcatctgttg-3 ′ ) and the reverse primer (5 ′ -tccaagcttattagcgcctgctac-3 ′ ) containing the xbai and hind iii sites (underlined), respectively, with the pmd18t-env as the template. pcr amplification was performed as follows: 95 • c for 5 min, 30 cycles of 94 • c for 30 s, 56 • c for 30 s and 72 • c for 1 min, and 72 • c for 5 min of final extension. the pcr product was confirmed by dna sequencing. electroporation was performed according to the method of josson et al. (22) . briefly, electrotransformation was performed in a 0.2 cm cuvette which was subjected to one single electric pulse (2.0 kv/cm, 200 , 25 µf) using a gene pulser (bio-rad, richmond, ca, usa). recombinant strains were selected on mrs medium plates with 5 µg/ml of erythromycin (ery; sigma, st. louis, mo, usa), which was incubated anaerobically at 37 • c for 36 h. the recombinant l. plantarum containing the plasmid of the pmg36e-pgsa-gp85 was identified via antibiotic selection and pcr experiments using the primers pgsa and gp85. sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and western blotting were used to analyze the expression of the target protein, and western blotting was developed using the gp85-specific mouse monoclonal antibody je9 as the primary antibody. horseradish peroxidase (hrp)-conjugated goat antimouse igg (sigma, 1:5,000) was used as the secondary antibody. for flow cytometry, l. plantarum cells were cultured in mrs broth overnight at 37 • c. the cell pellets were sequentially incubated with gp85-specific mouse monoclonal antibody (1:800) and fluorescein isothiocyanate (fitc)-conjugated antimouse igg secondary antibodies (1:5,000; sigma). finally, 3 × 10 4 cells were analyzed with a facs calibur platform (becton dickinson, oxnard, ca, usa) equipped with cellquest software. the layer chickens (120 in total) were randomly divided into three groups: control group, recombinant l. plantarum group and natural l. plantarum group. each group contained 40 chickens that were kept in separated isolators receiving filtered positive-pressure air. the recombinant l. plantarum group was immunized intragastrically with recombinant strains of l. plantarum (harboring plasmid pmg36e-pgsa-gp85), while the chickens in the control and negative control group were inoculated with pbs and natural l. plantarum, respectively. the suspensions of strains for administration were prepared as follows: recombinant strains cultured overnight were collected by centrifugation at 3,000 × g for 10 min, washed three times with sterile pbs, and then resuspended in sterile pbs to a concentration of 5 × 10 9 cfu/ml (400 µl/chicken). the control group and negative control group received equal doses of sterile pbs and natural l. plantarum. the immune protocol was administered on five consecutive days (days 1-5). a booster immunization was given between days 15 to 19, the second booster was given between days 29 to 33 and the third booster was given between days 43-47. blood samples were collected from the wing vein on days 1 (before immunization), 7, 14, 28, 35, 42, and 49. blood samples without additives were centrifuged and the serum was stored at −80 • c for subsequent analysis. to obtain bile and intestinal lavages samples, 3 chickens were randomly selected from each group, and were killed on day 1 (pre-immune), 7, 14, 28, 35, 42, and 49. moreover, using a previously described method based on that of wu and russell (23) , intestinal lavage fluids were obtained by flushing the excised small intestine with 3 ml of pbs containing 50 mm ethylenediaminetetraacetic acid (edta) and 0.1 mg /ml of soybean trypsin-chymotrypsin inhibitor (sigma). the contents were collected and retained on ice for processing, whereupon the fluids were vortexed and centrifuged at 650 × g for 10 min at 4 • c. a 30 µl volume of 100 mm phenylmethylsulfonyl fluoride (pmsf, sigma) was added to the supernatants before they were vortexed and spun at 27,000 × g for 20 min at 4 • c. a further 20 µl of pmsf, 100 µl of fetal bovine serum (fbs), and 20 µl of 1% sodium azide (sigma) were added to the supernatants before they were dispensed into aliquots and then stored at −80 • c. the anti-alv-j antibody in serum, bile and duodenal lavages were analyzed by elisa. briefly, the titers of igg were tested using an alv-igg elisa kit (idexx usa inc., beijing, china) according to the requirements of the specification. the optical density (od) of the igg were measured at 450 nm and determined by the sample-to-positive (s/p) method following the formula below: s/p = [(mean of od 450nm of sample-mean of od 450nm of negative control)]/[(mean of od 450nm of positive control-mean of od 450nm of negative control)]. the samples from each group were tested in three parallel trials, and the s/p values of any sample higher than 0.6 were considered as alv-j antibody positive. furthermore, the commercial alv-iga antibody test kit (lanpai bio., shanghai, china) was used to detect the positive ratio of the anti-alv-j iga in serum, bile and duodenal lavages according to the manufacture's protocol. the od of the iga was measured at 450 nm and determined by the critical value (cut off) method following the formula below: cut off = (mean of od 450nm of sample) + 0.15. the samples from each group were tested in three parallel trials, and the mean of od 450nm of each sample higher than cut off were considered to be alv-j antibody positive. in our study, the test was considered effective if the mean of the negative control wells was <0.10 and the mean of the positive control was >1.00. indirect elisa assay was used to assess the titers of anti-gp85 protein antibody. a 96-well elisa plate was coated overnight with gp85 protein of appropriate concentration (4 µg/ml) and kept at 4 • c. after blocking of the plates, the samples of serum, bile and duodenal lavages were used as the primary antibody and incubated at 37 • c for 1 h. then hrp-conjugated goat antichicken siga at a dilution of 1:2,000 (sigma) at 37 • c for 1 h was used to detect bound antibodies. following washing the plate thice with pbs-0.05% tween 20, tetramethylbenzidine (qiagen, germany) was added as the colorimetric substrate. finally, the optical density (od) was measured at 450 nm. alv-j nx0101 strain was selected as the challenge virus and transfected into chicken embryo fibroblasts (cef) for amplification and detection of virulence. the tcid50 of alv-j was judged following the method described by fadly (24) . all of the chickens were challenged intraperitoneally with 3 × 10 3 tcid50 of the alv-j nx0101 strain at day 50. the plasma samples from the chickens were aseptically collected and then inoculated into the cef cells in 24-wells plates. the cell supernatant of viremia was collected weekly to check for the presence of the virus using alv p27 antigen elisa test kits (idexx usa inc.). the relative antigen titer level was determined by calculating the s/p ratio using the formula mentioned above. the samples from each group were tested in triplicate, and plasma samples with s/p ratios higher than 0.2 were considered virus-positive. statistical analysis was performed using the spss software (version 11.0, spss inc., usa). meanwhile, one-way anova was used to identify the significant values. all values were expressed as mean ± standard error of the mean (sem). all measurements were replicated three times. the differences were considered significant when the p-value was <0.05. the amplification of the gp85 gene from the pmd18t-env recombinant vector (expected size 930 bp) is shown in figure 1a . the amplification of the pgsa gene from the t7-pgsbca recombinant vector (expected size 1,143 bp) is shown in figure 1b . the recombinant gene pgsa-gp85 was expressed in l. plantarum, and confirmed by sds-page (figure 2a) and western blotting using the gp85-specific mab (figure 2b) . flow cytometry was used to analyze the cell surface display of l. plantarum (figure 3a) . the cell surface-displayed pgsa-gp85 was performed using mouse je9 antibody as the primary antibody and fitc-conjugated goat anti-mouse igg as the secondary antibody. l. plantarum and l. plantarum cells harboring the plasmid pmg36e-gp85 were used as control for flow cytometry. the cells displaying pgsa-gp85 showed a significantly greater intensity of fluorescence signals than the control cells. this result is consistent with the data shown in figure 3b . one week after vaccination, the bw of chickens in recombinant l. plantarum group were significantly higher than control group at 49, 63, 70, 77, and 84 d (p < 0.05). meanwhile, bw of chickens in the natural l. plantarum group were also higher than control, but without significant differences to the recombinant group. results showed that the recombinant pmg36e-pgsa-gp85 l. plantarum significantly triggered specific igg and iga antibodies against alv-j, and enhanced the levels of igg and siga compared to the control group (figures 4, 5) . the level of systemic igg in serum increased significantly after oral inoculation as shown in figure 5 . the sera of 15 chickens in the orally immunized recombinant l. plantarum group were taken at random intervals each weekly after day 1 (pre-immune) and then analyzed by elisa. a low level of igg titers was detected after the primary immunization. the igg level increased slowly after the first booster immunization, and increased statistically after the second booster immunization on day 21 (3 weeks, p < 0.05). peak igg titers were reached after the third booster immunization on day 35 (5 weeks). however, no significant specific antibodies were observed in the negative l. plantarum group until day 49 the virus was challenged. samples were taken from three chickens in each group at random, at weekly intervals, from day 1 (pre-immune) until day 42 (6 weeks) and then analyzed by elisa. the specific alv-j iga antibodies in bile, duodenal lavages and sera were detected during each test period ( table 1) . the siga level increased consistently after oral inoculation in all samples of the recombinant l. plantarum group as shown in figure 5 . results of the siga levels in bile ( figure 5a) showed that, compared to the control groups (groups 1 and 3), the immunized group (group 2) started to increase rapidly after 7 days of the first immunization (p < 0.01). the profile showed a steady increase before day 21 (3 weeks) and achieved the highest level on the 35th day after the 3rd booster immunization (5 weeks, p < 0.01). there were no significant differences between the l. plantarum group and pbs control group (p > 0.05). the results of the siga level in duodenal lavages and sera (figures 5b,c) showed that, compared to the control groups (group 1 and 3), there was a significant increase in the immunized group (group 2) from the 21st day after 2nd booster immunization. it also reached the highest level on the 35th day (5 weeks, p < 0.01). in addition, there was no significant difference between the l. plantarum group and pbs control group (p > 0.05). therefore, these findings indicated that the recombinant pmg36e-pgsa-gp85 l. plantarum significantly enhanced the siga antibody response and produced local mucosal immune responses. viremia were determined weekly after challenge until the 77th day, the positive viremia ratios were calculated, and the results were shown in table 2 . the positive ratio of viremia in the natural l. plantarum+alv-j and pbs+alv-j groups were obviously higher than those in the recombinant l. plantarum+alv-j group. before 2005, alv-j was widely recognized and reported in many regions of the world; it was predominantly prevalent in white feather broiler chickens (1, 25, 26) . however, in recent years, alv-j has also occurred in commercial layer flocks (27) , and it has become a serious threat to local breeder flocks. for many pathogens, initial infection occurs in the mucosa of animals. chickens with alv-j can infect other healthy individuals via their excrements or cloacal secretions. therefore, our study was devoted to seeking an effective vaccine that would protect chickens from alv-j infection. compared to routine styles of antigen delivery, mucosal immunization still offers some advantages including those of being more convenient, lower cost, and less stress (14, 15) . in addition, lactic acid bacteria have been proposed as a live vehicle for the delivery of exogenous antigen proteins for mucosal immunization or for other therapeutic molecules. the biological functions of lactic acid bacteria are effectively combined with the immunogenicity of exogenous antigen genes (19, 20, 28) . l. plantarum, a lactic acid bacteria, that can be used as a probiotic for animal gastrointestinal tracts, has many functions, such as maintenance of intestinal flora, enhancement of immunity, and promotion of nutrient absorption. l. plantarum can also act as a potential delivery vehicle for mucosal vaccines because it is generally regarded as safe and is able to protect antigens from premature disintegration and degradation in animal gastrointestinal tract (19, 20) . however, the inherent immunogenicity of vaccine antigens in many cases is insufficient to elicit an effective immune response, which led to the suggestion that displaying antigens on the bacterial surface maybe a practical method for enhancing antigen immunogenicity (29) . to this end, alv-j gp85 protein was expressed on the surface of l. plantarum using the surface-display motif, pgsa, which was adopted from the poly-γ-glutamic synthetase complex. by using this approach, we hoped to address the aforementioned challenges. in the present study, the results indicated that pgsa effectively displayed the gp85 protein on the surface of l. plantarum (figures 3, 4) . l. plantarum as a carrier can successfully express exogenous antigens, resist damage from the extreme gastrointestinal environment, and significantly enhance the immunogenicity of pgsa-gp85 antigens. thus, it plays a double role of adjuvant and vector of the recombinant vaccine. because alv-j is able to cause growth retardation in infected chickens, body weight is considered as a valuable indicator to evaluate the protective effect of our established recombinant vaccine (30, 31) . as expected, in our study, body weight in the recombinant l. plantarum group was significantly higher than that in the control group (p < 0.05) at 49, 63, 70, 77, 84 d, indicating that vaccination with recombinant l. plantarum can prevent body weight loss caused by alv-j infection in chickens. this study described the mucosal and systemic immune responses specific to alv-j induced by an orally administered recombinant l. plantarum vaccine. efficient mucosal immunity is mediated predominately by secretory iga (32, 33) . some studies also show that recombinant l. plantarum can induce an intestinal mucosal immune response, promote the secretion of intestinal mucosal siga, and strengthen intestinal mucosal immunobarrier function (34) . iga is the predominant antibody on the mucosal surface, as it is produced locally at a level that exceeds those of all other immunoglobulins (35, 36) . therefore, an efficient gp85 oral vaccine can induce a specific mucosal immune response of iga (table 1) . in our study, we found that the siga levels in bile, duodenal lavages, and sera of the vaccinated chickens were dramatically increased after the first immunization compared to the natural l. plantarum and pbs groups, it reached the highest level on the 35th day after the third booster immunization (p < 0.01). these results indicated that recombinant pmg36e-pgsa-gp85 l. plantarum effectively enhanced the siga antibody response and produced strong local mucosal immune responses ( figure 5) . thus, l. plantarum could act as an excellent mucosal adjuvant and vector of recombinant vaccines (37) . moreover, mucosal delivery of vaccines should also elicit specific immunity in systemic lymphoid tissues because most infections that gain entrance through mucosal surfaces will become systemic (38) (39) (40) , and in this study recombinant l. plantarum can induce high igg in sera. igg is the main antibody produced by the humoral immune response, and it plays an important role against infection, including neutralizing toxins and internal conditioning in body defense mechanisms (41) . some researchers have succeeded in detecting the specificity of igg by using recombinant lactic acid bacteria to express exogenous genes (42, 43) . igg is an important indicator for assessing the immunity efficiency of exogenous vaccines. in our study, results showed that igg titers of the recombinant l. plantarum group were significantly higher than those in the natural l. plantarum and control group from day 7 to 49 (figure 4) . in addition, further study of alv viremia was performed to verify the protection created by this vaccine. compared to the natural l. plantarum and control groups, after challenge, better protection was observed in the recombinant l. plantarum group against alv-j. in conclusion, our study demonstrated that the alv-j gp85 protein was present on the surface of the non-pathogenic l. plantarum, this can be administered orally to animals, tolerate gastric acidity, elicit both mucosal and systemic immune responses, and effectively alleviate viremia. a novel subgroup of exogenous avian leukosis virus in chickens host range of rous sarcoma virus pseudotype rsv(hprs-103) in 12 avian species: support for a new avian retrovirus envelope subgroup, designated detection and localization of naturally transmitted avian leukosis subgroup j virus in eggtype chickens by in situ pcr hybridization molecular epidemiology of avian leukosis virus subgroup j in layer flocks in china isolation and characterization of emerging subgroup j avian leukosis virus associated with hemangioma in egg-type chickens epidemiological and pathological studies of subgroup j avian leukosis virus infections in chinese local "yellow" chickens avian leukosis virus subgroup j infection profiles in broiler breeder chickens: association with virus transmission to progeny pathogenicity of a viral strain (rpl 12) causing avian visceral lymphomatosis and related neoplasms. iii influence of host age and route of inoculation shedding of lymphoid leukosis virus in chickens following contact exposure and vaccination influence of strain, dose of virus, and age at inoculation on subgroup j avian leukosis virus persistence, antibody response, and oncogenicity in commercial meat-type chickens sequence of host-range determinants in the env gene of a full-length, infectious proviral clone of exogenous avian leukosis virus hprs-103 confirms that it represents a new subgroup (designated j) liposomes containing recombinant gp85 protein vaccine against alv-j in chickens gp85 protein vaccine adjuvanted with silica nanoparticles against alv-j in chickens overview of the mucosal immune system the mucosal immune system: from fundamental concepts to vaccine development the common mucosal immune system and current strategies for induction of immune responses in external secretions vaccination strategies for mucosal immune responses lactic acid bacteria as antigen delivery vehicles for oral immunization purposes comparison of eight lactobacillus species for delivery of surface-displayed mycobacterial antigen display of alpha-amylase on the surface of lactobacillus casei cells by use of the pgsa anchor protein, and production of lactic acid from starch characterization of a gram-positive broad-hostrange plasmid isolated from lactobacillus hilgardii induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin b subunit a laboratory manual for the isolation and identification of avian pathogenes detection of avian leukosis virus subgroup j using the polymerase chain reaction isolation and some characteristics of a subgroup j-like avian leukosis virus associated with myeloid leukosis in meat-type chickens in the united states tumors associated with avian leukosis virus subgroup j in layer hens during 2007 to 2009 in china interaction of lactic acid bacteria with the gut immune system surface display of n-terminally anchored invasin by lactobacillus plantarum activates nf-κb in monocytes high-level, inducible gene expression in lactobacillus sakei and lactobacillus plantarum using versatile expression vectors dynamic co-evolution and interaction of avian leukosis virus genetic variants and host immune responses. front microbiol comparison of the immune responses induced by local immunizations with recombinant lactobacillus plantarum producing tetanus toxin fragment c in different cellular locations immunoglobulin a-deficient mice exhibit altered t helper 1-type immune responses but retain mucosal immunity to influenza virus postbiotic l. plantarum rg14 improves ruminal epithelium growth, immune status and upregulates the intestinal barrier function in post-weaning lambs b cell superantigens in the human intestinal microbiota 11-function of mucosal immunoglobulins a2-ogra, pearay l immunogenicity in swine of orally administered recombinant lactobacillus plantarum expressing classical swine fever virus e2 protein in conjunction with thymosin α-1 as an adjuvant lactobacilli as live vaccine delivery vectors: progress and prospects mucosal vaccine made from live, recombinant lactococcus lactis protects mice against pharyngeal infection with streptococcus pyogenes mucosal immunization with surface-displayed severe acute respiratory syndrome coronavirus spike protein on lactobacillus casei induces neutralizing antibodies in mice structure and function of human and murine receptors for igg intragastric administration of lactobacillus casei expressing transmissible gastroentritis coronavirus spike glycoprotein induced specific antibody production induction of immune responses in mice after intragastric administration of lactobacillus casei producing porcine parvovirus vp2 protein jl and yl designed the study. sw, yx, and ng contributed analytic tools. sw, yq, and ng analyzed the data. jl, kl, and ms wrote the paper. dz, yl, and ms revised the manuscript. all authors reviewed the results and approved the final version of the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 wang, geng, zhou, qu, shi, xu, liu, liu and liu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-262375-1ex2ow07 authors: qun, sen; wang, yulan; chen, jun; huang, xiang; guo, hui; lu, zhaohui; wang, jinquan; zheng, changcheng; ma, yan; zhu, yuyou; xia, daqing; wang, yinzhong; he, hongliang; wang, yong; fei, mingming; yin, yihong; zheng, mao; xu, yehong; ge, wei; hu, fuyong; zhou, jian title: neutrophil-to-lymphocyte ratios are closely associated with the severity and course of non-mild covid-19 date: 2020-09-02 journal: front immunol doi: 10.3389/fimmu.2020.02160 sha: doc_id: 262375 cord_uid: 1ex2ow07 background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection is spreading worldwide. measuring the prevention and control of the disease has become a matter requiring urgent focus. objective: based on coronavirus disease 2019 (covid-19) clinical data from wuhan, we conducted an in-depth analysis to clarify some of the pathological mechanisms of the disease and identify simple measures to predict its severity early on. methods: a total of 230 patients with non-mild covid-19 were recruited, and information on their clinical characteristics, inflammatory cytokines, and t lymphocyte subsets was collected. risk factors for severity were analyzed by binary logistic regression, and the associations of neutrophil-to-lymphocyte ratios (n/lrs) with illness severity, disease course, ct grading, inflammatory cytokines, and t lymphocyte subsets were evaluated. results: our results showed that the n/lrs were closely related to interleukin (il)-6 and il-10 (p < 0.001, p = 0.024) and to cd3(+) and cd8(+) t lymphocytes (p < 0.001, p = 0.046). in particular, the n/lrs were positively correlated with the severity and course of the disease (p = 0.021, p < 0.001). compared to the values at the first test after admission, il-6 and il-10 were significantly decreased and increased, respectively, as of the last test before discharge (p = 0.006, p < 0.001). more importantly, through binary logistic regression, we found that male sex, underlying diseases (such as cardiovascular disease), pulse, and n/lrs were all closely related to the severity of the disease (p = 0.004, p = 0.012, p = 0.013, p = 0.028). conclusions: as a quick and convenient marker of inflammation, n/lrs may predict the disease course and severity level of non-mild covid-19; male sex, cardiovascular disease, and pulse are also risk factors for the severity of non-mild covid-19. at present, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection (1) is largely under control in china, but it is still spreading rapidly worldwide, seriously threatening and damaging global health (2) . the prevention and control of coronavirus disease 2019 (covid-19) is currently the most urgent issue facing the globe. many studies have confirmed that inflammatory storms are the key pathological basis determining the clinical symptoms, course, and prognosis of covid-19 (3) . according to the diagnosis and treatment protocol for covid-19 (seventh edition) (4), the pathologic anatomy of patients with covid-19 revealed that the lungs, spleen, cardiovascular system, liver, kidney, and other organs were markedly infiltrated by a large number of inflammatory cells, which resulted in functional damage and functional failure. many studies have confirmed that the impairment of immune function directly affects the course and prognosis of covid-19 (5) . by autopsy, a decrease was observed in the numbers of cd4 + and cd8 + t cells in the spleen and lymph nodes, and denaturation and necrosis of the spleen and lymph nodes were also found, in addition to the proliferation of spleen macrophages. it has been speculated that t lymphocytes and macrophages may also be important targets for covid-19 (6) . severe pneumonia and acute respiratory distress syndrome are the worst outcomes in patients with covid-19, resulting in cytokine release syndrome and multiorgan failure; the role of il-6 in the pathological development of patients with covid-19 has been a focus of research (7, 8) , but the function of il-6 in the course of the disease is still controversial (9) (10) (11) (12) . lymphocytopenia is a common feature of covid-19 and may be a key factor associated with disease severity and mortality (13) . it is critically important to identify at the early stage of the infection those patients who would be prone to developing the most adverse effects. based on clinical data from wuhan (which had a severe outbreak of covid-19 early in the pandemic), we conducted an in-depth analysis to clarify some of the pathological mechanisms of the disease and identify simple measures to predict its severity early on. this study was approved by the research ethics committee of the first affiliated hospital of the university of science and technology of china (ustc) and was therefore performed in accordance with the ethical standards laid down in the 1964 declaration of helsinki and its later amendments. all participants gave their informed consent prior to their inclusion in this study. consent was obtained directly from each patient or from a family member or other legal guardian. if a patient was considered incapable of giving informed consent, a family member or other legal guardian was contacted to give informed consent on behalf of the patient. all patients or their close relations were informed of the purpose of the study and signed informed consent. the consent procedures were approved by the ethics committee. we collected the clinical data of all inpatients with covid-19 in the cancer center of wuhan union hospital from february 2, 2020 to march 17, 2020. the diagnostic criteria for suspected cases of covid-19 pneumonia included the following (4): (1) epidemiological history included: (i) pre-onset history of travel or residence in wuhan or other areas where local cases continue to spread, (ii) fever or respiratory symptoms in patients who were from wuhan city or other areas with continued local spread and were exposed 14 days before onset, and (iii) a cluster or epidemiological association with covid-19 infection; (2) the following clinical features were present: (i) fever, (ii) imaging features indicative of pneumonia, and (iii) a normal or decreased total number of white blood cells in the early stage of the disease or a reduced lymphocyte count; or (3) if there was any epidemiological history, a suspected case could be diagnosed in the presence of any two of the listed clinical manifestations. the inclusion criteria were as follows: (1) the case met two criteria: (i) suspected covid-19 and (ii) detection of covid-19 nucleic acids by real-time fluorescent rt-pcr using sputum, pharyngeal swabs, or lower respiratory tract exudate; (2) nonmild severity. the exclusion criteria were as follows: mild cases were defined according to the national protocol: "the clinical symptoms were mild, and there was no sign of pneumonia on imaging" (4) . given the state of emergency at the time, only patients with moderate or severe cases were admitted to the cancer center of wuhan union hospital; patients with mild symptoms were admitted to the mobile cabin hospital (14) . clinical data on 230 patients with covid-19 pneumonia in the isolation ward of the cancer center of wuhan union hospital who met the above criteria were collected from february 2 to march 17, 2020. all confirmed patients were clinically classified according to the "novel coronavirus pneumonia treatment scheme of the national health commission of the people's republic of china (version 7)" at the time of admission (4), as follows: (1) general type: symptoms such as fever and respiratory tract complaints were present, and manifestations of pneumonia could be seen on imaging; (2) serious type: any of the following criteria were met: (1) respiratory distress, respiratory rate (rr) ≥ 30 times/min; (2) resting oxygen saturation ≤ 93%; or (3) arterial partial oxygen pressure (pao 2 )/oxygen absorption concentration (fio 2 ) ≤ 300 mmhg (1 mmhg = 0.133 kpa); (3) critical type: any of the following criteria were met: (1) respiratory failure and a need for mechanical ventilation, (2) shock, or (3) a combination of factors with other organ failure requiring icu care. the course of disease was defined as the time (days) between the onset of clinical symptoms of covid-19 and the time of discharge from the hospital (symptoms recovery). all images were independently read by three senior radiological specialists. the location, shape, number, and size of the abnormalities on chest cts were carefully observed and recorded. in case of discordant reading, consensus was reached during another reading session. ct grading was defined according to the "novel coronavirus pneumonia treatment scheme of the national health commission of the people's republic of china (version 7)"and chinese expert consensus (4, 15) . the confirmation of covid-19 was achieved by real-time reverse transcription polymerase chain reaction (rt-pcr) testing of throat sputum, pharyngeal swabs, and lower respiratory tract exudates of suspected patients. following the recommendation of the china national centre for disease control, two target genes were targeted as described previously (16) , namely, open reading frame 1ab (orf1ab) and nucleocapsid protein (n), and these genes were simultaneously amplified and tested during the real-time rt-pcr assay. the primers for target 1 (orf1ab) were forward primer ccctgtgggttttacacttaa; reverse primer acgattgtgcatcagctga; and probe 5 -fam-ccgtctgcggtatgtggaaaggttatgg-bhq1-3 . the primers for target 2 (n) were forward primer ggggaact tctcctgctagaat; reverse primer cagacattttgct ctcaagctg; and probe 5 -famttgctgctgcttgacag att-tamra-3 . a cycle threshold value (ct value) less than 37 was defined as a positive result, and a ct value exceeding 40 was defined as a negative test. venous blood was taken in the emergency department at admission and sent to the laboratory for examination of routine blood parameters and inflammatory cytokines. however, t lymphocyte subsets and some biochemical indicators were obtained by testing fasting blood the morning after admission. serum inflammatory cytokines were detected by enzyme-linked immunosorbent assays for il-2, il-4, il-6, il-6, il-10, and tumor necrosis factor-α (tnf-α). the tests were carried out strictly according to the instructions, and the test kits were purchased from ebioscience company (epx650-16500-901). in order to quantify t lymphocyte subsets, 100 µl of whole blood was incubated in 900 µl of tris-nh4cl buffer (thermo fisher scientific) at room temperature for 5 min to lyse erythrocytes. after two washes with phosphate-buffered saline (pbs), cells were incubated with cd3-apc, cd4-percp, and cd8-fitc antibodies (5 µg/ml each, all from bd biosciences) for 15 min on ice. after another two washes with pbs, cells were resuspended in 500 µl of pbs. samples were analyzed on a bd facs canto plus flow cytometer. among all collected events, single events were gated between forward scatter (fsc)-a and fsc-h. cell debris was excluded, and intact cells were then gated from single events based on fsc-a and side scatter (ssc). each cell population was then detected based on antibody staining. all statistical analyses were conducted with statistical package for the social sciences version 16.0 (spss, company, chicago, il, united states). all continuous data were tested for normal distributions using the kolmogorov-smirnov test. normally distributed variables were described as the mean ± standard deviation (m ± sd), and non-normally distributed variables were presented as the median and the interquartile range [median (iqr)]. categorical variables were expressed as constituent ratios and percentages in each category. one-way anova or kruskal-wallis analysis was applied to continuous variables, and the chi-square test or fisher's exact test was applied to categorical variables. differences in cytokine and t lymphocyte subsets tested twice for each patient were assessed by paired t-test or wilcoxon signed rank test. correlation analysis was used to determine the relationship between n/lrs and inflammatory cytokines, t lymphocyte subsets, severity of illness, course of disease, or ct grading. n/lrs in different patient condition groups were tested by kruskal-wallis analysis and the jonckheere-terpstra trend test. binary logistic regression analysis was used to test the risk factors for the severity level of patients with covid-19. a p value < 0.05 was considered statistically significant. (1) clinical characteristics of patients with covid-19. we collected 230 patients with covid-19; 99 (43%) patients were male, the average age was 62 (16) years, and the total course of disease was 27 (18) days. the distribution of severity categories was as follows: 190 (82.6%) patients had the general type, 35 (15.2%) patients had the serious type, and five (2.2%) patients had the critical type. according to ct grading, 99 (43.0%) patients were in the early stage, nearly half (106 patients, 46.1%) were in the progressive stage, and 25 (10.9%) were in the critical stage. the underlying (comorbid) diseases of the patients were as follows: 10 (4.3%) patients had respiratory system diseases, 51 (22.2%) patients had immune-related diseases, 79 (34.3%) patients had cardiovascular system diseases, 15 (6.5%) patients had liver or kidney diseases, and 127 (55.2%) patients had other comorbidities. the patients' initial symptoms were as follows: 154 (67.0%) patients had fever, 109 (47.4%) patients had respiratory system symptoms, 45 (19.6%) patients had general symptoms, nine (3.9%) patients had cardiovascular system symptoms, 21 (9.1%) patients had digestive system symptoms, 16 (7.0%) patients had nervous system symptoms, and five (2.2%) patients were asymptomatic. the damage to physiological systems was as follows (multiple system damage were directly caused by covid-19): 111 (48.3%) patients had respiratory system damage, 14 (6.1%) patients had cardiovascular system damage, 20 (8.7%) patients had liver function damage, 22 (9.6%) patients had renal function damage, 21 (9.1%) patients had digestive system damage, 16 (7.0%) patients had nervous system damage, and 14 (6.1%) patients had muscle damage. a total of 175 (76.1%) patients had general symptoms. of the patients, three (1.3%) were asymptomatic. the variables with statistical significance were identified by univariate analysis. these variables were sex (male) (p = 0.005), underlying disease (cardiovascular system diseases) (p = 0.019), multisystem damage (muscle) (p = 0.047), course of disease (p = 0.007), pulse (p = 0.049), systolic pressure (p = 0.044), l (p = 0. 014), and n/lrs (p = 0. 029) (see table 1 ). (2) there were significant differences between the first test and last test regarding the cytokines and classification of leukocytes. we collected the data regarding the cytokines and t lymphocyte subsets and classification of leukocytes at the time of admission (first test) and the time of discharge (last test), and there were statistically significant differences in cytokine levels between admission and discharge. for example, il-6 and il-10 were significantly decreased and increased, respectively (p = 0.006, p < 0.001). the il-6/il-10 ratio was also significantly different (p < 0.001). additionally, there were statistically significant differences in lymphocytes and n/lrs between admission and discharge (p = 0.002, p < 0.001). however, there were no statistically significant differences in t lymphocyte subsets between admission and discharge ( table 2) . (3) risk factors for the severity of covid-19. the risk factors for the severity of covid-19 were analyzed by binary logistic regression. due to the small number of critical-type samples, the critical type and the serious type were grouped together. variables with statistically significant differences among different groups of diseases by univariate analysis were substituted into the regression equation, and finally, four variables with statistical significance were selected: male sex, underlying disease (cardiovascular disease), pulse, and n/lrs were all closely related to the severity of the disease (p = 0.004, p = 0.012, p = 0.013, p = 0.028) ( table 3) . (4) n/lrs are closely related to severity level and disease course. we found that n/lrs were closely related to the severity level and disease course of covid-19 according to the kruskal-wallis test, p = 0.021 and p < 0.001, respectively ( table 1) . the difference between each pair of groups was statistically significant. patients with higher n/lrs had more severe disease. a jonckheere-terpstra trend test was performed and showed a statistically significant difference between each group; the more severe the disease, the higher the n/lr (figure 1) . patients with higher n/lrs had longer disease courses (figure 2 ) (p < 0.001). (6) n/lrs were closely related to il-6 and il-10. we found that n/lrs were positively related to il-6 and il-10, p < 0.001 and p = 0.024, respectively. however, there was no significant correlation with other cytokines (figure 3) . the trend of the relationship between n/lrs and different inflammatory cytokines is shown in figure 3 . (7) n/lrs are closely related to cd3 + and cd8 + cells (t lymphocyte subsets). we found that n/lrs were negatively related to cd3 + and cd8 + t lymphocytes (p < 0.001 and p = 0.046, respectively; figure 4 ). the trend of the relationship between n/lrs and different t lymphocyte subsets is shown in figure 4 . in this study, we analyzed the clinical data of 230 patients diagnosed with non-mild covid-19 in the cancer center of wuhan union hospital and summarized the clinical characteristics of this disease. we found the following: (1) n/lrs, male sex, underlying disease (cardiovascular disease), and pulse were all closely related to the severity of the disease; (2) n/lrs were positively correlated with the levels of cytokines (il-6, il-10); (3) n/lrs were negatively correlated with the proportion of cd3 + and cd8 + t lymphocyte subsets; (4) n/lrs were positively correlated with the severity of the disease, as the higher n/lrs were, the more serious the disease; (5) n/lrs were related to the course of disease, as the higher n/lrs were, the longer the course of disease; and (6) most patients had multisystem symptoms and injury and had underlying multisystem comorbidities. as the covid-19 epidemic continues to spread across the globe, effective treatment is urgent, and it is particularly crucial to explore the pathogenesis of the disease. covid-19 infection activates innate and adaptive immune responses. however, uncontrolled damage from the natural inflammatory response and adaptive immune response may lead to local and systemic tissue damage. lymphocytopenia is a common feature in patients with severe covid-19, with a dramatic decrease in the number of cd4 + and cd8 + t cells, b cells, and natural killer (nk) cells (5, (17) (18) (19) and lower proportions of monocytes, eosinophils, and basophils among leukocytes (18, 20) ; in particular, patients with higher n/lrs have poorer outcomes (20) . yang et al. found that nlrs can be considered independent biomarkers for indicating poor clinical outcomes (21) . consistent with the above data, our research found that the nlrs were closely related to the severity and course of patients with non-mild covid-19 and that higher n/lrs were related to longer courses and more severe non-mild covid-19. currently, n/lrs are a well-known marker of systemic inflammation and infection, and they have been studied as predictors of bacterial infection, showing superior predictive value over conventional inflammatory markers (22) (23) (24) . in addition, the n/lrs have displayed good predictive power for pneumonia, as well as dose-response information relating to the burden of community-acquired pneumonia, such as pneumonia severity or mortality (25, 26) . neutrophilia and lymphocytopenia are physiological responses of the innate immune system to systemic inflammation. lymphocytopenia consists of accelerated apoptosis and margination of lymphocytes within the reticuloendothelial system, liver, and splanchnic lymphatic system and of the redistribution of lymphocytes within the lymphatic system (27) (28) (29) . as immune changes and inflammation are the core pathological basis of covid-19, compared with imaging and biochemical examination, n/lrs may be a simpler and more specific way to determine the prognosis of covid-19. most patients with severe covid-19 show significantly elevated serum levels of inflammatory cytokines, such as il-6 and il-1, as well as il-2, il-17, g-gsf, gm-gsf, ip-10, mcp1, mip-1a (also known as ccl3), and tnf, known as a cytokine storm (5, (17) (18) (19) . in our research, we found that cytokines, such as serum il-2, il-4, il-6, il-10, tnf-α, and infγ, were significantly elevated at admission, and in some patients, these inflammatory cytokines were reviewed before discharge. there were statistically significant differences in cytokine levels at admission and discharge. interestingly, we found that in the process of treatment, the levels of some inflammatory cytokines were decreased, while others were increased. this phenomenon may be because some inflammatory cytokines are pro-inflammatory and others are anti-inflammatory; these changes may reflect the dynamic transformation process of inflammation from damage to repair during homeostasis and inflammation (30) . more importantly, correlation analysis indicated that n/lrs were closely related to il-6 and il-10. a recent study showed that m1 macrophages produce proinflammatory cytokines, such as il-6, and that m2 macrophages produce anti-inflammatory cytokines, such as il-10 (30). our research found that, compared to those at the first test, il-10 significantly increased and il-6 significantly decreased at the last test (p < 0.001), which described the patient's transition from the acute phase to the convalescent phase, and these phenomena may indicate that n/lrs were closely related to injury in the acute stage and repair in the recovery stage in patients with covid-19. the source, function, and interaction of various inflammatory cytokines are complex. severe acute respiratory syndrome coronavirus 2-infected macrophages demonstrate upregulation of il-6 production and low expression of interferons (31, 32) . th1 cell-derived ifn-γ is essential for an effective antiviral immune response. however, il-6 may inhibit th1 polarization by stimulating cd4 + cells to differentiate into th2 cells or by suppressing ifn-γ expression (33, 34) . however, il-6 can cooperate with transforming growth factor (tgf)-β to induce il-10 production in th17 cells (35) ; il-10 is also produced by regulatory t cells (tregs), and tgf-β is critical to enable human tregs to express il-10 (36). il-10 can stimulate lymphocytes, leading to a decrease in the secretion of il-2 and ifn-γ by t cells (37) . studying the complex inflammatory cytokine network after covid-19 infection is of great significance for the treatment and prediction of the disease. most patients with covid-19 exhibit mild to moderate symptoms, but approximately 15% progress to severe pneumonia, and approximately 5% eventually develop acute respiratory distress syndrome (ards), septic shock, and/or multiple organ failure (5, 17) . wei's group study showed that after covid-19 infection, cd4 + t lymphocytes are rapidly activated to become pathogenic t helper (th)1 cells and generate gm-csf. the cytokine environment induces inflammatory cd14 + cd16 + monocytes with high expression of il-6 and accelerates inflammation (3). these aberrant pathogenic th1 cells and inflammatory monocytes may enter the pulmonary circulation in large numbers and play an immune damaging role to cause lung functional disability and quick mortality (5, 38) . their team launched a clinical trial using tocilizumab to block inflammatory storms (il-6 receptor inhibitor) in severely ill patients and achieved some results (3) . partly corresponding to wei's team (3), our clinical study found that the serum level of il-6 decreased significantly before discharge, which may reflect the pro-inflammatory role of il-6 in the course of covid-19 from another perspective. however, our team did not find that inflammatory cytokines (such as il-6) were significantly different among different severity level groups. part of the explanation is that there are no mild cases in this study (see section "materials and methods"). more importantly, although the role of il-6 in the pathological development of covid-19 has attracted much attention (3), its specific role remains controversial (9) (10) (11) (12) , and several experimental models of viral lung infections suggest that il-6 demonstrates either pathogenic (39)or protective (40) effects in vivo; the consequences of il-6 induction in covid-19 may vary depending on the stage of infection and the immune status of the host. the role of these cytokines in sars-cov-2 infection should be carefully evaluated. severe acute respiratory syndrome coronavirus 2 might act mainly on lymphocytes, especially t lymphocytes (18) , and decreases in the levels of cd3 + and cd4 + t lymphocytes are associated with immunosuppression (41). our research found that n/lrs were negatively related to cd3 + and cd8 + t lymphocyte levels, suggesting that the elevated n/lrs reflect the degree of lymphatic impairment, which may support the hypothesis that n/lrs are a sensitive and simple biomarker of immune function in patients with covid-19. additionally, our results suggest that male sex, underlying disease (cardiovascular disease), and pulse are risk factors for covid-19. the risk associated with male sex raises a topic of great interest: the effects of estrogens on il-6 and on covid-19 progression. estrogens can suppress lipopolysaccharide (lps)-mediated il-6 expression in mouse macrophages by both blocking nuclear factor kappa b (nf-kb) activation (42, 43) and inhibiting p38 mitogen-activated protein kinase (mapk) phosphorylation (43) by acting on estrogen receptors to decrease the production of pro-inflammatory cytokines (42) . however, the immunomodulatory effects of estrogens in covid-19 require further study. cardiovascular disease and pulse are risk factors for covid-19, which may suggest that cardiovascular disease has a critical impact on covid-19 patient mortality (44) and requires more attention as a comorbidity. we found that there were statistically significant differences in cytokines and n/lrs at admission and discharge, while there were no differences in t lymphocyte subsets (cd3 + /cd4 + /cd8 + ) in the research. we wanted to know whether the indicators of lymphocyte subset recovery lagged relatively over time and what their significance was. further work is needed to understand the specific characteristics of the various inflammatory cytokines and t cell subsets in covid-19, the specific relationship between n/lrs and different inflammatory cytokines and t cell subsets in covid-19, and the prognostic value for the disease, especially during immunotherapy. post-covid-19 inflammation is a very complex network system that has a decisive influence on the prognosis of patients. some inflammatory cytokines (such as il-6), whose mechanisms and effects are still controversial, need further study. as a quick and convenient marker of inflammation, n/lrs may predict the disease course and severity level of non-mild covid-19; male sex, cardiovascular disease, and pulse are also risk factors for the severity of non-mild covid-19. first, the sample size of critical type patients was relatively small, which may have influenced the results. second, due to the emergency situation of the epidemic outbreak, there is a certain lack of clinical data, including inflammatory cytokines. we are also collating clinical data from other centers and investigating the mechanisms of inflammatory factors in animal models. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. a novel coronavirus outbreak of global health concern the outbreak of coronavirus disease 2019 (covid-19)-an emerging global health threat pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients national health commission of the people's republic of china. the guideline on diagnosis and treatment of the novel coronavirus pneumonia (ncp): revised version of the 7th edition pathological findings of covid-19 associated with acute respiratory distress syndrome cytokine release syndrome in severe covid-19: interleukin-6 receptor antagonist tocilizumab may be the key to reduce mortality cytokine storm in covid-19: pathogenesis and overview of anti-inflammatory agents used in treatment the role of il-6 in host defence against infections: immunobiology and clinical implications il-6 trans-signaling-dependent rapid development of cytotoxic cd8+ t cell function immuneinduced fever is mediated by il-6 receptors on brain endothelial cells coupled to stat3-dependent induction of brain endothelial prostaglandin synthesis fever and the thermal regulation of immunity: the immune system feels the heat a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster practice and reflection on the management of mobile cabin hospital during the covid-19 epidemic ct early signs and differential diagnosis of covid-19 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan, china dysregulation of immune response in patients with covid-19 in wuhan, china immunopathological characteristics of coronavirus disease 2019 cases in guangzhou immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19. medrxiv the diagnostic and predictive role of nlr, d-nlr and plr in covid-19 patients inflammation biomarkers in blood as mortality predictors in community-acquired pneumonia admitted patients: importance of comparison with neutrophil count percentage or neutrophil-lymphocyte ratio the neutrophil-lymphocyte count ratio in patients with community-acquired pneumonia neutrophil to lymphocyte ratio as a predictor of treatment response and mortality in septic shock patients in the intensive care unit neutrophil-to-lymphocyte ratio: an emerging marker predicting prognosis in elderly adults with community-acquired pneumonia neutrophil to lymphocyte ratio and platelet to lymphocyte ratio as diagnostic markers for pneumonia severity differential induction of apoptosis in lymphoid tissues during sepsis: variation in onset, frequency, and the nature of the mediators apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction differential lymphopenia-induced homeostatic proliferation for cd4+ and cd8+ t cells following septic injury macrophage plasticity, polarization, and function in health and disease the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes. medrxiv severe acute respiratory syndrome and the innate immune responses: modulation of effector cell function without productive infection the role of interleukin 6 during viral infections tlr ligand induced il-6 counter-regulates the anti-viral cd8(+) t cell response during an acute retrovirus infection interleukin-6 induces the generation of il-10-producing tr1 cells and suppresses autoimmune tissue inflammation requirements for growth and il-10 expression of highly purified human t regulatory cells ex vivo stimulation of lymphocytes with il-10 mimics sepsis-induced intrinsic t-cell alterations pulmonary pathology of earlyphase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer il-6 ameliorates acute lung injury in influenza virus infection t-lymphocyte subsets as a predictive biomarker for stroke-associated pneumonia crossroads of estrogen receptor and nf-kappab signaling estrogen inhibits lps-induced il-6 production in macrophages partially via the nongenomic pathway covid-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options sq was involved in the study design, data interpretation, and manuscript writing and was a recipient of the obtained funding. jz participated in the analysis, interpretation, and collection of the data. fh participated in the study design and the statistical analysis. zl, jw, jc, hg, cz, ym, yz, dx, yiw, hh, yow, mf, yy, mz, and yx were involved in the data collection. yuw and jc participated in the data analysis and data collection. xh participated in the data analysis. wg participated in the data analysis and manuscript writing. all authors contributed to the article and approved the submitted version. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 qun, wang, chen, huang, guo, lu, wang, zheng, ma, zhu, xia, wang, he, wang, fei, yin, zheng, xu, ge, hu and zhou. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003656-7mzsaz7a authors: wium, martha; jonker, hester isabella; olivier, adriaan jacobus; bellstedt, dirk uwe; botes, annelise title: dna vaccines against mycoplasma elicit humoral immune responses in ostriches date: 2019-05-14 journal: front immunol doi: 10.3389/fimmu.2019.01061 sha: doc_id: 3656 cord_uid: 7mzsaz7a in ostriches, the population densities resulting from intensive rearing increases susceptibility to pathogens such as mycoplasmas. in addition to good management practices, vaccination offers an attractive alternative for controlling mycoplasma infections in food animals, instead of using antibiotics, which often leave unacceptable residues. the use of live attenuated vaccines, however, carry the concern of reversion to virulence or genetic recombination with field strains. currently there are no commercially available vaccines against ostrich-infecting mycoplasmas and this study therefore set out to develop and evaluate the use of a dna vaccine against mycoplasma infections in ostriches using an oppa protein as antigen. to this end, the oppa gene of “mycoplasma nasistruthionis sp. nov.” str. ms03 was cloned into two dna vaccine expression vectors after codon correction by site-directed mutagenesis. three-months-old ostriches were then vaccinated intramuscularly at different doses followed by a booster vaccination after 6 weeks. the ability of the dna vaccines to elicit an anti-oppa antibody response was evaluated by elisa using the recombinant oppa protein of ms03 as coating antigen. a statistically significant anti-oppa antibody response could be detected after administration of a booster vaccination indicating that the oppa protein was successfully immunogenic. the responses were also both dose and vector dependent. in conclusion, the dna vaccines were able to elicit an immune response in ostriches and can therefore be viewed as an option for the development of vaccines against mycoplasma infections. the ostrich (struthio camelus var. domesticus) is the largest living, flightless bird species belonging to the ratite group (1) . although ostriches are traditionally endemic to africa, parts of arabia and the middle east (2), they are reared in several countries across the world. south africa, however, is currently the largest producer of ostrich products (meat, leather, and feathers) on international markets (3) . here, the majority of ostriches destined for slaughter are reared in free-range systems, but when environmental conditions are unfavorable for grazing, they are reared in grow-out camps from about 3 months of age (4) . mycoplasma, a genus of bacteria that belongs to the prokaryotic class mollicutes, is known to be highly infectious in these intensive rearing systems (5, 6) . characteristic features of mycoplasma include the lack of a cell wall, small at-rich genome and a minimal set of genes (7, 8) . the species found to specifically infect ostriches are "mycoplasma struthionis sp. nov." str. ms01, mycoplasma sp. str. ms02, and "mycoplasma nasistruthionis sp. nov." str. ms03 (9, 10) . they are typically associated with upper respiratory tract infections (9) , reduced growth rates, downgrading of carcasses and in extreme cases, chick mortalities (5, 9) . the severity of infections increases with exposure to environmentally induced stress, other bacterial and viral infections. of the three mycoplasma species, "mycoplasma nasistruthionis sp. nov." str. ms03 was found to have the highest prevalence (6) amongst ostriches and therefore was the focus of this study. mycoplasma infections are currently managed using a combination of biosecurity systems and antibiotics. with antibiotics there is the risk of resistance (11) as well as an accumulation of residues in meat with concomitant risks for consumers (12) . development of whole organism vaccines for use in ostriches is limited by the slow-growing nature of these organisms and the cost of medium required to sustain growth. dna vaccines, on the other hand, do not require large scale cultivation of the pathogen, can be produced at relatively low cost and are more temperature stable than other vaccines (13, 14) . it is therefore an attractive alternative for use in ostriches where the vaccine market is small, relative to e.g., poultry, and ostriches are usually farmed in semi-arid or arid regions where temperatures are frequently high and cold storage of vaccines can be problematic. similar to live vaccines, they are also able to stimulate humoral and cellular immunity as indicated by measuring antibody, t-helper cell and cytotoxic t-lymphocyte responses (15) . but unlike live vaccines cannot revert back to an infectious form (16) . el gazzar et al. (17) has reported the reversion to virulence of a commonly used live mycoplasma vaccine in poultry, ts-11, whilst another has reported possible genetic recombination between live vaccines and field strains upon long term use (18) . this exchange or transfer of genes could have a negative impact on either the pathogenicity or transmissibility of field strains. chromosomal integration of dna vaccines is of some concern, but preclinical and clinical studies have shown the rate of integration of plasmid dna into the host genome to be lower than that of spontaneous mutations (19, 20) . to date, dna vaccines have not been tested in ostriches or any other ratites. the aim of this study was to use the oppa protein of "m. nasistruthionis sp. nov." str. ms03 as antigen in the development and evaluation of dna vaccines in ostriches at different doses and using different vectors. a dna vaccine is a plasmid expression vector containing a gene that codes for a protein antigen. as a result of their parasitic lifestyle (7), mycoplasmas possess, and rely on, a wide range of transmembrane transport systems for their survival. the extracellular components of these transporters are ideal targets for vaccine development (21, 22) . in this study the extracellular oppa domain of an oligopeptide permease (opp) transporter was chosen as protein antigen (23, 24) . using mouse models, oppa has to date been evaluated as antigen in subunit vaccines against brachyspira pilosicoli (25) , moraxella catarrhalis (26) , and yersinia pestis (27) , as well as against haemophilus parasuis in pigs (28) . oppa has, however, not been evaluated in any organism as part of a dna vaccine. in this study we report, for the first time, that a dna vaccine can elicit a humoral immune response in ostriches using oppa as antigen. this response was both dose and vector dependent. site-directed mutagenesis of the oppa gene cultures of "m. nasistruthionis sp. nov." str. ms03 (genbank: km410300.1) were obtained from mr j.j. gouws (faculty of veterinary science, onderstepoort, university of pretoria). genomic dna (gdna) was isolated from these cultures using a method described by hempstead (29) . the type a oppa gene (24) pci-neo (promega) and vr1020 (vical inc.) were chosen as vaccine vectors (supplementary figure 1) . the pci-neo vector is a mammalian expression vector that contains a cmv enhancer/promoter region, chimeric intron and sv40 late polyadenylation signal sequence. the vr1020 vector also contains a cmv enhancer/promoter region, but in combination with a cmv intron, tissue plasminogen activator (tpa) signal peptide sequence and a bovine growth hormone polyadenylation signal sequence. the mutated oppa gene was sub-cloned into each vector using restriction digestion. for pci-neo, acci and mlui (fastdigest, thermo scientific) and for vr1020, bamhi (fermentas) restriction enzymes were used according to the manufacturer's instructions. cultures of the pci-neo_oppa and vr1020_oppa vaccine plasmid were prepared and the respective plasmids purified with an endotoxin-free plasmid dna purification kit (nucleobond r xtra midi plus ef, macherey-nagel, germany). yields were determined using a nanodrop spectrophotometer. prior to large scale plasmid isolation for vaccination, the nucleobond kit's ability to yield supercoiled pci-neo_oppa and vr1020_oppa plasmids was evaluated by digesting plasmids with the acci and bamhi restriction enzymes, respectively, followed by electrophoresis on a 1% (w/v) agarose gel. to prepare a sufficient amount of plasmid for vaccination, the plasmids were prepared in batches over several days. the purified plasmids were again evaluated as before to confirm the integrity and supercoiling of the plasmids prior to being used for vaccination. the isolated plasmids were diluted to 100, 600, and 1,200 µg/ml with sterile pbs (137 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo 4 , and 1.5 mm kh 2 po 4 , ph 7.2), respectively, a day before use. dilutions were prepared under aseptic conditions, in sterile serum glass bottles, closed with inert silicone stoppers and sealed with a tear away center aluminum cap (sigma-aldrich). ethical approval was obtained from the stellenbosch university animal ethics committee (su-acum 13-00019) as well as the south african department of agriculture, forestry and fisheries (reference: 12/11/1/1/3) in terms of section 20 of the animal disease act 1984 (act no. 35 of 1984). a group of 140 ostrich chicks of ±3-months-old were randomly selected from a chick rearing unit in the fraserburg district (northern cape province, south africa). since fraserburg is outside the major commercial ostrich production region, this limits pathogen exposure during the critical 3-4 months-old rearing phase (30) . as per standard practice, upon reaching a weight of about 40 kg (±4 months of age), trial ostriches were transported back (±280 km) to a commercial ostrich farm in the oudtshoorn district (western cape province, south africa). three-month-old chicks were chosen for vaccination to allow sufficient time for a primary immune response to develop before being transported to a higher stress environment with concomitant increase in pathogen load and increased risk of exposure to mycoplasma. the chicks were quarantined for 28 days after relocation and random testing was performed for avian influenza. this is required by avian influenza control measure before ostriches can be released and allowed to mix with other populations on any farm to which they have been transported. at the start of the trial, each ostrich was tagged with a unique number for identification under the right wing, in accordance with standard ostrich farming practices. the trial was conducted under similar conditions to which a commercial vaccine would be administered and therefore trial ostriches were at all times housed and treated in the same manner as non-trial ostriches on the farms. in fraserburg, the chick rearing units were open air camps (1,250 m 2 ) with 120-150 chicks per camp. in oudtshoorn, ostriches were kept in open air grow-out camps (25,000 m 2 ) with 100-120 ostriches per camp. the ostriches received food and water ad libitum and were only handled by trained and experienced farm personnel. trial birds were not kept separately but grouped with other ostriches of the same weight and age. the ostriches were randomly allocated to seven treatment groups of 20 ostriches each. the pci-neo_oppa and vr1020_oppa vaccine groups were vaccinated at a dose of 100, 600, and 1,200 µg, respectively. the last group was the control group that did not receive any vaccine. the dose typically administered to poultry ranges from 0.25 to 800 µg when injected intramuscularly, with a booster dose after 2-3 weeks (31-38). dunham (39) indicated that larger animals may require a larger dose of 500-2,500 µg, but that this needs to be optimized for individual vaccines since the dose required is influenced by the target species, its size and the efficiency of plasmid delivery. the different doses used in our study were therefore selected to compensate for the increase in weight of the ostriches during the course of the trial and the fact that no adjuvant was used in the vaccine formulation to limit possible skin reactions which can impact hide quality. vaccine doses were administered in a single 1 ml volume at week 0 and a booster injection administered at week 6 by intramuscular injection in the upper thigh. blood samples (4 ml) were drawn from the jugular vein using 18gx1/2 ′′ needles (vacuette r ) and collected in vacuette r z serum sep clot activator tubes at week 0, 3, 6, and 9. serum was separated by centrifugation at low speed for 10 min and stored at −20 • c. week 0 and 3 samples were collected in fraserburg, and week 6 and 9 samples in oudtshoorn. the ostriches were moved from fraserburg to oudtshoorn 12 days prior to the week 6 sampling and booster injections were therefore administered during the quarantine period. ostriches were monitored after vaccination for any adverse reactions. for analysis of anti-oppa antibody responses, recombinant oppa protein was produced for use as coating antigen in an enzyme-linked immunosorbent assay (elisa). to this end, the sdm corrected oppa gene was pcr amplified and sub-cloned into a pgex-4t-1 vector (ge healthcare life science, uk) using restriction digestion. primer sequences with bamhi and noti restriction sites are shown in supplementary table 1 . for protein expression, the pgex-4t-1_oppa plasmid was transformed into escherichia coli bl21(de3)plyss cells (promega) and freezer stocks prepared. an overnight culture (from a freezer stock) and expression cultures were prepared using terrific-broth (tb) medium. expression of the glutathione s-transferase (gst) oppa fusion protein was induced at an od 600 between 0.4 and 0.6 by the addition of 0.4 mm iptg, and cells harvested at 0 and 6 h by centrifugation (10,000 × g at 4 • c). the cells were resuspended in 1xten50 extraction buffer [10 mm tris-hcl, 1 mm edta, 50 mm nacl, 0.1% triton x-100, 0.2 m dithiothreitol and 10% glycerol (v/v)] at 100 µl extraction buffer per 1 ml culture used for centrifugation. protease inhibitor was also added to the ten50 buffer (1 tablet per 10 ml extraction solution of complete ultra tablets, mini, easypack tablet, roche). the gst-oppa protein was isolated using glutathioneagarose chromatography (sigma-aldrich) under gravity flow at 4 • c according to the manufacturer's instructions. a sample (8 ml) was prepared for loading of the column by treating the resuspended pellets with three freeze-thawing cycles (20 min at 37 • c followed by 10 min at −80 • c) and five cycles of 2 s sonication followed by 2 min on ice. the samples were then triturated three times through a 25gx5/8 ′′ needle (avacare) into a 1 ml injekt-f syringe (bbraun). this was repeated using a 23gx1/4" needle (nipro) followed by centrifugation at 10,000 × g for 10 min (4 • c) to obtain a clear supernatant which was loaded onto the column under gravity flow at 4 • c. fractions (1 ml) were collected and their protein concentration determined using a modified bradford assay (40) . expression and isolation products were analyzed using sds-page (40) and western blot analysis (41) . microtiter plates (maxisorp, nunc) were coated overnight at 4 • c with 100 µl of recombinant gst-oppa protein diluted to 10 µg/ml in carbonate buffer (50 mm nahco 3 , ph 9.6). to block non-specific binding, 200 µl/well casein buffer (10 mm tris-hcl, 154 mm nacl, 0.5% casein and 0.02% thiomersal, ph 7.6) was added and the plate incubated for 1 h at 37 • c. serum samples were diluted 1:100 with casein-tween [casein buffer containing 0.1% (v/v) tween r 20] and 100 µl/well added to the plate in triplicate before incubation for 1 h at 37 • c. biotinylated rabbit anti-ostrich ig polyclonal antibodies, prepared as previously described (42), were next added at a dilution of 1:100 in casein-tween and incubated for 1 h at 37 • c. a streptavidin horseradish peroxidase (hrp) conjugate mixture [2 ml streptavidin hrp (invitrogen), 38 ml 0.5% casein buffer, and 40 ml 50% glycerol], diluted 1:100 with casein-tween, was then added (100 µl/well) and the plate incubated for 1 h at 37 • c. finally, 100 µl/well substrate solution (0.5 mg/ml abts, 0.5 µl/ml h 2 o 2 , 0.1 m citrate buffer, ph 5) was added and absorbance measured at 405 nm with a thermo scientific multiskan ex plate reader after 30 min incubation at 37 • c. after each incubation step the content of the plate was first decanted followed by washing five times with pbs-tween [137 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo 4 and 1.5 mm kh 2 po 4 , ph 7.2, 0.1% (v/v) tween r 20] and washing three times with milli-q r water. between the coating and blocking steps washing was not applied. a first-row column blank (containing all components except ostrich serum) was used on each plate to blank absorbance values. all plates contained controls to monitor plate to plate variation. the controls were serum samples representing the week 0, 3, 6, and 9 sampling points of a single ostrich, randomly selected from the pci-neo_oppa 1,200 µg group based on high titers produced after vaccination. prior to the analysis of samples, the elisa was optimized with regard to coating concentration (1-10 µg/ml), serum dilution (1:40-1:640) as well as number of washing cycles between steps. non-specific binding of anti-oppa antibodies during elisa analysis was evaluated by coating the wells with carbonate buffer (100 µl/well) containing no capture antigen and serum from a vaccinated bird that gave a high absorbance value at a dilution of 1:100 when using the gst-oppa protein as capture antigen. the absorbance values obtained are referred to as elisa titres, which are a measurement of the antibody levels produced when using a serum dilution of 1:100 in the elisa. the weight of trial birds was monitored and recorded at weeks 0, 6, and 9. to determine the possible influence of existing mycoplasma infections on immune response data, trachea swabs were collected at weeks 0, 3, 6, and 9 and tested for the presence of mycoplasma infections by pcr. birds were, however, not excluded from the trial if they tested positive since ostriches are often naturally infected with mycoplasmas. swab samples were collected using dry sterile swabs (copan) which were then rinsed in 200 µl sterile pbs buffer. the eluate was tested by pcr using a generic primer pair specific for the mycoplasma genus (9). positive samples were further evaluated for the presence of the ostrich-infecting mycoplasmas ms01, ms02, and ms03 (9). statistical analysis of the elisa titer data was performed using the general linear model (glm) procedure in the agrobase generation ii r (agronomix software inc.) software. analysis of variance (anova) and least significant difference (lsd) values were calculated at a significance level of 0.05. the 3867 bp oppa gene was successfully amplified from the ms03 gdna and cloned into a pgem r -t easy vector as confirmed by sequencing. using sdm, all 16 mycoplasma tga codons in this plasmid were successfully mutated to universal tgg codons in seven consecutive steps of sdm as confirmed by sequencing (supplementary figure 2) . the mutated oppa gene was successfully sub-cloned into the pci-neo and vr1020 vaccine vector as confirmed by sequencing (supplementary figure 2) . large scale production of the dna vaccines was achieved and approximately 89 mg of plasmid was isolated for each of pci-neo_oppa and vr1020_oppa. the 260/280 absorbance ratio of the isolated plasmids ranged from 1.89 to 1.95. the integrity of the isolated plasmids was confirmed with agarose electrophoresis and most of the pdna was in the required supercoiled conformation. a supercoiled conformation has a higher transfection rate into mammalian cells, is less susceptible to intracellular degradation and is more effective at inducing an immune response than other plasmid conformations (43, 44) . ostriches were vaccinated with the prepared dna vaccines and no adverse reactions were observed at the injection sites (redness, swelling, inflammation, or allergic reaction) during and after the trial. ostriches resumed normal behavior such as eating, walking around and exploring immediately after vaccination (no lameness or loss of appetite). sub-cloning of the mutated oppa gene into the pgex-4t-1 vector was successful as confirmed by sequencing frontiers in immunology | www.frontiersin.org (supplementary figure 2) . sds-page and western blot analyses confirmed that the recombinant oppa protein was expressed successfully as an n-terminal gst-fusion protein with the predicted size of 170 kda (26 kda due to the gst tag) (figures 1a,b) . bradford analysis indicated that the oppa protein was eluted between fraction 7 and 12 with the highest concentration obtained in the 9th fraction. ostriches with more than two missing sampling points were excluded from elisa and subsequent statistical analysis. there was no non-specific binding of the anti-oppa antibodies in ostrich serum and the plate control samples indicated limited plate to plate variation. the titer values resulting from vaccination with the pci-neo_oppa and vr1020_oppa vaccines are shown in figures 2a,b , respectively. the titer values of the control group, which received no vaccine, did not show significant variation over time. however, there was a slight increase in the average titer at week 9. vaccination with the pci-neo_oppa vaccine (figure 2a ) resulted in significant treatment x time interactions (p = 0.0462), but only the 100 and 600 µg doses resulted in average elisa titers that differed significantly from the control. this was also only at week 9 after a booster vaccination was administered. the average titers of the different doses were, however, not significantly different from one another. vaccination with the vr1020_oppa vaccine (figure 2b ) resulted in a significant treatment × time interaction (p < 0.001) and all three doses resulted in average elisa titers that differed significantly (lsd = 0.2796) from the control. this was again only at week 9 after a booster vaccination was administered and all the doses differed significantly from one another. the vaccinated groups as well as the control group gained weight from weeks 0 to 6, but from weeks 6 to 9 there was an average weight loss of 0.05 kg ( table 1) . there was, however, no statistically significant difference between the treatment groups and the control group over the 9 weeks period (p = 0.9564 for pci-neo_oppa and p = 0.5290 for vr1020_oppa). the presence of mycoplasma infections was monitored with pcr during the trial ( table 2) . mycoplasma infections could not be detected in any of the groups at weeks 0, 3, and 6. infections were, however, detected at week 9. at this point the ostriches had been in oudtshoorn for 36 days, of which 8 days were out of quarantine. in the groups that received the vr1020_oppa vaccine the highest percentage of infections at week 9 was due to ms02 (10.5%) followed by ms03 (5.2%) and ms01 (3.5%) ( table 2) . compared to this, the groups that received the pci-neo_oppa vaccine had fewer mycoplasma infections. amongst these the highest percentage of infections was again due to ms02 (7.2%) followed by ms03 (3.6%) and ms01 (1.8%). for both vaccine groups, some of the birds were infected by more than one of these mycoplasma species. the only group that had no pcr-detectable mycoplasma infections at week 9 was the group that had received 1,200 µg of the vr1020_oppa vaccine and the control group. in this study, dna vaccines were developed for ostriches using the oppa gene of an ostrich-infecting mycoplasma (ms03) as vaccine antigen. the expression vectors used (pci-neo and vr1020), were selected based on dna vaccine studies in other birds, and on their immunostimulatory characteristics (45) (46) (47) . after vaccination of ±3-month-old chicks a statistically significant anti-oppa antibody response could not be detected at week 3, although a general trend of increases in the average titer values was observed from week 0 to 3 for groups that received the vr1020_oppa vaccines. based on previous studies using inactivated vaccines in ostriches (42, 48) , we expected a primary immune response about 3 weeks after the first vaccination (i.e., between weeks 0 and 6). after administering a booster vaccination, both dna vaccines were able to elicit a statistically significant anti-oppa antibody response. as these responses against oppa were significant in comparison to the negative responses following the first vaccinations, this is evidence of a secondary immune response as a result of immune memory. if memory cells are produced after the initial contact with an antigen, subsequent exposure to the same antigen will allow the host to recognize the antigen faster, and with greater magnitude (49, 50) . the responses elicited by the dna vaccines were, however, dose dependent as well as vector dependent since different results were produced by each vaccine when using the same dose. this highlights a possible role of the vaccine vector in the observed antibody responses. the vr1020_oppa vaccine, on average, elicited higher titer values compared to the pci-neo_oppa vaccine, which might be due to the tpa signal sequence of the vr1020 plasmid, which is situated upstream of the oppa gene, and assists with protein expression in mammalian cells and export of the protein out of the cell. this would in turn increase the immunogenicity of the antigen (45) . in addition to this, sequences in the plasmid backbone have intrinsic immunostimulatory activity which enhance the immune system's response to the expressed protein antigen (51, 52) . over the whole vaccination trial, the health of the ostriches was monitored by recording weight gain. all groups gained weight up to week 6, but toward week 9 their weight gain decreased. given that this trend was also observed for the control group ostriches, the weight loss cannot be ascribed to vaccination. the decrease in weight did, however, coincide with the movement of the birds to a new location. ostriches are known to be severely affected by stress and during this period of adapting to changes in social dynamic and housing environment, reduction in weight gain and even weight loss is normal amongst farmed ostriches (4) . the presence of existing mycoplasma infections at the start of the trial, as well as changes in infection status during the trial, were also monitored by pcr analysis of trachea swabs. mycoplasma infections could only be detected at week 9, which was after they were moved and exposed to the farm environment in oudtshoorn for more than 30 days. the absence of detectable mycoplasma infections at the earlier time points may be ascribed to fraserburg being outside of the commercial ostrich production region. the oudtshoorn district, on the other hand, has a higher incidence of mycoplasma infections especially during seasonal changes and our trial was conducted during late autumn. amongst the vaccinated groups, the number of detected ms03 and ms01 infections were low compared to ms02, with only the 1200 µg vr1020_oppa and control group having no detectable ostrichinfecting mycoplasmas. in future trials, careful consideration should be given to the timing of the primary and booster vaccination relative to the time of introduction to growout conditions. it is possible that the mycoplasma infections had an influence on the observed antibody responses. in a study done by yang et al. (26) using recombinant oppa protein as subunit vaccine against m. catarrhalis in mice, it was found that the elisa titer values were already raised at the start of the vaccination trial which they ascribed to the presence of existing systemic antibodies to oppa as a result of existing infections. despite the possible presence of antibodies due to mycoplasma infections, this did not mask the detection of a dose dependent antibody response to the oppa protein of ms03 during vaccination. in conclusion, this is the first study to show that dna vaccines are able to elicit an antibody response in ostriches against a mycoplasma antigen in spite of ostriches being prone to environmentally induced stress conditions that can suppress immune function. the mycoplasma oppa protein was sufficiently immunogenic to induce an anti-oppa antibody response and this response was firstly, dose dependent and secondly, required a booster vaccination. further trials are required to determine the extent of protection provided by this mycoplasma antigen relative to the timing of the primary and booster vaccination before introduction to grow-out conditions. this study (field trial) was carried out in accordance with the recommendations of the south african department of agriculture, forestry and fisheries (reference: 12/11/1/1/3) in terms of section 20 of the animal disease act 1984 (act no. 35 of 1984) . the protocol was approved by the stellenbosch university animal ethics committee (su-acum 13-00019). mw performed the sdm, prepared the dna vaccine and expression plasmids, optimized the expression of the recombinant oppa protein, and the initial optimization of the elisa. hj further optimized the elisa and evaluated the anti-oppa antibody responses during the vaccination trial. ao assisted with the vaccine trial. mw wrote the manuscript with support from ab. ab supervised the project with the assistance of db and ao. ab and db conceived the original idea. all authors provided critical feedback and helped shape the research, analysis and manuscript. this work was supported by the technology and human resources for industry programme (thrip), south africa (project reference tp50090719) and south african ostrich business chamber. we would like to thank vical inc., usa for supplying the vr1020 vector for research purposes and mr. j. j. gouws (faculty of veterinary science, onderstepoort, university of pretoria) for the culturing of the mycoplasma bacteria. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01061/full#supplementary-material ratite nonmonophyly: independent evidence from 40 novel loci molecular genetic relationships of the extinct ostrich, struthio camelus syriacus: consequences for ostrich introductions into saudi arabia profile of the south african ostrich market value chain bird handling, transportation, lairage, and slaughter: implications for bird welfare and meat quality ostrich diseases investigations into mycoplasma infections in south african ostriches. the third international ratite science symposium molecular biology and pathogenicity of mycoplasmas a systematic review of mycoplasma and ureaplasma in urogynaecology identification of three novel mycoplasma species from ostriches in south africa proposal for molecular tools for the epidemiology of contagious bovine pleuro pneumonia and classification of unknown mycoplasma sp. isolated from struthio camelus mycoplasmas and their host: emerging and re-emerging minimal pathogens antibiotic residues in food: the african scenario dna-antiviral vaccines: new developments and approaches-a review immunization of animals: from dna to the dinner plate dna vaccines: an historical perspective and view to the future dna vaccines: ready for prime time? characterization of a ts-11-like mycoplasma gallisepticum isolate from commercial broiler chickens the development and application of a mycoplasma gallisepticum sequence database detection of integration of plasmid dna into host genomic dna following intramuscular injection and electroporation biodistribution of dna plasmid vaccines against hiv-1, ebola, severe acute respiratory syndrome, or west nile virus is similar, without integration, despite differing plasmid backbones or gene inserts atp-binding cassette transporters are targets for the development of antibacterial vaccines and therapies bacterial surface proteins and vaccines oppa, the substrate-binding subunit of the oligopeptide permease, is the major ecto-atpase of mycoplasma hominis the identification of oppa gene homologues as part of the oligopeptide transport system in mycoplasmas evaluation of recombinant brachyspira pilosicoli oligopeptide-binding proteins as vaccine candidates in a mouse model of intestinal spirochaetosis characterization and evaluation of the moraxella catarrhalis oligopeptide permease a as a mucosal vaccine antigen the abc transporter protein oppa provides protection against experimental yersinia pestis infection immune response to oligopeptide permease a (oppa) protein in pigs naturally and experimentally infected with haemophilus parasuis an improved method for the rapid isolation of chromosomal dna from mycoplasma spp ostrich manual. elsenburg: western cape department of agriculture cross-protection among lethal h5n2 influenza viruses induced by dna vaccine to the hemagglutinin protective efficacy of dna vaccines against duck hepatitis b virus infection turkeys are protected from infection with chlamydia psittaci by plasmid dna vaccination against the major outer membrane protein dna vaccination in the avian a dna prime-protein boost vaccination strategy targeting turkey coronavirus spike protein fragment containing neutralizing epitope against infectious challenge protection of chickens against infectious bronchitis virus with a multivalent dna vaccine and boosting with an inactivated vaccine induction of a protective response in ducks vaccinated with a dna vaccine encoding engineered duck circovirus capsid protein sequential dna immunization of chickens with bivalent heterologous vaccines induce highly reactive and cross-specific antibodies against influenza hemagglutinin the application of nucleic acid vaccines in veterinary medicine evaluation of dna vaccines against mycoplasma nasistruthionis sp. nov. str. ms03 infections in ostriches and the production of iga heavy chain proteins. master's dissertation the development of a dna vaccine against mycoplasma nasistruthionis sp. nov. for use in ostriches antibody responses to la sota strain vaccines of newcastle disease virus in ostriches (struthio camelus) as detected by enzyme-linked immunosorbent assay impact of plasmid supercoiling on the efficacy of a rabies dna vaccine to protect cats transgene expression of transfected supercoiled plasmid dna concatemers in mammalian cells vaccinia expression of mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of m. tuberculosis ag85. microbes infect measuring the effects of an ever-changing environment on malaria control comparison of five expression vectors for the ha gene in constructing a dna vaccine for h6n2 influenza virus in chickens growth rate and hatching date in ostrich chicks reflect humoral but not cellmediated immune function generating memory with vaccination vaccination to gain humoral immune memory effect of plasmid backbone modification by different human cpg motifs on the immunogenicity of dna vaccine vectors cpg dna as a vaccine adjuvant the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 wium, jonker, olivier, bellstedt and botes. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-265855-zf52vl11 authors: mayor-ibarguren, ander; busca-arenzana, carmen; robles-marhuenda, ángel title: a hypothesis for the possible role of zinc in the immunological pathways related to covid-19 infection date: 2020-07-10 journal: front immunol doi: 10.3389/fimmu.2020.01736 sha: doc_id: 265855 cord_uid: zf52vl11 nan • zinc deficiency may be common and associated with severe infection. • zinc helps to enhance the interferon type 1 response to the virus and participates in many regulatory pathways. • low levels of zinc have been associated with higher il-6 responses. • il-6 plays an important role in severe lung injury due to covid-19 infection. • zinc inhibits sars-cov rna polymerase, and thus its replication capacity. • zinc may increase the efficacy of antimalarial agents, since they are zinc ionophores. • differences in mortality due to covid-19 infection may be explained to some degree by−174 il-6 gene polymorphism. zinc (zn) is the second most abundant trace metal in the human body after iron. however, unlike iron, there is no specialized zinc store (1) . zinc's functions can be classified as catalytic, structural, and regulatory (2) . for example, important zinc metalloenzymes include alkaline phosphatase, rna polymerases, and alcohol dehydrogenase (3) . zinc deficiency can precipitate an immune system imbalance, exemplified in severe deficiency by high susceptibility to infections, skin disorders, gastrointestinal disorders, weight loss, growth retardation and male hypogonadism, amongst other symptoms (4) . while severe zinc deficiency is rare, mild to moderate deficiency is more common worldwide (5) . there are very low levels of free zinc in plasma, since it is mostly bound to proteins such as albumin, alpha-2-macroglobulin (a2m), and transferrin. plasma zinc levels are therefore only around 1 µg/ml, equal to 0.1% of total body zinc, but are still the most important reservoir for zinc homeostasis, which requires "free" or "labile" zinc mobilization (6, 7) . kinetic studies suggest that only a small proportion of total body zinc (10%) represents the "functional pool" of zinc, located within the liver and other tissues, that exchanges rapidly with that found in the plasma (8, 9) . when this functional pool is depleted, zinc deficiency ensues (8) . intracellular zinc is distributed in zinc-storing vesicles called zincosomes, the nucleus and other organelles. in cytoplasm, zinc mostly binds zinc-chelating proteins called metallothioneins (mts). zinc homeostasis is understood to be the correct balance of zinc distribution. internal zinc homeostasis is regulated by the cooperative activities of two metal transporter protein families. one family consists of 10 solute-linked carrier 30 (slc30 or znt) exporters, and the other family consists of 14 solute-linked carrier 39 (slc39 or zip) importers (10, 11) . for instance, most labile zinc in the body is absorbed by intestinal epithelial cells via slc39a4 protein, and excessive zinc is excreted through the kidneys, and the intestine via slc39a5 (12) . we are currently experiencing an unprecedented covid-19 pandemic caused by a novel rna coronavirus called sars-cov-2, which can produce a severe acute respiratory distress syndrome (ards) (13) . it was first detected in wuhan province in china at the end of 2019 (14) , and on 11 march 2020, who characterized covid-19 as a pandemic (15) . the reported mortality rate for those infected varies between countries (0.5-7.7%) with the most important focus previously in italy and spain and currently in the usa, uk, and brazil (16) (17) (18) (19) . age, male sex, and pre-existing chronic metabolic diseases including diabetes, cardiovascular disease, and obesity are associated with greater severity of infection (20) . there is no specific treatment yet. many agents are being used with variable success, but none have had their efficacy demonstrated in clinical trials. examples include: antimalarial agents such as chloroquine and hydroxychloroquine, antivirals such as lopinavir/ritonavir and remdesivir, and tocilizumab as an anti-interleukin 6 (il-6) receptor antibody (21) (22) (23) (24) . remdesivir has shown good initial clinical outcomes in a clinical trial, when treatment started within 10 days of symptom onset (25). in contrast, in a double-blind randomized trial of 237 patients with severe covid-19 (hypoxia and radiographically confirmed pneumonia) in china, time to clinical improvement was not statistically different with remdesivir compared with placebo taken for 10 days (median time to improvement 21 vs. 23 days; hazard ratio for improvement 1.23 [95% ci 0.87-1.75]) (26) . mixed and controversial results have also been published regarding antimalarial agents (27) . thus, more robust data are needed before conclusions can be drawn regarding treatment. il-1 and il-6 may play an important role in severe lung inflammation, leading to acute respiratory distress syndrome, which can result in patient death (28, 29) . this pathway appeared relevant in sars-cov, producing severe acute respiratory syndrome (sars), and in mers-cov, producing middle east respiratory syndrome (mers) (30) . high serum levels of proinflammatory cytokines [il-1, il-6, il-12, interferon γ (ifnγ), and transforming growth factor-β] and chemokines (ccl2, cxcl9, cxcl10, and il-8) were found in patients with sars with severe disease compared with individuals with uncomplicated sars (31) . mers-cov infections of dendritic cells and macrophages result in robust and sustained production of pro-inflammatory cytokines and chemokines such as tnfα, il-6, cxcl-10, ccl-2, ccl-3, ccl-5, and il-8 (32) . the purpose of this article is to highlight the key roles that zinc can play in covid-19 infection (summarized in figure 1 ), based on pre-existing evidence of its role in immune system function and viral infections, as well as its estimated possible deficiency in at-risk populations. zinc deficiency may be present in up to 17% of the population worldwide. the elderly especially are at higher risk of zinc deficiency and its adverse effects (33) . impairment of zinc homeostasis has also been demonstrated in metabolic diseases including diabetes, obesity, and cardiovascular disease (34) . many antihypertensive drugs such as ace inhibitors, angiotensin 2 receptor antagonists, and thiazide diuretics are zinc chelators (35) . iron and calcium may interfere with zinc absorption too (36) . the us food and nutrition board recommends intake of 11 and 8 mg/day for adult men and women, respectively (37) . apart from calcium and iron, non-digestible plant ligands such as phytate, some dietary fibers, and lignin chelate zinc and inhibit its absorption. measurement of plasma zinc levels is the most useful clinical test for zinc deficiency, despite limited sensitivity, and specificity (38) . also, plasma zinc levels remain stable even with low dietary intake, due to homeostasis in the body, decreasing in blood only when deficiency is very prolonged (39) . the absence of a dedicated store for zinc repletion results in impairment of function when zinc status is compromised. homeostasis maintains a constant intracellular zinc concentration and a plasma concentration within the reference range of 11-25 µm (0.7-1.6 mg/l) (40) . low plasma zinc has been defined as <60 mcg/dl (<9.2 µm) (41). when zinc intake decreases, homeostatic mechanisms initially maintain the plasma concentration within the reference range, but when deficiency is severe or prolonged, the concentration decreases. however, although plasma zinc concentration moderately correlates to habitual intake, the test also has limited specificity because zinc levels are depressed during inflammatory disease states or pregnancy and increase with acute catabolic states (42) . in mild diseases, with c-reactive protein (crp) levels of 15 mg/l, a 10% decrease in zinc is observed. in severe infectious diseases, crp levels can reach 100-200 mg/l, with a much greater decrease in zinc levels (40-60%) (43) . if crp levels are normal, plasma zinc measurements are more reliable. moreover, the test has limited sensitivity since patients with mild zinc deficiency may have normal plasma levels (44) . the copper:zinc ratio may be an interesting marker for the diagnosis of zinc deficiency, since the latter leads to an increase in copper absorption (45) . to be reliable, this ratio must be higher than 1.5. however, critical patients may have high levels of copper, reflecting the effects of the systemic inflammatory response, thus not reliably representing their actual levels (46) . a marker that might be more sensitive to the nutritional status of zinc is the ratio of apo/holo activities of angiotensin converting enzyme (47) . zinc levels may also be measured in neutrophils, lymphocytes, or erythrocytes, but these assays generally have poor sensitivity (48, 49) . ruz m et al. reported that zinc levels in neutrophils do not change, even in the event of changes in plasma concentrations during experimentally-controlled zinc depletion (48) . metfah et al. found that zinc levels in lymphocytes, granulocytes, and platelets decreased significantly only during the late zinc depletion phase (49) . interestingly, plasma zinc levels did not change even during the late zinc depletion phase in this study. in contrast, they found that activity of ecto-5 ′ -nucleotidase (an integral zinc-dependent plasma enzyme located on most mammalian cells) was significantly decreased during mild zinc deficiency. when measured in neutrophils, zinc deficiency is defined as <42 mcg/10 10 cells (49) . when measured in lymphocytes, zinc deficiency is defined as <50 mcg/10 10 cells (49) . taking all this into account, we highlight that there is no good reliable definition for zinc deficiency, besides a low plasma concentration with respect to normal reference levels, which may not be representative, especially in acute states or mild grades. zinc homeostasis in immune system pathways is complex, since it participates both in pro-inflammatory and regulatory pathways, and much of the data comes from preclinical in figure 1 | legend: a schematic view of the involvement of zinc in various signaling pathways. green arrows: zinc-mediated activation. red t bar arrows: zinc-mediated inhibition. blue arrows: flow of activation pathway. obesity, cardiovascular disease, diabetes, and aging are associated with zinc deficiency. −174 gg polymorphism on the il-6 promoter gene is associated with zinc homeostasis impairment and elevated il-6 levels which contribute to lung damage. zinc deficiency may increase ace-2 receptor activity on type 2 pneumocytes and other cells that are infected by sars-cov-2, mainly in the lower respiratory tract. zinc inhibits rdrp, blocking viral rna replication. zinc-finger protein zcchc3 senses viral rna and activates through rig-1-like receptor a cascade that results in an increase in the interferon type 1 response. ifn type 1 stimulates synthesis of antiviral proteins such as rnasel and pkr. zinc helps to regulate the same kind of responses by activating the a20 protein that inhibits traf6 downstream activation, and by inhibiting pde, which results in increased levels of cgmp that will activate pka that will inhibit nf-κb. zinc also inhibits stat-3 dimerization, blocking active stat3 signaling from the il-6 receptor. acronyms: zcchc3: zinc finger cchc domain-containing protein 3. rig-1, retinoic acid-inducible gene i; mavs, mitochondrial antiviral-signaling protein; tank, traf family member-associated nf-κb activator; iκkε, i kappa b kinase epsilon; tbk1, tank binding kinase 1; irf3, interferon regulatory factor 3; tlr, toll-like receptor; myd88, myeloid differentiation primary-response protein 88; irak, interleukin-1 receptor-associated kinase; traf-6, tumor necrosis factor receptor-associated factor 6; tak1: ikk, i kappa b kinase; nfκb, nuclear factor kappa b; a20, zinc protein; pde, phosphodiesterase; cgmp, cyclic guanosine-monophosphate; gmp, guanosine-monophosphate; pka, protein kinase a; inf-1, interferon type 1; jak, janus kinase; stat, signal transducer and activator of transcription; rnase l, ribonuclease l; pkr, rna-activated protein kinase; ace-2, angiotensin-converting enzyme 2; rdrp, rna-dependent rna polymerase. vitro studies. despite this, it seems clear that deficient or excessive zinc levels can lead to malfunction of the adaptive and innate immune systems. zinc regulates the proliferation, differentiation, maturation and functioning of lymphocytes, and other leukocytes (6) . it also regulates the immune response, and its deficiency increases susceptibility to inflammatory and infectious diseases, including pneumonia (50) . zinc sulfate supplementation at 20 mg/day for 5 months reduced acute lower respiratory tract infection morbidity vs. placebo in a clinical trial (51) . zinc is essential in both the adaptive and innate immune systems (52) . for instance, the functionality of natural killer (nk) cells, which are essential for maintaining the immune response against viruses and tumors, is affected by low levels of zinc (53) . furthermore, zinc supplementation significantly increased nk cell numbers in whole blood cultures and nk cell activity in vivo (54, 55) . in this latter study, zinc supplementation in subjects with low or borderline-normal circulating zinc increased the concentration of this ion and improved nk lytic activity, as well as modulating plasma il-6. zinc homeostasis directly influences the formation of lymphocytes and the secretion of cytokines and indirectly alters their stimulation by the innate immune system (56) . there is also evidence that unregulated zinc homeostasis in macrophages impairs phagocytosis and results in an abnormal inflammatory response (57) . in a study performed in mice, a diet deficient in zinc was associated with more pronounced airway inflammation after agricultural organic dust exposure, compared with normal dietary zinc intake (58) . this was partially explained by the fact that macrophages maintained in a zinc-deficient environment exhibited increased cxcl1 and il-23 production, as a result of increased nf-kb activation. also, pulmonary zinc deficiency may be one of the mechanisms by which hiv-1 infection impairs alveolar macrophage immune function and facilitates severe pulmonary infection in these individuals (59) . zinc also has a role in viral recognition. the zinc-finger protein zcchc3 binds rna and facilitates the detection of intracellular rna viruses by activating retinoic acid-inducible gene-i (rig-1)-like receptors (rlrs), including rig-i and mda5 (60) . this action triggers the activation of the antiviral response mediated by downstream activation of antiviral genes (61) . in this process, kinases such as tbk1 and iκk further phosphorylate the interferon regulatory transcription factor 3 (irf3) and iκb-alpha, the nk-κb inhibitor, leading to activation of irf3 and nf-κb, which results in interferon type 1 upregulation (62, 63) (see figure 1) . interferon alpha-induced signaling results in upregulation of antiviral proteins (rnase l and pkr), known to degrade viral rna and inhibit its translation (64) . zinc also exerts an inhibitory effect on the activation of nf-κb, through the expression of the a20 protein. a20 is a zinc-finger protein that negatively regulates tumor necrosis factor receptor (tnfr) and toll-like receptor (tlr)-initiated nf-κb pathways (65) . furthermore, zinc acts as an inhibitor of cyclic nucleotide phosphodiesterase (pde). when pde is inhibited, cyclic nucleotide cgmp (cyclic guanosine monophosphate) is elevated, leading to the activation of pka (protein kinase a), and subsequent inhibition of nf-κb (66) . additionally, zinc supplementation has been shown to downregulate inflammatory cytokines by decreasing gene expression of il-1β, tnf-alpha, and by inhibiting nf-κb activation (67) . nutritional immunity is a process by which the host organism sequesters trace minerals during an infection so that their availability to pathogens is limited (1) . during infection and inflammation, there is a transient transfer of zinc from serum to the organs, causing temporarily low serum zinc levels, which normalize during resolution of the inflammatory response (6, 7). thus, a sufficient level of zinc is essential during responses to infection. zinc signals act in an anti-inflammatory manner during sepsis by regulating the pro-inflammatory response, due to cellular uptake of zinc by zip14 as shown in a polymicrobial model of sepsis in mice (68) . zinc deficiency was strongly associated with an elevated risk of exaggerated inflammation and mortality due to sepsis in a murine model (69) . in this study, mice with a zinc-deficient diet had a 50% reduction in plasma zinc levels compared with those with a normal diet, and had a significantly lower survival rate of 10% in the context of sepsis. based on the studies mentioned above, one could hypothesize that an initial chelation of zinc would trigger an antiviral response mediated by interferon type 1 (ifn-i). however, ensuring an adequate level of zinc would be necessary to regulate this response, since zinc participates as an inhibitory agent at many points in this pathway (see figure 1) . indeed, an early ifn-i response was shown to be optimal, while a delayed ifn-i response was associated with ards in a study with sars-covinfected mice (70) . ifn-1 subtypes were studied alone and in combination with other antiviral drugs for the treatment of sars and mers, in vitro and in vivo, with some beneficial reports, but later failed to improve outcomes in humans (71) (72) (73) . despite this, sars-cov-2 appears to be more sensitive than mers or sars-cov to ifn type 1, and its use as prophylaxis or treatment is also being studied (74) . although it is also hypothesized that it should be tested on the early phase of infection, late phase anti-ifn type 1 treatment could be beneficial for treating severe disease (75) . there is some evidence that sars-cov-2 infection triggers expression of numerous ifn-stimulated genes, which is thought to induce inadequate ifn responses (76) . although there are no specific data regarding zinc in this pathway for sars-cov-2, zinc may limit infection through upregulation of ifn-alpha production and an increase in its antiviral activity (77, 78) . in this latter in vitro study, when cultures of white blood cells from elderly subjects were supplemented with 15 µm zinc (the physiological concentration), they produced ifn in amounts comparable to those from the younger subjects. we hypothesize that transient zinc deficiency during infection could result in a hyperinflammatory state in those with prior zinc deficiency. also, zinc deficiency has been linked to a loss of taste and smell, symptoms recently attributed to infection by this virus (79, 80) . in our opinion, this could be a consequence of a transient acute zinc deficiency produced during infection. zinc deficiency may diminish protein synthesis in taste bud cells, reduce alkaline phosphatase activity in taste buds, alter a zinc-containing salivary protein, block the taste pore region of the taste bud or lead to central nervous system dysfunction (81) . il-6 appears to be important in triggering severe lung damage during sars-cov-2 infection. sustained elevation of il-6 is postulated as being responsible for severe immune-mediated lung damage as well as for macrophage activation syndrome (mas) that might overlap in patients with severe covid-19 (82) . there is much evidence for how this cytokine storm may be related to zinc levels. firstly, il-6 induces expression of metallothioneins (mt) and alpha-2-macroglobulin (a2m) (both zinc-binding proteins), which can reduce zinc bioavailability. il-6, mt, and a2m increase with age and impaired zinc availability contributes to immunosenescence (83) . secondly, zinc acts as an anti-inflammatory element, downregulating many pro-inflammatory signaling pathways, such as il-6-mediated activation of stat-3 (84) . thirdly, il-6 production seems to be increased in zinc-deficient elderly subjects. furthermore, obese patients with lower dietary intake of zinc present with lower plasma and intracellular zinc levels, along with upregulated gene expression of il-1 alpha, il-1 beta, and il-6, compared with patients with higher zinc intake (85) . in this in vivo study, 10 mg of pure zinc supplementation resulted in a significant 96.5% decrease in il-6 release from white blood cells in healthy elderly subjects. fourthly, a polymorphism has been described in the il-6 gene that is related to impaired zinc homeostasis. an il-6 promoter gene single nucleotide polymorphism (snp) at position −174 has been studied in several age-related diseases, such as cardiovascular disease, alzheimer's disease, diabetes, and cancer (86) (87) (88) . zinc deficiency induces a progressive demethylation of the il-6 promoter in thp1 cells, which correlated to increased il-6 expression (89) . genetic variation at the il-6-174g/c locus is involved in determining il-6 production and the immune response. elderly subjects with gg genotypes (called c-) have more risk of developing atherosclerosis due to higher il-6 production, impaired k cell cytotoxicity, increased mt gene expression, and low zinc ion availability compared with c+ carriers (90) . for instance, in elderly individuals aged 65-85 years, c+ polymorphism was associated with il-6 levels of 0.88 pg/ml and zinc levels of 82.2 µg/dl, whereas c-polymorphism was associated with il-6 levels of 1.21 pg/ml and plasma zinc of 77.5 µg/dl, these differences being statistically significant. in another study, c+ carriers had significantly higher plasma zinc levels, lower mt production, higher red blood cell zinc levels, and good nk cell cytotoxicity, as shown in an in vivo study performed in elderly subjects (91) . thus, patients with il-6-174 gg polymorphism (c-carriers) may be susceptible to developing a severe infection due to sars-cov-2, leading to an increase in il-6 levels that produce a cytokine storm related to impaired zinc homeostasis. interestingly, this polymorphism seems to be twice as common in people from italy (68.1%) and other mediterranean countries, compared with northern european countries such as germany (33.8%) (91) . this might explain, to some degree, the difference in mortality rates observed between these countries; as of the 21 march, italy recorded 53,578 confirmed cases and 4,825 deaths, while germany had 22,213 cases and 84 deaths (92) . to date, germany has one of the lowest case fatality rates at 4.10% as of the beginning of may, compared with italy (13.61%). it is probable that other factors, such as differences in early identification of cases and correct isolation, and differences in the proportion of the population that is elderly, may also have been important. nevertheless, studies on genetic susceptibility for developing covid-19 pneumonia and severe illness are underway (93, 94). there are no data regarding the prevalence of this polymorphism in other countries such as the uk or usa, which are known foci of the pandemic. the usa has almost 1,500,000 infected cases with more than 86,000 deaths, which translates to a fatality rate of 5.7% (92) . zinc has shown its ability to inhibit sar-cov rna polymerase (95) . zn 2+ cations, especially in combination with zn ionophore pyrithione, inhibited sars-cov rna-dependent rna polymerase, rdrp. a more than 50% reduction in overall rna synthesis was observed at zinc levels of 50 µm, while <5% activity remained at zinc levels of 500 µm. this finding would make zinc a potential antiviral agent for coronavirus diseases. additionally, chloroquine and hydroxychloroquine, among their other specific mechanisms, act as zinc ionophores and promote cellular uptake of zinc-a mechanism which may increase the effectiveness of these compounds in inhibiting the replicative capacity of the virus (96, 97) . sars-cov-2 and sars-cov require angiotensin-converting enzyme 2 (ace2) for entry into target cells. zinc exposure reduced recombinant human ace-2 activity in rat lung (98) . ace-2 is a zinc metallopeptidase that contains a hexxh motif that functions as the zincbinding domain at its active site. in this in vitro study, in the presence of 100 µm zinc, activity was significantly (p < 0.05) decreased in rat lung and rhace-2 compared with 0 or 10 µm zinc. in the presence of 1,000 µm zinc, activity was further reduced (p < 0.05) in all three preparations compared with 0, 10, and 100 µm zinc. thus, hypothetically, zinc deficiency could facilitate sars-cov-2 infection of target cells due to an increase in ace-2 activity that could facilitate binding with sars-cov-2. in conclusion, the world is facing a pandemic caused by a novel coronavirus, with some countries suffering a higher burden of disease. the infection is known to more severely affect older people with various chronic comorbidities such as obesity, hypertension, and diabetes. zinc has a known role in the regulation of immunity. a plausible biological mechanism for the involvement of zinc in this condition exists, which we summarize in figure 1 . its supplementation, alone or as an adjuvant to medicines that are currently being used to treat active infection, could be beneficial due to its effect on many key factors in the regulation of a severe immune response during infection. zinc supplementation could be a novel treatment for people at high risk of zinc deficiency who develop severe pneumonia due to covid-19. we believe there is enough evidence to further investigate how zinc status or homeostasis is involved in the pathogenesis of severe illness produced by sars-cov-2 infection, and its potential role as an active treatment should be assessed in clinical trials. zinc in infection and inflammation metal elements and gene expression acrodermatitis enteropathica and an overview of zinc metabolism discovery of human zinc deficiency: its impact on human health and disease zinc deficiency. a public health problem? zinc as a gatekeeper of immune function zinc signals and immunity assessment of zinc status size of the zinc pools that exchange rapidly with plasma zinc in humans: alternative techniques for measuring and relation to dietary zinc intake the slc30 family of zinc transporters -a review of current understanding of their biological and pathophysiological roles the slc39 family of zinc transporters the zinc transporter zip5 (slc39a5) regulates intestinal zinc excretion and protects the pancreas against zinc toxicity severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus covid-19): the epidemic and the challenges structure of the rnadependent rna polymerase from covid-19 virus coronavirus disease (covid-2019) the resilience of the spanish health system against the covid-19 pandemic covid-19 in italy: momentous decisions and many uncertainties introductions and early spread of sars-cov-2 in the new york city area clinical characteristics of coronavirus disease 2019 in china hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial in vitro antiviral activity and projection of optimized dosing design of hydroxychloroquine for the treatment of severe acute respiratory syndrome coronavirus a trial of lopinavirritonavir in adults hospitalized with severe covid-19 china approves use of roche drug in battle against coronavirus complications remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial efficacy of chloroquine and hydroxychloroquine in the treatment of covid-19 induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies clinical features of 69 cases with coronavirus disease 2019 in wuhan, china up-regulation of il-6 and tnf-alpha induced by sars-coronavirus spike protein in murine macrophages via nf-kappab pathway plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis infants and elderlies are susceptible to zinc deficiency zinc status is associated with inflammation, oxidative stress, lipid, and glucose metabolism pharmaco-nutrient interactions -a systematic review of zinc and antihypertensive therapy comparative effects of calcium deficiency and supplements on the intestinal absorption of zinc in rats dietary reference intakes for methods of assessment of zinc status in humans: a systematic review zinc: physiology, deficiency, and parenteral nutrition detection and monitoring of disorders of essential trace ele-ments trace elements in human and animal nutrition modern nutrition in health and disease effect of the inflammatory response on trace element and vitamin status assessment of marginal zinc status in humans zinc deficiency and clinical practice assessment of plasma and red cell trace element concentrations, disease severity, and outcome in patients with critical illness zinc deficiency in patients with idiopathic taste impairment with regard to angiotensin converting enzyme activity erythrocytes, erythrocyte membranes, neutrophils and platelets as biopsy materials for the assessment of zinc status in humans ecto 5' nucleotidase (5'nt) as a sensitive indicator of human zinc deficiency multiple impacts of zinc on immune function zinc supplementation for prevention of acute respiratory infections in infants: a randomized controlled trial zinc and the immune system alterations in human natural killer cell activity and monocyte cytotoxicity induced by zinc deficiency t-helper type 1 cytokine release is enhanced by in vitro zinc supplementation due to increased natural killer cells effect of zinc supplementation on plasma il-6 and mcp-1 production and nk cell function in healthy elderly: interactive influence of +647 mt1a and−174 il-6 polymorphic alleles zinc and immunity: an essential interrelation the role of zinc and zinc homeostasis in macrophage function insufficient zinc intake enhances lung inflammation in response to agricultural organic dust exposure hiv-1-transgene expression in rats decreases alveolar macrophage zinc levels and phagocytosis the zincfinger protein zcchc3 binds rna and facilitates viral rna sensing and activation of the rig-i-like receptors pathogen recognition and innate immunity glycogen synthase kinase 3β regulates irf3 transcription factor-mediated antiviral response via activation of the kinase tbk1 virus-triggered ubiquitination of traf3/6 by ciap1/2 is essential for induction of interferon-beta (ifn-beta) and cellular antiviral response zinc potentiates the antiviral action of human ifn-alpha tenfold functional significance of zinc-related signaling pathways in immune cells zinc-dependent suppression of tnf-alpha production is mediated by protein kinase a-induced inhibition of raf-1, i kappa b kinase beta, and nf-kappa b antioxidant effect of zinc in humans zinc dyshomeostasis during polymicrobial sepsis in mice involves zinc transporter zip14 and can be overcome by zinc supplementation zinc deficiency increases organ damage and mortality in a murine model of polymicrobial sepsis dysregulated type i interferon and inflammatory monocytemacrophage responses cause lethal pneumonia in sars-cov-infected mice. version 2 sars: systematic review of treatment effects treatment with lopinavir/ritonavir or interferon-β1b improves outcome of mers-cov infection in a nonhuman primate model of common marmoset treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial type 1 interferons as a potential treatment against covid-19 the use of antiinflammatory drugs in the treatment of people with severe coronavirus disease (2019). (covid-19): the perspectives of clinical immunologists from china heightened innate immune responses in the respiratory tract of covid-19 patients zinc ions inhibit replicaction of rhinoviruses zinc supplementation reconstitutes the production of interferon-alpha by leukocytes from elderly persons zinc and taste disturbances in older adults: a review of the literature anosmia and ageusia: common findings in covid-19 patients the effects of feeding a zinc-deficient diet on taste acuity and tongue epithelium in rats the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease zinc-binding proteins (metallothionein and alpha-2 macroglobulin) and immunosenescence zinc regulates the acute phase response and serum amyloid a production in response to sepsis through jak-stat3 signaling zinc supplementation in the elderly reduces spontaneous inflammatory cytokine release and restores t cell functions interleukin-6 gene alleles affect the risk of alzheimer's disease and levels of the cytokine in blood and brain interleukin-6-174 g > c polymorphism affects the association between il-6 plasma levels and insulin resistance in type 2 diabetic patients the interleukin-6−174 g>c promoter polymorphism is associated with a higher risk of death after an acute coronary syndrome in male elderly patients zinc deficiency enhanced inflammatory response by increasing immune cell activation and inducing il6 promoter demethylation the−174g/c polymorphism of il-6 is useful to screen old subjects at risk for atherosclerosis or to reach successful ageing zinc deficiency and il-6−174g/c polymorphism in old people from different european countries: effect of zinc supplementation. zincage study thüsen j, van der eerden m. histopathology and genetic susceptibility in covid-19 pneumonia human leukocyte antigen susceptibility map for severe acute respiratory syndrome coronavirus 2 zn(2+) inhibits coronavirus and arterivirus rna polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture chloroquine is a zinc ionophore new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? concentrationdependent effects of zinc on angiotensin-converting enzyme-2 activity (1067.4) all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 mayor-ibarguren, busca-arenzana and robles-marhuenda. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-267134-5gz2dotn authors: sallenave, jean-michel; guillot, loïc title: innate immune signaling and proteolytic pathways in the resolution or exacerbation of sars-cov-2 in covid-19: key therapeutic targets? date: 2020-05-28 journal: front immunol doi: 10.3389/fimmu.2020.01229 sha: doc_id: 267134 cord_uid: 5gz2dotn covid-19 is caused by the severe acute respiratory syndrome (sars) coronavirus (cov)-2, an enveloped virus with a positive-polarity, single-stranded rna genome. the initial outbreak of the pandemic began in december 2019, and it is affecting the human health of the global community. in common with previous pandemics (influenza h1n1 and sars-cov) and the epidemics of middle east respiratory syndrome (mers)-cov, covs target bronchial and alveolar epithelial cells. virus protein ligands (e.g., haemagglutinin or trimeric spike glycoprotein for influenza and cov, respectively) interact with cellular receptors, such as (depending on the virus) either sialic acids, dipeptidyl peptidase 4 (dpp4), or angiotensin-converting enzyme 2 (ace2). host proteases, e.g., cathepsins, furin, or members of the type ii transmembrane serine proteases (ttsp) family, such as transmembrane protease serine 2 (tmprss2), are involved in virus entry by proteolytically activating virus ligands. also involved are toll like receptor (tlr) family members, which upregulate anti-viral and pro-inflammatory mediators [interleukin (il)-6 and il-8 and type i and type iii interferons among others], through the activation of nuclear factor (nf)-kb. when these events (virus cellular entry and innate immune responses) are uncontrolled, a deleterious systemic response is sometimes encountered in infected patients, leading to the well-described “cytokine storm” and an ensuing multiple organ failure promoted by a downregulation of dendritic cell, macrophage, and t-cell function. we aim to describe how the lung and systemic host innate immune responses affect survival either positively, through downregulating initial viral load, or negatively, by triggering uncontrolled inflammation. an emphasis will be put on host cellular signaling pathways and proteases involved with a view on tackling these therapeutically. covid-19 is a respiratory disease whose aetiologic agent is a novel beta coronavirus (cov) called severe acute respiratory syndrome (sars)-cov-2/2019-ncov. the initial outbreak of the pandemic began in december 2019, and it is currently affecting the health and safety of the global community. indeed, on may 12, 2020, 4.5 million worldwide cases were confirmed (probably a significant under-estimation given the number of untested asymptomatic subjects), with a death toll exceeding 286,000. before the sars-cov-2 outbreak, two related highly pathogenic covs viruses, middle east respiratory syndrome (mers)-cov (1) and sars-cov (2) , provoked catastrophic epidemics and pandemics, respectively. unfortunately, no drugs nor vaccines have currently been approved to prevent or treat these viral episodes. the first anatomical/histological reports from the lungs of severely sars-cov-2-affected patients experiencing acute respiratory disease syndrome (ards) revealed excessive inflammatory activation and destruction of the bronchial and alveolar epithelium, features already observed during the first sars pandemics in 2003 (3, 4). indeed, in the latter pandemic, lung alveolar epithelial cells were identified as the most likely site of virus replication, and it was suggested that alveolar macrophages may be responsible for the dissemination of viruses within the lungs (3). in accordance, initial histological analyses of lung biopsies from patients positive for sars-cov-2 have shown exfoliation of the bronchial epithelium, which may induce altered mucociliary clearance and affect host immune responses (5). indeed, there is no doubt that the latter are involved in modulating disease onset and progression. for example, early studies report that, similarly with what was observed with sars-cov, lymphopenia [sometimes equivalent or more severe than that observed in human immunodeficiency virus (hiv) infection] is often observed in severely affected patients progressing to ards. despite, or maybe correlated with this, aberrant non-effective innate immune host responses seem associated with severe lung disease during sars (6) (7) (8) (9) (10) (11) (12) . the following sections will give an overview of the molecular and cellular mechanisms underpinning sars-cov virus infections and how lung and systemic host innate immune responses affect survival either positively, through downregulating the initial viral load, or negatively, by triggering uncontrolled inflammation. a particular emphasis will be put on the description of the host cellular signaling pathways and proteases involved with a view on tackling these therapeutically. covs are enveloped viruses with a positive-polarity, singlestranded rna genome encoding four structural proteins: the transmembrane trimeric spike glycoprotein (s, composed of two subunits s1 and s2), envelop (e), matrix (m), and nucleocapsid (n) (13) . the entry of cov viruses into host epithelial cells is mediated by the interaction between the viral envelope s protein homotrimers and the cell surface receptors. following proteolytic cleavage of the cov s protein ("priming"), the s1 ecto-domain recognizes a membrane receptor [angiotensinconverting enzyme 2 (ace2) for sars-cov and sars-cov-2 as well as dipeptidyl peptidase 4 (dpp4) for mers-cov], whereas the s2 c-terminal domain is involved in cell fusion and viral entry (14) (15) (16) . this mechanism of action is very similar to that used by influenza, except that the latter use sialic acids as the cognate receptor for its hemagglutinin (ha) ligand. importantly, many viruses (influenza, mers, cov, and paramyxoviruses such as hendra and nipah viruses) use similar host proteolytic enzymes for cleaving their ligands (ha and s), namely, mostly lysosomal (cathepsins b, l), furin, or trypsin-like proteases (17, 18) . indeed, it is believed that it is the cellular source of these proteases that may determine the infectivity spectrum of these viruses, with the lung and the gastro-intestinal tract being high producers (19, 20) . although a variety of these proteases have been studied and shown to be involved to varying degrees in virus activation, including neutrophil elastase (21) , proteases of the type ii transmembrane serine proteases (ttsp) family [hat, transmembrane protease, serine (tmprss)2, and tmprss4] have recently been demonstrated to be particularly important, albeit probably at different stages of the virus cell cycle (19, 20, 22, 23) . in particular, recent research on sars-cov-2 has focused on tmprss2 and has shown it to be important (although mostly using cell lines infected with pseudotyped virus particles bearing sars-cov-2 s protein) for virus entry (24, 25) . in that context, it has also been demonstrated that the serine protease inhibitor camostat (see also below section on therapeutic targets and conclusion) was protective (24, 25) . in contrast, dpp4 which is necessary for the entry of mers-cov (26, 27) is not involved in sars-cov-2 entry (24) . unlike other sars-covs, the s protein of sars-cov-2 has a furin cleavage site at the boundary between the s1 and s2 subunits, which is processed during biogenesis and which may explain cov-2 high infectivity (28) . although mechanistic studies are obviously still in their infancy, it is very likely that sars-cov and sars-cov-2 target mainly respiratory epithelial cells with similar mechanisms. indeed, as indicated above, initial work has shown that ace2 is the s receptor for both sars-cov (29) and sars-cov-2 viruses (24, 28, 30) , and structural studies using cryo-electron microscopy suggest a binding of two s protein trimer to an ace2 dimer (28, 30) . whether this is strictly dependent on ace2/protease expression is debatable since ace2 is present in other tissues in humans [such as the intestine, kidney, and testis (31) ]. indeed, "seasonal" low pathogenic covs (e.g., cov-229e, cov-oc43) infect mostly upper airways, whereas pathogenic covs (sars-cov/sars-cov-2 and mers) have a tropism for the distal lung and can cause severe pneumonia and ards (32) , as currently demonstrated again in the present pandemic. indeed, potentially explaining this is the fact that seasonal coronaviruses do not use ace2 as a receptor. in vitro, primary nasal and tracheobronchial epithelial cells as well as the calu-3 bronchial cell line were shown to express ace2 (the latter not colocalizing with cilia), and their infection with sars-cov was shown to be highly cytotoxic (33, 34) . in the distal lung, as hinted above, primary alveolar type ii epithelial (atii) cells are also permissive to sars-cov infection (35, 36) . sars-cov-2 has also been shown to infect various respiratory epithelial cell lines including a549 (alveolar origin), beas2-b (bronchial origin), calu-3 cells, as well as primary human bronchial epithelial cells (24) . besides the lung, ace2 is also highly expressed in the intestine (37) , and gastrointestinal symptoms have been recorded with covid-19 (38) . it was shown that sars-cov2 is able to infect enterocytes as well as intestinal organoids and induces a viral response characterized by the expression of mediators related to type i and iii ifn (39) . even if sars-cov2 is thought to originate from bats, the intermediate host between bats and humans is still unknown. sars-cov was previously shown to infect various wild and domestic animals, including cats, ferrets and pigs (40) (41) (42) . similarly, recent work reveals that domestic animals, including ferrets and cats, are permissive to sars-cov-2 infection. in contrast, the virus replicates poorly in pigs, ducks, chickens, and dogs (43) . given the described importance of host proteases in mediating infectivity of a number of viruses, it is no surprise that, upon virus infection, murine knock-out (ko) for some of these molecules has shown some protection. for example, tmprss2-ko mice were protected from pulmonary disease and death following h1n1 and h7n9 influenza infection, but not from that of the influenza h3n2 subtype, demonstrating some specificity and showing also that other ttsp proteases [such as desc1 (tmprss11e) and mspl (tmprss13)] or other factors may be important (44) (45) (46) (47) . similarly, tmprss2 ko mice showed reduced body weight and viral loads compared to wt mice in animals infected with sars-cov (48) . also, it was demonstrated that over-expression of the human dpp4 in mice promoted mers-cov infection, causing lethal disease (49) , and that tmprss2 was instrumental in that context (48) . the control of viral infection requires an optimal and innate coordinated host antiviral immunity. this response is activated by various sensors, including pattern recognition receptors (prr), which recognize pathogen-associated molecular patterns (pamps). although for many viruses, viral rna is a pamp classically detected by different sensors, including toll-like receptors (tlr)3 (which senses double stranded (ds)rna), tlr7 and tlr8 [which sense single stranded (ss)rna], rig-i (which senses short dsrna and ssrna specific motifs), and mda-5 (which senses long dsrna) (50), the sensors potentially recognizing sars-cov genomic material are still elusive. in addition, although, as mentioned above, distal peripheral lung alveolar epithelial cells seem to harbor sars-cov infection in vivo, and although respiratory epithelial cells are known to express tlr3, tlr7, and tlr8 (51, 52) and initiate innate immunity in the lung (53), the study of these cells in anti-cov responses has been hampered by their general poor permissibility to the virus in vitro (except for intestinal caco-2 and hek293 kidney epithelial cells) (54) . in that respect, although the specific prr involved was not identified, the m protein of sars-cov was indeed shown to induce interferon (ifn)-β in a tlrrelated-traf3-independent mechanism in hek293 cells (55) . regarding the lung, the differentiated calu-3 cell line [when cultured at the air-liquid interface (ali)] is the model of choice: in that set-up, sars-cov infection triggered an inflammatory response characterized by increased production of interleukin (il)-6, il-8, gamma interferon (ifn-γ), inducible protein 10 (ip-10), and activation of the transcription factor nf-κb (56) . however, the kinetics of this response was extremely slow, and importantly, type i ifn, an important mediator of anti-viral responses, was undetected. also, another study involving a549 cells demonstrated that the trimeric spike s glyprotein and virus-like particles were able to modestly upregulate ccl2, an important monocytic chemokine (57) . in addition to lung epithelial cells cultured at ali, precisioncut lung slices could also be an interesting tool to study sars-cov2-cells interactions (58) , as demonstrated in influenza infections with human (59) or animal-derived material (60). as mentioned above, ttsps can activate virus-ligands (ha and s protein), but they are also able to modulate cell signaling pathways. for example, recombinant hat is able to activate mucin gene expression in nci-h292 lung epithelial cells (61) . relatedly, we have shown both in vitro in epithelial cells and in a murine model that influenza h3n2 is able to upregulate mucin expression and that this is dependent on human (or mouse) hat upregulation and tace activity (62) . interestingly, haga et al. have shown that inhibiting tace prevents sars-cov cellular entry (63) . strengthening the signaling potential of the receptors, iwata-yoshikawa et al. demonstrated in vivo that poly ic (tlr3 ligand) induces the expression of a variety of pro-inflammatory mediators (ccl2, kc, and il-1) through the expression of tmprss2 (48) . in addition, although unclear as whether it is beneficial or detrimental to the host cell, sars-cov have been shown to activate host stress response, apoptosis, and autophagy (13) . these are also various pathways that may also need to be evaluated therapeutically in the context of the current pandemic. relatedly, we have shown that chloroquine, which also inhibits the autophagic cellular flux by decreasing autophagosomelysosome fusion, can inhibit influenza-mediated ccl5 production (64) . importantly, after having established a foothold in the epithelial compartment, sars-cov can disrupt the epithelial polarity, thereby getting access to the parenchyma tissue: for example, it has been shown that the virus membrane protein e binds to pals1 (protein associated with lin seven 1), a junction protein involved in epithelial polarity, and modifies its cellular distribution at the surface of hek-293 cells (65) . myeloid cells, e.g., alveolar and interstitial macrophages or dendritic cells (dcs), elicit different immune responses toward influenza viruses, according to their subtypes (66) . it is thus predictable that specificities may also exist with respect to sars-cov-2 infections. indeed, although studies are scant, these cells have generally been shown to be poorly permissive to sars-cov replication (54, 67, 68) . however, a few studies have shown that myeloid cells can respond to sars-cov infection. indeed, dosch et al. showed that the s protein could, through tlr2, trigger nf-κb activation and inflammatory responses in peripheral blood mononuclear cells (pbmc) (69) . also, in common with epithelial cells, it was shown that pbmcs and dcs infected with sars-cov produced cytokines and chemokines such as and c-c motif chemokine ligand (ccl)-2 and/or c-x-c motif chemokine ligand (cxcl)-10/rantes/tumor necrosis factor (tnf)/il-8/il-6, but, importantly, not ifn-β (67, 68) . by contrast, a study performed mostly on thp-1 macrophages suggest that mers s protein suppresses macrophages pro-inflammatory responses through dpp4-induction of irak-m and pparγ (70) . furthermore, in an interesting "2-way" system involving differentiated sars-permissive lung calu-3 cells and monocytederived macs and dcs, it was shown that mediators produced by calu-3 cells activate cytokine production by macrophages (il-1β, g-csf, mip-1, and tnf-α) and dcs (il-12p40, mip-1, ifn-γ, il-6, il-8, and mcp-1) but that some of these calu-3 derived mediators (in particular il-6 and il-8) compromised the ability of dcs and macs to activate naïve t cells and phagocytosis (4, 56). this echoes data obtained from patients suggesting that sars may in fact be partly caused by a "paralysis" of the adaptive immune system, characterized by a diminished number of immune cell types including t lymphocytes, dcs and macs (4). demonstrating that sars-cov can induce tlr-dependent host responses in vivo, tlr4, tlr3, and tram ko mice were shown to be more susceptible to mouse-adapted sars-cov, albeit without exhibiting extra mortality (71) . in comparison, mice deficient for the signaling molecule trif were highly susceptible to cov infections, exhibited diminished lung function, aberrant inflammatory responses, and importantly, higher mortality (71) . in addition, a mouse genetic study revealed that the tlr adaptor protein ticam2 was a susceptibility gene to sars-cov (72); mice ko for ticam2 (72), but also myd88 (73), another tlr adaptor protein, were highly susceptible to a mouse-adapted sars-cov lung infection. since polymorphisms of tlrs and myd88 have been associated in humans with heightened sensitivity to a variety of pathogens (74) , these studies, in addition to demonstrating the role of tlr pathways in the sars-cov infection, suggested a human genetic predisposition to sars-cov, and this could explain the variability of severity in patients with covid-19 disease. forthcoming human genetic studies from international collaborative efforts (https://www. covid19hg.org) could reveal genetic variants associated with sars-cov2 susceptibility, as in the gene encoding ace2 as recently suggested (75) . indeed, ace2 genetic variants may be associated with a modulated ace2 protein expression, the sars-cov-2 receptor, which may explain in part patients' susceptibility to infection. genes associated with tlr pathways also represent good candidates, as demonstrated in other respiratory viral infection (e.g., influenza) where tlr3 variants (76) were shown to modulate its virulence. maladaptive activation of innate immune responses (see figure 1b) as already mentioned above, aberrant maladaptive innate immune host responses, including "cytokine storm" events, have been associated with severe lung disease and the development of ards during sars and the covid-19 current episode. mechanistically, these events usually occur at a late stage of the disease, and several mechanisms have been proposed. in particular, a murine study has shown that a prolonged (albeit delayed, as demonstrated also in vitro, see above) type i ifn signaling was instrumental in triggering over-exuberant innate inflammatory monocytes-macrophages immune responses and an impaired virus-specific t-cell response (77) . in complement to the mechanism proposed above, increased lung inflammatory protease (neutrophil elastase and metalloprotease) activity has been demonstrated in ards (78, 79) , with a concomitant imbalance between protease and protease inhibitors activity (80) . in addition, although not yet measured, to our knowledge, in sars murine models, we and others have shown increased protease-mediated lung damage in mice infected with influenza (81-83). additionally, in a mers-cov murine model, it was shown that excessive complement activation was partly responsible for exacerbated lung inflammation (84) . lastly, "cytokines storm" may also results from socs (suppressors of cytokine signaling) inhibition (85) . indeed, upon influenza infection, socs1 and socs3 were shown to reduce type i ifn antiviral responses in human bronchial epithelial cells (86) . also, socs4-deficient mice exhibited heightened sensitivity to influenza infection (87) . studies about socs involvement during coronavirus infections are currently lacking and should therefore bring new interesting information. on may 12, 2020, using the term "covid, " an unbiased search of already registered trials on https://clinicaltrials.gov/ retrieved 1,409 hits, and, when refined with "double blind/placebo, " 119 hits were found. although the number of trials that are ongoing or "under recruitment" is expectedly very high, the range of molecules tested is relatively narrow and aimed at targeting mainly antivirals. these include remdesivir (21 hits), lopinavir/ritonavir (also used in aids), as well as interferons (46 hits) . also falling in that category are trials testing molecules aiming to block viral entry at the cellular surface by targeting ace-inhibitors (32 hits) or the membrane proteases of the ttsp family (see above) using camostat mesilate (5 hits). repurposing of non-antiviral drugs may offer new promising options, such as with ivermectin-an fda-approved anti-parasitic drug widely available and recently shown to inhibit sars-cov-2 in vitro (88) . because the virus load is not necessarily correlated with symptoms deterioration in sars (the latter being often caused by worsening of inflammation at day 7-10 post onset of clinical signs), it follows that anti-inflammatory drugs could/should be prescribed during that stage of the disease (8) . in that context, "classical" anti-inflammatory drugs are indeed currently being tested against covid-19 [e.g., methylprednisolone, budesonide, hydrocortisone, azithromycin, and non-steroïdal anti-inflammatory drugs (nsaids)]. in addition, more specific agents are also being investigated, targeting either il-β (anakinra, 13 hits), il-6 signaling (siltuximab/3 hits, tocilizumab/42 hits, sarilumab/13 hits), or cd24 (cd24fc) with the main objective to modulate the "cytokine storm." however, chloroquine/hydroxychloroquine has, so far, undoubtedly taken the lion's share (178 hits), and it has attracted a lot of media attention. in that respect, the results from an initial pan-european endeavor ("discovery"), now conducted largely in france because of enrollment difficulties, are eagerly awaited. this drug has a "mixed" mode of action. indeed, it acts as an anti-viral (presumably through inhibition of lysosomal enzymes requiring an acidic ph and of activation of endolysosomes, see above section "mechanisms of entry") and as an anti-inflammatory molecule, and it has notably been used in inflammatory rheumatic diseases (89) . despite a relative safe profile, having been administered to millions of people over the years, worries have nevertheless arisen about cardiac issues in many individuals with severe covid-19, and this will have to be properly assessed (90) . regardless, the ultimate prize in the fight against covid-19 (or further sars-cov infections) undoubtedly lies with the future generation of effective vaccines and the development of neutralizing antibodies (91, 92) . unfortunately, coronavirus vaccines in general have attracted less attention compared to the effort dedicated to vaccines against other potential pandemic viruses such as influenza. for example, from 2012 onwards, few sars-cov vaccines reached phase 1 clinical trials for lack of interest from the pharmaceutical industry when it became evident that the virus was not making a "comeback" after its initial appearance. however, although probably too late for affecting the current "first wave" of sars-cov-2 pandemic, many pharmaceutical companies and research laboratories are now working on a plethora of vaccine formulations [for a review, see (91) and https://clinicaltrials.gov, the latter reporting so far 83 clinical trials on vaccines]. indeed, in pre-clinical studies, the determination of cryo-em structures of the sars-cov-2 s ectodomain trimer is providing a blueprint for the design of vaccines and inhibitors of viral entry (28) . in this context, promising results show that murine polyclonal antibodies against s protein of sars-cov are able to elicit polyclonal antibody responses, preventing sars-cov-2 entry into cells, and thus indicating that cross-neutralizing antibodies targeting conserved s epitopes can be elicited upon vaccination (28) . in addition to testing the best sars-cov-2 specific epitopes from the most suitable proteins (s, n, etc.) and way of administration (best vectors, etc.), it is important to select the best animal models. although convincing murine studies are still pending, as indicated above in the section "mechanisms of entry. . . ", studies in other animals investigated the virus susceptibility of chickens, ducks, dogs, pigs, cats, and ferrets, with the latter two being the most permissive (43) . further up in the phylogenetic scale, a recent study reported that an inactivated vaccine candidate for sars-cov-2 was protective in macaques (93) . finally, large epidemiological studies have demonstrated that bacille calmette-guerin (bcg) can heterologously protect against virus infections [e.g., yellow fever virus (94) , probably by tapping on trained immunity mechanisms (95, 96) ]. using such adjuvant-mediated strategies against sars-cov viruses may therefore be an exciting avenue worthwhile pursuing (97) (98) (99) . all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. lg and j-ms received grants from the faculté de médecine sorbonne université (aap covid19 and université de paris (fonds d'urgence sars-cov-2 et covid19) , respectively. this manuscript has been released as a pre-print at osf (100 clinical and immunologic features in severe and moderate coronavirus disease complex immune dysregulation in covid-19 patients with severe respiratory failure clinical features of patients infected with 2019 novel coronavirus in wuhan clinical and virological data of the first cases of covid-19 in europe: a case series breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients human coronavirus: host-pathogen interaction host cell proteases: critical determinants of coronavirus tropism and pathogenesis structural insights into coronavirus entry the spike glycoprotein of the new coronavirus 2019-ncov contains a furinlike cleavage site absent in cov of the same clade type ii transmembrane serine proteases type ii transmembrane serine proteases in cancer and viral infections activation of influenza viruses by proteases from host cells and bacteria in the human airway epithelium influenza virus activating host proteases: identification, localization and inhibitors as potential therapeutics proteasemediated enhancement of severe acute respiratory syndrome coronavirus infection proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor enhanced isolation of sars-cov-2 by tmprss2-expressing cells molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc structure, function, and antigenicity of the sars-cov-2 spike glycoprotein a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury structural basis for the recognition of the sars-cov-2 by full-length human ace2 tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs sars-cov replication and pathogenesis in an in vitro model of the human conducting airway epithelium sars-cov replicates in primary human alveolar type ii cell cultures but not in type i-like cells innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus quantitative mrna expression profiling of ace 2, a novel homologue of angiotensin converting enzyme covid-19: gastrointestinal manifestations and potential fecal-oral transmission sars-cov-2 productively infects human gut enterocytes isolation and characterization of viruses related to the sars coronavirus from animals in southern china virology: sars virus infection of cats and ferrets sars-associated coronavirus transmitted from human to pig susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 tmprss2 is essential for influenza h1n1 virus pathogenesis in mice the host protease tmprss2 plays a major role in in vivo replication of emerging h7n9 and seasonal influenza viruses tmprss2 is a host factor that is essential for pneumotropism and pathogenicity of h7n9 influenza a virus in mice desc1 and mspl activate influenza a viruses and emerging coronaviruses for host cell entry tmprss2 contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection mouse-adapted mers coronavirus causes lethal lung disease in human dpp4 knockin mice rig-i and other rna sensors in antiviral immunity involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded rna and influenza a virus toll-like receptor expression and induction of type i and type iii interferons in primary airway epithelial cells respiratory epithelial cells orchestrate pulmonary innate immunity sars coronavirus and innate immunity the membrane protein of severe acute respiratory syndrome coronavirus functions as a novel cytosolic pathogen-associated molecular pattern to promote beta interferon induction via a toll-likereceptor-related traf3-independent mechanism severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocytederived macrophages and dendritic cells upregulation of the chemokine (c-c motif) ligand 2 via a severe acute respiratory syndrome coronavirus spike-ace2 signaling pathway applications and approaches for 3d precision-cut lung slices: disease modeling and drug discovery innate immune response to h3n2 and h1n1 influenza virus infection in a human lung organ culture model innate immune response to a h3n2 subtype swine influenza virus in newborn porcine trachea cells, alveolar macrophages, and precision-cut lung slices human airway trypsin-like protease increases mucin gene expression in airway epithelial cells influenza a induces the major secreted airway mucin muc5ac in a protease-eg, fr-extracellular regulated kinase-sp1-dependent pathway tace antagonists blocking ace2 shedding caused by the spike protein of sars-cov are candidate antiviral compounds silver nanoparticles impair retinoic acid-inducible gene i-mediated mitochondrial antiviral immunity by blocking the autophagic flux in lung epithelial cells the sars coronavirus e protein interacts with pals1 and alters tight junction formation and epithelial morphogenesis the contributions of lung macrophage and monocyte heterogeneity to influenza pathogenesis cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells sars coronavirus spike protein-induced innate immune response occurs via activation of the nf-kappab pathway in human monocyte macrophages in vitro middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp4-mediated induction of irak-m and ppargamma toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection allelic variation in the toll-like receptor adaptor protein ticam2 contributes to sars-coronavirus pathogenesis in mice. g3 myd88 is required for protection from lethal infection with a mouse-adapted sars-cov toll-like receptors in the pathogenesis of human disease comparative genetic analysis of the novel coronavirus receptor ace2 in different populations severe influenza pneumonitis in children with inherited tlr3 deficiency dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice elastolytic activity in pulmonary lavage fluid from patients with adult respiratory-distress syndrome matrix metalloproteinases and protein tyrosine kinases: potential novel targets in acute lung injury and ards secretory leukocyte proteinase inhibitor is preferentially increased in patients with acute respiratory distress syndrome role of host cellular proteases in the pathogenesis of influenza and influenzainduced multiple organ failure extracellular matrix proteolysis by mt1-mmp contributes to influenzarelated tissue damage and mortality influenza a virus pre-infection exacerbates pseudomonas aeruginosamediated lung damage through increased mmp-9 expression, decreased elafin production and tissue resilience blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov socs proteins in infectious diseases of mammals cutting edge: innate immune response triggered by influenza a virus is negatively regulated by socs1 and socs3 through a rig-i/ifnar1-dependent pathway suppressor of cytokine signaling 4 (socs4) protects against severe cytokine storm and enhances viral clearance during influenza infection the fda-approved drug ivermectin inhibits the replication of sars-cov-2 in vitro mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology risk of qt interval prolongation associated with use of hydroxychloroquine with or without concomitant azithromycin among hospitalized patients testing positive for coronavirus disease 2019 (covid-19) sars-cov-2 vaccines: status report neutralizing antibodies against sars-cov-2 and other human coronaviruses rapid development of an inactivated vaccine candidate for sars-cov-2 bcg vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity trained immunity and local innate immune memory in the lung induction of autonomous memory alveolar macrophages requires t cell help and is critical to trained immunity effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine could bcg vaccination induce protective trained immunity for sars-cov-2? front immunol innate immune memory of tissue-resident macrophages and trained innate immunity: re-vamping vaccine concept and strategies host signaling and proteolytic pathways in the resolution or the exacerbation of coronavirus (cov-2) infection in covid-19 disease: what therapeutic targets? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 sallenave and guillot. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-034310-izpt032p authors: chernomordik, fernando; cercek, bojan; lio, wai man; mihailovic, peter m.; yano, juliana; herscovici, romana; zhao, xiaoning; zhou, jianchang; chyu, kuang-yuh; shah, prediman k.; dimayuga, paul c. title: the role of t cells reactive to the cathelicidin antimicrobial peptide ll-37 in acute coronary syndrome and plaque calcification date: 2020-10-06 journal: front immunol doi: 10.3389/fimmu.2020.575577 sha: doc_id: 34310 cord_uid: izpt032p the human cationic anti-microbial peptide ll-37 is a t cell self-antigen in patients with psoriasis, who have increased risk of cardiovascular events. however, the role of ll-37 as a t cell self-antigen in the context of atherosclerosis remains unclear. the objective of this study was to test for the presence of t cells reactive to ll-37 in patients with acute coronary syndrome (acs). furthermore, the role of t cells reactive to ll-37 in atherosclerosis was assessed using apoe−/− mice immunized with the ll-37 mouse ortholog, mcramp. peripheral blood mononuclear cells (pbmcs) from patients with acs were stimulated with ll-37. pbmcs from stable coronary artery disease (cad) patients or self-reported subjects served as controls. t cell memory responses were analyzed with flow cytometry. stimulation of pbmcs with ll-37 reduced cd8+ effector t cell responses in controls and patients with stable cad but not in acs and was associated with reduced programmed cell death protein 1 (pdcd1) mrna expression. for the mouse studies, donor apoe−/− mice were immunized with mcramp or adjuvant as controls, then t cells were isolated and adoptively transferred into recipient apoe−/− mice fed a western diet. recipient mice were euthanized after 5 weeks. whole aortas and hearts were collected for analysis of atherosclerotic plaques. spleens were collected for flow cytometric and mrna expression analysis. adoptive transfer experiments in apoe−/− mice showed a 28% reduction in aortic plaque area in mcramp t cell recipient mice (p < 0.05). fifty six percent of adjuvant t cell recipient mice showed calcification in atherosclerotic plaques, compared to none in the mcramp t cell recipient mice (fisher’s exact test p = 0.003). recipients of t cells from mice immunized with mcramp had increased il-10 and ifn-γ expression in cd8+ t cells compared to controls. in conclusion, the persistence of cd8+ effector t cell response in pbmcs from patients with acs stimulated with ll-37 suggests that ll-37-reactive t cells may be involved in the acute event. furthermore, studies in apoe−/− mice suggest that t cells reactive to mcramp are functionally active in atherosclerosis and may be involved in modulating plaque calcification. adaptive immunity has a major role in atherosclerosis (1), the underlying cause of coronary artery disease (cad), associated with a variety of antigens that have been described (2) . however, other potential self-antigens remain unknown (3) . it has been proposed that in the chronic inflammatory state present in atherosclerosis, tolerance to self-antigens could be broken through several potential mechanisms, implicating an autoimmune component in atherosclerosis (4) . this is supported by the reported correlation between atherosclerosis and t effector memory cells (5) . furthermore, t effector memory cell density is associated with atherosclerotic plaque stage in humans (6) . rupture of the atherosclerotic plaque is the most common cause of acute coronary syndromes (acs). antimicrobial peptides are an important part of the innate immune system and they are active against pathogens directly through their antimicrobial properties, or through their immunomodulatory effects (7) . the human antimicrobial peptide ll-37, a cleavage product of the cathelicidin proprotein hcap-18, is present in human atherosclerotic plaques (8) , and is associated with platelet activation and induction of thrombosis (9) , with higher serum levels in coronary circulation compared to systemic levels in patients with st elevation myocardial infarction (stemi) (10) . moreover, ll-37 is present in neutrophil extracellular traps (nets), which have been implicated in atherogenesis (11) . they are associated with self-immunity as self-dna/ll-37 complexes that aggravate atherogenesis (12) . interestingly, net burden was associated with infarct size and negatively associated with st segment resolution in patients with stemi (13) . furthermore, ll-37 induced differentiation of human mononuclear cells into bone-forming cells (14) , suggesting a potential role in calcific mineralization. ll-37 is a t cell self-antigen in patients with psoriasis (15) , who have increased risk of cardiovascular morbidity and mortality (16, 17) . it is not known if there is a self-reactive t cell response to ll-37 as a self-antigen in the context of atherosclerosis and if so, whether it is beneficial or pathogenic. cramp is the mouse homolog of the human cathelicidin proprotein hcap-18, which like cramp, is proteolytically cleaved to generate a cathelin-like domain and the cationic anti-microbial domain ll-37, or mcramp in mice (figure 1 ). similar to ll-37 in humans, the proprotein cramp has been linked to atherogenesis in apoe−/− mice as suggested by experiments showing that cramp−/− apoe−/− mice have less atherosclerosis compared to apoe−/− mice (18) . our group has previously shown that immunization with a low dose of the murine proprotein cramp was associated with decreased atherosclerosis in apoe−/− mice (19) . the objective of this study was to test the role of ll-37 as a potential t cell self-antigen in patients with acs. furthermore, the role of t cells reactive to ll-37 in atherosclerosis was assessed using apoe−/− mice immunized with the ll-37 mouse ortholog, mcramp. the protocols were approved by the cedars-sinai institutional review board (irb). peripheral blood mononuclear cells (pbmcs) were isolated from blood collected from 10 patients with acs within 72 h of admission to the cedars-sinai cardiac intensive care unit. patients were consented under the approved irb protocol pro48880. exclusions were inability to give informed consent, age less than 18 years old, active cancer treated with chemotherapy or radiation, patients taking immune-suppressive drugs, and pregnant women. pbmcs from 10 stable cad patients were isolated from blood collected on the day of coronary angiography, consented under the approved irb protocol pro50839 with the same exclusions. consented data use was limited to age, sex, ldl levels, and use/non-use of cholesterol-lowering medication. pbmcs were isolated using ficoll density gradient centrifugation and cryo-preserved in commercially available cryogenic solution (immunospot) in liquid nitrogen. cryo-preserved pbmcs from self-reported controls (n = 15) were purchased from a commercial source (immunospot). cryo-preserved pbmcs were thawed, rinsed in anti-aggregation solution (immunospot), and seeded in culture plates at a density of 3 × 10 6 cells per ml of rpmi 1640 medium supplemented with 10% heat-inactivated pooled human serum and 1× antibiotic/antimycotic. peptides (lifetein) used for stimulation corresponded to ll-37 and the truncated cathelin domain of hcap-18 [cat-hcap-18 (aa 39-136)]. cells were stimulated with one of the following: 20 µg/ml ll-37 or cat-hcap-18 peptide, 0.5× t cell stimulation cocktail containing pma and ionomycin (thermo fisher). culture medium was added at 1 / 3 of the starting volume 48 h later to replenish the nutrients in the medium. cells were harvested 72 h after seeding, stained for viability (live/dead fixable aqua dead stain kit, thermo fisher), and subjected to cell surface staining for flow cytometry using the following antibodies: cd3, cd4, cd8, cd45ra, cd45ro, cd62l, and cd197 (ccr7). isotypes were used as staining control. cd4+ or cd8+ t effector cells were gated on cd45ro+cd62l(−)cd197(−). t effector memory cells were cd45ro+cd45ra(−) cd62l(−)cd197(−), t effector memory ra+ cells were cd45ro+cd45ra(+)cd62l(−)cd197(−). results were tabulated as response index using the following calculation (20) : the results are expressed as response index to account for inherent variations introduced by culturing cells in vitro over time, controlled for by assessing response relative to baseline cell phenotype (% no stimulation) and maximal stimulation (% cocktail stimulation) for each subject pbmc. each data point represents one subject. in samples with sufficient cell numbers, stimulated pbmcs cultured for 72 h were collected for rna extraction using trizol (thermo fisher) and subjected to qrt-pcr with sybr green and a primer pair for human pdcd1. cyclophilin a served as the reference gene. results were expressed as fold-change relative to non-treated cells of each sample using the ct method. conditioned medium was collected after 72 h of ll-37 stimulation of pbmcs and ifn-γ was measured using a commercially available elisa kit (abcam) according to manufacturer's instructions. pbmcs from control samples were stimulated with ll-37 alone or in the presence of either anti-human hla-a, b, c (clone w632) or anti-human hla-dr (clone l243) monoclonal antibody (biolegend; 30 µg/ml) for 72 h and stained for flow cytometry as described above. the studies were approved by the cedars-sinai institutional animal care and use committee. male apoe−/−mice were purchased from jackson laboratory at 6 weeks of age and housed in a specific pathogen-free facility, kept on a 12-h day/night cycle, and had unrestricted access to water and food. the mcramp peptide was purchased (anaspec) with >95% purity according to the manufacturer. donor mice were subcutaneously immunized with either 100 µg mcramp in adjuvant [adju-phos (brenntag, 12.5 µl of a 2% solution) and monophosphoryl lipid a (mpla-sm vaccigrade, invivogen, 10 µg)] or adjuvant alone in a final volume of 200 µl using pbs as vehicle. one group of mice was fed normal chow and immunized at 7, 10, and 12 weeks of age. mice were then euthanized and splenocytes isolated to assess t cell response at 13 weeks of age using flow cytometry. another group of mice was fed normal chow and immunized at 7, 10, and 12 weeks of age. the mice were then euthanized as t cell donors at 13 weeks of age. recipient mice were fed high fat diet consisting of 21% fat, 0.15% cholesterol (td.88137, envigo) starting at 7 weeks of age until euthanasia at 23 weeks of age. after collection of donor mouse splenocytes, red blood cells were lysed using rbc lysis buffer (biolegend), washed with pbs, and cells from the same group were pooled. splenocytes were enriched for t cells using a commercially available mouse t cell magnetic isolation kit (thermofisher) according to the manufacturer's protocol. t cells were enriched to ∼90% assessed by flow cytometry. after counting the isolated t cells, a suspension of 2 x10 6 t cells in pbs was injected by tail vein injection into 18 week-old recipient mice fed with high fat diet for 11 weeks. high fat diet feeding in recipient mice continued for an additional 5 weeks and mice were euthanized at 23 weeks of age. at 23 weeks of age, recipient mice were euthanized and spleens, aortas and hearts were collected. serum was collected for cholesterol level measurement. spleens were collected for flow cytometric staining and analysis. a small portion of the spleen was used for rna extraction. the aortas were dissected free of connective tissue and fat, and stained with oil red o for en face lipid staining (19) . aortic size and plaque content were quantified by a blinded observer using image analysis software (imagepro plus version 4.0, media cybernetics inc., rockville, maryland). heart bases were imbedded in oct (optimum cutting temperature, tissue-tek) and frozen for cryo-sectioning. ten-micron cryosections of the aortic sinus were collected. lipid was stained using oil-red-o. staining for macrophage was performed using cd68 antibody. collagen area was assessed using masson trichrome stain. three slides of approximately 0.1-millimeter intervals for each animal were used for each stain and averaged. image analysis was performed using imagepro. tissue calcification in aortic sinus plaques was confirmed by alizarin red-s stain. splenocytes were stained for viability and surface markers cd3, cd4, cd8, cd44, and cd62l. intracellular staining for foxp3 was performed after surface marker staining followed by fixation and permeabilization (ebioscience). for intracellular cytokine staining, cells were resuspended in 10% heat inactivated fbs-rpmi medium containing 1× monensin (invitrogen) and cultured at 37 • c and 5% co 2 for 4 h. after viability and surface marker staining, cell fixation and permeabilization was performed followed by intracellular staining for il-10 and ifn-γ for flow cytometry. for degranulation assay to measure cd8+ t cell cytolytic activity, splenocytes were incubated with 2.5 µg/ml fluorescent conjugated cd107a in rpmi for 1 h followed by the addition of monensin and incubation for another 4 h. cells were collected and stained for surface markers for flow cytometry. splenic rna was isolated using trizol and subjected to qrt-pcr using primer pairs for mouse il-1β, pdcd1, ctla4, wnt10b, runx2, rankl, and osteocalcin. gapdh was used as reference gene. data were analyzed using the ct method with one adjuvant sample as calibrator. results are expressed as fold change relative to adjuvant. mouse serum levels of soluble rankl and undercarboxylated osteocalcin, the active form of osteocalcin, were measured using commercially available elisa kits (thermofisher and mybiosource, respectively) according to manufacturers' instructions. statistical analysis was performed using r software version 3.5 (r core team, 2018) and graphpad prism version 7 (graphpad software, la jolla, california). data are presented as mean ± standard deviation. for multiple group analysis, significance for normally distributed samples was tested using anova followed by holm-sidak's multiple comparisons test. significance for non-normally distributed samples was tested using kruskal-wallis test followed by dunn's multiple comparisons test. correlation was analyzed using the spearman test with a two-tailed p-value. for two-group analysis, following normality testing, differences between groups were performed using t-test or wilcoxon test, accordingly. a p-value < 0.05 was considered significant, but data trends were also noted. given the role of anti-microbial peptides as potential selfantigens in atherosclerosis, and the possible association with acute events, we tested if the cleaved fragment of hcap-18, the cationic antimicrobial peptide ll-37, would induce differential t cell immune responses in patients with acs. peripheral blood mononuclear cells (pbmcs) from self-reported healthy controls (controls), patients with stable cad (stable) or acs were stimulated with ll-37 for 72 h. pbmcs stimulated with the cathelin domain of hcap-18 (cat-hcap-18) served as control. baseline characteristics of the subjects are detailed in table 1 . stimulation with ll-37 resulted in reduced cd8+ effector t cell response, in both t effector memory (tem) and t effector memory ra+ (temra) cells in pbmcs from controls and patients with stable cad, while pbmcs from patients with acs were resistant to this reduction (supplementary figure 1 and figures 2a-c) . cd4+ effector t cell responses were trending similar to cd8+ effector t cells (figures 2d-f) . there was no significant difference in cd8+ effector t cell response when cells were stimulated with cat-hcap-18 (figure 3) . there was reduced expression of programmed cell death protein 1 (pdcd1) mrna in pbmcs from patients with acs stimulated with ll-37 compared to pbmcs from controls ( figure 4a ), but no difference was noted between groups in pdcd1 mrna expression when cells were stimulated with cat-hcap-18 ( figure 4b ). the nature of the self-reactive responses to cat-hcap-18 and ll-37 were investigated further by testing the relationship between the t cell response to both antigens in each subject. there was significant correlation in cd4+ effector t cell response ( figure 4d ) to cat-hcap-18 and ll-37, but not in cd8+ effector response ( figure 4c) . the results suggest that there is potentially shared antigenic reactivity to cat-hcap-18 and ll-37 in cd4+ t cell responses. ifn-γ measured in conditioned medium from pbmcs stimulated with ll-37 showed qualitative difference in acs compared to control and stable cad patients (supplementary figure 2) . the results suggest that ll-37 reactive t cells may be involved in the acute event. additionally, effector t cell responses in control pbmcs stimulated with ll-37 was partially reversed by blocking with hla class-i antibody (supplementary figure 3) suggesting hla class-i mediated response. the t cell response in acs patients was further subdivided into patients with their first acs event and those who had a recurrent event. the cd8+ t effector response to ll-37 was consistent between first and recurrent acs patients, compared to patients with stable cad (supplementary figures 4a-c) . on the other hand, cd4+ t effector response was significantly higher only in the recurrent acs patients but not in the first acs event compared to stable patients (supplementary figure 4d ). this suggests that cd8+ t cell response to ll-37 persists in immunologic memory and is a common response in both first event acs and recurrent acs. cd4+ t cell effector response on the other hand seems to have evolved and is more prominent in the recurrent acs. no significant differences were observed in cat-hcap-18 response between first acs event and recurrent acs patients in cd8+ t effector (supplementary figures 5a-c) and cd4+ t effector (supplementary figures 5d-f) responses. to investigate the potential role of t cells that are self-reactive to the cationic antimicrobial peptide in atherosclerosis, we used the apoe−/− mouse model of atherosclerosis and immunization with the mouse ortholog of ll-37 called mcramp. immunization provokes a self-reactive t cell response to mcramp in apoe−/− mice to investigate the potential role of t cells reactive to the mouse cationic antimicrobial peptide mcramp in the male apoe−/− mouse model of atherosclerosis, we first tested if t cells were reactive to mcramp. apoe−/− mice fed with normal chow were immunized with mcramp at 7, 10, and 12 weeks of age and euthanized 1 week later for assessment of t cell response. mice injected with adjuvant alone served as control. there was no difference in cd8+ effector memory t cells (supplementary figure 6 and figure 5a ) but a significant increase in cd8+ central memory (cm) t cells (figure 5b ) in splenocytes from apoe−/− mice immunized with mcramp. additionally, there was decreased cd8+foxp3+ cells (supplementary figure 6 and figure 5c ) and increased cytolytic activity, assessed by cd8+cd107a+ t cells (supplementary figure 7 and figure 5d ), in splenocytes of mice immunized with mcramp compared to adjuvant. no differences were observed in cd4+ memory t cell subsets or in cd4+foxp3+ treg cells (figures 5e-g) . to assess whether mcramp-primed t cells are functionally involved in modifying atherosclerosis, adoptive transfer of donor t cells from apoe−/− mice immunized with mcramp or adjuvant alone was performed on apoe−/− recipient mice that had been fed high fat diet for 11 weeks prior to transfer. the 11week feeding with high fat diet assured that the recipient mice were already primed for atherosclerosis. recipient mice were euthanized 5 weeks after cell transfer. recipients of t cells from mcramp immunized mice had significantly reduced aortic plaque area compared to recipients of t cells from adjuvant mice (28% reduction, p < 0.05, figures 6a,b) . there was no significant difference between groups in mean body weight (mcramp = 42 ± 6 gr; adjuvant = 43 ± 6 gr) or mean serum cholesterol (mcramp = 1500 ± 294 mg/dl; adjuvant = 1270 ± 366 mg/dl). thus, atherosclerosis progression was reduced in the mcramp t cell recipient mice without differences in weight or serum cholesterol compared to adjuvant t cell recipient mice. aortic sinus plaque size, lipid content (oil red o staining; figures 7a-c) , macrophage (cd68 staining; figures 7d,e) , and collagen area (masson's trichrome staining; figures 7f,g) were not different between the t cell recipient groups. there were also no differences in il-1β (figure 8a) , pdcd1 (figure 8b ) or cytotoxic t-lymphocyte-associated protein 4 (ctla4, figure 8c ) splenic mrna expression between the t cell recipient groups. focal staining of hematoxylin was observed in several aortic sinus sections from recipient mice, suggesting calcification in the plaque. the presence of plaque calcification was assessed with the calcium-specific alizarin red s staining. calcification in atherosclerotic plaques occurred in 56% of adjuvant t cell recipient control mice, compared to none in the mcramp t cell recipient mice (figures 9a,b and table 2 ; fisher's exact test p = 0.003). immune pathways associated with t cell mediated tissue calcification were then investigated further. recipients of t cells from mcramp immunized mice had increased il-10 and ifnγ expression in splenic cd8+ t cells (supplementary figure 8 and figures 9c,d) , but not in cd4+ t cells (figures 9e,f) , compared to adjuvant t cell recipient control mice. there was increased expression of wnt10b mrna in splenocytes of mcramp t cell recipients, when compared to adjuvant t cell recipient control mice ( figure 10a) . however, there was no difference in the expression of runx2 mrna ( figure 10b) . furthermore, splenocytes from mcramp t cell recipients had increased rankl and osteocalcin mrna expression when compared to adjuvant (figures 10c,d) . however, no difference between groups was noted in serum rankl and undercarboxylated osteocalcin concentration (figures 10e,f) , which is the known active state of osteocalcin. in this study, we showed that: (a) ll-37 stimulation of pbmcs from patients with acs induced the persistence of cd8+ tem cell response compared to patients with stable cad or selfreported controls; (b) immunization of apoe−/− mice with mcramp, the cationic fragment of cramp, increased cd8 cm t cell activation and cytolytic activity; and (c) adoptive transfer of t cells from mice immunized with mcramp was associated with smaller atherosclerotic aortic plaque area, and absence of aortic sinus plaque calcification. our findings suggest that ll-37 self-reactive t cells may be important in an acute coronary event in the context of atherosclerotic disease. ll-37 is a t cell self-antigen in patients with psoriasis (15), who have increased risk of early cardiovascular disease (16, 17) . it has been proposed that psoriasis and atherosclerosis, both chronic inflammatory conditions, share a common inflammatory pathogenic basis (21, 22) that can potentially explain the increased risk of atherosclerosis and its complications associated with this condition. tem cells have rapid effector function upon reexposure to the antigen, but a limited proliferative potential (23) . memory t cells correlate well with atherosclerosis in both mice and humans (5, 24) , and with vulnerable and ruptured plaques in humans (6) . ll-37 was reported to decrease t cell proliferation in resting human pbmcs with increased cell viability without changes in cd4+foxp3+ t reg cell percentage suggesting that in the steady-state, ll-37 treatment results in a degree of immunemodulation apparently independent of increased t regs (25) . in the same report, t cells increased proliferation when co-activated with phytohaemagglutinin without affecting cell viability and with increased t reg cells. increased t cell proliferation coupled with increased t reg cells in their report suggests that t regs may be compensating for the increased proliferation, or that t regs may not have a significant role in ll-37 reactive t cells. our findings extend their report by demonstrating that t cell memory response to ll-37 was reduced in the control subjects and stable cad but persisted in samples from acs patients. this is supported by the qualitative difference in the ifn-γ secretion among the groups. these differences were associated with reduced immune checkpoint pdcd1 mrna expression in pbmcs from acs patients. the reduction in t cell response to ll-37 in the control pbmc was blocked by hla class-i antibody suggesting that the intrinsic response is at least partially mhc-i dependent. however, our results cannot rule out the possibility that adjuvant effects attributed to ll-37 are also involved (15) . the observed blocking of complementary cd4+ t cell memory response by anti-hla class-i antibody is consistent with the report by lande et al. (15) suggesting complementarity of t cell subset responses to ll-37. whether the complementary t cell response to ll-37 in the controls also extend to acs as was reported for some psoriasis patients (15) remains to be determined. thus, combined with other reports, our results suggest that the intrinsic t cell response to ll-37 is down-modulation but in the presence of co-activating factors such as those in the acs patients the t cell memory response persists that may be due in part to reduced checkpoint pdcd1 expression. these findings have potentially important clinical implications with the reported cases of acs associated with immune checkpoint inhibitor treatment (26) . the results in our report may be a response to the acute event but adaptive immune memory to neoantigens develop over a longer time frame than that in our study (within 72 h of admission for the acute event). it is notable that cd8+ tem response to ll-37 is consistent in patients whether it was their first acs event or a recurrent event suggesting the continued presence of memory t cells. it cannot be determined at this time whether the persistent t cell response in acs is pathogenic or a compensatory response in the acute stage of the disease. on the other hand, the cd4+ t effector response was altered in patients that had a recurrent event suggesting that the t cell response in acs evolved as the recurrent patients remained at risk. these observations are consistent with the notion of underlying inflammation in patients who remain at risk for a recurrent event. our results further show a correlation between the cd4+ t cell response to ll-37 and cat-hcap-18. this finding may be a manifestation of expanding antigenic determinants reported in antigen spreading, wherein the original reactive antigen determinant spreads to other regions of the same protein (27) , in this case hcap-18. although speculative, it is interesting that this was not observed in cd8+ t cell response. it remains to be determined what the nature of the involvement of the tem response to ll-37 is in the acute event. studies were performed in apoe−/− mice to investigate the role of t cells reactive to the cationic antimicrobial peptide in atherosclerosis. although the cathelin domain of the proprotein hcap-18/cramp is reported to be active (28) , functional activity is mostly attributed to the cationic antimicrobial peptide domain. our results show increased cd8+ cm t cells and cd8+ t cell cytotoxic activity in mice immunized with the self-peptide mcramp coupled with decreased cd8+foxp3+ t cells. these extend our previous report where immunization with the proprotein cramp resulted in increased cd8+ t cells and cytolytic activity, and reduced atherosclerosis (19) . cd8+ t regs have an important role in self-tolerance, and lower levels cd8+ t regs have been associated with increased immune activity (29) (30) (31) . we confirmed that t cells reactive to mcramp are functionally involved in atherosclerosis by the adoptive transfer of enriched t cells from mcramp-primed apoe−/− mice into recipient apoe−/− mice. to the best of our knowledge, this is the first report of reduced atherosclerosis attributed to t cells primed with mcramp. however, no differences were found in aortic sinus plaques consistent with the report of site-specificity for atherosclerosis in murine models of atherosclerosis (32) . some key signaling pathways involved in tissue calcification potentially regulated by t cells in the recipient mice were investigated. wnt10b, a member of the wnt family signaling pathway, is secreted by t lymphocytes (33, 34) and activates signal transduction cascades that regulate runx2, a transcription factor needed for osteoblast differentiation (35) . rankl activates osteoclasts and bone remodeling in adult mice (36) . t cell expression of rankl (37) is involved in the regulation of bone metabolism (38) . osteocalcin is secreted by osteoblasts associated with bone formation. splenocytes expressing osteocalcin induce atherosclerosis and vascular calcification in apoe−/− mice (39). although the dysregulation of calcification pathways is manifested in differential mrna expression of specific genes involved, the systemic levels detected in serum seemed to remain in equilibrium. this is further observed in the increase in both il-10 and ifn-γ expressing cd8+ t cells. the results suggest the involvement of factors that remain to be characterized. nevertheless, these observations suggest dysregulated tissue calcification pathways in atherosclerosis potentially mediated by memory t cells. interestingly, ll-37 is a t cell antigen in psoriatic disease (15) and altered bone remodeling through osteoblastosteoclast uncoupling has been proposed as an explanation for the concomitant dysregulated processes of both pathological bone formation and resorption usually found in patients with psoriatic arthritis (40) . in our study, plaque calcification was absent in mice that were recipient of t cells primed with mcramp suggesting a role in regulating the process. coronary artery calcification is a marker of atherosclerosis and its role in cad is nuanced (41) . on one hand, the widely used coronary artery calcium score is a strong independent risk factor of major adverse cardiovascular and cerebrovascular events (42, 43) . on the other hand, calcified atherosclerotic plaques may be more stable than non-calcified plaques (44) . the use of statins has been associated with progression of plaque calcification, in spite of their protective role in atherosclerosis (45) . the association found in our animal experiments between recipients of mcramp-primed t cells and the lack of plaque calcification is intriguing, but its significance remains to be determined. the role of t cells reactive to ll-37 in humans, and their potential influence in the pathways of atherosclerotic plaque calcification need to be investigated further. limitations of the study include the potential of ll-37 to be a "promiscuous" hla-binding peptide or to have intrinsic adjuvant properties (15) which coupled with the reduced pdcd1 expression in acs may explain the observed persistence of cd8+ effector cells. although anti-hla class-i antibody blocked the response to ll-37 in controls, our study cannot completely exclude these possibilities. the mouse studies demonstrating the involvement of t cells reactive to mcramp as a self-antigen in atherosclerosis is consistent with the observed tem cell response to ll-37 in acs patients, supporting the presence of a self-reactive tem cell population involved in atherosclerosis. however, the persistence of tem response to ll-37 in patients who suffered an acute event is not in complete alignment with the results of adoptive transfer of mcramp-primed t cells in our mouse studies. one might speculate that the controlled nature of immune priming in the mice skewed the response to be protective against atherosclerosis, even as the physiologic role of reduced calcification of mouse plaques remains to be clarified. it is also possible that the persistence of ll-37 reactive tem response in acs patients is a compensatory response to the inflammatory milieu that subsequently proved inadequate. these remain speculative handicapped by several limitations, including the lack of a reliable mouse model of spontaneous coronary artery plaque rupture which is the major cause of acs in humans. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. the studies involving human participants were reviewed and approved by cedars-sinai irb. the patients/participants provided their written informed consent to participate in this study. the animal study was reviewed and approved by cedars-sinai institutional animal care and use committee. fc, bc, wl, pm, and pd contributed to conception and design of the study. fc, wl, jy, pm, xz, jz, and pd contributed to the data acquisition and analysis. bc and rh contributed to patient recruitment. fc, bc, wl, k-yc, ps, and pd contributed to the interpretation of the data, drafting, and revising the manuscript. all authors contributed to the article and approved the submitted version. innate and adaptive immunity in the pathogenesis of atherosclerosis antigen-induced immunomodulation in the pathogenesis of atherosclerosis t cell subsets and functions in atherosclerosis autoimmunity in atherosclerosis: a protective response losing control? effector memory t cells are associated with atherosclerosis in humans and animal models a change in inflammatory footprint precedes plaque instability: a systematic evaluation of cellular aspects of the adaptive immune response in human atherosclerosis a comprehensive summary of ll-37, the factotum human cathelicidin peptide involvement of the antimicrobial peptide ll-37 in human atherosclerosis the endogenous antimicrobial cathelicidin ll37 induces platelet activation and augments thrombus formation acute stsegment elevation myocardial infarction is associated with decreased human antimicrobial peptide ll-37 and increased human neutrophil peptide-1 to 3 in plasma neutrophil extracellular traps in atherosclerosis and atherothrombosis auto-antigenic protein-dna complexes stimulate plasmacytoid dendritic cells to promote atherosclerosis coronary neutrophil extracellular trap burden and deoxyribonuclease activity in st-elevation acute coronary syndrome are predictors of st-segment resolution and infarct size generation of novel bone forming cells (monoosteophils) from the cathelicidin-derived peptide ll-37 treated monocytes the antimicrobial peptide ll37 is a t-cell autoantigen in psoriasis visualization of atherosclerosis as detected by coronary artery calcium and carotid intimamedia thickness reveals significant atherosclerosis in a cross-sectional study of psoriasis patients in a tertiary care center lack of neutrophil-derived cramp reduces atherosclerosis in mice the cathelicidin protein cramp is a potential atherosclerosis self-antigen in apoe(-/-) mice keratin 8 is a potential self-antigen in the coronary artery disease immunopeptidome: a translational approach psoriasis and systemic inflammatory diseases: potential mechanistic links between skin disease and co-morbid conditions psoriasis and atherosclerosis: two plaques, one syndrome? t cell responses: naive to memory and everything in between single-cell immune landscape of human atherosclerotic plaques ll-37 treatment on human peripheral blood mononuclear cells modulates immune response and promotes regulatory t-cells generation acute coronary syndrome with immune checkpoint inhibitors: a proof-ofconcept case and pharmacovigilance analysis of a life-threatening adverse event the role of antigen-spreading in the efficacy of immunotherapies antimicrobial and protease inhibitory functions of the human cathelicidin (hcap18/ll-37) prosequence recent advances in cd8+ regulatory t cell research description of cd8 + regulatory t lymphocytes and their specific intervention in graft-versus-host and infectious diseases, autoimmunity, and cancer regulatory t cell (treg) subsets return in patients with refractory lupus following stem cell transplantation, and tgf-beta-producing cd8+ treg cells are associated with immunological remission of lupus site specificity of atherosclerosis: siteselective responses to atherosclerotic modulators the mouse wnt-10b gene isolated from helper t cells is widely expressed and a possible oncogene in br6 mouse mammary tumorigenesis treatment with intermittent pth increases wnt10b production by t cells in osteoporotic patients targeted disruption of cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts soluble rankl contributes to osteoclast formation in adult mice but not ovariectomy-induced bone loss trance is a novel ligand of the tumor necrosis factor receptor family that activates c-jun n-terminal kinase in t cells tcell-mediated regulation of osteoclastogenesis by signalling cross-talk between rankl and ifn-gamma myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression altered bone remodeling in psoriatic disease: new insights and future directions coronary artery calcification: from mechanism to molecular imaging coronary artery calcium and long-term risk of death, myocardial infarction, and stroke: the walter reed cohort study nla/pcna guideline on the management of blood cholesterol: a report of the american college of cardiology/american heart association task force on clinical practice guidelines calcified carotid atherosclerotic plaque is associated less with ischemic symptoms than is noncalcified plaque on mdct impact of statins on serial coronary calcification during atheroma progression and regression the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.575577/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 chernomordik, cercek, lio, mihailovic, yano, herscovici, zhao, zhou, chyu, shah and dimayuga. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003378-0ozhye9q authors: yu, haijing; liu, yang; wang, hongwu; wan, xiaoyang; huang, jiaquan; yan, weiming; xi, dong; luo, xiaoping; shen, guanxin; ning, qin title: clara cell 10 kda protein alleviates murine hepatitis virus strain 3-induced fulminant hepatitis by inhibiting fibrinogen-like protein 2 expression date: 2018-12-13 journal: front immunol doi: 10.3389/fimmu.2018.02935 sha: doc_id: 3378 cord_uid: 0ozhye9q background: fulminant hepatitis (fh) is a serious threat to human life, accompanied by massive and rapid necroinflammation. kupffer cells, the major immune cell population involved in innate immune responses, are considered to be central for fh. fibrinogen-like protein 2 (fgl2) is a pro-coagulant protein that is substantially induced in macrophages upon viral infection, and fgl2 depletion represses murine hepatitis virus strain 3 (mhv-3) infection. clara cell 10 kda (cc10) protein is a secretory protein with anti-inflammatory properties in allergic rhinitis and asthma. however, its mechanisms of action and pathogenic roles in other disease are still unclear. in this study, we aimed to determine the role of cc10 in fh and the regulation of fgl2 by cc10. methods: a mouse fh model was established by peritoneal injection of mhv-3. the mice received cc10 protein through tail vein injection before viral infection. survival rate, liver function, liver histology, fibrin deposition, and necrosis were examined. the regulatory effect of cc10 on fgl2 expression was investigated using thp-1 cells and mouse peritoneal macrophages in vitro. results: in the mouse fh model induced by mhv-3, the survival rate increased from 0 to 12.5% in the cc10 group compared to that in the saline-only control group. meanwhile, the levels of alt and ast in serum were significantly decreased and liver damage was reduced. furthermore, hepatic fgl2, tnf-α, and il-1β expression was obviously downregulated together with fibrin deposition, and hepatocyte apoptosis was reduced after administration of cc10 protein. in vitro, cc10 was found to significantly inhibit the expression of fgl2 in ifn-γ-treated thp-1 cells and mhv-3-infected mouse peritoneal macrophages by western blot and real-time pcr. however, there was no direct interaction between cc10 and fgl2 as shown by co-immunoprecipitation. microarray investigations suggested that hmg-box transcription factor 1 (hbp1) was significantly low in cc10-treated and ifn-γ-primed thp-1 cells. hbp1-sirna treatment abrogated the inhibitory effect of cc10 on fgl2 expression in human umbilical vein endothelial cells (huvecs). conclusion:cc10 protects against mhv-3-induced fh via suppression of fgl2 expression in macrophages. such effects may be mediated by the transcription factor hbp1. fulminant hepatitis (fh) is a serious life-threatening disease characterized by massive hepatocyte necrosis, severe liver damage, and high mortality. the underlying mechanisms and the pathogenesis of fh are not clear. however, accumulating evidence suggests that, regardless of the pathogenesis of fh, the host's inflammatory responses contribute to liver microcirculatory disorders and injuries. accordingly, it has been shown that immune cell activation and inflammatory cytokines play an important role in fh (1) . in recent years, our laboratory has conducted extensive research on the pathogenesis of fh and found that immune cells play a key role in it. kupffer cells, natural killer (nk) cells (2, 3) , cytotoxic t-lymphocytes (ctls), and double negative t-cells (dnt) (4) (5) (6) in liver and the cytokines that are produced by these cells cause liver damage. prothrombinase fgl2 belongs to the fibrinogen superfamily and is produced by activated macrophages or endothelial cells, transforming prothrombin directly into thrombin, so as to quickly initiate the process of coagulation. this promotes the conversion of fibrinogen into fibrin, resulting in thrombosis (7) (8) (9) (10) (11) (12) . our study found that fgl2 was highly expressed in peripheral blood mononuclear cells (pbmcs) and in liver tissue of humans or mice with severe viral hepatitis, and was positively related to the severity of the disease (13, 14) . gene therapy targeting fgl2 silencing showed that the survival rate of fulminant hepatitis mice increased from 0 to 33.3% (15) . thus far, the discovery and related research involving fgl2 have provided new insights into the molecular mechanism of hepatocyte necrosis in fh. in view of the important role of fgl2 in severe viral hepatitis, investigations concerning the regulation of fgl2 will be beneficial in the search for new strategies for treatment of severe hepatitis. clara cell 10 kda protein (cc10), also considered to be uteroglobin, clara cell secretory protein, is one of members of secretoglobin superfamily. expressed in mucosal epithelial cells of organs (including lungs and nose) that communicated with the outside world (16) . cc10 has immunomodulatory and anti-inflammatory effects. compared to wild-type mice, cc10-knockout mice exhibited excessive airway inflammation abbreviations: fh, fulminant hepatitis; mhv-3, murine hepatitis virus strain 3; fgl2, fibrinogen-like protein 2; cc10, clara cell 10 kda protein; alf, acute liver failure; pfu, plaque-forming units; pbs, phosphate-buffered saline; alt, alanine aminotransferase; ast, aspartate aminotransferase; pca, pro-coagulant activity; hrp, horseradish peroxidase; tunel, terminal deoxynucleotidyl transferase dutp nick end labeling. caused by allergic reaction and bacterial and viral infections (17) . reduced levels of cc10 are associated with inflammatory and allergic airway diseases, including sinusitis, asthma and allergic rhinitis (18) (19) (20) (21) . previous studies and published articles show that cc10 protein can not only inhibit th17 cell responses by inhibiting expression of related molecules of dendritic cells and cytokines in mice with allergic rhinitis, but also can inhibit chitosan-3 like protein 1 (22, 23) . moreover, cc10 inhibits the expression of an important immune regulator, osteopontin (opn), in models of allergic rhinitis (21) . in this study, we investigated the role of cc10 in hepatitis virus strain 3 (mhv-3)-induced fh in mice and explored whether cc10 protein could regulate fgl2 in the disease process. female balb/cj mice (shanghai shilaike animal seed center, shanghai, china), 6-8 weeks of age, with a body weight of 18.0-20.0 g, were kept in tongji hospital with food and water. mice were divided into two groups: cc10 group (experimental group) and phosphate-buffered saline (pbs) group (control group). this study was carried out in accordance with the recommendations of the guidelines of the national institutes of health and the animal experiment committee of tongji hospital. this study was reviewed and approved by the animal experiment committee of tongji hospital. the human monocyte cell line thp-1 was purchased from the cell institute of the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) were obtained from the biology treasure center of wuhan university, china. the chinese hamster ovary (cho) cell line was acquired from the typical culture preservation commission cell bank, the chinese academy of sciences (shanghai, china). human umbilical vein endothelial cells (huvecs) and cho cells were cultured in dulbecco's modified eagle's medium (dmem), and thp-1 cells were maintained in rpmi 1,640 containing 10% heat inactivated fetal bovine serum (fbs, gibco life technologies, usa), 100 u/ml penicillin, and 100 mg/ml streptomycin and cultured at 37 • c, 50 ml/l co 2 and 95% humidity. peritoneal exudative macrophages (pems) were obtained from balb/cj mice. cells were resuspended in rpmi 1,640 supplemented with 10% fbs at 1-2 × 10 6 cells/ml in a 6-well plate and incubated for 4 h. they were then washed with rpmi 1640 medium and non-adherent cells discarded. the adherent cells were macrophages and were incubated for a further 12 h. peritoneal exudative macrophages (pems) were divided into two groups. one group was supplemented with cc10 protein (150 ng/ml) and in the other group, pbs was added. after 2 h of stimulation, 1,000 plaque forming units (pfus) of mhv-3 was added to the cells, which were then cultured for 4 h. peritoneal exudative macrophages (pems) were harvested and lysed for real-time pcr and western blotting analysis. cell apoptosis was detected by the terminal deoxynucleotidyl transferase dutp nick end labeling (tunel) method with a tunel apoptosis detection kit (roche, switzerland). briefly, 5 µm sections were deparaffinized, dehydrated through an alcohol series and incubated with proteinase k for 30 min at 37 • c. after stopping the proteinase k digestion reaction with pbs, the samples were incubated with terminal deoxynucleotidyl transferase end-labeling cocktail (a mixture of terminal deoxynucleotidyl transferase and dutp at a ratio of 2:29, respectively), for 2 h at 37 • c in an immunohistochemistry wet box. following washing and blocking, each section was supplemented with reagent (converter-pod) to cover the tissues and incubated for 30 min at 37 • c in a wet box. then, the liver tissue sections were washed with pbs, and colored with diaminobenzidine (dab) subsequently. hepatocytes with nucleus stained brownish yellow were considered to be apoptotic cells. the expression of fgl2 on thp-1 cells was measured by flow cytometry (bd facs canto ii, usa). briefly, cells (2 × 10 5 per tube) were incubated with human trustrain fcx (fc receptor blocking solution, biolegend, usa) for 10 min at room temperature and then incubated in the dark with mouse anti-fgl2 antibody (1:100, abnova,) or normal goat serum (an isotype control) at 4 • c for 40 min. cells were washed with pbs and incubated in the dark with pe-conjugated goat anti-mouse igg antibody (1:50, biolegend, usa) at 4 • c for 30 min. cells were then washed with pbs and resuspended in 300 µl pbs for study. liver slices were fixed in 4% paraformaldehyde and then embedded in paraffin. immunohistochemistry of liver tissues was performed using sp-9001 splink detection kits (biotin-streptavidin hrp detection systems) (zsgb-bio, beijing, china) according to the manufacturer's instructions. for immunohistochemistry staining, the expression of fgl2, fibrinogen, fas and tnf-receptor 1 in mouse liver tissues was detected with polyclonal rabbit anti-mouse fgl2 antibody (1:100, proteintech, usa), polyclonal rabbit anti-mouse fibrinogen antibody (1:1,000, abcam, england), polyclonal rabbit antimouse fas antibody (1:50, abcam, england), and polyclonal rabbit anti-mouse tnf-receptor 1 antibody (1:500, abcam, england), respectively. after incubation with an horseradish peroxidase (hrp)-labeled goat igg fraction to rabbit igg fc, the target protein was detected using a dab kit (zsgb-bio, beijing, china). the slides were then counterstained with hematoxylin and visualized under a microscope (olympus, tokyo, japan). liver tissue and cells were homogenized in ripa lysis buffer with phenyl methane sulfonyl fluoride (pmsf) protease inhibitor. protein lysates were separated by sds-page, and western blotting was performed using a monoclonal mouse antihuman/mouse fgl2 (1:750, abnova), a monoclonal mouse antihuman hbp1 (1:100, santa cruz, usa), and a monoclonal rabbit anti-human/mouse β-actin (1:1,000, cell signaling technology, usa). liver tissues were collected from mhv-3-infected balb/cj mice at 72 h, and total rna was extracted using trizol reagent (invitrogen, usa) and then reverse transcribed into cdna by using revertra ace qpcr rt kit (toyobo, japan). the cdna was then amplified by rt-pcr by using dream taq green pcr master mix (2 ×) (thermo scientific, usa). realtime quantitative pcr (qpcr) with sybr green real-time pcr master mix (toyobo, japan) was performed using a cfx96 real-time pcr detection system (bio-rad, usa) and mrna levels were normalized with reference to those of the house keeping gene gapdh. primer sequences for qpcr amplification were as follows: mtnf-α forward, 5 ′ -ttt gag atc cat gcc gtt gg-3 ′ ; mtnf-α reverse, 5 ′ -gcca cca cgc tct tct gt-3 ′ ; mil-1β forward, 5 ′ -tgt aat gaa aga cgg cac acc-3 ′ ; mil-1β reverse, 5 ′ -tct tct ttg ggt att gct tgg-3 ′ . mfgl2 forward, 5 ′ -gcc aaa tgt gag tcc ctg gaa-3 ′ ; mfgl2 reverse, 5 ′ -ttc cac cca aga gca cgt tta ag-3 ′ ; hfgl2 forward 5 ′ -aca gtt cag gct ggt ggt-3 ′ ; hfgl2 reverse, 5 ′ -ggc tta aag tgc ttg ggt-3 ′ ; hbp1 forward, 5 ′ -tga agc aga agc tgg gagt-3 ′ ; hbp1 reverse, thp-1 cells were treated with 100 ng/ml phorbol 12-myristate 13-acetate (pma) (sigma, usa) for 48 h to induce differentiation toward adherent macrophage-like cells as reported previously (24) . the cc10 group was supplemented with cc10 protein (150 ng/ml). after 2 h of stimulation, ifn-γ (10 ng/ml) was added to these cells, which were then cultured for 12 h before they were collected for western blotting and real-time pcr studies. the chinese hamster ovary (cho) cells were cultured in 10 cm cell culture dishes with dmem supplemented with 10% fbs until 80-90% confluence. next, 12 µg pcdna3.1-hfgl2 (constructed in our lab) was mixed with 12 µg pcdna3.1-hcc10 in serumfree dmem. the mixture was then combined with lipofectamine 2,000 (invitrogen, usa) and mixed gently. after incubation at 27 • c for 20 min, the solution was added to cho cells and incubated at 37 • c in 5% co 2 . four to six hour after transfection, the medium was removed and fresh medium containing 10% fbs was added. at 48 h after transfection, the cells were collected for co-immunoprecipitation analysis to evaluate the interaction of cc10 with fgl2. both huvec and thp-1 cells express fgl2. however, in the transfection experiments, it is difficult to transfect the thp-1 cells with sirna, so we use huvec instead of thp-1. human umbilical vein endothelial cells (huvecs) were cultured in figure 1 | cc10 protein increased survival rate and reduced liver damage in mice. (a) the survival rate of cc10 group is higher than the control group comprised of mhv-3-infected balb/cj mice treated with saline. cc10 protein (2 µg) or saline were injected into mice by tail vein. balb/cj mice then received 100 pfu of mhv-3 intraperitoneally 24 h later to develop fulminant viral hepatitis. then, cc10 protein (2 µg) or saline were injected into mice by tail vein following mhv-3 infection 24 h later. the survival rate was observed for 10 days (n = 24/group). representative data from three independent experiments are shown. the survival curve was analyzed by using the log-rank test. ***p < 0.001 compared with saline group. (b) histopathology of liver tissues (h&e staining; original magnification, ×400, n = 5/group) at 72 h post-mhv-3 infection was evaluated in the two groups of mhv-3-infected balb/cj mice. livers were collected from saline-treated (a) and cc10-treated (b) balb/cj mice at 72 h after mhv-3 infection. arrows point to inflammatory cell infiltration areas or necrotic regions with inflammation. (c) effect of cc10 on serum alt and ast levels (n = 6-8/group). values represent means and standard error of three independent experiments performed in triplicate. **p < 0.01 compared with the saline group. six-well plates with dmem supplemented with 10% fbs until 70-80% confluence. 50 pmol hbp1-sirna was mixed with 125 µl serum-free dmem. two microliter lipofectamine 2,000 was gently mixed with serum-free dmem. after incubation at 27 • c for 5 min, the solution was added to huvecs and incubated at 37 • c. four hour after transfection, the medium was removed and fresh medium containing 10% fbs was added. at 48 h after transfection, cells were collected for real-time pcr and western blot analysis to evaluate the effects of hbp1 on fgl2. at 24 h after transfection, the cc10 group was supplemented with the cc10 protein (150 ng/ml). after 4 h of stimulation, ifn-γ (10 ng/ml) was added to these cells. these cells were then cultured for 24 h before they were harvested for real-time pcr studies to evaluate the effects of cc10 on fgl2 by hbp1. negative control was used as a control. to detect whether there was a potential interaction between cc10 protein and fgl2, cho cells were transfected with pcdna3.1-hcc10 and pcdna3.1-hfgl2 for 48 h. cells transfected with empty plasmid pcdna3.1 (mock) were used as negative controls for cc10 gene transfection. immunoprecipitation and immunoblotting were performed by using pierce co-immunoprecipitation kit (pierce, usa). total cell proteins were extracted as previously described (25) . the proteins were immunoprecipitated by mouse anti-human fgl2 antibody (1:500, abnova). for co-immunoprecipitation experiments, western blotting was performed using both rat anti-human uteroglobin/scgb1a1 antibody (1:750, r&d, usa) frontiers in immunology | www.frontiersin.org and mouse anti-human fgl2 antibody (1:500, abnova). control isotype rat igg1 was used as a negative control for primary antibodies. the human cc10 coding region gene, including a 389 bp sequence, was amplified from homogenized human turbinate tissue by rt-pcr. in this study, the sequences of pcr primers for cc10 were as follows: hcc10-forward, 5 ′ -ccc tcc acc atg aaa ctcg-3 ′ ; hcc10-reverse, 5 ′ -tga gat gct tgt ggt tta ttg aag-3 ′ . the pcr products were cloned into peasy-t1 cloning vector (transgen, beijing, china) and then subcloned into hindiii/xbai site of pcdna3.1 vector (invitrogen, usa) to form eukaryotic expression plasmids pcdna3.1-hcc10. microarray analysis was used to screen changes in genome-wide gene expression patterns in thp-1 cells with or without cc10 protein. the changes in over 47,000 human gene expression patterns were assessed using affymetrix gene microarrays (human genome u133 plus 2.0) (capitalbio co.,ltd., beijing, china). three replicates were used for microarrays analysis. data obtained from the experiments are expressed as means ± sem. survival curve comparisons were performed with the log rank test. multiple group analyses for data were evaluated by one-way analyses of variance. analyses of two group results were performed using student's t-test to evaluate the statistical significance of differences. values of p < 0.05 indicated significance. to establish an animal model of mouse fh, mhv-3 was injected intraperitoneally to balb/cj mice (24 mice/group). to further study the role of cc10 in fh, recombinant mouse cc10 protein (2 µg/mouse) or saline was administrated into the tail vein 24 h prior to mhv-3 infection. the same dose of cc10 protein or saline was then administered 24 h later. the survival rate of the cc10 and saline groups was observed for 10 days. the results showed that mice in the two groups began to die at 48 h after injection of mhv-3 and exhibited symptoms of horripilation, slow activity, and reduced food consumption. in the cc10 group 24 mice were alive on day 3 after infection, 4 mice alive on day 4, and 3 of 24 (12.5%) mice recovered from fulminant viral hepatitis. at the same time, in saline treated group, there were 5 mice alive on day 3, 1 mice alive on day 4 after infection, and no mice survived to day 5. that is to say, the mice in the saline group died within 3 or 4 days. three of 24 (12.5%) mice of the cc10 group recovered from fulminant viral hepatitis ( figure 1a) . to better understand the mechanisms underlying the biological effects of the cc10 protein, liver function (alt and ast levels in serum) and liver histology in mice of mhv-3-infected was performed. liver tissues were harvested 72 h following mhv-3 infection, and liver histology was detected by h&e staining. these results showed that there was substantial inflammatory cell infiltration and widespread necrosis of hepatocytes in the liver tissue of the saline group mice (figure 1ba ). there were rare or no infiltrating inflammatory cells, and few or no hepatocyte necrosis in the livers of mice in the cc10 group 72 h after mhv-3 infection (figure 1bb) . serum alt and ast levels in mice were observed 72 h after mhv-3 infection. the results showed that serum alt and ast levels in the saline group reached a peak 72 h after mhv-3 infection, but there was no significant increase in the cc10 group compared to the levels in the control group (p < 0.01, figure 1c) . these results suggested that cc10 protein has a role in protection against mhv-3-induced liver injury in mice. to further elucidate the mechanisms of reduced liver injury following cc10 protein injection, we investigated the cytokines tnf-α and il-1β expression. because these two cytokines play a crucial role in the liver damage of fh. they are characterized by an increase in apoptosis. levels of tnf-α and il-1β in liver tissues were markedly reduced in the cc10 group (as shown in figure 2a) . hepatic apoptosis (figure 2b ) was significantly reduced in the cc10 group. we and collaborators have a long standing interest in studying the role of fgl2 in viral hepatitis. fgl2 has been verified to play an essential role in the progression of fulminant viral hepatitis as we appreciate from previous reports. we have provided liver pathology figures and liver function for mhv-3 infected mice with a fgl2 gene knockout as shown in supplementary figure 1 . the data was comparable with previous reports from our center and collaborators. from this current study we shown that cc10 plays a protective role in liver damage.to study the related molecules of cc10 in mhv-3-induced fh mice, we evaluated whether there was crosstalk between fgl2 and cc10. we found that the expression of fgl2 in the liver of mice was reduced 72 h after mhv-3 infection and treatment with cc10 protein (figures 3a,b) . furthermore, fibrin deposition, an indicator of liver injury associated with fgl2 expression in fh, was also decreased in the livers of cc10-treated mice compared to that in controls (figure 3c ). this indicates that cc10 treatment reduced liver injury after viral infection by inhibiting fgl2 expression. we examined the effect of increasing doses of cc10 protein (0, 50, 150, and 300 ng/ml) on ifn-γ-induced fgl2 expression in thp-1 cells. cc10 treatment showed a 10.1% decrease in thp-1 cells compared to that in control after stimulation with 10 ng/ml ifn-γ for 12 h. cc10 protein inhibited fgl2 expression between doses of 0 ng/ml and 300 ng/ml (figure 4a ). in particular, 150 ng/ml cc10 protein had the strongest inhibitory effect on fgl2 expression among the doses, and we chose this dose for the following experiments. we explored the effect of different time points of stimulation with a concentration of 150 ng/ml cc10 protein. after stimulation with cc10 protein for 6, 12, and 24 h compared to the pbs control, the strongest inhibitory effect on fgl2 expression was noted at 12 h; hence, we chose this time point for the following studies ( figure 4b ). an increasing number of studies suggest that macrophages are the primary source of fgl2. in order to ascertain that cc10 has a direct effect on macrophages, we treated thp-1 cells with recombinant cc10 and assessed the expression of fgl2. unlike in controls, ifn-γ induced a significant increase in fgl2 expression. this effect was attenuated when cells were treated with cc10 protein (figures 4c,d) , revealing that cc10 directly reduces the levels of fgl2 in macrophages. to further explore the possibility that cc10 protein directly acts on macrophages, we infected murine pems with mhv-3 in the presence of recombinant cc10 and determined fgl2 expression. compared to levels in the controls, mhv-3infected macrophages exhibited a significant increase in fgl2 production, and this effect was abolished by using cc10 protein (figures 5a,b) , indicating that cc10 directly modulates fgl2 production in macrophages. in order to determine genes that were downregulated after stimulation by cc10 protein, we used dna microarray analysis to screen for differentially expressed genes. thp-1 cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl2 was stimulated by ifnγ. the experimental group was treated with cc10 protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube2w, hectd1, mir612, atrx, sox4, hbp1, and fgl2 (supplementary table 1) . and then these genes were tested by qpcr. however, ube2w, hectd1, mir612, atrx, and sox4 was not differentially expressed by qpcr, while hbp1 and fgl2 were still down-regulated genes. dna microarray analysis identified hbp1 as a down-regulated gene involved in the pathological processes of the regulation of cc10. recently, very limited studies have explored the role of hbp1 in fh. nevertheless, the mechanistic functions of hbp1 in fh remain largely unexplored. therefore, we selected this gene for further study. qpcr analysis confirmed that mrna levels of hbp1 were significantly decreased in thp-1 cells after cc10 protein stimulation compared to that in the pbs control group (figure 6a ). we knocked down hbp1 using hbp1-sirna. then, transfection of hbp1-sirna into huvecs was detected by qpcr and western-blotting methods. as expected, hbp1 knockdown led to significantly decreased expression of hbp1 (figures 6b,c) . furthermore, hbp1 knockdown impaired expression of fgl2 (figure 6d ), suggesting that hbp1 was able to activate fgl2. hbp1-sirna was used to transfect huvecs. then, ifn-γ was added to induce the expression of fgl2 followed by stimulation with cc10 protein (150 ng/ml) after 2 h. finally, we explored the expression of fgl2 by qpcr. the results showed that hbp1-sirna treatment abrogated the inhibitory effect of cc10 on fgl2 expression in huvecs (figure 7) . that is to say, cc10 could suppress fgl2 expression in macrophages. such an effect may be mediated by the transcription factor hbp1. it is well-known that cc10 protein can suppress the immune response. in animal models of allergic diseases of the respiratory tract, most of evidences confirm this inhibition (26) . its function in fh has not been investigated yet. here, we used a murine fh model established by mhv-3 infection to explore the effects of cc10 in this disease process. to determine the role of cc10 in the pathogenesis of fh, cc10 protein was injected into a mouse fh model established by mhv-3 infection. mhv-3-induced liver injury in cc10-treated mice occurred rarely and the areas of lesions were much fewer than those in saline-treated control mice. in summary, these results suggested that cc10 could reduce pathological liver damage in this fh model together with lower mortality rates followed by mhv-3 infection. mhv-3 induced fulminant viral hepatitis progresses rapidly and infected mice die within 3-5 days. previous studies suggested fgl2 played a vital role in this process with a 15-40% increase of survival when fgl2 was deleted (12, 15, 27, 28) . multiple inflammatory factors or mediators including tnf-α and ifn-γ, il-1β and c5ar have been demonstrated to promote fh progression with significant discrepancies between liver damage and survival rate (29) (30) (31) (32) , which is accordant with our observation that cc10 substantially alleviated liver injury though survival rate improved mildly. the survival rate based on hours may be more accurate to examine the effect of cc10 on fh. it is speculated that fgl2 can mediate lethality in mhv-3-induced fh. this is due to the fact that fgl2 induces the deposition of fibrinogen, which leads to activation of the coagulation cascade and induction of procoagulant activity (15) . to determine whether the tissue necrosis was mediated by fgl2 in cc10-treated mice following infection, fgl2 expression was observed. results suggested that the expression of fgl2 was significantly increased in mhv-3-induced fh mice and cc10 treatment significantly reduced the production of fgl2 in the infected liver and serum. in addition, decreased fibrinogen deposition was also observed in the livers of cc10-treated mice. therefore, our research results strongly clarify that the lower mortality of cc10-treated mice after mhv-3 infection is due to the lower levels of fgl2 and decreased fibrinogen deposition. indeed, it has been reported that fgl2 is expressed on macrophages, and the expression of fgl2 is believed to be induced by ifn-γ and tnf-α (22) . cultured thp-1 cells activated by ifn-γ or il-2 have been demonstrated, with induction of fgl2 expression and enhanced activation of human prothrombin (23) . therefore, in this study, we explored this cell line to investigate the modulation of cc10 on fgl2. surprisingly, we found that cc10 directly inhibited ifn-γ-induced fgl2 expression in thp-1 cells. as we know, ifn-γ has proved to be the main cytokine that leads to the development and progression of fh. also, it was shown that ifn-γ might exert its own proinflammatory biological function through enhancing fgl2 expression. therefore, in our study, cc10 might counter the effect of ifn-γ in the setting of fh, which substantiates its role in fh. these results demonstrated that cc10 regulates the expression of fgl2 in macrophages. in the current study, we used co-immunoprecipitation to analyze binding between cc10 and fgl2. in this study, we investigated possible protein-protein interactions between cc10 and fgl2 in vitro. the chinese hamster ovary (cho) cells transfected with pcdna3.1-hcc10 and pcdna3.1-hfgl2. cellular proteins were immunoprecipitated with anti-cc10 antibody or anti-fgl2 antibody. immunoblotting was performed with anti-fgl2 and anti-cc10 antibodies. immunoprecipitation of protein extracts from pcdna 3.1-cc10 and pcdna3.1-fgl2 co-transfected cho cells with anti-fgl2 or anti-cc10 antibody followed by western blotting with fgl2 and cc10 antibodies indicated that cc10 did not co-immunoprecipitate with fgl2, showing that there is no direct relationship between cc10 and fgl2 (data not shown). the results showed that cc10 has no direct interaction with fgl2. from our previous study the gene of fgl2 contributed profoundly in mhv-3 induced fulminant hepatitis and is extensively expressed in macrophages and endothelium (12, 33) . our microarray indicated a cc10 down-regulated fgl2 expression and this is further confirmed by qpcr and western blotting in vivo (peritoneal macrophages) and in vitro (thp-1, macrophage cell line). therefore, it is reasonable to focus on macrophages to display the effect of cc10 on fgl2 expression and eventually mice survival. we entirely agree there may be other possibilities for a protective effect of cc10 to contribute to the disease process. this is worth further studies. the potential receptor of cc10 has not been revealed yet. our previous study have demonstrated that cc10 have effect of dendritic cells in allergic rhinitis (34) . in this research, we evaluated the effect of cc10 on macrophages functions and found fgl2 was substantially down-regulated upon cc10 treatment, therefore, we speculate that potential cc10 receptor may be also expressed on macrophages. the potential target of cc10 on other immune cells cannot be excluded. dna microarray analysis is one of the most powerful approaches for the potential identification of unexpected genes involved in pathogenic processes. by using this approach, hmgbox transcription factor 1 (hbp1) was found to be one of the most downregulated genes after cc10 treatment of thp-1 cells. hbp1 is a well-described transcriptional repressor that modulates expression of genes involved in cell cycle progression. in a recent study, it was found that hbp1 is a direct target of mir-21 and confirmed that hbp1 modulates the inhibitory function of mir-21-aso in hepatosteatosis and carcinogenesis simultaneously (23) . hbp1 is an endogenous inhibitor of the wnt signaling pathway in both normal and cancer cells. the tumor suppressor role of hbp1 has been reported in some malignancies, such as oral cancer and glioma (35) . however, an association between hbp1 and fgl2 has not been investigated yet. the current study clearly demonstrated that cc10 protects against mhv-3 induced fh via suppression of fgl2 expression. such effects might be mediated by hbp1. however, the functional status of hbp1 in the cc10 pathway requires further research, and such studies are conducting in our laboratory. in conclusion, we demonstrated that cc10 could limit the immunopathological damage in mhv-3-induced fh mice. our results suggest that enhancing cc10 expression by an immunotherapeutic approach might be an effective treatment for fh. hy performed all the described experiments and wrote the manuscript. yl assisted with some experiments, analyzed experimental results, and edited the manuscript. hw analyzed experimental results. xw reviewed and edited the manuscript. jh, wy, dx, xl, gs, and qn provided experimental help and design. immune mediated liver failure increased killing of liver nk cells by fas/fas ligand and nkg2d/nkg2d ligand contributes to hepatocyte necrosis in virus-induced liver failure kctd9 contributes to liver injury through nk cell activation during hepatitis b virusinduced acute-on-chronic liver failure cd4-cd8-t cells contribute to the persistence of viral hepatitis by striking a delicate balance in immune modulation a disparate subset of double-negative t cells contributes to the outcome of murine fulminant viral hepatitis via effector molecule fibrinogen-like protein 2 liver tcrγδ(+) cd3(+) cd4(-) cd8(-) t cells contribute to murine hepatitis virus strain 3-induced hepatic injury through a tnf-α-dependent pathway association of mouse fibrinogen-like protein with murine hepatitis virusinduced prothrombinase activity molecular and functional analysis of the human prothrombinase gene (hfgl2) and its role in viral hepatitis expression of the fgl2 and its protein product (prothrombinase) in tissues during murine hepatitis virus strain-3 (mhv-3) infection the nucleocapsid protein of murine hepatitis virus type 3 induces transcription of the novel fgl2 prothrombinase gene c5a/c5ar pathway is essential for the pathogenesis of murine viral fulminant hepatitis by way of potentiating fgl2/fibroleukin expression the fgl2/fibroleukin prothrombinase contributes to immunologically mediated thrombosis in experimental and human viral hepatitis hepatitis b virus-induced hfgl2 transcription is dependent on c-ets-2 and mapk signal pathway acute liver failure: mechanisms of immune-mediated liver injury novel mfgl2 antisense plasmid inhibits murine fgl2 expression and ameliorates murine hepatitis virus type 3-induced fulminant hepatitis in balb/cj mice uteroglobin: a novel cytokine? clara cell 10-kd protein suppresses chitinase 3-like 1 expression associated with eosinophilic chronic rhinosinusitis protein content in bronchoalveolar lavage fluid of patients with asthma and control subjects inverse relation between nasal fluid clara cell protein 16 levels and symptoms and signs of rhinitis in allergen-challenged patients with intermittent allergic rhinitis the expression of osteopontin and its association with clara cell 10-kda protein in allergic rhinitis fulminant viral hepatitis: molecular and cellular basis, and clinical implications microrna-21 is a potential link between non-alcoholic fatty liver disease and hepatocellular carcinoma via modulation of the hbp1-p53-srebp1c pathway salidroside attenuates lps-stimulated activation of thp-1 cell-derived macrophages through downreglation of mapk/nf-kb signaling pathways modulation of t-cell activation by the glucocorticoid-induced leucine zipper factor via inhibition of nuclear factor kappa-b clara cell 10-kd protein in inflammatory upper airway diseases combined adenovirusmediated artificial micrornas targeting mfgl2, mfas, and mtnfr1 protect against fulminant hepatic failure in mice the novel cd4+cd25+ regulatory t cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis cytokine-induced hepatic apoptosis is dependent on fgl2/fibroleukin: the role of sp1/sp3 and stat1/pu.1 composite cis elements intact type 1 immunity and immune-associated coagulative responses in mice lacking ifngamma-inducible fibrinogen-like protein 2 the nlrp3 inflammasome and il-1β accelerate immunologically mediated pathology in experimental viral fulminant hepatitis tnfα, and fgl2 contribute to coagulation and complement activation in virus-induced fulminant hepatitis fulminant hepatic failure in murine hepatitis virus strain 3 infection: tissue-specific expression of a novel fgl2 prothrombinase clara cell 10-kda protein inhibits t(h)17 responses through modulating dendritic cells in the setting of allergic rhinitis mir-155 promotes the growth of osteosarcoma in a hbp1-dependent mechanism the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2018.02935/full#supplementary-material table 1 | list of down-regulated genes by dna microarray. in order to determine genes that were downregulated after stimulation by cc10 protein, we used dna microarray analysis to screen for differentially expressed genes. briefly microarray analysis was used to screen changes in genome-wide gene expression patterns in thp-1 cells with or without cc10 protein. the changes in over 47,000 human gene expression patterns were assessed using affymetrix gene microarrays (human genome u133 plus 2.0) (capitalbio co. ltd., beijing, china). three replicates were used for microarrays analysis. thp-1 cells were cultured and pma was added to induce differentiation into macrophages. the production of fgl2 was stimulated by ifn-γ. the experimental group was treated with cc10 protein for microarray detection of differentially expressed genes. the results showed that the most obviously downregulated genes were ube2w, hectd1, mir612, atrx, sox4, hbp1, and fgl2. key: cord-030385-btf502ju authors: sun, zhiheng; pan, yuchen; qu, junxing; xu, yujun; dou, huan; hou, yayi title: 17β-estradiol promotes trained immunity in females against sepsis via regulating nucleus translocation of relb date: 2020-07-22 journal: front immunol doi: 10.3389/fimmu.2020.01591 sha: doc_id: 30385 cord_uid: btf502ju sepsis is more common among males than females, and the unequal estrogen levels have been suspected to play a vital role in gender differences. recently, trained immunity is reported to be a novel strategy for the innate immune system to fight infection. however, it has not been clarified whether β-glucan-induced trained immunity causes different responses to early sepsis between male and female mice. in this study, sepsis was induced in mice by intraperitoneal injection of escherichia coli (e. coli). the changes of inflammatory cytokines expression, and macrophage polarization in male, female, and ovariectomized c57bl/6 mice in sepsis model were investigated. for in vitro studies, different macrophages were treated with lps. the function of estradiol (e2) on macrophage cell lines was verified and the mechanism of e2 affecting trained immunity was explored. we demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. macrophage polarization toward the m1 phenotype, which exhibited enhanced trained immunity, was related to the difference in sepsis resistance between female and male mice. moreover, ovariectomized (ovx) mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. female bone marrow-derived macrophages (bmdms) were also apt to be polarized to the m1 phenotype in response to trained immunity in vitro. furthermore, e2 promoted trained immunity in macrophage cell lines j774 and raw264.7. e2 was also verified to facilitate trained immunity in primary bmdms from female and male mice. mechanistically, we found that e2 inhibited the nuclear translocation of relb, which is a member of non-canonical pathway of nfκb and contributes to macrophage polarization to change the intensity of trained immunity. this study is the first to indicate the role of e2 in the trained immunity induced by β-glucan to protect against e. coli-induced sepsis via the non-canonical nfκb pathway. these results improve our understanding of the molecular mechanisms governing trained immunity in gender differences. sepsis is a systemic reaction, which can even be caused by an ordinary infection (1) . the infection is most commonly bacterial but also can be fungal, viral, or parasitic. since many organs are affected, the mortality rate of sepsis is 30-50% (2) . with the development of modern medicine, the mortality rates have declined to about 30% (3), but this is still far from acceptable. the biomarker of sepsis is widely discussed by a number of reviews (4) (5) (6) (7) . several important signs of sepsis are damage to the kidney, liver, and lung, as well as an increased bacterial load in the kidney and elevated levels of transaminase and lactate in serum (8) . moreover, the common model of sepsis is an immune response to escherichia coli or endotoxin, lipopolysaccharide (lps), found in the cell wall of gram-negative bacteria. endotoxin is an excellent example of a pathogen-associated molecular pattern (pamp). of note, a number of studies indicate gender dimorphism in terms of response to sepsis (5) . the intuitive result is that the incidence of female sepsis is much lower than that of males (9, 10) . furthermore, some studies attributed these differences to the prevailing hormonal milieu of the victim (11) (12) (13) . thus, it is necessary to explore the mechanism of estrogen in modulating immunity, which allows females to resist sepsis. the nfκb (nuclear factor kappa-b) family of transcription factors constitutes five members (rela or p65, relb, crel, nfκb1 or p50, and nfκb2 or p52), all of which play important roles in cell homeostasis, especially in the immune process (14) . apart from the well-known canonical signaling pathway, the noncanonical pathway is also involved in vital immune processes, which could be triggered by signaling from a subset of tnfr members. this pathway mediates the persistent activation of relb/p52 complex with the ability to modulate a series of gene expression, including macrophage polarization-associated cytokines. to date, a growing number of studies indicated that estrogen is involved in some immune processes. estrogen can bind to and activate estrogen receptors (ers), which regulate the expression of downstream genes (15) . the interaction between ers and nfκb is mainly discussed in breast cancer articles (16) . a highly significant negative correlation between the expression of nfκb target genes and er activation was found. some studies abbreviations: e 2 , 17-β-estradiol; ti, trained immunity; lps, lipopolysaccharide; pbs, phosphate buffer saline; ast, aspartate aminotransferase; alt, alanine aminotransferase; il-6, interleukin-6; tnfα, tumor necrosis factor α; il-10, interleukin-10; il-4, interleukin-4; m-csf, macrophage colony-stimulating factor. demonstrated the interaction of er with nfκb; ers can compete with nfκb for binding to transcriptional coactivators (ie, creb) or ers to recruit co-suppressors into nfκb complexes (17) . ers can inhibit de novo relb synthesis in breast cancer tissues and cell lines. in addition, e 2 inhibits the nucleus translocation of p65, c-rel, and relb without affecting p50 in mouse splenocytes (18) . these data suggest that estrogen-er signaling regulates the nfκb pathway at the transcriptional level of its constituents. the host's immune defense mechanisms can be divided into innate immunity and adaptive immunity. adaptive immunity, although slower than the innate immune response, has good specificity and produces immunological memory (19) . recently, researchers discovered an ability in innate immunity, similar to immune memory in adaptive immunity, called trained immunity (20) . trained immunity is found in mammals and its basic features have been identified by several researchers. trained immunity mainly involves a set of cells (myeloid cells, natural killer cells, and innate lymphoid cells) (21) . antituberculosis vaccine bacillus calmette-guérin (bcg) is a wellknown immune modulator that induces trained immunity. it was reported that estrogen did not influence the induction of trained immunity by bcg, and did not induce training or tolerance in monocytes themselves, indicating that the estrogen is unlikely to explain the sex-differential effects after bcg vaccination (22) . in fact, β-glucan is a major cell wall component of c. albicans and can induce trained immunity in monocytes. the initiation of trained immunity is associated with enhanced signaling of the akt (protein kinase b)-mtor (mammalian target of rapamycin)-hif-1α (hypoxia-inducible factor-1α) pathway (23) , modifications in metabolic pathways (conversion to glycolysis), and epigenetic rewriting (24, 25) . however, the effect of estrogen on β-glucan-induced trained immunity to resist sepsis has not been clarified. β-glucan (g-59303) and β-estradiol (50-28-2) were purchased from xiensi biotechnology company (tianjin, china). m-csf (#cb34), ifn-γ (#c746), and tnfα (#cf09) were purchased from novoprotein (shanghai, china). e 2 and lps was purchased from sigma (st. louis, mo, usa). antibody against p-akt (ser473, #4060), akt (#4691), p-4ebp1 (#2855), 4ebp1 (thr37/46, #9644t), p-s6 (ser235/236, #4858), s6 (#2217), pcna (#13110), p65 (#8242), estrogen receptor α (13258s), gapdh (#5174), and β-actin (#3700) were purchased from cell signaling (boston, mo, usa). relb (sc-166416) was purchased from santa cruz biotechnology (dallas, tx, usa). f4/80-apc, f4/80-fitc, cd116-pe, cd206-apc, cd206-fitc, inos-pe were all from biolegend (san diego, ca, usa). raw264.7, j774, and hek293t cells were obtained from the type culture collection of the chinese academy of sciences, shanghai, china. cells were cultured in phenol red-free dmem with 10% fbs, 1% (100 u/ml penicillin and 100 ug/ml streptomycin) at 37 • c in an atmosphere of 95% air and 5% co 2 . cells were seeded onto different types of plates for further experiments when the cell density reached ∼80%. the cells were used within a maximum of five passages. the in vitro trained immunity model was established with raw264.7 and j774. cells were challenged with 5 µg/ml βglucan for 24 h. the cells were then washed and rested in culture medium for 5 days. next, cells were treated with 100 ng/ml lps for 6 h, and then the cytokines were measured in mrna level. 1 × 10 5 bmdms were seeded in 96-well plates (200 µl final volume; corning) and stimulated with 100 µg/ml β-glucan for 24 h. then cells were washed and rested for 3 days in culture medium. on day 4, bmdms were washed again and treated with 25 ng/ml ifn-γ for 24 h. on day 5, a final wash was performed, and cells were primed with 1 µg/ml lps. the supernatants were harvested for elisa assay after 24 h of lps stimulation. other experiments were all based on the same model but with different numbers of bmdms. to assess mrna expression and cell viability, 6 × 10 5 bmdms were plated in non-treated 24well plates (1,200 ml final volume; corning) and followed the training scheme described above. for western blotting (wb) assays 3 × 10 6 bmdms were plated in six-well plates (3 ml final volume; corning). male and female c57bl/b mice were purchased from the model animal research center of nanjing university. mice used in the model of trained immunity and sepsis were 10 weeks old. all animal procedures were performed in accordance with guidelines of the us nih with specific pathogen free conditions. soyfree standard rodent chow and water were provided ad libitum. female mice were anesthetized with 4% chloral hydrate and then underwent ovariectomy (ovx) at 6 weeks of age. when they were 10 weeks old, the same model of trained immunity and sepsis was established. peripheral blood mononuclear cells (pbmcs) were separated from mouse plasma by ficoll centrifugation using lymphocyte separation medium from mp biomedicals (solon, usa) according to the standard procedures. mice were sacrificed via cervical dislocation and sterilized by soaking in 75% ethanol. dissected the legs and bone marrow was extracted from tibia and femur bones by using a 25-gauge needle and a 1 ml syringe filled with pbs following removal of surrounding muscle. blow the bone marrow gently and spread it through a 70 µm cell strainer. the cell suspension was centrifuged at 300 g for 5 min at room temperature. cells were cultured in phenol red-free dmem with 10% ultra-low endotoxin fbs and m-csf (20 ng/ml). after changing the fresh medium on the third and fifth day, bmdm was induced. the e. coli 15597 strains were purchased from atcc, collected, and identified by the medical laboratory center of zhongda hospital in nanjing jiangsu, china, and stored at −80 • c. bacterial strains were prepared in lb medium. a nuclear protein extraction kit was purchased from biyuntian (wuhan, china) and used according to the manufacturer's instructions. sirna transfection sirna was transfected according to the product instructions (ruibo company, china). the concentration of sirna used in the study was 50 nm. the erα sirna target sequence is tgcacattgaagatgctga. the target sequence of non-coding (nc) sirna was a random sequence with no biological effects. to assess the effect of e 2 on cell viability, a cck-8 assay was used according to the manufacturer's instructions (bioss company, china). raw264.7/j774 were seeded onto 96-well plates at a concentration of ∼5 × 10 4 cells/well. different concentrations of e 2 were used to treat cells for different length of time as described in the article. total rna was extracted from cells using trizol reagent, and reverse transcriptions were performed in a 20 µl mixture with 1 µg of total rna according to the manufacturer's instructions (vazyme company, china). the oligonucleotide primers used for pcr amplification are listed in table 1 . pcr amplification consisted of 30 cycles of denaturation at 95 • c for 2 min, annealing at 60 • c for 45 s, and extension at 72 • c for 2 min. all reactions were run in triplicate. the gene expression levels were normalized to ß-actin. the protein samples were obtained from lysis buffer treated cells. cell lysates were put on ice for 15 min and then centrifuged at 12,000 × g for 10 min. subsequently, 30 µg of protein per lane was separated on 10% polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (millipore, billerica, ma, usa). membranes were blocked with 5% bovine serum albumin (bsa) in tris-buffered saline containing 0.1% tween 20, and then the membranes were incubated with specific antibodies. the values were normalized to the β-actin/gapdh intensity levels. mice were trained with two intraperitoneal (i.p.) injections of 1 mg β-glucan particles on days −7 and −4. sterile pbs was used as a control. on day 0, mice were challenged with 1.77 × 10 7 e. coli. the lung, kidney, and serum were harvested 24 h after e. coli treatment. the fresh lung tissues were fixed in 4% paraformaldehyde (pfa). then, the samples were gradually dehydrated and embedded in paraffin. after that, the samples were cut into 3 µm sections and stained with hematoxylin and eosin for further light microscopy observation. scores were evaluated by a pathologist based on the lung tissue integrity, alveolar integrity, and mononuclear infiltration (0 = none; 1 = mild; 2 = moderate; 3 = severe). cultured cells were seeded on glass coverslips in six-well plates. after three pbs washes, the samples were fixed for 15 min at room temperature with 4% paraformaldehyde. fixed cells were rinsed with pbs and then incubated for 10 min at 4 • c with 0.2% triton x-100 and 0.2% bsa in pbs. following permeabilization, non-specific binding in the cells was blocked by 5% bsa in pbs for 1 h at room temperature. cell samples were incubated with anti-erα, anti-relb, and anti-p65 primary antibodies at a 1:200 dilution for 2 h at room temperature. samples were further incubated with alexa fluor 488-conjugated and alexa 647conjugated secondary antibody at a 1:400 dilution for 1.5 h in the dark. after washed with pbs, the nuclei were stained by dapi. slides were visualized using a nikon eclipse ti-u fluorescence microscope equipped with a digital camera (ds-ri1, nikon). the protein concentration of il-6, lactate, tnfα, and estrogen in cell supernatant or mouse serum were detected using the corresponding mouse enzyme-linked immunosorbent assay (elisa) kit according to the manufacturer's instructions (biolegend, china). the kidney e. coli burden at indicated time points was measured by plating organ homogenates obtained mechanically over 70 µm cell strainers (bd biosciences) following slicing the tissue, in serial dilutions on lb agar plates; colony-forming units (cfus) were counted after growth at 37 • c for 24 h, and data are shown as cfus in total kidney. pbmcs or bmdms were filtered through a 70 µm cell strainer and then washed with complete rpmi medium to generate single-cell suspensions. an fc-receptor blocker (cd16/32, ebioscience) was used to reduce non-specific antibody binding. antibodies used in these experiments included f4/80-apc, f4/80-fitc, inos-pe, cd206-apc, and cd11b-pe. stained samples were detected by a facs calibur flow cytometer (bd bioscience) and data were analyzed using flowjo software (treestar, ashland, or). the statistical analysis was performed using prism (prism 5 for windows, graphpad software inc., usa). unless specified, statistical significance for comparison between two sample groups with a normal distribution (shapiro-wilk test for normality) was determined using two-tailed paired or unpaired student's t-test. differences were considered significant at p < 0.05 as indicated. except when specified, only significant differences are shown. as indicated in figure legends, either a representative experiment or a pool is shown, and the number of repetitions of each experiment and number of experimental units (either cultures or mice) is indicated. the results are presented as the means ± standard error (sem). several studies have shown that women have better survival and tolerance to sepsis than men (26, 27) . thus, we further verified this phenomenon by establishing a sepsis model in mice ( figure 1a) . the difference in lung injury between male and female mice in sepsis was determined by h&e analysis of lung sections (figure 1ba) . the histochemical scores (figure 1bb ) indicated that male mice had more severe lung damage than female mice; trained immunity prevented lung injury in mice and the protective effect of trained immunity was better for females than males. notably, we observed a decreased renal e. coli burden in females than males. in addition, in trained immunity mice, females had less renal e. coli burden than males (figures 1c,d) . in addition, liver damage markers, aspartate aminotransferase (ast), and alanine aminotransferase (alt) were increased in males than females; and trained immunity markedly decreased ast and alt concentration, with a greater decrease in females than in males (figures 1e,f) . we found that in the sepsis model, the serum lactate concentration of the male mice was higher than that of the female mice. in the trained immunity group, serum lactate was decreased, with the level in the females lower than males ( figure 1g ). as expected, male serum e 2 content is much lower than female mice ( figure 1h) . our results showed that females expressed higher il-6 and tnfα than males in sepsis, and trained immunity exacerbated this trend (figures 1i,j) . macrophages are the most significant cells in the body in regard to trained immunity (28) . the promoted trained immunity is related to the increased m1 phenotype macrophage. in order to understand the different response to sepsis between the different genders, we further investigated the polarization of macrophages during the process. thus, we focused on macrophage content and polarization. by detecting the percentage of macrophages in the spleen, we found that macrophages in female mice are more abundant than male mice, and trained immunity significantly increased the content of female macrophages (figure 2a) . sepsis promoted the polarization of macrophages to m1 and trained immunity aggravated this trend. notably, females showed more changes than males (figure 2b) . on the other hand, females inhibited the polarization of m2 macrophages ( figure 2c) . trained immunity simulated m1type polarization of macrophages, and this effect was more pronounced for females. by analyzing the experiments above, we found that the difference between male and female responses to sepsis was due to the difference in macrophage polarization in vivo, and the difference in estrogen content in the different genders was very significant. to verify that differences in the estrogen level play a vital role, ovx mice were made, since the ovary is the main organ to produce e 2 . the same sepsis model was established with ovx mice (figure 1a) . firstly, the serum e 2 concentration was significantly decreased (figure 3a) . the degree of lung injury was much higher than that of female mice as well as ti + e. coli group (figure 3b) . the kidney e. coli burden was also evaluated. the results showed that ovx mice also had more severe kidney injury ( figure 3c ) as well as lung injury, as measured by serum alt (figure 3d ) and ast ( figure 3e ) concentrations, in comparison to female mice. with a lower serum il-6 and tnfα concentration in ovx mice in the ti + e. coli group, the data suggested that ovx mice have a lower trained immunity response than female mice (figures 3f,g) . finally, similar to male mice, ovx mouse pbmcs began polarizing to m2-type at 24 h after i.p. e. coli ( figure 3h ). taken together, these results demonstrated that e 2 does facilitate trained immunity in the body to resist sepsis, and simultaneously inhibits macrophage polarization to m2. since the macrophages are mostly derived from bone marrow cells, and also the cytokines up-regulated in trained immunity are pro-inflammatory cytokines. although the upregulation of inflammatory cytokines after trained immunity of macrophages is determined, no research has studied the polarization of macrophages during this process. therefore, exploring the polarization of bmdm as an important primary macrophage in trained immunity is very significant and necessary to understand the nature of trained immunity. to determine whether bmdms are similar to macrophage polarization in pbmcs, we tested the polarization of microphages from male or female mice when challenged with lps or trained immunity plus lps. we considered f4/80 + inos + bmdms as m1-type bmdms and f4/80 + cd206 + bmdms as m2-type bmdms. the results demonstrated that female bmdms are more polarized to m1 in both the lps group (7.04-5.12%) and ti + lps group (28.8-17.9%) ( figure 4a) . also, male bmdms began to transform to m2 in 24 h, which did not occur in female bmdms. trained immunity also prevented both male and female bmdms from m2 polarization as well as pbmcs (figure 4b ). macrophage cell lines j774 and raw264.7 in order to examine the effect of estradiol on trained immunity, we decided to pre-treat the macrophage cell lines with estradiol before trained immunity model. since the concentration of estradiol that can stimulate the macrophage cell lines raw264.7 and j774 is unknown, we made a concentration gradient of estradiol on the activity of the macrophage cell lines. the results showed that the response of the macrophage cell lines to the stimulation of estradiol is not particularly sensitive, and the viability of j774 begins to increase significantly under the stimulation of 50 nm estradiol. thus, the concentration of e 2 used in cell experiments was verified as 50 nm (figures 5a,b) . the in vitro trained immunity model was established with raw264.7 and j774 ( figure 5c ) cell lines derived from male and female mice, respectively. using tnfα and il-1β as the marker of inflammation, our data suggested that e 2 can induce stronger trained immunity both in raw264.7 and j774 (figures 5d,e) . we also made a trained immunity model of bmdms (figure 6a ). m-csf was used to induce bmdm with a purity of over 95% from both male and female mice ( figure 6b) . then, the mrna levels ( figure 6c ) and protein levels ( figure 6d ) of tnfα and il-6 were measured. the results suggested that female bmdms can have a more intensive response to lps as well as a trained immunity response than male bmdms. phosphorylation of akt and mtor targets (s6 and 4ebp1) are considered to be key hallmarks of trained immunity (23) . to clarify the effect of e 2 on trained immunity, these hallmarks are checked by western blot, which indicated that e 2 as well as β-glucan induced trained immunity in bmdms ( figure 6e ). in addition, the data showed that e 2 further boosted the polarization of m1-type macrophage in trained immunity. consistently, this effect of e 2 is more apparent in female bmdms, and e 2 inhibited m2 polarization in trained immunity (figure 6f ). nfκb signaling pathway is involved in the regulation of many cellular physiological processes. previous articles have reported that relb −/− mice, or inhibiting the nucleus translocation of relb, led to a higher basal inflammatory level (29, 30) . other reviews declared that e 2 could regulate nuclear translocation of nfκb through ers. since it has been widely studied that tnfα can combine with tnfr on the cell membrane surface to promote nfκb translocate into the nucleus. also, because tnfα as a representative inflammatory cytokine is secreted by macrophages and will stimulate back to macrophage. we use tnfα as a positive control to study nfκb entry into the nucleus. we use immunofluorescence to explore the intracellular localization of nfκb protein. in immunofluorescence, the results indicated that when tnfα is used to stimulate cells, it does not affect the colocalization of erα and the nucleus but increases the colocalization area of p65 and relb with the nucleus. on the contrary, e 2 treatment appeared to induce nuclear positioning of erα and kept relb distribution in the cytoplasm in both hek293t and bmdms (figures 7a,b) . however, stimulation of e 2 did not affect the distribution of p65 in cells. next, we used the rna interference system to further verify the impact of e 2 on the nuclear translocation of relb. the silencing performance of interfering small rna sequence was evaluated both at the mrna level ( figure 7c ) and protein level ( figure 7d) . by extracting nuclear protein, we found that estrogen could reduce the content of relb protein in the nucleus; by silencing the expression of erα, the content of relb protein in the nucleus was also restored ( figure 7e ). as markers of m2 macrophage polarization, il-4 and il-10 are also the downstream genes regulated by relb (30, 31) . we found that e 2 inhibited the mrna expression level of il-10 and il-4 by regulating nuclear translocation of relb ( figure 7f ). in conclusion, these results indicated that e 2 could block relb translocation to the nucleus; to further inhibit m2-associated gene expression, e 2 suppressed bmdms m2 polarization. during the development of sepsis, many systemic organ abnormalities occur including damage to the lung, liver and kidney; the normal operation of the body's neurosensory system may even be affected (32) . since lps is a component in the cell wall of gram-negative bacteria, it is wildly used as an ideal stimulus to establish sepsis models. when tlr4 on figure 4 | female bmdms are apt to be polarized to m1 phenotype in responses to trained immunity in vitro. (a) female bmdms m1 polarization was stronger than male bmdms treated with lps or ti + lps groups. (b) very few female bmdms to polarized to m2 in lps group, while male bmdms showed the polarization of m2 phenotype. ti suppressed bmdms to m2 polarization from both male and female mice (n ≥ 3/group). ***p < 0.001, paired student's t-test comparing lps group and ti + lps group in the same gender. # p < 0.05, ### p < 0.001, paired student's t-test comparing same group between different genders. the macrophage cell membrane recognizes lps, a series of signaling pathways will be initiated to stimulate macrophages to produce pro-inflammatory cytokines, which can induce potent systemic inflammatory responses in vivo. therefore, tnfα, il-1β, and il-6 are currently considered to be markers of the early phase of sepsis (33) . the development of sepsis begins with systemic inflammatory response syndrome (sirs) (34) , which is considered as the early phase of sepsis. elevated expression of pro-inflammatory cytokines could help the body return to a normal state in early sepsis (35) . after sirs, the body enters the cars (compensatory anti-inflammatory response syndrome) stage. in the cars stage, the immune system will undergo the decline-regulation (36) . the hyper-inflammatory response of sirs will normally be restored by the subsequent cars stage. the patients can return to normal state after the cars stage (37) . however, if the cars phase fails, it can cause death from sepsis. as one of the important ways to protect the body from foreign microbes and antigens, the role of trained immunity is very significant. this study focused on the effects of e 2 on early sepsis by promoting trained immunity. in the development of early sepsis, estrogen induced a stronger trained immunity response of macrophages to remove antigens and microorganisms from the body. simultaneously, estrogen promoted the polarization of macrophages to m1 in vivo and inhibited the polarization of m2, which is considered anti-inflammatory in the early phase of sepsis. in summary, estrogen can maintain the higher proinflammatory state of macrophages. this also illustrates the reason why females are more tolerant to sepsis than males in one aspect. however, further research is needed to understand the role of estrogen in the later stages of sepsis. the mrna levels of tnfα and il-1β in raw264.7 were detected by qpcr to determine the trained immunity effect with or without e 2 (n ≥ 3/group). # p < 0.05, ### p < 0.001, paired student's t-test comparing β-glucan + lps group and lps group. *p < 0.05, ***p < 0.001, paired student's t-test comparing with control group. ∧p < 0.05, ∧∧p < 0.01, and ∧ ∧ ∧p < 0.001, paired student's t-test comparing between β-glucan + lps groups with or without e 2 . here, we demonstrated that β-glucan-induced trained immunity was more resistant to sepsis in female than male mice. ovx mice manifested serious sepsis consequences with a weaker trained immunity effect than female mice. macrophage polarization toward m1 phenotype is thought to exhibit the enhanced trained immunity. e 2 promoted trained immunity in vitro and in vivo. inhibition of e2 on the nucleus translocation of relb contributed to macrophage polarization to change the intensity of trained immunity. our results indicated that e 2 is involved in the trained immunity in gender differences. since trained immunity was discovered in 2014, although there are many articles about trained immunity, there is no article to find the fundamental way of trained immunity activation but only about the changes of immune cell characterization after trained immunity activation. such as epigenetic changes, upregulation of glycolysis, autophagy level, akt phosphorylation, and so on. in this study, we found that e 2 promotes trained immunity by suppressing relb entry into nucleus, while inhibiting macrophage polarization to m2 in inflammation (a way reverse the inflammatory response). it is possible that e 2 can change the epigenetics of macrophages through estrogen receptors to affect the trained immunity. this hypothesis needs further researches. trained immunity is a way of immunization that occurs in the innate immune cells through the first stimulation to fight the later secondary infections. by obtaining trained immunity, the body can fight various infections from bacteria, fungi and even viruses timely. it is worth mentioning that in countries and regions with underdeveloped scientific research capabilities, it may be an economical and quick means of protection against covid-19 to enhance people's trained immunity ability. sepsis is a clinically common infectious disease with obvious gender differences. in this article, sepsis model is used as an infection model to evaluate the role of estradiol in enhancing trained immunity. the mechanism and effects of estradiol in promoting trained immunity also need subsequent study in other types of infectious diseases. the purpose of this paper was to explore the reasons why different genders have different tolerance effects on sepsis. an article reported that estradiol and dht (dihydrotestosterone) did not influence bcg-induced trained immunity, and the article only focused on the inflammatory cytokines of monocytes induced by bcg in vitro (22) . apart from this, almost no articles study the effects of e 2 on trained immunity and its biological significance. our research attempted to explain its effects on trained immunity and understand how it causes women to be more tolerant of sepsis than men. however, in terms of gender differences, many factors, such as androgen concentration, genetic differences, metabolic differences, and physiological differences may all be the result of different tolerances for sepsis between genders (12, 38, 39) . gender differences are a very complex and exquisite scientific issue (40) , and all of the factors that contribute to gender differences are worth exploring for subsequent experiments. in summary, we demonstrated that as one of the key factors for gender differences, e 2 plays a vital role in the stronger tolerance of females to sepsis than males. one the one hand, e 2 can better facilitate trained immunity by increasing expression of inflammatory cytokines and inducing macrophage m1-type polarization; on the other hand, e 2 can inhibit or delay the macrophage m2-type polarization by regulating nucleus translocation of relb and its down-stream m2-associated genes. thus, this may provide a new idea for the clinical treatment of sepsis. drugs with a structure similar to estrogen may be developed to treat sepsis. more importantly, the treatment of some diseases may able to find better solutions by addressing the different characteristics of different genders. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by model animal research center of nanjing university. all authors reviewed the manuscript, contributed to data analysis, drafting and revising the article, gave final approval of the version to be published. zs conceived and designed the project. zs, yp, jq, and yx participated in the animal experiments. zs and jq wrote the manuscript. yh and yx revised it. biomarkers of sepsis two decades of mortality trends among patients with severe sepsis: a comparative meta-analysis incidence and trends of sepsis in us hospitals using clinical vs claims data biomarker cruises in sepsis: who is the captain? discussion on "circulating biomarkers may be unable to detect infection at the early phase of sepsis in icu patients: the captain prospective multicenter cohort study sepsis pathophysiology, chronic critical illness, and persistent inflammationimmunosuppression and catabolism syndrome penkid: a novel biomarker of reduced gfr in sepsis adaptation of a biomarker-based sepsis mortality risk stratification tool for pediatric acute respiratory distress syndrome reconsidering lactate as a sepsis risk biomarker gender differences in sepsis: genetically determined? shock effects of gender on the severity of sepsis sexual dimorphism in bacterial infections gender differences in trauma, shock and sepsis sex and severe sepsis nfkappab is a key player in the crosstalk between inflammation and cardiovascular diseases mutations in the estrogen receptor alpha hormone binding domain promote stem cell phenotype through notch activation in breast cancer cell lines nfkappab affects estrogen receptor expression and activity in breast cancer through multiple mechanisms an additive interaction between the nfkappab and estrogen receptor signalling pathways in human endometrial epithelial cells despite inhibition of nuclear localization of nf-kappa b p65, c-rel, and relb, 17-beta estradiol up-regulates nf-kappa b signaling in mouse splenocytes: the potential role of bcl-3 bcg-induced trained immunity in nk cells: role for non-specific protection to infection trained immunity: a new avenue for tuberculosis vaccine development targeting ship-1 in myeloid cells enhances trained immunity and boosts response to infection the impact of sex hormones on bcg-induced trained immunity mtor-and hif-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity epigenetic programming of monocyte-to-macrophage differentiation and trained innate immunity increased acetylation of h3k14 in the genomic regions that encode trained immunity enzymes in lysophosphatidylcholine-activated human aortic endothelial cells-novel qualification markers for chronic disease risk factors and conditional damps severe sepsis and septic shock epidemiology of severe sepsis in the united states: analysis of incidence, outcome, and associated costs of care trained immunity: a smart way to enhance innate immune defence differential involvement of relb in morphine-induced modulation of chemotaxis, no, and cytokine production in murine macrophages and lymphocytes relb regulation of chemokine expression modulates local inflammation relb cellular regulation and transcriptional activity are regulated by p100 year in review in intensive care medicine, 2008. i. brain injury and neurology, renal failure and endocrinology, metabolism and nutrition, sepsis, infections and pneumonia sepsis and septic shock: a review sepsis: a review of advances in management review: immunology of sinusitis, trauma, asthma, and sepsis review on sepsis in children did not mention important trial clinical outcome and risk factors of neonatal sepsis among neonates in felege hiwot referral hospital systematic review of gender differences in sepsis management and outcomes the influence of gender on the epidemiology of and outcome from severe sepsis age and sex influence the hippocampal response and recovery following sepsis the authors appreciate the warm-hearted help from all the co-authors and the professor. key: cord-003825-tkqxb1ql authors: toman, miroslav; celer, vladimir; kavanová, lenka; levá, lenka; frolichova, jitka; ondráčková, petra; kudláčková, hana; nechvátalová, kateřina; salat, jiri; faldyna, martin title: dynamics and differences in systemic and local immune responses after vaccination with inactivated and live commercial vaccines and subsequent subclinical infection with prrs virus date: 2019-08-06 journal: front immunol doi: 10.3389/fimmu.2019.01689 sha: doc_id: 3825 cord_uid: tkqxb1ql the goals of our study were to compare the immune response to different killed and modified live vaccines against prrs virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. in the experiment, we immunized four groups of piglets with two commercial inactivated (a1—progressis, a2—suivac) and two modified live vaccines (b3—amervac, b4—porcilis). twenty-one days after the final vaccination, all piglets, including the control non-immunized group (c5), were i.n., infected with the lelystad strain of prrs virus. the serum antibody response (igm and igg) was the strongest in group a1 followed by two mlv (b3 and b4) groups. locally, we demonstrated the highest level of igg antibodies in bronchoalveolar lavages (balf), and saliva in group a1, whereas low iga antibody responses in balf and feces were detected in all groups. we have found virus neutralization antibody at dpv 21 (days post vaccination) and higher levels in all groups including the control at dpi 21 (days post infection). positive antigen specific cell-mediated response in lymphocyte transformation test (ltt) was observed in groups b3 and b4 at dpv 7 and in group b4 at dpv 21 and in all intervals after infection. the ifn-γ producing lymphocytes after antigen stimulation were found in cd4(−)cd8(+) and cd4(+)cd8(+) subsets of all immunized groups 7 days after infection. after infection, there were obvious differences in virus excretion. the virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at dpi 3. significantly lower virus load was found in groups a1 and b3 at dpi 21. negative samples appeared at dpi 21 in b3 group in saliva. it can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. immunization with inactivated vaccine a1—progressis induces high levels of antibodies produced both systemically and locally. immunization with mlv-vaccines (amervac and porcilis) produces sufficient antibody levels and also cell-mediated immunity. after infection virus secretion gradually decreases in group b3, indicating tendency to induce sterile immunity. the goals of our study were to compare the immune response to different killed and modified live vaccines against prrs virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. in the experiment, we immunized four groups of piglets with two commercial inactivated (a1-progressis, a2-suivac) and two modified live vaccines (b3-amervac, b4-porcilis). twenty-one days after the final vaccination, all piglets, including the control non-immunized group (c5), were i.n., infected with the lelystad strain of prrs virus. the serum antibody response (igm and igg) was the strongest in group a1 followed by two mlv (b3 and b4) groups. locally, we demonstrated the highest level of igg antibodies in bronchoalveolar lavages (balf), and saliva in group a1, whereas low iga antibody responses in balf and feces were detected in all groups. we have found virus neutralization antibody at dpv 21 (days post vaccination) and higher levels in all groups including the control at dpi 21 (days post infection). positive antigen specific cell-mediated response in lymphocyte transformation test (ltt) was observed in groups b3 and b4 at dpv 7 and in group b4 at dpv 21 and in all intervals after infection. the ifn-γ producing lymphocytes after antigen stimulation were found in cd4 − cd8 + and cd4 + cd8 + subsets of all immunized groups 7 days after infection. after infection, there were obvious differences in virus excretion. the virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at dpi 3. significantly lower virus load was found in groups a1 and b3 at dpi 21. negative samples appeared at dpi 21 in b3 group in saliva. it can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. immunization with inactivated vaccine a1-progressis porcine reproductive and respiratory syndrome (prrs) is the most economically significant infectious disease currently affecting swine worldwide. typical clinical symptoms of prrs are mild to severe respiratory disease in infected newborn and growing pigs, and reproductive failure in pregnant sows. two genotypes of the prrs virus (prrsv) have been identified: european (type 1) and north american (type 2). there are considerable genetic and virulence differences between and within prrsv genotypes (1-3) correlated with a lack of cross-protection by vaccines (4) (5) (6) (7) (8) . highly pathogenic strains of prrsv (hp-prrsv) have been identified within both genotypes (9) (10) (11) . depending on viral strain and immune status of the host, some swine farms may have pigs subclinically infected, whereas others experience severe reproductive, and/or respiratory disease. infection with both "classical" and highly pathogenic strains is associated with aberrant host immune response (9, 12) . swine are the only known natural host of prrsv and the primary target cells for replication of prrsv are porcine alveolar macrophages (pams) (13) . the first stage is represented by acute infection, resulting in viremia 6-12 h post-infection (pi), and lasting for several weeks despite the presence of circulating antibodies. in the second, persistent stage of infection, the virus is no longer detected in blood and lungs, and pigs no longer exhibit signs of clinical disease. in this stage, viral replication is primarily localized in lymphoid organs, including tonsils, and lymph nodes (14) . infection with prrsv elicit poor innate and adaptive immune responses associated with immune modulation and incomplete viral clearance in most of the pigs, depending on their age, and immune status (12, (15) (16) (17) . infection with certain prrsv strains induced significant suppression of nk cell cytotoxic activity (18) . the quantity of pro-inflammatory cytokines is significantly lower than in other viral infections and is strain dependent (19) . prrsv is also a poor inducer of ifn-α. infection with prrsv induces an antibody response (production) by 7-9 dpi but with no evidence of protection against prrsv infection; serum neutralizing antibodies appear only later, typically ≥28 days pi (20) . the virus also evades host cell-mediated immunity most likely by the promotion of immunosuppressive cytokines il-10 and tgf-β resulting in delayed onset of th1 immune response (18) . similarly, an immunosuppressive function of prrsv was shown to probably be mediated by the cytokines il-10 and tgf-β and action of treg (21) (22) (23) . immunosuppression induced by prrsv facilitates other viral and bacterial infections (18, 24, 25) . vaccination is the principal means used to control and treat prrsv infection. several comprehensive review articles have been published recently. they critically evaluate different vaccination approaches against the prrs virus and indicate the main weaknesses of current vaccines and vaccination strategies (26) (27) (28) (29) . among others the problem are caused by high heterogeneity and occurrence of highly pathogenic strains and therefore efforts have been made to develop vaccines with a broad spectrum of effects (4, 5, 7, (30) (31) (32) (33) . however, the opinion still prevails that vaccination is more cost-beneficial over other health interventions (34) (35) (36) . our study had the following three aims: 1) to establish complex immune response characteristics using several methodological approaches; 2) to monitor the dynamics in different compartments and in a time-dependent manner after vaccination and the challenging infection; 3) to compare the types of immune responses after vaccination with inactivated or live attenuated vaccines and subsequent challenge using a homologous strain. twenty-five weaned piglets aged 8 weeks and weighing 8-12 kg of the large white breed from a prrsv negative herd were used. the negative status of the animals was confirmed by serology using commercial elisa kit (idexx labs). the use of animals was approved by the branch commission for animal welfare of ministry of agriculture of the czech republic (approval protocol no. mze-1487) as a part of project as a part of project respig (qj1210120). four commercial vaccines were used. their characteristics are in table 1 . the virus was clarified by centrifugation, and its concentration was determined by plaque assay. the concentration of stock virus used in experiments was 5 × 10 6 plaque forming units per ml. twenty-five piglets were used in the experiment. the piglets were assigned to five groups of five animals each according to weight and gender. the animals were housed in bsl2 isolation rooms, keeping animals from only one experimental group in each room. the animals were left to acclimate for 14 days after stocking. all piglets were clinically healthy at the time the experiment started. on day 0 (d0) two groups of piglets (a1 and a2) were immunized. each animal was administered 2 ml of inactivated vaccine by an intramuscular (i.m.) injection. after 21 days (d21), piglets in these groups were revaccinated with the same dose, and piglets from the other two groups (b3 and b4) were immunized with 2 ml of a mlv vaccine. the health status of piglets was monitored on a regular basis, including temperature measurements, and samples of blood and other body fluids were taken for respective examinations at preset time intervals. after an additional 21 days (d42), all pigs, including control group (c5), were infected with 2 ml of the live prrs virus. the piglets were monitored for another 21 days and then slaughtered (d63). euthanasia was performed by exsanguination after combined anesthesia with a tkx (telazol-ketamin-xylazin) mixture containing 12.5 mg/ml tiletamine and 12.5 mg/ml zolazepam (telazol, virbac, carros, france), 12.5 mg/ml ketamine (vetoquinol, lure, france), and 12.5 mg/ml xylazine (bioveta, ivanovice na hane, czech republic), administered intramuscularly in a final volume of 0.2 ml/kg body weight. as well as collection of blood and other body fluids (intestinal contents, bronchoalveolar lavage), an autopsy was performed and organs (lung parenchyma, spleen, lymph nodes,...) were collected for virological examination. blood samples for serum and heparin-treated blood samples were taken from the jugular vein. group saline samples were collected using ropes which were left in the hutch for 3 h. individual fecal samples were collected when handling the animals. bronchoalveolar lavage fluid (balf) sampling was performed for the first time on live animals and for the second time after slaughter. the intravital lavage was performed with the animals under general anesthesia (a mixture of xylazine and ketamin) without the use of an endoscope by a method described earlier (37) . pigs were positioned in the sternal recumbency. an endotracheal tube was inserted into the trachea and 20 ml of sterile pbs (ph 7.2) was injected into the distal parts of the airways, toward the bronchus. about 60% of the infused saline was recovered as balf aspirate and was filtered and centrifuged for 15 min at 200 g. supernatant was stored at −20 • c prior to serological analyses. total rna from experimental samples of sera, oral fluids, and bal (100 µl) was extracted using a nucleospin r rna ii kit (macherey-nagel), in accordance with the manufacturer's instructions (protocol for total rna preparation from biological fluids). the rna obtained was eluted in 60 µl rnase-free water and immediately used for qrt-pcr amplification. remaining rna was frozen at −80 • c for subsequent use. isolated rna was used for qrt-pcr amplification by ez-prrsv tm mpx 4.0 real time rt-pcr kit (tetracore), in accordance with the manufacturer's instructions. quantification of the virus genome copies was based on quantification standards included in the kit. for the evaluation of systemic and local antibody production two elisa methods were used. all swine sera tested were examined by commercially available elisa test ingezim prrs universal (ingenasa), in accordance with the manufacturer's instructions, to examine for the presence of n protein specific igg antibodies. all swine sera, oral fluids, and bal tested were examined by home-made indirect elisa test based on recombinant nucleocapsid protein n of prrs virus (developed previously in our department) for detection of specific igm, igg, and iga antibodies. optimal antigen, serum, and antibodies concentrations were determined by checkerboard titration of positive and negative porcine sera. the cut-off value was determined by defining the upper prediction limit based on the upper tail of the t-distribution of negative control od readings, at a confidence level of 99.5%. positive serum with an absorbance corresponding to the calculated cut-off was included in all test plates. the recombinant n protein diluted in 50 mm bicarbonate-carbonate buffer ph 9.6 to a final concentration of 1.5 µg/ml was coated on 96-well-microtiter plates (maxisorp ii r immunoplates, nunc, denmark) overnight at 26 • c. the wells were then blocked with 3% skimmed milk in pbs for 90 min at 37 • c and then washed with pbs. positive and negative controls were included in each test plate. each sample diluted in 3% skimmed milk in t-pbs (pbs with 0.1% tween 20 and 0.5 m nacl) was added in duplicates on antigen-coated wells with some differences among different types of samples. one hundred microliters of serum samples diluted 1:40 (for detection of igm antibodies) or 100 µl of non-diluted samples of bal (for detection of igg and iga antibodies) were incubated for 60 min at 37 • c. two hundred and fifty microliters of oral fluids diluted 1:2 were incubated 16 h at 4 • c (for detection of igg antibodies). subsequently the plates were washed three times with t-pbs and antibody binding was detected by incubation for 60 min at 37 • c with 100 µl of anti-pig igm peroxidase conjugate (1:10,000, bethyl), anti-pig igg peroxidase conjugate (1:30,000, sigma), or with anti-pig iga peroxidase conjugate (1:3,000, bethyl) separately (diluted in t-pbs with 3% skimmed milk). after washing the plates as described above, 100 µl per well of the tmb-complete (test-line) substrate was added. the optical density (od) was measured at 450 nm after an incubation time of 5-10 min at room temperature. the virus neutralization test for detection of prrsv neutralization antibodies was performed as follows. samples of sera were diluted 1:4 in dmem medium (sigma-aldrich) supplemented with 3% fbs. then, heat inactivated sera (56 • c for 60 min) were diluted 2-fold serially in flat-bottom 96-well-microplate (nunc). next, equal volume (50 µl) of media containing 50 prrsv pfu (lelystad-capm v-490) was added to each well. following incubation (60 min at 37 • c) marc-145 cells were added to each well (3 × 10 4 per well) in 100 µl media per well. after 5 days of cultivation (37 • c, 5% co 2 ), the cytopathic effect (cpe) of prrsv on marc-145 was evaluated by optical microscopy. the reciprocal value of the last sera dilution causing 50% reduction of cpe was defined as virus neutralization antibody titer. the lymphocyte transformation test was performed according to the method published earlier (38) . peripheral blood mononuclear cells (pbmc) were obtained by gradient centrifugation post infection d45 ++ ++ + + + + + + -+ + + + + + ++ + + + + + + ++ the end of experiment d63 + ++ ++ + ++ + + + ± -+ + ++ + frontiers in immunology | www.frontiersin.org (histopaque-1077, sigma-aldrich). concentration of the cells was adjusted to 200,000 cells in 200 µl of rpmi-1640 medium supplemented with 10% of autologous serum, 100 iu/ml penicillin, 100 µg/ml streptomycin, and 4 µg/ml gentamicin). they were incubated with the 20 µg of optimal concentration of the specific antigen (moi 1.0) for 5 days at 37 • c in 5% co 2 . negative controls were incubated with rpmi-1640 medium only. all samples were evaluated in triplicate. 3 h-thymidine was added on the last day of cultivation. subsequently, the cells were harvested (filtermate harvestor, packard bioscience company, usa), and 3 h-thymidine incorporation was measured by a microplate scintillation and luminescence counter (topcount nxt tm , packard bioscience company) in counts per minute (cpm). the results were expressed in terms of stimulation indexes (si), which were calculated as the ratio of cpm in stimulated samples vs. cpm in non-stimulated controls. the number of ifn-γ producing cells was calculated by elispot techniques. commercially available porcine ifn-γ elispot kit (3130-4hpw-10, mabtech) was used in accordance with the manufacturer's instructions. the number of cells used in the test was 5 × 10 5 /well. prrsv was used for stimulation of the antigen-specific response in multiplicity of infection (moi) 0.5. mitogen cona at a concentration 66 µg/ml was used as a positive control. cells without stimulation were used as a negative control. incubation lasted for 20 h. spots were detected using elispot reader system elro7tl (aid, germany). the results were recalculated to the number of cd3 + lymphocytes. the 5 × 10 5 of pbmc per well was stimulated with prrs virus in moi 0.5 for 20 h. the 5 × 10 5 of cultured pbmc were pelleted and 20 µl of primary monoclonal antibody cocktail containing anti-cd4 (igg2b, clone 74-12-4, wsu, monoclonal antibody center, usa), anti-cd8 (igg2a, clone 76-2-11, wsu, usa), and anti-γδ tcr (igg1, clone pgbl22a, wsu, usa) and 20 µl of heat-inactivated goat serum was added. the cells were incubated for 20 min at 4 • c and then rinsed twice with cell washing solution. then, 50 µl of goat anti-mouse secondary antibody cocktail (anti-igg2b: dylight 405, anti-igg2a: alexa fluor 647, and anti-igg1: pe-cy7) was added and the cells were incubated for another 20 min at 4 • c. the cells were rinsed and then 70 µl of anti-cd3 antibody (igg1, clone ppt3, southern biotech, pre-stained with alexa fluor 488 dye using zenon antibody labeling kit, invitrogen) was added and the cells were incubated, rinsed twice, and fixation and permeabilization for subsequent intracellular staining was performed by solutions a and b of intra stain kit (dako cytomation, usa) (39) . finally, 5 µl of rpe-conjugated anti-ifn-γ antibody (clone cc302, abd serotec uk) was added and the cells were incubated for 30 min. the cells were measured as soon as possible using bd lsr fortessa flow cytometer (becton-dickinson, usa). at least 100,000 events were acquired. the post-acquisition analysis of data was performed using the facs diva software (becton-dickinson, usa). the following lymphocyte subpopulations were identified: (cd3 + ) γδ + cd8 + , (cd3 + ) γδ + cd8 − , (cd3 + γδ − ) cd4 + cd8 + , (cd3 + γδ − ) cd4 − cd8 + , (cd3 + γδ − ) cd4 + cd8 − , (cd3 + γδ − ) cd4 − cd8 − , and cd3 − cd8 + . the percentage of ifn-γ-positive cells was established for each subpopulation. the normality of data distribution were confirmed. experimental groups were compared using non-parametric man-whitney test. data from different dates were compared using non-parametric wilcoxon test for paired samples. after vaccination with inactivated vaccines (a1 and a2) the first igm in the serum started to appear 14 days after the first dose in some piglets, and 7 days after the second dose in all animals of the a1 group ( figure 1a and table 2 ). igg antibodies appeared in all animals of both groups 7 days after the second dose ( figure 1b) . the level of antibodies in the a1 group was significantly higher than in the group given the a2 vaccine. in groups of piglets vaccinated with mlv vaccines (b3 and b4), both igm and igg antibodies appeared 14 days after vaccination. on day 21 after immunization, their antibody responses were comparable to that of the a1 group. after infection, we identified a further increase in antibodies in the vaccinated groups. for the a1 group, a further increase in serum igg antibodies was observed after 1 week and especially at 14 and 21 days after infection, when this antibody level significantly exceeded the values in the mlv immunized groups (b3 and b4) and the control one (c5). the a2 group showed a sharp increase on post infection days 14 and 21, and the level of serum igg antibodies at these intervals was comparable to a1. in groups immunized with mlv (b3 and b4), serum igg levels increased after 7, 14, and 21 days post infection, but did not reach the a1 group values. in the control, non-immunized group, the first igm antibodies appeared 3 days after infection, with a significant increase on day 7 and 14. igg antibody levels appeared 14 and 21 days after infection but were lower than in the immunized groups. the virus neutralization antibody was detected in sera of animals 21 days after vaccination (figure 1c) . these antibodies were detected in some animals in the groups a1 and a2 only. at the end of experiment (d63, 21 days post-infection) the high level of virus neutralization antibodies were detected in all groups except b3 group, in which significantly lower level was found ( figure 1c) . local antibodies in the balf performed 14 days post vaccination we detected low levels of iga in all immunized groups (figure 2a) and igg in a1, a2, and b3 (none in b4) with the level in the a1 group being significantly higher (figure 2b) . 21 days after infection we detected low levels of iga antibodies in a2, b3, b4, and control group (c5) and low levels of igg antibodies in all immunized groups (none in c5). local antibodies were detected in the saliva of the a1 group in low concentrations 14 and 21 days after the second vaccination and in all groups after infection with variability in individual intervals and with no statistically significant between-group difference ( figure 2c) . in the feces, local antibodies (iga) were detected from 1 week after the second immunization dose in groups a1 and a2, and from 1 week after mlv immunization in groups b3 and b4 ( figure 2d ) increased levels of antibody were detected in all groups including control after infection with no statistically significant betweengroup difference. a positive cell-mediated response after lymphocyte stimulation with specific antigen in vitro (stimulation index in ltt above 3) was observed in the b4 group after 7 and 21 days post vaccination and 7 and 21 days post-infection (figure 3) . a positive stimulation index was detected in the b3 group at 7 days post vaccination only as a non-specific basal stimulation occurred from 21 days post vaccination in this group (figure 3a) . groups immunized with inactivated vaccines a1 and a2 showed a marked non-specific stimulation of cells even without using antigen ( figure 3a ) and, therefore, it was impossible to demonstrate the effect of antigen addition and thus cell-mediated immune response. cell-mediated immune response after challenge infection was positive in all vaccinated groups and in the control group after 21 days post infection, using elispot in pbmc from bronchoalveolar lavages. the results were recalculated to the number of cd3 + lymphocytes. the differences between individual animals, but no significant differences between groups were detected ( figure 3d) . we detected ifn-γ producing lymphocytes after prrs antigen stimulation. the most marked differences from control were found in cd4 − cd8 + and cd4 + cd8 + (and partly also in cd3 − 8 + and γδ + 8 + ) subsets of all immunized groups 7 days after infection. in the groups vaccinated with live vaccines (b3 and b4) , the virus load was demonstrated in serum and saliva from day 3 after immunization, in balf 14 days after immunization (the only time point when the lavage was taken), and in feces occasionally 7 days after vaccination, then in all piglets 14 days after immunization (data not shown). no clinical signs were observed in piglets after infection. elevated body temperature was occasionally found in the first 2 days, independent of the experimental group. however, viral shedding was noted, with between-group differences. the virus appeared in serum, saliva, and feces in all groups including the control group 3 days after infection (figures 4a,b,d) . the virus was detected in balf 21 days after infection in a1, a2, and c5 groups (figure 4c) . virus shedding was decreased in immunized groups 14 and 21 days after infection with the level in the a1 and b3 group being significantly lower compared to control group 21 days after infection. negative samples appeared 21 days after infection in saliva (in b3 group) and in feces (b3 and b4 groups). the goals of our study were (1) to establish comprehensive immune response characteristics using several methodological approaches and monitor the dynamics in different compartments and in a time-dependent manner after vaccination and the challenging infection and (2) to compare the immune response to different killed and modified live vaccines against prrs using these methodological tools. in order to compare the immune response after vaccination with different vaccines, we used a model of vaccinations of young piglets (beginning at 8 weeks of age) and given vaccination intervals and subsequent infections, regardless of the fact that manufacturers' recommendations were different (especially in progressis). there are only a few papers published providing a comprehensive picture of immune response after vaccination against prrsv (40) (41) (42) (43) because the majority of the existing studies are based mainly on the evaluation of the vaccination effectiveness by monitoring the immune responses found in the blood (5, 30, 33, (44) (45) (46) . our results show that antibodies after immunization and infection, and the virus after infection, can be detected in all the monitored compartments (blood, respiratory tract, intestine). by repeated sampling and simultaneous monitoring of the antibody and cell-mediated immunity and virus shedding systematically and locally, we have managed to get comprehensive information about the dynamics of the immune response after vaccination or prrs virus infection. in practical diagnostics of field samples is an effort to seek simple approaches to obtain tentative information on the epidemiological situation of the herd. one current trend is the monitoring of antibody levels and shedding of the virus in the oral fluid (41) (42) (43) . in our experiment, the antibody detection rate in the oral fluid collected with ropes in pens was sufficient. the levels of antibodies detected after vaccination were low, but they increased after challenge infection. these findings confirms the possibility of using this approach for preliminary characteristics in the herd. it was interesting to observe the dynamics of antibody levels and viral shedding in feces too. this is an approach which is not often used for prrsv infection monitoring but is used in other situations where feces samples are more readily available than samples from other sources (47, 48). we were wondering, among other things, to what extent infections occurring systemically, or locally in the respiratory tract occur in this remote compartment. our findings show that fecal samples can also be used for prrsv infection monitoring. detection of both the viruses and antibodies is not entirely consistent, because they appear in individual animals, and cease at later intervals, therefore, it is necessary to consider these findings as approximate. they can be used for herd-or pen-level testing, but not for establishing a diagnosis in individual animals. it appeared to be technically difficult to demonstrate specific cell-mediated immunity. the partial results were provided by each of the three methods used and a comprehensive picture could be obtained by compiling this information. therefore, it was not possible to use only data of ifn-γ production in elispot, although it is currently the most commonly used method for cmi (5, 29, 37, 38, 49, 50) . a positive cell-mediated response after lymphocyte stimulation with specific antigen in vitro (in lymphocyte transformation test) was observed in mlv groups and especially in the b4 group as a non-specific basal stimulation occurred from 21 days post-vaccination in b3 group. the strong non-specific stimulation of pbmc without specific antigen were detected in groups a1 and a2 immunized with inactive types of vaccine. this non-specific stimulation of cells in vivo masks the overall picture, and thus specific cell-mediated immunity cannot be demonstrated. this effect is attributed to the use of strong adjuvants in inactivated types of vaccines. in the test of ifn-γ production and detection with elispot, which is very often used to identify cmi both in experimental studies (5, 37) , and in the field (49, 50) , we have shown an increase in both blood and cells acquired by lavage, but the individual variability among the animals was too high and, consequently, there were no differences found between the groups under study. we detected also ifn-γ producing lymphocytes after prrs antigen stimulation in all immunized groups 7 days after infection. the most marked differences from control were found in cd4 − cd8 + and cd4 + cd8 + (and partly also in cd3 − cd8 + and γδ + cd8 + ) subsets of lymphocytes. the cd4 − cd8 + subpopulation belongs to cytotoxic groups of cells, cd4 + cd8 + is considered a group of th1 memory cells (51) . in another study the expression of cytotoxic cd4 + cd8 + and cd4 + cd8 − was described which help to recover from prrs infection (52) . there were qualitative and quantitative differences in the immune responses to the inactivated vaccines and to mlv ones. after immunization with the inactivated vaccine (especially a1-progressis), high levels of antibodies were produced generally (serum), which were mostly of the igm, and igg isotypes, and also locally (saliva, balf), both igg and iga. nevertheless it should be noted that we have applied progressis to piglets in our study, while the manufacturers declare the use of this vaccine for gilts and sows. cell-mediated immunity was detected only after infection, high non-specific cell stimulation was detected after vaccination and therefore any specific response could not be demonstrated in these intervals. the antigen specific cell mediated immunity after inactivated vaccine is rarely described (50) . most work describes low or no cmi after vaccination with inactivated vaccine. after immunization with mlv vaccines, sufficient levels of antibodies in serum and balf (igg) were also produced, but lower than after the inactivated vaccine administration. the levels of iga antibodies in balf were comparable but low. low levels of virus neutralization antibodies after vaccination can be explained by a short interval between vaccination and infection, since neutralizing antibodies after vaccination or prrs infection occur within 28 days (42) . the dynamics of virus shedding after vaccination and infection is often used for monitoring vaccine efficacy (30, 40, 49) . the decrease in virus secretion was observed 14 days after mlv immunization and disappearance in 28 days (42) . in another study the excretion of virus was described still for 21 days after vaccination with for porcilis or amervac vaccine (53) . demonstration of cell-mediated immunity and reduction in viral load correlate with studies by other authors and support the preferred use of mlv vaccines in the control of prrs infection (29, 46) . the question is to what extent these results are influenced by the composition of vaccines from different manufacturers and to what extent different types of vaccines (inactivated vs. live attenuated). there was an obvious difference in the quality between the inactivated vaccines, whereas the character of the immune response to both mlv vaccines was similar with only partial differences in the time-related response dynamics. the vaccine b3 (amervac) showed a more pronounced decrease in virus secretion and a tendency to induce sterile immunity, while b4 (porcilis) vaccine had a more pronounced of cmi in lymphocyte transformation test. it should be noted that the strain used in porcilis had a higher genetic link with the lelystad strain compared to the strain of amervac (54, 55) which, however, probably did not significantly affect the above characteristics. despite the fact that many studies focused on prrs immunoprophylaxis have already been published and many procedures are implemented in the agricultural industry, a universal model does not yet exist (46) (47) (48) (49) (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60) . the use of live attenuated vaccines is generally preferred as was also confirmed in our field study (61) . in this study, we controlled the infection by a repeated blanket immunization with mlv vaccine (porcilis), followed by targeted immunization of gilts, and sows. the success of the strategy selected and evidence of virus eradication from the given herd were demonstrated by introducing sentinel animals into a fattening herd. based on this result, we believe that control programs can be adopted even in herds with continual throughput housing without interrupting production. however, in this case, vaccination is only one of the necessary preconditions and the introduction of very strict principles of good biosecurity is of no less importance. the use of animals was approved by the branch commission for animal welfare of ministry of agriculture of the czech republic (approval protocol no. mze 1487) as a part of project as a part of project respig (qj1210120). mt, vc, js, and mf designed the study. mt, ll, js, and kn performed the experiments. ll, hk, lk, po, and jf performed the lab work and analyzed the data. lk produced the figures and statistical analysis. mt wrote the manuscript. js and mf participated in manuscript preparation. all authors read and approved the final manuscript. the study was supported by projects no. qj1210120, qj1510108, and ro0518 of the ministry of agriculture of the czech republic and by project lo1218 with financial support from the ministry of education, youth, and sports of the czech republic under the npu i program. porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes definition of subtypes in the european genotype of porcine reproductive and respiratory syndrome virus: nucleocapsid characteristics and geographical distribution in europe genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype i strains strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus evaluation of the efficacy of a new modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine (fostera prrs) against heterologous prrsv challenge comparison of the efficacy of autogenous inactivated porcine reproductive and respiratory syndrome virus (prrsv) vaccines with that of commercial vaccines against homologous and heterologous challenges comparison of protection provided by type 1 and 2 porcine reproductive and respiratory syndrome field viruses against homologous and heterologous challenge cross efficacy of immune responses to porcine reproductive and respiratory syndrome virus infection aberrant host immune response induced by highly virulent prrsv identified by digital gene expression tag profiling pathogenesis and antigenic characterization of a new east european subtype 3 porcine reproductive and respiratory syndrome virus isolate pathogenesis and control of the chinese highly pathogenic porcine reproductive and respiratory syndrome virus innate and adaptive immunity against porcine reproductive and respiratory syndrome virus effects of origin and state of differentiation and activation of monocytes/macrophages on their susceptibility to porcine reproductive and respiratory syndrome virus (prrsv) porcine reproductive and respiratory syndrome virus: description of persistence in individual pigs upon experimental infection the challenge of prrs imunology immunopathogenesis of porcine reproductive and respiratory syndrome in the respiratory tract of pigs porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system porcine reproductive and respiratory syndrome virus-induced immunosuppression exacerbates the inflammatory response to porcine respiratory coronavirus differential production of proinflammatory cytokines in the pig lung during different respiratory virus infections: correlation with pathogenicity a modified serum neutralization test for the detection of antibody to porcine reproductive and respiratory syndrome virus in swine sera cytokine mrna profiles in bronchoalveolar cells of piglets experimentally infected with porcine reproductive and respiratory syndrome virus: association of sustained expression of ifn-γ and il-10 after viral clearance upregulation of il-10 gene expression in peripheral blood mononuclear cells by porcine reproductive and respiratory syndrome virus immune response of pigs after experimental infection with european strain of porcine reproductive and respiratory syndrome virus immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and haemophilus parasuis in vitro current infection of monocyte-derived macrophages and with porcine reproductive and respiratory syndrome virus and haemophilus parasuis: a role of ifnα in pathogenesis of co-infections challenges for porcine reproductive and respiratory syndrome virus(prrsv) vaccinology. vaccine immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) inactivated and subunit vaccines against porcine reproductive and respiratory syndrome: current status and future direction live porcine reproductive and respiratory syndrome virus vaccines: current status and future direction comparison of different vaccination schedules for sustaining immune response against porcine reproductive and respiratory syndrome virus modified-live prrsv subtype 1 vaccine unistrain prrs provides a partial clinical and virological protection upon challenge with east european subtype 3 prrsv strain lena efficacy and safety of simultaneous vaccination with two modified live virus vaccine against porcine reproductive and respiratory syndrome virus types 1 and 2 in pigs development of a broadly protective modified-live vaccine candidate against porcine reproductive and respiratory syndrome virus economic analysis of vaccination strategies for prrs control review on the transmission porcine reproductive and respiratory syndrome virus between pigs and farms and impact on vaccination cost of porcine reproductive and respiratory syndrome virus at farm level -an economic model significance of different types and levels of antigen-specific immunity to actinobacillus pleuropneumoniae infection in piglets cell-mediated immune response in swine infected with mycobacterium avium subsp intracellular cytokine detection by flow cytometry in pigs: fixation, permeabilization and cell surface staining assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters upon challenge comparison of serum and oral fluid antibody responses after vaccination with modified live (mlv) porcine reproductive and respiratory syndrome virus (prrsv) vaccine in prrs endemic farms detection of porcine reproductive and respiratory syndrome virus (prrsv)-specific igm-iga in oral fluid samples reveals prrsv infection in the presence of maternal antibody oral fluid samples used for prrsv acclimatization program and sow performance monitoring in endemic prrs-positive farms ability of elisas to detect antibodies against porcine reproductive and respiratory syndrome virus in serum of pigs after inactivated vaccination and subsequent challenge humoral immune response and viral shedding following vaccination with modified live porcine reproductive and respiratory syndrome virus vaccines long duration of immunity against a type 1 heterologous prrs virus challenge in pigs immunised with novel prrs mlv vaccine: a randomised controlled study new species of torque teno miniviruses infecting gorillas and chimpanzees adenovirus infection in savanna chimpanzees (pan troglodytes schweinfurthii) in the issa valley safety and immune response after intradermal application of porcilis prrs in either neck or the perianal region porcine reproductive and respiratory syndrome (prrs) virus-specific interferon-γ + t-cell responses after prrs virus infection or vaccination with inactivated prrs vaccine t-helper cells from naive to commited pig that recover porcine reproduction and respiratory syndrome virus infection develop cytotoxic cd4 + cd8 + and cd4 + cd8 − t-cells that kill virus infected cells safety of reproductive and respire syndrome modified live virus (mlv) vaccine strains in a young pig infection model molecular epidemiology of prrsv: a phylogenetic perspective genomic characterization and pathogenicity of a strain of type 1 porcine reproductive and respiratory syndrome virus elimination of prrs virus (eu-type) from farrow-to finish breeding farm by vaccination with ingel-vac r prrs mlv under unfavourable conditions microbial ecology of swine farms and prrs vaccine vaccination strategies development and control of an acute prrs field virus infection in an endemic prrs breeding herd after vaccination with modified live vaccine different porcine reproductive and respiratory syndrome (prrs) vaccine regimes and its effect on pig immunity status at south asia pig farms evaluation of the effect of a porcine reproductive and respiratory syndrome (prrs) modifiedlive virus vaccine on sow reproductive performance in endemic prrs farm successful elimination of prrs virus from an infected farrow-to-finish herd by vaccination key: cord-268501-z4oztgi0 authors: palatnik-de-sousa, clarisa b. title: what would jenner and pasteur have done about covid-19 coronavirus? the urges of a vaccinologist date: 2020-08-26 journal: front immunol doi: 10.3389/fimmu.2020.02173 sha: doc_id: 268501 cord_uid: z4oztgi0 nan vaccines are the best cost-benefit tools to control and eradicate infectious diseases. the live smallpox vaccination, called variolation, was the injection of the homologous virus and this promoted self-healing local lesions that guaranteed strong and long-lasting protection. however, since 3% of these variolations caused cases of smallpox in the vaccinated individuals, it was considered unsafe and was discontinued (1) (2) (3) (4) (5) . in 1796, edward jenner, who had been variolated, discovered the vaccination principle when he used the cowpox virus live-vaccine (vaccinia virus) to induce cross-immunity and prevent human smallpox in a child. his strong merit was to initiate the campaign that turned vaccination against smallpox obligatory and universal, and to discover that cross-protection promoted by a heterologous, although related, organism was sufficient to guarantee efficacy and reduce the safety issues of the homologous live-vaccine (6) . in 1967, due to the world health campaigns, smallpox was considered the first and only human viral infection ever eradicated (4, 5) . ironically, jenner never knew that smallpox was induced by a virus (6, 7) which suggests that, what is needed for the eradication of a disease is the systematic worldwide use of a potent and efficient vaccine. after the sabin anti-polio vaccine, which was launched in the 1960's, many other vaccines have been developed based on whole attenuated viruses. however, poliomyelitis was also induced by the sabin vaccine poliovirus 2 in healthy subjects (8) (9) (10) . this means that, nowadays, live-vaccination with whole wild or attenuated virus is no longer ethically possible, mainly because of the large population of immunocompromised subjects, in which a live-vaccine could cause the disease. due to these safety issues, whole virus and bacterial dead or inactivated vaccines have progressively substituted live-vaccines. we could guess that this is precisely what louis pasteur, the father of microbiology, would have done to control and eradicate covid19. although he worked initially with the attenuation of viruses and bacteria, after his successful work with rabies, fowl cholera and anthrax (11) , it became clear three steps were needed to develop a protective vaccine against infection. first, the organism should be isolated, then inactivated, and finally injected (5) . in 1885, pasteur's rabies vaccine employed an air dried fixed virus. semple improved the fixation by adding phenol (12) . currently β-propiolactone is considered to be better than phenol or formaldehyde. however, it is carcinogenic (13) . therefore, other methods like ultraviolet or gamma-irradiation, high pressure (14) , visible ultrashort pulsed laser, and low-energy electron irradiation have been suggested (15) . the most important advantage of whole inactivated vaccines is that, unlike the live or attenuated vaccines, they do not cause the disease ( table 1 ). in fact, inactivated vaccines preserve the intact structure of the antigens and their b-cell epitopes that enable them to interact with the antibody paratopes, and promote the synthesis of neutralizing antibodies. they can not only stimulate the humoral, but the cellular immune responses as well, in a manner similar to live viruses, since they preserve the virus structures during inactivation (15) . cross-presentation of conserved epitopes to the cytotoxic t cells (ctl), through the major hla class 1 histocompatibility system, in addition to the viral pathogen associated patterns (pamps), which use innate immune receptors such as toll like receptor 7, can induce t cell mediated responses. in fact, in the late endosome of the infected apc three different events can occur: (1) viral degradation following the exogenous pattern and presentation to cd4 t cells via the mhc class ii molecules, (2) cross presentation pathway to cd8 t cells via the mhc class i molecules, and (3) viral membrane fusion following the endogenous pathway. the above mentioned pathways along with the recognition of viral pamps, using prrs such as tlr7, as well as production of cytokines such as ifn-1 can promote potent cellular mediated immune responses (15) . however, in the case of influenza vaccines for instance, the inactivated formulations may not always induce t cell responses as potent as the live-vaccines. in fact, the inactivated vaccine may even prevent or suppress the induction of cross-reactive cd8 + t-cells (16) . pamps are constitutive components of the virus and bacteria (viral or bacterial nucleic acids, polysaccharides, lipopolysaccharides, lipid a, monophosphoryl lipid a, bacterial peptidoglycans, etc.). they are compounds universally recognized by the innate immune system of healthy subjects, who build a natural protective response against them. in contrast, modern purified, fractionated, recombinant, or synthetic vaccines gained in safety but lost potency because they lack the pamps (table 1) . while, vaccines using inactivated organisms with pamps have shown great success against polio, whooping cough, and tetanus (17, 18) . if we use fixation of the structures of some of the isolates that cause the disease, then inactivate them and preserve their whole structures, the possible deleterious effect of the high mutagenicity detected in a few of the proteins of the virus (19) would be overcome by the strong immune response generated against the whole virus structure. therefore, the mutagenicity should not be critical for the generation of protection, and would not damage the efficacy of the whole vaccine. furthermore, it might be that the whole virus inactivated vaccine could even induce some cross protection against other coronovirus agents of sars, which hold conservative structures (20) . furthermore, whole inactivated vaccines are considered good candidates for designing universal vaccines capable of giving protection against multiple strains of influenza virus (15) . it is true that an impressive amount of data about the dna sequencing of the virus has been gathered in a relatively short period of time and with that, the knowledge of its biological properties increased enormously (23,927 sequences in pubmed) (19, (21) (22) (23) (24) (25) (26) . however, for an urgent strategy, we could also take advantage of the lessons taught by the history of vaccinology in order to prevent the disease and save lives. furthermore, if we want to enhance the efficacy of the vaccine we should combine the inactivated virus with a good adjuvant. the adjuvant might contain saponins of quillaja saponaria molina, which induce antibodies of desired subtypes, promote both the cytotoxic antiviral cd8 + and cd4 + th1 cell responses against the infection and that been used with success in vaccines against leishmaniasis (7, 27, 28) , cancer (29) , malaria (30), herpes zoster (31) , and hiv (32) . in spite of the valuable guidelines from the work of jenner and pasteur, who with much fewer resources, developed vaccines that controlled and eradicated smallpox that showed a 40% mortality rate (33) and rabies with 100% mortality rate (34) ; and even with all the knowledge acquired since then, there is no urgent combined international effort to produce one unique vaccine. instead, 6 months after the description of the first covid19 cases in china, 147 vaccines are reported to be in development all over the world and, only two of them contain the inactivated virus (35, 36) . all the other formulations include live attenuated, non-replicating or replicating viral vector, recombinant protein, peptide base, virus-like particle, and virus dna and rna. most of these vaccines are focused on only one antigen of the coronavirus, therefore, these formulations will certainly be less potent than a vaccine made up of multiple antigens contained in the whole pathogen. furthermore, many of these formulations do not use the technology involved in any previously licensed vaccines (37) . cepi (coalition for epidemic preparedness innovation) estimated the development of phase i clinical trials of 8 vaccines, phase 2 and 3 trials for up to 6 vaccines and progression to regulatory approval and production of up to 3 candidates (38) . in fact, by may 11th, 2020 seven vaccines had already entered phase i clinical trials: (1) encapsulated mrna encoding protein s (moderna and niaid, usa); (2) adenovirus expressing protein s (cansino biologics, china); (3) dcs modified with lentivirus expressing several proteins and ctls (shenzen geno-immune medical, china); (4) an apc modified with lentivirus expressing several viral proteins (35); (5) inno 4800, sars cov2 dna injection (innovio, usa); (6) chadox1 vaccine from the jenner institute, oxford university, (uk) which is a genetically modified adenovirus expressing coronavirus proteins (39) , and is also being tested in a phase ii trial; and finally (7) the whole inactivated coronavirus with alum by sinovac, china (40) . furthermore, on july 2nd, 2020 the who communicates that there are 18 covid19 candidate vaccines in clinical evaluation and more 129 under pre-clinical assays (36) . only one of the vaccines under clinical trials is currently supported by a peer reviewed scientific publication in science (40). the results of its pre-clinical assays in the mouse, rat and non-human primate model were published before, without peer review on april 13th in the biorxiv. later on, on may 13th, the results of the chadox1 adenovirus vaccine of the jenner institute of oxford university were published with no peer review in the biorxiv (39) . until june 29th, there has been no peer reviewed publication of this vaccine. regarding the formulations, the inactivated whole virus sinovac vaccine is composed of one isolate of sars-cov2 (cn2) obtained from a patient of china and alum adjuvant (40) , while the chadox1 ncov19 vaccine of oxford is composed of a chimpanzee recombinant adenovirus, which expresses the s protein of sars-cov2 (39) . sinovac biotech (china) in collaboration with several universities, public health institutions and the medical academy of the army of china have been able to produce a whole virus inactivated vaccine adjuvanted by alum that was stable and showed 99.8 to 100% sequence identity to 10 other isolates also obtained from broncheoalveolar fluid (balf) of hospitalized patients (five in intensive care), from china, italy, united kingdom, switzerland and spain (40) . the virus was propagated in cultures of vero cells in vitro and inactivated with β-propiolactone (40) . the use of alum adjuvant is approved for human vaccines because it induces strong antibody responses, mainly of the igg1 and ige types that show efficacy against virus or bacterial diseases, which need neutralizing antibodies to be controlled. however, alum is a poor promoter of the cellular immune responses against pathogens (41) . in contrast, the chadox1 ncov-19 vaccine developed by oxford university and astrazeneca is composed of a recombinant non-replicant chimpanzee adenovirus, which expresses the s protein of sars-cov-2 (39) . different from the technology used for inactivated vaccines since the 1800's, this adenovirus platform was developed in 2012 (42) . the authors aimed to include an adenovirus in the vaccine that would not infect humans, in order to avoid its potential rejection by human antibodies. the chosen chimpanzee adenovirus was phylogenetically related to the human adenovirus. the inventors deleted the region e1 of the chimpanzee adenovirus genome in order to render the virus defective and non-replicant, while the e3 region was excluded to increase the insert capacity. in addition, a bacterial artificial chromosome (bac) containing a codon-optimized full-length spike protein of sars-cov-2 with a human tpa leader sequence (39) was added between the deleted e1 region and e4 to facilitate the genetic modifications. this approach has been reported to improve genetic stability (42) . however, additional modifications were needed to guarantee that the e4 region of the virus would express a human, instead of a simian protein, that would enable the virus recognition and propagation inside human cells in in vitro culture, for large-scale virus production (42) . regarding the number of samples, the sinovac inactivated vaccine was tested in groups of 10 mice and 10 rats and in 4 cohorts of 10 monkeys (macaca mulatta) (40), while the chadox1 ncov-19 vaccine was only tested in groups of 5-8 mice and in 6 rhesus monkeys using only 3 monkeys as controls ( table 2 ) (39) . regarding the antibody response in mice and rats, the sinovac vaccine promoted high igg antibody titers against protein s, against its receptor binding domain, and to a lower extent against protein n ( table 2 ) and also high titers of virus neutralizing antibodies. the cytokine expression induced by the inactivated vaccine in mice was not analyzed (40) . in contrast, the chadox1 vaccine induced anti-s1 and s2 protein igg antibody titers (igg2a, igg2b, and igg1) and neutralizing antibodies in only three of the five balb/c mice, but showed ifn-γ, tnf-α, and il-4 expressed by cd4 + and cd8 + t cells and ifn-γ, tnfα, il-2, and remarkably il-6 secreted to the supernatants (39) ( table 2) . furthermore, in vaccinated monkeys, seven days after infection, the sinovac inactivated vaccine at 6 µg/dose induced high titers of igg antibodies directed against the s, rbd and lower levels of anti-n protein antibodies, high titers of virus neutralizing antibodies with no detected antibodydependent enhancement of disease (ade) (40) . in contrast, anti-s protein igg and neutralizing antibodies were detected in the 6 rhesus monkeys vaccinated with chadox1 ( table 2 ) (39) . moreover, regarding the concern of the increased proinflammatory events and cytokine storm related to the severity of covid19, the sinovac vaccine was safe and did not promote any alteration in the frequencies of cd3 + , cd4 + , or cd8 t cells nor of secretion of ifn-γ, tnf-α, il-2, il-4, il-5, or il-6 ( table 2 ) (40) . in contrast, increased levels of ifn-γ, tnf-α, il-6, and il-10 were observed in monkeys vaccinated with the chadox1 vaccine (39) ( table 2 ) in which the frequencies of cytokine secreting t lymphocytes was not studied ( table 2) . regarding cross-protection to other sars cov2 isolates, the sinovac inactivated vaccine protected mice and rats against the challenge with 11 different virus isolates ( table 2) suggesting its potential use all over the world (40) . there is no available data concerning cross-protection for the chadox 1 vaccine (39) . besides, for a fair comparison of vaccine efficacies, the two sars-cov2 vaccines should be assayed in the same field trial, and the efficacy end-points should be determined prior to the assay. due to the urgency in saving lives, this might be not be feasible during the pandemic. however, for comparative purposes, early infection, disease, severe disease, and death due to covid19 or other causes should be recorded as vaccine efficacy end-points. for instance, reduction of the virus load in the nasal and pharynx mucosa indicates not only protection against early infection, but also the blockade of the transmission of infection by respiratory droplets. this means that this end point is particularly important when seeking a vaccine to interrupt the epidemic. in addition, clinical symptoms indicate disease, while pulmonary distress, cytokine storm, need of intensive care, intubation indicate severe disease. the number of deaths due to covid19 or other causes should also be recorded and compared in order to evaluate the reduction of mortality. notable, the sinovac inactivated vaccine reduced to zero the viral load in throat swabs (pharynx and crissum), anal swabs and all regions of both lungs of vaccinated and challenged monkeys (40) indicating that the inactivated vaccine prevents not only the early infection but also blocks the transmission of the disease by droplets curtailing the epidemics. in contrast, no viral sgrna indicative of viral replication, could be detected in bal fluids, and in the lungs of two of six monkeys vaccinated with chadox 1 and challenged (39) ( table 2 ). in fact, the lung viral load decreased by ∼60% in monkeys vaccinated with chadox1. however, viral grna was detected in nose swabs of all vaccinated and challenged animals ( table 2 ) (39) , indicating that the chadox1 vaccine would not prevent the sars cov2 human infection nor block its transmission and interrupt the epidemics. vaccinated and infected subjects will continue to be infectious and spread sars cov-2. however, the vaccine will probably, in most cases, reduce the pulmonary symptoms, and make the disease less severe. accordingly, the inventors of the chadox1 vaccine seem to be aware of the limitations of its efficacy when they describe it as a vaccine that prevents pneumonia in monkeys (39) . unfortunately, neither the investigations of the sinovac nor the chadox1 vaccine have disclosed if any of their formulations prevent or reduces mortality. furthermore, in a report that analyzes the first results of the vaccine trials, published in nature, peter hotez considered that the oxford vaccine induced very modest titers of neutralizing antibodies and that considerable higher titers would be needed to afford protection (43) . at the same time, hotez also says that the sinovac vaccine elicited a more promising antibody response in macaques monkeys (43) . in spite of that, who disclosed that this vaccine is in fact being tested in uk in phase i, ii and iii trials (36) and will be tested in a phase iii trial in brazil on 2,000 volunteers. consequently, contracts for large-scale fabrication have already been signed with the public laboratory of the brazilian ministry of health bio-manguinhos. in brazil, 30 million doses are intended to be produced by bio-manguinhos and another 100 million after the proven efficacy of the vaccine. at this point it is important to know which end-points of vaccine efficacy will be taken into consideration for such an important decision. on the other hand, the sinovac whole virus inactivated vaccine was also reported to have been successful in phase i and ii trials in 18-59 year olds (n = 422) and in healthy elderly adults >60 years old (n = 744) in china (36) although these results have not yet been published in detail. more than 90 % of the volunteers showed neutralizing antibodies (44) . a recent contract has been signed between sinovac and the public laboratory instituto butantan of são paulo, brazil, in order to produce doses of the vaccine to immunize 8,870 healthcare professionals for a double-blind randomized phase iii trial in brazil, where the incidence of cases and deaths due to covid19 is still high (45) . the results of the efficacy are expected in october 2020. in return, the instituto butantan will gain the transfer of the technology and the license to manufacture 60,000,000 doses for brazil. testing anti-covid19 vaccines in brazil became interesting because of the high morbidity and mortality and active expansion of the epidemics. an important warning is given by ewen callaway in his article published in nature (43) , in which he asks for caution about the potential success of vaccines that arise from small animal or human studies. this might be the case of the moderna-niaid vaccine composed of lipid nano-particle encapsulated synthetic mrna, which encodes the spike s protein and already underwent phase i and ii clinical trials in usa (36) . moderna company announced that phase iii trials are predicted to start in july 2020 and that studies in monkeys are underway in parallel. none of these mrna based vaccine has ever been licensed before (43) . we conclude that the first results of anti-covid19 vaccine candidates confirm that the whole virus inactivated vaccine, which preserves the immunogenicity of all the antigens of the virus and contains pamps (40) is more potent than the recombinant vaccines that have only the important s spicula protein, either expressed by an engineered adenovirus (39) or by lnp encapsulated mrna (43) . furthermore, the inactivated vaccine also contains the alum adjuvant. there are other examples support the superiority of whole inactivated vaccines above those expressing recombinants single antigens. for instance, 7.5 µg/dose of the trivalent inactivated influenza vaccine is as safe as, but more immunogenic than the 22.5 µg/dose of the recombinant baculovirus-expressed hemagglutinin flubok vaccine, in young children (46) . this is a fast moving scenario and several phase 1 clinical trials of covid vaccines have been published, either with or without peer reviews. two recombinant adenovirus vaccines expressing the s spike protein of sars-cov-2, the chadox1 and the cansino vaccines (47, 48) , and two other vaccines composed of mrna codifying for the s-protein (mrna1273, moderna vaccine) (49) or its rdb domain (mrna bnt162b1 pfizer-biontech vaccine) (50, 51) have been assayed for safety and immunogenicity in phase i-ii clinical trials in humans. there were no serious adverse events related to any of the four vaccines (47) (48) (49) (50) (51) . local and systemic reactions commonly including pain, feeling feverish, chills, muscle ache, headache, and malaise were recorded for all formulations (47) (48) (49) (50) (51) and were reduced, in the case of the chadox1 vaccine, with use of prophylactic paracetamol (47) . only the cansino vaccine was given as a single dose (48) while chadox1, moderna, and pfizer biontech vaccines were assayed in two-dose protocols (47, (49) (50) (51) . anti-s protein igg responses rose by day 14 (47) and peaked or increased by day 21-28, after the first (48) or second immunization dose, respectively (47, (49) (50) (51) . in addition, spike-specific t-cell responses detected by an ex-vivo interferon-γ enzyme-linked immunospot assay, peaked on day 14 for the chadox1 (47) and on day 28 for the cansino adenovirus vaccine (48) . moreover, the moderna mrna-1,273 vaccine induced a th1 response against the s-protein peptide pools (tnf-α >il-2 >ifn-γ), with a minimal th2 cytokine expression (il-4 and il-13) and with cd8 t-cell responses, only detected at low levels, after the second vaccination (49) . in agreement, most participants vaccinated with the pfizer-biontech mrna-rbd vaccine (bnt162b1) also had th1 skewed t cell immune responses with rbd-specific cd8 + and cd4 + t cell expansion and ifn-γ produced by a high fraction of rbdspecific cd8+ and cd4+ t cells (51) . furthermore, the levels of neutralizing antibodies raised by each one of the vaccines could be considered as correlates of their potential efficacy. while the mrna-1273 of moderna disclosed 50% ec values ranging between 256 and 512 (49), the maximal titer for the mrna rbd vaccine of pfizer-biontech was 308 (51) and for the chadox1 vaccine, from 256 to 512 (47) . the cansino vaccine expresses its results as gmt (4-55, 61) impeding an accurate comparison (48) . unfortunately, the results of phase i-ii clinical trial of the whole virus inactivated vaccine of sinovac have not yet been published in detail, therefore although the vaccine was tested in the largest number of individuals (n = 1,166) (36), a fair comparison of the safety and immunogenicity results is not yet possible. ultimately, only the results of the phase iii trials will disclose the potential impact of the vaccines on reduction of deaths, clinical cases, and virus particles or viral rna in nasopharynx and will allow their efficacy and capability to interrupt the epidemic to be evaluated. in the imminence of a pandemic involving high mortality and economic distress, several factors could speed up the development of vaccines. one of them would be the use of an already standardized methodology. it is worth noting that most of the molecularly defined vaccines now in development would meet severe restrictions for large-scale production and this could led to an enormous delay to deliver vaccines for mass vaccination of the public. in contrast to this, nowadays large industries and public laboratories are authorized to produce inactivated vaccines against influenza. it is also reasonable to hypothesize that generation of protection and immunological memory against a group of antigens will be more efficient than that generated against a single antigen, no matter, how important it is. two concerns could be considered as the downside of the inactivated vaccines for sars diseases. the first would be the fear of an incomplete inactivation of the virus that could cause outbreaks among the vaccine production workers or in vaccinated populations (52) . this concern is common to all vaccines produced with native antigens, which demand the production of large mass of pathogens. however, to guarantee safety, each batch of vaccine is submitted to validation of inactivation controls that include sequential passages assays of residual virus infectivity in embryonated eggs or tissue culture, and detection of live virus by tcid50 assays (14) . the whole virus sars-cov-2 inactivated vaccine of sinovac includes validation of inactivation controls (40) . the second concern would be the promotion of an antibody disease enhancement syndrome (ade) by the vaccine. this is usually related to non-neutralizing antibodies, which determine an increased lung pathology and it was observed before in vaccines against rsv and measles in the 1960's (53) . since sars-cov-1, mers-cov, and sars-cov-2 are phylogenetically related viruses that have caused epidemics over the last 16 years and ade pathology was present for some sarscov-1 and mers vaccine candidates in animal models, there is also a concern about the induction of ade syndrome in humans vaccinated with sars-cov-2 vaccine candidates (53) . however, ade pathology is not exclusive for inactivated vaccines and has been also demonstrated for the vectored vaccine expressing n protein, a replicon particle platform expressing s protein (53), the recombinant protein s with or without gold nanoparticles (54) and a mva vectored vaccine expressing s proteins (53, 55) . with the aim of preventing these safety issues in sars-cov-2 vaccines cepi and the brighton collaboration safety platform for emergency vaccines (speac) convoked an expert scientific meeting on march 12 and 13, 2020 in order to establish the assessment of the risk of ade during sars-cov-2 vaccine development (53) . in murine models, ade was observed for an inactivated whole virus vaccine against mers (56) and against sars-cov-1 (53, 57, 58) . in fact an inactivated mers-cov vaccine, with and without adjuvant, induced in mice neutralizing antibody, reduced the viral load in lungs but showed mononuclear infiltrates containing eosinophils and eosinophils secreting il-5 and il-13 cytokines (56) . a formalin double-inactivated sars-cov-1 (div) vaccine, adjuvanted or not, induced also immunopathology involving eosinophils in aged mice (53, 57) . in addition, a formalininactivated sars-cov-1 vaccine promoted ade in nhp with macrophage and lymphocyte infiltration in the lungs and fibrin and protein-rich edema in the alveolar cavity (58) . on the other hand, other inactivated vaccines against sars were reported as non-inducers of ade (59) . fortunately, other studies disclosed the absence of ade in hamsters and monkeys immunized with whole inactivated vaccine against sars-cov-1. these studies differed from the previous one in the use of β-propiolactone instead of formalin to inactivate the virus (60, 61) . the conclusion was that, nhps could be used to evaluate the anti-covid19 vaccines with or without adjuvants to select the formulations with desired efficacy and reduced risk of ade (53) . in addition, transgenic mice expressing the human ace receptor will be needed to evaluate the vaccine induced ade. the immunopathology was a consequence to a th2 type of response to the antigen and it was avoided in vaccines that drive the response to a th1 immunity, with or without adjuvants. also, it is known that the presence of fetal calf serum in the preclinical vaccine preparation may induce eosinophil influx to lungs (53) . for instance, the passive transfer in nhps of human antibodies generated during phase 1 trials, followed by viral challenge could be considered to assess the risk of disease enhancement (53) . it was recommended to challenge the vaccinated animals with close related species in order to evaluate cross protection for future epidemic (15, 53) . this has been done for the whole inactivated sinovac vaccine with other isolates of the sars-cov-2 (40) . experts also recommended including animals vaccinated with formalin inactivated, alum-adjuvanted whole virus sars-cov-1 or sars-cov-2, for immunopathology studies, as positive controls. this will help in establish accepted end-points to allow comparison (53) . the group of experts considers that continuous monitoring of this risk will be needed during clinical trials. each effect observed should be discussed by the developers with their regulators who will ultimately define the actual requirements for clinical studies (53) . if we consider that a whole virus inactivated vaccine with a potent qs21 saponin adjuvant is the ideal formulation for an anti-covid19 urgent first vaccine, the sinovac vaccine is not only the closest formulation to the ideal, only differing in the adjuvant, but also the one that can be developed the fastest. the potential use of alum, mf59, as03, as04, or as01, which contain qs21 saponin has been discussed (53, 62) . it was concluded that, the immunopathology of sars vaccines was a consequence to a th2 type of response to the antigen and it was avoided in vaccines that drive the response to a th1 immunity, with or without adjuvants (53) . time matters and is an extremely important factor considering the high daily rate of deaths worldwide. in fact, the assays of the sinovac vaccine in the mouse, rat, and macaque models seems to have been performed simultaneously from january to march, 2020. in addition, this is the only vaccine with results already published in a peer reviewed journal (40) . on the other hand, eradication of the pandemic or at least its control, as jenner knew in 1796, will only be possible by a universal and simultaneous use of the same vaccine. this is what took place with smallpox, rabies, yellow fever, influenza, h1n1, etc. in spite of that, we do not see the united international effort to gather together resources for the production of enough doses of one vaccine to vaccinate the world. the support given to 147 different research projects (36) and the deposit of hundreds of patents confirms that. again, the urgent formulation might already be known and waiting to be rediscovered from the history of vaccinology. if different vaccines with diverse degrees of efficacy values are used, even the countries that have low incidence of covid19 will not be safe and will not be able to open their frontiers. to support the production of one ideal vaccine should be the common focus worldwide. maybe the observed multiple individualistic efforts that have arisen are due to the lack of leadership from the developed nations, which have the highest capacity to produce vaccines. in the usa, for instance, the economic interest of the large vaccine industries in preventive vaccination has recently decreased. conversely, they have started to invest in immunotherapies or drug treatments. in addition, in the usa there are not large public laboratories for production of the vaccines under a governmental request. consequently, the governmental public health decisions are restricted by the interests of the private vaccine companies. in contrast, in some developing countries, where infectious diseases are often the most important causes of mortality, public laboratories can produce large amounts of vaccine doses without the need to make a profit, under the auspices of their ministries of health. this is the case of instituto butantan and bio-manguinhos in brazil, instituto biológico de la plata and the administración nacional de laboratorios e institutos de la salud (anlis-malbrán) in buenos aires, argentina, and of the serum institute of india. fortunately, instituto butantan will produce the sinovac inactivated vaccine and bio-manguinhos the adenovirus chadox1 vaccine of oxford in brazil. the serum institute of india will also produce the chadox1 vaccine of oxford. finally, the modern policies for vaccine regulations should be taken into consideration. these regulations demand that phase i, phase ii, and phase iii trials should be developed with success before a government licenses a vaccine and uses it on phase iv trials and for industrialization. this usually takes at least a decade. although these tests enhance the confidence of a product, one might think that if we are dealing with a vaccine produced by a technology that is already well-established in many licensed vaccines, such as a whole inactivated virus, more rapid or simultaneous tests would be accepted as proofs of concepts (38) . this would be another increased cost-benefit value of the vaccine, which takes into account the high mortality, worldwide incidence and impressive impact on the economy promoted by the quarantines. probably, this is what jenner and pasteur would have done. cp-s designed and wrote this article. smallpox vaccines: new formulations and revised strategies for vaccination smallpox: variolation anti-infectious human vaccination in historical perspective ann illustrated history of smallpox and its eradication from empiricism to rational design: a personal perspective of the evolution of vaccine development livro-introdução à virologia humana the delay in the licensing of protozoal vaccines: a comparative history the safety and immunogenicity of two novel live attenuated monovalent (serotype 2) oral poliovirus vaccines in healthy adults: a double-blind, single-centre phase 1 study development of thermostable lyophilized sabin inactivated poliovirus vaccine implications of a circulating vaccine-derived poliovirus in nigeria microbes infect rabies control and treatment: from prophylaxis to strategies with curative potential evaluation of different stabilizers and inactivating compounds for the enhancement of vero cell rabies vaccine stability and immunogenicity: in vitro study intranasal immunization with pressure inactivated avian influenza elicits cellular and humoral responses in mice inactivation methods for whole influenza vaccine production vaccination against human influenza a/h3n2 virus prevents the induction of heterosubtypic immunity against lethal infection with avian influenza a/h5n1 virus toward a modern synthesis of immunity: charles a. janeway jr. and the immunologist's dirty little secret recent advances in the discovery and delivery of vaccine adjuvants genetic diversity and evolution of sars-cov-2 human monoclonal antibodies against highly conserved hr1 and hr2 domains of the sars-cov spike protein are more broadly neutralizing genomic analysis of sars-cov-2 strains among indians returning from italy, iran & china, & italian tourists in india sars-cov-2: structural diversity, phylogeny, and potential animal host identification of spike glycoprotein two linear epitopes on the sars-cov-2 spike protein that elicit neutralising antibodies in covid-19 patients attenuated sars-cov-2 variants with deletions at the s1/s2 junction simulation of the clinical and pathological manifestations of coronavirus disease 2019 (covid-19) in golden syrian hamster model: implications for disease pathogenesis and transmissibility the proximal origin of sars-cov-2 immunogenicity assay of the leishmune vaccine against canine visceral leishmaniasis in brazil from mouse to man: safety, immunogenicity and efficacy of a candidate leishmaniasis vaccine leish-f3+gla-se thomsen-friedenreich (tf) antigen as a target for prostate cancer vaccine: clinical trial results with tf cluster (c)-klh plus qs21 conjugate vaccine in patients with biochemically relapsed prostate cancer vaccines against malaria immune responses to a recombinant glycoprotein e herpes zoster vaccine in adults aged 50 years or older qs-21 promotes an adjuvant effect allowing for reduced antigen dose during hiv-1 envelope subunit immunization in humans smallpox virus plaque phenotypes: genetic, geographical and case fatality relationships rabies: a medical perspective the covid-19 vaccine development landscape draft landscape of covid-19 candidate vaccines rapid covid-19 vaccine development ensuring global access to covid-19 vaccines chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques rapid development of an inactivated vaccine candidate for sars-cov-2 on vaccine's adjuvants and autoimmunity: current evidence and future perspectives a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity coronavirus vaccine trials have delivered their first results-but their promise is still unclear china's sinovac plots pivotal covid-19 vaccine trial in brazil after positive phase 2 clinical trial of efficacy and safety of sinovac's adsorbed covid-19 (inactivated) vaccine in healthcare professionals evaluation of the safety, reactogenicity and immunogenicity of flublok trivalent recombinant baculovirus-expressed hemagglutinin influenza vaccine administered intramuscularly to healthy children aged 6-59 months safety and immunogenicity of the chadox1 ncov-19 vaccine against sars-cov-2: a preliminary report of a phase 1/2, single-blind, randomised controlled trial safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial an mrna vaccine against sars-cov-2-preliminary report phase 1/2 study to describe the safety and immunogenicity of a covid-19 rna vaccine candidate (bnt162b1) in adults 18 to 55 years of age: interim report. medrxiv concurrent human antibody and t h 1 type t-cell responses elicited by a covid-19 rna vaccine. medrxiv sars vaccine development consensus summary report for cepi/bc march 12-13, 2020 meeting: assessment of risk of disease enhancement with covid-19 vaccines gold nanoparticle-adjuvanted s protein induces a strong antigenspecific igg response against severe acute respiratory syndrome-related coronavirus infection, but fails to induce protective antibodies and limit eosinophilic infiltration in lungs antispike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection immunization with inactivated middle east respiratory syndrome coronavirus vaccine leads to lung immunopathology on challenge with live virus a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunodominant sars coronavirus epitopes in humans elicited both enhancing and neutralizing effects on infection in non-human primates a double-inactivated whole virus candidate sars coronavirus vaccine stimulates neutralising and protective antibody responses immunogenicity and protective efficacy in monkeys of purified inactivated vero-cell sars vaccine immunogenicity and protective efficacy in mice and hamsters of a βpropiolactone inactivated whole virus sars-cov vaccine updated insights into the mechanism of action and clinical profile of the immunoadjuvant qs-21: a review thanks to prof. charles greenblatt of the hebrew university of jerusalem, for helpful comments and discussion. david straker was acknowledged for the english review of this manuscript. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 palatnik-de-sousa. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-004151-9815ikzg authors: pan, xiaocheng; zhang, nianzhi; wei, xiaohui; jiang, yinan; chen, rong; li, qirun; liang, ruiying; zhang, lijie; ma, lizhen; xia, chun title: illumination of prrsv cytotoxic t lymphocyte epitopes by the three-dimensional structure and peptidome of swine lymphocyte antigen class i (sla-i) date: 2020-01-08 journal: front immunol doi: 10.3389/fimmu.2019.02995 sha: doc_id: 4151 cord_uid: 9815ikzg to investigate ctl epitope applications in swine, sla-1(*)1502-restricted peptide epitopes matching porcine reproductive and respiratory syndrome virus (prrsv) strains were explored by crystallography, biochemistry, and the specific pathogen-free (spf) swine experiments. first, nine predicted prrsv peptides were tested by assembly of the peptide-sla-1(*)1502 (psla-1(*)1502) complexes, and the crystal structure of the sla-1(*)1502 complex with one peptide (nsp9-tmp9) was determined. the nsp9-tmp9 peptide conformation presented by psla-1(*)1502 is different from that of the peptides presented by the known psla-1(*)0401 and psla-3(*)hs0202 complexes. two consecutive pro residues make the turn between p3 and p4 of nsp9-tmp9 much sharper. the d pocket of psla-1(*)1502 is unique and is important for peptide binding. next, the potential sla-1(*)1502-restricted peptide epitopes matching four typical genetic prrsv strains were identified based on the peptide-binding motif of sla-1(*)1502 determined by structural analysis and alanine scanning of the nsp9-tmp9 peptide. the tetrameric complex of sla-1(*)1502 and nsp9-tmp9 was constructed and examined. finally, taking nsp9-tmp9 as an example, the ctl immunogenicity of the identified prrsv peptide epitope was evaluated. the spf swine expressing the sla-1(*)1502 alleles were divided into three groups: modified live vaccine (mlv), mlv+nsp9-tmp9, and the blank control group. nsp9-tmp9 was determined as a prrsv ctl epitope with strong immunogenicity by flow cytometry and ifn-γ expression. our study developed an integrated approach to identify sla-i-restricted ctl epitopes from various important viruses and is helpful in designing and applying effective peptide-based vaccines for swine. the development of new viral vaccines should be increasingly focused on biosafety, especially to remove the viral genetic material and to avoid the possibility of recombinant viruses developing due to the use of vaccines. in view of this central idea, diverse viral vaccines, such as cytotoxic t lymphocyte (ctl) and b cell epitope vaccines, have been experimentally researched in animals for the ongoing control of viral diseases and immunological deficiency diseases (1) . porcine reproductive and respiratory syndrome virus (prrsv) is one of the most important swine pathogens and has caused significant economic losses in the swine industry worldwide for two decades (2) . prrsv is an enveloped positive-strand rna virus with a viral genome of ∼15 kb in length and contains 11 open reading frames (orfs) (3) . orfs 1a and 1b are situated 5 ′proximal to the polycistronic genome and encode two large non-structural replicase polyproteins, pp1a, and pp1b, which are processed into at least 14 non-structural proteins (nsps). eight relatively small genes following orf1 in the 5 ′ to 3 ′ direction encode four membrane-associated glycoproteins, three membrane proteins, and a nucleocapsid protein. progress has been made in identifying nsp function related to rna synthesis (nsp9 and nsp10), subgenomic mrna synthesis regulation (nsp1), membrane-rearrangement (nsp2 and nsp3), replicative endonuclease (nsp11), major virulence factors (nsp38) , and viral pathogenesis and host immunity (nsp1, nsp2, nsp4, nsp7, and nsp11) (4) . the frequent mutation and recombination of the prrsv rna genome have resulted in the emergence of numerous variants (5, 6) . these phenomena can cause the emergence of some virulent strains, such as the highly pathogenic prrsvs that are causing enormous economic losses in asia (7) , and have led to the failure of vaccines against new emerging prrsvs. there are considerable challenges and specific requirements in the development of novel vaccines to prevent prrs, such as the ctl-epitope vaccine (8, 9) . the greatest challenge is that prrsv can markedly suppress the swine immune defense system (10) . the current evaluation of the prrsv vaccine is based on its induced antibody response. although high antibody titers can be produced after immunization, protection is not ideal because the key neutralizing antibodies (nabs) against prrsv appear late, typically >28 days post-infection (dpi), and usually at low levels (11) . furthermore, nabs are usually specific for the homologous prrsv strain and confer little cross-protection against heterologous strains (12, 13) . regarding ctl-mediated immunity, specific ctl responses have been observed in prrsv (10, 14, 15) . the virulent type 1 (lena) prrsv resulted in increased il-1α production and a higher percentage of cd8+ t cells and ifnγ-producing cells compared with controls. cross-reactivity against divergent prrsv is also associated with cytotoxic cd8+ifnγ and cd8-ifnγ+ cells to a different extent (9) . prrsv-specific t cells could be observed as early as 2 weeks after infection, with the viral loads decreasing in persistent infection (13) . modified live vaccines (mlvs) could induce ctl immune responses and confer better protection against heterologous prrsv strains than inactivated prrs vaccines (16) . these findings indicate that specific cd8 + ctl immunity may play an important role in controlling prrsv infection. however, there is limited clear and direct evidence of ctls eliminating prrsv infection, and more basic immune reagents, such as the tetramer of swine major histocompatibility complex (mhc) class i with prrsv peptide epitope, are required to address these important issues (9) . swine mhc class i has been referred to as swine lymphocyte antigen (sla-i). there are three classical sla-i loci (sla-1, sla-2, and sla-3) in the swine genome, and all are dominantly expressed (17) . sla-i molecules can present viral peptide epitopes to swine cd8 + t cells and induce the ctl response to kill the infected cells (18) . similar to human mhc (also known as human leukocyte antigen, hla), sla-i molecules are a highly polymorphic gene superfamily whose peptide-binding specificities are significantly influenced by highly variable sites (17) . thus, far, more than 100 sla-i genes have been cloned (ipd; http://www.ebi.ac.uk/ipd/index.html), and two three-dimensional (3d) structures of peptide-sla-i (p/sla-i) molecules have been determined, revealing the peptide presentation characteristics of sla-i molecules in swine (19, 20) . thus, the situation is favorable for the design and development of a novel viral ctl vaccine against swine prrs based on these 3d structures of sla-i molecules. in an attempt to identify anti-prrsv ctl epitopes in this study, first, predicted peptide epitopes derived from prrsv were synthesized, and a trimolecular complex, the structure of the epitope from prrsv-nsp9 (tmppgfely, termed nsp9-tmp9)-bound sla-1 * 1502 (psla-1 * 1502), was solved. next, the potential sla-1 * 1502-restricted peptide epitopes matching four typical genetic prrsv strains were identified. finally, the immunogenicity of the ctl epitope was identified. our results provide a novel strategy, i.e., the use of the mhc-restricted structural mechanism, to identify and validate ctl epitopes that could be used to develop a peptide-based vaccine against swine prrs. peptide epitopes were predicted by the netmhcpan 4.0 server (http://www.cbs.dtu.dk/services/netmhcpan/) based on the whole protein sequences of four typical prrsv strains (vr2332, genbank accession no. ef536003.1; hb-13.9, genbank accession no. eu360130.1; jxwn06, genbank accession no. ef641008.1; and chsx1401, genbank accession no. kp861625.1). these potential non-apeptides were predicted using the sla-1 * 1502 allele (genbank accession no. hq909439) and purified to >90% purity by analytical reverse-phase high-performance liquid chromatography (hplc) (scilight biotechnology) ( table 1) . these peptides were stored in lyophilized aliquots at −20 or −80 • c after synthesis and were dissolved in dimethyl sulfoxide (dmso) before use. refolding of the sla-1 * 1502 complex to assemble the psla-1 * 1502 complexes with each non-apeptide (table 1) , sla-1 * 1502 heavy chain (hc) and swine β2m(sβ2m) inclusion bodies were refolded (in a 1:1:1 molar ratio) via the gradual dilution method we described previously (21, 22) . the sla-1 * 1502 hc and sβ2m inclusion bodies were also refolded without peptides or with non-combined peptides as negative controls. in addition, the sla-1 * 0401 hc and sβ2m inclusion bodies were refolded with a positive peptide (amino acid sequence nsdtvgwsw) as a positive control. after 48 h of incubation at 4 • c, the remaining soluble portion of the complex was concentrated and then purified via chromatography in a superdex200 16/60 column, followed by resource-q anion-exchange chromatography (ge healthcare), as previously described (21) . crystallization and data collection of psla-1 * 1502 the purified complex (44 kda) of psla-1 * 1502 with the nsp9-tmp9 peptide (amino acid sequence tmppgfely, derived from residues 198-206 of the prrsv non-structural protein) was dialyzed against crystallization buffer (20 mm tris-hcl ph 8.0, 50 mm nacl) and concentrated to 12 mg/ml. the sample was then mixed with reservoir buffer at a 1:1 ratio and crystallized via the hanging-drop vapor diffusion technique at 277 and 291 k. index kits (hampton research, riverside, ca) were employed to screen the crystals. with a protein concentration of 12 mg/ml, crystals of psla-1 * 1502 were obtained in 10-14 days from index solution no. 65 (0.1 m bis-tris ph 5.5, 0.1 m ammonium acetate, 17% peg 10 000) at 4 • c. diffraction data were collected at a resolution of 2.2 å (psla-1 * 1502) with an in-house x-ray source (rigaku micro-max007 desktop rotating anode x-ray generator with a cu target operated at 40 kv and 30 ma) and an r-axis iv ++ imaging plate detector at a wavelength of 1.5418 å. the crystals were first soaked in reservoir solution containing 25% glycerol as a cryoprotectant and then flash-cooled in a stream of gaseous nitrogen at −173 • c (23). the collected intensities were indexed, integrated, corrected for absorption, scaled, and merged by using the hkl2000 package (24) . the structures of psla-1 * 1502 with nsp9-tmp9 were solved via molecular replacement using the molrep program with hla-a * 1101 (pdb code, 1q94) as the search model. extensive model building was performed by hand with coot (25), and restrained refinement was performed with refmac5. additional rounds of refinement were conducted by using the phenix.refine program implemented in the phenix package (26) with isotropic atomic displacement parameter (adp) refinement and bulk solvent modeling. the stereochemical quality of the final model was assessed with the procheck program (27) . data collection and refinement statistics are listed in table 2 . determination of the circular dichroism spectra and thermal unfolding of psla-1 * 1502 the thermostability of sla-1 * 1502 with six mutant peptides was examined via circular dichroism (cd) spectroscopy. cd spectra were measured at 20 • c in a jasco j-810 spectropolarimeter equipped with a water-circulating cell holder. far-uv cd spectra (180-260 nm) were collected at a protein concentration of 0.2 mg/ml in 20 mm tris (ph 8.0) buffer in a cuvette with a length of 1 mm at 0.1-nm spectral resolution. the ellipticity at 218 nm was continuously recorded during heating. thermal denaturation curves were obtained by monitoring the cd value at 218 nm in a cell with an optical path length of 1 mm as the temperature was raised from 25 to 90 • c at a rate of 1 • c/min. the temperature of the sample solution was directly measured with a thermistor. the fraction of unfolded protein was calculated from the mean residue ellipticity (θ ) by the standard method: the unfolded fraction (%) is expressed as (θ -θ n )/(θ u -θ n ), where θ n and θ u are the mean residue ellipticity values in the fully folded and fully unfolded states. the midpoint transition temperature (tm) was determined by fitting the data to the denaturation curves by using the origin 8.0 program (originlab), as described previously (28) . the tetrameric psla-1 * 1502 complex was constructed according to a previously described method (29) . briefly, a sequence containing a bira enzymatic biotinylation site was added to the c-terminus of the sla-1 * 1502 hc via pcr. the pcr primers and conditions were as described previously (30) . then, the entire construct was cloned into the pet-21a(+) vector, which was subsequently transfected into escherichia coli strain bl21(de3) for protein expression. the inclusion bodies of recombinant sla-1 * 1502 hc containing the bira site and of sβ2m were refolded with the nsp9-tmp9 peptide as described above. the psla-1 * 1502 complex was then purified and biotinylated by using the bira enzyme (avidity aurora, co). finally, the complex was purified and tetramerized by mixing psla-1 * 1502-bsp with pe-labeled streptavidin (biosource international, camarillo, ca) at a molar ratio of 4:1, after which the samples were separated by using 100 kda millipore tubes. sds-page electrophoresis was used to determine the efficiency of tetramerization. a total of nine specific pathogen-free (spf) swine (15 kg, 8-9 weeks old). beijing center of spf swine breeding and management) expressing the sla-1 * 1502 alleles were divided into three groups: mlv, mlv+nsp9-tmp9, and a blank control group. for initial immunization, the mlv and mlv+nsp9-tmp9 groups were injected with an attenuated prrsv vaccine according to the manufacturer's instructions (boehringer-ingelheim, ingelvac). after seven days, for the second immunization, the mlv + nsp9-tmp9 group was injected with the nsp9-tmp9 peptide mixed with complete freund's adjuvant (cfa, 1:3 emulsification). the mlv group was injected with the mlv peptide mixed with cfa. seven days later, peptide mixed with incomplete freund's adjuvant (ifa, 1:3 emulsification) was injected into the mlv+nsp9-tmp9 group. the mlv group was injected with mlv mixed with ifa. the immune dose of the peptide was 0.1 mg/kg body weight. the control group was injected with phosphate-buffered saline (pbs), deionized water mixed with cfa (1:3 emulsification), and deionized water mixed with ifa (1:3 emulsification) at the same time as the immunization group. equivalent volumes were used in the immunization group and the control group. blood was collected from the anterior vena cava, and peripheral blood mononuclear cells (pbmcs) were isolated by the kit according to the manufacturer's instructions (solarbio). the pbmcs were incubated for 30 min at 37 • c in staining buffer (pbs with 0.1% bsa and 0.1% sodium azide) containing the pe-labeled tetrameric complex and the fitc-labeled anti-cd8 monoclonal antibody. the cells were then washed once with staining buffer and detected via flow cytometry. more than 10 6 cell events were acquired for each sample. cells stained with pe-labeled tetramers and a fitc-labeled anti-cd8 monoclonal antibody were counted as ctl response cells (31) . the results for fluorescence-activated cell sorting (facs) data are presented as the mean ± standard error of the mean (sem) for the three animals in each group. statistical analysis was performed using graphpad prism 7 (https://www.graphpad.com) for windows. significant differences (p < 0.01) between means were tested by two-tailed student's t-test. immunization with nsp9-tmp9 one week after immunization with nsp9-tmp9 or deionized water, pbmcs were collected from immunized and control groups. these pbmcs were stimulated with nsp9-tmp9 peptide at a concentration of 2 µg/ml. pha was added at the same concentration to each positive control group, while an equivalent volume of pbs was added to the negative groups. swine ifn-γ in the supernatant was detected via an elisa kit according to the manufacturer's instructions (invitrogen) after the cells had been incubated at 37 • c for 18 h. six sla-i alleles were cloned from landrace pigs. sla-1 * 1502 showed better prrsv peptide-binding ability than the others (table s1 ) according to in silico prediction (http://www.cbs.dtu.dk/services/netmhcpan). nine prrsv peptides, all of which could be presented by sla-1 * 1502, were synthesized to test this prediction ( table 1) . all nine peptides could form complexes with sla-1 * 1502 and swine β2m (psla-1 * 1502) by in vitro refolding. the stable psla-1 * 1502 complexes were further used to screen the crystal structures. 3d structure of psla-1 * 1502 sla-1 * 1502 in complex with nsp9-tmp9 was crystallized in the p2 1 2 1 2 1 space group with a high resolution of 2.20 å ( table 2) . one asymmetric unit contains only one sla-1 * 1502 molecule. the psla-1 * 1502 complex displays a canonical p/mhc i structure, including the α1, α2, and α3 domains of the hc and the light chain sβ2m. nsp9-tmp9 is located in the peptide-binding groove (pbg) formed by the α1 and α2 domains ( figure 1a) . the root mean square differences (rmsds) between sla-1 * 1502 and two other solved p/sla i structures (sla-1 * 0401, pdb code: 3qq3; sla-3 * hs0202, pdb code: 5h94) were found to be 0.446 and 0.592, respectively, indicating similarities among the overall structures of the p/sla i molecules. the nsp9-tmp9 peptide is fixed by 15 hydrogen bonds with residues in the n-and ctermini of the pbg, and no hydrogen bonds were observed in the middle portion (p3-p7) (figure 1b) . based on the surface model, the p4 and p8 residues are located outside the pbg, and their side chains are solvent accessible, especially the p8 residue, which is at the top position of the nsp9-tmp9 peptide conformation ( figure 1c ). pocket composition of psla-1 * 1502 bound to nsp9-tmp9 peptide the compositions and polarities of the six pockets of psla-1 * 1502 are shown in figure 2 , and the interactions between the nsp9-tmp9 peptide and these pockets are listed in table 3 . the pockets of psla-1 * 1502, p/sla-1 * 0401, and p/sla-3 * hs0202 are compared in figure 3 . the a pocket of psla-1 * 1502, composed of leu 5 , tyr 7 , phe 33 , tyr 59 , glu 63 , tyr 159 , leu 163 , ser 167 , and tyr 171 , fixes p1-thr via hydrogen bonds and strong van der waals forces (vdws) (figure 2a ; table 3 ). the residues forming the a pockets of sla i molecules, including ser 167 , are highly conserved (figure 3) . in most mhc i molecules of other (figure 2b ; table 3 ). the residue composition of sla-1 * 1502 is similar to that of sla-1 * 0401, and only the residue at position 66 (lys/val) is different (figure 3) . the c, d, and e pockets usually form a large cavity in the middle portion of the pbg. the amino acid compositions of these three pockets in sla-1 * 1502 are shown in figures 2c-e. no hydrogen bonds or salt bridges were found in these structures; instead, many vdws were observed between the three pockets and the nsp9-tmp9 peptide ( table 3) . the d pocket is critical for the peptide selection of sla-1 * 0401 and sla-3 * hs0202 because of the charged residue at position 156 (19, 20) . the non-polar met 156 causes the d pocket of sla-1 * 1502 to be hydrophobic, in contrast to the charged d pocket of sla-1 * 0401 or sla-3 * hs0202 (figure 3) . the f pocket of psla-1 * 1502 consists of asn 77 , ala 80 , leu 81 , tyr 84 , leu 95 , asp 116 , tyr 123 , ile 124 thr143, and lys 146 and shows numerous interactions with p9-tyr, reflecting a key anchoring site ( figure 2f ). p9-tyr can form 6 hydrogen bonds and many vdws with the residues of the f pocket ( table 3 ). the f pockets of both psla-1 * 1502 and p/sla-1 * 0401 can accommodate p9-tyr, and only two different residues (asn/gly 77 and ala/thr 80 ) were found between the two f pockets (figure 3) . the nsp9-tmp9 peptide conformation presented by sla-1 * 1502 is different from that of the peptides presented by sla-1 * 0401 and sla-3 * hs0202 (figure 4) . because of the two consecutive pro residues, the turn between p3 and p4 of the nsp9-tmp9 peptide is much sharper than that in the other two peptides (figures 4a,b) . previous studies on sla-1 * 0401 and sla-3 * hs0202 showed that the residue at position 156 plays a key role in peptide binding by fixing the p3 residue with a salt bridge or hydrogen bond (figures 4c,d) . in contrast, no salt bridge or hydrogen bond forms between the p3-pro of nsp9-tmp9 and met 156 of sla-1 * 1502. to determine the peptide-binding motif of sla-1 * 1502, the peptide nsp9-tmp9 was mutated by alanine scanning (19) , and cd spectra were used to test the stability of psla-1 * 1502 complexes with these mutant peptides ( figure 5) . the in vitro refolding and cd results showed that the binding stabilities of p2-ala, p3-ala, and p9-ala mutant peptides are significantly lower than that of the wild-type nsp9-tmp9 peptide. although p3-pro cannot form a hydrogen bond or salt bridge with the d pocket of sla-1 * 1502, its ala mutant still impairs the stability of the psla-1 * 1502 complex. according to these results, the p2, p3, and p9 residues are the primary anchor residues of the epitope peptides presented by sla-1 * 1502. the b, d and f pockets accommodate these primary anchor residues and determine the peptide-binding motif of sla-1 * 1502, similar to sla-1 * 0401 and sla-3 * hs0202. the b and f pockets accommodate the p2 and p9 anchor residues of the binding peptide, respectively, and their preference for p2 and p9 anchor residues is determined by their amino acid composition. figure 2 shows the pocket composition of sla-1 * 1502. figure 3 shows that the amino acid composition of the b and f pockets of sla-1 * 1502 is very similar to that of sla-1 * 0401. differential amino acids are found only at one or two individual sites and do not form direct contacts with the side chains of the p2 or p9 residues of the binding peptide. because of the similar b and f pockets, the p2 and p9 residues of the sla-1 * 1502-binding peptides should be the same as in the sla-1 * 0401 peptides. the sla-1 * 0401 binding peptide (20) and the sla-1 * 1502 binding peptide have a large overlap at the p2 and p9 residues ( table 1 ). the b pocket of sla-1 * 1502 accommodates multiple uncharged residues, while the f pocket mainly binds phe, tyr and trp. the in vitro refolding results for the peptides supported this reasonable speculation ( table 1) . the uncharged d pocket of sla-1 * 1502 might accommodate various uncharged p3 residues, unlike those of sla-1 * 0401 and sla-3 * hs0202. peptides with p3-ala cannot provide sufficient affinity, unlike larger amino acids, such as l, m, f, s, n, and p ( table 1 ). in summary, the preliminary peptide-binding motif of sla-1 * 1502 is expected to contain the following combination: x-(s/m/f/w/t/v/i/l)-(l/p/m/f/s/n)-x-x-x-x-x-(f/y/w). the predicted peptide epitopes derived from whole protein sequences of four typical prrsv strains were screened: the first isolated strain, vr2332; the low-virulence strain hb-13.9; the highly pathogenic strain jxwn06; and the chsx1401 strain, which was responsible for a recent epidemic in china (figure 6) . most sla-1 * 1502-restricted prrsv peptides are located in the non-structural protein and the rna-dependent rna polymerase (rdrp) encoded by orf1a and 1b. although numerous 9mer sla-1 * 1502-binding peptides exist in each of these four prrsv strains (∼90 peptides), only 30 peptides were found to be completely conserved in all four strains (table s2) . rdrp contains 13 conserved peptides, which is a much greater number than in the other proteins. identification of nsp9-tmp9 as the ctl epitope by using the tetramer technique and the detection of swine ifn-γ the tetrameric psla-1 * 1502 complex was constructed (figures 7a-c) (33) . six landrace pigs expressing the sla-1 * 1502 genes were used to check the immunogenicity of nsp9-tmp9 peptide (figure 8) . a total of 10,000 events were recorded by the flow. the ratio of psla-1 * 1502 tetramer and cd8 double-positive cells was at a rate of ∼0.5-1% in the mlv+nsp9-tmp9-immunized group, which was significantly higher than in the control group (p = 0.0305). the mlvimmunized group was significantly higher than in the control group (p = 0.0355); however, there was no significant difference between the mlv+nsp9-tmp9-immunized group and the mlv-immunized group (p = 0.0538) (figure 8b) . additionally, swine ifn-γ expression in the peripheral blood of each pig was detected according to the methods used by kumar and walker (34, 35) . secreted ifn-γ was detectable in all of the immunized pigs but was lower than the lowest detectable limit in all control pigs ( figure 8c) . these data indicated that nsp9-tmp9, as the ctl epitope, could stimulate specific ctl immunity in swine. ctl epitopes might be a requirement of optimal prrsv immunity for the control and treatment of prrsv infection (36, 37) . in our study, a novel approach was used to select prrsv ctl epitopes, i.e., starting from a computer prediction for prrsv peptides, followed by in vitro complex refolding with sla-1 * 1502, the analysis of the complex crystal structure, the identification of sla-1 * 1502-restricted potential epitopes from whole genomes of different prrsv strains, and finally, verification of the immunogenicity of sla-1 * 1502-restricted prrsv epitope. the crystal structure of sla-1 * 1502 is the third to be solved for an sla-i allele. the crystal structure of sla-1 * 1502 exhibits the typical structural characteristics of an mhc i complex. the structure of sla-1 * 1502 is very similar to those of sla-1 * 0401 and sla-3 * hs0202, indicating that the overall combination of heavy chains, light chains, and peptides in the swine sla-i complex is highly conserved. however, in terms of peptide binding, the sla-1 * 1502 structure not only reflects the common features of sla-i alleles but also exhibits unique allelic-specific characteristics. similar to the previously resolved sla-1 * 0401 and sla-3 * hs0202, the n-terminus of the sla-1 * 1502 pbg is open because the amino acid at position 167 of the a pocket is a small ser (figure 2a) , but in other species such as humans and mice, the amino acid at this position is a large trp (19, 20) . the peptide-binding motif of sla-1 * 1502, like that of sla-1 * 0401 and sla-3 * hs0202, is determined by the three pockets b, d and f together, while the hla-i molecule is mostly determined by the two pockets b and f. these common characteristics indicate that sla-i has its own unique species features in binding peptides. in the b and f pocket composition, sla-1 * 1502 and sla-1 * 0401 are very similar, and only the non-critical amino acids in the individual positions are different (figure 3) , resulting in a large overlap of the anchoring residues accommodated in their b and f pockets (20) . in the structures of sla-1 * 0401 and sla-3 * hs0202, the d pocket plays a key role in fixing the bound peptides, with a strong salt bridge between the charged residue 156 and the p3 residue of the peptides (19, 20) . the uncharged met 156 of sla-1 * 1502 cannot form strong charge interactions with p3 residues similar to those observed for sla-1 * 0401 and sla-3 * hs0202 (figure 4) . nevertheless, the d pocket is still important in determining the peptide binding of sla-1 * 1502 and prefers uncharged residues of a certain size to form sufficient vdws. the three p/sla i structures indicate that regardless of its properties, the d pocket is critical in determining the peptide-binding motif of sla-i, and this phenomenon is expected to be a common feature among different sla-i alleles. sla-1 * 1502 was predicted in silico to present more prrsv peptide epitopes than other sla-i alleles cloned from landrace pigs, and the in vitro refolding results confirmed that most of the predicted prrsv peptides could be bound by sla-1 * 1502. four typical prrsv strains of the north american genotype were used to screen sla-1 * 1502-restricted binding peptides. according to the summarized peptide-binding motifs, approximately 90 peptides in each prrsv strain could be presented by sla-1 * 1502. these peptides are unevenly distributed in different regions, with the nsp3/4/5 proteins encoded by orf1a, nsp9/10/11 encoded by orf1b and gp2/3 exhibiting most of the candidate peptide epitopes. approximately one out of three peptides are conserved among the four prrsv strains, and approximately half of these peptides are encoded by orf1b of rdrp. ctl-epitope-based vaccines present advantages in terms of safety, specificity, and usability and are successfully used to control many viruses, such as hiv, hpv, and dengue virus (38) (39) (40) (41) . although studies aimed at developing an anti-prrsv epitope-based vaccine have been performed, no mature product is currently available (42) (43) (44) . our data indicated that rdrp (especially nsp9/10/11) may be the best target for developing a prrsv vaccine to induce a ctl response to genetically heterologous strains. tetramers of p/mhc i alleles are basic reagents that are used in immunological studies (11, 45, 46) . however, the absence of sla-i tetramers limits effective and convincing research on swine antiviral ctl responses, especially regarding accurate quantitative research. in this study, the crystallized nsp9-tmp9 peptide was used to produce the tetramer for evaluating sla-1 * 1502-restricted ctl responses. the nsp9-tmp9 epitope could induce cd8 and tetramer double-positive ctls at a rate of ∼0.5-1% in mlv+nsp-tmp9-immunized pigs, similar to the results obtained for other known efficiently protective viral ctl epitopes found in humans and mice by facs (47) (48) (49) . our results also showed that the mlv used (produced by the vr2332 strain) could induce a ctl response specific to prrs. somewhat disappointingly, we do not have live prrsv with which to challenge these swine groups and evaluate the protection of mlv and nsp-tmp9 epitope. however, nsp9-tmp9 was identified as an immunogenic epitope that could stimulate the proliferation of specific cd8 + ctls and the expression of ifn-γ in spf landrace pigs bearing sla-1 * 1502 alleles. immunization enhancement with nsp9-tmp9 produces a specific ctl response similar to that of immunization with mlv, indicating that the peptide vaccine can produce effective immunoprotection and thus that it is feasible to develop an effective prrsv polypeptide epitope vaccine. in conclusion, we solved the crystal structure of sla-1 * 1502 and described its prrsv peptide-binding map according to its preliminary peptide-binding motif determined via biochemical analyses. using the tetramer of sla-1 * 1502, the immunogenicity of nsp9-tmp9 was identified by facs and the expression of ifn-γ. the results increase our understanding of how to acquire a viral ctl vaccine against swine prrs disease. in addition, this study provides a complete and credible method for identifying sla-i-restricted viral epitopes, demonstrating the feasibility of peptide vaccines in antiviral immunity of swine. based on our experimental results, we encourage and promote the development of a safe peptide vaccine that can effectively activate ctl immune protection, solve the safety problems figure 8 | identification of the functional nsp9-tmp9 ctl epitope. (a) immunization program for spf pigs. nine pigs expressing sla-1*1502 were divided into three groups: the mlv group, mlv+peptide group and blank control group. the mlv and mlv+peptide groups were injected with an attenuated prrsv vaccine as the first immunization. seven days later, the mlv+peptide group was injected with the nsp9-tmp9 peptide mixed with cfa, and the mlv group was injected with mlv mixed with cfa as the second immunization. at day 14, the mlv+peptide group was injected with the nsp9-tmp9 peptide mixed with ifa, and the mlv group was injected with mlv mixed with ifa as the third immunization. pigs in control group were injected with pbs, deionized water mixed with cfa, and deionized water mixed with ifa. (b) nsp9-tmp9-specific ctls stained with pe-labeled sla-1*1502 tetramer and fitc-labeled anti-cd8 monoclonal antibody were detected via flow cytometry. (c) the secreted ifn-γ contents of the control group and immunized group were measured via elisa kit. the secreted ifn-γ content of the control group was less than the lowest detectable limit (78 pg/ml). caused by conventional attenuated vaccines, and provide new ideas for controlling not only prrs but also the extent of african swine fever. the coordinates and structural characteristics of psla-1 * 1502 have been deposited in the protein data bank under accession number 5ylx; the sequence of sla-1 * 1502 is available at the national center for biotechnology information (ncbi) database under accession number hq909439. the animal trials in this study were performed according to the chinese regulations for laboratory animals-the guidelines for the care of laboratory animals (ministry of science and technology of people's republic of china) and laboratory animal requirements for environment and housing facilities (gb14925-2010, national laboratory animal standardization technical committee). the license number associated with this research protocol is cau20140305-2, which was approved by the laboratory animal ethics committee of china agricultural university. the protocol adhered to the recommendations in the institute for laboratory animal research's guide for the care and use of laboratory animals. full protection of swine against foot-and-mouth disease by a bivalent b-cell epitope dendrimer peptide assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states the structural biology of prrsv the prrsv replicase: exploring the multifunctionality of an intriguing set of nonstructural proteins analysis of orf5 and full-length genome sequences of porcine reproductive and respiratory syndrome virus isolates of genotypes 1 and 2 retrieved worldwide provides evidence that recombination is a common phenomenon and may produce mosaic isolates complete genome sequence of a novel variant porcine reproductive and respiratory syndrome virus (prrsv) strain: evidence for recombination between vaccine and wildtype prrsv strains genetic characterization of 11 porcine reproductive and respiratory syndrome virus isolates in south china from 2014 to 2015 emergence of fatal prrsv variants: unparalleled outbreaks of atypical prrs in china and molecular dissection of the unique hallmark heterogeneity of porcine reproductive and respiratory syndrome virus: implications for current vaccine efficacy and future vaccine development porcine reproductive and respiratory syndrome virus (prrsv): pathogenesis and interaction with the immune system innate and adaptive immunity against porcine reproductive and respiratory syndrome virus immune evasion of porcine reproductive and respiratory syndrome virus through glycan shielding involves both glycoprotein 5 as well as glycoprotein 3 dna shuffling of the gp3 genes of porcine reproductive and respiratory syndrome virus (prrsv) produces a chimeric virus with an improved cross-neutralizing ability against a heterologous prrsv strain the level of virus-specific t-cell and macrophage recruitment in porcine reproductive and respiratory syndrome virus infection in pigs is independent of virus load lymphocyte activation as cytokine gene expression and secretion is related to the porcine reproductive and respiratory syndrome virus (prrsv) isolate after in vitro homologous and heterologous recall of peripheral blood mononuclear cells (pbmc) from pigs vaccinated and exposed to natural infection porcine reproductive and respiratory syndrome virus vaccines: immunogenicity, efficacy and safety aspects polymorphism and peptide-binding specificities of porcine major histocompatibility complex (mhc) class i molecules molecular genetics of the swine major histocompatibility complex, the sla complex structural and biochemical analyses of swine major histocompatibility complex class i complexes and prediction of the epitope map of important influenza a virus strains crystal structure of swine major histocompatibility complex class i sla-1 0401 and identification of 2009 pandemic swine-origin influenza a h1n1 virus cytotoxic t lymphocyte epitope peptides complex assembly, crystallization and preliminary x-ray crystallographic studies of rhesus macaque mhc mamu-a * 01 complexed with an immunodominant siv-gag nonapeptide complex assembly, crystallization and preliminary x-ray crystallographic studies of mhc h-2kd complexed with an hbv-core nonapeptide macromolecular crystal annealing: overcoming increased mosaicity associated with cryocrystallography refinement and reliability of macromolecular models based on x-ray diffraction data coot: model-building tools for molecular graphics the phenix software for automated determination of macromolecular structures main-chain bond lengths and bond angles in protein structures a role for the p1 anchor residue in the thermal stability of mhc class ii molecule i-ab screening and identification of severe acute respiratory syndrome-associated coronavirus-specific ctl epitopes phenotypic analysis of antigen-specific t lymphocytes hla-a * 0201 t-cell epitopes in severe acute respiratory syndrome (sars) coronavirus nucleocapsid and spike proteins major histocompatibility complex class i (fla-e * 01801) molecular structure in domestic cats demonstrates species-specific characteristics in presenting viral antigen peptides generation of mhc-peptide tetramers: a new opportunity for dissecting t-cell immune responses cell-mediated immunity to predict cytomegalovirus disease in highrisk solid organ transplant recipients ex vivo monitoring of human cytomegalovirus-specific cd8+ t-cell responses using quantiferon-cmv identification of cytotoxic t lymphocyte epitopes on swine viruses: multi-epitope design for universal t cell vaccine identification of cd8+ cytotoxic t lymphocyte epitopes from porcine reproductive and respiratory syndrome virus matrix protein in balb/c mice synthetic peptides containing b-and t-cell epitope of dengue virus-2 e domain iii provoked b-and t-cell responses a phase i trial of a human papillomavirus (hpv) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are hpv 16 positive safety and immunogenicity of a ctl multiepitope peptide vaccine for hiv with or without gm-csf in a phase i trial location and characterization of the antigenic portion of the fmdv immunizing protein assessment of the efficacy of commercial porcine reproductive and respiratory syndrome virus (prrsv) vaccines based on measurement of serologic response, frequency of gamma-ifn-producing cells and virological parameters of protection upon challenge vaccine efficacy of porcine reproductive and respiratory syndrome virus chimeras efficacy of a modified live porcine reproductive and respiratory syndrome virus (prrsv) vaccine in pigs naturally exposed to a heterologous european (italian cluster) field strain: clinical protection and cell-mediated immunity mhc-peptide tetramers to visualize antigen-specific t cells the numbers game for virus-specific cd8+ t cells identification of an hla-a * 0201-restricted t-cell epitope on the mpt51 protein, a major secreted protein derived from mycobacterium tuberculosis, by mpt51 overlapping peptide screening detection, phenotyping, and quantification of antigen-specific t cells using a peptide-mhc dodecamer identification and structural definition of h5-specific ctl epitopes restricted by hla-a * 0201 derived from the h5n1 subtype of influenza a viruses we thank the shanghai synchrotron radiation facility (ssrf), shanghai, people's republic of china, for the crystal diffraction data. we thank dr. lei zhou for his help in collecting information on prrsv strains. we thank prof. george f. gao of the chinese academy of sciences. cx: design of the study. xp and nz: data collection. xp, yj, and ql: analysis and interpretation of data. cx, nz, and xw: drafting the article. cx, xp, rl, lz, and lm: critical revision of the article. xp, nz, xw, yj, rc, ql, rl, lz, lm, and cx: final approval of the version to be published. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02995/full#supplementary-material key: cord-253108-p3wlw5d4 authors: olson, brian m.; sullivan, jeremy a.; burlingham, william j. title: interleukin 35: a key mediator of suppression and the propagation of infectious tolerance date: 2013-10-18 journal: front immunol doi: 10.3389/fimmu.2013.00315 sha: doc_id: 253108 cord_uid: p3wlw5d4 the importance of regulatory t cells (tregs) in balancing the effector arm of the immune system is well documented, playing a central role in preventing autoimmunity, facilitating graft tolerance following organ transplantation, and having a detrimental impact on the development of anti-tumor immunity. these regulatory responses use a variety of mechanisms to mediate suppression, including soluble factors. while il-10 and tgf-β are the most commonly studied immunosuppressive cytokines, the recently identified il-35 has been shown to have potent suppressive function in vitro and in vivo. furthermore, not only does il-35 have the ability to directly suppress effector t cell responses, it is also able to expand regulatory responses by propagating infectious tolerance and generating a potent population of il-35-expressing inducible tregs. in this review, we summarize research characterizing the structure and function of il-35, examine its role in disease, and discuss how it can contribute to the induction of a distinct population of inducible tregs. the immune system has evolved to establish multiple layers of defense against a variety of pathogens and diseases. however, concurrent with these effector responses are a robust network of regulatory responses that are able to keep the effector branch of the immune system in check and ensure that they do not lead to potentially harmful autoimmunity. these suppressive responses are mediated by a myriad of cell types, including myeloid-derived suppressor cells as well as macrophages with suppressive function [such as tumor-associated macrophages (1) ], but suppressive function is most commonly associated with regulatory t cells (tregs). t cells with potential suppressive activity were identified in the seminal research by gershon and kondo as well as nishizuka and sakakura more than 40 years ago, showing that lymphocytes can suppress t cell responses and that this tolerance could be transferred into naive mice (2) (3) (4) (5) . however, after this foundational work, research into tregs went through a period of controversy and conflicting results, with difficulty in identifying a molecular basis for their suppressive function leading some to question their existence. following more than a decade of studies aimed at elucidating the mechanisms that mediate treg activity, interest was rekindled in the mid-1990s with the transformational research of sakaguchi and colleagues, who specifically identified a population of cd4+cd25+ t cells that had suppressive function, which were coined as naturally occurring thymic-derived tregs, or natural abbreviations: apc, antigen-presenting cell; dc, dendritic cell; eae, experimental autoimmune encephalitis; ebi3, epstein-barr virus-induced gene 3; il, interleukin; itreg, induced regulatory t cell; nima, non-inherited maternal antigen; nk, natural killer; ntreg, natural regulatory t cell; pap, prostatic acid phosphatase; tgf, transforming growth factor; treg, regulatory t cell. tregs (ntreg) (6) . these tregs were later identified as also expressing the intracellular transcription factor foxp3, and were found to mediate suppression against a wide array of effector immune responses, including cd4+ and cd8+ t cells, b cells, natural killer (nk), and nk-t cells, and even inducing dendritic cells (dcs) and macrophages into a more tolerogenic phenotype. however, while ntregs play a central role in mediating tolerance to a variety of self antigens, they are recognized as not being the primary mediator of tolerance to pathogens and other antigens encountered in the periphery. this role belongs to a broad class of cells classified as peripherally derived or inducible tregs (itregs), which are cd4+ or cd8+ t cells which enter the periphery as naive t cells but encounter their antigen under conditions which are not conducive to the generation of productive effector responses, such as environments rich in immunosuppressive cytokines such as tgf-β. when activated in these conditions, itregs gain potent suppressive functions, inhibiting t-cell proliferation and effector functions in an antigen non-specific fashion, and play a central role in mediating regulation and propagating infectious tolerance in a variety of malignancies, including infectious diseases and cancer. inducible tregs are further divided into subclasses of itregs, which are classified based largely on the mechanisms they use to mediate regulation (though the functional mechanisms of suppression utilized by these various itregs are not strictly limited to each subpopulation -for example, multiple itreg populations use surface molecules such as ctla-4 or pd-1 to mediate infectious tolerance). tr1 induced regulatory cells mediate suppression primarily through the secretion of the immunosuppressive cytokine il-10, and are further characterized by their lack of foxp3 and cd25 expression which are expressed by ntregs (7) . the second class of itregs are th3 cells, which were one of the earliest populations of tregs and were identified as playing a role in mediating tolerance in experimental autoimmune encephalitis (eae). these cells express cd25 and foxp3 and predominantly utilize tgf-β to mediate suppression, with minimal expression of il-4 and il-10 (8). while the tr1 and th3 populations of itregs were long considered to be the only defined induced regulatory populations, research has identified another population of induced tregs that can are potent mediators of suppression as well as in the propagation of infectious tolerance: itr35 regulatory cells. these inducible regulatory cells were identified by dario vignali and colleagues and mediate suppression primarily through the expression of the regulatory cytokine il-35 (9) . in this review, we will discuss the identification and characterization of il-35, how it mediates suppression, the role it has been shown to play in disease, and the importance of il-35 and more specifically itr35 cells in propagating infectious tolerance. interleukin 35 belongs to the il-12 family of cytokines, which is a group of heterodimeric cytokines that are composed of one of five subunits [p19, p28, p35, p40, and epstein-barr virus-induced gene 3 (ebi3)] that come together in various combinations to form il-12, il-23, il-27, and il-35, as illustrated in figure 1a (10). despite their shared components, these cytokines run the spectrum of immunological effector functions. il-12 is a proinflammatory cytokine that is closely associated with the activation of th1 immune responses. it is predominantly expressed by monocytes and dcs, and its expression can be triggered by activated t cells. the inflammatory activity of il-12 is clearly seen in efforts to target its activity in a variety of diseases. in patients with malignancy, research has shown that recombinant il-12 can elicit anti-tumor responses in vivo (11) . alternatively, efforts to inhibit the inflammatory effects of il-12 have been developed, including il-12-blocking antibodies used to treat autoimmune disorders such as eae, where it has been shown to prevent uncontrolled immune responses (12, 13) . similar to il-12, il-23 also has inflammatory activity and can drive th1 responses, as well as promoting the activity of nk and th17 cells (14) . as opposed to il-12 and il-23, il-27 has a wide range of immunomodulatory activities. while it can promote th1 development, il-27 can also inhibit th2 responses and promote the suppression of t-cell responses depending on the microenvironment (15). which can signal through gp130 or il-12rβ2 homodimers, or through a unique gp130:il-12rβ2 heterodimer, which results in the formation of a novel stat1:stat4 heterodimer that has distinct effects on target cells: maximal suppression, il-35 expression, and their conversion into itr35 regulatory t cells. while il-12, il-23, and il-27 can all play a role in promoting inflammatory immune responses, the youngest member of the il-12 family, il-35, is a purely immunosuppressive cytokine. il-35 was identified in the mid-2000s, first reported by dario vignali and colleagues, and was soon after reported by his group and others to be a potent mediator of suppression (9, 16) . il-35 is a heterodimer composed of the p35 and ebi3 subunits, which were both identified as being over-expressed by tregs and not effector cells (9) . the potential of these two subunits coming together to form a novel heterodimer was first described in 1997 by devergne and colleagues, who found that cells transfected with p35 and ebi3 lead to the secretion of a p35-ebi3 heterodimer (17) . in this report, it was suggested that given the expression of ebi3 in many tissues replete with immune cells, it was likely that this heterodimer had immunomodulatory function -however, no functional studies were conducted for another 10 years. recent studies into the formation of this heterodimer found that subunits from human and mouse can bind to the opposite species, indicating that the protein-protein interactions that form il-35 are novel to the il-12 family and conserved between species (18) . furthermore, the protein binding sites were unique when compared to those used for il-12 and il-27, and that no single mutation could disrupt this interaction (18) . this is particularly significant, as the design of therapeutic interventions aimed at targeting the suppressive activity of il-35 could focus on small-molecule inhibitors of this interaction which would selectively target il-35 while leaving il-12 and il-27 unaffected. in addition to having a unique function when compared to the other il-12 family members, il-35 is also unique in that rather than being expressed primarily by antigen-presenting cells (apcs), il-35 is expressed primarily by tregs. since it was identified in 2007, dozens of reports have been published describing il-35 expression in both thymus-derived and peripheral tregs. this includes a subset of cd4+cd25+foxp3+ ntregs in humans, mice, and even pigs (9, 19, 20) , though this expression is thought to occur only in a subset of il-35+ ntregs and is not constitutive (21) . research has also identified expression of il-35 in a population of il-35-induced cd4+ tregs, defined as itr35 cells (22) . in addition to cd4+ tregs, il-35 has also been shown to be expressed and mediate antigen-specific suppression in a population of cd8+ tregs in patients with prostate cancer (23) . interestingly, other non-immune cell types have also been shown to express il-35, including tumor cells (24, 25) and potentially an even broader tissue expression profile in the course of inflammation (26) . however, in all of these cell types, it has been noted that il-35 expression is minimal in unactivated t cells -rather, these cells need to become activated for the induction of il-35, such as through engagement of their t-cell receptor or following inflammation (19, 22, 26) . this suggests that il-35 may be more associated with the suppressive activity of tregs in peripheral tissues rather than a constitutive marker of tregs. the suggested expression of il-35 by multiple cells types, including both natural and induced treg, emphasizes the need to further characterize the mechanisms that regulate the expression of il-35 in these populations. after being expressed and secreted by tregs, il-35 then acts on its target cells following binding to the il-35 receptor. however, as is the case with the subunits that make up the il-12 family of heterodimeric cytokines, the receptors for the il-12 family are also composed of five different subunits: il-12rβ1, il-12rβ2, il-23r, wsx, and gp130 (as illustrated in figure 1b) . the il-35 receptor is composed of il-12rβ2 and gp130, which are also associated with the il-12 and il-27 receptors, respectively (27, 28). following binding of il-35 to its receptor, its signal is transduced through stat1 and stat4, which can also form a unique heterodimer and result in the expression of target genes including p35 and ebi3, resulting in a feedback loop promoting increased il-35 expression (28). however, il-35 is also unique from the other members of the il-12 family in that it can also signal through a homodimer of its receptor subunits. however, when il-35 binds to one of its homodimeric receptors, only one branch of its signal transduction pathway is activated (either stat1 or stat4 for gp130:gp130 or il-12rβ2:il-12rβ2 homodimers, respectively), resulting in a partial loss of the suppressive activity of il-35 compared with signaling through the fully functional il-12rβ2-gp130 heterodimer receptor, as diagramed in figure 1b (28). the use of these subunits sheds some light onto the target of il-35 activity; gp130 is expressed in most cell types (29), whereas il-12rβ2 is expressed predominantly on activated t cells, nk cells, and to a lesser extent dcs and b cells (30). since its discovery, the predominant mechanism of suppression associated with the activity of il-35 is its ability to suppress t-cell proliferation and effector functions. foundational work into the activity of il-35 utilized il-12a −/− and ebi3 −/− mice, finding that cd4+ treg lacking il-35 expression had a significantly reduced ability to suppress t-cell proliferation (9) , an observation that has been repeated in numerous models by several groups (19, 22, (31) (32) (33) . the ability of il-35 to suppress t-cell responses has also been clearly illustrated in studies using recombinant il-35 (ril-35), where it can decrease t-cell proliferation as well as t-cell cytokine expression, though these studies have been somewhat complicated by the difficulty in generating an active heterodimeric form of il-35 (9, 16, 34, 35) . in related studies, the ectopic expression of il-35 by conventional cd4+ t cells (using a transfected il-35 expression construct) results in these conventional t cells gaining a regulatory phenotype, manifested by the ability to potently suppress t-cell proliferation (9, 22) . the suppressive activity of il-35 is not limited to cd4+ tregs, as a population of cd8+ctla-4+ tregs was also found to suppress the proliferation of autologous t cells in a contact-independent, il-35-dependent fashion (23) . while mechanistic studies into il-35 have focused on its ability to suppress cd4+ and cd8+ t-cell proliferation, il-35 has also been shown to have a role in suppressing th17 responses. tregs expressing il-35 have been shown to inhibit the differentiation of cd4+ t cells into th17 effector cells, and mice lacking ebi3 have a significant increase in the production of il-17 (32, [36] [37] [38] . this has also been reproduced in studies using ril-35, in which treatment with ril-35 reduces th17 differentiation as well as the function of th17 cells (16, 34) . in addition to its effects on th17 immunity, one report has even suggested that ril-35 can lead to decreased antibody titers (34). while this is the only report to our knowledge to associate il-35 activity with the suppression of humoral immunity, it has significant implications toward the precise mechanism of action of il-35. while in vitro studies have clearly shown that il-35 is able to directly act on effector t cells (supported by the expression profiles of the il-35 receptor subunits), the ability of il-35 to suppress antibody responses could suggest that il-35 is also able to act on other cell populations, though it could also be a reflection of the inhibition of helper t cell responses that contribute to humoral immunity. given the direct suppressive activity of il-35, there has been interest in evaluating the role that il-35 can play in the development of a variety of diseases (summarized in table 1 ). several diseases have been shown to be associated with increased il-35 expression, including multiple inflammatory diseases, coronary artery disease, and cancer. in individuals with acute myeloid leukemia, the development of disease has been shown to be associated with elevated plasma levels of il-35 (39) . this has been supported by results in lung cancer, where a study evaluating ebi3 levels in lung cancer patients found that ebi3 levels are elevated in patients with malignancy, predicts for poor outcome, and is an independent prognostic indicator of disease (although this study only examined ebi3, and not the p35 subunit of il-35) (40) . additionally, in murine models of melanoma and colorectal carcinoma, the establishment of tumors has been shown to lead to increased il-35 expression in cd4+ tumor-infiltrating lymphocytes, which are subsequently able to suppress t-cell proliferation (22) . this likely contributes to the inhibition of the anti-tumor effects of adoptively transferred cd4+ and cd8+ t cells in this melanoma model. the loss of il-35 has also been shown to be associated with the development and exacerbation of disease, including many inflammatory diseases such as encephalomyelitis and inflammatory bowel disease ( table 1 ). in multiple models of encephalomyelitis, wild-type tregs can prevent the onset and severity of disease. however, animals that lack functional il-35 were shown to have enhanced inflammatory immune responses and increased disease (9, 32, 33). similar observations have been shown in inflammatory bowel disease, liver fibrosis, and models of lethal autoimmune disease (9, 22, 35, 38) . conversely, given that the loss of il-35 is associated with increased incidence and severity of inflammatory diseases, the induction of il-35 expression has been shown to alleviate a variety of disease symptoms ( table 1 ). in models of inflammatory bowel disease, il-35 gene therapy and the adoptive transfer of il-35-expressing tregs have been shown to cure colitis symptoms (22, 35) . the same holds true in collagen-induced arthritis, where ril-35 reduces the frequency and severity of arthritis and a decrease in inflammatory immune responses (16, 34) . as opposed to these inflammatory diseases, tumor models have shown that il-35 contributes to tumorigenesis (22, 25) . these effects are mediated through both immune-directed and tumordirected effects, as il-35 can act to suppress tumor-infiltrating lymphocytes that may have anti-tumor activity, as well as potentially supporting the proliferation of tumor cells by promoting angiogenesis (22, 25) . while the direct suppressive activity of il-35 has been established in numerous reports in vitro and in vivo, research into immune tolerance has shown that the low frequency of individual regulatory populations alone are largely insufficient to control effector immunity. therefore, to expand the breadth of suppressive immunity, tregs are able to induce and mobilize additional regulatory immune cells. this concept of infectious tolerance is central to the ability of the immune system to maintain tight control of effector responses, whereby tregs can transfer suppressive function onto a nominally conventional t cell population. suppressive cytokines play a central role in the propagation of infectious tolerance, including il-35, which has been shown to play an important role in the expansion of regulatory immunity. the concept of infectious tolerance was first proposed by gershon and kondo in the early 1970s, where they showed that, "tolerance . . . can be spread from one cell to another" (4). this was further elucidated by benjamin and waldmann, who used antibodies blocking t cell populations to induce tolerance to skin grafts (46) , and later in elegant studies by qin and colleagues from the same group, who used congenically marked t cells to show that suppressive activity can be transferred from one cell population to another (47) . as additional molecular data regarding the suppressive mechanisms of treg has become available, it has become clear that tregs can secrete cytokines that can induce naïve and even effector t cells to gain a regulatory phenotype. this can occur by directly targeting effector t cells and causing them to gain a suppressive phenotype, as well as targeting dc populations and causing them to promote the conversion of effector cells into regulatory cells (48) . the most commonly thought of cytokines involved in this conversion are treg-produced il-10 and tgf-β, which can drive the generation of tr1 and th3 cells, respectively. however, the ability to transmit infectious tolerance is also a characteristic of il-35, the production of which can cause the conversion of conventional effector t cells into induced regulatory cells that are potent mediators of suppression in vitro and in vivo. some of the earliest reports of il-35 began to shed light on the potential role of this cytokine in infectious tolerance. in a report by niedbala and colleagues in 2007, they found that a ril-35 fusion protein induced the proliferation of a population of cd4+cd25+foxp3+ t cells, and which expressed il-10 and suppressed t-cell proliferation in vitro (16) . additionally, in another report utilizing a recombinant single-chain il-35, it was found that treatment of mice with ril-35 resulted in a significant increase in il-10 (but not tgf-β) production by cd4+ t cells in draining lymph nodes (34). when these mice were examined further for the impact of il-35 on treg function, they found that administration of il-35 led to an increase in the frequency of cd4+cd39+ tregs that expressed foxp3 and il-10, and that il-35 promoted the proliferation of these t cells (34). furthermore, when these cd4+cd39+ t cells were adoptively transferred they could protect animals from collagen-induced arthritis in an il-10dependent fashion (34). these data together suggest that il-35 is able to promote the expansion of il-10 producing itregs, and that these tregs are able to mediate suppression in vitro and in vivo. while these studies provided the earliest evidence that il-35 could play a role in the propagation of infectious tolerance, it remained unclear whether this induced regulatory population also expressed il-35, or whether il-35 played any role in mediating suppression in these induced regulatory cells. this was addressed frontiers in immunology | immunological tolerance (38) in an expansive report from by collison and colleagues in late 2010 that not only specifically studied the role that il-35 plays in the conversion of conventional t cells into induced tregs, but also evaluated the role that il-35 has in mediating the suppressive function of these itreg (22) . in this report, they show that treating either human or mouse conventional cd4+foxp3− t cells with il-35 causes these t conv to begin to express il-35 (but not il-10 nor tgf-β), and that these t conv cells can then suppress t-cell proliferation in a contact-independent fashion. further supporting the lack of a role for il-10 and tgf-β, blocking either of these cytokines did not affect the suppressive function of these cells whereas blocking il-35 significantly abrogated this suppression, suggesting that these il-35-induced regulatory cells represented a novel population of itregs rather than the conventional tr1 or th3 cells, which they defined as itr35 cells. the conversion of conventional t cells into an il-35-expressing inducible treg was also shown to occur when t conv were cultured with ntreg, which have been shown to express higher levels of il-35 when cultured with conventional t cells (49) . when mouse ntreg were cultured with t conv cells, the t conv cells began to express il-35 and were then able to suppress t-cell proliferation (22) . interestingly, this conversion was found to require il-35 and il-10 expression by ntregs; however, once these t conv cells had gained a regulatory phenotype, il-10 was not required for their suppressive activity. in a later report, the same group confirmed that this conversion of t conv into il-35-expressing itregs occurred in a contact-independent fashion through the activity of il-35, but did not require il-10 nor tgf-β (19) . furthermore, in this report they also show that maximal treg suppression requires not only il-35 expression but also contact with t conv that can subsequently be converted into itr35 cells (19) , further highlighting the importance of infectious tolerance in the overall suppressive activity of il-35. the generation of itr35 cells has also been shown to occur naturally following the onset of various diseases. in one model, mice were infected with an intestinal parasite that induces an inflammatory response followed by the expansion of treg responses in the intestine. following this infection, cd4+ foxp3+ ntregs in the spleen were found to have negligible levels of il-35 expression, whereas cd4+foxp3+ at the site of infection had a significant frontiers in immunology | immunological tolerance increase in il-35 (22) . interestingly, when cd4+foxp3− conventional t cells were examined for il-35, negligible levels were found in the spleen, whereas cd4+foxp3− t cells at the infection site had a significant increase in il-35 expression (22) . similar results were observed in two different tumor models (mc38 colorectal and b16 melanoma tumor cell lines), where tumor inoculation led to an increase in il-35 expression in cd4+foxp3+ and cd4+foxp3− t cells that infiltrated the tumor, whereas there was negligible il-35 expression in splenic t cells (22) . furthermore, these tumor-infiltrating cd4+foxp3− t cells were able to suppress t-cell proliferation in vitro in an il-35-dependent fashion, indicating that tumor establishment led to the generation of itr35 cells (22) . the observation that tumor formation leads to the generation of itr35 cells suggests that these cells may play a role in promoting tumor development, a characteristic that is associated with other induced treg populations. in a variety of malignancies, increased frequencies of tregs has been shown to correlate with a poor prognosis for patients, though this observation is not absolute (50) . the profoundly suppressive tumor microenvironment has been shown to promote the generation of regulatory immune responses, using factors such as tgf-β or adenosine to mediate the conversion of effector lymphocytes into itregs (51, 52) . furthermore, these tumor-infiltrating itreg have been shown to have greater suppressive activity that ntreg, both in terms of the levels of suppression as well as the mechanisms used (22, (53) (54) (55) . however, this does not appear to be the case with il-35, as tumorinfiltrating cd4+foxp3+ ntregs had higher levels of suppression then infiltrating foxp3− itr35 cells (22) . this likely reflects the multitude of suppressive mechanisms that ntreg are able to utilize to mediate suppression, as tumor-infiltrating ebi3 −/− ntreg were able to suppression t-cell proliferation at equal levels compared with wild-type ntreg (22) , and even treg that lack both il-35 and il-10 expression can still mediate suppression through factors such as trail (56). however, itr35 cells appear to lack this functional plasticity, as ebi3 −/− mice do not have tumor-infiltrating induced regulatory cells with suppressive function, and itr35 cells lacking ebi3 and/or il-12p35 lack efficacy in preventing autoimmune responses in a variety of models in addition to these tumor models (22) . despite this requirement for il-35, itr35 cells, and the role of il-35 expression on the propagation of infectious tolerance is an important component of the suppressive tumor microenvironment. when rag1 −/− mice are challenged with tumors and then receive adoptively transferred wild-type cd4+ and cd8+ t cells, these t cells are able to mediate an anti-tumor response and keep tumor growth in check (22) . when wild-type tregs are transferred as well, the tumors grow rapidly, reflecting the ability of treg to suppress the anti-tumor response associated with the transfer of the cd4+ and cd8+ t cells, both by directly suppressing t-cell proliferation as well as converting these conventional t cells into regulatory cells (22) . however, when tumor-bearing animals receive an adoptive transfer containing wild-type cd8+ t cells and ebi3 −/− cd4+ t cells, the growth of these tumors was reduced by approximately 50% (22) . this suggests that the conversion of t conv into itr35 cells is a significant contributor to the suppression of anti-tumor immunity, and that the therapeutic targeting of this regulatory population could promote anti-tumor responses. the dependence of itr35 cells on il-35 also suggests that these cells may have different characteristics regarding their long-term phenotypic and functional stability. given the nature of induced regulatory cells, which gain or lose immunosuppressive activity depending on the microenvironment in which they are activated, the stability of these induced populations is thought to be transient. however, data regarding the stability of itr35 cells in vivo suggests otherwise. in numerous models, the transfer of itreg was shown to mediate clinical efficacy for several weeks following a single adoptive transfer, suggesting that these cells retain their suppressive function for an extended period of time (22) . furthermore, when congenically marked itr35 or th3 cells were injected into mice and recovery was measured over time, it was found that 33% of the injected itr35 cells were recoverable after 1 week, 30% after 2 weeks, and 20% after more than 3 weeks, compared with only 12% of th3 cells that were recovered 1 week following transfer (22) . additionally, these cells retained their suppressive function; even 25 days following injection, adoptively transferred itr35 cells were able to suppress t-cell proliferation at the same levels as freshly isolated itr35 cells, whereas th3 cells had significantly reduced suppressive function (22) . this suggests that itr35 cells may represent more of a terminally differentiated regulatory population, and rely on the potent suppressive activity of il-35 to mediate suppression. however, further work is necessary to characterize these itr35 cells, and other suppressive mechanisms that they may gain or lose over time. as we look toward the future of immune regulation and infectious tolerance, it is essential to focus not only on identifying novel mediators of tolerance, but how these populations can be reliably identified. this is particularly relevant with the generation of induced tregs during the propagation of infectious tolerance, as the plastic nature of these populations makes them challenging to identify and track over time. even itr35 cells, which may represent more of a stable regulatory population than tr1 or th3 cells, are incredibly challenging to identify. while these cells are predominantly identified based on their robust expression of il-35, the detection of il-35 expression can be daunting. the nature of the il-12 family of cytokines requires the expression of all five subunits to be interrogated to ensure that it is il-35 that is being expressed by a putative regulatory population (even though tregs do not express significant levels of the other il-12 family members). this is also manifested in difficulty detecting il-35 protein levels using current commercially available reagents and the lack of an antibody that jointly recognizes a conformational epitope from il-35, thus requiring ebi3, p35, and the other il-12 subunits to be examined. additionally, the difficulty in generating functionally active ril-35 also delayed research and led to the necessity of multiple levels of evaluation in studying the suppressive effects of il-35. as these reagents are developed and become commercially available, it is to be hoped that increased research into il-35 will better define and characterize tregs that express this cytokine. while il-35 expression can be analyzed on fixed or lysed cell populations, what would be ideal would be a series of cell surface www.frontiersin.org markers that can be used to identify itr35 cells, allowing these cells to be isolated and further characterized. currently, itr35 cells are characterized only by their expression of ctla-4 and cd25, as well as a lack of intracellular foxp3; however, a gene microarray comparing itr35 cells with conventional treg found that there was no significant genetic signature that could be used to distinguish one regulatory population from the other (22) . furthermore, while treatment with ril-35 was shown to induce a population of cd4+cd39+cd25− tregs that express il-10, these cells were not evaluated for expression of il-35, causing cd39 to remain one of the potential markers of itr35 cells, though its expression is clearly not restricted to itr35 cells (34). this highlights the importance of elucidating markers for itr35 cells that have not yet been evaluated [such as gitr, pd-1, cd127, or even helios, whose expression is traditionally associated with ntreg but was recently suggested to also be present on itreg (57, 58) ] that can facilitate the identification of itr35 cells without expression analysis. as clearly illustrated in table 1 , il-35 plays an important role in a variety of diseases. furthermore, research from our group has identified that il-35-expressing tregs also play a central role in mediating tolerance in transplantation tolerance, immune responses to non-inherited maternal antigens (nimas), and prostate cancer [ref. (23) ; and olson et al., unpublished results], which we also found were dependent on the expression of ctla-4. interestingly, when we examined collagen type v-specific regulatory responses, we did not find a role for il-35 [contrary to results obtained from other groups (16, 34) ] nor ctla-4, though we did find that these responses relied heavily on tgf-β. this suggests that the regulatory responses we identified in the transplant, nima, and prostate cancer patients may have been relying on itr35 cells, whereas the responses we identified to collagen v were ntregs, and suggests that potentially ctla-4 and il-35dependency may be a technique that can be used to identify this population of inducible regulatory cells. in our research evaluating the role of il-35 expression in antigen-specific tolerance in prostate cancer patients, we identified cd8+ctla-4+ il-35-expressing t cells specific for the prostate tumor-associated antigen prostatic acid phosphatase (pap), which were present in some patients with prostate cancer (23) . immediately following immunization with a dna vaccine targeting pap, these antigen-specific cd8+ctla-4+ t cells prevented the detection of concurrent pap-specific effector responses; however, in long-term follow ups, we found that pap-specific effector responses could be detected in these individuals. these results raise questions regarding the nature of these cd8+ctla-4+ il-35-expressing regulatory cells; in particular, whether they represent a population of cd8+ t cells that have been induced in the periphery to gain il-35 expression and suppressive activity, or alternatively if they are an antigen-specific thymus-derived population of ntregs. if these pap-specific cd8+ regulatory responses represent a population of ntreg cells, their presence in the periphery and tumor microenvironment pre-and immediately post-immunization would suppress and prevent the detection of pap-specific effector responses (as well as potentially induce the generation of il-35-expressing tregs), whereas the long-term generation and expansion of effector responses could eventually outnumber these suppressive responses and ultimately lead to the desired goal: the generation of productive, antigen-specific anti-tumor immunity (figure 2a) . alternatively, if these papspecific cd8+ regulatory cells represent an induced population of cd8+ itr35 cells, then their presence pre-immunization would be able to convert antigen-specific effector responses into additional regulatory responses (thus furthering the propagation of infectious tolerance) until extended period following immunization when effector responses overcome these regulatory responses, either by simply outnumbering regulatory cd8 t cells or by preventing the conversion of effector cells to regulatory cells through the generation of a non-immunosuppressive microenvironment ( figure 2b) . regardless, both of these models suggest that the goal of antigen-specific immunotherapies is not simply to generate effector responses, but rather to tip the balance between antigen-specific effector and regulatory responses toward productive anti-tumor immunity. further research into these antigenspecific populations, how they are affected by antigen-specific vaccination, how they affect the generation of antigen-specific effector immune responses, and whether they have any predictive value toward the ultimate efficacy of anti-tumor vaccines, remains to be elucidated. while cd8+ tregs are not as well studied as their cd4+ treg counterparts, both cd8+ ntreg and itreg have been identified and characterized, including reports in individuals with cancer (50) . in our published studies, the reliance of pap-specific cd8+ctla-4+ t cells on il-35 for mediating contact-independent suppression (with no identifiable role played by il-10 nor tgf-β) suggests that these cells may be more akin to a population of cd8+ itr35 cells which are dependent on il-35 for contact-independent suppressive activity, as opposed to ntreg which are able to utilize multiple mechanisms of contactindependent suppression. furthermore, our observation that the suppressive function of il-35-expressing cd8+ctla-4+ treg is temporally regulated following antigen-specific immunization suggests that this population may be able to be modulated depending on the tumor microenvironment. however, additional research is required to determine how antigen-specific il-35-expressing treg are affected by antigen-specific immunization, as well as how these il-35+ treg responses are generated in tumor-bearing individuals. to better characterize the generation and fate of itr35 cells, it will be important to shed light onto the mechanisms that regulate the expression of il-35 by tregs. it is clear that expression of il-35 by ntreg and itreg requires activation, whether through inflammatory responses, non-specific stimulation of the t-cell receptor, or through encounter of antigen by antigen-specific tregs (19, 22, 23) . additionally, it appears that foxp3 does not directly play a role in the regulation of il-35 expression, providing further evidence that il-35 serves primarily as a potent mediator of suppression in induced regulatory populations rather than foxp3+ ntregs (59). however, the regulation of foxp3 does potentially open up new avenues of potential means of il-35 regulation. foxp3, along with other factors associated with tregs such as ctla-4, are specifically hypomethylated in ntreg cells, resulting in increased access to the transcriptional complex and higher expression levels compared to t conv cells, where these sequences are preferentially hypermethylated (60, 61) . additionally, the expression of various cytokines has been shown to be epigenetically regulated, including il-10 and frontiers in immunology | immunological tolerance if the observed pap-specific cd8+ctla-4+ t cells represent a population of natural tregs, pre-immunization samples (left panels) have pap-specific cd8+ ntregs present (red cells) that utilize il-35 to suppress the activity of pap-specific effector cells (dark green) both in the periphery (top panels) as well as in the tumor microenvironment (bottom panels), as well as the ability to induce a population of il-35-expressing tregs (light green). administration of a dna vaccine encoding pap leads to the presentation of pap-derived epitopes on the surface of apcs immediately post-immunization (center panels), leading to the expansion of antigen-specific effector cells. however, these cells are in small numbers, and when they traffic to the tumor site, they are outnumbered by pap-specific ntreg that are able to suppress their proliferation and effector functions. it is not until long-term follow up when these effector responses are able to outnumber antigen-specific ntreg, leading to the generation of productive anti-tumor immunity. (b) in a model where cd8+ctla-4+ t cells represent a population of novel cd8+ itr35 cells (light green cells), these itregs would be able to convert effector cells (dark green) into additional itreg through their expression of il-35, thus propagating infectious tolerance to prevent the generation of productive anti-tumor immunity both pre-immunization as well as immediately post-immunization. this process would be predicted to continue until long-term follow up, when antigen-specific effectors could expand to a sufficient level to outnumber these itreg responses, and potentially prevent the generation of induced antigen-specific treg by promoting tumor destruction and a non-suppressive tumor microenvironment. tgf-β, which can in turn induce epigenetic changes that can lead to the generation of itreg populations (62) (63) (64) (65) . this raises the possibility that the induction of il-35 expression in itr35 cells may be epigenetically regulated, which would permit the heritable transmission of il-35 expression into subsequent progeny itr35 cells while maintaining flexibility for altered expression levels based www.frontiersin.org on the immune profile of the microenvironment. to date, epigenetic regulation of il-35 expression has not been specifically evaluated -however, regions of the il-12p35 promoter have been shown to become methylated to regulate il-12 expression by dcs (66) and il-12p35 intronic regions can become demethylated in non-activated tregs (65) . additionally, the il-12rβ2 receptor has also been shown to be epigenetically regulated (67) , suggesting that il-35 could also be regulated using an epigenetic mechanism. many challenges also remain regarding the various populations of induced regulatory cells, and how these populations complement each other. clearly there is a role for il-10 in the generation of itr35 cells, though the converse does not appear to be true, with il-35 not appearing to play a central role in the generation of il-10-secreting tr1 or tgf-β-secreting th3 populations. this may suggest that these populations may have distinct roles in the suppression of inflammatory immune responses. alternatively, this could be a reflection of the redundancy of the immune system, having multiple layers of regulatory populations that can mediate similar effects, but that perhaps itr35 cells represent more of a terminally differentiated regulatory cell that is mobilized when high levels of immunosuppression are required. while studying the differentiation pattern of this population raises significant experimental challenges, these studies will be essential to better understand the nature of t-cell functional plasticity, as well as how il-35-expression fits into this process. doing so will allow for the identification of methods that can be used to control and guide these regulatory responses to design effective therapies for cancer, autoimmunity, and tissue transplantation. identification and manipulation of tumor associated macrophages in human cancers thymus and reproduction: sex-linked dysgenesia of the gonad after neonatal thymectomy in mice cell interactions in the induction of tolerance: the role of thymic lymphocytes infectious immunological tolerance immunoregulatory circuits among t-cell sets. identification of a subpopulation of t-helper cells that induces feedback inhibition immunologic self-tolerance maintained by activated t cells expressing il-2 receptor alpha-chains (cd25) weiner hl. induction and mechanism of action of transforming growth factor-beta-secreting th3 regulatory cells the inhibitory cytokine il-35 contributes to regulatory t-cell function il-12 family cytokines: immunological playmakers interleukin-12: biological properties and clinical application curcumin inhibits experimental allergic encephalomyelitis by blocking il-12 signaling through janus kinase-stat pathway in t lymphocytes functional blocking monoclonal antibodies against il-12p40 homodimer inhibit adoptive transfer of experimental allergic encephalomyelitis il-23: one cytokine in control of autoimmunity il-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory t cells and suppression of th17 cells epstein-barr virus-induced gene 3 and the p35 subunit of interleukin 12 form a novel heterodimeric hematopoietin distinct subunit pairing criteria within the heterodimeric il-12 cytokine family cutting edge: human regulatory t cells require il-35 to mediate suppression and infectious tolerance porcine t-helper and regulatory t cells exhibit versatile mrna expression capabilities for cytokines and co-stimulatory molecules human cd4+ cd25+ foxp3+ regulatory t cells do not constitutively express il-35 il-35-mediated induction of a potent regulatory t cell population human prostate tumor antigen-specific cd8+ regulatory t cells are inhibited by ctla-4 or il-35 blockade il-35 over-expression increases apoptosis sensitivity and suppresses cell growth in human cancer cells tumor-derived il-35 promotes tumor growth by enhancing myeloid cell accumulation and angiogenesis il-35 is a novel responsive anti-inflammatory cytokine -a new system of categorizing anti-inflammatory cytokines exacerbation of delayed-type hypersensitivity responses in ebv-induced gene-3 (ebi-3)-deficient mice il-35 production by inducible costimulator (icos)-positive regulatory t cells reverses established il-17-dependent allergic airways disease deletion of interleukin (il)-12p35 induces liver fibrosis in dominant-negative tgfbeta receptor type ii mice aberrant expression of treg-associated cytokine il-35 along with il-10 and tgf-beta in acute myeloid leukemia identification of epstein-barr virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer decreased plasma il-35 levels are related to the left ventricular ejection fraction in coronary artery diseases erythromycin enhances cd4+foxp3+ regulatory t-cell responses in a rat model of smoke-induced lung inflammation airway inflammation and ige production induced by dust mite allergen-specific memory/effector th2 cell line can be effectively attenuated by il-35 prevention of autoimmune diabetes by ectopic pancreatic beta-cell expression of interleukin-35 interleukin-35 enhances lyme arthritis in borrelia-vaccinated and -infected mice induction of tolerance by monoclonal antibody therapy infectious" transplantation tolerance the battle against immunopathology: infectious tolerance mediated by regulatory t cells regulatory t cell suppression is potentiated by target t cells in a cell contact, il-35-and il-10-dependent manner regulatory t cells in cancer cutting edge: tumor-specific cd8+ t cells infiltrating prostatic tumors are induced to become suppressor cells induced and natural regulatory t cells in human cancer expansion of human t regulatory type 1 cells in the microenvironment of cyclooxygenase 2 overexpressing head and neck squamous cell carcinoma increased ectonucleotidase expression and activity in regulatory t cells of patients with head and neck cancer targeting human inducible regulatory t cells (tr1) in patients with cancer: blocking of adenosineprostaglandin e(2) cooperation the plasticity of regulatory t cell function expanded subpopulation of foxp3+ t regulatory cells in renal cell carcinoma coexpress helios, indicating they could be derived from natural but not induced tregs expression of helios in peripherally induced foxp3+ regulatory t cells inducible reprogramming of human t cells into treg cells by a conditionally active form of foxp3 epigenetic mechanisms of regulation of foxp3 expression t cell receptor stimulation-induced epigenetic changes and foxp3 expression are independent and complementary events required for treg cell development epigenetic regulation of cytokine gene expression in t lymphocytes the regulation of il-10 production by immune cells epigenetic suppression of the tgf-beta pathway revealed by transcriptome profiling in ovarian cancer development and maintenance of regulatory t cells changes in il12a methylation pattern in livers from mice fed ddc aberrant methylation of il-12rbeta2 gene in lung adenocarcinoma cells is associated with unfavorable prognosis 28. collison frontiers in immunology | immunological tolerance key: cord-267237-wbwlfx7q authors: gómez-rial, jose; currás-tuala, maria josé; rivero-calle, irene; gómez-carballa, alberto; cebey-lópez, miriam; rodríguez-tenreiro, carmen; dacosta-urbieta, ana; rivero-velasco, carmen; rodríguez-núñez, nuria; trastoy-pena, rocio; rodríguez-garcía, javier; salas, antonio; martinón-torres, federico title: increased serum levels of scd14 and scd163 indicate a preponderant role for monocytes in covid-19 immunopathology date: 2020-09-23 journal: front immunol doi: 10.3389/fimmu.2020.560381 sha: doc_id: 267237 cord_uid: wbwlfx7q background: emerging evidence indicates a potential role for monocytes in covid-19 immunopathology. we investigated two soluble markers of monocyte activation, scd14 and scd163, in covid-19 patients, with the aim of characterizing their potential role in monocyte-macrophage disease immunopathology. to the best of our knowledge, this is the first study of its kind. methods: fifty-nine sars-cov-2 positive hospitalized patients, classified according to icu or non-icu admission requirement, were prospectively recruited and analyzed by elisa for levels of scd14 and scd163, along with other laboratory parameters, and compared to a healthy control group. results: scd14 and scd163 levels were significantly higher among covid-19 patients, independently of icu admission requirement, compared to the control group. we found a significant correlation between scd14 levels and other inflammatory markers, particularly interleukin-6, in the non-icu patients group. scd163 showed a moderate positive correlation with the time lapsed from admission to sampling, independently of severity group. treatment with corticoids showed an interference with scd14 levels, whereas hydroxychloroquine and tocilizumab did not. conclusions: monocyte-macrophage activation markers are increased and correlate with other inflammatory markers in sars-cov-2 infection, in association to hospital admission. these data suggest a preponderant role for monocyte-macrophage activation in the development of immunopathology of covid-19 patients. emerging evidence from sars-cov-2 infected patients suggests a key role for monocyte-macrophage in the immunopathology of covid-19 infection, with a predominant monocytederived macrophage infiltration observed in severely damaged lungs (1) , and morphological and inflammation-related changes in peripheral blood monocytes that correlate with the patients' outcome (2) . an overexuberant inflammatory immune response with production of a cytokine storm and t-cell immunosuppression are the main hallmarks of severity in these patients (3) . this clinical course resembles viral-associated hemophagocytic syndrome (vahs), a rare severe complication of various viral infections mediated by proinflammatory cytokines, resulting in multiorgan failure and death (4) . a chronic expansion of inflammatory monocytes and over-activation of macrophages have been extensively described in this syndrome (5) (6) (7) . viral-associated hemophagocytic syndrome has been identified as a major contributor to death of patients in past pandemics caused by coronaviruses (8) , including previous sars and mers outbreaks (9) , and currently suggested for sars-cov-2 outbreak (10) . cd14 and cd163 are both myeloid differentiation markers found primarily on monocytes and macrophages, and detection of soluble release of both in plasma is considered a good biomarker of monocyte-macrophage activation (11, 12) . elevated plasma levels of soluble cd14 (scd14) are associated to poor prognosis in vih-infected patients, are a strong predictor of morbidity and mortality (13, 14) , and associated with diminished cd4+-t cell restoration (15) . in addition, soluble cd163 (scd163) plasma levels are a good proxy for monocyte expansion and disease progression during hiv infection (16) . in measles infection, a leading cause of death associated with increased susceptibility to secondary infections and immunosuppression, scd14 and scd163 levels have been found to be significantly higher, indicating an important and persistent monocyte-macrophage activation (17) . we hypothesized that monocytes/macrophages may be an important component of immunopathology associated to sars-cov-2 infection. in this paper, we analyze serum levels of soluble monocyte activation markers in covid-19 patients and their correlation with severity and other inflammatory markers. we recruited 59 patients with confirmed pcr-positive diagnosis of sars-cov-2 infection, classified according to icu admission requirement (n = 22 patients), or non-icu requirement (n = 37), and age-matched healthy individuals (n = 20) as a control group. demographic data, main medication treatment and routine lab clinical parameters including inflammatory biomarkers were collected for all infected patients. leftover sera samples from routine analytical controls were employed for the analysis, after obtaining the corresponding informed consent. time elapsed from hospital admission to sample extraction was also recorded. to determine levels of soluble monocyte activation markers in serum specimens, appropriate sandwich elisa (quantikine, r&d systems, united kingdom) were used following manufacturer indications. briefly, diluted sera samples were incubated for 3 h at room temperature in the corresponding microplate strips coated with capture antibody. after incubation, strips were washed and incubated with the corresponding human antibody conjugate for 1 h. after washing, reactions were revealed and optical density at 450 nm was determined in a microplate reader. concentration levels were interpolated from the standard curve using a four-parameter logistic (4-pl) curvefit in prism8 graphpad software. final values were corrected applying the corresponding dilution factor employed. data are expressed as median and interquartile range. all statistical analyses were performed using the statistical package r. mann-whitney tests were used for comparison between icu and non-icu groups versus healthy controls. pearson's correlation coefficients were used to quantify the association between scd14 and scd163 concentration and other lab parameters in non-icu patients. data outliers, falling outside the 1.5 interquartile range, were excluded from the statistical analysis. the nominal significance level considered was 0.05. bonferroni adjustment was used to account for multiple testing. patients in the icu group showed significant differences when compared to non-icu group in several clinical laboratory parameters: lymphocytes, ferritin, d-dimer, lactate dehydrogenase (ldh), procalcitonin (pct), and interleukin-6 (il-6). the absolute value for circulating monocytes did not show significant differences between groups. however, these values may have been distorted by the use of tocilizumab, an il-6 blocking drug extensively employed in the icu group which interferes with monocyte function. age and time elapsed from admission to sample extraction did not show differences between groups. values are summarized in table 1 . median levels for scd14 in sera from icu patients were 2444.0 (95%ci: 1914.0-3251.0) ng/ml, compared to 2613.0 (95%ci: 2266.0-2991.0) ng/ml in non-icu patients. the healthy control group median value was 1788.0 (95%ci: 1615.0-1917.0) ng/ml. we observed significant statistical differences when comparing infected patients against controls (p-value < 0.0001), however no significant differences were observed between icu and non-icu groups. median levels for scd163 in sera from icu patients were 911.5 (95%ci: 624.7-1167.0) ng/ml, and 910.4 (95%ci: 733.1-1088.0) ng/ml in non-icu patients. the healthy control group value was 495.6 (95%ci: 332.5-600.7) ng/ml. as with scd14, we observed significant differences for values from infected patients compared to control group (p-value < 00001), but no differences between icu and non-icu infected patients. values are summarized in table 2 and figure 1 . we assessed the correlation between scd14 and scd163 levels and time elapsed from hospital admission to sample extraction (figure 2) . we found a significant positive correlation between scd163 levels and time elapsed (r 2 = 0.3246, p-value = 0.0156) we did not observe a significant correlation between scd14 levels and time elapsed from hospital admission to sample extraction. we found significant correlations between scd14 and scd163 levels and several clinical laboratory parameters in infected patients (in these analysis, adjusted significance under bonferrori correction is 0.01), but only in the non-icu group, possibly reflecting an interference of the use of tocilizumab or corticoids in the icu group. levels of scd14 showed a negative correlation with the absolute value of lymphocytes (r 2 = −0.5501, p-value = 0.0005) and a positive correlation with levels of ldh (r 2 = 0.5906, p-value = 0.0001), crp (r 2 = 0.6275, p-value < 0.0001); pct (r 2 = 0.4608, p-value = 0.0091), and ferritin (r 2 = 0.4414, p-value = 0.0090) (figure 3) . no other significative associations were found with other lab parameters. levels of scd163 did not show significant correlation with clinical laboratory parameters (figure 3) . particularly, il-6 also showed significant positive correlation with scd14 (r 2 = 0.6034, p-value = 0.0003) (figure 4) . we analyzed possible interference of different treatments on scd14 and scd163 serum levels for all patients. we found an interference of corticoid treatment on scd14, levels with median values of 2034 (95%ci: 1319-3159) ng/ml for treated group, and values of 2613 (95%ci: 2466-2913) ng/ml for nontreated group. values were significantly lower in corticoid-treated group (p-value = 0.0069) (figure 5) . no impact was found for corticoids on scd163 levels. likewise, hydroxychloroquine and/or tocilizumab were not found to have an impact on scd14 and scd163 serum levels. levels of scd14 and scd163 did not show association with length of hospital stay in both groups. also, these biomarkers did not show association with the number of days of onset of symptoms. we analyzed for possible age-dependence of scd14 and scd163 levels. values did not show association between these biomarker levels and the age of patients. our results show, for the first time, increased levels of scd14 and scd163 in sera from sars-cov-2 infected patients admitted to hospital. we did not observe statistical differences when comparing icu versus non-icu patients. this is probably due to the interference on monocyte function and scd14 levels produced by the use of corticoid treatment in icu patients, as shown here and previously by others (18, 19) . however, levels of scd14 showed a strong correlation with clinical laboratory parameters, including acute phase reactants (ferritin, ldh, c-reactive protein, procalcitonin) and a strong correlation with il-6 levels in the non-icu patient group, where no corticoids treatments were used. hydroxychloroquine and tocilizumab treatment did not show interferences on scd14 and scd163 levels. furthermore, scd163 levels showed a correlation with the time elapsed from hospital admission to sample extraction, suggesting a potential indicator of disease progression. monocytes and macrophages constitute a key component of immune responses against viruses, acting as bridge between innate and adaptive immunity (20) . activation of macrophages has been demonstrated to be pivotal in the pathogenesis of the immunosuppression associated to several viral infections (such as vih, measles), where expansion of specific subsets of monocytes and macrophages in peripheral blood are observed, and considered to be drivers of immunopathogenesis (21) . our results support the hypothesis of a preponderant role for monocytes in sars-cov-2 immunopathology, associated to an overexuberant immune response. increased levels of monocytemacrophage activation markers, and their correlation with other inflammatory biomarkers (particularly il-6), indicate a close relationship between monocyte activation and immunopathology in these patients. inflammatory markers are closely related to severity in covid-19 pathology (22) and selective blockade of il-6 has been demonstrated to be a good therapeutic strategy in covid-19 pathology (23). our results thus suggest that monocyte-macrophage activation can act as driver cells of the cytokine storm and immunopathology associated to severe clinical course of covid-19 patients. further, monitorization of monocyte activity trough these soluble activation markers and/or follow-up of circulating inflammatory monocytes in peripheral blood, could be useful to assess disease progression in the same way as in other viral infections (16) . in addition, our results identify monocyte-macrophage as a good target for the design of therapeutic intervention using drugs that inhibit monocyte-macrophage activation and differentiation. in this sense, anti-gm csf inhibitor drugs, currently under clinical trials for rheumatic and other auto-inflammatory diseases, might provide satisfactory results in covid-19 patients. other drugs targeting monocyte and/or macrophage could also be useful in covid-19, as in other inflammatory diseases (24) . the strategy of inhibiting monocyte differentiation has proved useful in avoiding cytokine storm syndrome after car-t cell immunotherapy (25), suggesting a possible therapeutic application to covid-19 immunopathology (26, 27) . the present study has several limitations, including a relatively low sample size and the interference of corticoids in icu patients' results. however, these preliminary results are strongly suggestive of an important implication of monocytemacrophage in covid-19 immunopathology, as highlighted by the correlations found between these biomarker levels and inflammatory parameters. further studies using broader series are needed to confirm our findings. in summary, our data underscore the preponderant role of monocyte and macrophage immune response in covid-19 immunopathology and provide pointers for future interventions in drug strategies and monitoring plans for these patients. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by comité de ética de la investigación con medicamentos de galicia (fast-track approval 18-march-2020). written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing. medrxiv covid-19 infection induces readily detectable morphological and inflammation-related phenotypic changes in peripheral blood monocytes, the severity of wich correlate with patient outcome. medrxiv speciality collaboration, covid-19: consider cytokine storm syndromes and immunosuppression virus associated hemophagocytic syndrome cd14(dim)/cd16(bright) monocytes in hemophagocytic lymphohistiocytosis how viruses contribute to the pathogenesis of hemophagocytic lymphohistiocytosis. front immunol recommendations for the management of hemophagocytic lymphohistiocytosis in adults virus-associated hemophagocytic syndrome as a major contributor to death in patients with 2009 influenza a (h1n1) infection is secondary hemophagocytic lymphohistiocytosis behind the high fatality rate in middle east respiratory syndrome corona virus? the pathogenesis and treatment of the 'cytokine storm' in covid-19 soluble cd14 is a nonspecific marker of monocyte activation differential expression of cd163 on monocyte subsets in healthy and hiv-1 infected individuals plasma levels of soluble cd14 independently predict mortality in hiv infection elevated levels of serum-soluble cd14 in human immunodeficiency virus type 1 (hiv-1) infection: correlation to disease progression and clinical events immunologic failure despite suppressive antiretroviral therapy is related to activation and turnover of memory cd4 cells increased monocyte turnover from bone marrow correlates with severity of siv encephalitis and cd163 levels in plasma persistent high plasma levels of scd163 and scd14 in adult patients with measles virus infection modulation of human monocyte/macrophage activity by tocilizumab, abatacept and etanercept: an in vitro study effects of corticosteroids on human monocyte function co-ordinating innate and adaptive immunity to viral infection: mobility is the key soluble cd163, a novel marker of activated macrophages, is elevated and associated with noncalcified coronary plaque in hiv-infected patients correlation analysis between disease severity and inflammation-related parameters in patients with covid-19 pneumonia. medrxiv the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality gm-csf inhibition reduces cytokine release syndrome and neuroinflammation but enhances car-t cell function in xenografts a strategy targeting monocyte-macrophage differentiation to avoid pulmonary complications in sars-cov2 infection role of monocytes/ macrophages in covid-19 pathogenesis: implications for therapy the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 gómez-rial, currás-tuala, rivero-calle, gómez-carballa, cebey-lópez, rodríguez-tenreiro, dacosta-urbieta, rivero-velasco, rodríguez-núñez, trastoy-pena, rodríguez-garcía, salas and martinón-torres. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-263141-n200x6z1 authors: zelaya, hortensia; alvarez, susana; kitazawa, haruki; villena, julio title: respiratory antiviral immunity and immunobiotics: beneficial effects on inflammation-coagulation interaction during influenza virus infection date: 2016-12-23 journal: front immunol doi: 10.3389/fimmu.2016.00633 sha: doc_id: 263141 cord_uid: n200x6z1 influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation–coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. influenza virus (ifv) is a major respiratory pathogen of global importance, and the cause of a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given its high capacity to change antigenically, acquired immunity is often not effective to limit ifv infection and therefore vaccination must be constantly redesigned to achieve effective protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatory-mediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. keywords: immunobiotics, influenza virus, inflammation, coagulation, respiratory immunity introduction influenza virus (ifv) is a member of the orthomyxoviridae family that contains a negative-sense, single-stranded, segmented rna genome protected by a capsid of viral ribonucleoproteins. this virus is categorized into subtypes based on the expression of hemagglutinin (ha) and neuraminidase on the surface of the viral envelope. influenza is a highly contagious viral infection that has a substantial impact on global health and ifv is a major respiratory pathogen that causes a high degree of morbidity and mortality, especially in high-risk populations such as infants, elderly, and immunocompromised hosts. given the high capacity of ifv to change antigenically, acquired immunity is often not effective to limit infection and therefore vaccination must be constantly redesigned to achieve protection. improvement of respiratory and systemic innate immune mechanisms has been proposed to reduce the incidence and severity of ifv disease. in the last decade, several research works have demonstrated that microbes with the capacity to modulate the mucosal immune system (immunobiotics) are a potential alternative to beneficially modulate the outcome of ifv infection. this review provides an update of the current status on the modulation of respiratory immunity by orally and nasally administered immunobiotics, and their beneficial impact on ifv clearance and inflammatorymediated lung tissue damage. in particular, we describe the research of our group that investigated the influence of immunobiotics on inflammation-coagulation interactions during ifv infection. studies have clearly demonstrated that hostile inflammation is accompanied by dysfunctional coagulation in respiratory ifv disease, and our investigations have proved that some immunobiotic strains are able to reduce viral disease severity through their capacity to modulate the immune-coagulative responses in the respiratory tract. the first barrier that protects the host against ifv infection is the respiratory epithelium through its capacity to recognize the viral attack. when ifv successfully overcomes the respiratory barrier constituted by the mucus layer and the ciliar movement, it mediates its attachment and internalization into respiratory epithelial cells to start its replication (1) . during the viral attack, several pathogen-associated molecular patterns (pamps) are exposed and recognized by pattern-recognition receptors (prrs) expressed in respiratory cells (figure 1 ). it is now well established that the most important prrs involved in the recognition of ifv are the toll-like receptor (tlr)-3 and tlr7 and the rna recognition protein rig-1 (2) . tlr3 is expressed in endosomes and is able to recognize viral double-stranded rna (dsrna) that is produced during viral replication; while endosomal tlr7 and cytoplasmic rig-i recognize single-stranded rna (ssrna). rig-i signals through mitochondrial antiviral signaling protein. the pamps-pprs interaction leads to the activation of several signaling pathways that induce the activation of nuclear factor κb (nf-κb) and interferon (ifn) regulatory factor 3 (irf3) and the production of type i and iii ifns and inflammatory cytokines (2) . type i ifns, especially ifn-β, produced and released during the earlier stages of ifv infection are key to develop an antiviral state in the respiratory tract. it was reported that human bronchial epithelial cells release preformed ifn-β in response to ifv challenge inducing a protective role (3) . ifns produced by infected cells are able to act in a paracrine or autocrine manner activating their receptors (ifnar) and increasing the expression of hundreds of genes that counteract viral replication. functional genomic studies have identified several of the ifn-induced factors that have important roles in controlling ifv replication (2) including the ifn-inducible transmembrane proteins 1, 2, and 3 (4), mx1 proteins (5) , and 2′,5′-oligoadenylate synthetase (oas)-rnaasel system (6) . proinflammatory cytokines and chemokines produced as a result of tlr3 and rig-i activation during ifv infection are also important for the generation of the respiratory antiviral innate immune response. infection of epithelial cells by ifv increases the expression of tnf-α, il-6, il-8, ccl2 (mip-1), ccl5 (rantes), ccl3 (mip-1α), and cxcl10 (ip-10) (7). the production of these cytokines is complemented by activity of inflammasomes that induce the activation of caspase-1 and promote the generation of the active forms of il-1β and il-18 (figure 1) . ifv has been shown to activate mainly the nlrp3 inflammasome which is essential for the protection against the virus since several studies demonstrated that mice lacking nlrp3 or caspase-1 have decreased il-1β and il-18 secretion and increased mortality after ifv challenge (8) (9) (10) . the proinflammatory cytokines and chemokines are responsible for the activation of resident immune cells such as innate lymphoid cells, alveolar macrophages, and dendritic cells (dcs) as well as for the recruitment of neutrophils, macrophages, and lymphocytes into the respiratory tract (2, 7) (figure 2) . respiratory cells infected with ifv express ha on their surface that is important for its recognition by nk cells (11) . it was established that ha expressed by the infected cells is recognized by nkp44 and pkp46 receptors of nk cells that then mediated the lysis of ifv-infected cells (12) . macrophages activated during ifv infection produce ifns, il-6, tnf-α, and nitric oxide synthase that amplify the inflammatory response. in addition, macrophages limit the viral spread by the elimination of apoptotic-infected cells and through phagocyte-mediated opsonophagocytosis of ifv (7) . the production of proinflammatory cytokines during the generation of the respiratory innate immune response against ifv also conditions the adaptive immune response, which includes the production of virusspecific systemic and mucosal antibodies as well as the induction of specific t cell responses (13) . after exposure to ifv there is an activation of antibody responses in the respiratory tract. upper airway exposure results primarily in an iga response while the contact of ifv with the deep lung induces an increased production of pathogen-specific igg (14) . following exposure to ifv in the airways there is an antigen uptake and processing by dcs, activation of cd4 + th cells, and generation of iga-producing plasma cells that populate airway lamina propria. secretory iga has a noninflammatory protective function since these antibodies can bind to virus without activating complement or stimulating the release of inflammatory mediators by innate immune cells (14, 15) . iga prevents ifv from adhering to the epithelial surface by inducing viral agglutination, and masking adhesion epitopes. in the deep lung, when ifv reach the alveolar space, there is a differentiation and expansion of antibody-secreting plasma cells that are committed to the production of igg. induction of neutralizing respiratory and serum igg antibodies is a key event in the defense against influenza infection since igg prevents systemic spread (16) . influenza infection in the lungs also activates the cellular adaptive immune response by stimulating the production of ifn-γ by th1 cells that effectively activate cd8 + t cells and macrophages, which clear virus and infected cells from the lungs (17) . therefore, during uncomplicated influenza, adaptive immune response ultimately results in clearance of ifv from the lungs through the activity of virus-specific antibodies and cd4 + and cd8 + t cells. the gut microbiome, which is defined as the collective group of microorganisms and their associated genes within the intestinal tract, is considered as a key player in the modulation of host intestinal immune responses (18, 19) . in fact, the impact of gut commensal bacteria on the innate and adaptive immune responses to enteric pathogens has been recognized conclusively (20) (21) (22) . however, the effect of gut microbiome on the immune responses in distal mucosal sites and its impact in the outcome of respiratory infections has recently been exposed. in this regard, some studies have demonstrated an important role for intestinal microbiota in maintaining respiratory antiviral immunity against ifv (23, 24) . iwasaki and colleagues observed that commensal bacteria within the gut, especially gram-positive bacterial populations, had an important role in supporting an appropriate immune response to ifv infection in the respiratory tract (23) . the work demonstrated that oral antibiotic treatments impaired the resistance of mice to the intranasal infection with ifv as noted by the elevated lung viral titers when compared to non-antibiotictreated animals. results indicated that gut gram-positive bacteria provided protection by triggering an adequate inflammatory response through inflammasomes activation. in antibiotictreated mice, synthesis of pro-il-1β, pro-il-18, and nlrp3 was impaired even at the steady state. in addition, depletion of grampositive bacterial populations in the gut resulted in an alteration of the distribution and activation of respiratory dcs at steady state as well as in a diminished dcs migration from the lung to the draining lymph nodes, resulting in reduced activation of cd8 + and cd4 + t cells after influenza challenge (23) . alteration of respiratory dcs activities also correlated with impaired expansion of influenza-specific b cells and reduced influenza-specific antibodies. by using germ-free and antibiotic-treated mice challenged with ifv, abt et al. (24) showed that the absence or the alteration of intestinal microbiota induced an exacerbated weight loss, a greater drop in blood oxygen saturation, increased mortality, and elevated lung viral titers indicating a weaker ability to resist influenza. even more, germ-free and antibiotic-treated mice infected with ifv experienced higher epithelial cell necrosis, peribronchiolar inflammation, severe bronchiole epithelial degeneration, and epithelial hyperplasia when compared to conventional animals (24) . interestingly, those effects were observed when both the pr8 strain and the x31-gp33 virus, a less pathogenic strain of ifv that causes minimal mortality and morbidity in conventional mice, were used. consistent with the work by ichinohe et al. (23) , germ-free and antibiotic-treated mice challenged with ifv had an impaired adaptive immune response as shown by the lower influenza-specific antibodies (serum igm and igg), fewer number of ifv-specific t cells present in lungs, as well as a reduced capacity of specific t cells to produce effector cytokines such as tnf-α, mip-1α, il-2, and ifn-γ (24) . moreover, authors demonstrated that the alterations of adaptive immune responses were related to defects in the early innate immune response mediated by macrophages. in fact, transcriptional profiling and computational analyses of macrophages from antibiotic-treated mice indicated a reduced expression of antiviral genes including ifnb, tnfa, il1b, irf7, mx1, and oas1a when compared to immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december 2016 | volume 7 | article 633 conventional mice. in addition, functional assays of macrophages from antibiotic-treated mice demonstrated that those cells had a defective response to type i ifns and an impaired capacity to limit ifv replication (24) . the cellular and molecular mechanisms through which the gut microbiome and their derived signals maintain and modulate immune responses in distal mucosal sites are poorly understood. two possible mechanisms that are not mutually exclusive have been proposed to explain this beneficial effect of the gut microbiome. one possibility is that distal mucosal and peripheral immune cells are directly exposed to bacterial products that activate prrs in the steady state and help to maintain the normal immune tone. there is evidence that bacterial products from gut commensals such as peptidoglycan can be absorbed and circulate throughout the host and help to modulate the normal development of immune cells (25) . in line with this hypothesis, iwasaki and colleagues speculated that bacterial products from gut commensals trigger prrs to stimulate immune cells systemically and that factors released by those cells supported steady-state production of pro-il-1β, pro-il-18, and nlr proteins. this idea was sustained by their observation that intestinal injection of tlr ligands restored immune responses to ifv in antibiotic-treated mice (23) . another possibility is that commensal bacteria may indirectly influence systemic and distal mucosal immune responses through immune factors released from the intestinal mucosa including cytokines, chemokines, and grow factors. these research works demonstrated that the gut microbiome provides signals to sustain antiviral innate immune defense mechanisms in the respiratory tract allowing robust and efficient effector responses upon challenge by viral pathogens such as ifv. therefore, the role of the gut microbiome in regulating respiratory antiviral immunity represents an exciting area of research that could help to provide the scientific basis for the development of novel prevention strategies for lung infectious diseases. however, several questions need to be answered to identify new alternatives to improve antiviral respiratory defenses by modulating the microbiota. how the different microbial species from the gut microbiota influence the common mucosal immune system? which prrs are activated by the gut microbiota to functionally modulate antiviral immunity locally and in distal mucosal sites? which cellular functions are modulated by the microbiota after prr activation? has the microbiota the ability to influence immune responses to other respiratory viruses? are similar immune mechanisms activated by the microbiota in high-risk populations (infants, elderly, immunocompromised hosts) in which respiratory viral infections are more frequent and severe? is it possible to beneficially modulate antiviral respiratory defenses by orally administering selected microorganisms with immunomodulatory capacities? research from the last years has provided some answers for the last question. the first studies that assessed the capacity of immunobiotics to favorably modulate the immune response against ifv focused on the humoral immunity ( table 1) . yasui et al. (26) reported that the oral administration of bifidobacterium breve yit4064 improved the production of anti-ifv igg antibodies in serum of ifv-infected mice. the yit4064 strain reduced viral titers, improved the survival rate, and decreased the severity of the symptoms associated to the influenza infection. similarly, it was shown that orally administered non-viable lactobacillus pentosus b240 (27) or viable lactobacillus brevis kb290 (28) were able to improve the levels of respiratory specific iga and igg antibodies of mice challenged with ifv. moreover, the improved humoral response induced by these strains correlated with significant reduction of viral titers, body weight loss, and a decrease of the alterations of physical conditions induced by ifv. more recently, kikuchi et al. (29) demonstrated a beneficial effect on the outcome to ifv infection related to an improved respiratory humoral response in lactobacillus plantarum ayatreated mice. in addition, the work proposed a mechanism for the distal immunomodulatory activity induced by orally administered immunobiotics. authors showed that l. plantarum aya fed to mice impacted in peyer's patches (pps) inducing an activation of antigen presenting cells (mainly cd11b + dcs) and increasing the production of il-6. those changes promoted an igm-to-iga class switch recombination, the differentiation of iga + b cells into plasma cells, and improved the production of mucosal iga in both the intestine and the respiratory tract. those studies show that immunobiotics are capable to modulate the production of systemic and mucosal antibodies against influenza and therefore, to enhance the humoral immune response (figure 2) . however, the precise mechanism by which orally administered immunobiotics induce iga production in distant mucosal sites remains unclear. it was also demonstrated that immunobiotics are able to improve cellular immune response against ifv (figure 2) . in this regard, it was reported that orally administered lactobacillus casei shirota improved the outcomes of ifv infection of aged (30) and infant mice (31) by increasing systemic and respiratory nk cell activity and improving the production of ifn-γ and tnf-α by respiratory lymphocytes. both studies also demonstrated that ifv titers were significantly reduced in aged and infant mice treated with the shirota strain (30, 31) . similar to the mechanism proposed to explain the improvement of humoral response, it was postulated that immunobiotic l. casei shirota stimulated th1 cells and nk cells in pps and induced a mobilization of those cells to lungs and respiratory-associated lymphoid tissues where they produced ifn-γ and enhanced the antiviral defenses. several other studies corroborated these findings by showing similar effects for orally administered lactobacilli (32, 33) . immunobiotic lactobacillus strains (l. gasseri tmc0356, l. rhamnosus gg, or l. plantarum 06cc2) beneficially modulated nk cells activity and th1 response against ifv, diminished virus titers and reduced lung pathological changes (32, 33) ( table 1) . more recently, kawahara et al. (34) described the improvement of respiratory antiviral response by an orally administered bifidobacteria strain. it was shown that bifidobacterium longum mm-2 increased respiratory nk cell activity and ifn-γ production resulting in improved clinical symptoms, reduced mortality, and decreased virus titers after ifv challenge. research work has also demonstrated that nasal administration of immunobiotics is an interesting alternative to improve cellular response against influenza infection (35-37) ( table 1) . hori et al. (35) observed that balb/c mice nasally treated with non-viable l. casei shirota had increased levels of il-12, ifnγ, and tnf-α in mediastinal lymphoid nodes and lungs. this improved cellular respiratory immunity correlated with a beneficial clinical outcome to ifv challenge. similar observations were performed with nasally administered l. pentosus s-pt84 (36) and l. rhamnosus gg (37). other recent studies have also demonstrated the ability of immunobiotics to improve respiratory innate antiviral defenses in the respiratory tract (table 1; figure 2) . it was reported that orally administered non-viable l. plantarum l-137 improved protection against ifv by increasing type i ifn production (39). the work clearly demonstrated that the increased production of ifn-β induced by the immunobiotic strain correlated with the reduction of viral loads in lungs as well as the improved survival of infected mice. more recently, it was shown that l. gasseri sbt2055 enhanced survival rates and reduced lung viral titers in mice infected with ifv (40). interestingly, authors observed that the lung expression of the antiviral genes mx1 and oas1a was enhanced in l. gasseri sbt2055-treated mice and that the inflammatory response triggered by ifv was differentially regulated inducing a lower inflammatory damage (40). our group has also reported a beneficial regulation of the ifv-triggered inflammatory response by immunobiotics. lung damage induced by ifv is known to be produced by virus replication as well as the uncontrolled inflammatory response that is characterized by a hypersecretion of proinflammatory cytokines, especially tnf-α, il-1β, and il-6 (45). the adequate production of inflammatory factors is necessary to protect against ifv infection together with an appropriate regulation with anti-inflammatory cytokines to prevent the damage of lung tissue. thus, the proper balance of cytokines is a key factor in determining the outcome of ifv infection. in this regard, we observed that orally (41) or nasally (42) administered immunobiotic l. rhamnosus crl1505 differentially regulated the levels and kinetics of inflammatory cells and cytokines in mice after ifv challenge. in our experimental model, we observed increased levels of respiratory tnf-α, il-6, neutrophils, and macrophages in crl1505-treated mice early after the challenge with ifv. later, proinflammatory cytokines and infiltrated cells started to decrease in immunobiotic-treated animals in contrast to control mice, in which those parameters continued increasing. the trend toward lower inflammatory factors and cells registered later during ifv infection in l. rhamnosus crl1505-treated mice correlated with a reduced severity of pulmonary damage when compared to control mice (41, 42). chen et al. (43) also investigated the ability of orally administered enterococcus faecalis kh2 to beneficially modulate the innate immune response to influenza infection. authors observed that kh2 strain protected c57bl/6 mice against ifv as observed by the reduced mortality, weight loss, and lung viral titers. as expected, ifv enhanced the levels of proinflammatory mediators in the respiratory tract including il-6, tnf-α, ifn-γ, il-1β, il-17, and mcp-1 while the treatment with e. it is not clear how immunobiotics initiates the cross-talk with the immune system in order to modulate the respiratory antiviral immunity. it is not known exactly which prrs are activated by immunobiotics in the intestinal or respiratory mucosa to functionally modulate antiviral immunity locally and in distal mucosal sites, respectively. neither it has been determined with exactitude which cellular functions are modulated by immunobiotics immediately after prr activation. research from the last decade has demonstrated that the immunomodulatory effects of probiotic bacteria are the consequence of complex interactions between several bacterial molecules and host receptors located in different immune and non-immune cells (46, 47). it has also been shown that the immunomodulatory properties of immunobiotics are dependent on the strains. therefore, studies carried out with certain strains cannot be easily extrapolated to other bacteria, even those of the same genus and species (48, 49). consequently, it is still necessary to carry out deeper studies to find out the molecular mechanisms by which immunobiotics beneficially influence the respiratory antiviral immunity. the studies mentioned before showed the potential of immunobiotics to be used for the reduction of the incidence and severity of ifv infections. however, in addition to deepening the knowledge of their mechanisms of action, several other points should be considered for the efficient application of immunobiotics in humans. for example, it is necessary to determine the best time as well as the most appropriate route for their administration. immunobiotics used as components of functional foods can be included in diets on a regular basis and thus help to improve respiratory defenses, especially in high-risk populations and during the seasons with the highest incidence of respiratory infections occurs. in this sense, in a randomized controlled trial we demonstrated that l. rhamnosus crl1505 (administered in a yogurt formulation) improved mucosal immunity and reduced the incidence and severity of intestinal and respiratory infection in children (50). hence, the incidence of infectious events was reduced from 66% in the placebo group to 34% in the group that received the probiotic yogurt. furthermore, there was also a significant reduction in the occurrence of indicators of disease severity such as fever and the need for antibiotic treatment in children receiving the probiotic yogurt. this immunobiotic yogurt (yogurito ® ) has been included into official national nutritional programs in argentina and is given daily to children at schools in several provinces thanks to the government actions. epidemiological studies in the schools receiving the immunobiotic product have shown a reduction in the incidence of infections and in the associated school absenteeism (alvarez et al., unpublished results). on the other hand, as mentioned earlier the nasal administration of immunobiotics is more efficient than the oral administration to enhance respiratory immunity. this route of administration poses a practical disadvantage considering that the treatments with immunobiotics showed favorable results when they were used before the infectious challenges. in this way, it would be necessary to predict the exact moment in which the viral pathogen will be in contact with the host in order to carry out the prophylactic immunobiotic treatment. this option could be used for example during a school or work outbreak in which cases of respiratory infections occur and it is desired to prevent or reduce the severity of infections in asymptomatic individuals. for an intervention of these characteristics, it would be also important to determine the exact time after the contact with the virus in which it is possible to administer immunobiotics to achieve the beneficial effect. in a recent study, percopo et al. (51) have defined this as "the window of opportunity. " the work evaluated the effect of the nasal administration of live or inactivated l. plantarum ncimb 8826 in a mice model of severe respiratory infection with the pneumonia virus of mice (pvm) and found that immunobiotic treatment promoted full survival from acute pvm infection when administered within 1 day after virus challenge (51) . similar studies would be of value in ifv infection models. another point of interest is related to the duration of the improvement of respiratory defenses after the last immunobiotic administration. our studies have showed that the immunomodulatory effect of some nasally administered immunobiotics persisted for at least 15 days (villena et al., unpublished results) . other studies have also reported short-term protection after nasal treatment with different immunobiotic strains (43). interestingly, garcia-crespo et al. (52) found that adult mice primed nasally with l. plantarum ncimb 8826 or lactobacillus reuteri f275 were completely protected against lethal pvm infection and that protection persisted for at least 5 months after the initial priming. these findings open an interesting challenge in the study of immunobiotics to improve the defenses against ifv, since it would be very useful to establish the duration of the protective effect for each strain and treatment, since in the majority of cases these long-term studies were not taken into account. ifv infections often result in mild to moderate lung infection; however, life-threatening disease can occur. it has been demonstrated that the most severe disease outcomes are associated with secondary bacterial pneumonia caused primarily by staphylococcus aureus or streptococcus pneumoniae (53) . taking into account the high incidence of viral infections and the frequency of associated secondary bacterial infections which contribute to aggravate the health status of the host and reduce its chance of recovery, various approaches for preventing and treating influenza and secondary bacterial pneumonia are been investigated. a wide range of antibiotics and anti-inflammatory drugs has been tested in mice [reviewed in ref. (54) ]. it would be of interest to evaluate the potential beneficial effect of immunobiotics on these circumstances. in this regard, preliminary studies from our laboratory showed that nasally administered l. rhamnosus crl1505 is able to improve survival, reduce bacterial cell counts in lung and blood, and limit lung inflammatory damage caused by s. pneumoniae infection in mice produced after the infection with ifv or respiratory syncytial virus (rsv) (villena et al., unpublished results) . these results opened an interesting topic for future investigations. finally, it would be also of interest to investigate whether immunobiotic treatments may influence other physiological systems involved in the defenses against viral respiratory infections such as the coagulation system. our group has made some progress in this regard, as mentioned below. coagulation is an extremely ordered process that involves the interaction of three key components: endothelial cells (ecs), platelets, and coagulation factors. tissue injury that activates ecs typically initiates coagulation that is characterized by the binding of platelets to activated ecs and the formation of the platelet plug. almost simultaneously, tissue factor (tf) released by ecs result in factor x activation, which induces thrombin and the generation of fibrin strands to strengthen the platelet plug leading to a stable platelet-fibrin clot. all these processes are tightly regulated by anticoagulant and fibrinolytic mechanisms to avoid thrombotic and/or haemorrhagic complications. a key role has been attributed to ecs in the temporal and special regulation of coagulation activation. resting ecs avoid the inappropriate plug formation by controlling platelet adhesion and activation and generating several anticoagulant factors providing a non-thrombogenic barrier (55, 56) . once activated or injured, ecs expose collagen to blood, increase platelet binding and aggregation, reduce the expression physiological anticoagulant factors, increase the expression of tf and von willebrand factor, and suppress the fibrinolytic activity (57, 58) . all these changes in the hemostatic system facilitate thrombosis in the infected or inflammated tissue. both hemorrhagic and thrombotic complications have been described during ifv infection. influenza is able to cause pulmonary hemorrhage and edema related to coagulopathy or induce uncontrolled thrombosis through an over-activated coagulation (figure 3 ) (55, 58) . animal models have helped to explain the mechanisms by which ifv infection activates coagulation and key role has been attributed to tf. it was described that ifv activates coagulation by enhancing tf production, thrombin generation and fibrin deposition in c57bl/6 mice (59) . in a mice model of ifv infection, it was recently shown that wild-type animals increased lung tf expression and activation of coagulation but presented alveolar hemorrhage (60) . moreover, selective deletion of tf in epithelial cells from lung significantly reduced tf expression after ifv infection and had higher alveolar hemorrhage and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december 2016 | volume 7 | article 633 reduced survival than controls. on the contrary, deficiency of tf in either respiratory myeloid cells or ecs did not enhanced alveolar hemorrhage or modified survival of ifv-infected mice (60) . these results indicate that an appropriate modulation in the production of tf in the lung during ifv infection is necessary to maintain tissue hemostasis avoiding hemorrhage and excessive fibrin deposition. production of tf by lung epithelial cells will be required to maintain alveolar hemostasis during ifv infection, while excessive release of tf by macrophages and ecs would contribute to pathology and lung tissue injury (59, 60) . it is considered that ecs may play an important role in the pathogenesis of ifv. influenza infection is able to induce alveolar edema and pulmonary hemorrhage through the alteration of ecs via several mechanisms, including direct damage and loss of tight junctions and apoptosis (61) . in addition, recognition of damageassociated molecular patterns such as hmgb1 or oxidized phospholipids through tlr4 activates ecs to drive lung injury (62) . direct stimulation of tlr3 by viral rna also results in the upregulation of tf and the downregulation of thrombomodulin (tm) in ecs (63) . at the same time, the inflammatory activation of ecs leads to the activation of the coagulation cascade. inflammation caused by ifv infection increases various proinflammatory cytokines such as tnf-α, il-1β, and il-6 that induce the secretion of tf by ecs and monocytes (58) . in addition to their roles in coagulation, activated proteins such as thrombin, fxa, and fviia also enhance the inflammatory response. the inflammatory potentiating abilities of coagulation factors are mediated through their activation of protease-activated receptors (pars) that are expressed in platelets, ecs, macrophages, and respiratory epithelial cells (58) . the tf/thrombin/par-1 pathway has been associated to the promotion of a deleterious innate inflammatory response to ifv infection in mice (64, 65) . therefore, both the hyper-inflammatory response and the aberrant activation of coagulation, which are potentiated with each other, are involved in severe influenza pneumonia and are key events that have to be controlled in order to reach a favorable resolution of the infectious process. considering the importance of the coagulative response in the outcome of influenza infection and the ability of immunobiotics to beneficially influence the immune response to this respiratory pathogen, we wonder whether some immunobiotic strains would be able to beneficially modulate the immuno-coagulative response triggered by ifv. for this purpose, we performed challenge-infection experiments in mice and evaluated the influence of viable and non-viable immunobiotic l. rhamnosus crl1505 strain on the respiratory immuno-coagulative response induced by ifv (41, 42) . our data demonstrated that oral administration of l. rham nosus crl1505 to mice significantly reduced lung viral titers and tissue damage after the challenge with ifv (41). we later explored the capacity of nasally administered l. rhamnosus crl1505, alive or heat killed, to reduce the influenza burden of disease (42). those treatments induced a significant decrease in ifv titers in lungs, lessened pulmonary damage, and increased survival. interestingly, a similar effect was achieved with the nasal administration of viable and non-viable crl1505 strain. moreover, the nasal route was more efficient than the oral administration to protect mice against ifv infection (41, 42). the protective effect achieved by the immunobiotic strain was related to its ability to modulate the respiratory antiviral immune response, particularly to its capacity to improve the levels of ifn-γ and ifn-β in the respiratory tract (figure 2) . type i ifns trigger the activation of the jak-stat pathway and increase the expression of antiviral genes. in addition, ifn-γ is produced by immune cells, especially th1 cells, and it further improves antiviral immune response by inducing activation of nk cells and macrophages. therefore, the modulation of type i ifns and ifn-γ would be responsible of the reduction of viral loads in ifv-infected mice previously treated with the crl1505 strain, similarly to other immunobiotic strains as mentioned before ( table 1) . we demonstrated that the crl1505 strain increased the levels of gut cd3 + cd4 + ifn-γ + t cells, induce a mobilization of these lymphocytes into the lung and enhanced the respiratory production of ifn-γ and the activity of local antigen presenting cells (41, 66, 67) . it was also noted that nasal administration was more effective than the oral route to increase pulmonary cd3 + cd4 + ifn-γ + t cells (41, 42). the mechanism by which nasally administered viable or heat-killed l. rhamnosus crl1505 improves ifn-γ + t cells population is not clear. however, our studies support the possibility that the immunobiotic strain l. rhamnosus crl1505 impact in the nasalassociated lymphoid tissue or bronchial-associated lymphoid tissue producing an innate imprinting in antigen presenting cells that contribute to the enhanced number and activity of cd3 + cd4 + ifn-γ + t cells. our studies also showed that immunobiotic treatments were able to beneficially modulate the activation of coagulation during respiratory viral infection, an effect that was not reported before (41, 42). then, our studies were the first in demonstrating a beneficial modulation of the immune-coagulative response during respiratory trl3 activation and ifv infection induced by immunobiotic microorganisms (figure 3) . although ifv is an ssrna virus, it generates dsrna replication intermediates that activate tlr3 and contribute to the initiation of the antiviral respiratory immune response. in fact, ifv triggers type i ifn secretion through tlr3 recognition in immune (myeloid dcs or macrophages) and non-immune (fibroblasts or pneumocytes) cells (68) . challenge-infection experiments with respiratory viruses in tlr3 −/− mice showed that tlr3 does not modify the clearance of viral pathogens but it is relevant for the modulation of the lung inflammatory response (69, 70) . it was showed that wild-type mice mount a robust inflammatory response in the lung after ifv infection and that this process is significantly diminished in tlr3 −/− animals (70) . tlr3 −/− mice showed a longer survival when compared wild-type animals and this effect was associated with a reduction of inflammatory cells recruitment and lower levels of inflammatory factors in the respiratory tract. other in vivo studies also demonstrated that tlr3 activation by poly(i:c) enhanced proinflammatory cytokines and immunobiotics for influenza virus infection frontiers in immunology | www.frontiersin.org december 2016 | volume 7 | article 633 antiviral factors expression (71), altered vascular permeability (72) , and incremented the levels of d-dimers indicating that coagulation and fibrinolysis were triggered. in line with these findings, it was observed that the levels of d-dimers in tlr3 −/− mice were significantly lower than in wild-type animals after poly(i:c) administration (63 (neutrophils and macrophages) and proinflammatory mediators (il-1β, tnf-α, il-8, and il-6) in the respiratory tract. moreover, tlr3 activation also induced an increase in tf expression and thrombin-antithrombin complex (tatc) levels in the lung while it reduced tm expression. these inflammatory-coagulative modifications were accompanied by respiratory tissue alterations and impairment of lung function (41, 42) . of interest, we demonstrated that orally (41) or nasally (42) administered immunobiotics before the challenge with poly(i:c) differentially modulated the inflammatory-coagulative response. l. rhamnosus crl1505 was able to reduce and increase the expression of tf and tm, respectively, after the respiratory activation of tlr3. thus, the crl1505 strain significantly diminished coagulation activation in blood and in the respiratory tract after the nasal stimulation with poly(i:c). we also evaluated pulmonary coagulation during ifv infection (41, 42). the respiratory virus induced activation of coagulation in the lungs of infected mice as demonstrated by the increased levels of respiratory tatc. these procoagulant changes were related to alterations in the expression of tm and tf in lungs. our findings are in line with previous studies in humans and animal models of influenza infection demonstrating increased lung fibrin deposition and enhanced numbers of intravascular thrombi in the respiratory tract (59, 73, 74) . we demonstrated that immunobiotic treatment is able to significantly diminish the activation of coagulation in ifv-challenged mice. in fact, lower levels of respiratory tatc and a reduced expression of tf was observed in l. rhamnosus crl1505-treated mice infected with ifv when compared to controls (41, 42). as mentioned before, ifv promote a procoagulant state directly through its capacity to infect ecs and monocytes stimulating the expression of tf (75, 76) . in addition, ifv induce activation of coagulation indirectly by the enhancement of proinflammatory factors such as il-6 (75, 76) . therefore, the ability of immunobiotics to modulate the ifv-triggered immune-coagulative response could be explained by their direct influence on viral replication related to the enhancement of the antiviral state in the respiratory mucosa, and indirectly through the modulation of the inflammatory response. considering this last point, we performed experiments using anti-il-10r blocking antibodies in order to evaluate the role of the regulation of the inflammatory response in the reduction of coagulation activation. results showed that il-10 is important for the regulation of coagulation induced by the immunobiotic l. rhamnosus crl1505 (41). blocking of il-10r abolished the capacity of the crl1505 strain to change the expression of tm and tf in the lungs. this was in line with our previous studies evaluating the ability of l. rhamnosus crl1505 to confer protection against inflammatory damage induced by tlr3 activation or rsv infection, which showed that il-10 is a key factor for the reduction of lung injury (67) . additionally, it was demonstrated that lethal disease caused by ifv infection is prevented by il-10 administration through the reduction of lung immunopathology (77) . moreover, tf expression and procoagulant activity of macrophages and ecs are reduced by il-10 (78, 79) . therefore, we demonstrated that immunobiotic administration induce an early increase in the levels of tnf and il-6 in the respiratory tract after poly(i:c), rsv, or ifv challenge, while the levels of those proinflammatory factors are significantly reduced later during infection (41, 42, 67) . the early increase of proinflammatory mediators and the augmented levels of ifn-γ explain the ability of l. rhamnosus crl1505 to diminish viral replication while the improved production of il-10 would lead to a beneficial modulation of the immune-coagulative response which results in a reduced severity of lung damage. it has been suggested that respiratory viral infections increase the risk of venous thromboembolism and ischemic heart disease through ecs perturbation, coagulation activation, reduction of anticoagulant factors, and inhibition of fibrinolysis (80) (81) (82) . then, our studies suggest that immunobiotics could be an interesting alternative not only to reduce the incidence and/or severity of respiratory viral infections, but in addition to reduce the risk of atherothrombotic alterations associated to respiratory viral infections. research from the last decade has clearly demonstrated that beneficial microorganisms are able to modulate respiratory tract immunity and promote the resolution and lessen the severity of respiratory infections caused by pathogens such as ifv. studies in animal models have demonstrated that orally or nasally administered immunobiotics are able to improve protection against ifv by three main mechanisms. first, immunobiotics increase the respiratory antiviral state by their capacity to improve levels of type i ifns, the number and activity of antigen presenting cells, nk cells, cd4 + ifn-γ + t, and iga + b lymphocytes, as well as the levels of systemic and mucosal specific antibodies. second, immunobiotics beneficially modulate the ifv-triggered respiratory inflammatory response by inducing changes in the levels and kinetics of proinflammatory factors and immunoregulatory cytokines such as il-10 that allow the clearance of virus with a minimal inflammatory lung tissue damage. finally, as demonstrated by our recent research works, immunobiotics modulate lung immune-coagulative response triggered by tlr3 activation or ifv infection, mainly by downregulating lung tf and restoring tm levels. studies in animal models suggest that immunobiotics would influence principally the innate immune response, modulating in that way the early antiviral inflammatory response and the subsequent cellular and humoral immune responses. therefore, immunobiotics would have mainly an adjuvant effect. however, the exact molecular mechanisms by which immunobiotics differentially modulate the innate antiviral immune response against ifv remain to be elucidated. additionally, a growing number of studies in humans have examined the effect of immunobiotics on the incidence and severity of ifv infection. considering the impact of immunobiotics in the innate immune response clinical studies have evaluated principally their potential adjuvant effects on ifv vaccination ( table 2) . although mechanistic studies have not been addressed in depth, there is promising evidence for beneficial effects of immunobiotics on human respiratory health and resistance against ifv. these observations might be helpful to propose new influenza a penetrates host mucus by cleaving sialic acids with neuraminidase the amazing innate immune response to influenza a virus infection critical role of constitutive type i interferon response in bronchial epithelial cell to influenza infection ifitm3 restricts the morbidity and mortality associated with influenza molecular signatures associated with mx1-mediated resistance to highly pathogenic influenza virus infection: mechanisms of survival regulation of ifn-alpha/beta, mxa, 2ʹ,5ʹ-oligoadenylate synthetase, and hla gene expression in influenza a-infected human lung epithelial cells induction of innate immunity and its perturbation by influenza viruses the intracellular sensor nlrp3 mediates key innate and healing responses to influenza a virus via the regulation of caspase-1 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna inflammasome recognition of influenza virus is essential for adaptive immune responses nkp46 o-glycan sequences that are involved in the interaction with hemagglutinin type 1 of influenza virus recognition of viral hemagglutinins by nkp44 but not by nkp30 immune responses to influenza virus infection exploiting mucosal immunity for antiviral vaccines secretory iga possesses intrinsic modulatory properties stimulating mucosal and systemic immune responses b cell responses to influenza infection and vaccination regulating the adaptive immune response to respiratory virus infection innate sensing of the gut microbiota: modulation of inflammatory and autoimmune diseases the commensal microbiota drives immune homeostasis interactions between the microbiota and pathogenic bacteria in the gut resurrecting the intestinal microbiota to combat antibiotic-resistant pathogens antibiotics and the intestinal microbiome: individual responses, resilience of the ecosystem, and the susceptibility to infections microbiota regulates immune defense against respiratory tract influenza a virus infection commensal bacteria calibrate the activation threshold of innate antiviral immunity recognition of peptidoglycan from the microbiota by nod1 enhances systemic innate immunity protection against influenza virus infection of mice fed bifidobacterium breve yit4064 oral administration of heat-killed lactobacillus pentosus strain b240 augments protection against influenza virus infection in mice oral administration of lactobacillus brevis kb290 to mice alleviates clinical symptoms following influenza virus infection oral administration of lactobacillus plantarum strain aya enhances iga secretion and provides survival protection against influenza virus infection in mice augmentation of cellular immunity and reduction of influenza virus titer in aged mice fed lactobacillus casei strain shirota reduction of influenza virus titer and protection against influenza virus infection in infant mice fed lactobacillus casei shirota oral administration of lactobacilli from human intestinal tract protects mice against influenza virus infection efficacy of oral administration of heat-killed probiotics from mongolian dairy products against influenza infection in mice: alleviation of influenza infection by its immunomodulatory activity through intestinal immunity immunobiotic lactobacillus administered post-exposure averts the lethal sequelae of respiratory virus infection lactobacillus priming of the respiratory tract: heterologous immunity and protection against lethal pneumovirus infection influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease the immunology of influenza virus-associated bacterial pneumonia influenza virus and endothelial cells: a species specific relationship endothelium -role in regulation of coagulation and inflammation endothelial activation and dysfunction in the pathogenesis of influenza a virus infection aberrant coagulation causes a hyper-inflammatory response in severe influenza pneumonia effects on coagulation and fibrinolysis induced by influenza in mice with a reduced capacity to generate activated protein c and a deficiency in plasminogen activator inhibitor type 1 tissue factor deficiency increases alveolar hemorrhage and death in influenza a virus-infected mice endothelial cells are central orchestrators of cytokine amplification during influenza virus infection damp molecule s100a9 acts as a molecular pattern to enhance inflammation during influenza a virus infection: role of ddx21-trif-tlr4-myd88 pathway a key role for toll-like receptor-3 in disrupting the hemostasis balance on endothelial cells par1 contributes to influenza a virus pathogenicity in mice par-1 contributes to the innate immune response during viral infection orally administered lactobacillus rhamnosus modulates the respiratory immune response triggered by the viral pathogen-associated molecular pattern poly(i:c) immunobiotic lactobacillus rhamnosus improves resistance of infant mice against respiratory syncytial virus infection toll-like receptor and rig-i-like receptor signaling deletion of tlr3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection frontiers in immunology | www.frontiersin.org december detrimental contribution of the toll-like receptor (tlr)3 to influenza a virus-induced acute pneumonia essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus toll-like receptor 3 mediates west nile virus entry into the brain causing lethal encephalitis pulmonary pathology of severe acute respiratory syndrome in toronto highly pathogenic h5n1 influenza virus causes coagulopathy in chickens procoagulant activity of endothelial cells after infection with respiratory viruses procoagulant and inflammatory response of virus-infected monocytes autocrine regulation of pulmonary inflammation by effector t-cell derived il-10 during infection with respiratory syncytial virus monocyte il-10 produced in response to lipopolysaccharide modulates thrombin generation by inhibiting tissue factor expression and release of active tissue factor-bound microparticles anticoagulant properties of the anti-inflammatory cytokine il-10 in a factor xa-activated human monocyte model risk of myocardial infarction and stroke after acute infection or vaccination risk of deep vein thrombosis and pulmonary embolism after acute infection in a community setting crosstalk between inflammation and thrombosis oral intake of lactobacillus fermentum cect5716 enhances the effects of influenza vaccination a probiotic fermented dairy drink improves antibody response to influenza vaccination in the elderly in two randomised controlled trials lactobacillus gg as an immune adjuvant for live-attenuated influenza vaccine in healthy adults: a randomized double-blind placebo-controlled trial daily intake of heat-killed lactobacillus plantarum l-137 enhances type i interferon production in healthy humans and pigs evaluation of the immune benefits of two probiotic strains bifidobacterium animalis ssp. lactis, bb-12(r) and lactobacillus paracasei ssp. paracasei, l. casei 431(r) in an influenza vaccination model: a randomised, double-blind, placebo-controlled study nonpathogenic lactobacillus rhamnosus activates the inflammasome and antiviral responses in human macrophages immunoprotective effects of oral intake of heat-killed lactobacillus pentosus strain b240 in elderly adults: a randomised, double-blind, placebo-controlled trial lactobacillus in jelly enhances the effect of influenza vaccination in elderly individuals the use of the probiotic lactobacillus rhamnosus gg and viral findings in the nasopharynx of children attending day care effects of probiotic lactobacillus brevis kb290 on incidence of influenza infection among schoolchildren: an open-label pilot study prebiotic and probiotic supplementation prevents rhinovirus infections in preterm infants: a randomized, placebo-controlled trial effects of oral intake of plasmacytoid dendritic cells-stimulative lactic acid bacterial strain on pathogenesis of influenza-like illness and immunological response to influenza virus key: cord-282336-zvc04s39 authors: choudhary, ishita; vo, thao; bathula, chandra s.; lamichhane, richa; lewis, brandon w.; looper, jayme; jeyaseelan, samithamby; blackshear, perry j.; saini, yogesh; patial, sonika title: tristetraprolin overexpression in non-hematopoietic cells protects against acute lung injury in mice date: 2020-09-02 journal: front immunol doi: 10.3389/fimmu.2020.02164 sha: doc_id: 282336 cord_uid: zvc04s39 tristetraprolin (ttp) is a mrna binding protein that binds to adenylate-uridylate-rich elements within the 3′ untranslated regions of certain transcripts, such as tumor necrosis factor (tnf) mrna, and increases their rate of decay. modulation of ttp expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. accordingly, we tested the role of ttp in lipopolysaccharide (lps)-induced acute lung injury (ali) in mice. lps-challenged ttp-knockout (ttp(ko)) mice, as well as myeloid cell-specific ttp-deficient (ttp(myeko)) mice, exhibited significant increases in lung injury, although these responses were more robust in the ttp(ko). mice with systemic overexpression of ttp (ttp(δare)) were protected from ali, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ali. interestingly, while irradiated wild-type (wt) mice reconstituted with ttp(ko) hematopoietic progenitor cells (hpcs) showed exaggerated ali, their reconstitution with the ttp(δare) hpcs mitigated ali. the reconstitution of irradiated ttp(δare) mice with hpcs from either wt or ttp(δare) donors conferred significant protection against ali. in contrast, irradiated ttp(δare) mice reconstituted with ttp(ko) hpcs had exaggerated ali, but the response was milder as compared to wt recipients that received ttp(ko) hpcs. finally, the reconstitution of irradiated ttp(ko) recipient mice with ttp(δare) hpcs did not confer any protection to the ttp(ko) mice. these data together suggest that non-hpcs-specific overexpression of ttp within the lungs protects against ali via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration. acute lung injury (ali) and its severe form, acute respiratory distress syndrome (ards), are serious health concerns due to a high rate of mortality (1) . ali is characterized by elevated levels of proinflammatory mediators, exaggerated neutrophil recruitment, and compromised pulmonary epithelial-endothelial barrier, resulting in increased vascular permeability (2) . non-cardiogenic pulmonary edema, characterized by excessive accumulation of protein-rich edematous fluid and inflammatory cells in the alveolar spaces, results in hypoxemia in ards that requires aggressive clinical management including mechanical ventilation (1) . despite significant health burdens posed by these diseases, the identity of key cellular and molecular players of host defense against ali remains unclear. zinc finger protein 36 (zfp36), commonly known as tristetraprolin (ttp), is an mrna binding protein that binds to adenylate-uridylate-rich elements (ares) within the 3 untranslated regions (3 utrs) of target mrnas and increases their rate of decay (3) . germline ttp-knockout (ttp ko ) mice exhibit the spontaneous development of a systemic inflammatory syndrome characterized by cachexia, erosive arthritis, myeloid hyperplasia, dermatitis, conjunctivitis, and autoimmunity (4) . these phenotypes were shown to be essentially completely prevented in ttp ko mice with either tnf receptor deficiency, or when ttp ko mice were treated with anti-tnf antibodies (5) . biochemical studies demonstrated that ttp binds to ares within the tumor necrosis factor (tnf ) mrna 3 utr and results in tnf mrna degradation under normal conditions (6, 7) . subsequent reports have shown that a number of other proinflammatory mediators including cxcl1 (8, 9) , cxcl2 (8), il-10 (10), il-17 (11) , ccl3 (12) , and il-23 (13) are also regulated by ttp (14) . recently, using a systemic ttp overexpression (ttp are ) mouse model, we demonstrated protective effects of enhanced ttp levels in chronic immune-mediated inflammatory diseases including mouse models of arthritis, psoriasis, and autoimmune encephalomyelitis (15) . ttp are mice lack ares in the 3 utr of the endogenous ttp gene (zfp36) that results in increased stability of ttp mrna and, in turn, moderately increased expression of ttp protein in essentially all the tissues (15) . together, these studies have indicated that ttp may be an endogenous anti-inflammatory protein and that enhancing its levels may be beneficial against various chronic inflammatory diseases. in the present study, we investigated the role of ttp in regulating lung inflammation in a mouse model of ali. using an oropharyngeal aspiration approach, lipopolysaccharide (lps)induced ali was modeled in adult mice, and the animals were monitored for signs of ali. to identify the protective role of ttp in a cell-specific manner, we performed bone marrow irradiation and reconstitution experiments in wild-type (wt), ttp ko (4) , and systemic ttp overexpression (ttp are ) mice (15) . our findings elucidate cell-specific roles of ttp in protection against ali, and indicate that ttp is an important modulator of endotoxin-induced ali. zfp36 floxed mice (zfp36 flox/flox ) (16) were crossed with lysmcre recombinase expressing mice (17) to generate mice for experimental (cre +/+ /zfp36 flox/flox ; ttp myeko ) and control (cre −/− /zfp36 flox/flox ; cre +/+ /zfp36 wt/wt ) groups. genotype status of progeny was determined by polymerase chain reaction (pcr) as described previously (16) . ttp knockout mice (ttp ko ) and ttp overexpression mice (ttp are ) have been described before (4, 15) . all the animal experiments were performed in accordance with principles and procedures outlined in the national institute of health guide for the care and use of laboratory animals and were approved by the louisiana state university animal care and use committee. both male and female adult (8-10-week-old) mice were used for experiments. mice were anesthetized with isoflurane/oxygen followed by administration of 10 µg lipopolysaccharide (lps) from escherichia coli o111:b4 (l4391, sigma-aldrich) per mouse dissolved in sterile endotoxin-free saline (50 µl total volume), or an equivalent volume of sterile endotoxin-free saline as a vehicle control, via oropharyngeal aspiration (18) . mice were observed for signs of distress including anorexia, weight loss, hunched posture, ruffled haircoat, labored breathing, and dehydration every 8-12 h post lps challenge. mice exhibiting at least four of these clinical signs were humanely euthanized before the end of the study. following saline or lps treatment, mice were anesthetized with 2,2,2-tribromoethanol (sigma-aldrich, st. louis, mo, united states) at the indicated time points, and mid-line laparotomy was performed. briefly, bronchoalveolar lavage fluid (balf) was harvested from the right lung. recovered balf was processed and analyzed for total and differential cell counts by routine methods (19) . unlavaged left lung lobes were fixed in 10% neutral buffered formalin (nbf) and used for preparation of slides for histopathological evaluation. right lung lobes were snap-frozen and stored at −80 • c. bronchoalveolar lavage fluid was harvested and centrifuged at 500 × g for 5 min, and the supernatant was stored at −80 • c for further analyses. the cell pellet was resuspended in 500 µl of pbs and total cell counts were determined using a hemocytometer (brightline, horsham, pa, united states). cytospins were prepared using 200 µl of cell suspension (statspin cytofuge 2; hemocue, brea, ca, united states) followed by differential staining (modified giemsa kit; newcomer supply, middleton, wi, united states). mouse cytokine and chemokine levels were assayed in cell-free balf supernatant using luminex-xmap-based assay (mcytomag-70k), according to the manufacturer's instructions (emd millipore, billerica, ma, united states). five micrometer sections of lung were stained with hematoxylin and eosin (h&e) for routine histology. histology: a semiquantitative histopathological scoring system was used to analyze the sections as follows: (1) consolidation (percent of total surface area of lung section affected); (2) bronchiolitis (0, no bronchioles affected; 1, one bronchiole affected; 2, between 2-4 bronchioles affected; 3, more than 4 bronchioles affected); (3) perivascular edema (1, minimal; 2, mild; 3, moderate; 4, severe); (4) perivascular inflammation/inflammatory cells (0, absent; 1, minimal; 2, mild; 3, moderate; 4, severe); (5) airspace edema (1, minimal; 2, mild; 3, moderate; 4, severe); (6) airspace hemorrhages (0, absent, 1, patchy, mild; 2, extensive, moderate; 3, extensive, severe). slides were graded in a blinded manner without knowledge of sex and treatment groups. bone marrow transplantation experiments were performed as described previously (20) . briefly, 8-10-week old recipient mice were irradiated with 6 megavolt x-rays from a linear accelerator (varian clinac 21ex) with two (dorsal and ventral) 525-rad (525 cgy) doses. to prepare bone marrow cells for transplantation, femur bones of donor mice were flushed to collect bone marrows, and single cell suspensions were prepared. a total of 8 × 10 6 cells were injected into the tail vein of lethally irradiated recipient mice. reconstituted recipient mice were given 0.2% neomycin sulfate dissolved in acidified water for the first 2 weeks post-transplantation. lps-challenge experiments were performed 8 weeks post bone marrow reconstitution, which has been previously shown to be an optimal period for repopulation of resident alveolar macrophages with donor cells following total body irradiation (21) . lung tissue was lysed using pierce tm ripa buffer (thermo fisher scientific, waltham, ma, united states) supplemented with pierce tm protease inhibitor mixture (thermo fisher scientific, waltham, ma, united states) and phosphatase inhibitors (10 mm sodium fluoride and 1 mm sodium orthovanadate). tissues were mechanically homogenized using a bead beater (thermo fisher scientific, waltham, ma, united states). tissue lysates were centrifuged (13,000 × g, 10 min, 4 • c) to remove insoluble material and protein concentration of the supernatants was measured through bradford assay (bio-rad laboratories, hercules, ca, united states). equivalent amounts of denatured protein was separated on a 4-12% bis-tris plus precast gels (invitrogen, carlsbad, ca, united states), transferred on to pvdf membrane (invitrogen, carlsbad, ca, united states) and probed with a 1:5000 dilution of rabbit antiserum raised against a recombinant mouse ttp-maltose binding protein fusion (15) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit igg (bio-rad). signal was determined using supersignal west pico chemiluminescent substrate (pierce) on x-ray film. significant differences among groups were determined by oneway analysis of variance (anova) followed by tukey's post hoc test for multiple comparisons except for cytokine assays where two-way anova was used. measurements from two groups were compared using student's t-test assuming unequal variance. all data were expressed as mean ± sem. a p-value < 0.05 was considered statistically significant. statistical analyses were performed using graphpad prism 7.0 (graphpad software, la jolla, ca, united states). in order to explore the role of ttp in ali, ttp ko and littermate control wt mice were subjected to ali through oropharyngeal aspiration of endotoxin (lps) (single dose; 10 µg lps/mouse) for a period of 72 h. while saline-treated ttp ko and wt groups had comparable numbers of total immune cells in the balf (saline-treated wt; 58 × 10 3 ± 15 × 10 3 , saline-treated ttp ko , 74 × 10 3 ± 19 × 10 3 ), lps administration resulted in increased infiltration of immune cells in both the ttp ko and the wt groups. the total number of recovered immune cells in lps-challenged ttp ko mice (4867 × 10 3 ± 1167 × 10 3 ) were ∼ fourfold higher as compared to lps-challenged wt (1184 × 10 3 ± 467 × 10 3 ) mice ( figure 1a) . increases in total cell counts in lps-challenged ttp ko mice were attributed to a significant increase in neutrophil ( figure 1b) , macrophage ( figure 1c) , and lymphocyte counts ( figure 1d ). these increases were associated with an increased injury to the pulmonary vascular barrier, as depicted by the presence of red blood cells in the cytospins prepared from the balf fluid of ttp ko mice ( figure 1e ; right panel, black arrow) versus control lps-challenged wt mice ( figure 1e ; left panel). histologically, the lungs of lps-challenged wt mice were characterized by mild to moderate consolidation (∼ 26% of total area of lung section), two-to fourfold increase in alveolar septal thickening (broken green arrow), moderate perivascular and airspace edema, and perivascular inflammation (figures 1f-h) . in contrast, the lung injury in lps-challenged ttp ko mice was characterized by severe consolidation (>90% of total area of lung section) (figures 1f,g) that included infiltration of neutrophils, edema, fibrin, and airspace hemorrhage within the airway and alveolar lumen, multifocal loss of bronchiolar epithelium with infiltration of neutrophils and red blood cells within the bronchiolar lumen, and moderate to severe perivascular edema and inflammation (figures 1f-h) . of note, ∼50% lps-challenged ttp ko mice succumbed to lps challenge before 72-h and these had to be excluded from the analysis. these data suggest that systemic loss of ttp results in extreme susceptibility of mice to lpsinduced ali. in order to explore the role of myeloid cell-specific ttp on inflammatory response in ali, myeloid cell-specific ttp deficient mice (ttp myeko ; cre +/+ /zfp36 flox/flox ) and control (cre control; cre +/+ /zfp36 wt/wt and flox control; cre −/− /zfp36 flox/flox ) mice were challenged with lps. similar to saline-treated wt and ttp ko groups, saline-treated control and ttp myeko groups had comparable numbers of total . data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparison test. *p < 0.05; **p < 0.01; ***p < 0.001. n = 3 each for wt and ttp ko saline controls; n = 6, wt group; n = 3, ttp ko group for lps-challenge group. three lps-challenged ttp ko mice succumbed to lps-challenge before 72 h and were not lavaged for further analyses. representative photomicrographs of wright-giemsa stained balf cytospins of lps-challenged wt (e; left panel) and lps-challenged ttp ko (e; right panel) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow), red blood cell (black arrow) (original magnification ×400). representative photomicrographs (f) from h&e-stained left lung lobe sections from adult lps-challenged wt (f; left panel) and lps-challenged ttp ko (f; right panel) mice. septal thickening (green broken arrow), intra-alveolar neutrophilic infiltrates (green arrow), intra-alveolar red blood cells (red arrow), bronchiolar lumen neutrophilic accumulation (blue arrow), and perivascular cellular infiltration (black arrow). asterisk represents alveolar space that is minimally affected (f; left) or severely consolidated with blood and neutrophils (f; right) (original magnification ×200). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged wt and lps-challenged ttp ko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, airspace hemorrhage, and airspace edema in lps-challenged wt and lps-challenged ttp ko mice (h). statistical analysis in g and h was performed using student's t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. immune cells (flox control; 46 × 10 3 ± 1 × 10 3 , ttp myeko ; 60 × 10 3 ± 5 × 10 3 ). lps administration resulted in increased numbers of immune cells in ttp myeko as well as both the control groups. this increase in total cell counts was, however, significantly greater in ttp myeko (1636 × 10 3 ± 136 × 10 3 ) compared to cre control (925 × 10 3 ± 109 × 10 3 ) mice. the increase in cellular recovery did not reach statistical significance in lps-challenged ttp myeko when compared to the lps-challenged flox control group (p = 0.07) (figure 2a ). this effect was comparable in both sexes (data not shown). of note, the increase in cellular infiltration was ∼ threefold less in lps-challenged ttp myeko (1636 × 10 3 ± 136 × 10 3 ) (figure 2a ) when compared to lps-challenged ttp ko mice (4867 × 10 3 ± 1167 × 10 3 ) ( figure 1a) . while neutrophil counts were comparable between lps-challenged ttp myeko and lpschallenged control mice (figures 2b,e) , macrophage counts were significantly elevated in the balf obtained from lps-challenged ttp myeko mice compared to the two groups of control mice (figures 2c,e) . lymphocyte counts did not differ between the lps-challenged ttp myeko and the two groups of control mice (figures 2d,e) . histopathological analysis revealed comparable levels of lung consolidation with widespread inflammatory cellular infiltrates within the airspaces of both lps-challenged ttp myeko and flox control mice; however, perivascular edema, perivascular inflammation, and airspace edema were somewhat exaggerated in lps-challenged ttp myeko mice compared to the flox control group (figures 2f-h) . unlike lps-challenged ttp ko mice, airspace hemorrhage was not observed in any of the groups. all the lps-challenged ttp myeko mice survived lps challenge, as compared to the lps-challenged ttp ko mice, in which ∼50% mortality was observed. these data indicate that myeloid cell-specific ttp is essential for protection against ali. mitigates lps-induced ali in mice during acute and sub-acute course of lung injury next, we examined whether the systemically ttp overexpressing (ttp are ) mice exhibit protection against ali. in experimental ali, while time points earlier than 72 h of lps-challenge represent acute phases of ali, later time points represent somewhat sub-acute to chronic or resolution phases of endotoxin-induced ali in mice (22) . therefore, we examined both lps-challenged wt and lps-challenged ttp are mice over time points representing acute to subacute phases, i.e., 12 ± 5.0 × 10 3 , 31.6 × 10 3 ± 7.9 × 10 3 , and 50.8 × 10 3 ± 3.6 × 10 3 cells at 12 h, 24 h, 72 h, 5 days, and 7 days, respectively) ( figure 3a) . lipopolysaccharide administration resulted in increased numbers of total cells in balf from both wt and ttp are mice when compared to saline administration ( figure 3a) . total cell counts in balf from lps-challenged wt mice were 748.9 × 10 3 ± 72.7 × 10 3 , 735.6 × 10 3 ± 28.0 × 10 3 , 1258 × 10 3 ± 121 × 10 3 , 270 × 10 3 ± 25.0 × 10 3 , and 115.4 × 10 3 ± 6.9 × 10 3 at 12 h, 24 h, 72 h, 5 days, and 7 days time points, respectively ( figure 3a) . interestingly, significantly reduced numbers of immune cells were recovered from the lungs of lps-challenged ttp are mice compared to lungs of lps-challenged wt mice at all time points examined post lpschallenge (264.6 × 10 3 ± 24.0 × 10 3 , 358.4 × 10 3 ± 62.1 × 10 3 , 633.1 × 10 3 ± 71.6 × 10 3 , 153.3 × 10 3 ± 9.4 × 10 3 , and 83.2 × 10 3 ± 4.3 × 10 3 at 12 h, 24 h, 72 h, 5 days, and 7 days, respectively) ( figure 3a) . the decrease in total cell counts in lps-challenged ttp are mice was contributed by significantly reduced numbers of neutrophils at 12, 24, and 72 h (figures 3b,e) . interestingly, however, the total numbers of macrophages were only significantly different between lps-challenged wt and lps-challenged ttp are mice at 12 h and 5 days time points (figures 3c,e) . lymphocyte counts were not significantly different in lps-challenged ttp are mice compared to lps-challenged wt mice (figures 3d,e) . cellular counts followed the same trend in both the sexes (data not shown). reduced cellular infiltration was also evident in cytological slides prepared from lps-challenged wt and lps-challenged figure 2 | myeloid-ttp deficiency exacerbates lps-induced ali in mice. total cell counts (a) in the harvested balf of adult saline-(white bars; n = 2 each for flox control and ttp myeko ) or lps-challenged cre control (cre +/+ /zfp36 wt/wt , gray bar; n = 3), flox control (cre -/-/zfp36 flox/flox , purple bar; n = 6), and ttp myeko (cre +/+ /zfp36 flox/flox , green bar; n = 6) mice. differential cell counts are shown for neutrophils (b), macrophages (c), and lymphocytes (d). data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparisons. ns, non-significant; *p < 0.05; ***p < 0.001; ****p < 0.0001. representative photomicrographs of wright-giemsa stained balf cytospins from lps-challenged flox control (e; left) and ttp myeko (e; right) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow) (original magnification ×400). representative photomicrographs from h&e-stained left lung lobe sections from post-72 h lps-challenged flox control (f; left) and ttp myeko (f; right) mice (original magnification ×200). asterisk represents alveolar spaces minimally obliterated in flox control (f; left) and severely obliterated in lps-challenged ttp myeko mice (f; right). bronchiolar lumen neutrophilic infiltrates (blue arrow), absent in lps-challenged flox control (f; left) but present in lps-challenged ttp myeko (f; right). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged flox control and lps-challenged ttp myeko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, and airspace edema in lps-challenged flox control and lps-challenged ttp myeko mice (h). n = 5 (flox control); n = 6 (ttp myeko ). statistical analysis in g and h was performed using student's t-test. *p < 0.05. ttp are bal fluid, which showed the sparsely present neutrophils, macrophages, and lymphocytes in lps-challenged ttp are mice compared to dense presence of these cells in lps-challenged wt mice ( figure 3e) . histologically, lpschallenged wt lungs exhibited ∼ 33% lung consolidation with significantly increased infiltration of immune cells within the airspaces, bronchiolitis, perivascular edema, and inflammation (figures 3f-h) . in contrast, bronchiolitis and perivascular edema were significantly attenuated in lps-challenged ttp are mice. lung consolidation, perivascular inflammation, and airspace edema were not significantly different between the two groups, and airspace hemorrhage was not observed in any group (figures 3f-h) . we next analyzed the changes in the protein expression levels of ttp over the course of ali in wt and ttp are mice whole lung tissue. as shown in figure 3i , basal expression levels of ttp were higher in ttp are versus wt lungs. upon lps administration, the expression levels of ttp increased in both wt and ttp are mice lungs by 12 h post lps administration. this increase was significant in ttp are mice lungs as compared to unchallenged wt mice lungs. the levels of ttp started reducing at subsequent time points in both wt and ttp are lungs. while the ttp expression decreased to basal levels in wt mice, the ttp expression remained at relatively higher levels in the ttp are mice lungs at all subsequent time points. these data show that the presence of higher levels of ttp under basal conditions, combined with a significant increase at 12 h post-lps challenge and maintenance of higher expression levels at later time points post lps challenge, protects ttp are mice from ali. cellular recruitment within the lung in response to lps challenge is mediated by the production of chemoattractants. therefore, next we sought to examine the levels of inflammatory cytokines and chemokines within the balf of lps-challenged wt and lps-challenged ttp are mice. of the 25 cytokines/chemokines analyzed, four cytokines/chemokines, including g-csf, kc, il-6, and il-12p40, were found to be significantly different between the lps-challenged wt and the lpschallenged ttp are mice. g-csf was significantly reduced in lps-challenged ttp are compared to lps-challenged wt mice at 12 and 72 h; kc and il-6 were significantly reduced at 12 h; and il-12 (p40) was significantly reduced at 72 h post-lps challenge (figures 4a-d) . interestingly, the levels of tnfα ( figure 4e ) and mip2 (figure 4f) , two known ttp targets, were not significantly different between the two groups. to determine the cell-specific role of ttp levels in ali, we modulated ttp levels in hematopoietic progenitor cells (hpcs) and non-hpcs. in order to test whether donor hpcs repopulate the recipient mouse lungs, we first made bone marrow chimeras in which total body irradiated wt mice were transplanted with hpcs from a mouse expressing green fluorescent protein (gfp) in their somatic cells. we found that greater than 90% of the cells recovered from the balf of these mice were gfp positive (data not shown). next, we generated three bone marrow chimeras in which irradiated wt recipient mice received hpcs from either wt (wt→wt), ttp are (ttp are →wt), or ttp ko (ttp ko →wt) mice ( figure 5a) . while saline-treated ttp ko →wt chimeras had no signs of cellular infiltration that included total cells (figure 5b) , neutrophils (figure 5c) , macrophages (figure 5d) , and lymphocytes (figure 5e) , lps-challenged wt→wt, ttp are →wt, and ttp ko →wt chimera mice had significant cellular recruitment, as indicated by balf total cellular counts (figure 5b) , neutrophils (figure 5c) , and macrophages ( figure 5d) . while lpschallenged ttp are →wt mice had somewhat reduced cellular infiltration as compared to lps-challenged wt→wt chimeras (figures 5b-d) , the lps-challenged ttp ko →wt chimera mice had remarkably higher number of bal cellular counts (figures 5b-d) . lymphocyte counts did not differ significantly between the three groups ( figure 5e) . histologically, ∼23, 26, and 47% of the lung parenchyma was consolidated in lps-challenged wt→wt, lps-challenged ttp are →wt, and lps-challenged ttp ko →wt group, respectively (figures 6a top panel, 6c) . consolidated parenchyma was characterized by the presence of large infiltrates of neutrophils and macrophages within the airspaces (alveolar and airway). other histological findings included the presence of edema and immune cells within the perivascular spaces, bronchial lumen cellular infiltrates, airspace edema, and occasional bronchoalveolar lymphoid aggregates (figures 6d-g) . these data suggest that while baseline expression of ttp in hpcs is essential for protection against exaggerated ali, its overexpression in these cells does not confer significant additional advantage. next, we generated bone marrow chimeras in which irradiated ttp are recipient mice received hpcs from either wt (wt→ttp are ), ttp are (ttp are →ttp are ), and ttp ko (ttp ko →ttp are ) mice ( figure 5a) . as compared to wt recipient chimera counterparts, lps-challenged wt→ttp are , lps-challenged ttp are →ttp are , and lps-challenged ttp ko →ttp are chimeras had significantly lower degrees of cellular recruitment, that included total cells ( figure 5b , blue solid bar, orange solid bar, green solid bar), neutrophils ( figure 5c , blue solid bar, orange solid bar, green solid bar), and macrophages ( figure 5d , blue solid bar, orange solid bar, green solid bar). lymphocyte counts were significantly reduced in lps-challenged ttp are →ttp are compared to the lps-challenged wt→wt chimeras ( figure 5e ). although the lps-challenged ttp ko →ttp are chimeras had significantly higher cellular recruitment as compared to lps-challenged wt→ttp are and lps-challenged ttp are →ttp are chimeras, the extent of recruitment was much diminished in lps-challenged ttp ko →ttp are chimera as compared to lps-challenged ttp ko →wt chimera ( figure 5b) . histologically, ∼11, 13%, 17% of the lung parenchyma was consolidated in lps-challenged wt→ttp are , lpschallenged ttp are →ttp are , and lps-challenged ttp ko →ttp are , respectively (figures 6a bottom panel, figure 6c ). histologically, mild increases in septal thickening with cellular infiltration, mild bronchiolitis, perivascular edema, inflammation, and airspace edema, and occasional balts were evident in all the three groups (figures 6d-g, solid blue, orange, and green bars). these data suggest that enhanced expression of ttp in lung non-hpc populations conferred significant protection against ali. further, this protection is somewhat compromised in the absence of baseline levels of ttp in hpcs. a tabular summary of differential cellular and ali responses in various chimeric mice is included in supplementary table 1 . finally, we generated bone marrow chimeras in which irradiated ttp ko recipient mice received hpcs from ttp are (ttp are →ttp ko ). as expected, the cellular counts were significantly higher than any of the other lps-challenged chimeras (figures 5b-e, red solid bar) . however, total cellular counts in lps-challenged ttp are →ttp ko chimera were ∼ twofold lower than the lps-challenged ttp ko mice (figure 1) . additionally, none of the lps-challenged ttp are →ttp ko chimeras succumbed to ali, as compared to 50% mortality in lps-challenged ttp ko mice (figure 1) . these data suggest that complete loss of ttp in lung non-hpc populations significantly exaggerates ali, and that overexpression of ttp in hpcs may provide partial protection in severe ali. histologically, this group exhibited the most severe lung injury, characterized by ∼70% lung consolidation with neutrophils, macrophages, fibrin, and edema, severe bronchiolitis, and moderate to severe bronchiolar and alveolar hemorrhages (figures 6b-g) . tristetraprolin knockout (ttp ko ) mice exhibit a systemic inflammatory syndrome that is characterized by cachexia, polyarticular synovial arthritis, dermatitis, and myeloid hyperplasia (4). however, the lungs of ttp ko mice exhibit very little spontaneous inflammation, characterized by the presence of rare foci of leucocytic infiltrates within the pulmonary parenchyma (8) . these rare leucocytic infiltrates are abrogated upon combined deletion of ttp and tnf receptors, indicating the role for tnf activity in leucocytic infiltration (8) . myeloid cell-specific loss of ttp (ttp myeko ) does not recapitulate the ttp ko phenotype, indicating that non-myeloid cells are required for the overall manifestation of the ttp deficiency syndrome (16) . however, ttp myeko mice were found to be hypersensitive to endotoxin-induced systemic inflammation, particularly through exaggerated tnf production, delineating the critical role of myeloid cell-specific ttp in protection against systemic injury and inflammation (16) . the role of ttp in localized lung inflammation, however, has remained unexplored. therefore, in this study we sought to explore the role of ttp in ali. we hypothesized that ttp modulates acute lung inflammation and that cell-specific modulation of ttp levels will differentially affect the outcome of acute lung inflammation. the unchallenged ttp ko mice display minor degrees of leukocytic infiltration in lung parenchyma that were thought to be contributed by tnf activity (8) . here, in lps-challenged ttp ko mice, immune cell infiltration was ∼ fourfold higher as compared to lps-challenged wt mice. in fact, the susceptibility of ttp ko mice to lps-induced ali was so severe that ∼50% ttp ko mice succumbed to lps-challenge within 24-72 h post-lps administration, while no mortality was observed in lpschallenged wt mice. the surviving ttp ko mice displayed severe pulmonary pathology with exaggerated airspace and interstitial neutrophilic infiltration, exaggerated edema, vascular congestion, and lung injury that included epithelial and endothelial damage. it is likely that the increased production of inflammatory mediators, that are otherwise regulated by ttp, in lpschallenged ttp ko mice, contribute to their worsened pulmonary pathology. one such mediator, tnf, is already established to be highly secreted in ttp ko mice (4, 6) . macrophages are the primary source of tnf in ali (23, 24) although alveolar epithelial cells have also been suggested to release tnf in lps-induced lung inflammation (25) . accordingly, we reasoned that, if macrophages are the primary source of tnf, deletion of ttp in myeloid cells would enhance its activity, leading to worsening of pulmonary pathology. although the lps-challenged ttp myeko mice had exaggerated pulmonary pathology as compared to lps-challenged wt mice, the extent of tissue damage and neutrophilic infiltration was not as severe as in lps-challenged ttp ko mice. these differences in the sensitivity of systemic versus myeloid cell-specific ttpdeficient mice indicate that ttp in cells other than myeloid-cells may play critical roles in modulating endotoxin-induced ali. these data suggest that while loss of myeloid cell-specific ttp worsens the ali, ttp expression in non-myeloid cells confers significant protection. clinically, the numbers of neutrophils within the balf of patients with ards have been shown to correlate with the severity of disease and poor outcome (26, 27) . despite being the first line of defense against pathogenic insults, excessive recruitment and activation of neutrophils leads to lung tissue damage and loss of lung function (28, 29) . therefore, targeting neutrophilic recruitment through suppressed release of key neutrophil chemoattractants may be an attractive therapeutic strategy against ali. we observed correlations between ttp deficiency or ttp overexpression and neutrophilic inflammation. for example, balf counts and tissue infiltration of neutrophils were overwhelming in the lps-challenged ttp ko mice. these outcomes were also exaggerated, but to a lesser degree, in lps-challenged ttp myeko mice. on the other hand, these outcomes were significantly attenuated in the lps-challenged ttp are mice. our findings are in line . data are represented as mean ± sem. statistical analysis in c-g was performed by one-way anova followed by tukey's correction for multiple comparisons within the three recipient groups and student's t-test between the three recipient groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. with previous reports in other tissues. for instance, massive infiltration of neutrophils has been shown to occur in the skin of ttp ko mice subjected to psoriasis-like inflammation (30) , whereas reduced airway neutrophilic infiltration was shown in mice genetically modified to express constitutively active endogenous unphosphorylated ttp following challenge with cigarette smoke (31) . a number of studies have shown that ttp is phosphorylated at multiple sites by p38-regulated kinase mapk-activated protein kinase 2 (mapkapk2) and that ttp activity is significantly affected by its phosphorylation status (32) (33) (34) . ttp phosphorylation has been shown to result in reduced ttp activity/reduced binding of ttp to ares, thus resulting in stabilization of its target mrnas (35) . regulation of ttp activity by p38 mapk was in fact shown to result in a biphasic response of tnfα-induced il-6 expression in human bronchial smooth muscle cells (36) . conversely, dephosphorylation of ttp resulted in reduced expression of il-6 and il-8 in a549 lung epithelial cells (37) . since lps is an inducer of both ttp and p38 mapk, ttp would be expected to undergo phosphorylation and inactivation shortly after lps challenge. however, the effect of ttp phosphorylation is transient and would be expected to be reversed upon dephosphorylation when p38 is turned off. although we did not track the phosphorylation status of ttp in various lung cells during ali, we speculate that the p38-mediated phosphorylation of ttp occurs well before 12h post lps challenge and is reversed by 12 h time point. consistent with our speculation, we found differential effect of ttp overexpression on late phase cytokines (kc) versus early phase cytokines (tnf) (figure 4) . tristetraprolin is a known regulator of key neutrophil chemoattractants including cxcl1/kc (8, 9) and cxcl2/mip2 (8, 38) . cxcl1/kc is a central chemokine in neutrophil recruitment into the airspace in ards (39) (40) (41) . clinically, increased concentrations of il-8 (cxcl1/kc homolog in human), disease severity, and neutrophil migration into airspaces have been shown to be correlated (42) (43) (44) . cxcl1 mrna 3 utr contains ttp-binding sites and has been previously demonstrated to be a direct target of ttp (8, 9) . consistent with this, the lps-challenged ttp are mice had significantly lower cxcl1/kc concentrations. in contrast, the two well characterized ttp targets, tnf and mip2/cxcl2, were not significantly different between lps-challenged wt and lpschallenged ttp are mice. these observations are in line with our previous report, in which no differences were observed in the levels of tnf and mip2/cxcl2 in the serum of wt and ttp are mice systemically challenged with lps (15). these two inflammatory mediators peak early (before 6 h in the serum) (15) , and we speculate that overexpressed ttp may be less effective at mrna decay due to its phosphorylation status at these early time points. g-csf, another neutrophil chemoattractant, is also consistently detected within the balf of ali and ards patients and has been suggested to be associated with the accumulation and activation of neutrophils in ards (45) . plasma g-csf levels have also been shown to correlate with clinical outcome in patients with ali (27, 46) . g-csf has also been shown to exacerbate bleomycin induced lung injury in rats through marked infiltration of activating neutrophils (47) . g-csf levels have been found to be increased in ttp ko mouse plasma (48) . although g-csf has not been shown to be a direct ttp target, g-csf mrna 3 utr in mouse possesses two ttp binding sites, uauuuau. the balf g-csf levels were significantly reduced in lps-challenged ttp are mice. g-csf levels were also found to be significantly reduced in ttp are mice in a previous study where we showed ttp are mice to be significantly protected from exhibiting collagen-antibody induced arthritis (15) . lipopolysaccharide-challenged ttp myeko mice exhibited milder ali as compared to lps-challenged ttp ko mice, indicating that total loss of ttp expression in non-myeloid as well as myeloid cells contributes to severe ali. on the other hand, systemic ttp overexpression conferred significant protection against lps-induced ali. here, we addressed two logical questions: (1) whether enhanced levels of ttp in non-hpcs would ameliorate endotoxin-induced ali, (2) whether enhanced levels of ttp in hpcs would confer protection against endotoxin-induced ali. towards this, employing various bone marrow chimeras, we investigated cell-specific roles of ttp in protection against ali. in these experiments, we modulated ttp levels in hpcs by using bone marrow donors that were either wt, ttp are , or ttp ko . to modulate ttp levels in non-hpcs, wt, ttp are , or ttp ko recipients were used. one limitation of this study was that since hpcs also include various non-myeloid populations, we were not able to specifically investigate the effect of ttp modulation in myeloid cells. the bone marrow irradiation and reconstitution experiments revealed somewhat unexpected but interesting findings. as compared to wt→wt chimera, the ali in ttp are →wt chimeras was somewhat attenuated, whereas the ali in ttp ko →wt chimeras had worsened significantly. these data suggest that, when the ttp expression in non-hpcs is genetically unaltered (wt ttp in the recipient's non-hpcs), the ttp overexpression in hpcs is partially protective against lps-induced ali. however, when the ttp expression was ablated in non-hpcs [ttp depleted in the recipient's non-hpcs (ttp are →ttp ko chimera)], the overexpression of ttp in the hpcs was not sufficient to confer protection against ali. as compared to ttp are →ttp ko chimeras, in ttp ko →ttp are chimeras, the mere overexpression of ttp in non-hpcs (ttp overexpression in the recipient's non-hpcs) provided significant protection, even though the hpcs were ttp-deficient. however, this protection was further enhanced when the normal levels of ttp were restored in the hpcs (wt→ttp are chimera). based on these data, we conclude here that the ttp levels in non-hpcs are critical in protection against ali. further, while the wt levels of ttp in hpcs are essential for additional protection, the overexpression of ttp in hpcs is not significantly advantageous. since the non-hpcs population in lungs consists of a multitude of cell types including epithelial cells, endothelial cells, and fibroblasts, the cell-specific role of ttp still remains elusive. contribution of non-hpc-specific ttp toward modulation of inflammatory responses has also been suggested in previous reports. for instance, ttp regulation of tnf in keratinocytes, but not in myeloid or dendritic cells, was shown to protect mice from exacerbation of psoriasis-like pathology, development of spontaneous systemic inflammation, and dactylitis (30) . another study suggested a role of intestinal epithelial cell-specific ttp in exacerbation of acute colitis (49) . along similar lines, ttp depletion in myeloid cells did not replicate the ttp-deficiency phenotype (16) , and the spontaneous reactive granulopoiesis seen in ttp ko mice was suggested to be caused by a non-cell autonomous mechanism likely contributed by liver cells (48) . all these studies indicated an essential role of ttp in non-hpcs in regulating inflammation. among various non-hpc cell types in the lung, alveolar epithelial cells produce pro-inflammatory cytokines and chemokines (50) . in in vitro conditions, lpschallenged pulmonary type ii epithelial cells have been shown to produce greater levels of neutrophil chemoattractants [il-8, a human homolog for cxcl1 (kc), cxcl2 (mip2), and cxcl5 (lix)] indicating that ttp in lung alveolar epithelial cells may play an important role in regulating ali (50) . consistent with these reports, while our data indicate critical roles of non-hpcs in lungs in mediating neutrophil recruitment to the airspaces, the exact identity of these non-hpcs remain unknown. in conclusion, our results show that (a) enhancing the levels of ttp is protective against endotoxin-induced ali; (b) the protective effect seen in ttp are mice is attributable to reduced neutrophilic recruitment and, in turn, reduced lung damage; (c) reduced neutrophilic recruitment is attributed to reduced secretion of chemoattractants, particularly kc and g-csf; and that, (d) ttp in non-hpcs plays an essential role in protection against endotoxin-induced ali. based on these results, we propose a model in which endotoxin damages epithelial cells within the lung, which then initiates a cascade of events leading to exaggerated release of proinflammatory mediators and neutrophilic chemoattractants, resulting in further lung damage and neutrophilic infiltration (figure 7) . in this process, ttp acts as an intracellular regulator for the expression of proinflammatory mediators and neutrophilic chemoattractants. ttp expression in the non-hpcs, i.e., most likely epithelial and endothelial cells, confers protection against endotoxin-induced ali via suppression of mrnas encoding proinflammatory mediators and neutrophilic chemoattractants. hence, strategies to increase ttp expression or activity in non-hpcs together with hpcs population may prove beneficial for patients with ali/ards. in future studies, ttp expression within the lung could also be investigated as a prognostic indicator for the severity of ali/ards. our studies could also have implications for the lung hyper-inflammation and potentially life-threatening cytokine storms in the severe coronavirus disease (covid-19). the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by lsu iacuc. sp conceived and designed the study, maintained the animal colony, and performed histopathological analyses. ic, tv, and bl conducted animal necropsies. ys, cb, and rl performed balf cellularity assays. rl performed the intravenous injections. ys, tv, ic, and cb performed cytokine assays. jl performed the irradiations. sj provided technical expertise on bone marrow transplantations and reviewed the manuscript. pb provided various transgenic and knockout mice and edited the manuscript. sp and ys wrote and reviewed the manuscript for intellectual contents. all authors contributed to the article and approved the submitted version. the acute respiratory distress syndrome: pathogenesis and treatment acute lung injury: a clinical and molecular review tristetraprolin as a therapeutic target in inflammatory disease a pathogenetic role for tnf alpha in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin (ttp) deficiency roles of tumor necrosis factor-alpha receptor subtypes in the pathogenesis of the tristetraprolin-deficiency syndrome feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin evidence that tristetraprolin binds to au-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mrna tristetraprolin (ttp) coordinately regulates primary and secondary cellular responses to proinflammatory stimuli tristetraprolin regulates cxcl1 (kc) mrna stability genome-wide analysis identifies interleukin-10 mrna as target of tristetraprolin tristetraprolin down-regulates il-17 through mrna destabilization zinc finger protein tristetraprolin interacts with ccl3 mrna and regulates tissue inflammation tristetraprolin regulation of interleukin 23 mrna stability prevents a spontaneous inflammatory disease tristetraprolin (ttp): interactions with mrna and proteins, and current thoughts on mechanisms of action enhanced stability of tristetraprolin mrna protects mice against immunemediated inflammatory pathologies myeloid-specific tristetraprolin deficiency in mice results in extreme lipopolysaccharide sensitivity in an otherwise minimal phenotype conditional gene targeting in macrophages and granulocytes using lysmcre mice a non-invasive and technically non-intensive method for induction and phenotyping of experimental bacterial pneumonia in mice early postnatal secondhand smoke exposure disrupts bacterial clearance and abolishes immune responses in muco-obstructive lung disease myeloid differentiation protein-2-dependent and -independent neutrophil accumulation during escherichia coli pneumonia optimal timing to repopulation of resident alveolar macrophages with donor cells following total body irradiation and bone marrow transplantation in mice an official american thoracic society workshop report: features and measurements of experimental acute lung injury in animals early tumor necrosis factor-alpha release from the pulmonary macrophage in lung ischemia-reperfusion injury alveolar macrophage-derived microvesicles mediate acute lung injury production of tumour necrosis factor alpha by primary cultured rat alveolar epithelial cells neutrophils and acute lung injury g-csf and il-8 but not gm-csf correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome contribution of neutrophils to acute lung injury lung neutrophils in the adult respiratory distress syndrome. clinical and pathophysiologic significance tristetraprolin expression by keratinocytes controls local and systemic inflammation enhancing tristetraprolin activity reduces the severity of cigarette smokeinduced experimental chronic obstructive pulmonary disease mitogenactivated protein kinase p38 controls the expression and posttranslational modification of tristetraprolin, a regulator of tumor necrosis factor alpha mrna stability mapkap kinase 2 phosphorylates tristetraprolin on in vivo sites including ser178, a site required for 14-3-3 binding the role of ttp phosphorylation in the regulation of inflammatory cytokine production by mk2/3 mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mrna stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/uridine-rich element temporal regulation of cytokine mrna expression by tristetraprolin: dynamic control by p38 mapk and mkp-1 activating protein phosphatase 2a (pp2a) enhances tristetraprolin (ttp) anti-inflammatory function in a549 lung epithelial cells bi-phased regulation of the post-transcriptional inflammatory response by tristetraprolin levels expression and biologic characterization of the murine chemokine kc il-8) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ards) and patients at risk for ards interleukin 8-related neutrophil elastase and the severity of the adult respiratory distress syndrome increased interleukin-8 concentrations in the pulmonary edema fluid of patients with acute respiratory distress syndrome from sepsis elevated levels of nap-1/interleukin-8 are present in the airspaces of patients with the adult respiratory distress syndrome and are associated with increased mortality alveolar granulocyte colony-stimulating factor and alpha-chemokines in relation to serum levels, pulmonary neutrophilia, and severity of lung injury in ards plasma granulocyte colony-stimulating factor levels correlate with clinical outcomes in patients with acute lung injury effects of granulocyte colony-stimulating factor (g-csf) on bleomycin-induced lung injury of varying severity deletion of tristetraprolin caused spontaneous reactive granulopoiesis by a non-cellautonomous mechanism without disturbing long-term hematopoietic stem cell quiescence tristetraprolin targets nos2 expression in the colonic epithelium differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type ii epithelial cells and macrophages we thank thaya stoufflet for assistance with multiplex cytokine assays, sherry ring for histological tissue processing, and deborah stumpo for helping with the ttp mutant mice and their genotyping. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.02164/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 choudhary, vo, bathula, lamichhane, lewis, looper, jeyaseelan, blackshear, saini and patial. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-298458-p7rvupjo authors: schmidt, megan e.; varga, steven m. title: the cd8 t cell response to respiratory virus infections date: 2018-04-09 journal: front immunol doi: 10.3389/fimmu.2018.00678 sha: doc_id: 298458 cord_uid: p7rvupjo humans are highly susceptible to infection with respiratory viruses including respiratory syncytial virus (rsv), influenza virus, human metapneumovirus, rhinovirus, coronavirus, and parainfluenza virus. while some viruses simply cause symptoms of the common cold, many respiratory viruses induce severe bronchiolitis, pneumonia, and even death following infection. despite the immense clinical burden, the majority of the most common pulmonary viruses lack long-lasting efficacious vaccines. nearly all current vaccination strategies are designed to elicit broadly neutralizing antibodies, which prevent severe disease following a subsequent infection. however, the mucosal antibody response to many respiratory viruses is not long-lasting and declines with age. cd8 t cells are critical for mediating clearance following many acute viral infections in the lung. in addition, memory cd8 t cells are capable of providing protection against secondary infections. therefore, the combined induction of virus-specific cd8 t cells and antibodies may provide optimal protective immunity. herein, we review the current literature on cd8 t cell responses induced by respiratory virus infections. additionally, we explore how this knowledge could be utilized in the development of future vaccines against respiratory viruses, with a special emphasis on rsv vaccination. introduction given its continuous exposure to the outside environment, the respiratory mucosa is highly susceptible to viral infection. the human respiratory tract can be infected with a variety of pulmonary viruses, including respiratory syncytial virus (rsv), influenza virus, human metapneumovirus (hmpv), rhinovirus (rv), coronavirus (cov), and parainfluenza virus (piv) (1) . the severity of disease associated with respiratory viral infection varies widely depending on the virus strain as well as the age and immune status of the infected individual. symptoms can range from mild sinusitis or cold-like symptoms to more severe symptoms including bronchitis, pneumonia, and even death. rsv is the leading cause of severe lower respiratory tract infection in children under 5 years of age (2) . rsv is commonly associated with severe lower respiratory tract symptoms including bronchiolitis, pneumonia, and bronchitis and is a significant cause of hospitalization and mortality in children, the elderly, and immunocompromised individuals (2) (3) (4) (5) (6) . similarly, piv commonly infects children and is a major cause of croup, pneumonia, and bronchiolitis (7, 8) . seasonal influenza infections, most often of the influenza a virus (iav) subtype, are responsible for 3-5 million cases of severe infection annually (9) . seasonal iav infections also result in approximately 290,000-650,000 deaths per year, most commonly in either young children or elderly populations (9) (10) (11) . however, infection with emerging pandemic iav strains, such as the 2009 h1n1 pandemic strain, primarily induces severe disease and mortality in otherwise healthy adults younger than 65 years of age (12) . in contrast, respiratory infection with hmpv, rv, and cov are most commonly associated with symptoms of the common cold (13) (14) (15) . two notable exceptions are severe acute respiratory syndrome (sars) cov and middle east respiratory syndrome (mers) cov, which cause acute respi ratory distress and mortality in infected individuals (16) (17) (18) . despite their profound impact on human health, most common respiratory viruses lack an approved vaccine. the strategy employed most often in vaccine development is the induction of robust neutralizing antibody responses. however, the hallmark of many respiratory viral infections, including rsv, hmpv, and rv, is the ability for reinfections to occur frequently throughout life (19) (20) (21) . this suggests that the antibody response to these respiratory viruses may wane over time. indeed, despite a correlation between pre-existing nasal iga and protection from reinfection, the development of long-lasting rsv-and rv-specific mucosal iga responses was poor in infected adults (22, 23) . although iav-specific neutralizing antibodies are elicited efficiently through either infection or vaccination, iav vaccine formulations must be redeveloped annually to account for the rapid mutations of ha and na genes in seasonal strains (24) . therefore, vaccinations that solely promote the induction of neutralizing antibodies may not be optimal in providing protection against many respiratory virus infections. the induction of cellular immune responses has thus far received little attention in respiratory virus vaccine development. cd8 t cells play a critical role in mediating viral clearance following many respiratory virus infections including rsv, iav, and hmpv (25) (26) (27) . in addition, recent murine studies utilizing cd8 t cell epitope-specific immunization strategies observed significantly reduced lung viral titers following iav, rsv, or sars challenges (28) (29) (30) . therefore, the induction of virus-specific cd8 t cell responses has the potential to improve upon the efficacy of current vaccination strategies. here, we review the current literature on cd8 t cell responses following respiratory virus infections and discuss how this knowledge may best be utilized in the development of future vaccines. following an acute respiratory infection, dendritic cells (dcs) that have taken up viral antigen stimulate the activation of naive cd8 t cells in the lung draining lymph node to induce robust virus-specific cd8 t cell responses [reviewed in ref. (31) (32) (33) ]. respiratory virus infection in mouse models results in an increase in the frequency and number of total and antigenspecific cd8 t cells in the lungs and airways. rsv-specific cd8 t cell responses typically reach peak numbers in the lung at approximately day 8 following an acute infection (34) (35) (36) (37) . the kinetics of virus-specific cd8 t cells are slightly more delayed following other respiratory virus infections with peaks occurring at approximately day 10 for iav, days 10-14 for hmpv, and days 12-14 for pneumonia virus of mice (pvm), a model respiratory virus (38) (39) (40) (41) (42) . the peak of the antigen-specific cd8 t cell response generally corresponds to lung viral clearance following rsv, iav, hmpv, and pvm infections (36, (40) (41) (42) (43) . human cd8 t cell kinetics following respiratory virus infections are less well known given the lack of identified cd8 t cell epitopes and the difficulty in obtaining respiratory tract samples from children following initial virus exposure. the frequency of total activated cd8 t cells in tracheal aspirates peaked at approximately 10 days after the onset of symptoms in children with rsv, iav, rv, or cov infections (44). rsv-specific cd8 t cells were detected in the tracheal aspirates of children; however, the evaluated epitopes were present at very low frequencies, comprising up to only 2% of the total cd8 t cell response (44). following peak expansion, cd8 t cell contraction occurs and a memory population of virus-specific cd8 t cells remains within the lung. the majority of virus-specific cd8 t cells are located within the lung parenchyma, rather than the pulmonary vasculature, following localized respiratory infections in mice (37) . similarly, human rsv-and iav-specific cd8 t cells were enriched within the lung compared to the blood (45). rsv-specific cd8 t cells in tracheal aspirates of children remain elevated during convalescence following a severe rsv infection, in contrast to murine studies (46). these studies suggest that cd8 t cell responses in the airways may be more prolonged following viral clearance in humans compared to mice. following respiratory virus infection, cd8 t cells become activated and develop the ability to produce inflammatory cytokines. virus-specific cd8 t cells in the lung and airways of mice upregulate expression of markers associated with activation including cd11a, cd25, nkg2a, and cd44 as well as downregulate expression of the lymphoid homing receptor cd62l (34, 35, 47, 48) . activated cd8 t cells also acquire effector functions following viral infection. virus-specific cd8 t cells rapidly produce cytokines including ifn-γ and tnf as well as degranulate, as measured by cd107a expression, following ex vivo peptide stimulation (35, 38, 41, 48) . human virusspecific cd8 t cells also acquire an activated phenotype and effector functions following a respiratory virus infection. cd8 t cells from the tracheal aspirates of children following rsv, rv, or cov infections expressed elevated levels of the activation markers cd38 and hla-dr and the proliferation marker ki-67 (44). expression of effector molecules such as granzyme b and perforin were also increased. similarly, cd8 t cells from bronchiolar lavage (bal) fluid samples exhibited increased expression of ki-67, granzyme b, cd38, and hla-dr following either experimental rsv infection of adults or severe, natural rsv infection of infants (46, 49). additionally, human virusspecific cd8 t cells produce cytokines following respiratory virus infection, as peripheral blood cd8 t cells secreted ifn-γ, tnf, and il-2 following stimulation with peptides derived from rsv, iav, hmpv, or rv (49-53). following contraction, a subset of virus-specific cd8 t cells remain in the host to form a long-lasting memory population that provides protection against subsequent infection. cd8 t cell contraction to form long-term memory populations in the lung is regulated in part by inflammatory chemokine signaling (54). mice deficient in either cxcr3 or cxcr3 and ccr5 exhibit a significant increase in the number of memory cd8 t cells following iav infection, suggesting that chemokine signaling through cxcr3 and ccr5 plays a critical role in t cell memory generation (54). following respiratory viral infections in mice and humans, virus-specific cd8 t cells can be detected up to several months post-infection (47, 49, 55, 56). however, respiratory virus-specific memory cd8 t cell populations decline in magnitude with age in the peripheral blood (57) . interestingly, adult rsv-specific cd8 t cell responses are significantly reduced compared to iav-specific cd8 t cell responses in the peripheral blood, suggesting that memory cd8 t cell responses to iav in humans may be more stable than rsv (57) . memory cd8 t cells rapidly expand in the lung following a secondary respiratory virus infection in both mice and humans (35, 38, 39, 44, 49) . the observed expansion is primarily due to the migration of circulating cd8 t cells into the lung and airways, rather than proliferation of resident cells (58) . the expansion of virus-specific cd8 t cells in the lung and airways following infection corresponds with an increase in cxcr3-and ccr5-binding chemokines, supporting a role for chemokine-mediated migration of cd8 t cells following secondary infection (59) . indeed, ccr5 expression on memory cd8 t cells is required for their early recruitment into the airways after secondary infection, but not to the lung parenchyma (59) . following secondary expansion, memory cd8 t cells rapidly produce effector cytokines such as ifn-γ and tnf (30, 38, 60) . additionally, virus-specific memory cd8 t cells express high levels of cd11a and produce cytolytic molecules, such as granzyme b, after infection (61, 62) . these effector functions of respiratory virus-specific memory cd8 t cells are critical for mediating viral clearance and protecting against infection, as discussed below. based on the expression of activation marker cd45ra and lymphoid homing receptor ccr7, human memory cd8 t cells have been broadly separated into four major subsets: (1) naive (cd45ra + ccr7 + ), (2) central memory (tcm; cd45ra -ccr7 + ), (3) effector memory (tem; cd45ra − ccr7 − ), and (4) late effector memory (temra; cd45ra + ccr7 − ) (63) . due to their expression of ccr7, tcm home primarily to secondary lymphoid organs, while tem migrate to peripheral tissues and rapidly exert effector functions. temra are a subset of tem cells that have re-expressed cd45ra. they exhibit reduced proliferative and functional capacity, and thus are considered to be terminally differentiated cells. human virus-specific memory cd8 t cell populations are typically composed of a combination of tem and temra within the peripheral blood (44, 46, 50, 52, 55). alternatively, rsv-specific memory cd8 t cells located in the airways in both adults and infants are primarily of tem phenotype and also express high levels of cd27, cd28, and ccr5 and low levels of cd62l (46, 49). together, these studies indicate that tem cd8 t cells are dominant following respiratory virus infection in humans. given the frequent exposure to viruses in the respiratory tract, tem cells may be critical for the rapid employment of cd8 t cell effector mechanisms following reinfection. recently, an additional population of memory cd8 t cells that persist within peripheral tissues has been identified, termed tissue-resident memory cd8 t cells (trm) (64) . trm have been observed within several peripheral organs including the intestine, skin, female reproductive tract, and lung. trm generated following a respiratory virus infection represent a non-circulating population of memory cd8 t cells that are maintained within the lung parenchyma (65) . virus-specific trm are located along the wall of large airways and within pulmonary tissue surrounding bronchioles and alveoli (66, 67) . respiratory virus infection also induces trm within the airway lumen (68, 69) . airway trm downregulate cd11a expression and can be distinguished from recently trafficked cd8 t cells that express high levels of cd11a (70, 71) . the localization of lung and airway trm following respiratory virus infection is distinctly different from that of tcm, which traffic through the pulmonary vasculature and accumulate in the lung-draining lymph node (72, 73) . virus-specific tem are also differentially located from trm residing primarily in the pulmonary vasculature or within the lung tissue near blood vessels, spacially distinct from regions that contain trm (67, 73). following either rsv or iav infection in mice, lung and airway trm are induced and can be identified by their expression of the canonical resident memory markers cd69 and cd103, which promote their migration to and retention within the lung tissue (37, 65, (74) (75) (76) . iav-and rsv-specific trm are also generated in the lungs of mice that have been locally vaccinated via an intranasal route, but not mice that have been immunized systemically (30, (77) (78) (79) . importantly, iav-specific trm expressing cd69 were also detected in human lung tissue sections but were absent from the spleen (65) . similarly, rsv-specific trm expressing both cd69 and cd103 were identified in the human bal but were not present in the peripheral blood (49). following secondary viral infection, trm expand prior to the recruitment of circulating memory cd8 t cell populations from the peripheral blood and rapidly produce ifn-γ (60, 66). thus, trm provide a crucial first-line of defense for protecting the host from re-infection with a respiratory virus. however, in contrast to other memory cd8 t cell subsets that remain stable for long periods of time, iavspecific trm exhibit limited longevity and enhanced apoptosis with time following infection (66, 80) . the loss of iav-specific trm corresponds to an increase in viral titers and weight loss following a heterosubtypic iav infection (66, 80) . interestingly, infant mice generate fewer lung trm following iav infection or vaccination and exhibit reduced heterosubtypic protection compared to mice initially infected as adults (79) . given the role of lung trm in providing protection against respiratory virus infections, identifying strategies to promote the generation of long-lived trm will be critical for future vaccines, particularly for infant populations. it has been well established that cd8 t cells are critical for viral clearance following an acute respiratory virus infection in mice. adoptive transfer of cd8 t cell clones resulted in significantly reduced viral titers in the lung following rsv, iav, or hmpv infections (25, 27, (81) (82) (83) (84) . similarly, the transfer of either rsv-or iav-immune splenic cd8 t cells accelerated viral clearance in the lung following infection (85) (86) (87) (88) (89) . accordingly, rsv infection of mice depleted of cd8 t cells resulted in significantly increased lung viral titers at day 7 post-infection, although the virus was ultimately cleared by day 11 (26) . in contrast, depletion of cd8 t cells alone did not alter clearance of hmpv (40). instead, depletion of both cd4 and cd8 t cells together elevated lung virus titers at day 7 following infection with hmpv. importantly, cd8 t cells have been shown to be sufficient to mediate viral clearance in the lung following acute respiratory infections (87, 88) . athymic nude mice, which lack t cells, fail to clear either rsv or iav resulting in persistent infections (87, 90) . however, the transfer of either rsv-or iav-immune splenic cd8 t cells into athymic mice resulted in significantly reduced lung viral titers by day 15 and 21, respectively (87, 88) . together, these studies indicate that cd8 t cells play a critical role in mediating viral clearance following acute respiratory infections in mice. although studies are limited, a role for cd8 t cells in the elimination of respiratory viruses has also been established in humans. early studies demonstrated that immunocompromised children with t cell defects experienced prolonged viral shedding following rsv, iav, or piv infections compared to immunologically normal children (3, 91, 92) . following bone marrow transplantation of an rsv-infected child with severe combined immunodeficiency, a marked reduction in nasal viral load was observed that correlated with an elevation of cd8 t cell counts (92) . recently, it has been demonstrated that the number of pre-existing virus-specific cd8 t cells in the airway of adults experimentally infected with rsv correlated with reduced overall viral load in the nasal cavity and bronchial brushings (49). in addition to pre-existing cd8 t cell numbers, cd8 t cell effector functions also correlate with reduced viral load. cd8 t cell target cell lysis activity measured by chromiumrelease assay correlated with a lack of viral shedding in the nasal washes of adults experimentally infected with h1n1 iav (93) . additionally, individuals with the lowest frequencies of ifn-γ + cd8 t cells exhibited the highest viral titers following natural h7n9 iav infection (94) . these studies support the role of cd8 t cells in respiratory virus clearance in humans, consistent with the numerous murine studies. cd8 t cells mediate viral clearance by utilizing a variety of effector mechanisms to induce the apoptosis of virus-infected cells (95) . cd8 t cells can use direct cell-cell contact to eliminate target cells through the interactions of surface molecules such as fas (cd95) and fasl (cd95l). additionally, trail expressed on cd8 t cells can interact with its receptors dr4 and/or dr5 to induce the destruction of infected cells. cd8 t cells can also secrete perforin and granzymes to cause membrane pore formation and induce apoptosis. lastly, cd8 t cells produce inflammatory cytokines, such as ifn-γ and tnf, which may either directly or indirectly promote the cell death of virus-infected cells. while the exact mechanism utilized is unclear, many of these effector functions have been associated with cd8 t cell-mediated clearance of respiratory viruses. fas/fasl interactions and the perforin pathway have been established as the primary mechanisms by which cd8 t cells eliminate infected cells following an iav infection (96, 97) . studies utilizing trail-deficient mice and antibody-mediated trail blockade have also demonstrated a role for trail in cd8 t cell-mediated clearance of iav (98, 99) . similarly, fas/ fasl and perforin pathways have also been associated with virus elimination following rsv infection. perforin-deficient and fasl-deficient gld mice exhibit significantly delayed viral clearance (100, 101) . however, both perforin-deficient and gld mice achieve complete viral clearance by day 10 post-infection, suggesting that cd8 t cells compensate for those deficiencies through alternative mechanisms. one such mechanism is likely tnf production, as neutralization of tnf in perforin-deficient and gld mice significantly increased viral titers compared to igg-treated controls (101) . this is in contrast to studies following pvm and iav infections, where viral clearance occurs independently of tnf (102, 103) . ifn-γ does not appear to play a prominent role in cd8 t cell-mediated viral clearance, as both ifn-γ-deficient mice and mice that received ifn-γdeficient cd8 t cells exhibit equivalent viral titers to wild-type mice following rsv, pvm, or iav infections (101, (103) (104) (105) . together, these studies demonstrate that cd8 t cells use multiple complementary mechanisms to eliminate virally infected cells following a respiratory virus infection. given the ability of cd8 t cells to mediate viral clearance following an acute viral infection, it is no surprise that memory cd8 t cells also play a critical role in protecting against secondary respiratory virus infections. the adoptive transfer of airway iav-specific memory cd8 t cells resulted in significantly reduced lung titers following iav challenge compared to pbs transfer controls (60) . similarly, transfer of airway rsv-specific memory cd8 t cells reduced lung viral load and weight loss following subsequent rsv infection (76) . these studies indicate that transferred memory cd8 t cells are capable of providing protection against secondary respiratory virus challenge. memory cd8 t cell-mediated protection against secondary infection has been shown more convincingly in mouse models through the use of vaccination strategies to generate virus-specific memory cd8 t cells. recombinant baculovirus or murine cytomegalovirus (mcmv) vectors expressing the rsv m2 protein induced m2-specific cd8 t cells that mediated the reduction of lung viral titers following rsv challenge (78, 106) . whole protein vaccination with hmpv virus-like particles containing f and m proteins elicited hmpv-specific cd8 t cells that reduced viral titers in μmt mice, which lack antibodies (107) . cd8 t cell epitope vaccines against either rsv or hmpv have also demonstrated cd8 t cell-mediated protection following challenge by reducing lung viral load and histopathology compared to unimmunized controls (108, 109) . a similar strategy utilizing dc-peptide vaccination to generate pre-existing pvm-specific memory cd8 t cells also resulted in enhanced viral control following pvm infection (42). recently, several studies have utilized dc-prime, recombinant listeria monocytogenes-(dc-lm) or vaccinia virus-boost (dc-vv) vaccination protocols to generate a high frequency of pre-existing antigen-specific memory cd8 t cells in the absence of virus-specific cd4 t cell memory and antibodies (28) (29) (30) . prime-boosted mice exhibited significantly reduced lung viral titers following rsv, iav, or sars infections compared to controls lacking virus-specific memory cd8 t cells. additionally, memory cd8 t cells were able to reduce weight loss and mortality following lethal challenges with either iav or sars. overall, these studies provide clear evidence that memory cd8 t cells provide protection against secondary respiratory virus infection by reducing viral titers. the most well studied example of memory cd8 t cellmediated protection from a secondary respiratory virus infection is heterosubtypic immunity to iav subtypes. iav-specific neutralizing antibody responses recognize the surface glycoproteins hemagglutinin (ha) and neuraminidase (na), which vary between subtypes as a result of genetic reassortment, known as antigenic drift. however, the internal proteins of the virus are often conserved between iav subtypes. therefore, memory cd8 t cells that recognize epitopes within conserved viral proteins may be capable of providing cross-protection between iav viruses of differing subtypes. evidence of heterosubtypic immunity was first demonstrated by the protection of h1n1 iav-immune mice from a lethal h2n2 iav challenge without the induction of a neutralizing antibody response (110) . since then, a memory cd8 t cell-mediated role in accelerating clearance of a heterosubtypic iav strain has been well-established in mouse, chicken and non-human primate models (66, (111) (112) (113) (114) . recently, it has been demonstrated that trm are essential in providing cross-protection against secondary iav infection with a heterosubtypic strain (60, 66, 80) . mice with cd103 + trm in the lung exhibit more efficient viral clearance and reduced weight loss following heterosubtypic challenge than mice lacking a trm response (66) . importantly, the protection was provided solely by lung-resident memory cd8 t cells, as blocking the ability of recently proliferated tcm cells from trafficking to the lung did not impact protection (66) . consistent with the limited lifespan of iav-specific trm, heterosubtypic protection by memory cd8 t cells wanes over time, with a decline observed as early as 60 days following the initial infection (80, 111) . interestingly, systemic immunization with cognate antigen is capable of boosting the trm pool by expanding the circulating tem population that seeds the lungs (80) . therefore, it is possible that trm-mediated heterosubtypic protection could be re-established by vaccination after a waning of the protective trm population in the lung. while protection in mouse models is well established, whether memory cd8 t cells play a critical role in protection following secondary respiratory infection in humans is currently unclear. similar to studies in murine models, evidence for heterosubtypic immunity mediated by memory cd8 t cells has also been demonstrated in humans. individuals lacking h1n1-specific neutralizing antibody titers exhibited an inverse correlation between memory cd8 t cell activity and viral shedding following their first exposure with h1n1 iav (93) . more recently, it was demonstrated that the frequencies of pre-existing crossreactive memory cd8 t cells correlated with reduced symptoms, including fewer patients with fever, sore throat, and cough, following infection with the 2009 pandemic h1n1 iav strain (50). similarly, a correlation between pre-existing h3n2-specific memory cd8 t cells and reduced risk of viral shedding following 2009 pandemic h1n1 iav infection was observed (115) . thus, memory cd8 t cell-mediated heterosubtypic protection is also likely to be critical in humans. following experimental rsv infection in humans, the frequency of pre-existing rsv-specific memory cd8 t cells in the airways correlates with a reduction in both cumulative and lower respiratory tract symptom scores, suggesting a possible role for memory cd8 t cells in protection against rsv in humans (49). however, evidence has also been provided suggesting that memory cd8 t cells may not contribute to protection following respiratory virus infections in humans. natural reinfection of infants with rsv did not result in a boosting of the cd8 t cell response (116) . similarly, the frequency of rsv-specific memory cd8 t cells in the peripheral blood of healthy adults is significantly reduced compared to iavspecific memory cd8 t cells (57) . therefore, the extent to which memory cd8 t cells play a role in providing protection against rsv infection in humans remains unclear. despite their beneficial role in mediating viral clearance and protecting against secondary infection, cd8 t cells have also been associated with the induction of immunopathology following respiratory virus infection. although mice depleted of cd8 t cells exhibited elevated lung viral titers, weight loss and symptom illness scores were significantly reduced in cd8 t cell depleted mice following acute rsv infection (26) . similarly, the adoptive transfer of cd8 t cell lines exacerbated weight loss following an acute rsv infection, despite accelerating viral clearance (82) (83) (84) . similar reduction in disease severity was also demonstrated following either hmpv or pvm infection of cd8 t cell depleted mice or mice genetically deficient in cd8 t cells, respectively (40, 103). in addition to the induction of immunopathology following acute respiratory virus infections, we recently demonstrated that memory cd8 t cells also mediate severe immunopathology following secondary rsv infection (30) . large frequencies of systemic, pre-existing rsv-specific memory cd8 t cells generated through dc-lm immunization induced significant weight loss, pulmonary dysfunction, and mortality following rsv challenge, despite a significant reduction in lung viral titers. this result was in contrast to studies using similar immunization strategies to either iav or sars, in which memory cd8 t cells mediated protection against lethal viral challenge in the absence of immunopathology (28, 29) . interestingly, the immunopathology induced by rsv-specific memory cd8 t cells occurred only in the context of an rsv infection, as mice challenged with a recombinant iav strain expressing an rsv-derived cd8 t cell epitope exhibited significantly reduced morbidity and were protected from mortality (30) . this result is consistent with several studies that demonstrate cd8 t cells enhance viral clearance while preventing mortality following iav infection (25, 81, 85, 87, 117) . together, these studies demonstrate a clear role for cd8 t cells in the development of immunopathology following primary and secondary infections with some respiratory virus infections, particularly rsv. antiviral mechanisms utilized by cd8 t cells to mediate viral clearance following respiratory virus infection also contribute to the development of immunopathology. removal of the fas/ fasl pathway in gld mice resulted in significant amelioration of weight loss and symptom illness scores following rsv infection (101) . similarly, rsv-infected perforin-deficient mice exhibited prolonged weight loss and symptom illness scores compared to wild-type mice (100) . tnf contributes substantially to immunopathology, as antibody-mediated depletion of tnf prior to either rsv or iav infection significantly reduced weight loss (101, 102, 118) . additionally, mice that are ifn-γ-deficient, depleted of ifn-γ, or received an adoptive transfer of ifn-γ-deficient cd8 t cells prior to rsv infection lost significantly less weight than controls (89) . cd8 t cell production of tnf and ifn-γ following pvm infection also induced pulmonary immunopathology by initiating a cytokine storm (119) . in addition to causing disease in acute respiratory infections, ifn-γ produced by memory cd8 t cells mediated the severe and fatal immunopathology following rsv infection of dc-lm prime-boosted mice (30) . the role of cd8 t cells in the development of pathology following respiratory infections in humans remains unclear. the best evidence supporting a pathogenic role for cd8 t cells in humans infected with respiratory viruses comes from a study evaluating an rsv-infected severe-combined immunodeficiency patient after bone marrow transplantation (92) . the patient exhibited increased cd8 t cell counts following bone marrow transplant, which corresponded to a sharp reduction in rsv nasal titers. however, the appearance of cd8 t cells also correlated with a marked increase in respiratory rate indicative of reduced pulmonary function. also supporting a pathogenic role of cd8 t cells is the finding that children requiring mechanical ventilation due to severe rsv infection expressed significantly increased levels of activated granzymes and more cd8 t cells producing granzyme b compared to healthy controls (120) . in contrast, a study of infants following either fatal iav or rsv infections revealed a near absence of cd8 t cells from affected lung regions by immunohistochemical staining (121, 122) . similarly, infants with severe rsv infection exhibited an underexpression of genes related to cd8 t cells in the peripheral blood (123) . in support of a protective, rather than pathogenic, role of cd8 t cells, correlations have been identified between increased cd8 t cell cytolytic activity and cytokine production with reduced symptom score, faster recovery, and fewer fatalities following h1n1 or h7n9 iav infections (93, 94) . therefore, whether cd8 t cells play a primary role in mediating pathology versus protection following human respiratory virus infection remains controversial and is an important topic of future investigation. given the potential of cd8 t cell effector functions to cause immunopathology following respiratory virus infection, the immune system has evolved critical regulatory mechanisms to prevent prolonged cd8 t cell effector activity following viral clearance. cd8 t cell effector functions, including production of ifn-γ and tnf, are suppressed in the lung following the resolution of iav and rsv infections (124) (125) (126) (127) . one of the primary mechanisms utilized to limit the cd8 t cell response is through suppression by regulatory cd4 t cells (tregs). tregs accumulate in the lungs following either rsv or iav infection peaking at approximately day 6 post-infection, prior to the peak of the cd8 t cell response (36, (128) (129) (130) . antibody-mediated depletion of cd25 + tregs prior to rsv infection resulted in exacerbated weight loss, pulmonary dysfunction, and lung inflammation (128) . this enhanced illness corresponded to an increased frequency of rsv-specific cd8 t cells and elevated levels of ifn-γ and tnf protein in the lung (36, 128) . consistent with the treg depletion studies, increasing rsv-specific tregs prior to rsv infection using rsv peptide-immunization resulted in an amelioration of weight loss and a reduction in cd8 t cell numbers in the blood and spleen, but not the lung (131) . tregs also can suppress cd8 t cell effector functions following a secondary infection with iav (130) . antibody-mediated cd25 + treg depletion prior to heterosubtypic iav challenge resulted in enhanced inflammation and pulmonary dysfunction corresponding to an increase in cd8 t cell numbers and ifn-γ production. one mechanism through which tregs may suppress cd8 t cell responses is through the production of the anti-inflammatory cytokine il-10. foxp3 + tregs secrete il-10 following primary infection with rsv or iav (130, (132) (133) (134) . infection of either il-10-deficient mice or mice treated with il-10 receptor blocking antibody resulted in increased numbers of either ifn-γ + or ifn-γ + tnf + cd8 t cells, suggesting that il-10 suppresses cd8 t cell effector functions following respiratory virus infection (132) (133) (134) . interestingly, il-10 production by foxp3 − cd4 t cells and cd8 t cells following either rsv or iav infection has also been reported, indicating that effector t cell responses may self-regulate their effector functions (132) (133) (134) (135) . together, these studies demonstrate that tregs and il-10 production play a critical role in regulating cd8 t cells following primary and secondary respiratory virus infections to prevent immunopathology. interactions between inhibitory receptors on cd8 t cells with their ligands represents another important mechanism mediating the inhibition of cd8 t cell effector functions following infection. regulation of cd8 t cells through the pd-1:pd-l1 pathway is a common inhibitory pathway utilized following respiratory virus infection. expression of pd-1 on pulmonary cd8 t cells is upregulated following rsv, iav, or hmpv infection in mice (41, 136, 137) . blockade of pd-l1 in primary rsv, iav, or hmpv and secondary hmpv infections results in enhanced cd8 t cell effector functions, including ifn-γ, tnf, and granzyme b production (41, [136] [137] [138] . cd8 t cell effector functions are also enhanced following either hmpv or iav infections in pd-1-deficient mice (41). importantly, the pd-1:pd-l1 pathway has also been associated with human cd8 t cell responses. human cd8 t cells in the nasal cavity significantly upregulated pd-1 following rsv infection compared to cd8 t cells from the blood of either healthy or rsv-infected individuals (137) . pd-1 and pd-l1 are also both upregulated in the lung figure 1 | critical factors for an optimal cd8 t cell-mediated respiratory syncytial virus (rsv) vaccine. a future rsv vaccine designed to elicit a cd8 t cell response will require a balance between cd8 t cell-mediated protection and immunopathology, which may be achieved through the consideration of three important aspects: (1) magnitude, (2) localization, and (3) regulation. an optimal magnitude of the cd8 t cell response will be one that achieves efficient viral clearance in the absence of immunopathology. the vaccination route will be critical in determining the localization of the cd8 t cell response. a pulmonary route of vaccination will induce trm in the lung that provides superior protection compared to a systemic immunization that would likely not generate protective trm. lastly, regulation of the cd8 t cell response generated through vaccination will be crucial, as uncontrolled effector functions, particularly ifn-γ production, can result in immunopathology. frontiers in immunology | www.frontiersin.org april 2018 | volume 9 | article 678 tissue following severe infections with either rsv or the 2009 h1n1 iav pandemic strain (41). in vitro human studies have demonstrated that pd-l1 is constitutively expressed on human airway and bronchial epithelial cells, but expression is significantly upregulated following either iav or rsv infection (136, 139) . similar to in vivo mouse studies, in vitro pd-l1 blockade resulted in significantly increased cd8 t cell production of ifn-γ, il-2, and granzyme b following rsv infection (139) . together, these studies demonstrate a critical role for pd-1 in the suppression of cd8 t cell-mediated immunopathology and cytokine production in both mice and humans. in the absence of pd-1 signaling following hmpv infection, cd8 t cell ifnγ production remains impaired, suggesting the involvement of compensatory inhibitory pathways (140) . antigen-specific lung cd8 t cells express inhibitory receptors tim-3, lag-3, and 2b4 following hmpv infection and exhibit enhanced cytokine production following in vitro blockade of each receptor individually (140) . in vivo blockade of lag-3 partially restored cd8 t cell ifn-γ production in pd-1-deficient mice following hmpv infection (140) . tim-3 has also been demonstrated to be critical in suppressing cd8 t cell responses in vivo, as tim-3 receptor (galectin-9)-deficient mice exhibited significantly enhanced cd8 t cell responses following both primary and secondary iav infections (141) . together, these studies indicate that multiple inhibitory receptor pathways are utilized following pulmonary virus infection to dampen the pathogenic cd8 t cell response and prevent immunopathology. successful vaccinations against the majority of respiratory viruses remain elusive. the goal of most vaccination strategies is to induce robust virus-specific neutralizing antibody responses. however, the antibody response generated by infection with many respiratory infections, including rsv and rv, wanes over time. therefore, neutralizing antibody responses as the sole mediator of a vaccine against most respiratory viruses may not provide long-term protection without yearly vaccination. vaccination strategies that include the induction of virus-specific cd8 t cell responses, either alone or in combination with humoral immunity, may be advantageous by providing many benefits associated with cellular immune responses. cd8 t cells are critical for the elimination of virusinfected cells, and viral clearance was prolonged in the absence of cd8 t cells following acute respiratory virus infections. additionally, robust memory cd8 t cell responses efficiently reduced lung viral titers in the absence of neutralizing antibodies following rsv, iav, or sars secondary infections. an important property of cd8 t cells is that they often recognize conserved viral proteins, allowing for cross-protection between different virus strains. this is particularly important for heterosubtypic protection of iav strains, as neutralizing antibodies are not capable of recognizing iav strains of differing subtypes. despite their benefits in mediating viral clearance and providing protection against secondary infections, memory cd8 t cell responses have been associated with the induction of immunopathology following respiratory virus infections. the same antiviral mechanisms employed by memory cd8 t cells to accelerate viral clearance also contribute to immunopathology, including the fas/fasl pathway-and perforin-mediated cytolysis and ifn-γ and tnf cytokine secretion. thus, the efficient elimination of respiratory viruses by memory cd8 t cells comes at a cost of disease for the host. cd8 t cell-mediated immunopathology appears to be virus-specific. although high frequency, systemic, antigen-specific memory cd8 t cells induced severe disease and mortality following rsv infection, no pathology was observed using similar systems for iav and sars infections. therefore, induction of memory cd8 t cells as the sole immune mediator may be particularly dangerous for an rsv vaccine, but significantly less so in either an iav or a sars vaccine. to be able to include cd8 t cell responses within a future respiratory virus vaccine, it will be extremely important to determine how to balance cd8 t cell-mediated protection versus immunopathology following respiratory infection. for rsv in particular, three critical aspects to consider in this balance include magnitude, localization, and regulation of the rsv-specific cd8 t cell response (figure 1) . dc-lm immunization generated m282-90-specific cd8 t cells at a frequency of approximately 20% in the peripheral blood, but induced fatal immunopathology following rsv challenge (30) . however, dc-prime only and trivax immunizations generated a much lower frequency of total m282-specific cd8 t cells, and rsv induced significantly reduced disease in these mice (30, 109) . thus, identifying the optimal magnitude of rsv-specific cd8 t cells for protection in the absence of immunopathology is crucial. it is also clear from recent studies in mouse models that localization of rsv-specific cd8 t cells is a significant factor for both their efficacy of mediating viral clearance and their ability to induce immunopathology following infection. intranasal immunization with mcmv-m generated trm within the lung tissue that accelerated viral clearance. in contrast, mice administered mcmv-m systemically did not generate trm and exhibited delayed viral clearance (78) . similar results were observed with local immunization with dc-iav-m282 (30) . m282-specific lung trm generated by pulmonary immunization did not induce immunopathology following rsv infection, in contrast to systemic dc-lm immunization, which resulted in severe pathology in the absence of trm cells. thus, vaccination strategies against rsv will likely be the most effective when administered through a pulmonary route to generate trm that will provide protection within the lung following reinfection without inducing immunopathology. lastly, identifying ways to regulate vaccine-generated cd8 t cell responses will likely reduce immunopathology following subsequent infection. ifn-γ produced by cd8 t cells was the primary mediator of immunopathology following rsv infection of dc-lm vaccinated mice (30) . however, neutralization of ifn-γ had no effect on lung viral titers, suggesting that cd8 t cells utilize other antiviral mechanisms to mediate viral clearance in this system. since cd8 t cells are able to reduce viral titers in the absence of ifn-γ, reducing the amount of ifn-γ produced by cd8 t cells would likely result in ameliorated disease following rsv infection. if vaccination strategies can identify mechanisms by which cd8 t cell cytokine production, particularly ifn-γ, can be attenuated without altering their ability to eliminate virusinfected cells, the pathology induced by cd8 t cells would likely also be decreased. the development of a cd8 t cell-mediated vaccine should be pursued given the limitations of antibody responses to respiratory viruses. it is possible that the ideal vaccine for respiratory virus infections will include the induction of both virus-specific cd8 t cells and neutralizing antibodies. a vaccination approach combining both arms of the adaptive immune response may allow for optimal viral control in the absence of disease symptoms. however, before cd8 t cells can be developed further as a mediator of protective immunity, the balance between protection and pathology must be achieved. future studies evaluating aspects of memory cd8 t cell magnitude, localization, and regulation will greatly assist in reaching this balance. both authors wrote and edited the manuscript. incubation periods of acute respiratory viral infections: a systematic review global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial viral infection in children with compromised immune function respiratory syncytial virus and influenza a infections in the hospitalized elderly respiratory syncytial virus infection in elderly and high-risk adults influenza-and respiratory syncytial virus-associated mortality and hospitalisations epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children < 5 years old parainfluenza viruses estimates of us influenza-associated deaths made using four different methods. influenza other respir viruses global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study viruses and bacteria in the etiology of the common cold human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children clinical disease in children associated with newly described coronavirus subtypes a novel coronavirus associated with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia risk of primary infection and reinfection with respiratory syncytial virus the role of human metapneumovirus in upper respiratory tract infec tions in children: a 20-year experience prolonged shedding of rhinovirus and re-infection in adults with respiratory tract illness changes in iga and igg concentrations in nasal secretions prior to the appearance of antibody during viral respiratory infection in man impaired antibody-mediated protection and defective iga b-cell memory in experimental infection of adults with respiratory syncytial virus antibody and th1-type cell-mediated immune responses in elderly and young adults immunized with the standard or a high dose influenza vaccine influenza nucleoprotein-specific cytotoxic t-cell clones are protective in vivo role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice mapping and characterization of the primary and anamnestic h-2d-restricted cytotoxic t-lymphocyte response in mice against human meta pneumovirus lung airway-surveilling cxcr3(hi) memory cd8+ t cells are critical for protection against influenza a virus virus-specific memory cd8 t cells provide substantial protection from lethal severe acute respiratory syndrome coronavirus infection memory cd8 t cells mediate severe immunopathology following respiratory syncytial virus infection central role of dendritic cells in shaping the adaptive immune response during respiratory syncytial virus infection differential roles of lung dendritic cell subsets against respiratory virus infection the effector t cell response to influenza infection visualization and characterization of respiratory syncytial virus f-specific cd8(+) t cells during experimental virus infection characterization of the cd8+ t cell responses directed against respiratory syncytial virus during primary and secondary infection in c57bl/6 mice regulatory t cells promote early influx of cd8+ t cells in the lungs of respiratory syncytial virus-infected mice and diminish immunodominance disparities the pulmonary localization of virus-specific t lymphocytes is governed by the tissue tropism of infection virus-specific cd8+ t cells in primary and secondary influenza pneumonia respiratory syncytial virus-specific cd8+ memory t cell responses in elderly persons nonspecific recruitment of memory cd8+ t cells to the lung airways during respiratory virus infections the chemokine receptor ccr5 plays a key role in the early memory cd8+ t cell response to respiratory virus infections airway-resident memory cd8 t cells provide antigen-specific protection against respiratory virus challenge through rapid ifn-gamma production cutting edge: effector memory cd8+ t cells in the lung airways retain the potential to mediate recall responses type i interferons regulate cytolytic activity of memory cd8+ t cells in the lung airways during respiratory virus challenge the who's who of t-cell differentiation: human memory t-cell subsets memory t cells in nonlymphoid tissue that provide enhanced local immunity during infection with herpes simplex virus lung niches for the generation and maintenance of tissue-resident memory t cells lung-resident memory cd8 t cells (trm) are indispensable for optimal cross-protection against pulmonary virus infection specific niches for lung-resident memory cd8+ t cells at the site of tissue regeneration enable cd69-independent maintenance long-term persistence and reactivation of t cell memory in the lung of mice infected with respiratory syncytial virus long-term maintenance of virus-specific effector memory cd8+ t cells in the lung airways depends on proliferation memory t cell populations in the lung airways are maintained by continual recruitment cutting edge: antigen is not required for the activation and maintenance of virus-specific memory cd8+ t cells in the lung airways in situ imaging reveals different responses by naive and memory cd8 t cells to late antigen presentation by lymph node dc after influenza virus infection smad4 promotes differentiation of effector and circulating memory cd8 t cells but is dispensable for tissue-resident memory cd8 t cells environmental and antigen receptor-derived signals support sustained surveillance of the lungs by pathogen-specific cytotoxic t lymphocytes enhanced survival of lung tissue-resident memory cd8+ t cells during infection with influenza virus due to selective expression of ifitm3 airway t cells protect against rsv infection in the absence of antibody vaccine-generated lung tissue-resident memory t cells provide heterosubtypic protection to influenza infection intranasal administration of rsv antigen-expressing mcmv elicits robust tissue-resident effector and effector memory cd8+ t cells in the lung reduced generation of lung tissue-resident memory t cells during infancy dynamics of influenza-induced lung-resident memory t cells underlie waning heterosubtypic immunity in vivo effector function of influenza virus-specific cytotoxic t lymphocyte clones is highly specific cytotoxic t cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus cd4+ t cells clear virus but augment disease in mice infected with respiratory syncytial virus. comparison with the effects of cd8+ t cells distinct types of lung disease caused by functional subsets of antiviral t cells the recovery of mice from influenza virus infection: adoptive transfer of immunity with immune t lymphocytes transfer of specific cytotoxic t lymphocytes protects mice inoculated with influenza virus recovery from a viral respiratory infection. ii. passive transfer of immune spleen cells to mice with influenza pneumonia clearance of persistent respiratory syncytial virus infections in immunodeficient mice following trans fer of primed t cells virus clearance and immunopathology by cd8(+) t cells during infection with respiratory syncytial virus are mediated by ifn-gamma cytotoxic lymphocytes in the lungs of mice infected with respiratory syncytial virus cellular response to respiratory viruses with particular reference to children with disorders of cell-mediated immunity quantitative effects of palivizumab and donor-derived t cells on chronic respiratory syncytial virus infection, lung disease, and fusion glycoprotein amino acid sequences in a patient before and after bone marrow transplantation cytotoxic t-cell immunity to influenza recovery from severe h7n9 disease is associated with diverse response mechanisms dominated by cd8(+) t cells cd8+ t cell effector mechanisms in resistance to infection cd8+ t cells clear influenza virus by perforin or fas-dependent processes multiple redundant effector mechanisms of cd8+ t cells protect against influenza infection role of tumor necrosis factor-related apoptosis-inducing ligand in immune response to influenza virus infection in mice cd8 t cells utilize trail to control influenza virus infection alternative mechanisms of respira tory syncytial virus clearance in perforin knockout mice lead to enhanced disease prolonged production of tnf-alpha exacerbates illness during respiratory syncytial virus infection inhibition of tumor necrosis factor reduces the severity of virus-specific lung immunopathology role of t cells in virus control and disease after infection with pneumonia virus of mice production of interferon-gamma by influenza hemagglutinin-specific cd8 effector t cells influences the development of pulmonary immunopathology the role of ifn in respiratory syncytial virus pathogenesis recombinant baculovirus-based vaccine expressing m2 protein induces protective cd8+ t-cell immunity against respiratory syncytial virus infection lung cd8+ t cell impairment occurs during human metapneumovirus infection despite virus-like particle induction of functional cd8+ t cells cytotoxic t-lymphocyte epitope vaccination protects against human metapneumovirus infection and disease in mice vaccine-elicited cd8+ t cells protect against respiratory syncytial virus strain a2-line19f-induced pathogenesis in balb/c mice induction of partial specific heterotypic immunity in mice by a single infection with influenza a virus heterosubtypic immunity to influenza type a virus in mice. effector mechanisms and their longevity profound protection against respiratory challenge with a lethal h7n7 influenza a virus by increasing the magnitude of cd8+ t-cell memory protective cross-reactive cellular immunity to lethal a/goose/guangdong/1/96-like h5n1 influenza virus is correlated with the proportion of pulmonary cd8+ t cells expressing gamma interferon cross-reactive t cells are involved in rapid clearance of 2009 pandemic h1n1 influenza virus in nonhuman primates natural t cell-mediated protection against seasonal and pandemic influenza. results of the flu watch cohort study natural reinfection with respiratory syncytial virus does not boost virus-specific t-cell immunity transgenic mice lacking class i major histocompatibility complex-restricted t cells have delayed viral clearance and increased mortality after influenza virus challenge the chemokine mip1alpha/ccl3 determines pathology in primary rsv infection by regulating the balance of t cell populations in the murine lung animal model of respiratory syncytial virus: cd8+ t cells cause a cytokine storm that is chemically tractable by sphingosine-1-phosphate 1 receptor agonist therapy activation of the granzyme pathway in children with severe respiratory syncytial virus infection severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection respiratory syncytial virus infection suppresses lung cd8+ t-cell effector activity and peripheral cd8+ t-cell memory in the respiratory tract functional impairment of cytotoxic t cells in the lung airways following respiratory virus infections impairment of the cd8+ t cell response in lungs following infection with human respiratory syncytial virus is specific to the anatomical site rather than the virus, antigen, or route of infection regulation of cytokine production by virus-specific cd8 t cells in the lungs foxp3+ cd4 regulatory t cells limit pulmonary immunopathology by modulating the cd8 t cell response during respiratory syncytial virus infection influenza a virus infection results in a robust, antigen-responsive, and widely disseminated foxp3+ regulatory t cell response antigen-specific memory regulatory cd4+foxp3+ t cells control memory responses to influenza virus infection epitope-specific regulatory cd4 t cells reduce virus-induced illness while preserving cd8 t-cell effector function at the site of infection effector t cells control lung inflammation during acute influenza virus infection by producing il-10 multiple cd4+ t cell subsets produce immunomodulatory il-10 during respiratory syncytial virus infection il-10 regulates viral lung immunopathology during acute respiratory syncytial virus infection in mice cd8+ treg cells suppress cd8+ t cell-responses by il-10-dependent mechanism during h5n1 influenza virus infection local blockade of epithelial pdl-1 in the airways enhances t cell function and viral clearance during influenza virus infection control of pathogenic effector t-cell activities in situ by pd-l1 expression on respiratory inflammatory dendritic cells during respiratory syncytial virus infection programmed death-1 impairs secondary effector lung cd8+ t cells during respiratory virus reinfection rsv-induced bronchial epithelial cell pd-l1 expression inhibits cd8+ t cell nonspecific antiviral activity multiple inhibitory pathways contribute to lung cd8+ t cell impairment and protect against immunopathology during acute viral respiratory infection t cell immunoglobulin and mucin protein-3 (tim-3)/ galectin-9 interaction regulates influenza a virus-specific humoral and cd8 t-cell responses key: cord-262673-j2ot35lt authors: ahmed-hassan, hanaa; sisson, brianna; shukla, rajni kant; wijewantha, yasasvi; funderburg, nicholas t.; li, zihai; hayes, don; demberg, thorsten; liyanage, namal p. m. title: innate immune responses to highly pathogenic coronaviruses and other significant respiratory viral infections date: 2020-08-18 journal: front immunol doi: 10.3389/fimmu.2020.01979 sha: doc_id: 262673 cord_uid: j2ot35lt the new pandemic virus sars-cov-2 emerged in china and spread around the world in <3 months, infecting millions of people, and causing countries to shut down public life and businesses. nearly all nations were unprepared for this pandemic with healthcare systems stretched to their limits due to the lack of an effective vaccine and treatment. infection with sars-cov-2 can lead to coronavirus disease 2019 (covid-19). covid-19 is respiratory disease that can result in a cytokine storm with stark differences in morbidity and mortality between younger and older patient populations. details regarding mechanisms of viral entry via the respiratory system and immune system correlates of protection or pathogenesis have not been fully elucidated. here, we provide an overview of the innate immune responses in the lung to the coronaviruses mers-cov, sars-cov, and sars-cov-2. this review provides insight into key innate immune mechanisms that will aid in the development of therapeutics and preventive vaccines for sars-cov-2 infection. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) reportedly emerged at a live animal market in the chinese city of wuhan is currently causing a pandemic and negatively affecting global health (1) (2) (3) . there are more than 11 million confirmed sars-cov-2 infections with an mortality rate that widely varies by country and region (4) . even in industrialized countries, sars-cov-2 led healthcare systems approach the brink of collapse by overwhelming their capacity and straining resources. governments and local leaders ordered the shutdown of cities, regions, countries leading to massive disruptions in the local and global economy. unlike previous coronavirus (cov) infections, the rapid global spread, high transmission rate, longer incubation time, and disease severity of sars-cov-2 requires a better understanding of the epidemiology and immunopathogenesis of this viral outbreak in order to learn from this experience and to manage future pandemics. sars-cov-2 is a highly pathogenic cov (5) (case-fatality rate of 3.6-3.8%) (4, 6) that is related to severe acute respiratory syndrome cov (sars-cov) (case-fatality rate of 14-15%) and the middle east respiratory syndrome cov (mers-cov) (case-fatality rate of 34.4%), see also table 1 (138, 139) . sars-cov, sars-cov-2 and mers-cov target the lower respiratory system, causing respiratory illnesses, including severe pneumonia, acute lung injury (ali) and acute respiratory distress syndrome (ards) (140, 141) . sars-cov-2 infection results in higher hospitalization rates in the elderly (>65) and persons with pre-existing conditions including hypertension, diabetes and obesity compared to rates among younger populations without pre-existing conditions ( table 1 ) (142, 143) . in addition to an age disparity, males with covid-19 appear to have higher risk for worse outcomes and death (143, 144) . epidemiological research of the sars and mers infections also showed that males had a higher mortality rate than females (144) (145) (146) . while sars-cov-2 is a novel coronavirus, several important insights have already been made about its basic mode of transmission. virus particles are inhaled in respiratory droplets expelled from the airways of infected individuals. angiotensinconverting enzyme 2 (ace2), expressed on the ciliated bronchial cells, endothelial cells, and type i and ii alveolar cells, is the host receptor for cell entry into the respiratory tract by both sars-cov-2 and sars-cov ( table 1 ) (147) (148) (149) (150) . the spike protein (s) of cov is responsible for the entry of the virus into the target cell (figure 1 ) (147, 151) . ace2 is a type i transmembrane metallocarboxypeptidase that plays a vital role in the renin-angiotensin system (ras) (152, 153) , which in turn is critical in maintaining blood pressure homeostasis as well as fluid and salt balance in mammals. ace2 is found in vascular endothelial cells, in the renal tubular epithelium, and in leydig cells of the testes (154) . studies have shown that ace2 is expressed in gastrointestinal (gi) tissues, making it a potential site for harboring sars-cov (155) . this may be one of the reasons for gi pathology reported in some patients with covid-19 and viral shedding in stool. in contrast, mers-cov uses dipeptidyl-peptidase 4 (dpp4) as an entry receptor, which is expressed on endothelial cells and some epithelial tissues ( table 1 ) (19, 156) . accumulating data suggest that the innate anti-viral response and adaptive immunity may contribute to a cytokine storm leading to systemic hyper inflammation and exacerbation of the disease in patients with (a) comorbidities (b) older than 65 years of age (c) of the male sex. the exact role of the innate immune system in disease pathogenesis and prevention between the sexes and the impact of age is not fully elucidated. in addition, the potential dysregulation of the innate immune response by sars-cov and sars-cov-2 is not completely understood which warrants further research. the cells of the airway epithelium are the first line of defense, providing a mechanical barrier (mucociliary escalator) that expels particles and pathogens via cilia, mucus, and induced coughing (157, 158) . this barrier includes cells of the pulmonary epithelium and tissue-resident macrophages and dendritic cells (dcs). the macrophages and dcs express pattern recognition receptors (prrs) that can detect molecules from pathogens (pathogen-associated molecular patterns-pamps) or molecules released from damaged cells (damage or danger-associated molecular patterns-damps) (158) (159) (160) . in the lung, there are two populations of macrophages, alveolar and interstitial macrophages (161) . in addition to these macrophages, dcs play a vital role in facilitating the host defenses against respiratory diseases (162) (163) (164) . dcs can be divided into plasmacytoid (pdc) and myeloid types (mdc) (165) (166) (167) . macrophages and the two dc subtypes trigger antiviral responses by generating a substantial amount of type i interferon and these cells play important roles in immune surveillance in the airways and the distal lung (74, (168) (169) (170) (171) (172) . during steady state, the dc density in lung associated tissue declines from the trachea to the alveoli (173) while the representation of cells in macrophage compartment seems more complex (174) . if a virus infects airway epithelial cells, the viral rna would be sensed via intrinsic innate receptors, including rig-1, mda5, nlrp3 inflammasome, and the rna sensing tlrs 7 and 8. in the case of influenza a infection, triggering the prrs causes a strong induction of type 1 interferon (ifn) in epithelial cells (175, 176) . in other viral infections, such as respiratory syncytial virus (rsv), alveolar macrophages are the predominant source of type 1 ifns (161). furthermore, respiratory epithelial cells and lung macrophages are capable of secreting a broad range of chemokines like il-8, macrophage inflammatory protein-1 (mip-1), rantes and cytokines including tnf-α, il-6, il-1β that influence the types of immune cells being recruited to the area in response to acute viral infections (177, 178) . macrophages, depending on their polarization status of either m1 or m2, and in a similar way as airway epithelial cells, can further elicit a th1 or th2 response (158, 161, 178) . in the case of influenza virus infection, the magnitude of epithelial cell response can be proportional to the amount of virus which result in paracrine induction of ifn λ (175) . not only can airway epithelial cells produce a large array of cytokines/chemokines in response to an acute viral infection, but, depending on the magnitude of prr engagement and the combination of pamps and damps triggered, these epithelial cells can modulate the type of chemokines/cytokines produced and influence the influx of innate and adaptive immune cells (158, 160) . the response to different viral infections is generally similar, however, the response can be tailored in timing, magnitude and the induced gene signatures in response to each virus (179) . unlike rsv and mers-cov, which both productively infect alveolar multiple cell types in the lower respiratory tract were found to be infected, including type i alveolar epithelium, macrophages, and putative cd34 + oct-4 + stem/progenitor cells in human lungs (15) (16) (17) ciliated bronchial epithelial cells and type ii pneumocytes (7, 18) un-ciliated bronchial epithelial cells and type ii pneumocytes (19) (20) (21) infect mostly human type i and type ii pneumocytes and alveolar macrophages (22) respiratory, nasal, corneal and intestinal epithelial cells (23) club cells, ciliated cells, type i and type ii alveolar cells (24) ciliated epithelial cells of the upper and lower respiratory tract (25) the ciliated cells of the human airway epithelium are the main target, it also infects basal cells (26) and immune cells, such as macrophages, b cells cd4 + and cd8 + t cells (27) upper and lower airways epithelial cell ( no specific antiviral drugs (134) antiviral drugs may be a treatment option (135) no antiviral agents symptomatic treatment supportive care (136) there are no approved antiviral medications (137) ace2 macrophages (73, 180) , seasonal influenza and sars-cov usually lead to non-productive infections in these cells (181) . in addition, sars-cov infection of primary monocytes yielded little virus, likely due to the suppressive effects of ifn-α (182) . thus, the initial cell type(s) involved in propagating a viral infection intensifies the complexity of the immune response. another key factor that determines the magnitude of the immune response is the induction and rate of cell death. although related, mers-cov induces widespread cell death when compared to sars-cov in human bronchial epithelial cell cultures (183) . however, the sars-cov open reading frame 3a (orf3a) protein can induced necrotic cell death in a variety of cell lines (184) . the same orf3a protein can activate the nlrp3 inflammasome, leading to activation of nf-κb and elevated secretion of active il-1β in cell culture (185) . cytokine (186), with slightly different cytokine/chemokine profiles. this delay in cytokine induction was confirmed in another study using the same epithelial cell lines (187) as well as in human alveolar type ii cells (18) . in both cell lines and primary alveolar type ii cells, sars-cov induced ifn-β, ifn-λ, cxcl10, cxcl11, il-6, ip-10, and tnf-α (18, 187) . mers-cov did not induce ifn-β but induced higher level of il-8 transcript in cell culture. however, no difference in il-8 production was observed between sars-cov and mers-cov at 48 h post-infection (186) . this was confirmed in-vivo using a non-human primate model comparing the immune responses to sars-cov infection between young adult cynomolgus macaques (3-5 yrs) and older macaques (10-19 yrs) (188) . interestingly, the high induction of il-8 was observed on a transcript level in the older animals, while the younger once showed higher levels of ifn-β transcript (188) . in all animals, the expression of ifn-β was inversely correlated with the pathology score, supporting the role of ifn-β in controlling disease severity (188) and introducing a potential area of research to define age disparity observed in patients infected with sars-cov-2. both older age and male sex are important factors associated with high mortality of sars-cov and sars-cov-2 infection (189, 190) . many viruses have evolved to disrupt and subvert the immune responses. a common virus that is well-known to affect the lower airway and counteract the immune response is rsv (178, 191) . the rsv genome encodes non-structural proteins ns1 and ns2 that can block type 1 ifn production and signaling in cell cultures (191) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat1 and stat2 signaling in cell lines (192) , whereas mers-cov m protein also suppresses type 1 ifn by inactivating irf3 (193) , leading to the low expression of ifn-β. in contrast to reports in epithelial cell lines or primary alveolar type ii cell culture and observations in non-human primates, sars-cov nucleocapsid (n) protein and membrane (m) protein, as well as nsp1, can suppress ifn response via various mechanisms in cell lines (194) (195) (196) . to bridge the dichotomy of inhibition of ifn signaling in cell lines, and the ifn expression in vivo, cells recruited by the infection need to be considered as a potential source. as previously discussed, infected epithelial cells via paracrine signaling to neighboring cells and resident macrophages, secrete chemokines and cytokines to attract other immune cells. in general, monocytes/macrophages are recruited by ccl3 (mip-1a), ccl2 (mcp-1), and neutrophils are recruited by il-8 (cxcl8), cxcl2, and cxcl5 chemokines, all of which can be secreted by airway epithelial cells (178, 197, 198) . both monocytes and neutrophils are also recruited by complement fragment (anaphylatoxin) c5a (figure 1) . both influenza and sars virus can induce acute lung injury (ali) which is accompanied by high levels of c5a, leading to the influx and activation of innate immune cells (199) (figure 1) . serum samples and lung tissue of sars patients showed high-level expression of cxcl10 (ip-10), which is also found to be induced by sars-cov in the epithelial cell line calu-3 (200) . significant neutrophil, macrophage and cd8 t-cell infiltration can be found in the lung of sars patients by immunohistochemistry (76, 77, 201) . in addition to post-mortem lung histology analysis in patients with sars-cov, experiments using rhesus macaques infected with sars-cov found monocyte and macrophage recruitment. the accumulation of pathogenic inflammatory monocytemacrophages (imms) was also observed in a sars-cov mouse model. the accumulation of imms resulted in heightened lung inflammatory cytokine/chemokine levels, extensive vascular leakage, and impaired virus-specific t cell responses (202) . a strong infiltration of cd68 and mac387 positive monocytes/macrophages were found in the human and animals lung samples (203, 204) . macrophages further stimulate fibroblasts to deposit extracellular matrix leading to pulmonary fibrosis (205) , which was also observed in patients who recovered from sars (206, 207) . autopsy samples acquired from patients with sars-cov-2 patients contained viral nucleocapsid protein (np) positive cd68 + macrophages in the capillaries of the spleen and in lymph nodes, indicating that sars-cov-2 might migrate into the spleen and lymph nodes through macrophages. this study also found that cd169 + macrophages appear to mediate sars-cov-2 into these tissue sites, contributing to virus dissemination (208) . similar to sars-cov-2, sars viral particles and genomic sequences were detected in monocytes, macrophages as well as within different organs of sars patients (15) . sars-cov was shown to infect both immature and mature human monocyte-derived dcs by electron microscopy and immunofluorescence. the detection of negative strands of sars-cov rna in dcs suggests viral replication, but no increase in viral rna was observed (209) . as mentioned above, there was no perceivable increase to sars-cov replication in primary monocytes (182) . another study looked at sars-cov and mers-cov replication in human macrophages, human monocyte-derived macrophages, and dendritic cells (mddcs) and found that both viruses replicated poorly. mers-cov induced ifn-λ1, cxcl10, and mxa mrnas in both macrophages and mddcs, however, sars-cov was unable to induce such responses (69) . interestingly, depletion of inflammatory monocyte-macrophages in the mouse model partially protected from lethal sars infection (210) . these data suggest that monocytes, macrophages and dendritic cells have essential roles in cov infection. the severity of disease and the response to these viruses seems to be dependent on the induced cytokine/chemokine profiles and the amplification of the immune response by the recruited cells. growing evidence of dysregulation of an innate anti-viral response originates from studies using clinical samples (211) and murine models (202, 212, 213) . in addition to dysregulated cellular responses, the complement system may play an important role in sars-cov-2 infection (figure 1) . evidence comes from sars infected patients who had lower levels of mannan binding lectin (mbl) in serum compared to healthy controls (214) . the sars patients with a higher frequency of mbl gene polymorphisms were associated with lower serum levels or deficiency of mbl (214) . it is still unknown if this is also true for covid-19 patients, which requires further investigation. in cell culture experiments sars-cov was able to bind and activate the complement cascade and block viral infection (214) . preliminary findings in a limited number covid-19 patients found significant deposits of the membrane attack complex (mac), c4d and mbl-associated serine protease (masp)2 in the microvasculature indicating sustained, systemic activation (215) . the sars-cov-2 spike protein was co-localized with c4d and mac (215) . in a non-peer reviewed publication by gao et al., mers-cov, sars-cov and sars-cov-2 n protein are able to bind to masp-2 leading to enhanced complement activation (216) (figure 1) . in a later phase of the infection, the complement system might be also triggered via antibodies bound to the virus (classic activation pathway, figure 1 ). this excessive complement activation might play a role in multi organ damage in severe covid-19 cases (217) . in a mers-cov mouse model the blockade of the c5a-c5ar axis alleviated not only lung damage but also spleen damage (218) . mice treated with a monoclonal antibody to c5a showed reduced lung infiltration of cd68 + cells and significantly lower cytokine levels of il-1 β, tnf-α, inf-γ and il-12 (218) . complement blockade might be an important way to curb part of the immune dysregulation associated with covid-19. overall, we need to look closer at the role of the complement system, the recruited innate immune cells and their combined role in pathogenesis, viral clearance and the eventual resolution of the infection. the most abundant leukocytes, neutrophils, play a critical role in clearing viral infections. neutrophils, attracted by chemokines/cytokines released by tissue-resident macrophages and dcs, swarm to the site of infection. they recognize and release bioactive compounds, including cytokines, chemokines and ros, as well as nos in the very early phase of the infection (219, 220) . neutrophils modulate other innate and adaptive immune responses via cytokine/chemokine release and cell death and, therefore, can ameliorate or exacerbate disease progression. neutrophils infiltrate tissues infected by cov, including sars-cov, rat coronavirus (rcov), and mouse hepatitis virus (mhv). a high neutrophil count in the blood of sars patients at the time of hospital admission is associated with a poor prognosis (221, 222) . gao et al. suggested that patients with sars-cov-2 pneumonia can be stratified by neutrophil to lymphocyte ratio (nlr) and age (216) . patients older than 50 years of age and having an nlr ≥ 3.13 had more severe illness, so rapid access to the intensive care unit is required (79, 223) . experiments in mice showed that sars-cov disease severity in older mice correlated with increased pulmonary inflammation and influx of neutrophils (224, 225) . infection of rats with rcov could lead to neutrophil infiltrating into the respiratory tract early after inoculation, followed by the recruitment of macrophages and lymphocytes (226) . infection of mice with a neurotropic murine cov (mhv-jhm) showed infiltration of neutrophils into the brain as early as the first day after inoculation, which then promoted the recruitment of other types of inflammatory cells into the brain, likely through the loss of the blood-brain barrier integrity (227) . gene expression analysis in experimentally infected rhesus macaques with mers-cov revealed the recruitment of neutrophils into infected lung tissue (228, 229) . angiotensin-converting enzyme inhibitors (ace-is) could serve as a potential risk for fatal covid-19 through the upregulation of ace2 (230) and may provide a direct linkage to neutrophils and disease progression. investigators found that ace2 modulates il-17-mediated neutrophil influx by impacting stat3 activity (231) . animal models used to study the pathogenesis of sars-cov-2 have revealed important roles of neutrophils in infection and confirmed findings observed in patients. a new aspect in sars-cov-2 infection is the potential role of neutrophil extracellular traps (nets). the process of net formation is a specific type of cell death that can be triggered under inflammatory conditions (232, 233) , such as gm-csf+c5a, il-8, ifn-α+c5a or other tlr response mediators; all conditions present in severe sars-cov-2 infection (232, 233) . the net formation has been observed in covid-19 patients and may contribute to thrombotic complications in covid-19 patients (234, 235) . microvascular injury and thrombosis have been reported in covid-19 patients, increasing the likelihood that neutrophil net formation might play a role (215, 236, 237) . net formation was reported to be involved in clot formation and thrombosis and can further increase inflammation (232, 233) . therefore, neutrophils can attract a second wave of immune cells to the site of infection by cytokine/chemokine secretion as well as via netosis (238, 239) , which included monocytes and natural killer cells. on the other hand aggregated nets were reported to limit inflammation by degrading cytokines and chemokines and disrupting neutrophil recruitment and activation (240) . despite the presence of neutrophils in sars-cov-2-infected tissues, their role in the clearance and/or immunopathology of the viral infection remains unclear. future studies on the responses of neutrophils to sars-cov-2-infection or infected cells in vitro may elucidate the role of neutrophils in the pathogenesis of respiratory cov infections. natural killer (nk) cells are a heterogenic immune cell subset that acts promptly to combat viral infections. they produce significant amounts of ifn-γ, kill virus-infected cells, provide direct support to other innate immune cells, and aid in the adaptive immune response to counter viruses. nk cells express activating receptors that detect viral antigens, enabling the destruction of infected cells (241) (242) (243) (244) . lectin-like receptor cd94 and killer immunoglobulin-like receptors, such as cd158b, regulate the function of nk cells. a study of 221 patients with sars explored the relationship of the number of nk cells and the expression level of their immunoglobulin-like receptor cd158b in the peripheral blood to the severity of sars (245) . the overall count of nk cells and cd158 + nk cells and the percentage of cd158 + nk cells in patients with sars were significantly lower than counts in healthy subjects (245) . a separate study that analyzed lymphocytes and lymphocyte subsets in a cohort of 38 patients with sars observed reduced nk cell counts in 21 patients (55%) (246) . clinical reports reveal that children appear to have a milder form of sars-cov-2, with peripheral blood lymphocyte levels remaining in the normal range, suggesting less immune dysfunction following the disease (247) . this could be attributed to healthy children expressing lymphocytes, especially nk cells, in a greater quantity compared to healthy adults (248) . interestingly, previous studies found rapid and significant restoration of lymphocyte subsets including, nk cells, in peripheral blood in patients recovering from the initial stages of sars infection (249) . although the primary mechanism for the decrease in nk cells and other subsets during disease onset remains unknown, their contribution to sars-cov-2 needs further study especially from a treatment perspective. innate lymphoid cells (ilcs) are a family of innate immune cells that include ilc1, ilc2 and ilc3. although ilc2 facilitates lung repair after injury, the role of ilcs during respiratory viral infection is not clearly defined (250) . evidence for the potential involvement of ilc2 cells in the lung during viral infection was reported in a murine model (251) . this study found a rapid accumulation of ilc2 cells in the lung after an influenza virus infection, however their initial contribution to exacerbation of the disease was limited (251) . a recent study identified an interaction between ace2-expressing sars-cov-2 target cells and ilcs in the colon (252) . thus, elucidating the role of ilc subsets will be important in understanding the pathogenesis of sars, sars-cov-2 and mers infections. there is distinct evidence indicating an important role of ifns in sars and other cov infections (201, 253) . the sera of patients with sars revealed the presence of high levels of il-1, il-6, infγ, ccl2, cxcl10, and il-8 and products of interferon stimulated genes (254, 255) . high expression levels of isgs such as cd58, ifnar1, and ifngr1 and ifn-stimulated chemokines cxcl10 and ccl2 were observed in another cohort of sars patients and were correlated with the severity of pathogenesis (256) . significant upregulation of cxcl10 gene expression was observed in the severe phase of patients who died from sars. this data is corroborated by studies in patients with mers that found upregulation of cxcl10 in the serum of patients who developed pneumonia (254) . cxcl10 and infα were also correlated with severity of disease (255) . the importance of ifn signaling in response to cov infection has been well-demonstrated in several knockout mouse models. type i, ii, and iii ifn signaling deficient mice have increased susceptibility to mouse-adapted sars-cov strains (257, 258) . studies using mice lacking the ifnar1 and ifnlr1 or stat1 identified higher sars-cov replication in the lungs and delayed virus clearance (259, 260) . another study with mers-cov in mice expressing the human dpp4 receptor showed a role for the ifnar1 in viral clearance and lung inflammation (112) . these mouse models suggest an important role of ifn response for cov clearance. this quick expanding medical literature is very suggestive of an important role of ifn responses for cov control and clearance. many viruses have evolved to disrupt and subvert the immune response. rsv counteracts the immune response (178, 191) ; as discussed earlier, the rsv genome encodes non-structural proteins (ns1 and ns2) that are able to block type 1 ifn production and signaling in cell cultures (191) . similar to rsv, the measle virus v protein blocks ifn-α and β signaling by inhibiting stat1 and stat2 signaling in cell culture lines (192) . covs have developed several ways to escape from innate immune pressure. mers-cov m protein suppresses type 1 ifn by blocking the irf3 activation (193) , explaining the low expression of ifn-β. in various cell lines, sars-cov nucleocapsid (n) protein, membrane (m) protein, as well as nsp1, were reported to suppress ifn response (194, 196, 261) . the nucleocapsid protein (n) of sars-cov interferes with the function of irf3. although it does not form a complex with rig-i or mda5, rna binding activity at the initial recognition stage of viral rna potentially contributes to immune evasion (261, 262) . aside from the hcov, structural proteins, accessory, and non-structural proteins (nsp) are involved in innate immunity modulation. in both sars-cov and mers-cov, host mrna endonucleolytic cleavage is promoted by nsp1 protein, which modifies capped non-viral rnas (263, 264) . nsp1 in sars-cov prevents host mrna translation by interacting with the 40s subunit of the ribosome; in turn, transcription and translation of viral rna is given preference over the host mrna (263) . another study found that additional sars-cov nsp1 residues interfered with ifn-dependent signaling (265) . ifn production is affected by nsp3 proteins in sars-cov and mers-cov. these proteins have both papain-like protease (plpro) and a plp2 domain, and the plpro domains in both sars-cov and mers-cov downregulate mrna levels of ccl5, infβ, cxcl10, and other pro-inflammatory cytokines (266) . the suppression of ifn responses by sars-cov plpro is due to the inhibition of phosphorylation of ifn-regulatory factor 3 (irf3) and its subsequent translocation to the nucleus where it enhances ifn gene transcription (267) . mers-cov plpro also suppresses rig-i and mda5 and antagonizes ifn induction (266, 268) . in hcov-229e and sars-cov suppression of ifn responses, the key molecule is a adp-ribose-1-monophosphatase macrodomain encoded within nsp3 (269) . accessory proteins are not key in viral replication; however, in human cov, this group of proteins are involved in diverse cellular signaling, including cell proliferation, apoptosis, and ifn signaling (270) . by downregulating phosphorylation and nuclear translocation of irf3, open reading frame orf3b and -6 interfere with ifnβ synthesis and prevent ifnβ-induced activation of ifnstimulated response element (isre) in the promoter of isg in sars-cov (262) . in cells transfected with orf4a, -4b, and -5 of mers-cov, ifnβ promoter-driven luciferase activity is significantly reduced, and it may follow a similar pattern of suppression of irf3 nuclear translocation (141) . therefore, the suppression of signaling events in infected immune and airway epithelial cells, as well as the magnitude of suppression due to elevated expression levels of these accessory proteins, needs to be further elucidated to understand delayed or hyperimmune responses and cytokine storm that occurs in cov infection. in addition to revealing our unpreparedness of handling a worldwide pandemic by a viral infection, covid-19 exposed our lack of understanding of the pathogenesis of diseases as well as the host immunity. the interaction of the host innate immune system and other factors including age, sex, and pre-existing conditions need further investigation regarding disease severity and morbidity of sars/mers and covid-19. disease severity and its related progression are further associated with dysregulation of multiple components of both innate and adaptive immune responses leading to a cytokine storm and severe pathology. for the development of a therapeutic intervention, it is vital to elucidate the interplay among the different layers of the innate immune response and their relation to the clinical factors associated with increased morbidity and mortality in cov infection. investments in basic science research are needed to help elucidate the roles of different immune cells, and their contribution to disease severity; it will pave the way to prevent or abrogate cov outbreaks and potentially new viruses. nl, td, dh, zl, and nf performed the literature search, analyzed the literature, and wrote the manuscript. ha-h, bs, yw, and rs performed the literature search and wrote the manuscript. all authors contributed to the article and approved the submitted version. a novel coronavirus outbreak of global health concern a novel coronavirus from patients with pneumonia in china korean society of epidemiology -nco vtft. an interim review of the epidemiological characteristics of 2019 novel coronavirus cross-country comparison of case fatality rates of covid-19/sars-cov-2. osong public health res perspect understanding of covid-19 based on current evidence current status of epidemiology, diagnosis, therapeutics, and vaccines for novel coronavirus disease 2019. (covid-19) angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structural basis for the recognition of sars-cov-2 by full-length human ace2 the mers-cov receptor dpp4 as a candidate binding target of the sars-cov-2 spike. iscience the sialic acid binding activity of human parainfluenza virus 3 and mumps virus glycoproteins enhances the adherence of group b streptococci to hep-2 cells cx3cr1 as a respiratory syncytial virus receptor in pediatric human lung rhinoviruses and their receptors multiple organ infection and the pathogenesis of sars a novel subset of putative stem/progenitor cd34+oct-4+ cells is the major target for sars coronavirus in human lung time course and cellular localization of sars-cov nucleoprotein and rna in lungs from fatal cases of sars innate immune response of human alveolar type ii cells infected with severe acute respiratory syndrome-coronavirus dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc anaesthetic and medico-legal problems in patients intoxicated by alcohol reverse genetics with a full-length infectious cdna of the middle east respiratory syndrome coronavirus comparative replication and immune activation profiles of sars-cov-2 and sars-cov in human lungs: an ex vivo study with implications for the pathogenesis of covid-19 sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes investigating influenza a virus infection: tools to track infection and limit tropism parainfluenza virus infection respiratory syncytial virus can infect basal cells and alter human airway epithelial differentiation respiratory syncytial virus (rsv) infects cd4+ t cells: frequency of circulating cd4+ rsv+ t cells as a marker of disease severity in young children the emerging role of rhinoviruses in lower respiratory tract infections in children -clinical and molecular epidemiological study from croatia summary table of sars cases by country q&a: influenza and covid-19 -similarities and differences epidemiology of acute respiratory infections in children of developing countries 2019 case definition coronavirus as a possible cause of severe acute respiratory syndrome epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia association between age and clinical characteristics and outcomes of covid-19 distribution of influenza virus types by age using case-based global surveillance data from twenty-nine countries human parainfluenza virus 4 outbreak and the role of diagnostic tests age-specific incidence rates and risk factors for respiratory syncytial virusassociated lower respiratory tract illness in cohort children under 5 years old in the philippines. influenza other respir viruses respiratory syncytial virus infection in adults human rhinovirus c: age, season, and lower respiratory illness over the past 3 decades who. consensus document on the epidemiology of severe acute respiratory syndrome (sars) who. contract mers transmission and risk factors: a systematic review estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature transmission of respiratory syncytial virus among children under 5 years in households of rural communities, the philippines lethal respiratory disease associated with human rhinovirus c in wild chimpanzees investigation of a nosocomial outbreak of severe acute respiratory syndrome (sars) in toronto comparison of incubation period distribution of human infections with mers-cov in south korea and saudi arabia incubation period and other epidemiological characteristics of 2019. novel coronavirus infections with right truncation: a statistical analysis of publicly available case data incubation periods of acute respiratory viral infections: a systematic review human parainfluenza viruses (hpivs) available online at transmission dynamics and control of severe acute respiratory syndrome q&as on covid-19 and related health topics clinical features and short-term outcomes of 144 patients with sars in the greater toronto area acute renal impairment in coronavirus-associated severe acute respiratory syndrome prevalence of comorbidities in cases of middle east respiratory syndrome coronavirus: a retrospective study respiratory tract cell-mediated immunity clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study prevalence of comorbidities and its effects in patients infected with sars-cov-2: a systematic review and meta-analysis hospitalized patients with 2009. h1n1 influenza in the united states severe morbidity and mortality associated with respiratory syncytial virus versus influenza infection in hospitalized older adults respiratory syncytial virus infection in elderly and high-risk adults clinical characteristics and outcomes of human rhinovirus positivity in hospitalized children detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients upper and lower respiratory tract infections by human enterovirus and rhinovirus in adult patients with hematological malignancies middle east respiratory syndrome coronavirus shows poor replication but significant induction of antiviral responses in human monocytederived macrophages and dendritic cells macrophages: a trojan horse in covid-19? depletion of alveolar macrophages during influenza infection facilitates bacterial superinfections activation of human macrophages by bacterial components relieves the restriction on replication of an interferon-inducing parainfluenza virus 5 (piv5) p/v mutant productive infection of isolated human alveolar macrophages by respiratory syncytial virus alveolar macrophage-derived type i interferons orchestrate innate immunity to rsv through recruitment of antiviral monocytes rhinovirus replication in human macrophages induces nf-kappabdependent tumor necrosis factor alpha production sars-cov regulates immune function-related gene expression in human monocytic cells modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro mers-cov pathogenesis and antiviral efficacy of licensed drugs in human monocyte-derived antigen-presenting cells dysregulation of immune response in patients with covid-19 in wuhan, china depressed human monocyte function after influenza infection in vitro infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3 human airway epithelial cells direct significant rhinovirus replication in monocytic cells by enhancing icam1 expression productive replication of middle east respiratory syndrome coronavirus in monocytederived dendritic cells modulates innate immune response heightened innate immune responses in the respiratory tract of covid-19 patients influenza a virus infection of human primary dendritic cells impairs their ability to cross-present antigen to cd8 t cells human parainfluenza virus type 2 vector induces dendritic cell maturation without viral rna replication/transcription innate immune components that regulate the pathogenesis and resolution of hrsv and hmpv infections human rhinoviruses inhibit the accessory function of dendritic cells by inducing sialoadhesin and b7-h1 expression complement activation contributes to severe acute respiratory syndrome coronavirus pathogenesis mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract innate immunity to influenza virus infection respiratory syncytial virus (rsv) and its propensity for causing bronchiolitis chemokines and inflammation in the nasal passages of infants with respiratory syncytial virus bronchiolitis elevated cytokine concentrations in the nasopharyngeal and tracheal secretions of children with respiratory syncytial virus disease haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways high levels of virus-specific cd4+ t cells predict severe pandemic influenza a virus infection infection and immunoregulation of t lymphocytes by parainfluenza virus type 3 protective and dysregulated t cell immunity in rsv infection rhinovirus has the unique ability to directly activate human t cells in vitro lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study middle east respiratory syndrome coronavirus (mers-cov): infection, immunological response, and vaccine development breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 a b cell population that dominates the primary response to influenza virus hemagglutinin does not participate in the memory response b-cell fate decisions following influenza virus infection respiratory syncytial virus infection in infants is associated with predominant th-2-like response innate immune signals modulate antiviral and polyreactive antibody responses during severe respiratory syncytial virus infection peripheral blood t and b lymphocyte subpopulations in infants with acute respiratory syncytial virus brochiolitis human rhinoviruses enter and induce proliferation of b lymphocytes neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection longitudinally profiling neutralizing antibody response to sars coronavirus with pseudotypes rapid generation of a mouse model for middle east respiratory syndrome persistence of antibodies against middle east respiratory syndrome coronavirus neutralizing antibodies against sars-cov-2 and other human coronaviruses protective antibodies against influenza proteins protective effect of antibody to parainfluenza type 1 virus respiratory syncytial virus neutralizing antibodies in cord blood, respiratory syncytial virus hospitalization, and recurrent wheeze the time course of the humoral immune response to rhinovirus infection dynamic changes in blood cytokine levels as clinical indicators in severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome the relationship between serum interleukins and t-lymphocyte subsets in patients with severe acute respiratory syndrome active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis contribution of cytokines to tissue damage during human respiratory syncytial virus infection induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies cytokines induced during influenza virus infection human parainfluenza virus serotypes differ in their kinetics of replication and cytokine secretion in human tracheobronchial airway epithelium human rhinovirus induced cytokine/chemokine responses in human airway epithelial and immune cells the deadly coronaviruses: the 2003. sars pandemic and the 2020 novel coronavirus epidemic in china evaluation and treatment coronavirus (covid-19) cdc. influenza vaccines -united states rhinovirus vaccination: the case against review of the clinical characteristics of coronavirus disease 2019. (covid-19) available online at respiratory syncytial virus: diagnosis, treatment and prevention update on human rhinovirus and coronavirus infections coronavirus-associated pneumonia in previously healthy children clinical disease in children associated with newly described coronavirus subtypes identification of a novel coronavirus in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia hospitalization rates and characteristics of patients hospitalized with laboratory-confirmed coronavirus disease presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area gender differences in patients with covid-19: focus on severity and mortality do men have a higher case fatality rate of severe acute respiratory syndrome than women do? the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor severe acute respiratory syndrome coronavirus infection of mice transgenic for the human angiotensin-converting enzyme 2 virus receptor human coronavirus nl63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis structure, function, and evolution of coronavirus spike proteins angiotensin-i-converting enzyme and its relatives receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus trilogy of ace2: a peptidase in the renin-angiotensin system, a sars receptor, and a partner for amino acid transporters a novel coronavirus associated with severe acute respiratory syndrome dipeptidyl-peptidase iv from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme dpp iv airway epithelial differentiation and mucociliary clearance respiratory epithelial cells orchestrate pulmonary innate immunity the role of toll-like receptors in the production of cytokines by human lung macrophages lung epithelial cells: therapeutically inducible effectors of antimicrobial defense pulmonary macrophages: key players in the innate defence of the airways the development and function of lungresident macrophages and dendritic cells contribution of dendritic cells in protective immunity against respiratory syncytial virus infection the contributions of lung macrophage and monocyte heterogeneity to influenza pathogenesis plasmacytoid dendritic cells: development, regulation, and function human dendritic cell subsets: an update the multifaceted biology of plasmacytoid dendritic cells role of dendritic cells in sars coronavirus infection plasmacytoid dendritic cells and type i ifn: 50 years of convergent history the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting macrophage polarization in virus-host interactions macrophages: sentinels and regulators of the immune system lung dendritic cells at the innate-adaptive immune interface lung interstitial macrophages: past, present, and future innate immune response to influenza virus at singlecell resolution in human epithelial cells revealed paracrine induction of interferon lambda 1 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna innate immunity in the respiratory epithelium the airway epithelium: soldier in the fight against respiratory viruses lung epithelial cells have virus-specific and shared gene expression responses to infection by diverse respiratory viruses helix 1 tryptophan variants in galleria mellonella apolipophorin iii synergy of il-27 and tnfalpha in regulating cxcl10 expression in lung fibroblasts sars-coronavirus replication in human peripheral monocytes/macrophages middle east respiratory syndrome coronavirus infection: virus-host cell interactions and implications on pathogenesis molecular mechanisms of cell death: central implication of atp synthase in mitochondrial permeability transition severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection exacerbated innate host response to sars-cov in aged non-human primates analysis on 54 mortality cases of coronavirus disease 2019 in the republic of korea from prevalence of sars-cov antibody in all hong kong patient contacts respiratory syncytial virus measles virus v protein blocks interferon (ifn)-alpha/beta but not ifn-gamma signaling by inhibiting stat1 and stat2 phosphorylation middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 severe acute respiratory syndrome coronavirus nsp1 suppresses host gene expression, including that of type i interferon, in infected cells severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ikkepsilon complex the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination neutrophil recruitment and function in health and inflammation host-pathogen interactions during coronavirus infection of primary alveolar epithelial cells the role of c5a in acute lung injury induced by highly pathogenic viral infections characterization of cytokine/chemokine profiles of severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) spatiotemporal interplay of severe acute respiratory syndrome coronavirus and respiratory mucosal cells drives viral dissemination in rhesus macaques location or origin? what is critical for macrophage propagation of lung fibrosis? thinsection ct in patients with severe acute respiratory syndrome following hospital discharge: preliminary experience pathogenesis of severe acute respiratory syndrome the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) directly decimates human spleens and lymph nodes. medrxiv chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome the pathogenicity of sars-cov-2 in hace2 transgenic mice pathogenesis of sars-cov-2 in transgenic mice expressing human angiotensin-converting enzyme 2 mannosebinding lectin in severe acute respiratory syndrome coronavirus infection complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection: a report of five cases highly pathogenic coronavirus n protein aggravates lung injury by masp-2-mediated complement over-activation. medrxiv the case of complement activation in covid-19 multiorgan impact blockade of the c5a-c5ar axis alleviates lung damage in hdpp4-transgenic mice infected with mers-cov neutrophils in viral infections: current concepts and caveats a role for neutrophils in viral respiratory disease. front immunol severe acute respiratory distress syndrome (sars): a critical care perspective a major outbreak of severe acute respiratory syndrome in hong kong neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019. novel coronavirus in the early stage mouse-passaged severe acute respiratory syndrome-associated coronavirus leads to lethal pulmonary edema and diffuse alveolar damage in adult but not young mice effect of heterophil depletion by 5-fluorouracil on infectious bronchitis virus infection in chickens neutrophils are needed for an effective immune response against pulmonary rat coronavirus infection, but also contribute to pathology neutrophils promote mononuclear cell infiltration during viral-induced encephalitis middle east respiratory syndrome coronavirus (mers-cov) causes transient lower respiratory tract infection in rhesus macaques clinical features and viral diagnosis of two cases of infection with middle east respiratory syndrome coronavirus: a report of nosocomial transmission sars-cov2: should inhibitors of the renin-angiotensin system be withdrawn in patients with covid-19? a dynamic variation of pulmonary ace2 is required to modulate neutrophilic inflammation in response to pseudomonas aeruginosa lung infection in mice neutrophil extracellular traps in immunity and disease neutrophil extracellular traps (nets) -formation and implications targeting potential drivers of covid-19: neutrophil extracellular traps the role of neutrophil extracellular traps in covid-19: only an hypothesis or a potential new field of research? autopsy findings and venous thromboembolism in patients with covid-19 coagulation abnormalities and thrombosis in patients with covid-19 mechanisms underlying neutrophil-mediated monocyte recruitment partners in crime: neutrophils and monocytes/macrophages in inflammation and disease aggregated neutrophil extracellular traps limit inflammation by degrading cytokines and chemokines selective reduction of natural killer cells and t cells expressing inhibitory receptors for mhc class i in the livers of patients with hepatic malignancy expression of killer cell inhibitory receptors is restricted to true nk cell lymphomas and a subset of intestinal enteropathy-type t cell lymphomas with a cytotoxic phenotype modulation of expression of the mhc class i-binding natural killer cell receptors, and nk activity in relation to viral load in hiv-infected/aids patients hla-cw7 zygosity affects the size of a subset of cd158b+ natural killer cells the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome expression of lymphocytes and lymphocyte subsets in patients with severe acute respiratory syndrome sars-cov-2 infection in children: transmission dynamics and clinical characteristics will children reveal their secret? the coronavirus dilemma significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus t cells and ilc2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice single cell rna sequencing of 13 human tissues identify cell types and receptors of human coronaviruses how the sars coronavirus causes disease: host or organism? comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity clinical progression and cytokine profiles of middle east respiratory syndrome coronavirus infection mobile group ii introns of yeast mitochondrial dna are novel site-specific retroelements resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires stat1 sars-cov pathogenesis is regulated by a stat1 dependent but a type i, ii and iii interferon receptor independent mechanism lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections combined action of type i and type iii interferon restricts initial replication of severe acute respiratory syndrome coronavirus in the lung but fails to inhibit systemic virus spread sars-cov nucleocapsid protein antagonizes ifn-beta response by targeting initial step of ifn-beta induction pathway, and its c-terminal region is critical for the antagonism severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists sars coronavirus nsp1 protein induces templatedependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin identification of residues of sars-cov nsp1 that differentially affect inhibition of gene expression and antiviral signaling mers-cov papain-like protease has deisgylating and deubiquitinating activities regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus proteolytic processing, deubiquitinase and interferon antagonist activities of middle east respiratory syndrome coronavirus papain-like protease the adp-ribose-1''-monophosphatase domains of severe acute respiratory syndrome coronavirus and human coronavirus 229e mediate resistance to antiviral interferon responses accessory proteins of sars-cov and other coronaviruses the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 ahmed-hassan, sisson, shukla, wijewantha, funderburg, li, hayes, demberg and liyanage. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003091-uvfppirt authors: gornati, laura; zanoni, ivan; granucci, francesca title: dendritic cells in the cross hair for the generation of tailored vaccines date: 2018-06-27 journal: front immunol doi: 10.3389/fimmu.2018.01484 sha: doc_id: 3091 cord_uid: uvfppirt vaccines represent the discovery of utmost importance for global health, due to both prophylactic action to prevent infections and therapeutic intervention in neoplastic diseases. despite this, current vaccination strategies need to be refined to successfully generate robust protective antigen-specific memory immune responses. to address this issue, one possibility is to exploit the high efficiency of dendritic cells (dcs) as antigen-presenting cells for t cell priming. dcs functional plasticity allows shaping the outcome of immune responses to achieve the required type of immunity. therefore, the choice of adjuvants to guide and sustain dcs maturation, the design of multifaceted vehicles, and the choice of surface molecules to specifically target dcs represent the key issues currently explored in both preclinical and clinical settings. here, we review advances in dcs-based vaccination approaches, which exploit direct in vivo dcs targeting and activation options. we also discuss the recent findings for efficient antitumor dcs-based vaccinations and combination strategies to reduce the immune tolerance promoted by the tumor microenvironment. introduction vaccines represent one of the most effective copernican revolutions for humankind and world health. this innovative discovery by edward jenner in the late years of the xviii century allowed for control or complete eradication of infectious diseases as smallpox (1979) and rinderpest virus (2011) (1) . this immunization strategy posed the bases for current remarkable therapeutic approaches against not only infections but also cancer. in evolutionary terms, pathogens have acquired the capability to circumvent the immune system with several evasion mechanisms, revised elsewhere (2) , that prevent pathogen clearance and the establishment of immune memory. vaccines represent the unique tool we have to impede pathogen spread; therefore, the urgent need for efficient vaccines is as relevant as before. mycobacterium tuberculosis, which causes tuberculosis, is currently one of the most feared infectious agent due to its capability to evade the immune system, leading to death of more than one million of people per year. unbelievably, the only licensed vaccine against mycobacterium tuberculosis is bacillus calmette-guérin (bcg) conceived about 100 years ago. nonetheless, bcg has displayed some degree of inefficacy in humans, thus raising the need for new tailored vaccination strategies that are currently under investigation (3) . moreover, every year, new cases of human immunodeficiency virus (hiv) infections lead to the necessity of a vaccine to control and prevent the spread of the virus. up to now, vaccines against hiv have not passed phase ii clinical trials due to poor protection conferred, requiring revision of delivered antigens (ags) and strategy to improve t cell response (4) . moreover, the recent outbreaks of ebola virus and zika virus infections clearly demonstrate that still nowadays more than few infectious diseases need to be overwhelmed, as reported by the world health organization. on the other hand, vaccines represent also a therapeutic tool against cancer. one of the hallmarks of cancer is the capability of tumor cells to evade immune-mediated destruction (5) by promoting a tolerant milieu. therefore, the immune system has to be pushed to respond specifically and robustly against tumors cells. to address this purpose, it is becoming more and more evident that dendritic cells (dcs) stand out as a potent tool in our hands, being the mediators of cellular and humoral responses (6) . dcs have been discovered in 1973 by r. steinman and z. cohn that divided phagocytic cells (discovered by e. metchnikoff in 1887) in macrophages and dcs on the basis of different effector functions: microbial scavenging activities for macrophages and antigen-presenting function for dcs (7, 8) . since then, dcs have emerged as the most potent antigen-presenting cells capable of shaping adaptive responses both during infections and cancer. moreover, the broad spectrum of dcs activation makes them suitable for fine shifting of the type of response the context needs. taking advantage of new adjuvants, innovative ags-delivery carriers and targeting strategies, it is now feasible to optimize the activation and ag presentation processes by the specific dcs subset that is the most effective in the initiation of the adaptive response needed in a given context. here, we discuss the diverse phenotypical and functional properties of dcs subtypes that are exploited by recently developed vaccine approaches, dealing with advances in the use of ags, adjuvants, carriers and dcs-expressed molecules, object of targeting. dendritic cells are the primary professional antigen-presenting cells (apcs) that reside in both lymphoid and non-lymphoid organs (9) (10) (11) . dcs encompass several heterogeneous subsets whose subdivision relies on ontogeny, expression of surface-receptors, and transcription factors (12) (13) (14) . much effort has been done in the identification and characterization of tissue-specific dc subsets to unravel the correlation between phenotype, localization, and functional properties, both in health and disease. initially, dcs have been classified into conventional dcs (cdcs) and plasmacytoid dcs (pdcs). briefly, cdcs prime naïve t cells and orchestrate ag-specific adaptive responses, while pdcs intervene during viral infections producing type i interferons (ifns). advanced approaches have extremely pushed our understanding of dc biology, resulting in a recent readapted taxonomy (12, 15, 16) . indeed, villani and colleagues identify six subsets of dcs and monocytes in human (figure 1 ): dc1 (clec9a + cd141 + dcs), dc2 and dc3 (cd1c + dcs), dc4 (fcgr3a/cd16 + dcs), dc5 (axl + siglec6 + dcs) and dc6 (pdcs). dc1 represent the cross-presenting cd141 + /bdca3 + dcs while d2 and d3 correspond to cdcs displaying antigen uptake and processing capabilities. dc4 seem to be more prone to respond to viruses and are phenotypically close to monocytes. dc5 represent a newly defined subset that share features with both pdcs and cdcs, even though they appear to be functionally different from pdcs and more similar to cdcs. indeed, dc5 localize in t cell zone of tonsils, probably promoting fast adaptive immunity. due to this fine clustering, dc6 correspond to a more pure pdcs population (12) . this precise classification opens the way for a more accurate view of dcs role in pathologies and provides cues for more specific targeting in immunotherapies. indeed, it is reasonable to assume that this extreme phenotypical diversity correlates with different intrinsic functional properties of dcs, as emerged in villani's work (12, 17, 18) . in addition, environmental cues dictate dc activation and drive specific t cell responses (19, 20) . indeed, dcs display a plethora of pattern recognition receptors (prr) that are specifically bound by microbe-or damage-associated molecular pattern (pamp and damp, respectively) (21) . upon receptors engagement in peripheral tissues, the transduction signals lead to dc maturation with the upregulation of co-stimulatory molecules (referred to as "signal 2") and the pivotal chemokine receptor ccr7 that allows dcs migration through afferent lymphatic vessels to the draining lymph node (ln) (22) (23) (24) . in parallel, dcs mediate ag proteolysis to present intracellular peptides on major histocompatibility complex (mhc) class i to cd8 + t cells and exogenous peptides on mhc ii to cd4 + t cells (referred as "signal 1"). dcs can present exogenous ags on mhc class i through the so-called cross-presentation, allowing them to induce cd8 + cytotoxic t lymphocytes (ctls) against viruses and tumor cells. indeed, once in the ln, mature dcs encounter cognate naïve t cells and initiate adaptive responses (25) . in the absence of maturation, as in steady-state conditions, the ag presentation and consequent migration to ln promote peripheral tolerance via t cell anergy or regulatory t cell formation (26) (27) (28) . depending on the receptors engaged, dcs display different maturation states and produce different inflammatory mediators (often referred to as "signal 3") that impact on the following cellular and humoral responses. the three signals released by dcs drive t helper (th) cell differentiation. briefly, dcs educate cd4 + t cells against intracellular bacteria by promoting their polarization into ifn-γ-producing th type 1 (th1) cells. upon infection by multicellular parasites, dcs, with the help of basophils, polarize cd4 + t cells into th type 2 (th2) cells that produce mainly il-4. for specialized mucosal and skin immunity, dcs drive the activation of th type 17 (th17) (29) . thus, polarization of t cells is a crucial event that provides mechanisms specifically orchestrated to restore physiological homeostasis. dcs undergo apoptosis once they have fulfilled their functions. the rapid dc turnover after activation is necessary to avoid excessive t cell activation (30) and to maintain self-tolerance (31, 32) . t lymphocyte activation culminates with the establishment of the immunological memory, providing the host with t cells more prone and efficient in responding to a reinfection by the same pathogen or upon tumor relapses (33) . besides, dcs are key players in humoral responses too. indeed, they directly interact with b cells and indirectly support them by activating cd4 + t cells, leading to humoral memory. all these notions strengthen the idea that dcs represent an optimal target for immunotherapies and vaccines, acting at the interface of innate and adaptive immunity. to harness robust responses through dc-targeting vaccinations, dc maturation is essential. adjuvants become compulsory complement of inactivated or subunit vaccines that may promote suboptimal responses. furthermore, they improve dc migration, ag availability, and specific targeting. although it seems clear that immunization could benefit from adjuvant uses, the solely adjuvant licensed in clinics, until recently, was alum (34) . despite alum has been used in vaccination practice since the beginning of the last century, the mechanism through which it activates innate immunity for the subsequent activation of adaptive immune responses remains elusive. the adjuvant properties of alum were initially attributed to the activation of nlrp3 inflammasome (35, 36) ; nevertheless, further studies have clearly shown the dispensability of nlrp3 and caspase-1 for the generation of responses in the presence of this adjuvant (37, 38) . tlr signaling is also dispensable for alum adjuvanticity (39) as well as mast cells, eosinophils, or macrophages (40) . recently, it has been proposed that upon contact with alum, dcs produce il-2 through the activation of src and syk kinases, ca 2+ mobilization, and nfat nuclear translocation. il-2, in turn, is required for optimal t cell priming, activation, and antibody production (41) . in addition to alum, other chemical adjuvants have been tested in preclinical models, showing a clear heterogeneity in the responses driven by different adjuvants, independently of the ag (42) . this underlies the need of deepening our knowledge on these powerful tools to drive immune responses. indeed, mf59, an oil-in-water emulsion adjuvant, that allows long-lasting ag retention in draining ln and enhanced ag uptake by ln-resident dcs, promotes robust humoral responses via follicular dc activation (43) and cd4 + t cell immunity induction (44) . conversely, ic31 adjuvant, which consists of an antibacterial peptide and a synthetic oligodeoxynucleotide (odn), elicits ifn-β release by human dcs via engagement of endosomal tlrs supporting immunity against intracellular pathogens and cancer (45) . in the last decades, attention has been focused on tlr ligands as adjuvants. currently, several compounds are under investigation: pam2csk4, pam3csk4, or analogs as tlr2/6 or tlr2/1 ligands (46, 47) , poly(i:c) and similar compounds acting on tlr3 (48, 49) , tlr4 agonists (50), flagellin acting on tlr5 (51), imiquimod and other tlr7 ligands (52, 53) , tlr8 agonists (54), cpg odn binding tlr9 (55, 56) . due to the possible reactogenicity that may be induced by administering tlr agonists, some compounds are chemically modified to reduce toxicity or are delivered specifically to the dc subsets of interest, avoiding tlr ligand dissemination. monophosphoryl lipid a, a low-toxicity molecule derived from lipopolysaccharide (lps), displays promising effects for vaccine design (57) even though it promotes terminal differentiation of cd8 + cells, leading to reduced memory protection (58) . another lps-analog is 7-acyl lipid a that has emerged as potent inducer of ifn-γ-mediated ag-specific responses when co-delivered with poorly immunogenic tumor ags (59) . to improve the effectiveness and strength of immunity, in addition to the efficiency of apc, activation and ag processing and presentation of other aspects should be taken into account. the importance of dc-derived il-2 in the activation of adaptive responses has been shown not only in alum-driven immune responses and in mouse models of infections (60, 61) but also in tests of human t cell priming in the presence of activatory dcs. during the first few hours after interaction with t cells, activatory monocyte-derived dcs (modcs stimulated with the cytokine cocktail, tnf-α, il-6, il-1β, and pge2) produce il-2 and cd25 (62) . dc-derived il-2 is, in turn, trans-presented to t cells at the immunological synapse via cd25. since naïve t cells start to express cd25 only many hours after ag encounter, the dc-mediated presentation of the il-2/cd25 complex is indispensable for an efficient t cell priming (62) . it has been proposed that this is the reason why approved therapies based on the use of anti-cd25 antibodies to avoid the acute phases of autoimmune diseases, or acute rejection of kidney, heart, and hand transplants, are so efficient in interfering with t cell priming or t cell reactivation (62) . since il-2 is produced in nfatdependent manner to improve the adjuvanticity of prr agonists for vaccination purposes, the capacity of selected prr agonists to induce nfat signaling pathway activation and il-2 production should be considered. many prr ligands have been shown to activate the nfat transcription factor family members in innate immune cells (63) . the nfat pathway is activated in neutrophils, macrophages, and dcs in response to curdlan (64, 65) , it is also activated in dcs in response to lps (30) downstream of cd14, it is activated in response to mannose-capped lipoarabinomannan (man-lam), a major lipoglican of mycobacterium tuberculosis (66) , and downstream of tlr9 in response to β-glucan bearing fungi (67) . the production of il-2 by innate immune cells during inflammatory responses is relevant not only for an efficient t cell priming but also for the skewing of t cell activation toward type i responses. in mice, dc-derived il-2 is one of the cytokines required to elicit ifn-γ production from nk cells both in lpsmediated inflammatory conditions and during fungal infections (68) (69) (70) . ifn-γ potently activates macrophages and favors th1 commitment of cd4 + t cells. therefore, early ifn-γ release by nk cells is not only crucial for controlling a variety of primary bacterial and fungal infections but also for the induction of type i immunity and memory, fundamental for the protection against bacterial, fungal, and viral infections and in antitumor immune therapies. another important reason for considering the capacity to activate the nfat pathway in adjuvant selection tests is represented by the fact that nfats regulate also the production of the prostanoid pge2 by activated dcs (71) . pge2 promotes activated dc migration (72) and sustains vasodilation and local edema formation during the inflammatory process. this is particularly relevant for vaccination purposes since the increase of the interstitial pressure generated by the edema forces the fluids into the afferent lymphatics and favors a first wave of antigen arrival to the draining ln (71) . intriguingly, ln drainage of proteins or antigens occurs very rapidly after subcutaneous, intradermal, and intramuscular immunization (73) (74) (75) , thus permitting an extremely fast uptake by phagocytes strategically localized in close proximity to the subcapsular sinus or lymphatic sinus of draining ln (76) (77) (78) (79) . antigen-presenting cells in ln then maintain the homeostasis of ln themselves and activate adaptive immune responses. in the last decades, the long-held paradigm of migratory dcs, resident in peripheral tissues as the skin, as unique apcs involved in t cell immunity has dramatically changed. indeed, cd169 + subcapsular sinus macrophages, medullary macrophages, and ln-resident dcs are ln sentinels that avoid excessive pathogen dissemination (80, 81) and mediators of immune responses (76) . concerning migratory dcs and considering the skin, which represents the site of utmost importance for vaccination strategies due to the ease accessibility and the extremely high presence of dcs, skin-resident dcs have been subdivided into epidermalresident langerhans cells (lcs), which are langerin + and two diverse subsets of dermal (d)dcs: langerin + cd103 + and langerin − cd103 − (14, 82) . upon infection, ddcs migrate to the ln within 10-24 h while lcs within 48-72 h, supporting long-lasting ag-presentation. several works reveal the intrinsic differences between the two subsets in inducing th or ctl responses, due to the particular cross-presenting capabilities of cd103 + ddcs, for instance (19, 20, 83) . once in the ln, whose strategical architecture enhances the probability of encounter between migratory dcs and cognate naïve t cell, adaptive immunity is initiated. of note, ln-resident dcs are sufficient to promote early adaptive responses independently of migratory dcs when pathogens or antigens directly access the lymphatic conduits (76, 84, 85) . in antiviral responses, cd8α + ln-resident dcs play a crucial role, thanks to their intrinsic capability of cross-presentation to cd8 + ctl (86, 87) that may be supported by pdcs (88) . in herpes simplex virus (hsv) skin infection, cd8α + ln-resident dcs uptake cargo-antigens, ferried by skinresident migratory dcs in order to elicit ctl (89) . indeed, lcs and ddcs synergize with cd8α + ln-resident dcs, which stand out as the most potent ctl inducers, preferentially sustaining cd4 + th responses both in influenza (90) and hsv cutaneous infections (91) . in addition to cd8α + ln-resident dcs, cd103 + ddcs display intrinsic capability of cross-presentation, as their human counterpart, clec9a + cd141 + dcs (92) (93) (94) (95) (96) . besides, some authors demonstrated that blocking dc migration from the skin hinders cd4 + t cell activation in response to subcutaneous bacterial (97) or soluble antigen challenge (98) . ablating langerin + ddcs reduced t cell immunity strength, corroborating the notion that migratory dc complement ln-resident dc effects on adaptive responses (99) (100) (101) . nonetheless, the roles of lcs in activating t cells are still uncertain, probably due to the controversial functional properties of this innate subset (102) (103) (104) . despite this, the synergic effects of ln resident and migratory dcs seem to be undoubted (25, 105) . indeed, allenspach and colleagues reported that ag presentation by ln-resident dcs few hours after the infection is required to entrap ag-specific t cells in the draining ln and to favor an optimal activation of t cells by migratory dcs that arrive at the ln many hours later (106) . it emerges, therefore, that another aspect to be considered for the identification of efficacious adjuvants concerns the type of dc subset to be targeted and the consequential effects that adjuvants imprint on that subset. adjuvants play a pivotal role in determining tissue-resident dc mobility to draining ln and efficiency of t cell polarization. indeed, ddcs acquire mobility after subcutaneous injection of th1-specific adjuvants as cpg and lps, but not with th2-specific ones, as papain, or following contact sensitization with dibutyl phthalate and acetone. moreover, ddcs are sufficient to promote th1 and th2 responses, while lcs are only supportive of th1 (107) . this evidence underscore that, in addition to the polarizing capabilities of adjuvants, also the targeted dc subset must be considered to elicit specific adaptive immunity. indeed, antonialli and colleagues reported differential immune responses when cd8α + and cd8α − dcs were targeted with the same ag and adjuvant, either cpg odn or flagellin (108) . in addition, to enhance the efficacy of vaccination, the coincident delivery of ag and prr adjuvants to apcs plays a crucial role. encouraging evidence highlights the importance of conjugation of ag with prr adjuvants, since it improves ag uptake, humoral and cellular responses when compared to vaccination with ag codelivered with free tlr ligands (109) . these findings strengthen the notion that adjuvants are formidable chiefs in shaping immune responses and must be selected for the outcomes they promote, in chemical association to the ag of interest. the traditional vaccination approaches consisted in the adminis tration of live or attenuated micro-organisms. up to now, several innovative strategies have emerged to address the need for efficient vaccines, especially against diseases that are critical to treat, as cancer and the infectious diseases already mentioned. the main purpose is to convey ag, adjuvant, and targeting-molecule in a unique compound to increase the efficacy of the ag-specific immune response. to address this issue, different approaches have been explored or are currently under investigation, as shown in figure 2 . recombinant antibody (rab) represents a feasible option. this approach exploits the possibility to chemically fuse peptide ag, adjuvant, and targeting-molecule to ab to tailor dcs targeting (110) (111) (112) . in addition to rab, single-chain fragments variable (scfv) revealed to be an appealing strategy due to their reduced size and enhanced infiltration into tissues, as in solid tumors (113) . other approaches involve the use of nano-carries as vehicles. the most promising solution to target phagocytes is indeed the use of particulate materials (114, 115) . nanoparticles (nps) are the best candidates as delivery system, since they can be manipulated to efficiently and predominantly target phagocytes. this is possible, thanks to the versatility of nps due to: (i) the large amounts of existing different nanomaterials; (ii) the possibility to adjust their size, morphology, and deformability with great precision; (iii) the possibility to load virtually any different type of drug molecules (116) . viral vectors-based vaccines or virus-like particles rely on the intrinsic capability of viruses to infect cells and exploit their protein-encoding machinery, allowing expression in the cytosol of the engineered plasmid-genes, as ag, costimulatory molecules, cytokines, and adjuvants, providing the bases for strong ctl induction (117) . on the other hand, naked dna can be directly injected or conjugated to nano-carriers to favor specific targeting. the easy designing of nano-carriers-based vaccines along with their multi-component loading feature improve targeting of specific subsets (118) and shape immune responses (119, 120) , favoring their application in several fields. in a cancer setting, nano-carriers allow to avoid killing of healthy cells, by delivering tumor ags or dna encoding these peptides to apcs, inducing specific antitumor responses. indeed, nps allow endocytosis and mhc presentation on both class i and ii (121) eliciting broad adaptive immunity, even against cancer cells. rosalia and colleagues designed a polymer-based biodegradable poly(lactic-co-glycolic acid) plga nps loaded with ag, pam3csk4, and poly(i:c) and coated with an agonistic αcd40-monoclonal ab (np-cd40). this multi-functional strategy resulted in efficient and selective delivery of nps to dcs in vivo upon s.c. injection and induced priming of cd8 + t cells against tumor associated ags, increasing tumor-bearing mice survival (109) . plga nps carrying the poorly immunogenic melanoma-derived antigen tyrosinase-related protein 2 along with 7-acyl lipid a, manage in breaking the immunotolerance acting against tumor-antigens. indeed, administration of the abovementioned nps resulted in antigen-specific cd8 + ctl responses, characterized by ifn-γ production and increase of pro-inflammatory cytokines in the tumor microenvironment (tme) (59) . another nano-carrier-based approach relies on liposome, self-assembled vesicles composed by lipid bilayers with high functionalizing properties. besides, maji and colleagues reported that after uptake by dcs, cationic liposomes localize in endosomal compartments that allow ag presentation preferentially on mhc i but do not exclude mhc ii ag presentation (122), suggesting a crucial role in antitumor or antiviral immunity supported by th responses. in addition to the use of nps, targeting dc-specific receptors has become an attractive strategy for vaccine development due to the enforced efficiency of immune responses when compared to generic-delivering approaches. here, we report the more characterized dcs receptors, currently under investigation in the scenario of tailored-vaccination, as shown in table 1 . clec9a or dngr1 is a c-type lectin receptor that mediates endocytosis, but not phagocytosis, with low ph endosomes promoting the drift toward cross-presentation. importantly, clec9a binding of antigens induces antigen presentation on both mhc i (cross-presentation) and mhc ii. it is highly and specifically expressed on cd11c + cd141 + xcr1 + cdcs and cd14 + cd16 − monocytes in human and in murine pdcs and xcr1 + cd8a + ln resident but not cd103 + xcr1 + migrating dcs (123, 124) . indeed, cd141 + xcr1 + dcs constitute the human counterpart of cd8α + xcr1 + murine dcs (125) . they share xcr1, the receptor of xcl1. xcl1 is released by activated t cells and the axis xcr1-xcl1 is necessary for robust ctl responses (126) . cd141 + xcr1 + dcs are the main cross-presenting dcs in human, thus they appear promising for ctl-mediated responses, in tumors and viral infections (127) . this specific subset is characterized by the expression of tlr3 that may be exploited to fully activate clec9a + xcr1 + dcs since antibody binding of clec9a leads to its rapid internalization but not tlr-pathway activation, preventing pro-inflammatory cytokine production and full maturation of dcs (127) . conversely, caminschi and li independently demonstrated the potentiality of targeting clec9a that resulted in enhanced humoral immunity independently of trif-myd88 or tlr4 pathway, even in the absence of adjuvants (128, 129) . targeting clec9a induces enhanced cd4 + t cell proliferation in vivo, which supports b cell immunity, when compared to the targeting of another endocytic receptor, discussed later, dec-205, independently of the use of adjuvants as cpg (130) . some years later, different authors demonstrated that this strong humoral response is endorsed by the establishment of follicular t helper cells memory, even upon vaccination with glycoprotein d of hsv, both in mice and non-human primates (128, 131, 132) . these promising results were confirmed also in a human in vitro setting, on cd141 + dcs (133) . finally, the efficacy of targeting clec9a has been evaluated in the delivery of poorly immunogenic virus-derived antigens. park and colleagues managed in conferring specific humoral response, protective upon reinfection (134) . thus, exploiting the specific expression of this receptor on the most specialized dcs in cross-presentation in combination with tlr3 ligands, will enhance antiviral and anticancer responses (135), combined with robust humoral immunity. dec-205 or cd205 is a 205 kda endocytic receptor that has a cysteine-rich domain, a fibronectin type ii domain, and 10 c-type lectin-like domains, as well as an internalization sequence in its cytoplasmic tail (136) . thus, it mediates cross-presentation through clathrin-and dynamin-dependent receptor-mediated endocytosis. indeed, it is expressed by the most professional cross-presenting dcs, the cd8α + dcs subtype, while cd8α − dcs display very low level of this receptor. in addition, dec-205 is found on dermal/interstitial dcs and lcs (137), thus guaranteeing ag delivery to both skin-resident and ln-resident professional apcs. in humans, dec-205 is shared among cdcs, monocytes, and b cells, while pdcs, granulocytes, nk cells, and t lymphocytes express low levels of this receptor (138) . in addition, dec-205 regulates molecule recycling through late endosomes, promoting also mhc ii presentation to cd4 + t cells in lcs (139) . steinman and nussenzweig have addressed this molecule to improve vaccine efficacy since 2000 (140) . by taking advantage of anti-dec-205 rab conjugated to ova peptide, they demonstrated that s.c. injections of this compound lead to a strong ifn-γ and il-2mediated immunity only when dcs activation was supported by αcd40 mab, otherwise, tolerance against the ova peptide occurs (141) . indeed, diversely from prr agonists, antibody crosslinking the dec-205 does not induce dcs maturation (142) . furthermore, few years later, the combined strategy of anti-dec-205 and αcd40 was reported to confer protection against melanoma and intranasal influenza infection (112) . in a viral setting, anti-dec-205 rab chemically coupled with hiv p24 gag protein tested in vitro on blood cells derived by 11 hiv-infected donors has revealed efficient expansion of ifn-γ-producing cd8 + t lymphocytes (143) from all the different donors. this indicated that dcs and cd205 can lead to the generation of different peptides from a single protein. moreover, vaccines based on the filamentous bacteriophage fd presenting an αdec-205 scfv, efficiently induce dcs maturation via the activation of the tlr9-myd88 pathway (144) , without adjuvants and further elicit potent antitumor responses when compared to non-tailored ag delivery (145) . intriguingly, dec-205, orphan of a specific ligand, has been proven to be necessary for cpg uptake and eventual dc activation (146) . cd40 is a molecule belonging to the tnf receptor family, expressed by several cell types and among these, dcs. it has emerged as a receptor for the human chaperone heat shock protein (hsp) 70 that mediates the internalization of peptides bound to hsp70 itself (147) . moreover, upon activation, t cell transiently expresses cd40l allowing cross-linking of cd40 on dcs and completing their maturation. from these notions, cd40 appeared an interesting molecule to target for dc-based vaccination strategies. indeed, by engineering antibody chemical structure, schjetne and colleagues demonstrated the efficacy of cd40 engagement conferring protection against myeloma-and lymphoma-derived ags (148) . moreover, through the co-administration of two dna-based vaccines encoding either cd40 and the foot-and-mouth disease-derived ags, the transient increase of endogenous αcd40 antibodies allows an efficacious dcs activation and an efficient development of ag-specific t cell immunity, if compared to the administration of dna encoding ags alone (149) . further promising results have been obtained in a vaccine against cyclin-d1 that is overexpressed by mantle cell lymphoma (mcl). thanks to algorithm analysis, chen and colleagues identified three cyclin-d1-derived peptides that efficiently bind to mhc class i of dcs, potentially overexpressed in all mcl patients. by generating a rab targeting cd40, they efficiently delivered these tumor associated ags to dcs and mounted ifn-γ-specific t cell responses in patients-derived peripheral blood mononuclear cells (150) . thus, cd40 represents a specific dc-targeting molecule that has been used in combination with other targeting approaches to support specific dcs activation, avoid tolerance, and induce robust t cell immunity (110, 141) . when evaluating vaccination strategies for cancer patients, it is compulsory to take into account one of the hallmarks of cancer: avoiding immune destruction by promoting tolerance and disarming the immune system (5) . the orchestration of antitumor responses involves multiple protagonists and mediators, among these, cytotoxic t cells and nk cells, whose activation is supported by dcs (151) . furthermore, dcs-based vaccines has emerged as more efficient in promoting t cell immunity if compared to peptide-based vaccination approaches (152) . thus, much effort has been made to improve strategies of dcs-based vaccination in neoplastic diseases, to ameliorate the prognosis or eradicate both primary tumor and metastases. up to now, two different approaches have been addressed: ex vivo generation of autologous pulsed dcs and direct in vivo targeting of dcs, as previously discussed. the former strategy provides a better control of the maturation and activation state of dcs and a specific load of the ag to the selected dcs subset. despite this, intense work is needed to generate this vaccine, since it is personalized for each patient and only few subsets of dcs are feasibly generated in vitro or collected ex vivo, limiting the access of ags to other more functionally driven subsets. diversely, the in vivo targeting methods allow the generation of large amount of vaccine in a one-step procedure, and the targeting of diverse dcs subsets in their natural environment. once the dcs-based vaccine is generated, the efficacy of antitumoral responses has to be evaluated. it is mainly related to (i) the capability to establish specific antitumor-associated ag (taa) immunity and (ii) the overcome of the tolerogenic status promoted by the tme. to select highly immunogenic ags, multiple solutions have been tested: whole tumor lysate or killed tumor cells, synthetic long peptides (slps), full length proteins, transfection or electroporation with dna or mrna coding for taa, transduction with lentiviral vectors and neoantigens. the availability of an elevated number of antigens through the incubation of dcs with whole tumor lysates or autologous tumor cells allows the presentation of multiple epitopes, loaded on both mhc class i or ii, which leads to th and cytotoxic responses. indeed, several clinical trials are currently evaluating the benefits obtained by using this approach (nct01875653; nct00045968; nct02496520). slps are 28-35 aa long peptides cross-presented by dcs (153), currently under investigation in both preclinical and clinical setting. compared to short synthetic peptides, the use of slps lacks the necessity to know the patients' hla haplotype, thus permitting their full exploitation in a larger cohort of people. moreover, slps administration to dcs leads to an enhanced cd8 + t cells activation since, once engulfed, they rapidly escape from the endolysosome to follow the path of mhc class i presentation, fundamental in antitumor responses. indeed, slps and dcs-based vaccines are showing promising results in terms of safety and immunogenicity, in both preclinical and clinical settings (154) . they have gained attention in the context of human papilloma virus cervical (155) , ovarian (156) , and colorectal cancer (157, 158) , displaying immunogenic capacities, in terms of antibody production and cd4 + and cd8 + t cell activation, when delivered with adjuvants, as poly iclc, montanide-isa-51 (nct02334735), and ifnα. when comparing slps and full length proteins, it has emerged that dcs process slps better that full length protein, due to the slower processing route the latter display (154) . concerning transfection or electroporation of dcs with mrna or dna encoding, not only for taa but also for costimulatory molecules and cytokines, to enforce adaptive immunity, has proven to be efficacious in inducing antitumor cd4 + and cd8 + t cells expansion, mediated by dcs targeting (159) . a similar approach regards in vivo lentiviral transduction of dcs, which displays versatility for gene delivery and efficient transduction for non-dividing cells, as dcs. indeed, bryson et al. conceived a multifunctional vaccine composed by a modified lentivirus, whose glycoproteins can directly target dc-sign on dcs, loaded with breast cancer ags, alpha lactalbumin, and erb-b2 receptor tyrosine kinase 2. single injections of the compound provided tumor self-ags-specific cd8 + t cell immunity, reducing tumor growth (160) . despite the improvements derived by these advanced strategies, in the last years, neoantigens are becoming more and more appealing (161) . during tumor progression, cancer cells give rise to neoantigens, novel ags different from the self-tumor ags, derived by the tumor-specific mutations. therefore, prediction tools, rna mutanome, and deep-sequencing have allowed the identification of specific non-self-ags that are fundamental in strong t cell immunity (162) (163) (164) . indeed, several clinical trials are currently investigating the potential of neoantigens (nct0235956; nct01970358; nct02149225; nct02348320; nct02316457). as emerged, different strategies of ags selection have been explored and, even though one strategy may result in a more enforced antitumor immunity if compared to another, still the issue of the tme negative influence on the immune system has to be faced. indeed, the tme actively suppresses the activation of the immune system. tumor cells secrete immunosuppressive cytokines, as vascular endothelial growth factor (165, 166) , macrophage colony-stimulating factor (167), transforming growth factor β (tgf-β) (168) , and il-10 (169, 170) . even though some of these cytokine display controversial roles, depending on the pathological context, they generally promote dcs tolerogenicity, by limiting their activation and increasing their expression of pro-tumor molecules, such as programmed cell death 1 (pd-1) and indoleamine 2,3-dioxygenase (ido). therefore, tolerogenic dcs lead to t cells anergy, tregs expansion, and th1 responses inhibition. phenotypical characterization of immune cells isolated from breast cancer patients, highlighted the functional alteration in dcs, t, and nk cells in promoting antitumor responses (171) . furthermore, tumor cells retain dcs into the tme, preventing their migration to draining lns and promoting metastatization (172) . to address this issue, some ex vivo generated dcs-based vaccines are directly administered intranodally, as for the cd1c + dcs pulsed with hla-a2.1-restricted tumor peptides administered to patients with stage iv melanoma (nct01690377), which generated tumor-specific cd8 + t cells responses and further improvement of survival (173) . to reduce the tolerogenic influence of the tme on dcs, the positive role of gm-csf in improving dcs survival and responsiveness is currently exploited in some clinical trials like a phase i/ii trial with a dc/tumor cell fusion vaccine administered in association with gm-csf to treat renal cancer (nct00458536). similarly, others are focusing their attention on fms-like tyrosine kinase 3-ligand (flt3l), another crucial dcs growth factor, in combination with other compounds (nct01811992; nct01976585; nct02129075; nct02839265). flt3l has, indeed, been shown to increase the efficacy of proteins-and rna-based vaccines, due to a maturation effect on dcs (174) (175) (176) . additional efforts made to counteract the tolerogenic influence of the tme include the use of pd-1 and ido inhibitors. co-administration of anti-pd-1 molecules increases the efficacy of dcs-based vaccines, in terms of enforced intratumoral cd8 + t cell responses and trafficking of cd8 + memory t cells, as observed in a preclinical model of glioblastoma (177) . in parallel, several clinical trials are aiming at evaluating the efficacy of dcs-based vaccines combined with anti-pd-1 agents (nct03014804; nct03325101; nct03035331). the other tolerogenic marker addressed in cancer immunotherapy and dcs-based vaccine is ido. indeed, silencing approaches to reduce the expression of ido in dcs for vaccination in preclinical models, have resulted in decreased t cell apoptosis, reduced numbers of tregs, decreased tumor size when compared to mice that had received ags-loaded dcs without ido silencing (178) . ido inhibitors in dcs vaccination are currently being tested in phase ii clinical trials (nct01560923; nct01042535). all these approaches have explored different scenarios to evaluate the more efficient therapeutic combination that seems to move toward personalized vaccinations for cancer patients. in this review, we have underscored the crucial role of dcs in orchestrating immune responses and; therefore, the great interest in targeting these cells in novel vaccination strategies. we have reported examples of different approaches aimed at amplifying the efficiency of immunizations against cancer or infectious diseases. indeed, the urgent need of vaccines is as relevant as before because of newly emerging diseases with ineffective current therapies. deepen the mechanisms underlying these pathologies may provide cues on the more appropriate design of vaccines and by merging diverse tailoring strategies we could enforce the immune system. as a matter of fact, it is suggested to act on different fronts when designing new vaccines, since several factors must be considered: (i) targeting dc subsets specialized in initiating the desired cellular or humoral immunity/memory; (ii) adjuvants that strengthen and drive t and b cell responses; (iii) fine and optimized selection of the immunogenic ags to drive enforced responses; (iv) novel strategies to convey ags and adjuvants to dcs; (v) route of administration. starting from these notions, in the last decades, enormous efforts have been made to tailor vaccination strategies. new technologies as well as recent advances have allowed extreme flexibility in designing vaccines and shaping the following outcomes. nowadays, researchers do have smart tools to manipulate immune responses with prophylactic or therapeutic vaccinations. the abovementioned findings pave the way for possible therapeutic approaches, theoretically applicable to all pathological contexts. despite this encouraging evidence, several limitations or issues still have to be overcome. indeed, more than a few vaccines do not pass phases i of clinical trials either for toxicity issues and lack of immunogenicity in some individuals. what is missing? part of the answer to this question could sit on human genetics and population variability. syngeneic animal models are ideal settings in which the systems are pushed although they constitute a necessary and useful step preceding clinical trials. moreover, when translating vaccine testing from in vivo experiments on animals to ex vivo on human cells, often the opted choice are blood human cells, while in most of the cases vaccines will be administered in the skin, having a complete different dcs-based milieu (15) . crucially, idoyaga and colleagues dissected the interindividual variability in skin-resident dcs, stressing the need of shedding light on the effects that genetics and environment imprint on dcs. it is compulsory to decode the complex scenario of human diversity to provide personalized therapies with increased efficacy. in the omics era, systems biology and computational modeling integrate huge data-sets to address the urgent need of information on the global behavior. indeed, genome-wide association studies have provided insights into human genetics variants associated to the immunogenicity of vaccines (179, 180) . therefore, integration of "wet" evidence and "dry" notions may fasten the designing process and provide both efficient vaccine strategies and their predictive efficacy. all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. eradicating infectious disease: can we and should we? front immunol anti-immunology: evasion of the host immune system by bacterial and viral pathogens why don't we have an effective tuberculosis vaccine yet? conserved hiv epitopes for an effective hiv vaccine hallmarks of cancer: the next generation dendritic cells and the control of immunity identification of a novel cell type in peripheral lymphoid organs of mice. i. morphology, quantitation, tissue distribution identification of a novel cell type in peripheral lympoid organs of mice. ii. functional properties in vitro dendritic cells display subset and tissue-specific maturation dynamics over human life unsupervised high-dimensional analysis aligns dendritic cells across tissues and species lymphoid organ-resident dendritic cells exhibit unique transcriptional fingerprints based on subset and site single-cell rna-seq reveals new types of human blood dendritic cells, monocytes, and progenitors human lymphoid organ dendritic cell identity is predominantly dictated by ontogeny, not tissue microenvironment the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting high-dimensional phenotypic mapping of human dendritic cells reveals interindividual variation and tissue specialization mapping the human dc lineage through the integration of high-dimensional techniques development and function of dendritic cell subsets characterization of resident and migratory dendritic cells in human lymph nodes candida albicans morphology and dendritic cell subsets determine t helper cell differentiation skin-resident murine dendritic cell subsets promote distinct and opposing antigen-specific t helper cell responses pattern recognition receptors and inflammation ccr7 and irf4-dependent dendritic cells regulate lymphatic collecting vessel permeability lymph node homing of t cells and dendritic cells via afferent lymphatics afferent lymph-derived t cells and dcs use different chemokine receptor ccr7-dependent routes for entry into the lymph node and intranodal migration distinct dendritic cell populations sequentially present antigen to cd4 t cells and stimulate different aspects of cell-mediated immunity migratory, and not lymphoid-resident, dendritic cells maintain peripheral self-tolerance and prevent autoimmunity via induction of itreg cells dendritic cells induce peripheral t cell unresponsiveness under steady state conditions in vivo tolerogenic dendritic cells th17 cells in mucosal immunity and tissue inflammation cd14 regulates the dendritic cell life cycle after lps exposure through nfat activation dendritic cell apoptosis in the maintenance of immune tolerance elimination of antigen-presenting cells and autoreactive t cells by fas contributes to prevention of autoimmunity most microbe-specific naïve cd4+ t cells produce memory cells during infection alum: an old dog with new tricks cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the nalp3 inflammasome cutting edge: inflammasome activation by alum and alum's adjuvant effect are mediated by nlrp3 the nlrp3 inflammasome is critical for aluminum hydroxide-mediated il-1β secretion but dispensable for adjuvant activity dna released from dying host cells mediates aluminum adjuvant activity adjuvantenhanced antibody receptor signaling alum induces innate immune responses through macrophage and mast cell sensors, but these sensors are not required for alum to act as an adjuvant for specific immunity the syk-nfat-il-2 pathway in dendritic cells is required for optimal sterile immunity elicited by alum adjuvants different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens vaccine adjuvant mf59 promotes retention of unprocessed antigen in lymph node macrophage compartments and follicular dendritic cells vaccine adjuvant mf59 promotes the intranodal differentiation of antigen-loaded and activated monocyte-derived dendritic cells the two-component adjuvant ic31 ® boosts type i interferon production of human monocyte-derived dendritic cells via ligation of endosomal tlrs pegylation of a tlr2-agonist-based vaccine delivery system improves anti gen trafficking and the magnitude of ensuing antibody and cd8+t cell responses the tlr2/6 ligand pam2csk4 is a th2 polarizing adjuvant in leishmania major and brugia malayi murine vaccine models defined tlr3-specific adjuvant that induces nk and ctl activation without significant cytokine production in vivo using 3 tlr ligands as a combination adjuvant induces qualitative changes in t cell responses needed for antiviral protection in mice the science of vaccine adjuvants: advances in tlr4 ligand adjuvants functional properties of flagellin as a stimulator of innate immunity novel adjuvant alum-tlr7 significantly potentiates immune response to glycoconjugate vaccines vaccine composition formulated with a novel tlr7-dependent adjuvant induces high and broad protection against staphylococcus aureus a formulated tlr7/8 agonist is a flexible, highly potent and effective adjuvant for pandemic influenza vaccines a tlr9 agonist enhances the anti-tumor immunity of peptide and lipopeptide vaccines via different mechanisms a dual tlr agonist adjuvant enhances the immunogenicity and protective efficacy of the tuberculosis vaccine antigen id93 the toll-like receptor 4 agonist monophosphoryl lipid a augments innate host resistance to systemic bacterial infection tlr4 ligands lipopolysaccharide and monophosphoryl lipid a differentially regulate effector and memory cd8+ t cell differentiation co-delivery of cancer-associated antigen and toll-like receptor 4 ligand in plga nanoparticles induces potent cd8+t cell-mediated anti-tumor immunity inducible il-2 production by dendritic cells revealed by global gene expression analysis regulation and dysregulation of innate immunity by nfat signaling downstream of pattern recognition receptors (prrs) a role for interleukin-2 trans-presentation in dendritic cell-mediated t cell activation in humans, as revealed by daclizumab therapy immunity and cancer: a transcription factor comes of age dectin-1 stimulation by candida albicans yeast or zymosan triggers nfat activation in macrophages and dendritic cells calcineurin regulates innate antifungal immunity in neutrophils dectin-2 is a direct receptor for mannose-capped lipoarabinomannan of mycobacteria phagocytosis-dependent activation of a tlr9-btk-calcineurin-nfat pathway co-ordinates innate immunity to aspergillus fumigatus skin infections are eliminated by cooperation of the fibrinolytic and innate immune systems il-15 cis presentation is required for optimal nk cell activation in lipopolysaccharide-mediated inflammatory conditions a contribution of mouse dendritic cell-derived il-2 for nk cell activation cd14 and nfat mediate lipopolysaccharide-induced skin edema formation in mice prostaglandin e2 is generally required for human dendritic cell migration and exerts its effect via ep2 and ep4 receptors a spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread capture of influenza by medullary dendritic cells via sign-r1 is essential for humoral immunity in draining lymph nodes conduits mediate transport of low-molecular-weight antigen to lymph node follicles strategically localized dendritic cells promote rapid t cell responses to lymph-borne particulate antigens histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to dendritic cell subset microanatomy in lymph nodes subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus the conduit system transports soluble antigens from the afferent lymph to resident dendritic cells in the t cell area of the lymph node lymph node macrophages restrict murine cytomegalovirus dissemination subcapsular sinus macrophages limit acute gammaherpesvirus dissemination tracking and quantification of dendritic cell migration and antigen trafficking between the skin and lymph nodes dynamics and function of langerhans cells in vivo: dermal dendritic cells colonize lymph node areasdistinct from slower migrating langerhans cells lymph-node resident cd8a+ dendritic cells capture antigens from migratory malaria sporozoites and induce cd8+ t cell responses lymph node resident rather than skin-derived dendritic cells initiate specific t cell responses after leishmania major infection cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens cross-presentation of cell-associated antigens by mouse splenic dendritic cell populations cd8+t cells orchestrate pdc-xcr1+dendritic cell spatial and functional cooperativity to optimize priming migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient ctl priming multiple dendritic cell populations activate cd4+ t cells after viral stimu lation cross-presentation of viral and self antigens by skin-derived cd103+ dendritic cells imaging of the cross-presenting dendritic cell subsets in the skin-draining lymph node resident cd8+ and migratory cd103+ dendritic cells control cd8 t cell immunity during acute influenza infection human tissues contain cd141hicross-presenting dendritic cells with functional homology to mouse cd103+nonlymphoid dendritic cells peripheral cd103 + dendritic cells form a unified subset developmentally related to cd8α + conventional dendritic cells selective expression of the chemokine receptor xcr1 on cross-presenting dendritic cells determines cooperation with cd8+ t cells ccr6-dependent recruitment of blood phagocytes is necessary for rapid cd4 t cell responses to local bacterial infection expression of the self-marker cd47 on dendritic cells governs their trafficking to secondary lymphoid organs tumor immunotherapy by epicutaneous immunization requires langerhans cells langerin expressing cells promote skin immune responses under defined conditions langerin + dermal dc, but not langerhans cells, are required for effective cd8-mediated immune responses after skin scarification with vaccinia virus langerhans cells: not your average dendritic cell langerhans cells suppress contact hypersensitivity responses via cognate cd4 interaction and langerhans cell-derived il-10 the balance between immunity and tolerance: the role of langerhans cells robust anti-viral immunity requires multiple distinct t cell-dendritic cell interactions migratory and lymphoid-resident dendritic cells cooperate to efficiently prime naive cd4 t cells selective and site-specific mobilization of dermal dendritic cells and langerhans cells by th1-and th2-polarizing adjuvants cpg oligodeoxinucleotides and flagellin modulate the immune response to antigens targeted to cd8α+and cd8α-conventional dendritic cell subsets cd40-targeted dendritic cell delivery of plga-nanoparticle vaccines induce potent anti-tumor responses intensified and protective cd4 + t cell immunity in mice with anti-dendritic cell hiv gag fusion antibody vaccine antigen targeting to dendritic cells elicits long-lived t cell help for antibody responses in vivo targeting of antigens to maturing dendritic cells via the dec-205 receptor improves t cell vaccination antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors magic bullets" for targeting the immune system drug nanocarriers to treat autoimmunity and chronic inflammatory diseases nanoparticles and innate immunity: new perspectives on host defence overall survival analysis of a phase ii randomized controlled trial of a poxviral-based psa-targeted immunotherapy in metastatic castrationresistant prostate cancer a novel antigen delivery system induces strong humoral and ctl immune responses vaccine nanocarriers: coupling intracellular pathways and cellular biodistribution to control cd4 vs cd8 t cell responses synthetic vaccine nanoparticles target to lymph node triggering enhanced innate and adaptive antitumor immunity plga microspheres for improved antigen delivery to dendritic cells as cellular vaccines a lipid based antigen delivery system efficiently facilitates mhc class-i antigen presentation in dendritic cells to stimulate cd8+t cells superior antigen cross-presentation and xcr1 expression define human cd11c + cd141 + cells as homologues of mouse cd8 + dendritic cells clec9a is a novel activation c-type lectin-like receptor expressed on bdca3+ dendritic cells and a subset of monocytes characterization of human dngr-1 + bdca3 + leukocytes as putative equivalents of mouse cd8α + dendritic cells the role of xcr1 and its ligand xcl1 in antigen cross-presentation by murine and human dendritic cells the c-type lectin receptor clec9a mediates antigen uptake and (cross-) presentation by human blood bdca3+ myeloid dendritic cells antibodies targeting clec9a promote strong humoral immunity without adjuvant in mice and non-human primates the dendritic cell subtype restricted c-type lectin clec9a is a target for vaccine enhancement targeting antigen to mouse dendritic cells via clec9a induces potent cd4 t cell responses biased toward a follicular helper phenotype targeting antigen to clec9a primes follicular th cell memory responses capable of robust recall evolution of b cell responses to clec9a-targeted antigen targeting clec9a delivers antigen to human cd141+ dc for cd4+ and cd8+t cell recognition enhancing vaccine antibody responses by targeting clec9a on dendritic cells tumor therapy in mice via antigen targeting to a novel, dc-restricted c-type lectin the receptor dec-205 expressed by dendritic cells and thymic epithelial cells is involved in antigen processing cd205 (dec-205): a recognition receptor for apoptotic and necrotic self expression of human dec-205 (cd205) multilectin receptor on leukocytes the dendritic cell receptor for endocytosis, dec-205, can recycle and enhance antigen presentation via major histocompatibility complex class ii -positive lysosomal compartments a monoclonal antibody to the dec-205 endocytosis receptor on human dendritic cells efficient targeting of protein antigen to the dendritic cell receptor frontiers in immunology | www.frontiersin.org in the steady state leads to antigen presentation on major histocompatibility complex class i products and peripheral cd8 + t cell tolerance dec-205 mediates antigen uptake and presentation by both resting and activated human plasmacytoid dendritic cells dec-205 receptor on dendritic cells mediates presentation of hiv gag protein to cd8+ t cells in a spectrum of human mhc i haplotypes antigen delivery by filamentous bacteriophage fd displaying an anti-dec-205 single-chain variable fragment confers adjuvanticity by triggering a tlr9-mediated immune response vaccination with filamentous bacteriophages targeting dec-205 induces dc maturation and potent anti-tumor t-cell responses in the absence of adjuvants dec-205 is a cell surface receptor for cpg oligonucleotides cd40, an extracellular receptor for binding and uptake of hsp70-peptide complexes delivery of antigen to cd40 induces protective immune responses against tumors cd40-expressing plasmid induces anti-cd40 antibody and enhances immune responses to dna vaccination a novel vaccine for mantle cell lymphoma based on targeting cyclin d1 to dendritic cells via cd40 hematology & oncology prolonged contact with dendritic cells turns lymph node-resident nk cells into anti-tumor effectors peptide-pulsed dendritic cells have superior ability to induce immune-mediated tissue destruction compared to peptide with adjuvant superior induction of anti-tumor ctl immunity by extended peptide vaccines involves prolonged, dc-focused antigen presentation dendritic cells process synthetic long peptides better than whole protein, improving antigen presentation and t-cell activation vaccination against hpv-16 oncoproteins for vulvar intraepithelial neoplasia phase i trial of overlapping long peptides from a tumor self-antigen and poly-iclc shows rapid induction of integrated immune response in ovarian cancer patients addition of interferon-α to the p53-slp v vaccine results in increased production of interferon-c in vaccinated colorectal cancer patients: a phase i/ii clinical trial induction of p53-specific immunity by a p53 synthetic long peptide vaccine in patients t reated for metastatic colorectal cancer vaccination with mrna-electroporated dendritic cells induces robust tumor antigen-specific cd4+ and cd8+ t cells responses in stage iii and iv melanoma patients breast cancer vaccines delivered by dendritic cell-targeted lentivectors induce potent antitumor immune responses and protect mice from mammary tumor growth neoantigens in cancer immunotherapy personalized cancer vaccine effectively mobilizes antitumor t cell immunity in ovarian cancer personalized rna mutanome vaccines mobilize poly-specific therapeutic immunity against cancer an immunogenic personal neoantigen vaccine for patients with melanoma vascular endothelial growth factor impairs the functional ability of dendritic cells through id pathways vascular endothelial growth factor inhibits the function of human mature dendritic cells mediated by vegf receptor-2 high co-expression of il-34 and m-csf correlates with tumor progression and poor survival in lung cancers tumor-derived tgf-β reduces the efficacy of dendritic cell/tumor fusion vaccine serum il-10 predicts worse outcome in cancer patients: a meta-analysis il-10: master switch from tumor-promoting in flammation to antitumor immunity immune cell dysfunctions in breast cancer patients detected through whole blood multi-parametric flow cytometry assay inhibition of dendritic cell migration by transforming growth factor-b 1 increases tumor-draining lymph node meta stasis effective clinical responses in metastatic melanoma patients after vaccination with primary myeloid dendritic cells classical flt3l-dependent dendritic cells control immunity to protein vaccine flt3 ligand enhances the cancer therapeutic potency of naked rna vaccines dendritic cell-specific delivery of flt3l by coronavirus vectors secures induction of therapeutic antitumor immunity pd-1 blockade enhances the vaccination-induced immune response in glioma silencing ido in dendritic cells: a novel approach to enhance cancer immunotherapy in a murine breast cancer model searching for the human genetic factors standing in the way of universally effective vaccines global analyses of human immune variation reveal baseline predictors of postvaccination responses key: cord-297790-tpjxt0w5 authors: mandl, judith n.; schneider, caitlin; schneider, david s.; baker, michelle l. title: going to bat(s) for studies of disease tolerance date: 2018-09-20 journal: front immunol doi: 10.3389/fimmu.2018.02112 sha: doc_id: 297790 cord_uid: tpjxt0w5 a majority of viruses that have caused recent epidemics with high lethality rates in people, are zoonoses originating from wildlife. among them are filoviruses (e.g., marburg, ebola), coronaviruses (e.g., sars, mers), henipaviruses (e.g., hendra, nipah) which share the common features that they are all rna viruses, and that a dysregulated immune response is an important contributor to the tissue damage and hence pathogenicity that results from infection in humans. intriguingly, these viruses also all originate from bat reservoirs. bats have been shown to have a greater mean viral richness than predicted by their phylogenetic distance from humans, their geographic range, or their presence in urban areas, suggesting other traits must explain why bats harbor a greater number of zoonotic viruses than other mammals. bats are highly unusual among mammals in other ways as well. not only are they the only mammals capable of powered flight, they have extraordinarily long life spans, with little detectable increases in mortality or senescence until high ages. their physiology likely impacted their history of pathogen exposure and necessitated adaptations that may have also affected immune signaling pathways. do our life history traits make us susceptible to generating damaging immune responses to rna viruses or does the physiology of bats make them particularly tolerant or resistant? understanding what immune mechanisms enable bats to coexist with rna viruses may provide critical fundamental insights into how to achieve greater resilience in humans. an estimated ∼60% of emerging infectious diseases are caused by pathogens which originate from a non-human animal source, referred to as zoonoses (1) (2) (3) . moreover, the frequency of outbreaks caused by zoonotic pathogens has been increasing over time in the human population, with viruses being the most successful at crossing the species barrier (2) (3) (4) . given the impact of viral zoonoses on global public health, considerable resources have been invested into better understanding patterns in their emergence to improve predictions of where they might arise. one key variable in such predictions is to determine the animal reservoir populations within which these novel viruses can be maintained indefinitely (with or without disease) and which therefore act as sources for transmission to humans (5) . in some instances, epidemiological associations may provide clues to identifying a reservoir host species, and the detection of natural infection through seroconversion or the virus itself provides further evidence. recently, phylogenetic analyses have also been used to investigate viral origins-with a presence of greater diversity and of strains ancestral to those in humans being indicative of a virus circulating within a particular natural host population (6) . once identified, viral reservoirs have historically been critical levers through which to reduce human cases (5) . however, reservoir hosts may also provide us with fundamental insights into host-pathogen interactions and are a rich opportunity to examine the immunological processes that contribute to patterns governing which pathogens cross into humans, cause disease and why (7, 8) . this can be particularly informative as in many instances, the zoonotic viruses that are so pathogenic in humans do not cause disease in the reservoirs with which they coexist. bats have been confirmed as reservoir hosts for many viruses, several of which are associated with fatality rates as high as 90% among diagnosed human cases. it has long been appreciated that rabies and other lyssaviruses causing lethal encephalitis can be transmitted from numerous bat species (9, 10) . live marburg virus (marv) has been isolated from rousettus aegyptiacus fruit bats which, jointly with epidemiologic evidence and detection of viral rna, strongly suggests that r. aegyptiacus is a reservoir host of this filovirus (11) . the related ebolavirus (ebov) likely also circulates in african fruit bats, with a few species having been implicated so far-the mobility of which accounts for the sudden appearance of ebola in west africa during the 2014 outbreak, a region where ebolavirus had not previously been detected (12, 13) . the highly pathogenic henipaviruses, of which hendra virus emerged in australia and nipah virus in south-east asia via horse and pig intermediate hosts respectively, have been shown to be transmitted from pteropus bats (14, 15) . in china, horseshoe rhinolophus bats have been identified as the reservoirs for sars coronavirus via palm civet intermediate hosts, the cause of a large outbreak of atypical pneumonia across several countries that began in 2002 in china (16) (17) (18) . more recently, mers coronavirus that has caused lethal respiratory infections mostly in saudi arabia, likely transmitted via dromedary camels, was shown to be closely related to several bat coronaviruses, including those sequenced from neoromicia capensis, pipistrellus abramus, and vespertilio superans bats (19, 20) . moreover, additional viruses may continue to emerge from bats, as in the single case of sosuga virus infection in a wildlife biologist collecting bats in south sudan (21) . in addition to these emerging zoonotic viruses, bats may be the source of a number of viruses with which humans have older evolutionary associations. for instance, bats harbor viruses closely related to both mumps (rubula virus) and measles (morbilli virus) and have likely been donors of these viruses to other mammalian groups, possibly including humans (6, 22) . furthermore, both old and new world bats carry diverse hepadnaviruses, some of which are related to hepatitis b virus and can infect human hepatocytes (23) . hepaciviruses that are related to hepatitis c virus and pegiviruses that are related to human gb viruses were detected in the sera of many different bat species, and given the basal position of these bat viruses in phylogenetic trees, may also represent strains ancestral to those found in humans (24, 25) . the preponderance of links between bat and human pathogens has led to a debate about whether bats disproportionately contribute to emerging viral infections crossing the species barrier into humans (26) (27) (28) (29) (30) . given the diversity of the chiroptera order (figure 1) , we may simply see more bat viruses because there are so many (>1,300) species of bats (31) . however, even when accounting for the fact that they make up ∼20% of extant terrestrial mammals, bats are overrepresented as reservoir hosts of pathogens with a high potential for spilling into human populations (32, 33) . in fact, no known predictors that have been described to impact the likelihood of crossing the species barrier, including reservoir host ecology, phylogenetic relatedness to humans or frequency of reservoir-human contact, explain this pattern (32) . thus, why bats are such a frequent source of pathogenic human viruses remains a tantalizing mystery. among viruses, those that have genomes encoded by rna generally jump across species boundaries more frequently, presumably due to their inherently greater mutation rates that facilitate the rapid adaptation to replicating within new hosts (34) . interestingly, all pathogenic viruses that have made the jump to humans for which bat species may be reservoirs share the common feature that they have single-stranded rna genomes (with the exception of hepadnaviruses which have a dna genome but replicate via an rna intermediate). so far, available evidence suggests that bats remain disease-free when infected with the rna viruses they carry-even those highly pathogenic to humans-and are able to coexist with them without detectable fitness costs using measures such as changes in temperature, loss of body weight, or overt signs of inflammation (35) . indeed, so far only one rna virus studied which circulates in a bat population has been shown to consistently cause significant morbidity and mortality: tacaribe virus in the jamaican fruit bat (artibeus jamaicensis), which recent evidence suggests is not a reservoir host for this virus (36) . data from experimental rabies and lyssavirus infections suggests that rhabdoviruses may also cause disease in bats, although experimental infection outcome is very dependent on the infection route. intracerebral infection with different strains and in different bat species invariably led to death (37, 38) . in contrast, intramuscular infection led to muscle weakness, paralysis and visible histological cns lesions in 30% of experimentally infected flying foxes (pteropus poliocephalus) (39) . similarly, a subset of vampire bats (desmodus rotundus) experimentally infected intramuscularly with a high dose of rabies virus remained healthy despite viral shedding in the saliva and survived (40) . naturally infected bats are thought to either die or remain healthy and seroconvert, but transmission in freeranging populations remains incompletely understood (41) . while bats seem to be frequent hosts for rna viruses, current available data indicates that primates and humans disproportionately harbor dna viruses such as herpesviruses (32) . interestingly, it is these dna viruses that can persist in an individual which can also be found in isolated, small indigenous groups-perhaps suggestive of humans having a more ancient relationship with such dna viruses (42) . it may even be the case that persistent dna viruses in humans impact immune responses specifically to rna viruses, but this has not yet been examined. it is likely that differences in evolutionary history of pathogen exposure between bats and humans have led to distinct adaptations in anti-viral immune responses and the ability to tolerate certain infections without disease while being susceptible to others. importantly, bats differ in many aspects of their physiology and behavior from humans that may have direct or indirect effects on immune function. bats are a monophyletic mammalian group traditionally divided by morphological data into two suborders, the megabats and microbats, which more recent molecular data has revised into the yinpterochiroptera and yangochiroptera suborders (figure 1 ). bats possess a suite of traits that make them distinct from other mammals in a number of ways. these unique life history traits may play a role in understanding which pathogens bats have evolved to coexist with and why. in particular, such traits may explain the ability of bat populations to maintain particular viral pathogens indefinitely, and may have effects on immune function through specific energetic or evolutionary trade-offs we have yet to better define. despite the diversity of viruses carried by bats, they are not typically known to cause mass bat die-offs or reduce bats' remarkable longevity. in this respect, bats represent a potential opportunity for long-term persistence of viruses within a population and across generations. bats live significantly longer than similarly-sized terrestrial mammals and, despite their small size, are characterized as "slow" mammals in the slow-fast continuum (43, 44) . although their weights range from 2 grams to 2 kilograms, with respect to longevity bats group with large mammals such as humans and non-human primates (45) . aerial living has an obvious advantage in avoiding predation, but bats outlive even birds. for example, the brandt's bat (myotis brandtii) lives up to 41 years, compared to selasphorus platycercus, a bird species of similar size that lives for ∼14 years (45, 46) . thus, flight can only partially account for their extraordinarily long lives. initially, the longevity of some bats was attributed to seasonal hibernation, as temperate-zone species enter continuous torpor of up to 75 days, with a dramatic drop in metabolic rate such that small fat reserves can sustain them throughout the entire hibernating season (43) . however, even non-hibernating bat species live three times longer, on average, than predicted by their size, and heterothermy is not an accurate predictor of lifespan in other mammalian orders, suggesting that the driving force behind their surprising longevity is intrinsic to bats as a group (47) (48) (49) . like other "slow" mammals, bat females typically only have one offspring per year, perhaps because the volant lifestyles of bats make it difficult to rear more than one offspring, as pregnant females and those with recent births must navigate and forage with added weight; on average, neonatal bat pups are ¼ of their mother's weight (50) . the physical and energetic constraints of rearing multiple offspring may necessitate small litters, which would in turn require prolonged reproductive capability and enhanced longevity to ensure maintenance of the population over generations. thus, in bats, the dependence of colony survival as a whole may depend upon enhanced individual survival and delayed senescence (51) . genetic analyses of several bat species have shown differences in the growth hormone (gh)/insulin-like growth factor 1 (igf1) axis which in humans is associated with aging, resistance to diabetes and cancer (52) . the determinants of adult survival in bats have been historically difficult to identify, as this requires tracking individuals over many years, and until recently longitudinal studies of bat mortality were conducted using tagged bats, of which only a fraction were recovered (53) . recently, a 19year study of a colony of bechstein's bats demonstrated that unlike terrestrial mammals, survival could not be predicted by common indicators such as season, age, and body size. instead, the only accurate predictor of mortality was a single cataclysmic weather event that affected multiple countries in north-central europe. additionally, even the oldest female bats were reproductively capable, indicating that bat survival is primarily affected by catastrophic natural events rather than factors that normally dictate an individual's fitness (45) . molecular phylogenetic studies of bats suggest that there are massive gaps in bat fossil records. as bats are the second most diverse order of mammals, outnumbered only by rodents, the number of species unrepresented in the fossil records is staggering. over half of microbat and nearly all of megabat fossil histories are missing (31, 54) . the enormous incompleteness of the fossil records has made it difficult to identify when specific morphological traits of bats arose. as molecular phylogeny groups two echolocation-reliant microbat species with megabats (also called old world bats or pteropodids), which do not rely on echolocation, there is some debate as to whether echolocation first arose in the common ancestor of bats and was subsequently lost in megabats, or whether it arose twice, independently (31) . pteropodids have adaptations that enhance visual acuity at night (55), and they do not require echolocation for foraging (56) . there are multiple types of echolocation that can be partially delineated by species, but are more clearly categorized by the type of environment. divergent species that inhabit the same type of environment, such as those that hunt in large, open spaces, often use the same form of echolocation, suggesting that habitat has a greater influence on echolocation than phylogeny (31) . importantly, echolocation can result in the production of droplets or small-particle aerosols of oropharyngeal fluids, mucus, or saliva, thus facilitating transmission of viruses between individuals in close proximity (57, 58) . the unique navigation tactic of many bat species may inadvertently facilitate virus transmission among bats in the same habitat. bats are the only mammal capable of powered flight, which likely evolved ∼65 million years ago alongside birds following radical ecological changes that resulted in the extinction of the dinosaurs (54, 59) . during flight, bats consume approximately four times as much oxygen, and they have a markedly higher concentration of red blood cells compared to small terrestrial mammals (60). bat flight is markedly different from that of birds and insects, whose wing surfaces are typically composed of inflexible material, such as feathers or chitin. bat wings are constructed from live skin stretched across elongated arm and finger bones, making them extraordinarily malleable and sensitive to environmental cues (59) . the plasticity of bats' wings allows them to navigate and inhabit diverse ecospheres, contributing to their extensive speciation. moreover, the capability of powered flight can allow the efficient spread of viruses and thus the introduction of pathogens to which colonies may otherwise have remained naïve. as flight is extremely metabolically demanding, in addition to evolving the physical mechanisms required for flight, bats have also evolved necessary underlying molecular mechanisms. the mitochondrial respiratory chain accounts for nearly all atp required for mobility in eukaryotes, and genetic analysis of both micro-and megabat species revealed an enrichment of genes specific to the oxidative phosphorylation (oxphos) pathway. specifically, 4.9% of nuclear-encoded and 23% of mitochondrial oxphos genes have evidence of positive selection in bats, which is markedly higher than the expected 2% of orthologous genes in previous genome-wide studies that show evidence of positive selection (61) . genomic analysis of pteropus alecto and m. davidii suggests positive selection for the dna damage checkpoint pathway and changes in overlapping aspects of this pathway with the innate immune system, indicating that evolutionary adaptations important for flight may have secondarily affected bat immunity (62). as a group, bats exhibit the greatest diversity of social systems in mammals. tropical species are primarily responsible for this diversity, as temperate species are more restricted in their social behavior. generally, however, bats are extremely social creatures that tend to form dense roosting colonies (63) , and almost all temperate-zone species live in closed societies with very little infiltration of foreign bats into established roosts (63, 64) . in particular, female bats form maternity colonies in which males do not take part. as bats are capable of longdistance flight, dispersal barriers cannot explain the philopatry of females. instead, benefits such as knowledge of foraging areas and social thermoregulation likely selected for these colony types. additionally, there is evidence that forming closed societies limits the potential invasion of new pathogens, thereby protecting colony members that would otherwise be vulnerable to infection. for example, pseudogymnaoscus destructans has decimated north american bat populations that do not live in the type of closed societies observed elsewhere (64) . dna analysis of a closed society of bechstein's bats revealed extraordinarily high conservation of mitochondrial dna and relatively low conservation of nuclear dna, suggesting stable maternal populations within colonies and gene flow between colonies via promiscuous mating with males. it is possible that the mating patterns of temperate-zone species may allow transmission of pathogens between colonies via traveling males while the frontiers in immunology | www.frontiersin.org more insular females may allow viruses to persist throughout generations within a colony. an important commonality among pathogenic rna viruses in humans presenting with disease is that the host response is an important contributor to the disease process, with dysregulated and excessive innate immune responses being particularly important drivers of tissue damage during infection (8) . given the general absence of clinical signs of disease in bats infected with the same viruses that are so lethal in humans or other non-natural hosts infected experimentally, a critical question has been to understand whether bats might establish effective disease tolerance, thus maintaining fitness despite pathogen replication, or whether bats are more resistant to infection through more successful control of pathogen replication and what the contribution of the immune response is (65, 66) . the lack of many fundamental immunological tools enabling the probing of bat immune responses has meant that truly mechanistic studies of bat immunity have been very limited, although recently there has been some progress in establishing approaches such as flow cytometry to identify distinct bat immune cell populations (67, 68) . so far, studies of bat immunity have primarily taken one of three approaches, whereby each comes with important strengths and weaknesses that have to be kept in mind: (i) comparative genome studies, (ii) in vitro cell culture assays, and (iii) experimental infections. comparative genome studies have confirmed that the critical components of the innate and adaptive immune system are conserved in bats at the gene level and that bats have the machinery for innate responses to pathogen-associated molecular patterns (pamps), the production of anti-viral effector molecules such as type i interferons (ifn), t cell responses (variable t cell receptors, mhci and mhcii), and b cell responses [reviewed in (35) ]. interestingly, based on the 10 bat genomes sequenced so far, the only family of genes lost entirely in all of them are pyhin genes (69) . members of the pyhin family are dna sensors capable of recognizing foreign dna, including dna viruses and damaged self dna which can be generated by rna viral infection. recognition of dna results in production of ifn through interaction with stimulator of interferon genes (sting). the pyhin family also encode the only identified class of dna sensors capable of activating the inflammasome. it has been hypothesized that the absence of the pyhin family may allow bats to limit activation of the innate immune response to damaged self-dna generated by rna viral infection, thus avoiding excessive inflammation (69, 70) . genome comparisons highlighting contractions or expansions of specific gene families, specific genes under positive selection, or nonconserved sequence differences in critical protein domains can thus provide the basis for hypotheses worth testing further. however, it is important to note that much can be missed in absence of data on gene regulation, especially during infection when gene expression kinetics can make a critical difference to the infection outcome. moreover, the absence of a gene or gene family does not rule out that other proteins have evolved to compensate for their loss of function. thus, while whole genome analyses can provide a context for specific questions or be hypothesis-generating, on their own they cannot distinguish tolerance from resistance mechanisms. the repeated identification of signatures of positive selection in innate immune genes in particular, does however lend credence to the idea that bats have specific adaptations as a result of a long co-evolutionary history with viruses. cell culture assays with bat cell lines, or, in some instances, primary bat cells, have been used to assess whether bats are permissive for viral replication and to determine whether particular immune receptor signaling pathways are intact. as discussed below, such studies have probed the type i ifn pathway in particular, revealing some possible species-specific differences among bats (71) (72) (73) (74) (75) (76) (77) (78) (79) (80) (81) (82) (83) . however, it is important to note that in some instances immortalized cells can behave differently from primary cells and that such cultures may miss additional differences imposed by changes in cell localization, cell recruitment or cell-cell interactions in a whole animal. careful experiments measuring the quality, magnitude, and kinetics of immune responses in bats during infection and upon administration with defined stimuli for which we have comparative information from humans remain to be done to provide additional evidence that specific innate immune pathways are wired differently. experimental infections come with the enormous challenge of having to house and/or breed colonies of bats and to have biosafety-level 4 facilities in place to perform infections with viruses lethal to humans. moreover, some trial and error is involved in determining which route and dose leads to viral replication, establishing a source of the virus (humanadapted strains tend to replicate less well in bats than strains obtained from naturally infected bats), and amplifying this viral stock without extensive tissue culture passaging. studies to date have examined the kinetics of viral replication by quantifying the extent of viremia and dissemination to other tissues, and assessing changes in white blood cell counts, body mass, and temperature. given the generally low levels of viral shedding and short infectious periods observed so far it remains poorly understood how transmission occurs in the wild to sufficient levels that cross-species jumps occur. some infection experiments have also provided evidence that a particular bat species is unlikely to be a reservoir despite epidemiological evidence, for example for r. aegyptiacus and ebolavirus. certainly, once good experimental infection models are established, such studies have the potential to be hugely informative with regard to anti-viral immune responses elicited using, for instance, comparative transcriptome analyses. one drawback may be that experimental infections do not mimic the impact of chronic stress arising from the disruption of wildlife populations, which bats are particularly sensitive to jones et al. (84) . comparison of either cave-roosting or foliage-roosting species in areas of malaysian borneo designated as actively logged forest, recovering forest, or fragmented forest revealed varying impacts of habitat disturbance on stress and circulating white blood cells (85) . overall, the limited studies of bat immunity that have been done have focused largely on 2 species: p. alecto and r. aegyptiacus. we summarize this work below, but comparisons of observations made across species suggest that although a number of species appear to be capable of avoiding the pathological effects of rna virus infection, each bat species may have achieved this through distinct pathways, possibly involving changes to both increase pathogen replication control and to mitigate any immunopathology through decreased inflammatory responses and hence increased disease tolerance. the most well studied bat species with regard to antiviral immune responses is the australian black flying fox (p. alecto). this interest has stemmed from the fact that pteropid bats have been identified as the natural reservoirs for the deadly hendra and nipah viruses (86) , which continue to cause outbreaks [such as most recently in india in may 2018 (87)]. to date, several studies have examined the kinetics of viral infection in pteropus bats and the nature of transmission and replication in other susceptible species (88) (89) (90) (91) . in australia, all four species of pteropid bats (p. alecto, p. poliocephalus, p. scapulatus, and p. conspicillatus) have antibodies to hendra virus but only p. alecto and p. conspicillatus are considered to be the primary reservoir hosts (14, 92, 93) . in south east asia, both pteropus spp. occurring in malaysia have been found to be seropositive for nipah virus neutralizing antibodies, and the virus has been isolated from p. hypomelanus and p. vampyrus (15, 94) . experimental infections of pteroid bats with hendra or nipah virus result in sub-clinical infection with short periods of virus replication and shedding, and low antibody titres (88) (89) (90) (91) . upon subcutaneous infection of p. poliocephalus with hendra virus, viral antigen was detected by immunohistochemistry at 10 dpi in blood vessels of spleen, kidney and placenta (89) . similarly, oronasal hendra virus infection of p. alecto led to the presence of viral genome in lung, spleen, liver and kidney 3 weeks later, but virus isolation was unsuccessful at this timepoint (89, 91) . the malaysian flying fox, p. vampyrus and the australian species, p. poliocephalus demonstrate similarly short periods of viremia upon infection with nipah virus. in subcutaneously infected p. poliocephalus, virus was isolated from the kidney and uterus of bats euthanized at 7dpi, but no virus was isolated at any of the other timepoints examined (3, 5, 10, 12, or 14 dpi) and there was no evidence of antigen in any tissue by immunohistochemistry, including tissues collected at 7 dpi. in this study, low neutralizing antibodies were detected in all bats with the exception of one individual that developed a significant neutralizing antibody titre -possibly reflecting the fact that p. poliocephalus is not the natural host for nipah virus (90) . in p. vampyrus challenged by oronasal nipah inoculation, viral genome was detected in a throat swab at 4 dpi and a rectal swab of the same individual at 8 dpi but virus was undetectable in tissues collected at postmortem from all individuals (49, 50, or 51dpi), consistent with a short period of viremia. similar to previous studies, antibody titres were low in all p. vampyrus bats (91) . overall, these results are consistent with bats controlling replication rapidly, at least following experimental infections which involve higher doses of virus compared to what bats would likely be naturally exposed to in the wild. the absence of a robust antibody response also appears to be typical of all experimental hendra and nipah virus infections performed to date. since antibody responses are the only immune parameter that has been measured during experimental infections of bats so far, it is difficult to speculate on the mechanisms responsible for control of viral infections in vivo. pteropus alecto was among the first bat species to have its genome described in detail. genomic studies provided initial clues for possible differences in the innate immune system of bats, with evidence for selection of key innate immune genes and the expansion or contraction of specific immune gene families (62, 68, 95) . the mhci region is contracted (96) , as is the type i ifn locus, which in p. alecto contains fewer ifn genes than any other mammalian species sequenced, with only three functional ifn-α loci (68) . in contrast, pteropid bats have the largest and most diverse family of apobec (apolipoprotein b mrna editing enzyme, catalytic polypeptide-like) proteins identified in any mammal (95) . apobecs interfere with the replication of retroviruses by deaminating cytosine residues in nascent retroviral dna. this is notable, as bats are an important source of mammalian retroviruses, many of which have been transmitted to other mammals (97, 98) . apobec diversification may therefore have occurred to counteract the effect of retroviruses and possibly other viruses, as apobecs have been shown to restrict the replication of other virus families including hepadnaviruses, and parvoviruses (99, 100) . members of the apobeca3 protein family exhibit direct antiviral activity through dna cytosine deamination which results in hypermutation of the nascent retroviral dna which is then degraded or rendered non-functional (101) . the mechanism of antiviral activity against non-retroviruses remains largely unknown. for parvovirus adeno-associated virus, apobec meditated inhibition has been speculated to involve direct interaction with the viral dna or the replication machinery (102) . whether the expanded family of abobecs in bats have evolved other mechanisms to control dna and rna viruses remains to be determined. as apobecs can be induced by even low levels of type i ifn (103) , one hypothesis to be tested is that bats, through their multiple apobecs, are able to restrict viral replication without causing inflammation. pteropus alecto is the only bat species to date in which apobec genes have been mapped, and whether the expansion of this gene family extends to other bat species remains to be determined. in addition to the identification of putative immune pathways distinct in p. alecto through genome studies, differences have been identified in the activation of innate immune effectors in p. alecto from studies performed in vitro, primarily using cell lines derived from tissues including the kidney and lung. ifns are the first line of defense following viral infection and unsurprisingly, because of this, they have been the most extensively studied group of genes in bats. both type i (ifna and ifnb) and iii (ifnl) ifns are detectable in bat cells. curiously, a unique characteristic of pteropid bats is the constitutive expression of mrna for ifna and the signaling molecule, ifn regulatory factor 7 (irf7) in unstimulated tissues and cells [75, 68a] . constitutively expressed ifna and irf7 may allow bats to respond more rapidly to infection, thus avoiding the lag time between pathogen detection and response. furthermore, viral infection or stimulation with synthetic ligands result in little ifna induction in pteropid bat cells (68) . the constitutive expression of ifna has been described in two species of pteropid bats (p. alecto and cynopterus brachyotis) and is a first for any species. ifnb and ifnl are activated following stimulation of cells from p. alecto and p. vampyrus with synthetic ligands such as polyic (71) (72) (73) (74) . moreover, bat ifns demonstrate antiviral activity (68, (71) (72) (73) (74) 104) . however, viral infection of p. alecto splenocytes results in induction of ifnl but not ifnb, hinting at differences in the function of type i and iii ifns (74) . in humans and mice, ifnl has recently been demonstrated to have a role not only in controlling virus replication, but also in dampening damage-inducing neutrophil functions and in modulating tissue-damaging, transcriptionindependent responses such as production of ros (77, 80) . a hypothesis yet to be tested is whether upregulation of ifnl rather than ifnb has a similar function in bats. the endoplasmic reticulum (er) membrane protein, sting, is involved in induction of type i ifn by cytosolic dna (105) . stimulation of bat splenocytes with gmp-amp, which is produced following sensing of cytosolic dna by cgas, results in little induction of ifn compared to responses observed in mouse splenocytes (83) . bat sting contains an amino acid substitution of the highly conserved and functionally important serine residue s358 which may be responsible for dampening sting-dependent ifn activation in bat cells in response to dna. however, comparable levels of ifn induction in mouse and bat cells in response to the rna viral mimic polyic indicate that sting-associated inhibition of the ifn response does not extend to rna viruses (83) , thus the relevance to rna viruses in bats remains unknown. downstream of the induction of ifns, novel subsets of ifn stimulated genes (isgs) have been detected in unstimulated and stimulated pteropid bat cells indicative of a response that is less damaging to the host. furthermore, the isg response is elevated for a shorter period of time in p. alecto compared to human cell lines which again may be a strategy to avoid tissue damage (78, 81) . the less inflammatory profile of isgs may be the key to the ability of bats to tolerate higher ifn expression without adverse consequences. the balance between resistance and tolerance may therefore be achieved through careful selection of the pathways that are activated and shorter periods of activation or limited activation to prevent inflammation. in this regard, studies of the regulation of ifn signaling in bats is likely to provide important additional insights. a second bat species whose host responses to viral infections has been studied more recently is the egyptian fruit bat (r. aegyptiacus). marburg virus (marv) has been repeatedly isolated from this species with demonstrated seasonal pulses of active marv replication in juvenile bats living in caves in uganda (11, 106) . moreover, r. aegyptiacus were a suspected reservoir for ebolavirus (ebov) based on epidemiological evidence and detected seroreactivity to ebov, but no infectious virus has been isolated thus far from wild rousettus bats (107) . indeed, while cell lines from r. aegyptiacus are equally susceptible to marv and ebov (79, 108) , experimental infections of r. aegyptiacus seem to confirm that it is a reservoir for marv, but is unlikely to be the source of ebov spillover to humans. subcutaneous ebov infection results in very low viral replication, no viremia, little dissemination to other tissues, and no viral shedding, although some animals seroconvert, suggesting that r. aegyptiacus are unlikely to perpetuate ebov in the wild (109, 110) . in contrast, experimental marv infection of r. aegyptiacus resulted in acute viremia that peaked on days 5-6 post-infection (although generally at lower levels than in humans), oral shedding that peaked on days 7-8 postinfection, and dissemination to other tissues including spleen, liver, kidney and salivary glands (109, (111) (112) (113) . interestingly, viral replication was not associated with increases in white blood cell counts, any clinical signs of infection such as changes in body temperature or body weight, and infected tissues showed little evidence of inflammatory infiltrates (109) . in all experiments, viremia was cleared by day 13 and oral shedding ceased by day 19. intriguingly, a cohousing experiment resulted in marv transmissions to uninfected bats 4-7 months after experimental infection, raising the question of whether persistent infection with intermittent shedding is possible or whether very long latent periods without detectable viral replication could follow exposure (114) . upon secondary challenge of previously marv-infected bats, none showed any detectable viral replication or shedding, providing evidence that protective immunity is established (115) . unlike for pteropus bats, no constitutive expression of type i ifns has been detected in r. aegyptiacus (79) , but type i ifns are induced in r. aegyptiacus cell lines upon stimulation with sendai virus as seen in other mammals (82) . furthermore, in r. aegyptiacus the type i ifn genes are expanded, again in contrast to p. alecto (82), but like for p. alecto a number of genes in the type i ifn pathway or involved in innate immune recognition of pamps show signs of having been under positive selection (82) . whether positive selection of genes in either bat species is associated with tolerance remains to be determined, especially given that innate immune genes in humans have also been under positive selection (116) . a transcriptome study which generated 20 rna sequencing libraries from 11 tissues taken from 1 female and 1 male r. aegyptiacus found a reduced coverage of nk cell related genes compared to other mammals, but confirmed that in these bats the predominant t cells had an αβ t cell receptor, and showed that ige, igg, igm, and iga, as well as a number of pro-and anti-inflammatory cytokines, were all detectable (117) . the recently sequenced r. aegyptiacus genome revealed substantial differences in the repertoire of nk cell receptors, with this bat species entirely lacking functional killer cell immunoglobulin receptors (kirs) and with all killer lectinlike receptors (klrs) encoding either activating and inhibitory interaction motifs, or inhibitory interaction motifs only (82) . nk cells are important immune cell players in an antiviral response but without assessment of the consequences of these genomic differences it is difficult to draw any specific conclusions with regard to viral control or the magnitude of inflammation elicited upon infection with viruses like marv. nonetheless, these genomic data provide some interesting hypotheses to be tested in the future. some additional studies probing the induction of cytokines upon stimulation of bat cells with defined innate immune stimuli provides some evidence that innate immune recognition of viruses may be altered, leading to a reduction in proinflammatory responses. stimulation of kidney and myeloid cells from the big brown bat (eptesicus fuscus) with polyinosinicpolycytidylic acid (polyi:c) resulted in only limited activation of the inflammatory cytokine, tumor necrosis factor alpha (tnfα) compared to human cells which display a robust tnfα response. induction of tnfα is controlled by transcription factors, including the nf-kappa b (nf-κb) family which consists of five members, [rela (p65), relb, c-rel, nfκb-1 (p50), and nfκb-2 (p52)] which form homo-or hetero-dimers that are bound by molecules of the inhibitor of nfκb (iκb) family and retained in the cytoplasm of the cell in an inactivated state (118) . in e. fuscus, a potential repressor (c-rel) binding motif was identified in the tnfα promoter region which may explain the difference in induction of tnfα in e. fuscus cells. consistent with this hypothesis, partial knockdown of c-rel transcripts significantly increased basal levels of tnfα transcripts in e. fuscus cells (104) . the transcription factor, c-rel has also undergone positive selection in the bat ancestor which may indicate that this mechanism is common to other species of bats (62) . of note, low levels of tnfα induction have also been associated with tolerance in european bank voles which are a natural reservoir for puumala hantavirus (puuv) (119) . stimulation of macrophages from the greater mouse eared bat (myotis myotis) suggested that this species may have also evolved mechanisms to avoid excessive inflammation caused by cytokines. while high levels of tnfα, il1β, and ifnβ were produced in response to in vitro challenge with lipopolysaccharides (lps) and polyi:c, there was also a sustained, high-level transcription of the anti-inflammatory cytokine il-10, which was not observed in mouse macrophages (120) . furthermore, unlike in the mouse, m. myotis macrophages did not produce the proinflammatory and cytotoxic mediator, nitric oxide, in response to lps. the same study also showed evidence of bat specific adaptations in genes involved in antiviral and proinflammatory signaling pathways through comparison with other mammalian taxa, including rig-i, il1b, il-18, nlrp3, sting, and casp1, further supporting the evolution of adaptations associated with reducing inflammatory responses in bats (120). even less is known about immune responses of bats to nonviral pathogens than to viral pathogens, but it is clear that while anti-inflammatory responses may be characteristic of antiviral responses in bats, they are susceptible to disease upon infection with particular pathogens-in some instances due to dysregulated and damaging immune responses. one particular example of this is the emerging infectious disease, white nose syndrome (wns), that has decimated north american bat populations beginning in 2006, in what will likely rank as one of the most devastating wildlife diseases in history (121) (122) (123) . for reasons that remain poorly understood, the psychrophilic fungus pseudogymnoascus destructans (formerly geomyces destructans) causes no mass mortality in european bats despite being abundantly detected (124, 125) . indeed, evidence suggests that a single p. destructans genotype was introduced to north american bat species from europe (125) . in north america, p. destructans infection is not specific to a particular bat genus, replicating in many different bat species during hibernation and targeting the furless skin of the wings, ears, and muzzle (126) . distinct hypotheses have been proposed for why p. destructans is so deadly in north american bats, ascribing the impaired tolerance to infection compared to european bat counterparts to either physiological or immunological factors. on the one hand, more frequent arousal, electrolyte depletion, and dehydration are thought to contribute to mortality following infection (127, 128) . the destruction of wing tissue in wns results in a marked electrolyte imbalance, as the wings play a critical role in maintaining water levels, especially during hibernation, during which bats are particularly vulnerable to dehydration (129, 130) . dehydration catalyzes arousal in hibernating bats, which is extraordinarily metabolically costly and rapidly depletes the fat reserves necessary to survive until spring (127) . an alternative hypothesis posits that the restoration of the immune system following emergence from hibernation induces the fatal pathology of wns. during hibernation, destruction of cutaneous tissue is limited and infiltrating immune cells are entirely absent, yet in the weeks following arousal, infected bats exhibit overt wing damage and corresponding neutrophilic and lymphocytic infiltration (131) . hibernation does not preclude a localized immune response to p. destructans at the site of infection and transcriptomic analysis of infected tissue showed upregulation of some acute inflammatory genes in infected tissue (132, 133) . however, the observed immune responses likely occur during arousal periods, which are more common in infected bats. ultimately, immunosuppression during torpor allows p. destructans to colonize infected bats relatively unchecked (124) , and upon emergence from hibernation, the exuberant immune response may result in deadly immunopathology during wns (131) . in addition to general studies of immune cell recruitment and transcriptional responses during wns, body mass and white blood cell counts were examined following lps administration in four bat species (134) (135) (136) (137) . subcutaneous lps challenge in of pallas's mastiff bats (molossus molossus) led to a loss of body mass of ∼7% within the first day, but did not result in changes in circulating white blood cell counts or body temperature (135) . seba's short-tailed fruit bat (carollia perspicillata) also showed a decrease in body mass following lps challenge, but this was associated with increases in white blood cell counts as well as increases in derivatives of reactive oxidative metabolites (drom) (134) . subdermal lps challenge of fish-eating myotis (myotis vivesi) led to body mass decreases, increased resting metabolic rate and skin temperature (136) , while intraperitoneal lps challenge of wrinkle-lipped bats (chaerephon plicatus) caused an increase in circulating leukocytes, but did not result in a reduction in body mass compared to controls (137) . the differential responses to lps challenge suggest that the immune response to bacterial infection varies across species. of note, postmortem examinations of ∼500 dead bats comprising 19 species from germany revealed inflammatory lesions, many of which had evidence of underlying bacterial or parasitic infections, particularly in the lung (138) . bats have an array of unique life history characteristics that not only allow them to be particularly good reservoirs for viruses that are highly pathogenic in other species, but also appear to have shaped their immune systems. although research on bat antiviral immunity has focused on only a few species to date, at the genomic level, selection on genes is concentrated on the innate immune system across both suborders of bats. however, while these studies have provided a rich source of hypotheses, the majority remain to be tested at the functional level and many questions remain that cannot be answered from comparative genome studies. experimental studies to date have demonstrated some functional differences between bat species, with the common emerging theme that the overall antiviral response appears to converge on a lower inflammatory profile, with tight regulation of the cytokine and inflammatory response key to clearing viral infection without the pathological outcomes typically associated with infection. however, whether this is due to specific tolerance mechanisms that are at play or increased resistance to rna virus replication still remains unclear. fewer studies have examined the adaptive immune system than those probing innate immune pathways, but experimental infections with bat borne viruses have demonstrated that bats generate low or absent antibody responses which often wane rapidly. this is reminiscent of the response of another reservoir host, the sooty mangabey which is the natural reservoir for simian immunodeficiency virus (siv) and for yellow fever virus. sooty mangabeys given an attenuated yellow fever virus vaccine strain generate much lower, transient antibody responses as compared to humans or rhesus macaques. changes to innate immune responses are also evident in sooty mangabeys (139) . thus, intriguingly, different reservoir hosts may have arrived at similar solutions to avoid the pathological consequences that follow viral infection in non-natural hosts. despite the ability of bats to avoid disease associated with viral infection, this trait does not extend to all pathogens, as evidenced by the severe consequences associated with infection of north american bats with the fungus that causes wns. thus, the pathways associated with the control of other pathogens have not been under the same selection pressures as those responsible for controlling infections with rna viruses-or there are immunological trade offs involved which lead to greater susceptibilities to some pathogens than others. overall, it is clear that studying host-pathogen interactions in reservoir hosts has considerable potential to provide novel insights into host tolerance mechanisms that eventually could assist in the treatment of diseases in humans and other susceptible hosts and may also offer solutions for the treatment of diseases that are a conservation threat to bats themselves. risk factors for human disease emergence global trends in emerging infectious diseases global rise in human infectious disease outbreaks prediction and prevention of the next pandemic zoonosis identifying reservoirs of infection: a conceptual and practical challenge bats host major mammalian paramyxoviruses studying immunity to zoonotic diseases in the natural host -keeping it real reservoir host immune responses to emerging zoonotic viruses host phylogeny constrains cross-species emergence and establishment of rabies virus in bats the spread and evolution of rabies virus: conquering new frontiers isolation of genetically diverse marburg viruses from egyptian fruit bats fruit bats as reservoirs of ebola virus comparative phylogeography of african fruit bats (chiroptera, pteropodidae) provide new insights into the outbreak of ebola virus disease in west africa isolation of hendra virus from pteropid bats: a natural reservoir of hendra virus isolation of nipah virus from malaysian island flying-foxes severe acute respiratory syndrome coronavirus-like virus in chinese horseshoe bats bats are natural reservoirs of sars-like coronaviruses isolation and characterization of a bat sars-like coronavirus that uses the ace2 receptor rooting the phylogenetic tree of middle east respiratory syndrome coronavirus by characterization of a conspecific virus from an african bat bat origin of human coronaviruses a recently discovered pathogenic paramyxovirus, sosuga virus, is present in rousettus aegyptiacus fruit bats at multiple locations in uganda isolation of multiple novel paramyxoviruses from pteropid bat urine bats carry pathogenic hepadnaviruses antigenically related to hepatitis b virus and capable of infecting human hepatocytes identification of gbv-d, a novel gb-like flavivirus from old world frugivorous bats (pteropus giganteus) in bangladesh bats are a major natural reservoir for hepaciviruses and pegiviruses what links bats to emerging infectious diseases? mass extinctions, biodiversity and mitochondrial function: are bats 'special' as reservoirs for emerging viruses? are bats exceptional viral reservoirs? a comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? bats as 'special' reservoirs for emerging zoonotic pathogens a molecular phylogeny for bats illuminates biogeography and the fossil record host and viral traits predict zoonotic spillover from mammals are disease reservoirs special? taxonomic and life history characteristics the evolutionary genetics of emerging viruses antiviral immune responses of bats: a review serological evidence of arenavirus circulation among fruit bats in trinidad experimental rabies infection in insectivorous bats pathogenesis of bat rabies in a natural reservoir: comparative susceptibility of the straw-colored fruit bat (eidolon helvum) to three strains of lagos bat virus pathogenesis studies with australian bat lyssavirus in greyheaded flying foxes (pteropus poliocephalus) salivary excretion of rabies virus by healthy vampire bats rabies virus distribution in tissues and molecular characterization of strains from naturally infected non-hematophagous bats infectious diseases in primitive societies generation time: a reliable metric to measure life-history variation among mammalian populations rare catastrophic events drive population dynamics in a bat species with negligible senescence. sci rep birds as long-lived animal models for the study of aging mammalian aging, metabolism, and ecology: evidence from the bats and marsupials fly now, die later: life-history correlates of gliding and flying in mammals reduced free-radical production and extreme longevity in the little brown bat (myotis lucifugus) versus two non-flying mammals constraints on reproduction by flying vertebrates: energy and calcium life histories and elasticity patterns: perturbation analysis for species with minimal demographic data genome analysis reveals insights into physiology and longevity of the brandt's bat myotis brandtii survival estimation in bats: historical overview, critical appraisal, and suggestions for new approaches bat flight comparative morphology of the tapetum lucidum (among selected species) the evolution of echolocation in bats rabies virus in nasal mucosa of naturally infected bats bats: important reservoir hosts of emerging viruses bat flight: aerodynamics, kinematics and flight morphology the physiology and energetics of bat fligth adaptive evolution of energy metabolism genes and the origin of flight in bats comparative analysis of bat genomes provides insight into the evolution of flight and immunity everyday bat vocalizations contain information about emitter, addressee, context, and behavior causes and consequences of living in closed societies: lessons from a long-term socio-genetic study on bechstein's bats two ways to survive infection: what resistance and tolerance can teach us about treating infectious diseases tolerance of infections phenotypic and functional characterization of the major lymphocyte populations in the fruit-eating bat pteropus alecto unlocking bat immunology: establishment of pteropus alecto bone marrow-derived dendritic cells and macrophages unique loss of the pyhin gene family in bats amongst mammals: implications for inflammasome sensing influenza infection induces host dna damage and dynamic dna damage responses during tissue regeneration establishment, immortalisation and characterisation of pteropid bat cell lines chiropteran types i and ii interferon genes inferred from genome sequencing traces by a statistical gene-family assembler interferon production and signaling pathways are antagonized during henipavirus infection of fruit bat cell lines type iii ifns in pteropid bats: differential expression patterns provide evidence for distinct roles in antiviral immunity irf7 in the australian black flying fox, pteropus alecto: evidence for a unique expression pattern and functional conservation contraction of the type i ifn locus and unusual constitutive expression of ifn-α in bats ifn-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function the ifn response in bat cells consists of canonical and non-canonical isgs with unique temporal expression kinetics. biorxiv innate immune responses of bat and human cells to filoviruses: commonalities and distinctions interferon (ifn)-λ takes the helm: immunomodulatory roles of type iii ifns. front immunol ifnar2-dependent gene expression profile induced by ifn-α in pteropus alecto bat cells and impact of ifnar2 knockout on virus infection the egyptian rousette genome reveals unexpected features of bat antiviral immunity dampened sting-dependent interferon activation in bats carpe noctem: the importance of bats as bioindicators habitat disturbance results in chronic stress and impaired health status in forest-dwelling paleotropical bats henipaviruses in their natural animal hosts outbreak of nipah virus encephalitis in kerala state of india transmission studies of hendra virus (equine morbillivirus) in fruit bats, horses and cats experimental hendra virus infection in pregnant guinea-pigs and fruit bats (pteropus poliocephalus) experimental nipah virus infection in pteropid bats (pteropus poliocephalus) pteropid bats are confirmed as the reservoir hosts of henipaviruses: a comprehensive experimental study of virus transmission serologic evidence for the presence in pteropus bats of a paramyxovirus related to equine morbillivirus spatiotemporal aspects of hendra virus infection in pteropid bats (flying-foxes) in eastern australia nipah virus infection in bats (order chiroptera) in peninsular malaysia differential evolution of antiretroviral restriction factors in pteropid bats as revealed by apobec3 gene complexity evolution and comparative analysis of the bat mhc-i region identification of diverse full-length endogenous betaretroviruses in megabats and microbats bats and rodents shape mammalian retroviral phylogeny apobec3a is a potent inhibitor of adeno-associated virus and retrotransposons apobec1 and apobec3 cytidine deaminases as restriction factors for hepadnaviral genomes in non-humans in vivo the apobec3 family of retroelement restriction factors deaminase-independent inhibition of parvoviruses by the apobec3a cytidine deaminase ifnalpha induces apobec3g, f, and a in immature dendritic cells and limits hiv-1 spread to cd4 + t cells lack of inflammatory gene expression in bats: a unique role for a transcription repressor sting an endoplasmic reticulum adaptor that facilitates innate immune signaling seasonal pulses of marburg virus circulation in juvenile rousettus aegyptiacus bats coincide with periods of increased risk of human infection large serological survey showing cocirculation of ebola and marburg viruses in gabonese bat populations, and a high seroprevalence of both viruses in rousettus aegyptiacus cell lines from the egyptian fruit bat are permissive for modified vaccinia ankara experimental inoculation of egyptian rousette bats (rousettus aegyptiacus) with viruses of the ebolavirus and marburgvirus genera experimental inoculation of egyptian fruit bats (rousettus aegyptiacus) with ebola virus virological and serological findings in rousettus aegyptiacus experimentally inoculated with vero cellsadapted hogan strain of marburg virus oral shedding of marburg virus in experimentally infected egyptian fruit bats (rousettus aegyptiacus) lack of marburg virus transmission from experimentally infected to susceptible in-contact egyptian fruit bats modelling filovirus maintenance in nature by experimental transmission of marburg virus between egyptian rousette bats egyptian rousette bats maintain long-term protective immunity against marburg virus infection despite diminished antibody levels. sci rep genomic signatures of selective pressures and introgression from archaic hominins at human innate immunity genes de novo transcriptome reconstruction and annotation of the egyptian rousette bat the nuclear factor nf-κb pathway in inflammation tnf-α expression and promoter sequences reflect the balance of tolerance/resistance to puumala hantavirus infection in european bank vole populations a potent anti-inflammatory response in bat macrophages may be linked to extended longevity and viral tolerance white-nose syndrome in bats: illuminating the darkness bacteria isolated from bats inhibit the growth of pseudogymnoascus destructans, the causative agent of white-nose syndrome united states geological survey skin lesions in european hibernating bats associated with geomyces destructans, the etiologic agent of white-nose syndrome white-nose syndrome without borders: pseudogymnoascus destructans infection tolerated in europe and palearctic asia but not in white-nose syndrome initiates a casacde of physiological disturbances in the hibernating bat host frequent arousal from hibernation linked to severity of infection and mortality in bats with white-nose syndrome electrolyte depletion in white-nose syndrome bats wing pathology of whitenose syndrome in bats suggests life-threatening disruption of physiology evaporative water loss is a plausible explanation for mortality of bats from white-nose syndrome pathology in euthermic bats with white nose syndrome suggests a natural manifestation of immune reconstitution inflammatory syndrome the white-nose syndrome transcriptome: activation of anti-fungal host responses in wing tissue of hibernating little brown myotis immune responses in hibernating little brown myotis (myotis lucifugus) with white-nose syndrome inflammatory challenge increases measures of oxidative stress in a free-ranging, long-lived mammal no fever and leucocytosis in response to a lipopolysaccharide challenge in an insectivorous bat metabolic cost of the activation of immune response in the fish-eating myotis (myotis vivesi): the effects of inflammation and the acute phase response simulated bacterial infection disrupts the circadian fluctuation of immune cells in wrinkle-lipped bats (chaerephon plicatus) diseases in free-ranging bats from germany distinctive tlr7 signaling, type i ifn production, and attenuated innate and adaptive immune responses to yellow fever virus in a primate reservoir host all authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.the handling editor declared a shared affiliation, though no other collaboration, with the authors jm and cs.copyright © 2018 mandl, schneider, schneider and baker. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-262944-9k64f0tw authors: parker, elaine l.; silverstein, rachel b.; verma, sonam; mysorekar, indira u. title: viral-immune cell interactions at the maternal-fetal interface in human pregnancy date: 2020-10-07 journal: front immunol doi: 10.3389/fimmu.2020.522047 sha: doc_id: 262944 cord_uid: 9k64f0tw the human decidua and placenta form a distinct environment distinguished for its promotion of immunotolerance to infiltrating semiallogeneic trophoblast cells to enable successful pregnancy. the maternal-fetal interface also successfully precludes transmission of most pathogens. this barrier function occurs in conjunction with a diverse influx of decidual immune cells including natural killer cells, macrophages and t cells. however, several viruses, among other microorganisms, manage to escape destruction by the host adaptive and innate immune system, leading to congenital infection and adverse pregnancy outcomes. in this review, we describe mechanisms of pathogenicity of two such viral pathogens, human cytomegalovirus (hcmv) and zika virus (zikv) at the maternal-fetal interface. host decidual immune cell responses to these specific pathogens will be considered, along with their interactions with other cell types and the ways in which these immune cells may both facilitate and limit infection at different stages of pregnancy. neither hcmv nor zikv naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. here, we will highlight new approaches using placenta-on-a-chip and organoids models that are providing functional and physiologically relevant ways to study viral-host interaction at the maternal-fetal interface. pregnancy is a unique immunological phenomenon in which the semiallogenic fetus is able to grow in the maternal uterine environment. in order for a successful pregnancy to occur, healthy placentation is necessary to create an environment that is protective for the developing fetus and promotes growth. how immune balance is maintained by maternal and fetal cells to promote the survival of the genetically distinct fetus, while preventing infection by a large number of pathogens, is yet to be fully elucidated (1) . this little understood enigma has been the subject of interest and research for decades (2) . fertilization leads to the creation of single celled embryo which undergoes several successive divisions to form a blastocyst. the blastocyst is made up of two types of cells: the outer trophoblast or trophoectoderm (te) layer forming the placenta and chorion, and the inner layer or inner cell mass (icm) forming the embryo proper and amnion (3) . the decidua underlying the embryo is called the decidua basalis, which composes the maternal side of the placenta. the maternal-fetal interface is made up of the maternal decidua and fetally-derived placenta. during implantation, the blastocyst attaches to the decidualized endometrium and the outer layer of the blastocyst differentiates into different lineages. the te gives rise to cytotrophoblast cells (ctbs) which follow villous and extravillous pathways to form the placenta. in the villous pathway, the mononuclear ctbs fuse, creating multinucleated syncytiotrophoblasts (stbs) that establish floating villi (fv). the fv are surrounded by maternal blood, with stbs aiding the provision of nutrients by enabling gas exchange and exchange of secreted pregnancy-related hormones (human chorionic gonadotropin, hcg, human placental lactogen, hpl) at the maternal-fetal interface. furthermore, ctbs act as anchoring villi for the attachment of the embryo to the uterus. the ctbs present in the cell column of the anchoring villi follow the extravillous pathway and differentiate into interstitial (ictbs) and endovascular extravillous trophoblast cells (ectbs). the ictbs further invade up to the inner third of the myometrium and ectbs remodel the spiral arteries in low resistance high blood flow to provide nutrients to the developing embryo (3) (4) (5) (6) . the invasion of trophoblast cells at the maternal-fetal interface occurs in the presence of a large population of maternal immune cells (7) . this includes 70% decidual natural killer (dnk) cells, 20%-25% macrophages, 3%-10% t cells and 1.7% dendritic cells (8) (9) (10) . the abundance of decidual cytotoxic t cells and macrophages can vary through the course of pregnancy (11) . the abundance of nk cells in the decidua during the first trimester, and through the pregnancy (albeit at lower abundance), implicates them as an essential element in both the promotion of an immunotolerant environment and the control of pathogenic infection during pregnancy ( figure 1) . thus, the paradoxical maternal-fetal interface is admired for both its immunotolerance to semiallogeneic trophoblastic invasion (leading to a successful pregnancy) while remaining remarkably resilient to pathogenic infections. nevertheless, several pathogen, termed torch pathogens (described below), successfully cross the placental barrier and cause devastating infection in the developing fetus (12) . in this review, we will look at the interactions between decidual immune cells and specific viral torch pathogens and review known mechanisms which may enable viral pathogenesis within the placental environment. torch is an acronym defining some of the most common infections associated with vertical transmission. initially described in 1971, this group contained just 4 pathogens; toxoplasmosis, rubella, cytomegalovirus (cmv) and herpes simplex type 1 and 2 (13) . since then this group has been broadened to comprise a host of other infections including listeria monocytogenes, syphilis, varicella zoster virus, human immunodeficiency virus (hiv), enteroviruses and parvovirus b19 (14) . most recently, following the zika virus (zikv) epidemic in south america resulting in observed congenital anomalies, this group has been further expanded to include zikv, with some suggesting renaming this group "torchz" (15) . the mechanism by which these "torchz" pathogens are able to circumvent typical clearance by groups of immune cells (e.g. nk cells, macrophages and others) has been studied by many groups over the last few decades in order to elucidate not only routes of pathogenicity but also roles of immune cells within this immune-privileged environment (12) . it remains to be proven whether the new emerging viral threat by sars-cov2 which causes covid-19 including in pregnant women, will be included in this group of vertically transmitted pathogens (16) . in this review, we will focus on maternal and fetal macrophages, t cells, and nk cells and their relationship with each virus. we will focus on the viruses human cytomegalovirus (hcmv) and zikv, which are known causes of adverse pregnancy outcomes and delve into how they interact with various decidual immune cells to promote their survival and replication. we will examine the timings of pregnancy that appear to be most permissive to pathogenic infection by these viruses and we will look at the role of various immune cells in this context ( figure 2 ). in the early first term decidua, 3%-10% of resident leukocytes are t cells with approximately 30%-45% of these t cells being cd4+ (t helper cells) and 45%-75% being cd8+ (cytotoxic) t cells (17) (18) (19) . further studies have estimated the decidual cd4+ population to be comprised of about 50% activated memory cd25dim t cells and 5% cd4+ cd25bright foxp3+ treg cells. unlike the peripheral circulation, the decidua has a higher ratio of cd8+ t cells to cd4+ t cells and an overall higher number of cd8+ t cells (20) . in addition, approximately 40% of the decidual cd8+ population are effector-memory t cells with reduced perforin and granzyme b in comparison to their peripheral counterparts (21) . one study published in 2016 described a small percentage of cd8+ t cells found in uncomplicated term decidua to be viral specific. though these populations of viral specific cd8+ t cells were 1.3% and 2.2% in the decidual basalis and decidual parietalis respectively, they demonstrated that this was higher than that seen in peripheral blood and postulated a role for their presence in the decidua as one of immunoprotection for the fetus. this study could not conclude upon the origin of these t cells, and whether they were recruited from the periphery or activated in the decidua. in addition, more work remains to be done to establish whether these virus specific cd8+ t cells exist early in pregnancy (22) . another study has described the presence of a small population of cd4+ hla-g+ t cells which are thought to acquire hla-g through trogocytosis from decidual dendritic cells. it is thought that these t cells promote immunotolerance at the maternal-fetal interface, and they have been shown to be downregulated in pathologies such as preeclampsia (pe) (23) . therefore, it appears t cells play specific roles in immunity and tolerance. to this end we will look at the role that various populations of t cells may play in either enabling or preventing infection by torch pathogens at the maternal-fetal interface. macrophages constitute 20-25% of all leukocytes in the first trimester decidua and play an important role in tissue remodeling, angiogenesis, host defense and immunotolerance (24) . macrophages are considered a key link between adaptive and innate immunity, communicating to other immune cells and modulating their activity (25, 26) . these cells are therefore vital throughout pregnancy, adapting their phenotype to address the changing requirements of the evolving decidua (27) . tissue resident decidual macrophages are thought to be recruited from monocytes in the peripheral circulation (28) . distinct subtypes of macrophages have been shown to be present in first-trimester decidual tissue exhibiting immunomodulatory, proinflammatory, and tissue remodeling phenotypes and play key roles in protective immunity as well as fetal tolerance (29) . decidual macrophages are known for their highly immunosuppressive phenotype at the maternal-fetal interface, expressing cd206, dc-sign and tim-3 among other receptor markers (30, 31) . in addition to these maternally derived macrophages exist fetal-derived macrophages called hofbauer cells (hcs), which sit in the stroma of the chorionic villi (32) . these hcs are resident in close proximity to fetal vessels and trophoblast cells from the first trimester until birth. hcs could serve as a portal of entry for pathogens from the infected mother (33) . initially during implantation, they appear to have an inflammatory m1 phenotype which has both microbicidal activity and promotes a cell-mediated th1 cytokine response. later, they shift to a mixture of both m1 and m2 phenotypes following trophoblastic invasion and remodeling (34, 35) . several studies have implicated hcs in host viral interactions. here, we look at the reciprocal interactions between hcs, maternal macrophages, and hcmv and zikv. the nk cell population in the peripheral circulation is predominately made up of cd56dim cd16+ cells, which are believed to have a more cytotoxic phenotype (36) . approximately 10% of the peripheral circulation is constituted by cd56bright nk cells, which have a more immunotolerant phenotype (37) . in the decidua, these nk cell proportions are reversed; 70-80% of the total lymphocytes are cd56bright cd16(36) . research has demonstrated a number of dnk subsets within the cd56+cd16-population. it is believed that this distinct immunotolerant population is fundamental to the maintenance of a successful pregnancy, with research postulating both an ability to enable the semiallogenic fetus to thrive while at the same time responding to pathogenic infections. these nk cells reside in the decidua basalis close to invading evts and express specific receptors (e.g. kir receptors, cd94/nkg2a, ilt2) to activate or inhibit evt function (38) . this large population of dnk cells are known to be sustained during the first and second trimester, with their numbers declining toward term (11, 39) . despite the unique immunotolerant phenotype demonstrated by dnk cells, it is evident that this cell population displays a high level of plasticity, gaining cytotoxic function in the presence of specific pathogens (39) . one way by which this happens is through activation of dnk cell cytotoxcity via killer cell ig-like receptor 2ds1 (kir2ds1). reduced expression of this receptor has been associated with adverse pregnancy outcomes such as miscarriages and fetal growth restriction and individuals with increased kir2ds1 expression have shown better outcomes post-viral infections (40) . we will explore further the role that nk cells play in specific viral infections in pregnancy torch pathogens hcmv human cytomegalovirus (hcmv) was first described in 1954 by margaret smith, who replicated a virus from two newborn babies who had died from cytomegalic inclusion disease (cid) (41) . what we now know as hcmv first came to the attention of ribbert et al. in 1881, where intranuclear inclusions within large cells were noted in renal and parotid gland cells of stillborn fetuses. these inclusions, often described as 'owl's eye inclusions', were noted to be surrounded by a clear halo (42) . hcmv was identified in the 1950s when smith, weller and rowe isolated and cultured hcmv from salivary glands, adenoid tissue and liver biopsies respectively (43, 44) . mechanisms of vertical transmission of hcmv can either be transplacental during gestation or transvaginal during parturition; additionally, there is some evidence for breastmilk transmission (45) . hcmv infection is most likely to occur in the third trimester, demonstrating a 30% risk of mother to child transmission in the first trimester compared to a 70% risk in the third trimester (46) (47) (48) . congenital hcmv has been estimated to affect 5-20 in every 1,000 live births, with 10% of hcmv positive infants suffering neurological consequences from birth (49) . hcmv infection during pregnancy therefore poses a substantial risk to the developing fetus, leading to congenital disease including cerebral abnormalities such as periventricular calcifications, microcephaly, visual impairment, sensorineural hearing loss, neurodevelopmental delay and hepatomegaly (45) . congenital hcmv affects 20,000-40,000 pregnancies annually in the united states and accounts for 25% of all incidents of pediatric sensorineural hearing loss (50) (51) (52) . it is estimated that the burden of morbidity associated with congenital hcmv infection is greater than that of other common congenital pediatric conditions such as down's syndrome or fetal alcohol syndrome (53) (54) (55) . hcmv is also associated with intrauterine growth restriction and miscarriage. there is a great need to understand maternal immunity pathways involved in hcmv infection to develop effective vaccines (56) . hcmv is associated with asymptomatic infection of most of the world's population and subclinical illness in pregnant mothers. in the us, an estimated 2% of unexposed pregnant women experience primary infection during pregnancy, resulting in congenital infection in 32% of cases from this population (53, (57) (58) (59) (60) (61) . however, vertical transmission of hcmv is not only seen in mothers with primary infection but also igg seropositive mothers, who exhibit a 1% rate of congenital hcmv infection. mechanisms of infection have been studied through analysis of placental tissue from all three trimesters of human gestation. in placental tissues from those suffering from hcmv, necrosis and oedema has been noted associated with severity of congenital disease symptoms. it has also been noted that hcmv infection is often associated with bacterial coinfection with a potentially pathogenic synergism (62) . hcmv resides in the chorionic villi, specifically infecting ctbs, stbs and hcs. it is believed that the ability to travel between stbs in the decidua is key to hcmv pathogenesis (63) . many studies have explored the role of the adaptive and innate immune system in hcmv infection. below we review established interactions between hcmv and immune cells (figure 3 ). hcmv's ability to infect different populations of macrophages has been demonstrated by several studies. hcmv has been shown to be sequestered by hcs, with placentas from confirmed cases of hcmv infection demonstrating significant hyperplasia of this cell population (64) (65) (66) . a study investigating vaccine development showed that when neutralizing antibodies are produced against hcmv, rates of hcs infection are decreased (67) . a different study utilizing placental explants showed hcmv-igg immune complexes to undergo fc receptor mediated transcytosis as a mechanism to traverse the syncytium to ctbs. hcmv is then taken up by hcs in the placental villi (68) . furthermore, another study by loenen et al., supports the idea that hcmv genes are able to increase fcr expression on infected cells (69) . another study suggested that hcmv replication in stbs is upregulated in the presence of macrophages (70) by analyzing hcmv replication in stbs alone or when infected stbs were cultured with uninfected placental macrophages. this study also demonstrated elevated levels of hcmv viral titres in co-cultured supernatants when compared to those from stbs cultured alone. this demonstrates that not only do macrophages have the capacity to be infected by hcmv, but also that they may amplify hcmv infection of surrounding cells in the decidua. some studies have depicted a role for latently infected maternal decidual macrophages in congenital hcmv infection, describing how microbial infections or insults in the placenta may reactivate these macrophages and in turn reactivate hcmv infection (71) (72) (73) . the maternal-fetal interface is unique in respect to allogenic interactions with cd8+ t cells. evts are known to invade the decidua, evading destruction despite the intrinsic ability of cd8+ t cells to recognize foreign antigen via mhc class i molecules. as discussed previously, one mechanism by which evts are believed to evade cd8+ t cell recognition is through a lack of expression of hla-a and hla-b, which are key to cd8+ cytotoxic activity. during pregnancy, many viruses have been shown to upregulate maternal cd8+ t cell activity, leading to migration of highly differentiated effector memory t cells to the decidua. despite many descriptions regarding the role of t cells in hcmv infection in the fetus and the mother, there are few studies identifying their tissue specific role at the maternal-fetal interface (74) . hcmv is thought to limit cd8+ t cell activity through restriction of mhc class ii expression on apcs, which in turn may prevent activation of cd8+ and cd4+ t cells (69) . this is thought to be mediated through the hcmv protein gpus2, which may degrade mhc class ii glycoproteins or disrupt downstream ciita/jak/stat signaling pathways (69) . crespo et al., in 2016 demonstrated that hcmv did not induce a significant difference in hla-g expression on either jeg-3 cells or primary evts. hla-g expression has been associated with immunotolerance, and therefore its persistence despite infection may act to protect infected trophoblast cells from cytotoxic destruction (40) . studies looking at the role of t cells in viral infection at the maternal-fetal interface demonstrated lower t-cell numbers and response in mothers who vertically transmitted hcmv to their offspring when compared to infected mothers who did not transmit hcmv, potentially suggesting an active role for t cells in vertical hcmv transmission (75) . more specifically, a reduction in the number of cd4+cd45ra +ifn-g+ treg cells and cd8+cd45ra+ifn-g+ t cells in mothers who transmitted hcmv to their fetus was noted when contrasted with mothers who were hcmv positive but did not transmit the infection. there was also a measurable blunted t cell response in hcmv infected mothers who vertically transmitted infection, compared to infected mothers who did not transmit the virus (76, 77) . in infected mothers, hcmv virus specific t cells have been shown to be elevated in the final trimester when compared to uninfected mothers (78) . congenital hcmv infection risk is highest for the fetus in the third trimester, with a 72% transmission risk compared to a 30% risk in first trimester (46) . this is despite the abundance of immune cells, specifically nk cells, in early pregnancy. dnk cells exposed to hcmv infected decidual fibroblasts are known to alter their phenotype to express higher levels of activating receptors (such as nkg2d and cd94/nkg2c or nkg2e). uniquely, utilizing in vitro studies, it was noted that decidual nk cells had targeted cytotoxic activity against hcmv infected autologous decidual fibroblasts and heterologous uninfected fibroblast cells, but appeared to spare trophoblast cells (79, 80) . this demonstrates a clear cytotoxic effector response by decidual nk cells to hcmv, switching from their typically immunotolerant phenotype with high levels of inhibitory receptor expression (cd94/nkg2a, lir-1, kirs), to a cytotoxic phenotype (79, 80) . this group also studied the interaction between dnk and hcmv-infected cells using hcmv positive and hcmv negative decidual villous explants. this investigation revealed through fluorescent staining of dnk cells that colocalisation of dnk cells to cells throughout the hcmv positive explant occurred, including synaptic connections which was not seen in hcmv negative explants. this was thought to suggest that the dnk cells were unable to connect with uninfected trophoblasts. this also demonstrates that dnk cells are able to localize and target hcmv infected cells while sparing fetal derived semiallogenic trophoblast cells (80) . dnk cells are unique in their function, both contributing to immunotolerance at the maternal-fetal interface, thereby enabling invasive trophoblastic activity, as well as controlling pathogenic infection (81) . this is thought to be mediated by secretion of specific cytokines (79, (82) (83) (84) (85) . the relatively limited vertical transmission of hcmv during the first trimester of pregnancy, when the population of nk cells is abundant, has led many to speculate about the role nk cells may play in hcmv control (10) . tilburgs and colleagues have recently demonstrated distinct cytotoxic responses in dnk cells to hcmv in first trimester versus at term wherein term pregnancy dnks harbor reduced efficacy in responding to hcmv-infected cells (86) . siewera et al., suggested that dnk cells undergo a phenotypic transformation to acquire cytotoxic function in the presence of hcmv-infected cells (80) . this study proved, through antibody mediated abrogation of the fas ligand (fasl) and tumor necrosis factor-related apoptosis-induced ligand (trail) on dnk cells, that death of hcmv infected cells is not initiated by dnk cells through these death receptor-ligand pathways. however, this study demonstrated that dnk cells form immunological synapses with hcmv infected fibroblasts, enabling the delivery of perforin/granzyme for cellular destruction. furthermore, the ability of dnk cells to degranulate in the presence of hcmv infected fibroblasts was demonstrated to be through high levels of cd107a expression, a key cell surface molecule in the mechanism of lytic granule release. dnk cells have also been found to secrete higher quantities of granulysin when compared to peripheral blood nk cells. upon incubation with infected fibroblast cells, it was noted that cd56bright nk cells decreased from 76.3% to 48%, while there was an elevation in cd16 expression by nk cells, denoting a transformation to a cytotoxic phenotype. hcmv infected cells have been noted to upregulate expression of natural cytotoxicity receptor (ncrs) nkp44 by almost 2-fold on dnk cells as well as increasing expression of nkg2c. ncrs are associated with activation of the cytotoxic profile of nk cells. accompanying this was a reduction in nkg2a, kir2dl1, kir2dl4, and ilt2 receptor expression, receptors aligned with nk effector inhibitory function (76) . activating dnk cell receptors such as kir2ds1, kir2ds2, kir2ds5 and kir3ds1 have been correlated with antiviral activity (40) . a study by crespo et al., demonstrated an increased population of kir2ds1 + nk cells in the decidua, suggesting an increased activating dnk cell capability in response to hla-c2, and thereby increased cytotoxic potential. these cells also displayed higher levels of cytolytic molecules when compared to peripheral nk cells. this study demonstrated that kir2ds1 + dnk cells showed increased cytotoxicity to hcmv infected decidual stromal cells (dscs) positive for hla-c2 when compared to kir2ds1-dnk cells. this was not the case for infected jeg-3 and primary evt cells, which did not appear to initiate degranulation or cytokine secretion from dnk cells. despite this, a reduction in the number of infected evts in the presence of co-cultured dnk cells was noted, suggesting that dnk may be clearing virus infected evts by other means (40) . hcmv has been seen to reduce expression of mhc class i, thereby potentially evading cd8+ t cell destruction (87) (88) (89) . one study reports an initial reduction in hla-c expression on evts in hcmv infection. the possible reason for this is not clear, however this study suggests it could prevent inhibition of nk cells through the hla-c/kir2dl1 route, with an additional suggestion of potentially other unknown ligands being upregulated for activation of kir2ds1, leading to cytotoxic action against infected cells (40) . another study showed that the potential effect of dnk cell activation on t-cell activation could be mediated via an upregulation in hla-dr expression upon exposure to hcmv infected fibroblasts (80) . therefore, dnk cells may play a role in congenital hcmv infection by potentially protecting the first trimester fetus from infection via activation of t cell function. collectively, these studies indicate varied interactions between dnk cells and hcmv, with many routes by which hcmv may evade clearance as well as a number of ways through which dnk cells may be activated in the presence of hcmv infected cells. additionally, dnk cells are seen specifically to modulate activity in the context of t cell activation. zika virus (zikv) was first isolated in 1947 in zika forest, uganda, from infected rhesus monkey serum during epidemiological yellow fever research (90, 91) . however, the first case of human infection was not reported until 1954, when three patients presented with jaundice and were later confirmed to have rising levels of zika antibodies (92) . initially, zikv was associated with innocuous prodromal illness on the african and indian subcontinents transmitted by the aedes aegypti mosquito, leading to an asymptomatic or self-limiting course of infection (93) . in 2007, a mild disseminated infection was identified to be zikv in over 70% of the population of the island of yap (94) . concerns regarding human zikv infections were not aroused until 2013 when incidences of neurological deficits associated with zikv infection were first described, with almost 30,000 recorded infections noted in french polynesia (95, 96) . shortly following this in 2015, a zikv epidemic began in south america where not only were neurological deficits such as guillain-barreå� yndrome seen, but also spontaneous abortion and congenital malformations such as microcephaly in infants from infected mothers (91) . by the end of the 2017 epidemic in brazil, there were more than 200,000 notifications of zikv cases (97) . estimates for infants born with congenital zika syndrome (czs) after the 2015-2016 epidemic ranged from 5 to50 in every 10,000 births (98) . the threat of a zikv epidemic lingers, with who reporting 61 countries affected by aedes aegypti mosquitoes, therefore carrying the potential for zikv infection and transmission (99) . zikv demonstrated continuing global epidemic capacity in india in 2018 (100) . zikv belongs to the flavivirus family alongside west nile virus, dengue virus, and yellow fever virus. zikv is an enveloped and icosahedral virus with a nonsegmented, 10.7 kb single stranded positive sense rna genomes (101) . this virus is composed of several proteins, categorized as three structural (capsid, pre-membrane and envelope) and seven nonstructural proteins. the seven nonstructural proteins (ns1, ns2a, ns2b, ns3, ns4a, ns4b, and ns5) are essential for viral replication and assembly, as well as being responsible for the pathogenicity of the virus by binding to transcription and restriction factors (95) . the biggest risk of congenital zikv infection is for mothers infected during their first trimester (102) . zikv infection demonstrates wide tissue tropism, with zikv successfully infecting the central nervous system, blood, retinal, genital and reproductive tissues including placenta (103) (104) (105) (106) (107) . zikv was thought to be exclusively arthropod transmitted until cases of human-human transmission emerged in nonendemic regions, illustrating a role for sexual transmission (108) (109) (110) . the presence of zikv rna has also been found in breast milk of zikv infected mothers (111) (112) (113) . however, there are reports which suggest that vertical transmission of zikv by breastmilk does not occur in most cases, which suggests the possibility that breastmilk does not have a high enough viral load to infect the newborn (114, 115) . despite some knowledge regarding zikv pathogenesis, its mechanism of infection in placental immune cell types remains limited (116) (117) (118) (119) . histopathology of zikv infected placentae has shown zikv infection in first trimester villous stromal tissue cells, which includes immune cells in the chorionic villi (117, 120, 121) . uniquely, zikv was also found to infect ctbs, endothelial cells, fibroblasts and hc in chorionic villi, as well as amniotic epithelial cells and trophoblast progenitor cells (103, (116) (117) (118) (122) (123) (124) (figure 4 ). similar to hcmv, zikv has also been shown to infect hcs and ctbs (90, 116, 117) . during the first trimester of pregnancy, zikv infects hcs, entering the fetal blood stream in order to reside in the placenta. zikv uses hcs as a "trojan horse". this strategy is utilized by several viruses in order to cross the blood brain barrier, where the virus infects leukocytes, leading to them being carried across barriers and thereby enabling the propagation and spread of infection (125) (126) (127) . hcs have been associated with zikv spread to the fetus through the "trojan horse" route (91) . the presence of zikv-specific antigen was demonstrated in hcs in confirmed maternal infection. multiple studies suggest hcs are a crucial step in vertical transmission of zikv to fetal cells, demonstrating that hcs are preferentially infected when compared to ctbs (116, 122, 128) . infection of hcs with zikv is thought to propagate infection through hyperplasia and proliferation of these cells, leading to persistence of this hc population into later trimesters (116, 122, 128) . a study performed on first trimester fetal and maternal tissue showed that zikv can replicate in different cell types, such as decidual fibroblasts and macrophages. it can also infect trophoblasts and hcs as well as umbilical cord mesenchymal stem cells, suggesting that the route of zikv infection may move from the decidua basalis to the anchoring villi (129) . a study performed using blood from 30+ asian zikv infected pregnant women shows that cd14+ monocytes are the primary target of zikv infection. these monocytes are resistant to change in m2 phenotype and downregulate type 1 ifn signaling, which induces the expression of different host genes involved in pregnancy complications (130) . in the decidua basalis, zikv infects evts, macrophages and stromal cells. zikv also targets proliferative ctbs in the anchoring villi, however is unlikely to infect stbs due to ifnl-mediated antiviral defense mechanisms (131) . zikv achieves replication within macrophages through fcr, tlr4 and dc-sign receptors (116, 132) . in vitro studies have demonstrated zikv infection to be augmented in hcs by igg from prior flavivirus exposure through antibody dependent enhancement (ade) (133) . there remain many gaps in knowledge regarding the role for macrophages targeted and infected by zikv. a study performed using decidual and chorionic villous tissue from early and mid-gestation human pregnancy shows that zikv appears to elevate type i and iii ifn expression, which does not occur in hcmv infection (131) . studies looking at the interaction between zikv and t cells in humans are scarce although zikv infection has been demonstrated to activate both cd4 and cd8 t cells (134) with specific increases in vd2 tcr+ cells which have been implicated in recurrent miscarriages but not associated with zikv-induced fetal complications. there have not been notable studies looking specifically at t cell zikv communication at the human maternal-fetal interface (135) . a recent study examined peripheral t cell responses of 45 confirmed cases of zikv infection that had been stimulated with pooled zikv peptides from all viral components (136) . this study demonstrated responses from both cd4+ and cd8+ t cells to both structural and nonstructural zikv components. however, this study particularly showed that cd4+ t cells exhibited a strong response to nonstructural proteins ns1, ns3 and ns5, and cd8+ t cells a strong response to cap and env proteins. this response was demonstrated by marked ifn-g production from both cell subtypes indicating cell activation (136) . another case looking at a zikv infected individual from the united states demonstrated interactions between the zikv ns2 and env proteins with cd4+ and cd8+ t cells, respectively. (137) . furthermore, in a different study, cd4+ t cells of two a b d c fibroblasts occurs through fcgr and results in increased expression of some interferons, such as ifna, and decreased expression of others, such as ifn-g and ifnb. infection is associated with increased secretion of proinflammatory cytokines such as ip-10, il-6, and mcp-1. ns5 viral proteins are thought to downregulate interferon-stimulated genes (isgs) and reduce interferon signaling via stat2 degradation, while the viral proteins ns1 and ns4b inhibit ifn signaling by downregulating tbk1. (c) cd4+ t cells exhibit a strong response to nonstructural ns1, ns3, and ns5 zikv proteins, while cd8+ t cells respond to cap and env zikv proteins. in both cases, response to zikv proteins was characterized by increased ifn-g production. (d) systemic dnk cells exhibit increased activation, including increased ifng production and cd107a expression when incubated with zikv infected monocytes. frontiers in immunology | www.frontiersin.org october 2020 | volume 11 | article 522047 zikv infected individuals showed activity in response to nonstructural proteins (ns1, ns3 and ns5). consistently, cd8+ t cells were seen to raise activity against the structural protein env (138, 139) . these studies demonstrate consistency in the response of cd4+ t cells and cd8+ t cells to zikv proteins, revealing cd4+ t cells to specifically respond to particular nonstructural proteins and cd8+ t cells to react to structural proteins, particularly cap and env (136, 138, 139) . several studies have looked at the ability of denv-specific cd4+ and cd8+ t cells to be stimulated by the presence of zikv peptides in humans (140, 141) . these studies showed viral epitopes for specific peptides located in similar regions and structurally conserved across flaviviruses; however, they displayed differences in their sequences (141) . nonetheless, these studies indicated cross-reactivity between the viruses regarding their cd4+ and cd8+ t cell activity. one study demonstrated that cd8+ and cd4+ t cells from denv positive donors reacted to zikv viral peptides, resulting in an upregulation of ifng secreting cells. this group also showed that stimulation with zikv peptides for those in acute phase of zikv infection resulted in recruitment of elevated levels of cd8+ ifn-g+ t cells (142) . a recent transcriptomics study investigated transcriptional signatures in cd4/cd8 t cells, b, and nk cells and plasmacytoid dendritic cells in patients (nonpregnant) infected with zikv (143) . interestingly, they did not note significance transcriptional changes in nk or cd8 t cells in a zikv infected background but noted significance alterations in pdcs. whether pregnancy plus zikv infection would affect the immune cell transcriptome in humans remains to be determined. studies specifically analyzing interactions between dnk cells and zikv in humans once again are lacking. however, studies have looked at zikv and its communication with peripheral nk cells. one such study postulated crosstalk between monocytes and nk cells in zikv infected patients. the activation of nk cells was associated with the presence of monocytes, which induced expression of ifn-g and cd107a, key markers of nk cell function. depletion of monocytes in the peripheral blood reduced the levels of these markers and thus the activation of nk cells (144) . there are few studies showing the interaction between zikv with nk cells. glasner et al., showed that zikv infection led to activation of mhc class i, which was somehow not sensed by dnk cells and their activating receptors, allowing the virus to escape nk cell-mediated killing. mhc class i expression is triggered through the ifn-b pathway via activation of rigi-irf3 (145) . however, the mechanism by which nk cells may promote an immunosuppressive environment in the face of zikv infection is not clear. some studies have indicated that interactions between other aspects of the innate immune system and nk cells may be at play in zikv pathogenesis. there are several studies suggesting that pathogenesis of zikv is not mediated through decidual immune cells alone but rather conducted, at least in part, through the activation of interferonstimulated genes (isgs), which in turn leads to activation of innate host cell immunity (131) . isgs act to specifically target viral replication. multiple studies have indicated zikv stimulation of interferons (ifn) to vary depending on the type of ifn. while type i and iii ifns have been shown to be inhibited by zikv, specifically the ns5 component of the pathogen, type ii ifns have been shown to be upregulated by the virus (146, 147) . one study demonstrated that when type iii ifns were upregulated, specifically ifn-l1, trophoblast cells were infected with zikv at a lower rate. further, ns1, ns4a and ns4b have been demonstrated to inhibit ifn type i response. this leads to suppression of the tank binding kinase 1 (tbk1)/irf3 and jak-stat pathway, which in turn results in reduced activation of innate immune responses (148) . interferon induced transmembrane protein 1 (ifitm1) and ifitm3 specifically are isgs which act as restriction factors to inhibit zikv replication. the mechanism by which the inhibition and activation of innate immunity impacts the recruitment of innate immune cells to the site of infection is not clear. little is known about the role of nk cells in human zikv infection. one study has noted interactions between tlr7, cd81 and ifitm1, postulating that the restriction of zikv is associated with inhibitory activity of ifitm1, potentially through activation of nk cells (149, 150) . another group looking at isgs showed that viperin played a role in zikv pathogenesis, with data revealing that when viperin levels were high, zikv mrna levels were low and vice versa (148) . ns4 is seen to target directly the akt-mtor pathway, leading to reduced signaling from this pathway and subsequent activation of autophagy in host cells (151) . zikv has been shown to co-opt the autophagy pathway for post-rna replication capacity and survival (152, 153) . importantly, the ns2b-ns3 protease activity of zikv can be blocked by an inhibitor of autophagy, hydroxychloroquine (hcq) (154) . hcq is an fda approved drug considered safe to use during pregnancy and could serve as an effective treatment for preventing zikv congenital syndrome (124) . the relationship between zikv infected cells and attenuated ifn production has been extensively reported, leading to questions regarding the mechanism underlying this association. it has been proposed that zikv may infect cells through ade of infection. many cells express the fcg receptor, and it is thought that viral particles may complex with antibodies and thereby enter into cells via fcg receptors (133) . host cells (such as trophoblasts and fibroblasts) infected with zikv demonstrate innate immune system activation with a rise in specific ifns (e.g. ifn-a), but falling levels of others such as ifn-l1 and ifn-b (155) . the elevated levels of proinflammatory cytokines and chemokines, namely il-6, mcp-1 and ip-10 which are linked to recruitment of immune cells such as monocytes and t cells (155) . zikv has been shown in multiple studies to downregulate type i ifn signaling and to be active in suppression of antiviral signaling. zikv nonstructural proteins ns1 and ns4b inhibit ifn signaling by downregulating levels of tbk1. however, ns2b3 downregulates the jak-stat pathway and inhibits apoptosis of zikv, and hence inhibits innate antiviral responses (150) . one study specifically has implicated the role of the nonstructural zikv protein ns5 in promoting zikv propagation by targeting stat2 for degradation, thereby reducing isg levels (156) . this is thought to promote viral replication through a dampened host innate immune cell response. there remains much to be elucidated in terms of zikv infection in human pregnancy. new studies are identifying metabolic reprogramming pathways underpinning innate immune responses to zikv which opens additional avenues of investigation (157) . we refer readers to recent reviews highlighting zikv-immune interactions in adverse pregnancy outcomes (119) and ade (158) . the limited availability of placental tissues during early pregnancy has always been a challenge for the reproductive biologists, hampering the study of placental physiology and cell to cell interactions. in vitro cell line models can often be biologically distinct and therefore unable to demonstrate enough similarity to replicate the conditions of human pregnancy. in addition, the use of cell line models can fail to reproduce the complexity of the number of cell types and cell interactions present within the decidua. therefore, functional in vitro 3d models being are developed, for example placenta-on-a chip and organoid cultures, which can mimic in vivo conditions and would be useful to understand the mechanisms of viral host interactions. the 'placenta-on-a-chip' is a microfluidics model utilizing human trophoblast cells (bewo) and fetal derived cells (huvecs and hpvecs) (159, 160) . these cell lines are cultured and separated by a semipermeable membrane within flow conditions with the purpose of understanding placental mechanisms and barrier function (159) . recent reports have described the faithfulness of placenta-on-a-chip model to in vivo placental conditions (161) . for example, glucose transport using a placenta-on-a-chip model was demonstrated by lee et al., and blundell et al., highlighting significant similarity to in vivo glucose transport in the human placenta (159, 160) . placenta-on-a-chip models have also been used to investigate the transport of heparin and anti-hyperglycemic agents such as glyburide using bewo and human placental villous endothelial cells (162) . recently, the transport of the xenobiotic compound caffeine across the placenta has been studied using this model system, providing new insights into the extent of caffeine transfer from mother to fetus (163) . bacterial infections have also been studied using this model. zhu et al., showed that in the presence of escherichia coli (e. coli), trophoblast cells (bewo) activated the circulating macrophages on the "maternal" side of the chip to secrete several inflammatory cytokines that mimicked in vivo conditions during pregnancy (164) . the impact of common environmental exposures such as titanium dioxide nanoparticles (tio 2 -nps) has also been studied using this 3d placental model showing a series of different placental responses (barrier permeability, oxidative stress, cell apoptosis, and maternal immune cells behavior (165) . they showed placental barrier permeability and maternal immune cells to be influenced by even low concentration of nps (165) . therefore, this simple in vitro model can prove useful in understanding the environmental exposure of nps during pregnancy and can help in a range of biological studies (165) . recent studies report generation of an organ-on-a-chip model, wherein decidualized human endometrial stromal cells and macrophage cell lines are co-cultured in a microfluidic device and shown to inhibit secretion of tnf-a in response to lps stimulation (166) . these devices have also been used to determine the impact of cytokine secretion by dnk cells on the migration of primary trophoblast cells. these studies illustrate the functionality of microfluidic organ on chip devices to elucidate importance of maternal immune cells in the placenta (167) . thus, the use of fetal membrane on organ-ona-chip provides a suitable model to explore the impact of pathogenic infections during pregnancy (168, 169) . the use of in vitro trophoblast organoids as a 3d culture model also provides a new tool to understand the mechanism of implantation at the maternal-fetal interface. recent studies have shown the characterization of these organoids derived from 1 sttrimester ctbs (6 to 8 weeks) and suggest their resemblance to primary trophoblast cells (170) (171) (172) . due to similarity with the placental architecture, these organoids could be used to study physiological, metabolic and hormonal changes that occur during pregnancy. the viruses we highlighted in this review, hcmv and zikv, do not naturally infect commonly used animal models [e.g., mice] which makes it challenging to understand disease pathogenesis. in particular, there remains a paucity of understanding zikv-immune cell interactions during pregnancy. thus, the employment of placenta-on-a-chip or organ-on-a-chip, and organoid models will be pivotal in providing functional and physiologically relevant ways to study the interaction of immune cells at the maternal-fetal interface with viral pathogens that affect pregnancy. both hcmv and zikv can be sequestered into fetal macrophages. hcmv implicates hcs in the potential infection of other decidual cells, leading to the promotion of hcmv transcytosis in trophoblasts. zikv preferentially infects hcs, persisting in this cell population and potentially mediating infection of other fetal-derived cells. more poignant is the suggestion that decidual macrophages may mediate reactivation of hcmv by acting as a latent reservoir for infection. these studies collectively indicate a central role for macrophages in the pathogenesis of torch viruses. dnk cells have been seen to alter their phenotype to express higher levels of various activating receptors when in the presence of infected decidual fibroblast cells. they also are known for their plasticity in the face of specific pathogens, acquiring more cytotoxic function. kir2dl1/hla-c2 has been identified as a mechanism by which dnk cells are activated and display cytoxicity toward hcmv infected cells. it has also been suggested that dnk cell activation may trigger activation of t-cells through upregulating hla-dr expression on infected fibroblast cells. zikv viral components demonstrate capacity to elicit strong responses from peripheral cd4+ and cd8+ t cells, with ns1, ns3, and ns5 being associated with cd4+ stimulation whereas cap and env proteins being associated with cd8+. we also see in this review the importance of interferon stimulating genes in the restriction of zikv replication. thus, the implications and outcomes of viral interactions with immune cells at the maternal-fetal interface are varied. we see the importance of the host immune response and recognize the importance of studying mechanisms of pathogenesis in detail to enable targeted therapeutic interventions including vaccines to mitigate the adverse outcomes of viral infections during pregnancy ( figure 5) . finally, we posit that better understanding of the immunological underpinnings of infections at the maternal fetal interface can support the inclusion of pregnant women in trials testing vaccines and therapeutics to compact existing and emerging viral infections. ep, sv, and im wrote the manuscript. rs generated the figures. all authors contributed to the article and approved the submitted version. this work was funded in part by a grant from the national institutes for health/national institute for child health and development r01hd091218 to im. rs was supported by a marc ustar fellowship. regulatory t cells and the immune pathogenesis of prenatal infection immune mechanisms at the maternal-fetal interface: perspectives and challenges what is the placenta? defective implantation and placentation: laying the blueprint for pregnancy complications the effects of oxygen concentration and gestational age on extravillous trophoblast outgrowth in a human first trimester villous explant model single-cell reconstruction of the early maternal-fetal interface in humans maternal allo-recognition of the fetus inflammation and pregnancy: the role of the immune system at the implantation site granulated lymphocytes of pregnancy the unique properties of uterine nk cells decidual leucocyte populations in early to late gestation normal human pregnancy congenital viral infection: traversing the uterine-placental interface the torch complex-perinatal infections associated with toxoplasma and rubella, cytomegol-and herpes simplex viruses zika virus -reigniting the torch sars-cov2 and pregnancy: an invisible enemy? t cell behavior at the maternal-fetal interface fetal-maternal hla-c mismatch is associated with decidual t cell activation and induction of functional t regulatory cells foxp3+ regulatory t cells and t helper 1, t helper 2, and t helper 17 cells in human early pregnancy decidua expression of nk cell receptors on decidual t cells in human pregnancy human decidual tissue contains differentiated cd8+ effectormemory t cells with unique properties the possible role of virus-specific cd8(+) memory t cells in decidual tissue expansion of cd4(+) hla-g(+) t cell in human pregnancy is impaired in pre-eclampsia the contribution of macrophages to normal and pathological pregnancies endometrial nk cells are special immature cells that await pregnancy human decidual macrophages suppress ifn-gamma production by t cells through costimulatory b7-h1:pd-1 signaling in early pregnancy role of macrophages in pregnancy and related complications monocyte and macrophage heterogeneity two unique human decidual macrophage populations macrophages at the fetal-maternal interface express markers of alternative activation and are induced by m-csf and il-10 tim-3 regulates innate immune cells to induce fetomaternal tolerance hofbauer cells: placental macrophages of fetal origin the phenotype of human placental macrophages and its variation with gestational age macrophage activation and polarization v-atpase upregulation during early pregnancy: a possible link to establishment of an inflammatory response during preimplantation period of pregnancy natural killer cells and pregnancy cd56bright natural killer (nk) cells: an important nk cell subset the role of decidual immune cells on human pregnancy features of human decidual nk cells in healthy pregnancy and during viral infection expression of kir2ds1 by decidual natural killer cells increases their ability to control placental hcmv infection propagation in tissue cultures of a cytopathogenic virus from human salivary gland virus (sgv) disease the history of cytomegalovirus and its diseases human cytomegalovirus: taking the strain the pathogenesis of human cytomegalovirus treatment of congenital cytomegalovirus infection: implications for future therapeutic strategies intrauterine transmission and clinical outcome of 248 pregnancies with primary cytomegalovirus infection in relation to gestational age increased risk of cytomegalovirus transmission in utero during late gestation hiv in pregnant women and their offspring: evidence for late transmission the march towards a vaccine for congenital cmv: rationale and models cytomegalovirus viral and antibody correlates in young children cytomegalovirus shedding and delayed sensorineural hearing loss: results from longitudinal follow-up of children with congenital infection congenital cytomegalovirus (cmv) infection as a cause of permanent bilateral hearing loss: a quantitative assessment washing our hands of the congenital cytomegalovirus disease epidemic congenital cytomegalovirus infection: audiologic outcome newborn hearing screening-a silent revolution advancing our understanding of protective maternal immunity as a guide for development of vaccines to reduce congenital cytomegalovirus infections primary cytomegalovirus infection in pregnancy. incidence, transmission to fetus, and clinical outcome cytomegalovirus seroconversion rates and risk factors: implications for congenital cmv awareness of and behaviors related to child-to-mother transmission of cytomegalovirus review and meta-analysis of the epidemiology of congenital cytomegalovirus (cmv) infection cytomegalovirus seroprevalence and childhood sources of infection: a population-based study among pre-adolescents in the united states models of vertical cytomegalovirus (cmv) transmission and pathogenesis human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis histologic correlates of viral and bacterial infection of the placenta associated with severe morbidity and mortality in the newborn modeling of human cytomegalovirus maternal-fetal transmission in a novel decidual organ culture characterization of the fetal inflammatory response to cytomegalovirus placentitis. an immunohistochemical study cytomegalovirus vaccines: current status and future prospects maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal fc receptor-mediated transcytosis immune evasion by human cytomegalovirus: lessons in immunology and cell biology placental macrophage contact potentiates the complete replicative cycle of human cytomegalovirus in syncytiotrophoblast cells: role of interleukin-8 and transforming growth factor-beta1 cytomegalovirus remains latent in a common precursor of dendritic and myeloid cells reactivation of latent human cytomegalovirus in cd14(+) monocytes is differentiation dependent intra-abdominal bacterial infection reactivates latent pulmonary cytomegalovirus in immunocompetent mice cd8+ effector t cells at the fetal-maternal interface, balancing fetal tolerance and antiviral immunity lymphoproliferative response in primary human cytomegalovirus (hcmv) infection is delayed in hcmv transmitter mothers development of human cytomegalovirus-specific t cell immunity during primary infection of pregnant women and its correlation with virus transmission to the fetus kinetics of effector functions and phenotype of virus-specific and gammadelta t lymphocytes in primary human cytomegalovirus infection during pregnancy cytomegalovirus sero positivity dramatically alters the maternal cd8+ t cell repertoire and leads to the accumulation of highly differentiated memory cells during human pregnancy the human decidual nk-cell response to virus infection: what can we learn from circulating nk lymphocytes? human cytomegalovirus infection elicits new decidual natural killer cell effector functions cytotoxic potential of decidual nk cells and cd8+ t cells awakened by infections human decidual natural killer cells are a unique nk cell subset with immunomodulatory potential decidual nk cells regulate key developmental processes at the human fetal-maternal interface tgfbeta promotes conversion of cd16+ peripheral blood nk cells into cd16-nk cells with similarities to decidual nk cells critical and differential roles of nkp46-and nkp30-activating receptors expressed by uterine nk cells in early pregnancy human term pregnancy decidual nk cells generate distinct cytotoxic responses human cytomegalovirus immunity and immune evasion viral immune evasion: lessons in mhc class i antigen presentation kir2ds1-mediated activation overrides nkg2a-mediated inhibition in hla-c c2-negative individuals zika virus: new clinical syndromes and its emergence in the western hemisphere maternal-fetal transmission of the zika virus: an intriguing interplay zika virus: a report on three cases of human infection during an epidemic of jaundice in nigeria immune response to dengue and zika zika virus outbreak on yap island, federated states of micronesia zika virus in the americas: early epidemiological and genetic findings women's reproductive health knowledge, attitudes and practices in relation to the zika virus outbreak in northeast brazil the zika virus epidemic in brazil: from discovery to future implications countries and territories with current or previous zika virus transmission zika virus disease in india -update molecular biology of flaviviruses zika virus infection during pregnancy and congenital abnormalities zika virus pathogenesis and tissue tropism prolonged detection of zika virus in vaginal secretions and whole blood presence of zika virus in conjunctival fluid zika virus in the female genital tract zika virus infects human cortical neural progenitors and attenuates their growth probable non-vector-borne transmission of zika virus zika virus: history, epidemiology, transmission, and clinical presentation cross-reactive dengue virus-specific cd8(+) t cells protect against zika virus during pregnancy zika virus shedding in human milk during lactation: an unlikely source of infection? evidence for mother-to-child transmission of zika virus through breast milk infectious zika viral particles in breastmilk breast milk transmission of flaviviruses in the context of zika virus: a systematic review transmission of zika virus through breast milk and other breastfeeding-related bodily-fluids: a systematic review zika virus infects human placental macrophages zika virus targets different primary human placental cells, suggesting two routes for vertical transmission zika virus infection of hofbauer cells maternal-fetal interplay in zika virus infection and adverse perinatal outcomes zika virus infection of first-trimester human placentas: utility of an explant model of replication to evaluate correlates of immune protection ex vivo syncytiotrophoblast of placentae from women with zika virus infection has altered tight junction protein expression and increased paracellular permeability zika virus productively infects primary human placenta-specific macrophages viral infection, proliferation, and hyperplasia of hofbauer cells and absence of inflammation characterize the placental pathology of fetuses with congenital zika virus infection host and viral mechanisms of congenital zika syndrome equine herpesvirus type 1 enhances viral replication in cd172a+ monocytic cells upon adhesion to endothelial cells osteopontin facilitates west nile virus neuroinvasion via neutrophil "trojan horse a trojan horse mechanism for the spread of visna virus in monocytes placental pathology of zika virus: viral infection of the placenta induces villous stromal macrophage (hofbauer cell) proliferation and hyperplasia zika virus reveals broad tissue and cell tropism during the first trimester of pregnancy asian zika virus strains target cd14(+) blood monocytes and induce m2-skewed immunosuppression during pregnancy zika virus infects early-and midgestation human maternal decidual tissues, inducing distinct innate tissue responses in the maternal-fetal interface host-virus interaction of zika virus in modulating disease pathogenesis enhancement of zika virus pathogenesis by preexisting antiflavivirus immunity human zika infection induces a reduction of ifn-gamma producing cd4 tcells and a parallel expansion of effector vdelta2 t-cells protective to a t: the role of t cells during zika virus infection virologic, and immunologic characteristics of zika virus infection in a cohort of us patients: prolonged rna detection in whole blood ontogeny of the b-and t-cell response in a primary zika virus infection of a dengue-naive individual during the 2016 outbreak in miami, fl biphasic zika illness with rash and joint pain pericarditis associated with acute zika virus infection in a returning traveler cross-reactivity and anti-viral function of dengue capsid and ns3-specific memory t cells toward zika virus structural influence on the dominance of virus-specific cd4 t cell epitopes in zika virus infection prior dengue virus exposure shapes t cell immunity to zika virus in humans immune-profiling of zikv-infected patients identifies a distinct function of plasmacytoid dendritic cells for immune cross-regulation zika virus infection preferentially counterbalances human peripheral monocyte and/or nk zika virus escapes nk cell detection by upregulating major histocompatibility complex class i molecules type iii interferons produced by human placental trophoblasts confer protection against zika virus infection selective activation of type ii interferon signaling by zika virus ns5 protein zika virus inhibits type-i interferon production and downstream signaling predicted protein interactions of ifitms may shed light on mechanisms of zika virus-induced microcephaly and host invasion zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro zika virus ns4a and ns4b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy inhibition of autophagy limits vertical transmission of zika virus in pregnant mice differential and convergent utilization of autophagy components by positivestrand rna viruses hydroxychloroquine inhibits zika virus ns2b-ns3 protease hiv-1 at the placenta: immune correlates of protection and infection zika virus targets human stat2 to inhibit type i interferon signaling metabolic reprogramming by zika virus provokes inflammation in human placenta cross-reactive antibodies during zika virus infection: protection, pathogenesis, and placental seeding placenta-on-achip: a novel platform to study the biology of the human placenta a microphysiological model of the human placental barrier drug transport across the human placenta: review of placenta-on-a-chip and previous approaches placental drug transport-on-a-chip: a microengineered in vitro model of transporter-mediated drug efflux in the human placental barrier placentaon-a-chip: in vitro study of caffeine transport across placental barrier using liquid chromatography mass spectrometry placental barrier-on-a-chip: modeling placental inflammatory responses to a 3d human placenta-on-achip model to probe nanoparticle exposure at the placental barrier decidual stromal cell-derived pge2 regulates macrophage responses to microbial threat a microfluidics assay to study invasion of human placental trophoblast cells instrumenting a fetal membrane on a chip as emerging technology for preterm birth research amnion membrane organon-chip: an innovative approach to study cellular interactions self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta trophoblast organoids as a model for maternal-fetal interactions during human placentation derivation of trophoblast stem cells from naive human pluripotent stem cells conflict of interest: im serves on the scientific advisory board of luca biologics.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright â© 2020 parker, silverstein, verma and mysorekar. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-262575-06i2nv0t authors: caracciolo, massimo; macheda, sebastiano; labate, demetrio; tescione, marco; la scala, stefano; vadalà, eugenio; squillaci, rosalba; d’aleo, francesco; morabito, antonella; garreffa, cristina; marciano, maria concetta; oliva, esther n. title: case report: canakinumab for the treatment of a patient with covid-19 acute respiratory distress syndrome date: 2020-08-25 journal: front immunol doi: 10.3389/fimmu.2020.01942 sha: doc_id: 262575 cord_uid: 06i2nv0t severe cases of covid-19 present with serious lung inflammation, acute respiratory distress syndrome and multiorgan damage. sars-cov-2 infection is associated with high cytokine levels, including interleukin-6 and certain subsets of immune cells, in particular, nk, distinguished according to the cell surface density of cd56. cytokine levels are inversely correlated with lymphocyte count, therefore cytokine release syndrome may be an impediment to the adaptive immune response against sars-cov-2 infection. canakinumab, a monoclonal antibody targeting il-1β is under investigation for the treatment of severe sar-cov-2 infection. an 85 year old male presenting in our hospital with covid-19, whose condition was complicated by acute respiratory distress syndrome and cardiac and renal failure (with oliguria) after 25 days of hospitalization, was intubated and received canakinumab for compassionate use. on the next day, diuresis recovered and conditions improved: high il-6 levels and nk cells expressing cd56(bright) (associated with cytokine relase) were significantly reduced giving rise to nk cd56(dim). patient died on day 58 with pulmonary bacterial superinfection and persistent sars-cov-2 positivity. in conclusion, canakinumab rescued a high risk, very elderly patient, from multiorgan damage complicating covid-19. it may represent an useful treatment in severe cases. introduction sars-cov-2 is responsible for the current pandemic of coronavirus disease 2019 . patients present with fever, dry cough, dyspnea, and pneumonia (1) . some patients (approximately 15%), prevalently elderly and with comorbidities, develop serious multiple organ inflammation and acute respiratory distress syndrome (ards) (2) (3) (4) and require intensive care unit (icu) admission (3, 4) . the immune response, including the release of pro-inflammatory cytokines and activation of t cells, are essential for controlling the viral spread, inflammation, and tissue renewal (5, 6) . the damged host cell releases proteins induce the production of pro-inflammatory cytokines by nearby cells. monocytes, macrophages and t cells are attracted to the site of infection, establishing a pro-inflammatory feedback circuit. when the immune response is hampered, the excessive proinflammatory cytokines in the lungs are responsible for lung tissue damage and the cytokine storm (cs), or macrophage activation syndrome (mas), leading to multi-organ damage. severe cases with cs progress to ards (7, 8) . the mechanisms leading to such complications are complex and still under investigation. many features of covid-19 resemble mas triggered by viral infection (8, 9) . in fact, sars-cov-2 infection is associated with high levels of cytokines and, interestingly, levels of il-6 have been reported to be correlated with mortality (10) . furthermore, in severe cases, a reduction of natural killer cells and other t lymphocytes, has been observed. cytokine levels are inversely correlated with lymphocyte count, therefore cs may be an impediment to the adaptive immune response against sars-cov-2 infection (6, 11) . moreover, the severity of covid-19 is correlated with nk subsets distinguished according to the cell surface density of cd56. in fact, cd56 bright subset increases with severity. cd56 dim nk cells physiologically comprise around 90% of nk cells in peripheral blood and are frequently described as the most cytotoxic, whereas cd56 bright nk cells are abundant cytokine producers (12, 13) . interleukin-1 (il-1) activates the expression of several pro-inflammatory genes. il-1β induces inflammation during infection and autoimmunity (14) . il-1β is released by various cell types, including macrophages (15, 16) . canakinumab, a monoclonal antibody targeting il-1β, is approved for use in rheumatologic disorders 1 . based on the mechanism of action of canakinumab, the drug is under investigation for the treatment of severe sar-cov-2 infection. we present a case of an 85 year old male presenting with covid-19, complicated by ards and cardiac and renal failure, rescued by canakinumab. an indication for compassionate use for covid-19 during the current pandemic and approval from the local ethics committee was obtained in our center. patient was admitted to hospital on march 23, 2020, presenting with fever (38.5 • c), hypoxemia (p02 = 61 mmhg), cough, and dyspnea. medical history revealed only mild arterial hypertension treated with amlodipine and prostatic hypertrophy not requiring treatment. sars-cov-2 swab was positive. chest x-ray showed an interstitial lung pattern and small left pleural effusion. renal and liver biochemistry were normal. noteworthy, the patient presented lymphopenia. reactive c protein (rcp) was 139 mg/l. coagulation tests were normal except for fibrinogen 619 mg/dl and d-dimers 409 ng/ml. he was at first treated in the covid ward and received broad spectrum antibiotics, hydroxychloroquine, and oxygen therapy with venturi mask with 1 https://www.drugs.com/pro/ilaris.html#s-34067-9 30% fio2 setting. on day 3, a chest computerized tomography (ct) without contrast showed severe lung injury (figure 1) . on day 4, though the fever had subsided, his respiratory condition deteriorated and continuous positive airway pressure (cpap) non-invasive ventilation with 40% fio2 setting and positive end-expiratory pressure (peep) 10 cmh 2 0 was initiated, together with azitromicin, enoxaparin sodium and lopinavir/ritonavir. on day 5, tocilizumab 8 mg/kg was administered intravenously (within a clinical trial) repeated after 12 h, while continuing hydroxychloroquine, azitromicin and enoxaparin. on day 23, his conditions precipitated with presentation of ards, a pao 2 /fio 2 ratio (pf) of 103 (fi0 2 setting 60%, p0 2 62 mmhg) and severe arterial hypertension. he was transferred to the intensive care unit (icu) in an obnubilated and non-collaborative condition, so that he was sedated with dexmedetomidine while continuing cpap ventilation. on day 24, patient presented oliguria with acute renal and cardiac failure and progressive respiratory failure. he was intubated and received assisted mechanical ventilation together with furosemide continuous intravenous infusion and vasopressor amines. on day 25, the patient's son was informed of the severity of the patient's clinical conditions and of the risks and benefits of canakinumab treatment. he signed informed consent to administer treatment, to process and publish all relevant clinical research data and potentially identifying information. canakinumab was administered at a single 300 mg s.c. dose on days 25 and 31. to evaluate the biochemical effects of canakinumab, general laboratory chemistry, il-6, and immunophenotype were collected before and after first and second administration. the drug was well tolerated in the short term, and on the day following the first administration, the patient's diuresis normalized and renal function improved gradually without full recovery (on day 53, creatinine level reached 88 µmol/l). the findings are summarized in table 1 . during hospitalization, the patient underwent periodical microbiological surveillance tests. sars-cov-2 genome was evaluated by the microbiology and virology laboratory of our hospital. samples from upper (nasopharyngeal) and lower (bronchoalveolar, bronchoaspirate, and tracheal aspirate) airways were collected and processed within 24 h. rna-covid 19 was evaluated using an allplex 2019-ncov assay that identifies three different target genes: e (envelope), rdrp (rna-dependent rna polymerase), and n (nucleoprotein gene). based on the interpretation criteria, detection of one or more genes was interpreted as positive covid-19. there was a high viral replication persisting on day 43. on day 31, as the respiratory conditions did not improve significantly, the film array pneumonia detected the presence of bacterial infection caused by acinetobacter c.b. complex -107 copies/ml -and pseudomonas aeruginosa -106 copies/ml, treated initially with the association of piperacillin + tazobactam together with cotrimoxazole provided intravenously qid, followed by colistin 3000000 aerosol bid, then with ceftazidime/avibactam intravenous tid and finally with doxycycline 100 mg intravenous bid (the latter ongoing). the initial chest ct scan on day 13 and x-rays performed before first administration (day 23) and after second administration (day 38) are shown in figure 2 . canakinumab is an il-1 antagonist indicated to treat autoinflammatory disorders. severe covid-19 cases show symptoms associated with an excessive release of cytokines (17, 18) . the il-1 cytokine family plays an important role in regulating inflammation and is produced in response to inflammatory stimuli and infections. il-1 production requires inflammasome/caspase-1-dependent processing. it mediates its effects by binding to its receptor to activate downstream signaling which activates mapks and nf-kappa b, leading to the expression of pro-inflammatory mediators that drives the il-6 signaling pathway. il-6 significantly contributes to mas. its levels increase with the severity of covid-19 (9, 19, 20) and the area of pulmonary infiltration (>50%) in patients with ards, together with specific lymphocyte subsets (21) . though the present case had received tocilizumab prior to canakinumab, il-6 level remained high, postulating that tocilizumab be insufficient to rescue the patient from the subsequent cytokine storm. in our patient, il-6 and nk cd56bright both decreased after treatment with canakinumab, suggesting that canakinumab, by interfering with il-1, reduces il-6. following the second administration, il-6 and crp levels increased which can be explained with the development of superimposed pulmonary bacterial infection. several immunotherapeutic drugs are promising for the treatment of the cytokine storm associated with covid-19 (22) . amongst these, anakinra, an il-1 receptor antagonist, saltuximab, an il-6 antagonist, and sarilumab, an il-6 receptor antagonist, are undergoing phase 3 stage development (23) . however, canakinumab treatment is associated with adverse events, mainly an increased incidence of serious infections. in fact, il-1β physiologically contributes to host defense against infection by enhancing the antimicrobial action of phagocytes and inducing th1 and th17 adaptive immune responses (24) . in our case, the patient survived the mas, but developed bacterial pulmonary superinfection, which was the final cause of death on day 58. this case represents the first published report of canakinumab for the treatment of multiorgan damage associated with covid-19. excessive cytokine release induces severe complications and worsens the prognosis in covid-19. there are numerous ongoing trials to find treatments that target virus and/or inflammation. until an effective treatment is found, in this scenario, canakinumab due to its blocking action of proinflammatory activity by il-1β can constitute a potential useful treatment in the modulation of hyperinflammatory symptoms, for a subgroup of patients with covid-19, that resemble the cytokine storm in patients with mas. the active involvement of immunologists in the clinic and in clinical trials may improve patient outcomes. the studies involving human participants were reviewed and approved by comitato etico sezione sud -regione calabria. the patients/participants provided their written informed consent to participate in this study. mc, eo, and dl drafted the initial manuscript. cg performed flow cytometry. fd'a performed microbiological testing. mm performed cytokine tests. all authors reviewed and revised the manuscript and have read and agreed to the published version of the manuscript to the work reported. a pneumonia outbreak associated with a new coronavirus of probable bat origin pathological findings of covid-19 associated with acute respiratory distress syndrome clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan clinical characteristics of coronavirus disease 2019 in china regulation of type i interferon responses coronavirus infections and immune responses hemophagocytic lymphohistiocytosis: review of etiologies and management dysregulation of immune response in patients with covid-19 in wuhan, china characteristics of lymphocyte subsets and cytokines in peripheral blood of 123 hospitalized patients with 2019 novel coronavirus pneumonia (ncp) the anti-viral facet of anti-rheumatic drugs: lessons from covid-19 sars-cov-2: a storm is raging coronavirus infections and immune responses the biology of human natural killer-cell subsets functions of natural killer cells therapeutic strategies to reduce il-1 activity in treating local and systemic inflammation glial proinflammatory cytokines mediate exaggerated pain states: implications for clinical pain role of interleukin-1beta during pain and inflammation clinical course and risk factors for mortality of adult in patients with covid-19 in wuhan, china: a retrospective cohort study hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression secretion of il-1β from imatinib-resistant chronic myeloid leukemia cells contributes to bcr-abl mutation-independent imatinib resistance the immunology of macrophage activation syndrome the definition and risks of cytokine release syndrome-like in 11 covid-19-infected pneumonia critically ill patients: disease characteristics and retrospective analysis adjunct immunotherapies for the management of severely ill inflammasome activation and il-1β and il-18 processing during infection novartis provided compassionate use of canakinumab for the case described. conflict of interest: eo reports personal fees from novartis, during the conduct of the study.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 caracciolo, macheda, labate, tescione, la scala, vadalà, squillaci, d'aleo, morabito, garreffa, marciano and oliva. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003685-jcvrqeew authors: gelain, maria elena; bonsembiante, federico title: acute phase proteins in marine mammals: state of art, perspectives and challenges date: 2019-05-29 journal: front immunol doi: 10.3389/fimmu.2019.01220 sha: doc_id: 3685 cord_uid: jcvrqeew the term “acute phase response” (apr) is referred to a nonspecific and complex reaction of an organism that occurs shortly after any tissue damage, such as infection, trauma, neoplasia, inflammation, and stress. the apr can be identified and monitored with some laboratory tests, such as the concentration of several plasma proteins, the acute phase proteins (apps). the apps are components of the non-specific innate immune response, and their plasma concentration is proportional to the severity and/or the extent of tissue damage. the evaluation of health status of marine mammals is difficult because the classical clinical signs of illness used for human and domestic animals are difficult to recognize and understand. for this reason, in the past years, several efforts were done to identify laboratory markers of disease in these animals. the apps have demonstrated their role as early markers of inflammation in veterinary medicine, thus several apps were tested in marine mammals, such as c-reactive protein (crp), serum amyloid-a (saa), and haptoglobin (hp). however, the difficulty to extrapolate the knowledge about apps in one species to another, the lack of specie-specific reagents, the absence of data about negative apps have hampered their extent use in marine mammals. herein, the state of art of apps in marine mammals is reviewed, with particular attention to pre-analytical and analytical factors that should be taken into account in validation and interpretation of apps assays. moreover, the current application, potential utility and the future developments of apps in marine mammals is highlighted and discussed. the mammalian immune system includes innate or nonspecific immunity as well as adaptive or specific immunity. the responses of these two different pathways are distinct, but highly interconnected. the first reaction of an organism to different pathological conditions is an innate, non-specific response (1), a more conserved response during evolution which aim is the immediate reaction against pathological stimuli (2) . after the initial recognition of pathogens or tissue damages by the tissue-resident macrophages, which express the pattern recognition receptors (prrs), a variety of different inflammatory mediators are produced by leukocytes, endothelial cells, tissue cells or are derived from plasma proteins. these mediators include different chemokine, cytokine, vasoactive amines and products of the arachidonic acid: their primary effect is to elicit inflammatory response locally and to recruit leukocytes and plasma proteins in the site of injury (3) . glycoprotein (agp) (9) , in ruminants is haptoglobin (hp) (14) ; in horse is serum amyloid a (saa) (15) and in pigs are hp, saa, and major acute phase protein (map) (10) . recently, in veterinary medicine, studies on the role of apps as markers of infectious, inflammatory and neoplastic diseases have proliferated (16) and at least 40 different plasma proteins have been identified as apps (8) . their use as marker of homeostasis perturbation provides some advantages compared with traditional parameters like the white blood cell (wbc) counts. compared to wbc count, the diagnostic sensitivity of apps is higher and the change in concentration is faster (17) . moreover, their stability in serum/plasma is high, so it is possible to measure apps in frozen samples (18) . one limitation of the apps is the poor diagnostic specificity, for this reason they cannot be used as primary diagnostic test for a specific disease, but they were successfully used to detect subclinical diseases and to monitor clinical evolution and to assess the response to treatment (5) . additionally, the combined measurement of several apps provides more information than the evaluation of a single protein: in the "app value index, " proposed by gruys et al. (1) , sensitivity and specificity are improved by combining response of both positive and negative major and minor apps. marine mammals are a group of around 130 mammalian species which depend on water environment for most of their needs. they include 3 orders: carnivora (pinnipeds, otters, and polar bear), cetacea (dolphins, whale, and porpoise) and sirenia (manatee and dugogons). marine mammals are differently adapted to life in water, with some species, which are fully aquatic (cetaceans and sirenia), and others that spend part of their life on land (pinniped and polar bears) (19) . from an immunological point of view, aquatic adaptation caused few differences in distribution and function of immune system between marine and terrestrial mammals (20) . however, nowadays the marine mammal' immune system is deeply exposed to environmental pollution because they are a long-lived animals placed at the top of food chain, thus they are exposed to a progressive bioaccumulation of fat-soluble pollutants, such as pcbs, which affect both innate and adaptive immune function (21) . for these reasons, increasing knowledge in cellular and humoral immune response is continuously required to understand their immune system and in particular its relationship with infectious pathologies and the environmental pollution (22) . furthermore, marine mammals live also in controlled environment like aquaria, rehabilitation facilities and research center where health assessment is fundamental to evaluate the correct management of animals and to monitor the response to therapy during rehabilitation. from this perspective, the availability of new markers to asses immune functions is fundamental both for medical care and research purpose (20) . innate immune response represent the first line of response against pathological stimuli, it's very fast and it's based primarily on effector cells (e.g., mast cells, macrophages, neutrophils) and antimicrobial substances (e.g., complement, reactive oxygen, and nitrogen species). on the other hand, adaptive immune system is antigen-specific, it takes more days to be effective and it's based on different t-cells response and on b-lymphocytes which are responsible of humoral response mediated by the different subclass of immunoglobulin (igg, igm, iga) (20) . several assays were proposed to evaluate both immune response in marine mammals, generally based on isolated leucocytes with the aim to evaluate the leukocytes response against in vitro stimuli (23) . to assess the response pattern of cetaceans' cellular innate immune system, the phagocytosis and the generation of reactive oxygen species of polymorphonuclear leukocytes were investigated. in particular, in vitro ingestion of latex beads and hydrogen peroxide production have been evaluated in beluga whales (delphinapterus leucas) and in bottlenose dolphins (tursiops truncatus) (24, 25) whereas phagocytosis and respiratory burst assay, using whole blood from bottlenose dolphins, were used to assess the antimicrobial activity (26) . in addition, the investigation of apr by analyzing the cytokine expression gives important information on the functionality of lymphoid cells. the production of specie-specific antibodies allows the development of immunological assays for the quantification of cytokine expression useful to investigate the inflammatory response in whales and dolphins. the coding regions of il-2, il-1β, il-6, and tnf-α gene of the beluga whale have been sequenced, and a cytokine-specific rabbit antisera have been produced (27) (28) (29) . in harbor porpoise (phocoena phocoena), the quantification of mrna of il-1β, il-2, il-4, il-6, il-10, and tnf-α have been performed by rt-pcr (30) , and the increase of il-10 was seen in harbor porpoises suffering from long lasting infectious (31) . also in bottlenose dolphins, pacific white-sided dolphins (lagenorhynchus obliquidens), and beluga whales, il-2, il-4, il-10, il-12, il-13, il-18, tnf-α, tgf-β, and interferon (ifn)-γ quantification was performed using rt-pcr (32) . an il-2 receptor expression assay and an il-6 elisa were developed in bottlenose dolphins and killer whale (orcinus orca), respectively (33, 34) . similarly, both innate and the cell-mediated response were studied in pinnipeds. to better understand the innate response, phagocytic activity of isolated peripheral blood leukocytes was evaluated in harbor seal (phoca vitulina), gray seal (halichoerus grypus), and harp seal (phoca groenlandica) pups, in harbor seal female during lactation and in harbor seal pups admitted to rescue center (35, 36) . the authors found an age-related variation in both pups and adults: phagocytosis increased with age in gray and harbor seal pups, while in female harbor seals decreased from sub-adult to adulthood. at the same time, pups after rehabilitation showed a decreased phagocytic activity, probably due to the decreased stimulation of innate response after therapy. also cytokine response was evaluate in harbor seal. pro-inflammatory cytokine mrna (il-1β, il-6, il-8, and il-12) in pups in a rehabilitation center were higher at admission whilst il-4 was higher before the release (37), demonstrating the recovery from inflammation. recently, a multiplex canine cytokine assay was validate in harbor, gray and harp seal to measure proteins levels in cell culture supernatant of peripheral blood mononuclear cells (pbmc) (38) . however, all these techniques are not generally applicable in a clinical setting in which the primary goal is a sensitive diagnostic tool with a rapid turnaround, even if give us important information on factors affecting cetaceans' immune system. for this reason, in the past years, several efforts were made to identify laboratory markers of disease in these animals. first parameters tested were wbc and erythrocyte sedimentation rate (39) . however, even if they are inexpensive and rapid, they lack specificity and sensitivity. moreover, changes in wbc occur after several hours after inflammatory stimuli. thus, efforts were directed to identify inflammation at earlier stage (40) . to examine the humoral response, species-specific antibodies against igg were produced and used to evaluate serum igg levels in killer whale by radial immunodiffusion assay (41) and by competitive elisa in bottlenose dolphins (42, 43) . the determination of igg baseline values in free-ranging and in managed dolphins revealed higher levels of immunoglobulin in the first group with several values over the accurate range of the assay, probably due to the higher parasitic load in free-ranging dolphins (43) . serum total protein, albumin, globulin and albumin:globulin ratio (a:g) are undoubtedly among the most measured markers in basic health assessment in domestic animals as well as in marine mammals. serum protein electrophoresis is also broadly applied in veterinary medicine and it has the advantage to produce an accurate measurement of albumin and the visualization of globulin fractions (44) . the interpretation of total proteins values and electrophoretic pattern of serum proteins is receiving increased attention also in marine mammals in which a typical pathologic pattern could be identified in inflammatory diseases (40) . reference intervals for these markers are available for free-ranging bottlenose dolphins (45) and, compared to these, recently data on managed dolphins showed slightly lower values of tp, α-globulins, and γ-globulins and higher albumin and albumin/globulins ratio (46) . it's interesting to note that hp, α1-antitrypsin, α1antichymotripsin, and α2-macroglobulin migrate in the α-globulins fraction, while the igg and crp migrate in the γ-globulins fraction. albumin acts as a negative acute phase protein since the synthesis of this protein is decreased during an inflammation (47) . thus, the lower concentration of positive apps associated to a higher concentration of albumin and the consequent higher albumin/globulins ratio could reflect lower antigenic stimuli in managed population compared to the free-ranging populations (36) . serum total protein analysis were used to assess health status in several cetaceans species such as pantropical spotted dolphins (stenella attenuata) (48), beluga (49) , minke whales (balaenoptera acutorostrata) (50) and killer whales (51) as well as in other marine mammals, like harbor seals (phoca vitulina) (52) and walruses (odobenus rosmarus) (53) . in all these species, serum total protein analysis was demonstrated to be one of the most used and commonly accepted marker of inflammation. however, specific apps have demonstrated their superior role as early markers of inflammation, so based on the results obtained in humans and companion animals, several positive apps were tested in marine mammals ( table 1) . published works had the primary aims to evaluate the feasibility of the assays to measure the apps, to validate the antibody-based assay and to determine the ris. in bottlenose dolphins three apps (crp, saa, and hp) were tested, even if not always complete validation studies were performed (54, 61) . for these apps, the authors established the ris in free-ranging and managed dolphins using automated assays (54) and they found significantly lower saa and higher hp levels in free ranging animals. the only clinical significance of these alteration was a higher ability to detect chronic inflammation for hp. regarding hp, segawa and colleagues validated commercially available hp-elisa and hp-hemoglobin binding assay in bottlenose dolphins with ''acceptable" intra-and inter-assay imprecision (cv: 3.3% healthy dolphins and 3.5% inflamed dolphins; cv: 10.4% healthy dolphins and 21.7% inflamed dolphins) and demonstrated that hp levels in the serum increase under inflammatory conditions (62) . positive apps were tested also in florida manatees (trichechus manatus latirostris) to define the more accurate marker of inflammation. five different apps were tested: agp, crp, hp, fibrinogen, and saa. saa showed the highest diagnostic sensitivity and specificity (90% for both sensitivity and specificity) in the detection of inflammatory diseases, the diagnostic specificity of hp and fibrinogen were 93 and 95%, respectively, while their diagnostic sensitivity were 60 and 40%, respectively, (56) . when used in stranded manatee suffering from cold stress and trauma, saa showed 93% of sensitivity and 98% of specificity in detecting diseased animals (57) . by contrast, the abs used for the determination of agp and crp did not cross-react in this species (56) . in harbor seal, an ab anti-crp and a competitive immunoassay was produced (63), but hp is probably the app most used in pinnipeds. a multispecies assay based on hemoglobin binding capacity was used to demonstrate as hp is a sensitive marker of the health vs. disease status in harbor seal (64) . in seal pups admitted in a rescue center. hp, total protein, igg and globulin values correlated positively, but hp levels increased during the hospitalization, probably reflecting age-related changes (35) . hp is considered a health marker also in steller sea lions (eumetopias jubatus): significantly higher levels of hp were found in declining population compared to more stable ones (58) . however, also genetic differences between distant and isolated population of wild animals could be the causes of this difference, not only a pathological condition. if some data on marine mammals positive apps are available in literature, quite surprising are the lack of data available on negative apps. for these reasons, the possibility to evaluate the usefulness of an "app value index" is still far from being applied. the availability of sensitive markers of inflammation both for free-ranging and managed marine mammals is nowadays considered fundamental to evaluate the health status and, in rehabilitation setting, to monitor the response to therapy and to define the prognosis. as serum markers, the apps have several advantages: they have longer stability compared to other blood component such as wbc; they can be performed on frozen serum, thus the samples can be shipped to references laboratories; some assays can be automated to obtain results in an excellent turnaround time. however, is important to consider that the knowledge about apps in one species cannot be readily generalized to another species, in which healthy levels, response to inflammation or infection, and prognostic significance may be different (65) . moreover, the evolution of marine mammals and their adaptation throughout the millennia to an aquatic environment had led to a different physiology and metabolism compared to terrestrial mammals. thus, the understanding of the genetic, phenotypical and biochemical properties of marine mammals apps are essential prior to using them as a new biomarker. an example of how the biochemical properties influence the analytic method is paroxonase-1 (pon1), a hdl-bound esterase which protects against organophosphate compounds, acts as negative app and as oxidative stress marker. pon1 is usually assessed by enzymatic method and, based on the different pon1 functions, several substrates have been identified to evaluate serum pon1 activities. nevertheless, both in humans as in some terrestrial mammals, pon1 gene polymorphisms highly influence the enzymatic activity toward different substrate: the single-nucleotide polymorphisms (snps) leu55met and gln192arg increase the paraoxonase activity (66) in humans and different pon1 genotypes influence activities toward paraoxon and phenyl-acetate in rabbit (67) . also in cows, some snps in the promotor region of pon1 gene are associated with serum pon1 activity (68) . recently, a phylogenetic study on convergent functional losses across marine mammals, has identified a pon1 functional loss in marine mammals, probably related to their different lipid metabolism and fatty acid oxidation due to adaptation to the marine environment and a high concentration of ω-3 fatty acids on their diet. as a consequence, in several marine mammals species paroxonase activity is very low, while enzymatic activity against other pon1 substrates is still present, such as arylesterase activity (69) . for these reasons, the use of classical enzymatic assays is hampered in these animals and further studies are needed to elucidate the role of pon1 as possible negative app, oxidative stress marker and the consequences of its inability to detoxify organophosphates compounds. from an analytical point of view, another challenge in the evaluation of apps in marine mammals is the need of speciesspecific assays, especially for the immunological assay, such as elisa or immunoturbidimetry. this means the development of a de-novo method, often a time-consuming and expensive approach, or the validation of a commercial available assay used in other species (65) . the latter approach is surely the most used in veterinary medicine, in which some human assays were validated for dogs, cats and horses (62) . however, even if some apps appeared highly conserved among species, an accurate validation of antibody cross reactivity is needed as well as species specific standards and control material (54) . among positive apps, saa is the most used across different species: it appeared as the most conserved app in mammals even if some difference in circulating isoforms were reported (61) and it's considered a major app in all the mammals in which it was investigated (65) . some commercial saa assays showed good results also in marine mammals, such as bottlenose dolphin, manatee and striped dolphin (stenella ceoreloualba) (54, 56, 70) and its use as diagnostic and prognostic marker appears nowadays the most promising. to obtain accurate data, all the pre-analytical factors that could influence the results should be taken into consideration. the effect of storage, temperature and different anticoagulant had to be evaluated in a correct validation process as well as the interference of hemolysis and lipemia, as done in other species (5) . the application of a novel biomarker required a full evaluation of all the analytical performances and the clinical value. this process is usually divided in 4 steps: the assessment of analytical features (precision, accuracy, detection limits), the overlap performance (the ability to detect difference between healthy and diseased animals), the assessment of diagnostic capacity (sensitivity, specificity, accuracy, positive, and negative predictive values) and, at the end, the evaluation of the outcome of the new methods (which is the advantage of the test and its influence in the patient management) (65) . in veterinary medicine, the validation studies do not always follow all these steps, mainly due to the lack of resources or technical limitation (44, 65) . also in marine mammals, the majority of studies had performed only some steps (44, 54, 56, 61, 62) . this is mainly due to the limitation in species-specific reagents, the number of samples from animal with known health status and, last but not least, the capability to generate appropriate reference intervals, hampered the possibility to perform complete validation studies. population-based reference intervals derived from an appropriate group of reference individuals are usually required for diagnostic purpose (71) . however, a number of biological factors have to be taken in consideration to select the appropriate reference population. surely, age, sex and pregnancy could be used for partitioning (45, 72) , but in marine mammals greater attention should be given to the difference between wild and managed animals. serum protein electrophoresis values obtained in managed bottlenose dolphins showed lower total proteins and higher albumin levels compared to reference intervals derived from free-ranging (46) while 9 wild-caught manatees, apparently clinically healthy, had saa level above reference limit (56) . these data could indicate a trend to an inflammatory status or the presence of subclinical inflammation in free-ranging animals which are more exposed to immunological stimuli. in any case, this highlights the need to define appropriate reference intervals for animals living in different environment to have an accurate toll for the evaluation of clinical condition. compared to human and companion animals, the use of apps in marine mammals is just getting started. the increasing need of knowledge on immune system and its response against infectious diseases or chemical pollutants and the request of more sensitive inflammation markers have increased the effort of researchers to study the apr and apps. even if apps are considered a sensitive, but non-specific marker of inflammation, some studies revealed that, in some infectious diseases, apps showed a specific behavior and biochemical features. one example is the modification of the glycan moiety of agp in feline infectious peritonitis, fiv and felv, influencing the host-pathogens interaction and the immune response (73) (74) (75) . currently, some of the greatest threats for wild marine mammals is pathogens, like morbillivirus, herpesvirus, brucella ceti, and toxoplasma gondii (76): the evaluation of apr and apps patterns during these infectious diseases could lead to the identification of a distinctive response of the immune system and increase the understanding of hostpathogen interaction. secondly, for managed or rescued animals, the forthcoming needs are the increase of automated assays, the standardization of procedures across laboratories and the discovery of new markers, for example negative apps, to generate an app index also in marine mammals. these new tools will certainly increase the diagnostic and prognostic skills for health assessment and, especially for stranded animals, the development of new "health status" markers will provide valuable resources in evaluating the response to treatment and rehabilitation prior to the release into the wild. mg and fb analyzed the literature review, designed, and wrote the review. monitoring health by values of acute phase proteins inflammation and factors that may regulate inflammatory response origin and physiological roles of inflammation inflammatory mechanisms: the molecular basis of inflammation and disease acute phase proteins in dogs and cats: current knowledge and future perspectives initiation of acute phase response and synthesis of cytokines acute phase proteins: biomarkers of infection and inflammation in veterinary medicine current research on acute phase proteins in veterinary diagnosis: an overview the feline acute phase reaction application of acute phase protein measurements in veterinary clinical chemistry acute phase proteins as promising biomarkers: perspectives and limitations for human and veterinary medicine applications of acute phase reactants in infectious diseases an immunoturbidimetric assay for canine c-reactive protein bovine acute phase response following turpentine injection serum amyloid a protein (saa) in horses: objective measurement of the acute phase response the final hurdles for acute phase protein analysis in small animal practice study on biological variability of five acute-phase reactants in dogs haptoglobin and ceruloplasmin as determinants of inflammation in dogs chapter 1 -introduction marine mammal immunology immunotoxic effects of environmental pollutants in marine mammals cetacean host-pathogen interaction(s): critical knowledge gaps immunology of whales and dolphins evaluation of the polymorphonuclear cell functions of bottlenose dolphins immune functions in beluga whales (delphinapterus leucas): evaluation of mitogen-induced blastic transformation of lymphocytes from peripheral blood, spleen and thymus simultaneous measurement of phagocytosis and respiratory burst of leukocytes in whole blood from bottlenose dolphins (tursiops truncatus) utilizing flow cytometry molecular cloning and characterization of beluga whale (delphinapterus leucas) interleukin-1beta and tumor necrosis factoralpha molecular cloning, phylogenetic analysis and expression of beluga whale (delphinapterus leucas) interleukin 6 molecular cloning and phylogenetic analysis of beluga whale (delphinapterus leucas) and grey seal (halichoerus grypus) interleukin 2 development of a lymphocyte-transformation-assay for peripheral blood lymphocytes of the harbor porpoise and detection of cytokines using the reverse-transcription polymerase chain reaction increased blood interleukin-10 mrna levels in diseased free-ranging harbor porpoises (phocoena phocoena) quantitation of leukocyte gene expression in cetaceans development of an interleukin-2 receptor expression assay and its use in evaluation of cellular immune responses in bottlenose dolphin (tursiops truncatus) expression and functional characterization of killer whale (orcinus orca) interleukin-6 (il-6) and development of a competitive immunoassay immune status and function in harbor seal pups during the course of rehabilitation phagocytosis in pup and adult harbour, grey and harp seals cytokine and acute phase protein expression in blood samples of harbour seal pups validation of a commercial canine assay kit to measure pinniped cytokines clinical pathology crc handbook of marine mammal medicine measurement of serum immunoglobulin concentration in killer whales and sea otters by radial immunodiffusion development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin g of bottlenose dolphins (tursiops truncatus) baseline circulating immunoglobulin g levels in managed collection and freeranging bottlenose dolphins (tursiops truncatus) acute phase response in animals: a review hematologic and serum biochemical reference intervals for freeranging common bottlenose dolphins (tursiops truncatus) and variation in the distributions of clinicopathologic values related to geographic sampling site detection of hereditary bisalbuminemia in bottlenose dolphins (tursiops truncatus fundamentals of veterinary clinical pathology hematological, serum, and plasma chemical constituents in pantropical spotted dolphins (stenella attenuata) following chase, encirclement, and tagging serum chemistry of freeranging white whales (delphinapterus leucas) in svalbard serum chemistry of the minke whale from the northeastern atlantic hematological and serum biochemical analytes reflect physiological challenges during gestation and lactation in killer whales (orcinus orca) hematology and serum chemistry in stranded and wild-caught harbor seals in central california: reference intervals, predictors of survival, and parameters affecting blood variables serum chemistry reference values in free-ranging north atlantic male walruses (odobenus rosmarus rosmarus) from the svalbard archipelago acute phase protein quantitation in serum samples from healthy atlantic bottlenose dolphins (tursiops truncatus) evaluation of immune and stress status in harbour porpoises (phocoena phocoena): can hormones and mrna expression levels serve as indicators to assess stress? comparison of methods used to diagnose generalized inflammatory disease in manatees (trichechus manatus latirostris) assessement of serum amyloid a levels in the rehabilitation setting in the florida manatee (trichechus manatus latirostris) plasma haptoglobin levels in threatened alaskan pinniped populations characterization of haptoglobin in the blood plasma of harbor seals (phoca vitulina) fibrinogen concentrations in captive bottlenose dolphins during pregnancy characterization of the circulating serum amyloid a in bottlenose dolphins molecular characterization and validation of commercially available methods for haptoglobin measurement in bottlenose dolphin harbor seal (phoca vitulina) c-reactive protein (c-rp): purification, characterization of specific monoclonal antibodies and development of an immuno-assay to measure serum c-rp concentrations acute phase protein haptoglobin in blood plasma samples of harbour seals (phoca vitulina) of the wadden sea and of the isle helgoland assay validation and diagnostic applications of major acute-phase protein testing in companion animals human paraoxonase-1 (pon1): gene structure and expression, promiscuous activities and multiple physiological roles rabbits possess a serum paraoxonase polymorphism similar to the human q192r characterization of single nucleotide polymorphisms in the promoter region of the bovine paraoxonase 1 (pon1) gene affecting serum enzyme activity in dairy cows ancient convergent losses of paraoxonase 1 yield potential risks for modern marine mammals clinico-pathological findings in a striped dolphin (stenella coeruleoalba) affected by rhabdomyolysis and myoglobinuric nephrosis effects of age and sex on clinicopathologic reference ranges in a healthy managed atlantic bottlenose dolphin population association between faecal shedding of feline coronavirus and serum alpha1-acid glycoprotein sialylation hyposialylated α1-acid glycoprotein inhibits phagocytosis of feline neutrophils glycan moiety modifications of feline alpha1-acid glycoprotein in retrovirus (fiv, felv) affected cats emerging infectious diseases in cetaceans worldwide and the possible role of environmental stressors clinico-pathological investigation of serum proteins in odontocetes the literature review presented in the manuscript is partially included in ph.d. thesis of bonsembiante (77), supported by a ph.d. grant from the university of padova. key: cord-280605-2i4gk7et authors: bachmann, maría consuelo; bellalta, sofía; basoalto, roque; gómez-valenzuela, fernán; jalil, yorschua; lépez, macarena; matamoros, anibal; von bernhardi, rommy title: the challenge by multiple environmental and biological factors induce inflammation in aging: their role in the promotion of chronic disease date: 2020-10-14 journal: front immunol doi: 10.3389/fimmu.2020.570083 sha: doc_id: 280605 cord_uid: 2i4gk7et the aging process is driven by multiple mechanisms that lead to changes in energy production, oxidative stress, homeostatic dysregulation and eventually to loss of functionality and increased disease susceptibility. most aged individuals develop chronic low-grade inflammation, which is an important risk factor for morbidity, physical and cognitive impairment, frailty, and death. at any age, chronic inflammatory diseases are major causes of morbimortality, affecting up to 5–8% of the population of industrialized countries. several environmental factors can play an important role for modifying the inflammatory state. genetics accounts for only a small fraction of chronic-inflammatory diseases, whereas environmental factors appear to participate, either with a causative or a promotional role in 50% to 75% of patients. several of those changes depend on epigenetic changes that will further modify the individual response to additional stimuli. the interaction between inflammation and the environment offers important insights on aging and health. these conditions, often depending on the individual’s sex, appear to lead to decreased longevity and physical and cognitive decline. in addition to biological factors, the environment is also involved in the generation of psychological and social context leading to stress. poor psychological environments and other sources of stress also result in increased inflammation. however, the mechanisms underlying the role of environmental and psychosocial factors and nutrition on the regulation of inflammation, and how the response elicited for those factors interact among them, are poorly understood. whereas certain deleterious environmental factors result in the generation of oxidative stress driven by an increased production of reactive oxygen and nitrogen species, endoplasmic reticulum stress, and inflammation, other factors, including nutrition (polyunsaturated fatty acids) and behavioral factors (exercise) confer protection against inflammation, oxidative and endoplasmic reticulum stress, and thus ameliorate their deleterious effect. here, we discuss processes and mechanisms of inflammation associated with environmental factors and behavior, their links to sex and gender, and their overall impact on aging. the inflammatory response is different in men and women. adult females develop stronger innate and adaptive immune responses than males. these sex-related differences can determine the ability of immune cells to generate an effective inflammatory response, which translates into epidemiological differences on the prevalence of various pathologies, including allergies (22), asthma (23, 24), autoimmune diseases (25), anaphylaxis (26), neonatal sepsis (27), and cancer (28), among several pathologies. the immune response of women is polarized towards an increased production of th2 cells, t regulatory cells (treg), m2 macrophages, il4, il10, and gata-3 cytokines, and decreased th1, th17, tbet, and rorgt lymphocytes (29-31). on the contrary, men show an immune response that depends on th1 lymphocytes (32, 33), high il33 production (34) and low levels of reactive mast cells (35) . men have also an increased response of microglia in the central nervous system (cns) and an increased presence of tnfa and prostaglandins in response to inflammatory stimuli (36). differences in inflammatory response between men and women vary among specific tissues. in the cns inflammation, women show greater levels of b-cell (cd19+, cd5+, cd1d hi b10) migration from the spleen to the site of injury than men, followed by an increase of macrophages/microglia (cd11b+, cd206), which appears to generate a lower neuroinflammatory response in female compared with male mice (37). in addition, women develop an increased immunoreactivity due to high numbers of ifn-producing dendritic cells (38, 39). female mice tend to have m2 phenotype and activated eosinophils and mast cells show a higher reactivity than in male mice (35, 40) . however, in response to an acute inflammatory stimulus, males produce higher amounts of inflammatory cytokines, cd8a+ neutrophil and t cells infiltration of the injury site (41). conversely, the inflammatory microenvironment in female mice is characterized by an increased production of antibodies (42, 43) and a differential pattern migration of antibody-secreting cells (42). the immune system responds differently in men and women not only because of the influence of sex hormones, but also differences in the patterns of autosomal methylation and x chromosome methylation, which determine distinctive profiles of gene expression (43, 44). sex hormones exert antagonist effects on the immune system: both estradiol and testosterone have a suppressive effect on the immune response (45). estrogen is the sex hormone with the greatest impact on the immune response, being described as one of the non-modifiable regulators of the immune system, due to its immunoregulatory and protective effects in many inflammatory models (46). however, this is contradictory with the fact that women have a higher prevalence of autoimmune diseases than men, although estrogens should be a protective condition (47). the sex-dependent difference in the immune response is time-, and estrogen dose-dependent (29). variations on the estrogen concentration during the ovulatory cycle, puberty or menopause, can promote the development of immune-related diseases (48). mice exposed to chronic estrogen-treatment generate hormone resistance, decreasing the clonal expansion of treg lymphocytes in autoimmune diseases (49, 50) . estrogen regulates immune response primarily through aand b-estrogen receptors (era/b), mitogen-activated protein kinase (mapk) pathways, estrogen-dependent 3′-5′-cyclic adenosine monophosphate (camp) response element-binding (creb), and modifications in the production of camp in immune cells (51). in addition to estrogen receptors, the presence of il receptors influences the type of immune response; female macrophages express greater amounts of il4 receptors than males. il4 receptors favor the m2 phenotype when stimulated by estrogen. in agreement with that, estrogen induces an increased expression of il4 on naive cd4+ t cells (40, 52) . for a better general view of estrogen´s mechanisms and effect on the innate immune system cells we recommend reviews that have extensively covered those topics (53-56). sex regulates gene expression in multiple human tissues, in fact, one third of the autosomal genes that are expressed in a sexbiased manner exhibit androgen or estrogen hormonal response elements (57, 58). sex hormones play a strong role in sexually dimorphic gene networks (59), inducing aberrant expression in immune response genes via differential methylation ccl18 cxcl5 il5 (60) . there are changes in the methylation pattern of sex-dependent immune response genes during embryonic development, which are reinforced in puberty by the estrogenmediated induction of active forms of chromatins that are maintained during adulthood (61) . immune response-related genes located in chromosomes 3 and x are differently expressed in b lymphocytes depending on the sex of the individual (62). among the differentially expressed genes that are relevant for the immune/inflammatory response, can be mentioned the toll-like signaling, cytokine receptors, jak-stat pathway and genes related to the activation of t-cell receptors (63) . phenotypically, the different pattern of gene expression may explain the greater female t-cell expandable capacity when exposed to an antigen (64) . female t cells present higher activation and division capacities than their male counterparts. however, male t cells can develop greater infiltration potential and a lower self-reactive phenotype than female ones (65, 66) . these differences could be due to the high expression of peroxisome proliferator-activated receptors (ppars) (64) , prostaglandins, and cyclooxygenase-2 (cox-2) in males (67) . the influence of sex on the immune response is observed throughout life and is accentuated with aging. in the neonatal stage, women have a lower concentration of regulatory t lymphocytes than men (68) . during childhood, men develop a more intense immune response and are more likely to develop infections by various pathogens compared with women (69, 70) . with increasing age, the dynamics and proportion of lymphocytes and myeloid cells differ depending on the sex due to the differential expression of 144 genes of the immune response in men and women (71) . also, in aged individuals, epigenomic changes generate a more robust innate and pro-inflammatory response in men and an increased activity in the adaptive immune response in women (72, 73) . in recent times, during the covid-19 pandemic, it has been observed that the infection by sars-cov-2 in older adults shows conspicuous differences; men have elevated plasma levels of il8 and il18 and a high amount of monocytes whereas women develop a robust activation of t lymphocytes (74) . this differences in the immune response could explain the higher mortality of covid19 in men than in women (75, 76) . to recapitulate, sex hormones and genetic expression patterns in men and women can generate distinct immune and inflammatory responses that determine singularities in the epidemiological distribution of immune diseases. research protocols in immune response and inflammation must be redefined to avoid results biased by sex. furthermore, research in women is urgently needed to define the efficacy for women of several therapies that were originally tested in men. the increase in noncommunicable diseases (ncds), such as obesity, hypertension and cancer as well as the low-grade chronic inflammation that characterizes most ncds (77) can be affected by environmental factors that change the immune response. lifestyle factors like nutrition can modulate the immune system. it has been reported in mice that western diet-induced systemic inflammation and reprogramming of myeloid cell precursors is mediated by the activation of the nlrp3 inflammasome, which is a key sensor of the innate immune system for metabolic danger signals, such as uric acid and cholesterol (78) . metabolic regulation appears to be very robust and long lasting, being reported that proper nutrition during pregnancy can reduce the risk for ncds in the offspring even at adult age (79, 80) . the impact of the diet on the immune response and inflammation some diet types can result in metabolic and epigenetic changes that affect immune function (81) , as reported in populations that consume a high-fat and low-fiber western diet, who show a prevalence of ncds higher than populations that consume a mediterranean diet or a diet based on bioactive compounds, like the hydroxytyrosol in olive oil (82) (83) (84) . there is evidence supporting the anti-inflammatory activity of phenolic extracts from olive oil, such as their ability to reduce lipopolysaccharide (lps)-stimulated nitric oxide (no) production by the raw-264.7 macrophage cell line. the hydroxytyrosol stearate and the hydroxytyrosol oleate decrease no production in a concentration-dependent manner (85) . in addition, olive oil extracts increase total plasma glutathione concentration (86) , increasing the antioxidative response of the individual. nordic diet has many similarities with the mediterranean diet, but its effects on low-grade chronic inflammation are less known. both diets include abundant fruits, vegetables, whole grain products, fish and vegetable oil, but restrict saturated fat and red and processed meats (87, 88) . observational (89, 90) and interventional (91, 92) studies report an inverse association between the adherence to nordic diet and the concentration of high sensitivity c-reactive protein (hscrp). single intervention studies reported beneficial effects, reducing il1 receptor a (il1ra) (87) and cathepsin s (93) , and downregulation of inflammatory mediators in the adipose tissue (94) and peripheral blood mononuclear cells (pbmcs) (95) . a key nutrient in fish are the n3 polyunsaturated fatty acids (pufas) (88) . the greenland inuit population, which has a high dietary intake of n3-pufas, have a lower incidence of myocardial infarction than the danish population (96) . numerous studies associate the cardioprotective effects of n-3 pufas to their effect on immunomodulation (97) (98) (99) , and control of inflammation, including neuroinflammation during aging (100) . the mechanism of the anti-inflammatory effects of n3-pufas n3-pufas can regulate the transcription and expression of inflammatory mediators such as cytokines, chemokines and adhesion molecules in cardiomyocytes, fibroblasts, endothelial cells, and monocyte-macrophages (101) (102) (103) (104) . anti-inflammatory effect of eicosapentaenoic acid (epa), docosahexaenoic acid (dha) and their biologically active metabolites (d and e resolvinsmediators derived from omega-3 fatty acids, primarily epa and dha that block the production of proinflammatory mediators and regulate leukocyte trafficking to inflammatory sites) can be mediated through one of the mechanisms capable of reducing inflammation of raw-264.7 cells and of primary intraperitoneal macrophages (105) . one of the mechanisms is the activation of g-protein coupled receptors (gpr), ea. gpr120 inhibition of toll-like receptor 4 (tlr4)-mediated inflammatory response, which blocks nfkb activation. the other is mediated by nuclear receptors, particularly ppars-a/g. dha binds to ppars with high affinity resulting in the activation of anti-inflammatory cascades (106) , which appears to be responsible for the beneficial health effects (97) . the inhibition of nfkb-mediated pro-inflammatory activity (107) is the common mechanism of immunomodulation by n3-pufas, being dha more effective than epa in reducing lps-n3-pufas induced inflammatory cytokine production by macrophages (108) . n3-pufas are incorporated into phospholipid bilayers and in human atherosclerotic plaques. their incorporation is associated with a reduction in the number of foam-and t cells, and a decrease in inflammation (109) . the increased incorporation of n3-pufas in membranes affects both the innate and adaptive immune responses, impairing the maturation of dendritic cells and the function of macrophages, as well as the polarization and activation of t and b cells (110) (111) (112) . it is well known that n3pufas compete with n6-pufas for being incorporated into cell membranes and for the active sites of cox-2 and lipoxygenase, resulting in the production of less potent pro-inflammatory or even anti-inflammatory mediators, such as the 3-series of prostaglandin and thromboxane (113) . resolvins reduce also neutrophil-derived ros production, favoring neutrophil apoptosis and clearance by macrophages, and inhibit chemokine signaling (114) . the partial agonist/antagonist activity of resolvin e1 (rve1) on the leukotriene b4 receptor on polymorphonuclear cells (pmns), inhibits nfkb activation, reduces release of pro-inflammatory cytokines and reduces infiltration by pmn (115) . moreover, rve1 reduces tnfa and ifng presence in the aortic wall, decreases the levels of the inflammatory marker crp and reduces macrophage infiltration of the intima. thus, rve1 attenuates atherosclerosis and atherosclerotic plaque formation (116) . aging is associated with the activation of inflammatory signaling pathways (117, 118) , which can be targeted by specific nutrients with anti-inflammatory effects, such as n3-pufas (119, 120) . in the brain, the main n3-pufa is dha, representing 12-14% of total fatty acids (121) . aging and neurological disorders are associated with decreased levels and turn-over rate of brain n3-pufas (122) (123) (124) (125) . in aged mice, n3-pufa supplementation and diets enriched in dha have been reported to revert age-induced spatial memory deficits and impairment on learning and memory (126) (127) (128) . in older adults, a low consumption of n3-pufas and decreased erythrocyte dha levels are associated with cognitive impairment (129, 130) . dietary supplementation with dha is positively correlated with an improvement in declarative memory test performance, improved cognitive function (131, 132) and a lower risk of developing neurological disorders (133) . the probable mechanisms by which n3-pufas mediate their effects in the resolution of age-related neuroinflammation are the increased synthesis of n3-pufa-derived rvd1 and decreased n6-pufaderived oxylipins, displaying an anti-inflammatory profile (134, 135) . to recapitulate, the evidence indicates that n3-pufas and their bioactive metabolites have immunomodulatory and antiinflammatory properties. potential cardioprotective lipid mediators, through multiple mechanisms, including changes in cell membranes composition, and modification of both cell signaling and gene expression, shift the pattern of lipid metabolites toward a more anti-inflammatory metabolite profile. dietary habits may be essential regulators of the inflammatory profile and promote healthy aging, reinforcing the recommendation of a n3-pufa rich diet. the long term chronic psychological stress is increasing among the world's population (136) . its circuit arises at high cortical centers through the limbic system to the hypothalamus, where corticotropin-releasing factor (crf) is produced, which is responsible for inducing the pituitary gland to liberate adrenocorticotropic hormone (acth) that signals the adrenal cortex to synthesize and secrete glucocorticoids (gcs) (137) . stress also activates the sympathetic nervous system (sns), particularly the adrenal medulla, activating chromaffin cells to produce epinephrine (epi), a main stress hormone along with gcs. the latter plays a key regulation feature inhibiting the hypothalamic-pituitary-adrenal (hpa) axis through negative feedback at the pituitary gland, hypothalamus, and medial prefrontal cortex, reducing crf secretion [rewieved in (138) ]. the interplay of social and environmental stressors induces inflammation through multiple biological mechanisms, including epigenetic factors (139) . studies in rats show that the methylation patterns of genes involved in the stress response, such as the glucocorticoid receptor (nr3c1) and crf, can be modified by psychosocial factors from early childhood (140) . similarly, early life adversity induces acute and long-lasting epigenetic modifications in nr3c1 genes, regulating hpa axis and cytokine production, reinforcing the importance of the activation inputs during critical periods of development (137, 141) . acute short-term emotional stress, such as speaking in public, leads to a transient increase in circulating inflammatory biomarkers and natural killer (nk) cells by the sns catecholaminergic activity (142) . on the contrary, chronic stress results in a reduction of cytotoxic nk activity, determining a poorer response to cytokines (143) . therefore, stress appears to have short term beneficial immune effects, whereas chronic stress in the absence of immune challenge has the opposite effect (138, 144) , activating constantly the hpa axis with the consequent persistent elevation of systemic gcs and reduction of nk cell responsiveness to cytokines (143) , affecting the balance of the t helper cell type 1/type 2 (th1/th2) cytokine networks, predisposing to a wide range of diseases (145) . the stress magnitude has been associated with il1b mrna overexpression in peripheral pbmcs, providing a molecular mechanism by which psychological stress is translated into an immune system response (146, 147) . when stress becomes chronic, such as in depression, there is a maintained overproduction of inflammatory cytokines, which have been associated with gcs resistance. immune cells become less sensitive to their anti-inflammatory effects because of their persistent secretion, leading to chronic low-grade inflammation (147, 148) . activation of the innate and adaptive immune system by chronic mild stressors increases inflammatory cytokines gene expression, maturation and trafficking of dendritic cells (dc), increased macrophage number and t cells recruitment and activation. social stressors can induce an increase in inflammatory responses and a state of gcs resistance at different levels (144, 149) . the acute repeated social defeat stress (rsds) and chronic restraint stress (crs) models induce an inflammatory response that results in neuroinflammation and depressive behavior (150) . stress activates the hpa axis and the sympatho-adrenomedullar (sam) axis causing neuroinflammation by circulating cytokines that crossed the blood-brain barrier (bbb) at the circumventricular organs and by cytokine bbb transporters. an inflammatory response that promotes bbb permeability, allowing more inflammatory factors entering the brain, including crf, metalloproteinase-9, il6, and tnfa (150) . additionally, microglia produce chemokines that attract monocytes into the brain (150) . activation of sns and hpa axis through continuous psychological stress dysregulate cytokine production, and together with the stress hormones corticosteroids and catecholamines, can affect endothelial adhesion molecules, causing endothelial damage (138) . corticosteroids could facilitate the infiltration of monocytes by increasing the expression of il1 and il6 receptors on endothelial cells. these monocytes and lymphocytes, after attaching to such sites, would commence the process of infiltration into the wall vessels, leading to foam cell formation and thrombotic events (138, 151) . chronic unpredictable mild stress (cums) decreases body mass and impairs the metabolism of carbohydrates and lipids. a model for cums showed an increased liver and pancreas protein-lipid peroxidation and protein oxidation (152) . high ros production in both organs could be a result of a response mechanism to stress at the cellular level. in the liver, protein oxidation can be due to the regulation of metabolic impairments by gcs and epi (152). the antioxidant system of the liver is in general more efficient than the pancreas. however, it is insufficient to clear the reactive species increased as consequence of chronic stress, which could be due to alterations in the antioxidant enzymatic activity (138) . altogether, stress appears to have short term beneficial effects on the immune function, whereas chronic stress (138, 144) activates persistently the hpa, elevating systemic gcs, and impairing the cytokine balance. the overproduction of inflammatory cytokines lead to gcs resistance driven by immune cells that lose their sensitivity to gcs, leading to a state of chronic low-grade inflammation (138, 145) . this gcs imbalance, shares common features with aging, mediating an enhanced neuroinflammatory priming (153) . the presence of psychological stress potentiates the defective immune response observed in aging, which at the same time conditionate an exaggerated sickness response to immune challenges (such as chronic stress). thus, chronic stress contributes to the phenomenon of inflammaging, which promotes the development of several age-related pathologies, including atherosclerosis and diabetes among others [reviewed in (154) ]. additionally, there is an impairment of the antioxidant defense system to manage ros production after chronic stress, resulting in the damage of various tissues (138) . in addition, people exposed to chronic stress age rapidly, showing a faster telomere shortening in their cells (155) (156) (157) . on the other hand, epigenetic changes acquired during critical developmental stages could shape chronic stress-response along the lifespan, either promoting or reducing pathological aging (139, 140) . substance abuse, such as alcohol and drugs, are important triggers of chronic inflammatory processes (158, 159) . the effects of alcohol on human health are complex and depend on multiple factors. however, many of those factors are associated with the generation of immunosuppression and increased morbimortality in heavy users. those effects, which have been previously reviewed by goral et al. (160) will not be discussed in this review. here, we will describe the effect of cocaine and methamphetamine abuse. both drugs are potent psychostimulants that, when repeatedly consumed, significantly disrupt the functioning of the cns, and modify the regulation of the immune response, leading to a chronic neuroinflammatory state (161) . in general, it is known that drug abuse, among other factors, increases nfkb transcription of multiple proinflammatory genes that spread across brain cell types further amplifying of nfkb transcription, as has been reviewed by crews et al. (162) . cocaine (benzoylmethylecgonine according to the international common denomination) is a strong stimulant tropane alkaloid that acts by modulating the catecholaminergic neurotransmitter dopamine. studies of the striatum of mice after the administration of various drugs showed that 1 h after administration of 25 mg/kg cocaine, there is a significant increase in gene arrays for hypoxia-inducible factor 1 (hif-1), transcription factors, and cytokine receptors (il6r, tnfa). two hours after cocaine administration, there is an increased gene expression for various tnf receptors, inducible no synthase (inos) and adhesion molecules (163) . in the nucleus accumbens of mice stimulated with cocaine, there is a significant increase in matrix metalloproteinase 28 (mmp28), macrophage colony stimulating factor (mcsf) and major histocompatibility complex ii (mhc-ii) (164) . the brain of human subjects consuming cocaine shows an increased density of macrophages and activated microglia (165) . cocaine induces the activation of microglia through the endoplasmic reticulum stress and autophagy pathways (166) . studies of human and rodent immune cell populations after cocaine administration show decreased numbers of t lymphocytes, modulation of nk activity and cytokine production (167) . among brain glial cells, astrocytes are the most abundant, and perform critical functions, being involved in neurogenesis, promotion of neuronal survival, elimination of free radicals, and the production of no to maintain neuronal homeostasis (166) . nevertheless, astrocytes can also be activated by toxic stimuli, leading to a new phenotype called "reactive astrocytes", similar with the changes observed after inflammatory activation. this phenomenon has been described in various neuropsychiatric disorders, such as alzheimer's and parkinson's disease, amyotrophic lateral sclerosis and multiple sclerosis (166) . the reactivity of astrocytes to toxic stimuli, such as cocaine, infection or disease, potentiates the neuroinflammatory process (168) . methamphetamine (desoxyephedrine; meth) is a synthetic adrenergic agonist with psychostimulatory effects, structurally related to the ephedrine alkaloid and adrenaline. studies on the effect of meth are limited. however, it has been determined that its abuse affects the immune response. animals exposed to both acute and chronic meth use show alkalization of normally acidic organelles in immune cells, inhibition of antigen presentation, and impairment of phagocytosis (169) . meth also generates mitochondrial oxidative damage, dysfunction of t lymphocytes and decreased production of antibodies and cytokines (159) . meth has effects in various tissues (170) . in the lungs, the number of t lymphocytes decreases compared with that of untreated animals, indicating a reduction in circulating cd3+ cells, and levels of il6 and il10 increases. in the spleen, recruitment of pmn and the number of ly-6g+ and f4/80+ are increased, whereas cd3+ cells are significantly reduced. in addition, levels of tnfa, ifng, il6, and il12 are higher than those of control mice. in the liver, there is an increase of t lymphocytes and macrophages, hepatocellular atrophy, and increased levels of ifng, tnfa, il1b, -4, -6, -10, and -12 in the group exposed to meth compared with control animals (170) . in the cns, meth can induce the activation of calpains and caspases; the production of ros with the subsequent induction of oxidative stress, and the release of high amounts of glutamate, causing excitotoxicity (171) . recently, raineri et al. reported that meth induces activation of astrocytes and microglia, increasing the levels of il6 and tnfa mrna and its receptor (tnfr1) in the mouse striatum and hippocampus (172, 173) . medical advances have resulted in the increment of the average life expectancy in developed countries. the aging of the population is associated with an increase in the number of older people using drugs of abuse. from 2000 to 2012, the number of cocaine users aged 55 or older that required treatment for drug addiction in the us increased by 63% (174, 175) . aging is associated with low-grade basal inflammation that can be compounded by substance use. as cocaine exposure is associated with elevated inflammation and altered immune functioning, the presence of cocaine use disorder might exacerbate inflammatory processes in aging adults (176) . a recent report by soder et al, compared the levels of inflammation (through the neutrophil to lymphocyte ratio) in older adults with cocaine use disorder (cud) and in healthy older adults, finding that the group with cud had a significantly higher baseline level of inflammation (176) . the use of illegal drugs such as cocaine or methamphetamine has not been shown to affect cognitive function in older adults at the clinical level. however, the evaluation of the cognitive function of young drug users reveals a decreased performance compared with healthy young people. in fact, the cognitive function of young drug users is similar to that of adults older than 60 years of age (174, 177, 178) . in summary, both cocaine and meth can directly impair the immune response, induce the activation of glial cells and stimulate the release of pro-inflammatory mediators in the cns. all those effects cause relevant changes in glial cell regulation and inflammatory activation, triggering chronic neuroinflammation and potentiating pathological aging. air pollution has become an important threat to public health. air pollutants consider a mixture of gases such as nitrogen oxides (nox), sulphur oxides (sox), tropospheric ozone (o3), volatile organic compounds (vocs), and particulate matter (pm) (179) . pm can enter the respiratory tract leading to severe in situ damage as well as inducing additional systemic effects (180) . the world health organization (who) suggests a maximum annual exposure of 10 μg/m³ of pm2.5, however, the exposure of 90% of the world's population exceeds the proposed limit (181) . exposure to air pollutants is associated with increased morbimortality associated with respiratory, cardiovascular, metabolic, neurological, carcinogenic and autoimmune diseases (17, (182) (183) (184) . inflammation is the main pathophysiological mechanism induced by air pollutants. in terms of the molecular and cellular mechanism induced by pollutants, pm and sox can generate ros, inducing oxidative stress, together with mitochondrial dysfunction and the consequent energy deprivation (185) (186) (187) . as a direct consequence, nfkb and mapk inflammatory pathways are activated, triggering an innate immune activation (188, 189) . despite the attempts to resolve the inflammatory event, the outcome appears to be an imbalance in lymphocyte homeostasis and immune system dysregulation, with inhibition of th1 and treg lymphocytes (190) . there is also an increase of th2 lymphocytes and recruitment of eosinophils, resulting in respiratory disorders such as asthma (186, 191, 192) . in parallel, pm deactivates the nuclear factor erythroid 2 pathway (nrf2), involved in antioxidant regulation and prevention of oxidative stress, a necessary process for the resolution of inflammation. therefore, to maintain oxidation-reduction reactions becomes impossible, becoming a breaking point towards increased ros production and the non-resolution of the inflammatory event (193) . another mechanism of action of pollutants is the activation of the aryl hydrocarbon receptor (ahr) by toxic agents. the binding of pm to ahr increases circulating th17 and decreases treg lymphocytes. increase in th17 associates to the release of il17, promoting an abrupt increase of th2 lymphocyte response. these changes promote the dysregulation of the immune response associated with the development of autoimmune processes (193) . aberrant increases in th17 may result in increased inflammation, with consequences such as asthma and acute respiratory failure syndrome (ards), due to neutrophil infiltration and tissue damage (194) . studies suggest the existence of a decline in treg levels and, therefore, an inability to suppress th1, th2 and phagocyte responses (195, 196) . in addition, exposure to pm has been associated with fibrotic events, where il17 increases synthesis and secretion of collagen in the lung parenchyma (197, 198) . in addition, it has been described that pm also induces the expression of tgfb, directly promoting fibroblast differentiation, which could also induce collagen deposition followed by a lower antifibrotic process in the liver (199) . pollutants may promote direct dna damage through oxidation of nitrogenous bases. hu and yu described in a 2019 paper different mechanisms and changes in mirna expression that comprise specific targets of dna methyltransferases, which can impair the methylation of tumor suppressor genes (200) . furthermore, urban populations show increased levels of mitochondrial methylation genes due to pm exposure (201) . there is evidence of the existence of methylation, acetylation and phosphorylation of histones h3 and h4, markers found in genes involved in the activation of immune cells and cardiovascular diseases (200, (202) (203) (204) (205) . altogether, air pollutants can generate dna adducts promoting carcinogenesis and deteriorate telomerase activity, as reviewed by martens and nawrot (2016) , and contributing to continuous dna damage and premature aging (206, 207) . in vivo studies suggest that the inflammatory activation is doseand time-dependent. mice exposed to pm show that both variables are determinant for the outcome. however, inflammatory effects and major genetic changes appear to be especially dependent on the exposure to high concentrations of pm. one possible explanation is that a prolonged exposure could induce an adaptive response of the inflammatory activation (208) , which may be mediated by the inactivation of the nrf2 pathway, generating a loss of antioxidant capacity and deregulation of the immune system (193) . the resolution appears to depend on the exposure context. acute exposure would result in high levels of ros and damage, whereas prolonged stimulation, even a low-grade one, generates a constant production of ros and chronic low-grade inflammation (187) , consequent with the potentiation of disease risk and an epigenetic age acceleration (206) , promoting pathological aging. direct causes of the deregulation of the inflammatory resolution process resulting from inhaled contaminants are still unknown, however, the burden of associated chronic diseases is expected to increase. it is mandatory to intensify environmental policies specifically in lower-middle-income countries in prevention of the development of inflammatory conditions and the subsequent chronic diseases. aging, characterized by a progressive loss of cellular functions, is an inevitable physiologic process inherent to all living beings (209) . the number of older adults is increasing. during the next 30 years, up to 22% of the world population will be older than 60 years (210) . this demographic change is accompanied by a higher incidence of ncds accumulated in the aging population (211) . therefore, various strategies have been proposed to improve the health and quality of life of older adults (212), along with recommendations for the development of public policies that support the fiscal expenditure resulting from ncds (213) . one of the most studied events of aging is the impairment of the immune system, characterized by an aberrant-increased activation of the innate immunity (214, 215) , and high levels of circulatory inflammatory mediators that establish an inflammatory environment, and a decrease of the adaptive immune response (216, 217) and a decrease of the adaptive immune response (214) due to this low-grade chronic inflammation (214, 218) , which together would promote the inflammaging phenomenon (219) . interestingly, it is proposed that age would not be the cause per se of these diseases associated with aging (214) . thus, there is a deterioration of the immune system's response to external stimuli, which depends on the individual's history (218) . also, several epigenetic mechanisms can modulate the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events (220) . on the other hand, it is described that epigenetic clocks would be useful to analyze mechanisms associated with this environmental influence (221) . finally, they would be capable of modulating the immune response in aging, enhancing changes in intercellular communication that could perpetuate inflammatory events (220). multiple age-dependent changes play important roles in the promotion of ncds, with increased oxidative stress standing out as one of the main mechanisms. over the last two decades, evidence has revealed that increased oxidative stress and inflammation are involved in various ncds such as alzheimer's disease (219) , rheumatoid arthritis (222) , cardiovascular diseases (223, 224) , and cancer (225), among others. also, recent studies propose that the activation of nfkb signaling pathways could be the main driver of these associations (226) (227) (228) (229) . interestingly, de almeida et al. showed different sources of low-grade chronic inflammation that promote cardiovascular disease (226) . in the cns, high levels of ros lead to the activation of astrocytes and microglia, further increasing the overproduction of ros and proinflammatory cytokines that promote the development of neurodegenerative changes (217, 230, 231) . in fact, several systemic biomarkers appear to be associated with neuroinflammation and the development of cns diseases associated with aging (230) . these modifications trigger the phenotype of senescent or aged cells characterized as sasp (216, 232) extensively studied in the context of the deleterious effects of aging. however, sasp is also essential for remodeling and promoting wound healing, which requires a strict control of the inflammatory response, thus avoiding the induction of cell aging phenotypes that contribute to the development of chronic inflammatory diseases (233) . the immune imbalance in aging occurs due to various alterations in cellular behavior and phenotype, which cause functional deficiencies in immune cells (3) . for example, this context induces polarization of macrophages towards an inflammatory phenotype characterized by strong activation of the inflammasome (234) . thus, these events could induce il1b and tnfa release, changes in the chemoattraction of neutrophils mediated by the reduction of the intercellular adhesion molecule 1 (icam-1) expression, and the aberrant activation of the phosphoinositide lipid kinase-3 (pi3k) (235) . also, there is a decrease in the expression of pattern recognition receptors (prr), which leads to the activation of proinflammatory signaling promoting tissue damage (215, 216) . finally, the reduced level of certain hormones due to the impaired hypothalamic function causes the loss of muscle mass and an increase in adipose tissue, further contributing to the release of inflammatory cytokines and changes in metabolism (236) . despite the remarkable effort being made to understand the basis of the processes underlying the inflammatory imbalance during aging, it is not fully understood. in aging, there are cumulative epigenetic changes that promote low-grade inflammation (220, 237) , including a decrease in the global genome methylation, with increased methylation in specific regions, as those with repressive histone marks of cd8+ and cd4+ t cells (238) and bivalent chromatin domains (239) and histone acetylation and methylation. however, the influence of genomic methylation during aging remains undetermined (237) . several studies correlate the methylation of multiple sites on cpg islands with the increase of the low-grade inflammation marker, crp (220, 232, 240) . nonetheless, stevenson et al. propose that the dna methylation could be better associated with the low-grade chronic inflammation than crp (237) . in addition, the age-related mitochondrial dysfunction, with the resulting oxidative stress and decreased atp production (241) , affect the expression and activity of dna methyltransferases, which are responsible for maintaining the methylation pattern of dna (242) . the reduced methylation results in the demethylation of the tnfa promoter in leukocytes and macrophages (243) and the adhesion of immune cells to the endothelium (244) . also, many epigenetic events contribute to the differentiation of proinflammatory t cells, th17 (220) , which can compromise immunocompetence, associated with repression of differentiation of immune cells, loss of treg function (240) and the alteration of the hematopoietic stem cells differentiation (245) . thus, epigenetic mechanisms appear to have a major role in the inflammatory imbalance, which are associated with the accumulation of damage in time that ultimately leads to the perpetuation of a constant inflammatory response. according to the who, 60% of the world population is sedentary, lacking the benefits of physical exercise (246) . conditions such as sedentarism, unhealthy diet, overweight, obesity and aging induce chronic low-grade inflammation. physical exercise increases the anti-inflammatory potential and reduces the pro-inflammatory effect (247) . this equilibrium is partly modulated through tlrs (248) , which are fundamental for the recognition of prrs, including the damage-associated molecular patterns (damps) and the induction of an inflammatory response in the absence of pathogens. there is evidence that in young people, physical exercise decreases tlrs expression, co-stimulatory molecules cd80/ cd86, and mhcii (248, 249) in cd14+ monocytes. physical exercise also affects the adipose tissue. exercising reduces tlr4 mrna expression and tnfa production in adipocytes (250, 251) in obese mice. chronic physical exercise decreases tnfa and tlr4 gene expression in the skeletal muscle (252) . the evidence suggests that obesity-or cerebral ischemiainduced neuroinflammation, which are associated with the overexpression of tlr2 and tlr4, may be reduced by physical exercise through the reduction of tlrs expression as well as their downstream signaling molecules (tnfa, il1b, myd88, traf6, 552 tak1, and nfkb), together with the reduced microglial activation (253, 254) . there is evidence that cigarette smoking induces inflammatory status [reviewed in (255) ]. however, exercise training reduces smoke-induced inflammation. in that sense, training for 30 min with endurance exercise for 5 days in smoke-exposed mice demonstrated that therapeutic exercise training significantly reduces the expression of il1b and tnfa mrna in rectus femoris (256) . physical exercise has been used as a therapeutic tool in chronic pathological conditions. in that sense, obese older adults (body mass index 38 ± 2 kg/m2; 69 ± 1 years) undergoing an exercise program consisting in physical therapy, endurance, and resistance for 90 min, 3 days per week, show a reduced expression of tlr4, il6, and tnfa mrna in skeletal muscle (257) . in older adults, 8-week physical exercise reduces the expression of tlr4 and tlr2, as well as tlrs downstream mediators, such as myd88, p65, pp38, trif, ikki/ikkϵ, irf3, and pirf7 in pbmcs (258) . similarly, dendritic cells from multiple sclerosis patients undergoing an exercise (endurance and resistance) program for 12 weeks reduce tnfa and mmp9 secretion when stimulated with a tlr4 ligand (lps in combination with ifng, or a tlr7 ligand) (259) , suggesting that long-term physical exercise decrease tlr responsiveness. on the other hand, high-intensity physical exercise in untrained individuals induces inflammation, resulting in the increased expression of tlr4, ap1, nfkb, and p65 in mice myocardium and in adipose tissue (260) (261) (262) . physical exercise associated with eccentric contractions causes expression of tlr and nfkb in skeletal muscle and liver in rats (263, 264) . furthermore, this phenomenon induces muscle damage, which can increase chemotaxis, attracting nk, cd8+ 559 t cells, macrophages and neutrophils to the site of injury, promoting the production of cox560 2, inos, monocyte chemotactic protein-1 (mcp-1), tnfa, il6, and il1b, in addition to the production of ros and the activation of nfkb (265, 266) . in healthy young males, one session of intense endurance exercise (1 h intense cycling immediately followed by 1 h intense running), increases plasmatic concentrations of il6 and il10, in addition to increased gene expression of proinflammatory il1 receptor (il1r) and tlr signaling pathways. moreover, plasma myoglobin changes in correlation with neutrophil tlr4 gene expression (r= 0.74), suggesting that their transcriptional activity was particularly induced by damps (267) . therefore, inflammation and muscle damage are mainly associated with the type and intensity of the exercise, with loads that exceed individual physical abilities. chronic physical exercise generates epigenetic modifications. the physical exercise associated with an energy expenditure >500 kilocalories per week, results in hypomethylation of the il10 gene and hypermethylation of the tnfa gene (268) , with an inverse correlation between tnfa methylation and tnfa mrna expression (269) . the methylation of the caspase recruitment domain (asc) of the apoptosis-associated specklike protein gene, the main regulator of inflammasome and promoter of the activation of il1b and il18, decreases with aging. however, older adults who maintain physical exercises regularly express higher levels of asc methylation than subjects not exercising, which would imply a decreased release of inflammatory cytokines (270) (271) (272) (273) . similarly, in review a 6-month walk training can induce hypermethylation of the nfkb-2 gene, suppressing inflammation through the inhibition of the nfkb pathway (274) . as life expectancy increases, age-related diseases thrive. aging is a complex multifactorial process of molecular and cellular decline that renders individuals susceptible to disease and death. maintenance of cell integrity, cell metabolism and host-defense mechanisms are tightly regulated by the surrounding microenvironment. a growing body of evidence in different biological models has contributed towards identifying biological mechanisms that ward off structural and functional deterioration. these data offer us insights into healthy aging. molecular integrity of the genome, telomere length, epigenetic stability, and protein homeostasis are all features linked to more youthful stages (regardless of the age), associated with mitochondrial fitness, metabolic regulation, efficient intercellular communication, stem cell renewal, and regenerative capacity in tissues. a good understanding of the environmental and endogenous mechanisms that underlie agerelated normal and deleterious changes, and how these pathways interconnect, remains a major challenge for slowing pathological aging while extending older adults' healthy lifespan. the study of the environmental influence on the development of complex-chronic diseases shows that in addition to genetic predisposition, the pathogenesis is promoted by changes in metabolism and behavior, cellular environment, and epigenetic regulation patterns. the type of nutrient, or environmental cytokine milieu dramatically affects not only the homoeostasis of tissues but also of complete organs and even of the whole individual. thus, tissue stress, malfunction, and damage may induce inflammation alarm responses, which result either in resolution of tissue damage, restoration of normal cell function or development of chronic disease ( figure 1 ). older adults often present inflammaging, characterized by increased levels of proinflammatory cytokines il1, il6, il8, tnfa/crp (275) . however, the cellular sources of these cytokines are partially unknown. the increased inflammatory cytokines have been proposed to be a driver of unsuccessful aging (increased morbidity, degenerative processes, or frailty) and shortened health-span. the inflammatory scenario is complex and occurs in response to various internal and environmental stimuli ( figure 1 ) mediated mainly by a high level of proinflammatory cytokines. indeed, in healthy aging, increased production of the anti-inflammatory cytokines tgfb and il10, can regulate the pro-inflammatory state (276, 277) . research into the impact of environmental factors on inflammaging is at an early stage and the involved mechanisms are not completely understood. several hypotheses have been developed to explain the chronic inflammation: aging-related increase of stress (278) and oxidative stress (279) , dna damage in senescent cells [reviewed in (280) ], and stem cell aging (281) . the proposed mechanisms are likely interdependent, resulting in the generation of ros causing oxidative damage and amplification of the cytokines secretion, thus perpetuating a vicious circle of systemic inflammation where tissue injury and healing mechanisms proceed in parallel while damage slowly accumulates over the lifespan of the individuals. endocrine and metabolic alterations are linked to the shift towards a pro-inflammatory profile, which could explain some age-related pathologies, such as alzheimer's and parkinson's disease, osteoporosis, diabetes, cancer, and frailty (282, 283) . regarding stress-induced immune modifications, new evidence suggests that cross talk signals between the cns, endocrine and immune system are required for optimal response to stress [discussed in (284) ]. various stressors can affect the activity and regulation of immune cells via direct regulation by the autonomic and peptidergic system or through the release of neuroendocrine mediators. moreover, neuronal catecholamines modulate immune cell functions. these interactions are bidirectional, cytokines produced by immune cells, such as il1, can modulate the production of corticotropin-releasing hormone (crh) by the hypothalamus. chronic diseases are favored by some modern living conditions, such as the intake of high-caloric foods and the low level of physical activity, or endogenous signals produced by the chronic stress of modern life. there are many challenges in conducting research on biosocial processes, which will define novel disease-trigger factors. tailor-made approaches will depend on genetics, epigenetics and a constellation of factors depending on the historical as well as the present exposure to the environment. although environmental factors also express themselves as epigenetic changes, the combinatorial effect of the multiple factors generates complex patterns of epigenetic regulation, and the concomitant exposure to environmental factors can further modify the individual response. all authors contributed equally on the conception of the work, the analysis of literature and preparing the content of the review. rbe drafted and organized the manuscript. all authors contributed to the article and approved the submitted version. endogenous and environmental factors can be mostly beneficial (in green) and deleterious (in red) or can have both beneficial and deleterious effects depending on the specific context. the interplay of lifespan endogenous and environmental factors regulates the aging phenotype depending on dna damage, epigenetic changes, and inflammation. these drivers can induce functional aging hallmarks: changes in endocrine and metabolic regulation, and defective immune regulation that will further determine the response of the individual. in yellow we show processes that can participate in both protection and damage. exposure to various alarm signals induce an acute inflammation that, when associated with deleterious environmental and biological factors, potentiates chronic inflammation, which can be further promoted by excess ros production and oxidative stress that results from mitochondrial dysfunction or nox2 activity, leading to inflammaging and eventually to age-related disease. on the contrary, in the presence of protective environmental and biological factors, the initial inflammatory activation will be resolved and lead to a healthy aging process. ros, reactive oxygen species. sarcopenic obesity and inflammation in the inchianti study physiological aging: links among adipose tissue dysfunction, diabetes, and frailty immunosenescence: the potential role of myeloid − derived suppressor cells ( mdsc ) in age − related immune deficiency the hallmarks of aging circulating c1q complement/tnf-related protein (ctrp) 1, ctrp9, ctrp12 and ctrp13 concentrations in type 2 diabetes mellitus: in vivo regulation by glucose aging, inflammation and the environment both genetic and dietary factors underlie individual differences in dna damage levels and dna repair capacity pro12ala polymorphism of the pparg2 gene interacts with a mediterranean diet to prevent telomere shortening in the predimed-navarra randomized trial micrornas linking inflamm-aging, cellular senescence and cancer metabolic control of longevity immunobiography and the heterogeneity of immune responses in the elderly: a focus on inflammaging and trained immunity epidemiology of parkinson disease mitochondrial dysfunction and longevity in animals: untangling the knot. sci cardiac oxidative stress in diabetes: mechanisms and therapeutic potential nadph oxidase and mitochondria are relevant sources of superoxide anion in the oxinflammatory response of macrophages exposed to airborne particulate matter evaluation of oxidative damage and nrf2 activation by combined pollution exposure in lung epithelial cells emerging role of air pollution in autoimmune diseases inhibitory cross-talk upon introduction of a new metabolic pathway into an existing metabolic network child development and the physical environment psychological stress in childhood and susceptibility to the chronic diseases of aging: moving towards a model ofbehavioral and biological mechanisms sex steroidinduced dna methylation changes and inflammation response in prostate cancer integrative analysis of methylome and transcriptome in human blood identifies extensive sex-and immune cell-specific differentially methylated regions gender differences of b cell signature related to estrogen-induced ifi44l/baff in systemic lupus erythematosus gender differences of b cell signature in healthy subjects underlie disparities in incidence and course of sle related to estrogen peroxisome proliferator-activated receptor (ppar)alpha expression in t cells mediates gender differences in development of t cell-mediated autoimmunity sex-specific t-cell regulation of angiotensin ii-dependent hypertension. hypertens (dallas tex 1979 peroxisome proliferator-activated receptor (ppar)alpha and -gamma regulate ifngamma and il-17a production by human t cells in a sexspecific way sex differences in prostaglandin biosynthesis in neutrophils during acute inflammation sex disparity in cord blood foxp3+ cd4 t regulatory cells in infants exposed to malaria in utero sex differences in pediatric infectious diseases correlation between female sex, il28b genotype, and the clinical severity of bronchiolitis in pediatric patients sex differences in the blood transcriptome identify robust changes in immune cell proportions with aging and influenza infection sexual-dimorphism in human immune system aging sex differences in older adults' immune responses to seasonal influenza vaccination sex differences in immune responses that underlie covid-19 disease outcomes gender differences in patients with covid-19: focus on severity and mortality sex-specific clinical characteristics and prognosis of coronavirus disease-19 infection in wuhan, china: a retrospective study of 168 severe patients noncommunicable diseases country profiles western diet triggers nlrp3-dependent innate immune reprogramming the next innovation cycle in toxicogenomics: environmental epigenetics transgenerational effects of maternal diet on metabolic and reproductive ageing nutrition and epigenetics: an interplay of dietary methyl donors, one-carbon metabolism and dna methylation population differences in associations between c-reactive protein concentration and adiposity: comparison of young adults in the philippines and the united states diet and the epigenome effects of olive oil and its minor components on cardiovascular diseases, inflammation, and gut microbiota identification of hydroxytyrosyl oleate, a derivative of hydroxytyrosol with anti-inflammatory properties, in olive oil by-products olive phenolics increase glutathione levels in healthy volunteers effects of an isocaloric healthy nordic diet on insulin sensitivity, lipid profile and inflammation markers in metabolic syndrome -a randomized study (sysdiet) what is a healthy nordic diet? foods and nutrients in the nordiet study associations of the baltic sea diet with cardiometabolic risk factors-a meta-analysis of three finnish studies associations of the baltic sea diet with obesity-related markers of inflammation health effect of the new nordic diet in adults with increased waist circumference: a 6-mo randomized controlled trial a diet high in fatty fish, bilberries and wholegrain products improves markers of endothelial function and inflammation in individuals with impaired glucose metabolism in a randomised controlled trial: the sysdimet study influence of a prudent diet on circulating cathepsin s in humans healthy nordic diet downregulates the expression of genes involved in inflammation in subcutaneous adipose tissue in individuals with features of the metabolic syndrome healthy nordic diet modulates the expression of genes related to mitochondrial function and immune response in peripheral blood mononuclear cells from subjects with metabolic syndrome-a sysdiet sub-study eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? marine omega-3 fatty acids and inflammatory processes: effects, mechanisms and clinical relevance omega-3 fatty acids and inflammatory processes: from molecules to man cardioprotective mechanism of omega-3 polyunsaturated fatty acids neuro-immune dysfunction during brain aging: new insights in microglial cell regulation n-3 polyunsaturated fatty acids (pufa) modulate the expression of functionally associated molecules on human monocytes polyunsaturated fatty acids inhibit the antigenpresenting function of human monocytes effects of omega-3-rich harp seal oil on the production of pro-inflammatory cytokines in mouse peritoneal macrophages dietary fish oil diminishes lymphocyte adhesion to macrophage and endothelial cell monolayers gpr120 is an omega-3 fatty acid receptor mediating potent anti-inflammatory and insulin-sensitizing effects human dendritic cell activities are modulated by the omega-3 fatty acid, docosahexaenoic acid, mainly through pparg:rxr heterodimers: comparison with other polyunsaturated fatty acids eicosapentaenoic acid prevents lps-induced tnf-a expression by preventing nf-kb activation docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharide-stimulated human thp-1 macrophages more effectively than eicosapentaenoic acid eicosapentaenoic acid (epa) from highly concentrated n-3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque epa is associated with decreased plaque inflammation and increased stability docosahexaenoic acid prevents dendritic cell maturation, inhibits antigen-specific th1/th17 differentiation and suppresses experimental autoimmune encephalomyelitis regulatory activity of polyunsaturated fatty acids in t-cell signaling n-3 pufa improves fatty acid composition, prevents palmitate-induced apoptosis, and differentially modifies b cell cytokine secretion in vitro and ex vivo effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils resolvin e1 and protectin d1 activate inflammation-resolution programmes resolvin e1 selectively interacts with leukotriene b 4 receptor blt1 and chemr23 to regulate inflammation resolvin e1 (rve1) attenuates atherosclerotic plaque formation in diet and inflammation-induced atherogenesis human cns immune senescence and neurodegeneration microglial senescence: does the brain's immune system have an expiration date? ageassociated changes in the content and fatty acid composition of brain glycerophospholipids the aging human orbitofrontal cortex: decreasing polyunsaturated fatty acid composition and associated increases in lipogenic gene expression and stearoyl-coa desaturase activity modulation of brain pufa content in different experimental models of mice dha-enriched phospholipid diets modulate age-related alterations in rat hippocampus docosahexaenoic acid-induced changes in phospholipids in cortex of young and aged rats: a lipidomic analysis from inflammation to sickness and depression: when the immune system subjugates the brain fatty acid composition of brain phospholipids in aging and in alzheimer's disease chronic administration of docosahexaenoic acid improves the performance of radial arm maze task in aged rats docosahexaenoic acid-rich phospholipid supplementation: effect on behavior, learning ability, and retinal function in control and n-3 polyunsaturated fatty acid deficient old mice dha improves cognition and prevents dysfunction of entorhinal cortex neurons in 3xtg-ad mice association between mediterranean diet and late-life cognition red blood cell omega-3 fatty acid levels and markers of accelerated brain aging dietary intake of eicosapentaenoic and docosahexaenoic acids is linked to gray matter volume and cognitive function in elderly cognitive aging, childhood intelligence, and the use of food supplements: possible involvement of n-3 fatty acids elevated levels of proinflammatory oxylipins in older subjects are normalized by flaxseed consumption dietary n-3 long chain pufa supplementation promotes a pro-resolving oxylipin profile in the brain systematic review of the association between chronic social stress and telomere length: a life course perspective the developmental origins of chronic physical aggression: biological pathways triggered by early life adversity psychological stress, immune response, and atherosclerosis the epigenome at the crossroad between social factors, inflammation, and osteoporosis risk epigenetic programming by maternal behavior the role of dna methylation in stress-related psychiatric disorders selective mobilization of cytotoxic leukocytes by epinephrine immune dysregulation and chronic stress among older adults: a review chronic stress and age-related increases in the proinflammatory cytokine il-6 depression, mood, stress, and th1/th2 immune balance in primary breast cancer patients undergoing classical massage therapy nuclear factor-kb is a critical mediator of stress-impaired neurogenesis and depressive behavior social signal transduction theory of depression inflammation as a psychophysiological biomarker in chronic psychosocial stress repeated social defeat activates dendritic cells and enhances toll-like receptor dependent cytokine secretion neuroinflammation caused by mental stress: the effect of chronic restraint stress and acute repeated social defeat stress in mice anger emotional stress influences vegf/vegfr2 and its induced pi3k/akt/mtor signaling pathway chronic unpredictable mild stress generates oxidative stress and systemic inflammation in rats stress and aging act through common mechanisms to elicit neuroinflammatory priming the link between chronic stress and accelerated aging psychological wellbeing and healthy aging: focus on telomeres toward a deeper understanding of a triad of early aging tired telomeres: poor global sleep quality, perceived stress, and telomere length in immune cell subsets in obese men and women alcohol and epigenetic changes: summary of the 2011 alcohol and immunology research interest group (airig) meeting methamphetamine decreases cd4 t cell frequency and alters proinflammatory cytokine production in a model of drug abuse exposure-dependent effects of ethanol on the innate immune system cocaine induced inflammatory response in human neuronal progenitor cells induction of innate immune genes in brain create the neurobiology of addiction the dissection of transcriptional modules regulated by various drugs of abuse in the mouse striatum histone deacetylase 5 epigenetically controls behavioral adaptations to chronic emotional stimuli decreased brain dopamine cell numbers in human cocaine users cocaine induces astrocytosis through er stress-mediated activation of autophagy immune system inflammation in cocaine dependent individuals: implications for medications development psychostimulant abuse and neuroinflammation: emerging evidence of their interconnection methamphetamine and its immune-modulating effects methamphetamine administration modifies leukocyte proliferation and cytokine production in murine tissues causes and consequences of methamphetamine and mdma toxicity modafinil abrogates methamphetamine-induced neuroinflammation and apoptotic effects in the mouse striatum methamphetamine-induced neuroinflammation and neuronal dysfunction in the mice hippocampus: preventive effect of indomethacin trends in substance use admissions among older adults elevated neutrophil to lymphocyte ratio in older adults with cocaine use disorder as a marker of chronic inflammation age-and sex-dependent effects of methamphetamine on cognitive flexibility and 5-ht2c receptor localization in the orbitofrontal cortex of sprague-dawley rats crack-cocaine dependence and aging: effects on working memory air pollution prevention and control policy in china air pollution and allergy: you are what you breathe air pollution associated epigenetic modifications: transgenerational inheritance and underlying molecular mechanisms biomarkers of the health outcomes associated with ambient particulate matter exposure the effects of exposure to air pollution on the development of uterine fibroids role of oxidative damage in toxicity of particulate mechanisms of heightened airway sensitivity and responses to inhaled so2 in asthmatics exposure scenario: another important factor determining the toxic effects of pm2.5 and possible mechanisms involved in vitro toxicity of particulate matter (pm) collected at different sites in the netherlands is associated with pm composition, size fraction and oxidative potential -the raptes project exposure to ultrafine particulate matter induces nf-kb mediated epigenetic modifications diesel exhaust particle exposure in vitro impacts t lymphocyte phenotype and function acute nitrogen dioxide (no2) exposure enhances airway inflammation via modulating th1/th2 differentiation and activating jak-stat pathway diesel exhausts particles: their role in increasing the incidence of asthma. reviewing the evidence of a causal link air pollution-derived pm2.5 impairs mitochondrial function in healthy and chronic obstructive pulmonary diseased human bronchial epithelial cells acute respiratory distress syndrome effects of pm2.5 exposure on the notch signaling pathway and immune imbalance in chronic obstructive pulmonary disease the impact on tregulatory cell related immune responses in rural women exposed to polycyclic aromatic hydrocarbons (pahs) in household air pollution in gansu, china: a pilot investigation pm 2.5 induced pulmonary fibrosis in vivo and in vitro blocking il-17a promotes the resolution of pulmonary inflammation and fibrosis via tgf-b1-dependent and -independent mechanisms effects of sub-chronic exposure to atmospheric pm2.5 on fibrosis, inflammation, endoplasmic reticulum stress and apoptosis in the livers of rats genetic and epigenetic alterations in normal and sensitive copd-diseased human bronchial epithelial cells repeatedly exposed to air pollution-derived effects of airborne pollutants on mitochondrial dna methylation prenatal particulate air pollution and dna methylation in newborns: an epigenomewide meta-analysis dose-and time-effect responses of dna methylation and histone h3k9 acetylation changes induced by traffic-related air pollution air pollution and dna methylation: effects of exposure in humans epigenetic response profiles into environmental epigenotoxicant screening and health risk assessment: a critical review air pollution, particulate matter composition and methylation-based biologic age air pollution stress and the aging phenotype: the telomere connection the effect of exposure time and concentration of airborne pm2.5 on lung injury in mice: a transcriptome analysis facing up to the global challenges of ageing coming of age: molecular drivers of aging and therapeutic opportunities find the latest version: review series introduction coming of age: molecular drivers of aging and therapeutic opportunities disability incidence and functional decline among older adults with major chronic diseases quality of life assessment instruments for adults: a systematic review of population-based studies comparative financing analysis and political economy of noncommunicable diseases the integration of inflammaging in age-related diseases scavenger receptor-a deficiency impairs immune response of microglia and astrocytes potentiating alzheimer's disease pathophysiology source of chronic inflammation in aging microglial cell dysregulation in brain aging and neurodegeneration aging and the immune system: an overview cellular senescence and alzheimer disease: the egg and the chicken scenario the epigenetics of inflammaging: the contribution of age-related heterochromatin loss and locus-specific remodelling and the modulation by environmental stimuli wandering along the epigenetic timeline myeloperoxidase as an active disease biomarker: recent biochemical and pathological perspectives the role of signaling pathways of inflammation and oxidative stress in development of senescence and aging phenotypes in cardiovascular disease oxidative stress and advanced lipoxidation and glycation end products (ales and ages) in aging and age-related diseases how the ageing microenvironment influences tumour progression unveiling the role of inflammation and oxidative stress on age-related cardiovascular diseases nf-kb-mediated neuroinflammation in parkinson's disease and potential therapeutic effect of polyphenols epigenetic upregulation of fkbp5 by aging and stress contributes to nf-kbdriven inflammation and cardiovascular risk nf-kb signaling in astrocytes modulates brain inflammation and neuronal injury following sequential exposure to manganese and mptp during development and aging neuroinflammation in frontotemporal dementia immunosenescence in aging: between immune cells depletion and cytokines up-regulation back to the future: epigenetic clock plasticity towards healthy aging immunity and inflammation: from jekyll to hyde update of inflammasome activation in microglia/macrophage in aging and aging-related disease aging of the immune system: focus on inflammation and vaccination inflammaging: a new immune-metabolic viewpoint for age-related diseases characterisation of an inflammation-related epigenetic score and its association with cognitive ability agerelated profiling of dna methylation in cd8+ t cells reveals changes in immune response and transcriptional regulator genes human aging-associated dna hypermethylation occurs preferentially at bivalent chromatin domains abnormal epigenetic regulation of immune system during aging do you remember mitochondria? mitochondrial turnover and aging of long-lived postmitotic cells: the mitochondriallysosomal axis theory of aging agerelated loss of cpg methylation in the tumour necrosis factor promoter dysregulation of c-x-c motif ligand 10 during aging and association with cognitive performance understanding intrinsic hematopoietic stem cell aging world health organization's global strategy on diet, physical activity and health: the process behind the scenes interleukin-10 responses from acute exercise in healthy subjects: a systematic review the physiological regulation of toll-like receptor expression and function in humans the influence of prolonged cycling on monocyte toll-like receptor 2 and 4 expression in healthy men exercise training inhibits inflammation in adipose tissue via both suppression of macrophage infiltration and acceleration of phenotypic switching from m1 to m2 macrophages in high-fat-diet-induced obese mice the anti-inflammatory effects of exercise: mechanisms and implications for the prevention and treatment of disease chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and tlr4 mrna in rat skeletal muscle exercise therapy downregulates the overexpression of tlr4, tlr2, myd88 and nf-kb after cerebral ischemia in rats neuroprotective effects of endurance exercise against high-fat diet-induced hippocampal neuroinflammation ten hacken nht. acute effects of cigarette smoke on inflammation and oxidative stress: a review exercise training reverses inflammation and muscle wasting after tobacco smoke exposure exercise but not dietinduced weight loss decreases skeletal muscle inflammatory gene expression in frail obese elderly persons role of toll-like receptor 2 and 4 signaling pathways on the inflammatory response to resistance training in elderly subjects 12 weeks of combined endurance and resistance training reduces innate markers of inflammation in a randomized controlled clinical trial in patients with multiple sclerosis efecto del ejercicio agudo sobre la expresioń del receptor tipo toll-4 y los mecanismos inflamatorios en corazoń de rata acute exercise activates myocardial nuclear factor kappa b exhaustive exercise increases inflammatory response via toll like receptor-4 and nf-kbp65 pathway in rat adipose tissue diclofenac pretreatment effects on the toll-like receptor 4/nuclear factor kappa b-mediated inflammatory response to eccentric exercise in rat liver diclofenac pretreatment modulates exercise-induced inflammation in skeletal muscle of rats through the tlr4/nf-kb pathway exercise-induced increase in serum inferleukin-6 in humans is related to muscle damage honokiol protects rats against eccentric exercise-induced skeletal muscle damage by inhibiting nf-kb induced oxidative stress and inflammation transcriptome analysis of neutrophils after endurance exercise reveals novel signaling mechanisms in the immune response to physiological stress exercise and inflammation-related epigenetic modifications: focus on dna methylation impact of aerobic exercise and fatty acid supplementation on global and genespecific dna methylation role of physical exercise in the regulation of epigenetic mechanisms in inflammation, cancer, neurodegenerative diseases, and aging process epigenetic regulation on gene expression induced by physical exercise regulatory molecules involved in inflammasome formation with special reference to a key mediator protein exercise effects on methylation of asc gene nfkb2 gene as a novel candidate that epigenetically responds to interval walking training inflammaging and anti-inflammaging: a systemic perspective on aging and longevity emerged from studies in humans role of tgf b signaling in the pathogenesis of alzheimer's disease age-dependent changes in the activation and regulation of microglia stress responses and innate immunity: aging as a contributory factor oxidative stress, inflamm-aging and immunosenescence dna damage response (ddr) and senescence: shuttled inflamma-mirnas on the stage of inflamm-aging emerging models and paradigms for stem cell ageing from inflamm-aging to immune-paralysis: a slippery slope during aging for immune-adaptation aging and parkinson's disease: inflammaging, neuroinflammation and biological remodeling as key factors in pathogenesis impact of stress on aged immune system compartments: overview from fundamental to clinical data the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 bachmann, bellalta, basoalto, goḿez-valenzuela, jalil, lépez, matamoros and von bernhardi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003368-f8f2utzx authors: lutterberg, karina; kleinwort, kristina j. h.; hobmaier, bernhard f.; hauck, stefanie m.; nüske, stefan; scholz, armin m.; deeg, cornelia a. title: a functionally different immune phenotype in cattle is associated with higher mastitis incidence date: 2018-12-06 journal: front immunol doi: 10.3389/fimmu.2018.02884 sha: doc_id: 3368 cord_uid: f8f2utzx a novel vaccine against bovine viral diarrhea (bvd) induced pathogenic antibody production in 5–10% of bvd-vaccinated cows. transfer of these antibodies via colostrum caused bovine neonatal pancytopenia (bnp) in calves, with a lethality rate of 90%. the exact immunological mechanisms behind the onset of bnp are not fully understood to date. to gain further insight into these mechanisms, we analyzed the immune proteome from alloreactive antibody producers (bnp cows) and non-responders. after in vitro stimulation of peripheral blood derived lymphocytes (pbl), we detected distinctly deviant expression levels of several master regulators of immune responses in bnp cells, pointing to a changed immune phenotype with severe dysregulation of immune response in bnp cows. interestingly, we also found this response pattern in 22% of non-bvd-vaccinated cows, indicating a genetic predisposition of this immune deviant (id) phenotype in cattle. we additionally analyzed the functional correlation of the id phenotype with 10 health parameters and 6 diseases in a retrospective study over 38 months. the significantly increased prevalence of mastitis among id cows emphasizes the clinical relevance of this deviant immune response and its potential impact on the ability to fight infections. vaccines are the most effective and also affordable disease-prevention tools (1) and maternal antibodies protect the offspring from infections in man (2) and animals (3) . in cattle, pre-partum vaccination with various virulence factors of bacterial or viral infectious diseases is a standard procedure which effectively stimulates the production of specific antibodies (4, 5) . a novel production technology using an allogeneic cell line (6) and the addition of a highly potent adjuvant to pregsure bvd, a new vaccine against bovine viral diarrhea (bvd), induced fatal immune reactions with production of alloreactive antibodies in 5-10% of vaccinated cows (7) (8) (9) . these alloantibodies were transmitted to calves, regardless of kin, via the colostrum of these dams (8, 10) and caused bovine neonatal pancytopenia (bnp) (7, (11) (12) (13) , even years after the vaccine has been taken off the market (14) . bnp calves suffered from hemorrhagic diathesis, thrombo and leukocytopenia and bone marrow depletion, which resulted in a deadly bleeding disorder (10, 15, 16) . several authors hypothesized alloimmune reactions against the major histocompatibility complex class i (mhc i) as causal for bnp (17) (18) (19) (20) . this hypothesis is still controversial since mhc i alloantibodies would be expected to bind all nucleated cells, not only to blood-derived and hematopoietic progenitor cells (12, 16) . also transcriptome analyses provided evidence that mhc i can be excluded as single causal agent for bnp-associated alloantibodies (21) . as other authors claimed the possibility of a genetic predisposition of bnp dams for production of bnp inducing alloantibodies (22) (23) (24) , our studies focused on investigating a general difference in immune responses between pregsure bvd vaccinated control cows and bnp donors. we wanted to analyze if there is evidence for differential expression of master transcription factors of cellular immune response in lymphocyte proteome of controls and bnp dams. interestingly, we found a markedly different immune phenotype in bnp dams, characterized by significant hyperproliferation of lymphocytes and a deviant immune signaling pathway compared to controls. we were next interested to see if we could find evidence for this immune phenotype in cows that were never vaccinated with pregsure bvd. further testing showed that this altered immune phenotype was also detectable in 22% of cows from a pregsure bvd unvaccinated cohort. this confirmed its natural, not vaccineinduced, appearance in cattle and gave more evidence for immune deviant (id) phenotype as a genetic predisposition. in further in-depth experiments we found that interleukin-2 (il-2) plays an important role in diverging immune response of id lymphocytes. in addition to these immune response studies, we analyzed the functional correlation of the id phenotype with overall 10 health parameters and 6 diseases in a retrospective study over 38 months. here, a significantly increased mastitis incidence in cows with an id phenotype became apparent, highlighting the clinical impact of altered immune response. in this study, peripheral blood derived lymphocytes (pbl) of a total of 132 cows were used. eleven of these cows had previously been vaccinated with pregsure bvd, of which six did not produce bnp-inducing antibodies (bvd vaccinated control cows) and five showed alloreactive antibody production (bnp dams) resulting in their calves dying from hemorrhagic diathesis with confirmed thrombocytopenia, leukocytopenia and bone marrow depletion. the remaining 121 cows were healthy controls (female, age: 2-10 years, first to eight lactation period) from one dairy farm never vaccinated with pregsure bvd. no experimental animals were killed through this study. withdrawal of blood was permitted by the local authority regierung von oberbayern, munich, permit no. 55.2-1-54-2532.3-22-12. bovine venous blood samples were collected in tubes with heparin sodium 25.000 i.u.. blood was diluted with equal parts staining of 5 × 10 5 cells per well was performed with anti-bovine cluster of differentiation (cd) 4 (mouse igg1 monoclonal, bio-rad abd serotec, puchheim, germany; 1:100), anti-bovine cd8 (mouse igg2a monoclonal, bio-rad abd serotec; 1:50) and fitc-conjugated anti-bovine igm (bio-rad abd serotec, puchheim, germany; 1:50) antibodies, diluted in staining buffer (1% bsa + 0,001% nan 3 in pbs). respective secondary antibodies anti-mouse igg1 fitc and anti-mouse igg2a fitc (both santa cruz biotechnology; heidelberg, germany 1:200) were added. all antibodies were incubated for 30 min on ice. cells were washed with staining buffer between primary and secondary antibody staining steps (200 × g, 4 • c, 1 min). for all stainings with secondary antibodies respective isotype controls were used. cells were fixed in 1% pfa diluted in staining buffer and stored at 4 • c until analysis. on facs canto ii, measurement of cells was performed with facs diva software (bd biosciences). lymphocytes were gated according to forward scatter (cell size) and side scatter (intercellular granularity) properties of cells. per staining, between 5 × 10 3 and 1 × 10 4 cells were measured. pbl (2.2 × 10 7 cells) of two pregsure bvd vaccinated control cows and two bnp dams were stimulated with pwm and cona (5 µg/ml) for 48 h. for lc-ms/ms analysis, peptides were separated on a reversed chromatography column (75 µm id × 15 cm, acclaim pepmap 100 c18. 100å/size, lc packings, thermo fisher scientific, bremen, germany) and the analysis was conducted with an ultimate 3000 nano-hplc system (dionex, idstein, germany). nano-hplc was connected in a linear quadruple ion trap-orbitrap (ltq orbitrap xl) mass spectrometer (thermo fisher scientific, bremen, germany). the mass spectrometer was operated in the data-dependent mode to automatically switch between orbitrap-ms and ltq-ms/ms acquisition. up to 10 most intense ions were selected for fragmentation on the linear ion trap using collision induced dissociation at a target value of 100 ions and subsequently dynamically excluded for 30 s. elevated spectra were imported into progenesis software (version 2.5). after comparison and normalization, spectra were exported as mascot generic files and searched against the ensembl bovine database (version 80) with mascot (matrix science, version 2.4.1). peptide assignment was reimported to progenesis software. all unique peptides allocated to a protein were considered for quantification. only proteins quantified with at least two peptides were included for further analysis. for hierarchical cluster analyses of normalized protein abundances, arithmetic means of two respective vaccinated control and bnp samples were generated and proteins with similar expression patterns were clustered with perseus software algorithm (version 1.6.1.1; computational systems biochemistry, martinsried, germany) (25) . proteins with a ratio of at least 2fold in normalized abundance between control and bnp samples were defined as differentially. for protein expression analyses with western blots, pbl were first lysed in lysis buffer (9 m urea, 2 m thiourea, 65 mm dithioerythritol, 4% chaps). proteins were then separated by sds-page on 8% gels (7 µg protein/slot) and blotted semidry onto 8.5 × 6 cm pvdf membranes (ge healthcare, freiburg, germany) and blocked with 4% bsa (1 h). blots were incubated overnight with respective primary antibodies: rabbit anti-pstat1 tyr701, rabbit anti-pstat3 tyr705 (cell signaling, darmstadt, germany, 1:500) or mouse anti-beta actin (sigma, taufkirchen, germany, 1:5000). as secondary antibodies, either hrp-coupled goat anti-rabbit igg (h+l) antibody (cell signaling, darmstadt, germany, 1:5000) or goat anti-mouse igg (h+l) antibody (sigma-aldrich, taufkirchen, germany, 1:5000) were used (1 h). signals were detected by enhanced chemiluminescence on xray film (super-2000g ortho, fuji; christiansen, planegg, germany). films were scanned on a transmission scanner and densitometric quantification of western blot signals was performed using imagej software (open source: http:// imagej.nih.gov/ij/). abundances of pstat1 and pstat3 were subsequently normalized to beta actin. milk production and health parameters (observed by veterinarians) of 100 non-pregsure bvd vaccinated cattle from one dairy farm were analyzed for 38 months retrospectively. results were then correlated to immune response data of in vitro assays. seventy-three control cows with low proliferation rate after cona stimulation and 27 id cows, which showed a bnp-like hyperproliferation to cona, were included in this study. ten health parameters [daily milk yield, average lactation performance (300 days), milk structure (lactose, fat, urea, somatic cell count), fertility parameters (amounts of inseminations, calving-to-conception intervals, medicinal induction of oestrus, ovarian cysts)] and 6 diseases [diseases of musculoskeletal system, claws, digestive tract, respiratory system, and metabolic disorders (ketosis, hypocalcemia)] were analyzed. all parameters were recorded and listed by the same veterinarians and the statistical analysis was performed using the odds ratio and chi-square distribution. data were analyzed in prism software (graphpad, version 5.04) with kolmogorow-smirnow (ks) test. if ks test was significant (p < 0.05; normal distribution), student's t-test was used for statistical analysis, if ks test was not significant (p > 0.05; no normal distribution) statistics were performed using mann-whitney test. for statistical analysis of health parameters, we used odds ratio and chi-square distribution. differences were considered statistically significant with the following p-values: * p < 0.05, * * p < 0.01, * * * p < 0.001, and * * * * p < 0.0001. after in vitro stimulation (48 h) with t and b cell mitogen pwm (26) and t cell mitogen cona (27), a clearly divergent reaction of lymphocytes from pregsure bvd vaccinated cows became evident. cells from cows known to produce alloreactive bnp antibodies after vaccination (bnp lymphocytes) proliferated significantly stronger (4.5-fold) than cells from vaccinated control dams after pwm stimulation ( figure 1a , bnp to ctr, * * * * p < 0.0001) and cona stimulation (8-fold stronger; figure 1b , bnp to ctr, * * * * p < 0.0001). thus, in vitro proliferation assays revealed a highly significant hyperproliferation of bnp lymphocytes demonstrating an increased reaction to polyclonal immune stimulation in these cows. to exclude differences in lymphocyte subset percentages between control and bnp dams as a possible cause for the differential responses toward polyclonal immune stimulation, we analyzed the distribution of cd4 + , cd8 + , and b cells in control and bnp pbl. there were no significant differences in proportional composition of subpopulations between pregsure bvd vaccinated control and bnp animals (figure 2 ). to investigate if observed deviant reactions to in vitro stimulation originated from differences in immune cell regulation, we executed a differential proteomics experiment with pwm and cona treated lymphocytes as well as unstimulated cells. overall, we detected 5,471 proteins. hierarchical cluster analysis of complete proteomes already visualized fundamental differences in protein abundances between pregsure bvd vaccinated control and bnp cows in constitutive protein tyr705 signals were normalized to beta-actin and quantified using image-j software. protein expression is shown in mean ± sd. for statistical analyses student's t test was performed. levels (ce, figures 3a,b) . differences in protein expression were also confirmed after immune stimulation (pwm and cona, figures 3a,b , hierarchical clustering of all identified proteins). these proteome analyses therefore revealed substantial quantitative differences on protein level. next, we used our dataset to perform more in-depth analyses on part of the proteome involved in transcription pathways of immune cell regulation. to identify possible differences in master transcription factor expression, we compared lymphocytes from vaccinated control and bnp cows after polyclonal in vitro stimulation. signal transducers and activators of transcription (stat) induce different t helper (th) responses in mice and man (24) as master transcription factors. therefor we analyzed all identified stats from both proteomic approaches and characterized usage of different stat pathways as a response to immune stimulation in both cow groups. after polyclonal activation of lymphocytes with pwm, stat3 and stat5a were selectively upregulated at least 2-fold in bnp lymphocytes ( figure 3c) . cona stimulation of control cells, however, caused upregulation of stat1 and stat6 expression levels (figure 3d) , whereas bnp pbl showed enhanced stat3 in immune response ( figure 3d) . thus, control and bnp pbl upregulated different master transcription factors as response to stimulation pointing to usage of different immune pathways. since we decided to further analyze the differential activation of stats through t cell mitogen cona, we determined figure 5 | stat3 inhibitor wp1066 selectively inhibits hyperproliferation of lymphocytes from bnp cows. stat3 inhibitor did not inhibit proliferation of control pbl (white bar, n = 4), but significantly inhibited cona stimulated bnp cells (black bars, n = 4, ***p < 0.001). percentage of inhibition rate shown in mean ± sd. student's t-test was used for statistical analyses. phosphorylation of stat1 and stat3 in response to immune stimulation. in lymphocytes of vaccinated controls, phosphorylation of stat1 tyr701 significantly increased after in vitro stimulation with cona compared to bnp pbl, where stat1 tyr701 phosphorylation decreased after cona stimulation (figures 4a,b , ctr to bnp after cona stimulation, * * p < 0.01). in contrast, lymphocytes of bnp dams phosphorylated stat3 tyr705 comparatively stronger than pbl of vaccinated control cows after cona (figures 4c,d , bnp to ctr after cona stimulation, * p < 0.05). thus, these experiments ascertained a qualitative difference in immune reactions between the cow groups to a t cell stimulus. since bnp lymphocytes responded to polyclonal stimulation with hyperproliferation and activation of stat3, we next tested if inhibition of stat3 reversed respective hyperproliferation. wp1066 (stat3 inhibitor) significantly reduced proliferation of bnp lymphocytes after cona stimulation (figure 5 , inhibition of bnp compared to ctr, * * * p < 0.001), but not of control lymphocytes. this verified the importance of stat3-pathway for the hyperproliferation in bnp pbl after polyclonal t cell stimulation. since we first detected the deviant immune phenotype in bnp cows, we next tested cows that were never vaccinated with pregsure bvd in order to clarify whether the deviant phenotype was induced through vaccination or if it occurs naturally in a certain subset of cows. therefore, we examined response of lymphocytes to different polyclonal stimuli in a large group of pregsure bvd unvaccinated cows. to exclude differences caused by environmental factors, cows all came from the same farm. in in vitro proliferation assays with t cell mitogen cona we observed that 22% of the unvaccinated cows reacted similar to bnp dams by showing a hyperproliferative reaction to cona (immune deviant (id) phenotype; figure 6a , reaction difference id to controls or bnp to controls, * * * * p < 0.0001). lymphocytes from id cows with a hyperproliferative immune phenotype did not show significant differences to lymphocytes of bnp cows in these assays (figure 6a , reaction difference bnp to id). these data confirmed the natural occurrence of this id phenotype in cattle and gave stronger evidence for genetic predisposition instead of vaccine-induced appearance. in order to further characterize the pathway targeted by the polyclonal activators pwm and cona we tested additional mitogens. the t cell mitogen banlec (28) , which activates human t cells through the il-2 pathway (29, 30), led to similar hyperproliferation of pbl from bnp dams as observed with cona ( figure 6a , reaction difference factor: 5.0, bnp to controls, * * * * p < 0.0001). pbl of id cows responded to banlec stimulation with a similar immune response intensity as bnp cows (figure 6b , reaction difference factor: 3.0, id compared to controls, * * * * p < 0.0001). the reaction of lymphocytes showed no significant differences between id and bnp cows. since the results with cona and banlec pointed to a response via il-2 pathway, we next tested stimulation with purified bovine il-2 only. after il-2 stimulation, lymphocytes of id cows also reacted excessively (figure 6c , reaction difference factor: 3.1, id to controls, * * * * p < 0.0001) and did not show significant differences to bnp lymphocytes ( figure 6c , reaction difference factor: 3.7, bnp to controls, * * * * p < 0.0001). finally, we tested further signature cytokines for different th immune responses, bovine interferon gamma (ifnγ; th1) and interleukin 4 (il-4; th2), to analyze differentiation of t helper subsets. we detected no significant differences between lymphocytes of vaccinated and unvaccinated controls and bnp cases after ifnγ or il-4 stimulation in vitro (figures 6d,e) . this proves a crucial role for il-2 in the deviant immune responses, but not for ifnγ or il-4. in this study, the proliferative phenotype of controls and id cows could be repeatedly tested during an overall observation period of 38 months. all of the sampled immune deviant cows showed hyperproliferative reaction to cona at least three figure 6 | immune deviant phenotype also detectable in unvaccinated cows. (a) in in vitro proliferation assays, 22% of unvaccinated cows showed a bnp-like hyperproliferative reaction to cona [****p < 0.0001 vs. ctr (white bar, n = 121)]. lymphocytes from id cows (gray bars, n = 27) with a hyperproliferative immune phenotype showed no significant (ns) differences to lymphocytes of bnp cows (black bars, n = 4, technical replicates n =86). (b) after banlec stimulation, id lymphocytes reacted hyperproliferative in comparison to control lymphocytes (****p < 0.0001) and the proliferation rates of these id lymphocytes did not show significant differences (ns) compared to bnp lymphocytes. (c) after il-2 stimulation, lymphocytes of id cows also reacted excessively (****p < 0.0001 vs. ctr) just like lymphocytes from bnp donors (ns). (d/e) after bovine ifnγ (d) and il-4 (e) stimulation, no significant differences between lymphocytes of unvaccinated and bnp cows were determined (ns). proliferation rate shown in mean ± sd. student's t-test was used. times. overall, two-thirds of the id animals consistently showed significant differences in their immune response compared to the unvaccinated control cows (figure 7a ). since we wanted to know if this newly discovered immune deviant phenotype related to impaired health parameters in respective cows, we retrospectively analyzed milk production, health parameters and different diseases of control and id cows from the same farm never vaccinated with pregsure bvd. from a total of 16 parameters tested, there was no difference in 15 parameters. however, mastitis incidence was significantly different between both cow groups. we detected that id cows suffered from mastitis twice as often as control cows (figure 7b , * p < 0.05). at the moment of blood sampling, all cows were clinically healthy and showed no signs of mastitis which could potentially influence adverse effects to immune stimulation. thus, this clearly indicates that the hyperproliferative phenotype of id cows has clinical relevance apart from the response to the bvd vaccine. the hyperproliferative phenotype of bnp cows (black) and id cows (gray) as well as the low proliferation of control cows (white) were consistently detectable over the entire observation period (38 months). three representative control cows and id cows are shown and two bnp cows, white symbols = controls, gray symbols = id, black symbols = bnp. (b) id cows (gray, n = 27) suffered twice as often (difference in incidence *p < 0.05) from mastitis than the control cows (white, n = 73). incidence of mastitis shown in percent. odds ratio and chi-square distribution was used for statistical analyses. bnp was the unwanted result of a fatal immune response to vaccination with pregsure bvd (8, 9) . five to ten percentage of vaccinated cattle produced disease inducing antibodies which were transferred via colostrum, killing 90% of receiving calves, regardless of kin (15, 31) . it is still unclear, why a certain subgroup of cows responded differently to the vaccination. we hypothesize that the origin of this altered immune response lies in the existence of a naturally occurring deviant immune phenotype. in this study, we could effectively confirm a quantitative difference in immune cell activation of bnp pbl through hyperproliferative responses in vitro (figures 1a,b) . a shift in lymphocyte subpopulations (cd4 + , cd8 + , b cells) as a potential reason for these differential responses toward polyclonal immune stimulation could be excluded (figure 2) . the hierarchical clustering of identified lymphocyte proteins from our shotgun proteomics experiment substantiated a general quantitative difference in constitutive expression of proteins in pbl of controls and bnp dams (ce, figures 3a,b) . after immune stimulation, these differences increased even further (pwm/ cona, figures 3a,b) . in order to perform more indepth investigations of potential functional differences, we subsequently focused on the analysis of master transcription factors of immune cell regulation from our dataset. as a result, we detected several differentially expressed stats. stats initiate the differentiation of various th cell subsets (24) as firstresponse master regulators and provide lineage specificity by promoting the differentiation of a given th cell subset while opposing the differentiation to alternative th cell subsets (32) . this makes them especially interesting to us, since expression of different stats in controls and bnp specimen indicates divergent immune response pathways in these groups. in control lymphocytes we identified stat1 with increased expression ( figure 3d ) and significantly higher activation (figures 4a,b) after t cell stimulation with cona. in mice and man, stat1 is important for the differentiation of naive cd4 + t cells to th1 cells (33) or to type 1 regulatory (tr) t cells (34) . from these findings, we conclude that after immune stimulation, bovine control lymphocytes use the stat1 pathway and we hypothesize that control cows react with a th1 or tr immune response in polyclonal stimulation assays. the effect of stat1 inhibition could not be tested, since we found no stat1 specific inhibitor that was commercially available. in contrast to controls, pbl proteome of bnp dams showed higher expression levels of stat3 ( figure 3d) , with increased phosphorylation (figures 4c,d) in bnp lymphocytes after immune stimulation. stat3 could therefore be a possible master regulator for the deviant immune response in bnp dams. in mouse and man, stat3 induces the development of follicular th (tfh), th17, and th22 cells (32, 35, 36) . our findings proved a stat3 pathway dependent immune response of bnp lymphocytes after polyclonal stimulation, but so far it is unknown which th subsets exactly are regulated by stat3 in cows. the dependence of the hyperproliferative phenotype of immune deviant pbl on stat3 was shown by inhibition of respective factor using wp 1,066, a cell permeable tyrphostin analog which blocks the stat3 pathway through inhibition of janus kinase 2 (jak2) protein tyrosine kinase (37) . as a result, we observed a significantly reduced proliferation to 37% in bnp pbl, but no effect in control pbl (figure 5 ). our findings therefore show, that the deviant immune response of bnp donors is associated with stat3/jak2 pathway. further studies will be needed to clarify whether immune cells from control cows differentiate to th1 or tr subsets and if bnp donor cows develop tfh, th17, or th22 cells after immune stimulation. hyper-reactivity of bnp pbl and the stat3 driven differentiation of tfh cells after immune stimulation are supported by findings of other research groups, describing higher alloantibody levels in bnp cows compared to pregsure bvd vaccinated control dams (22) . this group also hypothesized, that differences between bnp and control dams are likely due to genes controlling the quantitative alloantibody response after vaccination (22) . to find evidence for a genetic predisposition of this immune deviant bnp phenotype in cows (22, 23) , we additionally analyzed the immune phenotypes of 121 non-pregsure bvd vaccinated cows. in our study, 22% of cows (id cows) showed a bnp-like hyperproliferative response to t cell stimulation in vitro (id, figures 6a,b) . this percentage fits to the findings of benedictus et al., who hypothesized a heritability of 19% in dams for the development of bnp in the calf (22) . this proves the existence of an immune deviant phenotype among a certain subpopulation of cattle, which is not triggered by vaccination with pregsure bvd but occurs naturally. the immune deviant response described here in these id cows clearly depends on il-2 ( figure 6c) , which we could prove through hyperproliferative response of bnp and id lymphocytes to stimulation with banlec ( figure 6b) , a mitogen that activates human t cells (28, 30) via the il-2 pathway (29). for bovine t cells, the specific impact of il-2 itself was not described so far. in man and mice, however, il-2 has pleiotropic autocrine or paracrine functions that regulate proliferation of t cells (38) . it also plays a key role in promoting development, homeostasis and function of regulatory t cells though il-2/stat5 signals (39) and balances expression of different stats in th cells of mice (40) . therefore, the association of il-2 with the immune deviant response in cows is a very interesting finding which merits further in-depth investigations in future studies. to determine whether the newly detected deviant immune phenotype is of clinical relevance, we analyzed 10 clinical parameters and 6 diseases in 100 cows from the sampled dairy farm and compared incidence between controls (n = 73) and id animals (n = 27). interestingly, we found a significantly increased mastitis incidence in id cows compared to controls ( figure 7b ). this highly important correlation points to an altered immune reaction of id cows to classical mastitis pathogens, facilitating mastitis onset in these animals. in conclusion, we could prove the existence of an immune deviant phenotype in 22% of cattle, regardless of the vaccination status. the id phenotype shows altered reactions to immune stimulation identical to bnp dams, such as hyperproliferation of lymphocytes, stat3/jak2 regulated pathway and association with il-2. these features potentially triggered alloantigenproduction after maternal pregsure bvd vaccination, causing bnp. the correlation of the id phenotype with high mastitis incidence ( figure 7b ) underlines its clinical relevance. simply put: vaccination saves lives vaccines and pregnancy: past, present, and future pros and cons of vp1-specific maternal igg for the protection of enterovirus 71 infection vaccination of pregnant cows with espa, espb, gamma-intimin, and shiga toxin 2 proteins from escherichia coli o157:h7 induces high levels of specific colostral antibodies that are transferred to newborn calves serological, colostral and milk responses of cows vaccinated with a single dose of a combined vaccine against rotavirus, coronavirus and escherichia coli f5 (k99) the risks of using allogeneic cell lines for vaccine production: the example of bovine neonatal pancytopenia case control study to investigate risk factors for bovine neonatal pancytopenia (bnp) in young calves in southern germany bovine neonatal pancytopenia: is this alloimmune syndrome caused by vaccine-induced alloreactive antibodies? incidence of bovine neonatal pancytopenia in 243 farms in germany pancytopenia and haemorrhage in young beef calves calf-level factors associated with bovine neonatal pancytopeniaa multi-country case-control study effect of the vaccination scheme on pregsure(r) bvd induced alloreactivity and the incidence of bovine neonatal pancytopenia bovine neonatal pancytopeniacomparative proteomic characterization of two bvd vaccines and the producer cell surface proteome (mdbk) pregnancy boosts vaccine-induced bovine neonatal pancytopenia-associated alloantibodies ingestion of colostrum from specific cows induces bovine neonatal pancytopenia (bnp) in some calves demonstration of early functional compromise of bone marrow derived hematopoietic progenitor cells during bovine neonatal pancytopenia through in vitro culture of bone marrow biopsies vaccineinduced antibodies linked to bovine neonatal pancytopenia (bnp) recognize cattle major histocompatibility complex class i (mhc i) alloantibodies against mhc class i: a novel mechanism of neonatal pancytopenia linked to vaccination haematopoietic depletion in vaccine-induced neonatal pancytopenia depends on both the titre and specificity of alloantibody and levels of mhc i expression pathogenicity of bovine neonatal pancytopenia-associated vaccineinduced alloantibodies correlates with major histocompatibility complex class i expression a novel rnaseqassisted method for mhc class i genotyping in a non-model species applied to a lethal vaccination-induced alloimmune disease bovine neonatal pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the mhc haplotype bovine neonatal pancytopenia (bnp): novel insights into the incidence, vaccination-associated epidemiological factors and a potential genetic predisposition for clinical and subclinical cases a genetic predisposition for bovine neonatal pancytopenia is not due to mutations in coagulation factor xi the perseus computational platform for comprehensive analysis of (prote)omics data in vitro activation of human t and b lymphocytes by pokeweed mitogen mitogenic stimulation of t cells reveals differing contributions for b7-1 (cd80) and b7-2 (cd86) costimulation banana lectin: a brief review musa acuminata (del monte banana) lectin is a fructose-binding lectin with cytokine-inducing activity isolation and characterization of banlec-i, a mannoside-binding lectin from musa paradisiac (banana) sera from dams of calves with bovine neonatal pancytopenia contain alloimmune antibodies directed against calf leukocytes transcriptional and epigenetic regulation of t-helper lineage specification t-bet and gata3 orchestrate th1 and th2 differentiation through lineagespecific targeting of distal regulatory elements the competitive nature of signal transducer and activator of transcription complex formation drives phenotype switching of t cells genes associated with t helper 17 cell differentiation and function stat3 regulates cytotoxicity of human cd57 + cd4 + t cells in blood and lymphoid follicles wp1066 disrupts janus kinase-2 and induces caspase-dependent apoptosis in acute myelogenous leukemia cells the role of interleukin-2 during homeostasis and activation of the immune system interleukin-2 and stat5 in regulatory t cell development and function opposing regulation of the locus encoding il-17 through direct, reciprocal actions of stat3 and stat5 a functional different immune capacity in cattle is associated with higher mastitis incidence the authors thank sven reese for statistical analyses and roxane degroote for critical discussions. the manuscript was released as a pre-print at https://www.biorxiv.org/content/early/2018/05/13/ 311316 (41) . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-020770-wpub7krf authors: benmamar-badel, anouk; owens, trevor; wlodarczyk, agnieszka title: protective microglial subset in development, aging, and disease: lessons from transcriptomic studies date: 2020-04-03 journal: front immunol doi: 10.3389/fimmu.2020.00430 sha: doc_id: 20770 cord_uid: wpub7krf microglial heterogeneity has been the topic of much discussion in the scientific community. elucidation of their plasticity and adaptability to disease states triggered early efforts to characterize microglial subsets. over time, their phenotypes, and later on their homeostatic signature, were revealed, through the use of increasingly advanced transcriptomic techniques. recently, an increasing number of these “microglial signatures” have been reported in various homeostatic and disease contexts. remarkably, many of these states show similar overlapping microglial gene expression patterns, both in homeostasis and in disease or injury. in this review, we integrate information from these studies, and we propose a unique subset, for which we introduce a core signature, based on our own research and reports from the literature. we describe that this subset is found in development and in normal aging as well as in diverse diseases. we discuss the functions of this subset as well as how it is induced. the term "microglia" was brought to the scientific community's attention a century ago with its first use by pio del rio-hortega (1), who strived to distinguish them from oligodendrocytes. his early work also highlighted their phagocytic ability, as well as their potential to undergo morphological changes. this early description led the community to consider microglial cells as a homogeneous population, even though the first description of a microglial subset ("satellite microglia") appeared as early as 1919 (1) . microglia originate from yolk-sac progenitors that start migrating toward the fetus around midpregnancy. these progenitors reach the embryonic brain around embryonic day (e) 9.5-e10.5 (2, 3) until the formation of the blood-brain barrier around e13.5-e14.5 in the mouse, and between the 4th gestational week to the 24th gestational week in the human (4, 5) . as such, they are among the first cells to colonize the developing brain, and they participate in central nervous system (cns) development. for instance, they contribute to refine brain wiring through enhancing both synapse formation (6, 7) and elimination (8, 9) , they modulate axonal growth (10, 11) , they secrete factors promoting neuronal progenitors survival (12) helping with neuronal positioning (11, 13) , and they participate in the clearance of live and apoptotic cells during development (14) . microglia also take on physiological functions in the adult cns, as they constantly sense their immediate environment, in a so-called "never-resting state" (15, 16) . our knowledge of microglial physiology and process motility relies heavily on studies in anesthetized animals. understanding of microglial functions in the steady state is challenged by a recent study showing that microglial process motility and morphology are affected by the wakefulness state of mice (17) . aside from this surveillance immune function, they are also fundamental for regulation of social behavior, learning, and memory, as these functions are impaired upon their depletion and restored after repopulation (18) . microglial roles in injury and disease contexts have been investigated extensively, with new advances contributing to deepen our understanding of microglia and their effect on other glial cells [reviewed in greenhalgh et al. (19) ]. these physiological functions advanced our view of microglia, from being initially thought of as exclusively sentinel cells reacting in the context of injury. this dated view on microglia led to the superposition of macrophage m1/m2 phenotypes onto them (20), which was an early attempt to grasp the extent of microglial diversity. this classification is however mostly obsolete nowadays, as it was proved to be simplistic and disconnected from in vivo reality (21) . indeed, the variety of functions microglia take on in space, time, and health states along with reports of sex differences in microglial function have led the community to infer a greater microglial heterogeneity than initially thought. with the progress of technology, investigating such diversity has become possible, notably through the development of high-throughput techniques such as mass cytometry and with the recent advances in transcriptomic studies with single-cell rna-sequencing (rnaseq). these technologies allowed the identification of microglial signatures linked to their "activation state." in 2014, butovsky et al. described a "homeostatic" microglial signature, comparing microglia with monocytic populations and other cns cells (22) . this signature includes genes such as p2ry12, fclrs, tmem119, hexb, mertk, cx3cr1, csf1r, etc. that have been used in numerous studies thereafter to identify microglial cells. this was a fundamental step in distinguishing resident microglia from other tissue-resident macrophages and infiltrates in disease context. this "homeostatic" signature was more recently revised and extended to developmental stages in addition to adulthood by matcovitch-natan et al. (23) . in this study, single-cell rna-seq helped associate the microglial signature identified at each different age to the potential functions these cells take on during life. they pinpointed three different temporal stages of development, each linked to a particular signature: early microglia associated with proliferation and differentiation, pre-microglia related to neuronal development, and adult microglia. it has recently been suggested that microglial heterogeneity peaks early during development and then reaches a minimum in the homeostatic adult brain, only to regain diversity in old age (24) . in addition, some microglial subtypes have been based on surface markers and sometimes function [discussed in stratoulias et al. (25) ]. this has been mostly achieved through systematic transcriptional investigation of microglia in different contexts. however, because every study is done with different techniques (microarrays, bulk rna-seq, single-cell rna-seq, etc.), on different kinds of samples (whole brain, sorted microglia based on different gating strategies, microdissected microglia, sorted nuclei, etc.), and in different animal models, there is a risk for confusion of data. we believe that there is a need for an overview-by looking at the big picture, common patterns can be identified between studies that might otherwise have been overlooked. in this review, we summarize and interpret transcriptomic studies on microglia from development, homeostasis, and disease states to bring to light a subpopulation common to all these different states. we discuss the factors inducing this subpopulation and its functional importance in all of the studied conditions. finally, we provide a core signature for this subset and propose to systematize and unify the naming of this microglial subpopulation to clarify the literature and avoid redundancy in future studies. we propose to use a name already used in numerous studies and that accounts for these cells' expression signature: cd11c+ microglia. for long, microglia have been considered simply as macrophages, due to the belief that all macrophages emerged from the bone marrow. consensus that a subset of microglia expressed cd11c was therefore at first difficult to achieve. cd11c was widely accepted as a marker for dendritic cells (dcs), to the extent that some studies have used it as the sole identifier for dcs. added to this was the constant difficulty of discriminating cnsresident parenchymal microglia from blood-derived myeloid cells, with which they share many markers [reviewed in amici et al. (26) ]. until recently, it was indeed not possible to reliably discriminate microglia, especially activated microglia, from blood-derived monocytic myeloid cells, using morphology or routine myeloid markers. panels of differentially expressed genes that can be used to distinguish microglia including tmem119 (27) and the homeostatic marker p2ry12 (22) were however recently identified and validated in both homeostatic and disease conditions (28) . to our knowledge, the first observation of microglia expressing cd11c was made in human multiple sclerosis (ms) tissue by immunohistochemical analysis (29) . one, however, cannot be completely certain of the exclusive microglial nature of the cells identified in this study based on the markers used and our current knowledge of myeloid cell marker expression patterns. the first report to explicitly identify cd11c+ cells in the cns as microglia came from butovsky et al. in 2006 (30) . they identified populations of cd11c+ cells in a mouse model for alzheimer's disease (ad) as microglia, based on their location and co-expression of isolectin b4 and cd11b, although these cells showed a dendritic morphology. the major point of interest in that study was the observation that all mhc-ii+ microglia that engulfed amyloid β in the brain of glatiramer acetate (ga)vaccinated transgenic (tg)-ad mice co-expressed cd11c. also, relevant to our subsequent studies, these cells could be stained with an antibody specific for insulin-like growth factor 1 (igf1). a "gold standard" for microglial identification remains their relatively low level of expression of cd45 in flow cytometry analyses (31) . in the course of study of glial responses in the dentate gyrus to axonal transection in the entorhinal cortex (the perforant path lesion model), we noted a subpopulation of cd45 low cd11b+ cd11c+ cells in flow-cytometry profiles of cells isolated from lesion-reactive hippocampus. their functional significance and whether they derived intraparenchymally or by immigration from bone marrow were not determined (babcock and owens, unpublished). exactly similar cells were then observed in cuprizone-demyelinated corpus callosum (32, 33) . these were described to express slightly higher levels of cd45 than their cd11c− counterparts, while remaining within the cd45 low gate (33, 34) . in addition, they did not express ccr2 characteristic for infiltrating leukocytes and expressed high levels of cx3cr1 supporting their microglial status (33) . further analysis showed that cd11c+ microglia were also induced in experimental autoimmune encephalomyelitis (eae) (33) (34) (35) ) and a mouse model for neuromyelitis optica (nmo) (33) , as well as during postnatal development (24, (35) (36) (37) . in older studies, ambiguity in assigning cd45 levels resulted in cd11b+ cd11c+ populations in cns of mice with eae or infected with toxoplasma gondii being identified as dcs (38) , although, with hindsight, consideration of bimodal cd45 profiles allows that at least some of them may have been microglia. the fact that cd11c+ microglia express slightly higher cd45 levels than resting microglia may have contributed to uncertainty, and claims that dcs derived from microglia (38, 39) may need re-evaluation. relative cd45 levels as detected by flow cytometry are not as useful for histological discrimination. depending on the antibodies and staining protocols used, microglia may even not be detected as cd45+ cells, or else cannot be distinguished from other cd45 hi cells. similarly, cd11c promoter-driven fluorescent reporter transgenic mice cannot discriminate between the many cell types that can express or upregulate cd11c without co-staining for lineage-specific markers. identification of cd11c+ microglia in such mice relies on interpretation of sometimes fortuitous observations that include consideration of a cell's morphology and location. using an eyfp-cd11c transgenic strain, bulloch et al. identified a small fraction of cd11c+ microglia that were immunoreactive for mac-1, iba1, cd45, and f4/80 (40) . the parenchymal juxtavascular iba1+ cd11b+ gfp-cd11c+ cells described by prodinger et al. in a cd11c-gfp reporter mouse likely included microglia, although in a non-diseased mouse, they would only account for around 2% of them (41) . flow-cytometric analysis confirmed cd45 low gfp-cd11c+ cells in the cns of these mice (42) . the fact that they were mhc ii-negative likely reflects that they derived from non-diseased tissue, unlike the eae-derived cells that we described (34) . typical microglia markers and their functions are listed in table 1 . even before microglia were formally identified, the presence of fat-laden cells had been reported and suggested to be a part of (49) the normal developing cns (50) (51) (52) , and to participate in either cell death processes (53) or myelin formation (54) (55) (56) . early after the initial description of microglial cells, neuroanatomists began to track and map microglia in the cns. del rio-hortega was the first to describe "fountains of microglia" in the developing brain, having amoeboid morphology and being preferentially located in the white matter (57) . already in 1925, penfield reported that what he describes as "neuroglia of mesodermal origin" "were variously considered to be normal and having to do with myelination or to indicate an abnormal inflammatory process" (58) . in the mid-to late 1970s, with del rio-hortega's "fountains of microglia" in mind, these cells were investigated again using light and electron microscopy. most studies describe round, amoeboid, highly vacuolated cells with fat-containing granules, which are found in developing white matter, particularly along unmyelinated axonal tracts in the corpus callosum of rabbits (59) , rats (60), mice (61) , birds (62) , fish (63) , and humans (64) , as opposed to more highly ramified cells present in the gray matter. in all these studies, amoeboid or ovoid-shaped microglia invade the white matter before disappearing when increasing numbers of ramified microglia colonize the gray matter (peaking around postanatal day (p) 5 and disappearing around p10 to p15 in rodents). multiple studies support this finding and extrapolate their potential function, stating either that they have enhanced phagocytic abilities for the elimination of apoptotic material coming from normal developmental cell death or that they participate in myelination (59, 60, (65) (66) (67) (68) . this involvement in myelination was reinforced by a study by pont-lezica et al. showing that microglial alteration early in development leads to impaired corpus callosum fasciculation (11) . their phagocytic abilities along with their morphology provoked debates regarding their origin (68) , their fate (66), and even their microglia status with some studies modifying the nomenclature by referring to them as "brain macrophages" rather than "amoeboid microglia" (67, 68) . with the new notion of microglial phenotypes emerging, these early amoeboid microglia were hypothesized to have higher "activation" levels before becoming "deactivated" in a controlled manner, as this was believed to be temporarily helpful to scavenge debris coming from developmental cellular death. to corroborate this hypothesis, hristova et al. attempted the first phenotypic analysis of these cells, and reported expression of high levels of integrins alpha x (itgax, cd11c), alpha 4 (itga4), alpha 5 (itga5), and beta 2 (itgb2) in microglia from periventricular white matter in comparison to cortical microglia at p7 by staining quantification in iba1+ cells (37) . in addition, in situ hybridization clearly showed transient igf1 and colonystimulating factor 1 (csf1) mrna expression within microglial cells in the corpus callosum and periventricular white matter until approximately two postnatal weeks (37) . in this study, expression of igf1 and csf1 by microglia were hypothesized to play a protective role, preventing axonal damage for instance, which has since then been confirmed in a study by ueno et al. (12) . this finding was reinforced by our own study showing that microglial cells expressing high levels of itgax and igf1 are present in the white matter (cerebellum and corpus callosum) of developing mouse brains particularly between p3 and p5 where they make up almost 20% of all microglia and decrease in numbers already at p7 before being almost completely undetectable by p28 (35) . presence of igf1-expressing microglia in these locations in p5 brains was further confirmed by in situ hybridization (69) . we performed rna-seq on these cells between p3 and p5 after facs-sorting based on cd45 dim cd11b+ cd11c+ gating comparing them to their cd11c− counterparts. we identified a robust neurodevelopmental gene signature for developmental cd11c+ microglia, including factors involved in astrocyte and neuronal differentiation, tissue remodeling, and myelinogenesis accompanied by downregulation of immune function-related genes. of note, itgax, itga4, csf1, and igf1, which were highlighted in the hristova study, were also part of this signature. importantly, we demonstrated that igf1 expression by cd11c+ microglia during development is crucial for primary myelination. indeed, selective deletion of igf1 specifically from cd11c+ cells led to myelination defects in p21 brains (35) . interestingly, all neonatal microglia expressed neuroectodermal genes including nestin. a concomitant study by hagemeyer et al. similarly identified amoeboid microglia in the developing white matter of the corpus callosum and cerebellum particularly between p1 and p8 before being almost undetectable by p14 (70) . interestingly, they used a mac-3 staining to identify these cells, reminiscent of a study by valentino and jones who reported mac-3 expression in "fountain microglia" in a footnote (68) . they identified a signature akin to the one we found (38 genes in common out of 61 upregulated genes including itgax, csf1, and igf1) by comparing "fountain microglia" from corpus callosum at p7 with cortical microglia at the same age by whole-genome microarray (70) . of note, the study underscores that many of the most upregulated genes were related to a primed or activated microglial phenotype and they confirmed cd11c expression in the "fountain of microglia" cells with a reporter mouse. in addition, by depleting all microglia during the critical period of the first postnatal week, they showed that the number of oligodendrocyte progenitor cells was reduced and a long-lasting effect on myelination was induced into adulthood (70) , in line with our own results. two recent studies used single-cell rna-seq to elucidate microglial heterogeneity during development (24, 36) . the barres lab study used deep single-cell rna-seq on microglial cells sorted based on cd11b+ gating and cd45 levels from six different brain regions at e14.5, p7, and p60 (24). they found a cluster of cells they named "proliferative region-associated microglia" (pam), mainly found at p7 in the white matter, that have an amoeboid morphology and phagocytose newly formed oligodendrocytes (24) . in addition, they reported enhanced expression of igf1 and itgax in this cluster compared to any other at p7 or other time points. these cells were observed as early as e17.5 in the embryonic brain, their numbers peaking around p7 and were almost absent from p14 brains (24) . all these features fit with cd11c+ microglia from our study and the historical "fountain of microglia" cells. the stevens lab used high-throughput rna-seq on microglial cells from the whole brain sorted based on a cd45 dim cd11b hi cx3cr1 hi gating at e14.5, p4-5, p30, p100, and p540 and in injury contexts, prioritizing high numbers of cells over depth of sequencing (36) . they identified a cluster of cells exclusive for the p4-5 time point, which have an amoeboid morphology, express phagocytosis-related genes, and are restricted to the corpus callosum and cerebellum, associating closely with axonal tracts, which they named "axon tractassociated microglia" (atm) (36) . again, the features of this subset resembled closely the features of cd11c+ microglia and "fountain of microglia" cells described above. interestingly, their study showed no evidence for a sex bias, the number of cells associated to this cluster being similar for neonatal female and male pups (36) . in addition, anderson et al. (71) described gene signatures of retinal microglia in p7 mice, 60% of which were found to express cd11c. the microglial signature in the p7 retina fit the signature associated to developmental cd11c+ microglia as itgax, lpl, clec7a, and igf1 were enriched in sorted cd11c hi vs. cd11c low cells at p7, whereas p2ry12 and tmem119 were downregulated (71) . we therefore hypothesize that cd11c+ microglia, fountains of microglia, pams, and atms, although described in different studies by different methods under different names, actually represent the same population of cells. comparison of the transcriptomic signature found in each of these studies leads to a core signature of 11 genes found in all four studies (gpnmb, itgax, spp1, fam20c, fabp5, hpse, igf1, folr2, csf1, and anxa5) and 28 additional genes found in at least three of these studies (atp6v0d2, slpi, cd28, crip1, lgals1, anxa2, vat1, ifitm2, gm1673, plaur, s100a1, colec12, clec7a, atf3, atp1a3, ephx1, nceh1, lpl, pld3, plin2, aplp2, ccl3, bnip3, ccl9, gpx3, slc16a3, lag3, and lilrb4) (figure 1) . interestingly, csf1, one of the genes of the core signature, has been identified as one of the prominent genes characteristic of the pre-microglia homeostatic signature (23) . these 39 genes constitute the "developmental signature" of the microglial population described in this section. of note, homeostatic microglia markers, such as tmem119, p2ry12, sall1, tgfbr1, fcrls, and cx3cr1, have been shown to be expressed by this subset, although in most reports at slightly lower levels than in adult microglia or other neonatal microglia (24, 35, 36, 70) . later in this review, we will refer to this population as "developmental cd11c+ microglia". features of this population include peak numbers between p3 and p7, amoeboid morphology, phagocytic abilities, and location in white matter (figure 1 ). in addition, studies mentioned in this section clearly reveal a critical functional role of developmental cd11c+ microglia in the myelination process. their presence in high numbers in the white matter makes them strategically placed in both space and time to take on that role. the aforementioned data support their involvement in phagocytosis of newly formed oligodendrocytes, probably linked to the proper establishment of primary myelination (24, 35, 36, 70) . two of the studies show the long-term importance of these cells on oligodendrocytes and myelination later in life (35, 70) . although the number of common genes in the developmental signature might appear low, we would argue that this is probably due to discrepancies in the transcriptomic techniques used (microarray, bulk rna-seq, high-throughput single-cell rna-seq, deep single-cell rna-seq), as well as the isolation techniques used (facs-sorting based on various gatings, presence or absence of perfusion, whole brain dissection, or region microdissection) (see table 2 ) [discussed in (76) ]. however, similarities in the localization, colonization kinetics, morphology, and functional role leave little room for doubt regarding the uniqueness of the population described. recent studies have described the homeostatic adult brain as the state with lowest microglial heterogeneity (24) . in addition, most high-throughput studies investigating adult microglia in steady state generally report very homogeneous populations in the homeostatic clusters, whether by mass cytometry (77) or single-cell rna-seq (36) , characterized by robust expression of classical microglial homeostatic markers. however, in a cd11c-eyfp reporter mouse, yfp-expressing cells have been found throughout the brain and retina in adulthood. although initially thought to be dcs (40), they have since then been shown to exhibit a phenotype resembling microglia (41, 78) . interestingly, a particular abundance of these cells is found in ventral areas of the brain, white matter tracts, and areas of adult neurogenesis (78) . this is in line with a report that clec7a+ microglia are found in neurogenic niches in the adult mouse (24) , showing that in the homeostatic adult brain, microglia with a phenotype similar to developmental cd11c+ microglia could remain in low number in selected areas. consistent with this, a subset of microglia (also positive for tmem119 and p2ry12) expressing higher levels of cd11c was found in the human subventricular zone and thalamus (79) . in reporter mice, expression of cd11c has been shown to not always follow the expression of the yfp reporter and should therefore be taken cautiously (78) . the existence of cd11c-expressing microglia has however been confirmed in the adult homeostatic brain (around 2% of total microglia) (33-35, 42, 80, 81) . similarly, a small population of cells from the choroid plexus of adult mice was shown to be transcriptionally distinct from other choroid plexus cells and border-associated brain macrophages. this population named "kolmer's epiplexus cells" closely resembles microglial cells and was associated with enriched expression of spp1, apoe, and igf1 (82). although itgax was not among the significantly upregulated genes in this study, cd11c+ cells expressing low levels of cd45 have previously been described in the choroid plexus of adult mice (78) . change in microglial gene expression and phenotype in steadystate aging has been studied extensively. although reports agree on the changes in morphology and general phenotype of microglia toward dystrophic microglia (deramification, cytorrhexis, and fragmentation) in aging [reviewed in (83) ], genomic studies have given discrepant results, with some arguing for shift toward neuroprotection (84) and others highlighting a "primed phenotype" with higher immune activation (85) . that said, having a second look at datasets from various studies brings to light common highly expressed genes in aged microglia compared to young microglia: spp1, clec7a, igf1, lpl, axl, apoe, lgals3, itgax, cst7, etc. are indeed found across several studies (84) (85) (86) , although not all and not always in the same range of upregulation (87) . in a later study, holtman et al. related the "primed" microglial signature they found from two aging models (one physiological aging model and one accelerated aging model) to the study by hickman et al. and found a high correlation between the datasets (88) . high-throughput single-cell methods are a good way to decipher complex populations with mixed subsets. a masscytometry study revealed that a specific subset of microglia emerges during aging that overexpresses surface cd11c and cd14, clec7a, and cd68 as compared to other microglia figure 1 | cd11c+ microglia signature in developmental stages. during development, cd11c+ microglia have an amoeboid morphology and localize close to white matter tracts, essentially in the corpus callous and cerebellum. they are present early during embryonic development and their numbers peak between p3 and p7. comparison of genes upregulated in four studies (24, 35, 36, 70 ) reveals a common signature for developmental cd11c+ microglia of 39 genes upregulated in at least three of the studies (bold dark outline). genes shared with the disease signature in figure 2 are in bold. the venn diagram was generated using the online tool venny (72). at the same age, although they downregulate cx3cr1 and mertk (77, 89) . cd11c expression of microglia in the white matter and caudal areas of the cns of aged mice was also shown using immunohistochemistry (90) . this study also reports expression of clec7a in white matter tracts of aged animals and reports numerous changes in white matter microglia associated with aging. similarly, single-cell rna-seq revealed that several populations of microglia that were present in younger age at very low numbers become increasingly prevalent with aging. one of these populations (referred to as oa2) is characterized by genes from the developmental signature and genes classically associated with neurodegeneration (spp1, lpl, lgals3, lilrb4, (98) , possibly indicating the presence of cd11c+-microglia-like cells in the repopulating clusters they described. this is reinforced by our study in which cd11c+ microglia could be found in repopulating microglia clusters after genetic microglial depletion (35) . however, in contrast to zhan et al. our analysis did not show neonatal-like, neurodevelopmental gene signature in repopulated microglia (35) . the low extent of overlap between these studies and our newly defined neonatal cd11c+ microglia could be explained by heterogeneity of repopulating microglia, diluting the signal from cd11c+ microglia in bulk rna-seq studies. microglia activation is a common feature in many neurological disorders including inflammatory, demyelinating, and degenerative diseases, as well as glioma and injury. although microglia activation may have deleterious consequences, it has also been shown in many instances to exert protective and regenerative effects. it is now becoming clear that there is an emergence of cd11c+ microglia population in pathological conditions. in this section, we will discuss the importance and the role of this cell subset in several neurological diseases. for decades, it has been known that microglia localize around aβ plaques, and engulf aβ in ad, showing their importance in the disease. in recent years, interest in these cells has increased, largely due to a wave of transcriptomic and genome-wide association (gwas) studies. in addition, a majority of ad risk genes are related to microglia, including triggering receptor induced on myeloid cells 2 (trem2) [reviewed in mcquade and blurton-jones (105)]. despite the enormous amount of data generated, no consensus has yet been reached on whether microglia are protective or detrimental in neurodegeneration. some of the attempts to resolve this issue involved comparing transcriptomes of microglia sorted from healthy, aged, and diseased brains. the study by holtman et al. cited above identified a microglial signature found not only in aging models but also in disease models including the app/ps1 ad model and the sod1 model for amyotrophic lateral sclerosis (als) (88) . the common genes included itgax, clec7a, axl, lgals3, and apoe, indicating the presence of a cd11c-expressing microglial population in these models. the gene module described in this study mostly contained genes related to phagocytosis and cell proliferation, with tissue protective elements (88) . with a similar strategy, other studies demonstrated that microglia from aging brains and from amyloidosis (app/ps1) and tauopathy (aav-tau p301l) shared a common gene signature including cst7, itgax, gpnmb, clec7a, lpl, lgals3, apoe, and spp1 (86). similar results were also obtained by krasemann et al. in the app/ps1 model. such shared microglial characteristics led to the term "microglial neurodegenerative phenotype (mgnd) signature" (75) . this is also in line with the presence of cd11c-expressing microglia in these models, with a phenotype similar to the one found in physiological aging. the presence of cd11c+ microglia around aβ plaques has been shown in several studies (30, 73, 74, 106, 107) . a recent study by kamphuis et al. extensively investigated the localization, proliferation status, and transcriptome of cd11c+ vs. cd11c− microglia in app/ps1 mice (73) . importantly, this study also highlighted a steady increase in cd11c transcripts in brains of app/ps1 and 3xtg-ad mice with aging as plaques appear, as well as in hippocampal samples from ad patients, although it declines in the later stages of the disease (73) . the transcriptomic signature of cd11c+ microglia, when compared to their cd11c− counterparts, showed increased expression of gpnmb, fabp5, spp1, igf1, itgax, gm1673, cst7, cox6a2, apoe, ch25h, clec7a, lilrb4, csf1, axl, lpl, sulf2, egr2, anxa5, cd68, timp2, and ctsb among others. many of these genes are common with the developmental signature of cd11c+ microglia described above or with the signatures found in whole brain "primed" microglial signatures (73) . these findings further support that the "primed" microglia phenotype described in many studies recapitulates the cd11c+ microglia signature diluted among cd11c− counterparts. the robustness of the signature is hardly surprising, considering that cd11c+ microglia make up for 23% of all iba1+ cells in the aged app/ps1 brain (73) . of note, strong upregulation of some cd11c+ microglia signature genes, including itgax, clec7a, and cst7, was even detectable in whole tissue samples from cortex and hippocampus in ad models (73, 108) . high-throughput single-cell studies also contributed to our understanding of microglial populations in ad rodent models. the same study that identified cd11c and cd14 surface expression by mass cytometry on a microglia population emerging in aging also identified a similar population in app/ps1 brains (77) . single-cell rna-seq studies identified three microglial signatures in neurodegeneration models: the disease-associated microglia (dam) signature (74), the late response microglia signature (109) , and the activated response microglia (arm) signature (80) that emerge in the 5xfad, ck-p25, and app nl−g−f models for ad, respectively. all three studies described cell clusters showing nearly identical microglia populations, similar to the cd11c+ microglia signature observed in the kamphuis study. importantly, all of the dam cells were cd11c+ (74) with highly overlapping gene signatures uncovered by bulk sequencing of sorted cd11c+ microglia (73) . microglia with characteristics from the arm cluster are present in low numbers (ca. 2%) even in wild-type mice at young age, increasing as part of normal aging to reach up to about 12% of all microglia (80), consistent with observations discussed above of cd11c+ microglia in the steady state in adult and aging mice. arm microglia are however most evident in app nl−g−f mice where they outnumber all other microglial clusters reaching 52% of all microglia at 21 months of age (80) . this is in line with increases in cd11c+ microglia reported in other studies. importantly, the signature observed in cd11c+/dam/mgnd/arm microglia is enriched for known ad risk genes (80) . of note, this transcriptomic signature is similar to that induced by retinal degeneration (110) . cd11c+ microglia have been demonstrated to be beneficial for and to correlate with increased aβ uptake and induction of igf1-mediated neurogenesis in an animal model of ad (30) . in addition, abundance of igf1-expressing microglia around aβ plaques was recently confirmed by in situ hybridization in an ad model (69) . functional analyses led to discrepant results suggesting either protective, immunosuppressive function as well as enhanced capacity for uptake and lysosomal degradation of aβ (73) , or pathogenicity via possible contribution to local arginine deprivation and subsequent neurodegeneration (111) . butovsky's group also proposed a detrimental role for these cells due to ameliorated aβ deposition in 4-month-old trem2-deficient mice that lack cd11c+ microglia (75) . however, the role of trem2 is not clear, since other data show either protective or detrimental roles for this protein depending on the age of the animals (75, (112) (113) (114) . nonetheless, all these studies demonstrate lack of microglial proliferation and clustering around plaques in trem2-deficient animals, thus allowing for more dispersed aβ localization in ad models (75, (112) (113) (114) (115) (116) . this can be detrimental due to aβ spreading that is not limited by microglia clusters, ultimately leading to severe axonal dystrophy (114) . moreover, it has been demonstrated that in trem2-deficient animals older than 8 months, the aβ burden is enhanced as compared to 4-month-old animals, suggesting that trem2 signaling is necessary for limiting advanced stage pathology (117) . thus, cd11c+ microglia may actually be beneficial and protective in later stages of the disease as proposed by keren-shaul et al. (74) . human data further support this hypothesis since loss-of-function mutations in trem2 have been identified as a strong risk factor for the development of ad and other neurodegenerative diseases [reviewed in mcquade and blurton-jones and ulland and colonna (105, 118) ]. collectively, cd11c+ microglia (also referred to as primed microglia, late response microglia, dam, arm, or mgnd) are a well-defined population of cells that show adaptation predominantly for phagocytic clearance of apoptotic/necrotic neurons and limiting aβ spreading. given that ad risk genes are enriched in this population (80) , mutations in such genes may have an impact on the ability of cd11c+ microglia to cope with aβ plaque burden, either promoting or limiting ad pathology. als is a disease affecting motor neurons leading to their degeneration. microglial contribution to the disease has been established since a robust microglial activation has been found in both patient and transgenic mouse tissue (119, 120) . in addition, many risk factors for the disease have been shown to be expressed by microglia in the cns, reinforcing the idea of an involvement of these cells in the disease (121) . microglial activation in the disease arises from accumulation of misfolded protein, and, similarly to observations made in other disease contexts, microglia have been reported to play a beneficial role in the pre-symptomatic phase of the disease before shifting to detrimental roles in the advanced disease state (122) . however, microglial depletion in the context of als has not been found to increase survival (123) , leading to the idea that both functions might be concomitant, constantly counteracting each other. interestingly, a study from 2013 analyzed the transcriptome of microglia sorted from mice carrying an als-associated mutation and found a particular signature for these cells at the end stage of the disease compared to microglia from healthy brains (124) . once again, among the top regulated genes were genes related to huntington's disease, ad, and parkinson's disease (mapt, psen2, apoe, etc.). the signature found in this study includes both factors reported to be beneficial in the context of als (igf1, grn, trem2, tyrobp, etc.), and factors known to be detrimental (mmp12, optn, cybb, etc.), as well as some like spp1, gpnmb, and itgax recurrently found in neurodegenerative diseases. microglia were also found to upregulate surface cd11c. microglia from sod1 mice were also found to fit the abovementioned mgnd signature, in addition to expressing clec7a levels increasingly during disease progression (75) . neuron degeneration and nerve injury have been linked to microglia in various models for traumatic brain injury (tbi) (125) , spinal cord injury (sci) (126) , nerve injury (93) , and ischemic stroke (127) . much like in inflammation models, microglial contribution in all of these models is still rather unclear and they may play a double role considering their association with both beneficial and detrimental effects. studying microglia in context of inflammation can get quite complicated due to massive infiltration of peripheral immune cells, notably monocytes and macrophages, occurring subsequently to tbi (128) , sci (129) , and stroke (127, 130, 131) . in a study comparing the transcriptomics of microglia and macrophages after ischemia in rats, it was reported that microglia played a detrimental role and macrophages played a beneficial role with regard to recovery, based on their expression of classical inflammation markers (132) . investigation of the genes enriched in microglia three days after middle cerebral artery occlusion compared to sham controls, however, revealed spp1, gpnmb, lgals3, fabp5, and axl among others, fitting with the potential presence of cd11c+ microglia-like cells in this context, diluted among other microglia. consistent with this, ccl2 mrna was found to be increased in microglia and macrophages at this time point (132) , an aspect that has been associated with the emergence of cd11c+ microglia (81) . another study, conducted in a model of phototrombic stroke on whole tissue, actually showed upregulation of gpnmb, itgax, and clec7a in a cluster associated with early response (133) , which the authors related to the dam phenotype (74) . in a study of facial nucleus axotomy, the authors also related the observed microglial phenotype (134) to the dam phenotype, as well as to a phenotype found in the ck-p25 model (109): 72 genes were regulated in common between all three studies representing almost 75% of all genes upregulated in the facial nucleus axotomy model. interestingly, in an sci transcriptomic study, a profile of microglia reminiscent of the cd11c+ phenotype was identified (with upregulation of gpnmb, spp1, lpl, apoe, igf1, lgals3, and itgax among others) and persisted in a full transection model, whereas it contracted concomitantly to recovery in a hemisection model (135) , indicative of the transitory nature of this subset. conversely, in tbi, the microglial signature was further from the cd11c+ microglia signature, although itgax was among the upregulated genes 14 and 60 days post-injury, possibly indicating once again a dilution of the signature in all microglia (136) . in addition, considering the difficulty associated with gating out macrophages from microglia in a context of extensive infiltration, macrophage contamination of the sorted samples cannot be excluded in these studies, potentially complicating interpretation of the observed transcriptomes. ms is an inflammatory, demyelinating disease of the cns that can be modeled by eae or toxin-induced demyelinating models. recent advancement in our understanding of the disease points toward important roles for microglia in the pathomechanism. although the evidence supporting their implication in initiation and facilitation of the disease is strong (95) , there is a growing body of evidence for their protective functions including involvement in remyelination (137) . we have identified cd11c+ microglia during eae accounting for around 10% of total microglia in whole cns (33, 34) . of note, this subset is even more abundant in the spinal cord at the peak of the diseases reaching up to 60% of total microglia (wlodarczyk, unpublished) . the emergence of the cd11c+ microglia is a dynamic process starting at the onset, reaching a maximum at the peak and contracting in the chronic phase of eae (77, 138) . these cells are localized in the demyelinated spinal cord lesions (33) . cd11c+ microglia from eae again showed upregulation of similar genes as in neurodegenerative models including itgax, gpnmb, spp1, etc. (35) . a similar signature was confirmed by krasemann et al. (75) . in addition, deep analysis of genes that were upregulated in cd11c+ microglia population pointed to their involvement in immune responses (35) . a key aspect of neuroinflammation in eae is the recruitment and reactivation of encephalitogenic t cells to express their effector functions. many cell types are implicated in this process, including blood-derived dcs and monocytes/macrophages but also parenchymal microglia (139) . in eae, cd11c+ microglia express mhci, mhcii, and costimulatory molecules cd80/cd86 (34, 140) , which is in line with recent highthroughput mass-cytometry reports (77, 138) . we have provided evidence that cd11c+ microglia are able to induce similar proliferative response of encephalitogenic cd4+ t cells as blood-derived professional antigen-presenting cells (32, 34) . interestingly, in contrast to cd11c+ blood-derived cells and cd11c− microglia, cd11c+ microglia completely lacked mrna expression for il-23 (34) that is known to induce gm-csf-producing cd4+ t cells, critical for eae pathology (141) . this indicates that although cd11c+ microglia alone might contribute to t cell expansion, they are unlikely to induce pathogenic t cell responses. importantly, a subsequent study showed that they were a major source of message for myelinogenic igf1, suggesting that they might exert protective roles in eae (33) . this is supported by our recent study showing that stimulation of csf1r with its ligands during symptomatic eae significantly reduced demyelination and ameliorated disease progression most likely through induction of cd11c+ microglia (81) . moreover, decreasing cd11c+ microglia by blocking of trem2 signaling (as discussed below) led to increased severity of eae and exacerbated demyelinating lesions in the spinal cord (142) , further supporting protective roles of cd11c+ microglia. microglia are known to contribute to remyelination by creating an environment supporting opc recruitment and differentiation by phagocytosing myelin debris, secreting growth factors and modulating extracellular matrix [reviewed in lloyd and miron (137) ]. circumstantial evidence for remyelinating properties of cd11c+ microglia includes our first demonstration of the expansion of these cells in cuprizone-demyelinated corpus callosum (32) . a microarray study by olah et al. identified a pro-remyelinating microglial signature that includes several genes reminiscent of the cd11c+ microglia characteristics described above (itgax igf1, clec7a, apoe, spp1) (143) . moreover, cd11c immunoreactive microglia were present in remyelinating corpus callosum (32) . a similar microglial signature was later confirmed in both demyelination and remyelination phases (144) . conversely, microglia expressing the cd11c+ microglia signature including apoe, axl, igf1, lyz2, itgax, and gpnmb were identified by single-cell transcriptomics in both deand remyelinated lesions (145) . recently, cuprizone-mediated demyelination was shown to be alleviated in mice lacking microglial sirpα that have increased numbers of cd11c+ microglia, pointing to their protective role (89) . in line with the induction of cd11c+ microglia (81) , stimulation of csf1r ameliorated cuprizone-induced demyelination (146) . another line of evidence comes from the influence of trem2 deficiency, which leads to absence of cd11c+ microglia in adult mice (74, 75) , on remyelination after cuprizone demyelination. the data indicate that trem2 deficiency had no impact on the initial demyelination, but affected subsequent remyelination when the cuprizone treatment was prolonged, most likely by impairing myelin removal as well as myelin regeneration, which further supports a protective role for cd11c+ microglia in this paradigm (144, 147) . additionally, it was reported that microglial necroptosis in circumstances of lysophosphatidylcholine demyelination leads to repopulation by pro-regenerative cd11c+ microglia, as blocking of this mechanism prevented remyelination (148) . of note, demyelination induced by mouse hepatitis virus also led to enrichment of cd11c+ microglial gene signature in the spinal cord (149) . taken together, association of cd11c+ microglia to white matter (89) as well as their role in primary myelination strongly support their importance in induction and facilitation of remyelination. this opens the possibility for induction of innate repair programs in diseased cns via promotion of the emergence of cd11c+ microglia. very early studies identified microglial cells close to gliomas to resemble the amoeboid form described during development and to take on phagocytic functions (58) . more recent studies have shown that parenchymal microglia are attracted to the tumor in glioma-affected brains, representing up to 30% of the tumor mass (150) . microglia associated to the tumor have been termed glioma-associated microglia/macrophages (gam). these cells initially exhibit beneficial anti-tumor abilities but have been found to be hijacked by the tumor to exert tumor-promoting functions [reviewed in li and graeber (151) ]. a study from 2015 identified a signature for gams, and emphasized their high expression of spp1 and gpnmb (152) . they compared this signature to classical macrophage activation markers (m1/m2) and concluded a lack of overlap between the gam signature and these classical phenotypes. of note, the signature also includes genes such as itgax, fabp5, and clec7a among others recurrently found in disease signatures (152) . considering the similarities observed in gene expression from the different studies aforementioned, we compared the transcriptomic signatures obtained in studies comparing specifically microglia sorted based on a typical marker for this specific subset of microglia or from single-cell rna-seq (three of the ad studies and one eae study, figure 2 ). we found a core disease signature for microglia consisting of 89 genes shared between all four studies (figure 2) . itgax being once again a part of this signature and with clarity in mind, we will refer to this signature as the "cd11c+ microglia disease signature" henceforth. once again, the microglial nature of this subset is supported by expression, although slightly lower than in homeostatic microglia, of tmem119, cx3cr1, p2ry12, sall1, and tgfbr1 among other homeostatic genes (35, (73) (74) (75) . over the years, advancements in technology have allowed the scientific community to investigate cells and cell populations in increasingly detailed ways, particularly at the molecular level. this investigation has been done using a multiplicity of different conditions and models, leading to increasing amounts of data generated. although invaluable, this work has also led to redundancy in the microglial profiles that were identified (154) . our investigation led us to define two particularly strong signatures for cd11c+ microglia in development (figure 1 ) and in disease (figure 2) . interestingly, li et al. (24) as well as anderson et al. (71) related the developmental microglia signature observed in their studies to the dam microglial signature. these similarities prompted us to compare the signatures we identified from the literature. comparison of the developmental signature and the disease signature resulted in defining of a "core" signature common to cd11c+ microglia across all contexts, which consists of 22 genes: ank, anxa5, aplp2, atp1a3, clec7a, colec12, csf1, ephx1, fabp5, fam20c, gm1673, gpnmb, hpse, igf1, itgax, lilrb4, lpl, nceh1, plaur, pld3, plin2, and spp1 (figure 3 and supplementary table 1) . interestingly, the protein network linked to these genes had significantly more links than what can be expected, indicating at least a partial biological connection between these genes (figure 3) . further investigation of the physiological function of the proteins related to the genes present in the core signature revealed their involvement in lipid metabolism, cell migration and proliferation, and, to a lesser extent, immune function (supplementary table 1) . as expected, all of these proteins had been associated with various brain diseases (supplementary table 1 ). of note, many of these proteins assume similar function or have been found to interact directly or indirectly with each other (supplementary table 1 ). further investigation of these genes and proteins in link with one another would most likely unveil interesting mechanisms underlying cd11c+ microglia function. although described previously as different microglial subsets, we argue that the robust core signature we have identified can be found for this subset across all these different stages. we suggest that the differences in this subset observed between conditions reflect methodological discrepancies ( table 2) or microenvironment-linked context-specific changes and the subset's own phenotypic plasticity in coping with these variations, rather than fundamental differences in cell lineage. the dynamics of cd11c+ microglia seem tightly spatiotemporally regulated. they first emerge during the first postnatal week, peaking at p5 and gradually decreasing as animals age, being barely detectable in the healthy adult cns (33-35, 42, 80, 81) to increase again in aging or disease (33, 73, 81, 85, 89) . importantly, none of the studies that have investigated induction of inflammation by means of lipopolysaccharide, poly(i:c), or other immune challenges could recapitulate the robust cd11c+ signature found in steady state and disease and injury contexts (86, 88, 90, 124, 155, 156) . below, we present factors that participate in controlling the induction of this population (figure 4) . one candidate that has been extensively studied with regard to cd11c+ microglia is the trem2 pathway. trem2-deficient animals were shown to downregulate the cd11c+ microglia signature in cuprizone-induced demyelination (144) and in an ad model (113) . in addition, in the study from the amit lab, trem2 deficiency in an ad mouse model led to an arrest of microglia in an intermediate state between the homeostatic state and the cd11c+ microglia stage. barely any microglia in these mice exhibited the cd11c+ microglial signature (74) . this suggests that cd11c+ microglia induction is a two-step process, where the first step, to leave the homeostatic state, is trem2-independent and the second step, to reach the complete cd11c+ microglia phenotype, is trem2-dependent. these observations were confirmed by krasemann et al. in another trem2-deficient ad model (75) . similarly, apoedeficient mice exhibit lower numbers of cd11c+ microglia in ad, als, and ms mouse models (75, 80) . this is suggestive of a positive feedback loop, as this population itself strongly upregulates apoe (75) . surprisingly, the barres lab showed that induction of cd11c+ microglia during postnatal development in contrast to adulthood is trem2-apoe-independent (24) . a similar trem2 independence of cd11c+ microglia induction was shown in the developing retina (71). figure 2 | cd11c+ microglia signature in disease states. in diseased cns, cd11c+ microglia adopt an amoeboid, reactive morphology. in ad, they are found surrounding aβ plaques. similarly, in ms and als models and in injury, they are found around and in the lesions. in glioma, they are found mixed with tumor cells. cd11c+ microglia numbers in diseased cns vary considerably, ranging from 10 to 50% of all microglia. comparison of genes upregulated in four studies (35, (73) (74) (75) reveals a common signature for cd11c+ disease microglia of 89 genes upregulated in at least three of the studies (bold dark outline). genes shared with the developmental signature in figure 1 are in bold. raw data for the krasemann study were obtained using the gene expression omnibus database and the differential gene expression analysis was performed using the debrowser package in r (153) . the venn diagram was generated using the online tool venny (72). krasemann et al. highlighted phagocytosis of apoptotic neurons and monocytes as a trigger for the induction of the cd11c+ microglia phenotype (75) . of note, induction of this phenotype was not observed upon microglia exposure to escherichia coli, zymosan particles (75), or microparticles (marczynska et al., unpublished), suggesting that induction of cd11c+ microglia is a tightly controlled reaction to local cell damage or apoptosis, rather than to phagocytosis itself. interestingly, microglial necroptosis in demyelination models leads to brain repopulation with cd11c+ microglia from nestin+ resident microglia (148) . similarly, nestin+ microglia colonizing the brain after microglia ablation expressed surface cd11c (98) . the gene expression in repopulating microglia highly overlapped with the cd11c+ microglia signature. we showed that genetic or toxin-induced ablation of neonatal cd11c+ cells led to their instant repopulation (35) . whether the observed concomitant decrease of cd11c− microglia (35) reflects induction of cd11c+ phenotype in cd11c− cells by phagocytosis of dying microglia has not been determined. interestingly, a dramatic decrease in cd11c+ microglia was observed in the postnatal retina of mice deficient in bax, a pro-apoptotic gene that is essential for developmental death of neurons (71) . this emphasizes that apoptotic cells are a strong and common inducer of cd11c+ microglia regardless of age and condition. this is also in line with several studies where developmental cell death has been linked figure 3 | core cd11c+ microglia signature. considering similarities between the transcriptomic signatures and functions in the cd11c+ microglia subset in development and in disease, we compared both signatures to obtain a core of genes upregulated in this subset across all conditions. we observe overlap of 20% of the genes between both signatures, corresponding to 22 shared genes. upon interrogating the string database (szklarczyk d, gable al, lyon d, junge a, wyder s, huerta-cepas j, simonovic m, doncheva nt, morris jh, bork p, jensen lj, von mering c. string v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. nucleic acids res. 2019 nov; 47:d607-613.), we observed that the network formed by the proteins corresponding to the genes in the core signature had significantly more interactions than expected from a similar set of random proteins, indicating that these proteins related to the genes in the core signature are at least partially biologically connected. the thickness of the edges linking the different genes is proportional to the strength of the evidence linking the two proteins. the venn diagram was generated using the online tool venny (72) . to microglial entry in the developing cns (61) . in addition, retinal cd11c+ microglia were resistant to depletion induced by either csf1r deficiency or blocking, contrary to their cd11c− counterparts. in line with this, our own data showed that despite using several depletion regimens, cd11c+ microglia could not be depleted from postnatal brain as they were immediately repopulated (35) . we have shown that both populations of adult microglia (cd11c+ and cd11c−) express equal levels of csf1r (33) . importantly, stimulation of this receptor by its ligands, interleukin (il)-34 and csf1, induced a significant increase in cd11c+ microglia numbers, with faster kinetics for il-34 (81) . moreover, such stimulation induced ccl2 in the brain, and we showed that overexpression of ccl2 leads to a dramatic expansion of cd11c+ microglia in a ccr2independent manner (81). butovsky et al., on the other hand, showed that another cytokine, il-4, can induce cd11c+ expression on aβ pretreated microglia (30, 157) . moreover, they demonstrated that ga vaccination leads to an increase of cd11c+ microglia surrounding aβ plaques and suggested that this was induced by t-cell-derived il-4 (30). recently, the emergence of cd11c+ microglia in the adult brain has been shown to be homeostatically controlled by sirpα/cd47 interaction. genetic ablation of sirpα in microglia or global lack of cd47 equally resulted in increased numbers of cd11c+ microglia, suggesting that microglial sirpα suppresses cd11c expression in the same cells (89) . here, we have demonstrated that the subpopulation of microglia described in many recent studies (and named pam, atm, fountain of microglia, dam, arm, mgnd, and late response microglia) indeed reflects the characteristics of cd11c+ microglia, originally identified over a decade ago. thus, we believe that a unification of the nomenclature by referring to the microglial subset expressing the described signature, from development to old age, as cd11c+ microglia is a necessary step to progress our understanding of microglia biology. this subset emerges in development before contracting during adulthood but is triggered to re-emerge in aging as well as in the context of disease or tissue injury (figure 4) . the summary of the data that mentioned microglia showing the aforementioned signature strongly points to the importance figure 4 | cd11c+ microglia as a subset of microglia present through life and across conditions. our investigation leads us to believe that cd11c+ microglia represent a subset of microglia characterized by a robust signature of 22 genes expressed by this subset at any age and in various disease states. emergence of this subset is induced by various factors including signaling through the trem2-apoe pathway, cell death, il-4 signaling, and cytokine signaling through csf1r inducing ccl2, and is inhibited by cd47/sirpα signaling. in physiological conditions, cd11c+ microglia account for around 15% of all microglia, before contracting to 2% in adulthood and being re-induced by aging at levels similar to development. in disease states, their numbers oscillate between 10 and 50%. we argue that despite the numerous names given to this subset across conditions, it is unique and should be referred to as "cd11c+ microglia". of cd11c+ microglia in primary myelination during cns development as well as their protective, remyelinative, and regenerative capacities in cns pathology. this opens new perspectives for therapeutic targeting of microglia in neurological conditions. aw and ab-b designed the manuscript. ab-b analyzed the transcriptomic data and prepared the figures. ab-b, aw, and to wrote and approved the manuscript. tercer elemento" de los centros nerviosos. i. la microglía en estado normal. ii. intervención de la microglía en los procesos patológicos (células en bastoncito y cuerpos gránulo-adiposos). iii. naturaleza probable de la microglía fate mapping analysis reveals that adult microglia derive from primitive macrophages microglia derive from progenitors, originating from the yolk sac, and which proliferate in the brain entry and distribution of microglial cells in human embryonic and fetal cerebral cortex microglial dynamics during human brain development microglia contact induces synapse formation in developing somatosensory cortex microglia promote learning-dependent synapse formation through brain-derived neurotrophic factor synaptic pruning by microglia is necessary for normal brain development microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction microglia modulate wiring of the embryonic forebrain microglia shape corpus callosum axon tract fasciculation: functional impact of prenatal inflammation layer v cortical neurons require microglial support for survival during postnatal development neural progenitor cells orchestrate microglia migration and positioning into the developing cortex microglia regulate the number of neural precursor cells in the developing cerebral cortex atp mediates rapid microglial response to local brain injury in vivo resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo neuronal network activity controls microglial process surveillance in awake mice via norepinephrine signaling dynamic microglial modulation of spatial learning and social behavior immune cell regulation of glia during cns injury and disease a polarizing question: do m1 and m2 microglia exist? identification of a unique tgf-β-dependent molecular and functional signature in microglia microglia development follows a stepwise program to regulate brain homeostasis developmental heterogeneity of microglia and brain myeloid cells revealed by deep single-cell rna sequencing microglial subtypes: diversity within the microglial community molecular mechanisms modulating the phenotype of macrophages and microglia. front immunol new tools for studying microglia in the mouse and human cns comprehensive gene expression meta-analysis identifies signature genes that distinguish microglia from peripheral monocytes/macrophages in health and glioma phenotypic differences between human monocytes/macrophages and microglial cells studied in situ and in vitro glatiramer acetate fights against alzheimer's disease by inducing dendritic-like microglia expressing insulin-like growth factor 1 normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive cd4+ t cells compared microglial recruitment, activation, and proliferation in response to primary demyelination pathologic and protective roles for microglial subsets and bone marrow-and blood-derived myeloid cells in central nervous system inflammation comparison of microglia and infiltrating cd11c+ cells as antigen presenting cells for t cell proliferation and cytokine response a novel microglial subset plays a key role in myelinogenesis in developing brain single-cell rna sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes activation and deactivation of periventricular white matter phagocytes during postnatal mouse development brain dendritic cells and macrophages/microglia in central nervous system inflammation dendritic cells and dendritic-like microglia in focal cortical ischemia of the mouse brain cd11c/eyfp transgene illuminates a discrete network of dendritic cells within the embryonic, neonatal, adult, and injured mouse brain cd11c-expressing cells reside in the juxtavascular parenchyma and extend processes into the glia limitans of the mouse nervous system cd11c-positive cells from brain, spleen, lung, and liver exhibit site-specific immune phenotypes and plastically adapt to new environments modulation of immune cell signalling by the leukocyte common tyrosine phosphatase microglia sculpt postnatal neural circuits in an activity and complement-dependent manner fractalkine signaling and microglia functions in the developing brain microglia/macrophage-specific protein iba1 binds to fimbrin and enhances its actin-bundling activity non-identical twins: different faces of cr3 and cr4 in myeloid and lymphoid cells of mice and men dectin-1: a signalling non-tlr pattern-recognition receptor the multiple functions and mechanisms of osteopontin études sur les diverses formes d'encéphalite: (anatomie et physiologie pathologiques) paris: a. delahaye studien über die encephalitis und myelitis des ersten kindesalters untersuchungen über die morphologie und biologie der abraümzellen im zentralnervesystem étude de la stéatose interstitielle diffuse de l'encéphale chez le nouveau-né die histologie und histogenese der nervösen centralorgane über die entwicklung des menschlichen rückenmarks und seiner formelemente cytology & cellular pathology of the nervous system microglia and the process of phagocytosis in gliomas round and amoeboid microglial cells in the neonatal rabbit brain amoeboid microglial cells in the corpus callosum of neonatal rats immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain first appearance, distribution, and origin of macrophages in the early development of the avian central nervous system zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a m-csf receptor-dependent invasive process colonisation of the developing human brain and spinal cord by microglia: a review radioautographic investigation of gliogenesis in the corpus callosum of young rats ii. origin of microglial cells transformation of monocytes into amoeboid microglia in the corpus callosum of postnatal rats, as shown by labelling monocytes by carbon particles differential immunochemical markers reveal the normal distribution of brain macrophages and microglia in the developing rat brain morphological and immunocytochemical identification of macrophages in the developing corpus callosum microglia express insulin-like growth factor-1 in the hippocampus of aged appswe/ps1 e9 transgenic mice microglia contribute to normal myelinogenesis and to oligodendrocyte progenitor maintenance during adulthood developmental apoptosis promotes a disease-related gene signature and independence from csf1r signaling in retinal microglia an interactive tool for comparing lists with venn's diagrams transcriptional profiling of cd11c-positive microglia accumulating around amyloid plaques in a mouse model for alzheimer's disease a unique microglia type associated with restricting development of alzheimer's disease. cell the trem2-apoe pathway drives the transcriptional phenotype of dysfunctional microglia in neurodegenerative diseases microglia responses in acute and chronic neurological diseases: what microglia-specific transcriptomic studies taught (and did not teach) us. front aging neurosci high-dimensional single-cell mapping of central nervous system immune cells reveals distinct myeloid subsets in health, aging, and disease a case of mistaken identity: cd11c-eyfp+ cells in the normal mouse brain parenchyma and neural retina display the phenotype of microglia, not dendritic cells human microglia regional heterogeneity and phenotypes determined by multiplexed single-cell mass cytometry the major risk factors for alzheimer's disease: age, sex, and genes modulate the microglia response to aβ plaques csf1r stimulation promotes increased neuroprotection by cd11c+ microglia in eae a single-cell atlas of mouse brain macrophages reveals unique transcriptional identities shaped by ontogeny and tissue environment neuroglia in ageing the microglial sensome revealed by direct rna sequencing increased white matter inflammation in aging-and alzheimer's disease brain microglial translational profiling reveals a convergent apoe pathway from aging, amyloid, and tau acute isolation and transcriptome characterization of cortical astrocytes and microglia from young and aged mice induction of a common microglia gene expression signature by aging and neurodegenerative conditions: a co-expression meta-analysis microglial sirpα regulates the emergence of cd11c+ microglia and demyelination damage in white matter age related changes in microglial phenotype vary between cns regions: grey versus white matter differences microglia turnover with aging and in an alzheimer's model via long-term in vivo single-cell imaging coupled proliferation and apoptosis maintain the rapid turnover of microglia in the adult brain a new fate mapping system reveals context-dependent random or clonal expansion of microglia the lifespan and turnover of microglia in the human brain microglia in physiology and disease microglia mediate postoperative hippocampal inflammation and cognitive decline in mice microglial repopulation resolves inflammation and promotes brain recovery after injury genetic cell ablation reveals clusters of local self-renewing microglia in the mammalian central nervous system microglial repopulation model reveals a robust homeostatic process for replacing cns myeloid cells enforced microglial depletion and repopulation as a promising strategy for the treatment of neurological disorders characterizing newly repopulated microglia in the adult mouse: impacts on animal behavior, cell morphology, and neuroinflammation repopulated microglia are solely derived from the proliferation of residual microglia after acute depletion competitive repopulation of an empty microglial niche yields functionally distinct subsets of microglia-like cells proximal recolonization by self-renewing microglia re-establishes microglial homeostasis in the adult mouse brain microglia in alzheimer's disease: exploring how genetics and phenotype influence risk brain microglia constitutively express β-2 integrins neutralization of granulocyte macrophage colony-stimulating factor decreases amyloid beta 1-42 and suppresses microglial activity in a transgenic mouse model of alzheimer's disease temporal gene profiling of the 5xfad transgenic mouse model highlights the importance of microglial activation in alzheimer's disease temporal tracking of microglia activation in neurodegeneration at singlecell resolution microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration arginine deprivation and immune suppression in a mouse model of alzheimer's disease trem2 deficiency eliminates trem2+ inflammatory macrophages and ameliorates pathology in alzheimer's disease mouse models trem2 lipid sensing sustains the microglial response in an alzheimer's disease model trem2 haplodeficiency in mice and humans impairs the microglia barrier function leading to decreased amyloid compaction and severe axonal dystrophy trem2 deficiency impairs chemotaxis and microglial responses to neuronal injury trem2-mediated early microglial response limits diffusion and toxicity of amyloid plaques disease progression-dependent effects of trem2 deficiency in a mouse model of alzheimer's disease trem2 -a key player in microglial biology and alzheimer disease relationship of microglial and astrocytic activation to disease onset and progression in a transgenic model of familial als inflammatory processes in amyotrophic lateral sclerosis implications of microglia in amyotrophic lateral sclerosis and frontotemporal dementia microglia in als: the good, the bad, and the resting ablation of proliferating microglia does not affect motor neuron degeneration in amyotrophic lateral sclerosis caused by mutant superoxide dismutase a neurodegeneration-specific gene-expression signature of acutely isolated microglia from an amyotrophic lateral sclerosis mouse model neuroimmunology of traumatic brain injury: time for a paradigm shift repertoire of microglial and macrophage responses after spinal cord injury inflammation and brain injury: acute cerebral ischaemia, peripheral and central inflammation the far-reaching scope of neuroinflammation after traumatic brain injury recruitment of beneficial m2 macrophages to injured spinal cord is orchestrated by remote brain choroid plexus immature monocytes recruited to the ischemic mouse brain differentiate into macrophages with features of alternative activation monocyte-derived macrophages contribute to spontaneous long-term functional recovery after stroke in mice dissecting functional phenotypes of microglia and macrophages in the rat brain after transient cerebral ischemia the spinal transcriptome after cortical stroke: in search of molecular factors regulating spontaneous recovery in the spinal cord unique microglia recovery population revealed by single-cell rnaseq following neurodegeneration rna-seq analysis of microglia reveals time-dependent activation of specific genetic programs following spinal cord injury time-dependent changes in microglia transcriptional networks following traumatic brain injury the pro-remyelination properties of microglia in the central nervous system singlecell mass cytometry reveals distinct populations of brain myeloid cells in mouse neuroinflammation and neurodegeneration models antigen presentation in eae: role of microglia, macrophages and dendritic cells rna sequencing of microglia and monocyte-derived macrophages from mice with experimental autoimmune encephalomyelitis illustrates a changing phenotype with disease course the encephalitogenicity of t(h)17 cells is dependent on il-1-and il-23-induced production of the cytokine gm-csf blockade of trem-2 exacerbates experimental autoimmune encephalomyelitis identification of a microglia phenotype supportive of remyelination trem2 sustains microglial expansion during aging and response to demyelination spatial and temporal heterogeneity of mouse and human microglia at singlecell resolution mcsf-induced microglial activation prevents myelin loss and promotes its repair in a mouse model of multiple sclerosis trem2 regulates microglial cell activation in response to demyelination in vivo central nervous system regeneration is driven by microglia necroptosis and repopulation analysis of the host transcriptome from demyelinating spinal cord of murine coronavirus-infected mice the brain tumor microenvironment the molecular profile of microglia under the influence of glioma glioma-associated microglia/macrophages display an expression profile different from m1 and m2 polarization and highly express gpnmb and spp1 debrowser: interactive differential expression analysis and visualization tool for count data the kaleidoscope of microglial phenotypes microglia transcriptome changes in a model of depressive behavior after immune challenge single-cell transcriptomics reveals distinct inflammation-induced microglia signatures microglia can be induced by ifn-γ or il-4 to express neural or dendritic-like markers the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.00430/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 benmamar-badel, owens and wlodarczyk. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-268438-bjs5oliw authors: jin, yilin; jia, kuntong; zhang, wanwan; xiang, yangxi; jia, peng; liu, wei; yi, meisheng title: zebrafish trim25 promotes innate immune response to rgnnv infection by targeting 2card and rd regions of rig-i for k63-linked ubiquitination date: 2019-12-03 journal: front immunol doi: 10.3389/fimmu.2019.02805 sha: doc_id: 268438 cord_uid: bjs5oliw rig-i-like receptors (rlrs) play important roles in response to virus infection by regulating host innate immune signaling pathways. meanwhile, the rlr signaling pathway is also tightly regulated by host and virus to achieve the immune homeostasis between antiviral responses and virus survival. here, we found that zebrafish trim25 (zbtrim25) functioned as a positive regulator of rlr signaling pathway during red spotted grouper nervous necrosis virus (rgnnv) infection. post-rgnnv infection, zbtrim25 expression was obviously inhibited and ectopic expression of zbtrim25 led to enhanced expression of rlr signaling pathway-related genes. overexpression and knockdown analysis revealed that zbtrim25 promoted zebrafish rig-i (zbrig-i)-mediated ifn signaling and inhibited rgnnv replication. mechanistically, zbtrim25 bound to zbrig-i; in particular, the spry domain of zbtrim25 interacted with the tandem caspase activation and recruitment domains (2card) and repressor domain (rd) regions of zbrig-i. zbtrim25 promoted the k63 polyubiquitination of 2card and rd regions of zbrig-i. furthermore, zbtrim25-mediated zbrig-i activation of ifn production was enhanced by k63-linked ubiquitin, indicating that zbtrim25-mediated zbrig-i polyubiquitination was essential for rig-i-triggered ifn induction. in conclusion, these findings reveal a novel mechanism that zbtrim25 positively regulates the innate immune response by targeting and promoting the k63-linked polyubiquitination of zbrig-i. the innate immune system recognizes pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) as against microbial pathogen invasion (1). retinoic acid inducible gene-i (rig-i)-like receptors (rlrs), as intracellular prrs, composed of rig-i, mda5, and lgp2, recognize non-self signatures of viral rnas in the cytosol of cells. after activated by viral rna, rig-i and mda5 recruited the downstream adaptor molecule, mavs, to their n-terminal caspase-recruitment domains (cards). then, tumor necrosis factor receptor-associated factors (traf) and tank-binding kinase 1/iκ-b kinase ε interacted with mavs, which in turn leads to the phosphorylation and cytoplasm-to-nucleus translocation of interferon (ifn) regulatory factor 3 (irf3), and the activation of type i ifn. subsequent ifns activated a variety of ifn-stimulated genes (isgs) to limit the virus replication (2) . nervous necrosis virus (nnv) is a non-enveloped, singlestranded rna virus belonging to the family nodaviridae. increasing evidence has shown that nnv can infect more than 120 fish species and causes mass mortalities of infected fish worldwide (3) . it has been revealed that rlrs respond in vivo or in vitro to the stimulation of nnv and possess capacities in the induction of ifns and isgs in a variety of fish species. for example, in zf4 cells, expression of rlrs was significantly enhanced post-nnv infection and rig-i knockdown significantly restrained group ii type i ifn activation (4) . our previous studies also suggested that rlr signaling pathway was activated during red spotted grouper nervous necrosis virus (rgnnv) infection in sea perch and its key components possessed anti-rgnnv activities (5, 6) . however, regulation mechanisms of rlr signaling pathway during rgnnv infection is still unclear. rlr-mediated antiviral signaling pathway is tightly regulated at multiple steps in the signaling cascade. several studies demonstrated that posttranslational modifications, including ubiquitination, isgylation, and phosphorylation, were important mechanisms that regulated the rlr signaling pathway, of which ubiquitination was a key regulatory mechanism for rlr pathway (2) . for instance, rnf122 negatively regulated rlr signaling pathway by targeting rig-i (7) . mda5 and mavs were targeted for k48-linked ubiquitination by trim13 and rnf5, respectively, which induced mda5 and mavs degradation and rlrs signal termination (8, 9) . trim25 e3 ubiquitin ligase induced the k63linked ubiquitination of rig-i, which activated rlr signaling pathway to elicit host antiviral innate immunity (10) . trim25, an ifn-inducible e3 ligase, is associated with all kinds of cellular processes, such as the immune response, cancer, and so on (11) . it is becoming evident that trim25 has a dual role in rig-i regulation, since trim25 not only induces k63-linked ubiquitination of rig-i to positive regulate rlr signaling activation but also negatively regulates rig-i activation through inhibiting hla-f adjacent transcription 10 degradation, a negative regulator of rig-i-mediated inflammatory response (12) . multiple fish trim25 homologs have been reported, including rhodeus uyekii (13), epinephelus coioides (14) , and larimichthys crocea (15) . increasing evidence showed that fish trim25 was involved in antiviral immunity and played a pivotal role in rlr antiviral signaling pathway (14) . however, the mechanism by which fish trim25 regulates rlr signaling pathway has not been explored. in the present study, zebrafish trim25 (zbtrim25) was involved in rgnnv infection and was identified as a positive mediator of rlr signaling pathway by binding to and ubiquitinating the caspase activation and recruitment domain (2card) and repressor domain (rd) regions of rig-i, which is different with the findings in mammals. our findings reveal a novel mechanism of trim25 to activate rlr signaling pathway and will help to develop new treatments for viral nervous necrosis disease. all procedures with zebrafish were approved by the ethics committee of sun yat-sen university and the methods were carried out following the approved guidelines. zebrafish wild-type ab line was purchased from china zebrafish resource center. fish were raised with 10 h darkness and 14 h light at 28 • c and were fed with commercial pellets twice a day. all embryos were obtained by natural spawning and staged as previously reported (16) . zbe3 cells derived from zebrafish embryos were cultured at 28 • c as previously described (17) . hek 293t cells were cultured in dmem (invitrogen) enriched with 10% fbs (invitrogen) at 37 • c under a humidified atmosphere of air containing 5% co 2 . rgnnv was propagated in zbe3 cells and stored at −80 • c until use. anti-flag (m20008), anti-myc (m20002), anti-his (m20001l), and anti-ha antibodies (m20013) were purchased from abmart. anti-α-tubulin (ab15246) and anti-gfp antibodies (g1544) were purchased from abcam and sigma, respectively. goat anti-rabbit igg-hrp, goat anti-mouse igg-hrp, alexa fluor 488-labeled goat anti-mouse igg, and alexa fluor 555-labeled goat anti-rabbit igg secondary antibodies were purchased from invitrogen. for in vitro infection, zbe3 cells were challenged with rgnnv [multiplicity of infection (moi) = 1] for 6, 12, and 24 h, respectively. subsequently, rna from cells was extracted to detect the expression of zbtrim25 mrna by quantitative realtime pcr (qrt-pcr). for in vivo infection, rgnnvs (10 8 tcid 50 /ml) were injected into the egg yolk of 50 embryos at the single-cell stage in the experimental group. in the mock group, 50 embryos were injected with dmem. a total of 1 nl of solution was microinjected into each embryo using a microinjector. rna from zebrafish embryo was extracted to detect the expression of zbtrim25 mrna by qrt-pcr at 24 h post-injection. knockdown of zbtrim25 by sirna zbtrim25 sirna (5 ′ -gaatccagttgaagagaaa-3 ′ ) and control sirna were synthesized by ribobio company (guangzhou, china). zbe3 cells were transfected with zbtrim25 sirna or control sirna according to the manufacturer's protocol using lipofectamine 3000 as previously described (18) . twenty-four hours after transfection, zbe3 cells were infected with rgnnv (moi = 1) for 24 h and total rnas were extracted for qrt-pcr analysis. the orf of zbtrim25 (genbank accession no. nm200175.1) was sub-cloned into pcmv-flag or pcmv-myc vectors (invitrogen) to generate recombinant plasmid pcmv-flag-zbtrim25 or pcmv-myc-zbtrim25, respectively. full-length zbrig-i and zbrig-i deletion mutant cdnas encoding amino acids 1-187 (zbrig-i-2card), 188-937 (zbrig-i-2card), 812-927 (zbrig-i-rd), and 188-811 [zbrig-i-(2card+rd)] were inserted into the pegfp-n3 vectors. full-length zbrig-i was inserted into the pet-32a(+) (clontech) vector to generate recombinant plasmid pet-32a(+)-zbrig-i. zbtrim25 deletion mutant zbtrim25-spry and zbtrim25-spry were generated using the pcmv-flag-zbtrim25 plasmid as a template. primers used for amplifying these genes are listed in table s1 . ha-k63ub plasmid was purchased from rebio (shanghai, china). rna extraction and cdna synthesis were performed using trizol (invitrogen) and primescript tm 1st strand cdna synthesis kit (takara) according to the manufacturer's instructions. qrt-pcr analyses of zbtrim25, zbrig-i, rna dependent rna polymerase (rdrp), rlr signaling pathway related genes (mavs, traf3, irf3, and ifn 1), and isg15 were performed as previously described (19) . relative expression levels of target genes were normalized to 18s rrna using 2 − ct methods. data represent the mean ± sd from three independent experiments, each performed in triplicate. primers sequences used for qrt-pcr are listed in table s1 . hek 293t cells, pre-seeded in 24-well plates, were transfected with 250 ng of pgl3-drifn 1-pro-luc plasmid or pgl3-basic empty vector with 25 ng of prl-tk vector (promega) together with pcmv-myc-zbrig-i or pcmv-myc and pcmv-flag or pcmv-flag-zbtrim25 (250 ng per well) for 24 h. then, cells were incubated with poly i:c for 48 h and lysed. luciferase activities were measured using the dual-luciferase reporter assay system (promega). relative luciferase activities were expressed as the ratio of firefly to renilla luciferase activity. the results were the representative of three independent experiments in triplicate. hek 293t cells, pre-seeded in 24-well plates, were transfected with 250 ng of pgl3-drifn1-pro-luc plasmid or pgl3-basic empty vector with 25 ng of prl-tk vector (promega). meanwhile, pcmv-myc-zbtrim25, mutant zbrig-i or empty control plasmids were co-transfected. after being incubated with poly i:c for 48 h, cells were lysed for luciferase assay as described above. at least three independent experiments were performed. hek 293t cells, seeded on glass cover slips, were transfected with pcmv-myc-zbtrim25 and pcmv-flag-zbrig-i plasmids. twenty-four hours post-transfection, cells were washed with pbs three times and fixed with prechilled methanol and then permeabilized using 1% triton x-100 in pbs for 10 min and blocked with 5% normal goat serum for 30 min at room temperature (rt). cells were incubated with anti-myc and anti-flag antibodies for 60 min at rt. finally, cells were washed with pbs and incubated with the appropriate alexa fluor 488 or 555 conjugated secondary antibodies for 1 h. after cell nucleus was stained with hoechst 33342, cells were observed by a confocal microscope (zeiss, germany). co-ip and western blotting experiments were performed as described previously (18) . hek 293t cells in 75-cm 2 flasks were co-transfected with 10 µg of different plasmid combinations for 48 h. then, the cells were lysed on ice with lysis buffer for 15 min and were immunoprecipitated with the indicated antibodies. the precipitated samples and whole-cell lysates (input) were analyzed by immunoblotting with the indicated antibodies. escherichia coli bl21(de3) cells were transformed with pet-32a(+)-zbrig-i or pet-32a(+) plasmids, respectively. then, cells were grown in 50 ml of lb medium (beyotime) containing 0.5 mm isopropyl-1-thio-β-d-galactopyranoside (iptg) (sigma) at 18 • c overnight with shaking at 120 rpm. cells were pelleted by centrifugation at 4,500 rpm for 30 min and lysed in 10 ml of lysis buffer (100 mm sodium phosphate, ph 8.0, 600 mm nacl, and 0.02% tween-20) (beyotime) via sonication on ice. the sonicated mixture was centrifuged at 15,000 rpm at 4 • c for 20 min, and then the supernatant was affinity-purified with dynabead his-tag magnetic beads (invitrogen) according to the manufacturer's instruction. his pull-down assays were performed as described previously with some modifications (20) . his-zbrig-i-magnetic beads were incubated with the lysates of hek 293t cells transfected with pcmv-flag-zbtrim25 or pcmv-flag empty vectors on a roller, respectively. after incubation at 4 • c overnight, the magnetic beads were washed three times with lysis buffer to remove unbound his-zbrig-i and then analyzed via western blotting using anti-flag or anti-his antibodies. his tag protein alone was served as a negative control. ubiquitination assays were performed as described previously with some modifications (21) . hek 293t cells, pre-seeded in 75-cm 2 flasks overnight, were co-transfected with 10 µg of different plasmid combinations. cells were lysed at 24 h after transfection, and then gfp-zbrig-i mutants were immunoprecipitated with anti-gfp antibodies as described above. immunoprecipitates or input were analyzed by immunoblotting with the indicated antibodies. all statistics were calculated using spss version 20. differences between control and treatment groups were assessed by one-way anova. p < 0.05 is considered statistically significantly different. p < 0.01 was considered highly significant. as shown in figure 1a , mrna level of zbtrim25 was downregulated within 24 h after rgnnv infection. meanwhile, we also investigated the expression of zbtrim25 in rgnnvinfected zebrafish embryos at 24 h, and the results were concordant with zbe3 cells (figure 1b) . these data indicated a potential role of zbtrim25 in innate immune response to rgnnv infection. in mammals, trim25 has been suggested to promote ifnβ production by functioning as a key upstream activator of rig-i to activate the rlr signaling pathway (10) . to investigate whether zbtrim25 regulated rlr signaling pathway in zebrafish, the expression of several rlr signaling pathwayrelated genes was measured in zbtrim25 overexpressing zbe3 cells. as shown in figure 2a , overexpression of zbtrim25 markedly enhanced the expression of rig-i, mavs, traf3, irf3, ifn 1, and isg15 during rgnnv infection. similar results were detected in zbtrim25 overexpressing zbe3 cells treated with poly i:c ( figure 2b) . furthermore, coexpression of zbtrim25 with zbrig-i induced a dosedependent increase in ifn activation compared with the zbrig-i overexpression alone (figure 2c) . overexpression of zbtrim25 dose-dependently inhibited rgnnv replication ( figure 2d) . on the contrary, knockdown of zbtrim25 using sirna increased the level of rdrp in rgnnv-infected zbe3 cells (figures 2e,f) . all these results demonstrate that zbtrim25 is a positive regulator of rlr signaling pathway and functions as an antiviral factor during rgnnv infection in zebrafish. to elucidate the mechanism by which zbtrim25 participates in rlr signaling pathway in zebrafish, the interaction of zbtrim25 and zbrig-i was investigated. we co-expressed myc-zbtrim25 and flag-zbrig-i plasmids in hek 293t cells, and immunofluorescence imaging showed zbtrim25 and zbrig-i colocalized in the cytoplasm of hek293t cells (figure 3a) . co-ip against the myc tag revealed that zbtrim25 could interact with full-length zbrig-i but not with flag-vector ( figure 3b) . his pull-down analysis showed that zbtrim25 was directly bound to zbrig-i ( figure 3c ). all these data suggest that zbtrim25 interacts with zbrig-i. to identify the region involved in the zbrig-i/zbtrim25 interaction, firstly, zbrig-i deletion mutants [pegfp-zbrig-i-2card, pegfp-zbrig-i-2card, pegfp-rig-i-rd, and pegfp-rig-i-(2card+rd)] were constructed and cotransfected with flag-zbtrim25 in hek 293t cells ( figure 4a) . zbrig-i-2card, zbrig-i-2card, and zbrig-i-rd could bind to zbtrim25 individually (figures 4b-d) ; however, zbrig-i-(2card+rd) failed to co-precipitate with zbtrim25 ( figure 4e) . these results indicate that zbrig-i binds to zbtrim25 through its n-terminal 2card region and the c-terminal rd region. furthermore, we constructed two truncations of zbtrim25 (zbtrim25-spry and zbtrim25-spry) co-transfected with zbrig-i-2card or zbrig-i-rd in hek 293t cells, respectively ( figure 4f) . we found that the spry domain of zbtrim25 interacted with 2card and rd regions of zbrig-i (figures 4g-j) . collectively, these results indicate that the spry domain of zbtrim25 is responsible for its interaction with 2card and rd regions of zbrig-i. to investigate whether the e3 ligase activity of zbtrim25 is involved in the regulation of zbrig-i, the ubiquitination of zbrig-i was tested in zbtrim25 overexpressing cells. we found that zbtrim25 markedly promoted the k63 polyubiquitination of zbrig-i ( figure 5a) . furthermore, hek 293t cells were transfected with flag-tagged zbtrim25, zbrig-i deletion mutants [gfp-zbrig-i-2card, gfp-zbrig-i-(2card+rd), and gfp-zbrig-i-rd], and ha-tagged k63 ubiquitin, and our results showed that zbtrim25 obviously enhanced the ubiquitination of zbrig-i-2card and zbrig-i-rd (figures 5b,c) , but not zbrig-i-(2card+rd) (figure 5d) . these data suggest that zbtrim25 ubiquitinates both n-terminal 2card and c-terminal rd regions of zbrig-i. it has been reported that ubiquitination of rig-i by trim25 is vital for ifn signaling. thus, the effect of zbtrim25-mediated zbrig-i ubiquitination on zbrig-i's ifn-inducing activities was assessed. our results showed that ectopic expression of zbrig-i-2card and zbrig-i-rd could enhance ifn promoter activity (figure 6a) , and this activation was markedly enhanced by zbtrim25 overexpression (figures 6b,c) . furthermore, overexpression of k63-linked ubiquitin dose-dependently increased the promotion effect of zbtrim25 on zbrig-i-2card and rd mediated ifn 1 promoter activation (figures 6b,c) . these data confirm the importance of zbtrim25-mediated k63 ubiquitination in the n-terminal 2card region and c-terminal rd region of zbrig-i for zbrig-i-mediated ifn induction. rlr signaling pathway plays crucial roles in recognizing viral infections and initiating the antiviral immune response. rig-i, as an important component of rlr signaling pathway, can detect viral dsrnas in the cytoplasm and induce type i ifn production and the secretion of pro-inflammatory cytokines to suppress virus spread during virus infection (22) . multiple studies have demonstrated that the ubiquitination of rig-i plays an important role in the rig-i-mediated antiviral signaling pathway. for instance, trim25, trim4, and mex3c positively regulate rig-i-mediated signaling by targeting rig-i for k63linked polyubiquitination (23, 24) . trim25, well-known as an ubiquitin e3 ligase and an isg15 e3 ligase, is widely involved in the regulation of innate immunity (10, 25) . in mammals, previous reports showed that trim25 enhanced rlrs antiviral pathway by binding viral rna-activated rig-i to induce its k63-linked polyubiquitination and subsequent ifns and isgs production (26) . in teleost fish, several trim25 homologs were reported to play a pivotal role in innate immunity (14, 15) ; however, the mechanisms by which fish trim25 modulates the innate immune response against viruses remain elusive. here, we found that zbtrim25 positively regulated rlr signaling pathway and facilitated zbrig-i-mediated ifn 1 promoter activation, and overexpression of zbtrim25 inhibited rgnnv infection, indicating the conservative antiviral properties of trim25 in fish and mammals. several reports showed that trim25 was involved in the regulation of antiviral innate immunity by targeting rig-i (10, 27) . the mammal rig-i protein contains two n-terminal cardlike domains, a c-terminal rd region and an rna helicase region (28) . in zebrafish, rig-ia (an insertion variant of rig-i) and rig-ib (the typical rig-i) were identified as two transcripts of rig-i, and overexpression of rig-ib in cultured fish cells, but not rig-ia, activated zebrafish type i ifn and induced antiviral response (29) . thus, in this report, we investigated the interaction of zbtrim25 and zbrig-i (rig-ib), and our results showed that zbtrim25 was directly associated with zbrig-i and especially the 2card or rd region of zbrig-i was sufficient for its interaction with zbtrim25. trim25 is characterized by an n-terminal region containing a catalytic ring domain, one or two b-box domains, a coiled-coil dimerization domain, and a c-terminal spry domain (30) . among these domains, spry was associated with protein-protein interactions and/or rna binding (31) . gack et al. reported that the c-terminal spry domain of trim25 interacted with the first card of rig-i, but not the helicase region and rd of rig-i, and this interaction delivered the k63-linked ubiquitin moieties to the second card of rig-i, which facilitated the dimerization of rig-i and subsequent interaction with mavs to induce antiviral signal transduction (10) . we further investigate whether the spry domain of zbtrim25 was responsible for its interaction with zbrig-i. unlike previous studies, we found that the spry domain of zbtrim25 interacted not only with 2card but also with rd regions of zbrig-i. in non-infected cells, rd covered the rna-binding and helicase domains and cards folded over one another, which made rig-i to exist in an auto-repressed conformation. upon virus infection, viral rnas interacted with the rd and the helicase domain of rig-i, which in turn exposed the cards for mavs interaction, thereby triggering antiviral responses (32, 33) . considering the interaction between rd of rig-i and viral rnas, we speculated that the interaction of zbtrim25 and zbrig-i rd might inhibit zbrig-i sensing of viral rnas. meanwhile, it has been known that card domains of rig-i are widely involved in its interaction with other proteins, such as mavs, trim40, and virus proteins (27, 34, 35) . thus, the interaction of zbtrim25 and zbrig-i rd might also make room for other proteins to bind to 2card of zbrig-i, zbtrim25, and other proteins and will work cooperatively in regulation of rlr signaling pathway. the differences between the findings for zbtrim25 and trim25 in mammals indicate that zbtrim25 may regulate rlr signaling pathway in various ways. ubiquitination is a vital post-translational modification for the modulation of rig-i activity. several e3 ubiquitin ligases that mediate k63-linked ubiquitination of rig-i for its activation have been identified. for instance, mex3c overexpression caused the k63-linked ubiquitination of rig-i-2card but not rig-i-2card, and lysines 48, 99, and 169 of rig-i were required for rig-i ubiquitination by mex3c (23) . rnf135 mediated the k63-linked polyubiquitination of rig-i-rd, and lysines 849 and 851 residues of rig-i were crucial for rnf135-mediated ubiquitination (36) . in contrast to rnf135, trim25 mediated the k63-linked polyubiquitination of rig-i-2card, but not rig-i-2card, and the lysine 172 residue of rig-i was critical for efficient trim25-mediated ubiquitination and the ability of rig-i to activate antiviral signal transduction (10) . our results indicated that zbtrim25 mediated k63-linked polyubiquitination of both 2card and rd regions of zbrig-i, which is distinct from the findings in mammals that trim25 only targeted and promoted the k63-linked polyubiquitination of rig-i 2card. in addition, our reporter analysis showed that overexpression of zbrig-i-2card led to the activation of ifn 1 promoter, which is similar with other reports (37) . overexpression of zbrig-i-rd also resulted in the activation of ifn 1 promoter. furthermore, k63-linked ubiquitin is essential for the zbtrim25-mediated enhancement of zbrig-i 2card and rd-dependent ifn 1 promoter activation. zbrig-i possessed capacities in the induction of ifns and isgs to enhance the antiviral response (38) . taken together, these findings suggest that zbtrim25mediated ubiquitination of 2card and rd regions of zbrig-i is crucial for its antiviral innate immune response. however, due to the lack of trim25 or rig-i-knockout zebrafish, we cannot assess the impact of the zebrafish trim25/rig-i pathway at the in vivo level. a recent study demonstrated that zebrafish rnf135 also interacted with and ubiquitinated zbrig-i (39) . further studies will be performed to determine the precise architecture of the zebrafish trim25/rnf135/rig-i protein complex and the mechanism by which zbtrim25 and zbrnf135 worked together to regulate ubiquitination of zbrig-i. it was known that several virus proteins could positively or negatively regulate rlr signaling pathway by targeting its key components or regulatory proteins (22) . for instance, paramyxovirus v proteins interacted with the rig-i/trim25 regulatory complex and inhibited rig-i signaling (27) . influenza a virus ns1 protein bound to trim25 to block ubiquitination of the rig-i (40) . severe acute respiratory syndrome nucleocapsid inhibited trim25-mediated rig-i ubiquitination, causing the inhibition of ifn production (41) . the rgnnv genome encodes a structural (capsid protein, cp) and a nonstructural (rna-dependent rna polymerase, rdrp) protein (3). huang et al. reported that rdrp from ognnv induced ifn by activating irf3, the key regulatory component of rlrs-ifn signaling (42) , indicating that rdrp might be a positive rlr signaling pathway. whether rdrp targets the key components of rlr signaling pathway to exert its positive regulation role is a question that deserves further research. in addition, some mirnas could target critical regulatory proteins of rlr pathway for immune evasion (43, 44) ; whether rgnnv infection-related mirna was also involved in the regulation of rlr signaling pathway needs to be further investigated. in summary, zbtrim25 is identified as a positive regulator of rlr signaling pathway by targeting zbrig-i. the spry domain of zbtrim25 is required for its interaction with 2card and rd regions of zbrig-i. zbtrim25 promotes k63 polyubiquitination of both zbrig-i 2card and rd regions, which subsequently induces the activation of downstream signaling event via mavs and thereby inhibits viral infection (figure 7) . these findings represent a new mechanism underlying the regulation of rlr signaling pathway. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by the ethics committee of sun yat-sen university. shaping the landscape of host immunity regulation of rlr-mediated innate immune signaling -it is all about keeping the balance understanding the interaction between betanodavirus and its host for the development of prophylactic measures for viral encephalopathy and retinopathy rig-i specifically mediates group ii type i ifn activation in nervous necrosis virus infected zebrafish cells identification and characterization of the melanoma differentiation -associated gene 5 in sea perch, lateolabrax japonicus characterization and expression analysis of laboratory of genetics and physiology 2 gene in sea perch rnf122 suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation trim13 is a negative regulator of mda5-mediated type i interferon production the e3 ubiquitin ligase rnf5 targets virus-induced signaling adaptor for ubiquitination and degradation trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity trim25 in the regulation of the antiviral innate immunity. front immunol ubiquitin-like modifier fat10 attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform molecular characterization of tripartite motif protein 25 (trim25) involved in erα-mediated transcription in the korean rose bitterling rhodeus uyekii ring domain is essential for the antiviral activity of trim25 from orange spotted grouper the two trim25 isoforms were differentially induced in larimichthys crocea post poly (i:c) stimulation. fish shellfish immun stages of embryonic development of the zebrafish establishment of a cell line with high transfection efficiency from zebrafish danio rerio embryos and its susceptibility to fish viruses mandarin fish caveolin 1 interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection interferon regulatory factor 3 from sea perch (lateolabrax japonicus) exerts antiviral function against nervous necrosis virus infection the eukaryotic translation initiation factor 3 subunit e binds to classical swine fever virus ns5a and facilitates viral replication ubiquitination is essential for avibirnavirus replication by supporting vp1 polymerase activity rig-i-like receptor regulation in virus infection and immunity pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination the ubiquitin-specific protease usp15 promotes rig-i-mediated antiviral signaling by deubiquitylating trim25 mechanism of trim25 catalytic activation in the antiviral rig-i pathway paramyxovirus v proteins interact with the rig-i/trim25 regulatory complex and inhibit rig-i signaling structural features of influenza a virus panhandle rna enabling the activation of rig-i independently of 5'-triphosphate higher antiviral response of rig-i through enhancing rig-i/mavs-mediated signaling by its long insertion variant in zebrafish trim/rbcc, a novel class of 'single protein ring finger' e3 ubiquitin ligases structure and function of the spry/b30.2 domain proteins involved in innate immunity structural insights into rna recognition and activation of rig-i-like receptors the structural basis of 5 ' triphosphate double-stranded rna recognition by rig-i c-terminal domain the e3 ubiquitin ligase trim40 attenuates antiviral immune responses by targeting mda5 and rig-i. cell rep identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection involvement of zebrafish rig-i in nf-kappab and ifn signaling pathways: insights into functional conservation of rig-i in antiviral innate immunity retinoic acid-inducible gene i (rig-i)-like receptors (rlrs) in fish: current knowledge and future perspectives rnf135 is a positive regulator of ifn expression and involved in rig-i signaling pathway by targeting rig-i. fish shellfish immun influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination protein a from orange-spotted grouper nervous necrosis virus triggers type i interferon production in fish cell downregulation of microrna mir-526a by enterovirus inhibits rig-i-dependent innate immune response microrna-146a feedback inhibits rig-i-dependent type i ifn production in macrophages by targeting traf6, irak1, and irak2 yj and kj performed all experiments with assistance from yx, wz, pj, and wl analyzed data. kj and my conceived the study and designed experiments. kj and my wrote the manuscript. all authors read and approved the final manuscript. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02805/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 jin, jia, zhang, xiang, jia, liu and yi. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-284867-p4jgyusp authors: schöler, lara; le-trilling, vu thuy khanh; eilbrecht, mareike; mennerich, denise; anastasiou, olympia e.; krawczyk, adalbert; herrmann, anke; dittmer, ulf; trilling, mirko title: a novel in-cell elisa assay allows rapid and automated quantification of sars-cov-2 to analyze neutralizing antibodies and antiviral compounds date: 2020-10-09 journal: front immunol doi: 10.3389/fimmu.2020.573526 sha: doc_id: 284867 cord_uid: p4jgyusp the coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is currently the most pressing medical and socioeconomic challenge. constituting important correlates of protection, the determination of virus-neutralizing antibodies (nabs) is indispensable for convalescent plasma selection, vaccine candidate evaluation, and immunity certificates. in contrast to standard serological elisas, plaque reduction neutralization tests (prnts) are laborious, time-consuming, expensive, and restricted to specialized laboratories. to replace microscopic counting-based sars-cov-2 prnts by a novel assay exempt from genetically modified viruses, which are inapplicable in most diagnostics departments, we established a simple, rapid, and automated sars-cov-2 neutralization assay employing an in-cell elisa (icelisa) approach. after optimization of various parameters such as virus-specific antibodies, cell lines, virus doses, and duration of infection, sars-cov-2-infected cells became amenable as direct antigen source for quantitative icelisa. antiviral agents such as human sera containing nabs or antiviral interferons dose dependently reduced the sars-cov-2-specific signal. applying increased infectious doses, the icelisa-based neutralization test (icnt) was superior to prnt in discriminating convalescent sera with high from those with intermediate neutralizing capacities. in addition, the icnt was found to be specific, discriminating between sars-cov-2-specific nabs and those raised against other coronaviruses. altogether, the sars-cov-2 icelisa test allows rapid (<48 h in total, read-out in seconds) and automated quantification of virus infection in cell culture to evaluate the efficacy of nabs and antiviral drugs using reagents and equipment present in most routine diagnostics departments. by the time of writing, more than 29 million people experienced a laboratory confirmed infection with the severe acute respiratory syndrome coronavirus (sars-cov)-2 and more than 930,000 people died while having coronavirus infectious disease . first surveillance studies and calculations of excess mortality rates indicate that the precise number of infections and the true number of fatalities exceed above-mentioned numbers by far. coronaviruses (covs) are positive strand rna viruses widespread among various vertebrate hosts including bats and rodents (1) . together with four seasonal human covs (hcovs) and the two other emerging hcovs sars-cov-1 and mers-cov, sars-cov-2 is the seventh hcov causing widespread human diseases (2) . in december 2019, sars-cov-2 was first recognized in the hubei province in china (3) from where it rapidly spread throughout the world. in addition to its genetic similarity, sars-cov-2 not only shares some clinical characteristics with sars-cov-1 (4) but also exhibits some highly relevant particularities such as an increased spreading efficacy and the length of the course of disease (5) . on january 31, the who declared the sars-cov-2 outbreak a public health emergency of international concern. on march 11, 2020 , who started to denote the outbreak a global pandemic. the center of the pandemic shifts between regions, posing danger of repetitive local and temporal reintroduction circles. thus, even countries that managed the first wave must prepare in terms of diagnostics capacities for potential future re-emergences. most sars-cov-2 infections lead to mild or moderate illnesses. however, a considerable fraction of cases proceeds to severe pneumonia or life-threatening acute respiratory distress syndrome. elderly individuals and people with pre-existing comorbidities such as impaired immunity, chronic respiratory diseases, cardiovascular diseases, and cancer are more prone to suffer from severe covid-19. the case fatality rate (cfr) is difficult to calculate in the midst of the pandemic. depending on the age, cfr estimates of up to 18.4% for individuals older than 80 years and 1.38% (range 1.23-1.53) for the general population have been reported (6) . given the extent, pace, and severity of the covid-19 pandemic, diagnostics departments even in countries with highly developed medical systems struggle to provide sufficient and timely test capacities. since nucleic acid-based pathogen detection has a very short window of opportunity, assays detecting long-lasting immune responses such as antibodies are required to monitor virus spread and to estimate potential herd immunity. with some delay, most infected individuals raise a detectable humoral immune response including specific immunoglobulins (ig) m, iga, and igg (7) (8) (9) . neutralizing antibodies (nabs) bind and abrogate the function of viral proteins such as the sars-cov-2 spike (s) protein that are essential for virus entry into host cells, e.g., through recognition of the cognate receptor ace2 (10) . accordingly, monoclonal nabs exhibit strong therapeutic and prophylactic efficacies in sars-cov-2-infected human ace2-transgenic mice (11) . a recent vaccination study conducted in non-human primates identified nabs as correlate of protection (12) , indicating that a human sars-cov-2 vaccine should also be capable to elicit potent nab responses. accordingly, first human vaccine studies focussed on the question if nabs were induced (13) . additionally, monoclonal nabs and nab-containing hyper-immunoglobulin preparations may be applicable as treatment against covid-19 (14) . nabs are also the backbone of convalescent plasma (cp) therapies (15) (16) (17) that are one of seven clinical recommendations of the idsa (18) . based on the havoc covid-19 causes to the global economy, immunity passports and vaccination certificates, documenting protection through nabs, have been discussed [e.g., (19) ]. taken together, sars-cov-2-specific nabs and their quantification appear to be of central importance for the medical and socio-economic management of the pandemic. different types of neutralization tests (nt) have been developed for sars-cov-2. however, to our knowledge, these assays rely on laborious microscopic counting of virus plaques or antibody-stained foci by trained personnel (20) (21) (22) or on genetically modified viruses such as transgenic sars-cov-2 mutants (23) or pseudo-typed viruses [e.g., vesicular stomatitis virus (vsv) or human immunodeficiency virus (hiv)] expressing the s protein of sars-cov-2 (24, 25) . genetically modified viruses are generally prohibited in routine diagnostics laboratories and usually not applicable in less developed regions. due to the central importance of nabs and the limitations of the currently available methodology, we established a cheap, simple, fast, reliable, and automatable in-cell elisa (icelisa)-based icnt applicable in routine diagnostics departments with access to bsl3 laboratories. caco-2 (atcc htb-37) and vero e6 (atcc crl-1586) were cultivated in roswell park memorial institute 1640 [rpmi-1640 (gibco 21875-034)] and high glucose dulbecco`s minimal essential medium [dmem (gibco 41966-029)], respectively, supplemented with 10% (v/v) fcs, penicillin, and streptomycin at 37°c in an atmosphere of 5% co 2 . sars-cov-2 was isolated from a patient sample using vero e6 and confirmed by sars-cov-2 diagnostic qrt-pcr. viral titers were determined by tcid50 titration. human ifna2 and ifnb were purchased from pbl assay science (#11101) and peprotech (#300-02bc), respectively. the collection of serum samples and the virus isolation have been approved by the ethics committee of the medical faculty of the university of duisburg-essen (20-9208-bo, 20-9511-bo, and 20-9512-bo). anti-sars-cov-2 igg antibodies were detected using the elisa detecting antibodies recognizing the sars-cov-2 spike protein (euroimmun medizinische labordiagnostika, germany) according to manufacturer's instructions. for the analysis of neutralizing antibodies, serum samples were inactivated at 56°c for 30 min. a detailed icelisa/icnt protocol is provided in supplementary file 1. briefly, defined doses of sars-cov-2 were incubated with different serum dilutions for 1 h at 37°c prior to vero e6 infection. at 16-24 h p. i., cells (~5 × 10 4 /well of a 96-well plate) were fixed with 4% (w/v) paraformaldehyde/pbs. cells were permeabilized with 1% (v/v) triton-x-100/pbs and blocked with 3% (v/v) fcs/pbs. the primary antibody was added and incubated for 2 h at room temperature or overnight at 4°c. peroxidase-labelled secondary antibody was incubated for 1-2 h. washing steps were performed with 0.05% (v/v) tween-20/pbs. tetramethylbenzidin (tmb) substrate was added to visualize the enzyme reaction. the reaction was stopped with 0.5 m hcl. subsequently, the absorbance was measured using a microplate multireader (mithras2 lb 943; berthold technologies). the a-n mab1 (abin6952435), a-n mab2 (abin6952433), a-e ab (abin1031551), and pod-coupled secondary antibodies (dianova) were used. in the case that an absolute quantification is necessary, the relative quantification of the icelisa can be transformed into an absolute quantification in terms of the virus dose using virus calibration curves (like in figure 1 ) or using commercially available recombinant n preparations. immunoblotting was performed as described previously (26) using antibodies recognizing the sars-cov-2 n protein (abin6952435) or gapdh (sc-25778, santa cruz). lysates were inactivated by sequential heat incubation (10 min at 70°c and 10 min at 95°c) before they were discharged from the bsl3 laboratory. proteins were visualized using peroxidase-coupled secondary antibodies (dianova) and the ecl chemiluminescence system (cell signaling technology). we hypothesized that virus-encoded proteins expressed by infected cells should be amenable as source of viral antigens for the detection and quantification by elisa. we optimized the experimental conditions such as cell type, virus dose, infection period, cell fixation method, blocking reagent, amd type and concentrations of primary and secondary antibodies ( figure 1 and data not shown). as described in the materials and methods section and provided as detailed laboratory protocol in the supplementary information (supplementary file 1), we compared different commercially available antibodies for the icelisa-based quantification of sars-cov-2 proteins. based on existing data on virus entry (10), we infected human caco-2 cells with graded virus doses (ranging from 0.03125 to 2 pfu/cell). at 3 days post infection (d p.i.), we fixed and stained the cells with different antibodies either recognizing the e or the n protein of sars-cov-2. in accordance with high expression level of the n protein (27, 28) , certain n-specific mouse iggs exhibited a signalto-noise ratio favourable for icelisa ( figure 1a) . a comparison of vero e6 and human caco-2 cells revealed that the icelisa is applicable to different cells ( figure 1b) . since the icelisa signal directly correlated with viral replication and viral antigen expression, we tested its ability to determine antiviral effects. human caco-2 cells were treated with graded concentrations of human interferon (ifn) a2 or ifnb and infected 3 h later with sars-cov-2. at 3 d p.i., viral antigen amounts were quantified by icelisa. in accordance with a recent clinical phase 2 trial (29), ifnb exhibited strong and dosedependent antiviral activity against sars-cov-2 in human cells ( figure 1c) , indicating that the icelisa is applicable for future experiments addressing the efficacy of potential antiviral drugs in human cells. despite different start mois, similar icelisa signals were observed at 72 h p.i. in vero e6 ( figure 1b) , indicating multiple rounds of virus amplification and extraordinary fast replication kinetics of sars-cov-2 in vero e6 cells, consistent with previous studies (30) . to test if shorter infection periods might result in virus dose-dependent signals, we analyzed infected vero e6 cells after 6, 15, and 22 h. sars-cov-2 was readily detectable in vero e6 cells by icelisa already at 6 h p.i. (figures 1d-f) . to evaluate if the icelisa faithfully reports on the amounts of sars-cov-2-expressed antigens, we infected cells with graded infectious doses and performed immunoblots and icelisas in parallel. as expected, icelisa and immunoblot signals correlated very well ( figure 2a ). since we observed icelisa signals already at 6 h p.i. (figures 1a, d) and the n protein is an abundant component of sars-cov-2 particles (31) (32) (33) , the n protein derived from input viruses could have contributed to icelisa signals. to evaluate this, we infected cells in the presence and absence of the translation inhibitor cycloheximide ('chx'). consistent with the notion that the icelisa signal is dominated by de novo n protein expression, the signal was significantly diminished by chx ( figure 2b) . taken together, these data indicate that the icelisa allowed rapid identification and relative quantification of sars-cov-2 replication in vero e6 and human cells and its inhibition by antiviral compounds. since the icelisa allowed simple and automated quantification of sars-cov-2-dependent antigen expression, we tested if an icelisa-based neutralization test (icnt) can be established. we infected vero e6 cells for 6, 15, and 24 h with graded sars-cov-2 infection doses (500, 5,000, or 50,000 pfu/well) in the absence or presence of two convalescent sera in three different concentrations (1/20, 1/40, and 1/80 dilution). using the high infectious dose, viral antigens became dose dependently detectable by icelisa as early as 6 h p.i. ( figure 3a ). both infection. even the strong icelisa signal at 24 h p.i. was dose dependently neutralized by both sera ( figure 3f ). please note that the neutralizing capacity of given sera dilutions was less pronounced at higher virus doses as compared to lower virus doses, as indicated by residual icelisa signals. taken together, the icelisa resulted in a time-and virus dose-dependent signal constituting a surrogate for sars-cov-2 infection and replication. the fact that the infection and the resulting icelisa signal were neutralized by nabs present in immune sera indicated that the fast and automated icelisa format is applicable for icnts. although most sars-cov-2 nts have not been formally validated and certified, classic plaque reduction neutralization tests (prnt) are currently considered to represent the gold standard for the detection of sars-cov-2-specific nabs. various commercially available igm, iga, and igg elisas have been compared to prnts [e.g., (30) ]. to validate the novel icnt, 53 sera-24 positive for sars-cov-2-specific igg as determined using the euroimmun elisa (elisa ratio: 1.13-9.92) and 29 elisa-negative sera (elisa ratio < 0.9)-were compared side-by-side by icnt and standard prnt using 200 pfu/well ( figure 4a ). one set was processed by icnt, while for the other set virus foci were stained using an antibodybased aec staining method and manually counted by microscopy. standard sars-cov-2 nts base on microscopic counting of plaques or antibody-stained virus foci. to enable plaque/foci recognition and individual counting by trained personnel, a countable number of pfu must be applied in prnts. depending on the prnt protocol, 100 (20) to 400 pfu (21) are used to infect each well. based on previous experiences with virus neutralization experiments (34, 35) , we suspected that lower infectious doses might be more susceptible to nabs than higher virus doses, e.g., through altered ratios of nabs and antigenic regions determining neutralization, such as the receptor binding domain (rbd) of the s protein of sars-cov-2. accordingly, graded infectious doses (1,250-10,000 pfu per well) showed virus dose-dependent susceptibilities to the same serum sample (supplementary figure s1 ). before we assessed clinical specimens, we compared icnt signals upon a high moi infection with viral progeny as determined by parallel tcid50 titration. as expected, we observed that icnt signals and residual virus numbers correlated very well (supplementary figure s2) . figures 5a-c) , the neutralizing capacity of the same sera considerably differed at the more restrictive high virus dose ( figures 5d-f infections of cell cultures with well-defined virus preparations in the presence or absence of compounds that may impair the infection. thus, the nt specificity results from the choice of virus applied to the cells. however, antiviral compounds can either be specific for one particular virus species (e.g., most monoclonal neutralizing antibodies), broadly active against virus families (e.g., receptor blocking agents) or even bigger genera (e.g., hyperimmunoglobulin preparations). in addition to sars-cov-2, other hcovs such as the alphacoronaviruses hcov-229e and hcov-nl63 and the betacoronaviruses hcov-oc43 and hcov-hku1 circulate autochthonously in the human population, resulting in high sero-prevalence rates (36) . to address if the experimental sars-cov-2 infection in the icnt might be diminished by cross-reactive antibodies, we tested sera of individuals who have had infections with the alphacoronaviruses hcov-229e or hcov-nl63 or betacoronaviruses such as hcov-hku1 ( figure 6a ) or a combination of hcov-229e and hcov-oc43 ( figure 6b) . in contrast to the positive control containing sars-cov-2-specific nabs, all tested serum samples recognizing other coronaviruses did not neutralize sars-cov-2. although the limited sample size prevents definitive conclusions regarding crossneutralizing capacities of antibodies raised by other hcovs, in conjunction with consistent publications (37, 38) it indicates the specificity of the icnt assay. we established a novel icelisa-based test principle for detection and relative quantification of sars-cov-2. given the excellent signal-to-noise ratio between infected and uninfected cells, the test was applicable to quantify the efficacy of antiviral compounds, here shown for ifnb, and sars-cov-2-specific nabs present in immune sera. compared to icelisa and icnt, standard virus titrations and prnts are far more laborious, time consuming, and expensivenot to speak from subjective and expectation biases upon usage for research. the entire icnt can be processed in less than 2 days including the infection period. given that the protocol includes an early fixation step using 4% (w/v) paraformaldehyde which inactivates sars-cov-2, all subsequent steps can be processed outside the biosafety level 3 laboratory. the actual data acquisition is conducted within seconds, using standard elisa plate readers present in most routine diagnostics departments. we believe that this is advantageous compared to other nt assays that rely on more sophisticated and more expensive devises, e.g., for imaging cytometry (39) . the icelisa and icnt provide increased data quality and precision by generating continuous data sets. since the detection antibodies can be applied in icelisa and icnts in relatively high dilutions (1/5,000 to 1/10,000 and 1/2,000 of primary and secondary antibody, respectively), the assay is relatively cheap (in our case, around 0.10 € per well for both antibodies and the tmb peroxidase elisa substrate). the specificity of the icelisa and icnt is provided by the defined sars-cov-2 added to the cell cultures on purpose. the primary and secondary detection antibodies just serve to visualize and quantify viral antigens. thus, sars-cov-2-specific antibodies can be applied for icelisa detection notwithstanding potential cross-recognition of other covs such as hcov-hku1 or hcov-oc43 -simply because these viruses are not present in the culture. obviously, such antibodies recognizing conserved residues cannot be used for classic antigen-recognizing elisas due to their inability to discriminate coronaviruses. more virus (>1pfu/cell) can be applied to icnts. such high pfu icnts scrutinize the virusneutralizing capacity of sera more strictly, enabling a higher resolution compared to prnt assays that all rely on low virus numbers. it is tempting to speculate that sera exhibiting superior neutralization in high pfu icnt might be more beneficial in cp therapies. nts based on pseudo-typed viruses using heterologous expression of the sars-cov-2-encoded s by non-related viruses may have certain advantages. however, genetically modified viruses are inapplicable by law in various routine diagnostics departments and usually unavailable in less developed countries. recently, an elegant surrogate assay has been designed that determines if antibodies are capable to prevent the interaction of immobilized hace2 with a reporter enzyme-coupled receptor binding domain (rbd) of s (40) . this assay showed a good correlation with conventional nts (r 2 = 0.8591; p value < 0.0001) and nts based on pseudo-particles (r 2 = 0.8374). however, without the use of genuine infectious viruses, assays relying on pseudo-typed viruses do not fully interrogate the full spectrum of antiviral effects for example if other viral proteins influence the system, e.g., by complement activation (41) . accordingly, the icnt showed a superior correlation compared to prnt (r 2 = 0.987; p value 5.81e-50). taken together, we propose the icelisa and icnt for the quantification of sars-cov-2 replication and its inhibition by nabs and antiviral compounds. by changing the detection antibody and, if necessary, the cells according to the viral infection system, the test principle is transferable to all other viruses. all datasets presented in this study are included in the article/ supplementary material. the studies involving human participants were reviewed and approved by ethics committee of the medical faculty of the university of duisburg-essen (20-9208-bo, 20-9511-bo, and 20-9512-bo). the patients/participants provided their written informed consent to participate in this study. ls, vtkl-t, me, and dm performed research. oa, ak, and ah provided essential reagents. ls, vtkl-t, and mt analyzed the data. ls, vtkl-t, ud, and mt interpreted the data. vtkl-t and mt supervised the project. ls, vtkl-t, and mt wrote the manuscript. all authors contributed to the article and approved the submitted version. the authors received support by the stiftung universitätsmedizin essen, kulturstiftung essen, the else-kröner promotionskolleg elan, and the deutsche forschungsgemeinschaft (dfg) through grants rtg 1949/2, tr1208/1-1, and tr1208/2-1. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. vtkl-t and ls contributed equally to this work. we thank benjamin katschinski and kerstin wohlgemuth for excellent technical assistance. mt was supported by the stiftung universitätsmedizin essen, kulturstiftung essen, the else-kröner promotionskolleg elan, and the deutsche forschungsgemeinschaft (dfg) through grants rtg 1949/2, tr1208/1-1, and tr1208/ 2-1. this manuscript has been released as a pre-print at biorxiv, (42) . the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.573526/ full#supplementary-material supplementary figure 1 | the infectious virus dose strongly influences the neutralization capacity of serum samples. graded amounts of sars-cov-2 were incubated with indicated dilutions of a serum sample (chosen to exhibit a low neutralizing capacity) for 1 h before vero e6 cells were infected. neutralization was evaluated by icelisa. the results of the high moi icnt correlate with residual virus after neutralization. sars-cov-2 was incubated with indicated dilutions of serum samples for 1 h before vero e6 cells were infected. (a) neutralizing capacity was evaluated by icelisa. (b) neutralizing capacity was evaluated by back titration of residual virus by tcid50 in 2-fold serial dilutions. supplementary file 1 | detailed sars-cov-2 icnt and icelisa laboratory protocol. origin and evolution of pathogenic coronaviruses emerging coronaviruses: genome structure, replication, and pathogenesis a novel coronavirus from patients with pneumonia in china overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and 2019-ncov clinical characteristics of refractory covid-19 pneumonia in wuhan, china estimates of the severity of coronavirus disease 2019: a model-based analysis antibody responses to sars-cov-2 in patients of novel coronavirus disease 2019 antibody detection and dynamic characteristics in patients with covid-19 early detection of sars-cov-2 antibodies in covid-19 patients as a serologic marker of infection sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells dna vaccine protection against sars-cov-2 in rhesus macaques an mrna vaccine against sars-cov-2 -preliminary report a noncompeting pair of human neutralizing antibodies block covid-19 virus binding to its receptor ace2 convalescent plasma to treat covid-19: possibilities and challenges effectiveness of convalescent plasma therapy in severe covid-19 patients treatment of 5 critically ill patients with covid-19 with convalescent plasma infectious diseases society of america guidelines on the treatment and management of patients with covid-19 covid-19 immunity passports and vaccination certificates: scientific, equitable, and legal challenges evaluation of sars-cov-2 neutralizing antibodies using a cpe-based colorimetric live virus micro-neutralization assay in human serum samples severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease serological assays for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) a high-throughput neutralizing antibody assay for covid-19 diagnosis and vaccine evaluation detection of sars-cov-2-specific humoral and cellular immunity establishment and validation of a pseudovirus neutralization assay for sars-cov-2 gamma interferon-induced interferon regulatory factor 1-dependent antiviral response inhibits vaccinia virus replication in mouse but not human fibroblasts targeted proteomics for the detection of sars-cov-2 proteins the coding capacity of sars-cov-2 triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial sars-coronavirus-2 replication in vero e6 cells: replication kinetics, rapid adaptation and cytopathology structures and distributions of sars-cov-2 spike proteins on intact virions sars-cov-2 structure and replication characterized by in situ cryoelectron tomography sars-cov-2 (covid-19) by the numbers highly individual patterns of virus-immune igg effector responses in humans exploitation of herpesviral transactivation allows quantitative reporter gene-based assessment of virus entry and neutralization a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease no sars-cov-2 cross-neutralization by intravenous immunoglobulins produced from plasma collected before the 2020 pandemic lack of cross-neutralization by sars patient sera towards sars-cov-2 virus reduction neutralization test: a single-cell imaging high-throughput virus neutralization assay for dengue a sars-cov-2 surrogate virus neutralization test based on antibody-mediated blockage of ace2-spike protein-protein interaction highly pathogenic coronavirus n protein aggravates lung injury by masp-2-mediated complement over-activation. medrxiv (2020) a novel in-cell elisa assay allows rapid and automated quantification of sars-cov-2 to analyse neutralizing antibodies and antiviral compounds the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 schöler, le-trilling, eilbrecht, mennerich, anastasiou, krawczyk, herrmann, dittmer and trilling. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-267567-w39f584z authors: pombo, joao palma; sanyal, sumana title: perturbation of intracellular cholesterol and fatty acid homeostasis during flavivirus infections date: 2018-06-04 journal: front immunol doi: 10.3389/fimmu.2018.01276 sha: doc_id: 267567 cord_uid: w39f584z cellular lipid homeostasis is maintained through an intricately linked array of anabolic and catabolic pathways. upon flavivirus infections, these are significantly altered: on the one hand, these viruses can co-opt lipid metabolic pathways to generate atp to facilitate replication, or to synthesize membrane components to generate replication sites; on the other hand, more recent evidence suggests counter strategies employed by host cells, which actively modulate several of these networks in response to infection, enhancing interferon signaling by doing so, and thus creating an antiviral environment. in this review, we discuss recent data on mechanisms of alteration of lipid metabolic pathways during infection by flaviviruses, with a focus on cholesterol and fatty acid biosynthesis, which can be manipulated by the invading viruses to support replication, but can also be modulated by the host immune system itself, as a means to fight infection. metabolic reprogramming in immune cells is a recurrent phenomenon when exposed to proinflammatory stimulants in the form of pathogens or cytokines. macrophages and dendritic cells in particular are well-equipped to sense and respond to impending danger by pathogens, thus establishing the frontline of host defenses. recent studies have highlighted the extraordinary contribution that multiple host metabolic pathways confer toward the ability of innate immune cells to respond to infections (1) . not surprisingly, some of the very same pathways that function to eradicate infection are often rewired by the invading pathogen. most viruses are known to induce aerobic glycolysis akin to the warburg effect (2, 3) . more recently, perturbation in lipid metabolic pathways has also been reported for several classes of pathogens (4, 5) . intracellular lipid homeostasis is achieved through a balance in biosynthetic, transport, and degradation processes. current evidence increasingly points toward an intricate relationship between host lipid metabolism and intracellular pathogens, including bacteria, viruses, and parasites. while the mechanistic details are yet to be unraveled, it is hypothesized that these pathogens, on account of their limited genome sizes, co-opt the host metabolic network to meet the energy demands and procure precursors for their anabolic processes including replication and intracellular transport. in addition, viruses alter lipid metabolism to facilitate amplification and evade the host immune response. this has been decidedly observed in cases of positive strand rna virus infections, such as dengue, west nile, hepatitis c, and several coronaviruses (6) (7) (8) (9) (10) . marked alterations in cholesterol and fatty acid biosynthesis occur upon infection, accompanied by the appearance of distinctive compartments, believed to be their replication sites (11) (12) (13) (14) . despite diversity in their genome organization, many viruses share certain salient features, primary of which is their dependence on host factors to undergo replication, assembly, intracellular transport, and release (15) (16) (17) (18) (19) (20) . the intracellular life cycle of positive strand rna viruses is largely confined to the cytosol, within or on the surface of virus-induced organelle-like structures regarded as replication compartments (21) (22) (23) (24) (25) (26) . notwithstanding differences in transmission, host cell tropism, and pathogenesis, these viruses employ similar strategies for replication and assembly, often accompanied by reorganization of the host secretory pathway (13, 24, 25, 27, 28) . the replication sites serve multiple purposes that function in a concerted fashion to facilitate efficient virus propagation. primarily, they offer spatial segregation of the different steps in the intracellular life cycle, such as rna translation, replication, and packaging of the viral genome into virions during assembly. viral replication compartments also enable a high local concentration of the necessary components-both viral and host-in a physically constrained space, ensuring efficient rna amplification. an equally important feature of these replication sites is to limit exposure of viral rna to the hostile cytoplasmic environment that contains cellular nucleases and sensors of the innate immune surveillance. degradation of dsrna replication intermediates is minimized by protection in membrane-delimited compartments. although lipid metabolism has received particular attention with gram-negative bacterial infection, several recent reports highlight their function in viral infections (29) . analogous to lipopolysaccharide (lps)-mediated downregulation of sterol synthesis in case of viral infections, limiting cholesterol biosynthesis in human macrophages and fibroblasts via genetic knockdown of sterol regulatory element-binding proteins [sterol-regulatory element-binding proteins (srebps), discussed in a later section], was reported to spontaneously engage type i ifn signaling and restrict infection (30) (31) (32) (33) . initiation of anti-viral immunity thus displays a clear link with intracellular cholesterol biosynthesis, in a way that the induction of cholesterol synthesis would allow subversion of host immune responses and facilitate viral multiplication. with the advent of omics-based studies, it is increasingly becoming obvious that viruses induce large-scale alterations in host cellular metabolism (3, (34) (35) (36) (37) . among other examples are the induction of fatty acid synthesis by hepatitis c virus (hcv) in human hepatocytes, and the utilization of cellular lipid stores of hepatocytes by dengue virus. the effects of these events have been experimentally demonstrated by genetic and pharmacological inhibition of lipid biosynthetic pathways that attenuate viral pathogenesis (5, 38) . these viral adaptation strategies can effectively increase available energy for virus replication and assembly, provide specific components for progeny particles, and for creating replication sites while suppressing antiviral signaling cascades. these reports highlight the intricate link between viruses and lipid metabolism. in the following sections, we discuss emerging data on fatty acid and cholesterol biosynthetic pathways that are upregulated by certain viruses to facilitate infection. fatty acid synthase (fasn) intracellular contents of fatty acids and cholesterol contribute to fuel storage as well as a source of components necessary for increased membrane production. the core reaction of fatty acid synthesis is catalyzed by fasn starting from acetyl coa and malonyl coa. once synthesized, palmitate can have several different fates, including further elongation to long chain fatty acids, which can be used for membrane production or storage in lipid droplets (lds) in the form of triacylglycerols and esterified cholesterol. lds are storage organelles consisting of triacylglycerols and steryl esters, and function as inert storage depots of excess cellular lipids. abundance and size of lds could be indicative of increased fatty acid synthesis, which might poise the cell for rapid membrane generation if needed and also maintains energy reserves (39) . according to cellular states and their corresponding energy demands, fatty acids undergo β-oxidation to generate acetyl coa and nadh and fadh2 molecules in the mitochondrial matrix, for atp production via oxidative phosphorylation. viruses induce and require availability of fatty acids at several stages of their lifecycle-either to supplement energy requirements for their anabolic processes or to generate viral replication compartments, most notably observed during infection by positive strand rna viruses (40, 41) . this is primarily due to the process of replication-confined to the cytoplasm-where such viruses alter the host intracellular lipid composition to create a beneficial environment. this phenomenon is exemplified by hcv, where all aspects of the viral lifecycle, including entry, replication, assembly, and release are host lipid associated (8) . hcv requires low density lipoprotein receptor as a co-factor for entry into target cells (42) . its replication occurs in membranous web-like compartments referred to as double membrane vesicles (13, 43) and they assemble using lds as platforms (18, 44) . to generate replication sites, hcv triggers synthesis of fatty acids, cholesterol, and lds (45) (46) (47) (48) . another member of the flaviviridae family, dengue, has also been reported to induce production of fatty acids (49, 50) . fasn and acc1 were identified through a targeted sirna screen as necessary factors for efficient dengue virus replication (38, 51) . drugs that inhibited fasn activity resulted in a significant attenuation in virus replication (49) . infection with dengue virus does not affect fasn expression levels, but rather its redistribution to virus-triggered structures referred to as convoluted membranes (50) . this phenomenon appears to be rab18-mediated, a member of the gtpase family that typically resides in the er and lds. upon infection dengue ns3 was found to interact with rab18, which allowed recruitment of fasn to viral replication sites, thus promoting fatty acid biosynthesis to increase their local concentration (51, 52) . inhibiting fasn activity has a similar effect in mosquito cells with loss of infectious progeny virion production (53). 3-hydroxy-3-methylglutaryl-coa reductase is the rate-limiting enzyme for cholesterol biosynthesis and is regulated via a negative feedback mechanism mediated by products of the mevalonate pathway. in mammalian cells, hmgcr activity is suppressed by cholesterol imported through receptor-mediated endocytosis of low density lipoproteins (54) . dengue infection inhibits phosphorylation of hmgcr at an inactivating site, generating a cholesterol-rich environment in the process (55, 56) . this was further corroborated through inactivation of ampk and a subsequent increase in hmgcr activity, respectively (56) . in comparison, west nile virus infection has a more direct impact on intracellular cholesterol distribution. infection was accompanied by redirecting cholesterol from the plasma membrane to virus replication sites (12) . in mammalian cells cholesterol homeostasis is tightly regulated in a feedback mechanism via transcription factors that sense intracellular cholesterol levels (57, 58) . these transcription factors are termed srebps that associate tightly with the sterol-sensing srebp cleavage-activating protein (scap) within the er membrane, via an additional interaction with the er-resident protein insig, which functions as an inhibitor of srebp (59, 60) . scap has an additional role as a chaperone that mediates transport of the srebp-scap complex to the golgi network, where srebp is proteolytically cleaved by two resident golgi proteases (s1p and s2p) to release the transcriptionally active fragment of srebp from the membrane. the released forms of srebps are transported to the nucleus and activate transcription of target genes required for cholesterol and fatty acid biosynthesis, including hmgcr and fasn, respectively. when cholesterol levels are high, scap binds to cholesterol in the er, promoting an association with insig, and retains the complex within the er, thus reducing the synthesis of cholesterol. conversely, when cholesterol levels are low, binding of scap to insig is disrupted, and cholesterol synthesis is initiated (61) (62) (63) . the authors of these studies postulated that de-enrichment of cholesterol from sites harboring sensory molecules, such as the scap-srebp-insig complex, results in activation of this signaling pathway, enabling the host cell to increase cholesterol levels to accommodate proliferation of intracellular membranes. lipid droplets are multifunctional organelles present in most organisms from bacteria to eukaryotes (64) (65) (66) . these structures are particularly abundant in mammalian adipocytes and insect fat body cells. lds are mainly composed of a phospholipid monolayer and structural proteins, such as perilipins, which are involved in ld biogenesis and degradation. despite previous notions on a rather static role of lds in the maintenance of lipid homeostasis, more recently, it has become evident that lds are also present in immune cells, such as neutrophils and macrophages, where they regulate inflammatory or infectious processes (65, 67) . upon stimulation with different challenges, they display an increase in abundance and thereby serve as reliable markers of immune cell activation. autophagy dependent degradation of lds has been reported for dengue virus infection in human hepatocytes (38) . a similar activation of the autophagy pathway was recently described for zika virus infection as well (68) . our own data (accepted, queued for publication) support a drastic upregulation of ld consumption through induction of autophagy upon both dengue and zika virus infections. this pathway appears to operate in an ancient ubiquitous protein 1 (aup1)-dependent manner, and is dictated by its ubiquitylation status. unmodified aup1 enabled dispersion of lds, which underwent lipophagy upon infection. this virus-triggered pathway is essential for assembly and production of newly synthesized progeny virions (in press). current consensus, therefore, supports a model where mobilization of lds in combination with increased synthesis of fatty acids and cholesterol provides a proviral environment for production of progeny virions (53) (figure 1 ). the interdependence of innate immune signaling processes and the regulation of sterols and fatty acid metabolism is increasingly being consolidated through emerging data (30) . their role in production of inflammatory mediators has been reported by several groups (69) (70) (71) . interferons (ifns) modulate the expression of a multitude of ifn-stimulated genes including viperin, which has been observed to be highly upregulated in response to bacterial lps, double-stranded dna, and rna analogs, and also possesses antiviral activity against a range of viruses including hcv and dengue virus (72) . in a similar vein, inhibition of cholesterol biosynthesis also exerts an antiviral effect (12, 73, 74) . srebps are involved in coordinating the regulation of the sterol and fatty acid biosynthesis pathways; ifns effectively inhibit srebp2 at both mrna and protein levels. interestingly, wnv-induced redistribution of cellular cholesterol was found to downregulate ifn-stimulated jak-stat antiviral signaling response to infection, potentially by removing cholesterol from their usual microenvironment. recent evidence suggests that alterations to cellular lipid metabolism have a more direct role in host defense, through positive regulation of the type i ifn-mediated antiviral response: for example, activation of type i ifn signaling can induce upregulation of β-oxidation and inhibition of cholesterol synthesis, in order to create a hostile cellular environment for viruses (31, 75) . intracellular pathogens are known to stimulate de novo lipid and cholesterol biosynthesis to ensure their own survival. accordingly, repressing these anabolic pathways can inhibit the evolution of intracellular infections. activation of type i interferon receptors has been correlated to inhibition of cholesterol biosynthesis; however, repression of lipid metabolism in this manner is accompanied by an increase in the influx of environmental lipids, which maintain intracellular lipids and cholesterol at normal levels. thus, type i ifn signals reprogram cellular lipid metabolism, but this does not function to limit lipid availability to pathogens. it, therefore, remains unclear whether ifn-i linked repression of cholesterol biosynthesis, in the context of intracellular infection, is meant to limit nutrient availability to pathogens, or if it serves a different purpose. the scap protein acts as a sterol-sensing element, as well as a chaperone, which associates with immature srebp transcription factors in the er membrane. by knocking out or knocking down scap in macrophages, srebp activity is lowered, as well as expression of genes involved in lipid metabolism. as anticipated, de novo synthesis of cholesterol and fatty acids went down in the absence of scap, but total intracellular lipid levels remained unchanged. loss of scap also correlated with heightened resistance to viral infection in in vitro and in vivo models, confirming the functional equivalence between activation of type i interferon pathway and inhibition of lipid metabolism. culture medium supernatants from scap −/− macrophage cultures were enough to markedly increase resistance to viral challenge, when supplied to wild-type bmdms, suggesting that the higher type i interferon-mediated viral resistance was a causal effect of a secreted effector molecule, such as interferonbeta (ifnβ). in light of this, qpcr analysis revealed that both scap −/− bmdms and alveolar macrophages extracted from scap −/− mice constitutively express higher levels of ifnβ and interferon-stimulated genes (isgs), compared to wild-type macrophages. finally, blocking the interferon alpha/beta receptor (ifnar) was enough to restore interferon and isgs expression back to normal levels, as well as losing resistance to viral infection. these data strongly suggested that the absence of scap activity spontaneously triggers type i interferon production, which translates into a constitutive state of higher resistance to viral infection in macrophages (31) . this suggests that a higher type i interferon response is specifically caused by an inhibition of cholesterol metabolism (31, 32) . in support of this hypothesis, cells (immune and non-immune) with deficiency in the mevalonate pathway showed a constitutively exacerbated type i interferon response. also, addition of free cholesterol to srebp2-deficient cells, or to cells with genetically impaired cholesterol metabolism, causes the exaggerated type i interferon response to decrease to basal levels (31, 71) . stimulator of interferon genes (sting) is an er resident kinase, which activates interferon regulatory factor 3 (irf3) through phosphorylation of tank binding kinase-1 (tbk1) (76) . sting kinase activity is stimulated by cyclic dinucleotides, which are synthesized by cyclic gmp-amp synthase (cgas). cgas, sting, and phosphorylated tbk1 (ptbk1) exist in higher basal levels in srebp2 −/− cells compared to wild-type cells; in addition, knocking down either cgas or sting in enough to drastically lower ptbk1 presence in srebp2-deficient cells. also, knockdown of cgas, sting, or tbk1 in srebp2 −/− cells caused the expression of ifnb1 and isgs to decrease to levels similar to those in wild-type cells (31) . addition of free cholesterol to srebp2-knockout cells significantly decreased ptbk1, while blocking ifnar had no effect on ptbk1 levels, reinforcing the idea that cholesterol directly influences sting-mediated activation of tbk1. these data support a model in which a lack of cholesterol in the cell makes sting more sensitive to cyclic dinucleotides, upregulating the sting-ptbk1-irf3 signaling axis, and ultimately increasing expression of ifnb1 and isgs, conferring an intrinsic proinflammatory phenotype to cholesterol-deficient cells (77) . admittedly, most of these experiments used mhv68; however, these conclusions may very well be relevant in other virus infections. repressing the cholesterol biosynthetic pathway through inhibitors of hmgcr is a common treatment for cardiovascular diseases (78) . the clinical success of these inhibitors for human disorders provides strong support that targeting lipid metabolism can effective for human therapy. elucidating the specific alterations incurred upon virus infections would allow novel therapeutic approaches to emerge through targeted inhibition of such metabolic pathways. ifns or viral infections often result in induction of 25-hydroxycholesterol in macrophages-an antiviral effector, which broadly inhibits many enveloped viruses by interfering with membrane fusion (79) . whether it has an additional impact on activating the interferon signaling pathway is to be seen in future studies. different strategies can be employed to interfere with virus infection, including those involving lipid utilization; notwithstanding, it is tempting to speculate that drugs already in clinical use against cholesterol and fatty acid metabolic pathways might be repurposed to boost antiviral immunity and provide resistance to infection. jp and ss discussed and wrote the manuscript. metabolic reprogramming in macrophages and dendritic cells in innate immunity from glioblastoma to hepatitis c: it's a metabolism thing nuclear receptors control pro-viral and antiviral metabolic responses to hepatitis c virus infection emerging role of lipid droplets in host/pathogen interactions enabling hcv's need for fat viral rewiring of cellular lipid metabolism to create membranous replication compartments multifaceted roles for lipids in viral infection hepatitis c virus and host cell lipids: an intimate connection the lipid mediator protectin d1 inhibits influenza virus replication and improves severe influenza positive-strand rna viruses stimulate host phosphatidylcholine synthesis at viral replication sites cholesterol is required for surface transport of influenza virus hemagglutinin cholesterol manipulation by west nile virus perturbs the cellular immune response flaviviridae replication organelles: oh, what a tangled web we weave hepatitis c virus and lipid droplets: finding a niche virus-host interactions: from unbiased genetic screens to function genetic dissection of flaviviridae host factors through genome-scale crispr screens discovery of insect and human dengue virus host factors a genome-wide genetic screen for host factors required for hepatitis c virus propagation type i interferon imposes a tsg101/isg15 checkpoint at the golgi for glycoprotein trafficking during influenza virus infection a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection the endoplasmic reticulum provides the membrane platform for biogenesis of the flavivirus replication complex sciencedirectcoupling of replication and assembly in flaviviruses pathogenesis of flavivirus infections: using and abusing the host cell viral rna replication in association with cellular membranes composition and three-dimensional architecture of the dengue virus replication and assembly sites assembly and maturation of the flavivirus kunjin virus appear to occur in the rough endoplasmic reticulum and along the secretory pathway, respectively viral reorganization of the secretory pathway generates distinct organelles for rna replication early dengue virus protein synthesis induces extensive rearrangement of the endoplasmic reticulum independent of the upr and srebp-2 pathway bacterial membrane lipids: where do we stand? crosstalk between reverse cholesterol transport and innate immunity limiting cholesterol biosynthetic flux spontaneously engages type i ifn signaling how low cholesterol is good for anti-viral immunity cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus gc/ms-based metabolomics reveals fatty acid biosynthesis and cholesterol metabolism in cell lines infected with influenza a virus cell surface proteomic map of hiv infection reveals antagonism of amino acid metabolism by vpu and nef systems virology: host-directed approaches to viral pathogenesis and drug targeting type i interferons in anticancer immunity dengue virus-induced autophagy regulates lipid metabolism regulation of lipid droplet size and phospholipid composition by stearoyl-coa desaturase probing the interaction between vesicular stomatitis virus and phosphatidylserine the lipid droplet binding domain of hepatitis c virus core protein is a major determinant for efficient virus assembly expression profiles of genes associated with viral entry in hcv-infected human liver modification of intracellular membrane structures for virus replication the lipid droplet is an important organelle for hepatitis c virus production activation of sterol regulatory element-binding protein 1c and fatty acid synthase transcription by hepatitis c virus non-structural protein 2 hepatitis c virus nonstructural 4b protein modulates sterol regulatory element-binding protein signaling via the akt pathway hepatitis c virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress hepatitis c virus core protein induces hepatic steatosis in transgenic mice plos pathogens: dengue virus capsid protein usurps lipid droplets for viral particle formation dengue virus nonstructural protein 3 redistributes fatty acid synthase to sites of viral replication and increases cellular fatty acid synthesis involvement of fatty acid synthase in dengue virus infection rab18 facilitates dengue virus infection by targeting fatty acid synthase to sites of viral replication dengue virus infection perturbs lipid homeostasis in infected mosquito cells regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in cultured human fibroblasts. comparison of cells from a normal subject and from a patient with homozygous familial hypercholesterolemia the increase in cholesterol levels at early stages after dengue virus infection correlates with an augment in ldl particle uptake and hmg-coa reductase activity denv up-regulates the hmg-coa reductase activity through the impairment of ampk phosphorylation: a potential antiviral target transcriptional activities of nuclear srebp-1a, -1c, and -2 to different target promoters of lipogenic and cholesterogenic genes sterol-regulated transport of srebps from endoplasmic reticulum to golgi: insig renders sorting signal in scap inaccessible to copii proteins proteolytic activation of sterol regulatory element-binding protein induced by cellular stress through depletion of insig-1 regulated endoplasmic reticulum-associated degradation of a polytopic protein: p97 recruits proteasomes to insig-1 before extraction from membranes identification of complexes between the cooh-terminal domains of sterol regulatory element-binding proteins (srebps) and srebp cleavage-activating protein cleavage of sterol regulatory element-binding proteins (srebps) at site-1 requires interaction with srebp cleavage-activating protein. evidence from in vivo competition studies insig required for sterol-mediated inhibition of scap/srebp binding to copii proteins in vitro the biogenesis and functions of lipid bodies in animals, plants and microorganisms emerging roles for lipid droplets in immunity and host-pathogen interactions the life of lipid droplets hepatitis c virus infection activates an innate pathway involving ikk zika virus ns4a and ns4b proteins deregulate akt-mtor signaling in human fetal neural stem cells to inhibit neurogenesis and induce autophagy 25-hydroxycholesterol acts as an amplifier of inflammatory signaling macrophage fatty acid oxidation and its roles in macrophage polarization and fatty acid-induced inflammation control of the innate immune response by the mevalonate pathway the interferon-inducible protein viperin inhibits influenza virus release by perturbing lipid rafts the greasy response to virus infections the antiviral effector ifitm3 disrupts intracellular cholesterol homeostasis to block viral entry type 1 interferons induce changes in core metabolism that are critical for immune function phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas controlling cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-coa reductase (hmgcr) 25-hydroxycholesterols in innate and adaptive immunity key: cord-033488-du8heorx authors: ho, thuong thi; nguyen, giang thu; pham, ngoc bich; le, van phan; trinh, thi bich ngoc; vu, trang huyen; phan, hoang trong; conrad, udo; chu, ha hoang title: plant-derived trimeric co-26k-equivalent epitope induced neutralizing antibodies against porcine epidemic diarrhea virus date: 2020-09-16 journal: front immunol doi: 10.3389/fimmu.2020.02152 sha: doc_id: 33488 cord_uid: du8heorx porcine epidemic diarrhea virus (pedv) is a causative agent of a highly infectious disease with a high mortality rate, especially in newborn piglets in asian countries resulting in serious economic loss. the development of a rapid, safe, effective and cost-efficient vaccine is crucial to protect pigs against pedv infection. the coe antigen is regarded to be a major target for subunit vaccine development against pedv infection. the naturally assembled coe protein forms a homotrimeric structure. in the present study, we successfully produced a trimeric coe protein as a native structure by fusion with the c-terminal isoleucine zipper trimerization (gcn4pii) motif in nicotiana benthamiana, with a high expression level shown via semi-quantified western blots. trimeric coe protein was purified via immobilized metal affinity chromatography (imac), and its trimeric structure was successfully demonstrated by a cross-linking reaction, and a native page gel. a crude extract containing the coe trimer was used for evaluating immunogenicity in mice. after 1 and 2 booster immunizations, the crude extract containing trimeric coe elicited elevated pedv-specific humoral responses, as demonstrated by elisa and western blot analyses. notably, a virus-neutralizing antibody assay indicated that the neutralization activities of sera of mice vaccinated with the crude extract containing coe-gcn4pii were similar to those of mice vaccinated with a commercial vaccine. these results suggest that crude extract containing trimeric coe is a promising plant-based subunit vaccine candidate for pedv prevention. porcine epidemic diarrhea (ped) is a highly infectious disease identified by dehydration, acute watery diarrhea, and a high mortality rate, especially in newborn piglets (1) (2) (3) . pedv, the disease causative agent of ped, spreads to several countries in the world, and resulting in serious economic loss to the swineproduction (4) (5) (6) . the pedv genome comprises five open reading frames (orfs) encoding four structural proteins [the spike (s), envelope, membrane and nucleocapsid proteins] and three non-structural proteins [the replicases orf1a and 1b, and orf3; (7, 8) ]. among the structural proteins, the s protein locating on the surface of pedv virion, plays a key role in the attachment of pedv to host cell receptors (9) (10) (11) . in addition, the s protein is a target for neutralizing antibody induction because it harbors virusneutralizing epitopes and is the principle antigenic determinant (8) . the s protein is naturally assembled in homotrimeric form with a number of predicted glycosylation sites (12) . the co-26k-equivalent epitope (coe epitope) is one of the various neutralizing epitopes on the s protein of pedv which have been recognized (10) . coe is the antigen epitope motif that was identified by the monoclonal antibody 2c10 at the cterminal end of the s protein (13) and the s1d domain (14) . the coe protein is regarded as a critical target for the subunit vaccine development against pedv infection (15) . the neutralizing epitope region of coe contains 139 amino acids within the s1 domain extending from amino acid 499-638 (10, 15) . coe has been expressed as monomer or pentamer structures in various plants including tobacco, rice, and lettuce (15) (16) (17) (18) (19) . mice fed transgenic plants or immunized transgenic rice calli protein extracts containing the monomeric coe protein were found to have both systemic and mucosal immune responses against the coe antigen (15, 16) . the immunogenictity tests of pentameric coe have not been tested. to date, the expression of trimeric coe as a native coe structure in plants and immunogenicity of trimeric coe in animals have not been reported. gcn4, known as the gcn4 leucine zipper, is a yeast transcription factor that is responsible for the reductive reaction of amino acid deficiency (20) . gcn4 can be switched from native dimer form to multimeric states by mutations in the a-and dpositions (21) . gcn4pii is generated by a core that was formed entirely of beta branched residues. gcn4pii has been used for the successful production of trimeric ha proteins of h5n1 viruses (22, 23) , and trimeric s protein of pedv (12) . in which, the gcn4pii was used to trigger trimerization of the proteins of interest, and increase protein stability and solubility. the development of a rapid, valid, safe, and cost-effective vaccination strategy to protect swine against pedv is urgently needed, especially in developing asian countries producing pigs. plant-based subunit vaccines have been reported with several advantages including low manufacturing cost, effortlessness of scaling, high stability and long shelf life [for a review, see (24) ]. in addition, low profit margins in the industrial vaccine development are provided from plants with beneficial and economical platforms (25) . agro-infiltration methods can offer various advantages in production of substantial amounts of recombinant proteins in short times (a few days) after completing vector construction process, and therefore, this system generally exhibits as a very fast and efficient method to produce subunit vaccines (26) . in this study, we generated a plant vector containing a dna sequence encoding the coe protein of the pedv dr13 strain fused gcn4pii as a vector model to investigate the immunogenicity of a plant-based coe antigen in mice compared to that of a commercial vaccine. interestingly, the neutralization activities of sera of mice vaccinated with the crude extract containing coe trimer were similar to those of mice vaccinated with the commercial vaccine. our first successful initial results are expected to provide an alternative strategy to generate a plantbased trimeric coe vaccine against pedv infection for national rapid response. the coe nucleotide sequence encoding for amino acid 499-638 of the coe in the s protein of the attenuated pedv dr13 strain (ncbi accession number jq023161.1) was synthesized, codon optimized commercially in tobacco (genebank accenssion number bankit2361779 optimized mt761690), and then inserted in a pez cloning vector (poch, life science missouri city, texas 77489). the coe gene was amplified from the vector, and replaced for the h5 sequence present in prtra-camv35s-h5-gcn4pii-cmyc-his-kdel (22) to via the bamhi and bsp120i sites. the resulting expression cassette ( figure 1a ) was inserted into the expression vector pcb301-kan (27) via hindiii cleavage, then transformed into the agrobacterium tumefaciens [pgv2260 in c58c1; (28) ] strain via electroporation at 2.5 kv, 25 µf capacitance, and 400 ohm resistance. to express the recombinant protein in planta, the agroinfiltration protocol was performed as described by pham et al. (29) , with some modifications. briefly, bacteria containing the expression vector pcb301-coe-gcn4pii-cmyc-his-kdel and plant vector including hcpro (23) , that was used as a gene silencing suppressor for enhancing the expression levels of targeting proteins in plants (30, 31) , were mixed and diluted in an infiltration buffer [10 mm 2-(n-morpholino) ethanesulfonic acid (mes), 10 mm mgso 4, ph 5.6]. n. benthamiana plants (5 weeks old) were completely infiltrated in an agrobacterium solution, and maintained in a greenhouse. six days after agro-infiltration, plant leaf samples were collected and stored at −80 • c. recombinant coe-gcn4pii protein was purified via immobilized metal affinity chromatography (imac) as described by pham et al. (29) . the oligomeric form of purified coe-gcn4pii protein was determined by a cross-linking reaction that was described by weldon et al. (32) and a native page. leaf samples were ground in liquid nitrogen, mixed with pbs buffer (137 mm nacl, 2.7 mm kcl, 10 mm na 2 hpo4, 1.8 mm kh 2 po4, ph 7.4). the crude extract was clarified by centrifugation twice at 13,000 rpm for 30 min at 4 • c. the coe-gcn4pii protein in leaf crude extract and a number of known concentrations of anti-tnfα-nanobody-elp standard protein [100, 200, 400, 800 ng, (33) ] were separated in a 4-12% sds-page gel and transferred to a pvdf membrane (millipore). the protein expression was determined via western blot that was performed as described by pham et al. (29) using monoclonal anti-c-myc antibody. the coe-gcn4pii protein expression level in the leaf crude extract was semi-quantified via western blotting by comparison of the western blot signal intensities and the coe sequence encoding the coe protein of pedv was fused with the c-terminal trimeric motif (gcn4pii) for generating trimeric coe protein. the recombinant protein was fused to a c-myc tag and his-tag for downstream detection by western blot and protein purification by imac, respectively. the legumine b4 signal peptide and the kdel motif were used to target protein retention in the er. camv35s pro, cauliflower mosaic virus 35s ubiquitous promoter; camv 35s term, cauliflower mosaic virus 35s terminator; asterisk, the molecular weight of proteins was calculated for unglycosylated monomers. (b) a total of 11.25 µg of total soluble proteins in crude extract containing coe-gcn4pii or wild type crude extract and a series of known concentrations of the anti-nanobody-elp standard (100, 200, 400, and 800 ng) were separated in 4-10% gel polyacrylamide. an anti-cmyc tag antibody was used as a primary antibody. hrp-linked goat anti-mouse igg was used as a secondary antibody. the coe content in the plant crude extract was determined by comparing the blot signal intensities and those of the standard protein using imagej software. (c) sds-page analysis of the purification procedure of coe-gcn4pii from total soluble protein extracts using imac; re, raw extract; ft, flow through; w, wash fraction and p, purified fraction in sds-page. (d) western blot analysis of the purification procedure of coe-gcn4pii from total soluble protein extracts using imac. coe-gcnpii was immunologically detected via an anti-his monoclonal antibody. those of the anti-tnfα-nanobody-elp standard protein using imagej software. the study was approved by the ethical committee of the institute of biotechnology, academic of science and technology vietnam (vast), hanoi, vietnam. the crude extracts after 6 days of the storage were mixed with the emulsigen r -d adjuvant (mvp technologies, 4805 g street, omaha, ne 68117, usa) with a ratio of 4:1 (v/v), respectively. three groups of 6-8-week-old female balb/c mouse (five per group) respectively numbered g1, g2, g3 were subcutaneously vaccinated at days 0, 14 and 28 with 200 µl of emulsigen r -d adjuvant-formulated crude plant extracts of non-transgenic plants as negative control) or 200 µl of the commercial vaccine against the pedv dr13 strain (4 × 10 6 tcid50/dose, ctc vacc ped, korea) as positive control or 200 µl of emulsigen r -d adjuvant-formulated crude plant extracts containing 18.76 µg of coe-gcn4pii protein that was semiquantified by western blotting. the bloods of mice were collected at seven days after the second and the third immunization via the retro-orbital sinus. all mouse sera were collected separately by centrifugation. to inactivate the non-specific complement, all mouse sera were incubated at 56 • c for 30 min before being stored at −20 • c until used. pedv propagation and purification were carried out as described by hofmann et al. (34) , with modifications. the vero e6 cell line (atcc r crl-1587tm) was propagated and incubated at 37 • c in dulbecco's modified eagle medium (dmem) including 10% fetal bovine serum (fbs) and antibiotics (100 µg/ml penicillin/streptomycin). the cells were cultured in a 5% co 2 at 37 • c. then, the pedv-dr13 strain was propagated in vero cells with 10 µg/ml trypsin treated-tosyl phenylalanyl chloromethyl ketone (tpck) (worthington, lakewood, nj, usa). after 36 h of cultivation, when all cells showed 100% cytopathogenic effects with morphological changes using cell morphology evaluation by inverted light microscopy, the infective culture fluid was harvested andfreeze-thawed three cycles. next, cellular debris was pelleted by centrifugation at 10,000 ×g for 30 min. the clarified supernatant was then enriched by ultracentrifugation at 30,000 ×g. sucrose density gradient centrifugation (20, 40, 60%) was then used to purify pedv. to detect pedv-specific igg mouse antibodies, 1 µg of purified pedv dr13 was loaded onto 3-wells of one sds-page gel. two-fold serial dilutions of serum samples after the 2nd immunization were prepared in α-minimum essential medium (mem) including 1% antibiotic-anti-mycotic solution (invitrogen, usa). then, 10 3 tcid50/0.1 ml of pedv dr13 was added with an equal volume of diluted serum, and the virus-serum mixture was maintained at 37 • c for 1 h. next, 100 µl of each virus-serum mixture was introduced onto vero cell monolayers in 96-well plates. the virus-serum mixture was removed after adsorption at 37 • c for 1 h. the plates were then washed for 10 min with pbs. finally, 200 µl of serum-free α-mem medium containing trypsin were placed into each well and maintained at 37 • c for 6 days. for controlling this assay, the virus control, positive serum control, negative serum control and blank control were used. the serum neutralization titres (sn titres) were defined as the highest serum dilution and consequent on inhibition of the cytopathic effect. statistical analyses for the elisa test and virus neutralization assay were carried out in sigma plot software using a t-test. the difference between sample data mean was compared and is showed as the x ± standard deviation (sd). p-values that were <0.05 were determined to be significantly different. the expression of the coe-gcn4pii protein in planta was successful demonstrated via separation of sds-page under reducing conditions, blotted and detected by western blot using an anti-c-myc monoclonal antibody ( figure 1b) . the apparent band shown in figure 1b with coe molecular weight was larger than the expected coe size predicted from the coe polypeptide sequence (26 kda). one of the possible reasons for the increase in the molecular weight of coe protein is that the n-glycosylation sites located within the coe-s protein of pedv at amino acids 511 and 533 may influence the electrophoretic behavior during the page separation (16, 17) . no coe protein was detected in wild type crude extract. the coe-gcn4pii protein expression level in leaf crude extract was semiquantified by western blotting. the coe-gcn4pii protein was quantified with a high expression level of ∼4% of the total soluble protein. the amount of plant-produced coe-gcn4pii protein was found to be 234 mg/kg wet weight. several publications have reported the accumulation of coe in transgenic plants (15, 17, 19) ; however, the expression level was still lower than that in our report. we demonstrated that the accumulation of coe in tobacco leaves could be significantly improved by codon optimization for plant expression by using a strong expression system, such as agro-infiltration. the purification process was validated by collecting samples from each step of the purification procedure to analyse via sds-page and western blot using a monoclonal anti-his antibody (figures 1c,d) . these results indicate the enrichment and successful purification of coe-gcn4pii protein from n. benthamiana leaves. the oligomeric state of the coe-gcn4pii protein was successfully determined by a cross-linking reaction with bs3 and a separation under native condition by native-page. a band with a molecular weight of approximately 100 kda corresponding to molecular weight of trimeric coe form was detected (figures 2a-c) . these results revealed that the trimeric coe protein was successfully generated in planta by the fusion of coe with gcn4pii motif. since animal vaccine development should minimize downstream processing in pig immunizations, the crude plant extract was chosen for testing immunogenicity in mice. interestingly, after storing the crude extract containing trimeric coe at 4 • c for six days, the coe content was still stable, as revealed by western blotting (see supplementary file) . the antibody-mediated humoral immune responses from vaccinated mice were first examined against the purified pedv dr13 strain by western blot (figure 3a) . before vaccination, pedv-specific igg antibody responses were not detected in mice. the western blots in figure 3a showed that there was a band with molecular weight of over 245 kda detected in mice groups g2 (vaccinated with the commercial vaccine against pedv) and g3 (vaccinated with crude extract containing coe-gcn4pii) that was larger than the expected size of s protein pedv (151.38 kda). the larger band size obtained in western blot might be explained that might be due to the influence on electrophoretic behavior by the 29 potential n-glycosylation sites locating within the s protein of pedv during the page separation (36) . the results indicate that pedv-specific igg antibody responses were induced in mice groups g2 and g3 after the 2nd immunization and the 3rd immunization, and the antibody responses were strongly increased in both mice groups g2 and g3 after the 3rd immunization. in contrast, no pedv-specific igg antibody response was detected in mice group g1 vaccinated with crude extracts of non-transgenic plants. different levels of pedv-specific igg, iga and igm antibodies in each mouse sera group were calculated as the reciprocals of the geometric mean titer of the five mice of each group. end-point titer of each mouse sera group was compared by the t-test. the results showed that after the 2nd immunization, crude extract containing coe-gcn4pii (g3) elicited elevated levels of igg, iga and igm antibody responses against pedv (figures 3b-d) , figure 3 | determination of the levels of pedv-specific igg, iga, and igm antibody responses via a western blot and elisas. (a) sera from five mice from each group immunized with a negative control (wild-type crude extract, g1) or a positive control (commercial vaccine, g2) or the crude extract containing coe-gcn4pii (g3) were mixed, diluted 200 times and used as a primary antibody for detecting 1 µg of purified pedv antigen. hrp-linked goat anti-mouse igg was used as a secondary antibody. in total, 500 ng of purified pedv per well was coated on a plate. each serum was measured in triplicate. the sera were serially diluted and analyzed via elisa. different levels of pedv-specific igg (b), iga (c), and igm (d) antibodies in each mouse sera group were calculated as the reciprocal of the geometric mean titer of the five mice of each group vaccinated with the negative control (wild-type crude extract, g1) or the positive control (commercial vaccine, g2) or the crude extract containing coe-gcn4pii (g3). end-point titer of igg, iga, or igm antibodies against pedv of each mouse sera group was compared using the t-test (sigmaplot), and is presented. *p< 0.05 was defined as a statistically significant difference. reaching end-point antibody titres of 1:216 178, 1:45 429, 1:37 801, respectively. the levels of igg, iga and igm antibody responses in mice group g3 were strongly enhanced after the 3rd immunization, having end-point antibody titres of 1:383 711, 1:195 985, 1:65 198, respectively. notably, no statistically significant difference in pedv-specific igg antibody responses was obtained between mice group g3 and those in mice group g2, with p-values of 0.056 after the third immunization (p < 0.05). therefore, plant crude extract containing the trimeric coe protein had the level of igg antibodies similar to that of commercial vaccines against the pedv dr13 strain after the third injection. however, level of pedv-specific iga and igm antibody responses in mice group g3 were lower than those in mice group g2 after the second and the third immunization. levels of igg, iga and igm antibody responses against pedv found in the sera of negative control mice group g1 were very poor. the cytopathic effect caused by the wild-type pedv dr13 virus of all serum samples was determined using a microscope and a representative cytopathic effect result observed under microscope of a single dilution of a serum from each group was presented (figure 4a) . the highest dilutions of sera that caused cytopathic effect inhibition were defined as the serum figure 4 | virus neutralization assay. (a) cytopathic effect caused by wild-type pedv dr13 virus at the dilution of 1:32 of a single serum from a mouse from the groups vaccinated with the negative control (wild-type plant extract, g1) or the positive control (commercial vaccine, g2) or the crude extract containing coe-gcn4pii (g3) was determined by using a microscope. (b) determination of serum neutralization titer. serum samples were firstly incubated to inactivate for 30 min at 56 • c. two-fold serial dilutions (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256) were applied. a pedv dr13 strain (200 µl 10 3 tcid50/0.1 ml) was mixed with an equal volume of diluted sera. the virus control, positive serum control, blank control, negative serum control, and were used for controlling the assay. the serum neutralizing titres (sn titres) were expressed as the highest serum dilution and consequence on the inhibition of cytopathic effect. statistical analyses were performed using the t-test (sigmaplot) and are shown. a single dot indicates the sn value of a single mouse serum. the sd is included on a single dot that corresponds to the sn data variation of a single mouse serum with three replications. the bars indicate the average value of the test groups. *p < 0.05 was defined as a statistically significant difference. neutralizing antibody titres. the serum neutralizing antibody (sn) titer of every single mouse serum was presented in each dot ( figure 4b ). as expected, cytopathic effect inhibition of sera from the negative control group was observed in a low serum dilution, which produced neutralizing geometric mean titres of this group as low as 1. in contrast, sera of mice vaccinated with the crude extract containing coe-gcn4pii and the commercial pedv vaccine (the positive control) had the ability to neutralize pedv with high neutralizing geometric mean titres: 57.6 and 61.86, respectively. these titres were significantly different from those of the negative control group. notably, the serum neutralization titer in the crude extract containing coe-gcn4pii (g3) was not significantly different compared to that of sera from mice vaccinated with the commercial vaccine, with a p-value of 0.187. in this study, the immunogenicity in mice group g3 vaccinated with the coe trimer in crude extract was compared to those of mice positive control group g2 vaccinated with commercial pedv vaccine and mice negative control group g1 vaccinated with crude extracts from non-transgenic plants. as expected, western blot results demonstrated that pedvspecific igg antibodies were presented in mice group g3 and mice group g2 after the second and the third immunization. more interestingly, the elisa analyses illustrated that crude extract containing coe-gcn4pii (g3) elicited strong levels of igg antibody responses against pedv, and especially the level of pedv-specific igg antibody responses found in mice group g3 was similar to that in mice group g2 after the third immunization. mucosal immune responses play an important role in the defense of pedv, and mucosal iga antibody response was found to correlate with the protection against pedv infection (37, 38) , however igg antibody-mediated humoral response is also essential to protect the neonatal pig against pedv infection (37, 39) . early publications showed the important role of igg in protection of gastrointestinaltract (40, 41) . in this study, trimeric coe antigen (g3) was vaccinated in mice via subcutaneous route that is a conventional vaccination route widely used for various human and animal vaccines to elicit igg antibody-mediated humoral responses. interestingly, beside pedv-specific igg, pedv-specific iga and igm antibodies were presented in mice group g3, however they were lower as compared to those found in mice group g2. our results are comparable to several recent evidence studies that subcutaneous route can induce both antigenspecific igg antibody, and antigen-specific iga and igm antibodies in sera (42) (43) (44) (45) (46) . moreover, su and his co-workers proposed that under some circumstances (antigen, adjuvant, delivery vehicle) systemic routes may induce systemic immune responses and muscosal immune responses against infectious diseases (46) . in addition, the ability of the neutralizing antibodies induced in mouse sera to neutralize pedv via binding to coe epitopes related to pedv neutralization was further determined. after virus neutralization, there was an inhibition generated by neutralizing antibodies to the virus's infection cycle, containing surface binding, fusion, entry, endocytosis, and replication (47) . the assessment of pedv-neutralizing activity illustrated that there was no statistically significant difference between the serum neutralization titres of mice vaccinated with the crude extract containing trimeric coe (g3) and that of mice vaccinated with the commercial vaccine (g2), with a p-value of 0.187. therefore, we concluded that plant crude extract containing the trimeric coe protein had a strong immunogenicity and induced a neutralizing antibody titer similar to that of the commercial vaccine against the attenuated pedv dr13 strain. the enhancement of neutralizing antibody responses induced in animals after vaccination is an important requirement for vaccine development due to the powerful correlation of vaccine efficacy with neutralizing antibodies for numerous commercial vaccines (48) . the crude extract containing trimeric coe (g3) showed neutralizing activity, with a geometric mean titer of 1:57.6. a high neutralizing antibody titer indicated that mice subcutaneously administered the crude extract containing trimeric coe possessed a strong ability to neutralize pedv. to increase the immune response, especially mucosal immune responses against pedv, oral mucosal vaccination with coe but not subcutaneous administration in animals has been previously presented to induce anti-pedv mucosal immune responses (15, 49) . in addition, since pedv causes mainly intestinal infections, the coe antigen was fused with dendritic cell-targeting peptide (dcpep) and m celltargeting peptide (col) for targeting intestinal microfold (m) cells and dendritic cells (dcs) (15, 49, 50) . the neutralizing activity mouse sera orally provided genetically engineered lactobacillus but not plant extracts expressing coe targeting m cells or dcs or both has been previously reported (49, 50) . when compared to the publication of ma and coworkers, the neutralizing antibody titres obtained by oral administration in mice with the recombinant lactobacillus casei strains expressing the pedv coe antigen on the cell surfaces by fusion with dcpep or col or both dcpep and col were 1:24, 1:24, and 1:36, respectively (49) , which were lower than the neutralizing antibody titres observed in this study. these results showed that crude extract containing trimeric coe can be a promising vaccine candidate against pedv infections. in summary, the trimeric coe protein was successfully produced in plants with high expression levels. crude extract containing trimeric coe elicited strong humoral immune responses and elevated neutralizing antibody titres against pedv. in particular, the neutralizing activities of mice vaccinated with the crude extract containing coe-gcn4pii were similar to those of mice vaccinated with the commercial vaccine. these results suggest that crude extract containing trimeric coe might be a potential subunit vaccine antigen against pedv infection. further studies will focus on investigating immune efficacy and protection against pedv in piglets. all datasets presented in this study are included in the article/supplementary material. the animal study was reviewed and approved by the principles of the basel declaration and recommendations of arrive guidelines, ethical committee of institute of biotechnology, academic of science and technology vietnam (vast), hanoi, vietnam on the use of animals for research. hc, np, and th designed the research. th and gn constructed vectors and performed transient expression. th purified protein and performed the cross-linking reaction, performed elisa and western blotting analyses, and performed the calculations, all data analysis and wrote the manuscript. vl and tt purified the pedv dr13 strain and carried out the virus-neutralizing antibody assay. uc, hp, np, tv, and hc revised the manuscript. hc is the corresponding author and holds all the responsibilities related to this manuscript. porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis emergence of porcine epidemic diarrhea virus in the united states: clinical signs, lesions, and viral genomic sequences identification of neutralizing monoclonal antibodies targeting novel conformational epitopes of the porcine epidemic diarrhoea virus spike protein outbreak-related porcine epidemic diarrhea virus strains similar to us strains, south korea ped virus reinfecting us herds. virus estimated to have killed 7 million-plus pigs phylogenetic analysis of the spike (s) gene of the new variants of porcine epidemic diarrhoea virus in taiwan diseases of swine porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines efficacy of inactivated variant porcine epidemic diarrhea virus vaccines in growing pigs identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus coronavirus spike proteins in viral entry and pathogenesis cryoelectron microscopy structure of a coronavirus spike glycoprotein trimer isolation and characterization of a new porcine epidemic diarrhea virus variant that occurred in korea in 2014 deciphering key features in protein structures with the new endscript server immunogenicity of a neutralizing epitope from porcine epidemic diarrhea virus: m cell targeting ligand fusion protein expressed in transgenic rice calli induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen expression of the synthetic neutralizing epitope gene of porcine epidemic diarrhea virus in tobacco plants without nicotine expression of a synthetic neutralizing epitope of porcine epidemic diarrhea virus fused with synthetic b subunit of escherichia coli heat labile enterotoxin in rice endosperm expression of a cholera toxin b subunitneutralizing epitope of the porcine epidemic diarrhea virus fusion gene in transgenic lettuce (lactuca sativa l) gcn4 protein, a positive transcription factor in yeast, binds general control promoters at all 5' tgactc 3' sequences a switch between two-, three-, and four-stranded coiled coils in gcn4 leucine zipper mutants elpylated haemagglutinins produced in tobacco plants induce potentially neutralizing antibodies against h5n1 viruses in mice neutralizing immune responses induced by oligomeric h5n1-hemagglutinins from plants the case for plantmade veterinary immunotherapeutics plant-based oral vaccines against zoonotic and non-zoonotic diseases plant-made vaccines for humans and animals a mini binary vector series for plant transformation efficient octopine ti plasmid-derived vectors for agrobacteriummediated gene transfer to plants nanodiamond enhances immune responses in mice against recombinant ha/h7n9 protein optimization of elastin-like polypeptide fusions for expression and purifcation of recombinant proteins in plants a chemically inducible cucumber mosaic virus amplicon system for expression of heterologous proteins in plant tissues enhanced immunogenicity of stabilized trimeric soluble influenza hemagglutinin elpylated anti-human tnf therapeutic single-domain antibodies for prevention of lethal septic shock quantitation, biological and physicochemical properties of cell culture-adapted porcine epidemic diarrhea coronavirus (pedv) a statistically defined endpoint titer determination method for immunoassays sequence of the spike protein of the porcine epidemic diarrhoea virus mucosal and systemic isotypespecific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus cross protective immune responses in nursing piglets infected with a us spike-insertion deletion porcine epidemic diarrhea virus strain and challenged with an original us pedv strain immune responses to porcine epidemic diarrhea virus (pedv) in swine and protection against subsequent infection role of maternally derived circulating antibodies in protection of neonatal swine against porcine group a rotavirus serum and intestinal isotype antibody responses to wa human rotavirus in gnotobiotic pigs are modulated by maternal antibodies bovine respiratory syncytial virus iscoms-immunity, protection and safety in young conventional calves immunogenicity of a vaccine formulated with the chlamydia trachomatis serovar f, native major outer membrane protein in a nonhuman primate model retinoic acid induces homing of protective t and b cells to the gut after subcutaneous immunization in mice immunization with recombinant macaque major histocompatibility complex class i and ii and human immunodeficiency virus gp140 inhibits simianhuman immunodeficiency virus infection in macaques induction of mucosal immunity through systemic immunization: phantom or reality occupancy and mechanism in antibody-mediated neutralization of animal viruses maternal antibodies, childhood infections, and autoimmune diseases oral recombinant lactobacillus vaccine targeting the intestinal microfold cells and dendritic cells for delivering the core neutralizing epitope of porcine epidemic diarrhea virus oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus coe antigen: a promising vaccine strategy against pedv all authors contributed to the article and approved the submitted version. this study was financed by vast through the project: study on the expression of s1 oligomer of porcine epidemic diarrhea virus (pedv) in nicotiana benthamiana for the next generation of vaccine development, listed in scientific program: biotechnology, code: vast02.02/18-19. we would like to thank dr. song daesub for supplying the pedv dr13 strain. we also appreciate dr. do thi thao from ibt, hanoi, vietnam for the mouse experiments. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.02152/full#supplementary-material key: cord-257116-6td3efjw authors: zhou, yanrong; wu, wei; xie, lilan; wang, dang; ke, qiyun; hou, zhenzhen; wu, xiaoli; fang, ying; chen, huanchun; xiao, shaobo; fang, liurong title: cellular rna helicase ddx1 is involved in transmissible gastroenteritis virus nsp14-induced interferon-beta production date: 2017-08-09 journal: front immunol doi: 10.3389/fimmu.2017.00940 sha: doc_id: 257116 cord_uid: 6td3efjw transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor 3 (irf3) and nuclear factor-kappab (nf-κb) in porcine kidney (pk-15) cells. screening of tgev-encoded proteins demonstrated that non-structural protein 14 (nsp14) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf3. further analysis showed that nsp14 interacted with ddx1, a member of the dexd/h helicase family. knockdown of ddx1 by specific small interfering rna (sirna) significantly decreased nsp14-induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx1-specific sirna, indicating the vital role of ddx1 to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx1 to the induction of ifn-β response initiated by tgev and demonstrated that nsp14 is an important ifn inducer among the tgev-encoded proteins. transmissible gastroenteritis virus (tgev), an enteropathogenic coronavirus (cov) of porcine, causes lethal watery diarrhea and severe dehydration in piglets and leads to severe economic losses in the swine industry. unlike most covs that antagonize type i interferon (ifn) production, previous studies showed that tgev infection induces ifn-i production both in vivo and in vitro. however, the underlying mechanism(s) remain largely unknown. in this study, we found that tgev infection significantly facilitated ifn-β production as well as activation of the transcription factors ifn regulatory factor 3 (irf3) and nuclear factor-kappab (nf-κb) in porcine kidney (pk-15) cells. screening of tgev-encoded proteins demonstrated that non-structural protein 14 (nsp14) was the most potent ifn-β inducer and induced ifn-β production mainly by activating nf-κb but not irf3. further analysis showed that nsp14 interacted with ddx1, a member of the dexd/h helicase family. knockdown of ddx1 by specific small interfering rna (sirna) significantly decreased nsp14-induced ifn-β production and nf-κb activation. furthermore, tgev-induced ifn-β production and ifn-stimulated gene (isg) expression were decreased in cells transfected with ddx1-specific sirna, indicating the vital role of ddx1 to tgev-induced ifn-β responses. in summary, our data revealed a potential coactivator role of host rna helicase ddx1 to the induction of ifn-β response initiated by tgev and demonstrated that nsp14 is an important ifn inducer among the tgevencoded proteins. keywords: transmissible gastroenteritis virus, non-structural protein 14, ddx1, interferon-beta, innate immune response, pattern-recognition receptors introduction transmissible gastroenteritis virus (tgev) is a member of the alphacoronavirus genus within the family coronaviridae in the order nidovirales. tgev infection mainly causes acute enteric disease characterized by lethal watery diarrhea, severe dehydration, and high mortality in suckling piglets less than 3 weeks old, which has led to severe economic losses in the global swine industry since gene name sirna sequence hddx1 (sense) ggagcuucugauaauuggagguguu hddx1 (anti-sense) aacaccuccaauuaucagaagcucc pddx1 (sense) gaaagaccuuggucuggcauuugaa pddx1 (anti-sense) uucaaaugccagaccaaggucuuuc the first outbreak in 1933 in ilinois, usa (1, 2) . tgev contains a single-stranded, positive-sense rna genome of about 28.5 kb (3) , including at least nine open reading frames (orfs). two slightly overlapping orfs, orf1a and orf1b, located at the 5' two-thirds of the viral genome, encode a replicase complex that is proteolytically processed into 16 non-structural proteins (nsp1 to 16) (4). structural proteins, nucleocapsid (n) protein, membrane (m) glycoprotein, spike (s) glycoprotein, a small envelope (e) glycoprotein, and accessory proteins 3a, 3b, and 7 are encoded by genes located at the 3′ end (5) . as early as 1981, the presence of high levels of type i interferon (ifn-i) activity in the digestive tract of tgev-infected newborn piglets was first observed (6) . thereafter, charley et al. reported that tgev infection in human or bovine peripheral blood mononuclear cells also induced high ifn-α production (7) . subsequently, bosworth et al. demonstrated that 2′,5′-oligoadenylate synthetase (oas), a well-known ifn-stimulated gene (isg), was increased in tgev-infected pigs (8) . our previous quantitative proteomics analysis revealed that tgev infection induced canonical ifn-i signaling through janus kinase signal transducer and activator of the transcription 1 (jak-stat1) pathway, and eight tested isgs, including ifninduced protein with tetratricopeptide repeats 1 (ifit1), ifit2, ifit3, oas1, oas2, mx1, mx2, and isg15 were upregulated after tgev infection (9) . these early results indicated that tgev infection activated the ifn-i pathway in vitro and in vivo. however, the underlying mechanism(s) utilized by tgev to induce ifn-i, and especially which viral protein(s) contribute to it, remain largely unclear. the ifn-i response is a well-known innate immune reaction that occurs in response to virus infection and considered as an important bridge between innate and adaptive immunity. nuclear factor-kappab (nf-κb) and ifn regulatory factor 3 (irf3) are two critical transcription factors for the regulation of ifn-i production. secreted ifn-i then stimulates the jak-stat1 signaling pathway to induce the expression of numerous isgs, which collaborate to regulate the replication of virus (10) . many viruses antagonize ifn responses to benefit their propagation, and some viruses such as human immunodeficiency virus-type 1 (11), vesicular stomatitis virus (vsv) (12), influenza a virus (iav) (13) , encephalomyocarditis virus (14), reovirus (15), herpes simplex virus 2 (hsv2) (16), respiratory syncytial virus (17), newcastle disease virus (18) , and sendai virus (sev) (19) initiate innate immune responses. different viruses employ different mechanisms to regulate innate immune responses. for example, hsv2 induces ifn-α/β production through toll-like receptor 9 (tlr9), dna-dependent activator of ifn regulatory factors (dai), and ifn-inducible 16 (16) . for iav infection, at least tlr3, tlr7, retinoic acid-inducible gene i (rig-i), and pyrin domain-containing 3 (nlrp3) are responsible for the detection of iav and subsequent innate immune responses (13) . elucidating the mechanisms through which viruses regulate innate immune responses will help us understand the interactions between virus and host. this study sought to identify tgev-encoded protein(s) involved in the induction of ifn-β production. our results revealed that tgev nsp14 was the best inducer of the ifn-β pathway among the tgev-encoded proteins. mechanistically, nsp14 activates nf-κb but not irf3, and it interacts with rna helicase ddx1, which in turn activates ifn-β production. porcine kidney (pk-15) cells and hek-293t cells were cultured in dulbecco's modified eagle's medium (invitrogen, carlsbad, ca, usa) supplemented with 10% fetal bovine serum in a humidified incubator with 37°c/5% co2. tgev strain wh-1 (genbank accession no. hq462571) was propagated and titered in pk-15 cells. recombinant vsv-expressing green fluorescent protein (vsv-gfp) was generously provided by prof. zhigao bu from the harbin veterinary research institute, china. rabbit polyclonal antibodies against p65, irf3, phosphorylated irf3 (p-irf3), and ddx1 were purchased from abclone (wuhan, china). rabbit polyclonal antibody against phosphorylated p65 (p-p65) was purchased from cell signaling technology (beverly, ma, usa). anti-β-actin antibody was purchased from beyotime (nantong, china). mouse monoclonal antibodies (mabs) against hemagglutinin (ha) and flag were purchased from medical and biological laboratories (mbl, nagoya, japan). mab against tgev n protein was prepared by our laboratory. horseradish peroxidase (hrp)-conjugated goat anti-rabbit antibody and hrp-conjugated goat anti-mouse antibody were purchased from mbl. alexa fluor 594-conjugated donkey anti-rabbit igg, alexa fluor 594-conjugated donkey anti-mouse igg, and alexa fluor 488-conjugated donkey antimouse igg were obtained from santa cruz biotechnology inc. (santa cruz, ca, usa). expression plasmids of tgev-encoded proteins used in this study were constructed by rt-pcr amplification from the genomic rna of tgev strain wh-1 and cloned into expression vector pcaggs-ha. the details of primers used for pcr clone are available on request. the p65 gene was derived from human rela cdna and cloned into pegfp-c1 vector. the ddx1 expression plasmid was constructed by rt-pcr amplification from the cdna of pk-15 cells and cloned into pcmv-tag2b vector. luciferase reporter plasmids p125-luc (ifn-β-luc), 4 × prdiii/i-luc (referred to as irf3-luc), 4 × prdii-luc (referred to as nf-κb-luc), and the internal control plasmid prl-tk have been described previously (20) . small interfering rna (sirna) targeting ddx1 or negative control sirna (sinc, invitrogen) were each transfected at a final concentration of 50 nm. the sirna sequences used here are listed in table 1 . np-40, 20 nm phenylmethylsulfonyl fluoride], and protein concentration was measured and adjusted. for each immunoprecipitation, 500 µg of cell lysate protein was incubated with 2 µg of indicated antibody and 25 µl of protein a/g-agarose (beyotime) overnight at 4°c. after three washes with 1 ml lysis buffer, precipitates were subjected to 10% sds-page and subsequently analyzed with immunoblot analysis using the indicated antibodies. porcine kidney (pk)-15 cells were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with pre-cooled methanol for 10 min, blocking with 5% bovine serum albumin for 45 min, incubated with the indicated primary antibodies for 1 h, followed by staining with specific alexa fluor-conjugated secondary antibodies for 1 h. the cells were subsequently stained with 4',6-diamidino-2-phenylindole (beyotime) for 15 min. after washing with pbs, fluorescent images were obtained using an olympus fv10 laser scanning confocal microscope (olympus, japan). total rnas were extracted using trizol reagent (invitrogen). real-time rt-qpcr was performed using sybr green real-time pcr master mix (toyobo biologics, osaka, japan) in the abi prism 7000 sequence detection system (applied biosystems). individual transcripts in each sample were assayed three times. the fold change in gene expression relative to normal was calculated using the delta-delta cycles to threshold (δδct) method. primers ( table 2) were designed using primer express software (version 3.0; applied biosystems, carlsbad, ca, usa). all experiments were performed at least three times with reproducible results. data are presented as the mean ± sd. statistical analysis was performed using one-way anova without interaction terms followed by dunnett's for multiple comparisons. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number 00024534) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. previous studies demonstrated that tgev infection potently induced ifn-α (6, 21, 22) , as well as ifn-β in ipec-j2 and swine testicular (st) cells (23) (24) (25) . however, whether tgev infection induces ifn-β in pk-15 cells remains unknown. to explore the effect of tgev on ifn-β, dual luciferase assays were performed. pk-15 cells were transfected with ifn-β-luc and prl-tk. after the induction of ifn-i is reliant on the co-regulation of transcription factors irf3 and nf-κb. to investigate the potential mechanism(s) involved in the ifn-β production by tgev infection, the effect of tgev on irf3 and nf-κb promoters were also tested. as displayed in figures 1c,d , tgev infection also upregulated irf3 and nf-κb promoter activity dosedependently, indicating that irf3 and nf-κb are involved in the ifn-β production by tgev infection. because of the high sensitivity of vsv-gfp to ifn, vsv-gfp expression is commonly monitored for ifn detection. to further evaluate the ifn-β response in tgev-infected cells, a vsv-gfp-based ifn detection assay was performed. pk-15 cells were infected or mock infected with sev or tgev (moi = 0.02, 0.1, 0.5). then, cell supernatants were collected, uv-irradiated, and then transferred onto fresh pk-15 cells. after 24 h, cells were infected with vsv-gfp and observed under a fluorescence microscope at 16 hpi. as a positive control, supernatants collected from sev-treated cells suppressed vsv-gfp replication prominently compared with the negative control group (mock). in accordance with the results of dual luciferase assays described above, tgev infection limited the replication of vsv-gfp in a dose-dependent manner (figure 1e) . these results suggested that tgev infection increased ifn-β production. transmissible gastroenteritis virus encodes 16 non-structural proteins (nsp1-16), four structural proteins (n, m, s, e), and three accessory proteins (orf7, orf3a, orf3b) . to identify the key viral protein(s) involved in ifn-β induction, an ifn promoterreporter system was employed to screen tgev-encoded proteins for their relative capacities to activate the ifn-β promoter. hek-293t cells were cotransfected with ifn-β-luc, prl-tk, and different tgev protein expression vectors. as shown in figure 2a , nsp14 was the most significant inducer of ifn-β production. furthermore, similar to sars-(coronavirus) cov (26), tgev m glycoprotein also potently mediated ifn-β induction. because nf-κb and irf3 are necessary transcription factors for ifn-β production, we examined the effects of all tgev-encoded proteins on irf3 and nf-κb promoter activity. interestingly, nsp14 upregulated an approximately 8.8-fold change in nf-κb promoter activity, but only an approximately 1.8-fold change in irf3 promoter activity (figures 2c,e) . m glycoprotein induced a higher fold change in irf3, but a lower fold change in nf-κb compared with nsp14. because m glycoprotein has been investigated previously (27), we focused on nsp14. to confirm the ability of nsp14 to induce ifn-β production, large-scale screen experiment was also conducted in pk-15 cells, a permissive cell line of tgev infection. as shown in figures 2b,d,f, nsp14 was also a potent ifn inducer that mainly induced activation of nf-κb but not irf3 promoter in pk-15 cells. to confirm the above large-scale screen results, increasing doses (0.2, 0.4, 0.8 µg) of pcaggs-ha-nsp14 and ifn-β-luc, irf3-luc or nf-κb-luc together with prl-tk were cotransfected into hek-293t cells. in line with the results in figure 2 , nsp14 enhanced the activation of ifn-β and nf-κb in a dose-dependent manner (figures 3a-c) . interestingly, nsp14 induced the activation of nf-κb to a greater extent than that of irf3, indicating nf-κb has a fundamental role in nsp14induced ifn-β activation. similar results were also obtained in pk-15 cells (figures 3d-f) . nf-κb and irf3 activation are characterized by the phosphorylation and subsequent translocation of p65 (nf-κb subunit) or irf3 to the nucleus, respectively (28) (29) (30) . next, we investigated the role of nsp14 in the phosphorylation of p65 and irf3. hek-293t cells were transfected with increasing amounts of pcaggs-ha-nsp14, and cell lysates were examined for the expression levels of p-p65 or p-irf3 and total p65 or irf3 at 30 h post-transfection. as shown in figure 3g , nsp14 overexpression had no significant effect on the amount of p65, irf3, and p-irf3; however, markedly increased p65 phosphorylation levels, indicating the activation of nf-κb, rather than irf3 is associated with nsp14-induced ifn-β production. therefore, the subcellular location of p65 was further investigated after nsp14 overexpression. as shown in figures 3h,i , ectopic expression of nsp14 resulted in the nuclear translocation of overexpressed p65 in pk-15 cells ( figure 3h ) and endogenous p65 in hek-293t cells (figure 3i ). early studies suggested that nsp14 of cov ibv and sars-cov interacts with host protein ddx1 (31) . therefore, we investigated whether tgev nsp14 also interacts with ddx1 by co-ip. hek-293t cells were cotransfected with pcaggs-ha-nsp14 and pcmv-tag2b-ddx1. because ddx1 is a dexd/h-box helicase and is associated with rna metabolism, the lysates were treated with rnase to avoid the effect of rna on the co-ip experiment. as shown in figure 4a , flag-tagged ddx1 was coprecipitated by ha-tagged nsp14, indicating the interaction between nsp14 and ddx1. reversed ip with flag antibody further confirmed this interaction ( figure 4b) . to test whether the colocalization of nsp14 and ddx1 occurs, a prerequisite for the interaction, pk-15 cells were transfected with pcaggs-ha-nsp14 or empty vector and fixed at 30 h posttransfection. ha-tagged nsp14 protein was detected with mouse anti-ha antibody, and ddx1 was detected with rabbit anti-ddx1 antibody. the results revealed that nsp14 was colocalized with ddx1 and distributed both in the cytoplasm and nucleus (figure 4c) , which further confirmed the interaction between tgev nsp14 and ddx1. because nsp14 activates ifn-β and interacts with ddx1, we investigated whether ddx1 is involved in nsp14-induced ifn-β production. synthesized sirna targeting human ddx1 (hsiddx1), which efficiently decreases the expression of endogenous ddx1 mrna ( figure 5a ) and protein (figure 5b) , was selected. next, hek-293t cells were transfected with hsiddx1 or sinc, followed by co-transfection of ifn-β-luc or nf-κb-luc, prl-tk, along with pcaggs-ha-nsp14 or empty vector. the results revealed that knockdown of ddx1 significantly decreased nsp14-induced promoter activity of ifn-β ( figure 5c ) and nf-κb ( figure 5d) . moreover, mrna expression levels of nsp14-induced ifn-β were downregulated values are the mean ± sd of three independent tests. **p < 0.01 or ***p < 0.001 compared with empty vector group. (g) hek-293t cells were transfected with increasing quantities (1, 2, 4 µg) of pcaggs-ha-nsp14 or empty vector for 30 h, and then subjected to immunoblotting with antibodies specific for endogenous irf3, phosphorylated irf3 (p-irf3), p65, or p-p65. anti-ha mouse antibody was used to confirm the expression of nsp14. β-actin expression was used as a loading control. the ratio of phosphorylated/total p65 and phosphorylated/total irf3 was analyzed using imagej software. (h) pk-15 cells were cotransfected with plasmids encoding ha-tagged nsp14 protein or empty vector together with plasmids encoding egfp-tagged p65 protein. then, the cells were fixed and immunostained with anti-ha monoclonal antibodies (mabs) to observe the nuclear translocation of overexpressed p65 using confocal microscopy. (i) hek-293t cells were transfected with plasmids encoding ha-tagged nsp14 protein or empty vector. then, the cells were fixed and immunostained with anti-p65 antibody to observe the nuclear translocation of endogenous p65 using confocal microscopy. by ddx1 deficiency (figure 5e) , suggesting the involvement of ddx1 in ifn-β induction by nsp14. to determine whether ddx1 is involved in tgev-induced ifnβ activation, we designed three pairs of sirnas targeting porcine ddx1 (psiddx1) and selected one with the best knockdown efficiency as demonstrated by rt-qpcr ( figure 6a ) and western blot assay (figure 6b) , for subsequent experiments. pk-15 cells were cotransfected with ifn-β-luc or nf-κb-luc, prl-tk, together with psiddx1 or sinc. at 12 h post-transfection, cells were infected with tgev for 24 h, followed by dual luciferase assay. ddx1 depletion had no effect on the basal activity of ifn-β and nf-κb promoter, but significantly decreased tgev-induced activation of ifn-β and nf-κb (figures 6c,d) . we also detected the expression level of p-p65 in tgev-infected pk-15 cells when ddx1 was silenced. as shown in figure 6e , knockdown of ddx1 reduced tgev-induced p65 phosphorylation. these results suggested that ddx1 is associated with tgev-induced ifn-β and nf-κb activation. interferon-i initiates a series of signaling cascades through the jak/stat pathway, resulting in the expression of numerous isgs (32) . furthermore, nf-κb activation plays a pivotal role in regulating the transcription and expression of many proinflammatory cytokines. because ddx1 is involved in tgev-induced ifn-β and nf-κb activation, theoretically, it should have an impact on tgev-induced isg and pro-inflammatory cytokine expression. the expression levels of some isgs (ifit1, ifit2, ifit3) and proinflammatory cytokines (il-6, il-8) in ddx1-knockdown cells were analyzed after tgev infection. as expected, ddx1 depletion inhibited the expression of ifit1, ifit2, ifit3, as well as il-6 and il-8 to some degree, compared with that in cells transfected with sinc (figures 6f-j) . the innate immune response characterized by the synthesis of ifn and proinflammatory cytokines is the first line of antiviral defense. multiple studies have reported the involvement of covs in the regulation of innate immune responses. the majority of covs decreased dsrna-mediated ifn-β production, including porcine epidemic diarrhea virus (pedv) (33), severe acute respiratory syndrome coronavirus (sars-cov) (34) , and infectious bronchitis virus (ibv) (35) . interestingly, mouse hepatitis virus (mhv)-induced ifn-α/β and established an antiviral state in plasmacytoid dendritic cells and macrophages, but failed to produce ifn in neurons, astrocytes, and hepatocytes (36, 37) , indicating that mhv induction of ifn-α/β is cell type dependent. a previous study showed that tgev-induced ifn-α secretion in vitro and in vivo (38) . here, we found that tgev infection increased the production of ifn-β in a dose-dependent manner in pk-15 cells, in line with previously reported data (25) . the significant roles of many cov proteins in the regulation of innate immune response have been identified. for example, nsp1, nsp7, nsp15, papain-like protease (plpro), orf3b, orf6, (25) . this indicated the potential effect of tgev nsp14 on ifn-β induction, which is in line with our data and further confirms our conclusion using a reverse genetic system. although nsp14 was identified as a key ifn-β activator among tgev-encoded proteins, it remains unclear which prr(s) is involved in its detection and induction of ifn-β production. indeed, viral proteins, similar to viral nucleic acids or replication intermediates, can in some cases also function as pamps, specifically recognized by certain host prrs, such as tlr2 and tlr4, to modulate the ifn responses during viral infection (46, 47) . for example, the m glycoprotein of sars-cov has been reported to function as a novel cytosolic pamp to promote ifn-β production by activating a non-canonical tlr signaling cascade (26) . in addition to the four major prr groups reported previously, including tlrs, rig-i-like receptors, nod-like receptors and cytoplasmic dna receptors (48) , multiple dexd/h-box helicases, such as ddx1, ddx3, ddx9, and ddx41, were reported recently to act as prrs and sense viral pamps to activate the nf-κb signaling pathway and induce ifn-β production (49) (50) (51) (52) (53) . previous study revealed that ddx1, ddx21, and dhx36 form a complex with the adaptor molecule trif to sense dsrna in dendritic cells (53) . in this paper, ddx1 interacted with tgev nsp14 in a rna-independent manner and enhanced both tgevand nsp14-induced activation of ifn-β responses. however, the direct interactions between nsp14 and ddx21 or dhx36 were not observed. we speculated that nsp14 may be sensed by ddx1/ ddx21/dhx36 complex by interacting with ddx1. these results suggest that nsp14 may be recognized as pamp by ddx1, (e) pk-15 cells were transfected with psiddx1 or sinc, and 12 h later, cells were mock infected or infected with tgev (moi = 0.5) for 24 h. then, the cells were collected for western blot assay with specific antibodies against p65, p-p65, ddx1, or tgev n protein, using β-actin expression as a loading control. the ratio of phosphorylated/total p65 was analyzed using imagej software. (f-j) pk-15 cells were treated as described for (e) and collected at 24 hpi. cell rnas were extracted for rt-qpcr to examine the mrna expression levels of ifit1 (f), ifit2 (g), ifit3 (h), il-6 (i), and il-8 (j). the mrna expression levels were normalized to porcine gapdh transcripts. values are the mean ± sd of three independent tests. *p < 0.05 or **p < 0.01 compared with the sinc group. which triggers an antiviral response. further studies are required to investigate this in more detail. ddx1 is a dexd/h helicase family member composed of the dead-box and related deah, dexh, and dexd protein family and is involved in multiple cellular processes of rna metabolism (54, 55) . besides these traditional roles, it appears that multiple proteins of the dexd/h-box helicase family are associated with viral components and/or have alternative effects on viral propagation (56) (57) (58) . for instance, ddx3 shows antiviral functions against vaccinia virus, denv, and hbv (59-61), but is of benefit for hcv and hiv infection (62, 63) . in addition, ddx1 interacts with the nsp3 of venezuelan equine encephalitis virus and enhances viral multiplication (64) . the interaction of ddx1 with human immunodeficiency virus type 1 rev protein is involved in the regulation of virus replication (65) . in the present study, the knockdown and ectopic expression of ddx1 demonstrated that ddx1 had antiviral activity against tgev replication (data not shown). interestingly, earlier studies also showed an interaction between ddx1 with coronavirus (ibv and sars-cov) nsp14, and in contrast to tgev, this interaction might enhance the replication of ibv (31) . this difference suggests that ddx1 is not likely to be a general target against cov infection. furthermore, it should be noted that the effect of ddx1 on progeny tgev production was moderate (data not shown). difference from other covs, such as sars-cov and mers-cov which antagonize ifn-i, tgev infection induces ifn-i production, and a most recent paper showed that poly(i:c)induced ifn-i responses could only inhibit tgev replication in the early infection stage, but failed in the late infection stage (23) . they also demonstrated that the activation of ifn-i responses by tgev infection cannot inhibit viral replication. our results are consistent with the conclusion proposed by zhu and colleagues. in addition, it is surprising that the expression levels of ifn-β are paralleled with the increase of viral rna during tgev infection. these may explain why the effect of ddx1 on tgev replication was moderate accompany with its significant role in ifn-β induction by tgev. however, more studies are required to investigate the complex interaction between tgev and ifns. in conclusion, our data demonstrate that tgev infection induces ifn-β production and nsp14 is the most significant ifn-β inducer among the tgev-encoded proteins. nsp14 interacts with cellular dexd/h helicase ddx1 to activate ifn-β in a nf-κb dependent manner, and ddx1 is associated with tgev-induced ifn-β production, revealing a potential coactivator role of host rna helicase ddx1 on virus and viral protein induced innate immune responses. all animal experiments were approved by the hubei administrative committee for laboratory animals (permission number 00024534) and complied with the guidelines of hubei laboratory animal welfare and ethics of hubei administrative committee of laboratory animals. author contributions yz, ww, and lf designed research; yz, ww, and lx performed research; yz, yf, dw, and sx analyzed data; yz and lf wrote the first draft of the manuscript. hc, qk, zh, and xw contributed to modify the manuscript. all the authors read and approved the manuscript. acknowledgments simulation of the economic impact of transmissible gastroenteritis on commercial pig production in australia effects of virulent and attenuated transmissible gastroenteritis virus on the ability of porcine dendritic cells to sample and present antigen complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus identification of protease and adp-ribose 1"-monophosphatase activities associated with transmissible gastroenteritis virus non-structural protein 3 strategy for systematic assembly of large rna and dna genomes: transmissible gastroenteritis virus model high interferon titer in newborn pig intestine during experimentally induced viral enteritis induction of alpha interferon by transmissible gastroenteritis coronavirus: role of transmembrane glycoprotein e1 induction of the 2-5a system by interferon and transmissible gastroenteritis virus quantitative proteomic analysis reveals that transmissible gastroenteritis virus activates the jak-stat1 signaling pathway convergence of the nf-kappab and irf pathways in the regulation of the innate antiviral response interferonalpha is the primary plasma type-i ifn in hiv-1 infection and correlates with immune activation and disease markers vesicular stomatitis virus glycoprotein g activates a specific antiviral toll-like receptor 4-dependent pathway influenza a virus infection, innate immunity, and childhood encephalomyocarditis virus disrupts stress granules, the critical platform for triggering antiviral innate immune responses reovirus activates human dendritic cells to promote innate antitumor immunity herpes simplex virus 2-induced activation in vaginal cells involves toll-like receptors 2 and 9 and dna sensors dai and ifi16 respiratory syncytial virus induced type i ifn production by pdc is regulated by rsv-infected airway epithelial cells, rsv-exposed monocytes and virus specific antibodies newcastle disease virus induces pro-inflammatory conditions and type i interferon for counter-acting treg activity interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures molecular cloning and functional characterization of porcine ifn-beta promoter stimulator 1 (ips-1) interferon-alphaproducing cells are localized in gut-associated lymphoid tissues in transmissible gastroenteritis virus (tgev) infected piglets coronavirus pseudoparticles formed with recombinant m and e proteins induce alpha interferon synthesis by leukocytes transmissible gastroenteritis virus does not suppress ifn-beta induction but is sensitive to ifn in ipec-j2 cells alphacoronavirus protein 7 modulates host innate immune response mutagenesis of coronavirus nsp14 reveals its potential role in modulation of the innate immune response the membrane protein of severe acute respiratory syndrome coronavirus functions as a novel cytosolic pathogen-associated molecular pattern to promote beta interferon induction via a toll-like-receptor-related traf3-independent mechanism single amino acid changes in the viral glycoprotein m affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus nf-kappab regulation in the immune system the diverse and complex roles of nf-kappab subunits in cancer rig-i like receptors and their signaling crosstalk in the regulation of antiviral immunity the cellular rna helicase ddx1 interacts with coronavirus nonstructural protein 14 and enhances viral replication mechanisms of type-i-and type-ii-interferon-mediated signalling porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat1 sars and mers: recent insights into emerging coronaviruses infectious bronchitis coronavirus inhibits stat1 signaling and requires accessory proteins for resistance to type i interferon activity murine coronavirus induces type i interferon in oligodendrocytes through recognition by rig-i and mda5 murine coronavirus cell type dependent interaction with the type i interferon response in vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero tgev coronavirus injection the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 the glycosylation status of the murine hepatitis coronavirus m protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain the membrane protein of sars-cov suppresses nf-kappab activation severe acute respiratory syndrome coronavirus m protein inhibits type i interferon production by impeding the formation of traf3.tank.tbk1/ ikkepsilon complex small interfering rna effectively inhibits the expression of sars coronavirus membrane gene at two novel targeting sites rotavirus nsp4 triggers secretion of proinflammatory cytokines from macrophages via toll-like receptor 2 dengue virus ns1 protein activates cells via toll-like receptor 4 and disrupts endothelial cell monolayer integrity pattern recognition receptors and the innate immune response to viral infection dhx9 pairs with ips-1 to sense double-stranded rna in myeloid dendritic cells the helicase ddx41 senses intracellular dna mediated by the adaptor sting in dendritic cells dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-beta-inducing potential aspartate-glutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells ddx1, ddx21, and dhx36 helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells the dead-box protein family of rna helicases dead-box proteins: the driving forces behind rna metabolism rna interference screen for human genes associated with west nile virus infection dexd/h-box rna helicases as mediators of anti-viral innate immunity and essential host factors for viral replication cellular rna helicases and hiv-1: insights from genome-wide, proteomic, and molecular studies ddx3 dead-box rna helicase inhibits hepatitis b virus reverse transcription by incorporation into nucleocapsids viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation dead-box rna helicase ddx3x inhibits denv replication via regulating type one interferon pathway understanding the interaction of hepatitis c virus with host dead-box rna helicases requirement of ddx3 dead box rna helicase for hiv-1 rev-rre export function venezuelan equine encephalitis virus non-structural protein 3 (nsp3) interacts with rna helicases ddx1 and ddx3 in infected cells ddx1 is an rna-dependent atpase involved in hiv-1 rev function and virus replication key: cord-003545-corvd5cs authors: li, chen; liu, jiaxin; zhang, xin; wei, shina; huang, xiaohong; huang, youhua; wei, jingguang; qin, qiwei title: fish autophagy protein 5 exerts negative regulation on antiviral immune response against iridovirus and nodavirus date: 2019-03-19 journal: front immunol doi: 10.3389/fimmu.2019.00517 sha: doc_id: 3545 cord_uid: corvd5cs autophagy is an important biological activity that maintains homeostasis in eukaryotic cells. however, little is known about the functions of fish autophagy-related genes (atgs). in this study, we cloned and characterized atg5, a key gene in the autophagy gene superfamily, from orange-spotted grouper (epinephelus coioides) (ecatg5). ecatg5 encoded a 275-amino acid protein that shared 94 and 81% identity to seabass (lates calcarifer) and humans (homo sapiens), respectively. the transcription level of ecatg5 was significantly increased in cells infected with red-spotted grouper nervous necrosis virus (rgnnv). in cells infected with singapore grouper iridovirus (sgiv), ecatg5 expression declined during the early stage of infection and increased in the late stage. fluorescence microscopy revealed that ecatg5 mainly localized with a dot-like pattern in the cytoplasm of grouper cells. overexpression of ecatg5 significantly increased the replication of rgnnv and sgiv at different levels of detection, as indicated by increased severity of the cytopathic effect, transcription levels of viral genes, and levels of viral proteins. knockdown of ecatg5 decreased the replication of rgnnv and sgiv. further studies showed that overexpression ecatg5 activated autophagy, decreased expression levels of interferon related cytokines or effectors and pro-inflammatory factors, and inhibited the activation of nuclear factor κb, ifn-sensitive response element, and ifns. in addition, ectopic expression of ecatg5 affected cell cycle progression by hindering the g1/s transition. taken together, our results demonstrated that fish atg5 exerted a crucial role in virus replication by promoting autophagy, down-regulating antiviral ifn responses, and affecting the cell cycle. autophagy is a conserved cell biological pathway that delivers cytoplasmic components to lysosomes for degradation and elimination of useless or harmful substrates to maintain cell homeostasis in all eukaryotic cells (1). this fundamental process involves formation of double membrane autophagosomes, which fuse with lysosomes to degrade the sequestered cargo (2, 3) . viruses depend on the host cell's machinery to replicate the genome and generate progeny virus particles. autophagy, as a cell steward, has been reported to play an important role in virus replication. many studies have reported that viruses cause accumulation of autophagosomes and exploit these membrane structures as "virus factories" (4) . in addition, several studies have confirmed that autophagy interacts with the innate antiviral immune response (5) (6) (7) . autophagy can amplify the innate immune response that is mediated by nucleic acid-sensing toll-like receptors and enhance delivery of cytosolic pathogen-associated molecular patterns (8) . additionally, some autophagy factors can downregulate rig-i (retinoic acidinducible gene i)-like receptors and type i interferon (ifn) signaling and suppress inflammasome activation (9, 10) . the process of autophagosome formation is regulated by several autophagy-related genes (atgs) (11) . in the autophagy gene superfamily, atg5 is a key gene that plays an important role in early autophagosome formation. enhanced or reduced atg5 levels affect the occurrence and alteration of autophagy pathways. on one hand, atg5 protein can conjugate to atg12 to form a complex with the multimeric protein atg16, and the atg12-atg5-atg16 complex facilitates extension of the autophagosome (12, 13) . on the other hand, the combination of atg5 complex and autophagic vesicle membrane can promote recruitment of lc3 (atg8) to autophagic vesicles (14) . atg5 is involved in various physiological and pathological processes. in lipid metabolism, silencing or knocking out atg5 can lead to lipid deposition. atg5 also plays a role in regulating ifn immune and inflammation responses (7) . the grouper (epinephelus spp.) is a well-known mariculture species that is widely distributed in south china and southeast asia. in 2016, the scale of grouper breeding in china was 108,319 t, which was 8.31% higher than that in 2015. however, outbreaks of viral diseases have caused heavy economic losses in the grouper aquaculture industry. two representative pathogens are singapore grouper iridovirus (sgiv) and red-spotted grouper nervous necrosis virus (rgnnv) (15, 16) . current research on the prevention and control of viral diseases in grouper is mainly focused on exploration of the anti-virus immune network and key immune genes. although numerous immune regulatory molecules have been found to play vital roles in the grouper antiviral response (17) (18) (19) (20) (21) , the roles of atgs in the replication of sgiv or rgnnv have not been reported. in other studies of aquatic viruses, proliferation of svcv was significantly reduced in beclin-1(atg6) and lc3(atg8)-depleted endothelial progenitor cells. however, references to atg5 in aquatic animal viruses are limited (22) . in this study, we cloned a key autophagy related gene (atg5) from orange-spotted grouper (e. coioides) (ecatg5) and investigated the roles of ecatg5 in autophagy, innate immunity, and cell cycle. our results provide new insights into the roles of fish atg5 in virus infection. based on several expressed sequence tag sequences of ecatg5 from the grouper spleen transcriptome (23), primers (table 1) were designed to amplify the full-length open reading frame (orf) of ecatg5. identity analysis between ecatg5 and other atg5 sequences was performed using blastp searches of the ncbi database. amino acid alignments were conducted using mega5.0 software and edited with the genedoc program. the phylogenetic analysis was carried out using the boot-strapped neighbor joining method in clustalx 2.1 software. orange-spotted groupers (30-40 g) used in this study were purchased from a local farm in hainan province and kept in a laboratory recirculating seawater system as described previously (20) . the relative expression level of ecatg5 was examined using quantitative real-time pcr (qrt-pcr) in selected tissues, including liver, spleen, head kidney, kidney, heart, intestine, brain, gill, stomach, muscle, fin, and skin. all tissues were collected from three fish. a grouper spleen (gs) cell line was established in our lab (24) , and cells were propagated and maintained at 28 • c in leibovitz's l-15 medium (gibco, usa) supplemented with 10% fetal bovine serum (gibco, usa). sgiv and rgnnv were isolated in our laboratory and propagated in gs cells with titer of 10 5 tcid 50 /ml as described previously (15, 16) . to clarify the molecular function of ecatg5 in vitro, ecatg5 was subcloned into the vectors pegfp-c1 and pcdna3.1-3×ha using the primers listed in table 1 . all recombinant plasmids were confirmed by dna sequencing. gs cells were transfected with ecatg5 sirna (siecatg5:5 ′ -gaaagagauguacccugcugcuuua-3 ′ ) or same volume negative control for 24 h, and then infected with sgiv or rgnnv for 12, 24, and 36 h. at the end of each incubation period, the total rna or protein of cells were extracted for detection. gs cells were seeded onto cover slips (10 × 10 mm) in 24-well plates. after allowing the cells to adhere for 24 h, pegfp-c1 and pegfp-ecatg5 plasmids were transfected into gs cells using the transfection reagent lipofectamine 2000 (invitrogen, carlsbad, ca, usa). at 24 h post-transfection, cells on the cover slips were washed with phosphate buffered saline (pbs), fixed with 4% paraformaldehyde for 1 h at room temperature, and then stained with 4,6-diamidino-2-pheny-lindole (dapi) for 10 min. finally, cells were observed under a fluorescence microscope (leica, wetzlar, germany). total rna isolation was performed using the sv total rna isolation system (promega, usa) according the manufacturer's instructions, and reverse transcription was carried out using revertra ace (toyobo, osaka, japan). qrt-pcr was performed in an abi quant studio 5 device (applied biosystems, carlsbad, ca, usa). the expression levels of viral genes and host immune genes were detected. the relative expression ratio of the selected gene vs. β-actin (reference gene) was calculated using the 2 − ct method. reactions of sybr green were performed in a 10 µl volume containing 5 µl of 2 × sybr r premix ex taq tm , 0.3 µl of each forward and reverse primer (10 µm), 3.4 µl of water, and 1 µl of cdna. all experiments were performed in triplicate, and the cycling parameters were chosen according to the manufacturer's instructions. cells were collected and lysed in ripa buffer. proteins were separated by 12% sds-page and transferred onto immobilon-p polyvinylidene difluoride membranes (millipore, temecula, ca, usa). blots were incubated with the indicated primary antibody: anti-ha (1:1,000 dilution), anti-β-actin (1:1,000 dilution), anti-atg5 (1:500 dilution), anti-lc3 (1:1,000 dilution), anti-sgiv major capsid protein (mcp) (1:1,000 dilution), anti-rgnnv capsid protein (cp) (1:1,000 dilution). subsequently they were incubated with horseradish peroxidase (hrp)-conjugated goatanti-rabbit igg (1:5,000 dilution). mouse monoclonal anti-ha antibody was purchased from sigma (usa). mouse monoclonal anti-β-actin antibody and rabbit polyclonal atg5 antibody were purchased from proteintech (rosemont, il, usa). rabbit monoclonal anti-lc3 antibody was purchased from abcam (usa). hrp-conjugated goat anti-rabbit and anti-mouse antibodies were purchased from kpl (usa). the polyclonal anti-mcp antibody of sgiv and the polyclonal anti-cp antibody of rgnnv were prepared in our lab. immunoreactive proteins were visualized using an enhanced hrp-dab chromogenic substrate kit (tiangen, beijing, china). to evaluate the promoter activity regulated by ecatg5, luciferase reporter plasmids, including interferon-sensitive response element (isre)-luc, ifn3-luc, and nuclear factor (nf)-κb (clontech, usa), were used for co-transfection. briefly, gs cells were transiently transfected with the luciferase plasmids together with the indicated ecatg5 expression vectors using lipofectamine 2000 reagent. the prl-sv40 renilla luciferase vector was used as an internal control. luciferase activity of total cell lysates was measured using a luciferase reporter assay system (promega). statistical analysis was performed using spss version 13. oneway anova was used to evaluate the variability between treatment groups ( * p < 0.05, * * p < 0.01). the full-length orf of ecatg5 was obtained using pcr amplification. sequence analysis indicated that ecatg5 encoded a 275-amino acid protein that shared 94% and 81% identity to seabass (lates calcarifer) and humans (homo sapiens), respectively ( figure 1a) . phylogenetic analysis indicated that ecatg5 was closely related to the fish subgroup, followed by amphibians, birds, and mammals ( figure 1b) . to analyze the tissue distribution, qrt-pcr was conducted in different tissues of healthy juvenile orange-spotted grouper. frontiers in immunology | www.frontiersin.org ecatg5 was constitutively expressed in all the analyzed tissues in healthy grouper, and it was relatively high mrna levels in the brain, liver, and fin (figure 2a) . to analyze the gene expression profiles in response to different viral infections, the transcription levels of ecatg5 were examined in rgnnv or sgiv infected cells. the transcription levels of ecatg5 were significantly increased in rgnnv infected cells. in sgiv infected cells, the expression levels of ecatg5 first decreased within 24 h post-injection and then increased after 36 h ( figure 2b ). to demonstrate the subcellular localization of ecatg5, pegfp-ecatg5 was transfected into grouper cells, and fluorescence was observed under fluorescence microscopy. green fluorescence was observed in the cytoplasm in ecatg5 transfected grouper cells, and most of these cells exhibited fluorescence aggregation (figure 3) . in pegfp-c1 transfected cells, fluorescence was distributed both the cytoplasm and nucleus. the results showed that ecatg5 was a cytoplasmic protein. to clarify the function of ecatg5, the eukaryotic expression vector of pcdna3.1-3×ha-ecatg5 was constructed, and the recombinant plasmid successfully expressed ha-ecatg5 protein after being transfected into gs cells ( figure 4a ). on the contrary, ecatg5 protein level was decreased after sirna silencing ( figure 4b ). autophagy is characterized by the formation of autophagosomes. conjugation of the essential lc3 to phosphatidylethanolamine is required for autophagosome biogenesis. therefore, lc3 lipidation is used as a faithful marker of autophagy activation (22, 25) . to assess whether ecatg5 overexpression affected gs autophagy, we investigated the level of lc3 lipidation in cells overexpressing or silencing ecatg5. the lc3-ii (the lipidated form) level was higher in cells transfected with ecatg5 compared with control cells (figure 4a) , and ecatg5 knockdown reduced the lc3-ii level (figure 4b) , which suggests that ecatg5 might activate autophagy by promoting lc3 lipidation in gs cells. to clarify the effects of ecatg5 overexpression on virus infection, ecatg5 transfected cells were infected with sgiv or rgnnv, and then viral replication was investigated. severity of the cytopathic effect (cpe) induced by sgiv infection evoked at 24 h (figure 5a) . the amount and severity of vacuoles induced by rgnnv infection also increased in ecatg5 overexpressing cells compared to the empty vector transfected cells. at the transcription level, the expression of sgiv mcp, icp18, vp19, and litaf increased in ecatg5 overexpressing cells after sgiv infection ( figure 5b) . the transcription of rgnnv cp and rdrp genes also increased compared with control cells after rgnnv infection ( figure 5c) . consistently, ectopic expression of ecatg5 also increased the protein levels of sgiv mcp and rgnnv cp (figure 5d) . meanwhile, the effects of silencing ecatg5 on sgiv and rgnnv replication were also studied. quantitative analysis results showed that the transcription level of sgiv mcp, icp18, vp19, and litaf decreased after ecatg5 knockdown ( figure 6a) . the transcription of rgnnv cp and rdrp genes also decreased compared with negative control cells ( figure 6b) . consistently, ecatg5 knockdown decreased the protein levels of sgiv mcp and rgnnv cp in gs cells ( figure 6c) . together, ecatg5 was speculated to promote sgiv and rgnnv replication in gs cells. to explore the potential mechanism involved in the action of ecatg5 in fish virus infections, the roles of ecatg5 on the host interferon immune and inflammation response were evaluated. the pcdna3.1-3×ha-ecatg5 and the empty vector were transfected into gs cells, and cells were harvested at 24, 36, and 48 h. the transcription levels of host immune factors and pro-inflammatory cytokines were detected using qrt-pcr. as shown in figure 7 , expression levels of interferon related cytokines or effectors, including irf3, irf7, mda5, isg15, lgp2, mxi, ifp35, myd88, and traf6, were all decreased in ecatg5 overexpressing cells compared with control vector transfected cells. in addition, we also found that the expressions of pro-inflammatory cytokines such as interleukin (il)-1β, il-6, il-8, and tumor necrosis factor alpha, were all significantly decreased in ecatg5 overexpressing cells (figure 8 ). to further explore the roles of ecatg5 during fish virus infection, the promoter activity of reporter genes in typical antiviral pathways, including isre, type i ifn, and nf-κb, were measured using the plasmids isre-luc, inf-luc, and nf-κb-luc. as shown in figure 9a , ecatg5 overexpression suppressed the promoter activity of these genes. in addition, siecatg5 was co-transfected with the plasmids isre-luc, inf-luc, and nf-κb-luc, and the promoter activity of three reporter genes were measured. the results showed that sirna-mediated atg5 knockdown increased the promoter activity of three reporter genes ( figure 9b) . thus, we proposed that ecatg5 negatively regulates nf-κb and the ifn immune responses. mammalian atg5 plays a causal role in regulating cell cycle progression (26, 27) . whether ecatg5 has the similar effects on cell cycle remains uncertain. to explore the role of ecatg5 on cell cycle progression, gs cells were transfected with pcdna3.1-3×ha or ecatg5. ectopic expression of ecatg5 clearly inhibited the g1/s transition compared to the empty vector overexpressing cells. the percentages of g1 phase cells in pcdna3.1-3×ha and ecatg5 overexpressing cells were 65.34 and 72.28%, respectively (figure 10) . those results indicated that ecatg5 may affect cell cycle progression from the g1 to the s phase and arrest cells in the g1 phase. autophagy is a highly conserved pathway, and it plays an important role in resistance to intracellular viruses and other pathogens (28) . autophagosome formation relies on the atg family (29) , and atg5 has been studied in detail in mammals. atg5 is involved in autophagic membrane extension and curvature, lc3 (atg8) recruitment, and lysosome and late intracellular regeneration. it also has been implicated in the ifn immune response, inflammation response, and lipid metabolism (25, 30) . in fish, the roles of atg5 gene in zebrafish neurogenesis and organogenesis has been reported, and the results showed that the formation of the atg5-atg12 conjugate may depend on atg5 protein generation and its splicing (31) . twelve autophagyrelated genes from yellow catfish pelteobagrus fulvidraco and their transcriptional responses to waterborne zinc exposure were also characterized (32). however, little has been reported about the roles of atg5 in virus replication and its relationship with the innate antiviral immune response in aquatic animals by far. in the present study, an atg5 homolog from orange-spotted grouper (ecatg5) was cloned and its roles during fish virus infection were investigated. ecatg5 encoded a 275-amino acid protein that shared 94% and 81% identity to seabass (lates calcarifer) and humans (homo sapiens), respectively. atg5 functions as an e3 (b) knockdown of ecatg5 decreased rgnnv gene transcription including cp and rdrp. one-way anova was used to evaluate the variability between treatment groups (*p < 0.05, **p < 0.01). (c) virus protein level after transfection with siecatg5. the level of sgiv-mcp or rgnnv-cp was detected by western blot, and β-actin was used as the internal control. band intensity was calculated using quantity-one software and ratios of mcpor cp/β-actin was assessed (p < 0.05). ligase-like enzyme (33) . ecatg5 possesses all the characteristic features of canonical ubiquitin ligase, including two ubiquitinlike domains, a helix-rich domain, and the conserved calpain cleavage sites (32, 33) . as a preliminary step to unravel the physiological role of ecatg5, the mrna tissue distribution was determined. the present results indicated that the mrna expression of ecatg5 was ubiquitous within all the tested tissues. the ubiquitous distribution suggested that autophagy was implicated in many metabolic pathways among the tissues. originally autophagy was identified as a response to nutrient deficiency, so it is thought to be a receptor of cellular energy and metabolism (34) . however, it is now evident that autophagy can be induced by a variety of factors, including starvation, reactive oxygen figure 7 | ecatg5 overexpression decreased expression of interferon related cytokines or effectors. gs cells were transfected with ecatg5, then cells were harvested at the indicated time. expression levels of host ifn associated genes were determined using qrt-pcr. one-way anova was used to evaluate the variability between treatment groups (*p < 0.05, **p < 0.01). species, endoplasmic reticulum stress, microbial invasion and so on (35) . based on this, we detected the expression of ecatg5 under the stimulation of two viruses. transcription levels of ecatg5 increased from the early stage of rgnnv infection, suggesting that rgnnv infection may significantly induce autophagy activity to facilitate its proliferation. in sgiv infected cells, the expression levels of ecatg5 firstly decreased within 24 h post-infection and then increased after 36 h. this pattern might be caused by the lack of cellular nutrition. with the cell growth, metabolism and virus replication, the nutrient deficiency in cells will increase autophagy activity over time. autophagy is an important cellular process by which atg5 initiates the formation of double membrane vesicles (dmvs). recently, the contribution of an autophagy protein, atg5, to viral replication has been demonstrated (36) , and atg5 was identified as an interacting protein for the hepatitis c virus ns5b (37) . the altered expression level of ecatg5 in gs cells infected with sgiv and rgnnv suggested that ecatg5 might play an essential role in the grouper response to fish virus infection, so the impact of ecatg5 overexpression on virus proliferation were investigated. overexpression of ecatg5 promoted sgiv and rgnnv replication, evidenced by the severity of cpe, the increased transcription levels of viral genes, and the increased levels of viral proteins. knockdown of ecatg5 decreased sgiv and rgnnv replication by assessing transcription and protein levels of viral genes. the results suggested that atg5 might share conserved function to viral replication from fish to mammals. studies of mammals suggested that atg5-atg12 promotes viral replication by negatively regulating the ifn response (38) . the atg5-atg12 conjugate interacts directly with the mitochondrial antiviral-signaling protein (mavs) and retinoic acid-inducible gene i (rig-i) through the n-terminal caspase recruitment domain (card), resulting in inhibition of type i ifn production (38) . n-terminal fragments of rig-i and ifn-α possess great capacity to activate ifn-β and isre promoters in atg5 deficient cells (39) . when the formation of autophagosomes was promoted, the activity of the ifnβ promoter was decreased so that autophagy contributed to sustained hepatitis c virus infection (40) . here, overexpression of ecatg5 in grouper cells not only decreased the expression levels of several interferon related cytokines or effectors, but also negatively regulated the expression of pro-inflammatory factors. moreover, the ectopic expression of ecatg5 significantly decreased isre, ifn, and nf-κb promoter activities, and figure 8 | overexpression of ecatg5 decreased the transcription of proinflammatory factors. gs cells were transfected with ecatg5, then cells were harvested at the indicated time. expression levels of proinflammatory factors were determined using qrt-pcr. one-way anova was used to evaluate the variability between treatment groups (*p < 0.05, **p < 0.01). knockdown of ecatg5 increased promoter activities of nf-κb, ifn, and isre. the promoter activity was measured using the luciferase reporter gene assay. one-way anova was used to evaluate the variability between treatment groups (*p < 0.05, **p < 0.01). knockdown of ecatg5 raised promoter activities of these reporter genes. atg5-atg12 inhibits the production of ifn in canonical autophagy while playing the opposite role in alternative autophagy (41) . thus, overexpression of ecatg5 might activate canonical autophagy in gs cells. overexpression of ecatg5 up-regulated the level of lc3-ii, indicating that ecatg5 can activate autophagy. taken together, we speculated that ecatg5 decreased interferon immune response and activated autophagy might contribute greatly to its promoting effect on sgiv and rgnnv replication. in mammals, atg5 can induce cell cycle arrest at the g1/s phase by up-regulating expression of p21 (a cyclindependent kinase inhibitor) at the level of post-transcription in response to challenges such as nutrient deficiency (42, 43) . considering that atg5 is a key and relatively conserved protein, we speculated that fish atg5 might play a similar role in cell cycle progression. in the present study, ecatg5 affected cell cycle progression from the g1 to the s phase and arrested cells in the g1 phase. it was also reported that the replication level and virus titer of rgnnv were greater in cells released from the g1 phase or s phase of the cell cycle compared to cells released from the g2 phase (44) . those results suggested that overexpression of ecatg5 may facilitate rgnnv replication. however, whether fish atg5 affects the cell cycle by regulating p21 requires further investigation. in conclusion, a key autophagy related gene (atg5) from orange-spotted grouper (e. coioides) (ecatg5) was cloned, and the roles of ecatg5 in autophagy, innate immunity, and cell cycle were investigated in this study. the results showed that ecatg5 plays crucial roles in virus replication via promoting autophagy, down-regulating antiviral ifn responses, and affecting cell cycle. this study identified a link between the autophagic machinery and innate immune signaling against viral infection. the datasets for this manuscript are not publicly available because this data has not been published. requests to access the datasets should be directed to jingguang wei, weijg@scau.edu.cn. all animal-involving experiments of this study were approved by the animal care and use committee of college of marine sciences, south china agricultural university, and all efforts were made to minimize suffering. qq and jw designed the experiments. cl performed the majority of the experiments, analyzed data, and wrote the manuscript. jl and xz contributed experimental suggestions. sw, yh, and xh helped to design the experiments. all authors revised the manuscript. national natural science foundation of china (31572643, 31772882, and 41506176) autophagosome formation: core machinery and adaptations virus-induced double-membrane vesicles viruses and the autophagy pathway contribution of autophagy to antiviral immunity autophagy is involved in regulating the immune response of dendritic cells to influenza a(h1n1) pdm09 infection autophagy in infection, inflammation, and immunity noncanonical autophagy is required for type i interferon secretion in response to dna-immune complexes atg9a controls dsdna-driven dynamic translocation of sting and the innate immune response regulation of inflammasome signaling development by self-digestion: molecular mechanisms and biological functions of autophagy structure of atg5,atg16, a complex essential for autophagy the evolving functions of autophagy in ocular health: a double-edged sword loss of atg12, but not atg5, in pro-opiomelanocortin neurons exaerbates diet-induced obesity characterization of a novel ranavirus isolated from grouper epinephelus tauvina sin ym. characterization, pathogenicity and neutralization studies of a nervous necrosis virus isolated from grouper, epinephelus tauvina expression and functional characterization of trif in orange-spotted grouper (epinephelus coioides) traf6 is a critical factor in fish immune response to virus infection fish trim32 functions as a critical antiviral molecule against iridovirus and nodavirus molecular cloning and characterization of fadd from the orange-spotted grouper (epinephelus coioides) grouper viperin acts as a crucial antiviral molecule against iridovirus spring viraemia of carp virus induces autophagy for necessary viral replication transcriptome analysis of orange-spotted grouper (epinephelus coioides) spleen in response to singapore grouper iridovirus characterization of two grouper epinephelus akaara cell lines: application to studies of singapore grouper iridovirus (sgiv) propagation and virus-host interaction molecules and their functions in autophagy atg5-mediated autophagy deficiency in proximal tubules promotes cell cycle g2/m arrest and renal fibrosis atg5 can regulate p53 expression and activation autophagy and virus infection the autophagosome: origins unknown, biogenesis complex autophagy fights disease through cellular self-digestion expression pattern and functions of autophagy related gene atg5 in zebrafish organogenesis characterization of twelve autophagy-related genes from yellow catfish pelteobagrus fulvidraco and their transcriptional responses to waterborne zinc exposure structure of the human atg12-atg5 conjugate required for lc3 lipidation in autophagy er stress negatively regulates akt/tsc/mtor pathway to enhance autophagy porcine reproductive and respiratory syndrome virus induces autophagy to promote virus replication coronavirus replication complex formation utilizes components of cellular autophagy autophagy protein atg5 interacts transiently with the hepatitis c virus rna polymerase (ns5b) early during infection the atg5-atg12 conjugate associates with innate antiviral immune responses autophagy: a novel guardian of hcv against innate immune response interplay between autophagy and innate immunity in antiviral responses footand-mouth disease virus infection suppresses autophagy and nf-κb antiviral responses via degradation of atg5-atg12 by 3cpro autophagy regulates ros-induced cellular senescence via p21 in a p38 mapkα dependent manner effect of atg5-mediated anti-nutritional crisis in hepatocellular carcinoma rgnnv-induced cell cycle arrest at g1/s phase enhanced viral replication via p53-dependent pathway in gs cells the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 li, liu, zhang, wei, huang, huang, wei and qin. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-268511-dx2cqqt5 authors: kunkl, martina; amormino, carola; frascolla, simone; sambucci, manolo; de bardi, marco; caristi, silvana; arcieri, stefano; battistini, luca; tuosto, loretta title: cd28 autonomous signaling orchestrates il-22 expression and il-22-regulated epithelial barrier functions in human t lymphocytes date: 2020-10-14 journal: front immunol doi: 10.3389/fimmu.2020.590964 sha: doc_id: 268511 cord_uid: dx2cqqt5 il-22 is a member of the il-10 cytokine family involved in host protection against extracellular pathogens, by promoting epithelial cell regeneration and barrier functions. dysregulation of il-22 production has also frequently been observed in acute respiratory distress syndrome (ards) and several chronic inflammatory and autoimmune diseases. we have previously described that human cd28, a crucial co-stimulatory receptor necessary for full t cell activation, is also able to act as a tcr independent signaling receptor and to induce the expression of il-17a and inflammatory cytokines related to th17 cells, which together with th22 cells represent the main cellular source of il-22. here we characterized the role of cd28 autonomous signaling in regulating il-22 expression in human cd4(+) t cells. we show that cd28 stimulation in the absence of tcr strongly up-regulates il-22 gene expression and secretion. as recently observed for il-17a, we also found that cd28-mediated regulation of il-22 transcription requires the cooperative activities of both il-6-activated stat3 and rela/nf-κb transcription factors. cd28-mediated il-22 production also promotes the barrier functions of epithelial cells by inducing mucin and metalloproteases expression. finally, by using specific inhibitory drugs, we also identified cd28-associated class 1a phosphatidylinositol 3-kinase (pi3k) as a pivotal mediator of cd28-mediated il-22 expression and il-22–dependent epithelial cell barrier functions. il-22 has been discovered in 2000 as a member of the il-10 family that affects the functions of several non-hematopoietic cells, such as epithelial cells, keratinocytes, and hepatocytes (1) . il-22 is mainly produced by th17 cells, a subset of cd4 + t helper (th) cells that regulate efficient immune defense against extracellular pathogens (2) and by a unique cd4 + th cell subset, named th22 (3) (4) (5) . other cell types have been described to produce il-22 such as natural killer (nk) cells, ilc3 subset of innate lymphoid cells and at lower extent macrophages (6) (7) (8) . il-22 elicits its main effect on epithelial cells by stimulating their regeneration and proliferation, and by promoting epithelial barrier functions essential for the host defense against extracellular microbes at mucosal surfaces (9) (10) (11) (12) (13) (14) . however, high levels of il-22 and il-17 were detected in plasma of patients with sepsis-induced acute respiratory distress syndrome (ards) and were correlated with poor prognosis (15) . more recently, a novel severe acute respiratory syndrome-related coronavirus 2 (sars-cov2) has been identified as the etiological agent of covid-19 (16) , a respiratory pandemic disease that, in several patients, causes ards associated with high levels of pro-inflammatory cytokines (17) including th17 cytokines such as il-22, il-17, and il-6 (18, 19) . moreover, an excessive production of il-22 together with other cytokines of the th17 signature has also been involved in maintaining and amplifying the chronic state of several inflammatory and autoimmune diseases (20) (21) (22) (23) (24) (25) (26) . therefore, the identification of stimulatory molecules and associated signaling pathways regulating and/or amplifying il-22 production may provide new insight into immune defense against pathogens and novel therapeutic targets for inflammatory and autoimmune diseases. cd28 is one of the most important costimulatory receptor required for full t cell activation and differentiation (27) . cd28 interacts with b7.1/cd80 or b7.2/cd86 on the surface of professional antigen presenting cells (apcs) and lowers the threshold of the t cell receptor (tcr), thus enhancing the early signaling events necessary for optimal cytokine production, cell cycle progression and survival (28, 29) . human cd28 is also able to deliver tcr-independent signals, which leads to the production of pro-inflammatory cytokines and chemokines (30, 31) in cd4 + t cells. we also evidenced that cd28 autonomous stimulation of peripheral blood cd4 + t cells from either healthy donors (hd) or type 1 diabetes (t1d) or multiple sclerosis (ms) patients promotes the production of inflammatory cytokines and chemokines related to the th17 cell phenotype (32) (33) (34) , including il-22 (35) . in this work, we elucidated the role of cd28 autonomous stimulation in the regulation of il-22 expression and il-22mediated effector functions. we found that il-22 gene expression and secretion was strongly up-regulated by cd28 in human cd4 + t cells. similar to il-17a, cd28-induced il-22 gene expression was regulated by both rela/nf-kb and il-6-regulated stat3 transcription factors. co-culture experiments of cd28-activated t cells with caco-2 epithelial cell lines evidenced a critical role of il-22 in regulating the expression of mucins and metalloproteases. finally, we also evidence a pivotal role of cd28-associated class 1a phosphatidylinositol 3-kinase (pi3k) in regulating both il-22 expression and il-22-dependent epithelial barrier functions. human primary cd4 + t cells were enriched from pbmc by negative selection using an easyseptm isolation kit (#17952, stemcell technology) and cultured in rpmi 1640 supplemented with 5% human serum (euroclone, uk), lglutamine, penicillin and streptomycin. the purity of the sorted population was 95% to 99%, as evidenced by staining with anti-cd3 plus anti-cd4 abs. human naïve cd4 + cd45ra + and effector/memory cd4 + cd45ro + t cells were sorted using a high speed cell sorter (moflo astrios eq, beckman coulter). purity of sorted cells was consistently > 98%, and was acquired on a cytometer (cytoflex, beckman coulter). pbmcs were derived from buffy coats or anonymous healthy blood (hd) donors provided by the policlinico umberto i (sapienza university of rome, italy). written informed consent was obtained from blood donors and both the informed consent form and procedure was approved by the ethics committee of policlinico umberto i (ethical code n. 1061bis/2019, 13/09/2019). caco-2 epithelial cell line from human colon was provided by atcc (maryland, usa) and cultured in dmem supplemented with 10% fbs (euroclone, uk), l-glutamine, penicillin and streptomycin. the following antibodies were used: anti-human cd28 (cd28.2, #555726, 2 mg ml −1 ), anti-human cd3 (ucht1, #555330, 2 mg ml −1 ), goat anti-mouse (gam, # 553998, 2 mg ml −1 ), anti-human cd3-pe (#555333), anti-human cd45ra bv421 (#562885), mouse anti-human stat3 (#610189), antihuman cd45ro pe (#555493) (bd biosciences); rabbit antihuman lck (#sc-13, 2 mg/chip), rabbit anti-human rna polymerase ii (n-20, #sc-899, 2 mg/chip), anti-human relb (c-19, #sc-226, 2 mg/chip), anti-human gapdh (#sc-25778), antihuman mucin 1 (muc1) (#sc-7313) (santa cruz biotechnology, usa), rabbit anti-human rela (#8242s) (cell signaling technology, usa), mouse anti-human il-6 (#mab206, 10 mg ml −1 ), mouse anti-human il-22 (#mab7822, 10 mg ml −1 ) (r&d systems, usa), anti-human cd4 fitc (#130-114-531, miltenyi biotec, italy). human recombinant il-6 (ril-6) was from miltenyi biotec (#130-093-931, 50 ng ml −1 ). the following inhibitory drugs were used: ps1145 (#p6624, sigma aldrich), s31-201 (#sc-204304, santa cruz biotechnology), as605240 (#a0233, sigma aldrich). caco-2 cells were plated at 2.5 × 10 5 ml −1 and co-cultured with cd4 + t cells (2 × 10 6 ml −1 ) in 24 trans-well plates as indicated at 37°c. at the end of incubation, total cell extracts were obtained by lysing cells for 30 min on ice in 1% nonidet p-40 lysis buffer (150 mm nacl, 20 mm tris-hcl (ph 7,5), 1 mm egta, 1 mm mgcl 2 , 50 mm naf, 10 mm na 4 p 2 o 7 ) in the presence of inhibitors of proteases and phosphatases (10 mg ml −1 leupeptin, 10 mg ml −1 aprotinin, 1 mm navo 4 , 1 mm pefablock-sc). proteins were resolved by sds-page and blotted onto nitrocellulose membranes. blots were incubated with the anti-muc1 or anti-gapdh (1:400 dilution), extensively washed and after incubation with horseradish peroxidase (hrp)-labeled goat anti-rabbit (#na934v, 1:5000 dilution) or hrp-labeled goat anti-mouse (#na931v, 1:5000 dilution) developed with the enhanced chemiluminescence's detection system (ge healthcare life sciences, italy). protein levels were quantified by densitometric analysis using the imagej 1.50i program (national institute of health, usa). cd4 + t cells were plated at 2 × 10 6 ml −1 in 24-well plate or 24well plate inserts in the experiments of co-culture with caco-2 cells (2.5 × 10 5 ml −1 ) and stimulated for the indicated times with control isotype abs (ig) or crosslinked anti-cd28 (2 mg ml −1 ), or anti-cd3 (2 mg ml −1 ) or anti-cd3 plus anti-cd28 abs (2 mg ml −1 ). secretion of human il-22, il-6, and mmp9 was measured from the supernatants of cd4 + t cells cultured alone or with caco-2 cells and stimulated with control isotype abs or crosslinked anti-cd28.2 (2 mg ml −1 ), by using human il-6 (#dy206), il-22 (#dy782), and mmp9 (#dy911) elisa kits (r&d systems). data were analyzed on a bio-plex (bio-rad, hercules, ca, usa). the assays were performed in duplicate. the sensitivity of the assay was 9.4 pg ml −1 for il-6 and 31.2 pg ml −1 for il-22 and mmp9. cd4 + t cells were plated at 2 × 10 6 ml −1 in 24-well plate or 24well plate inserts in the experiments of co-culture with caco-2 cells and stimulated for the indicated times with control isotype matched abs (ig) or crosslinked anti-cd28 (2 mg ml −1 ), or anti-cd3 (2 mg ml −1 ) or anti-cd3 plus anti-cd28 abs (2 mg ml −1 ). total rna was extracted by either cd4 + t cells or caco-2 cells using trizol according to the manufacturer's instructions and was reverse-transcribed into cdna by using moloney murine leukemia virus reverse transcriptase (thermo fisher scientific ca, usa). taqman universal pcr master mix and human il-6, il-22, mmp9, muc1, and gapdh primer/probe sets were from thermo fisher scientific. the relative quantification was performed using the comparative c t method. the results were expressed as arbitrary units (au) by using the mean value of cells stimulated with control ig as c t calibrator or fold induction (f.i.) over control ig-stimulated cells after normalization to gapdh. the pil-22 (-644)-gfp construct containing the gfp under the control of the −644 bp region upstream of the transcriptional start site and the +156 region upstream the translational start site was generated as previously described (36) . briefly, human il-22 promoter fragment (−1069/+156) was amplified from genomic dna isolated from human pbmcs by using amplitaq (applied biosystem). the primers used (excluding additional flanking cloning/restriction sites) were as follow: forward 5′-ggtatttgcattttgatacttgcatttgct-3′ and 5′-tgcagacaattctaactcgag-3′. by using an internal asei restriction site located at position − 644 of the human il-22 promoter, the pil-22 (-644)-gfp construct was obtained by subcloning the pcr fragment into asei-hind iii sites within the cmv promoter of pegfp-n1 vector (clontech, uk). the sequence of human il-22 promoter fragment (−644/+156) was verified by dna sequencing and was as follow: 5′-attaata plasmid vectors expressing ha-tagged human rela, ikka, ikkb and nik were previously described (37, 38) . cd28-positive jurkat t cells were electroporated (at 260 v, 960 mf) in 0.45 ml rpmi-1640 supplemented with 10% fbs with 1 mg of pil-22(-644)-gfp together with pcdna3 control vector 10 mg ha-rela, or ha-ikka or ha-ikkb or 20 mg ha-nik. twenty-four hours after transfection, the cells were analyzed by using a bd facscan flow cytometer (bd biosciences, italy). mean fluorescence intensity was calculated on a total of 5 × 10 3 gfp-positive events by gating for ssc and fsc. chromatin immunoprecipitation 10 7 cd4 + t cells were stimulated as indicated and chromatin immunoprecipitation (chip) assays were performed as previously described (37) . briefly, after fixing in 1% formaldehyde, t cells were lysed for 5 min in 50 mm tris, ph 8.0, 2 mm edta, 0.1% np-40, 10% glycerol supplemented with proteases inhibitors. nuclei were suspended in 50 mm tris, ph 8.0, 1% sds, and 5 mm edta. chromatin was sheared by sonication, centrifuged and diluted 10 times in 50 mm tris, ph 8.0, 0.5% np-40, 0.2 m nacl, 0.5 mm edta. after pre-clearing with a 50% suspension salmon sperm-saturated protein-a or protein g sepharose beads (amersham), lysates were incubated at 4°c overnight with anti-rela (1:100 dilution), anti-relb (2 mg), anti phoshotyr705 stat3 (pstat3, 1:100 dilution), anti-rna-polymerase ii (pol ii, 2 mg), or control anti-lck abs (2 mg). immune complexes were collected with sperm-saturated protein-a or protein g sepharose beads, washed three times with high salt buffer (20 mm tris, ph 8.0, 0.1% sds, 1% np-40, 2 mm edta, 500 mm nacl), and five times with 1× tris/edta (te). immune complexes were extracted in 1× te containing 1% sds, and protein-dna cross-links were reverted by heating at 65°c overnight. dna was extracted by phenol-chloroform and about 1/30 of the immunoprecipitated dna was analyzed by real-time pcr. quantitative real-time pcr with sensimix ™ sybr ® hi-rox kit (bioline, uk) was performed for the human il-22 promoter. specific enrichment was calculated as previously described (39) by using the cycle threshold (ct): 2 (ct of control chip-ct of control input)/ 2 (ct of specific chip -ct of specific input) . the human il-22 promoter primers used for each specific chip were as follow: rela i, relb i and pstat3 i, 5′-gcttactcagcc actataggagatca-3′ and 5′-ccggagggtatttta c a g a c a a a t c c -3 ′ ; rela ii, 5′ -a c c c t c c g g g ctctaatagtgac-3′ and 5′-agaacgacctaaccacca cagga-3′; pstat3 ii and pol ii 5′-cctgtggtggtta ggtcgttct-3′ and 5′-gctgcttttatagcccccag gat-3′. the cytotoxicity of as605240 on caco-2 cells was evaluated by propidium iodide (pi) staining (10 mg ml −1 ). caco-2 cells were plated at 2.5 × 10 5 cells ml −1 in 24-well plates and treated with 10 mm as605240 or dmso, as vehicle control, for 48 h. cytotoxicity was analyzed by a bd biosciences facscalibur (mountain view, ca) by quantifying the percentage of pi positive cells. results were calculated from at least three independent experiments. the sample size was chosen based on previous studies to ensure adequate power. parametrical statistical analysis (mean and sem) was performed to evaluate differences between continuous variables through prism 5.0 (graph pad software, san diego, ca) using student t test. for multiple group comparisons, significant differences were calculated using nonparametric mann-whitney u or wilcoxon tests, and linear regression analyses were performed using the pearson chi-squared test. for all tests, p values < 0.05 were considered significant. we have recently found that cd28 stimulation induces the expression of th17 related cytokines in cd4 + t cells from hd, relapsing-remitting ms (rrms) and t1d patients (32) (33) (34) . more recent preliminary data also evidenced that cd28 stimulation up-regulated il-22 gene expression in peripheral cd4 + t cells from stable rrms patients (35) . in order to verify whether cd28-induced il-22 expression was also effective in hd and clarify the molecular basis of this activity, we performed a kinetic analysis of il-22 gene expression by stimulating human cd4 + t cells from hd with an agonistic anti-cd28 ab (cd28.2) binding the same epitope recognized by b7 molecules (40) or anti-cd3 (ucht1) or anti-cd3 plus anti-cd28 abs. cd28 stimulation of cd4 + t cells from one representative hd induced a significant increase (p < 0.01) of il-22 gene expression within 6 h that further increased 24 to 48 h and decreased 72 h after stimulation ( figure 1a) . no significant differences in neither il-22 mrna levels nor kinetics were observed between cd28 and cd3 plus cd28 stimulation, whereas cd3 stimulation alone induced only a slight increase of il-22 gene expression after 48 h ( figure 1a) . similar results were obtained by analyzing il-22 gene expression in cd28-stimulated cd4 + t cells from a larger sample size (n = 7) (figures 1b, c) . cd28-induced il-22 gene expression was also associated with a strong increase of il-22 cytokine secretion after 24 to 48 h from stimulation ( figures 1d, e) . in the human system, il-22 gene expression has been described to be induced by il-6 (41). consistently with our previous data (33) , cd28 stimulation induced a strong il-6 secretion ( figure 1f ). il-6-mediated signaling alone was not sufficient for inducing il-22 gene expression ( figure 1g) . however, the blockade of il-6-mediated signaling by a neutralizing anti-il-6 ab strongly inhibited cd28-induced il-22 gene expression ( figure 1h ) and secretion ( figure 1i ) in cd4 + t cells from hd. as we previously observed for il-17a (33) and other pro-inflammatory cytokines (31) , the analysis of cd28-induced il-22 secretion in purified naïve cd4 + cd45ra + (figure 2a ) or effector/memory cd4 + cd45ro + ( figure 2b ) evidenced no significant differences between the two population ( figure 2c) . these data evidence that cd28 intrinsic signaling regulates il-22 gene expression and secretion in both naïve and effector/ memory cd4 + t cells in a il-6-dependent manner. the human il-22 gene promoter contains two putative stat3 responsive elements (stat3 i and ii) and two nf-kb binding sites (nf-kb i and ii) upstream of the transcription start site that have been involved in il-22 promoter trans-activation (42, 43) ( figure 3a) . we have previously demonstrated that human cd28 individual stimulation induces the activation and nuclear translocation of rela (31, 44) and, more recently, we evidenced that cd28 induces the phosphorylation on tyr705 of stat3 (pstat3) and its nuclear translocation in a il-6dependent manner. pstat3 and rela/nf-kb in turn cooperate for inducing the promoter trans-activation and transcription of il-17a (33) . to assess if pstat3 and rela were also involved in cd28-mediated up-regulation of il-22, we performed chromatin immunoprecipitation (chip) assays in peripheral cd4 + t cells stimulated with agonistic anti-cd28.2 abs. to do that, three distinct oligonucleotide probes were used; probe i (−338 to −203) for both stat3 i and nf-kb i binding sites, probe ii (−213 to −110) for nf-kb ii binding site and probe iii (−131 to −20) for both stat3 ii binding site and tata box ( figure 3a) . the kinetic analysis of the specific recruitment to the il-22 promoter evidenced that rela efficiently bound nf-kb i but not nf-kb ii consensus sequences within 6 h from stimulation and persisted over 24 h, whereas no significant recruitment of relb was observed ( figure 3b ). pstat3 was significantly recruited on both stat3 i and stat3 ii consensus sequences on the il-22 promoter with similar kinetics ( figure 3c ). the specific recruitment of both rela on the nf-kb i and pstat3 on both i and ii binding sites was confirmed in cd4 + t cells from a larger sample size (n = 9) and was associated to cd28-induced transcriptional activation of il-22 promoter, as evidenced by rna polymerase ii (pol ii) promoter occupancy ( figure 3d ). the significant inhibition of il-22 gene expression exerted by the stat3 inhibitor s31-201 (45, 46) and the nf-kb inhibitor ps1145 (47) (figure 3e ), confirmed the mutual cooperation of both transcription factors in regulating cd28induced il-22 expression. the main functional effects of il-22 are exerted on epithelial cells, such as intestinal cells, where il-22 promotes the production of anti-microbial peptides, mucins and matrix metalloproteases (mmps) required for epithelial barrier functions and protection against extracellular pathogens (8, 9) . we next analyzed the functional role of cd28-mediated activation of il-22-producing t cells on intestinal epithelial cells. to do that, cd4 + t cells were stimulated with control isotype matched abs (ig), or anti-cd28, or anti-cd3 or anti-cd3 plus anti-cd28 abs and co-cultured in trans-well plates with caco-2 cells for different times. the gene expressions of mucin 1 (muc1), mmp9 and s100a9 anti-microbial peptide were then analyzed. the kinetic analysis evidenced that both mmp9 ( figure 4a ) and muc1 ( figure 4b ) gene expressions were strongly up-regulated in caco-2 cells after 48 h of coculture with cd28-stimulated cd4 + t cells, whereas no significant increase in s100a9 was observed ( figure 4c ). similar results were obtained by analyzing mmp9 ( figure 4d ) and muc1 ( figure 4e ) gene expression in caco-2 cells cocultured with cd28-stimulated cd4 + t cells from a larger sample size (n = 9). moreover, the up-regulation and muc1 gene expression induced by co-culturing caco-2 cells with cd28-stimulated cd4 + t cells was also associated with a strong increase of muc1 protein content ( figure 4f ) after 48 h from stimulation. in contrast to the gene expression data ( figure 4d ), when we looked at mmp9 secretion we found high levels of mmp9 in culture supernatants from caco-2 cells cocultured with cd4 + t cells stimulated with control ig that further increased following cd28 stimulation (supplementary figure s1a) , thus suggesting that cd4 + t cells also produced mmp9, as previously reported (48) . cd4 + t cells secreted, indeed, high levels of mmp9 in culture supernatant and no significant differences were observed following cd28 stimulation (supplementary figure s1b) . figure 1h ) and secretion ( figure 1i ), muc1 gene expression was strongly impaired by anti-il-6 neutralizing abs in caco-2 cells cocultured with cd28-stimulated cd4 + t cells ( figure 4h ). cd28 pro-inflammatory functions depend on the ability of its intracytoplasmic tail to recruit and activate class 1a pi3k (49) (50) (51) . we have recently demonstrated that class 1a pi3k activity is crucial for cd28-induced up-regulation of pro-inflammatory cytokines related to th17 cell phenotype in both hd and rrms patients (33, 35) . consistently, a non-cytotoxic dose of the class 1a pi3k inhibitor as605240 (52) (figures 5a, b) , significantly impaired mmp9 and muc1 gene expression in caco-2 cells co-cultured with cd28-stimulated cd4 + t cells ( figure 5c ) as well as cd28induced il il-22 ( figure 5d ) and il-6 secretion ( figure 5e) . moreover, the inability of recombinant il-6 to restore impaired il-22 gene expression in as605240-treated cd28-stimulated cd4 + t cells, strongly support a role of class 1a pi3k downstream cd28. altogether these data support a crucial role of cd28associated pi3k in il-22-regulated epithelial barrier functions. frontiers in immunology | www.frontiersin.org october 2020 | volume 11 | article 590964 discussion il-22 is an important cytokine that strengthen epithelial barrier functions against extracellular pathogen and regulates tissue homeostasis and repair. however, excessive production of il-22 has been also associated with severe complications in several pathogen infections (12) , including the recently identified sars-cov2 (18) as well as with inflammatory bowel diseases such as crohn's disease (23) , neurological disorders such as ms and guillain-barrè syndrome (25, 26) , and rheumatoid arthritis (53, 54) . therefore, the identification of receptors and associated signaling mediators regulating il-22 expression could represent an important goal of the ongoing research in inflammation and autoimmunity. in mouse t cells, il-22 is mainly regarded as a th17 cytokine (41, 55, 56) , whereas in human cd4 + t cells il-22 expression often does not correlate with the expression of il-17 or the master transcriptional regulator of th17 cells retinoic acid receptor-related orphan nuclear receptor gt (rorgt) (57, 58) and is produced by a unique memory th22 cell subset (3, 4) . herewith, we evidence that cd28 stimulation induced il-22 expression and production in the absence of tcr engagement (figure 1) . the up-regulation of il-22 transcription by cd28 autonomous stimulation of peripheral cd4 + t cell followed a kinetic ( figures 1a, b ) similar to il-17a (33) and no significant differences were observed between naïve (cd45ra) and effector/ memory (cd45ro) cd4 + t cells ( figure 2) . moreover, we found that cd28-induced up-regulation of il-22 depended on cd28-induced il-6 ( figure 1f) , as demonstrated by the strong inhibitory effects exerted by neutralizing anti-il-6 abs ( figures 1h, i) . however, although il-6 itself is able to prime il-22 production in activated human cd4 + t cells (41) , stimulation of cd4 + t cells with il-6 alone is not sufficient for up-regulating il-22 gene expression ( figure 1g ) but requires a second signal delivered by cd28. in this contest, among the most promising therapeutic strategies for dampening excessive inflammatory responses and ards in sars-cov2 infection, tocilizumab, an anti-il-6r mab inhibitor that interferes with il-6-mediated signaling and has already approved for ra treatment (59, 60) resulted effective in preventing the cytokine storm, ards and to reduce mortality in covid-19 patients (61) (62) (63) . il-6 regulates the expression of both il-17a and il-22 through il-6 receptor-associated stat3 (41, 55, (64) (65) (66) (67) . we have recently demonstrated that cd28 stimulation induced a strong and persistent stat3 phosphorylation on tyr705 and its nuclear translocation in cd4 + t cells. we also found that activated stat3 was also recruited to the proximal human il-17a promoter and induced its trans-activation in response to cd28 stimulation (33) . in t cells from stat3 deficient mice, il-22 expression is impaired (42, 68) , whereas the overexpression of constitutively active stat3 up-regulates il-22 expression (66) . for instance, two functional stat3 binding sites have been identified upstream of the transcription start site within the mouse il-22 promoter (42) . the human il-22 promoter contains similar high consensus sequences ( figure 3a ) and we found that cd28 stimulation promoted the recruitment of stat3 on both binding sites in human cd4 + t cells ( figures 3c, d) . these data together with the strong inhibition of cd28induced il-22 expression by the stat3 inhibitor s31-201 ( figure 3e) , support a direct role of stat3 in regulating il-22 gene expression in cd28-stimulated cd4 + t cells. in the human il-22 promoter, two putative nf-kb responsive elements (43) , nf-kb i and ii (figure 2a) , containing the consensus binding sequence gggrnnyycc (where r represents a or g, n represents any nucleotide, and y represents c or t) has been also identified (69) . the nf-kb family comprises five members: p50/nf-kb1, p52/nf-kb2, rela/p65, relb and c-rel. rela, c-rel and relb contain a transactivation domain and form transcriptionally active heterodimers in association with p50 and/or p52 (70) . we have demonstrated that cd28 autonomous stimulation by either b7.1/cd80 or anti-cd28.2 agonistic abs activates a non-canonical nf-kb2-like cascade (27) by recruiting and activating nik and ikka (71, 72) , thus leading to the nuclear translocation of rela-containing nf-kb dimers and to the transactivation of pro-inflammatory target cytokines, including il-17a (31) (32) (33) 44) . consistently, rela overexpression strongly upregulated human il-22 promoter-gfp construct [hil-22 prom (-644)-gfp], containing the gfp construct under the control of the il-22 5′-flanking region (−644) upstream of the transcriptional start point (supplementary figure s2) . moreover, as observed in ilc3 cells (43) , rela/nf-kb binds the high homology consensus sequence ii at position −184 of the human il-22 promoter ( figure 3a) and cooperates with stat3 for inducing il-22 transcription in cd28-stimulated cd4 + t cells (figures 3b, d) . although both ikka and ikkb induced a significant (p < 0.05) trans-activation of hil-22 promoter at similar levels (1.6 fold inductions) when overexpressed in jurkat cells, a strongest up-regulation (4 fold inductions) of hil-22 promoter was induced by nik overexpression (supplementary figures s1d, e) . these data are consistent with those from rudloff et al., who identified ikka as an important regulator of il-22 gene expression (36) . il-22 regulates essential functions in host defense against pathogens by mediating the crosstalk between the immune system and epithelial cells. in fact, il-22 regulates the expression of several genes that maintain the barrier functions of intact epithelium (9) . these include molecules involved in tight and gap junctions such as claudins that keep epithelial cells together and regulate the permeability of intestinal epithelial barrier to water, nutrients and ions (73) , anti-microbial proteins such as s100a (55, 74) and mucins. muc1, a membrane-bound mucin, is the main component of the intestinal mucus layer that provides a physiological barrier to prevent close contacts between epithelial cells and microbes (75) . in addition to its physiological barrier functions, muc1 has been described to modulate the functions of inflammatory t cells. in muc1-deficient mice, th17 cells and il-17-producing ilc3 cells were expanded and exacerbated colitis (76) . similarly, a reduced glycosylation of muc1 increased both susceptibility to colitis and progression to colon cancers (77, 78) . in both human and mouse colonic epithelial cell lines, il-22 has been reported to promote muc1 expression (79, 80) . we extend these data by evidencing an important role of cd28 in regulating epithelial barrier functions. during cd28 stimulation, rela/nf-kb and il-6-associated stat3 cooperate for inducing il-22 expression and secretion ( figure 3 ) that in turn acts on epithelial cells by promoting the up-regulation of muc1 ( figure 4) . moreover, il-22 secreted by cd28-activated cd4 + t cells also stimulate mmp9 expression in caco-2 cells (figure 4) . interestingly, pujada et al. recently showed that mmp9 expression in colonic epithelium was associated with a more efficient epithelial barrier functions by maintaining epithelial integrity, microbiota and immune homeostasis (81) . however, mmp9 is also up-regulated in inflamed intestine of ibd patients and has been recently suggested as a predictor marker of disease exacerbation in cd (82, 83) . moreover, high levels of mmp9 were also found in the serum and cerebrospinal fluid of ms patients and increase during relapses (84, 85) . thus, anti-inflammatory compounds able to dampen excessive production of mmps might represent potential therapeutic strategies for inflammatory diseases. our findings on the ability of class 1a pi3k inhibitor to impair both cd28-induced il-6 and il-22 production as well as il-22-dependent mmp9 and muc1 expression in caco-2 cells ( figure 5 ) are consistent with data from yan et al. who identified the pi3k/akt/mtorc1 as a crucial pathway in regulating both th17 cell-mediated inflammation and lung injury in ards (15) . our results provide novel insights on the role of cd28 and associated signaling mediators in il-22 regulation in human cd4 + t cells and may provide the biological bases for the development of new therapeutic strategies to dampen inflammatory and autoimmune disorders. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by ethical committee of the policlinico umberto i (ethical code n. 1061bis/2019, 13/09/2019). the patients/ participants provided their written informed consent to participate in this study. mk performed most of the experiments, analyzed the data, interpreted the results, and helped in writing the manuscript. ca, sf, sc, ms, and mb performed parts of the experiments and data analyses. sa contributed with human samples for the study. mk, ca, sf, ms, and lb were involved in the discussion about the data. lt designed the study, coordinated the work, and wrote the manuscript. all authors contributed to the article and approved the submitted version. il-tif/il-22: genomic organization and mapping of the human and mouse genes differentiation and function of th17 t cells production of interleukin 22 but not interleukin 17 by a subset of human skinhoming memory t cells identification of a human helper t cell population that has abundant production of interleukin 22 and is distinct from t(h)-17, t(h)1 and t(h)2 cells th22 cells represent a distinct human t cell subset involved in epidermal immunity and remodeling interleukin-22: a cytokine produced by t, nk and nkt cell subsets, with importance in the innate immune defense and tissue protection a human natural killer cell subset provides an innate source of il-22 for mucosal immunity interleukin-22: immunobiology and pathology signaling at the epithelial-microbiota interface il-22-producing cd4+ t cells: middle-men between the immune system and its environment il-17 and il-22: siblings, not twins il-22: there is a gap in our knowledge directing traffic: il-17 and il-22 coordinate pulmonary immune defense il-22 mediates mucosal host defense against gram-negative bacterial pneumonia rapamycin attenuates acute lung injury induced by lps through inhibition of th17 cell proliferation in mice a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in wuhan th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib marked t cell activation, senescence, exhaustion and skewing towards th17 in patients with covid-19 pneumonia emerging role of interleukin-22 in autoimmune diseases clinical importance of il-22 cascade in ibd interleukin-22 in human inflammatory diseases and viral infections the interleukin-20 cytokines in intestinal diseases exploring the role of interleukin-22 in neurological and autoimmune disorders the interleukin-10 family of cytokines and their role in the cns the modulators of inflammation in multiple sclerosis. cells cd28 costimulatory signals in t lymphocyte activation: emerging functions beyond a qualitative and quantitative support to tcr signalling cd28 and ctla-4 coreceptor expression and signal transduction signaling amplification at the immunological synapse nf-kappab family of transcription factors: biochemical players of cd28 co-stimulation a non-conserved amino acid variant regulates differential signalling between human and mouse cd28 cd28 ligation in the absence of tcr stimulation up-regulates il-17a and pro-inflammatory cytokines in relapsing-remitting multiple sclerosis t lymphocytes cd28 individual signaling up-regulates human il-17a expression by promoting the recruitment of rela/nf-kb and stat3 transcription factors on the proximal promoter isa-2011b, a phosphatidylinositol 4-phosphate 5-kinase alpha inhibitor, impairs cd28-dependent costimulatory and pro-inflammatory signals in human t lymphocytes cd28 autonomous signaling up-regulates c-myc expression and promotes glycolysis enabling inflammatory t cell responses in multiple sclerosis mechanisms of rapid induction of interleukin-22 in activated t cells and its modulation by cyclosporin a rela/nf-kappab recruitment on the bax gene promoter antagonizes p73-dependent apoptosis in costimulated t cells mitogen-activated kinase kinase kinase 1 regulates t cell receptor-and cd28-mediated signaling events which lead to nf-kappab activation epigenetic remodeling of the il-2 and ifn-gamma loci in memory cd8 t cells is influenced by cd4 t cells differential recognition by cd28 of its cognate counter receptors cd80 (b7.1) and b70 (b7.2): analysis by site directed mutagenesis interleukin-22, a t(h)17 cytokine, mediates il-23-induced dermal inflammation and acanthosis il-21 induces il-22 production in cd4+ t cells il-18 drives ilc3 proliferation and promotes il-22 production via nf-kappab cd28 delivers a unique signal leading to the selective recruitment of rela and p52 nf-kappab subunits on il-8 and bcl-xl gene promoters isoform selective inhibition of stat1 or stat3 homo-dimerization via peptidomimetic probes: structural recognition of stat sh2 domains selective chemical probe inhibitor of stat3, identified through structurebased virtual screening, induces antitumor activity effects of ikk inhibitor ps1145 on nf-kappab function, proliferation, apoptosis and invasion activity in prostate carcinoma cells human t lymphocytes synthesize the 92 kda type iv collagenase (gelatinase b) phosphatidylinositol 4-phosphate 5-kinase alpha activation critically contributes to cd28-dependent signaling responses phosphatidylinositol 4-phosphate 5-kinase alpha and vav1 mutual cooperation in cd28-mediated actin remodeling and signaling functions cutting edge: cd28-mediated transcriptional and posttranscriptional regulation of il-2 expression are controlled through different signaling pathways functional redundancy of class i phosphoinositide 3-kinase (pi3k) isoforms in signaling growth factor-mediated human neutrophil survival emerging cell and cytokine targets in rheumatoid arthritis the il-20 cytokine family in rheumatoid arthritis and spondyloarthritis interleukin (il)-22 and il-17 are coexpressed by th17 cells and cooperatively enhance expression of antimicrobial peptides expression and regulation of il-22 in the il-17-producing cd4+ t lymphocytes cd28-mediated regulation of il-22 frontiers in immunology | www.frontiersin.org october a critical function for transforming growth factor-beta, interleukin 23 and proinflammatory cytokines in driving and modulating human t(h)-17 responses multiparametric analysis of cytokine-driven human th17 differentiation reveals a differential regulation of il-17 and il-22 production tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (adacta): a randomised, double-blind, controlled phase 4 trial a new era for the treatment of inflammatory autoimmune diseases by interleukin-6 blockade strategy tocilizumab treatment in covid-19: a single center experience tocilizumab, an anti-il6 receptor antibody, to treat covid-19-related respiratory failure: a case report tocilizumab in patients with severe covid-19: a retrospective cohort study il-6 programs t (h)-17 cell differentiation by promoting sequential engagement of the il-21 and il-23 pathways il-21 is produced by th17 cells and drives il-17 production in a stat3-dependent manner stat3 regulates cytokine-mediated generation of inflammatory helper t cells diverse targets of the transcription factor stat3 contribute to t cell pathogenicity and homeostasis essential autocrine regulation by il-21 in the generation of inflammatory t cells interactions of nf-kappab with chromatin: the art of being at the right place at the right time shared principles in nf-kappab signaling a novel association between filamin a and nf-kappab inducing kinase couples cd28 to inhibitor of nf-kappab kinase alpha and nf-kappab activation vav-1 and the ikk alpha subunit of i kappa b kinase functionally associate to induce nf-kappa b activation in response to cd28 engagement il-22 increases permeability of intestinal epithelial tight junctions by enhancing claudin-2 expression il-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis structure and function of the cell surface (tethered) mucins the membrane-bound mucin muc1 regulates t helper 17-cell responses and colitis in mice cutting edge: transgenic expression of human muc1 in il-10-/-mice accelerates inflammatory bowel disease and progression to colon cancer increased susceptibility to colitis and colorectal tumors in mice lacking core 3-derived o-glycans il-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis expression of il-24, an activator of the jak1/stat3/socs3 cascade, is enhanced in inflammatory bowel disease matrix metalloproteinase mmp9 maintains epithelial barrier function and preserves mucosal lining in colitis associated cancer ecm remodelling in ibd: innocent bystander or partner in crime? the emerging role of extracellular molecular events in sustaining intestinal inflammation serum mmp-9: a novel biomarker for prediction of clinical relapse in patients with quiescent crohn's disease, a post hoc analysis interplay between matrix metalloproteinase-9, matrix metalloproteinase-2, and interleukins in multiple sclerosis patients imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrier the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020.590964/ full#supplementary-material key: cord-254192-86ksgl5t authors: li, liang; xue, mei; fu, fang; yin, lingdan; feng, li; liu, pinghuang title: ifn-lambda 3 mediates antiviral protection against porcine epidemic diarrhea virus by inducing a distinct antiviral transcript profile in porcine intestinal epithelia date: 2019-10-17 journal: front immunol doi: 10.3389/fimmu.2019.02394 sha: doc_id: 254192 cord_uid: 86ksgl5t type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ3 exerts a more potent effect than ifn-λ1. however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ3 has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ3 and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j2) cells by rna-seq. our results showed that the pretreatment of ipec-j2 cells with ifn-λ3 resulted in the differential expression of 983 genes. in contrast, ifn-α only modified the expression of 134 genes, and 110 of these genes were also observed in the response to ifn-λ3. a transcriptional enrichment analysis indicated that ifn-λ3 or ifn-α regulates multiple cellular processes and that ifn-λ3 activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j2 cells and porcine enteroids. moreover, transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by ifn-λ3 significantly inhibited pedv infection. collectively, the data showed that ifn-λ3 induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ3. these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. type iii interferon-lambda (ifn-λ) plays a critical role against infection, particularly in mucosal infection in the respiratory and gastrointestinal tract. our study and other previous studies have shown that porcine ifn-λ more efficiently curtails the infection of porcine epidemic diarrhea virus (pedv) in the intestine epithelia than type i ifn, whereas ifn-λ3 exerts a more potent effect than ifn-λ1. however, the underlying mechanism remains elusive, and in particular, the transcriptional profile induced by ifn-λ3 has not been reported. here, to resolve the mechanism responsible for the disparity between ifn-λ3 and type i ifn in anti-mucosal virus infection, we compared the transcription profiles induced by the two ifns in porcine intestinal epithelial (ipec-j2) cells by rna-seq. our results showed that the pretreatment of ipec-j2 cells with ifn-λ3 resulted in the differential expression of 983 genes. in contrast, ifn-α only modified the expression of 134 genes, and 110 of these genes were also observed in the response to ifn-λ3. a transcriptional enrichment analysis indicated that ifn-λ3 or ifn-α regulates multiple cellular processes and that ifn-λ3 activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, than ifn-α. furthermore, we verified the rna-seq results through an rt-qpcr analysis of ipec-j2 cells and porcine enteroids. moreover, transient expression of the porcine rsad2 and mx2 genes among the top 10 genes induced by ifn-λ3 significantly inhibited pedv infection. collectively, the data showed that ifn-λ3 induces a unique transcriptional profile that does not completely overlap with that induced by ifn-α and strongly elicits a set of genes responsible for the antiviral activity of ifn-λ3. these findings provide important knowledge regarding the elicited isgs of type i and iii ifns in restricting porcine intestinal viral infection. the surface epithelia of the mucosa are the major entry site of most pathogens in the host and serve as a first line of defense against invading pathogens. one of the most important antiviral cytokines in the host is interferons (ifns), which perform key roles in inhibiting viral infection (1, 2) . the ifn family is categorized into three different types: type i ifn (ifn-α/β), type ii ifn (ifn-γ), and type iii ifn (ifn-λ). type ii ifn, which is primarily produced by t cells and natural killer cells, exerts limited direct antiviral activity and plays a key role in modulating the host immune response (3) , whereas type i ifns (α/β) and the more recently discovered type iii ifns induce a strong antiviral state in responsive cells and play crucial roles in controlling viral infection (4) (5) (6) (7) (8) . although type i ifns have generally been thought to be a key element against systemic infections, recent research has shown that ifn-λ plays a critical role in mucosal infections, such as enteric infection (9, 10) . unlike type i ifns that are secreted by a wide range of different cell types upon stimulation, type iii ifns are primarily produced by epithelial cells, nk cells, and dendritic cells (dcs) (8, (11) (12) (13) . ifn-λ acts primarily on the mucosal epithelium, which might result in fewer side effects compared with type i ifn treatment (8) . these features make ifn-λ a potentially superior antiviral therapeutic candidate against local mucosal infection (7) . although the receptors for type i and iii ifns are different, the binding of both type i and iii ifns to their corresponding receptors stimulates a janus kinase (jak)-signal transducer of transcription (stat) pathway, and the stimulation of this pathways subsequently drives the transcription of ifnstimulated genes (isgs) and prompts cells toward an antiviral status (14) . consistent with the similarity of the induced signaling pathways, the spectrum of genes elicited by the two types of ifns show a high overlap (2) . however, recent studies have demonstrated that type iii ifns are critical non-redundant antiviral mediators of type i ifns in the gi tract (2) . to date, numerous studies in humans or mice have taken advantage of rna-seq or chip assays to show that ifn-λ and ifn-α elicit distinct downstream signaling events, even though many genes are induced by both type i and iii ifns (15, 16) . mice with type i ifn or iii ifn receptor knockout experience more severe viral intestinal infections, but ifnl −/− mice show higher viral loads and more serious clinical symptoms than ifnar −/− mice (17, 18) . studies conducted by pott et al. showed that intestinal epithelial cells exhibit stronger responses to ifn-λ compared with ifn-α/β in vivo (19, 20) . a comprehensive understanding of the unique signaling profiles of type i and iii ifns has become increasingly important for understanding host-virus interactions and the development of ifn-λ therapeutics. however, thus far, no direct comparative analyses of the transcriptional profiles induced by porcine type i vs. type iii ifns in swine intestinal epithelia have been performed. the piglet diarrhea caused by enteric coronavirus porcine epidemic diarrhea virus (pedv) is a highly contagious disease characterized by watery diarrhea, dehydration, and causes up to 100% mortality in neonatal piglets. we and other research groups previously reported that porcine ifn-λ results in better suppression against pedv infection compared with ifn-α and that ifn-λ3 more efficiently inhibits pedv than ifn-λ1 (21) (22) (23) . however, the mechanisms underlying the difference among ifn-λ1, ifn-λ3, and ifn-α in inhibiting enteric coronavirus remain less clear. previous studies have largely focused on the gene profiles induced by human or mouse ifn-λ1 and ifn-α, but the ifn-λ3-and ifn-α-elicited genes have not been compared. in this study, we comprehensively compared the transcriptional profiling of ifn-λ3-and ifn-α-induced genes in a porcine intestinal epithelial cell line (ipec-j2) and verified the rna-seq results by reverse transcriptase quantitative pcr (rt-qpcr) in vitro, and further confirmed the transcriptional profile difference in crypt-derived porcine enteroids. the intestinal porcine epithelial cell line j2 (ipec-j2; kindly provided by dr. anthony blikslager, north carolina state university, raleigh, nc, usa) was maintained in dulbecco's modified eagle's medium nutrient mixture f-12 (dmem/f12) supplemented with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), 0.1 mm hepes (gibco, usa), and 10% heat-inactivated fetal bovine serum (fbs) (gibco). african green monkey kidney cells (vero e6) were grown and maintained in dmem supplemented with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin) and 10% heat-inactivated fbs (gibco). pedv strain cv777 of genotype 1 (genbank accession no. kt323979) was maintained at the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. the biological antiviral activity of e. coli-derived recombinant porcine ifn-lambda 3 was prepared in our laboratory and evaluated in mdbk cells using a recombinant vesicular stomatitis virus (vsv) with a gfp reporter as described previously (22, 24) . the weight-activity unit (u/ml) of samples was calculated using porcine prokaryotic-derived ifn-α (4.0 × 10 8 u/mg) (prosit sole biotechnology, co., ltd., beijing, china) as a reference. porcine intestinal crypts were prepared from specific pathogenfree piglets using previously described protocols (21) . in brief, the intestine was flushed with cold pbs with antibiotics (100 units/ml penicillin and 100 µg/ml streptomycin), cut into 2mm segments, and washed with cold pbs with antibiotics until the supernatant was clear. the washed intestinal pieces were suspended in 15 ml of gentle cell dissociation reagent (stemcell, canada) and shaken at 100 rpm for 25 min to disassociate the crypts at room temperature (rt). the pellets of the intestinal pieces were suspended in 10 ml of cold pbs with 0.1% bovine serum albumin (bsa) and antibiotics (pen-strep) and passed through a 70-µm cell mesh. the crypt pellets were harvested by centrifugation at 200 × g at 4 • c for 5 min and resuspended in 10 ml of cold dmem/f12. after counting, the intestine crypts were resuspended in 25 µl of intesticult organoid growth medium (stemcell, canada) and 25 µl of matrigel (bd biosciences, usa) per 50 crypts and seeded into a 48-well plate at 50 crypts per well. the plate was incubated at 37 • c for 10 min until the matrigel solidified. the plate was filled with complete intesticult organoid growth medium and then incubated at 37 • c in a 5% co 2 incubator. the culture medium was exchanged every 3-4 days. the institutional animal care and use committee of the harbin veterinary research institute approved all the protocols related to the animal experiments performed in this study. expanded 3d enteroids were recovered from the matrigel after 7-11 days of growth by the addition of ice-cold dmem/f12 medium, transferred into 15-ml tubes, and centrifuged at 250 × g at 4 • c for 5 min. the pellet of enteroids was incubated in 0.25% trypsin (gibco) for 5 min at 37 • c and dissociated by repeated pipetting to obtain a single-cell suspension. dmem-f12 with 10% (v/v) fbs was added into the single-cell suspension, and the mixture was centrifuged at 800 × g for 5 min. the cell pellets were resuspended in complete intesticult organoid growth medium at rt and seeded at 50 enteroids per well in a matrigel-precoated 96-well plate. after differentiation for about 3-4 days, planar monolayers of 2d enteroids were ready for use in experiments. total cellular rna was extracted using the simply p total rna extraction kit (bioflux, china) according to the manufacturer's instructions. total rna (1 µg) was reverse-transcribed to cdna using the primescript tm ii first-strand cdna synthesis kit (takara, china). the synthesized cdna was subjected to qpcr performed in triplicate using a lightcycler r 480 ii real-time pcr instrument (roche, switzerland) and sybr green pcr mix (life technologies, usa) according to the manufacturer's instructions. all the data were acquired and analyzed using lightcycler r 480 ii software 1.5 based on the cycle threshold ( ct) method (25) . gapdh served as the internal control. the amplification efficiency of qpcr primers ranged from 85.83 to 106.38%. the primers used in this assay were designed using primer premier 5 software and are listed in table 1 . three biological replicates of each of the three groups, namely, untreated ipec-j2 cells (mock control), ipec-j2 cells treated with ifn-λ3 (1,000 ng/ml) for 24 h, or ipec-j2 cells treated with ifn-α (1,000 ng/ml) for 24 h, were prepared for rna sequencing. total rna was purified using the trizol reagent according to the manufacturer's instructions (thermo fisher scientific, usa). the total rna from each sample was quantified and qualified using an agilent 2100 bioanalyzer (agilent technologies, usa), a nanodrop instrument (thermo fisher scientific, inc.), and a 1% agarose gel. one microgram of total rna with a rin value >7 was used for subsequent library construction. next-generation enzyme mix to repair both ends and add a da tail through one reaction, and the product was subjected to t-a ligation to add adaptors to both ends. size selection of adaptor-ligated dna was then performed using an axyprep mag pcr clean-up kit (axygen), and fragments of ∼360 bp (with an approximate insert size of 300 bp) were recovered. each sample was then amplified by pcr for 11 cycles using the p5 and p7 primers carrying sequences that can anneal during bridge pcr with a flowcell and the p7 primer carrying a six-base index to allow multiplexing. the pcr products were cleaned up using the axyprep mag pcr clean-up kit (axygen), validated using an agilent 2100 bioanalyzer (agilent technologies, usa), and quantified with a qubit 2.0 fluorometer (invitrogen, usa). libraries with different indices were then multiplexed and loaded on an illumina hiseq instrument (illumina, usa) according to the manufacturer's instructions. sequencing was performed using a 2 × 150-bp paired-end (pe) configuration and image analysis and base calling were conducted using hiseq control software (hcs) + olb + gapipeline-1.6 (illumina) provided with the hiseq instrument. rna-seq was performed and analyzed using genewiz9 (suzhou, china). the analysis of the microarray data to identify differentially expressed genes was performed using edger software. the analysis starts with the count, and the data are standardized by tmm and then subjected to differential expression analysis. we selected | log 2 (fold change) (logfc) | > 1 and fdr < 0.05 as the screening criteria for the identification of differentially expressed genes. ipec-j2 or vero e6 cells were fixed with 4% paraformaldehyde for 30 min at rt. the fixed cells were permeabilized with 0.2% triton x-100 for 20 min at rt and then blocked with blocking buffer (pbs with 5% fbs and 5% skim milk) for 30 min at 37 • c. the cells were then incubated with pedv n protein antibody at 37 • c for 2 h and then labeled with alexa fluor 546 goat anti-mouse igg antibody (thermo fisher scientific, usa) at 37 • c for 1 h. dapi (sigma, usa) was used for the staining of cellular nuclei. the stained cells were visualized using an amg evos f1 fluorescence microscope (thermo fisher scientific, usa). the porcine rsad2 or mx2 coding region was amplified by rt-pcr, and cdna was prepared using the primescript tm ii 1st strand cdna synthesis kit (takara, china). rsad2 or mx2 was then amplified using a pair of primers specific for porcine rsad2 or mx2 (table 1) , respectively. the pcr products were purified, digested, and cloned into pcaggs-ha through ecor i and kpn i. construction of the prsad2/pmx2-ha expression plasmid was confirmed by sequencing. vero e6 cells were grown in 48-well plates to 70-80% confluence and then transfected with the prsad2/pmx2-ha expression plasmid using the attractene transfection reagent (qiagen), and the expression of prsad2/pmx2 was verified by anti-ha ifa. graphpad prism software version 7 was used to analyze the experimental results. the results are expressed as scatter plots in which the mean is indicated by a line. the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p-values are expressed as follows: * p < 0.05, * * p < 0.01, * * * p < 0.005, and * * * * p < 0.001. the inhibition of pedv in ipec-j2 cells by exogenous ifn-λ3 is superior to that achieved with ifn-α according to our study and other previous studies (21) (22) (23) , ifn-λ1, ifn-λ3, and ifn-α all significantly inhibit pedv infection, but ifn-λ3 shows the strongest antiviral activity against pedv. to confirm this disparity in the antiviral activities of ifn-λ3 and ifn-α against pedv, ipec-j2 cells were primed with ifn-α (1,000 ng/ml) or ifn-λ3 (1,000 ng/ml) for 24 h and then infected with pedv (moi = 1). consistent with our previous results, both ifn-α and ifn-λ3 substantially suppressed pedv infection in ipec-j2 cells, as demonstrated by measurements of the viral genomes and titers by rt-qpcr ( figure 1a ) and tcid 50 titration (figure 1b) , respectively. ifn-α reduced the number of pedv genomes by 24-fold, whereas ifn-λ3 decreased the number of pedv genomes by approximately 342-fold. the virus titers were consistent with results of pedv genomes: pedv titers decreased 10and 100-fold after pretreatment with ifn-α or ifn-λ3, respectively. the inhibition of pedv infection by both ifnα and ifn-λ3 was further verified by pedv n protein ifa ( figure 1c) . the virus-infected cells were significantly decreased after pretreatment with ifn-α or ifn-λ3, whereas the number of pedv-infected cells in the ifn-λ3-pretreated group was significantly decreased, as demonstrated by only a few sporadically distributed cells, compared with those in the ifnα-pretreated group. thus, ifn-λ3 restricted pedv infection in ipec-j2 cells more effectively than ifn-α, regardless of the quantification of viral rna, infectivity, or viral protein (figure 1 ). to assess the underlying mechanisms of the disparity between the anti-pedv responses induced by ifn-α or ifn-λ3, we performed an rna-sequencing (rna-seq) analysis of total cellular rna isolated from ipec-j2 cell cultures stimulated with ifn-α or ifn-λ3 for 24 h and ifn untreated ipec-j2 mock control. this duration of stimulation (24 h) was selected based on the efficacy of viral inhibition after exposure to ifn-λ3 and ifn-α (figure 1 ) (22) . each of the triple replicate yielded more than 3.89 × 10 7 clean reads and has more than 95% q20 (%), indicating the reliability of the rna-seq data. the ifn-λ3stimulated cells showed a total of 997 differentially expressed genes, including 983 upregulated genes and 14 downregulated genes, compared with the mock control, whereas ifn-α only upregulated the expression of 122 genes among the total of 126 differentially expressed genes and reduced the expression of four genes (figure 2a) . the number of ifn-λ3-modified genes was approximately 10-fold higher than that of ifn-αmodified genes. these results indicate that the intestine epithelia respond better to ifn-λ3 than to ifn-α. we further grouped the corresponding genes through supervised partitioning clustering and combined the differentially expressed genes induced by ifn-λ3 and ifn-α to obtain the heat map ( figure 2b ) and the venn map ( figure 2c ). ifn-λ3 yielded different gene expression profiles compared with that induced by ifn-α, even though these showed substantial overlap ( figure 2b) . one hundred ten genes were upregulated in both the ifn-λ3-and ifn-α-treated cells, whereas 873 and 12 genes were uniquely upregulated in the presence of ifn-λ3 and ifn-α, respectively ( figure 2c) . none of the coexpression genes were downregulated in both the ifn-λ3-and ifn-α-treated cells, whereas 14 and 4 genes were only downregulated in the presence of ifn-λ3 and ifnα, respectively. these results demonstrated that ifn-λ3 induces a unique gene transcriptional profile in the intestine epithelia compared with ifn-α. to further clarify the functional consequences of the gene profiles elicited by either ifn-λ3 or ifn-α, we performed a gene ontology (go) enrichment analysis using a database established by the gene ontology consortium, which aims to define and describe the functions of genes and proteins in various species (figure 3 ) (26) . among the 983 genes upregulated by ifn-λ3, 221 and 134 genes (22.17 and 13.44% of the total genes, respectively) were associated with a binding function and catalysis, respectively, and these two functions account for the main molecular functional changes. the dominant functions of the ifn-α-regulated genes are cellular transporter activity and nucleic acid-binding transcription factor activity, and these functions were associated with 40 genes (31.75% of total genes) and 22 genes (17.46% of total genes), respectively. the analysis of cellular components revealed that 221 differentially expressed genes in the ifn-λ3-treated group affected the cell part, and 36 genes associated with this cellular component were differentially expressed in the ifn-α-treated group. with respect to biological processes, the ifn-λ3-treated group included 200, 173, 169, 142, and 120 differentially expressed genes associated with the cellular process, the single-organism process, biological regulation, the metabolic process, and response to stimulus, respectively. the pattern obtained for the ifn-α-treated group was similar to that of the ifn-λ3-treated group, and the differentially expressed genes were also concentrated on the following five functions: the cellular process, the single-organism process, the biological regulation, the metabolic process, and the response to stimulus. however, the numbers of differentially expressed genes after ifn-α treatment were notably lower than those obtained after ifn-λ3 treatment. these results indicated that both ifn-λ3 and ifn-α are involved in the regulation of multiple cellular processes in ipec-j2 cells, such as cellular components and molecular functions, but ifn-λ3 exerts more potent effects than ifn-α. to further investigate the function of genes specifically induced by ifn-λ3, we extracted the transcriptional profiles of ifn-λ3 and ifn-α regarding biological processes (p < 0.01) and combined them based on the -log 10 (p) values to obtain a heat map that showed the enrichment of biological processes ( figure s1 ). the data demonstrated that in ipec-j2 cells, ifn-λ3 stimulation triggered more biological reaction processes than ifn-α, and these processes mainly involved the modulation of metabolic processes, including cellular metabolic processes, macromolecular metabolic processes, and primary metabolic regulation. taken together, these results indicate that the differentially expressed genes induced by ifn-λ3 are involved in more intracellular biological processes than those induced by ifn-α. to explore the clustering of the ifn-induced differential expression genes in anti-viral signaling pathways, we performed a kegg enrichment analysis of the differentially expressed genes using kobas 3.0. the kegg enrichment analysis revealed that the differentially expressed genes induced by ifn-λ3 involved in much broader signal pathways compared with ifn-α though they both primarily modified the nf-κb signaling pathway, the jak-stat signaling pathway, the phosphoinositide-3-kinase-akt (pi3k-akt) signaling pathway, the mitogen-activated protein kinase (mapk) signaling pathway, and the cgmp-pkg signaling pathway ( figure 4a) . to determine the signaling pathways involving the differentially expressed genes induced by ifn-λ3 or ifn-α, we combined the differentially expressed genes and analyzed their expression patterns ( figure 4b) . the results showed that the differentially expressed genes were most abundantly involved in the jak-stat signaling pathway, followed by the pi3k-akt signaling pathway and the mapk signaling pathway, which is consistent with the finding that the jak-stat signaling pathway primarily mediates ifn-induced antiviral responses. among the proteins encoded by the differentially expressed genes, sos2, pik3ca, jak2, il-6, socs4, il-28b, stat1, il22ra1, and socs1 play a major role in the jak-stat signaling pathway, and kras, pik3ap1, prkaa1, enssscg00000021148, enssscg00000022362, and map3k2 play a major role in the pi3k-akt signaling pathway. to predict the protein interactions between differentially expressed genes, we extracted the union of differentially expressed genes and introduced these into the web-based tool string (http:// www.string-db.org/) to generate protein-protein interaction networks ( figure 4c) . the results of the string analysis indicated that jak2, stat2, pten, pik3ca, irs1, kras, and il6st are closely related to other proteins and displayed significant differential expression compared with the ifn-αinduced proteins, which play critical roles in the innate immune response induced by ifn. collectively, the results showed that the ifn-λ3-induced differentially expressed genes are involved in more signaling pathways, particularly those associated with innate immunity, than those induced by ifn-α, which indicates that ifn-λ3 exhibits stronger antiviral activity in intestinal epithelial cells. to further elucidate the mechanisms underlying the antiviral activity discrepancy between ifn-λ3 and ifn-α, we focused on the top 100 ifn-λ3-induced genes (fold change compared with the mock control) and divided them into three subgroups (classical isgs, weakly ifn-λ3-induced genes, and strongly ifn-λ3-induced genes) as in a previous study (16) . the expression of the 100 top genes in all three subgroups induced by ifn-λ3 was significantly higher compared with that induced by ifn-α ( figure 5) . the isgs in the classic isg subgroup are primarily the classical isgs induced by ifn reported in the literature (16, 27) . the levels of isgs in this subgroup induced by ifn-λ3 were from 3-to 23-fold higher than those induced by ifnα. the weakly ifn-λ3-induced gene subgroup contained 25 genes, including three unknown genes (being denoted un1, un2, and un3). the analysis of this subgroup showed that both ifn-λ3 and ifn-α induced a more than 4-fold increase in the expression of isgs compared with mock control. the ifn-λ3-induced expression levels of ifit3, oasl, oas1, and gbp4, which are important innate immunity factors, were 10fold higher than those induced by ifn-α. the expression of 16 of the genes in the strongly ifn-λ3-induced gene subgroup was strongly induced by ifn-λ3, whereas ifn-α did not induce a substantial increase in expression (<2-fold compared with the mock control). interestingly, radical s-adenosyl methionine domain containing 2 (rsad2), a multifunctional protein with broad antiviral activity that can inhibit both dna and rna viruses, is the top gene upregulated by ifn-λ3 (28) . in contrast, ifn-α did not substantially upregulate rsad2 expression. protein interaction prediction analysis of differentially expressed genes by string database. the relevant genes were input into string (http://www.string-db. org/), the active interaction sources were selected as homology, and the experimentally determined interaction and database annotated were used as predictive conditions for protein interaction prediction. the thickness of the line represents combined score, which is the combined score of the prediction results of the three prediction conditions, indicating the strength of data support. several other genes (ifit2, parp14, and gbp6) in this subgroup are also important innate viral restriction factors (27, (29) (30) (31) . thus, ifn-λ3 induces a stronger antiviral innate immune response compared with ifn-α. to verify the unique antiviral gene expression profile induced by ifn-λ3 that was detected by rna-seq, as shown in figure 5 , we randomly selected three genes from each group to be verified by rt-qpcr. the analysis of the three subgroups confirmed that ifn-λ3 most substantially upregulated the expression of isgs (figure 6) . specifically, ifn-λ3 upregulated the expression of large distinct classes of isgs in the classical isg subgroup, such as mx2, isg15, and ifit3, the isg fold changes induced by ifn-λ3 were up to nearly 10-fold higher than those induced by ifn-α ( figure 6a ). the analysis of the weakly ifn-λ3induced gene subgroup showed that the expression of oasl, oas1, and isg12 (a) was induced by both ifn-λ3 and ifnα to significantly different levels. the oasl and oas1 fold changes induced by ifn-λ3 were up to 8-fold greater than those induced by ifn-α ( figure 6b ). more substantial fold changes in the expression of isgs in the strongly ifn-λ3-induced gene subgroup were obtained with ifn-λ3 compared with ifn-α, which resulted in only slight changes in expression ( figure 6c ). the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p values are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. rsad2, plac8, and ifit2 were more substantially upregulated by ifn-λ3 than by ifn-α. collectively, the in vitro rt-qpcr results confirmed that ifn-λ3 induces stronger isg expression compared with ifn-α. enteroids derived from intestinal crypt stem cells, which mimic the diverse cellular nature and physiological activity of the small intestine while also maintaining the genetic identity of the host, constitute a unique ex vivo model for studying the intestine (21, 27) . to further confirm whether the ifn-λ3induced gene expression profile in enteroids was the same as that in ipec-j2 cells, we stimulated enteroids with ifn-λ3 or ifn-α under the same conditions as ipec-j2 cells and evaluated their gene expression by rt-qpcr. in all three groups, the expression pattern of the genes induced by ifn-λ3 and ifn-α in porcine enteroids was consistent with that found in the ipec-j2 cells (figure 7) . in the classical isg subgroup, the upregulated levels of mx2 and ifit3 elicited by ifn-λ3 were ∼3-to 5-fold higher than those induced by ifn-α ( figure 7a ). in contrast, the upregulated levels of oasl and oas1, which belong to the weakly ifn-λ3-induced gene subgroup, induced by ifn-λ3 were 2-fold higher than those induced by ifn-α, and the fold change difference was moderate ( figure 7b) . for the strongly ifn-λ3-induced gene rsad2 and plac8, just as we observed in the other subgroups, enteroids were or were not primed with ifn-λ3 (1,000 ng/ml) or ifn-α (1,000 ng/ml) for 24 h, and the total rna from the enteroids was extracted and used for rt-qpcr. the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p values are expressed as follows: *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. ifn-λ3 significantly induced higher expression than ifn-α does ( figure 7c ). in summary, ifn-λ3 induces higher expression of isgs than ifn-α in porcine enteroids, which suggests that ifn-λ3 exerts a greater effect in gastrointestinal epithelial cells than ifn-α. pmx2 and prsad2 inhibit pedv infection in vero e6 cells we selected mx2 (classical isg) and rsad2 (strongly ifn-λ3induced gene) among the top 10 genes induced by ifn-λ3 to evaluate the antiviral effect of the ifn-λ3-induced expression of isgs against pedv infection. we cloned porcine mx2 or rsad2 into the eukaryotic expression vector pcaggs-ha, and the transient expression of pmx2 or prsad2 in vero e6 cells was verified by ifa (data not shown). as expected, pmx2 or prsad2 transient overexpression significantly inhibited pedv infection in vero e6 cells, as demonstrated by measuring the viral rna (figures 8a,c) and pedv n protein expression by ifa (figures 8b,d) . thus, these data indicate that the differentially upregulated pmx2 or prsad2 serves as an important ifn-λ3elicited antiviral host factor against pedv. type i and type iii ifns, which establish a cellular antiviral state and restrict viral infection in the host, are key players at the earliest stages of innate immunity against viral infection. despite the similarities between the effects of the two types of ifns, increasing evidence demonstrates that each class of ifn is essential for antiviral host defense and is not functionally redundant (2) . a previous study in mice demonstrated that the role of ifn-λ in functionally redundant intestinal viral infections cannot be compensated by ifn-α/β (20) . therefore, clarification of the induction of cell-specific ifn signaling profiles is essential for understanding the non-redundant roles of ifn-λ and ifn-α in viral infection at the tissue and organism levels. in this study, we found that the transcriptional profile induced by type iii ifn in the intestinal epithelia the differences between groups were compared using an unpaired mann-whitney test. p < 0.05 was considered significant, and the p values are expressed as follows: *p <0.05, **p <0.01, ***p <0.005, and ****p < 0.001. was unique compared with that induced by type i ifn. compared with ifn-α, stimulation with ifn-λ3 not only resulted in higher levels of most isgs but also substantially increased diversity in the gene profiles involved in various cellular functions. type i and iii ifns each signal through different heterodimeric complex receptors to initiate multiple downstream signaling pathways. in addition to activation of the jak-stat signaling pathway, ifns also activate the pi3k and mapk signaling cascades (5, 32) . the combined use of these signaling pathways by ifns and many other cytokines might help explain the different roles of ifns in regulating the antiviral and immune responses in a variety of environments and locations. we have very limited knowledge on the pathways activated by ifn-λ, which are important for understanding the immune-modulating activities of ifn-λ (8) . in this study, we found that ifn-λ3 not only activates the classical antiviral response jak-stat signaling pathway but also primarily activated the nf-κb signaling pathway, the camp signal pathway, the pi3k-akt signaling pathway, and mapk signaling pathway (figure 4) . ifn-λ3 activated much more signaling pathways in epithelia than ifn-α, and this finding might provide new insights into the mechanism through which ifn-λ modulates other cellular functions beyond its direct antiviral activity. ifn-λ3 largely stimulated wnt signaling pathway in iec compared with ifn-α, which plays critical roles in maintaining the homeostasis of intestinal epithelia in vivo (33) . further studies of different signaling pathways induced by ifn-λ3 or ifn-α will help dissect the complex interaction of ifn-induced signaling pathways with similar or overlapping intracellular signaling pathways stimulated by other cytokines. most somatic cells can induce and respond to type i ifns, but certain specialized cells, such as those in the mucosal epithelia, selectively produce and respond to type iii ifns during various virus infections. we demonstrated that the porcine intestinal epithelia respond to both type i and iii ifns, even though these ifns induced different isg expression levels. type iii ifns comprise four functional and highly homologous subtypes, namely, ifn-λ1, ifn-λ2, ifn-λ3, and ifn-λ4, in humans. our study and other studies have revealed differences in antiviral activity among different ifn-λ subtypes (22, 23, 34) . ifn-λ3 is superior or equal to ifn-λ1 in terms of its antiviral activity in the intestinal epithelia (22) and the immortalized liver cell line hepg2 in vitro (34) . the different kinetics and magnitude of isg induction after stimulation might account for the variation in the antiviral activity among different types or subtypes ifns. previous studies that attempted to identify the gene profiles induced by type iii ifns primarily focused on the comparison of ifn-λ1 with type i ifn (15) , and most of these studies were performed in immortalized human or mouse cell lines. the gene transcription profile induced by ifn-λ3, particularly the gene transcription profile induced by ifn-λ3 in porcine intestinal epithelial cells, has not been reported. here, we performed comparative analyses of the transcriptional profiles induced by ifn-λ3 and ifn-α in porcine intestinal epithelia cells. more importantly, in this study, we verified the rna-seq results in primary swine crypt-derived enteroids, an in vitro system that well recapitulates the complicated cellularity of the gi tract, which exhibits a potent response to both type i and iii ifns (21, 27, 35) . the differential expression in ipec-j2 or enteroids detected by qpcr is not related to the selection of housekeeping gene gapdh since we observed the same pattern when using another housekeeping gene actin (data not shown). therefore, studying the differences among ifn-λ3-and ifn-α-induced gene expression in enteroids is more realistic and clinically significant. the different antiviral activities of ifn-λ3 and ifn-α are likely due to the different degrees of isg induction by these two cytokines, and ifn-λ3 induced increased isg expression. the functions of some of the genes that were strongly induced by ifn-λ3 have been reported, but the functions and mechanisms of more genes remain unclear. rsad2, a host viral restrictor gene, showed the highest upregulated levels after ifn-λ3 rather than ifn-α stimulation, whereas mx1 was the most highly upregulated gene after ifn-α stimulation. a previous study demonstrated that porcine rsad2 effectively inhibited csfv replication in vitro, and this effect might occur via the interaction of rsad2 with csfv e2 protein in the cytoplasm (28) . our study found that rsad2 transient expression also substantially curtailed pedv infection in vero e6 cells in vitro, but the mechanism is unclear. mx2, an ifn-induced gtpbinding protein (36, 37) , reportedly inhibits the replication of lentivirus hiv-1, simian immunodeficiency virus (siv), and equine infectious anemia virus (eiav) in vitro (38, 39) . the function of porcine mx2 has been poorly studied. we confirmed that porcine mx2 inhibits pedv infection in vero e6 cells. we also discovered some unknown genes that were differentially expressed in response to ifn-λ3 or ifn-α stimulation, and this finding might provide a foundation for further elucidation of the different mechanisms of action of ifn-λ3 and ifn-α. in summary, we compared the transcriptional profiles of ifn-λ3 and ifn-α in ipec-j2 cells and identified a unique set of genes that were strongly induced by ifn-λ3 compared with ifn-α. the transcriptional enrichment analysis indicated that ifn-λ3 or ifn-α is involved in the regulation of cellular processes, such as cellular components and molecular functions, in ipec-j2 cells, and that ifn-λ3 activates more robust signaling pathways, particularly the antiviral jak-stat signaling pathway, compared with ifn-α. ifn-λ3 preferentially upregulates antiviral genes in epithelial cells compared with ifn-α. these results indicate that ifn-λ3 selectively targets small intestinal epithelial cells and induces a non-redundant antiviral host response at the enteric mucosal site. all datasets generated in this study are included in the manuscript/supplementary material. the animal study was reviewed and approved by the harbin veterinary research institute of the chinese academy of agricultural sciences, harbin. written informed consent was obtained from the owners for the participation of their animals in this study. pl, ll, and lf designed the research studies and analyzed and interpreted the data. ll, mx, ff, and ly conducted experiments and acquired data. ll and pl drafted the manuscript and all authors contributed revisions. funding support for this work was provided by grants from the national key r&d program of china (2017yfd0501102) and the national natural science fund (31772718). the content is solely the responsibility of the authors and does not necessarily represent the official views of the funding resources. ifn-lambda decreases murid herpesvirus-4 infection of the olfactory epithelium but fails to prevent virus reactivation in the vaginal mucosa type iii interferons in antiviral defenses at barrier surfaces cell type-specific signaling in response to interferon-gamma interferon-lambdas: frontline guardians of immunity and homeostasis in the respiratory tract interferon lambda genetics and biology in regulation of viral control. front immunol lambda interferon is the predominant interferon induced by influenza a virus infection in vivo interferon-lambda: a potent regulator of intestinal viral infections. front immunol type iii interferons in viral infection and antiviral immunity interferon lambda protects the female reproductive tract against zika virus infection induction and function of type i and iii interferon in response to viral infection interferon induction and function at the mucosal surface ifn-alpha receptor-1 upregulation in pbmc from hcv naive patients carrying cc genotype possible role of ifn-lambda interferon-lambda: immune functions at barrier surfaces and beyond type iii interferon (ifn) induces a type i ifn-like response in a restricted subset of cells through signaling pathways involving both the jak-stat pathway and the mitogen-activated protein kinases distinct and overlapping genomic profiles and antiviral effects of interferon-lambda andalpha on hcv-infected and noninfected hepatoma cells identification of a predominantly interferon-lambda-induced transcriptional profile in murine intestinal epithelial cells commensal microbes and interferon-lambda determine persistence of enteric murine norovirus infection type iii interferon-mediated signaling is critical for controlling live attenuated yellow fever virus infection in vivo diverse intracellular pathogens activate type iii interferon expression from peroxisomes ifn-lambda determines the intestinal epithelial antiviral host defense porcine intestinal enteroids: a new model for studying enteric coronavirus porcine epidemic diarrhea virus infection and the host innate response ifn-lambda preferably inhibits pedv infection of porcine intestinal epithelial cells compared with ifn-alpha type iii interferon restriction by porcine epidemic diarrhea virus and the role of viral protein nsp1 in irf1 signaling establishment of a stable cho cell line with high level expression of recombinant porcine ifn-beta analyzing real-time pcr data by the comparative c(t) method gene ontology: tool for the unification of biology. the gene ontology consortium a family of ifn-gamma-inducible 65-kd gtpases protects against bacterial infection porcine viperin protein inhibits the replication of classical swine fever virus (csfv) in vitro decreased ifit2 expression promotes gastric cancer progression and predicts poor prognosis of the patients ifit2 is a restriction factor in rabies virus pathogenicity structure, function and inhibition of poly(adp-ribose)polymerase, member 14 (parp14) regulation of type i interferon responses sustained in vitro intestinal epithelial culture within a wnt-dependent stem cell niche human interferon-lambda3 is a potent member of the type iii interferon family expression of ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of interferon lambda against norovirus and reovirus cdna structures and regulation of two interferon-induced human mx proteins human mxb protein, an interferon-alpha-inducible gtpase, contains a nuclear targeting signal and is localized in the heterochromatin region beneath the nuclear envelope equine myxovirus resistance protein 2 restricts lentiviral replication by blocking nuclear uptake of capsid protein the interferon-inducible mxb protein inhibits hiv-1 infection the authors thank dr. xiangxi pei (northeast agricultural university, harbin, china) for assistance and advice in rna-seq data analysis. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02394/full#supplementary-material figure s1 | the heat map of ifn-λ3 and ifn-α transcriptional profile biological process enrichment analysis. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 li, xue, fu, yin, feng and liu. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-268781-6l74rrlm authors: poh, chek meng; zheng, jian; channappanavar, rudragouda; chang, zi wei; nguyen, thi h. o.; rénia, laurent; kedzierska, katherine; perlman, stanley; poon, leo l. m. title: multiplex screening assay for identifying cytotoxic cd8(+) t cell epitopes date: 2020-03-11 journal: front immunol doi: 10.3389/fimmu.2020.00400 sha: doc_id: 268781 cord_uid: 6l74rrlm the cytotoxicity of epitope-specific cd8(+) t cells is usually measured indirectly through ifnγ production. existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of cd8(+) t cells in a single reaction. this can greatly reduce the amount of starting clinical materials for a systematic screening of cd8(+) t cell epitopes. in addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of cd8(+) t cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. this technique is shown to be useful to study both human and mouse cd8(+) t cells. besides, our results from human pbmcs and three independent infectious animal models (mers, influenza and malaria) further reveal that ifnγ expression by epitope-specific cd8(+) t cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). overall, our approach opens up new possibilities for comprehensive analyses of cd8(+) t cell cytotoxicity in a practical manner. the cytotoxicity of epitope-specific cd8 + t cells is usually measured indirectly through ifnγ production. existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of cd8 + t cells in a single reaction. this can greatly reduce the amount of starting clinical materials for a systematic screening of cd8 + t cell epitopes. in addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of cd8 + t cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. this technique is shown to be useful to study both human and mouse cd8 + t cells. besides, our results from human pbmcs and three independent infectious animal models (mers, influenza and malaria) further reveal that ifnγ expression by epitope-specific cd8 + t cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). overall, our approach opens up new possibilities for comprehensive analyses of cd8 + t cell cytotoxicity in a practical manner. after priming and activation by antigen-presenting cells in secondary lymphoid organs, epitope-specific cd8 + t cells expand in numbers, migrate to sites of infection, and kill infected cells (1) . cd8 + t cells also form part of the memory response. they can rapidly expand in number and eliminate pathogens after secondary challenge. also, cd8 + t cells can recognize tumor antigens presented by cancer cells and eradicate them before they metastasize (2) . the cytotoxicity of cd8 + t cells requires direct contact with target cells, although the capability of each cd8 + t cell to eliminate target cells remains uncertain (3, 4) . regardless, these cell-to-cell contacts enable cd8 + t cells to release cytotoxic granules and death ligands, thereby killing the target cells (5) . interferon-gamma (ifnγ) is one of the critical cytokines for establishing effective immune responses. in the context of t cells, this cytokine helps to drive the polarization of cd4 + t cell response to t h 1, which directs the overall t cell response toward cell-mediated immunity. in cd8 + t cells, activated subsets start to produce ifnγ rapidly, with the dominant epitope-specific cd8 + t cells expanding preferentially over subdominant ones (6, 7) . ifnγ production by activated cd8 + t cells also promotes t h 1 polarization (8) , enhances cell motility and cytotoxicity (9) , and exerts antiviral immunity in a wide variety of cells through ifnγ receptor signaling (10) . ifnγ also plays a vital role in regulating the contraction of effector cd8 + t cells after the clearance of primary infection (11, 12) . despite its multiple functions, ifnγ produced by activated epitope-specific cd8 + t cells is widely used as an indirect marker to demonstrate the functional efficacy of epitope-specific cd8 + t cells. for example, as assays for measuring ifnγ produced by cd8 + t cells can be highly robust (e.g., intracellular cytokine staining, ics), ifnγ is often used as a surrogate marker in screening assays for identifying potential cytotoxic cd8 + t cell epitopes. however, one should note that the relationship between these two parameters is not necessarily correlated. the cytotoxicity of cd8 + t cells against their cognate targets can be measured directly, which was initially assayed through radioactive labeling of target cells, mostly using chromium-51 (13) . fluorescence-based methods have since supplanted the radioactive labeling methods due to their higher sensitivity and better safety profile (14) . however, these assays are typically used in a single target format, allowing measurements of cytotoxic potential of one or two cd8 + t cell attributes. this approach is not highly robust, which limits its potential use in large-scale screening studies. when there is a need to test many cd8 + t cells epitopes, the amount of starting materials (mice or human pbmcs) that are needed will inevitably increase substantially, which may pose difficulties for conducting comprehensive analyses. in this study, we demonstrated the feasibility of measuring cytotoxicity activities of up to twenty-four specificities using just three fluorescent dyes in a single reaction, thereby increasing the multiplexity and capability to screen multiple cd8 + t cell epitopes simultaneously. we used cd8 + t cells from human pbmcs to analyze previously known hla-a11 * 01-restricted epitopes of influenza a viruses in vitro, showing that only some of these epitopes can elicit a robust cytotoxic response. in mouse models, we demonstrated that this method allows for screening of cd8 + t cells harvested from different anatomical sites and that our approach allows accurate measurement of cell killing that is attributable to cognate cd8 + t cells. we further demonstrated that ifnγ expression by epitope-specific cd8 + t cells does not necessarily correlate with its cytotoxic potential. our results highlight that this multiplex assay has potential uses in identifying potent cytotoxic cd8 + t cell epitopes. besides, epitopes that have differential effects on the ifnγ production and cytotoxic activity of cd8 + t cells can be identified by this approach in a rapid manner. peptides of >90% purity were purchased from genscript (piscataway, nj), reconstituted at 10 mg/ml with dimethyl sulphoxide (dmso), and stored at−20 • c. all healthy donors and patients provided written, informed consents to participate in the study. ethics approvals were obtained from the human research ethics committee of the university of hong kong and monash health (hrec/15/monh/64), royal melbourne hospital (local reference number: 2016/196). blood samples from the subjects were screened for blood-borne pathogens and sent for human leukocyte antigen (hla) typing. patient blood samples were collected in sodium heparin tubes and pbmcs isolated as previously described (15) . briefly, blood was diluted 1:1 (v/v) with pbs, overlaid on histopaque-1077 (sigma-aldrich, st. louis, mo) and centrifuged at 400 g for 30 min without brake. isolated pbmcs were washed with pbs and resuspended in fbs+10% dmso for liquid nitrogen storage. six-to eight-week-old balb/cj, c57bl/6j, and human dipeptidyl peptidase-4 knock-in (hdpp4-ki) mice on c57bl/6j background (16) were used in these experiments. mice were bred under specific-pathogen-free conditions. all animal experiments and procedures were approved by the following: a vaccinia virus carrying h5n1 np, ha, na, m1, m2 genes, and human il-15 previously reported by us was used as an experimental vaccine (17) . mice were anesthetized and given 1 x 10 7 pfu of this vaccine through the intranasal route twice at 3 weeks apart. three weeks after the second vaccination, mice were challenged with 10 mld 50 h1n1 (a/puerto rico/8/34) through the intranasal route. weight loss and survival of mice were followed for up to 2 weeks. plasmodium berghei anka clone 15cy1 (pba) (18) and plasmodium berghei nk65 (pbnk65) (19) parasites were passaged in c57bl/6j mice, and infected erythrocytes were resuspended in alsever's solution and stored in liquid nitrogen. to infect mice, 1 × 10 6 infected erythrocytes were injected through the intraperitoneal route. parasitaemia was monitored by flow cytometry (20) . all mers-cov experiments were carried out in the university of iowa absl-3 facility. mice were infected with 1 × 10 5 pfu of human isolate of mers-cov (mers-cov-emc) and these mice were challenged after 4 weeks with 2 × 10 3 pfu of a mouse-adapted strain of mers-cov. spleens from naïve mice were dissociated using a 70 µm cell strainer with a syringe piston to release splenocytes in rpmi complete medium, supplemented with 10% fetal bovine serum (fbs) and 100 u/ml penicillin-streptomycin (thermofisher scientific, waltham, ma). splenocytes were resuspended with ack lysis buffer (155 mm nh 4 cl, 10 mm khco 3 , 0.2 mm edta; all chemicals from sigma-aldrich) for at least a minute before washing with rpmi complete medium. the splenocytes were split into up to 24 groups and pulsed with relevant peptides at a final concentration of 10 mg/ml. treated cell were labeled with unique combinations of celltracker cmfda, cmtmr, and deep red dyes (thermofisher scientific; table s1 ) and were then washed with rpmi complete media. equal numbers of labeled cells from each group were combined and transferred into recipient mice at a total volume of 30 µl and 200 µl pbs for intranasal and retro-orbital routes, respectively. after 16-20 h, recipient mice were sacrificed to harvest donor splenocytes and bronchoalveolar lavage, which were labeled with live/dead fixable violet stain (thermofisher scientific) before acquisition by flow cytometry. thawed pbmcs were washed twice with rpmi complete medium, resuspended in 10 ml fresh medium in 50 ml falcon tube and left to recover at 37 • c, 5% co 2 overnight at about 5 • horizontal tilt with loose cap (21) . after recovery, cells were split into two groups: one group was treated with cd8 + t cell isolation kit and the other group treated with cd8 + nanobeads for depletion (biolegend, san diego, ca). the target cells obtained from the negative fraction from the latter group were split into groups for peptide pulsing and dye labeling as described earlier. equal numbers of cells from each group were then combined together and split into two groups: one group to be mixed with the isolated cd8 + t cells and the other group without. cells were seeded in a 96-well flat-bottom plate and incubated at 37 • c, 5% co 2 overnight. the next day, cells were labeled with live/dead fixable near ir stain (thermofisher scientific) before acquisition using a flow cytometer. splenocytes from mice at up to 5 × 10 6 cells were seeded together with 5 mg/ml mouse il-2 (biolegend), 1 µl bd golgiplug (becton dickinson, franklin lakes, nj) or brefeldin a (thermofisher scientific) and 10 µg peptide in 96-well tissue culture plates, followed by incubation at 37 • c, 5% co 2 for 5 h. for plasmodium studies, the mouse il-2 addition was omitted. they were then stained with zombie aqua fixable viability kit (biolegend) for 30 min, washed and followed by antibody stainings for 30 min, selected from the following panel: purified cd16/32 (clone 93); cd3 pe/dazzle 594 (clone 17a2); cd3ε brilliant violet 421 (clone 145-2c11); cd4 apc/cy7 (clone gk1.5); cd8a percp/cy5.5 (clone 53-6.7); cd8b percp/cy5.5 (clone yts 156.7.7, all from biolegend). after washing, cells are fixed in 4% formaldehyde, permeabilized with 0.1% saponin (sigma-aldrich) or per buffer (bd bioscience), followed by staining with ifnγ fitc (clone xmg1.2, biolegend/ebioscience). the formula for the calculation of cytotoxicity of antigen-specific cd8 + t cells are as follows: (1) mann-whitney u-test was used to analyse for statistical significance between two groups. for comparisons between more than two groups, one-way analysis of variance (anova) was used if sample data follow a normal distribution. otherwise, kruskal-wallis test and dunn's post-test were used. dunnett's or dunn's multiple comparisons were performed with the irrelevant control (csp280, ova, mog, or ebo580-588) designated as the control group. flow cytometric analysis was carried out with flowjo (treestar, ashland, or) and calculations were performed with prism 6 (graphpad, la jolla, ca). in contrast to the conventional method of using a single cell labeling dye (e.g., cmfda) to create two distinct target populations during flow acquisition (figure 1a) , we first explored the possibility of combining three different labeling dyes to exponentially increase the number of target populations that can be distinguished ( figure 1b) . this strategy was inspired by the use of multicolor-coded mhc multimers to test different antigen-specific t-cell responses in a single sample (22) . by modifying the concentrations of each dye, up to 24 different groups of donor cells can be created (table s1 ). when equal numbers of cells from each group were combined and transferred into recipient mice through the intravenous route, all 24 different groups of donor cells could be identified by flow cytometry (figure 1b) . the clear demarcation of each donor group raises the possibility of directly measuring the cytotoxicity of multiple cd8 + t cell specificities simultaneously in a single mouse (see below). this possibility is in sharp contrast to the conventional method, which requires one mouse for testing a single cd8 + t cell epitope. we previously developed a vaccinia-based influenza a virus (iav) vaccine (henceforth called vax a) that can express five h5n1 iav genes and human interleukin-15 gene in mice (17). this experimental vaccine is known to induce robust cd8 + responses in mice. mice vaccinated twice with vax a via intranasal route could be fully protected by a lethal heterologous influenza virus challenge (a/pr/8/34; 10 mld 50 ), whereas all unvaccinated mice succumbed to the challenge (figure 2a) . we used this animal model to evaluate our multiplex in vivo cytotoxicity assay. we divided mice into three groups, and they were subjected to different treatments ( figure 2b . naïve: no treatment; vax a + pr8: vaccination followed by a virus challenge; pr8: virus challenge only). at day 5 post-challenge, we prepared donor splenocytes from unrelated naïve mice and divided them into eight groups. each group of donor cells were pulsed and labeled with a specific combination of peptide and dye ( table s2 ). the treated donor cells were then transferred into these mouse groups via intravenous or intranasal routes (figure 2c , upper and lower panel), and they were recovered from these recipient mice the following day. as shown in figure 2c , we could identify these labeled cells and distinguish them from each other in all experimental conditions. furthermore, donor cells pulsed with influenza epitopes np147 (cmfda+) and ha518 (cmtmr+) experienced high levels of cytolysis in vaccinated mice upon challenge with influenza ( figure 2c , highlighted by * in the middle panels, and figure 2d ), but not in unvaccinated mice. however, the csp280 (cmtmr++) control group experienced significant cytolysis in both the spleen and bronchoalveolar lavage of the unvaccinated mice, with this effect more pronounced in latter tissue compartment (figure 2d , left vs. right panels). this observation suggested that the inflammatory microenvironment contributed to substantial non-specific killing in these unprotected mice. the proinflammatory milieu is known to induce fas receptor expression in different cells (23) (24) (25) , and others have shown that bystander cell lysis by activated cd8 + t cells occurs in a tnfα-or fasl-dependent manner (26) (27) (28) . thus, we believe that the non-specific cytolysis by activated cd8 + t cells in our model could occur similarly. we also believe that the bystander killing of target cells due to dysregulated inflammation resulting from iav infection can also extend to cells labeled with iav epitopes. thus, the inclusion of irrelevant epitopes in this multiplex assay provides a unique opportunity to quantitate this bystander killing effect. consequently, we used csp280 data points from individual mice to normalize against the rest of the corresponding data points to obtain figure 2e . these normalized data clearly showed that cd8-mediated cytolysis against influenza epitopes are also elevated in the lung mucosa of vaccinated mice. analyzing cytotoxicity of multiple epitope-specific human cd8 + t cells simultaneously in vitro next, we tested the potential of this multiplex approach using cd8 + t cells harvested from human pbmcs. we obtained frozen pbmc samples from hla-a * 11:01-positive healthy individuals and influenza-infected patients to purify cd8 + t cells. we also harvested homologous non-cd8 + cells from the corresponding samples and use them as target cells in the assay. these target cells were divided into groups and pulsed with different hla-a * 11:01-restricted influenza virus epitopes (table s3 ). these epitopes are experimentally known to induce influenza-specific cd8 + t cell responses in hla-a * 11:01-positive individuals (influenza research database and immune epitope database). peptide-pulsed target groups were differentially labeled with unique combinations of fluorescent markers, pooled together and split equally into two groups, with one group incubated together with homologous cd8 + t cells and the other group without. the latter group serves as the naïve population, which is essential for the calculation of epitope-specific cd8 + t cell cytotoxicity. table s2 , then inoculated into recipient mice and re-harvested the next day to analyze remaining target cells. representative flow plots of mice from different experimental groups were shown, depicting the gating strategies to identify corresponding donor cell groups. corresponding positions for target cells treated with csp280, ha518, np147, and dmso are highlighted in the top right panel for naïve mice as examples. (d-e) the analysis of cytolytic activity of specific cd8 + t cells, before (d) and after (e) normalization, were shown. *p < 0.05, mann-whitney u-test. of sixteen tested iav epitopes, six (h1n1 m1 178-187, h5n1 pa 104-113, h1n1 pb2 322-331, h3n2 pb2 690-699, h1n1 np 91-99, and h1n1 np 188-198) could evoke significant levels of cytotoxicity from their cognate cd8 + t cells, both in healthy individuals ( figure 3a ) and in hospitalized patients with acute influenza (figure 3b ). for these six cytotoxic cd8+ t cell epitopes, infected individuals consistently elicited stronger responses than the healthy individuals did (one-way anova with holm-sidak's post-comparison; p < 0.0001). hence, apart from identifying multiple epitope-specific cd8 + t cells that elicited cytotoxicity toward their targets simultaneously, the results of the multiplex in vitro cytotoxicity assay correlated with the immune status of individuals, i.e., the rapid proliferation of influenza-specific cd8 + t cells in infected individuals led to elevated levels of cytotoxic activities. ebola does not circulate in the region where our pbmc donors reside. we reasoned that hla-a * 11:01-restricted ebola virus epitope, ebo 580-588 (29) could serve as an appropriate irrelevant epitope to demonstrate the specificity of our multiplex cytotoxicity assay, as well as setting a baseline for comparison. as expected, no cytotoxic activity against ebo 580-588 could be detected, proving the specificity of this multiplex assay for testing human cd8 + t cells from the blood. all influenza virus epitopes used in the above human study were previously shown to stimulate cd8 + cell responses in various immune assays (table s3) . we were surprised to find that only a minority of these epitopes can elicit cytotoxic killing from their cognate cd8 + t cells. as hla-typed pbmc samples were highly limited, it was difficult for us to study this phenomenon comprehensively. therefore, we used different animal models to test whether cytotoxicity of epitope-specific cd8+ t cells can differ from corresponding ifnγ production. we first used the vaccinated mouse model described above to check for this phenomenon. we expanded the number of epitopes to be tested by searching through the influenza research database for h2-k d -binding pr8 h1n1 epitopes and their variants found in h3 or h5 subtype (if available). we selected a total of 24 epitopes for a 3-color combination screening (table s4) . we also chose to perform the multiplex in vivo cytotoxicity assay and ifnγ-ics assay (figure 4a) in separate experiments in order to avoid issues of spillover signals from brightly labeled donor splenocytes. both results were then overlaid onto a single graph to allow for better visual comparison ( figure 4b ; non-h1n1 viral epitopes are highlighted in gray and variant epitopes are in the same dotted box). in vaccinated mice subjected to a lethal pr8 challenge, the immunodominant epitopes np147 and ha518 consistently produced robust responses in both assays (figure 4b , left 2 and 3). however, the following epitopes did not give concordant results: flunp-8, fluha-4, fluha-6, and flupb1f2-1 (figure 4b, arrows) . especially for flunp-8, the peptide elicited a very significant positive response in cytotoxicity assays but not in assays measuring ifnγ expression. interestingly, with the exception of fluha-3, iav variant epitopes that were present in h3n2 or h5n1 subtypes elicited similar cytotoxicity and ifnγ expression by cd8 + t cells as the corresponding homologs present in pr8, suggesting that pr8specific cd8 + t cells may tolerate small changes in the epitope sequence, enabling them to target cells infected by other iav subtypes. in unvaccinated mice, cytotoxicity against np147 and flunp-8 were significantly elevated upon lethal pr8 challenge, albeit not at levels seen in vaccinated mice ( figure 4c ). more importantly, intracellular ifnγ staining did not show any significant difference between any of the tested peptides. in the above mouse work, we observed apparent discrepancies between ifnγ production, a common marker of t cell activation, and actual cytotoxicity in some iav-specific cd8 + t cells. we wondered if we could observe this phenomenon in other disease models. to answer this, we used the pathogenic malaria model for evaluation. the malaria parasite plasmodium berghei anka (pba) causes a severe neurological complication termed experimental cerebral malaria in susceptible c57bl/6j mice. at 6 days post-infection, mice infected with pba were subjected to both assays with the eleven previously known h-2 b malaria epitopes (table s5 ). in pba-infected mice, pb1-, f4-, and pbt1-specific cd8 + t cells displayed significantly elevated killing activities. interestingly, only pb1and pbt1-specific cd8 + t cells, but not those specific for the f4 epitope, registered significant increases in ifnγ production ( figure 5a) . a similar result also occurs when a different parasite strain, pbnk65, was used to infect mice ( figure 5b) . additionally, we investigated this phenomenon in a mouse model of mers-cov infection (16) . in this model, hdpp4-ki mice were infected with mers-cov-emc. after 4 weeks, these mice were challenged with a 4ld 50 (2000 pfu) mouseadapted strain of mers-cov. at the peak of the t cell response, splenocytes were harvested and assayed for epitopespecific cd8 + t cell activity, with the epitopes shown in table s6 , by ifnγ expression and in vivo cytotoxicity assays. as shown in figure 6 , cd8 + t cells recognizing all the tested figure 3 | feasibility of performing multiple in vitro cytotoxicity assay in a single well. frozen pbmcs from (a) healthy and (b) influenza-infected hla-a*11:01 individuals were rested in a 37 • c/5% co 2 incubator overnight. non-cd8 + t cells (target cells) were isolated from cd8 + t cells by macs, split into equal groups and pulsed with corresponding peptides (including one additional group that is incubated with dmso; peptide sequences are shown in table s3 ). pulsed target cells are combinatorically labeled with cell labeling dyes (celltrace violet, celltrace yellow, celltracker cmfda and celltracker deep red), mixed in equal numbers and split into two groups: one group incubated with cd8 + t cells and the other group without. cells are incubated at 37 • c/5% co 2 overnight and analyzed the next day by flow cytometry to calculate the specific lysis of target cells by cognate cd8 + t cells. ****p < 0.0001, one-way anova with holm-sidak's multiple comparison test against ebo 580-588 (control). cd8 + t cell epitopes show elevated ifnγ levels in challenged mice, but this was not corroborated in cytotoxic assays. when comparing s395-and m156-specific cd8 + t cell activities, the former showed potent cytotoxicity while the latter had no cytotoxicity, despite both of them having comparable levels of ifnγ expression (figure 6 ). in this study, we expanded the conventional in vivo cytotoxicity assay from two parameters to twenty-four parameters. this functional assay allows the direct assessment of multiple cd8 + t cell specificities simultaneously in a single mouse or human pbmc sample, leading us to the finding that not all epitopespecific cd8 + t cells that express ifnγ exhibit concomitant direct cytotoxicity. importantly, we demonstrate that this phenomenon is not limited to three different pathogen mouse models: iav, malaria and mers-cov, but it also appears on human pbmc screening against experimentally known iav cd8 + epitopes. these observations raise concerns about t cell studies in applications such as validating epitope candidates for vaccine studies. currently, ifnγ-ics remains widely used for such a purpose, with those eliciting ifnγ production regarded as responding positively to a pathogen insult, leading them to be selected as potential targets of vaccine design. however, this may not mean the t cells induced by this kind of epitope-based vaccines can always control the disease, as has been shown in figure 4 | discrepancy between direct and indirect measurements of cd8 + t cell effector functions in an influenza mouse model. splenocytes from vaccinated mice challenged by a lethal pr8 infection were used to measure epitope-specific cd8 t cell effector functions through direct (in vivo cytotoxicity) and indirect (ifnγ-ics) assays. twenty-three different peptides were employed for the screening (table s4 ) to fully utilize the capability of our multiplex assay. (a) the representative gating strategy for ifnγ-ics assay is shown. (b-c) the results for vaccinated (b) and unvaccinated (c) mice following challenge are shown. epitopes grouped within dashed boxes are homologs of each other, with those in gray originating from h3n2 or h5n1. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, kruskal-wallis test with dunn's post-test against irrelevant control (csp280). red and black asterisks refer to corresponding data from multiplex in vivo and ifnγ-ics assays respectively. frontiers in immunology | www.frontiersin.org table s3 ) that were published in the literature were employed in the screens, with the results as shown. ***p < 0.001, **p < 0.01, *p < 0.05, kruskal-wallis test with dunn's post-test against irrelevant control (ova). red and black asterisks refer to corresponding data from multiplex in vivo and ifnγ-ics assays respectively. a simian immunodeficiency virus vaccination/challenge study (30) , and a human iav trial (31) . although no cytotoxicity data were obtained in those studies, we argue that the possible discrepancies between ifnγ production and actual cytotoxic potential of the epitope-specific cd8 + t cells may be one of the causes. in this respect, we believe our multiplex cytotoxicity assay can further validate the epitope candidates in terms of the cytotoxicity potential of the cognate cd8 + t cells. in particular, our approach can be a robust tool for revealing a more comprehensive picture of the protective role of cd8 + t cells stimulated by t cell-based vaccines. furthermore, the multiplex cytotoxicity assay is also useful for evaluating cd8 + t cell cytotoxicity responses in t cell therapies such as those involving chimeric antigen receptor (car)-t cells or cancer vaccines. the multiplex cytotoxicity assay described here produces results that are comparable to that when the conventional single-plex cytotoxicity assay was performed instead (data not shown), which offers several advantages. firstly, the number of recipient mice needed in a given experimental setup can be reduced, resulting in lower costs, better compliance with animal ethics (the 3r rule), and data that are more reproducible across multiple epitopes. secondly, cd8 + t cell killing activities in different compartments can be analyzed simultaneously, which will provide data useful for evaluating the efficacy of a vaccine in inducing both systemic and mucosal figure 6 | discrepancy between direct and indirect measurements of cd8 + t cell effector functions in a middle east respiratory syndrome (mers) mouse model. the hdpp4-ki c57bl/6j mice were infected with 1 × 10 5 mers-cov-emc via intranasal route. after 4 weeks, these mice were challenged with 2 × 10 3 pfu of mouse-adapted strain of mers-cov via intranasal route. spleens from these mice were harvested and stimulated with the corresponding peptides in the presence of brefeldin a. ifnγ expression from cd8 t cells in the splenocytes were quantified (black). in parallel, splenocytes from healthy donor mice were used for multiplex in vivo cytotoxicity assay with the corresponding peptides and transferred into transduced/challenged recipients at day 5 post-infection. the recipient splenocytes were harvested and analyzed by flow cytometry (red). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, kruskal-wallis test with dunn's post-test against irrelevant control (ova). red and black asterisks refer to corresponding data from multiplex in vivo and ifnγ-ics assays respectively. adaptive immunity. thirdly, the expansion of parameters available for screening allows one to include one or more irrelevant epitopes in the cytotoxicity assay. by definition, irrelevant epitopes should not elicit destruction by cd8 + t cells. however, it has been shown that uninfected cells also undergo apoptosis and necrosis during iav infection (32) . hence, we argue that such non-specific killing can apply to intended target cells, and the destruction of irrelevant target cells acts as a control to measure non-specific killing activity, permitting the calculation of actual cd8 + t cell-mediated cytotoxicity levels. the expression of ifnγ in epitope-specific cd8 + t cells has been widely used to measure cell activation and hence represents a marker to evaluate the efficacy of cd8 + t cell epitopes. this expression and other commonly used maker staining methods (e.g., tetramer staining) are indirect methods of assessing t cell functionality. one would expect that cd8 + t cells that upregulate ifnγ production should be cytolytic against its cognate targets. however, in an hiv setting, single-cell analysis of individual hiv-specific cd8 + t cells in vitro revealed discordance between ifnγ expression and cytolytic potential (33) . in three different mouse models of infection tested in our study, we showed that such discrepancies also occur in some epitope-specific cd8 + t cells. in most cases, the discrepancies exist in specific cd8 + t cells that were ifnγ-negative but possess vigorous cytolytic activity. however, this can also happen vice versa. continuous ifnγ signaling by activated epitope-specific cd8 + t cells was shown to lead to their reduction of cytolytic potential (34) , suggesting that these cells may downregulate ifnγ production in order to maintain their cytolytic potential. on the other hand, the finding of substantial ifnγ production by cd8 + t cells that possess low cytotoxic potential, such as the m64-and m156-specific cd8 + t cells in our mers-covinfected mice, was also unusual. similar phenomena could also be found in humans (35) , but the mechanism behind this is yet to be determined. the mechanisms regulating the ifnγ production and cytolytic potential of epitope-specific cd8 + t cells may be highly complex, and future research on this topic is needed. nevertheless, our results point out the disadvantage of relying solely on ifnγ production as a marker to evaluate cd8 + t cell activation, particularly in screening of cd8 + t cell epitopes for vaccine candidates, as one may miss out cd8 + epitopes that are useful in generating protective cd8 + t cells. besides, evaluations of pathogen-specific cd8 + t cell activities in diseases settings should be cautious when ifnγ is the only marker used in the assessments. we believe that our multiplex assay might able to fill these gaps. the datasets generated for this study are available on request to the corresponding author. the studies involving human participants cd8(+) t cells: foot soldiers of the immune system recent advances in targeting cd8 t-cell immunity for more effective cancer immunotherapy in vivo killing capacity of cytotoxic t cells is limited and involves dynamic interactions and t cell cooperativity mechanisms and dynamics of t cellmediated cytotoxicity in vivo how do cytotoxic lymphocytes kill cancer cells? immunodominance in virus-induced cd8(+) t-cell responses is dramatically modified by dna immunization and is regulated by gamma interferon the rapidity with which virus-specific cd8+ t cells initiate ifn-gamma synthesis increases markedly over the course of infection and correlates with immunodominance ifn-gamma production by cd8+ t cells depends on nfat1 transcription factor and regulates th differentiation interferon-gamma derived from cytotoxic lymphocytes directly enhances their motility and cytotoxicity interferon-gamma: an overview of signals, mechanisms and functions regulation of antigen-specific cd8+ t cell homeostasis by perforin and interferon-gamma role of direct effects of ifn-gamma on t cells in the regulation of cd8 t cell homeostasis quantitative assay of the lytic action of immune lymphoid cells on 51-cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs carboxyfluorescein fluorochromasia assays. i non-radioactively labeled cell mediated lympholysis circulating tfh cells, serological memory, and tissue compartmentalization shape human influenza-specific b cell immunity rapid generation of a mouse model for middle east respiratory syndrome il-15 adjuvanted multivalent vaccinia-based universal influenza vaccine requires cd4+ t cells for heterosubtypic protection stable transfection of malaria parasite blood stages studies on sporozoite-induced infections of rodent malaria. i the pre-erythrocytic tissue stage of plasmodium berghei a rapid and robust tri-color flow cytometry assay for monitoring malaria parasite development standardization of cryopreserved peripheral blood mononuclear cells through a resting process for clinical immunomonitoring-development of an algorithm parallel detection of antigen-specific t-cell responses by multidimensional encoding of mhc multimers virally induced inflammation triggers fratricide of fas-ligandexpressing beta-cells roles of proinflammatory cytokines and the fas/fas ligand interaction in the pathogenesis of inflammatory myopathies deletion of fas in adipocytes relieves adipose tissue inflammation and hepatic manifestations of obesity in mice involvement of two distinct killing mechanisms in bystander target cell lysis induced by a cytotoxic t lymphocyte clone fas ligand-mediated lysis of self bystander targets by human papillomavirus-specific cd8+ cytotoxic t lymphocytes perforin-independent cd8(+) t-cell-mediated cytotoxicity of alveolar epithelial cells is preferentially mediated by tumor necrosis factor-alpha: relative insensitivity to fas ligand mapping hla-a2, -a3 and -b7 supertype-restricted t-cell epitopes in the ebolavirus proteome attenuation of simian immunodeficiency virus sivmac239 infection by prophylactic immunization with dna and recombinant adenoviral vaccine vectors expressing gag a synthetic influenza virus vaccine induces a cellular immune response that correlates with reduction in symptomatology and virus shedding in a randomized phase ib live-virus challenge in humans cellular inhibitor of apoptosis protein ciap2 protects against pulmonary tissue necrosis during influenza virus infection to promote host survival a high-throughput single-cell analysis of human cd8(+) t cell functions reveals discordance for cytokine secretion and cytolysis regulation of ifn-gamma signaling is essential for the cytotoxic activity of cd8(+) t cells correlation between interferon-gamma secretion and cytotoxicity, in virusspecific memory t cells we thank mike richards, jeni mitchell, and teresa so for their technical supports. we acknowledge the assistance from faculty core facility in hku medicine and flow cytometry platform in singapore immunology network for flow cytometry analyses. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.00400/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 poh, zheng, channappanavar, chang, nguyen, rénia, kedzierska, perlman and poon. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-003319-u88gznxq authors: huang, weishan; solouki, sabrina; carter, chavez; zheng, song-guo; august, avery title: beyond type 1 regulatory t cells: co-expression of lag3 and cd49b in il-10-producing t cell lineages date: 2018-11-19 journal: front immunol doi: 10.3389/fimmu.2018.02625 sha: doc_id: 3319 cord_uid: u88gznxq type 1 regulatory cd4(+) t (tr1) cells express high levels of the immunosuppressive cytokine il-10 but not the master transcription factor foxp3, and can suppress inflammation and promote immune tolerance. in order to identify and obtain viable tr1 cells for research and clinical applications, co-expression of cd49b and lag3 has been proposed as a unique surface signature for both human and mouse tr1 cells. however, recent studies have revealed that this pattern of co-expression is dependent on the stimulating conditions and the differentiation stage of the cd4(+) t cells. here, using an il-10(gfp)/foxp3(rfp) dual reporter transgenic murine model, we demonstrate that co-expression of cd49b and lag3 is not restricted to the foxp3(−) tr1 cells, but is also observed in foxp3(+) t regulatory (treg) cells and cd8(+) t cells that produce il-10. our data indicate that il-10-producing tr1 cells, treg cells and cd8(+) t cells are all capable of co-expressing lag3 and cd49b in vitro following differentiation under il-10-inducing conditions, and in vivo following pathogenic insult or infection in the pulmonary mucosa. our findings urge caution in the use of lag3/cd49b co-expression as sole markers to identify tr1 cells, since it may mark il-10-producing t cell lineages more broadly, including the foxp3(−) tr1 cells, foxp3(+) treg cells, and cd8(+) t cells. the mammalian immune system has evolved both effector and regulatory immune axes to protect the host from invading pathogens, along with a control mechanism to tune the level of immune reactivity against self-and non-self-agents to prevent host tissue damage. interleukin-10 (il-10) is a regulatory cytokine with a demonstrated anti-inflammatory function and plays an essential role in preventing allergic inflammation (1), autoimmunity (2) , and pathogen-induced immunopathology (3, 4) , but can also promote the establishment and maintenance of chronic infection (5, 6) . il-10 has been reported as a product of activation of multiple immune cell lineages. innate immune cells including dendritic cells (dcs) (7) , macrophages (8) , neutrophils (9) , and innate lymphoid cells (ilcs) (10) have been reported to express il-10 in vivo and in vitro. il-10 is also expressed by many cell subsets of the adaptive immune system, including b cells (11) and t cells comprising the foxp3 − cd4 + (12), foxp3 + treg (13) , and cd8 + t cell subsets (14) . regulatory t cells are defined by their immunosuppressive function, and the three aforementioned subsets of il-10-producing t cells have been reported as phenotypically distinct regulatory t cell subsets, playing important roles in promoting immune tolerance and/or suppressing inflammation in both mouse and human (15) (16) (17) (18) (19) (20) . among the il-10-producing t cells, the foxp3 − cd4 + t cell subset, also known as type 1 regulatory t cells (tr1 cells), are inducible in the periphery and have a pivotal role in limiting inflammation (15, (21) (22) (23) . tr1 cells have been shown to prevent allergic asthma (24) and atopic dermatitis (25) in murine models. in both mouse models and humans, induction of tolerance via specific antigen immunotherapy (sit) is accompanied by induction of tr1 cells (26, 27) . therefore, tr1 cells have strong promise as a potential therapeutic approach for inflammatory diseases. tr1 cells can be differentiated from naïve cd4 + t cells upon tcr engagement in the presence of il-27 in vitro (28) , and in order to identify and obtain viable tr1 cells for clinical application, co-expression of lag3 and cd49b has been recently proposed to be a cell surface signature of the foxp3 − il-10 high tr1 cells (15) . lag3 is a structural homolog of the cd4 molecule and can bind to mhc class ii with high affinity (29, 30) . lag3 is highly expressed by il-10 + cd4 + t cells (31) , as well as by activated effector t cells (32) and foxp3 + treg cells (33) . cd49b is the α2 integrin subunit, highly expressed by nk cells (34) . cd49b is up-regulated in t cells that may produce il-10 and/or pro-inflammatory cytokines (35) (36) (37) . in addition to foxp3 − tr1 cells, il-10 can be highly up-regulated in activated foxp3 + treg and cd8 + t cells under inflammatory conditions and/or upon tcr activation. given the importance of being able to identify foxp3 − tr1 cells, including under clinical conditions, and to gain a better understanding of the selectivity of co-expression of lag3 and cd49b as a cell surface signature for il-10-producing cells, we sought to determine whether co-expression of lag3 and cd49b can mark a broader range of t cell subsets that are actively producing high levels of il-10. using a murine model carrying an il-10 gfp /foxp3 rfp dual reporter system, we find that co-expression of lag3 and cd49b is a generic feature of the il-10-producing foxp3 − cd4 + , foxp3 + cd4 + , and cd8 + t cell subsets. the capacity of co-expression of lag3 and cd49b in marking il-10 high t cell subsets is dependent on the disease conditions and anatomical location of the cells. furthermore, co-expression of lag3 and cd49b is also a shared feature of human il-10-producing foxp3 − cd4 + , foxp3 + cd4 + , and cd8 + t cell subsets. our data reveal that co-expression of lag3 and cd49b is a generic signature of il-10-producing t cells, which is broader than previously appreciated. abbreviations: nb, nippostrongylus brasiliensis; tr1 cell, type 1 regulatory t (cd4 + tcrβ + foxp3 − il-10 + ) cell; treg cell, foxp3-expressing regulatory t cell; hdm, house dust mite; sr, saccharopolyspora rectivirgula; wsn, influenza a/wsn/1933 (h1n1). all mice were on the c57bl/6 background. rag1 −/− (b6.129s7-rag1 tm1mom /j), il-10 gfp (b6(cg)-il10 tm1.1karp /j) (38) , and foxp3 rfp (c57bl/6-foxp3 tm1flv /j) (39) reporter mice were from the jackson laboratory (bar harbor, me). single reporter strains were crossed to generate an il-10 gfp /foxp3 rfp dual reporter strain as we recently reported (40) . human peripheral blood samples were procured from the new york blood center collected from healthy cohorts. all experiments were approved by the office of research protection's institutional animal care and use committee and institutional review board at cornell university. all fluorescent antibodies are listed in "fluorochrome-target (clone; annotation if desirable)" format below. purified anti-cd16/32 (93; fc block), cd3ε (145-2c11), cd28 (37.51), ifn-γ (xmg1.2), and il-12 (c17.8) antibodies were from biolegend (san diego, ca); pacific blue-cd90 (53-2.1), fitc-tcrβ (h57-597), apc-lag3 (c9b7w), pe-cy7-cd49b (hmα2), and pe-cy7-cd62l (mel-14) were from biolegend; efluor 450-cd4 (gk1.5); alexa fluor 700-cd4 (gk1.5) were from ebioscience; bd horizon v500-cd44 (im7), pe-cd44 (im7), and apc-cy7-tcrβ (h57-597) were from bd biosciences; percp-cy5.5-cd8α (2.43) was from tonbo biosciences. purified anti-cd3ε (otk3) and cd28 (28.2), efluor 450-cd8α (rpa-t8) fitc-cd4 (okt4), and apc-foxp3 (236a/e7) were from ebioscience; pe-il-10 (jes3-19f1), alexa fluor 647-lag3 (11c3c65), and percp-cy5.5-lag3 (11c3c65) were from biolegend; fitc-cd49b (ak-7) and alexa fluor 700-cd4 (rpa-t4) were from bd biosciences. human trustain fcx (fc receptor blocking solution) was from biolegend; cell fixable viability dye efluor 506 was from ebiosciences. cells from various organs were isolated as we recently described (40) . briefly: blood cells were collected through cardiac puncture, and red blood cells were lysed before analysis; lungs were minced and digested in 0.2 mg/ml liberase tl (sigma, st. luis, mo) in 37 • c for 15-30 min, then filtered and red blood cells were lysed before analysis; intestines were flushed, opened longitudinally, and inner contents removed with the blunt end of scissors, then cut into 0.5-cm fragments, followed by digestion in 100 u/ml collagenase viii (sigma) in 37 • c for 1 h, filtered, and lymphocytes isolated using gradient separation by 40% and 80% percoll (ge healthcare, wilkes-barre, pa) solutions; perigonadal adipose tissues were minced and digested in 500 u/ml collagenase i (worthington biochemical corp., lakewood, nj) in 37 • c for 30 min, filtered and red blood cells were lysed before analysis. 50-150 u/ml dnase i (sigma) were added during digestion to reduce cell death triggered by free dna. foxp3 rfp il-10 gfp dual reporter mice were injected with 15 µg/mouse anti-cd3ε (145-2c11) intraperitoneally on day 0 and 2, and analyzed on day 4, as previously described (23) . mice were given 500 l3 nb larvae per mouse through subcutaneous injection, as we previously described (40) . cells from the lungs were analyzed 7 days post infection (7 dpi). mice were given daily intranasal exposures of 10 µg house dust mite (dermatophagoides pteronyssinus) protein extract (xpb82d3a2.5 from greer) in pbs, for 10 consecutive days. cells from the lungs were analyzed 24 h post the last treatment. mice were intranasally exposed to 150 µg saccharopolyspora rectivirgula (sr, atcc 29034) extract on 3 consecutive days each week as previously described (41) , for 4 weeks. cells from the lungs were analyzed on the last day of the fourth week. mice were intranasally infected with 1 ld 50 (10 4 pfu) wsn per mouse, as we previously described (40) . cells from the lungs were analyzed 7 days post infection (7 dpi). mouse tcrβ + foxp3 rfp− cd44 − cd62l + splenic naïve t cells were sorted on bd facs aria ii or fusion systems (bd biosciences, san jose, ca), then cultured with mitomycin-c (sigma, 50 µg/ml) treated antigen-presenting cells (apcs; rag −/− splenocytes) at 1:2 ratio in the presence of anti-cd3ε (1 µg/ml), anti-cd28 (1 µg/ml), recombinant murine (rm) il-27 (r&d systems, 20 -25 ng/ml), anti-ifn-γ and anti-il-12 (10 µg/ml) for 3 days. human peripheral blood mononuclear cells (pbmcs) were isolated from blood (new york blood center, long island, ny) using gradient separation in ficoll-paque plus (ge healthcare). pbmcs were cultured in full rpmi-1640 medium for 30 min in 37 • c, then non-adherent cells were used to enrich for cd4 + t cells using anti-human cd4 microbeads (miltenyl biotec, san diego, ca) or cd8 + t cells using a human cd8 isolation kit (biolegend). adherent cells were treated with mitomycin-c (sigma, 50 µg/ml) in 37 • c for 30 min and used as apcs. anti-human cd3ε (1 µg/ml) and cd28 (cd28.2, ebioscience, 1-3 µg/ml), recombinant human (rh) il-2 (peprotech, 200 u/ml), il-10 (peprotech, 100 u/ml), il-27 (r&d system, 25 ng/ml), and ifn-α2b (r&d system, 10 ng/ml) were added to differentiate human il-10-producing t cells. three days after cultures were set up, cells were stimulated with pma (100 ng/ml, sigma-aldrich), ionomycin (0.5 µm, sigma), brefeldin a (5 µg/ml), and golgistop (0.5 µl/ml, bd biosciences) for 4 h as we previously described (42) , and subjected to surface staining and intracellular staining (see details below). surface staining of live cells were done in the presence of fc block and fixable viability dye. to detect human cd4 in activated t cells, anti-cd4 antibody was added into the intracellular staining panel. for intracellular cytokine staining, cells were fixed with 2% paraformaldehyde (electron microscopy sciences, hatfield, pa), permeabilized and stained with anti-cytokine antibodies in pbs/0.2% saponin (sigma). staining for human transcription factor foxp3 was performed with a foxp3 staining buffer kit (ebioscience). flow cytometry data were acquired on lsrii, facs aria ii or fusion systems (bd biosciences), and analyzed in flowjo (tree star, ashland, or). all analyses were performed on fixable viability dye negative singlet population. non-parametric mann-whitney tests and one-way anova were performed using graphpad prism v5.00 (graphpad, san diego, ca), with p ≤ 0.05 considered statistically significant. "ns" refers to "no significance." both il-10-producing cd4 + and cd8 + t cells lag3 and cd49b co-expression was previously reported to be a cell surface signature for both mouse and human il-10-producing cd4 + t cells that lack the expression of foxp3 (also known as type 1 regulatory t cells, tr1 cells) (15) . we and others have previously reported that co-culturing murine naïve cd4 + t cells with antigen presenting cells (apcs) in the presence of anti-cd3, anti-cd28, anti-ifn-γ, anti-il-12, and il-27 can efficiently induce the differentiation of tr1 cells (28, 40, 43) , which express high levels of lag3 and cd49b. our recent data also demonstrated that this protocol can induce il-10 production in bulk t cell populations that include both cd4 + and cd8 + t cells ( figure 1a) . surprisingly, the resultant il-10-producing cd8 + t cells induced in vitro through this protocol also exhibited high levels of lag3/cd49b co-expression ( figure 1a , last plot). in fact, il-10-producing cd8 + t cells can express higher levels of lag3 and cd49b than their il-10producing cd4 + counterparts induced in the same cell culture ( figure 1b) . these data suggest that co-expression of lag3 and cd49b is not an exclusive cell surface signature of the tr1 cells, and may be a shared feature of both il-10-producing cd4 + and cd8 + t cells. both il-10-producing tr1 and treg cells il-10 production can be significantly elevated in the pulmonary mucosa during the late stages of parasitic infection by nippostrongylus brasiliensis (nb), predominantly by cd4 + t cells that are lag3/cd49b double positive (15, 44) . we recently observed that both foxp3 − and foxp3 + t cells are capable of producing il-10 in the pulmonary tissues post nb infection (40) . to determine whether co-expression of lag3 and cd49b is exclusive to foxp3 − tr1 cell subset, we infected il-10 gfp /foxp3 rfp dual reporter mice with nb, and analyzed the il-10-producing t cells. we found that, as previously described, a large majority of the il-10-producing t cells in the lungs of nb-infected mice express high levels of both lag3 and cd49b (figures 2a,b) , and are predominantly cd4 + t cells ( figure 2c) . however, interestingly, these il-10-producing lag3 + cd49b + cd4 + t cells included both foxp3 + and foxp3 − cd4 + t cells subsets (figure 2c , last plot; and figure 2d ). to further determine whether lag3/cd49b co-expression is correlated with il-10 and/or foxp3 expression in cd4 + t cells, we compared the percentage of lag3/cd49b double positive populations in foxp3 − il-10 − , foxp3 − il-10 + , foxp3 + il-10 − , and foxp3 + il-10 + cd4 + t cells isolated from the lungs of nb-infected mice. we found that regardless of expression of foxp3, nb infection did not lead to significant up-regulation of lag3/cd49b co-expression on il-10 − cd4 + t cells (figures 2e,f) . however, the percentage of the lag3 + cd49b + population of il-10 + cd4 + t cells is significantly higher than their il-10 − counterparts ( figure 2f) . moreover, the levels of lag3 and cd49b expression are similar between the foxp3 + and foxp3 − counterparts of il-10 + cd4 + t cells (figure 2g) . therefore, il-10-producing cd4 + t cells, regardless of foxp3 expression, have a high capacity of coexpressing lag3 and cd49b. together with the data shown in figure 1 , these data suggest that co-expression of lag3 and cd49b is a generic feature of il-10-producing t cells, including foxp3 − tr1 cells, foxp3 + treg cells and cd8 + t cells. the composition of lag3 + cd49b + il-10-producing t cells differs in different disease models il-10 plays an essential role in pulmonary inflammatory diseases, which has been reported in multiple murine models of lung disease, including allergic asthma (45) , hypersensitivity pneumonitis (hp) (46) and influenza pneumonia (20) . we examined whether the expression of lag3 and cd49b would differ based on the inflammatory response in three mouse models of lung inflammation. mice carrying the il-10 gfp /foxp3 rfp dual reporters were exposed intranasally to house dust mite (hdm) protein extract (as a model of allergic asthma), saccharopolyspora rectivirgula (sr) (as a model of hp/farmer's lung disease), or infected intranasally with wsn/flu virus (as a model of influenza infection). we observed significant percentages of il-10-producing t cells in the lung tissue of mice exposed to hdm (figure 3a) , sr (figure 3c) , or wsn/flu virus ( figure 3e) . these il-10-producing t cells all co-expressed high levels of lag3 and cd49b, and include foxp3 + cd4 + , foxp3 − cd4 − , and cd8 + subsets in all disease models analyzed (figure 3) . however, the relative proportions of il-10-producing foxp3 + cd4 + , foxp3 − cd4 − , and cd8 + subsets differed in the various disease models (figure 3) . in contrast to the composition of il-10-producing lag3 + cd49b + t cells induced by nb infection, in which the foxp3 − cd4 + subset is the majority (72% in figure 2d ), in hdm-induced allergic asthma model, the largest subset of the il-10-producing lag3 + cd49b + t cells in the lungs are foxp3 + cd4 + t cells (60%), followed by foxp3 − cd4 + t cells (32%), while cd8 + t cells are only around 1.4% (figure 3b ). in the sr-triggered farmer's lung disease model, foxp3 + cd4 + t cells are the majority of the il-10producing lag3 + cd49b + t cell subset in the lungs, however, there are similar percentages of foxp3 − cd4 + and cd8 + t cells (16% each) (figure 3d) . strikingly in the murine model of influenza infection, cd8 + t cells are the largest subset of the il-10-producing lag3 + cd49b + t cells in the lungs (86%), followed by foxp3 − cd4 + t cells, while foxp3 + cd4 + are a minority ( figure 3f) . these data compared the composition of il-10-producing lag3 + cd49b + t cells in various murine models of pulmonary inflammatory diseases. along with the model of parasitic infection shown in figure 2 , our data suggest that co-expression of lag3 and cd49b marks all il-10-producing t cell lineages in the pulmonary system, and relative abundance of the marked t cell subsets is dependent on the type of immune response as shown in the disease models. the composition of lag3 + cd49b + as discussed above, we demonstrated that co-expression of lag3 and cd49b is a generic feature of il-10-producing t cells in vivo in pulmonary tissues under multiple inflammatory conditions (figures 2, 3) . to determine whether this feature is applicable to il-10-producing cells in other organs, we injected il-10 gfp /foxp3 rfp dual reporter mice with an anti-cd3ε antibody that has been shown to stimulate pronounced il-10 production by t cells through tcr activation in vivo (23, 40) . we analyzed il-10-producing t cells in blood, lymph nodes (ln), lung, fat and small intestine, and found that coexpression of lag3 and cd49b marked a portion of the il-10producing t cells following tcr activation in vivo, which again included foxp3 + cd4 + , foxp3 − cd4 + , and cd8 + t cell subsets ( figure 4a ) in all organs analyzed. an interesting note is that the relative abundance of foxp3 + cd4 + , foxp3 − cd4 + , and cd8 + subsets among the il-10-producing lag3 + cd49b + t cells vary significantly in different organs of the same mice ( figure 4b) . upon tcr activation in vivo, foxp3 + cd4 + t cells are the major population that are il-10 + lag3 + cd49b + in the blood, lymph nodes and lungs, while cd8 + t cells are the majority of il-10 + lag3 + cd49b + t cells in the perigonadal fat and small intestine ( figure 4b) . these data suggest that co-expression of lag3/cd49b marks all three il-10-producing t cell subsets in figure 3 | composition of il-10-producing lag3 + cd49b + t cells in lung inflammatory and infectious disease models. all experiments were performed using mice carrying the il-10 gfp /foxp3 rfp dual reporter system, and cells were isolated from the lungs and gated on live singlets for analysis as shown in figure 2a . representative facs plots of lag3/cd49b co-expression by il-10-producing cells, and among these cells, ratio of cd4 + vs. cd8 + t cells, and foxp3 − vs. foxp3 + cd4 + subpopulations are shown, followed by pie chart showing the proportions of cd4 + foxp3 + , cd4 + foxp3 − , and cd8 + t cells in il-10-producing lag3/cd49b double positive t cell population. mice were exposed intranasally to: (a,b) house dust mite (hdm) protein extract for 10 consecutive days, and analyzed 24 h post the last treatment; (c,d) saccharopolyspora rectivirgula (sr) for 3 consecutive days every week for 4 weeks, and analyzed in the end of the fourth week; (e,f) influenza a (wsn) viruses, and analyzed 7 dpi. n ≥ 3, combined from three independent experiments. data presented as mean ± s.e.m. multiple organs and the relative abundance of the foxp3 + cd4 + , foxp3 − cd4 + and cd8 + t cell subsets in il-10-producing lag3 + cd49b + t cells is dependent on the anatomical location of the cells. human il-10-producing cd4 + and cd8 + t cells exhibit a lag3 + cd49b + phenotype in murine models, we demonstrated that co-expression of lag3 and cd49b marks il-10-producing foxp3 + cd4 + , foxp3 − cd4 + , and cd8 + t cells under different inflammatory conditions in the lungs (figures 2, 3) , as well as in different organs when tcr is activated in vivo (figure 4) . to further determine whether different subsets of il-10-producing t cells exhibit the shared feature of co-expression of lag3 and cd49b in humans, we isolated cd4 + and cd8 + t cells from human peripheral blood and cultured them under il-10-inducing conditions. we found that as in the mouse, human il-10producing foxp3 + cd4 + , foxp3 + cd4 + , and cd8 + subsets all up-regulated both lag3 and cd49b expression, with a significant lag3/cd49b double positive population (figure 5 ). our data presented in this report demonstrated that, as previously reported (15) , co-expression of lag3 and cd49b identifies foxp3 − il-10 high tr1 cells, but that these markers are not exclusive for tr1 cells in human and mouse. furthermore, we find that the il-10-producing lag3 + cd49b + t cell population is composed of foxp3 + cd4 + , foxp3 − cd4 + , and cd8 + t cell subsets, and this composition varies depending on the disease conditions and anatomical locations of the cells. the importance of this work is emphasized by the need to specially identify foxp3 − tr1 cells, especially under clinical conditions, and our findings suggest that the use of lag3 and cd49b should be combined with other markers to uniquely identify these cells. using tr1 cell clones derived from human naïve cd4 + t cells and purified by an il-10 secretion assay, gagliani, roncarolo and colleagues identified co-expression of lag3, cd49b, and cd226 as cell a surface signature of il-10-producing cd4 + t cells, and demonstrated that co-expression of lag3 and cd49b is sufficient to distinguish foxp3 − il-10 high tr1 cells from t helper and/or regulatory subsets that expressed lower levels of or no il-10 in both mouse and human (15) . however, it was recently reported that il-10-producing t cells derived from human cd4 + memory t cells exhibit low levels of surface expression of lag3 and cd49b (47) , suggesting that the pattern of co-expression of lag3 and cd49b may vary in human il-10-producing cd4 + t cells, which may be associated with whether they were derived from naïve precursors vs. memory cells. indeed, it is clear that co-expression of lag3 and cd49b can mark il-10-producing tr1-like cells, and serve to help eliminate those t cells lineages that are not capable of producing il-10 for potential clinical interest. however, other non-tr1-like il-10-producing t cells can also co-express these markers. whether co-expression of lag3 and cd49b allows efficient recovery of il-10 high t cells may be dependent on the proportion of il-10 high t cells that are co-expressing lag3 and cd49b, which may differ depending on whether the relevant il-10-producing cells had different origins and/or underwent different activation regimes. both lag3 and cd49b can individually be up-regulated in activated t cells, regardless of their production of the anti-inflammatory il-10 or pro-inflammatory cytokines (31) (32) (33) (35) (36) (37) . co-expression of lag3 and cd49b is more restricted to il-10-producing subsets, as previously described in foxp3 − cd4 + t cells (15) . our data here demonstrated that this is a generic feature of il-10-producing cells, including foxp3 − cd4 + , foxp3 + cd4 + , and cd8 + subsets. the cooccurrence of il-10 production and lag3 + cd49b + may be explained in two ways. the first explanation is that il-10 stimulation through autocrine and/or paracrine may induce the expression of lag3 and cd49b in the stimulated cells, therefore, il-10-capturing t cells [cells detected by roncarolo's group in ref (15) ] exhibited high levels of lag3/cd49b coexpression. this hypothesis would place il-10 up-stream of lag3 and cd49b expression, which is unlikely, as recent studies by flavell and huber's groups showed that the il-10 receptor is dispensable for tr1 cell differentiation, including the lag3/cd49b double positive feature; instead, il-10 receptor signaling is critical for maintaining the cell fate commitment and functional performance of the differentiated tr1 cells (48) . therefore, it is more likely that lag3 and cd49b signaling pathways function cooperatively to activate the expression of il-10. this hypothesis is more reasonable, given our data that lag3 and cd49b co-expression is a generic feature of il-10 producing cells in multiple subsets. lag3 is a structural homolog of the cd4 molecule, and can bind to mhc class ii with higher affinity than cd4 (29, 30) . in unstimulated t cells, lag3 is retained in the intracellular compartment and degraded in the lysosome. upon t cell activation, lag3 traffics from the lysosomal compartment to the cell surface through a protein kinase c (pkc) dependent pathway (49) . cd49b is the integrin α2 subunit and plays a critical role in cell-cell interaction and adhesion (50) . the cd49b pathway activates multiple downstream effector signaling pathways, among which is the ras/mapk signaling cascade (51, 52) . we recently reported that the ras/mapk signaling pathway functioning downstream of the tcr is indispensable for il-10 production by foxp3 − cd4 + cells, through activation of the expression of the transcription factor interferon regulatory factor 4 (irf4) (40) . pkc can regulate ras signaling to downstream effectors, and can activate mapk signaling in both ras-dependent and independent manners (53, 54) . the interplay between pkc and ras may regulate signals that connect lag3 and cd49b downstream pathways, leading to up-regulation of il-10. given the complexity of these pathways, comprehensive understanding of the mechanism(s) underlying the co-expression of these markers with il-10 will require significantly more in-depth analyses. regardless of the mechanism, our findings indicate that lag3/cd49b co-expression does not uniquely identify foxp3 − tr1 cells, but is a more general indicator of il-10 production in t cell lineages. despite the shared feature of co-expression of lag3 and cd49b by il-10-producing foxp3 + cd4 + , foxp3 − cd4 + , and cd8 + t cells, we also observed interesting discrepancies in the proportional composition of these three il-10 high t cell subsets that are all lag3/cd49b double positive in the lung mucosa of different pulmonary inflammatory disease models, as well as in different anatomical locations in the same mice upon tcr activation in vivo. for example, parasite infection (nb) and mite allergen (hdm) induced predominantly il-10producing lag3 + cd49b + t cells that are cd4 + with very few that are cd8 + . in the nb infection model, the majority of il-10 + lag3 + cd49b + cd4 + t cells are foxp3 − tr1 cells, while in hdm-exposed mice, foxp3 + treg cells are the majority. in the bacterial exposure (sr) and viral infection (flu) models, we observed an increased proportion of il-10 + lag3 + cd49b + t cells that are cd8 + , which is the predominant population in the flu model ( figure 3f ). this discrepancy in the composition of il-10-producing lag3 + cd49b + t cells may be due to the difference of the microenvironment in which the il-10producing cells are being induced. factors that may affect the different composition of the il-10 high lag3 + cd49b + t cells may include the type of immune response, abundance and affinity of the tcr ligands, the cytokines induced and orchestrated by the stimuli, and the nutritional microenvironments that favor different subsets of the t cells. for example, type i ifn can facilitate the preferential induction of il-10-producing effector cd8 + t cells through inducing and sustaining expression of the irf4 and blimp1 transcription factors (55) . type i interferon is significantly elevated during influenza infection (56) but much less so by hdm exposure (57) . another possible orchestrator could be the cytokine il-27, which has been reported to be directly required for il-10 induction in cd8 + t cells (58) and cd4 + foxp3 − (59) but not cd4 + foxp3 + t cell subsets (60) . transcription factors and their interacting molecular networks that exhibit differential functions for il-10 induction in different t cell lineages might provide an answer as well. for example, blimp-1 is indispensable for il-10 induction in foxp3 + and foxp3 − cd4 + t cells (61, 62) , as well as in cd8 + t cells (63) . ahr interaction with cmaf is critical in il-10 induction in foxp3 − tr1 cells in response to an il-27-supplemented environment, but is insufficient in inducing il-10 production in foxp3 + treg cells under the same conditions (43); ahr expression was not critical in il-10-producing cd8 + t cells either (64) , suggesting that ahr signaling has a multifaceted function in regulating the level of il-10 expression in different t cell lineages. a more comprehensive understanding of the t cellintrinsic molecular features that are shared or distinct among the il-10-producing cd8 + , foxp3 − cd4 + and foxp3 + cd4 + t cells awaits further investigation. our data reported here demonstrate that co-expression of lag3 and cd49b marks il-10 high t cell subsets that are foxp3 + cd4 + , foxp3 − cd4 + , or cd8 + in both human and mouse, and thus does not uniquely identify foxp3 − tr1 cells. however, this finding does not negate the feasibility of utilizing co-expression of lag3 and cd49b in marking a broader range of immunosuppressive il-10 high t cell populations that have potential therapeutic effects for clinical application. further investigations are required to determine the levels of regulatory and pro-inflammatory cytokine production in the bulk lag3 + cd49b + t cells, and to determine and compare the ability of the foxp3 + cd4 + , foxp3 − cd4 + , and cd8 + subsets of the lag3 + cd49b + t cells in suppressing effector immunity and inflammation in vivo. wh and aa conceived research, designed experiments, analyzed and interpreted data, and wrote the manuscript. wh, ss, and cc performed experiments; s-gz contributed reagents and intellectual input. potential role of interleukin-10-secreting regulatory t cells in allergy and asthma interleukin-10-deficient mice develop chronic enterocolitis in the absence of endogenous il-10, mice acutely infected with toxoplasma gondii succumb to a lethal immune response dependent on cd4+ t cells and accompanied by overproduction of il-12, ifn-gamma and tnf-alpha a defect in interleukin-10 leads to enhanced malarial disease in plasmodium chabaudi chabaudi infection in mice resolution of a chronic viral infection after interleukin-10 receptor blockade interleukin-10 determines viral clearance or persistence in vivo pathogen-specific t regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective t helper type 1 responses by bordetella pertussis interleukin-10 derived from macrophages and/or neutrophils regulates the inflammatory response to lps but not the response to cpg dna coactivation of syk kinase and myd88 adaptor protein pathways by bacteria promotes regulatory properties of neutrophils regulatory innate lymphoid cells control innate intestinal inflammation a regulatory b cell subset with a unique cd1dhicd5+ phenotype controls t cell-dependent inflammatory responses interleukin-10-secreting type 1 regulatory t cells in rodents and humans regulatory t cells expressing interleukin 10 develop from foxp3+ and foxp3-precursor cells in the absence of interleukin 10 cytokine-induced il-10-secreting cd8 t cells represent a phenotypically distinct suppressor t-cell lineage coexpression of cd49b and lag-3 identifies human and mouse t regulatory type 1 cells tr1 cells and the counter-regulation of immunity: natural mechanisms and therapeutic applications strategies for use of il-10 or its antagonists in human disease regulatory t cells and mechanisms of immune system control regulatory t cells and immune tolerance effector t cells control lung inflammation during acute influenza virus infection by producing il-10 roles of lag3 and egr2 in regulatory t cells the role of different subsets of regulatory t cells in immunopathogenesis of rheumatoid arthritis th17 cells express interleukin-10 receptor and are controlled by foxp3(-) and foxp3+ regulatory cd4+ t cells in an interleukin-10-dependent manner in vivo induction of type 1-like regulatory t cells using genetically modified b cells confers long-term il-10-dependent antigen-specific unresponsiveness nonpathogenic bacteria alleviating atopic dermatitis inflammation induce il-10-producing dendritic cells and regulatory tr1 cells il-10 and regulatory t cells cooperate in allergen-specific immunotherapy to ameliorate allergic asthma birch pollen immunotherapy leads to differential induction of regulatory t cells and delayed helper t cell immune deviation suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated t cells cd4/major histocompatibility complex class ii interaction analyzed with cd4-and lymphocyte activation gene-3 (lag-3)-ig fusion proteins characterization of the major histocompatibility complex class ii binding site on lag-3 protein cd4+cd25-lag3+ regulatory t cells controlled by the transcription factor egr-2 lag-3, a novel lymphocyte activation gene closely related to cd4 lag-3 expression defines a subset of cd4(+)cd25(high)foxp3(+) regulatory t cells that are expanded at tumor sites cutting edge: the mouse nk cell-associated antigen recognized by dx5 monoclonal antibody is cd49b (alpha 2 integrin, very late antigen-2) immature dendritic cells suppress collagen-induced arthritis by in vivo expansion of cd49b+ regulatory t cells alpha2beta1 integrin is the major collagen-binding integrin expressed on human th17 cells functional specialization of memory th cells revealed by expression of integrin cd49b expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse identifying foxp3-expressing suppressor t cells with a bicistronic reporter itk signalling via the ras/irf4 pathway regulates the development and function of tr1 cells th17-polarized immune response in a murine model of hypersensitivity pneumonitis and lung fibrosis dendritic cell-mhc class ii and itk regulate functional development of regulatory innate memory cd4+ t cells in bone marrow transplantation the aryl hydrocarbon receptor interacts with c-maf to promote the differentiation of type 1 regulatory t cells induced by il-27 an essential role for th2-type responses in limiting acute tissue damage during experimental helminth infection regulatory t cells and il-10 in allergic inflammation interleukin-10 modulates the severity of hypersensitivity pneumonitis in mice tr1-like t cells -an enigmatic regulatory t cell lineage il-10 receptor signaling is essential for tr1 cell function in vivo trafficking of lag-3 to the surface on activated t cells via its cytoplasmic domain and protein kinase c signaling the role of integrins alpha 2 beta 1 and alpha 3 beta 1 in cell-cell and cell-substrate adhesion of human epidermal cells integrin-mediated ras-extracellular regulated kinase (erk) signaling regulates interferon gamma production in human natural killer cells integrin signalling during tumour progression regulation of ras signaling specificity by protein kinase c protein kinase c activates the mek-erk pathway in a manner independent of ras and dependent on raf type i ifn signaling facilitates the development of il-10-producing effector cd8(+) t cells during murine influenza virus infection tlr7 stimulation in human plasmacytoid dendritic cells leads to the induction of early ifn-inducible genes in the absence of type i ifn type i interferon is required for t helper (th) 2 induction by dendritic cells persistent loss of il-27 responsiveness in cd8+ memory t cells abrogates il-10 expression in a recall response cutting edge: il-27 induces the transcription factor c-maf, cytokine il-21, and the costimulatory receptor icos that coordinately act together to promote differentiation of il-10-producing tr1 cells tregspecific il-27ralpha deletion uncovers a key role for il-27 in treg function to control autoimmunity the transcription factors blimp-1 and irf4 jointly control the differentiation and function of effector regulatory t cells role of blimp-1 in programing th effector cells into il-10 producers cd4+ t cell help and innate-derived il-27 induce blimp-1-dependent il-10 production by antiviral ctls highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirus-induced encephalitis we thank a. redko for animal care and l. zhang the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 huang, solouki, carter, zheng and august. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-000647-uofygmeu authors: stäger, simona; rafati, sima title: cd8(+) t cells in leishmania infections: friends or foes? date: 2012-01-24 journal: front immunol doi: 10.3389/fimmu.2012.00005 sha: doc_id: 647 cord_uid: uofygmeu host protection against several intracellular pathogens requires the induction of cd8(+) t cell responses. cd8(+) t cells are potent effector cells that can produce high amounts of pro-inflammatory cytokines and kill infected target cells efficiently. however, a protective role for cd8(+) t cells during leishmania infections is still controversial and largely depends on the infection model. in this review, we discuss the role of cd8(+) t cells during various types of leishmania infections, following vaccination, and as potential immunotherapeutic targets. cd8 + t cells play a major role in protective immunity to a wide variety of pathogens, including viruses, bacteria, and protozoan parasites. however, the protective role of cd8 + t cells during leishmania infections has been controversial, mainly because of the discrepancy among infections with different leishmania species. different leishmania species have different tropisms and their diversity is reflected in the various clinical manifestations they induce. hence, it is not surprising that the contribution of cd8 + t cells to the immune response against the parasite depends on the clinical form and the species that is causing it. here, we discuss the literature on the contribution of cd8 + t cells to the immune response against leishmania, taking into account the various clinical forms and experimental models. cd8 + t cells recognize peptides that are presented in the context of major histocompatibility complex (mhc) class i molecules via the t cell receptor (tcr). although peptides presented via mhci mainly derive from endogenous antigens, various exogenous cellassociated antigens have also been shown to be uploaded onto the mhci pathway, by a process referred to as cross-presentation. leishmania antigens were also shown to be cross-presented (bertholet et al., 2006) . during in vivo infections, cross-presentation of leishmania antigens may result from several internalization pathways, such as direct infection, receptor-mediated uptake (woelbing et al., 2006) , or internalization of apoptotic vesicles (winau et al., 2006) . thus far, two different processing pathways have been proposed. an early work demonstrated that a surface antigen of l. amazonensis was processed in a proteasome-dependent manner within the cytosol (kima et al., 1997) . in contrast, a more recent study showed that cross-presentation of secreted leishmanial antigens is confined to an intraphagosomal processing pathway that is tap-and proteasome-independent (bertholet et al., 2006) . after activation, antigen-specific cd8 + t cells differentiate into effector cells and acquire the capacity to kill target cells, and produce several cytokines and chemokines (kaech et al., 2002; harty and badovinac, 2008) . among the various cd8 + t cell subsets, tc1 were shown to play a major role in the fight against several protozoan parasites (jordan and hunter, 2010) . the hallmark of this subset is the production of ifn-γ and tnf, and cytotoxic capacity (woodland and dutton, 2003) . the precise mechanism underlying cytotoxic t lymphocyte (ctl) killing of microbes is still poorly understood. ctls can exert cytotoxicity through various mechanisms: via exocytosis of lytic granula containing perforin, granzyme a/b, and/or granulysin; through the interaction between fasl and fas expressed on targets cells; via tnf; or via trail (trapani and smyth, 2002) . a study has also shown that reactivated memory cd8 + t cells efficiently killed listeria monocytogenes via a mechanism mediated by ccl3 and involving the induction of radical oxygen intermediates (narni-mancinelli et al., 2007) . direct killing of extracellular pathogen by ctls has also been described. for example, ctls can mediate killing of mycobacterium tuberculosis through the release of anti-bacterial products (stenger et al., 1998; canaday et al., 2001) . however, direct killing of schistosoma mansoni (ellner et al., 1982) and entamoeba histolytica (salata et al., 1987) is thought to be contact-dependent. ctls have also been reported to directly kill extracellular toxoplasma gondii (khan et al., 1990) . interestingly, killing in this case appeared to be antigen-specific. to date there is no evidence that cd8 + t cells can mediate protection against leishmania parasites through their cytotoxic activity. however, since ctls have been www.frontiersin.org observed in various mouse models and also in human patients, a possible protective role for leishmania-specific cytotoxic t cells should not be excluded. in addition to killing and releasing cytokines and chemokines, recent studies have ascribed a novel regulatory role for cd8 + t cells (sun et al., 2009; palmer et al., 2010; trandem et al., 2011) . regulatory cd8 + t cells represent a transient state of effector cd8 + t cells (trandem et al., 2011) , which is possibly induced by potent tcr stimulation, which promotes the production of the immunosuppressive cytokine il-10 (zhang and bevan, 2011) . not only do these cells produce il-10, but they are also excellent killers and produce normal to higher amounts of ifn-γ and tnf (sun et al., 2009; palmer et al., 2010; trandem et al., 2011) . the main function of these cells is thought to lie in the prevention of immunopathology during infection without affecting the kinetics of pathogen clearance. il-10-producing cd8 + t cells have also been observed in human patients infected with l. guyanensis (bourreau et al., 2007) and in patients suffering from post-kalaazar dermal leishmaniasis (pkdl; ganguly et al., 2008) . the role of regulatory cd8 + t cells in the immune response against parasitic infections is still unknown. the role of cd8 + t cells in the immunity to l. major has always been controversial. early studies in balb/c mice reported that cd8 + t cells were the main mediators of protection following cd4 + t cell depletion in mice infected with l. major (titus et al., 1987; hill et al., 1989; muller et al., 1991) . interestingly, depletion of cd4 + t cells was rendering susceptible balb/c mice resistant to l. major infection. the results obtained using the cd4 + t cell depletion model suggested that cd8 + t cells could potentially control l. major infection in mice. a few years later, experiments in β2-microgobulin-deficient mice contradicted these findings and revealed that cd8 + t cells were not essential in mediating protection in l. major-infected balb/c mice (wang et al., 1993) . moreover, a study in cd8 + t cell-deficient mice demonstrated that cd8 −/− mice were able to control l. major infection for at least 1 year, suggesting that cd8 + t cells were not required for long-lasting immunity (huber et al., 1998) . the contribution of cd8 + t cells in the control of primary l. major infection became less important also because of the strong evidence that th1 cells were the primary cells involved in mediating protection against cutaneous leishmaniasis (reiner and locksley, 1995; louis et al., 1998; sacks and noben-trauth, 2002) . several studies have demonstrated that th1 cells producing ifn-γ were essential in controlling l. major infection, and that failure to develop a th1 response resulted in susceptibility to the diseases. hence, a consensus was reached in that if a mouse generates th2 responses, this will lead to susceptibility; in contrast, th1 responses were successfully controlling infection without the help of cd8 + t cells. this paradigm was later challenged when new findings arose from a more natural model of infection, where 100 metacyclic promastigotes were inoculated intradermally in the ears of c57bl/6 mice. in this model, cd8 −/− and cd8 + t cell-depleted mice fail to control l. major infection, and cd8 + t cells were thought to be necessary for supporting th1 responses (belkaid et al., 2002) . the discrepancy between the findings in the low-and the highparasite dose model was clarified by another work that compared the requirements of cd8 + t cells in both systems (uzonna et al., 2004) . interestingly, in the low infection model cd8 + t cells producing ifn-γ were essential for modulating cd4 + t cell responses toward a th1 response. in contrast, c57bl/6 mice inoculated with a high l. major dose did not require cd8 + t cell help to generate protective th1 responses. the cd8 + t cell requirement for optimal ifn-γ production by th1 cells was also proposed in a high-dose l. major infection model in balb/c mice (herath et al., 2003) . moreover, cd8 + t cell-derived ifn-γ was reported to contribute to the induction of nitric oxide production in macrophages during experimental cutaneous leishmaniasis (stefani et al., 1994) . although the role of cd8 + t cells-derived ifn-γ has been clarified, little is known about the involvement of cytotoxic cd8 + t cells in cutaneous leishmaniasis. in a low-dose model of l. major infection, cd8 + t cell responses were shown not only to be protective, but also to mediate pathology (belkaid et al., 2002) . hence, it is possible that ctls may be involved in the ulceration of skin lesions through tissue disruption. this suggests that perhaps two types of cd8 + t cell effectors are generated during l. major infection: antigen-specific cd8 + t cells that produce ifn-γ but lack cytotoxic activity; and ctls that are potent killers but produce little to no ifn-γ and promote pathology. although the role of cd8 + t cells during primary immune responses is controversial, these cells appear to play a prominent role in protecting mice from a secondary challenge (muller et al., 1993 . indeed, antigen-specific cd8 + t cells were expanding up to 50-fold in the spleen and lymph nodes of reinfected balb/c mice . this expansion correlated with a substantial production of ifn-γ, which is thought to be essential for controlling leishmania infections. these observations have major implications for vaccine design. in summary, during experimental cutaneous leishmaniasis, cd8 + t cells are necessary to support protective th1 responses through ifn-γ production, but they are also involved in the development of immunopathology. further investigations are needed to better identify various subtypes of cd8 + t cells that arise during cutaneous leishmaniasis. in contrast to the cutaneous models, cd8 + t cells have always been thought to play a major role in experimental visceral leishmaniasis (vl). over 20 years ago, stern et al. (1988) demonstrated for the first time that cd8 + t cells significantly contribute to the formation of granulomas in the liver of l. donovani-infected mice. indeed, cd8 + t cell depletion resulted in impaired granuloma formation and exacerbation of liver disease. in agreement with these results, kaye et al. (1992) also reported a delayed onset and a decrease of the liver granulomatous response in non-obese, diabetic mice expressing transgenic i-e molecules, suggesting that antigen-specific cd8 + t cells are required for proper granuloma formation. cd8 + t cells appear to participate in controlling parasite growth in the spleen as well, since cd8 + t cell depletion during chronic vl significantly increased splenic parasite burden (stäger, unpublished) . this observation was underscored by the fact that adoptive transfer of antigen-specific cd8 + t cells during frontiers in immunology | microbial immunology chronic l. donovani infection resulted in 90% reduction in the splenic parasite burden (polley et al., 2006) . moreover, therapeutic vaccination aimed at reactivating cd8 + t cells during chronic vl ensued in the control of parasite growth in the spleen (joshi et al., 2009) . interestingly, cd8 + t cells do not only participate in primary responses to l. donovani, but are also the major mediators of resistance upon reinfection (stern et al., 1988) . indeed, protection was abrogated following cd8 + t cell but not cd4 + t cell depletion. a prominent function for cd8 + t cells was also described in l. infantum-infected mice. using an intradermal infection model, ahmed et al. (2003) demonstrated that cd8 + t cells contribute to parasite clearance in the skin of l. infantum-infected mice. another study also showed that cd8 + t cells purified from l. infantum-infected mice expressed ifn-γ and tnf, and displayed considerable cytotoxic activity against cells expressing leishmania antigens (tsagozis et al., 2003) . interestingly, killing of infected target cells was mediated by both the perforin and fas/fasl pathways (tsagozis et al., 2003) . the fas/fasl pathway has also been implicated in the defense against l. donovani (alexander et al., 2001) . indeed, gld and lpr mice, which lack a functional fas/fasl pathway, were shown to be more susceptible to l. donovani. additionally to the classical cytotoxic pathways, a novel counterregulatory function for a subset of cytotoxic cd8 + t cells has recently been proposed in the l. donovani infection model (martin et al., 2010) . in this study, cd3 + cd8 + cd40 + t cells are shown to suppress regulatory t cells via cd40/cd40l interaction during the early stages of infection in balb/c mice. cd40 signals through ras, pi3k, and protein kinase c, leading to the induction of granzyme and perforin, and ultimately to the killing of tregs. cd8 + t cells may not be merely participating in the primary immune response by secreting ifn-γ and possibly killing infected target cells and/or tregs, but they could also be involved in the recruitment of inflammatory cells and in the maintenance of granulomas. indeed, a study using l. infantum demonstrated that cd8 + t cells expressed rantes and mip-1α (tsagozis et al., 2003) , two chemokines that are involved in the recruitment of t cells at the inflammatory site and in the formation and maintenance of granulomas (mackay, 2001) . the authors proposed that cd8 + t cells may thus be involved in granuloma formation. this hypothesis is in agreement with the depletion data (stern et al., 1988) , showing that depletion of cd8 + t cells results in impaired granuloma formation and ultimately in disease exacerbation. despite the documented evidence that cd8 + t cells strongly participate in the immune response to l. donovani and l. infantum, our recent findings suggest that l. donovani induces defective antigen-specific cd8 + t cell responses (joshi et al., 2009 ). interestingly, mice infected with l. donovani generate cd8 + t cell responses with limited clonal expansion. the extension of the clonal expansion is thought to be correlated with the effectiveness in eliminating pathogens. it was calculated that a naïve cd8 + t cell may go through 19 cell divisions in the first week after pathogen inoculation (badovinac et al., 2007) . massive clonal expansions have not only been observed during viral infections, but also following the injection of irradiated plasmodium berghei sporozoites (sano et al., 2001) . during l. donovani infection, cd8 + t cells underwent at least 8-9 rounds of division, but failed to accumulate in the spleen (joshi et al., 2009) . moreover, only 10% of cd8 + t cells during clonal expansion expressed markers typically associated with end-differentiated effector cells, such as klrg1, unpublished) . the cause of this limited expansion is yet unknown and may depend on several factors. one of the possible explanations is limited antigen availability that may result from poor antigen-processing and presentation. processing of leishmania antigens is thought to be confined to a tap-independent, intraphagosomal pathway that is less efficient and requires higher amounts of antigen than the endoplasmic reticulum-based, tap-dependent cross-presentation pathway (bertholet et al., 2006) . furthermore, the major surface protease of leishmania, gp63, was shown to cleave epitopes within the parasitophorous vacuole, further reducing antigen availability (garcia et al., 1997) . hence, leishmania antigens may be poorly presented and this poor presentation may not be enough to induce and sustain a massive clonal expansion of antigen-specific cd8 + t cells. nonetheless, antigen may be suddenly available in large amounts later on during l. donovani infection, since cd8 + t cells undergo a second round of activation, become dysfunctional, and ultimately die by "exhaustion" (joshi et al., 2009) ; high antigen levels have been described as a cause of cd8 + t cell "exhaustion" during chronic viral infections (mueller and ahmed, 2009 ). further research is needed to clarify the mechanisms involved in cd8 + t cell exhaustion during chronic vl. in conclusion, cd8 + t cells are required to control parasite growth during experimental vl and reactivation of these responses results in a dramatic reduction in parasite burden. therefore, immune interventions that target cd8 + t cell responses may have great therapeutic potential against vl. the role of cd8 + t cells in human leishmaniasis patients is still unclear and seems to depend on the various species of parasites and the disease they cause. few studies have been conducted with human vl patients. however, most of the studies ascribe a protective role for cd8 + t cells, in agreement with results obtained from experimental models. indeed, the control of l. infantum infection was shown not only to be associated with ifn-γ-producing cd4 + t cells, but also with cd8 + t cells (mary et al., 1999) . interestingly, during active vl, cd8 + t cells are less responsive to stimulation and a greater percentage stains positive for annexin v compared to healthy controls (clarencio et al., 2009 ). these observations correlate very well with what we observed in mice experimentally infected with l. donovani, where cd8 + t cells became increasingly dysfunctional during chronic infection and died by exhaustion (joshi et al., 2009) . whether human cd8 + t cells also display signs of exhaustion during active vl still remains to be tested. another study investigating cd8 + t cell responses in patients infected with l. chagasi has revealed that the frequency of cd18 + cd45ro + cd8 + t cells is significantly decreased in the spleen of patients with active vl (clarencio et al., 2009 ). in contrast, cd18 + cd8 + t cells seem to be retained in the bone marrow of vl patients. cd18, or integrin β-2, is the β subunit of lfa-1 (cd11a), cd11b, cd11c, and cd11d. in humans, lack of cd18 www.frontiersin.org causes leukocyte adhesion deficiency, a disorder characterized by lack of leukocyte extravasation from blood into the tissue (bunting et al., 2002) . with exception of the fact that cd18 + cells appear in the granulomas of dogs with asymptomatic vl (sanchez et al., 2004) , very little is known about the function of cd18 + cd8 + t cells during vl and whether cells lacking cd18 expression have similar migratory capacity and effector functions to their cd18 + counterparts. not only is the frequency of cd18 + cd8 + t cells reduced in l. chagasi patients, but also, the level of circulating memory t cells is significantly decreased during active vl (hailu et al., 2005; clarencio et al., 2009 ). this observation is in agreement with our findings in the experimental model of vl, where the majority of the cd8 + t cells displayed an effector phenotype during chronic infection (joshi et al., 2009) . although cd8 + t cells positively correlate with cure of vl patients, one report suggested that these cells may contribute to the immunopathogenesis of pkdl (ganguly et al., 2008) . indeed patients suffering from pkdl showed a significant increase in the percentage of cd8 + t cells producing il-10, which disappeared after cure (ganguly et al., 2008) . il-10-secreting cd8 + t cells are thought to play a regulatory role in different viral infection models. this cd8 + t cell subset was shown to display great cytotoxicity and produce granzyme b, ifn-γ, and tnf (sun et al., 2009; palmer et al., 2010; trandem et al., 2011) . il-10 + cd8 t cells seem to represent a transient and reversible state of cd8 + effector t cell differentiation. its primary function is to balance pathogen clearance with bystander tissue damage (zhang and bevan, 2011) . interestingly, in viral model, this subset disappears after the infection is cleared. hence, it is possible that the il-10-producing cd8 + t cells in pkdl patients are actually killing parasites and protecting patients from tissue damage, rather than suppressing protective responses. further studies are needed in order to define the nature of these cells. cd8 + t cells also actively participate in the immune response to cutaneous infections in human. as observed in the low-dose model in mice (belkaid et al., 2002; uzonna et al., 2004) , l. major also induces th1 and cd8 + t cells in human patients and both responses are associated with disease resolution (nateghi rostami et al., 2010) . cd8 + t cells were not only observed in large numbers in the lesions of l. major patients during the acute phase, but also during the healing process (da-cruz et al., 1994 gaafar et al., 1999) . the exact role of cd8 + t cells in l. major infections in humans is not yet known. a major correlate of protection appears to be the high amounts of ifn-γ produced by cd8 + t cells after restimulation (nateghi rostami et al., 2010) . in vitro studies have also demonstrated that leishmania-specific ctls are generated upon co-culturing human naïve t cells with antigens from l. amazonensis promastigotes and il-12 (russo et al., 1999) , or with l. major parasites (da . moreover, increased granzyme b activity was also found in patients with an active infection and was associated with a good prognosis (bousoffara et al., 2004) . in this study, in vitro cytotoxicity by peripheral blood lymphocytes on l. major-infected macrophages appeared to be mediated by granzyme b, suggesting that ctl activity may be involved in controlling parasite growth. it is possible, though, that the cytotoxic activity not only contributes to disease clearance, but also to the development of skin ulceration, as observed in l. major-infected mice (belkaid et al., 2002) . a strong cd8 + t cell expansion has also been observed in l. mexicana patients during the healing process (salaiza-suazo et al., 1999) . interestingly, lesions of patients with localized cutaneous leishmaniasis (lcl) harbor a higher number of cd8 + t cells compared to patients with diffuse cutaneous leishmaniasis (dcl; hernandez-ruiz et al., 2010) . as already observed in vl patients, cd8 + t cells in dcl patients, unlike lcl patients, show a reduced capacity to respond to antigen-specific stimulation during active infection. in fact, these cells displayed low cytotoxicity and only produced little ifn-γ upon stimulation, therefore showing typical signs of functional exhaustion (hernandez-ruiz et al., 2010) . strikingly, effector functions could be restored in vitro after stimulation with tlr2 agonists, highlighting the potential therapeutic benefit of the revival of cd8 + t cell functions in dcl patients. in contrast to the cutaneous and visceral forms of leishmaniasis -where cd8 + t cells seem to correlate with cure and contribute to the immune response -in mucocutaneous infections (ml) cd8 + t cells seem to be implicated in the pathogenesis of the disease. indeed, high numbers of cytotoxic cd8 + t cells were observed in ml patients (barral-netto et al., 1995; brodskyn et al., 1997) . moreover, the recruitment of granzyme a + cd8 + t cells is associated with lesion progression (faria et al., 2009) , suggesting that ctls may contribute to immunopathology. the development of ml is not only associated with the presence of ctl, but also with a high frequency of activated cd4 + t cells, an extreme ifnγ and tnf production, and a reduced control of inflammation due to low expression of the il-10 receptor (gaze et al., 2006; faria et al., 2009) . furthermore, il-17-secreting cd4 + and cd8 + t cells were also found in ml patients (boaventura et al., 2010) . consequently, neutrophils, which are typically recruited during a th17-mediated inflammatory response, were also detected in necrotic and perinecrotic areas (boaventura et al., 2010) . this suggests that neutrophils, together with ctls, may be involved in tissue injury and in the development of immunopathology. taken together, the literature shows that cd8 + t cells actively participate in the fight against most leishmania infections in humans and their presence correlates with cure. in contrast, cd8 + t cells in ml patients contribute to disease exacerbation. vaccination of humans with heat-killed leishmania or recombinant parasite proteins has so far failed to induce long-term immunity and only recovery from natural or experimental infection has provided proper protection. several trials have analyzed the protective effect of autoclaved l. major plus bacillus-calmette-guérin (bcg) versus bcg alone assessing the cumulative incidence of cutaneous leishmaniasis caused by l. tropica (sharifi et al., 1998) or l. major (momeni et al., 1999) , or of vl (khalil et al., 2000) caused by l. donovani. although no trial showed a significant effect on disease incidence, the vaccination induced skin test conversion and provided limited protection. additionally, a study showed that immunization of colombian soldiers with three doses of l. amazonensis alone was non-protective (velez et al., 2005) . in the human disease, there is evidence that mixed t helper cytokine profiles are present, while healing and protection against reinfection are associated with dominant th1 and cd8 + t cells. these findings suggest that it is the cytokine balance that activates or suppresses activation of macrophages harboring leishmania parasites. this, in turn, determines the outcome of the infection. thus treatments or antigen/adjuvant formulations that can alter the type of t helper response may change the course of disease (da-cruz et al., 2002; rogers and titus, 2004; mohajery, 2007) . for this purpose, different vaccination strategies have been examined in animal models including leishmanization (modabber, 1990) , killed parasite (grimaldi, 1995) , live attenuated parasite (titus et al., 1995) , and subsequently, subunit vaccines composed of recombinant or native proteins from different stages of the parasite's life cycle, and dna vaccines (skeiky et al., 1998; webb et al., 1998; stager et al., 2000; bottrel et al., 2001; campos-neto et al., 2001; rafati et al., 2001; coler et al., 2002) . the latter two strategies encompass candidates such as gp63, gp46, lack, cpb, cpa, kmp11, lmsti1, tsa, leif, haspb1, and lpg, and have shown promising results in murine models. nonetheless, only leish111f (a recombinant fusion protein of lmsti1, tsa, and leif) progressed through phase i and ii clinical trials (llanos-cuentas et al., 2010; chakravarty et al., 2011) . nowadays, it is clear that cd8 + t cells play an important role in the mechanisms for cure of and resistance to leishmania infection, either by production of ifn-γ and activation of macrophages, or by direct killing of parasitized macrophages, or a combination of both effects. cd8 + t cells have been associated with protection against leishmania reinfection in murine models; however, the induction of these t cell subsets in humans seems to be also related to the healing process. today, there are several reports about different leishmanial antigens eliciting ctl responses such as p8, gp46 , haspb1 (stager et al., 2000) , kmp11 (basu et al., 2007) , cpb (rafati et al., 2002) , nucleosomal histones (iborra et al., 2004) , lmacin (farajnia et al., 2005) , lmsti1, and tsa (coler et al., 2002) . the essential point to be considered in vaccine design for a heterogeneous population, such as that of humans, is the hla polymorphism. effective vaccination against a complex parasitic infection such as leishmania would require a multivalent vaccine composed of several antigens to enhance the possibility of covering a good number of mhc types. this is possible either through recombinant fusion proteins encompassing the whole antigen or through vaccines composed of peptides from different antigens (campos-neto et al., 2001; rafati et al., 2001; mendez et al., 2002) . the latter strategy, called polytope vaccine, is finding its way in vaccinology because of its extraordinary properties, especially the ability to direct the immune response toward the induction of ctls (sbai et al., 2001; schirmbeck et al., 2003; robinson and amara, 2005) . as ctl responses play a pivotal role in defense against viruses and tumor cells, polytope vaccines have found their way in these fields but there is still no report on leishmaniasis even it has been shown that ctls could be very important in protection and long-lasting resistance to infection. recently, we took advantage of the potential of immunoinformatics tools to screen for l. major epitopes that could be presented in hla a2, which is the most prevalent hla supertype in the iranian population. in vitro stimulation to recall memory cd8 + t cells from leishmania-infected individuals and intracellular cytokine assays for ifn-γ-producing cells confirmed that hla a2 positive individuals that recovered from an l. major infection successfully generated cd8 + t cell responses against peptides derived from lmsti1 and lpg-3 (seyed et al., 2011) . furthermore, walden and co-workers have mapped the t cell epitopes from kinetoplastid membrane protein of l. major (kmp11) via classical mapping for different human hla class i alleles (basu et al., 2007) . gazzinelli and co-workers have studied cd8 + t cell responses against the leishmania a2 antigen and mapped the cd8 + t cell epitopes in balb/c mice (resende et al., 2008) . laouini and co-workers (guerfali et al., 2009 ) and dumonteil and co-workers (dumonteil, 2009 ) started genomewide screenings for novel epitopes. using a combination of t cell epitope prediction tools, they successfully validated such epitopes in both balb/c and c57bl/6 mice. although understudied, cd8 + t cells appear to play an important role in the immune response to most leishmania infections. pilot studies in the murine model of vl have also demonstrated that adoptive transfer of antigen-specific cd8 + t cells (polley et al., 2006) or reactivation of cd8 + t cell responses through a therapeutic vaccine (joshi et al., 2009) results in the control of parasite growth. a better understanding of the mode of activation, the specificity, and effector functions of the various cd8 + t cell subsets generated during leishmania infections could ameliorate the design of vaccines and of novel therapeutic interventions. for the early control of parasite burden in the liver of leishmania donovani-infected mice. eur. j. immunol. 31, 1199-1210. badovinac, v. p., haring, j. s., and harty, j. t. (2007) . initial t cell receptor transgenic cell precursor frequency dictates critical aspects of the cd8(+) t cell response to infection. immunity 26, 827-841. barral-netto, m., barral, a., brodskyn, c., carvalho, e. m., and reed, s. g. (1995) . cytotoxicity in human mucosal and cutaneous leishmaniasis. parasite immunol. 17, 21-28. basu, r., roy, s., and walden, p. (2007). hla class i-restricted t cell epitopes of the kinetoplastid membrane protein-11 presented by leishmania donovani-infected human macrophages. j. infect. dis. 195, 1373 -1380 s., lira, r., caler, e., bertholet, s., udey, m. c., and . cd8+ t cells are required for primary immunity in c57bl/6 mice following low-dose, intradermal challenge with leishmania major. j. immunol. 168, 3992-4000. www.frontiersin.org intradermal infection model for pathogenesis and vaccine studies of murine visceral leishmaniasis leishmania antigens are presented to cd8+ t cells by a transporter associated with antigen processingindependent pathway in vitro and in vivo human mucosal leishmaniasis: neutrophils infiltrate areas of tissue damage that express high levels of th17-related cytokines flow cytometric determination of cellular sources and frequencies of key cytokine-producing lymphocytes directed against recombinant lack and soluble leishmania antigen in human cutaneous leishmaniasis il-10 producing cd8+ t cells in human infection with leishmania guyanensis analysis of granzyme b activity as a surrogate marker of leishmania-specific cellmediated cytotoxicity in zoonotic cutaneous leishmaniasis parasite-driven in vitro human lymphocyte cytotoxicity against autologous infected macrophages from mucosal leishmaniasis leukocyte adhesion deficiency syndromes: adhesion and tethering defects involving beta 2 integrins and selectin ligands protection against cutaneous leishmaniasis induced by recombinant antigens in murine and nonhuman primate models of the human disease cd4(+) and cd8(+) t cells kill intracellular mycobacterium tuberculosis by a perforin and fas/fas ligand-independent mechanism a clinical trial to evaluate the safety and immunogenicity of the leish-f1+mpl-se vaccine for use in the prevention of visceral leishmaniasis could the lower frequency of cd8+cd18+cd45ro+ lymphocytes be biomarkers of human vl? immunization with a polyprotein vaccine consisting of the t-cell antigens thiol-specific antioxidant, leishmania major stress-inducible protein 1, and leishmania elongation initiation factor protects against leishmaniasis perforin and gamma interferon are critical cd8+ tcell-mediated responses in vaccineinduced immunity against leishmania amazonensis infection leishmania major infection in mice primes for specific major histocompatibility complex class i-restricted cd8+ cytotoxic t cell responses flow cytometric analysis of cellular infiltrate from american tegumentary leishmaniasis lesions t-cell-mediated immune responses in patients with cutaneous or mucosal leishmaniasis: long-term evaluation after therapy leishmania-reactive cd4+ and cd8+ t cells associated with cure of human cutaneous leishmaniasis vaccine development against trypanosoma cruzi and leishmania species in the postgenomic era destruction of the multicellular parasite schistosoma mansoni by t lymphocytes mononuclear cells from patients recovered from cutaneous leishmaniasis respond to leishmania major amastigote class i nuclease with a predominant th1-like response recruitment of cd8(+) t cells expressing granzyme a is associated with lesion progression in human cutaneous leishmaniasis characterization of the local and systemic immune responses in patients with cutaneous leishmaniasis due to leishmania major increased levels of interleukin-10 and igg3 are hallmarks of indian post-kala-azar dermal leishmaniasis epitope cleavage by leishmania endopeptidase(s) limits the efficiency of the exogenous pathway of major histocompatibility complex class i-associated antigen presentation mucosal leishmaniasis patients display an activated inflammatory tcell phenotype associated with a nonbalanced monocyte population meetings on vaccine studies towards the control of leishmaniasis an in silico immunological approach for prediction of cd8+ t cell epitopes of leishmania major proteins in susceptible balb/c and resistant c57bl/6 murine models of infection t cell subset and cytokine profiles in human visceral leishmaniasis during active and asymptomatic or sub-clinical infection with leishmania donovani shaping and reshaping cd8+ tcell memory cross-talk between cd8(+) and cd4(+) t cells in experimental cutaneous leishmaniasis: cd8(+) t cells are required for optimal ifngamma production by cd4(+) t cells cd8 cells of patients with diffuse cutaneous leishmaniasis display functional exhaustion: the latter is reversed, in vitro, by tlr2 agonists elimination of cd4+ suppressor t cells from susceptible balb/c mice releases cd8+ t lymphocytes to mediate protective immunity against leishmania effective and longlasting immunity against the parasite leishmania major in cd8-deficient mice vaccination with a plasmid dna cocktail encoding the nucleosomal histones of leishmania confers protection against murine cutaneous leishmaniosis regulation of cd8+ t cell responses to infection with parasitic protozoa b7-h1 blockade increases survival of dysfunctional cd8(+) t cells and confers protection against leishmania donovani infections effector and memory t-cell differentiation: implications for vaccine development altered course of visceral leishmaniasis in mice expressing transgenic i-e molecules autoclaved leishmania major vaccine for prevention of visceral leishmaniasis: a randomised, double-blind, bcgcontrolled trial in sudan induction of antigenspecific human cytotoxic t cells by toxoplasma gondii presentation via the class i pathway by leishmania amazonensisinfected macrophages of an endogenous leishmanial antigen to cd8+ t cells a clinical trial to evaluate the safety and immunogenicity of the leish-f1+mpl-se vaccine when used in combination with sodium stibogluconate for the treatment of mucosal leishmaniasis regulation of protective immunity against leishmania major in mice chemokines: immunology's high impact factors cd40 signaling in cd8 + cd40+ t cells turns on contra-t regulatory cell functions control of leishmania infantum infection is associated with cd8(+) and gamma interferon-and interleukin-5-producing cd4(+) antigen-specific t cells optimization of dna vaccination against cutaneous leishmaniasis development of vaccines against leishmaniasis tc1 cells percentage in patients with cutaneous leishmaniasis before and after treatment with glucantime a randomised, doubleblind, controlled trial of a killed l. major vaccine plus bcg against zoonotic cutaneous leishmaniasis in iran high antigen levels are the cause of t cell exhaustion during chronic viral infection gamma interferon response in secondary leishmania major infection: role of cd8+ t cells expansion of gamma interferon-producing cd8+ t cells following secondary infection of mice immune to leishmania major establishment of resistance to leishmania major infection in susceptible balb/c mice requires parasitespecific cd8+ t cells memory cd8+ t cells mediate antibacterial immunity via ccl3 activation of tnf/roi+ phagocytes cd8+ t cells as a source of ifn-gamma production in human cutaneous leishmaniasis ifngamma-producing, virusspecific cd8+ effector cells acquire the ability to produce il-10 as a result of entry into the infected lung environment adoptive immunotherapy against experimental visceral leishmaniasis with cd8+ t cells requires the presence of cognate antigen recombinant cysteine proteinases-based vaccines against leishmania major in balb/c mice: the partial protection relies on interferon gamma producing cd8(+) t lymphocyte activation a protective cocktail vaccine against murine cutaneous leishmaniasis with dna encoding cysteine proteinases of leishmania major the regulation of immunity to leishmania major epitope mapping and protective immunity elicited by adenovirus expressing the leishmania amastigote specific a2 antigen: correlation with ifn-gamma and cytolytic activity by cd8+ t cells t cell vaccines for microbial infections characterization of the early cellular immune response to leishmania major using peripheral blood mononuclear cells from leishmania-naive humans leishmania: naive human t cells sensitized with promastigote antigen and il-12 develop into potent th1 and cd8(+) cytotoxic effectors the immunology of susceptibility and resistance to leishmania major in mice treatment of two patients with diffuse cutaneous leishmaniasis caused by leishmania mexicana modifies the immunohistological profile but not the disease outcome the interaction of human t-lymphocytes and entamoeba histolytica: killing of virulent amoebae by lectin-dependent lymphocytes organ-specific immunity in canine visceral leishmaniasis: analysis of symptomatic and asymptomatic dogs naturally infected with leishmania chagasi swift development of protective effector functions in naive cd8(+) t cells against malaria liver stages use of t cell epitopes for vaccine development enhanced priming of multispecific, murine cd8+ t cell responses by dna vaccines expressing stress protein-binding polytope peptides in silico analysis of six known leishmania major antigens and in vitro evaluation of specific epitopes eliciting hla-a2 restricted cd8 t cell response randomised vaccine trial of single dose of killed leishmania major plus bcg against anthroponotic cutaneous leishmaniasis in leif: a recombinant leishmania protein that induces an il-12-mediated th1 cytokine profile immunization with a recombinant stage-regulated surface protein from leishmania donovani induces protection against visceral leishmaniasis leishmania major-specific cd8+ t cells are inducers and targets of nitric oxide produced by parasitized macrophages an antimicrobial activity of cytolytic t cells mediated by granulysin role of l3t4+ and lyt-2+ cells in experimental visceral leishmaniasis effector t cells control lung inflammation during acute influenza virus infection by producing il-10 development of a safe live leishmania vaccine line by gene replacement involvement of specific lyt-2+ t cells in the immunological control of experimentally induced murine cutaneous leishmaniasis highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirusinduced encephalitis functional significance of the perforin/granzyme cell death pathway cd8(+) t cells with parasite-specific cytotoxic activity and a tc1 profile of cytokine and chemokine secretion develop in experimental visceral leishmaniasis low dose leishmania major promotes a transient t helper cell type 2 response that is downregulated by interferon gammaproducing cd8+ t cells failure of a killed leishmania amazonensis vaccine against american cutaneous leishmaniasis in colombia targeted activation of cd8 cells and infection of beta 2-microglobulin-deficient mice fail to confirm a primary protective role for cd8 cells in experimental leishmaniasis human and murine immune responses to a novel leishmania major recombinant protein encoded by members of a multicopy gene family apoptotic vesicles crossprime cd8 t cells and protect against tuberculosis uptake of leishmania major by dendritic cells is mediated by fcgamma receptors and facilitates acquisition of protective immunity heterogeneity of cd4(+) and cd8(+) t cells cd8(+) t cells: foot soldiers of the immune system this work was funded by the start up funds from the inrs-iaf to ss, and the national science foundation of iran (grant no. 87020176) to sima rafati. we would like to thank guillermo arango duque for editing the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-026866-0hlre9i6 authors: perruzza, lisa; jaconi, stefano; lombardo, gloria; pinna, debora; strati, francesco; morone, diego; seehusen, frauke; hu, yue; bajoria, sakshi; xiong, jian; kumru, ozan selahattin; joshi, sangeeta bagai; volkin, david bernard; piantanida, renato; benigni, fabio; grassi, fabio; corti, davide; pizzuto, matteo samuele title: prophylactic activity of orally administered flid-reactive monoclonal siga against campylobacter infection date: 2020-06-09 journal: front immunol doi: 10.3389/fimmu.2020.01011 sha: doc_id: 26866 cord_uid: 0hlre9i6 campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. secretory immunoglobulin a (siga) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. in this study, we describe the prophylactic activity of orally delivered recombinant siga generated from two human monoclonal antibodies (caa1 and ccg4) isolated for their reactivity against the flagellar-capping protein flid, which is essential for bacteria motility and highly conserved across campylobacter species associated with severe enteritis. in an immunocompetent weaned mouse model, a single oral administration of flid-reactive siga caa1 or ccg4 at 2 h before infection significantly enhances campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration in the cecum lamina propria. our data indicate that the prophylactic activity of caa1 and ccg4 is not only dependent on the specificity to flid but also on the use of the siga format, as the immunoglobulin g (igg) versions of the same antibodies did not confer a comparable protective effect. our work emphasizes the potential of flid as a target for the development of vaccines and supports the concept that orally administered flid-reactive siga can be developed to prevent or mitigate the severity of campylobacter infections as well as the development of post-infection syndromes. campylobacter infection is one of the most common causes of bacterial gastroenteritis worldwide and a major global health threat due to the rapid development of antibiotic resistance. currently, there are no vaccines approved to prevent campylobacteriosis, and rehydration is the main form of therapy. secretory immunoglobulin a (siga) is the main antibody class found in mucous secretions, including human milk, and serves as the first line of defense for the gastrointestinal epithelium against enteric pathogens. in this study, we describe the prophylactic activity of orally delivered recombinant siga generated from two human monoclonal antibodies (caa1 and ccg4) isolated for their reactivity against the flagellar-capping protein flid, which is essential for bacteria motility and highly conserved across campylobacter species associated with severe enteritis. in an immunocompetent weaned mouse model, a single oral administration of flid-reactive siga caa1 or ccg4 at 2 h before infection significantly enhances campylobacter clearance at early stages post-infection, reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration in the cecum lamina propria. our data indicate that the prophylactic activity of caa1 and ccg4 is not only dependent on the specificity to flid but also on the use of the siga format, as the immunoglobulin g (igg) versions of the same antibodies did not confer a comparable protective effect. our work emphasizes the potential of flid as a target for the development of vaccines and supports the concept that orally administered flid-reactive siga can be developed to prevent or mitigate the severity of campylobacter infections as well as the development of post-infection syndromes. keywords: secretory iga, monoclonal antibodies, prophylaxis, campylobacter, flagellar-capping protein, flid campylobacter is an established cause of diarrhea worldwide and has recently been added to the who list of bacteria whose antibiotic resistance might pose a global threat to human health (1) . campylobacter's epidemiology differs between high-income countries, where infections are sporadic, and low-and middle-income countries, in which the infection is common in early childhood and a major cause of lifethreatening acute watery diarrhea in infants (2) . considered as a leading zoonosis, campylobacter infection is mainly associated with the consumption of contaminated undercooked animal meat (poultry being the primary bacteria reservoir), water, or unpasteurized milk (3). campylobacter jejuni and campylobacter coli are the major causes of campylobacter enteritis in humans, and despite the clinical relevance of other emerging species (4), they will unlikely be eclipsed in terms of prevalence and magnitude of the disease. campylobacteriosis typically results in an acute, gastrointestinal illness characterized by watery or bloody diarrhea, fever, weight loss, and cramps that last on average 6 days (3, 5). although considered a self-limiting disease, the severe dehydration associated with campylobacter enteritis represents a significant cause of death among newborns and children particularly in developing countries (6) . furthermore, c. jejuni infections has been consistently linked with the onset of autoimmune conditions such as guillain-barré syndrome (gbs) (7, 8) and inflammatory bowel disease (ibd) (9) . despite a yet incomplete understanding of campylobacter pathogenesis, flagellum-mediated motility is widely presumed pivotal for virulence and pathogenicity, as demonstrated in both experimental animal models and in human healthy volunteer studies (10) . nevertheless, flagellin (flaa), the major constituent of the flagellum, is not highly conserved even within the same c. jejuni species, and its heavy glycosylation pattern varies greatly depending on the strain and growth phase (11, 12) . in addition, a recombinant non-glycosylated form of c. jejuni flagellin was shown to be poorly immunogenic in clinical trials (2) . moreover, the possibility to use c. jejuni as vaccines has been limited by the risk of gbs development associated with ganglioside mimicry of bacterial lipo-oligosaccharide (los) (2) . due to these shortcomings, there are currently no vaccines approved to prevent campylobacter infection. rehydration is the main form of therapy, and albeit antibiotics have been shown to be beneficial in severe infections, they are often not recommended due to the rapid development of antibiotic resistance (1) . despite recovering from infection, continuous exposure of infants in low-income countries to intestinal pathogens, including campylobacter, has been linked to environmental enteropathy (ee)/environmental enteric dysfunction (eed), a subclinical chronic inflammation of the small intestine. this inflammation is associated with malabsorption of nutrients, growth faltering, impaired cognitive development, changes in microbiota, and reduced responsiveness to oral vaccination (13) . secretory immunoglobulin a (siga) represents the most abundant class of antibodies produced in humans and serves as the first line of defense at the level of intestinal mucosa against enteric toxins and pathogens (14) . typically, a siga consists of two monomeric iga linked tail to tail by disulfide bonds via a 15-kda protein called joining (j) chain and further complexed with a heavily glycosylated secretory component (sc) (15) . while iga monomers and the j chain are synthesized, assembled, and secreted by plasma cells in the lamina propria, the sc consists of the ectodomain of the polymeric immunoglobulin receptor (pigr) that remains bound to polymeric iga (piga) following transcytosis across the enterocyte (16) . the sc protects the antibody from proteolytic degradation, confers innate recognition functions, and contributes to appropriate tissue localization by anchoring the immunoglobulin to the mucus lining the epithelial surface (17, 18) . siga acts primarily by preventing adhesion of pathogens to epithelial cells through receptor blockade, steric hindrance, and/or immune exclusion, eventually facilitating their clearance from the intestinal lumen via peristaltic and mucociliary activities (14, 19) . furthermore, antigen-bound siga can be transported back into peyer's patches (pps) subepithelial dome (sed) by microfold (m) cells in a process known as reverse transcytosis, which promotes migration and activation of follicular dcs, thus conditioning the inflammatory response associated to infection by enteric pathogens (14, 20) . siga is also the main antibody class found in mucous secretions, including human milk, in which the ig portion of the molecule is produced by mammary gland plasma cells that originate in the small intestine (entero-mammary pathway) (21) . the provision of siga by breastfeeding has been reported to be critical for the protection against enteric pathogens especially in early infancy, when the contribution of endogenous production is limited (22) (23) (24) . in this study, an immunocompetent mouse model of campylobacter infection was used to evaluated the prophylactic activity of orally delivered recombinant siga generated from human monoclonal antibodies isolated and selected for their reactivity against the flagellar-capping protein flid, which is pivotal for bacteria motility (25), and highly conserved across campylobacter species frequently associated with severe neonatal enteritis (26) . we demonstrated that, regardless of the iga isotype, prophylactically administered flid-reactive siga can enhance campylobacter clearance at early stages post-infection, dramatically reducing the levels of inflammation markers associated with epithelial damage and polymorphonuclear (pmn) cells infiltration. our data indicate that the prophylactic activity of these antibodies is not only dependent on the specificity to flid but also on features strictly associated with the siga format as the igg versions of the same antibodies did not produce a comparable beneficial effect. flid amino acid sequences from c. jejuni (n = 38) and c. coli (n = 17) isolates were retrieved from genbank and aligned using mega software to obtain a consensus sequence. condon optimization and synthesis of the corresponding nucleotide sequence was outsourced (genscript), and the construct was subcloned in pet21a in frame with the c-term his tag. the plasmid was transformed to escherichia coli rosetta strain. gene expression was induced by the addition of 1 mm isopropyl-beta-d-thiogalactopyranoside (iptg) to log-phase grown bacteria. after 5 h of induction at 37 • c, bacteria were collected by centrifugation and stored at −20 • c for 16 h before being resuspended in sonication buffer (50 mm nah 2 po 4 , 300 mm nacl, 10 mm imidazole, and ph 7.4) and being sonicated 4 × 30 s in ice. lysed bacteria were spun (14,000 × g, 1 h, and 4 • c), and the supernatants were filtered and then loaded on a nickel column (hitrap chelating hp, ge healthcare). column was washed with washing buffer (50 mm nah 2 po 4 , 300 mm nacl, 10 mm imidazole, and ph 7.4) followed by washing buffer + 0.1% triton-x114 and washing buffer again. bound protein was eluted from the column with elution buffer (50 mm nah 2 po 4 , 300 mm nacl, 500 mm imidazole, and ph 7.4) and dialyzed for 16 h at 4 • c in dulbecco's phosphate-buffered saline (dpbs). protein size was measure on size exclusion column (hiload 16/600 superdex 75 pg; ge healthcare), and the concentration was measured before storage at −20 • c. flid-reactive monoclonal antibodies caa1 and ccg4 were isolated, as previously described (27, 28) , from iga + and igg + memory b cells derived from tonsillar donors who had given written informed consent, following approval by the cantonal ethical committee of cantone ticino, switzerland (ce3536 2019-02061). the sequences for the variable regions of the mabs were obtained from memory b cell clone messenger rnas (mrnas) following reverse transcription pcr (rt-pcr) and sequencing. the vh region of each mab was cloned into a plasmid encoding iga1, iga2 allotype m(2) (29), or igg1 heavy chains. the constructs were transiently transfected in expi293 cells together with those for the corresponding light chains and, in the case of siga, also with those encoding for the joining chain (j) and the ectodomain of pigr (sc) (29) . after 48 h, cells were supplemented with supplement medium and further incubated for 5 days under shaking. next, cells were spun down, and the supernatant was filtered before being purified using captureselect tm iga affinity matrix (thermo fisher). siga assembly was appraised by ultrahigh performance liquid (uhpl) chromatography using acquity beh sec guard column (waters) and by denaturing nonreducing gel using siga purified from human milk (mp biomedicals) as positive control. the purified immunoglobulins were quantified with pierce tm rapid gold bca protein assay kit (life technologies) and then aliquoted and store at −80 • c. n-glycan oligosaccharide analysis was performed as previously described (30) . briefly, a glycoworks rapifluor-ms n-glycan kit (waters corporation, milford, ma) was used to identify and quantify n-linked glycans following the manufacturer's instructions. fluor-ms n-glycan analysis was performed using an agilent 1,260 infinity ii hplc system equipped with a 1,260 fld detector (agilent, santa clara, ca) and an agilent 6,230 electrospray ionization time-of-flight mass spectrometer (agilent, santa clara, ca). a hilic advancebio glycan mapping column (120 å, 2.1 × 150 mm, 2.7 µm), operated at 45 • c, was used to separate various n-glycans. fifty microliters of prepared samples was injected into liquid chromatographymass spectrometry (lc-ms) system, with a flowrate of 0.6 mg/ml and a gradient run time of 55 min. fluorescence was obtained using excitation and emission wavelengths of 265 and 425 nm, respectively. ms was acquired simultaneously from 400 to 2,000 m/z at a constant scan rate of one spectrum per second. n-glycans were assigned based on m/z values using the n-glycan glycobase database, and nglycan quantification was calculated on integration of the fluorescence chromatogram. total carbohydrate analysis was performed as previously described (30) . briefly, a glycoprotein carbohydrate estimation kit (thermo-fisher #23260) was used to determine the total carbohydrate content (both n-and o-linked oligosaccharides) in mab samples as a percentage of total protein mass. lysozyme, bovine serum albumin (bsa), ovalbumin, apo-transferrin, fetuin, and α1-acid glycoprotein were used as glycoprotein standards to construct a standard curve. size exclusion chromatography (sec) was performed as previously described (30) . briefly, a shimadzu prominence ultrafast liquid chromatography hplc system equipped with a diode array detector (with absorbance detection at 214 nm) was equilibrated at 0.5 ml/min flowrate in 0.2 m sodium phosphate buffer, ph 6.8 for at least 2 h. ten microliters of each mab (10 µg total protein) was injected and separated by a tosoh tskgel g4000swxl column (8 µm particle size, 7.8 mm id × 30 cm) with the corresponding guard column operated at ambient temperature (tosoh biosciences) using a 30-min run time. gel filtration molecular weight standards (bio-rad, hercules, ca) were injected before and after the mab samples to ensure integrity of the column and hplc system. potential presence of larger aggregates was determined by running mab samples with and without the sec column (i.e., protein percentage recovery). data were analyzed using lc-solution software (shimadzu, kyoto, japan). sedimentation velocity analytical ultracentrifugation (sv-auc) was performed as described previously (30) using a proteome lab xl-i (beckman coulter) analytical ultracentrifuge equipped with a scanning ultraviolet-visible optical system. all experiments were performed at 20 • c after at least 1 h of equilibration after the rotor reached 20 • c. sv-auc was performed at a rotor speed of 40,000 rpm and with detection at 280 nm. the data were analyzed using sedfit software (dr. peter schuck, nih). intrinsic tryptophan fluorescence spectroscopy measurements as a function of temperature and polyethylene glycol (peg) relative solubility measurements were both performed as described previously (30) . for fluorescence spectroscopy measurements, 20 µl of each mab sample was diluted to 0.2 mg/ml in pbs, ph 7.2 and loaded into a 384-well plate (hard-shell 384-well pcr plates) and overlaid with 2 µl of silicon oil (thermo fisher scientific, waltham, ma). samples were excited at 295 nm, and the emission spectra were recorded from 300 to 450 nm with an integration time of 100 ms using a fluorescence plate reader equipped with a charge-coupled device (ccd) detector (fluorescence innovations, minneapolis, mn). temperature ramps were programmed from 10 to 100 • c with an increment of 2.5 • c per step. the mean center of spectra mass peak algorithm was used to analyze the data to determine the shift in fluorescence peak position as a function of temperature. with respect to relative solubility measurements, various concentrations of peg 10,000 solutions ranging from 0 to 25% w/v were prepared with a final mab concentration of 0.2 mg/ml. samples were incubated overnight at room temperature in the dark in 384-well plates with 30 µl in each well. the next day, the plates were centrifuged for 15 min at ∼1,200×g, and the supernatant was transferred to a clean 384-well plate. relative protein concentration in each well was then measured at 214 nm using a spectramax m5 plate reader (molecular devices). thermal melting temperature values (t m ) and % peg midpoint calculations were performed using origin v 9.1 software (originlab, northampton, ma). quantification of recombinant siga and igg by elisa was performed by coating 96-well plates with goat anti-human iga or igg (southernbiotech). two-fold serial dilutions of the mabs in duplicates were incubated for 1 h at room temperature. in the case of siga, detection was performed using human pigr biotinylated antibody biotinylated anti-human sc antibody (r&d system) followed by incubation with streptavidin-ap, while goat anti-human igg-ap (southernbiotech) was used for igg. siga purified from human milk (mp biomedicals) and rituximab (mabthera) were used as quantification standards for siga and igg, respectively. binding of caa1, ccg4 siga, and igg counterparts to the recombinant antigen was measured by elisa using flidcoated 96-well-plates following the same detection protocol described above. epitope binning was tested by biolayer interferometry (bli) using octet red96 (fortebio) immobilizing recombinant flid antigen (5 µg/ml) onto aps biosensor (pall fortebio) for 7 min. after incubation with blocking buffer (bb), the sensor was incubated with caa1 siga2 (7.5 µg/ml) for 7 min before being further incubated with ccg4 siga2, caa1 siga2, or bb for additional 7 min. binding of siga2 to flid antigen in denaturing and reducing conditions was tested by western blot. briefly, 5 µg of recombinant flid was loaded on sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) nupage 12% bis-tris protein gel (thermo fisher scientific) in the presence of a reducing agent. blotting was performed using iblot system (invitrogen), and the membrane was blocked with 5% milk (biorad) in tris-buffered saline with tween 20 (tbst) for 1 h at room temperature (rt). the mabs were biotinylated by incubation with 20 mm biotin using the ez-link nhs-peo solid phase biotinylation kit followed by desalting with zeba spin desalting columns (thermo scientific). the membrane was incubated with a 1% blocking grade blocker (biorad) in tbst containing 20 µg/ml of biotinylated mabs followed by washes in tbst and by incubation with streptavidin-horseradish peroxidase (hrp). detection was performed using ecl detection kit (ge healthcare) on fusion fx7 (witec ag). sensitivity to igase was evaluate by incubating caa1 siga1 and siga2 with recombinant iga protease from neisseria gonorrhoeae (igase pro-pro-y-pro-mo bi tec). briefly, 100 µg of caa1 siga1 and siga2 were incubated at 37 • c for 18 h with 1.5 µg of igase or with reaction buffer alone. the post-incubation products were visualized in a denaturing non-reducing gel with himark prestained protein standard (thermo fisher). c. jejuni 81-176 and c. coli nctc 11437 strains were purchased from the american type culture collection (atcc) and public health england bacteria collection, respectively. the campylobacter species were grown from glycerol stocks in mueller-hinton agar (sigma aldrich) (15 g/l) supplemented with vancomycin and trimethoprim (10 µg/ml) (sigma-aldrich) for 96 h at 40 • c in microaerophilic jar with oxoid tm campygen tm 2.5 l sachet (fisher scientific). in vitro binding of the different flid-reactive siga to a pure culture of c. jejuni was measured by flow cytometry. fifty microliters of each mabs (20 µg/ml) was incubated with 50 µl of c. jejuni 81-176 (10 7 cfu/ml) for 1 h at 4 • c. after washing with pbs 2% fetal bovine serum (fbs), bacteria were incubated for 30 min with biotin-conjugated anti-human iga (southern biotech, cat. no. 2050-08). immunoglobulin-bound bacteria were then stained with streptavidin-pb at 4 • c for 30 min. samples were washed with pbs 2% fbs, resuspended in 2% paraformaldehyde and acquired on lsr fortessa flow cytometer (bd biosciences, franklin lakes nj, usa) using forward scatter (fcs) and side scatter (ssc) parameters in logarithmic mode. data were analyzed using the flowjo software (treestar, ashland, or, usa) or facs diva software (bd biosciences, franklin lakes nj, usa). binding of caa1 and ccg4 siga on the poles of c. jejuni was also confirmed by microscopy. briefly, 30 µg of caa1, ccg4, and hgn194 siga2 were incubated with 10 6 cfu of c. jejuni 81-176 for 1 h at 37 • c in microaerophilic conditions. next, bacteria-mabs complexes were washed and further incubated with antihuman iga-af647 conjugated (1:200, jackson immunoresearch, cat. no. 109-606-011) and sytobc (1:1,000, thermo fisher scientific, cat. no. s34855) for additional 30 min before being poured in microscope slides. images were acquired with a leica sp5 laser scanning confocal microscope with a 488-and 633-nm excitation. the resulting fluorescence emission was collected using 490-627-nm (for sytox green) and 636-770 nm (for alexa 647) windows. samples were imaged with a 63× objective (n.a. 1.4) in xy optical sections of 61.51 µm (1,024 × 1,024 pixels) with a pixel size of 60.1 nm. to improve lateral resolution, the pinhole was set to 0.5 au. animal experiments were performed in accordance with the swiss federal veterinary office guidelines and authorized by the cantonal veterinary office (ti-09-2019). twentyone-day-old c57bl/6 female mice were purchased from charles river laboratories. mice were housed, five per cage, under standardized conditions (20 ± 2 • c, 55 ± 8% relative humidity, 12 h light/dark cycle), at the institute for research in biomedicine. food and water were available ad-libitum, and mice were monitored daily. in order to measure c. jejuni specific antibody binding to the mouse microbiota, stools were collected and homogenized in sterile pbs (0.01 g of stools in 100 µl pbs). samples were centrifuged for 5 min at 400 g to pellet large debris, and then, the supernatant were centrifuged at 8,000 g for 5 min to pellet bacteria. the supernatant was removed, and the bacterial pellet was resuspended in 150 µl (50 µg/ml) of the different anti-campylo mabs and incubated at 4 • c for 30 min. after washing, bacteria were incubated at 4 • c for 30 min with biotin-conjugated anti-human iga (southern biotech, cat. no. 2050-08) and syto bc (1:1,000, thermo fisher scientific, cat. no. s34855). bacteria were washed once and then incubated with streptavidin-pb and, at the end, resuspended in 2% paraformaldehyde in pbs for flowcytometry analysis. all the samples were acquired on an lsr fortessa flow cytometer (bd biosciences, franklin lakes nj, usa) using fcs and ssc parameters in logarithmic mode. data were analyzed using the flowjo software (treestar, ashland, or, usa) or facs diva software (bd biosciences, franklin lakes nj, usa). differences in localization and persistence of siga vs. igg in the gastrointestinal (gi) tract of 21-day-old c57bl/6 mice were evaluated by testing the pk of the two species in the three gi subcompartments: small intestine, cecum, and colon. weaned mice were administered with a pbs solution containing 150 µg of campylobacter-irrelevant antibody hgn194, which targets hiv gp120 and presents no cross-reactivity with the murine microbiota, in the form of siga2m(2) or igg. a control group was administered only with pbs. at 2, 4, and 8 h postoral administration, five animals per group were euthanized, and the content of the three different intestinal tracts was collected and resuspended in 1.5 ml pbs. the samples were then homogenized, and after centrifugation, the supernatants were collected and stored at −20 • c. the detection of human iga or igg in the different parts of the gi tract was performed by elisa. briefly, 96-well plates were coated with goat antihuman iga or igg antibodies (southern biotechnology: 2050-01 and 2040-01) and blocked with 1% bsa blocking buffer. plates were then incubated with 2-fold serial dilutions (1:2 to 1:256) of each intestinal sample for 1 h at room temperature. after four washes with pbs + 0.05% tween 20, the presence of the antibodies was detected by incubation with goat anti-human lambda antibody ap conjugated (southern biotechnology: 2070-04) for 1 h at room temperature followed by 15 min incubation with p-nitrophenylphosphate (pnpp) and detection at 405 nm. taking into account the different numbers of lambda light chains possessed by the polymeric immunoglobulin, to quantify the amount of siga or igg in the samples, two distinct standard curves were built using hgn194 siga and hgn194 igg as standards, respectively. no signals were detected testing the samples from the control animals administered only with pbs. the inflammation status of mice was evaluated by measuring the levels of lipocalin-2 (lcn-2) in fecal supernatants via elisa assay (r&d systems, duoset elisa mouse lipocalin-2/ngal) 24, 48, and 72 h post-infection. briefly, 0.01 g feces was resuspended in 100 µl in pbs, centrifuged for 10 min at maximum speed, and diluted before performing the elisa assay, according to manufacturer's instructions. ceca from all animals were examined at necropsy, fixed in 10% neutral buffered formalin for at least 48 h prior to embedding in paraffin, and stained with hematoxylin and eosin (h&e). pathological scores were determined in a blinded manner using a previously described scoring scheme (31) . briefly, each tissue section was assessed for (1) submucosal edema (0, no change; 1, mild; 2, moderate; 3, severe); (2) crypt hyperplasia (0, no change; 1, 1-50%; 2, 51-100%; 3, >100%); (3) goblet cell depletion (0, no change; 1, mild depletion; 2, severe depletion; 3, absence of goblet cells); (4) epithelial integrity [0, no pathological changes detectable; 1, epithelial desquamation (few cells sloughed, surface rippled); 2, erosion of epithelial surface (epithelial surface rippled, damaged); 3, epithelial surface severely disrupted/damaged, large amounts of cell sloughing; 4, ulceration [with an additional score of 1 added for each 25% fraction of tissue in the cross-section affected up to a maximum score of 8 (4 + 4) for a tissue section that had entirely lost its crypt structure due to epithelial cell loss and immune cell infiltration]; (5) mucosal mononuclear cell infiltration (per 400× magnification field) (0, no change; 1, <20; 2, 20-50; 3, >50 cells/field); (6) submucosal pmn and mononuclear cell infiltration (per 400× magnification field) (1, <5; 2, 21-60; 3, 61-100; 4, >100 cells/field). a maximum score under this scale is 24. statistical analyses were performed using graphpad prism v7.04 (graphpad software, la jolla, ca, usa). mann-whitney u test was used to determine the statistical significance of the results. a p < 0.05 was considered significant in all cases. different campylobacter's proteins were evaluated as potential targets to prevent infection on the basis of (i) the presence of surface exposed epitopes, (ii) role in the bacteria pathogenesis, (iii) ability to stimulate an immune response in the context of natural infection, and (iv) level of amino acid sequence conservation within but not limited to the c. jejuni species. the flagellar-capping protein flid, also known as hookassociated protein 2 (hap2), is a 70-kda protein with high sequence conservation across the c. jejuni species (26) (supplemental figures 1a,b ). based on structures and selfoligomerization mechanisms characterized for other flagellated pathogens (25, 32), campylobacter flid oligomers form the cap protein complex located at the tip of the flagellum, which controls the distal growth of the filament by regulating the assembly of the flagellin molecules. due to its functional role in filament elongation, flid-deficient mutants exhibit defects in bacterial motility (25). the potential of flid as a promising target is strengthened by the proposed involvement in cell adherence (33) and the immunogenicity in chickens during natural infection. accordingly, it has been suggested as a vaccine candidate for the prevention c. jejuni in broilers (26, 34) . flid was therefore selected as a candidate target antigen for monoclonal antibody (mab) development. the frequencies of igg + and iga + flid-reactive memory b cells in 50 tonsillar samples of swiss origin were evaluated using the antigen-specific memory b cell repertoire analysis (ambra) (35) (supplemental figure 2) . igg + and iga + memory b cells from selected tonsils were then immortalized under clonal conditions (28) , and culture supernatants were screened using a 384-well plate-based high-throughput platform to identify b-cell clones expressing flid-reactive antibodies. using this approach, memory b cell clones producing two human monoclonal antibodies, namely caa1 and ccg4, were isolated and selected based on their specificity and affinity for recombinant flid. caa1 was originally isolated as an iga1 encoded by v h 3-48/d2-15/j h 3 with a 21-amino acid hcdr3 and v k 1-39/j k 5, whereas ccg4 was isolated as an igg3 encoded by v h 3-9/d1-7/j h 1 bearing a shorter 11-amino-acid hcdr3 and v l 3-27/j l 3. humans present two different iga subclasses that differ mainly for the length and glycosylation of the hinge region. iga1 possesses a 13-amino-acid longer hinge than iga2 containing up to five o-linked glycans at serine and threonine residues. the longer hinge not only confers to iga1 antibodies higher flexibility and a longer fab reach but is also linked to the sensitivity to a specific class of bacterial proteases known as iga1 proteases. bearing a shorter hinge region lacking proline-serine and/or proline-threonine peptide bonds, iga2 is instead resistant to these proteases (36) . in addition, when compared to igg, igm, and iga1, the iga2 isotype has the unique characteristic to undergo reverse transcytosis by contacting dectin-i receptor on the surface of pps m cells (20) . both the cα1 region and the glycosylation pattern appear pivotal for the interaction at the basis of this mechanism, which has been suggested as a strategy to boost adaptive immunity against pathogens (37) . the recombinant siga1, siga2, and diga2 versions of the flid-reactive mabs were generated by transient transfection of expi293 cells and were then purified by affinity chromatography. various physicochemical measurements were performed to monitor key structural attributes of recombinant flid-reactive polymeric igas as summarized in supplemental figure 3a . the conformational stability of the mabs was determined by monitoring their overall tertiary structure as a function of temperature (intrinsic tryptophan fluorescence spectroscopy), and a 6-7 • c lower thermal melting temperature (tm) value was observed for siga1 when compared to siga2 and diga2 (supplemental figure 3a) . the difference in conformational stability could be due to structural differences in the hinge region between the two subtypes. the relative solubility of the mabs was evaluated by a peg precipitation assay, and diga2 was found to be relatively more soluble compared to siga1 and siga2, a result of which suggests that the presence of the secretory component protein may affect mab solubility at neutral ph. molecular size analysis by sedimentation velocity analytical ultracentrifugation (sv-auc) revealed a heterogeneous distribution of species for each mab (supplemental figure 3a) . we observed that siga2 had a considerably higher relative percent composition of larger molecular weight species than the other two mabs (as compared to the percent main peak). as expected, diga2 had a later elution time when compared to siga1 and siga2 in size exclusion chromatography (supplemental figure 3b) . each of the caa1 iga molecules were heavily glycosylated with carbohydrates ranging from ∼31-39% of the total mass of each mab. as expected by sharing the same iga backbone, the nlinked oligosaccharide structural types were overall very similar in siga2 and diga2 antibodies (supplemental figure 3c ). differently to these antibodies, caa1 siga1 lacked g0f + gn, g1f -gn, gif + gn and g1f + nana, while it contained g2f2 and man4g12 + nana (supplemental figure 3c) . finally, incubation of caa1 siga1 and siga2 with recombinant igaase from n. gonorrhoeae confirmed the differences in sensitivity to bacterial iga proteases of the two isotypes, which have been associated to the length and sequence of the hinge regions (supplemental figure 3d) . based on its resistance to bacterial iga proteases and the ability to undergo reverse transcytosis, the iga2 scaffold was initially selected over the iga1 for our studies, and both caa1 and ccg4 siga2 were further characterized in vitro. to include a control antibody that was not reactive with the antigen in our experiments of campylobacter infection, we also generated the siga2 version of the hgn194 mab, which targets an epitope on the v3 loop of hiv-1 env glycoprotein (38) . caa1 and ccg4 siga2 molecules maintained the original flid binding activity, displaying similar ec 50 (caa1 = 0.08; ccg4 = 0.12 µg/ml) for the flagellar protein ( figure 1a) . epitope binning studies by bli indicated that the binding of one antibody to the antigen did not prevent the binding of the other mab (figure 1b) , pointing to the recognition of two distinct epitopes. in addition, caa1, but not ccg4, recognized flid by western blot analysis under denaturing and reducing conditions, confirming the binding to structurally different antigenic determinants (figure 1c) . accessibility of the two mabs to these epitopes in the flagellum was assessed by flow cytometry using two flid-divergent historical isolates c. jejuni 81-176 and c. coli nctc 11437. caa1 and ccg4 stained both pathogens, thus confirming epitope accessibility and breadth ( figure 1d) . consistent with flid localization, confocal imaging on c. jejuni 81-176 confirmed that the binding of the two mabs occurred at the cell poles ( figure 1e ). the murine intestine is highly resistant to campylobacter due to intense competition exerted by the rich gut microbiota and to a certain level of tolerance, which limits inflammation (39) (40) (41) . to overcome the challenge represented by the resident microbiota, pre-treatment via oral gavage with vancomycin, for which campylobacter species are inherently resistant (42) , is largely adopted. although the pretreatment allows robust bacterial colonization in the cecum, it does not appear to enhance the pathology in adult wild-type mice since minimal signs of inflammation were previously observed (31) . higher susceptibility to c. jejuni infection of infant wild-type mice in comparison to adult animals has been previously reported and linked to significant differences in the microbiota composition (43) . to set up a model that would allow us to evaluate the prophylactic activity of our mabs under immunocompetent conditions, we evaluated the sensitivity to c. jejuni 81-176 infection of pups (12 days old), weaned (21 days old), and adult (56 days old) c57bl/6 mice. animals were pre-treated with vancomycin via oral gavage to deplete the murine microbiota before infection with c. jejuni 81-176 at 10 8 cfu/mouse. campylobacter isolation from stools at 6 days post-infection revealed a ∼2 log higher increase in shedding from 21-day-old animals than from 12-and 56-day-old mice (figure 2a) . in line with these results, weaned mice displayed significantly higher intestinal inflammation as measured by the levels of lipocalin-2 in the stools and histological score values in the cecum in comparison to pups and adult mice (figures 2b,c) . since antibiotic pretreatment should provide comparable ecological niches for infection in the different animals, other factors could be accountable for the different sensitivity to c. jejuni infection displayed by the three groups of mice. analysis of murine iga in the stools of 12-, 21-, and 56-day-old animals revealed different concentrations among the groups (figure 2d ). a phylogenetic tree built using neighbor-joining method with jukes-cantor distance measurement is shown to provide the amino acid distance of flid between the two historical isolates tested (in red). one representative experiment out of three is represented. (e) binding of caa1 siga to c. jejuni as observed in confocal microscopy. bacteria were stained using syto bc (green), whereas caa1 was detected using anti-human iga af647 conjugated (red). interestingly, weaned mice presented negligible levels of iga in the stools in comparison to both pups and adult mice. this observation is consistent with the transition between the exogenous supply of maternal antibodies provided along with the milk (12-day-old mice) and the beginning of the endogenous production that is established in adult animals (i.e., 56-day-old mice). therefore, a likely important factor in the susceptibility of weaned mice to c. jejuni infection could be the lower concentration of secretory iga due to the relative immaturity of intestinal immune system and the depletion of maternal antibodies in these animals. based on these observations, weaned mice were selected as an immune competent animal model to study the prophylactic activity of flid-reactive mabs. since off-target binding of caa1 and ccg4 to the murine microbiota would result in reduced bioavailability and thus activity against pathogens in a prophylactic setting, we evaluated potential cross-reactivity of the recombinant siga with the microbiota of weaned mice. stools from animals orally infected with c. jejuni, c. coli, or pbs (mock infected) were collected 24 h post-infection and incubated with the two flid-reactive mabs and the control hgn194. ccg4 displayed limited levels of cross-reactivity with the murine microbiota (6.62%), which were comparable with those observed for the control antibody (1.11%) (supplemental figure 4a) . conversely, caa1 was binding the microbiota to a higher extent (22%), suggesting a possible fabmediated recognition of antigens in the commensal bacteria that present structures similar to flid (supplemental figure 4a) . nevertheless, for both flid-reactive mabs, the percentage of igacoated bacteria dramatically increased in the stools of infected animals confirming the prompt recognition of the c. jejuni and c. coli species (caa1, 79.2 and 73.9%; ccg4, 70.7 and 55.4%). interestingly, we also observed an increase in hgn194 binding to the bacteria in the stools of these animals (26.5 and 26.4%) that could be consistent with the innate binding activity of iga and secretory component against various pathogens (44, 45) . finally, to examine the conditions for testing the prophylactic efficacy of the mabs, we evaluated the pharmacokinetics of orally administered siga in different gastrointestinal tracts of weaned mice. the campylobacter-irrelevant hgn194 siga2, which displayed negligible cross-reactivity with the murine microbiota (supplemental figure 4a) , was administered as a single oral gavage of 150 µg in pbs (≈15 mg/kg), and its concentration in the different intestinal subcompartments was measured at 2, 4, and 8 h post-administration (supplemental figure 4b) . hgn194 siga concentration in the cecum was maintained almost constant within the first 4 h post-administration and then tended to rapidly decrease by 8 h. the human antibody was not detectable at 12 or 24 h post-administration (data not shown). we next evaluated the prophylactic activity of orally administered flid-reactive recombinant siga2 in immunocompetent-weaned mice infected with c. jejuni 81-176. as such, vancomycin pretreated animals were administered with a single oral gavage of 200 µg/mouse of caa1, ccg4, hgn194 siga2, or pbs 2 h before oral infection with 10 8 cfu/mouse of c. jejuni 81-176. treated animals and the corresponding control groups were monitored for 72 h, during which the severity of infection and degree of inflammation were recorded. of note, analysis of the stools from treated mice revealed a trend characterized by higher campylobacter shedding at 24 h post-infection followed by a significant decrease over time. conversely, untreated and hgn194-treated groups presented lower shedding at early time points followed by a consistent cfu increase at 48 h post-infection ( figure 3a) . these results suggest that that caa1 and ccg4 could prevent or reduce the ability of the pathogen to adhere to the surface of the mucosal epithelium, thus facilitating the clearance of bacteria via peristalsis or mucociliary activity at early stages postinfection. this hypothesis is further supported by the significant lower levels of lipocalin-2, a marker of intestinal inflammation linked to epithelial damage and neutrophil infiltration, recorded at 72 h post-infection in the stools of caa1-and ccg4-treated animals in comparison to the control groups (infected/nontreated and infected/hgn194-treated groups) ( figure 3b ). in addition, animals treated with a single administration of flidreactive mabs presented levels of pmn cells infiltration and histological score values in the cecum significantly lower than the hgn194-treated group (figures 3c,d) , which indicates that the activity exerted by caa1 and ccg4 is largely fab mediated. similar findings were observed in animals administered 2 h before infection with higher or lower campylobacter infection doses (10 7 or 10 9 cfu/mouse). indeed, in both cases caa1-administered animals presented significantly higher bacterial clearance than the untreated group (supplemental figures 5a,b) and levels of intestinal inflammation similar to those of noninfected mice (supplemental figures 5c,d) . furthermore, no significant differences were observed in the reduction in bacterial shedding, pmn infiltration in the cecum lamina propria, and lipocalin-2 levels in the stools when caa1 siga2 was provided 2 h before infection or when it was premixed with c. jejuni 81-176 before oral administration to the animals (supplemental figures 5e-g) . taken together, these results indicate that prophylactic administration of flid-specific caa1 and ccg4 siga2 increase protection from campylobacter infection by accelerating bacterial clearance at early stages after infection, thus significantly reducing inflammation. since longer fab reach, higher flexibility, and different glycosylation may affect the cross-linking ability and/or persistence of the polymeric ig in the intestine, we next investigated whether the two iga isotypes may exert different prophylactic activities in our mouse model of campylobacter infection. caa1 siga1 and siga2 displayed comparable binding to flid (supplemental figure 6a) , and no significant differences in reactivity to campylobacter species in vitro or with murine microbiota ex vivo were observed between the two formats (supplemental figures 6b,c) . the prophylactic activity of the two isotypes was then tested in the murine model of campylobacter infection. in line with our previous findings, animals administered with the flid-reactive mabs displayed higher campylobacter shedding at early time postinfection followed by decrease over time, while infected non-treated animals produced an opposite trend ( figure 4a) . although caa1 siga2 accelerated shedding faster than siga1 as confirmed also by the percentage of human iga-coated bacteria in the stools (supplemental figure 6d) , both subclasses were equally capable to limit inflammation in infected animals as shown by the levels of lipocalin-2 in the stools, pmn infiltration, and the corresponding histological score in the cecum at 72 h postinfection (figures 4b-d) . overall, our results indicate that differences in flexibility and glycosylation associated to the two human iga subclasses do not have an impact in the prophylactic activity exerted by caa1 in the in vivo model. although siga are the most abundant antibodies expressed in association with the intestinal mucosa and the first line of defense against enteric pathogens, they are characterized by a complex structure, and their development as drugs might result challenging in comparison to igg-based monoclonal antibodies. because the activity of the campylobacter-reactive mabs was previously shown to be dependent on the specificity for flid, we generated caa1, ccg4, and the campylobacterirrelevant antibody hgn194 as human igg1 and evaluated their prophylactic activity in comparison to their corresponding siga2 counterparts. since glycosylation might affect the ability of mabs to interact with mucin on the mucosal surface, the localization and persistence of hgn194 igg1 in the murine intestinal tract were appraised by administering the antibody with a single oral gavage to weaned mice and then by measuring the its concentration in the different intestinal subcompartments after 2, 4, and 8 h (supplemental figure 4c) . as in the case of hgn194 siga2, at every time point, the highest concentration of the igg1 was detected in the cecum; however, in this intestinal subcompartment, the igg1 concentration tended to decrease faster than for siga2, with a significant reduction already at 4 h post-administration (supplemental figures 4b,c) . next, we evaluated the prophylactic activity of the flidreactive mabs caa1 and ccg4 as igg1 or siga2 both administered to weaned mice 2 h before infection with c. jejuni 81-176. interestingly, while animals treated with siga2 antibodies displayed the previously observed pattern characterized by higher shedding at 24 h postinfection followed by a significant decrease at 48 and 72 h, the groups treated with the igg version of the same antibodies revealed trends similar to the non-treated groups ( figure 5a and supplemental figure 7a ). the importance of the siga format for the in vivo efficacy was further confirmed by the lower ability of caa1 and ccg4 igg to limit inflammation in comparison to their polymeric counterparts, as shown by pmn cells infiltration in the cecum and lipocalin-2 levels in the stools of the infected animals at 72 h post-infection (figures 5b,c and supplemental figures 7b,c) . overall, no significant differences in the histological scores were observed between the mice treated with the flid-reactive igg antibodies and the non-treated animals ( figure 5d and supplemental figure 7d) . conversely, the siga2 versions of the antibodies were able to replicate the beneficial effect previously observed, maintaining the histological score in the cecum to values significantly lower than both non-treated and igg-treated mice (figure 5d and supplemental figure 7d) . taken together, these data indicate that the caa1 and ccg4 igg have limited prophylactic activity when orally administered prior to campylobacter infection. the reduced activity of caa1 and ccg4 igg might be dependent on both the lower stability and persistence in the gastrointestinal tract and/or on the decreased "bonus effect of multivalency" at the basis of the cross-linking activity associated with the polymeric format. the aim of this study was to evaluate the ability of orally administered recombinant siga, derived from selected flidreactive monoclonal antibodies, to prevent infections by campylobacter species frequently associated with severe neonatal gastroenteritis. despite its pivotal role in bacteria motility, epithelial cells adherence (25, 33) , and the high level of conservation among c. jejuni (supplemental figure 1) , flid has never been assessed to date as a potential target for therapeutic mabs. consistent with the immunogenicity previously reported for this antigen (26, 34) , our results indicate that flid-reactive mabs can be isolated from the human memory b cell repertoire. further development of these antibodies, namely, caa1 and ccg4, resulted in recombinant siga that preserved the ability to bind flid on the campylobacter's flagellum of both c. jejuni and c. coli species (figure 1d) . genetically manipulated animals characterized by an exacerbated inflammatory response to bacteria, such as sigirr or il10 − / − , have been proposed as models to study campylobacter pathogenesis (31, 46) . these mutations, however, dramatically alter the murine immune system to an extent that even the presence of commensal microbes can potentially result in spontaneous enterocolitis (47) . the sensitivity of wild-type mice to campylobacter infection has consistently been linked to age-dependent variations in the composition of the resident microbiota (43) . of note, our findings indicate that different levels of iga in the murine intestine also play a role in campylobacter pathogenesis in mice. in particular, we observed that weaned mice represent the most permissive wild-type age group and that this is likely due to the transition between the exogenous iga supply provided by the maternal milk and iga endogenous production ( figure 2d) . moreover, giallourou et al. described a weaned mouse model of c. jejuni that can recapitulate enteropathy and diarrhea (48) . in the current study, we established a weaned mouse model of campylobacter infection to evaluate the prophylactic activity of the mabs based on bacterial shedding and inflammatory biomarkers (figures 2a-c) . in this animal model, we documented a consistent effect following a single prophylactic administration of flid-reactive siga 2 h before c. jejuni infection, which is characterized by significantly higher campylobacter shedding at the early stage of infection followed by a rapid decline at later time points (figure 3a) . our hypothesis is that by binding flid, the siga can limit bacteria motility and ability to adhere to the surface of the mucosal epithelium, thus accelerating their clearance via peristalsis. this idea is further strengthened by the fact that animals administered with flid-reactive mabs display considerably lower levels of inflammatory biomarkers in the cecum and stools than the control groups, which present an opposite shedding trend. flid-reactive mabs efficacy relies on the specific recognition of the bacterial surface antigen, as an irrelevant antibody in the same format does not produce similar beneficial effects (figures 3a-d) . further studies will elucidate whether these antibodies may act primarily by limiting bacteria motility (cross-linkage), by limiting attachment to cell receptors (steric hindrance), by limiting secretion via the t3ss of proteins that contribute to host cell invasion (e.g., ciab) or via a combination of all these activities. interestingly, similar prophylactic activity was observed when caa1 was premixed with c. jejuni or administered 2 h before infection, thus confirming siga stability during the passage through the small intestine and their persistence in the cecum (supplemental figures 5e-g) . despite the fact that siga2 showed a slightly higher ability to accelerate bacterial shedding at early stage of infection in comparison to the siga1 counterpart (figure 4a) , differences in length and glycosylation of the hinge region between the two human iga isotypes do not appear to significantly undermine the ability of the polymeric immunoglobulins to prevent inflammation (figures 4b-d) . however, iga2 might represent the most promising backbone for development of orally deliverable mabs due to resistance to iga proteases expressed by different pathogens (supplemental figure 3d ) and to the unique ability to undergo reverse transcytosis (20) , which in turn could contribute to boost a potential vaccinal effect (37) . the prophylactic activity of flid-reactive caa1 and ccg4 was significantly decreased when these mabs were administered as igg1. indeed, in the igg1 format, the mabs were neither able to boost campylobacter shedding at early time postinfection nor able to limit inflammation to an extent comparable with their corresponding siga (figure 5 and supplemental figure 7) . the superior efficacy of caa1 and ccg4 as siga might be related to higher resistance to proteases in the small intestine (30, 49) , higher localization and better persistence in the cecum (17, 18) , and/or to the mechanisms of action (i.e., cross-linking) associated with the polymeric immunoglobulin structure (14, 19) . in summary, our results indicate that the flagellar-capping protein flid represents a promising target for the development of campylobacter-reactive mabs capable to exert a prophylactic activity dependent on both their affinity to the antigen and on features strictly related to the siga format. although improvement in water sanitation and healthcare conditions are pivotal to reduce campylobacter's impact in infants in middle-and low-income countries, these findings suggest that the development of orally deliverable recombinant siga targeting flid might represent a novel strategy to prevent or mitigate both disease severity and the development of postinfection syndromes. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by swiss federal veterinary office guidelines and the cantonal veterinary office (ti-09-2019). who publishes list of bacteria for which new antibiotics are urgently needed status of vaccine research and development for campylobacter jejuni the clinical importance of emerging campylobacter species the global view of campylobacteriosis: report of an expert consultation pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (mal-ed) guillain-barre syndrome-related campylobacter jejuni in bangladesh: ganglioside mimicry and cross-reactive antibodies carbohydrate mimicry between human ganglioside gm1 and campylobacter jejuni lipooligosaccharide causes guillain-barre syndrome increased short-and long-term risk of inflammatory bowel disease after salmonella or campylobacter gastroenteritis motility as an intestinal colonization factor for campylobacter jejuni identification of the carbohydrate moieties and glycosylation motifs in campylobacter jejuni flagellin the genome sequence of the food-borne pathogen campylobacter jejuni reveals hypervariable sequences environmental enteropathy: elusive but significant subclinical abnormalities in developing countries secretory iga's complex roles in immunity and mucosal homeostasis in the gut the structure and dynamics of secretory component and its interactions with polymeric immunoglobulins receptor-mediated transcellular transport of immunoglobulin: synthesis of secretory component as multiple and larger transmembrane forms the polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces high-avidity iga protects the intestine by enchaining growing bacteria dectin-1 is essential for reverse transcytosis of glycosylated sigaantigen complexes by intestinal m cells breastfeeding: maintaining an irreplaceable immunological resource protection of breast-fed infants against campylobacter diarrhea by antibodies in human milk protection against campylobacter diarrhea: role of milk iga antibodies against bacterial surface antigens self-oligomerizing structure of the flagellar cap protein flid and its implication in filament assembly evaluation of flagellum-related proteins flid and fspa as subunit vaccines against campylobacter jejuni colonisation in chickens rapid development of broadly influenza neutralizing antibodies through redundant mutations an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus mucosal immune defense: immunoglobulin a preformulation characterization and stability assessments of secretory iga monoclonal antibodies as potential candidates for passive immunization by oral administration a novel mouse model of campylobacter jejuni gastroenteritis reveals key pro-inflammatory and tissue protective roles for toll-like receptor signaling during infection bacterial flagellar capping proteins adopt diverse oligomeric states host cell binding of the flagellar tip protein of campylobacter jejuni characterization and antigenicity of recombinant campylobacter jejuni flagellar capping protein flid clonal dissection of the human memory b-cell repertoire following infection and vaccination the iga1 proteases of pathogenic bacteria secretory iga as a vaccine carrier for delivery of hiv antigen to m cells analysis of memory b cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from hiv-1-infected individuals novel murine infection models provide deep insights into the "menage a trois" of campylobacter jejuni, microbiota and host innate immunity modification of intestinal microbiota and its consequences for innate immune response in the pathogenesis of campylobacteriosis campylobacter jejuni colonization of mice with limited enteric flora mechanisms of antibiotic resistance in campylobacter species campylobacter jejuni infection of infant mice: acute enterocolitis is followed by asymptomatic intestinal and extra-intestinal immune responses glycosylation of human iga directly inhibits influenza a and other sialic-acidbinding viruses binding of lactoferrin and free secretory component to enterotoxigenic escherichia coli the role of serine protease htra in acute ulcerative enterocolitis and extra-intestinal immune responses during campylobacter jejuni infection of gnotobiotic il-10 deficient mice c57bl/6 and congenic interleukin-10-deficient mice can serve as models of campylobacter jejuni colonization and enteritis a novel mouse model of campylobacter jejuni enteropathy and diarrhea increased risistance of immunoglobulin a dimers to proteolytic degradation after binding of secretory component genomic characterization of the guillain-barre syndrome-associated campylobacter jejuni icdccj07001 isolate campylobacter jejuni strain cg8421: a refined model for the study of campylobacteriosis and evaluation of campylobacter vaccines in human subjects campylobacter jejuni lipopolysaccharides in guillain-barre syndrome: molecular mimicry and host susceptibility the authors would like to thank dr. antonio pellanda, dr. elena scotti, dr. stefano ermanni (eoc-ospedale regionale di lugano, switzerland), and dr. nicola melik (eoc-ospedale regionale di locarno, switzerland) for providing tonsillar samples from patients undergoing tonsillectomy. we would also like to thank omar vandal and jeremy blum for helpful advice throughout the execution of the study. lp, fg, dc, and mp conceived the study. lp and mp initiated the study design. sj, gl, dp, fst, rp, and fb helped with implementation. dm and fse provided expertise in microscopy and immunohistochemistry, respectively. yh, sb, jx, ok, sbj, and dv performed the physiochemical characterization of the recombinant mabs. lp, fg, dc, and mp outlined and wrote the manuscript, whereas ok, sbj, and dv contributed to its finalization. all authors approved the final manuscript. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 perruzza, jaconi, lombardo, pinna, strati, morone, seehusen, hu, bajoria, xiong, kumru, joshi, volkin, piantanida, benigni, grassi, corti and pizzuto. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-032953-qy4b2l2f authors: picard-sánchez, amparo; estensoro, itziar; perdiguero, pedro; del pozo, raquel; tafalla, carolina; piazzon, m. carla; sitjà-bobadilla, ariadna title: passive immunization delays disease outcome in gilthead sea bream infected with enteromyxum leei (myxozoa), despite the moderate changes in igm and igt repertoire date: 2020-09-11 journal: front immunol doi: 10.3389/fimmu.2020.581361 sha: doc_id: 32953 cord_uid: qy4b2l2f passive immunization constitutes an emerging field of interest in aquaculture, particularly with the restrictions for antibiotic use. enteromyxum leei is a myxozoan intestinal parasite that invades the paracellular space of the intestinal epithelium, producing a slow-progressing disease, leading to anorexia, cachexia and mortalities. we have previously demonstrated that gilthead sea bream (gsb, sparus aurata) that survive e. leei infection become resistant upon re-exposure, and this resistance is directly related to the presence of high levels of specific igm in serum. thus, the current work was aimed to determine if passive immunization could help to prevent enteromyxosis in gsb and to study in detail the nature of these protective antibodies. serum from a pool of resistant (sur) or naïve (nai) animals was intracoelomically injected 24 h prior to the e. leei-effluent challenge and at 9 days post-challenge (dpc). effluent challenge lasted for 23 days, and then the injected groups were allocated in separate tanks with clean water. a non-lethal parasite diagnosis was performed at 56 dpc. at the final sampling (100 dpc), blood, serum and tissues were collected for histology, molecular diagnosis and the detection of circulating antibodies. in parallel, we performed an immunoglobulin repertoire analysis of the fish generating sur and nai sera. the results showed that, fish injected with parasite-specific antibodies (spabs) became infected with the parasite, but showed lower disease signs and intensity of infection than the other groups, indicating a later establishment of the parasite. repertoire analysis revealed that e. leei induced a polyclonal expansion of diverse igm and igt subsets that could be in part an evasion strategy of the parasite. nonetheless, gsb was able to produce sufficient levels of parasite-spabs to avoid re-infection of surviving animals and confer certain degree of protection upon passive transfer of antibodies. these results highlight the crucial role of spab responses against e. leei and set the basis for the development of effective treatment or prophylactic methods for aquaculture. pathogens are an important cause of economic losses in aquaculture, and the partial effectiveness of the available treatments increases the chances of drug-resistant pathogens in fish and in the aquatic environment (1) . the therapeutic use of specific antibodies (spabs) is an attractive alternative to provide immunity against pathogens. opposite to chemicals and antibiotics, which have a broad-spectrum of action, antibodies constitute a very specific defense mechanism. antibody mediated immunity can be active or passive. active immunity depends on the production of spabs after direct contact with the pathogen or antigen (vaccination or random encounter), it takes days/weeks to develop and results in the formation of immunological memory that can last for months or years. in passive immunity, spabs obtained from a previously infected or immunized donor, are introduced in a naïve individual to confer resistance or to combat a specific pathogen. passive immunity is faster, shortlived and does not involve memory (2) . passive immunization can be allogeneic or xenogeneic, if the origin of the transferred abs is the same or different than the recipient species, respectively. this technique has been applied successfully in human medicine (3) . it has also been tested in fish-parasite models showing promising results. allogeneic therapies have partially protected carp (cyprinus carpio) against trypanoplasma borreli (4), or tomato clownfish (amphiprion frenatus) against amyloodinium ocellatum (5) . however, passive immunization failed to protect in other fish-parasites models like rainbow trout (oncorhynchus mykiss) against gyrodactylus derjavini infection (6) . of note, the first two experiments required repeated doses of serum to induce protection, whereas in the latter, only one dose was administered. xenogeneic therapies have also been applied successfully. mouse monoclonal antibodies against surface proteins of ichthyophthirius multifiliis protected channel catfish (ictalurus punctatus) against infection (7) . clearly, a key aspect for the success of the procedure is a deep knowledge about the host-parasite model, especially the time for parasite spreading in the host and the time that transferred antibodies remain in the circulation of the recipient animal (8) . teleost fish have three different immunoglobulin (ig) isotypes: igm, igt/z, and igd (9) . the basic structure of fish igs is the same as that of mammals. the heavy chain is constituted of an isotype-specific constant domain (ch) and a variable domain (vh) which, together with the variable domain of the light chain (vl), are responsible for the great diversity of antigen binding sites. each v domain has three hyper variable regions termed complementarity-determining regions (cdr), which constitute the majority of the antigen binding sites of the ig molecule. different combinations of cdr in vl and vh are responsible for the amazing diversity in binding sites among the millions of antibodies in an individual (10) . in fish, igm is the highest expressed ig in all organs and it is essential for immune protection against different pathogens upon different routes of infection (11) (12) (13) (14) (15) . igm is highly abundant in fish serum with concentrations between 800 and 9,000 µg/ml (16) . the teleost specific isotype igt is considered the most important ig in mucosal surfaces, but is also found in serum at much lower concentrations than igm, so its role in systemic responses should not be discarded (17) (18) (19) (20) . the role of igd is still not well defined in mammals or fish, however, recent studies performed in fish have established that it is also secreted (21) and might have a relevant role in some mucosal surfaces such as gills (18) or intestine (22) . like mammals, fish can develop immunological memory. a secondary exposure to an antigen will induce faster and greater responses than primary response, with larger numbers of antigen-specific b cells originated from the expansion of the pool of memory b cells. detailed repertoire analyses have helped to define fish ig responses upon infections of different etiology (17, 23) . enteromyxum leei is a myxozoan intestinal endoparasite, that can be transmitted from fish-to-fish and causes a slow progressing catarrhal enteritis in gilthead sea bream (gsb, sparus aurata) (24, 25) . the parasite lives and divides in the paracellular space between enterocytes causing chronic infections with intestinal inflammation leading to dysfunctional absorption and anorexia, reflected in weight loss and decreased specific growth rate (sgr), condition factor (cf), and fat deposits in liver (25) . currently, this parasite cannot be cultured in vitro and there are no preventive or curative measures against this disease. however, there is plenty of background knowledge on the host response against the parasite (25) (26) (27) (28) (29) (30) (31) . we have recently demonstrated that gsb is able to develop acquired immunity against e. leei. fish that survived an infection did not get re-infected upon re-exposure, even 16 months after the initial exposure, and this resistance was correlated with high levels of spabs (igm) in serum (29) . therefore, the aim of the present study was to demonstrate the protective role of spabs against enteromyxosis in gsb by passive immunization of naïve animals. we also aimed to characterize the type of ig response produced by this parasite, analyzing and comparing the ig repertoire in naïve and resistant animals reexposed to the parasite. the results obtained in this study provide novel information to understand the role of antibodies during the response of gsb to enteromyxosis, and will lead us a step further toward developing successful vaccines and/or treatments against this economically important disease in aquaculture. specific-pathogen-free (spf) and clinically healthy gsb juveniles from a commercial fish farm, were kept in 5 µm-filtered and uv irradiated sea water (salinity 37.5 g/l) between 18 and 21.8 • c, with the natural photoperiod at latitude 40 • 5 n; 0 • 10 e. the spf status was confirmed by qpcr according to the protocol previously described (29) . fish were fed ad libitum once a day a commercial diet (biomar, palencia, spain). all experimental protocols involving fish were approved by the ethics and animal welfare committee of iats, csic, and generalitat valenciana. they were carried out in a registered installation facility in accordance with the principles published in the european animal directive (2010/63/eu) and spanish laws (royal decree rd53/2013) for the protection of animals used in scientific experiments. all efforts were made to minimize the suffering of animals. fish sera used for the passive immunization trial were obtained from a previous experimental infection (29) . briefly, gsb that had survived and cleared an infection with e. leei were reexposed to the parasite 9 months after the beginning of the first infection by exposure to parasite-infected water effluent. these fish showed to be resistant to re-infection (sur) and tested negative for the presence of the parasite 61 and 86 days after the second exposure. sera from the four fish that showed the highest levels of parasite-specific igm (immunoreactivity = 6, see below) were selected and pooled. sur serum and tissue samples were obtained 86 days after the second exposure to the parasite. in parallel, sera from four naïve (nai) fish from the same batch were tested to be negative for parasite-specific igm and pooled to be used as a control. before injection, the complement was heat-inactivated (42 • c 30 min). from the same eight animals, rna from the anterior intestine was extracted as previously described (29) . the anterior intestine was chosen because it is the tissue where immunoglobulin gene expression was significantly increased upon re-exposure to the parasite in sur animals (29) . fifty naïve gsb (25.5 g) were individually tagged with passive integrated transponders (pit-tags) and kept together in the same tank during the first part of the trial involving serum injections and effluent challenge. one day before and nine days after the challenge, 20 fish per group were injected intracoelomically (i.c.) with 10 µl/g of nai or sur serum. the remaining 10 fish were injected with 10 µl/g of sterile pbs. in addition, 10 naïve fish from the same stock were kept separately, constituting the nonchallenged control group. a preliminary trial was conducted to determine the duration of injected antibodies in the recipient fish blood circulation. for that purpose: 10 µl/g of rabbit serum (dako) were i.c. injected and rabbit igs were detected by dot blot in serum as soon as 1 h post-injection and remained in circulation at least 9 days (supplementary figure s1). one day (24 h) after the first injection, fish were challenged with e. leei by exposure to infected water effluent, mimicking one of the natural routes of infection in farmed fish (32) . briefly, the tank holding 50 fish, already injected with pbs, nai or sur serum, received water effluent from a donor tank holding 33 heavily e. leei-infected gsb, as previously described (33) . at 23 days post-challenge (dpc), fish were weighed and measured, and the different injected groups were moved in groups of 10 to individual tanks receiving clean water, ending the effluent challenge. serum-injected fish (n = 20 for each type of serum) were moved to two replicate tanks (n = 10) for each group. the 23-day exposure was selected as the minimum time needed for all fish to be challenged with the parasite without exerting a too high infection pressure that will mask the results at the end of the trial (34) . at 56 dpc, fish were non-lethally sampled to evaluate the progression of the infection and the final lethal sampling was performed at 100 dpc. a schematic representation of the trial and samplings can be found in figure 1 . before all samplings (7 days before challenge, 23, 56, and 100 dpc), fish were starved for two days and biometric parameters measured. additionally, at the second sampling (56 dpc), fish were also sampled for parasite diagnosis by non-lethal rectal probing and qpcr with specific primers for the parasite 18s rrna gene, as previously described (29) . finally, in the last sampling (100 dpc), fish were killed by overexposure to anesthetic (ms-222, 0.1 g/l; sigma), and blood and tissue samples for molecular and histological procedures were taken. blood samples (1 ml) were drawn from the caudal vessels by puncture with heparinized sterile needles and serum was obtained after overnight clotting at 4 • c and centrifugation at 3,000 × g for 30 min, and maintained at -80 • c until further use. intestine (anterior-immediately after the pyloric caeca-, middle and posterior-immediately before the anal ampoule), head kidney, spleen, and liver samples from all fish were taken in 10% buffered formalin for standard histological procedures. condition factor (cf = [100 × body weight]/fork length 3 ) and specific growth rate (sgr = [ln (final weight) -ln (initial weight) × 100]/days) were calculated for each animal individually. infection intensity was semiquantitatively evaluated on giemsa stained histological sections of anterior (ai), middle (mi), and posterior intestine (pi) using a scale from 1 (lowest) to 6 (highest) (28) . the remaining intestinal tissue was processed for molecular diagnostic (qpcr), as previously described (29) . in addition, pas-staining was carried out on liver sections. one fish of the nai group that died between the scheduled sampling points was checked postmortem for the presence of the parasite and was no longer included in the results. total igm and igt were measured by elisa as previously described (35) . specific igm against e. leei in serum samples was immunohistochemically detected using paraffin embedded sections of e. leei-infected gsb intestines obtained from previous and independent infection trials, following an immunohistochemical sandwich elisa protocol, as described previously (29) . intensity of immunoreactivity of each fish serum against the parasite was evaluated by microscopic examination of the immunolabelled tissue sections according to a semiquantitative scale ranging from 0 to 6 (scaling: 0 = no immunoreactivity against the parasite; 1 = very slight reactivity; 2 = slight reactivity; 3 = medium reactivity; 4 = medium-intense reactivity; 5 = intense reactivity; 6 = very intense reactivity). the detection of t cells (zap70 + ) in the intestine of experimental individuals was performed by immunohistochemistry (ihc), as previously described (28) . in the former study, we have reported that intestinal t cells increase in numbers upon e. leei infection and these cells are one of the key mechanisms to clear the parasite. briefly, paraffin sections (4 µm thick) were deparaffinized, hydrated and the endogenous peroxidase activity was blocked by incubation in hydrogen peroxide (0.3% (v/v) for 30 min). an antigen retrieval step was performed by boiling the samples in citrate buffer ph 6 for 20 min. incubations were performed in a humid chamber at room temperature and all washing figure 1 | schematic representation of the passive immunization trial. seven days before the challenge by effluent exposure, fish were tagged, weighted, measured and allocated together in the same tank. one day before the challenge and nine days post-challenge (dpc) fish were injected with the different sera or pbs. at 23 dpc, the effluent challenge was terminated and fish were separated in three different tanks (10 fish/tank, sur, nai, and pbs injected groups). at 56 dpc, a non-lethal diagnosis of the parasite by pcr was performed, and at 100 dpc all fish were sacrificed and samples for histology and diagnosis were collected. i.c., intracoelomic. procedures consisted of successive 5 min immersions in ttbs (20 mm tris-hcl, 0.5 m nacl, 0.05% tween 20, ph 7.2) and tbs (20 mm tris-hcl, 0.5 m nacl, ph 7.2). slides were washed, blocked for 30 min with 1.5% normal goat serum (vector laboratories). after washing, samples were then incubated with a monoclonal rabbit anti-zap70 antibody (99f2 cell signaling technologies) diluted 1:50 in tbs 1% bsa for 1 h and washed again. slides were incubated with a biotinylated goat anti-rabbit antibody (vector labs.) diluted 1:200 in tbs 1.5% normal goat serum for 1 h, washed and the avidin-biotin-peroxidase complex (abc) (vector labs.) was applied for 1 h before washing slides again. bound peroxidase was developed by adding 3,3'diaminobenzidine tetrahydrochloride chromogen (sigma) for 2 min and the reaction was stopped with deionized water. tissue sections were counterstained with gill's hematoxylin, dehydrated and mounted with di-n-butyl phthalate in xylene. incubation of tissue sections with abc alone served as control to discard the presence of endogenous biotin-binding proteins. negative controls omitting the primary antibodies, the secondary antibody and the abc were carried out and were negative. to perform repertoire analysis of gsb immunoglobulins, genomic regions potentially encoding ighv genes were identified along gsb draft genome (36) , searching for conserved domains using the online tool https://www.ncbi.nlm.nih.gov/ structure/cdd/wrpsb.cgi from ncbi. transcriptional activity of identified regions were further confirmed by blasting against a gsb transcriptome from a previous study (37) . nucleotide sequences were aligned using clustalw 1 in order to identify conserved regions among genes to design primers which may amplify all potential genes. a total of seven forward primers, recognizing the fr3 region, were designed and used in pcr amplifications in combination with reverse primers designed in igm or igt constant region (cµ2 and cτ1, accession numbers kx599199 and kx599201, respectively) (supplementary table s1 ). 1 https://www.genome.jp/tools-bin/clustalw to study the igm and igt repertoire in the fish used as serum donors for passive immunization, cdna from anterior intestine of sur and nai donor fish were used as template in pcr amplifications for each primer combination (f + r). mix of reagents for pcr reaction was: 1 µl of cdna as a template, 0.2 µm of each forward and reverse primer combination, and 2.5 u/µl dna polymerase (accuprime tm taq polymerase high fidelity, invitrogen) in 1 × accuprime pcr buffer i, containing 2 mm mgcl 2 and a mix of 0.2 mm of each dntp (invitrogen). the thermal cycling regime was as follows: 94 • c for 2 min, followed by 40 cycles (94 • c for 45 s, 55 • c for 1 min, and 72 • c for 1 min) and the final extension at 72 • c for 10 min. negative controls without cdna were also included. for the visualization of pcr products, a 10 µl aliquot from each pcr product was mixed with 2 µl of loading buffer and loaded into a 1% agarose with ethidium bromide staining. at the same time, samples for sequencing were composed by 2 µl from each reaction belonging to the same individual pooled together. samples were quality checked using an agilent 2100 bioanalyzer (agilent technologies) and sequenced by life sequencing (valencia, spain). one library per individual was constructed with the truseq dna pcr-free library prep kit (illumina, san diego, ca, united states) according to the manufacturer's protocol. libraries were pooled together, and paired-end sequencing was performed on an illumina miseq with a miseq reagent kit v3 (2 × 300 cycles) cartridge (illumina, san diego, ca, united states). raw sequencing data were demultiplexed and sequencing adapters and barcodes were removed from the sequences using the miseq analysis pipeline. the first 20 nucleotides from the reverse primers were used as a barcode for the identification of 3' ends corresponding to each constant ig gene. reads that matched a primer sequence with a maximum of three mismatches allowing an overlap of two nucleotides (-mismatches 3 -partial 2) were classified into the corresponding igm or igt isotype using the fastq/a barcode splitter tool 2 . the opposite paired end reads, corresponding to the 5 end of pcr products, were extracted with the fastq interlacer tool implemented in galaxy (38) . the paired forward and reverse reads were merged using pear software (39) with a minimum overlap size of 10 nucleotides. finally, a quality filter was applied keeping sequences with a phred base quality ≥20 in at least the 90% of sequence. in the current study, in absence of a v-d-j germline for our species, sequences for each isotype from the eight individuals were annotated by comparative genomics according the germline of zebrafish (danio rerio), used as model species, which is available at international immunogenetics information system databases (40) . thus, gsb repertoire was annotated according the best match identified for each gene (v,d, or j) from zebrafish using the imgt/highv-quest tool (41) . imgt/highv-quest results were parsed using convert tool from vdjtools software (42) . non-functional clonotypes were filtered out using filternonfunctional tool from vdjtools software. retained functional clonotypes were further analyzed using the repexplore, repclonality, repdiversity, geneusage, and getkmers tools from the r package immunarch (v0.5.5) (43) . prior to analysis, data were normalized by subsampling. this analysis allowed a first preliminary glimpse on the diversity of the clonotypes and the type of response that is being elicited by the parasite by comparison of two sampling groups (nai vs. sur). differences among groups were evaluated with one-way anova followed by tukey test. data which failed the normality or equal variance test were analyzed with kruskal-wallis anova-i on ranks followed by dunn's method. differences between two groups were determined by student's t-test or mann-whitney test when normality conditions were not met. a chi-square test was run to determine differences in the presence/absence of liver fat and glycogen deposits. in all cases differences were considered significant at p < 0.05. statistics and visualizations were performed with the r package immunarch (v0.5.5) (43) and graphpad prism (v6.01). the typical clinical signs of enteromyxosis include weight loss and decreased cf and sgr (25) . the current results show that while cf of some challenged fish was not affected until the final sampling at 100 dpc, sgr was already affected at the intermediate sampling (56 dpc) in the pbs-injected group (figures 2a-d) . fish injected with pbs or nai serum always showed lower cf and sgr than non-challenged fish. interestingly, fish injected with sur serum did not show significant differences with the nonchallenged controls and always displayed intermediate values. no significant differences were found in the prevalence of infection among the different challenged groups in any of the sampling points. however, the prevalence of infection in fish injected with parasite-spabs (sur serum) was always the lowest ( figure 2e) . histological diagnosis allowed assessing the percentage of fish with a progressed infection, that is, fish that have more than one intestinal segment parasitized. progression of infection also showed lower values in fish injected with sur serum (figure 2e) . interestingly, all infected passively immunized fish (sur serum) showed low intensity of infection (ct values > 27), whereas, roughly half of the individuals from the other two groups had high intensity of infection (ct values < 27) . the intensity of infection of sur-injected fish was significantly lower than that of the nai serum group (figure 2f , higher ct values correspond to lower intensity of infection). all parasite-challenged fish showed the typical intestinal reaction against e. leei, although some differences could be observed among groups. overall, 70, 73.7, and 85% of the pbs, nai, or sur fish, respectively, had abundant lymphocyte-like cells at the base of the intestinal epithelium, particularly at the anterior segment ( figure 3a) . ihc revealed that most of these cells were zap70 + , identifying, at least part of this increased cell population, as t cells (figures 3c,d) . all challenged groups showed a noteworthy infiltration of eosinophilic granular cells in the epithelial layer ( figure 3b) . interestingly, the prototypical hyperplasia of the submucosa upon e. leei infection was observed in fewer animals injected with parasite-spabs (35% in sur vs. 59% in nai) (figures 3e,f) . the most striking difference was found in the liver. the liver of heavily infected fish, due to the anorexia and the nutrient absorption impairment induced by the parasite, had clearly less accumulation of fat and glycogen deposits. this was the case for most pbs (80%) and nai (42%) challenged fish, whereas all sur challenged fish, except one (5%), had normal liver histological features with abundant fat and glycogen deposits (figures 3g,h,i,j) . this difference was statistically significant (chi-squared test, p = 0.02). total serum igm, igt and parasite specific igm of the different groups were measured at 100 dpc. total circulating igm was significantly higher in the group injected with serum with parasite-spabs than that of all the other groups, which did not show differences among them (figure 4a) . circulating igt was higher in the pbs injected group than in the control non-challenged group. the serum injected groups showed intermediate values (figure 4b) . no parasite-specific igm was found in the non-challenged group, while all the other groups showed no differences among each other ( figure 4c ). the next step we took was an attempt to characterize the type of ig response elicited by the parasite. thus, we studied the igm and igt repertoire in the gsb that acted as sur and nai serum donors for the passive immunization trial. by in silico analysis using immunoglobulin conserved domains, up to 46 regions showing homology with v genes were identified along the gsb genome (supplementary data s1). following this identification, intensity of infection is shown as ct values (mean ± sd) obtained from a parasite specific qpcr of the intestine at 100 dpc (f). low ct values indicate high infection intensity. pbs is the group injected with pbs, whereas nai represent the fish injected with serum without specific antibodies and sur the fish injected with parasite-specific antibodies. the negative control (ctrl-) was the group not challenged with the parasite. different letters indicate significant differences among groups (p < 0.05). seven forward primers were designed in v regions which in combination with reverse primers located in igm or igt constant regions allowed for the first time the amplification of a global gsb repertoire (supplementary figure s2) . using illumina miseq (2 × 300), an average of 2.2 million paired end reads per sample were obtained and assembled. using the isotype specific primers as a barcode, 72.4 and 15.9% of the sequences were cataloged as igm and igt, respectively. due to the absence of gsb reference sequences for vdj genes, imgt/highv-quest analysis was performed using zebrafish as a reference. this analysis resulted in the identification of productive sequences as well as their cdr3, and served to assign vdj genes and unique clonotypes according to homology to zebrafish, which allowed the comparison between experimental groups. after analysis, 93.2% of the igm sequences and 84.7% of the igt sequences were classified as productive (supplementary table s2 ). analysis with immunarch revealed igt (e) was also evaluated using the chao1 diversity index. the differential usage of vh-gene families for igm (c) and igt (f) was calculated with the assignment against zebrafish sequences. bars represent the average number (mean ± sd) on unique sequences for n = 4 fish. asterisks represent statistical differences between nai and sur groups at p < 0.05. that the number of unique clonotypes was significantly higher for both isotypes in immunized (sur) fish than in naïve (nai) individuals (figures 5a,d) . the clonotype diversity (estimated with the chao1 index) was also always significantly higher in sur animals than in nai fish (figures 5b,e) . sequences classified as similar to ighv5 and ighv10 zebrafish subgroups were the most expressed in both isotypes. ighv10 like sequences were significantly overexpressed in igt, whereas ighv5 like sequences were overrepresented in both isotypes in sur fish (figures 5c,f) . around 90% (igm) and 85% (igt) of the different clonotypes were found one to five times, indicating a broad diversity of ig repertoire in gsb intestines. interestingly, the percentage of igt clonotypes found one to five times decreased in sur animals, whereas igt clonotypes, which were represented more than 100 times, increased in sur fish. these results demonstrate a higher frequency of expanded igt clonotypes in sur fish, indicating a clear response. no significant changes were found for igm (figures 6a,b) . when analyzing the cdr3 amino acids length of the different clonotypes, a shift to longer cdr3 lengths was observed for igm, but no clear differences appeared in igt distribution (figures 6c,d) . none of the cdr3 length histograms fitted the expected gaussian bell distribution bars show the average percentage (mean + sd) of clonotypes observed n times in the datasets. asterisks represent statistical differences between nai and sur groups at p < 0.05. cdr3 length distributions for all detected igm (c) and igt (d) clonotypes. the graphs represent the average number of clonotypes of a given amino acid (aa) length (+sd) for sur and nai fish. kolmogorov-smirnoff tests reported that none of the curves followed a bell-shaped gaussian distribution (p > 0.05). normally expected for naïve animals (kolmogorov-smirnov normality test p > 0.05). analysis of the five most frequent k-mers (k = 5) from the cdr3 sequences revealed that the k-mer yfdyw is very frequent in both, igm and igt sequences, with a frequency increase in sur individuals. the other detected k-mers were not common for both isotypes (figure 7) . in mammals, passive immunization represents a prophylactic strategy when vaccination is not possible, such as in immunosuppressed or very young individuals. also, in the case of certain cancers or emerging human diseases, when vaccines or treatments have not yet been developed (44, 45) . in fish, passive transfer of spabs by injection has been successful against some infectious diseases, including parasites (4, 5, 7) . opposite to other animal production sectors, aquaculture animals live in the aquatic environment, which hinders individual treatment approaches. thus, passive immunization through injection is not the most feasible strategy for fish. however, research is nowadays being conducted on encapsulated antibodies to be delivered in-feed (46) , as it was previously described for pigs (47) . in any case, experimental injection trials, such as the one presented in this manuscript, allow to test whether spabs could be effective for treating a particular infection and can serve as a base to develop future control strategies such as vaccination or in-feed approaches (8) . in passive immunization trials two main considerations are important: the time of the injected antibodies to reach the bloodstream, and the half-life of these antibodies in circulation (8) , which will indicate the time-frame of protection. in salmonids, i.c. injected allogeneic igm against vibriosis were detected in serum 10 min post-injection and the uptake was complete after 8 h. these antibodies remained protective for up to 60 days (48, 49) . in carp, the half-life of allogeneic igm was much shorter, 22.5 days (50) . it is clear that the half-life of serum antibodies varies depending on the species, but teleost igs are assumed to remain in circulation from 12 to 16 days still retaining the antigen binding efficiency (9) . in the current study, we performed a preliminary test that revealed that i.c. injected rabbit igg could be detected in gsb serum from 1 h to 9 days post-injection in high concentrations. however, igg besides being xenogeneic, which could elicit a host response against the injected molecules, is also a monomer, so faster absorption times could be expected. in any case, these results, together with literature data, allowed us to plan the injection timings. fish were challenged with the parasite 24 h after serum injection, which is a safe window to ensure complete uptake of the antibodies. a second injection was performed 9 dpc to ensure that injected antibodies remained in circulation during the whole challenge period (23 days). the current passive immunization trial did not show significant protection of gsb against e. leei. however, the results indicate that injection with parasite-spabs delayed the initial propagation of the parasite in naïve gsb. the group that received serum with parasite-spabs (sur) showed minor signs of infection, particularly in terms of sgr and intensity of infection than fish injected with serum without spabs or pbs. e. leei invades and proliferates in the intestinal epithelium, causing anorexia, impaired nutrient absorption and weight loss, leading to emaciation (25) . decreased cf and sgr have been linked to e. leei infection in several studies (25, 51, 52) . this effect was evident in the pbs and nai-injected groups, showing a higher prevalence of infection. sur-injected fish also were infected, reaching 55% of prevalence of infection 100 dpc. however, these animals showed less disease signs than the other two challenged groups, in coincidence with lower prevalence and progression of the infection. e. leei starts invading the posterior part of the intestine, slowly progressing to the anterior and finally to the middle segment of the intestine (25, 53) . hence, the number of intestinal segments parasitized serves as a measure of the progression of the infection and provides a hint of the time a fish has been parasitized. in fact, the number of intestinal segments parasitized was significantly correlated (negatively) with disease signs such as cf (34) . in the same study, intensity of infection, measured as ct values of the parasite 18s rrna, was significantly correlated with cf, sgr and extension of the infection. lower ct values indicate more copies of parasite 18s rrna and coincided with stronger disease signs and more intestinal segments parasitized (34) . the current results are in agreement with this previous observation. enteromyxum leei induces a massive hyperplasia of the intestinal lamina propria-submucosa due to recruitment and proliferation of heterogeneous leukocytes (54) . igm + cells massively proliferate in the submucosa and within the epithelium (27) , whereas zap70 + t cells aggregate mainly at the base of the epithelium (28) . this inflammatory response, together with enterocyte apoptosis and necrosis, hamper intestinal barrier integrity producing nutrient malabsorption and osmotic intestinal failure (31) . as expected, these signs were also observed in the current experiment, where challenged fish showed an inflammatory response in the submucosa with abundant zap70 + t cells at the base of the epithelium and infiltrated in it. nutrient absorption impairment was also reflected in the reduction of lipid and glycogen deposits in the liver, which were probably used as an energy source to compensate the low energy intake. again, fish injected with parasite-spabs, even though being infected, showed lower signs than the other groups, probably denoting a later onset of the disease. antibodies have already been described as main actors in gsb immune response against e. leei (29) , and in other fish-myxozoan models (55) (56) (57) (58) (59) . the presence of high levels of circulating parasite-specific igm in gsb that survived an infection with e. leei has been linked to their resistance to re-infection (29) . e. leei induces igm and igt gene expression both at local (intestine) and systemic levels (head kidney), with a potent local effect. this increased gene expression has been correlated with increased serum levels of parasite-specific igm (29, 31) . regretfully, no information is available on levels of parasite-specific igt or whether these spabs can be found in circulation, as igt has been described mainly as a mucosal ig (20) . b cell responses against e. leei have been detected from 40 dpc onward (26, 27, 29, 35) , and this response is temperature dependent (34, 60) . delayed b cell responses against myxozoan parasites have already been described, with spabs being detectable only after 6-8 weeks (61) . in the current challenge, we did not observe major differences on circulating igm and igt among injected groups. the higher presence of total circulating igt in the pbs injected group could be related to a higher reaction to the antigen, as this group was the one showing the higher prevalence and progression of the infection. however, the mechanism behind this and the specificity of the antibodies should be checked in future studies. fish injected with specific-antibodies showed an increase in total igm, but this increase was not reflected as a parasite-specific response, therefore, this difference remains to be explained. in any case, as sur injected fish seem to have been protected in the first weeks of infection, a lower amount of parasite-spabs would support the latter onset of the disease. however, all groups challenged with the parasite showed an increase in parasite-specific igm in serum after 100 days, with no difference among groups. the overall low antibody response detected in the current experiment could be attributed to the low temperature as previously described (34) . the current results suggest that the injected antibodies may be acting at the initial steps of parasite invasion probably by blocking the parasite host-specific receptors (opsonization) and/or promoting phagocyte activity (62, 63) . however, probably due to the long-term nature of the infection, the injected antibodies were not enough against the parasite pressure. similar results have been found in other fish slow-progressing parasites models. passively immunized rainbow trout showed a lower, although not significant, abundance of the monogenean gyrodactylus derjavini one month after infection (6) . also in rainbow trout, passive immunization against the microsporidian parasite loma salmonae was not enough to protect the fish against infection, but delayed its arrival at the heart by one week (64) . in our experiment, fish were challenged with parasite containing effluent water for 23 days and then each group was separated to tanks with clean water to avoid excessive parasite pressure. nonetheless, cohabitation is also an effective infection route for e. leei (34) . thus, even with one animal getting infected during the challenge phase, the disease can still be transmitted to the cohabitants at later time points, when passively transferred antibodies are no longer viable. future experiments using repeated injections of serum throughout the whole period, injecting higher amounts of immune serum, and/or separating the fish individually after the challenge phase, will help to determine the exact degree of protection that can be acquired by passive immunization against e. leei. the current results support the hypothesis from previous studies that a spab response is key against enteromyxosis in gsb (29) . thus, we further analyzed the antibody response being elicited in the animals that acted as serum donors for the passive immunization trial. these animals had high levels of circulating parasite-specific igm and were resistant to reinfection. the repertoire analysis was performed on the anterior intestine, as previous studies revealed that in this particular organ igm and igt transcript expression increased upon re-exposure to the parasite in resistant fish (29) . the cdr3 of the vh domain was targeted because this region is the key determinant of specificity in antigen recognition in antibodies and the t cell receptor, whereas cdr1 and cdr2 sequences are much more cross-reactive (65) . a strong and specific response against a pathogen would induce a decrease in the clonotype diversity in favor of the expansion of a specific pathogen-specific subset. this phenomenon has been described in fish in some viral infections (17) . parasites, however, are much more complex organisms, in particular metazoans with different life stages like myxozoans. parasites can harbor vast amounts of antigenic peptides and some even demonstrated specific strategies to avoid the host's antibody responses. for instance, some parasites are known to induce hypergammaglobulinemia, resulting in a polyclonal expansion with significantly increased antibody titers but a limited proportion of parasite-spabs. this has been reported for human and fish protozoan parasites, such as trypanosoma cruzi (66) or trypanoplasma borreli (67), but also in some myxozoan infections caused by sphaerospora molnari (68) or tetracapsuloides bryosalmonae (23) . the current results, showing a significant increase in clonotype diversity for both igm and igt, together with previous findings on this host-parasite model (27, 35) , seem to indicate a similar strategy for e. leei. close to 90% of igt and igm clonotypes were found 1-5 times, indicating a high degree of repertoire diversity in gsb anterior intestine. this high clonotype diversity, with very few clonotypes expressed more than 50 times, has been described in naïve rainbow trout spleen and, upon viral (17) or parasitic (23) infection, having certain clonotypes a high frequency, and indicating a clear response against the pathogens. the results of the current study deal with the ig repertoire of intestinal b cells, thus the pattern is expected to be different from that found in spleen. intestine is a major mucosal tissue that is in close contact with the environment and harbors a very diverse community of symbiont microorganisms, the microbiota. hence, intestinal cells are constantly exposed to a myriad of antigens. this probably explains the different patterns found in naïve animals in our study with more abundant high frequency clonotypes and non-gaussian cdr3 length distributions. the cdr3 length distribution analysis is an estimate of the overall diversity and deviations from a bell-shaped gaussian distribution are indicative of clonal expansions (69) . the different distribution of igm cdr3 lengths, and the significantly more abundant igt clonotypes with frequencies of 101-500 in sur animals indicate that e. leei is inducing a clear response that involves both isotypes but probably through different mechanisms. a complete characterization of a gsb specific reference germline throughout the definition of the gsb igh locus following the identification of all segments encoding v, d and j genes will allow further deep analyses of this response at gene level. thus, the definition of a gsb specific germline will allow a better dissection of clonotypes specifically selected during the response to myxozoans as well as will allow to further explore somatic hypermutation patterns in selected clonotypes. to conclude, e. leei induces a differential igm and igt response in gsb characterized by a polyclonal expansion of diverse ig subsets. this could be part of an immuneevasion strategy elicited by the parasite aiming to dilute parasite-spabs by increasing the proportion of irrelevant antibodies, as has been described in similar host-parasite models (23, 68) . however, increased levels of parasite-spabs (igm) have been detected in the serum of gsb when challenged with the parasite, and the presence of high levels of these antibodies has been related with acquired resistance. in addition, the current results show that passive transfer of parasite-spabs can delay the establishment of the infection and the appearance of disease signs. taken together, these findings indicate that, regardless of the probable immuneevasion strategy of the parasite, antibodies are key molecules against enteromyxosis in gsb. future studies should be conducted to further evaluate the role of specific igt, a specialized mucosal immunoglobulin, in this model. also, more in depth studies on the protective antibody response will be key to develop effective treatments and prophylactic methods against e. leei. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found here: https://www.ncbi.nlm. nih.gov/, prjna644800. the animal study was reviewed and approved by ethics and animal welfare committee of iats, csic and generalitat valenciana. the authors thank p. boudinot (inrae) for his help in designing and interpreting the immunoglobulin repertoire study and results, j. pérez-sánchez (iats-csic) for providing access to the gilthead sea bream genome sequences to perform the repertoire analysis, and j. monfort and l. rodríguez (iats-csic) for histological processing. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.581361/full#supplementary-material antimicrobial drug resistance in fish pathogens active and passive immunity, vaccine types, excipients and licensing passive immunity in the prevention of rabies genetic variation in susceptibility to trypanoplasma borreli infection in common carp (cyprinus carpio l.) acquired immunity to amyloodiniosis is associated with an antibody response acquired resistance in rainbow trout against gyrodactylus derjavini surface antigen cross-linking triggers forced exit of a protozoan parasite from its host passive immunization of farmed fish mucosal immunoglobulins and b cells of teleost fish fish immunoglobulins systemic and mucosal immune response of rainbow trout to immunization with an attenuated flavobacterium psychrophilum vaccine strain by different routes common carp have two subclasses of bonyfish specific antibody igz showing differential expression in response to infection gene expression analyses of immune responses in atlantic salmon during early stages of infection by salmon louse (lepeophtheirus salmonis) revealed biphasic responses coinciding with the copepod-chalimus transition distribution of igm, igd and igz in mandarin fish, siniperca chuatsi lymphoid tissues and their transcriptional changes after flavobacterium columnare stimulation zebrafish immunoglobulin igd: unusual exon usage and quantitative expression profiles with igm and igz/t heavy chain isotypes the teleost humoral immune response teleost fish mount complex clonal igm and igt responses in spleen upon systemic viral infection early immune responses in rainbow trout liver upon viral hemorrhagic septicemia virus (vhsv) infection discovery of a unique ig heavy-chain isotype (igt) in rainbow trout: implications for a distinctive b cell developmental pathway in teleost fish igt, a primitive immunoglobulin class specialized in mucosal immunity discovery and characterization of secretory igd in rainbow trout: secretory igd is produced through a novel splicing mechanism teleost igd+ igm-b cells mount clonally expanded and mildly mutated intestinal igd responses in the absence of lymphoid follicles dysregulation of b cell activity during proliferative kidney disease in rainbow trout myxidium leei n. sp., a pathogenic myxosporean of cultured sea bream sparus aurata enteromyxum species modulation of leukocytic populations of gilthead sea bream (sparus aurata) by the intestinal parasite enteromyxum leei (myxozoa: myxosporea) modulation of the igm gene expression and the igm immunoreactive cell distribution by the nutritional background in gilthead sea bream (sparus aurata) challenged with enteromyxum leei (myxozoa) hints on t cell responses in a fish-parasite model: enteromyxum leei induces differential expression of t cell signature molecules depending on the organ and the infection status acquired protective immune response in a fish-myxozoan model encompasses specific antibodies and inflammation resolution effects of enteromyxum spp. (myxozoa) infection in the regulation of intestinal e-cadherin: turbot against gilthead sea bream disruption of gut integrity and permeability contributes to enteritis in a fish-parasite model: a story told from serum metabolomics experimental transmission of myxidium leei in gilthead sea bream sparus aurata effect of host factors and experimental conditions on the horizontal transmission of enteromyxum leei (myxozoa) to gilthead sea bream, sparus aurata l., and european sea bass, dicentrarchus labrax (l.) sitjà-bobadilla a. water temperature, time of exposure and population density are key parameters in enteromyxum leei fish-to-fish experimental transmission differential modulation of igt and igm upon parasitic, bacterial, viral, and dietary challenges in a perciform fish genome sequencing and transcriptome analysis reveal recent speciesspecific gene duplications in the plastic gilthead sea bream (sparus aurata) acting locally-affecting globally: rna sequencing of gilthead sea bream with a mild sparicotyle chrysophrii infection reveals effects on apoptosis, immune and hypoxia related genes manipulation of fastq data with galaxy pear a fast and accurate illumina paired-end read merger imgt, the international immunogenetics information system r tools for the nucleotide analysis of immunoglobulin (ig) and t cell receptor (tr) v-(d)-j repertoires, polymorphisms, and ig mutations: imgt/v-quest and imgt/highv-quest for ngs vdjtools: unifying post-analysis of t cell receptor repertoires immunarch: an r package for painless bioinformatics analysis of t-cell and b-cell immune repertoires towards physiologically and tightly regulated vectored antibody therapies coronavirus drugs: using plasma from recovered patients as a treatment for covid-19 protective effect of in-feed specific igm towards yersinia ruckeri in rainbow trout natural pig plasma immunoglobulins have anti-bacterial effects: potential for use as feed supplement for treatment of intestinal infections in pigs metabolism of coho salmon ig: intraperitoneal absorption properties of coho salmon tetrameric ig humoral factors important in resistance of salmonid fish to bacterial disease. ii. anti-vibrio anguillarum activity in mucus and observations on complement freshwater fish trypanosomes: definition of two types, host control by antibodies and lack of antigenic variation the nutritional background of the host alters the disease course in a fish-myxosporean system under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets novel horizontal transmission route for enteromyxum leei (myxozoa) by anal intubation of gilthead sea bream sparus aurata fish immune responses to myxozoa protective acquired immunity to enteromyxum scophthalmi (myxozoa) is related to specific antibodies in psetta maxima (l.) (teleostei) whirling disease: reemergence among wild trout humoral immune response of carp cyprinus carpio to myxobolus artus (myxozoa: myxobolidae) infection antigenic and biochemical study of pkx, the myxosporean causative agent of proliferative kidney disease of salmonid fish development of immunohistochemistry and enzyme-linked immunosorbent assays for the detection of circulating antibodies against enteromyxum scophthalmi (myxozoa) in turbot (scophthalmus maximus l.) impacts of low temperature on the teleost immune system fish immune response to myxozoan parasites influenza prevention and treatment by passive immunization the role of opsonization by antibody and complement in in vitro phagocytosis of microsporidian parasites by turbot spleen cells altered tissue distribution of loma salmonae: effects of natural and acquired resistance diversity in the cdr3 region of v(h) is sufficient for most antibody specificities trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic b-cell response which is a high source of non-parasite-specific antibodies the immune response of carp to trypanoplasma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation the kinetics of cellular and humoral immune responses of common carp to presporogonic development of the myxozoan sphaerospora molnari statistical analysis of cdr3 length distributions for the assessment of t and b cell repertoire biases key: cord-261367-i1n8x0uc authors: hwang, ji young; silva-sanchez, aaron; carragher, damian m.; garcia-hernandez, maria de la luz; rangel–moreno, javier; randall, troy d. title: inducible bronchus–associated lymphoid tissue (ibalt) attenuates pulmonary pathology in a mouse model of allergic airway disease date: 2020-09-25 journal: front immunol doi: 10.3389/fimmu.2020.570661 sha: doc_id: 261367 cord_uid: i1n8x0uc inducible bronchus associated lymphoid tissue (ibalt) is an ectopic lymphoid tissue associated with severe forms of chronic lung diseases, including chronic obstructive pulmonary disease, rheumatoid lung disease, hypersensitivity pneumonitis and asthma, suggesting that ibalt may exacerbate these clinical conditions. however, despite the link between pulmonary pathology and ibalt formation, the role of ibalt in pathogenesis remains unknown. here we tested whether the presence of ibalt in the lung prior to sensitization and challenge with a pulmonary allergen altered the biological outcome of disease. we found that the presence of ibalt did not exacerbate th2 responses to pulmonary sensitization with ovalbumin. instead, we found that mice with ibalt exhibited delayed th2 accumulation in the lung, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production. the presence of ibalt did not alter th2 priming, but instead delayed the accumulation of th2 cells in the lung following challenge and altered the spatial distribution of t cells in the lung. these results suggest that the formation of ibalt and sequestration of effector t cells in the context of chronic pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology. inducible bronchus associated lymphoid tissue (ibalt) is an ectopic lymphoid tissue that forms in the lung following inflammation or infection (1, 2) . large b cell follicles that often contain germinal centers (gcs) and a dense network of follicular dendritic cells (fdcs) are the most prominent features of ibalt (3) . unlike conventional lymph nodes, ibalt is not encapsulated, but is instead embedded in the lung tissue, most often along large bronchi or filling the peri-vascular space of pulmonary artieries (4, 5) . despite being in a mucosal tissue, ibalt often lacks a welldefined m cell-containing dome epithelium (6) and its high endothelial venules (hevs) express peripheral lymph node addressin (pnad) (7) rather than mucosal addressin cell adhesion molecule (madcam). thus, although ibalt clearly participates in pulmonary mucosal immune responses, it lacks some of the features often associated with classic mucosal lymphoid organs. unlike conventional lymphoid organs, which develop independently of antigen during embryogenesis (8, 9) , ibalt forms following pulmonary infection or inflammation (10, 11) . although ibalt may develop at any time after birth given the proper pulmonary insult, it most easily forms in neonatal mice due to the relative paucity of tregs and the relative abundance of il-17-producing γδt cells (12) (13) (14) . similarly, infant humans also seem to develop (or maintain) ibalt more easily than adults, as the frequency of ibalt areas in the lungs of healthy subjects progressively declines with age (15, 16) . interestingly, the neonatal period is the same developmental window when microbial exposure, or lack thereof, imprints the immune system to be more or less susceptible to developing atopic responses to foreign antigens (17, 18) or developing autoreactive responses to self antgens-the so-called hygiene effect. given that it functions as a lymphoid tissue (19) , it is not too surprising that ibalt plays an important role in immunity to pulmonary infections. for example, mice lacking conventional lymphoid organs develop ibalt following infection with influenza and are able to resist doses of virus that would normally be lethal to mice lacking ibalt (19) . conversely, mice lacking the chemokines that promote ibalt formation succumb to much lower doses of influenza virus (7) . those same chemokines are essential for the proper formation of pulmonary granulomas, the recruitment of antigen-specific t cells to the lung and the development of protective immunity to mycobacterium tuberculosis (20, 21) . moreover, the pulmonary administration of inflammatory agents that trigger ibalt formation also leads to enhanced resistance to influenza, sars corona virus, pneumovirus, francicella tulerensis and coxiella burnetti (12, 19, (22) (23) (24) . thus, the presence of ibalt is clearly beneficial in the context of pulmonary infections. in contrast, ibalt is a feature of pulmonary pathology that is associated with a variety of chronic lung diseases, including chronic obstructive pulmonary disease (copd) (25, 26) , rheumatoid lung disease (3), hypersensitivity pneumonitis (27) , and asthma (28, 29) , suggesting that ibalt may exacerbate these clinical conditions. for example, copd patients make autoantibodies that react with pulmonary antigens (30) and patients with advanced disease have reactive ibalt areas with well-developed gcs that show evidence of antigendriven selection (31) . similarly, patients with rheumatoid lung disease develop large gc-containing areas of ibalt that are surrounded by plasma cells that produce autoantibodies (3) . thus, many investigators have concluded that the presence of ibalt contributes to pathology in the context of chronic inflammatory diseases. however, it is not clear whether ibalt is a cause or consequence of pulmonary inflammation or whether it contributes in any way to pulmonary pathology (32) . given the link between ibalt and pulmonary pathology, we tested whether pre-existing ibalt would affect acute allergic responses to a pulmonary allergen. surprisingly, we found that the presence of ibalt did not exacerbate allergic responses to repeated pulmonary sensitization with ovalbumin (ova). instead, mice with ibalt had reduced th2-associated mrna expression, less eosinophil recruitment to the lungs and airways, attenuated goblet cell hyperplasia and reduced mucus production following pulmonary sensitization and challenge with ova. interestingly, the presence of ibalt did not alter the initial priming of th2 cells, but instead delayed their recruitment to the lung and altered their spatial distribution following challenge-th2 cells were preferentially sequestered in ibalt, which reduced the numbers of th2 cells in the lung parenchyma. these results suggest that the formation of ibalt and sequestration of effector t cells in the context of pulmonary inflammation may be a mechanism by which the immune system attenuates pulmonary inflammation and prevents excessive pathology. to test the role of ibalt in a mouse model of allergic lung inflammation, we intranasally administered 10 µg lps 5 times over the course of 10 days starting on day 2 after birth and analyzed the lungs by histology on day 70 ( figure 1a) . as expected (14) , we observed ibalt structures with separated b and t cell zones and distinct fdc networks in the lpstreated lungs, but not in the control lungs ( figure 1b) . we also quantified the number of gc b cells by flow cytometry and found that gc b cells were present in lps-treated lungs, but not in control lungs (figures 1c,d) . given that gcs only form in organized lymphoid tissues, we conclude that pulmonary exposure of neonates to lps promotes the formation of functional ibalt areas. to test the effect of ibalt on the immune response to a pulmonary allergen, we administered lps (or pbs) to neonatal mice as described above, allowed the mice to rest until they were 7 weeks old, then intranasally sensitized the ibalt and control groups with 100 µg ova in combination with low dose (0.1 µg) lps on days 49, 50, and 51 and challenged them on days 63, 64, 67, and 68 with 25 µg ova ( figure 1e ). we first examined the gc b cell response in the lungs of sensitized and challenged mice and found that gc b cells were abundant in the lungs of mice with ibalt, but were rare in control mice and almost undetectable in completely naïve mice (figures 1f,g) . we also examined the inflammatory response in the lung and airways by cytospin and found that the proportion (figures 1h,i) and number (figures 1j,k) of eosinophils were elevated in mice that had been sensitized and challenged with ova compared to those in naïve mice, however, the proportion (figures 1h,i) and number (figures 1j,k) of eosinophils in mice with ibalt were reduced compared to those in control mice. reductions in eosinophils were observed in both the lungs (figures 1h,j) and the airways (figures 1i,k) of mice with ibalt compared to those in control mice. the proportions of macrophage/monocytes, lymphocytes, and neutrophils are also shown in figure 1h and the numbers of those cells are shown in supplementary figure 1 . we also found that total serum ige concentration was elevated in control mice, but less ige was detected in mice with ibalt ( figure 1l) . upon a histological examination of lung sections, we found that ovaexposed mice with pre-existing ibalt had densely-packed areas of lymphocytes to one side of the bronchi, but relatively clear airspaces, whereas ova-exposed control mice had extensive areas of diffuse inflammation that surrounded the bronchi and extended into the alveolar airspaces ( figure 1m) . thus, contrary to our expectations, the presence of ibalt did not exacerbate ova-induced pulmonary inflammation, but rather reduced the inflammatory response. the presence of ibalt could have altered t cell priming so that fewer th2 cells were initially generated after antigen exposure. to test this possibility, we next used il-4 reporter (4get) mice to enumerate th2 cells. the 4get mice have the enhanced green fluorescent protein (egfp) targeted into the il-4 locus with an internal ribosome entry site (ires) separating the il-4 coding sequence from the egfp coding sequence (33) . thus, any cells that express il-4 mrna will also express egfp and can be easily identified using flow cytometry (34) . therefore, we treated neonatal 4get mice with lps to initiate ibalt formation, waited until they were adults and then intranasally sensitized ibalt and control mice with 100 µg ova and 0.1 µg lps on days 49, 50, and 51 (figure 2a) . two days after the last sensitization, we enumerated il-4 reporter (egfp)-expressing cd4 + t cells. we found a significant expansion of egfp + cd4 + t cells in both ibalt and control mice relative to naïve mice, but did not observe any difference in the frequencies ( figure 2b ) or numbers ( figure 2c ) of egfp + cd4 + t cells between control and ibalt groups. these data suggested that ibalt did not dramatically affect th2 priming in the lung. although the previous assay evaluated th2 priming, we had no way of enumerating antigen-specific cells. therefore, to test this possibility in another way, we transferred 1 x 10 6 naïve ovaspecific cd4 + t cells (ot-ii cells) into control and ibalt mice on day 48 after birth, sensitized the recipient mice with ova on days 49, 50, and 51, challenged them on days 63, 64, 67, and 68 and enumerated the responding cells 2 days later in the lung ( figure 2d ). we found that the frequency ( figure 2e ) and number ( figure 2f ) of ot-ii cells that accumulated in the lungs of sensitized and challenged mice were the same in control and ibalt groups. these data suggest that the presence of ibalt does not affect ova-specific t cell accumulation in the lung after sensitization and challenge. although th2 priming and expansion appeared normal in mice with ibalt, it was not clear whether the final effector cells could actually make th2 cytokines. therefore, to determine whether cd4 t cells primed by ova sensitization and challenge could actually produce th2 cytokines, we sensitized and challenged mice with and without pre-existing ibalt and measured the mrna expression of th2 cytokines in the lungs 48 h after the last ova challenge ( figure 3a) . we found that mrnas encoding il-4, il-5, and il-13 were increased in ova-exposed and challenged lungs relative to their expression in naïve lungs, but we did not observe a significant difference between the control and ibalt groups ( figure 3b) . we next tested whether cd4 + t cells from ova sensitized and challenged control and ibalt mice were equally capable of making th2 cytokines. to test this possibility, we collected cells from the lung tissue, restimulated them with pma and calcimycin for 4 h and assessed the production of il-4, il-5, and il-13 by intracellular staining. we found that the total number of activated cd44 hi cd4 + t cells was increased in control and ibalt mice relative to that in naïve mice (figures 3c,d) , but there was little difference between the control and ibalt groups. we also found that the numbers of cd44 hi cd4 + t cells that made il-4, il-5, or il-13 were increased in control and ibalt mice relative to those in naïve mice ( figure 3e ), but there was little difference between the control and ibalt groups. these data suggest that the presence of ibalt does not significantly alter the number of t cells capable of producing th2 cytokines following sensitization and challenge. th2 cytokines act on other cells in the lung, such as epithelial cells and macrophages, and promote their activation and differentiation, which changes gene expression. for example, genes like chitinase-3-like-4 (chi3l4) and mucin 5ac (muc5ac) are expressed in in the lung following il-13 signaling (35) . thus, we next examined the expression of mrnas encoding these genes in the lungs of ibalt and control mice after ova sensitization and challenge as in figure 3a . we found that mrnas encoding chi3l4 and muc5ac were increased in ova-exposed and challenged lungs relative to their expression in naïve lungs, but their expression in the ibalt group was significantly less than that in the control group ( figure 3f) . thus, the expression of genes that are responsive to th2 cytokines were reduced in the lungs of ibalt mice relative to those in control mice. these data are more consistent with the reduced histopathology that we see in the lungs of ibalt mice compared to those in the lungs of control mice, suggesting that the difference between the groups is not in th2 priming and expansion, but in the response to th2 cytokines. in the experiments above, we were unable to independently control the initial steps of t cell priming and expansion. to rigorously show that these steps were unaffected by the presence or absence of ibalt, we next generated ova-specific th2 effector t cells in vitro and showed that, upon restimulation, a high frequency of these cells made il-13, whereas less than 1% made ifnγ and almost none of them made il-17 ( figure 4a ). we next adoptively transferred 1 x 10 6 of these th2 cells into 48-day-old control or ibalt mice and challenged them with ova on days 49, 50, 53, and 54 ( figure 4b ). one day after the final challenge, we enumerated donor t cells in the lungs and found that a similar frequency ( figure 4c ) and number ( figure 4d ) of donor t cells had accumulated in the lungs of both groups. we also enumerated gc b cells in the lungs and found that a high frequency ( figure 4e ) and number ( figure 4f ) of gc b cells accumulated in the lungs of mice in the ibalt group, but not in the lungs of control or naïve mice. despite having similar numbers of donor th2 cells in the lungs of each group, the histology of the lungs was dramatically different, with the control group exhibiting extensive goblet cell hyperplasia and diffuse inflammatory infiltrate throughout the lungs, whereas the ibalt mice exhibited minimal goblet cell hyperplasia and dense lymphoid accumulations (ibalt) adjacent to, but not surrounding, the airways and in the perivascular space ( figure 4g) . we also quantified the amount of th2 cytokines in the bal fluid 6 h after the last challenge and found that, although the amount of il-4, il-5, and il-13 was slightly less than in the bal fluid of ibalt mice than in control mice, the differences were not significant ( figure 4h ). together, these data demonstrate that, despite starting with the same number of th2 cells in the lung, the biological outcome of inflammation was dramatically different in control and ibalt mice. given that the biological outcome of disease was so different despite starting with similar numbers of fully differentiated th2 cells, we reasoned that ibalt must engage some sort of regulatory mechanism. one important cell type that has the ability to powerfully attenuate inflammatory responses is the treg population (36) . tregs are immunosuppressive cd4 + t cells that have differentiated in a way that promotes the expression of the transcription factor, foxp3 (37) . cd4 + foxp3 + tregs can reduce eosinophilia (38) , impair humoral responses and secret an inhibitory cytokines, such as il-10 and tgfβ (39) , which suppress the effector functions of activated t cells and reduce inflammation. thus, changes in treg number or activity can have dramatic consequences on immune responses regardless of the number of antigen-specific effector t cells. given the immunosuppressive capacity of tregs, we enumerated cd4 + foxp3 + t cells in the lungs of ibalt and control mice following ova sensitization and challenge. we found that although the frequency ( figure 5a ) and number (figure 5b ) of tregs increased in the lungs following ova sensitization and challenge, there was no difference in tregs between ibalt and control groups. thus, alterations in treg numbers do not explain why mice with ibalt exhibit attenuated allergic responses in their lungs. previous reports show that pulmonary administration of lps to the lung conditions epithelial cells in a way that promotes the over-expression of a20 (40) , an ubiquitinmodifying enzyme that blunts nf-kb signaling thereby reducing expression of pro-th2 cytokines like il-33 (41) . thus, we were concerned that the exposure of neonatal mice to lps may permanently suppress pulmonary inflammatory responses. to test this possibility, we treated neonatal mice with lps or pbs to trigger ibalt formation and, when the mice were 7-weeks old, transferred ova-specific th2 effector cells and challenged the recipients 4 times with either ova or pbs ( figure 5c) . we then enzymatically digested the lung tissue, sorted epcam + cd31 − sca1 − cd45 − bronchial epithelial cells and performed qpcr for tnfaip3, the gene encoding the a20 enzyme. we found that tnfaip3 expression was similar in all groups of bronchial epithelial cells (figure 5d) , regardless of whether they came from mice that received lps as neonates or whether they came from mice that were challenged with ova as adults. we also performed qpcr to quantify the expression of the inflammatory cytokines, il-33 and tslp. we found that although il-33 expression was strongly increased following ova challenge, it was not altered by previous exposure to lps (figure 5e ). we also found that tslp expression was not changed by either ova challenge or previous exposure to lps ( figure 5e) . thus, the pulmonary exposure of neonatal mice to lps did not permanently alter the ability of bronchial epithelial cells to express inflammatory cytokines. despite the overall reduction in th2-driven pathology in the mice with ibalt, the number of th2 effector cells seemed to be similar at the endpoint of the experiment. however, we didn't know whether they accumulated in the lung at the same rate. to determine whether the kinetics of t cell recruitment was the same in mice with and without ibalt, we generated th2 effectors from naïve otii cells in vitro and then adoptively transferred 1 x 10 6 th2 effectors 24 h prior to a series of 4 ova challenges ( figure 6a) . we then enumerated the transferred th2 cells and the recruited eosinophils in the lungs and bal 24 h after each ova challenge. not surprisingly, the numbers of donor t cells and eosinophils increased over time following each challenge (figures 6b-e) . unexpectedly however, we observed significant reductions in the numbers of th2 effectors (figures 6b,d) and eosinophils (figures 6c,e) in both the lungs (figures 6b,c) and airways (figures 6d,e) of ibalt mice relative to controls after the first 3 challenges. however, by the fourth challenge, there was no difference in the numbers of t cells. these data suggest that the presence of ibalt slows the accumulation of th2 effectors as well as eosinophils following pulmonary allergen exposure. these data may also explain why we saw little difference in t cell numbers at the endpoints of earlier experiments, even though we observed significant differences in lung pathology. the most unique attribute of ibalt is its dense accumulations of spatially ordered lymphocytes in a tissue (the lung) that normally has relatively few lymphocytes and rarely has them so densely organized. therefore, we next asked whether the specialized environment of ibalt alters the spatial organization of t cells in the lung following ag exposure. to test this possibility, we generated ova-specific th2 effector cells in vitro using otii cells. these cells were subsequently transferred into ibalt and control mice and the recipients were challenged with ova 4 times before we examined the lungs by histology (figure 7a) . we performed immunofluorescence looking for the donor t cell marker (cd45.1), b cells (b220), and fdcs (cd21). we found that the donor effector th2 cells preferentially accumulated in the ibalt areas of ibalt mice and were relatively dilute in the rest of the lung (figure 7b ). in contrast, the donor th2 effector cells were distributed more evenly in the control mice. these data were quantified in figure 7c . importantly, we found that the total number of donor th2 effector cells in the lungs of mice with and without ibalt were indistinguishable at this time ( figure 7d) . we also examined cd4+foxp3+ tregs in tissue sections from mice with and without ibalt. we found that tregs were densely clustered in areas of ibalt, but were relatively dilute in the rest of the lung (figures 7e,f) , despite the similar numbers of tregs in the lungs of mice with and without ibalt (figures 5a,b) . together, these data suggest that the spatial distribution of effector th2 cells and tregs is affected by the presence of ibalt (they cluster together), which may explain how ibalt and control mice can have similar numbers of th2 cells in their lungs, but have so profoundly different outcomes in terms of eosinophil accumulation and histopathology. the above experiments used mice that had been administered lps as neonates in order to form ibalt. however, one concern with these experiments is that neonatal exposure to lps might have altered systemic immunity in some way that ameliorated subsequent inflammatory responses independently of ibalt formation. to rule out this possibility, we generated in vitrodifferentiated otii th2 cells and transferred 1 x 10 6 th2 effector cells into control mice, mice that received pulmonary lps as neonates (ibalt mice) and mice that received peritoneal lps as neonates (i.p. lps mice). the following 2 days, we administered 25 µg ova intranasally to all groups and measured germinal center b cells and eosinophils in the lungs 24 h later ( figure 8a) . when we enumerated th2 effectors ( figure 8b ) and eosinophils ( figure 8c ) in the lung tissue, the numbers were reduced in the presence of ibalt compared to control mice, but in mice treated with lps intraperitoneally, the number of effector t cells and eosinophils were the same as in the control mice (figures 8b,c) . thus, the exposure of neonates to pulmonary lps dramatically decreased the inflammatory response in adults, whereas the exposure of neonates to peritoneal lps did not. we also observed that mice that receive pulmonary lps as neonates (ibalt mice) had a significantly higher frequency ( figure 8d ) and number (figure 8e ) of gc b cells in the lungs, whereas mice that received lps intraperitoneally as neonates (i.p. lps mice) did not accumulate gc b cells and were comparable to control mice (figures 8d,e) . these data indicate that systemic exposure to lps does not lead to ibalt formation (as monitored by the germinal center response in the lungs). these data are consistent with the conclusion that lps-mediated ibalt formation alleviates th2-dependent inflammatory responses in the lung, independently of any systemic effect of lps on immunity. our data show that the presence of ibalt in the lungs prior to pulmonary sensitization and challenge with ova does not exacerbate th2 responses, but rather attenuates th2-driven pulmonary pathology. in fact, mice with ibalt exhibit delayed th2 accumulation, reduced eosinophil recruitment, reduced goblet cell hyperplasia and reduced mucus production compared to their control counterparts. although the initial priming of th2 cells is not affected by the presence of ibalt, the accumulation of th2 cells in the lung is delayed following pulmonary challenge. more importantly, the spatial distribution of effector t cells is altered by the presence of ibalt, such that effector cd4 t cells as well as tregs become concentrated in ibalt areas and relatively diluted in the rest of the lung. these results suggest that ibalt functionally sequesters effector t cells, thereby limiting the exposure of the lung parenchyma to t cellproduced inflammatory cytokines, which attenuates pulmonary inflammation and prevents excessive pathology. ibalt is associated with a wide variety of inflammatory lung diseases, including copd (26), hypersensitivity pneumonitis (27) and rheumatoid lung disease (3), all of which are the result of chronic exposure to antigens or inflammatory agents. although asthma is also a chronic lung disease, the mouse model of repeated sensitization and challenge with ova is more like an acute allergic response. however, our data demonstrate that the mere presence of ibalt does not necessarily lead to an increased inflammatory response or pulmonary pathology in the context of pulmonary allergen exposure as one might expect if ibalt was facilitating antigen presentation and t cell activation. therefore, despite participating in local immune responses, ibalt may be beneficial in the context of inflammatory diseases by sequestering activated lymphocytes. the idea of reducing inflammation by sequestering t cells in lymphoid tissue is consistent with the activities of the immunosuppressant drug, fty720, which prevents t cells from exiting lymphoid organs-effectively sequestering them and preventing their recirculation through peripheral tissues (42, 43) . in fact, treatment with fty720 reduces airway remodeling and pulmonary inflammation in a rat model of ova-induced asthma (44) . interestingly, ibalt areas are expanded in the lungs of fty720-treated animals, suggesting that the fty720-mediated sequestration of lymphocytes can occur in either conventional lymphoid organs or in areas of ibalt in the lung (13) . another physical change that occurs in the lung concomitantly with the development of ibalt is the formation of additional lymphatic vessels surrounding the ibalt follicles (45) . live imaging of ibalt suggests that these lymphatic vessels may facilitate the collection of dcs from the airways (46) . static imaging also shows that ibalt areas collect inhaled antigens and particulates (47) , suggesting that ibalt may sequester antigen as well as t cells. in this context, it is interesting to note that mice that spontaneously develop ibalt in the context of rheumatoid lung disease are highly resistant to developing fibrosis following pulmonary challenge with bleomycin (48) . if ibalt areas efficiently collect and sequester intratracheally administered bleomycin or remove it from the lung via lymphatics, then one would expect to observe reduced fibrosis. similarly, if ibalt areas collect and sequester pulmonary antigens like ova and remove them from the lung parenchyma, one would expect reduced pulmonary inflammation. thus, ibalt may protect the lung by collecting antigens and inflammatory substances as well as t cells. our data are also consistent with observations that ibalt attenuates inflammatory responses and improves clinical outcomes following infection with a variety of pulmonary pathogens. for example, the presence of ibalt promotes the survival of mice that are challenged with normally lethal doses of influenza virus, pneumovirus and sars corona virus (24) and attenuates the pulmonary pathology associated with these infections. much of the clinical pathology, such as weight loss, associated with pulmonary viral infections is linked to the production of inflammatory cytokines by t cells. thus, if ibalt sequesters virus-specific effector t cells, it may limit the exposure of the rest of the lung to inflammatory cytokines and chemokines and reduce the recruitment of additional inflammatory cells-thereby attenuating pathology. however, ibalt-mediated resistance to pneumovirus is associated with impressive increases, rather than decreases, in the mrna expression of ifnγ, cxcl10, and ccl3 in the lung (12) . these responses were measured by pcr using mrna extracted from whole lungs, so it is difficult to know whether the increases were predominantly confined to ibalt areas or not. in addition, it is not clear whether the reported increase is due to an overall increased magnitude of the response or altered kinetics, such that more t cells are responding earlier. this last possibility seems plausible, given that accelerated antibody responses are also observed in mice with ibalt (24) . interestingly, we also observe altered kinetics of effector t cell recruitment to the lungs following pulmonary challenge with allergen. however, we find that fewer effector th2 cells accumulate in the lungs at early times after allergen exposure. the differences between our results and those reported for pneumovirus may be due to the types of antigens (replicating virus vs. inert allergen) or to the types of t cells (virus-specific cd8 and th1 cells vs. allergen-specific th2 cells). in any case, the presence of ibalt clearly has an impact on the kinetics of pulmonary immune responses. t cells are often imprinted with particular effector functions that differ depending on the secondary lymphoid organ in which they were primed (49, 50) . thus, one could envision that ibalt skews cd4 t cell differentiation away from a th2 pathway and thereby attenuates the symptoms of allergic disease. however, we found equivalent numbers and frequencies of th2 cells primed in mice with or without ibalt, suggesting that cd4 t cell differentiation is not deviated toward an alternative effector function. moreover, we observed reduced pathology in mice with ibalt following the adoptive transfer of in vitro differentiated th2 effector cells, demonstrating that even when t cell priming occurs in vitro, the functional outcome of the effector response is altered beneficially in mice with ibalt. interestingly, naïve t cells primed in aortaassociated lymphoid tissues in apoe −−/−− mice preferentially differentiate into tregs that suppress atherosclerosis (51) . although the preferential formation of tregs in ibalt areas would help to explain the reduced inflammation and pulmonary pathology in mice with ibalt following ova sensitization and challenge, we did not observe changes in either the number and frequency of tregs in the lung, regardless of whether ibalt was present. nevertheless, we did find that tregs were preferentially concentrated in ibalt areas and co-localized with effector t cells, suggesting that tregs in ibalt may more effectively suppress the inflammatory functions of effector t cells in this location. thus, in the context of pulmonary sensitization with allergens such as ova, ibalt seems to alter the placement of t cells rather than changing their differentiation pathways. considering that many of the adoptively transferred th2 cells are being sequestered in the ibalt areas and seem to be located in the b cell follicle, it is possible that they are converting to il-4-expressing tfh cells (52) . this possibility would be consistent with the large gcs and high frequencies of gc b cells that we observe in the lungs following adoptive transfer of th2 cells and sensitization with antigen. in addition, ibalt-induced resistence to influenza and sars corona virus is associated with accelerated antibody responses (24) , suggesting that local tfh cells are promoting b cell differentiation. although th2-tfh cells in b cell follicles are potent producers of il-4 (53), they are directing cytokine secretion toward b cells rather than pulmonary epithelial cells, which would likely lead to accelerated antibody secretion, but reduced pulmonary pathology. thus, ibalt may be sequestering t cells and directing cytokine secretion toward alternative cell types, both of which should attenuate pulmonary pathology. in summary, our data show that the presence of ibalt does not exacerbate pulmonary pathology following sensitization and challenge with an allegen, but rather attenuates th2induced inflammatory responses. this same mechanism may also explain the ability of ibalt to ameliorate pulmonary pathology in the context of infection and may be a general mechanism by which ectopic follicles in a variety of tissues attempt to cope with chronic inflammatory responses. thus, we may want to develop treatments that promote, rather than prevent, ectopic follicle formation in the context of chronic inflammatory conditions. to induce ibalt, pups were intranasally administered 10 µg lps from escherichia coli (055:b5; sigma) in 10 µl pbs every other day starting on day 2 after birth for a total of 5 times. in control experiments, pups were intraperitoneally injected with 10 µg lps in 10 µl pbs every other day starting on day 2 after birth for a total of 5 times. control mice received 10 µl pbs or nothing according to the same schedule. to induce allergic airway inflammation, adult mice were sensitized intranasally with 100 µg ova (sigma) plus 0.1 µg lps in 100 µl pbs and subsequently challenged intranasally with 25 µg ova in 100 µl pbs according to the schedule indicated in each experiment. in the dc labeling experiments, we intratracheally administered 25 µg ova labeled with alexa flour r 647 (invitrogen). cd4 + t cells were purified from the spleens of naive cd45.1 + b6 congenics using ls columns and anti-cd4 macs beads (miltenyi biotec) according to the manufacturer's instructions. single t cell preparations were > 95% pure as determined by flow cytometry. th2 effectors were generated by activating naïve t cells for 48-72 h with plate-bound anti-cd3 (2 µg/ml; 145-2c11; bioxcell) and anti-cd28 (0.5 µg/ml; 37.51; ebioscience) in the presence of il−4 (50 u/ml; dnax) and anti-ifnγ (10 µg/ml; xmg1.2; ebioscience) followed by feeding with il−2 (20 u/ml; peprotech) for additional 48 h. naïve and effector cd4 + (cd45.1 + ) t cells (1 x 10 6 /mouse) were injected intravenously into no ibalt and ibalt recipients that were subsequently immunized with ova. lungs were cut into small fragments and digested with 0.6 mg/ml collagenase a (sigma) and 30 µg/ml dnase i (sigma) in rpmi 1640 medium (gibco) at 37 • c for 45 min to obtain single-cell suspensions. digested tissues were mechanically disrupted by passage through a metal strainer. cell suspensions from lungs and mediastinal lns were centrifuged and resuspended in 150 mm nh 4 cl, 10 mm khco 3 , and 0.5 mm ethylenediaminetetraacetic acid (edta; lonza) for 5 min to lyse red cells. cell suspensions were then filtered through a 70 µm nylon cell strainer (bd biosciences), washed, and resuspended in phosphate-buffered saline (pbs) with 5% donor calf serum and 10 µg/ml fc block (2.4g2; trudeau institute) on ice for 10 min before staining with fluorochromeconjugated antibodies against the following antigens: cd4 (rm4-5), cd11b (m1/70), cd25 (pc61), cd45r (b220; ra3-6b2), cd95 (fas; jo2), cd138 (281-2), siglec-f (e50-2440), and i-a b (af6-120.1; all from bd biosciences); cd11c (n418), cd44 (im7), cd45.1 (a20), cd103 (2e7), and foxp3; (fjk−16s); all from ebioscience; cd19 (6d5); from biolegend; cd38 (nimr−5); from southernbiotech; and goat anti-rabbit-alexa flour r 647 (invitrogen). for intracellular staining, single-cell suspensions from the lungs were stimulated on plates coated with anti-cd3 (2 µg/ml; 145-2c11; bioxcell) in the presence of brefeldin a (12.5 µg/ml; sigma) for 4 h. the restimulated cells were surface stained, then fixed in 4% paraformaldehyde, made permeable with 0.1% triton tm x−100 (sigma), and stained with anti-il−4 (11b11), anti-il−5 (trfk4; all from bd biosciences) or anti-il−13 (13a; ebioscience). cells were analyzed with a lsr ii (bd biosciences) or facscanto ii (bd biosciences) located at the university of rochester and university of alabama at birmingham flow cytometry core facility. to isolate lung epithelial cells, we perfused the lungs with pbs containing 0.05 mm edta (lonza), instilled with 1 ml dispase (160 µg/ml; corning), and incubated them in a shaker at 37 • c for 15 min. using forceps, lungs were torn into small pieces, and put back in the shaker at 37 • c for an additional 30 min in dispase (160 µg/ml; corning) and dnasei (250 µg/ml; worthington biochemical). digested tissues were red cell lysed, filtered through a 70 µm nylon cell strainer (bd biosciences), resuspended in pbs with 5% donor calf serum, 10 µg/ml fc block (2.4g2; trudeau institute) and anti-cd45 macs beads (miltenyi biotec) on ice for 10 min. cell suspensions were depleted of cd45-expressing cells using ls columns (miltenyi biotec) according to the manufacturer's instructions. the flow through cells were stained with anti-cd31 (390; invitrogen); anti-cd45.2 (104) and anti-cd326 (epcam; g8.8; all from biolegend) and anti-sca-1 (ly6a/e; d7; invitrogen). lung epithelial cells were sorted with facsaria ii (bd biosciences) located at university of alabama at birmingham flow cytometry core facility. lungs were instilled with 3 ml hank's balanced salt solution (hbss; corning) containing 0.05 mm edta (lonza) and cell suspensions (≈10,000 cells in 100 µl) were centrifuged at 800 rpm for 5 min in a shandon cytospin 3 cytocentrifuge (cell preparation system). the cytospin pellets were air dried on glass slides, stained with shandon kwik-diff tm stain kit (thermo scientific) and were mounted with richard-allan scientific tm cytoseal tm 60 (thermo scientific). total ige levels were determined by coating plates with 1 µg/ml purified rat anti-mouse ige (r35-72; bd biosciences). standard curve was prepared with purified mouse ige κ (c38-2; bd biosciences) and bound abs in serum samples were detected with biotin rat anti-mouse ige (r35-118; bd biosciences) and streptavidin-alkaline phosphatase (invitrogen). alkaline phosphate substrate (moss, inc.) was added, and color development was detected with spectramax r m2 (molecular devices) at 405 nm. paraffin-embedded sections (5 µm in thickness) were stained with hematoxylin and eosion (h&e) for standard histology and periodic acid-schiff (pas) for airway mucus production. frozen sections (8 µm in thickness) were prepared from lungs perfused with a mixture of optimum cutting temperature (oct) compound (sakura finetek) in pbs at a 1:1 ratio. sections were fixed in cold acetone for 10 min and blocked with fc block (10 µg/ml) and 5% (vol/vol) normal donkey serum in pbs at 25 • c for 30 min. sections were then stained with the following primary abs in pbs at rt for 30 min: goat anti-cd3ε (m−20; santa cruz biotechnology), anti-cd11c (hl3), and anti-cd45r (b220; ra3-6b2; all from bd biosciences); anti-cd21/cd35 (7e9; biolegend); and peanut agglutinin (pna; sigma). primary abs were detected with donkey antigoat-alexa flour r 568, rabbit fluorescein-alexa fluor r 488, streptavidin-alexa fluor r 555, and streptavidin-alex flour r 488 (all from invitrogen) at rt for 30 min. slides were mounted with slowfade r gold antifade mountant with 4 ′ ,6diamidino−2-phenylindole (dapi; invitrogen). images were collected with a zeiss axiocam digital camera (zeiss) or nikon andor clara camera (nikon). the images were obtained with a 20x objective for a final magnification of x200. t cells in immunofluorescent images were quantified by counting cd45.1 + cells in ibalt areas (defined manually using the outline tool) and by counting cd45.1 + cells in non-ibalt areas (excluding large empty airways). the mean number of t cells per µm 2 was determined using a mosaic of images obtained from control and ibalt-containing lungs that were sectioned at a similar depth and orientation. data were obtained from multiple sections from each mouse and 5 mice per group. total rna was extracted from lungs with trizol according to the manufacturer's specifications (invitrogen) and was repurified with an rneasy mini kit (qiagen). ribonucleic acid (rna; 2.2 µg) was reverse-transcribed with superscript ii and random primers (invitrogen) and complementary deoxyribonucleic acid (cdna; 25 ng) was amplified with following primers and probes: glyceraldehyde−3-phosphate dehydrogenase (gapdh; trudeau institute), il4, il5, il13, chi3l4, muc5ac. tnfaip3, and tslp (all from applied biosystems), with taqman r gene expression master mix (applied biosystems) and all reactions were run on a lightcycler 480 realtime pcr system (roche). the relative level of messenger rna (mrna) expression for each gene was first normalized to the expression of gapdh and then compared to the average level of mrna expression in lungs from naïve b6 mice. data is expressed as logarithmic fold changes in mrna expression. lungs were instilled with 1 ml hbss (corning) containing 0.02 mm edta (lonza) and sigmafast tm protease inhibitor cocktail tablets (sigma). total protein levels of cytokines il−4, il−5, and il−13 in lavage were quantified by mousespecific milliplex r multi-analyte kits (emd millipore) using a magpix r instrument platform and related xponent r software (luminex corporation). the readouts were analyzed with the standard version of the milliplex analyst software (emd millipore). the difference in mean values between two groups was analyzed with a two-tailed student's t-test. if three or more groups were compared, tukey's multiple comparison test was used (graphpad prism version 6.0). p-values of less than 0.05 were considered statistically significant. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by institutional animal care and use committee, university of alabama at birmingham. bronchus-associated lymphoid tissue bronchus-associated lymphoid tissue (balt) structure and function inducible bronchus-associated lymphoid tissue (ibalt) in patients with pulmonary complications of rheumatoid arthritis bronchial lymphoid tissue. i morphologic characteristics anatomical features of anti-viral immunity in the respiratory tract the response of rat bronchus-associated lymphoid tissue to local antigenic challenge pulmonary expression of cxc chemokine ligand 13, cc chemokine ligand 19, and cc chemokine ligand 21 is essential for local immunity to influenza organogenesis of lymphoid tissues ectopic lymphoid tissues and local immunity effects of microbial stimulation on the number, size and activity of bronchus-associated lymphoid tissue (balt) structures in the pig regulatory t cells prevent inducible balt formation by dampening neutrophilic inflammation regulatory t cells interfere with the development of bronchus-associated lymphoid tissue the development of inducible bronchusassociated lymphoid tissue depends on il-17 bronchus-associated lymphoid tissue (balt) in human fetal and infant lung bronchus-associated lymphoid tissue (balt) in the lungs of children who had died from sudden infant death syndrome and other causes exposure to environmental microorganisms and childhood asthma farm living: effects on childhood asthma and allergy role of inducible bronchus associated lymphoid tissue (ibalt) in respiratory immunity in a murine tuberculosis model, the absence of homeostatic chemokines delays granuloma formation and protective immunity cxcr5(+) t helper cells mediate protective immunity against tuberculosis bronchus-associated lymphoid tissue (balt) and survival in a vaccine mouse model of tularemia persistence and responsiveness of immunologic memory in the absence of secondary lymphoid organs inducible bronchus-associated lymphoid tissue elicited by a protein cage nanoparticle enhances protection in mice against diverse respiratory viruses increased number of b-cells in bronchial biopsies in copd the nature of small-airway obstruction in chronic obstructive pulmonary disease development of bronchus-associated lymphoid tissue in chronic hypersensitivity pneumonitis aggregations of lymphoid cells in the airways of nonsmokers, smokers, and subjects with asthma interleukin-5 expression in the lung epithelium of transgenic mice leads to pulmonary changes pathognomonic of asthma autoantibodies in patients with chronic obstructive pulmonary disease role of lymphotoxin-alpha in cigarette smoke-induced inflammation and lymphoid neogenesis lymphoid follicles in (very) severe copd: beneficial or harmful? analysis of type 2 immunity in vivo with a bicistronic il-4 reporter a two-step process for cytokine production revealed by il-4 dual-reporter mice interleukin-13: central mediator of allergic asthma regulatory t cells and immune tolerance foxp3+ regulatory t cells in the human immune system cd4+cd25+ t cells regulate airway eosinophilic inflammation by modulating the th2 cell phenotype antigen-specific regulatory t cells develop via the icos-icos-ligand pathway and inhibit allergen-induced airway hyperreactivity farm dust and endotoxin protect against allergy through a20 induction in lung epithelial cells house dust mite allergen induces asthma via toll-like receptor 4 triggering of airway structural cells fty720: sphingosine 1-phosphate receptor-1 in the control of lymphocyte egress and endothelial barrier function fty720 blocks egress of t cells in part by abrogation of their adhesion on the lymph node sinus treatment with a sphingosine-1-phosphate analog inhibits airway remodeling following repeated allergen exposure preferential lymphatic growth in bronchus-associated lymphoid tissue in sustained lung inflammation induced bronchus-associated lymphoid tissue serves as a general priming site for t cells and is maintained by dendritic cells inhalation of diesel exhaust for three months affects major cytokine expression and induces bronchus-associated lymphoid tissue formation in murine lungs autoreactive t and b cells induce the development of bronchus-associated lymphoid tissue in the lung a functionally specialized population of mucosal cd103+ dcs induces foxp3+ regulatory t cells via a tgf-beta and retinoic aciddependent mechanism selective imprinting of gut-homing t cells by peyer's patch dendritic cells artery tertiary lymphoid organs control aorta immunity and protect against atherosclerosis via vascular smooth muscle cell lymphotoxin beta receptors t follicular helper cells differentiate from th2 cells in response to helminth antigens il-4-producing cd4+ t cells in reactive lymph nodes during helminth infection are t follicular helper cells the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.570661/full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 hwang, silva-sanchez, carragher, garcia-hernandez, rangel-moreno and randall. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-255578-0ltb9dpa authors: li, xiangru; hao, zhenhua; liu, xiaorong; li, wei title: deficiency of mouse fhr-1 homolog, fhr-e, accelerates sepsis, and acute kidney injury through enhancing the lps-induced alternative complement pathway date: 2020-06-19 journal: front immunol doi: 10.3389/fimmu.2020.01123 sha: doc_id: 255578 cord_uid: 0ltb9dpa alternative complement pathway (ap) plays an important role in the development of sepsis, which is life threatening. deficiency of factor h-related protein 1 (fhr-1), which is a regulator of ap, has been considered as a susceptible factor for atypical hemolytic uremic syndrome (ahus) and other types of nephropathy when an inducer such as infection exists. however, the underlying mechanism of the disease development is largely unknown. there is no report on cfhr1 gene knockout in any animal infection model and its function in vivo is still unclear. here, a cfhr1 knockout mouse was generated for investigating ap in sepsis and sepsis-induced acute kidney injury (aki). we found that murine fhr-1 homolog (fhr-e) deficiency enhanced lipopolysaccharide (lps)-induced ap activation both in vitro and in vivo and that cfhr1 knockout mice exhibited more severe sepsis and aki in response to lps challenge. these results indicated that fhr-e deficiency promoted lps-induced sepsis and aki through ap over-activation, providing a mouse model for studying ap regulation and sepsis. this study revealed the function of fhr-e in vivo, which may further provide hints to the pathogenesis of fhr-1 deficiency-related diseases by enhancing lps-induced ap activation. sepsis is a critical health condition with high mortality rate (1, 2) and acute kidney injury (aki) is a common severe complication of sepsis (3) . gram-negative bacteria infection is a predominant cause of severe infection-triggered sepsis (4) . sepsis is an intricate process during which complement system is activated and proved to be double-edged with benefits and harms (5) . the complement system is crucial in immune surveillance (6) and has extensive cross-talk with coagulation system and inflammation for homeostasis (7) . it is triggered through three pathways among which the alternative complement pathway (ap) activation is responsible for more than 80% of terminal complement activation (8, 9) . ap plays an important role in endotoxin clearance during the process of sepsis (10) . lack of ap activation in individuals predisposes to infection (11) (12) (13) . however, it is also life-threatening when complement is excessively activated, and inhibition of its over-activation prevents organ injuries (14) . therefore, uncovering the precise regulatory mechanism of ap during sepsis may shed light on sepsis intervention. however, the mechanism of ap regulation during sepsis remains elusive. factor h (fh) and its related proteins (fhrs) are regulators of ap (15) and responsible for determining complement activating surfaces (16) . fh, the major regulatory factor of ap (17) , has been widely investigated, while functions of fhrs are unclear or controversial. fh and fhrs consist of different number of complement control protein modules (ccps). all fhrs contain homologous ccps to ccp19, 20 of fh which are responsible for ligand recognition (18) . in general, fh functions as a cofactor of factor i (fi) to cleave c3b and accelerate the decay of c3 convertase (19, 20) , while fhrs act as the competitor of fh (16) . however, different fhrs may have different functions. fhr-2 inhibits c3 convertase (21) . fhr-3 displays the cofactor activity for fi (22) . fhr-5 inhibits c3 convertase in fluid phase and displays cofactor activity for fi (23) . nevertheless, it remains elusive for the physiological functions of fhrs. mutations in fh and fhrs are associated with various diseases (24) . cfhr3-cfhr1 deletion increased the risk of atypical hemolytic uremic syndrome (ahus) and systemic lupus erythematosus (sle) (25) (26) (27) . cfhr3-cfhr1 deletion was proved to protect against iga nephropathy (igan) (28) and age-related macular degeneration (amd) (29) . the precise mechanism of this contradictory effect is unclear. in vitro studies of factor h-related protein 1 (fhr-1) have shown that fhr-1 interferes with the regulation of fh by competing with fh and inhibits the activity of c5 convertase and the formation of terminal complement complex (tcc) (30) . no obvious c3 and c5 regulatory activity at physiological concentration has been found in fhr-1 (16) , albeit it is the most abundant fhr protein (18, 31) . healthy individuals with cfhr3-cfhr1 deletion showed higher frequency of patrolling monocytes, plasmacytoid dendritic cells (dcs), and lower frequency of classical monocytes, myeloid dcs. monocytes with cfhr3-cfhr1 deletion secreted higher level of il-1β in response to lps challenge (32) . necrotic cells bound to fhr-1 promotes the secretion of tnf-α, il-1β, il-18, and il-6 by monocytes (33) . these reports suggest multiple effects of cfhr1 deletion and highlight its complexity. the existence of relatively high frequency of healthy individuals with cfhr3-cfhr1 deletion (34) indicates that other triggers are essential to amplify the effect of fhr-1 deficiency, such as infections (35, 36) . however, there is no report on fhr-1 deficiency in any animal infection model. three genes, fhrb, fhrc, and fhre (alias cfhr1), and two pseudogenes, fhra and fhrd, were identified in mouse (37, 38) . recombinant fhr-a, fhr-b, and fhr-c were reported to interact with human c3d, suggesting that murine fhrs function as homologs of human fhrs (39) . none of fhr-a, fhr-b, and fhr-c contain the critical dimerization domain that exists in human fhr-1, fhr-2, and fhr-5 (39), while fhr-e is predicted to contain it (37) . thus, fhr-e is more likely the murine homolog of human fhr-1 or fhr-2 (40) . nomenclature of human and mouse fhrs differs and there exist discrepancies in literature and existing database (41) . inferred from the homology analysis (figure 1) , we use mouse fhr-1 homolog fhr-e (np_056595) for the protein encoded by cfhr1 (refseq# at ncbi: nm_015780, and a locus at mgi database: 2138169) . homology analysis has shown the difference between human and murine fhrs (41, 42) , suggesting that murine fhrs may function differently from their human counterparts. fhr-b, fhr-c, and fhr-e are functional fhrs in mouse. fhr-b and fhr-c were detected in mouse plasma with anti-fh antibody (38) . mouse fhr-b interacts with c3b and pentraxin 3 to enhance complement activation and necrotic cells promoted this activation (43) . fhrc mrna was found completely absent in the liver of three lupus-prone mouse strains and one diabetic-prone mouse strain (44) . fhr-c may function on specific surfaces (39) . it is unknown whether fhr-e exists in murine plasma and how it functions in ap. its human homolog, fhr-1, was reported to function as a competitor of fh (22, 30, 45) and its deletion was associated with various autoimmune diseases. however, its function in vivo and whether it has a regulatory role alone in the activation of ap remain unclear. in this study, cfhr1 was deleted on c57bl/6 mouse to study the function of fhr-e on ap and the effect of fhr-e deficiency on lps-induced sepsis. we found that fhr-e deficiency increased the mortality rate of lps-induced sepsis and potentiated kidney injury through enhancing ap activation. our data demonstrated a protective role of fhr-e during lps-induced sepsis in vivo and highlighted its importance in ap regulation. cfhr1 was deleted on c57bl/6 mouse by using the crispr/cas9 system developed by biocytogen co. (beijing, china). the heterozygous mouse was backcrossed with wild-type c57bl/6 for more than six generations. all the mice were bred in the animal facility of the institute of genetics and developmental biology (igdb), chinese academy of science. all procedures were approved by the institutional animal care and use committee of igdb. the age-matched wild-type (wt) or heterozygous littermates were used as controls. all experiments were conducted on the offspring of mice backcrossed more than six generations. the polyclonal antibody anti-fhr-e was generated in new zealand white rabbit. the dna fragment of the last three ccp domains (amino acids 146 to 343) of fhr-e was inserted into the expression vector pgex-4t-1 and expressed in e. coli bl21 host cells. the recombinant protein was expressed as insoluble protein and separated by sds-page. the separated recombinant protein was used as an antigen to immunize the new zealand white rabbit every other week. the rabbit was sacrificed 1 week after the fourth immunization and the sera was used as the anti-fhr-e polyclonal antibody. the rat anti-c3 (ab11862), rat anti-c5 (ab194637), sheep anti-fh (ab8842), and rabbit anti-fibrinogen (ab27913) were obtained from abcam (cambridge, uk). purified the upper bands were amplified with primers f1 and r1. these bands can only be amplified in wild-type and heterozygous mice and cannot be amplified in homozygous mice because of the absence of the sequence of primer r1. the lower bands were amplified with primers f1 and r2. these bands can only be amplified in heterozygous and homozygous mice. the sequence between primers f1 and r2 is too long to be amplified in wild-type mice. (c) verification of cfhr1 deletion from rna level. hepatic rna of mice of different genotypes was extracted and reversely transcribed. primers spanning the open reading frame (orf, 1,032 bp, nm_015780) of cfhr1 were used for rt-pcr. actb was used as a semi-quantitative control. (d) verification of fhr-e deficiency at the protein level. plasma proteins of mice with different genotypes were separated by 8% sds-page gel. proteins were transferred to the membrane and analyzed by western blotting using anti-serum to mouse fhr-e, which was generated in rabbit immunized with a recombinant peptide of ccp 3-5 of fhr-e. an about 50 kda specific fhr-e band was detected in wt and heterozygous mice, but not in homozygous mice. "unknown" indicates an unknown protein recognized by anti-fhr-e antibody. western blotting of fh was regarded as an internal control. rat anti-mouse c5a (558027) and biotin rat anti-mouse c5a (558028) were obtained from bd pharmingen (california, usa). mice were marked by cutting different toes and the toes were lysed by 250 µl of lysis buffer (tris-hcl ph 8.0 100 mm, edta ph 8.0 5 mm, nacl 200 mm, 0.2% sds, and 0.1 mg/ml proteinase k) at 55 • c for 3 h. after transient centrifugation, 150 µl of 5 m nacl was added and mixed thoroughly. the mixture was centrifuged at 12,000 rpm for 15 min and the supernatant was collected by adding double volume of ethanol and mixed thoroughly. the mixture was centrifuged at 12,000 rpm for 15 min and then the supernatant was discarded. the pellet was washed with 70% ethanol. after the liquid was evaporated, 100 µl of ddh 2 o was added to dissolve the precipitated dna. one forward primer located on the upstream of the deleted sequence, one reverse primer located on the deleted region, and the other reverse primer located on the downstream of the deleted sequence were used for genotyping pcr assays. the primer's sequences are 5 ′ -cagtaagactgcaagagacatatg-3 ′ , 5 ′ -ctaagagcaacaggcgacag-3 ′ , and 5 ′ -cattttaa aagaaaaataagccagcca-3 ′ , respectively. the amplified products are depicted in figure 2a . mouse hepatic rna was extracted using rneasy mini kit (qiagen, germantown, germany) and reversely transcribed with bio-rad transcript cdna synthesis kit (bio-rad, california, usa). the cdna was used as templates to amplify cfhr1 and actb (gene for β-actin). primers of cfhr1 were 5 ′ -atggggtt ctgtcgcctgttgc-3 ′ and 5 ′ -tcaatgaataaacgtattg tga-3 ′ . primers of actb were 5 ′ -tgatggtgggaatggg tcaga-3 ′ and 5 ′ -ccgctcgttgccaatagtgat-3 ′ . immunoblotting equal volume of serum or plasma of mice was diluted 1:10 with pbs (hyclone, utah, usa). equal volumes of diluted samples were separated with 8% sds-page gel and transferred onto polyvinylidene difluoride (pvdf) membrane (millipore, massachusetts, usa). the membrane was blocked with 5% defatted milk (bd, california, usa) for 1 h at room temperature and immunoblotted with specific antibody overnight at 4 • c followed by incubation with hrp-conjugated secondary antibody (zsgb-bio, beijing, china) for 1 h at room temperature. the membrane was visualized by ecl (thermo fisher scientific, massachusetts, usa or ge healthcare, usa) and exposed with chemiluminescence apparatus (beijing sage creation, beijing, china). protein bands' gray values were measured by nih image j. serum was diluted by ap buffer (2.5 mm barbital, 1.5 mm sodium barbital, 144 mm nacl, 7 mm mgcl 2 , and 10 mm egta, ph 7.2-7.4). ten-fold diluted serum was incubated with 0.125 mg/ml lps o111:b4 (sigma-aldrich, missouri, usa) or equal volume of pbs as control at 37 • c for 30 min. the reactions were stopped by 50 mm edta. the samples were analyzed with 8% sds-page under reducing condition and protein bands' gray values were measured by nih image j. lps-stimulated ap activity was performed as previously published by elisa (46) . briefly, lps (2 µg/well) was coated on plates. ten-fold diluted serum with ap buffer was incubated on plates at 37 • c for 1 h. serum diluted with edta buffer (2.5 mm barbital, 1.5 mm sodium barbital, 144 mm nacl, and 50 mm edta, ph 7.2-7.4) was used as a negative control. after washing, the plates were incubated with rat anti-mouse c3 antibody and followed by incubation with hrp-conjugated antirat igg. tetramethylbenzidine (tmb, solarbio, beijing, china) was used as substrate and 1 m h 2 so 4 was used as a stop solution. the absorbance was measured with multiskan go microplate spectrophotometer (thermo scientific, massachusetts, usa). for the detection of lps-induced c5a production, 10-fold diluted serum was incubated with 0.125 mg/ml lps or equal volume of pbs as control at 37 • c for 2 h and the reactions were stopped by 50 mm edta. the reaction mixtures were added to the plate, which was coated with purified rat anti-mouse c5a antibody (1 µg/ml) and incubated at 37 • c for 2 h. the mixtures were discarded and biotin rat anti-mouse c5a (0.5 µg/ml) was added into the according plates as detection antibody. after washing, streptavidin hrp (1:1,000) (bd pharmingen, california, usa) was added and incubated at 37 • c for 1 h. tmb (solarbio, beijing, china) was used as substrate and 1 m h 2 so 4 was used as a stop solution. the absorbance was measured with multiskan go microplate spectrophotometer (thermo scientific, massachusetts, usa). six-to eight-week-old male mice were injected intraperitoneally (i.p.) with 10 mg/kg lps dissolved in pbs and the control group mice were injected i.p. with equal volume of pbs. the edta anticoagulant blood was extracted from the tail tip at different time points and the plasma was collected to analyze with western blotting. mice were sacrificed at different time points and the edta anticoagulant blood was harvested from heart puncture. partial whole blood was used for complete blood counting using tek-ii mini full-automatic animal blood analyzer (tecom science, jiangxi, china). the plasma was collected for c3a, c5a, il-1β and tnf-α concentration detection using elisa kit as described below. plasma c3a, c5a, il-1β, and tnf-α measurements lps challenged mice were anesthetized and edta anticoagulant blood was collected by heart puncture. the whole blood was centrifuged at 4,000 rpm 4 • c for 5 min and the supernatant was collected. plasma levels of c3a and c5a were determined using elisa kits (mybiosource, california, usa) according to the manufacturer's instructions. the dilution factor of c3a and c5a was 1:4. plasma il-1β and tnf-α were measured with elisa kits (r&d systems, minnesota, usa) according to the manufacturer's instructions. about 8-week-old mice were injected i.p. with 10 mg/kg lps. sixteen hours later, these mice were observed every half hour and the death of mice was recorded. the data were analyzed by graphpad prism version 5.0 using the log-rank (mantel-cox) test. mice were sacrificed at 12 h after lps challenge and the left kidneys were harvested. half of the kidney was fixed in 4% paraformaldehyde for 24 h at 4 • c, dehydrated using graded ethanol, cleared with xylene, and embedded in paraffin. paraffin blocks were sliced into 3-µm sections. hematoxylin and eosin (he) staining was performed on the sections using hematoxylin and eosin staining kit (solarbio, beijing, china). pictures were captured with nicon ci-l microscope (nicon, tokyo, japan) at 200× magnification. tubular damage was assessed by tubular dilation, necrosis, and apoptosis. tubular dilations were counted in every non-overlap field and averaged. cell necrosis and apoptosis were determined using in situ cell death detection kit (roche, basel, switzerland). the fluorescent pictures were captured with an lsm 710 confocal fluorescence microscope (zeiss, oberkochen, germany) at 100× magnification. only dots overlapped with nuclei were counted and the whole cell number of each field was counted by image j. proportion of apoptotic and necrotic cells was computed. paraffin slides that were processed by xylene de-waxing and gradient ethanol hydrating were blocked by ready-to-use goat serum (boster, wuhan, china) at 37 • c for 1 h and incubated with rabbit anti-fibrinogen (1:400) overnight at 4 • c. after washing three times with pbs, the slides were incubated with alexa fluor 488 goat anti-rabbit igg (1:1,000) (invitrogen, california, usa) at 37 • c for 1 h. the slides were washed three times and mounted with mounting medium with dapi (zsgb-bio, beijing, china). images were captured using leica tcs sp5 confocal laser scanning microscope (leica microsystems, wetzlar, germany) at 200× magnification. the fluorescence intensity and fluorescence positive area were analyzed by image j. the protein sequences were downloaded from ncbi-protein (https://www.ncbi.nlm.nih.gov/protein/). amino acid sequences were aligned by clustalw with mega 7.0 (47). the alignment diagram was drawn with genedoc. the evolutionary tree was constructed using the neighbor-joining method with mega 7.0 (47, 48) . the domain structures of fhrs were analyzed by smart (http://smart.embl-heidelberg.de/). domain similarities among different fhrs were computed with pblast. data were presented as mean ± sem. student's t-test was used to compare the two groups. time course of factors of different time points after lps challenge was analyzed with one-way anova by spss software. comparison of survival curves was analyzed by mantel-cox test. significant differences were considered when the p-value was less than 0.05 and extremely significant differences were considered when the p-value was less than 0.01. to ascertain the mouse fhr-e homolog of human fhr proteins, homology analysis between mouse and human fhrs was conducted. the evolutionary tree of fhrs showed that fhr-e converged with human fhr-1, fhr-2, and fhr-5 ( figure 1a) . the overall homology between mouse fhr-e and human fhr-1/2/5 is about 70%, while fhrb and fhrc have about 40% homology to fhr-1/2/5 (40) . structure analyses of fhr-e and human fhr-1, fhr-2, and fhr-5 by smart showed that they all consist of ccp modules. amino acid sequence of each ccp in fhr-e was compared with human fhr-1, fhr-2, and fhr-5 by pblast and amino acid sequence alignment was analyzed by mega 7.0. results showed that fhr-e shared more similarity with human fhr-1 (figures 1b-e) . with this regard, we selected the mouse cfhr1 gene that encodes the fhr-e protein for the generation of a gene knockout colony. the exons from 1 to 5 of cfhr1 were deleted by using the crispr/cas9 system on c57bl/6 mouse (figure 2a) . verification of cfhr1 deletion was conducted at dna, mrna, and protein levels (figures 2b-d) . the results demonstrated that fhr-e was detectable in murine plasma and was depleted in the knockout mice. our homemade anti-fhr-e antibody recognized murine fh as well due to the high epitope identities to scrs 19 and 20 of fh (data not shown). this is the first report of fhr-e in murine plasma. fhr-b and fhr-c has been detected in mouse plasma by using fh-specific antiserum (38) . the unrecognized fhr-e in their experiment may be because of the antibody specificity. both heterozygotes (cfhr1 −/+ ) and homozygotes (cfhr1 −/− ) were viable and healthy bred in spf (specific pathogen free) mouse facility. under physiological condition, ap keeps at a low level of activation by spontaneous hydrolysis of c3 (49) . to study whether cfhr1 deletion can trigger complement dysregulation under physiological condition, the plasma levels of c3, c5, and fh, central molecules of complement and main soluble regulator of ap, were detected on 6-to 8-week-old male sibling mice. western blotting results showed no obvious changes among mice of different genotypes (figure 3a) . the relative gray values of 18 mice were analyzed and results showed that there were no significant differences (figures 3b-d) . furthermore, the concentrations of c3a and c5a, which are small cleaved fragments generated by complement activation, and the key mediators of inflammation were measured. neither the level of c3a nor c5a had significant differences among different genotypes (figures 3e,f) . these results suggested that under physiological condition, the deletion of cfhr1 was not sufficient to impact the ap regulation. ap activation is enhanced in cfhr1 −/− mice in response to lps treatment in vitro lps, the principal component of gram-negative bacteria cell wall, is an activator of ap (50) and responsible for gramnegative bacteria induced sepsis (51) . to test the effect of fhr-e deficiency on lps-induced ap, in vitro lps-induced ap activation experiments were performed. lps was incubated with serum from mice of different genotypes and the mixtures were analyzed by western blotting. a higher amount of c3activated fragment in cfhr1 −/− mice was observed compared to the wild-type group (figures 4a,b) . ap activity measured by elisa assay (46, 52) was also conducted to verify this effect. as expected, cfhr1 −/− mice showed more deposited c3b on lps ( figure 4c) . furthermore, lps-induced c5a production, an ensuing event of ap activation, was determined and significantly higher concentration of c5a was detected in cfhr1 −/− mice compared to wild-type mice ( figure 4d ). an increasing tendency was observed in the heterozygotes, but there was no significant difference between wt and heterozygotes, suggesting that dose effects of fhr-e in heterozygotes may exist. thus, fhr-e deficiency promoted lps-induced ap activation in vitro. to further study the regulatory function of fhr-e, in vivo challenge of lps was applied. edta anticoagulant blood of different time points was collected from the tail tip to monitor the content changes of main complement or coagulation factors using western blotting. our results showed that the level of c3 decreased over time and that the level of c5 increased at first and decreased from 1 h after lps challenge. no significant difference was observed among different genotypes at different points. however, more significant decrease of c5 content over time was observed in cfhr1 −/− mice compared with wild-type mice (figures 5a-c) . the levels of fh and von willebrand factor (vwf), which is involved in hemostasis, were also determined. the content of vwf increased at first and decreased from 3 h post lps challenge (figures 5a,e) while the content of fh did not have any observable change over time (figures 5a,d) . this demonstrated that the hemostasis was promptly triggered and gradually vanished as the exhaustion of clotting factor, and that fhr-e deficiency and lps stimulation did not affect the level of fh. the contents of fhr-e in wild-type and heterozygous mice were also measured. interestingly, we found that fhr-e increased significantly at 3 h post-lps challenge (figures 5a,f) . the data in figure 5 were obtained from the same batches of mice. furthermore, another group of mice were challenged with lps and edta anticoagulant blood of different time points was harvested from heart puncture. the contents of plasma c3a and c5a of cfhr1 −/− mice were significantly higher than wild-type controls at 3 h post-challenge (figures 6a,b) . these results demonstrated that fhr-e deficiency accelerated lps-induced ap. the plasma il-1β and tnf-α contents at 3 h post-challenge were detected and (c) detection of ap activation by plate bounded lps in diluted serum with elisa method. equivalent lps was coated on the plate. ap buffer and edta buffer diluted serum was added into the plate and incubated for 1 h at 37 • c. edta buffer diluted serum was used as a negative control for background subtraction. after washing, the lps-bound c3b was detected with c3 antibody. twelve mice in each group were used. (d) measurement of the concentration of c5a produced at 2 h after lps in vitro stimulation. ap buffer diluted serum was incubated with lps or pbs for 2 h at 37 • c. equal volume of pbs was used as a negative control for background subtraction. the quantity of c5a was detected with sandwich enzyme-linked immunosorbent assay. ten pairs of mice were used. n.s., not significant. *p < 0.05. elevated concentrations of il-1β and tnf-α were found in cfhr1 −/− mice (figures 6c,d) . subsequently, we observed that the proportion of granulocytes significantly increased and lymphocytes mildly decreased at 6 h after challenge, which suggests that cfhr1 −/− mice had more severe inflammation (figures 6e,f) . the quantity of red blood cells in cfhr1 −/− mice at 12 h after lps challenge was dramatically lower than the wild-type mice, which indicates that much more congestion could happen in cfhr1 deletion mice (figure 6g) . in summary, the cfhr1 −/− mice had features of enhanced complement activation and inflammation, which may lead to organ injury. to explore the endpoint effect of fhr-e deficiency on lpsinduced sepsis, the mortality assay was performed. cfhr1 −/− and wild-type mice were administrated i.p. injection of 10 mg/kg lps. the survival data were recorded and analyzed. compared to the wild-type mice, the average survival time of cfhr1 −/− mice was significantly shorter and the mortality rate of cfhr1 −/− mice was significantly higher (figure 6h human cfhr1 deletion was associated with nephropathy (27, 53) and kidney injury can be induced by complement overactivation (54) . urea and creatinine, which are indicators of renal function, were tested at 12 h post-challenge and significant increases were observed in both wt and ko groups (figures 7a,b) . however, we did not observe an apparent increase in the ko group compared with the wt group after lps treatment. this suggests that renal function was not worsened dramatically in fhr-e deficiency at the time of measurement. we then performed histological examination of kidney. renal tissue sections of 12 h after lps challenge were prepared and he staining was conducted. kidney injury degree was assessed by the tubular dilations, apoptosis, and necrosis. tubular dilations were quantified, and cell apoptosis and necrosis were determined through tunel assay. more tubular dilations (figures 7c,d) and more cell apoptosis and necrosis (figures 7e,f) were exhibited in cfhr1 −/− mice. fibrin, which is the marker of vascular injury, was also stained to testify tissue injury. we found that cfhr1 −/− mice had significantly more fibrin deposition than wild-type mice (figures 8a-c) . plasma fibrin was also analyzed by western blotting and higher concentration of plasma fibrin was found in cfhr1 −/− mice ( figure 8d ). twenty non-overlapped fields were counted and averaged. (c) comparison of fluorescence intensity between wild-type and cfhr1 −/− mice. fluorescence intensity of each field was calculated by image j. twenty non-overlapped fields were counted and averaged. (d) western blotting analysis of plasma fibrin of 12 h after lps challenge. western blotting of fhr-e was performed to determine the genotypes. equal volumes of plasma were loaded. the western blotting of fh on the same blot was regarded as an internal control. *p < 0.05. in this study, cfhr1 knockout mice were generated to further investigate its role in ap regulation. no obvious changes of c3a and c5a concentration in plasma were found in cfhr1 knockout mice. these results suggest that the basal level of fhr-e plays negligible roles in spontaneously activated ap pathway. this may explain why many individuals with cfhr1 homozygous deletion are healthy (34) . surprisingly, with fhr-e deficiency, increased c3 and c5 cleavage were found after lps stimulation in vitro, suggesting that fhr-e itself inhibits lps-induced ap activation. this may be explained by the conformation switch model in which soluble fh exists in a low-affinity latent conformation and transits to high-affinity activated conformation by interacting with self-surface targets (55) . based on this model, fh may play negligible inhibition on lps-induced ap and fhr-e may compete c3b with positive regulators of ap, like properdin, which plays an essential role in lps induced ap activation (46) . when fhr-e is absent, ap activity is enhanced by the positive regulators. the inhibitory effect of fhr-1 on c5 convertase has been reported (30) and was regarded as a therapeutic target (56) . the result that more c5a production after lps challenge in cfhr1 −/− mice was observed supports the idea that fhr-e has functional homology to human fhr-1. cfhr1 deletion has been reported to be associated with renal disease in an incomplete penetrance and a second hit such as infection is considered to be essential for disease development (57) . it is unknown whether cfhr1 homozygous deletion alone would trigger the onset of these conditions upon infection. one reported case showed that shiga toxin was a potential trigger of cfhr1 deletion-related thrombotic microangiopathy (58) . here, we chose lps, which is the main constituent of the wall of gram-negative bacteria as a stimulator to study the effect of cfhr1 deletion in mice for possible pathological changes. both in vitro and in vivo assays showed that fhr-e deficiency significantly promoted lps-induced ap activation. these results suggested that fhr-e may play a coordinated role with fh in determining complement activation. however, the mechanism of selective activation of ap remains enigmatic. in many ahus patients with cfhr1 deletion, fh autoantibody was detectable (27) . it was believed that ahus may result from the blocking of fh function by fh auto-antibody. however, this cannot explain those ahus patients with cfhr1 deletion without fh auto-antibody (59) . our results suggest that fhr-e deficiency promotes infection-induced damage through enhancing ap activation. this may provide a hint for fhr-1 deficiency-related nephropathy. nevertheless, how fhr-e functions in this complex process warrants further investigation. in addition, we did not see much difference of fh level in fhr-e deficiency mice. to the best of our knowledge, there has been no report of fhrs on any inflammatory disease mouse models. our results revealed that cfhr1 knockout mice challenged with lps served as a good model to study the intricate network of complement, inflammation, and coagulation in sepsis and aki. we found that cfhr1 −/− mice showed higher content of c3a and c5a, more severe inflammation, more blood coagulation, and more severe renal injury after lps challenge compared with the wild-type group. however, we did not see significantly higher levels of urea and creatinine in cfhr1 −/− mice compared to the wildtype mice upon lps challenge. c3a and c5a, which are two main products of complement activation, have been shown to have anti-inflammatory (60, 61) and pro-inflammatory function (62) , respectively. administration of c5a receptor antagonist peptide in thrombotic glomerulonephritis model significantly reduced leucocyte accumulation and thrombus formation in glomeruli (63) and improved survival of mice with sepsis (64), while administration of c3a receptor antagonist exacerbated mortality of mice with sepsis (64) . the regulation mechanism of c3a and c5a on sepsis development, which seemed to have converse effect, is still unclear. in our study, the deficiency of fhr-e promoted complement activation with more c3a and c5a production, which in turn induced more inflammation and eventually accelerated coagulation and tissue injury. these results suggested that c5a may play a more potent role on sepsis development. our cfhr1 −/− mouse model provides a unique tool to study the underlying mechanism in which ap is activated upon lps challenge to induce tissue injury. beyond the scope of this study, it is speculative that patients with fhr deficiency may be susceptible to severe conditions under sars-cov-2 infection when sepsis is induced in covid-19 patients. the datasets generated for this study are available on request to the corresponding author. the animal study was reviewed and approved by institutional animal care and use committee of igdb (institute of genetics and developmental biology, chinese academy of science). xli, zh, and wl designed the experiments, analyzed the data, and supervised the project. xliu contributed to the initiation and design of the project. xli and wl wrote the manuscript. xli and zh performed the assays. this study was partially supported by grants from the ministry of science and technology of china (2016yfc1000306) and from national natural science foundation of china (31830054; 91854110). severe sepsis in pre-hospital emergency care: analysis of incidence, care, and outcome incidence and trends of sepsis in us hospitals using clinical vs claims data acute renal failure and sepsis epidemiology of sepsis and infection in icu patients from an international multicentre cohort study complexity of complement activation in sepsis complement: a key system for immune surveillance and homeostasis haemostasis and innate immunity -a complementary relationship: a review of the intricate relationship between coagulation and complement pathways the quantitative role of alternative pathway amplification in classical pathway induced terminal complement activation the alternative complement pathway revisited the role of complement system in septic shock deficient alternative complement pathway activation due to factor d deficiency by 2 novel mutations in the complement factor d gene in a family with meningococcal infections properdin deficiency in a boy with fulminant meningococcal septic shock deficiency in complement factor b complement inhibition decreases the procoagulant response and confers organ protection in a baboon model of escherichia coli sepsis the complement factor h-related proteins factor h-related proteins determine complement-activating surfaces the human complement factor h: functional roles, genetic variations and disease associations self-damage caused by dysregulation of the complement alternative pathway: relevance of the factor h protein family human complement c3b inactivator: isolation, characterization, and demonstration of an absolute requirement for the serum protein beta1h for cleavage of c3b and c4b in solution control of the amplification convertase of complement by the plasma protein beta1h human factor h-related protein 2 (cfhr2) regulates complement activation an imbalance of human complement regulatory proteins cfhr1, cfhr3 and factor h influences risk for age-related macular degeneration (amd) human factor h-related protein 5 has cofactor activity, inhibits c3 convertase activity, binds heparin and c-reactive protein, and associates with lipoprotein complement factor h related proteins (cfhrs) association of genetic variants in complement factor h and factor h-related genes with systemic lupus erythematosus susceptibility deletion of complement factor h-related genes cfhr1 and cfhr3 is associated with atypical hemolytic uremic syndrome association among complement factor h autoantibodies, deletions of cfhr, and the risk of atypical hemolytic uremic syndrome fine mapping implicates a deletion of cfhr1 and cfhr3 in protection from iga nephropathy in han chinese a common cfh haplotype, with deletion of cfhr1 and cfhr3, is associated with lower risk of age-related macular degeneration factor h-related protein 1 (cfhr-1) inhibits complement c5 convertase activity and terminal complex formation factor h-related (fhr)-1 and fhr-2 form homo-and heterodimers, while fhr-5 circulates only as homodimer in human plasma altered peripheral blood leucocyte phenotype and responses in healthy individuals with homozygous deletion of fhr1 and fhr3 genes serum fhr1 binding to necrotic-type cells activates monocytic inflammasome and marks necrotic sites in vasculopathies determining the population frequency of the cfhr3/cfhr1 deletion at 1q32 the high frequency of complement factor h related cfhr1 gene deletion is restricted to specific subgroups of patients with atypical haemolytic uraemic syndrome atypical ahus: state of the art identification and sequence analysis of four complement factor h-related transcripts in mouse liver two factor h-related proteins from the mouse: expression analysis and functional characterization modulation of the alternative pathway of complement by murine factor h-related proteins of mice and men: the factor h protein family and complement regulation structure of the murine complement factor h gene the murine factor h-related protein fhr-b promotes complement activation. front immunol new insights into disease-specific absence of complement factor h related protein c in mouse models of spontaneous autoimmune diseases dimerization of complement factor h-related proteins modulates complement activation in vivo activator-specific requirement of properdin in the initiation and amplification of the alternative pathway complement mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets the neighbor-joining method: a new method for reconstructing phylogenetic trees acquisition of c3b-like activities by spontaneous hydrolysis of the putative thioester in native c3 acylation of the lipid a region of a klebsiella pneumoniae lps controls the alternative pathway activation of human complement endotoxins and other sepsis triggers murine systemic thrombophilia and hemolytic uremic syndrome from a factor h point mutation mini review: a unique case of crescentic c3 glomerulonephritis role of c5a in multiorgan failure during sepsis functional anatomy of complement factor h the mfhr1 fusion protein is a novel synthetic multitarget complement inhibitor with therapeutic potential rare functional variants in complement genes and anti-fh autoantibodies-associated ahus shiga toxin as a potential trigger of cfhr1 deletion-associated thrombotic microangiopathy characterization of genetic predisposition and autoantibody profile in atypical haemolytic-uraemic syndrome cutting edge: targeted disruption of the c3a receptor gene demonstrates a novel protective anti-inflammatory role for c3a in endotoxin-shock is the complement activation product c3a a proinflammatory molecule? re-evaluating the evidence and the myth in vitro and in vivo dependency of chemokine generation on c5a and tnf-alpha the role of c5a in the development of thrombotic glomerulonephritis in rats carboxypeptidase b2 deficiency reveals opposite effects of complement c3a and c5a in a murine polymicrobial sepsis model the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 li, hao, liu and li. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-255034-x100xo2t authors: theresine, maud; patil, neha d.; zimmer, jacques title: airway natural killer cells and bacteria in health and disease date: 2020-09-25 journal: front immunol doi: 10.3389/fimmu.2020.585048 sha: doc_id: 255034 cord_uid: x100xo2t natural killer (nk) cells are innate lymphoid cells at the interface between innate and adaptive immunity and mostly studied for their important roles in viral infections and malignant tumors. they can kill diseased cells and produce cytokines and chemokines, thereby shaping the adaptive immune response. nowadays, nk cells are considered as a strong weapon for cancer immunotherapy and can for example be transduced to express tumor-specific chimeric antigen receptors or harnessed with therapeutic antibodies such as the so-called nk engagers. whereas a large body of literature exists about the antiviral and antitumoral properties of nk cells, their potential role in bacterial infections is not that well delineated. furthermore, nk cells are much more heterogeneous than previously thought and have tissue-characteristic features and phenotypes. this review gives an overview of airway nk cells and their position within the immunological army dressed against bacterial infections in the upper and predominantly the lower respiratory tracts. whereas it appears that in several infections, nk cells play a non-redundant and protective role, they can likewise act as rather detrimental. the use of mouse models and the difficulty of access to human airway tissues for ethical reasons might partly explain the divergent results. however, new methods are appearing that are likely to reduce the heterogeneity between studies and to give a more coherent picture in this field. historically, human natural killer (nk) cells have mostly been harvested from and studied in peripheral blood (pb), which is an easy way to access them, and where they usually represent 5-20% of all lymphocytes (1) (2) (3) . two different subsets have been initially described, called cd56 bright cd16 − (up to 10% of pb nk cells) and cd56 dim cd16 bright (at least 90% of pb nk cells). phenotypic and functional (cytokine production for the former and cytotoxic activity for the latter) characteristics distinguish both populations (1) (2) (3) . however, things are not that simple, as four additional subpopulations have been identified, which are (i) cd56 bright cd16 dim , (ii) cd56 dim cd16 − , (iii) cd56 − cd16 bright and finally (iv) cd56 dim cd16 dim (4) , the latter still being almost systematically overlooked in the literature (5) . human nk cell functions are governed by a balance between the messages transmitted through inhibitory receptors (ir), such as kir, cd94/nkg2a, ilt2, tigit, and activating receptors (ar), such as particularly nkg2d and the natural cytotoxicity receptors (ncr) nkp46, nkp30, and nkp44 (6). when stimulated, nk cells exert natural cytotoxic activity against tumor cells and virally infected cells, antibody-dependent cellular cytotoxicity (adcc) toward antibody-coated target cells via the fcγ receptor cd16, and cytokine and growth factor production (2, 6) . most of the ligands of the ir are represented by human leukocyte antigen (hla) class i molecules, so that target cells lacking those molecules in part or in total, become killed by the nk cells. the ir nevertheless have another important function, as they are responsible for nk cell education. indeed, before a developing nk cells becomes functional, its self-specific ir must interact with their ligands expressed by cells in their micro-environment (7, 8) . nk cells without such ir, which can represent up to 20% of all pb nk cells, remain uneducated, and hyporesponsive (7, 8) . however, they can be activated under certain conditions, such as some viral infections (9) . a hot topic in the nk cell field is of course their potential use as immunotherapeutic anticancer agents. to reach this aim, several approaches are studied, and for example the chimeric antigen receptor (car) nk cells, which allow the specific targeting of a tumor antigen (10) , or the use of multi-specific antibody constructs directed simultaneously at several nk cell ar and tumor surface molecules (6), appear as particularly promising. it has also been discovered that nk cells, which had been previously considered as exclusively innate immune cells, can develop a memory-like behavior (11) . finally, nk cell metabolism, which appears to be different between educated and uneducated cells, is extensively studied (12, 13) . another aspect that has changed our view on nk cells in recent years is the observation of a broad heterogeneity of this population. not only are there many subsets in pb based on the clonal distribution of several ir, immature, partly mature and completely mature fractions based on the relative expression of cd56, cd16 and the ir nkg2a and kir (14) , conventional and adaptive nk cells (14, 15) , but there are also heterogeneous aspects between pb and various tissues (15, 16) . very recent data by dogra et al. (17) suggests a model in which the phenotype, the degree of maturity and the functions of nk cells are closely dependent on the anatomic location, with no influence of age and gender. it is quite difficult to find a substantial amount of references regarding upper airway nk cells. in human, the articles were mostly reporting on the investigation of nk cells in chronic rhinosinusitis, an inflammatory state of the mucosa of the nose and the sinuses (18) with a significant impact on quality of life. two different forms, one with nasal polyps and one without nasal polyps, are distinguished (19, 20) . bacterial pathogens are considered as one of the etiological factors in this disease (18) . however, as the bacteriology of ethmoidal biopsies was the same regardless of the presence or absence of polyps, niederfuhr et al. questioned the bacterial role in the pathogenesis of the polyps as well as a systematic antibiotic treatment (19) . in a study of 18 patients, further subdivided into those responding and those resistant to treatment, and 19 healthy controls, kim et al. investigated exclusively pb nk cells. the authors demonstrated that the pb nk cells from the patients had decreased effector functions compared to the healthy controls, with the treatmentresistant individuals being most severely affected (18) . the recent manuscript by kaczmarek et al. (20) reported not only on pb nk cells, but also on those from nasal mucosa and from nasal polyps. however, the exploitation of the material was limited to cd3 − cd56 + cd16 + events, which excluded the population of cd56 bright cd16 − nk cells that might be numerically well represented in these tissues. the phenotypic investigations of this subset in the nose revealed a predominance of relatively immature, cd27 + nk cells. furthermore, the ar nkg2d was expressed at lower frequencies (lower percentages of nkg2d + cells) and lower density of expression in the nasal mucosa and the polyps compared to pb (populations negative for nkg2d were identified in the tissues). finally, the percentage of nk cells among lymphocytes (mean: 33%) was significantly higher in the polyps than in pb (20) . okada et al. published a paper about nk cells in the nasal mucosa of the mouse on the c57bl/6 background (21) , in which they showed that these nk cells were more immature (according to the relative levels of expression of cd27 and cd11b) and phenotypically more activated (reduced expression of cd62l, higher percentage of cd69 + cells) than those from spleen and lung. around 12% expressed the tissue residency and activation marker cd69 and one third of those also cd103. the pattern of expression of the ly49 receptor family was different between the three tissues. functionally [cd107a staining and interferon (ifn)-γ production], nasal nk cells appeared to be hyporesponsive compared to their spleen, but not their lung counterparts (21) , which might be related to the possibility that the fraction of cd69 + nk cells was not sufficient to significantly activate the global nk cell population in the chosen experimental readouts. although this dataset is interesting per se, it should not be ignored that casadei and salinas (22) , in a review about different animal models of nasal infections and immunity, cited several anatomic (functional vomeronasal organ in contrast to human, no waldeyer's ring) and physiologic (macrosmatic, no coughingsneezing reflex, lower sensitivity to human viruses) limitations of the mouse in this context, so that such results should always be considered with care before extrapolating to the human situation. lung nk cells have recently been extensively reviewed in this journal (23, 24) , so that a summary of their most important features might be sufficient. lungs are constantly exposed to microparticles from the environment. particularly, as the mucosal lung epithelium is at the interface between the outside world and the organism, it can become the entry site for infectious pathogens, be they bacterial, fungal, or viral in nature. therefore, an extensive and sophisticated local immune response is waiting to be triggered at this anatomic location, and human nk cells, which represent around 5-20% of lung lymphocytes (24) , are a part of it. the work of marquardt et al. has established that most human lung nk cells represented the circulating subset and had the mature cd56 dim cd16 bright phenotype (25) . they expressed more frequently the differentiation marker cd57 as well as educating kir than blood nk cells from the same donors but were relatively hyporesponsive upon stimulation with hla class i-negative target cell lines. in addition, however, a putative tissue-resident subset (around 20% of all lung nk cells), further subdivided into relatively immature cd56 bright cd16 − and cd56 dim cd16 − cells (24, 25) , expressed the tissue residency markers cd69, cd49a, and cd103. these cells were characterized in detail again by marquardt et al. (26) , who showed that they were functional, especially after stimulation with the cytokine interleukin (il)-15 and displayed a unique transcriptional profile. several subpopulations could be distinguished based on the relative expression of cd49a and cd103 (24, 26) . natural killer cells have likewise been investigated in mouse lungs, particularly by wang et al. (27) and michel et al. (28) . both groups found that lung nk cells were more mature than those from the spleen (28) or other organs (27) according to the relative expression of cd27 and cd11b. whereas the former authors described a higher expression level of the ir cd94/nkg2a and a lower level of the ar nkg2d, the second paper could confirm this data only regarding nkg2d in terms of mean fluorescence intensities. lung nk cells proliferated less, degranulated less (cd107a assay) and were less cytotoxic than splenic nk cells (28) , but these functions were rapidly up-regulated upon bacterial lung infection (27) . this suggests that at homeostasis, lung nk cells are inhibited to avoid damage to normal autologous cells, but that they can quickly intervene in case of an infectious insult (27) . michel et al. showed in in vitro co-culture systems that both spleen and lung macrophages could significantly up-regulate the cytotoxic activity of lung nk cells through a contact-dependent mechanism (28) . regarding the homeostatic situation, research in recent years has revealed that in contrast to the older view of the lungs as sterile organs, a lung microbiota is present in the lower airways which exerts significant effects in health and disease, although it is not as abundant as in the gut (29) (30) (31) (32) . the term "microbiota" refers to all the microorganisms present, namely bacteria, fungi, protozoans, and viruses (29) , but here we will only consider the role of bacteria. six phyla are predominantly represented in the lower airways: firmicutes, proteobacteria, bacteroidetes, actinobacteria (31, 32) , acidobacteria, and fusobacteria (32) . this microbiota is supposed to be transient in healthy donors and to be established from micro-aspiration and inhalation (32) and its composition at any given time point submitted to the parameters of bacterial arrival, bacterial removal, and local immune responses (32, 33) . in this way, an equilibrium state is reached that depends also strongly on the gut microbiota through various bacterial metabolites and contributes to the maintenance of homeostasis in the lower airways (gut -lung axis) (32) (33) (34) . everything that disturbs this balance, such as some medications and particularly antibiotics, increases in nutrients (high fat diet, low fiber diet), cigarette smoke, infectious agents, chronic inflammation, can disturb the gut as well as the lung microbiota and lead to a state of dysbiosis, characterized by an increased number of airway bacteria and a change in its composition. the dysbiosis is profoundly linked to several severe lung diseases [asthma, chronic obstructive pulmonary disease (copd), infections, cancer] (29) (30) (31) (32) (33) (34) (35) . natural killer cells have, to our knowledge at least, not been investigated in detail in the context of a normal lung microbiota to date. as most lung nk cells are not activated nor tissueresident (as illustrated by their negativity for cd69), they might not react very strongly to a normal microbiota. however, as they are expressing several bacteria-specific toll-like receptors (tlrs) that signal in the presence of bacterial pathogens (36) , it might be conceivable that they could also mount an immune response toward microbiota components and that this would contribute to homeostasis. the overall immunosuppressed state of lung nk cells at baseline would help to avoid aggression of harmless and useful bacteria and of uninfected autologous cells (31) . yang et al. (31) , as well as fuchs and colonna (37) , discuss data claiming that at steady state, alveolar macrophages secrete immunosuppressive cytokines which keep nk cells in respect. this is in contrast with the results of michel et al. (28) , discussed above. however, the macrophages and dendritic cells (dc) switch to pro-inflammatory cytokine production in case of a bacterial or viral infection and thereby activate the nk cells. this entity is the third cause of mortality in the united states of america (3) and worldwide (38) and is in most cases the consequence of prolonged cigarette smoking (39) . it is characterized by airflow obstruction, emphysema, recurrent infections (24, 39) , chronic inflammation, and overproduction of mucus (40) . acute exacerbations significantly limit the quality of life of the patients (38, 39) . the exacerbations are in principle caused by viral or bacterial infections, the latter most frequently due to haemophilus influenzae, streptococcus pneumoniae, and moraxella catarrhalis (39) . pseudomonas aeruginosa is another bacterium frequently involved and one of the most dangerous ones, based on its highly pathogenic properties (39) , and its remarkable level of resistance to antibiotics. natural killer cells have been investigated in human copd as well as in animal models of this disease. motz et al. demonstrated that exposure of pulmonary leukocytes to viral pathogenassociated molecular patterns (pamp) induced higher functional properties (degranulation measured with the cd107a assay, and ifn-γ production) ex vivo in chronic cigarette smoke exposed than in non-exposed c57bl/6 mice (40) . interestingly, bacterial pamp appeared to be less efficient in this model, as among five molecules tested, only fsl-1 (bacterial lipopeptide, tlr2/6 agonist) and lipopolysaccharide (lps, tlr4 ligand) increased the percentage of ifn-γ + nk cells above the one of the non-exposed mice. in contrast, other papers reported that nk cell functions are diminished in copd (41) . it has been further repeatedly demonstrated that in copd or relevant animal models, nk cell cytotoxic activity is increased relative to non-copd smokers and healthy individuals (23, 24) . based on the model of lung nk cell hypo-responsiveness at baseline, cigarette smoke and even more the inflammatory state of the lower airways in copd would activate the alveolar macrophages and induce their production of pro-inflammatory cytokines. these would, in turn, unleash the nk cells and increase their cytotoxic activity, cytokine and chemokine expression, leading to a further aggravation of the inflammation and the clinical status of the patients. indeed, in accordance with this concept, freeman et al. (42) showed that cd56 + cells (in fact a mixture of nk cells and cd56 + t lymphocytes) isolated from lung parenchymal samples of non-copd smokers and copd patients with a smoking history, although similar in terms of frequencies between the cohorts, had a different cytotoxic activity toward autologous lung epithelial cells. the cd56 + lymphocytes from the copd patients were more cytotoxic than the cells from the non-copd smokers, in an experimental setup without additional stimulation. the target cells were supposed to be mostly epithelial cells based on their positivity for cd326, their size, and their negativity for the hematopoietic cell marker cd45. the cytotoxicity was measured as the percentage of annexin v + target cells after the co-culture with the effectors and was around 10% in most samples. this was not a lot, but the nk cells and cd56 + t cells were not otherwise activated. most of the parenchymal lung nk cells were cd56 + cd16 + and the minor rest cd56 + cd16 − (42). another study was provided by the same group in 2018 (43) . it showed that isolated, purified lung nk cells induced apoptosis in autologous epithelial cells. this time, the mean level of cytotoxicity was rather high compared to the previous paper, and it was very significantly stronger in copd patients than in non-copd smokers. the nk cells, but not the target cells, determined this increased cytotoxic activity, because k562, a hla class i-negative myeloid leukemia cell line used as the standard nk cell target, was also lysed more efficiently by copd nk cells than by their non-copd counterparts. the authors confirmed their data in a mouse model and then showed that the nk cells were primed by dc via trans-presentation of il-15 (43), a phenomenon first described in 2007 by andreas diefenbach and his group (44) . this would nicely explain the higher level of nk cell cytotoxicity observed in copd. along the same line, okamoto et al. administered il-2 and il-18 to normal mice and observed a lethal effect within 4 days, selectively involving the lungs, with a profound interstitial infiltration of lymphocytes dominated by nk cells (45) . high levels of various cytokines and chemokines were found in serum and lungs. depletion of the nk cells by antibodies completely abrogated the lethal injury, which is a convincing demonstration of the potentially destructive power of nk cellactivating cytokines and nk cells themselves (45) . this work was intended as a contribution to the elucidation of the pathogenesis of interstitial pneumonia, but similar mechanisms, in the presence of high levels of pro-inflammatory cytokines in bacterial infections, might contribute to copd. in human cancer patients, administration of high dose il-2 induced a vascular leakage syndrome where the so-called lymphokine activated killer cells (equivalent to highly activated nk cells) destroyed endothelial cells, causing a generalized edema, and damaging several organs (46) . hodge et al. demonstrated a higher number of nk cells in the bronchoalveolar lavage fluid (balf) of copd patients (the cohort was composed of current smokers and of ex-smokers) than in healthy smokers (47), a higher content of the cytolytic molecule granzyme b and, most importantly, a significantly increased cytotoxic activity against k562. they also found a reduction in the percentage of balf nk cells expressing cd94 (which they consider as ir, although it is more a chaperone molecule for the true ir nkg2a). nevertheless, this indirect measure of a down-regulation of nkg2a could indicate that it contributes to the higher nk cell cytotoxic activity observed in copd (47) . recently, osterburg et al. presented a multiparameter flow cytometry study of pb nk cells from copd patients compared with smokers and never smokers (38) . in contrast to those, copd patients and smokers highly expressed the maturation marker cd57 as well as the ar nkp46 and nkp44 (normally only present on activated but not on baseline nk cells), but lower levels of cd56. certain nk cell subpopulations were indicative of prior exacerbations (38) . the ar nkg2c, which is significantly present only on adaptive nk cells from human cytomegalovirus (cmv)-infected individuals, was not differentially expressed in pb of copd patients with a smoking history and healthy volunteers, but present on a higher percentage of nk cells in the patients with the most frequent exacerbations and the most reduced lean mass (48). a relationship with the bacterial burden cannot be excluded in this context, as there might be a correlation between the viral reactivations and the bacterial colonization, contributing together to the higher number of exacerbations. most of the papers discussed above investigated the nk cell cytotoxicity toward autologous cells or conventional nk target cells, but what about a potential direct bacterial killing? nk cells, upon appropriate stimulation, release cytolytic molecules called perforin, granzymes and, in human but not in mice, granulysin, which have an additive or synergistic cytolytic effect toward bacteria (49) . they can form pores in the target cell walls and thereby eliminate the microorganisms, but in addition they are able to eliminate some types of eukaryote cells infected by bacteria (41, 49, 50) . furthermore, in addition to the direct effect, nk cells are embedded in the immunological network and react (through an increased cytotoxic activity and cytokine production) to the immune cells and the cytokines/chemokines in their environment (50) , which is strongly shaped in case of a bacterial infection ["cellular crosstalk" (50) ]. data about chronic rhinosinusitis, nasal polyposis and copd are summarized in table 1 . as mentioned above, this ubiquitous gram-negative pathogen is part of those colonizing the lower airways in copd, but it is also a major problem in cystic fibrosis and in nosocomial infections, with a high morbidity and mortality (51) . the role of nk cells in the host defense against this bacterium has been quite extensively studied by the team of michael t. borchers (51, 52) in consequences of the indicated diseases on nk cell phenotype and functions. copd, chronic obstructive pulmonary disease; balf, bronchoalveolar lavage fluid; and ifn-γ , interferon-gamma. mouse models. in the chronologically first work, outbred cd-1 mice were intranasally infected with the p. aeruginosa laboratory strain pao1 (52) and evaluated 24 h later. the findings can be summarized as follows: (i) the infection increased the expression of ligands for the ar nkg2d, present on almost all nk cells but also on a subpopulation of cd8 + t lymphocytes, in vivo; (ii) similarly, these ligands increased in an infected human lung epithelial cell line in vitro; (iii) the inhibition of the ar nkg2d with a monoclonal antibody significantly reduced the clearance of p. aeruginosa from the lungs; (iv) antibody-mediated nkg2d blockade down-regulated the amount of the cytokines il-1β, ifn-γ and tumor necrosis factor (tnf)-α and in addition of nitric oxide; and finally, (v) the same experiment also revealed a threefold reduction of epithelial cell damage, measured as shedding of these cells into the balf (52) . the latter point brings us again to the recurrent theme of lung cell damage that can be induced by activated nk cells, whereby it would have to be determined if this is beneficial (elimination of infected cells by nk cells) or deleterious (exaggerated damage to the epithelium). the follow-up paper (51) then presented a conditional mouse model with an inducible expression of nkg2d ligands on lung epithelial cells. here, the bacterial clearance was significantly higher in those mice that overexpressed the nkg2d ligands. moreover, the survival up to 96 h post-infection and the level of phagocytosis were significantly increased in the latter group. similarly, in in vitro experiments, where the nk cells were stimulated with lps, the percentage of nk cells producing ifnγ was much higher in the mice with the increased expression of nkg2d ligands. as expected, this percentage dropped (but was not completely abolished) in nk cells from infected mice treated with an anti-nkg2d antibody (51) . however, the p. aeruginosa-derived exotoxin a, which in combination with il-1α may induce a dangerous inflammatory state with tissue damage in the host, has also been shown to inhibit nk cell cytotoxic activity against k562, even in the presence of usually stimulating cytokines such as il-2 (53). the inhibition was almost complete with a high dose of the toxin and still partial with a low dose (53) . the effector cells were not purified nk cells but peripheral blood mononuclear cells (pbmc), so that an indirect effect on the nk lymphocytes might play a role in this readout. furthermore, pedersen and kharamzi described already in 1987 that the p. aeruginosa-derived alkaline protease and elastase inhibited nk cell cytotoxic activity against k562, presumably due to a reduction in the effector-target conjugate formation (54) . in addition, these molecules strongly reduced the binding of an anti-cd16 (called leu-11 at that time) antibody (54) . this group of pathogenic gram-negative bacteria is composed of several species, of whom some are dangerous for cystic fibrosis patients, as they are highly resistant to multiple antibiotics (55). li et al. (55) investigated the interaction between burkholderia cenocepacia and nk cells, and first demonstrated that the nklike leukemia cell line yt (56) , as well as primary purified human nk cells, significantly reduced the number of living bacteria (measured as cfu) after a co-incubation of 2 to 4 h. the results were confirmed with life cell imaging techniques and bacterial uptake of propidium iodide (pi). the authors then wanted to know if the killing activity was contact-dependent or not, and first showed that yt cells bound the fluorochromelabeled bacteria. then, they could demonstrate that a direct contact was needed for the killing activity, as nothing happened to the bacteria when they were separated from the nk cells by a porous membrane, allowing passage of soluble molecules but not of cells (55) . most bacteria remained extracellular and were not taken up by the yt cells. killing was almost completely abrogated after treatment with strontium chloride (srcl 2 ), which is known to deplete nk cells from their cytotoxic granules (57) . finally, it was established that src family kinases were activated in yt cells after the contact with b. cenocepacia (55) . this is a very nice demonstration that nk cells are able to directly kill certain extracellular bacterial species through nk cell -bacteria contact, although the precise mechanism is still unknown. other possible mechanisms of nk cell-mediated elimination of bacteria are the lysis of intracellular pathogens within the infected cells and the activation of other immune cells, and particularly of macrophages, via nk cell-derived cytokines (such as ifn-γ) (55) , and most likely also the killing of bacteria-infected cells expressing ligands for nk cell ar. this is another gram-negative pathogen which poses a major problem due to its frequent causative involvement in nosocomial infections (particularly in pneumonia) and the steady increase of strains multi-resistant to antibiotics (58). chalifour et al. (59) demonstrated that the outer membrane protein a (kpompa) from this microorganism, known to signal via tlr2, induced ifn-γ and α-defensin (an antimicrobial peptide) synthesis and release in human nk cells. in the mouse, both nk cell-derived ifn-γ (58) and il-22 (60) have been described to be necessary for bacterial clearance (58, 60) . in the paper from xu et al., it was nicely shown with genetic controls and depletion experiments that the immune defense against this pathogen indeed deeply involved nk cells and that a subset of them produced il-22. the nk cells had a conventional and mature phenotype (less cd27 + , more klrg1 + ) distinct from other innate lymphoid cells (ilc) (60). ivin et al. focused on the fact that ifn-γ production by nk cells, likewise necessary for the elimination of the bacteria through a network with alveolar macrophages, was dependent on the nk cell-intrinsic stimulation by type i ifn, in turn induced by k. pneumoniae (58) . in contrast to the crucial role of nk cell-derived cytokines, their granzymes (a and b) , one of the constituents of the lytic granules, did not seem to play a major role in this model (61) . however, this does not rule out that in human, granulysin and perforin together might have a cytotoxic effect on these bacteria. in the case of helicobacter pylori, responsible for chronic gastric inflammation with the potential to lead to ulcers or cancer, preincubation with fixed bacteria increased the cytotoxic activity of nk cell-enriched pbmc toward k562 and other tumor target cells, as well as the release of ifn-γ (62). furthermore, rudnicka et al. showed that the bacterial glycine acid extract induced nk cell expansion and ifn-γ production, whereas the lps from the same bacteria inhibited these parameters, and instead favorized the apparition of il-10-producing nk cells (63) . although this might just marginally be relevant for nk cells in the lungs, it nevertheless shows to which extent these cells can react to bacteria and how the latter try to manipulate them. legionella pneumophila, the agent of legionnaires' disease, is replicating intracellularly in macrophages. here again, nk cell production of ifn-γ, induced probably through direct tlr messages (64) one of the most frequent culprits in community-acquired pneumonia is s. pneumoniae. regarding the role of nk cells in this infection, their beneficial or detrimental action depended on the pathogen's serotype (68). thus, the control of serotype 1 depended on nk cells, as demonstrated by baranek et al. in a mouse model (68). these authors investigated the consequences of a defect in the transcriptional cofactor four-and-a-half lim-only protein 2 (fhl-2) on nk cells in general and on pneumococcal infection particularly. it had been previously established that ifn-γ was, once more, the crucial factor in host defense in this context, and that nk cells were one of its major producers (69) . in the spleen and the lungs of fhl-2 knockout (ko) mice, the number of nk cells and their expression of the ar nkg2d and nk1.1 (cd161c) were down-regulated and a negative effect of the deficiency on nk cell maturation was observed. mortality to s. pneumoniae lung infection was strongly increased in the ko mice but could be rescued by the adoptive transfer of wildtype nk cells. finally, the authors showed that ifn-γ production by nk cells was severely reduced and that less neutrophils were recruited to the lungs of the ko animals (68). the role of the mostly immunosuppressive cytokine il-10 in dampening the immune response to pneumococcal infection was shown in 1996, when van der poll et al. administered the pathogen intranasally together with il-10 and observed early mortality and reduced levels of the pro-inflammatory factors ifn-γ and tnf. conversely, all this was restored when the mice were pre-treated with an anti-il-10 antibody (70). these results were very recently confirmed by clark et al. (71) , who worked with il-10 reporter and il-10-ko mice to observe that s. pneumoniae induced il-10 production by nk cells (around 50% of total lung nk cells) with a negative effect on animal survival, and that the bacterial burden was diminished in the lungs of the ko mice compared to wildtype animals. nk cell depletion in the latter induced a strong reduction in the bacterial lung counts and in il-10. furthermore, il-10-deficient mice had significantly more neutrophils and monocytes in the infected lungs. finally, the virulence protein spr1875 from s. pneumoniae was identified as the il-10-inducing factor (71) . none of these papers investigated the potential balance between the pro-inflammatory and anti-inflammatory effects of ifn-γ and il-10, respectively, on the outcome of this infection, which would anyhow have been technically challenging. one might suppose that il-10 is there to down-regulate an overwhelming immune response that would damage lung tissues, but on the other hand, it might also be counterproductive to dampen it too much and thus to lose control over the pathogens (72) . other groups have described that human as well as mouse nk cells could produce and release il-10 (73, 74), although, according to perona-wright et al., this only occurred in the case of a systemic, but not a localized, pulmonary infection (with the gram-negative bacterium yersinia pestis) (74) . in the case of systemic infections with listeria monocytogenes and y. pestis, approximately 50% of blood nk cells became il-10 + , and the cytokine was produced by a nk cell subset circulating in blood prior to the infection (74) . before studying s. pneumoniae (71), clark et al. had already shown that l. monocytogenes elicits il-10 production by nk cells via the virulence factor p60 (with, as a consequence, an inhibition of the recruitment and the activation of myeloid cells) in a mouse model of systemic infection, where the lungs were not further investigated (75) . another frequently encountered nosocomial and multiresistant infectious agent is staphylococcus aureus. small et al. could demonstrate the fundamental role of nk cells in the response to these bacteria in the case of mouse lung infections (76) , as (i) nk cell numbers in the airways increased; (ii) in vitro contact with products from the pathogens activated nk cells; (iii) co-culture of nk cells with alveolar macrophages increased the phagocytic activity of the latter, (iv) il-15-ko mice were much more susceptible to the infection than wildtype mice, whereas they had much more neutrophils and macrophages in the lungs; and (v) nk cell depletion rendered even wildtype mice highly sensitive, despite a conserved il-15 production (76). these findings demonstrate indeed once again the important role of nk cells in immune defense against extracellular bacteria. in accordance with this model, zhao et al. showed that particular matter, associated epidemiologically with enhanced numbers of lung infections, diminished the amount of nk cells migrating to rat lungs in case of infection with s. aureus, whereas adoptive nk cell transfer restored a vigorous nk cell response (77) . in ex vivo experiments, nk cells improved, as in the previous study, the phagocytosis of the pathogens by alveolar macrophages. it is well known that after influenza, recovering patients are very susceptible to bacterial superinfection, notably by s. pneumoniae and s. aureus (78) . the contribution of nk cells to this phenomenon was demonstrated in a mouse model of h1n1 influenza virus infection followed by intratracheal instillation of s. aureus. the sequentially double-infected mice were much more susceptible to the infection (weight loss, survival rate) than those receiving pbs or bacteria only. this went hand in hand with severe changes in the histopathological aspect of the lungs and a marked reduction of local nk cell numbers and tnfα + nk cells. furthermore, the concentrations of tnf-α and of the chemokines ip-10 and mip-1α were diminished in the balf. adoptive transfer of naive nk cells could restore the immune response. the nk cells needed tnf-α to perform their antibacterial effect and this was organized via an interaction with alveolar macrophages and increased phagocytosis (78) . the conclusion that might be drawn from all these papers is that nk cells are very important, at least in mouse models, for the immune response to and the defense against pulmonary infections due to gram-positive bacteria, with, on the other hand, a detrimental influence of these lymphocytes in case they produce too much immunosuppressive factors [the same old story (72) ]. mycobacterium tuberculosis is the agent of tuberculosis (tb), an infectious disease that puts a high burden on the populations in developing but also in developed countries and increasingly shows resistance to conventional antibiotics. it latently infects about 25% of the total population and becomes clinically apparent in ten million patients per year, according to estimations from the world health organization (who) (79, 80) , rendering it a major public health issue. as recently reviewed by cong and wei (23) , nk cells could interact with this intracellular pathogen through the ar nkp46, nkp44, and nkg2d, as well as tlr2. although they became activated under these conditions, they seemed to play only a negligible protective role, according to junqueira-kipnis et al. (81) . these authors showed in a mouse model that lung nk cells augmented in number within the first 3 weeks after exposure to aerosols containing the mycobacteria and up-regulated cd69, ifn-γ and perforin. however, their depletion did not at all change the kinetics of the infection. human pb nk cells likewise up-regulate ifn-γ after contact with m. tuberculosis (23) . barcelos et al. (82) compared pb nk cells in cohorts of patients with active tb, isolated tuberculin + skin tests, and tuberculin − healthy donors. they found a different subset distribution according to the cohorts, with putative tb-exposed but-resistant individuals (defined as those with a positive tuberculin test) having overall less nk cells but an increased percentage of cd56 − cd16 + , cd56 + cd16 − and especially cd56 bright cd16 −/+ nk cell subsets compared to the other two donor groups. in contrast, tb patients displayed lower frequencies of cd56 + cd16 + cells. the authors speculated that this different subset distribution might have been related to the resistance or sensitivity to active tb, but of course, as the cells stem from pb, this dataset might have to be interpreted with some caution. surprisingly, however, roy chowdhury et al. (80) , by following a cohort of adolescents from an endemic region in south africa, could demonstrate with mass cytometry and functional experiments, that latent tb was associated with increased responses of pb nk cells, with a particular role for the ar cd16. indeed, the percentages of nk cells among total living cells, of total cd16 + cells, of granzyme b + and of perforin + cells were significantly higher in patients with latent tb than in healthy, non-infected donors. in addition, adcc (mediated via cd16) against p815 target cells was also higher in latent tb. by following further cohorts, the authors found that the percentage of pb nk cells was dynamically regulated during latency, progression of the disease and responses to antibiotic medication. this level of nk cells in pb even correlated inversely with inflammation in the lungs of patients with active tb (80) . such observations push nk cells again at the forefront of immune defenses in tb and at a possible role in the maintenance of latency. with a similar cohort-based approach, harris et al. (79) evaluated nk cell phenotype and functions in individuals with latent tb compared with healthy controls. furthermore, participants were separated in infected and non-infected in a tb-endemic region in kenya and a healthy volunteer cohort from the united states. among the three groups, the persons from the united states had the significantly lowest percentage of cd56 − cells, which are known to expand in chronic human immunodeficiency virus (hiv) infection and other viral diseases and to be dysfunctional (83) . the kenyan volunteers displayed, among cd56 dim nk cells, a higher expression of granzyme b and of the non-mhc class i-specific ir tigit, with the highest levels found in the healthy cohort. furthermore, these individuals had an increased expression of the ar nkp46. within the cd56 bright subpopulation, the kenyan participants showed increased expression of nkg2d but again decreased levels of nkp46, compared to the cohort from the united states. functionally, degranulation (cd107a assay), ifnγ production (intracellular flow cytometry) and cd69 expression were compared between the three cohorts after co-culture with k562 cells (evaluation of natural cytotoxicity) and p815 mouse cells plus anti-mouse antibody (evaluation of adcc). whereas for the latter parameter, no significant differences were observed, frequencies of cd69 + , cd107a + , and ifn-γ + nk cells turned out to be significantly higher in the united states study population, such as if an environment endemic for tb would impact the "missing self "-recognition capacities of nk cells (79) . the same regional discrepancies were observed after stimulation of total pbmc with three different antigen extracts from m. tuberculosis, and the reactivity to these antigens was shown to be at least partially dependent on the presence of il-12 and il-18, supposed to be derived from accessory cells. conradie et al. (84) described that the level of activation of pb nk cells (frequency of cd69 + and cd69 + hla-dr + events) allowed, among other parameters, to discriminate between m. tuberculosis-induced immune reconstitution syndrome, hiv infection and co-infection with both pathogens. although these three papers suggest some influence of m. tuberculosis on pb nk cells, it is not clear yet to which extent nk cells really intervene in the immune defense against this pathogen that persists in the lungs. an investigation on tuberculous pleurisy (85) revealed a large predominance of cd56 bright cd16 − nk cells in the pleural fluid, and an apoptotic effect of soluble factors from this environment predominantly on cd16 + nk cells. m. tuberculosis induced ifn-γ production from cd56 bright nk cells in the absence of monocytes, t cells and b cells, leaving open the possibility of a direct productive interaction between the bacteria and the nk cells. lai et al. (86) presented a work on nontuberculous mycobacterial lung infections, which means due to other mycobacterial species, such as mycobacterium abscessus and mycobacterium kansasii. as the latter become more and more prevalent in developed countries, these authors performed a study in c57bl/6 mice that were infected intratracheally with m. kansasii. they found that nk cell depletion increased bacterial burden, mortality, and pathogenetic postinfectious changes (macrophage phagocytosis, dc activation, cytokine production, and development of granuloma). the same observations were made in ifn-γ-ko animals and restored after transfer of wildtype nk cells. these cells were also the most important producer of ifn-γ in this model (86) . lai et al. further cited papers that had demonstrated a similar protective effect of ifn-γ produced by nk cells in the infections with bordetella pertussis, francisella tularensis, and chlamydia muridarum in mouse models of respiratory infection. previous publications by the same group had shown that nk cells can directly lyse m. tuberculosis and m. kansasii via the cytotoxic proteins granulysin and perforin in a contactdependent manner disrupting mycobacteria cell wall integrity (87) , and that in some patients with mycobacterial infections, anti-ifn-γ autoantibodies were detected (88) . the killing process involved signaling through nkg2d and ncr as well as map kinases, suggesting that similar mechanisms are involved for the killing of bacteria and of eukaryotic target cells (87) . this is potentially a very important observation, as it strongly suggests that both conventional cytotoxic mechanisms and cytokine production might be relevant in anti-mycobacterial defense. some studies were also performed on mycobacterium bovis bacillus calmette-guérin (bcg), an attenuated mycobacterial strain used as an anti-tuberculous vaccine (89) . for example, it was demonstrated in vitro that cd56 bright nk cells reacted to this microorganism by proliferation and ifn-γ production, whereas their cd56 dim counterparts better up-regulated the cytolytic proteins perforin and granzyme a (90), all of which was largely expected based on what is known about the functional specialization of these two nk cell subsets (1) (2) (3) . in a mouse in vivo model, where bcg was directly administered (intratracheally) into the lungs, nk cell-mediated production of ifn-γ rapidly increased in the first days after infection, similarly to the number of lung nk cells (89) . after nk cell depletion, the reduction of body weight was less pronounced compared to non-depleted mice, whereas the bacterial load remained identical. importantly, inflammation and injury of the pulmonary structures was much less pronounced in the nk cell-depleted animals, suggesting a pathogenic role for these lymphocytes. indeed, the level of pro-inflammatory cytokines and chemokines was also reduced in the absence of nk cells, and the percentages of ifn-γ + cd4 + and ifn-γ + cd8 + t cells was significantly increased in these mice. bacillus calmette-guérin-infected macrophages up-regulated nkg2d ligands, which induced their lysis via this receptor-ligand interaction. finally, the blocking of nkg2d with a monoclonal antibody restored the survival of the macrophages and the t cell-mediated immune response (89) . it is difficult to make a coherent synthesis of all these observations on nk cells and mycobacteria, but it is nevertheless quite appealing that again positive, negative and neutral aspects are described, which may vary according to the models and the experimental setup. this shows that nk cells still hide a lot of secrets regarding their function in anti-mycobacterial infections as well as in bacterial pathogenesis overall. a summary of the relationships between the bacteria discussed and nk cells is presented in table 2 . these are obligate intracellular pathogenic bacteria that are responsible for several types of human and mouse diseases. various studies dedicated to this type of microorganisms illustrated the concept that nk cells usually do not respond as a pure population as may be the case for in vitro experiments, but that in vivo they are part of a tightly controlled immune network composed of cells, cytokines, chemokines and exosomes. thus, in mouse models, nk cells influenced the interaction between dc, t helper (h)1 and th17 t lymphocytes in c. muridarum lung infection (91) , modulated the balance between th1 and th17 t cells and t regulatory cells (treg) in the same type of infection (92) , and again positively regulated the interactions between dc and t lymphocytes against chlamydophila pneumoniae (93) . in all these situations, nk cells exerted a protective and disease-controlling effect via their influence on the bridge between innate and adaptive immune responses. with the ambitious aim to experimentally investigate the famous "hygiene hypothesis, " han et al. (94) studied mice infected with c. muridarum and rendered allergic to ovalbumin (ova). they observed that prior infection could inhibit at least certain parameters of allergy. however, nk cell depletion partly suppressed the "beneficial" effect of the lung infection. adoptive transfer of nk cells from infected mice inhibited partially the development of an allergic response in non-infected recipients. nk cell-devoid mice coherently produced more th2 type cytokines ("pro-allergic" th2 cytokines, il-4, and il-5) than ifn-γ ("anti-allergic" th1 cytokine). a detrimental effect of nk cells had been shown for the immune response to the respiratory rodent pathogen mycoplasma pulmonis, related to the human infectious agent mycoplasma pneumoniae. indeed, in a quite complicated experimental setup, bodhankar et al. demonstrated that nk cell depletion interfered positively with the development of a protective adaptive immunity after nasal-pulmonary immunization with bacterial antigens (95) . this could be explained because nk cells shaped the t cell cytokine response toward more il-4, il-13, and il-17 but away from ifnγ production. wurzel et al. presented large cohort studies of children with protracted bacterial bronchitis (pbb) and mild bronchiectasis, associated or not with human adenovirus co-infection (96, 97) . besides typical socio-economic and clinical factors, an elevated nk cell number relative to the values of healthy children of the same age was observed in the pb of diseased children in general and with adenovirus species c particularly. nk cell phenotype and function were not further investigated. recently, sim et al. (98) made the important discovery that the hla-c-specific activating kir2ds4 did recognize a conserved bacterial peptide presented by hla-c, and more precisely by hla-c * 05:01. the sequence of the peptide required for this recognition was a "rare" self-peptide, but the epitope of interest is conserved in the recombinase a (reca) of many bacterial species (more than 1000 according to the authors' claims), most of them belonging to serious human pathogens, such as helicobacter pylori, brucella, campylobacter jejuni, and chlamydia trachomatis (98) . interestingly, activation of resting nk cells via kir2ds4 alone was sufficient to induce degranulation and cytokine production, whereas all other known ar, except cd16, need at least one co-activating molecule engaged at the same time (99) . there was, furthermore, an inverse correlation between the frequency of the kir2ds4 full length gene and the hla-c * 05:01 allele. thus, it appears that the kir family is not only involved in nk cell licensing and in multiple disease associations, but also, most likely, in antibacterial defense. this paper received an accompanying commentary by peter parham, which places the findings in the broader context of kir and hla class i molecules (100) . to sum up, nk cells might be directly activated by various bacteria via contact-dependent mechanisms whose modes of functioning are still unknown, via tlr, via kir2ds4, or more indirectly via the up-regulation of ligands for their ar, such as nkg2d, by infected cells. they might also react to cytokines released into the microenvironment by antigen-presenting cells (macrophages, dc). dietert et al. published a plea for the histopathological evaluation of the consequences of different infectious lung diseases in mouse models and described the pathogen-specific features characteristic for each of them (101) . indeed, many variations were observed between the infecting microorganisms, be they bacterial or viral in nature. the authors emphasized that histopathology remains the "most conclusive and practical read out" for the evaluation of the effects of the various infectious models on mouse lungs. although this is true, more "modern" and state-of-theart methods are being developed and are about to enter the laboratories, as a consequence of general scientific and technical progress but also of the "3r" approach regarding experiments with animals. in 2019, the team of hans clevers described the generation of human airway organoids derived from surgical material or from balf (102) . these were long-term proliferating structures that recapitulated a normal airway with different types of cells that are physiologically present in vivo. the beauty of the system per se was already an accomplishment, but it could be used for the study of various lung diseases, such as cystic fibrosis, cancer, or viral infections (102) . therefore, it is likely that bacterial infections could similarly be investigated in this system, and the data obtained would probably be more relevant to human pathology than the mere mouse models (and save the life of many mice by the way). the same year, ross et al. (103) published a review on the "ex vivo human lung." they worked with donor lungs not retained for transplantation, extracted primary cells from them and developed an "ex vivo-perfused single human lung" that would allow the investigation of different lung diseases. the system seems at first sight less elegant than the lung organoids and is maybe also more limited in the spectrum of possible pathologies that can be investigated. the advantage would be that an entire, complete organ is available and not just an organoid. yet another option is the "alveolus-on-a-chip, " developed by deinhardt-emmer et al. (104) . it was a three-dimensional structure with an air phase and a liquid phase, where endothelial cells, epithelial cells and macrophages could be co-cultured. in the presented work, a primary influenza virus infection, followed by a s. aureus superinfection, were investigated, and it was shown that the endothelium was seriously damaged under these conditions. likewise, single cell transcriptomics is a powerful tool that can reveal huge amounts of details about all kinds of immune cells, and among them nk cells, as exemplified by lung cancerinfiltrating immunocytes in human and mouse (105) . one aspect of nk cells is their putative potential for a dual role as "pro-inflammatory" and "regulatory" effectors, which might be mediated by different subsets (106) . our group has previously touched the problem that nk cells are in fact a double-edged sword, meaning that they might have sometimes beneficial but sometimes rather deleterious effects (72) . this has again become clear throughout this review, although the models and studies presented and discussed were all but homogeneous. it might be expected that this will change in the coming years if more and more teams will use the organoid and organ on-a-chip technologies and go into various "omics." overall, given the current and justified hype for nk cells as efficient agents for cancer immunotherapy, it would be difficult to convince the nk community that their favorite cells might also have a dark side. we emphasize that several methods to improve nk cell antitumoral efficiency, such as particularly car-nk cells (10) and nk cell engagers, recently described by the vivier group (107) , should be sufficient to stand up for the use of nk cells in this indication. however, what about the therapeutic indication of nk cells in infectious diseases in general and in the lungs particularly? due to the current covid-19 pandemic, this question has gained increased interest (108, 109) , and in addition, most of the papers discussed here that describe an influence of nk cells on the disease course in the airways conclude with the statement that nk cells should be targeted in respiratory infections. but it has clearly been shown that these lymphocytes can have detrimental side effects and cause significant damage to the airways, at least in viral diseases (108, 109) . available literature does not give clear indications regarding bacterial pathogenesis, but the issue was already discussed in 2012, with the question if nk cells are angels or devils in bacterial infectious diseases (50) . this problem is, in our opinion, not yet resolved and a lot of research work will be necessary in the field, keeping in mind that the number of multi-resistant bacterial strains is increasing at a terrifying rate and that alternatives to antibiotics must be discovered and developed. finally, a general problem in the field and a caveat to many of the presented studies is the difficulty of distinguishing nk cells reliably form ilc1, which also produce ifn-γ as a signature cytokine and have a partially overlapping phenotype. a high plasticity within the ilc family renders even possible the conversion of nk cells, in certain microenvironments, into ilc1like cells (110) (111) (112) . however, whereas both nk cells and ilc1 require the transcription factor t-bet for their development and function, nk cells need and express eomes in addition. ilc1 are preferentially located in tissues and are very rare in peripheral blood, in contrast to nk cells. thus, one can be confident that the studies discussed here that worked with blood (human) and blood or spleen (mouse) nk cells really investigated nk cells and not ilc1. for tissue-based studies, the differences might be more blunted, although ilc1 are considered as non-cytotoxic cells (110) (111) (112) . these difficulties are in line with the increasing number of "new" cell types that are currently discovered [for example mr1 t cells (113) ], as a consequence of the ever growing diversification and performance of the experimental tools in immunology. mt and np conceived the article, participated to bibliographic research, and critically reviewed the final revised manuscript. jz conceived the article and wrote the manuscript. all authors contributed to the article and approved the submitted version. the biology of human natural killer-cell subsets human natural killer cells human cd56 bright nk cells: an update human cd56 dim cd16 dim cells as an individualized natural killer cell subset. front immunol cd56 dim cd16 dim natural killer (nk) cells: the forgotten population harnessing innate immunity in cancer therapy nk cell education via nonclassical mhc and non-mhc ligands natural killer cell education and the response to infection and cancer therapy: stay tuned unlicensed' natural killer cells dominate the response to cytomegalovirus infection use of car-transduced natural killer cells in cd19-positive lymphoid tumors nk cell-mediated recall responses: memory-like, adaptive, or antigen-specific? natural killer cell education is associated with a distinct glycolytic profile education-dependent activation of glycolysis promotes the cytotoxic potency of licensed human natural killer cells deciphering natural killer cell homeostasis emerging insights into natural killer cells in human peripheral tissues the broad spectrum of human natural killer cell diversity tissue determinants of human nk cell development, function, and residence natural killer cells from patients with chronic rhinosinusitis have impaired effector functions the bacteriology of chronic rhinosinusitis with and without nasal polyps nk cells in patients with chronic rhinosinusitis show decreased maturity and limited expression of functional receptors identification and analysis of natural killer cells in murine nasal passages comparative models for human nasal infection and immunity natural killer cells in the lungs nk cells in the human lungs human lung natural killer cells are predominantly comprised of highly differentiated hypofunctional cd69 − cd56 dim cells unique transcriptional and protein-expression signature in human lung tissue-resident nk cells lung natural killer cells in mice: phenotype and response to respiratory infection mouse lung and spleen natural killer cells have phenotypic and functional differences, in part influenced by macrophages role of microbiota on lung homeostasis and diseases. sci china life sci the lung microbiome: clinical and therapeutic implications the impact of lung microbiota dysbiosis on inflammation the role of lung and gut microbiota in the pathology of asthma emerging pathogenic links between microbiota and the gut-lung axis dysbiosis of the gut and lung microbiome has a role in asthma the influence of lung microbiota on lung carcinogenesis, immunity, and immunotherapy toll-like receptors in natural killer cells and their application for immunotherapy natural killer (nk) and nk-like cells at mucosal epithelia: mediators of anti-microbial defense and maintenance of tissue integrity unique natural killer cell subpopulations are associated with exacerbation risk in chronic obstructive pulmonary disease chronic obstructive pulmonary disease (copd): evaluation from clinical, immunological and bacterial pathogenesis perspectives chronic cigarette smoke exposure primes nk cell activation in a mouse model of chronic obstructive pulmonary disease human cd56+ cytotoxic lung lymphocytes kill autologous lung cells in chronic obstructive pulmonary disease lung dendritic cells drive natural killer cytotoxicity in chronic obstructive pulmonary disease via il-15rα dendritic cells prime natural killer cells by trans-presenting interleukin 15 il-18) in synergy with il-2 induces lethal lung injury in mice: a potential role for cytokines, chemokines, and natural killer cells in the pathogenesis of interstitial pneumonia observations on the systemic administration of autologous lymphokine-activated killer cells and recombinant interleukin-2 to patients with metastatic cancer enhanced cytotoxic function of natural killer and natural killer t-like cells associated with decreased cd94 (kp43) in the chronic obstructive pulmonary disease airway microbial killing by nk cells natural killer (nk) cells in antibacterial immunity: angels or devils nkg2d is critical for nk cell activation in host defense against pseudomonas aeruginosa respiratory infection the nkg2d-activating receptor mediates pulmonary clearance of pseudomonas aeruginosa effect of pseudomonas aeruginosa exotoxin a on ifn-γ synthesis: expression of costimulatory molecules on monocytes and activity of nk cells inhibition of human natural killer cell activity by pseudomonas aeruginosa alkaline protease and elastase natural killer cells kill burkholderia cepacia complex via a contact-dependent and cytolytic mechanism natural killer cell line yt exerts cytotoxicity against cd86+ myeloma cells cryptococcus neoformans directly stimulates perforin production and rearms nk cells for enhanced anticryptococcal microbicidal activity natural killer cell-intrinsic type i ifn signaling controls klebsiella pneumoniae growth during lung infection direct bacterial protein pamp recognition by human nk cells involves tlrs and triggers α-defensin production conventional nk cells can produce il-22 and promote host defense in klebsiella pneumoniae pneumonia granzymes a and b regulate the local inflammatory response during klebsiella pneumoniae pneumonia contact of lymphocytes with helicobacter pylori augments natural killer cell activity and induces production of gamma interferon helicobacter pylori-driven modulation of nk cell expansion, intracellular cytokine expression and cytotoxic activity myd88-dependent ifngamma production by nk cells is key for control of legionella pneumophila infection innate immunity against legionella pneumophila during pulmonary infections in mice a novel role for neutrophils as critical activators of nk cells role of gamma interferon in induction of natural killer activity by legionella pneumophila in vitro and in an experimental murine infection model fhl2 regulates natural killer cell development and activation during streptococcus pneumoniae infection. front immunol nk and nkt cell depletion alters the outcome of experimental pneumococcal pneumonia: relationship with regulation of interferon-γ production interleukin-10 impairs host defense in murine pneumococcal pneumonia il-10-producing nk cells exacerbate sublethal streptococcus pneumoniae infection in the lung revisiting the functional impact of nk cells production of il-10 by human natural killer cells stimulated with il-2 and/or il-12 systemic but not local infections elicit immunosuppressive il-10 production by natural killer cells bacterial manipulation of nk cell regulatory activity increases susceptibility to listeria monocytogenes infection nk cells play a critical protective role in host defense against acute extracellular staphylococcus aureus bacterial infection in the lung exposure to particular matter increases susceptibility to respiratory staphylococcus aureus infection in rats via reducing pulmonary natural killer cells influenza infection leads to increased susceptibility to subsequent bacterial superinfection by impairing nk cell responses in the lung distinct human nk cell phenotypes and functional responses to mycobacterium tuberculosis in adults from tb endemic and non-endemic regions a multi-cohort study of the immune factors associated with m. tuberculosis infection outcomes nk cells respond to pulmonary infection with mycobacterium tuberculosis, but play a minimal role in protection natural killer cell subpopulations in putative resistant individuals and patients with active mycobacterium tuberculosis infection cd56 negative nk cells: origin, function, and role in chronic viral disease natural killer cell activation distinguishes mycobacterium tuberculosis-mediated immune reconstitution syndrome from chronic hiv and hiv/mtb coinfection increased susceptibility to apoptosis of cd56 dim cd16 + nk cells induces the enrichment of ifn-γ-producing cd56 bright cells in tuberculous pleurisy nk cell-derived ifnγ protects against nontuberculous mycobacterial lung infection nk cells kill mycobacteria directly by releasing perforin and granulysin identification of a major epitope by anti-interferon-γ autoantibodies in patients with mycobacterial disease nk cells inhibit anti-mycobacterium bovis bcg t cell responses and aggravate pulmonary inflammation in a direct lung infection mouse model human cd56 bright and cd56 dim natural killer cell subsets respond differentially to direct stimulation with mycobacterium bovis bacillus calmette-guérin natural killer cells regulate th1/treg and th17/treg balance in chlamydial lung infection nk cells modulate the lung dendritic cell-mediated th1/th17 immunity during intracellular bacterial infection nk cells modulate t cell responses via interaction with dendritic cells in chlamydophila pneumoniae infection nk cells contribute to intracellular bacterial infection-mediated inhibition of allergic responses nk cells interfere with the generation of resistance against mycoplasma respiratory infection following nasal-pulmonary immunization adenovirus species c is associated with chronic suppurative lung diseases in children prospective characterization of protracted bacterial bronchitis in children human nk cell receptor kir2ds4 detects a conserved bacterial epitope presented by hla-c activation, coactivation and costimulation of resting human natural killer cells a natural killer cell receptor takes sharp aim at the world of bacteria spectrum of pathogen-and model-specific histopathologies in mouse models of acute pneumonia long-term expanding human airway organoids for disease modeling the ex vivo human lung: research value for translational science co-infection with staphylococcus aureus after primary influenza virus infection leads to damage of the endothelium in a human alveolus-on-a-chip model single-cell transcriptomics of human and mouse lung cancers reveals conserved myeloid populations across individuals and species regulatory nk cells suppress antigen-specific t cell responses multifunctional natural killer cell engagers targeting nkp46 trigger protective tumor immunity flattening the covid-19 curve with natural killer cell based immunotherapies the promise and peril of natural killer cell therapies in pulmonary infection innate lymphoid cells: diversity, plasticity, and unique functions in immunity nk cells and ilcs in tumor immunotherapy plasticity of innate lymphoid cell subsets mr1-restricted t cells are unprecedented cancer fighters the authors would like to thank prof. dr. markus ollert, md, the director of the department of infection and immunity of the luxembourg institute of health, for his continuous support. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 theresine, patil and zimmer. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-265005-e6rpryrh authors: tomasello, elena; pollet, emeline; vu manh, thien-phong; uzé, gilles; dalod, marc title: harnessing mechanistic knowledge on beneficial versus deleterious ifn-i effects to design innovative immunotherapies targeting cytokine activity to specific cell types date: 2014-10-30 journal: front immunol doi: 10.3389/fimmu.2014.00526 sha: doc_id: 265005 cord_uid: e6rpryrh type i interferons (ifn-i) were identified over 50 years ago as cytokines critical for host defense against viral infections. ifn-i promote anti-viral defense through two main mechanisms. first, ifn-i directly reinforce or induce de novo in potentially all cells the expression of effector molecules of intrinsic anti-viral immunity. second, ifn-i orchestrate innate and adaptive anti-viral immunity. however, ifn-i responses can be deleterious for the host in a number of circumstances, including secondary bacterial or fungal infections, several autoimmune diseases, and, paradoxically, certain chronic viral infections. we will review the proposed nature of protective versus deleterious ifn-i responses in selected diseases. emphasis will be put on the potentially deleterious functions of ifn-i in human immunodeficiency virus type 1 (hiv-1) infection, and on the respective roles of ifn-i and ifn-iii in promoting resolution of hepatitis c virus (hcv) infection. we will then discuss how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the subtypes and dose of ifn-i produced, (ii) the cell types affected by ifn-i, and (iii) the source and timing of ifn-i production. finally, we will speculate how integration of this knowledge combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments in patients. specifically, we will discuss how induction or blockade of specific ifn-i responses in targeted cell types could promote the beneficial functions of ifn-i and/or dampen their deleterious effects, in a manner adapted to each disease. type i interferons (ifn-i) were the first cytokines discovered, over 50 years ago, based on their potent anti-viral effects (1, 2) . ifn-i play a crucial, non-redundant role in vertebrate anti-viral defenses (3) (4) (5) . ifn-i also mediate protective effects in other physiopathological contexts, including cancer (6) and multiple sclerosis (ms) (7) . on the contrary, ifn-i responses can be deleterious in a number of circumstances, including bacterial or fungal infections (8-10), many autoimmune diseases (11) , and, paradoxically, certain chronic viral infections (12) (13) (14) . it is only recently that an integrated picture has emerged of the cellular and molecular mechanisms regulating the production of ifn-i and underlying their functions. much knowledge was gained recently on another class of potent innate anti-viral interferons, the lambda, or type iii ifns (ifn-iii). we will review knowledge on ifn-i/iii (ifns) and discuss how it could be harnessed to develop innovative therapeutic strategies aimed at surgically tuning ifn activity toward protective responses in a manner adapted to each disease. we will focus on ifn-α/β/λ because they are the best characterized ifns and already used therapeutically. recent reviews are covering information on other ifn-i subsets including ifn-ε, which is produced at mucosal sites and promotes local anti-viral defenses (15, 16) . dendritic cells (dcs) are rare heterogeneous mononuclear phagocytes functionally characterized by their unique efficacy for antigen-specific activation of naïve t lymphocytes. dcs are sentinel cells of the immune system, able to sense and integrate a variety of danger input signals for delivery of output signals instructing the activation and functional polarization of effector immune cells. in mammals, five major dc subsets exist, which differ in their expression of innate immune recognition receptors (i 2 r 2 s) and in their functional specialization: monocyte-derived dcs (modcs), langerhans cells, cd11b + dcs, xcr1 + dcs, and plasmacytoid dcs (pdcs) (17) . a recurrent theme of this review will be the intricate relationships between ifns and dcs, since these cells can be a major source and/or target of these cytokines under various conditions. www.frontiersin.org the first section will synthesize current knowledge on ifn production and protective anti-viral functions. the i 2 r 2 s and downstream signaling pathways responsible for ifn-i production during viral infection will be listed. the roles of different cell types for this function will be discussed. the two main mechanisms through which ifn-i promote anti-viral defense will be reviewed, succinctly for direct anti-viral effects and in greater details for immunoregulatory functions. the second section will focus on the detrimental functions of ifn-i. selected diseases will be discussed to illustrate how different, and sometimes opposite, processes underlie deleterious ifn-i responses depending on the physiopathological contexts. ifn-i induction of unbridled inflammatory responses causing lethal tissue damage will be discussed as a major pathological mechanism during bacterial encounters secondary to influenza infection or in some autoimmune diseases. inappropriate functional polarization of immune responses by ifn-i will be discussed as one potential cause for enhanced susceptibility to bacterial or fungal infections. the complex and disputed role of ifn-i in chronic viral infections will be reviewed, with emphasis on the physiopathology of the infections by human immunodeficiency virus type 1 (hiv-1) and human hepatitis c virus (hcv), with an outlook for the development of novel immunotherapeutic strategies to combine with anti-viral drugs. the third section will recapitulate how the balance between beneficial versus deleterious ifn-i responses is modulated by several key parameters including (i) the source and timing of ifn-i production, (ii) the cell types affected by ifn-i, and (iii) the signaling pathways activated by ifn-i. in the last section, we will speculate how integration of all the knowledge discussed before combined with advanced biochemical manipulation of the activity of the cytokines should allow designing innovative immunotherapeutic treatments, based on induction or blockade of specific ifn-i responses in targeted cell types. this"activity-by-targeting"concept is based on the design of novel "immuno-ifns" consisting in covalent association between a mutated ifn-i with decreased affinity for its receptor and an antibody with high avidity for a molecule specifically expressed on target cell types (18) . this design ensures lack of activity of the immuno-ifns on all cell types but those targeted, contrary to previous strategies using ifns with close to maximal potency that were still able to mediate strong off-target effects despite their coupling to cell-type specific antibodies and/or their local delivery. type i interferons expression is not detectable under steady state conditions in vivo using classical methods such as gene expression analysis by rt-pcr or protein titration by elisa or bioassays. however, mice deficient for the expression of the alpha chain of the ifn-i receptor (ifnar1) harbor alteration in the ontogeny or functions of various cell types (19) (20) (21) (22) (23) (24) (25) (26) . hence, extremely small or localized but functionally relevant quantities of ifn-i must be produced under steady state conditions (27) . indeed, the existence of steady state responses to ifn-i in various organs in vivo was demonstrated by using reporter mice expressing the firefly luciferase under the control of the promoter of ifnb1 (28) or of mx2 (29) , a canonical ifn-i-stimulated gene (isg). steady state ifn-i responses are promoted by gut commensals (30) . early and transiently after many viral infections, large amounts of ifns can be detected, in blood and spleen in the case of systemic infections or locally in the case of confined infections. ifn induction during viral infections results from the detection of specific danger signals by specialized i 2 r 2 s. this includes the detection of pathogen-associated molecular patterns as well as the sensing of stress signals or damage-associated molecular patterns (31, 32) . based on the nature and intracellular location of the danger signals that induce the production of the cytokines, the cellular sources of ifns during viral infection can be classified in two main groups. infected cells often contribute to ifn production as a response to their sensing of endogenous viral replication, or consecutive to the metabolic stress induced during massive translation of viral structural proteins, or as a result of plasma membrane perturbations upon viral entry. specific subsets of uninfected cells can also significantly contribute to ifn production upon engulfment of material containing viral-derived nucleotide sequences and sensing of these molecules in endosomes by specific i 2 r 2 s. all sensing pathways leading to ifn induction converge on the activation of interferon response factors 3 or 7 (irf3/7), which are the master transcription factors inducing ifn genes. most cell types constitutively express irf3 but not irf7 or only at low levels. irf7 expression requires ifn-i stimulation. ifn-β can directly be induced by irf3. all but one of the ifn-α subtypes require irf7 for their induction. hence, ifn-β secretion promotes its own production and that of ifn-α in an autocrine manner (33, 34) . this positive feedback loop strongly amplifies ifn production during viral infections, promoting fast and widespread induction of cell-intrinsic anti-viral defenses in uninfected cells to prevent virus dissemination. other feedback loops tightly regulate ifn-i production positively or negatively. this section reviews different mechanisms controlling ifn production and how they could play different roles in host/virus interactions. different innate immune recognition receptors are involved in sensing various types of viral nucleic acids in distinct categories of cells during viral infections, which may promote different types of anti-viral defenses. for each selected sensor shown, the types of viral nucleic acids recognized and the downstream signaling cascade induced are represented in a simplified, schematic manner. the potential specific role of each cell type in anti-viral defenses is also indicated at the bottom of each panel. (a) potentially all types of infected cells can detect endogenous viral replication through cytosolic sensors triggering their local production of ifn-β/λ to control viral replication in an autocrine and paracrine fashion in infected tissues. (b) uninfected xcr1 + dcs selectively produce high levels of ifn-λ and ifn-β upon engulfment of materials containing dsrna and the consecutive triggering of tlr3 in endosomes. the receptor of ifn-λ is mostly expressed by epithelial cells. hence, xcr1 + dcs might be involved in inducing local ifn responses in virally infected epithelial tissues. since xcr1 + dcs are especially efficient at producing ifn-iii upon hcv stimulation, they might contribute to local or systemic ifn production during infection with this virus, to promote ifn-λ-mediated protection of hepatocytes. uninfected xcr1 + dcs and other uninfected cells may produce some ifn-β upon engulfment of materials containing ssrna and the consecutive triggering of tlr8 in endosomes. the contribution of this pathway to anti-viral defense is not well understood yet, in part because mouse tlr8 is deficient for this function. (c) uninfected pdcs selectively produce high levels of all subsets of ifns upon engulfment of materials containing ssrna or cpg dna and the consecutive triggering of tlr7/9 in endosomes. however, pdcs seem to be activated for this function only in lymphoid tissues. hence, pdc might contribute to systemic ifn production during blood-borne viral infections or as a failsafe mechanism activated upon abnormal widespread dissemination of a viral infection once it has escaped local confinement at its portal of entry. cm, cell membrane; nm, nuclear membrane. particular nucleotide sequences or tertiary structures, their signaling pathways and their physiological significance have been recently reviewed (31, 32) . cytosolic rna sensors encompass dexd/h helicases among which the retinoic-acid-inducible gene (rig)-i-like receptors (rlrs) have been the most studied, namely rig-i and melanoma differentiation associated gene 5 (mda5). rig-i recognizes rna with a 5 -ppp or 5 -pp (38) (uncapped) moiety, or double-stranded rna (dsrna), both structures being present in viral, but not in cytosolic eukaryotic, rna molecules. mda5 might specifically recognize long dsrna fragments. both rig-i and mda5 contain a dexd/h box-containing rna helicase domain, and 2 caspase recruitment domains (card1/2), which bind to mitochondrial anti-viral signaling protein (mavs). rna/rlr/sting molecular complexes initiate a signaling cascade leading to irf3/7-dependent induction of ifns (figure 1 ). other dexd/h helicases can promote ifn-i production in dcs, www.frontiersin.org although their physiological roles for in vivo immune defenses against viral infections remain to be established (32) . cytosolic dna sensors able to induce ifn-i (mostly ifn-β) and ifn-iii encompass molecules belonging to different protein families, including dexd/h helicases, the inflammasome component ifn-γ-inducible protein 16 (ifi16) , the z-dna binding protein 1 (zbp1), and the cyclic gmp-amp (cgamp) synthase (cgas) (31, 32) . most of the cytosolic dna sensors activate sting and lead to irf3/7-and nfκb-dependent induction of ifn-β and ifn-iii. many cell types express zbp1 and are able to produce ifn-i upon triggering of this molecule, including macrophages, dcs, and fibroblasts following an hsv-1 infection (39, 40) . upon dna binding, cgas catalyzes the production of cgamp. cgas is critical for the detection of lentiviruses including hiv-1/2 (41, 42) and can contribute to sensing of, and protection against, other rna viruses, including in vivo in mice (43) . cgamp also acts as a secreted second messenger signal alerting uninfected cells to directly induce their expression of intrinsic immune anti-viral defenses. the cgas/sting/irf3 signaling cascade and the irf1 transcription factor are "master" inducers of cell-intrinsic immunity able to control the replication of most dna and some rna viruses at least in part independently of ifns (43) . infected cells become a factory for production of viral particles. hijacking of the translation apparatus of the host cell for massive production of viral structural proteins leads to an overload of the capacity of the er for correct folding of newly synthesized proteins. er overload induces a homeostatic response of the cell, the unfolded protein response (upr). upr aims at restoring normal er functions by inhibiting translation. upr activation in infected cells contributes to prevent viral replication, including through inhibition of the production of viral proteins, promotion of ifn-i production, and induction of cell suicide (44) . toll-like receptors (tlrs) are among the first and best characterized i 2 r 2 s. tlrs are transmembrane proteins with a leucine-rich repeat extracellular domain involved in ligand recognition and an intracellular toll/interleukin-1 receptor domain essential for signaling (45) . among the nine tlrs conserved between mouse and human, tlr3, tlr7, tlr8, and tlr9 are located in endosomes where they can detect the abnormal presence of nucleic acids such as occurs upon endocytosis of virions or of virally infected cell material. tlr3 recognizes dsrna, tlr7/8 ssrna, and tlr9 dna sequences containing unmethylated cytidinephosphate-guanosine (cpg) motifs. tlr fine specificity and signaling pathways have been reviewed recently (32) and are summarized in figure 1 . we will discuss the expression patterns and functions of endosomal tlrs with regards to ifn production in uninfected specialized immune cell types, pdcs and xcr1 + dcs. plasmacytoid dcs uniquely produce very large amounts of ifns in response to in vitro stimulation with many viruses, without being infected (46) . ifn-i mrnas represent up to 40% of all mrnas in pdcs at the peak of their activation (47) . in vitro, upon exposure to influenza virus, herpes virus type 1, cytomegaloviruses, or vesicular stomatitis virus, individual pdcs produce 100-1000 times more ifns than total pbmcs, monocytes, modcs, cdcs, neutrophils, and fibroblasts (47) (48) (49) (50) (51) (52) . however, in vitro, high molarity infection of cdcs with certain viruses unable to inhibit ifn-i production in their target cells can also induce massive ifn-β secretion (53) . pdcs produce high levels of all subtypes of ifns, contrary to many other cell types including infected cells, which often preferentially produce ifn-β (46, 47) . in vivo, pdc depletion during systemic viral infections leads to over 95% decrease of ifn-i production, while the total number of pdcs producing ifn-i (<100,000 in one mouse) is much lower than the total number of infected cells (54) (55) (56) (57) (58) (59) . this shows that in vivo also individual activated pdcs produce much more ifn-i/iii than most other cell types, including virus-infected cells. the professional ifn-producing function of pdcs largely results from their high constitutive and selective expression of irf7, tlr7, and tlr9 (figure 1) . these molecules are pre-associated in readyto-signal complexes located in specialized endosomes specific to pdcs (60, 61) . pdcs must also be equipped for efficient sensing and up-take of virions and virus-infected cells. the corresponding cell surface i 2 r 2 s remain to be identified. selective expression of tlr3 in xcr1 + dc endows them with a unique ability to produce very high amounts of ifn-β and ifn-iii upon stimulation with dsrna or hcv irrespective of their own infection. xcr1 + dcs are very potent for antigenspecific activation of cd8 + t cells, in particular through crosspresentation of exogenous antigens that they have captured from other cells and processed for association with class i major complex histocompatibility (mhc-i) molecules (62) . in mice, xcr1 + dcs are crucial for the initiation of protective adaptive immune responses against tumors and a variety of viruses (63) . mouse and human xcr1 + dcs constitutively and selectively express high levels of tlr3 (figure 1) . they produce large amounts of ifn-iii and ifn-β upon stimulation with a synthetic mimetic of dsrna, polyinosinic:polycytidylic acid (polyi:c) (64, 65) . human xcr1 + dcs uniquely respond to stimulation with hcv by producing large amounts of ifn-iii in a tlr3-dependent manner (66, 67) , irrespective of their own infection. positive feedback loops. in addition to irf7 induction, other positive feedback mechanisms exist to amplify the production of ifns rapidly after initiation of a viral infection as illustrated by the following selected examples. ifns induce the expression of many cytosolic rna/dna sensors and of tlr7. this broadens the spectrum of host's cell types able to detect endogenous viral replication for ifn induction. induction of oasl by ifns in human cells enforces rig-i signaling, counteracting viral immune frontiers in immunology | microbial immunology evasion genes interfering with this sensing pathway (68) . the ifninducible ribonuclease l (rnasel) generates viral and cellular rna degradation products, which engage rlrs for amplification of ifn production (69, 70) . the ifn-inducible protein kinase r (pkr) stabilizes ifn-i mrna (71) . to prevent unbridled responses deleterious for the host, ifn activity must be tightly controlled including during viral infections. several negative feedback loops exist to terminate ifn production, after anti-viral defenses have been activated. the isg ubiquitin specific peptidase 18 (usp18) binds to ifnar2, preventing it from recruiting signal transducer and activator of transcription 1 (stat1). ifns induce the expression of tam receptor tyrosine kinases in dcs, monocytes, and macrophages. tam receptors associate and signal in part through ifnar1. they activate the suppressors of cytokine signaling-1/3 (socs-1/3). socs inhibit tlr and rlr signaling, thereby terminating ifn production (72) . tam receptor ligands, gas6 and pros, bind phosphatidylserine on dying cells and are produced by activated dcs and monocytes/macrophages. thus, ifn induction of tam inhibitory receptors on uninfected phagocytic immune cells could limit their propensity to produce the cytokines upon engulfment of dying virally infected cells. ifns induce tetherin on most cell types. pdcs express a receptor for tetherin, leukocyte immunoglobulin-like receptor, subfamily a (with tm domain), member 4 (lilra4). lilra4 triggering on pdcs inhibits their production of ifn-i. hence, through lilra4 engagement by tetherin, pdcs can monitor their efficacy at inducing an antiviral gene expression program in neighboring cells through ifns, and timely terminate their ifn production. how positive and negative feedback loops integrate in time and space to promote optimal kinetics and intensity of ifn production in order to efficiently control viral infection without causing severe immunopathology is not completely understood. positive feedback loops may occur very rapidly after initiation of viral infection to allow rapid secretion of high levels of the cytokines for fast and strong induction of anti-viral cell-intrinsic immunity. negative feedback loops occur likely later to terminate the response and thus avoid chronicity of cytokine production and its ensuing deleterious effects. pdcs do not constitute the major source of ifn production upon local infections by several viruses in the lung or in the female reproductive tract. pdcs are dispensable for resistance against these infections (56, 73, 74) . during pulmonary infection by newcastle disease virus (ndv), ifn-i are produced locally in the lungs mainly by infected alveolar macrophages. lung pdcs do not express the cytokines (73) . selective depletion of lung alveolar macrophages leads to systemic dissemination of ndv, and to a strong activation of pdcs for ifn-i production specifically in the spleen. even in the case of systemic viral infections such as caused by intravenous injection of ndv or intraperitoneal injection of mouse cytomegalovirus (mcmv), pdc ifn production is confined to the spleen. it is not detected in other organs even those with high viral replication (59, 73) . hence, splenic pdcs are especially prone to high level ifn production upon systemic acute viral infections. pdcs located in non-lymphoid organs, in particular mucosal barrier tissues, appear to be inhibited for ifn production. thus, ifn production by infected cells serves as first line of defense to block virus replication at its portal of entry in the body. ifn production by uninfected pdcs might constitute a failsafe mechanism mainly activated in the spleen when viral infection gets systemic (75) . under these conditions, to promote health over disease, the benefits for the host of producing high circulating levels of ifns in order to induce widespread cell-intrinsic anti-viral defenses might prevail over the deleterious effects that this could cause on certain cell types or tissues. indeed, pdcs are required for protection against hsv-2 and hsv-1 in mice only in systemic but not local infections (56) . this observation is consistent with the crucial role of pdcs for protection of mice against systemic infection by mouse hepatitis virus (mhv), a fast replicating coronavirus (55) . conflicting results have been obtained on the role of pdcs during intranasal influenza infection (74, (76) (77) (78) . a possible explanation is that pdc ifn production contributes to resistance to highly pathogenic influenza strains that might systemically spread from the lung early after infection, even if at low levels. another intriguing observation is that ifns are critical for host resistance to mcmv and that pdcs are the major source of ifns in this infection model but are dispensable for virus control (54) . studies are ongoing to understand this apparent paradox. patients bearing genetic mutations disrupting endosomal tlr signaling do not appear to suffer from life-threatening viral infections (79, 80) , contrary to patients impaired in ifnar signaling (4, 81) . a notable exception is the specific susceptibility to severe herpes virus encephalitis in individuals' deficient for tlr3 signaling (82, 83) . however, contrary to extracellular tlr, endosomal tlr have evolved under strong purifying selection in human beings (84) . hence, while pdcs and endosomal tlr might have been required for protection of our species against viral infections in the past, this appears not to be the case anymore perhaps due to improved social, hygiene, and health care in modern society (75) . attesting to the importance of ifns for anti-viral defense in vertebrates, many mammalian viruses encode immune evasion genes specifically inhibiting the production of ifns in infected cells (39, 85) . pdcs or xcr1 + dcs might be essential for ifndependent host protection against these viruses, because they are spared from the intracellular functions of viral immune evasion genes (75) . to the best of our knowledge, mcmv does not encode for immune evasion genes inhibiting ifn production. however, mcmv manipulates ifn-i responses through specific inhibition of stat1 functions in infected cells. thus, pdcs might be dispensable for resistance against systemic mcmv infection due to sufficient levels of ifn production by infected cells locally in all infected tissues. hepatocyte responses to ifn-iii appear to play a www.frontiersin.org critical role in human resistance to hcv. in infected hepatocytes, hcv induces the expression of cellular micrornas binding to ifn-iii mrna and leading to its degradation. uninfected xcr1 + dcs produce high levels of ifn-iii in vitro upon hcv stimulation (66, 67) . hence, during acute hcv infection in vivo, xcr1 + dc may be a strong and early source of ifn-iii not subjected to virus immune evasion strategies, therefore, contributing to protect naturally resistant individuals. in secondary lymphoid organs, a subset of macrophages is critical for the clearance of viruses from the lymph (86) . these macrophages are located on viral entry routes, near to subcapsular sinuses where the afferent lymph drained from non-lymphoid tissues flows. contrary to other subsets of macrophages, subcapsular sinus macrophages are highly susceptible to viral infection, because they constitutively express only low levels of effector molecules of cell-intrinsic anti-viral immunity and because their responses to ifns are inhibited by their high constitutive expression of usp18. subcapsular sinus macrophages rapidly become infected by viruses incoming from the lymph and produce large amounts of ifns. this altruistic suicide prevents virus dissemination to other adjacent cell types and promotes the induction of innate and adaptive anti-viral immunity (87) . upon instruction by ifns, cells express a wide variety of viral restriction factors, whose combined action blocks pathogen invasion by interfering with the different stages of viral life cycle (figure 2a ). this has been extensively reviewed recently (88) and will only be described succinctly here. virus fusion with host cell membrane can be blocked by cholesterol-25hydrolase (ch25h) that inhibits sterol biosynthesis. some viruses enter cells by escaping from endosomes/lysosomes, which can be blocked by interferon inducible transmembrane (ifitm) proteins. virus uncoating can be blocked by tripartite motif (trim) proteins, such as trim5α, which bind to hiv-1 capsid thus promoting its degradation, and by myxoma resistance gtpases, mx1, and mx2, which efficiently trap viral structural proteins at an early stage following virus entry into the cell. mx1 inhibits a number of viruses, including influenza virus through sequestration of its nucleocapsid. mx2 associates with host cyclophilin a and hiv-1 capsid protein. virion assembly can be blocked at transcriptional, translational, and posttranslational levels. the adenosine deaminase acting on rna 1 (adar1) and the apolipoprotein b mrna editing enzyme, catalytic polypeptide-like (apobec) deaminases induce viral rna destabilization and hypermutation (89, 90) . the sterile alpha motif and histidine-aspartic domain (hd) containing protein 1 (samhd1) blocks reverse transcription by hydrolyzing dntps (91) . adar1, apobec, and samhd1 functions have been mainly studied in infections by hiv-1 and other retroviruses. the 2 ,5 -oligoadenylate synthase (oas) proteins, the ifn-induced proteins with tetratricopeptide repeats (ifit), and pkr inhibit viral and host protein translation by using complementary mechanisms (88) . the major post-translation modification induced by ifns is the binding of the ubiquitinlike modifier isg15 to several viral and host proteins, a process called isgylation. most of the known isgylated proteins are targeted for degradation, with few exceptions that are on the contrary stabilized like irf3 (88) . finally, the egress and budding of virions of many enveloped viruses can be inhibited by tetherin or by viperin (88) . many anti-viral isgs have been functionally characterized only recently, largely thanks to large-scale screening approaches. they display a variable degree of viral specificity (43, 92) that might inversely relate to the extent of their side effects on host cells ( figure 2b ). anti-viral effectors acting on a broad spectrum of viruses often target key metabolic pathways that are also crucial for host cell functions. this is the case for the control of cholesterol metabolism by ch25h (93) or of protein translation by pkr, oas, or ifits (88) . other anti-viral restriction factors such as mx2 may specifically target one molecule of a very restricted set of viruses with no apparent side effects on host cells. some anti-viral isgs target specific functions critical for only a restricted array of viruses and might similarly exert side effects only on a subset of host cell types. for example, samhd1 inhibits retrovirus replication through dntp depletion, which might more specifically affect proliferating host cells. hence, the infected host must balance the intensity, breadth, and location of isg induction to circumvent viral replication while preventing life-threatening damages to vital cell types or tissues. one of the mechanisms contributing to this balance is translational control of the expression of isgs, especially those with pro-apoptotic or anti-proliferative functions (94) . while many anti-viral isgs are transcriptionally activated in most ifn-stimulated cells, their translation can be specifically blocked in uninfected cells by cellular microrna. this inhibition is relieved upon cell infection through negative regulation of the function of the rna-induced silencing complex. hence, ifn stimulation of uninfected cells prepares them for rapid and strong induction of cell-intrinsic anti-viral defenses upon viral infection while avoiding their unnecessary exposure to the toxic effects of certain isgs. further knowledge on the functions and the dynamic regulation of isgs is essential to develop novel therapeutic strategies against viral infections aiming at modulating ifn responses to promote their protective anti-viral cell-intrinsic functions over their deleterious toxic effects. a better understanding of the immunoregulatory effects of ifns will also help. type i interferon can modulate the functions of a broad spectrum of immune cells ( figure 3a ). we will review this knowledge, focusing on the functions of dcs, nk cells, t cells, and b cells, since they are involved in the control of most viral infections. we will discuss the hypothesis that dcs play a central role in ifn-i orchestration of innate and adaptive immunity for the induction of optimal anti-viral defenses (figure 3) . during viral infections and cancer immunosurveillance, ifn-i constitute one of the most important input signal acting on dcs to promote their delivery of appropriate output signals to t cells, b cells, and nk cells for protective immunity ( figure 3a ). dcs deliver three types of signals to activate and functionally polarize t cells. signal 1 is the triggering of the t cell receptor by viral peptide-mhc complexes. signal 2 is the triggering of activating t cell co-stimulation receptors such as cd28 or cd27 by the cd80/86 and cd70 co-stimulation molecules expressed on dcs. signal 3 corresponds to cytokines, which can promote t cell proliferation and acquisition of specific effector functions. under steady state conditions, most dcs are in an immature state characterized by low level expression of mhc-ii (signal 1) and co-stimulation molecules (signal 2) and by the lack of production of t cell-activating www.frontiersin.org cytokines (signal 3). upon activation, including early after viral infections in vivo, dcs up-regulate their expression of signal 1 and activating signal 2 and secrete t cell-activating cytokines. this process is called dc maturation. gene expression profiling of dcs stimulated by microbial stimuli identified a core set of genes upregulated in mature dcs irrespective of the stimulus they receive, irrespective of the subset they belong to, and conserved across evolution (95) . most of these genes are induced during dc maturation in part through cell-intrinsic ifn-i signaling (95) . consistently, cell-intrinsic ifnar signaling in dcs is required in many circumstances for the induction of protective immunity, including efficient cd8 t cell responses during viral infection or tumor development (96) (97) (98) , th1 responses upon polyi:c injection independently of il-12 or ifn-γ effects (99, 100) , as well as follicular helper t cell and humoral responses (101, 102) . mechanistically, ifn-i promote dc immunogenicity for efficient t cell activation through a variety of effects ( figure 3b) . it drives dc up-regulation of signal 2 in vivo during viral infections (103) and boosts their capacity to cross-present antigens for increased delivery of signal 1 to cd8 t cells (96) (97) (98) . it shapes their delivery of activating signal 3, in particular inducing il-15 and promoting or inhibiting il-12 depending on experimental conditions (58, 104) . finally, it is necessary to induce their metabolic shift from mitochondrial oxidative phosphorylation to aerobic glycolysis, which fuels the increased needs in energy and the expansion of the intracellular organelles required for the production and proper intracellular routing of the signal 1 and 2 proteins (100, 105). selective inactivation of ifnar on cdcs compromises mouse resistance to mcmv and mhv infections (103, 106) . in contrast, ifnar expression is not required on nk cells for protection against mcmv and on pdcs, t cells, and b cells for early control of mhv replication (103, 106) . although cell-intrinsic ifn-i signaling in nk cells can promote their activation (107) (figure 3a) , ifn-i-induced il-15 trans-presentation by dcs plays a more prominent role for this function in many conditions including in vivo during mcmv infection (103, 108) ( figure 3c) . cell-intrinsic ifn-i signaling in cd4 t cells (109), cd8 t cells (110, 111) , and b cells (112) can also contribute to their efficient activation and functional polarization (figure 3 ). this depends on experimental settings. cd8 t cell-intrinsic ifn-i responses are crucial for mounting efficient cytotoxic cd8 t cell responses against lcmv but are less critical against vaccinia virus and vesicular stomatitis virus (110, 113, 114) . mechanistically, cell-intrinsic ifn-i signaling in cd8 t cells can promote their survival during their antigen-induced proliferation (110) . cell-intrinsic signaling in dcs and cd8 t cells may act in a synergistic manner. indeed, conditional inactivation of ifn-i responsiveness was required to occur simultaneously in both of these two cell types to dramatically affect cd8 t cell expansion upon vaccination with a modified ankara vaccinia virus (115) . in summary, ifn-i generally play a crucial, beneficial, role in immune defenses against viral infections, both through the induction of cell-intrinsic anti-viral defenses and through the orchestration of innate and adaptive immunity. however, if these responses are not properly regulated, they can contribute to diseases as we will now discuss. a frequent side effect of ifn-i treatment against cancer or chronic viral infections is the induction of autoimmune reactions. consistently, isg expression is a hallmark of many spontaneous systemic or tissue-specific autoimmune diseases, including systemic lupus erythematosous (sle), sjogren's syndrome, psoriasis, and other skin disorders (11) . the dysregulation of ifn-i responses observed in patients with these autoimmune diseases likely results from both genetic and environmental factors. genome-wide association studies show that polymorphisms in genes involved in ifn-i responses strongly correlate with increased susceptibility to many autoimmune diseases (11) . diverse environmental factors can also contribute to the onset of autoimmune diseases. microbial infections often precede first clinical manifestations of autoimmune diseases. whether infections (116) and/or alterations in the commensal microbiota of the affected barrier tissues (117, 118) are the cause or rather the consequence of autoimmunity is still matter of debate. infection-or dysbiosis-induced tissue damages and unbridled ifn-i responses can contribute to initiate autoimmune reactions. gender is another prominent factor affecting susceptibility to autoimmune diseases. women are more prone to autoimmunity, which may result from endocrine regulation of ifn-i responses. pdc ifn-i production is enhanced in human and mouse females, due at least in part to cell-intrinsic enhancement of tlr7/9 responses by the female hormone estradiol (119). in autoimmune diseases, different mechanisms could operate to initiate the dysregulation of immune responses leading to a vicious circle of reciprocal activation between innate ifn-i responses and adaptive self-reactive lymphocyte responses (figure 4) . adaptive immune cells are educated to spare "self." this occurs through negative selection of potentially autoimmune b and t cells during their development in the bone marrow or thymus, respectively, a process called central tolerance. self-reactive b or t cells that have escaped this pruning can be either deleted or functionally inactivated once they have egressed in secondary lymphoid organs or non-lymphoid tissues, a process called peripheral tolerance. in some individuals, polymorphisms in genes involved in the promotion of central or peripheral tolerance lead to a higher number, diversity, and/or responsiveness of self-reactive lymphocytes in the periphery, in particular of b cells secreting anti-dna or anti-rnp antibodies (120, 121) . mammalian dna or rna are poor inducers of pdc ifn-i induction under normal conditions. however, pre-existing anti-dna or anti-rnp autoantibodies can break this innate tolerance of pdc. indeed, antibodies binding to self nucleic acids can protect them from degradation and compact them into nanoparticles that are very effective for the induction of ifn-i in pdc (figure 4) . dna-containing immune complexes (ics) are frequently found in the serum of sle patients (sle-ics) and can activate pdc ifn-i production (122) . in turn, pdc ifn-i activate cdcs, monocytes (123) , and b cells, leading to a vicious circle of reciprocal activation between dcs and frontiers in immunology | microbial immunology figure 4 | a simplified model of the deleterious role of ifn-i in several autoimmune diseases. when exposed to different kinds of injuries (microbial infection, commensal microbiota dysbiosis, chemical or physical insults), healthy tissues can undergo cell damage and death. these events induce the release of apoptotic bodies encompassing self rna or dna. neutrophil recruitment and activation in inflamed tissues can also constitute a potent source of self nucleic acids, through the release of neutrophil extracellular traps (net). self rna or dna can associate with cationic peptides (e.g., ll37) as shown in psoriatic patients or with inflammatory molecules (e.g., high mobility group box 1, hmgb1) to generate nanoparticles that are extremely efficient for ifn-i production by pdc and eventually other cell types. pdc can also be efficiently activated for ifn-i production by immune complexes (ics) generated by the association between self nucleic acids and auto-antibodies as frequently found in the serum of systemic lupus erythematosus patients. ifn-i promote the differentiation and/or the maturation of antigen-presenting cells, in particular different subsets of dc. activated dc can then present self-antigens for activation of auto-reactive t cd4 + cells, including follicular helper lymphocytes, which in turn activate auto-reactive b cells for auto-antibody secretion, leading to a vicious circle of reciprocal activation between innate and auto-reactive adaptive immune cells. idc, immature dc; mdc, mature dc; mo-dc, monocyte-derived dc. see main text for further details. self-reactive lymphocytes and to the exacerbation of autoimmune responses (figure 4) . certain infections or dysbiosis of the commensal microbiota of the affected barrier tissues could promote chronic production of host amphiphatic peptides able to combine with eukaryotic dna or rna, likely released from dying cells, thus forming pdc-activating nanoparticles. indeed, in psoriatic skin, both a high expression of ll37 and a massive infiltration of pdcs is observed (124) (figure 4) . hence, to treat many autoimmune diseases, novel therapeutic strategies could be designed to target dysregulated pdc ifn-i production or b cell activation by ifn-i. one of the most common complications of primary infections by many respiratory viruses, in particular influenza virus, is a lifethreatening pneumonia due to secondary pulmonary infections by bacteria, such as streptococcus pneumoniae, staphylococcus aureus, or haemophilus influenza (125, 126) . these pathologies affect especially infants, elderly, and immunocompromised patients. retrospective studies indicate that secondary bacterial pneumonia was highly recurrent in lung tissues isolated from patients who died during last century influenza pandemics, independently of antibiotic availability (127, 128) . influenza virus induces high ifn-i responses in human beings and mice. in both hosts, secondary bacterial infections are lethal only when they occur in a limited time window following primary viral infection (3-7 days), around the peak of ifn-i responses, before complete virus clearance. mouse models of viral/bacterial coinfections are being used to dissect disease mechanisms (129) . ifnar1-deficient mice appear more resistant to secondary pulmonary bacterial infections, showing that ifn-i responsiveness contributes to disease (130) . similarly, after lymphochoriomeningitis virus (lcmv) infection, wild-type but not ifnar1-deficient mice are more susceptible to lpsinduced septic shock (131) . several mechanisms may contribute to the detrimental role of ifn-i in secondary bacterial infections ( figure 5) . early during viral infection, ifn-i decrease the host ability to control bacterial replication, by dominantly polarizing immune responses toward anti-viral functions, simultaneously inhibiting the development of the types of immune responses required for protection against most bacterial infections. ifn-i can inhibit the production of chemokines required for the recruitment to the respiratory tract of antibacterial effector innate immune cells, in particular neutrophils or monocytes/macrophages (132, 133) (figure 5) . depending on the experimental models used, ifn-i can on the contrary induce a ccr2-dependant recruitment of classical monocytes (134) . in infected tissue, ifn-i might skew the functional polarization of resident or infiltrating monocytic cells toward immunosuppression, because it does limit their antibacterial functions by inhibiting their il-1 production (135) (136) (137) while it might promote their production of il-10 and nitric oxygen intermediates. the exact nature of infiltrating monocytic cells is not clear and could correspond to activated classical monocytes, modcs, monocyte-derived macrophages, or myeloidderived suppressor cells (mdscs). the boundaries between these putatively different cell types are currently ill-defined (138) . these cells could fuel local replication of monocyte/macrophage-tropic bacteria (134) , be immunosuppressive (139) or contribute to local immunopathology (140) . the role of ifn-i on monocytes/macrophages is complex and will require further investigations to determine when it is protective versus deleterious and what the underlying mechanisms are. depending on the context, ifn-i can either promote or inhibit the induction of th1 cytokines such as il-12 and ifn-γ, and myeloid cell responses to ifn-γ (10, (141) (142) (143) . ifn-i can also polarize cd4 t cell responses toward th1 at the expense of th17, while the th17-type cytokines il-17a and il-22 are required for host defense against pulmonary www.frontiersin.org bacteria by inducing the production of anti-microbial peptides and of tissue repair molecules ( figure 5 ) (141) (142) (143) . ifn-i may not only affect host resistance to bacterial infection, but also host tolerance, i.e., the ability of the host to tolerate a given burden of pathogen without undergoing excessive tissue damages (143, 144) . hence, to counter ifn-i deleterious effects during secondary bacterial infections, it will be important to better delineate the respective contribution of lung tissue tolerance modulation and of immune-mediated resistance weakening. another well documented example of deleterious effects of ifn-i due to their inappropriate functional polarization of immune responses is the enhanced susceptibility to fungal infections of patients with genetically determined hyperactive ifn-i responses, as exemplified in the hereditary disease chronic mucocutaneous candidiadis (cmc) (figure 5 ) (145) . patients with cmc have a significant deficit in th17 cd4 t cells, at least in part as a consequence of altered responsiveness to il-6 or il-21. several stat1 mutations were identified in patients with autosomal dominant cmc. gain-of-function stat1 mutations were found to hard wire cd4 t cell responses to cytokines toward stat1 signaling, compromising their stat3-dependent ability to produce il-17 upon il-6 or il-21 stimulation. this was associated to induction of a global ifn-i transcriptomic signature in blood (145) . deleterious ifn-i effects on immunity to candida might not only occur in cmc patients but also in other types of individuals upon secondary fungal infections occurring shortly after a primary viral infection, likewise to the situation discussed above for secondary bacterial infections. indeed, polyic induced ifn-i abrogate innate immunity to systemic candidiasis in mice (146) , and ifnar-deficient mice can be more resistant to candida infection under certain experimental settings (147) . however, the role of ifn-i in the modulation of the ability of immunocompetent hosts to control fungal infection is disputed (148, 149) . the inhibition of th17 responses by ifn-i could be protective in at least one important human pathology, ms (figure 5) . ms represents a striking exception to the previously discussed detrimental role of ifn-i in autoimmune diseases. indeed, a large proportion of ms patients have low serum ifn-i activity and low isg levels. these ms patients present a significant reduction of ms relapse upon ifn-β administration (150) . the underlying mechanisms are not yet completely unraveled. however, in the experimental autoimmune encephalitis mouse model of ms, th17 responses bear a major contribution to nervous system damages and are inhibited by the il-10 and il-27 induced upon ifn-i administration (151) . in summary, ifn-i responses can be deleterious in autoimmunity by promoting a vicious circle of reciprocal activation between innate immune cells and auto-reactive cd4 t or b lymphocytes. ifn-i responses can also be deleterious upon secondary bacterial or fungal infections in the lung or the kidneys occurring shortly after a primary viral infection, by compromising the recruitment of anti-microbial innate effector cells and/or by preventing the proper functional polarization of immune responses. we will now discuss how ifn-i responses can also compromise host immune defenses against certain viruses and promote chronic infections. different lcmv strains such as armstrong and clone-13 (cl13), respectively, lead to acute versus chronic infections in mice. a hallmark of chronic lcmv infection is the loss of the proliferative potential and effector functions of anti-viral cd8 t cells, a process called exhaustion. exhausted cd8 t cells are characterized by a high expression of the inhibitory receptors pd-1, ctla4, and lag-3 (152) . in vivo blockade of these inhibitory receptors can reverse t cell exhaustion and allow resolution of the chronic infection (152) . ifn-i and isgs are induced early after infection with all strains of lcmv, albeit to lower levels with those leading to chronic infection. this early ifn-i production is critical to limit viral replication (3). in models of acute infection, ifn-i responses rapidly return to normal, undetectable, levels, before viral replication is completely controlled. in contrast, isg induction is maintained in chronic infection, including the expression of pd-1 ligands on apcs and of the immunosuppressive il-10 cytokine, consistent with a prolonged expression of ifn-i albeit at low levels (13, 14) . in vivo neutralization of ifn-i by antibody administration promoted resolution of chronic lcmv cl13 infection, allowing the frontiers in immunology | microbial immunology restoration of functional anti-viral cd8 t cell responses at least in part through cd4 t cell-and ifn-γ-dependent mechanisms (13, 14) . during persistent lcmv cl13 infection, chronic low level ifn-i production polarizes cd4 t cell responses toward t follicular helper (tfh) rather than th1 functions. thus, chronic ifn-i responses promote enhanced anti-viral b cell responses but facilitate cd8 t cell exhaustion due to deficient cd4 t cell help, therefore contributing to host failure to prevent chronic infection (153) . strikingly, establishment of chronic infection by lcmv cl13 could also be prevented by early administration of two shots of a high dose of exogenous ifn-i, at days 2 and 5 post-lcmv inoculation. this treatment allowed viral clearance by rescuing anti-viral cd8 t cell from exhaustion (154) . altogether, these studies show that the timing and duration of ifn-i production during viral infections is critical in determining how this response will impact the balance between the virus and the host. an early and robust but transient production of ifn-i promotes strong induction of cell-intrinsic viral restriction mechanisms as well as adequate polarization of adaptive anti-viral immune responses, which combined effects lead to viral clearance. in contrast, if the production of ifn-i is too low and/or too late, both viral replication and low ifn-i responses become chronic, their combined action leading to induction of immunosuppressive effects and to inadequate functional polarization of cd4 t cells. this results in cd8 t cell exhaustion and maintenance of chronic infection. chronic viral replication and cd8 t cell exhaustion is also a hallmark of hiv-1 infection. we will now discuss the complex and disputed role of ifn-i in this disease. both in hiv-1 infection and in its most relevant animal model, infection of non-human primates with simian immunodeficiency virus (siv), disease progression after the acute phase of the infection is associated with high and chronic expression of isgs while ifn-i production is inconsistently detected (155) (156) (157) . in contrast, the individuals that do not progress toward disease despite persistent high viral loads show much lower immune activation, in particular low isg expression, after the acute phase of the infection (158) (159) (160) (161) . hence, chronic low levels of ifn-i are associated to disease progression independently of the level of viral replication. therefore, an outstanding question still open for a better understanding of the physiopathology of hiv-1 infection is whether chronic ifn-i responses are merely a marker of progression, or whether they are implicated in driving disease development. in addition to mechanisms similar to those uncovered in the mouse model of chronic lcmv infection, during hiv-1 infection other effects of ifn-i could promote a vicious circle of reciprocal activation between chronic viral replication and sustained, deleterious immune responses (figure 6 ). very early after hiv-1 infection, in most individuals, ifn-i production might be too weak or too late to induce a combination of cell-intrinsic defense mechanisms and of immune responses efficient enough to prevent later establishment of chronic infection. on the contrary, as demonstrated in the case of the mouse model of lcmv infection, ifn-i responses could favor cd8 t cell exhaustion, either by direct cell-intrinsic effects on cd8 t cells (figure 6 , ) or by contributing to deprive them from cd4 t cell help (figure 6, ) . several effects of ifn-i might compromise anti-hiv-1 th1 responses or more generally contribute to the global depletion of cd4 t cells. these mechanisms include functional polarization of anti-hiv-1 cd4 t cells toward tfh rather than th1 responses, cxcl10 production leading to enhance recruitment of memory cd4 t cells to the sites of viral replication where they fuel chronic viral replication with new hiv-1 target cells (figure 6, to ) , direct pro-apoptotic and anti-proliferative effects on cd4 t cells (figure 6 , ), as well as trail induction on pdcs licensing them for killing cd4 t cells irrespective of their infection (figure 6 , ) (162, 163) . altogether, these mechanisms entertain chronic viral replication and continuous depletion of cd4 t cells, leading to the dramatically enhanced susceptibility to opportunistic infections (figure 6 , ) characteristic of the acquired immunodeficiency syndrome (aids) (figure 6, ) . other lines of evidences have been reported to support a deleterious role of pdc activation during hiv-1 infection. women undergo faster hiv-1 disease progression than men with similar viral loads, which may result in part from the highest ifn-i production of women's pdcs including in response to hiv-1 stimulation (164) . pdc recruitment and activation in the vaginal mucosa of female macaques early after local siv inoculation contribute to attract and activate cd4 t cells, which can then be infected and promote virus dissemination from its portal of entry (165) . however, in vivo blockade of pdc ifn-α production by administration of tlr7/9-antagonistic oligonucleotides early after siv infection of macaques did not decrease t lymphocyte activation, which suggests that additional sources of ifn-i likely contribute to the immune dysfunction observed in siv/hiv-1 infections. targeting dysregulated ifn-i responses during hiv-1 infection might represent an interesting adjuvant therapeutic strategy to highly active antiretroviral treatments. administration of ifn-i in the non-pathogenic siv infection model of sooty mangabeys was not sufficient to switch it into a pathogenic model. no cd4 t cell depletion ensued, no hyperactivation of immune responses were observed. viral loads were even significantly decreased. however, this could be consistent with the positive impact of early and high dose ifn-i administration in chronic lcmv infection (154) . indeed, during the review process of this manuscript, it was reported that, early during primary siv infection in the pathogenic rhesus macaque model, a high dose injection of ifn-i was protective while neutralization of endogenous ifn-i was deleterious. in contrast, in the same animal model, prolonged ifn-i administration accelerated disease development in the chronic stage of the infection (166) . in mice with a humanized immune system, pdc depletion strongly decreased isg induction and enhanced viral replication both in the acute and chronic phases of hiv-1 infection. however, pdc depletion during chronic infection decreased infection-induced t cell apoptosis and increased t cell numbers in lymphoid organs (167) . these results further emphasize the dual role of ifn-i and pdcs in the physiopathology of hiv-1 infection. a strong and transient production of ifn-i early after infection benefits the host by lowering the set-point of viral replication during chronic infection. sustained production of low levels of ifn-i during chronic infection contributes to immune dysregulation and cd4 t cell depletion. further studies will be necessary to examine whether complementing standard-of-use antiretroviral drugs with pdc www.frontiersin.org depletion, ifn-i neutralization, or selective inhibition of t cell responses to ifn-i could yield additional benefits to chemotherapy in non-human primates during chronic siv infection. ifn-i administration has been used for many years to treat another human chronic viral disease, hcv infection. roughly, half of the patients do not show sustained virological responses (svr). the treatment causes severe side effects in many individuals. new chemotherapeutic drugs very potent at blocking hcv replication in vivo have recently become available. hence, whether ifn-i administration still constitutes a viable treatment against chronic hcv infection is being questioned (168, 169) . we will now discuss this issue. chronic hcv infection is the main cause of liver cirrhosis and hepatocellular carcinoma. there is currently no vaccine against hcv. the most common therapy for chronic hcv patients is the administration of recombinant pegylated ifn-α (peg-ifn-α) combined with the anti-viral drug ribavirin. however, because of ifnar pleiotropic expression, ifn-α administration induces severe side effects including flu-like syndrome, fever, fatigue, myalgia, and nervous depression ( figure 7a ) (170) . moreover, only about half of treated patients harbor svr (171) . prior-totreatment high hepatic isg expression is a negative predictor of svr upon peg-ifn-α therapy. high isg expression in untreated patients likely results from chronic but low ifn-i production triggered by persistent hcv replication. indeed, hepatocytes from non-responder patients were found to be infected at a greater frequency and to exhibit dampened antiviral and cell death responses (172) . what the cellular sources of ifn-i production are and why they persist only in non-responder patients still remain to be established. in chronic hcv infection, cytotoxic effector lymphocytes may contribute to the development of hepatocarcinoma by causing low level but sustained hepatocyte death and renewal. in contrast, local production of ifn-γ in the liver by nk and t lymphocytes could promote resistance to disease through non-cytolytic control of viral replication. as discussed previously for lcmv and hiv-1, low chronic production of endogenous ifn-i in hcv patients could compromise both innate and adaptive anti-viral immune responses. chronic exposure to ifn-i could dampen the ability of frontiers in immunology | microbial immunology utr of ifnl3 mrna to promote their degradation. the favorable ifnl3 allele associated with responsiveness to peg-ifn-α treatment may allow endogenous expression of sufficient levels of ifnl3 for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes. this process is, however, hampered by the limited expression of the receptor for this cytokine (ifnλr1) in these patients. peg-ifn-α treatment might promote resolution of the infection by inducing ifnλr1 in these patients, potentiating their response to their endogenous production of ifnl3. in the patients that do not respond to peg-ifn-α treatment, endogenous levels of ifnl3 are insufficient for efficient induction of cell-intrinsic anti-viral defenses in hepatocytes, due to the degradation of the corresponding mrna in infected hepatocytes. in these patient's hepatocytes, however, ifnλr1 is already expressed to high levels prior to treatment due to their high endogenous ifn-i responses. administration of exogenous ifn-λ might cure these patients. see main text for further details. nk and cd8 t cells to produce ifn-γ (173, 174) and promote cd8 t cell exhaustion (175) . it could also induce an antagonist form of cxcl10, a chemokine required for recruitment to the liver of anti-viral nk and cd8 t cell effectors (176) . it may also polarize monocytes toward immunosuppressive functions (177) . therefore, better understanding ifn-i effects in hcv infection is critical to improve care of both responders and non-responder patients to peg-ifn-α. for responder patients, the issue is to modify the treatment to favor beneficial antiviral and immunoactivating effects over side effects strongly affecting patient's quality of life (figure 7a ). this might be achieved by specific delivery of ifn-i to targeted cell types as discussed later. for non-responder www.frontiersin.org patients, the issue is to understand the mechanisms underlying treatment failure to determine whether alternative therapies could be designed (figure 7a) . genome-wide association studies identified various single nucleotide polymorphisms (snp) in the gene encoding il-28b/ifn-λ3, one of the ifn-iii, as well in its 5 and 3 non-coding regions (178) (179) (180) (181) . one snp, called rs12979860, is located 3 kb upstream of the ifnl3 gene. patients harboring the cc genotype have a favorable prognosis to ifn-i treatment. patients with the tt genotype are at high risk of treatment failure (178, 179) . in europeans, the favorable cc genotype is the major, most common, ifnl3 allele. the unfavorable tt snp is the minor allele. the frequency of these alleles is reversed in africans. the favorable allele allows escape of ifnl3 mrna from degradation by cellular microrna induced upon hcv infection (181) . until recently, ifnl3 genotypes and hepatic isg expression were considered as independent predictors of response to peg-ifn-α treatment in hcv patients (171) . here, we propose a potential explanation, which integrates both factors in a relatively simple model ( figure 7b ). our main hypothesis is that efficient control of hcv infection depends on hepatocyte response to ifn-λ rather than ifn-α. this is supported by reports that ifn-λ induces a stronger and more sustained isg expression in hepatocyte cell lines in vitro (182) , and that polyi:c-induced control of hcv replication in humanized liver in chimeric mice is correlated to the induction of ifn-λ but not ifn-i in human hepatocytes (183) . responder patients harboring the favorable ifnl3 allele preventing the degradation of the corresponding rna in infected cells might express significant levels of endogenous ifn-λ3, although this is disputed. however, they express only low levels of ifn-λr1, which limits ifnλ3 efficiency ( figure 7b ) (184) . how these patients benefit from peg-ifn-α treatment could be that it induces ifn-λr1 expression on hepatocytes thus boosting endogenous ifn-λ3 effects (184) . in contrast, high isg-expressing non-responder patients harboring the unfavorable ifnl3 allele might not express enough ifn-λ3 for virus control. however, they do express ifn-λr1 as a result of their endogenous production of ifn-i. hence, peg-ifn-α might be ineffective in these patients because they already express ifn-λr1 but fail to produce endogenous ifn-λ3 due to the degradation of its mrna in infected hepatocytes (figure 7b) . these patients may be good candidates for peg-ifn-λ therapy, currently undergoing clinical development. since the expression of ifn-λr1 is mainly restricted to epithelial cells, melanocytes, and hepatocytes, some of the side effects related to ifn-i treatment might be strongly attenuated in peg-ifn-λ therapy. however, as ifn-i are key to induce anti-viral immune responses, it will be critical to determine whether, beside viral clearance, peg-ifn-λ therapy can also induce long-term immune protection against hcv. ifn-i transduce intracellular signals through a single receptor, ifnar, but via a multitude of downstream signaling pathways. the janus activated kinase (jak)/stat pathway was the first to be identified (185) . ifnar is composed of two distinct subunits, ifnar1 and ifnar2, which are constitutively associated with members of the jak family, tyrosine kinase 2 (tyk2) and jak1, respectively (186) . the binding of ifn-i to their receptor leads to the phosphorylation of jak1 and tyk2, which in turn induce the phosphorylation and activation of the stat proteins (186) . different stat complexes can form upon triggering of ifnar (figure 8) . a transcriptional complex that forms in most conditions of ifn-i stimulation and induces the expression of many molecules of cell-intrinsic anti-viral immunity is interferonstimulated gene factor 3 (isgf3), a heterotrimer composed of pstat1, pstat2, and irf9 (187) (figure 8) . following its translocation into the nucleus, isgf3 binds to isre regulatory sequences in target genes. many molecules playing a key role in the function of innate or adaptive immune leukocytes are also induced by isgf3, including cd80, cd86, or il-15 in dc, and granzyme b in nk cells. isgf3 is generally composed of stat1 phosphorylated on tyr701 and ser727 and of stat2 phosphorylated on tyr689. however, alternative isgf3 complexes have been described in various contexts which could participate to the diversity of ifn-i effects (188) . the pstat1 homodimer also plays a prominent role in cell-intrinsic ifn-i-dependent gene induction. it binds ifnγactivated sequences (gas) and controls the expression of many pro-inflammatory molecules (187) . pstat1 homodimers can form upon stimulation with either ifn-i or ifn-γ. many gasregulated genes can be induced by either cytokines. depending on cell types, jak signaling downstream of ifnar can lead to the activation of virtually all stat proteins and to their combinatorial association into a variety of complexes with different affinities for specific gas elements (189) (190) (191) (figure 8) . this diversity contributes to ifn-i induction of different transcriptional programs in distinct cell types (39) . stat complex formation depends in part on the relative abundance of stat molecules in the cell (192) . while stat1, stat2, stat3, and stat5 can be activated in most cell populations, stat4 and stat6 are mainly activated in lymphocytes (193) . for example, quiescent nk cells express more stat4 than stat1, leading to constitutive association of ifnar to stat4 in these cells. hence, quiescent nk cells mount pstat4 homodimer-dependent responses to ifn-i stimulation, including ifn-γ production and t-bet-driven proliferation (figure 8 ) (194, 195) . changes in stat levels can also occur upon the differentiation/activation of a given cell type and lead to a shift in its functional response to the cytokines (196) . upon activation, nk cells decrease their expression of stat4 and increase that of stat1, shifting their ifn-i response from stat4-dependent in a quiescent state to stat1dependent in pre-activated cells. this translates into opposite ifn-i effects on ifn-γ production and proliferation for quiescent versus pre-activated nk cells (194) . however, this outcome can be modulated by simultaneous exposure to other cytokines such as il-15 or il-12/18. a reverse stat1-to-stat4 shift occurs in dc during their maturation, shifting their functional responses from inhibition to activation of il-12 production in response to combined stimulations with ifn-i and cd40l (197) . this frontiers in immunology | microbial immunology ifn-i binding to ifnar triggers the phosphorylation of tyk2 and jak1, which in turn phosphorylate a variety of stat proteins. activated stats are able to form complexes, as homo-or hetero-dimers. the heterodimer stat1-stat2 binds to a third partner, ifn-regulatory factor 9 (irf9), in order to form the isgf3 complex. this complex translocates into the nucleus and binds to specific regulatory sequences, ifn-stimulated response elements (isre), to activate the expression of many interferon-stimulated genes (isgs). in particular, isgf3 induces most, if not all, of the isgs encoding effector molecules of cell-intrinsic anti-viral defenses such as oas or mx1. alternative jak/stat pathways include the formation of stat1 or stat4 homodimers, which may drive different functional responses to ifn-i. stat1 homodimers bind to ifnγ-activated sequences (gas) in the promoter of certain isgs, which may promote inflammatory, anti-proliferative, and pro-apoptotic responses. stat4 homodimers also bind to gas but promote ifn-γ production and pro-proliferative responses. enables mature dc to efficiently activate cd8 t cells. other yet unknown mechanisms control the formation of different stat complexes in distinct cell types. the nature and dynamics of the signaling pathways triggered by ifn-α or -β were evaluated in bulk cultures of human blood leukocytes using flow cytometry (191) or high throughput mass cytometry (190) . a diversity of phosphorylation patterns of stat1/3/5 was observed upon ifn-i stimulation. ifn-α activation induced phosphorylation of stat1, stat3, and stat5 in most cell types, peaking at 15 min (190) . ifn-β-induced stat1 phosphorylation was found to be poor in b cells as compared to monocytes and t cells (191) . however, the underlying mechanism remains to be identified since b cells did not express lower amount of ifnar2 or stat1 or enhanced levels of the inhibitory socs1 molecule. the high stat1 activation in monocytes led to their induction of ifn-i-dependent pro-apoptotic genes while this was not the case in b cells. these results strikingly differ from those obtained in the other study upon ifn-α stimulation, where stat1 phosphorylation was on the contrary lower in cd14 + monocytes and was prolonged in b cells and nk cells (190) . the differences between these two studies might have resulted from the use of different subsets and doses of ifn-i. in any case, both studies consistently reported that cd4 t cells showed the highest activation of stat5. all cd4 t cells but not all cd8 t cells activated stat5 and for a longer time (190) . ifn-β activation of stat3 was delayed in cd4 t cells and b cells as compared to cd8 t cells and monocytes (191) . different stat complexes may lead to distinct transcriptional programs linked to different biological functions (figure 8) . more systematic studies are needed to understand this complexity. besides changing stat levels between cell types or www.frontiersin.org activation states, the processes controlling differential formation of stat complexes downstream of ifnar triggering remain to be identified. in addition to jak/stat signaling, other pathways can be activated downstream of ifnar, including those involving the phosphatidylinositol 3-kinase (pi3k), mitogen-activated protein kinases (mapk), and the crk adaptor molecules (39, 198) . this leads to the activation of other transcription factors such as irf, nf-κb, or pu.1, which contribute to orchestrate cell responses to the cytokines by regulating both distinct and overlapping sets of genes as compared to stat (199, 200) . in summary, ifnar signals through a remarkable diversity of pathways, including but not limited to diverse combinations and kinetics of stat phosphorylations. this explains at least in part the diversity of ifn-i effects, including their induction of opposite responses depending on the physiopathological contexts and/or the nature of the principal responding cell types (200, 201) . ifn-iii induce the same signaling pathways as ifn-i, although they engage a different heterodimeric receptor, composed of the il-28ra and il-10rb chains and preferentially expressed on epithelial cells including hepatocytes. in mice and human beings, numerous ifn-i subtypes exist. functional and population genetic analyses showed that these ifn-i subtypes significantly differ in their functions (202) (203) (204) (205) (206) (207) . hence, one of most extraordinary feature of ifn-i biology is how ifn-i subtypes can elicit so many pleiotropic and diverse functions by interacting with the same receptor complex (208) . both ifnar1 and ifnar2 are required for the initiation of ifn-i-dependent signals, as mice deficient in either one are highly susceptible to viral infections (3, 5) . the assembling of the ifnreceptor ternary complex is a two-step process. first, a binary complex is formed by the binding of one side of the ifn molecule to ifnar2. then, a single binary complex interacts with ifnar1 via the other side of the ifn molecule. the stability of the ternary complex will be determined in part by the association and dissociation kinetics between the cytokine and the two receptor chains, as well as by ifnar expression levels since the cell surface concentrations of the receptor subunits are relatively low. hence, both the affinity of ifn-i subsets for ifnar and the amounts of ifn-i, ifnar1, and ifnar2 will regulate their biological effects ( figure 9a ) (209, 210) . cell membrane density of ifnar1 and ifnar2 is also involved in differential ifn-β-versus ifn-α-induced functional activities, such as anti-proliferative function (211) . a variety of cell-intrinsic parameters can also impact the lifetime of the ifn-receptor ternary complex, such as the rate of endocytosis/degradation/recycling of signaling complexes, and negative isg regulators such as usp18 that decrease the affinity of ifn-ifnar1 binding (203, 212) . based on a definition of a prototypic cytokine-receptor binding module and by analogy with the epo receptor system, ifn-i subtypes were originally postulated to form ternary complexes of differing architectures, resulting in distinct geometry and assembling of intracellular signaling components (213) . experimental evidence rejected this hypothesis. rather, the differential activities of ifn-i subtypes are determined by the stability of the ligand/receptor ternary complex (207, 212) . differential affinities of the ifn-i subtypes for ifnar1 and ifnar2 extracellular domains generate subtype-specific signaling cascades and biological outcomes ( figure 9a ) (210, 214) . crystal structure of ternary ifn-i/ifnar1/ifnar2 complex illuminated the biochemical complexity of ifn-i interaction with their cognate receptors (215) . the main conformational features of ifn-i/ifnar1/ifnar2 ternary complexes are conserved among the different ifn-i, but are quite different from the other cytokine receptors (214, 215) . in the formation of the binary ifn-i/ifnar2 complex, ifn-i ligand discrimination resides on differential energetics during the interaction of anchor points with ifnar2, shared by all ifn-i, as well as on key amino acid substitution among ifn-i subtypes (215) . ifnar1 then performs major conformational changes to interact with ifn-i associated in the binary complex, thus displaying an optimized functional plasticity (215) . these differences in the chemistry of ifn-i subtype interaction with ifnar2 and ifnar1 thus explain the different affinities of ifn-α versus ifn-β within ternary complex and their differential activities (210) . the functions regulated by ifn-i strongly depend on the main responding cell types ( figure 9b ). this has been studied in vitro by examining the functional consequences of the stimulation of different cell types with ifn-i, and in vivo by determining the contribution of cell-intrinsic ifn-i responses of different cell types to resistance or susceptibility to various diseases. an emerging concept is the central role of dc responses to ifn-i for induction of protective immunity against viral infections or tumors (figure 3) . the development of mutant mice allowing conditional genetic inactivation of ifnar1 in a cell-type specific manner using the cre-lox system (216) has been instrumental in accelerating our understanding of how different cell types respond to ifn-i in vivo and what their respective contribution is to protective or deleterious ifn-i responses. this has been investigated most extensively in viral infections (106, 111, 112, 115, 217) but also in cancer (97, 98) , bacterial infections (218), autoimmunity (216, 219) , sepsis (220), or inflammatory diseases (221) . efforts are being pursued to better understand which cell types respond to ifn-i in a manner promoting protective versus deleterious effects in different physiopathological settings. that knowledge will considerably help to develop novel strategies to modulate ifn-i functions for promoting health over disease. the development of mutant mice allowing conditional genetic inactivation of stat1, stat3, and stat5 (222-226) will help better understanding how different signaling pathways in different cell types determine the outcome of ifn-i response in vivo in various conditions. this knowledge might lead to the development of strategies aiming at targeting a given cell type with a specific subset of ifn-i, or in the presence of antagonists of certain signaling pathways, to surgically tune ifn-i responses in vivo toward the most desirable outcome. frontiers in immunology | microbial immunology for example, the affinity of ifn-β for ifnar1 is 100-times higher than that of ifn-α2, and ifnβ is much more potent in inhibiting cellular proliferation or (continued ) ifn-i can also determine distinct functional outcomes. for example, during viral infections, early and transient high levels of ifn-i promote protective dc and t cell responses, while delayed, chronic and low level ifn-i production compromises host immune defenses and promotes chronic viral infections. within a given cell type, the outcome of ifn-i stimulation also depends on time of exposure to these cytokines relative to other modulatory signals (timing relative to other stimuli). for example, in naïve cd8 t cells, tcr signaling prior to ifn-i stimulation leads to increased expression of stat4 and promotes ifn-γ production and proliferation, while ifn-i stimulation prior to tcr triggering leads to stat1-dependent anti-proliferative and pro-apoptotic effects. the formation of specific stat complexes is a highly dynamic process. it depends not only on the cell type but also on its specific state at the time it sees ifn-i. hence, major parameters controlling the effects of ifn-i in a given cell type also include its microenvironment ( figure 9c ) and the timing of its exposure to the cytokines both in terms of duration of the stimulation and of previous activation history ( figure 9d) . the tam receptor ligand gas6 is expressed within tumor cells in various solid cancers (227, 228) . elevated gas6 expression is of bad prognosis in different cancers (228, 229) . in a mouse model of ovarian cancer, early during tumorigenesis tumor-infiltrating dcs were found to be immunogenic and promote antitumor immunity, but they were later altered in the course of tumor development to acquire immunosuppressive properties beneficial to the tumor (230) . one may thus hypothesize that expression of tam soluble ligands in certain tumors and of tam receptors on tumor-infiltrating dcs might contribute to dampen dc response to ifn-i and therefore facilitate their polarization by the tumor microenvironment into immunosuppressive cells (figure 9c) . acute versus chronic exposure to ifn-i can lead to strikingly opposite effects on a given cell type (13, 14, 231) . in addition to duration, the time when a cell is exposed to ifn-i can also dramatically impact its functional response, depending on its previous activation history ( figure 9d) . in vitro stimulation of dcs with ifn-β can lead to opposite outcomes depending whether it occurs simultaneously to, or after, tnfα-induced maturation. ifn-β polarizes dcs toward th1 induction in the former case, and toward il-10-secreting t cells in the latter case. these opposite effects result at least in part from the differential expression of il-12/18 by dcs (232) . similarly, ifn-i effect on the functional polarization of cd4 t cells is strongly modulated by the other cytokines present in the lymphocyte microenvironment at the same time (233) . ifn-i can also mediate opposite effects on cd8 t cells depending whether it occurs before or after cognate engagement of the t cell receptor. indeed, while cd8 t cells have the potential to respond to ifn-i by inducing both stat1-and stat4-dependent genes, this depends upon their activation history. naïve cd8 t cells respond mostly to ifn-i through stat1 signaling, leading to the inhibition of their proliferation and eventually to the induction of their apoptosis. however, cognate triggering of the t cell receptor causes a decrease in stat1 and an increase in stat4 expression in cd8 t cells. this leads to a shift of their ifn-i response from stat1-to-stat4 signaling, resulting in the promotion of their proliferation and ifn-γ production. during lcmv infection, this mechanism promotes stat4-dependant expansion of anti-viral cd8 t cells, but stat1-dependant inhibition of naïve cd8 t cell proliferation (234) . since the late 70s the clinical potential of ifn-i for the treatment of patients suffering of viral infection or cancer diseases has been widely acknowledged (235) . today, this expectation is tempered because ifn-i treatment can induce severe side effects and sufficient doses cannot be administered in patients. therefore, there is a strong need to create tuned ifn molecules devoid of side effects. based on our current understanding of ifn-i responses as reviewed above, many parameters could be tuned individually or in a combined manner to modulate ifn-i activity to promote their beneficial effects over the deleterious ones in a number of diseases. these parameters include modifying the affinity of ifn-i for its receptor, playing with the local quantity/concentration of ifn-i and with the duration of its delivery, and modulating the nature of the cells that are responding to ifn-i. we will discuss here novel strategies being developed to deliver ifn-i to, or block ifn-i responsiveness of, a specific target cell type in vivo (figure 10 ). if ifn-i-induced side effects are a consequence of the pleiotropic nature of ifn-i, and if the bioactivities mediating deleterious effects have some degree of independence from those mediating beneficial effects, one could mutate the ifn-i molecules in order to skew their activity toward a desired bioactivity. indeed, introducing key mutation in ifn-α2 allowed increasing its affinity to ifnar1 by a factor of 100. accordingly, this ifn-α2 mutant is 100times more potent in inhibiting cell proliferation, but as potent as wt ifn-α2 in inducing an anti-viral state (236) (237) (238) . hence, it is possible to tune ifn activity by modifying its binding to ifnar. however, translating such an approach for the design of molecules for clinical application is severely hampered by the poor understanding we have on the ifn-i bioactivities mediating the side effects. furthermore, we are far from having established the list of bioactivities that could be differentially modulated by changing the stability of the ifn-i/ifnar complex. we know more about the frontiers in immunology | microbial immunology cell types that mediate beneficial versus deleterious ifn responses in various diseases. hence, we will now discuss strategies aimed at focusing ifn activity to specific cell types to promote health over disease. several strategies have been developed to specifically target ifns on tumor cells, tumor-infiltrated immune cells or infected tissues. these strategies include intra-lesional injection (239, 240) , adenoviral-mediated gene transfer (241) (242) (243) , engineered tumorinfiltrating monocytes (244) , and fusion of ifns with a cleavable protecting shell (245) . another strategy to increase cytokine accumulation within the tumor or infected tissue is antibody-mediated targeting of cytokine delivery, where a cytokine moiety is fused to an antibody directed against a specific cell surface marker (figure 10) . the fusion molecule retains both antigen-binding and ifn-i bioactivities, and is enriched at the targeted site upon in vivo injection (246) (247) (248) (249) . when targeted to human cd20, ifn-i inhibited the proliferation of lymphoma cells engrafted in immunodeficient mice (250) . an ifn-i targeted to a tumor antigen can also amplify the therapeutic effect of the antibody by acting on tumor-infiltrated dcs, thus increasing antigen cross-presentation and antitumor cytotoxic t cell responses (249) . on non-targeted cells, the antibody conjugation negatively impacts ifn-i potency, but only modestly (18, 248, 251) (figure 10a) . fusion molecules generally retain full ifn-i biological activity on the cells expressing the antibody target ( figure 10b) . hence, this difference only leads to a modest ratio between the ifn-i specific activity measured on target and non-target cells (figure 10b) . such a targeting efficiency is definitely too low to reduce the toxic effect of ifn-i administration, because it will not specifically focus ifn-i activities on "beneficial cells" without stimulating "deleterious cells." the engineering of immuno-ifn-i must be improved to reach the very high targeting efficacy required to significantly diminish the treatment side effects. we recently reported an innovative strategy reaching this goal (18) . it is based on the postulate that the antibody moiety of an immuno-ifn-i stabilizes the ifn-i/receptor-complex by avidity. it also takes into account the fact that the biological potency of an ifn-i is proportional to the stability of the ifn-i/receptor complex up to a certain threshold beyond which increasing the stability does not increase its potency (238, 252) . ifn-α2 and ifn-β are used in most immuno-ifn-i studies. they have evolved to retain close to maximal potency. hence, their targeting by an antibody that only provides a modest gain in terms of biological potency. however, it is expected that decreasing the affinity of the ifn-i for its receptor, by introducing a mutation, would increase the targeting effect of the antibody (figures 10 c,d) . this is indeed the case. using an ifn-i with a single point mutation that dramatically decreases its affinity for ifnar2 ( figure 10c ) allows engineering immuno-ifns that are up to 1000-fold more potent on cells expressing the antibody target ( figure 10d) . the three log targeting efficiency of these novel types of immuno-ifns is found for various activities measured in vitro or in vivo when delivered in mice. if the toxic side effect experienced by the patients treated with ifn-i is due to systemic ifn-i activity, this targeting technology may find considerable clinical applications since such engineered immuno-ifns are virtually inactive while "en route" and are activated only after binding of the fused antibody to the desired target. it remains to define the useful targets according to pathologies, for example, tumor cells themselves and professional cross-presenting xcr1 + dcs for cancer (97, 98, 249) , or hepatocytes for chronic hcv infection. to treat autoimmune diseases, novel therapeutics targeting ifn-i have been developed, including two ifn-α-neutralizing monoclonal antibodies currently in clinical trials (sifalimumab and rontalizumab) (253, 254) . however, long-term systemic neutralization of ifn-i activity may increase susceptibility to viral infection and tumor development. alternative strategies are needed to specifically inhibit ifn-i deleterious effects in these diseases without globally compromising ifn-i anti-viral and www.frontiersin.org anti-tumoral functions. the sequential nature of the assembling of the ifn-i/receptor complex opens the possibility to design ifn-i antagonists specifically targeting the cell subsets responsible for ifn-i deleterious effects. an ifn-α2 carrying a single amino acid substitution that blocks the ifn-i/ifnar1 interaction engages ifnar2 in a complex, which cannot bind ifnar1 (255) . since the binary ifn-i/ifnar2 complex is devoid of any ifn-i activity, such mutant behaves as a potent ifn-i antagonist. when linked to an antibody specific for a cell surface marker, the antagonistic activity of the mutant ifn-i should be significantly reinforced specifically on the cells expressing the target. hence, it should be possible to design and construct targeted antagonists that inhibit responsiveness to endogenous ifn-i specifically on the cell subsets on which the cytokines act to promote autoimmunity or severe side effects, leaving the other cells fully responsive. for example, in chronic hcv patients treated with peg-ifn-α, one of the most deleterious side effects is nervous depression, which might be prevented by co-administration of an ifn-i antagonist specifically targeting neurons or other cells of the central nervous system. in the last decade, several major technological breakthroughs and the generation of novel animal models have remarkably advanced our understanding of the mode of action of ifns. in vitro high throughput screening allowed systematically studying the functions of isgs by ectopic expression or knock-down. advance biophysical investigation of the interactions between ifn-i and the ifn-i receptor allowed to rigorously investigate the mechanistic basis for the differential bioactivities of ifn-i subtypes. the analyses of the responses of different cell types to ifns or to viral infection, in vitro but also in vivo in various pathologies, demonstrated that ifn-i often mediate beneficial versus deleterious roles by acting on different cell types. from integrative analysis of these data, a picture is now emerging suggesting that it will be possible to segregate protective from deleterious ifn-i effects, based (i) on their differential induction depending on ifn-i subsets or on the magnitude/timing of ifn-i production, (ii) on their conditioning in different tissues, (iii) or on their occurrence in different cell types. hence, innovative immunotherapeutic treatments are being designed to tune ifn-i activity toward desired effects in order to promote health over disease in a manner adapted to each physiopathological condition. in particular, a proof-of-concept has been made in vitro that it will be possible to target ifn-i activity on given cell types or tissues to administer to patients sufficiently high doses of the cytokine at the site of interest while limiting unwanted effects in other tissues or cell types. the next steps will be to demonstrate efficacy of this strategy in vivo in preclinical animal models. importantly, to foster the development of these innovative immunotherapies, major efforts are still warranted to continue delineating which cell types are mainly responsible for the protective versus deleterious effects of ifn-i in different diseases. combining high throughput technologies and systems biology approaches will also advance our understanding of the molecular mechanisms dynamically controlling ifn-i responses in health and diseases, which should reveal potentially novel therapeutic targets. virus interference. i. the interferon virus interference. ii. some properties of interferon functional role of type i and type ii interferons in antiviral defense inborn errors of interferon (ifn)-mediated immunity in humans: insights into the respective roles of ifn-alpha/beta, ifn-gamma, and ifn-lambda in host defense suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type i interferon activity new insights into cancer immunoediting and its three component phases -elimination, equilibrium and escape how type i interferons work in multiple sclerosis and other diseases: some unexpected mechanisms role of type i interferons in inflammasome activation, cell death, and disease during microbial infection does type i interferon limit protective neutrophil responses during pulmonary francisella tularensis infection? front immunol type i interferons in bacterial infections: taming of myeloid cells and possible implications for autoimmunity fueling autoimmunity: type i interferon in autoimmune diseases hiv-1 immunopathogenesis: how good interferon turns bad persistent lcmv infection is controlled by blockade of type i interferon signaling blockade of chronic type i interferon signaling to control persistent lcmv infection unraveling the convoluted biological roles of type i interferons in infection and immunity: a way forward for therapeutics and vaccine design interferons, interferon-like cytokines, and their receptors dendritic cell maturation: functional specialization through signaling specificity and transcriptional programming high efficiency cell-specific targeting of cytokine activity critical role of constitutive type i interferon response in bronchial epithelial cell to influenza infection critical role of interferon-alpha constitutively produced in human hepatocytes in response to rna virus infection neutrophils responsive to endogenous ifn-beta regulate tumor angiogenesis and growth in a mouse tumor model critical role for constitutive type i interferon signaling in the prevention of cellular transformation self-priming determines high type i ifn production by plasmacytoid dendritic cells irf7-dependent ifn-beta production in response to rankl promotes medullary thymic epithelial cell development cell-intrinsic role for ifn-alpha-stat1 signals in regulating murine peyer patch plasmacytoid dendritic cells and conditioning an inflammatory response absence of ifn-beta impairs antigen presentation capacity of splenic dendritic cells via down-regulation of heat shock protein 70 a weak signal for strong responses: interferonalpha/beta revisited novel reporter mouse reveals constitutive and inflammatory expression of ifn-beta in vivo temporal and spatial resolution of type i and iii interferon responses in vivo priming of natural killer cells by nonmucosal mononuclear phagocytes requires instructive signals from commensal microbiota immune sensing of dna cytosolic sensing of viruses differential viral induction of distinct interferonalpha genes by positive feedback through interferon regulatory factor-7 positive feedback regulation of type i ifn genes by the ifn-inducible transcription factor irf-7 virus-cell fusion as a trigger of innate immunity dependent on the adaptor sting novel paradigms of innate immune sensing of viral infections breaking the barrier: membrane fusion triggers innate antiviral immunity antiviral immunity via rig-i-mediated recognition of rna bearing 5 -diphosphates interferons: signaling, antiviral and viral evasion a role for dnadependent activator of interferon regulatory factor in the recognition of herpes simplex virus type 1 by glial cells cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells rice: pan-viral specificity of ifn-induced genes reveals new roles for cgas in innate immunity mapping the crossroads of immune activation and cellular stress response pathways tlr-mediated activation of type i ifn during antiviral immune responses: fighting the battle to win the war plasmacytoid dendritic cells and the control of herpesvirus infections specialization, kinetics, and repertoire of type 1 interferon responses by human plasmacytoid predendritic cells plasmacytoid monocytes migrate to inflamed lymph nodes and produce large amounts of type i interferon the nature of the principal type 1 interferon-producing cells in human blood mouse type i ifn-producing cells are immature apcs with plasmacytoid morphology virus-induced interferon alpha production by a dendritic cell subset in the absence of feedback signaling in vivo tlr9-dependent recognition of mcmv by ipc and dc generates coordinated cytokine responses that activate antiviral nk cell function viral infection switches non-plasmacytoid dendritic cells into high interferon producers plasmacytoid dendritic cell ablation impacts early interferon responses and antiviral nk and cd8(+) t cell accrual control of coronavirus infection through plasmacytoid dendritic-cell-derived type i interferon plasmacytoid dendritic cells contribute to systemic but not local antiviral responses to hsv infections dendritic cell responses to early murine cytomegalovirus infection: subset functional specialization and differential regulation by interferon alpha/beta interferon alpha/beta and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo individual plasmacytoid dendritic cells are major contributors to the production of multiple innate cytokines in an organ-specific manner during viral infection pattern recognition receptors and inflammation intracellular toll-like receptors deciphering the role of dc subsets in mcmv infection to better understand immune protection against viral infections the dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting mouse cd8alpha+ dcs and human bdca3+ dcs are major producers of ifn-lambda in response to poly ic human xcr1+ dendritic cells derived in vitro from cd34+ progenitors closely resemble blood dendritic cells, including their adjuvant responsiveness, contrary to monocyte-derived dendritic cells human type 2 myeloid dendritic cells produce interferon-lambda and amplify interferon-alpha in response to hepatitis c virus infection human blood dendritic cell antigen 3 (bdca3)(+) dendritic cells are a potent producer of interferon-lambda in response to hepatitis c virus antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor activation of ifn-&#946; expression by a viral mrna through rnase l and mda5 rnase l releases a small rna from hcv rna that refolds into a potent pamp protein kinase r contributes to immunity against specific viruses by regulating interferon mrna integrity tam receptors are pleiotropic inhibitors of the innate immune response alveolar macrophages are the primary interferon-alpha producer in pulmonary infection with rna viruses differential type i interferon induction by respiratory syncytial virus and influenza a virus in vivo crosstalk between components of the innate immune system: promoting anti-microbial defenses and avoiding immunopathologies plasmacytoid dendritic cells and toll-like receptor 7-dependent signalling promote efficient protection of mice against highly virulent influenza a virus plasmacytoid dendritic cells are dispensable during primary influenza virus infection clearance of influenza virus from the lung depends on migratory langerin+cd11b− but not plasmacytoid dendritic cells human tlr-7-, -8-, and -9-mediated induction of ifn-alpha/beta and -lambda is irak-4 dependent and redundant for protective immunity to viruses pyogenic bacterial infections in humans with myd88 deficiency impaired response to interferon-alpha/beta and lethal viral disease in human stat1 deficiency herpes simplex virus encephalitis in a patient with complete tlr3 deficiency: tlr3 is otherwise redundant in protective immunity herpes simplex encephalitis in children with autosomal recessive and dominant trif deficiency evolutionary dynamics of human toll-like receptors and their different contributions to host defense type 1 interferons and the virus-host relationship: a lesson in detente subcapsular sinus macrophages prevent cns invasion on peripheral infection with a neurotropic virus enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus interferon-stimulated genes: a complex web of host defenses protein kinase pkr and rna adenosine deaminase adar1: new roles for old players as modulators of the interferon response dna deamination mediates innate immunity to retroviral infection samhd1 host restriction factor: a link with innate immune sensing of retrovirus infection a diverse range of gene products are effectors of the type i interferon antiviral response the transcription factor stat-1 couples macrophage synthesis of 25-hydroxycholesterol to the interferon antiviral response reciprocal inhibition between intracellular antiviral signaling and the rnai machinery in mammalian cells plasmacytoid, conventional, and monocyte-derived dendritic cells undergo a profound and convergent genetic reprogramming during their maturation crosspriming of cd8+ t cells stimulated by virus-induced type i interferon type i interferon is selectively required by dendritic cells for immune rejection of tumors host type i ifn signals are required for antitumor cd8+ t cell responses through cd8{alpha}+ dendritic cells dendritic cells require a systemic type i interferon response to mature and induce cd4+ th1 immunity with poly ic as adjuvant direct type i ifn but not mda5/tlr3 activation of dendritic cells is required for maturation and metabolic shift to glycolysis after poly ic stimulation type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo johansson-lindbom b. type i interferon signaling in dendritic cells stimulates the development of lymph-noderesident t follicular helper cells differential responses of immune cells to type i interferon contribute to host resistance to viral infection a type i interferon autocrine-paracrine loop is involved in toll-like receptorinduced interleukin-12p70 secretion by dendritic cells tlrdriven early glycolytic reprogramming via the kinases tbk1-ikkvarepsilon supports the anabolic demands of dendritic cell activation type i ifn-mediated protection of macrophages and dendritic cells secures control of murine coronavirus infection direct action of type i ifn on nk cells is required for their activation in response to vaccinia viral infection in vivo dendritic cells prime natural killer cells by trans-presenting interleukin 15 cutting edge: the direct action of type i ifn on cd4 t cells is critical for sustaining clonal expansion in response to a viral but not a bacterial infection type i interferons act directly on cd8 t cells to allow clonal expansion and memory formation in response to viral infection direct stimulation of t cells by type i ifn enhances the cd8+ t cell response during cross-priming cutting edge: enhancement of antibody responses through direct stimulation of b and t cells by type i ifn cd8 t cells specific for lymphocytic choriomeningitis virus require type i ifn receptor for clonal expansion innate inflammatory signals induced by various pathogens differentially dictate the ifn-i dependence of cd8 t cells for clonal expansion and memory formation concomitant type i ifn receptor-triggering of t cells and of dc is required to promote maximal modified vaccinia virus ankara-induced t-cell expansion antiviral immune responses: triggers of or triggered by autoimmunity? is chronic plaque psoriasis triggered by microbiota in the skin? genetic dysbiosis: the role of microbial insults in chronic inflammatory diseases the tlr-mediated response of plasmacytoid dendritic cells is positively regulated by estradiol in vivo through cell-intrinsic estrogen receptor alpha signaling accumulation of peripheral autoreactive b cells in the absence of functional human regulatory t cells cvid-associated taci mutations affect autoreactive b cell selection and activation human lupus autoantibody-dna complexes activate dcs through cooperation of cd32 and tlr9 ifn priming is necessary but not sufficient to turn on a migratory dendritic cell program in lupus monocytes plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide the pathology of influenza virus infections how do viral infections predispose patients to bacterial infections? predominant role of bacterial pneumonia as a cause of death in pandemic influenza: implications for pandemic influenza preparedness pathology of human influenza revisited immune dysfunction and bacterial coinfections following influenza type i interferon: friend or foe? a role for ifn-alpha beta in virus infection-induced sensitization to endotoxin type i ifns mediate development of postinfluenza bacterial pneumonia in mice synergistic stimulation of type i interferons during influenza virus coinfection promotes streptococcus pneumoniae colonization in mice intranasal poly-ic treatment exacerbates tuberculosis in mice through the pulmonary recruitment of a pathogen-permissive monocyte/macrophage population innate and adaptive interferons suppress il-1alpha and il-1beta production by distinct pulmonary myeloid subsets during mycobacterium tuberculosis infection host-directed therapy of tuberculosis based on interleukin-1 and type i interferon crosstalk inflammation. 25-hydroxycholesterol suppresses interleukin-1-driven inflammation downstream of type i interferon dendritic cells, monocytes and macrophages: a unified nomenclature based on ontogeny cytomegalovirus impairs antiviral cd8+ t cell immunity by recruiting inflammatory monocytes tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice influenza a virus exacerbates staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production role of tissue protection in lethal respiratory viral-bacterial coinfection disease tolerance as a defense strategy gainof-function human stat1 mutations impair il-17 immunity and underlie chronic mucocutaneous candidiasis type i interferon inhibits interleukin-1 production and inflammasome activation type i interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during candida infections ifnalpha/beta signaling is required for polarization of cytokine responses toward a protective type 1 pattern during experimental cryptococcosis interferonbeta production via dectin-1-syk-irf5 signaling in dendritic cells is crucial for immunity to c. albicans aberrant type i interferon regulation in autoimmunity: opposite directions in ms and sle, shaped by evolution and body ecology janus-like effects of type i interferon in autoimmune diseases coregulation of cd8+ t cell exhaustion by multiple inhibitory receptors during chronic viral infection type i interferon suppresses de novo virus-specific cd4 th1 immunity during an established persistent viral infection timing and magnitude of type i interferon responses by distinct sensors impact cd8 t cell exhaustion and chronic viral infection distinct transcriptional profiles in ex vivo cd4+ and cd8+ t cells are established early in human immunodeficiency virus type 1 infection and are characterized by a chronic interferon response as well as extensive transcriptional changes in cd8+ t cells chronic cd4+ t-cell activation and depletion in human immunodeficiency virus type 1 infection: type i interferon-mediated disruption of t-cell dynamics host genes associated with hiv-1 replication in lymphatic tissue global genomic analysis reveals rapid control of a robust innate response in siv-infected sooty mangabeys nonpathogenic siv infection of african green monkeys induces a strong but rapidly controlled type i ifn response downregulation of robust acute type i interferon responses distinguishes nonpathogenic simian immunodeficiency virus (siv) infection of natural hosts from pathogenic siv infection of rhesus macaques comparative transcriptomics of extreme phenotypes of human hiv-1 infection and siv infection in sooty mangabey and rhesus macaque hiv turns plasmacytoid dendritic cells (pdc) into trail-expressing killer pdc and down-regulates hiv coreceptors by toll-like receptor 7-induced ifn-alpha plasmacytoid dendritic cells express trail and induce cd4+ t-cell apoptosis in hiv-1 viremic patients sex differences in the toll-like receptor-mediated response of plasmacytoid dendritic cells to hiv-1 glycerol monolaurate prevents mucosal siv transmission type i interferon responses in rhesus macaques prevent siv infection and slow disease progression plasmacytoid dendritic cells suppress hiv-1 replication but contribute to hiv-1 induced immunopathogenesis in humanized mice chronic hepatitis c: future treatment host-targeting agents in the treatment of hepatitis c: a beginning and an end? the interferons: 50 years after their discovery, there is much more to learn immune responses to hcv and other hepatitis viruses interferon lambda alleles predict innate antiviral immune responses and hepatitis c virus permissiveness altered interferon-alpha-signaling in natural killer cells from patients with chronic hepatitis c virus infection natural killer cells are polarized toward cytotoxicity in chronic hepatitis c in an interferon-alfa-dependent manner coexpression of pd-1, 2b4, cd160 and klrg1 on exhausted hcv-specific cd8+ t cells is linked to antigen recognition and t cell differentiation evidence for an antagonist form of the chemokine cxcl10 in patients chronically infected with hcv monocyte activation by interferon alpha is associated with failure to achieve a sustained virologic response after treatment for hepatitis c virus infection genetic variation in il28b and spontaneous clearance of hepatitis c virus genetic variation in il28b predicts hepatitis c treatment-induced viral clearance a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus the favorable ifnl3 genotype escapes mrna decay mediated by aurich elements and hepatitis c virus-induced micrornas distinct and overlapping genomic profiles and antiviral effects of interferon-lambda and -alpha on hcv-infected and noninfected hepatoma cells targeted induction of interferon-lambda in humanized chimeric mouse liver abrogates hepatotropic virus infection ifn-lambda receptor 1 expression is induced in chronic hepatitis c and correlates with the ifn-lambda3 genotype and with nonresponsiveness to ifn-alpha therapies how cells respond to interferons jak-stat pathways and transcriptional activation in response to ifns and other extracellular signaling proteins regulation of type i interferon responses stat2 and irf9: beyond isgf3. jakstat (2013) single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators major differences in the responses of primary human leukocyte subsets to ifn-beta critical role for stat4 activation by type 1 interferons in the interferongamma response to viral infection immunomodulatory functions of type i interferons high basal stat4 balanced by stat1 induction to control type 1 interferon effects in natural killer cells type 1 interferon induction of natural killer cell gamma interferon production for defense during lymphocytic choriomeningitis virus infection changing partners at the dance: variations in stat concentrations for shaping cytokine function and immune responses to viral infections dendritic-cell maturation alters intracellular signaling networks, enabling differential effects of ifn-alpha/beta on antigen cross-presentation mechanisms of type-i-and type-ii-interferon-mediated signalling type i interferon [corrected] gene induction by the interferon regulatory factor family of transcription factors complex modulation of cell type-specific signaling in response to type i interferons alternate interferon signaling pathways ifn-beta induces serine phosphorylation of stat-1 in ewing's sarcoma cells and mediates apoptosis via induction of irf-1 and activation of caspase-7 usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response interferon-alpha and -beta differentially regulate osteoclastogenesis: role of differential induction of chemokine cxcl11 expression differential activity of type i interferon subtypes for dendritic cell differentiation evolutionary genetic dissection of human interferons the receptor of the type i interferon family interferons as biomarkers and effectors: lessons learned from animal models protection against progressive leishmaniasis by ifn-beta multifaceted activities of type i interferon are revealed by a receptor antagonist receptor density is key to the alpha2/beta interferon differential activities structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation the type i interferon receptor: structure, function, and evolution of a family business differential receptor subunit affinities of type i interferons govern differential signal activation structural linkage between ligand discrimination and receptor activation by type i interferons distinct and nonredundant in vivo functions of ifnar on myeloid cells limit autoimmunity in the central nervous system type i interferons protect t cells against nk cell attack mediated by the activating receptor ncr1 lymphadenopathy in a novel mouse model of bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/ beta cytosolic rig-i-like helicases act as negative regulators of sterile inflammation in the cns expression of type i interferon by splenic macrophages suppresses adaptive immunity during sepsis myeloid type i interferon signaling promotes atherosclerosis by stimulating macrophage recruitment to lesions stat3 activation is responsible for il-6-dependent t cell proliferation through preventing apoptosis: generation and characterization of t cell-specific stat3-deficient mice generation of mice with a conditional stat1 null allele conditional stat1 ablation reveals the importance of interferon signaling for immunity to listeria monocytogenes infection loss of stat1 from mouse mammary epithelium results in an increased neu-induced tumor burden inactivation of stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation meta-analysis of microarray data identifies gas6 expression as an independent predictor of poor survival in ovarian cancer axl and growth arrest-specific gene 6 are frequently overexpressed in human gliomas and predict poor prognosis in patients with glioblastoma multiforme gas6 expression identifies high-risk adult aml patients: potential implications for therapy ovarian cancer progression is controlled by phenotypic changes in dendritic cells ifnalpha activates dormant haematopoietic stem cells in vivo timing of ifn-beta exposure during human dendritic cell maturation and naive th cell stimulation has contrasting effects on th1 subset generation: a role for ifn-beta-mediated regulation of il-12 family cytokines and il-18 in naive th cell differentiation combinatorial flexibility of cytokine function during human t helper cell differentiation regulating type 1 ifn effects in cd8 t cells during viral infections: changing stat4 and stat1 expression for function interferons at age 50: past, current and future impact on biomedicine inquiring into the differential action of interferons (ifns): an ifn-alpha2 mutant with enhanced affinity to ifnar1 is functionally similar to ifn-beta an interferon alpha2 mutant optimized by phage display for ifnar1 binding confers specifically enhanced antitumor activities the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities intra-lesional low-dose interferon alpha2a therapy for primary cutaneous marginal zone b-cell lymphoma intratumoral injection of interferon-alpha and systemic delivery of agonist anti-cd137 monoclonal antibodies synergize for immunotherapy delivery of interferon alpha using a novel cox2-controlled adenovirus for pancreatic cancer therapy the efficacy of radiotherapy relies upon induction of type i interferondependent innate and adaptive immunity a trial of intrapleural adenoviral-mediated interferon-alpha2b gene transfer for malignant pleural mesothelioma tumortargeted interferon-alpha delivery by tie2-expressing monocytes inhibits tumor growth and metastasis targeting cytokines to inflammation sites livertargeting of interferon-alpha with tissue-specific domain antibodies antibody-based targeting of interferon-alpha to the tumor neovasculature: a critical evaluation targeting ifn-alpha to b cell lymphoma by a tumor-specific antibody elicits potent antitumor activities targeting the tumor microenvironment with interferon-beta bridges innate and adaptive immune responses targeted delivery of interferon-alpha via fusion to anti-cd20 results in potent antitumor activity against b-cell lymphoma targeted delivery of interferon-alpha to hepatitis b virus-infected cells using t-cell receptor-like antibodies variations in the unstructured c-terminal tail of interferons contribute to differential receptor binding and biological activity safety and pharmacodynamics of rontalizumab in patients with systemic lupus erythematosus: results of a phase i, placebo-controlled, double-blind, dose-escalation study safety profile and clinical activity of sifalimumab, a fully human anti-interferon alpha monoclonal antibody, in systemic lupus erythematosus: a phase i, multicentre, double-blind randomised study mutation of the ifnar-1 receptor binding site of human ifn-alpha2 generates type i ifn competitive antagonists the studies performed in the laboratories are supported by funding from inserm, cnrs, aix-marseille university, the labex mabimprove, and the european community's seventh framework programme fp7/2007-2013 (grant agreement 223608 for gilles uzé, european research council starting grant agreement number 281225 for marc dalod including salary support to emeline pollet and thien-phong vu manh). we thank past and present laboratory members for their contribution to studies on dcs or ifns. we apologize for not quoting certain studies because of space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-003122-a3f4l6iu authors: dou, dan; revol, rebecca; östbye, henrik; wang, hao; daniels, robert title: influenza a virus cell entry, replication, virion assembly and movement date: 2018-07-20 journal: front immunol doi: 10.3389/fimmu.2018.01581 sha: doc_id: 3122 cord_uid: a3f4l6iu influenza viruses replicate within the nucleus of the host cell. this uncommon rna virus trait provides influenza with the advantage of access to the nuclear machinery during replication. however, it also increases the complexity of the intracellular trafficking that is required for the viral components to establish a productive infection. the segmentation of the influenza genome makes these additional trafficking requirements especially challenging, as each viral rna (vrna) gene segment must navigate the network of cellular membrane barriers during the processes of entry and assembly. to accomplish this goal, influenza a viruses (iavs) utilize a combination of viral and cellular mechanisms to coordinate the transport of their proteins and the eight vrna gene segments in and out of the cell. the aim of this review is to present the current mechanistic understanding for how iavs facilitate cell entry, replication, virion assembly, and intercellular movement, in an effort to highlight some of the unanswered questions regarding the coordination of the iav infection process. influenza viruses belong to the orthomyxoviridae family and are classified as either type a, b, c, or the recently identified type d (1, 2) . influenza a viruses (iavs) and type b viruses (ibvs) contain 8, negative-sense, single-stranded viral rna (vrna) gene segments ( figure 1a ) (3, 4) , which encode transcripts for 10 essential viral proteins, as well as several strain-dependent accessory proteins ( figure 1b) . in comparison, influenza type c and d viruses only possess seven vrna gene segments, as the hemagglutinin-esterase fusion protein vrna replaces the hemagglutinin (ha or h) and the neuraminidase (na or n) vrnas (1, 2) . iavs will be the main focus of this review since they are the primary agents responsible for influenza pandemics, and a major contributor to the annual influenza epidemics in the human population (5) . the natural reservoir for iavs is wild aquatic birds, but they commonly infect other species, including humans, and have even been isolated from penguins in antarctica (12) (13) (14) (15) . the ability to adapt to multiple species is a major reason why iavs are more diverse than ibvs, which are essentially exclusive to humans. despite the host-range differences, many similarities do exist between these two viruses. both possess a host-derived lipid membrane, referred to as an envelope, which is decorated on the surface with the viral membrane proteins ha, na, and to a lesser extent the matrix 2 (m2) protein ( figure 1c ) (16) (17) (18) . the envelope is supported underneath by the matrix 1 (m1) protein, and inside, the eight vrnas are found as individual viral ribonucleoprotein (vrnp) complexes ( figure 1c , bottom). each vrnp is comprised of a vrna that is wrapped around numerous red circles represent the 5′ m 7 pppg cap, black lines denote the 10-13 nucleotide, host-derived primers that are obtained by the cap-snatching mechanism of the viral polymerase. a(n) corresponds to the 3′ poly-a tail produced by reiterative stuttering of the viral polymerase. the smaller mrnas (empty boxes) represent transcripts that encode nonessential accessory proteins found in many strains, whereas those that are less prevalent (pb2-s1, m42, and ns3) are not illustrated (6) (7) (8) (9) (10) (11) . (c) diagram of an influenza a or b virus. the viral membrane proteins ha, na, and m2 are shown, along with the eight viral ribonucleoproteins (vrnps), and the matrix protein m1 that supports the viral envelope. to highlight the vrnp components, the illustration beneath the virus is not to scale. a single vrna gene segment is shown wrapped around multiple nucleoprotein (np) copies with the conserved promoter regions in the 5′ and 3′ utrs forming a helical hairpin, which is bound by a single heterotrimeric viral rna-dependent rna polymerase (pb1, pb2, and pa). (d) top view of an influenza virus cross-section showing the vrnp "1 + 7" configuration. vrnps are depicted with black circles as it is not known if the positioning of a particular vrnp is conserved or interchangeable. 2 frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 copies of the viral nucleoprotein (np) and bound by a single copy of the heterotrimeric viral polymerase, consisting of pb1, pb2, and pa (19) (20) (21) . the polymerase binds the vrnas at a helical hairpin that results from the base pairing between the conserved semi-complimentary 5′ and 3′ ends (21) (22) (23) . morphologically, iavs can either form spheres with a diameter of ~100 nm or filaments that can reach up to 20 µm in length [reviewed in ref. (24) ]. however, upon passaging in eggs, or mdck cells, the filamentous form is generally lost (25, 26) . several studies have attributed the morphology change to m1, presumably through its function in supporting the envelope (27) (28) (29) . regardless of the virion shape, ha is the most abundant viral envelope protein, followed by na, and m2 (30) . recent work has shown that the viral envelope also contains host membrane proteins (30, 31) . these proteins are likely recruited based on the lipid composition at the plasma membrane budding site, which can differ between cell types (32, 33) . through possible interactions with each other and m1, the eight vrnps typicallly form a 1 + 7 configuration inside the virus ( figure 1d ) (34, 35) . the 1 + 7 configuration may have a mechanistic function, as it is also conserved in type c and d viruses that only possess 7 vrnas (36) . further supporting the mechanistic concept, it was recently shown that iavs can package cellular ribosomal rna (as a vrnp) when one of the vrnas is made unavailable (37) , possibly explaining how type c and d viruses acquire their "eighth" vrna. the classification of iavs into subtypes is based on the genetic and antigenic properties of the surface antigens ha and na, which mediate viral entry and release, respectively (17, 18) . to date, 16 ha (h1-16) and 9 na subtypes (n1-9) have been found in iavs isolated from aquatic birds (13) . two additional subtypes for ha (h17 and h18) and na (n10 and n11) have recently been identified in bats (38, 39) , but in contrast to the ha and na subtypes from the more traditional avian iavs, these do not appear to recognize sialic acid (sa) (40) (41) (42) . despite the numerous possible subtype combinations, only three have consistently persisted in the human population, causing the following pandemics in the process: 1918 and 2009 (h1n1), 1957 (h2n2), and 1968 (h3n2) (43) . currently, only the h1n1 and h3n2 subtypes, as well as the two antigenically distinct ibv lineages (victoria and yamagata), are endemic in the human population (44) , which is why many iav vaccines include two representative iav and ibv strains (5) . a significant challenge in battling iavs is the constant evolution of the surface antigens (ha and na) in response to pressure from the host immune system, which is referred to as antigenic drift and antigenic shift. antigenic drift is most evident in circulating seasonal iavs, where substitutions by the polymerase that cause mutations in the surface antigen epitopes have continuously been selected to enable reinfection of the same host (45) . antigenic shift is responsible for the development of the iav pandemics, and it relies on the less the terminal sialic acid residues are displayed with an α-2,3 linkage, as well as an α-2,6 linkage, to illustrate the "linear" and "bent" presentations. (b) illustration of iav cell entry. (i) iavs initiate cell entry by using the ha receptor-binding domain (located in the ha1 region) to associate with sialylated glycoconjugates on a host "receptor." binding to the "receptor" triggers endocytosis. (ii) the virus then traffics to the endosome where the lower ph facilitates a conformational change in ha, exposing the fusion peptide (located in the ha2 region) for insertion into the endosomal membrane. (iii) the ha pre-hairpin conformation begins to collapse, forming a six-helix bundle that promotes hemifusion of the viral envelop with the endosomal membrane. at some point, the m2 channel opens to release the viral ribonucleoproteins (vrnps) from m1 by acidifying the viral interior. (iv) ha further collapses into a trimer of hairpins to promote the formation of the fusion pore, which (v) releases the vrnps into the cytosol. (vi) the exposed nuclear localization signals (nls) on the vrnps are recognized by the adaptor protein importin-α, leading to the recruitment of importin-β that (vii) facilitates the transport through the nuclear pore complex (npc) and into the nucleus. frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 frequent process of reassortment, which involves the exchange of vrnas between two iavs during co-infection of a cell (43, 46, 47) . while reassortment can happen between two related iavs, antigenic shift occurs when the reassortment process yields a new iav subtype. iavs are also under constant negative selection due to the functional requirements of the viral proteins, and the constraints of the limited genome. several roles have been reported for most of the iav proteins. these include the function of ha in receptor binding, as well as membrane fusion, and viral release by the sialidase activity of na. to perform these functions, the proteins need to correctly fold, oligomerize, and as for the genome itself, they have to be properly trafficked and packaged into new virions. thus mutations that benefit one property may hinder another. the goal of this review is to highlight these functional requirements by providing a summary of the mechanisms iavs have evolved to facilitate cell entry, replication, virion assembly and movement, with particular attention to how iavs coordinate the infection process. iavs initiate the infection process by using the ha molecules on the viral envelope. upon reaching a potential host cell, the ha receptor-binding site attaches the virus to surface glycoconjugates that contain terminal sa residues (figure 2a ) (18, 48, 49) . iavs then scan the cell surface for the proper sialylated "receptor" by using the sialidase function of na to remove local sas and liberate nonproductive ha associations (50) . currently, the "receptor's" identity remains unknown, but it is generally thought that has from avian iavs have higher specificity for receptors with α-2,3-linked sas that have a "linear" presentation (51, 52) , whereas has from human iavs prefer an α-2,6 linkage, which results in a more "bent" presentation ( figure 2a ) (53, 54) . while these preferences correlate with sa linkages in the respective hosts (55) , several studies have shown that matching ha receptor binding preferences with the sa linkages in a particular host is not essential for infection, but is more critical for transmission (56) (57) (58) (59) . this implies that the iav "receptor" either displays significant cell tropism in the airways or that iavs can potentially use more than one receptor. despite the unknown identity of the receptor, it is clear that ha-mediated binding to the receptor triggers endocytosis of the virion ( figure 2b , step i). the endocytosis can either occur in a clathrin-dependent manner, involving dynamin and the adaptor protein epsin-1 (60-62), or by macropinocytosis (61, 63, 64) . once inside the cell, the virus is trafficked to the endosome, where the low ph activates the m2 ion channel (61, 65, 66) , and causes a large conformational change in ha that exposes the fusion peptide ( figure 2b , step ii) (67) (68) (69) . opening of the m2 ion channel acidifies the inside of the viral particle, releasing the packaged vrnps from m1 ( figure 2b , step iii), which enables the transfer of the vrnps to the host cytoplasm following ha-mediated fusion (70, 71 fusion of the viral-endosomal membranes by ha occurs through multiple steps [reviewed in refs. (72, 73) , and requires cleavage of ha by host cell proteases into two subunits, ha1 and ha2 (55, 74, 75) ]. the cleavage (see ha proteolytic activation at the golgi or plasma membrane) is required to enable the exposure of the fusion peptide on the n-terminus of the ha2 upon the ph change in the endosome (69). once exposed, the fusion peptide inserts into the endosomal membrane, while the c-terminal transmembrane domain (tmd) anchors ha2 in the viral membrane, creating a pre-hairpin conformation (see figure 2b , step ii "box"). the ha2 trimers then fold back on themselves creating a hairpin that begins to position the two membranes in close proximity to each other (see figure 2b , step iii "box"). the hairpin bundles then further collapse into a six-helix bundle, and in doing so, the two membranes come closer together enabling the formation of the lipid stalk, and the subsequent fusion of the two membranes ( figure 2b , step iv). to date, not all of these stages have been observed with ha and some have been inferred based on observations of related fusogens from other viruses. in contrast to the early steps in iav entry, vrnp trafficking to the nucleus following the fusion event is highly dependent on the host cell machinery and transport pathways [reviewed in ref. (76) ]. supported by numerous studies, the current model is that the newly released cytoplasmic vrnps use the importin-αimportin-β nuclear import pathway to gain entry to the host cell nucleoplasm ( figure 2b , steps vi and vii) (77) (78) (79) (80) (81) (82) (83) . to initially engage this pathway, it is thought that the vrnps use the surface exposed nuclear localization sequences from the numerous np molecules to recruit the adapter protein importin-α (80) (81) (82) . upon binding to the vrnp, importin-α is recognized by the importin-β transport receptor, which directs the vrnp to the nuclear pore complex, where it is transported into the nucleoplasm. recent improvements in imaging and rna labeling techniques have made it possible to monitor the entire entry process in single cells (61, 62, (83) (84) (85) . the cumulative results from these studies show that iavs can deliver their vrnps from the cell surface to polyadenylation "reiterative stuttering" the nucleus in approximately 1 h, with entry and fusion occurring rather quickly (~10 min), and nuclear import requiring the bulk of the time (85) . a striking observation from these studies is the efficiency with which the eight vrnas reach the nucleus, indicating how effectively vrnps recruit the host nuclear import factors. supporting this observation, it was shown that np adaptation to the importin-α isoforms of a particular species is crucial for productive iav infections (86) . while the bulk of the vrnp trafficking work has been carried out using various immortalized cell lines, the potential species related differences, and the essential role of vrnp trafficking in reassortment, emphasize the need for further methodology development to examine the details of iav entry in primary cells and tissue explants. inside the nucleus, the heterotrimeric viral rna-dependent rna polymerase carries out the transcription and replication of the vrnas [reviewed in refs. (87, 88) ]. the replication of the influenza genome involves two steps: transcription of complimentary rna (crna), followed by transcription of new vrna copies using the crnas as templates. the crnas are produced by an unprimed process that relies on the correct complementation of free rntps (generally gtp and atp) with the 3′ end of the vrna template ( figure 3a ) (89, 90) . the nucleotide complementation locks the vrna template into the polymerase active site within the pb1 subunit and results in the formation of an a-g dinucleotide from which the crna is elongated (91) . upon exiting the polymerase, the crna associates with newly synthesized np molecules and a single copy of the viral polymerase to assemble into a crnp (90) . currently, it is thought that the newly produced viral polymerases, which are incorporated into the crnps, generate multiple vrna copies in a manner similar to crna transcription ( figure 3b) . however, there is one distinction related to the difference in the positioning of the longer 3′ end of the positivesense crna. due to the increased length, the crna is positioned in the polymerase such that the rntp annealing and dinucleotide formation is likely to occur at the nucleotides located 4 and 5 bases from the crna 3′ end (figure 3b , pathway i) (90, (92) (93) (94) . the dinucleotide primer then has to dissociate and reanneal to the nucleotides at the 3′ end prior to elongation ( figure 3b) . alternatively, the crna 3′ end could reposition within the polymerase due to rntp bin ding, resulting in the generation of full-length vrna transcripts directly ( figure 3b , pathway ii). the transient nature of the rntp annealing and dinucleotide formation makes it technically challenging to exclude either possibility. the remaining task of assembling a vrnp is analogous to crnp formation. viral mrna transcription from the vrna templates is primed, making it significantly more efficient than crna and vrna transcription (95) . the viral polymerase obtains the primers through a mechanism termed cap snatching (96) , which is aided by the association with the cellular rna polymerase ii c-terminal domain (figure 4 ) (97) (98) (99) . for cap snatching, the viral polymerase uses the pb2 subunit to bind to 5′ caps of nascent host transcripts (100) and the pa subunit endonuclease domain to cleave 10-13 nucleotides downstream of the 5′ cap (101) (102) (103) . the pb2 cap-binding domain then rotates to position the newly acquired capped primer into the pb1 catalytic center where it is extended using the vrna as a template (95) . finally, each transcript is polyadenylated through a reiterative stuttering′ process, which occurs when the polymerase encounters the short poly-u sequence at the vrna 5′ end (figure 4 "box") (104, 105) . this process likely involves multiple cycles of dissociation, repositioning, and reannealing of the mrna to this template region of the vrna to achieve polyadenylation. during the course of infection, mrna synthesis occurs before crna and vrna transcription, and mrna transcription is much more abundant because the use of primers significantly increases the initiation efficiency (106) . the initial mrnas are transcribed by the vrnp-associated polymerases and exported from the nucleus for translation by cytoplasmic ribosomes (93) . however, the m and ns transcripts also possess donor and acceptor splice sites that match well with those in human transcripts (107) . these sites recruit the cell spliceosome, which produces the spliced transcripts that encode for the m2 and ns2 proteins, respectively (108) (109) (110) (111) (112) . the ns transcript has been reported to maintain a similar ratio of non-spliced and spliced transcripts throughout infection (113) , whereas the ratio of the spliced m transcripts (encoding m2) have been shown to increase during infection (114) . these observations imply that ns1 and ns2 are always equally expressed, while m2 expression is more biased toward the later stages of infection. however, it is likely that the splicing efficiency of the ns and m transcripts differs between iav strains (115, 116) . iav protein synthesis is entirely dependent on the translation machinery of the host cell. following nuclear export [reviewed in ref. (117) ], the translation of the viral mrnas is divided between cytosolic ribosomes (for pb1, pb2, pa, np, ns1, ns2, and m1) and endoplasmic reticulum (er)-associated ribosomes for the membrane proteins ha, na, and m2 (figure 5 , steps i and ii). nuclear localization sequences on the newly synthesized np proteins and polymerase subunits (pb1, pb2, and pa) target these proteins into the nucleus by recruiting the importin-α-importin-β pathway that is utilized for vrnp nuclear import (figure 5 , step iii). the np and pb2 proteins are imported individually, whereas the pb1 and pa proteins are imported as a heterodimer (81, 118) . in the nucleus, these newly synthesized proteins assist in viral mrna transcription and vrna replication. np monomers bind to 12 nucleotide stretches with a partial g bias in vrnas, and presumably crnas, to assemble vrnps and crnps through a process that may be regulated by the np phosphorylation (figure 5 , steps v and vii) (119) (120) (121) . the heterotrimeric polymerase assembles and binds to the newly formed crnps to transcribe vrnas ( figure 5 , step vi) that upon formation into vrnps can generate additional viral mrna (figure 5, step viii) , or crna transcripts (figure 5 , step ix) (90, 93) . the viral rna-binding protein ns1 is synthesized early and also imported into the nucleus, where it can act as an inhibitor of interferon signaling [reviewed in ref. (122) ]. in addition, ns1 may contribute to viral mrna export from the nucleus by linking the viral transcripts to the cellular nuclear export components tap/nxf1, p15, rae1, e1b-ap5, and the nucleoporin nup98 (123) . ns2 (alternatively known as the nuclear export protein) and m1 are imported into the nucleus as well. multiple studies have implicated these two proteins in the nuclear export of vrnps (70, 71, (124) (125) (126) (127) . while the mechanism remains unclear, current data support a model where m1 acts as an adaptor protein linking ns2 to vrnps (figure 5 , step x) (128, 129) . through established interactions with crm1, ns2 is then able to target the vrnp complex to the crm1 nuclear export pathway for transport to the cytoplasm (127), where m1 potentially prevents the re-import of vrnps by blocking access to the np nuclear localization sequences (figure 5 , step xi) (71) . within the cytoplasm the vrnps are trafficked toward the plasma membrane for viral assembly by rab11. rab11 facilitates the interaction by associating with the viral polymerase pb2 subunit (130) , potentially providing a quality control mechanism that ensures new virions incorporate vrnps carrying a polymerase. earlier studies proposed that vrnps specifically associate with rab11 on recycling endosomes, which use microtubules for transport toward the cell surface ( figure 5 , step xiia) (130) (131) (132) ). an alternative model has recently been proposed where infection causes tubulation of the er membrane network and the vrnps bind to rab11 molecules that have localized to this network for trafficking toward the plasma membrane ( figure 5 , step xiib) (133) . currently, it is not known how vrnps are transferred to the plasma membrane in either model, or how iavs incorporate all eight of the different vrnps in a "1 + 7" configuration. while several studies have indicated that specific vrnp associations likely contribute to the packaging of the eight vrnps (35, 134, 135) , the underlying mechanisms remain to be established. the iav membrane proteins, which are ultimately destined for the viral envelope, are synthesized by ribosomes associated with the er membrane. similar to cellular secretory proteins, ribosome-nascent chain complexes containing na, ha, or m2 are co-translationally directed to the er by interactions of their hydrophobic targeting sequences with the signal recognition particle (srp) (figure 6 , step ii) (136) (137) (138) (139) . the cleavable signal sequence on ha facilitates the interaction with srp, whereas na and m2 use their respective tmd as an er targeting sequence. once bound, srp targets the ribosome-nascent chain complexes to the srp receptor in the er membrane (figure 6, step iii) , which transfers the ribosome to a sec61 protein-conducting channel known as the translocon (140-142) . linked to the dependence on srp, mutations that alter the targeting sequence hydrophobicity of cellular secretory proteins have been shown to decrease their er targeting and subsequent synthesis (143, 144) . although this aspect has not been examined for the iav membrane proteins, there is evidence that the hydrophobicity of their er-targeting sequences change (138, 148) , which suggests iavs potentially use this mechanism to titrate na and ha expression. the translocon enables passage of the elongating na, ha, and m2 polypeptides into the er lumen and facilitates the membrane partitioning of their respective tmd segments through a lateral gate (145, 146) . to activate the membrane integration, the tmd segments have to be of the appropriate length and hydrophobicity (146, 147) . in human h1n1 and h3n2 viruses, these criteria are conserved in the tmds of ha and m2, but not in the tmd of na, as it has become progressively less hydrophobic in the h1n1 viruses (148) . the uncharacteristic hydrophobicity loss was shown to be possible because of the na tmd being positioned at the n-terminus (138) . the positioning (~435 amino acids from the c-terminus), combined with the slow rate of ribosomal translation (~5 amino acids per second), likely provides these nontypical tmds with significant time to properly orientate and facilitate membrane insertion during the co-translational translocation process. during translocation, the n-terminus of ha and m2 is directly translocated into the er lumen, whereas na inverts, positioning the c-terminus in the er lumen (137, 138) . in addition, ha and na receive multiple n-linked glycans. the glycans are transferred by the oligosaccharyltransferase to asn-x-ser/thr sequences, and vary in number as well as positioning based on the strain, or subtype (149) . one function of the glycans is to increase the folding efficiency of na and ha by recruiting the lectin chaperones (calnexin and calreticulin) and the associated oxidoreductase erp57, which aids in disulfide bond formation (136, (150) (151) (152) . this is especially crucial for the ha and na proteins that possess a significant number of intramolecular disulfide bonds (e.g., six in has, eight in n1, and nine in n2) (153) (154) (155) . by contrast, m2 possesses two intermolecular disulfide bonds in its tetrameric conformation (156) . depending on the subtype, na tetramers also possess 2 or more intermolecular disulfide bonds. oligomerization of ha involves the trimerization of independently folded monomers, whereas na tetramerization has been proposed to result from the pairing of two co-translationally formed dimers, which assemble through a process involving the n-terminal tmd of na (150, 157) . in line with this model, it has been shown that the tmd is essential for proper na folding, and that the decreasing hydrophobicity in the n1 tmds functions to frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 support the folding and oligomerization of the enzymatic head domain (158, 159) . iavs easily achieve the protein concentrationdependent requirement for oligomerization due to the abundance of ha and na that is synthesized during an infection. however, these high synthesis levels at the er can also be deleterious by activating the er-stress response. indeed, several studies have shown that iav replication does activate the er-stress induced unfolded protein response (160, 161) , but this response is also mitigated by the inhibition of the eif2α-kinase and stress granule formation through the functions of other viral proteins (162) . despite everything that is known about the synthesis and assembly of the iav membrane proteins, several aspects have yet to be addressed. these include obtaining atomic structures of fulllength ha and na in a membrane, something that should become easier to address with the advances in cryo-electron microscopy structure determination. identifying if the na protein removes sa residues directly from substrates within the golgi, as this could decrease the effectivity of nonmembrane permeable na inhibitors. it is also unclear how iavs regulate the timing and expression levels of the viral proteins as viral mrna transcription shows little temporal variation (163, 164) . while it is likely that m2 is regulated in part by splicing (112, 114) , this does not apply to ha and na. recent work has linked na and ha regulation to the nucleotide composition of the 5′coding regions for their er-targeting sequences, which dramatically differ from the profile of corresponding regions in human secretory protein mrnas (165, 166) . an obvious candidate for post-transcriptional regulation is the viral rna-binding protein ns1. indeed, many studies have shown that ns1 can increase translation of particular mrnas, possibly by enhancing the translation initiation rate through the recruitment of eif-4g to the 5′region of viral mrnas (165, (167) (168) (169) (170) (171) . however, a clear mechanistic picture for influenza protein regulation is lacking. ha traffics from the er as a fusion incompetent precursor termed ha0. to gain its fusion function, ha must be cleaved into the subunits ha1 and ha2 (74, 172, 173) . the cleavage occurs in either a monobasic, or a multibasic, cleavage site (55) . multibasic sites are commonly found in highly pathogenic avian iavs and are cleaved by furin, a calcium-dependent serine endoprotease that is located within the trans-golgi network (174) . furin is also ubiquitously expressed (175) , which is one of the major reasons why avian iavs with a multibasic cleavage site are generally more pathogenic. by contrast, human (and low pathogenic avian) iavs encode for has with a monobasic cleavage site, which have been shown to be processed by different proteases in human respiratory epithelial cells. these include the transmembrane protease serine s-1 member 2 (tmprss2), human airway trypsin-like protease (hat), and possibly tmprss4 (176, 177) . hat localizes at the plasma membrane where it can either cleave newly synthesized ha or the ha found in cell-associated virions (178, 179) . similar to furin, tmprss2 resides in the trans-golgi network, where it cleaves ha en route to the plasma membrane. the m2 ion channel is thought to prevent the premature activation of ha following cleavage by equilibrating the slightly acidic ph of the golgi (180, 181) . distinct from furin, tmprss2 expression has been found to be more restricted to the upper and lower respiratory tract, whereas hat was mainly shown to be expressed in the upper respiratory tract (182) . these cell tropisms suggest that lower respiratory infections are likely mediated by tmprss2, and could be one of the primary reasons human iavs are confined to the epithelial layer of the respiratory tract. compared with the bulk lipid profile of the plasma membrane, iav envelopes are enriched in cholesterol and sphingolipids (32) , indicating that they bud from distinct apical plasma membrane regions often referred to as "rafts" (183) . however, infectious iavs must possess mechanisms to target the eight vrnps, m1, ha, na, and m2 to these sites in the membrane (184, 185) . ha is believed to localize to these distinct regions based on fatty acid modifications of the c-terminal cysteine that occur in the golgi (186) (187) (188) (189) , whereas na enrichment has previously been attributed to a property in the c-terminus of the tmd (190) . in contrast, m2 has been shown to accumulate at the boundaries of these budding domains (191) , and the cytosolic protein m1 has been proposed to localize to the budding region by associating with the short cytoplasmic tails of ha and na (192) . however, it is equally plausible that na and ha create membrane domains with a unique lipid profile that have a high affinity for m1. finally, the vrnps, delivered to the cell periphery by rab11, are thought to localize to the budding site by binding to m1 (193, 194) . in addition to orchestrating the assembly of the correct viral components at the apical budding site, iavs also have to remodel the membrane to induce bud formation, and ultimately scission of the viral envelope from the plasma membrane. to promote bud formation, the virus must first induce significant curvature in the membrane and then constrict the two opposing membranes of the viral envelope to help to facilitate membrane scission. curvature can be induced by (i) protein or "molecular" crowding on one leaflet of a bilayer, (ii) association of curved or "bending" proteins with the bilayer, (iii) biased accumulation of cone shaped lipids in one leaflet of the bilayer, or (iv) the cytoskeleton (195) . based on cumulative data regarding budding, iavs appear to induce membrane curvature through a combination of these mechanisms. indicative of using molecular crowding and bending proteins, several studies have demonstrated that ha and na expression is sufficient to induce budding, and that the efficiency and shape uniformity benefit from the presence of m1 (196) (197) (198) (199) . these results indicate that the abundance of ha and na on one side of the membrane can contribute to curvature. it also is intriguing to speculate that the asymmetric (154) shape of na plays a role in this process as it is often seen clustering in the viral membrane (16, 199) . by contrast, m1 appears to be analogous to a membrane-bending protein as it recruited to the cytosolic side of the membrane budding site, oligomerizes upon reaching the membrane, and these oligomers have been modeled to form curved structures (200) (201) (202) . based on these properties, it is plausible that m1 significantly influences the membrane curvature at the budding site, potentially explaining its role in discerning whether iavs form spheres or filaments (27, 203) . the ion channel m2 localizes to the budding site boundary and has also been shown to contribute to iav scission by functioning as a membrane-bending protein (191, 204) . the membrane-bending property of m2 is localized in an amphiphilic α-helix that can incorporate the amino acid side chains from its hydrophobic face into a leaflet of the bilayer. with this domain positioned in the cytosol, the intercalation results in negative membrane curvature, which has been proposed to facilitate viral bud neck formation and scission, presumably by decreasing the distance between the two opposing membranes of the viral envelope (204) . while much of the framework concerning iav budding has been established, it has been difficult to identify the details of the budding process, in part due to the mobility and heterogeneity of the plasma membrane. the lack of strong phenotypes from domains proposed to contribute to budding could also imply that iavs have built redundancy into the budding process (205) (206) (207) . the possibility of redundancy is certainly plausible, as iavs contain the necessary components to allow for a combination of lipid recruitment, molecular crowding, and a membrane-bending protein. iav cell release and movement once the newly assembled iavs bud, their release is highly dependent on the sialidase activity of na. na is a homotetramer, and each subunit is comprised of a short n-terminal cytoplasmic tail (six amino acids), followed by a tmd, a length variable stalk, and a globular enzymatic head domain (208) . the globular head domain forms a 6-bladed propeller structure, where each blade is comprised of four antiparallel β-sheets that are stabilized by disulfide bonds (155, 209, 210) . the catalytic tyr residue is found in a highly conserved active site that forms a deep pocket in the center of each monomer (211) . all of the residues necessary for catalysis exist within each monomer (212) , which has made it difficult to reconcile why na evolved to function as a tetramer (208, 213, 214) . structures of the enzymatic head domain indicate that na tetramers bind up to five calcium ions and calcium has been shown to contribute to na activity (155, 208, 215) . however, it remains unclear why influenza na has evolved to position a calcium ion at the tetrameric interface. na facilitates viral release by catalyzing the hydrolysis of the glycosidic linkage that attaches sa to underlying sugar molecules (216) (217) (218) . by removing local sa residues, na prevents ha binding at the cell surface, which facilitates the release of the virus during budding (figure 6a and step vi) (219, 220). na has also been shown to promote the separation of iavs by removing sa residues from the n-linked glycans located on the ha and na molecules in the viral envelope ( figure 6b and step vii) (221) . in contrast to ha, nas from human iavs show a general preference for α2,3-linked sa with variable abilities to cleave α2,6-linked sa residues (208, 222, 223) . however, a thorough analysis of na sa preference is lacking. more recent studies have found that some strains possess nas that are inefficient enzymes, but still capable of sa binding, raising the question of whether a poor na enzyme could contribute to, or replace, the ha receptor-binding function (224, 225) . the movement of iavs from cell to cell in the respiratory epithelium is significantly different from that in immortalized cell lines grown in liquid culture due to the presence of different cell types and a mucus layer. the mucus layer provides a protective barrier for the epithelium and is rich in heavily glycosylated mucins that can interact with iavs and limit cell binding (226, 227) . studies measuring viral movement through mucus and respiratory epithelial cells have shown that na-mediated cleavage of sas from mucins enhances iav movement through the mucus layer and infectivity ( figure 6c and step viii) (226, 228, 229) . recent work showed that this function may also apply to transmission, as iavs that possess low na activity, and are inhibited by mucus, are deficient in aerosol and contact transmission (230) . iavs are constantly exposed to negative and positive selection pressure, which shapes how the virus evolves. the functional requirements of each iav protein, such as enzyme catalysis, substrate binding, oligomerization, and domains that perform essential interactions with host proteins all combine to create substantial negative selection pressure that often manifests in the form of sequence conservation. negative pressure can also come from functions within the vrna sequences. these include promoters and "packaging signals, " but are also likely to involve aspects such as the formation of structural elements, or possibly mediating vrnp interactions that generate the 1 + 7 assembly in viral particles. in addition, the exposure of iavs to the immune response and constantly changing environments such as host, temperature, ph, cell type, and antivirals result in positive selection pressure. experimentally, addressing each type of selection has its caveats, but clearly a holistic picture of both iav and host functions are required to begin predictions of evolutionary constraints on the virus. most studies on the influenza evolutionary process focus primarily on antigenic drift and antigenic shift. however, all the viral transcribed rnas are subject to replication errors by the viral polymerase, which are estimated at 1 per 2,000-10,000 nucleotides (231) (232) (233) . consequently, both the viruses and the viral proteins are likely to exist as large heterogeneous populations during an infection. as many iav proteins are homo-oligomers this can potentially generate heterogeneity within individual protein complexes that could have functional advantages. by applying single particle and single cell analysis, these types of aspects are beginning to be investigated (234) . another interesting approach is deep mutational scanning, which has been used to examine the site-specific amino acid tolerance of iav proteins in general, and in the context of different selection pressure (235) (236) (237) (238) . currently, the best characterized protein in iavs is ha, which has two primary functions, (i) to initiate binding to the host cell and (ii) to deliver the vrnps to the host cell cytosol by fusing the viral and endosomal membranes. these functions are efficiently divided between the two domains of ha (ha1 and ha2), created by proteolysis. the receptor-binding site responsible for entry is located in the considerably larger ha1 subunit that is known to be immunodominant, explaining the high sequence variability in this region (239) . by contrast, the smaller ha2 subunit, containing the fusion peptide that is necessary to deliver the viral genome to the host cell, shows considerably higher sequence conservation. this organization is logical from the viral perspective as the large ha1 subunit likely blocks antibody recognition of ha2. the viral downside is the need to escape antibodies that inhibit the receptor-binding pocket without losing specificity and the binding function. based on this knowledge, several exciting new strategies are being developed to elicit the production of antibodies that target the more conserved region of ha (240) (241) (242) . the hope is that these strategies will generate broadly neutralizing antibodies that recognize multiple ha subtypes from iavs and the distinct lineages in ibvs, providing longer lasting immunity and alleviating the threat of potential pandemics. a similar approach using na would likely provide additional benefits. however, our knowledge of na lags behind ha. currently, it is still not known why na has evolved to function as a tetramer, which is relevant because this property presumably restricts the potential antigenic drift (mutations) it can accommodate and still function. iav replication and spreading frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1581 a relatively overlooked feature in the replication process is the contributions of host rna-binding proteins (rbps). human cells are predicted to encode over 1,500 rbps, 700 of which are predicted to interact with mrnas (243) . as a rna virus, it is highly likely that iavs have evolved to utilize this enormous network of rbps, which is supported by observations that some rbps inhibit iav replication, whereas others contribute (244) (245) (246) . it should also be considered that changes in rbps have been associated with various cancers, which could possibly influence the susceptibility to influenza infections (247, 248) . with the growing interest in rna biology, this aspect of iav infections is likely to receive considerable attention in the future. in terms of iav antivirals, the recent progress in determining the structures and mechanisms of the viral polymerase should significantly aid in the current development of drugs aimed at inhibiting different aspects of iav transcription (249) . through continued progress in defining the fundamental mechanisms that are necessary for iav infections, replication and intercellular movement, it should become possible to minimize the annual burden caused by iavs. rd wrote the review with input from dd, rr, hö, and hw. dd, rr, hö, and hw put together the figures and wrote the figure legends. characterization of a novel influenza virus in cattle and swine: proposal for a new genus in the orthomyxoviridae family mapping of the influenza virus genome: identification of the hemagglutinin and the neuraminidase genes influenza virus genome consists of eight distinct rna species influenza vaccines: challenges and solutions an overlapping protein-coding region in influenza a virus segment 3 modulates the host response a complicated message: identification of a novel pb1-related protein translated from influenza a virus segment 2 mrna identification of a novel viral protein expressed from the pb2 segment of influenza a virus influenza research database: an integrated bioinformatics resource for influenza virus research identification of novel influenza a virus proteins translated from pa mrna a novel influenza a virus mitochondrial protein that induces cell death evolution and ecology of influenza a viruses evolution and ecology of influenza a viruses detection of evolutionarily distinct avian influenza a viruses in antarctica evidence for the introduction, reassortment, and persistence of diverse influenza a viruses in antarctica influenza virus pleiomorphy characterized by cryoelectron tomography morphology of influenza b/lee/40 determined by cryo-electron microscopy influenza hemagglutinin and neuraminidase membrane glycoproteins the structure of native influenza virion ribonucleoproteins organization of the influenza virus replication machinery structure of influenza a polymerase bound to the viral rna promoter photochemical cross-linking of influenza a polymerase to its virion rna promoter defines a polymerase binding site at residues 9 to 12 of the promoter genomic rnas of influenza viruses are held in a circular conformation in virions and in infected cells by a terminal panhandle filamentous influenza viruses genetic studies of influenza viruses. i. viral morphology and growth capacity as exchangeable genetic traits. rapid in ovo adaptation of early passage asian strain isolates by combination with pr8 spherical influenza viruses have a fitness advantage in embryonated eggs, while filament-producing strains are selected in vivo reverse genetics studies on the filamentous morphology of influenza a virus the m1 and m2 proteins of influenza a virus are important determinants in filamentous particle formation structural organization of a filamentous influenza a virus conserved and host-specific features of influenza virion architecture cellular proteins in influenza virus particles quantitative analysis of the lipidomes of the influenza virus envelope and mdck cell apical membrane lipid composition of viral envelope of three strains of influenza virus -not all viruses are created equal architecture of ribonucleoprotein complexes in influenza a virus particles a conserved influenza a virus nucleoprotein code controls specific viral genome packaging influenza c and d viruses package eight organized ribonucleoprotein complexes importance of the 1+7 configuration of ribonucleoprotein complexes for influenza a virus genome packaging a distinct lineage of influenza a virus from bats new world bats harbor diverse influenza a viruses crystal structures of two subtype n10 neuraminidase-like proteins from bat influenza a viruses reveal a diverged putative active site hemagglutinin homologue from h17n10 bat influenza virus exhibits divergent receptor-binding and ph-dependent fusion activities characterization of the glycoproteins of bat-derived influenza viruses the persistent legacy of the 1918 influenza virus cocirculation of two distinct evolutionary lineages of influenza type b virus since 1983 integrating influenza antigenic dynamics with molecular evolution the effective rate of influenza reassortment is limited during human infection constraints, drivers, and implications of influenza a virus reassortment structure of the influenza virus haemagglutinin complexed with its receptor, sialic acid influenza virus-mediated membrane fusion: determinants of hemagglutinin fusogenic activity and experimental approaches for assessing virus fusion influenza a virus hemagglutinin and neuraminidase act as novel motile machinery receptor determinants of human and animal influenza virus isolates: differences in receptor specificity of the h3 hemagglutinin based on species of origin comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza a viruses early alterations of the receptor-binding properties of h1, h2, and h3 avian influenza virus hemagglutinins after their introduction into mammals specification of receptor-binding phenotypes of influenza virus isolates from different hosts using synthetic sialylglycopolymers: nonegg-adapted human h1 and h3 influenza a and influenza b viruses share a common high binding affinity for 6'-sialyl(n-acetyllactosamine) the hemagglutinin: a determinant of pathogenicity a two-amino acid change in the hemagglutinin of the 1918 influenza virus abolishes transmission experimental adaptation of an influenza h5 ha confers respiratory droplet transmission to a reassortant h5 ha/h1n1 virus in ferrets airborne transmission of influenza a/h5n1 virus between ferrets identification, characterization, and natural selection of mutations driving airborne transmission of a/h5n1 virus early stages of influenza virus entry into mv-1 lung cells: involvement of dynamin assembly of endocytic machinery around individual influenza viruses during viral entry epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway visualizing infection of individual influenza viruses the m2 proton channels of influenza a and b viruses haemagglutinin of influenza virus expressed from a cloned gene promotes membrane fusion uncoating of influenza virus in endosomes structure of influenza haemagglutinin at the ph of membrane fusion nuclear transport of influenza virus ribonucleoproteins: the viral matrix protein (m1) promotes export and inhibits import effect of m1 protein and low ph on nuclear transport of influenza virus ribonucleoproteins viral membrane fusion fusion of enveloped viruses in endosomes activation of influenza a viruses by trypsin treatment enhancement of the infectivity of influenza a and b viruses by proteolytic cleavage of the hemagglutinin polypeptide at the centre: influenza a virus ribonucleoproteins transport of incoming influenza virus nucleocapsids into the nucleus nuclear import of microinjected influenza virus ribonucleoproteins nuclear import of influenza virus rna can be mediated by viral nucleoprotein and transport factors required for protein import the npi-1/npi-3 (karyopherin alpha) binding site on the influenza a virus nucleoprotein np is a nonconventional nuclear localization signal an unconventional nls is critical for the nuclear import of the influenza a virus nucleoprotein and ribonucleoprotein ultrastructural analysis of the nuclear localization sequences on influenza a ribonucleoprotein complexes colocalization of different influenza viral rna segments in the cytoplasm before viral budding as shown by single-molecule sensitivity fish analysis influenza a virus assembly intermediates fuse in the cytoplasm analysis of iav replication and co-infection dynamics by a versatile rna viral genome labeling method differential use of importin-alpha isoforms governs cell tropism and host adaptation of influenza virus the rna polymerase of influenza a virus: mechanisms of viral transcription and replication structural insights into rna synthesis by the influenza virus transcription-replication machine interaction of the influenza a virus nucleocapsid protein with the viral rna polymerase potentiates unprimed viral rna replication isolation and characterization of the positive-sense replicative intermediate of a negative-strand rna virus single-molecule fret reveals the pre-initiation and initiation conformations of influenza virus promoter rna different de novo initiation strategies are used by influenza virus rna polymerase on its crna and viral rna promoters during viral rna replication genetic trans-complementation establishes a new model for influenza virus rna transcription and replication internal initiation of influenza virus replication of viral rna and complementary rna in vitro structural insight into cap-snatching and rna synthesis by influenza polymerase a unique cap(m7gpppxm)-dependent influenza virion endonuclease cleaves capped rnas to generate the primers that initiate viral rna transcription association of the influenza a virus rnadependent rna polymerase with cellular rna polymerase ii rna-free and ribonucleoproteinassociated influenza virus polymerases directly bind the serine-5-phosphorylated carboxyl-terminal domain of host rna polymerase ii structural basis of an essential interaction between influenza polymerase and pol ii ctd the structural basis for cap binding by influenza virus polymerase subunit pb2 the cap-snatching endonuclease of influenza virus polymerase resides in the pa subunit crystal structure of an avian influenza polymerase pa(n) reveals an endonuclease active site the rna exosome syncs iav-rnapii transcription to promote viral ribogenesis and infectivity polyadenylation sites for influenza virus mrna direct evidence that the poly(a) tail of influenza a virus mrna is synthesized by reiterative copying of a u track in the virion rna template an in vitro fluorescence based study of initiation of rna synthesis by influenza b polymerase influenza viruses and mrna splicing: doing more with less sequence of interrupted and uninterrupted mrnas and cloned dna coding for the two overlapping nonstructural proteins of influenza virus mapping of the two overlapping genes for polypeptides ns1 and ns2 on rna segment 8 of influenza virus genome sequences of mrnas derived from genome rna segment 7 of influenza virus: colinear and interrupted mrnas code for overlapping proteins spliced and unspliced rnas encoded by virion rna segment 7 of influenza virus influenza virus mrna trafficking through host nuclear speckles differences in the control of virus mrna splicing during permissive or abortive infection with influenza a (fowl plague) virus frontiers in immunology | www.frontiersin.org july regulated m1 mrna splicing in influenza virus-infected cells the accumulation of influenza a virus segment 7 spliced mrnas is regulated by the ns1 protein inefficient splicing of segment 7 and 8 mrnas is an inherent property of influenza virus a/brevig mission/1918/1 (h1n1) that causes elevated expression of ns1 protein biogenesis, assembly, and export of viral messenger ribonucleoproteins in the influenza a virus infected cell nuclear import and assembly of influenza a virus rna polymerase studied in live cells by fluorescence cross-correlation spectroscopy genomewide analysis of influenza viral rna and nucleoprotein association nucleotide resolution mapping of influenza a virus nucleoprotein-rna interactions reveals rna features required for replication phosphorylation at the homotypic interface regulates nucleoprotein oligomerization and assembly of the influenza virus replication machinery the ns1 protein: a multitasking virulence factor influenza virus targets the mrna export machinery and the nuclear pore complex the influenza virus nep (ns2 protein) mediates the nuclear export of viral ribonucleoproteins a nuclear export signal in the matrix protein of influenza a virus is required for efficient virus replication influenza a virus ns2 protein mediates vrnp nuclear export through nes-independent interaction with hcrm1 a second crm1-dependent nuclear export signal in the influenza a virus ns2 protein contributes to the nuclear export of viral ribonucleoproteins crucial role of the influenza virus ns2 (nep) c-terminal domain in m1 binding and nuclear export of vrnp crystal structure of the m1 protein-binding domain of the influenza a virus nuclear export protein (nep/ns2) a rab11-and microtubule-dependent mechanism for cytoplasmic transport of influenza a virus viral rna rab11a is essential for transport of the influenza virus genome to the plasma membrane apical transport of influenza a virus ribonucleoprotein requires rab11-positive recycling endosome influenza virus genome reaches the plasma membrane via a modified endoplasmic reticulum and rab11-dependent vesicles interaction of the influenza virus nucleoprotein with the cellular crm1-mediated nuclear export pathway a functional sequence-specific interaction between influenza a virus genomic rna segments n-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin nh2-terminal hydrophobic region of influenza virus neuraminidase provides the signal function in translocation type ii transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion integration of a small integral membrane protein, m2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic nh2 terminus a yeast mutant defective at an early stage in import of secretory protein precursors into the endoplasmic reticulum protein translocation across the endoplasmic reticulum. ii. isolation and characterization of the signal recognition particle receptor inefficient srp interaction with a nascent chain triggers a mrna quality control pathway substratespecific translocational attenuation during er stress defines a pre-emptive quality control pathway solving the membrane protein folding problem x-ray structure of a protein-conducting channel molecular code for transmembrane-helix recognition by the sec61 translocon polar residues and their positional context dictate the transmembrane domain interactions of influenza a neuraminidases protein translocation across the rough endoplasmic reticulum the cotranslational maturation program for the type ii membrane glycoprotein influenza neuraminidase n-linked carbohydrates act as lumenal maturation and quality control protein tags the number and location of glycans on influenza hemagglutinin determine folding and association with calnexin and calreticulin structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 a resolution structure of the influenza virus glycoprotein antigen neuraminidase at 2.9 a resolution the 2009 pandemic h1n1 neuraminidase n1 lacks the 150-cavity in its active site influenza virus m2 integral membrane protein is a homotetramer stabilized by formation of disulfide bonds steps in maturation of influenza a virus neuraminidase assembly of subtype 1 influenza neuraminidase is driven by both the transmembrane and head domains the influenza virus neuraminidase protein transmembrane and head domains have coevolved influenza a viral replication is blocked by inhibition of the inositolrequiring enzyme 1 (ire1) stress pathway influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-jun n-terminal kinase-mediated transforming growth factor-beta release in lung epithelial cells influenza a virus host shutoff disables antiviral stress-induced translation arrest real-time rt-qpcr assay for the analysis of human influenza a virus transcription and replication dynamics strand-specific real-time rt-pcr for distinguishing influenza vrna, crna, and mrna translational regulation of viral secretory proteins by the 5' coding regions and a viral rna-binding protein the signal sequence coding region promotes nuclear export of mrna the ns1 protein from influenza virus stimulates translation initiation by enhancing ribosome recruitment to mrnas major contribution of the rna-binding domain of ns1 in the pathogenicity and replication potential of an avian h7n1 influenza virus in chickens eukaryotic translation initiation factor 4gi is a cellular target for ns1 protein, a translational activator of influenza virus influenza virus ns1 protein stimulates translation of the m1 protein influenza virus ns1 protein enhances the rate of translation initiation of viral mrnas influenza viruses cause hemolysis and fusion of cells interaction of influenza virus hemagglutinin with target membrane lipids is a key step in virus-induced hemolysis and fusion at ph 5.2 influenza virus hemagglutinin with multibasic cleavage site is activated by furin, a subtilisin-like endoprotease fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas proteolytic activation of influenza viruses by serine proteases tmprss2 and hat from human airway epithelium proteolytic activation of the 1918 influenza virus hemagglutinin cleavage of influenza a virus hemagglutinin in human respiratory epithelium is cell associated and sensitive to exogenous antiproteases cleavage of influenza virus hemagglutinin by airway proteases tmprss2 and hat differs in subcellular localization and susceptibility to protease inhibitors amantadine selection of a mutant influenza virus containing an acid-stable hemagglutinin glycoprotein: evidence for virus-specific regulation of the ph of glycoprotein transport vesicles influenza virus m2 protein ion channel activity stabilizes the native form of fowl plague virus hemagglutinin during intracellular transport influenza and sars-coronavirus activating proteases tmprss2 and hat are expressed at multiple sites in human respiratory and gastrointestinal tracts lipid rafts as a membrane-organizing principle influenza virus assembly and budding association of influenza virus proteins with membrane rafts mutations at palmitylation sites of the influenza virus hemagglutinin affect virus formation acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity s acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine influenza virus hemagglutinin concentrates in lipid raft microdomains for efficient viral fusion role of transmembrane domain and cytoplasmic tail amino acid sequences of influenza a virus neuraminidase in raft association and virus budding influenza virus m2 protein mediates escrt-independent membrane scission influenza virus assembly: effect of influenza virus glycoproteins on the membrane association of m1 protein influenza virus assembly and lipid raft microdomains: a role for the cytoplasmic tails of the spike glycoproteins identification of the domains of the influenza a virus m1 matrix protein required for np binding, oligomerization and incorporation into virions membrane curvature in cell biology: an integration of molecular mechanisms influenza virus hemagglutinin and neuraminidase, but not the matrix protein, are required for assembly and budding of plasmid-derived virus-like particles formation of virus-like particles from human cell lines exclusively expressing influenza neuraminidase budding capability of the influenza virus neuraminidase can be modulated by tetherin structural analysis of the roles of influenza a virus membrane-associated proteins in assembly and morphology the crystal structure of the influenza matrix protein m1 at neutral ph: m1-m1 protein interfaces can rotate in the oligomeric structures of m1 influenza virus matrix protein m1 preserves its conformation with ph, changing multimerization state at the priming stage due to electrostatics influenza a matrix protein m1 multimerizes upon binding to lipid membranes the m1 matrix protein controls the filamentous phenotype of influenza a virus viral membrane scission the influenza virus hemagglutinin cytoplasmic tail is not essential for virus assembly or infectivity the cytoplasmic tail of the neuraminidase protein of influenza a virus does not play an important role in the packaging of this protein into viral envelopes mutations in the membrane-proximal region of the influenza a virus m2 protein cytoplasmic tail have modest effects on virus replication influenza neuraminidase influenza virus neuraminidase: structure, antibodies, and inhibitors the 2.2 a resolution crystal structure of influenza b neuraminidase and its complex with sialic acid mechanism-based covalent neuraminidase inhibitors with broad-spectrum influenza antiviral activity structure of the catalytic and antigenic sites in influenza virus neuraminidase conversion of a class ii integral membrane protein into a soluble and efficiently secreted protein: multiple intracellular and extracellular oligomeric and conformational forms a 2 (n2) neuraminidase of the x-7 influenza virus recombinant: determination of molecular size and subunit composition of the active unit influenza virus sialidase: effect of calcium on steady-state kinetic parameters mucin as substrate of enzyme action by viruses of the mumps influenza group mucins and mucoids in relation to influenza virus action; the destruction of francis inhibitor activity in a purified mucoid by virus action neuraminidase: the specific enzyme of influenza virus and vibrio cholerae preparation and properties of antibody directed specifically against the neuraminidase of influenza virus inhibition of influenza virus replication in tissue culture by 2-deoxy-2,3-dehydro-n-trifluoroacetylneuraminic acid (fana): mechanism of action characterization of temperature sensitive influenza virus mutants defective in neuraminidase mismatched hemagglutinin and neuraminidase specificities in recent human h3n2 influenza viruses oligosaccharide specificity of influenza h1n1 virus neuraminidases neuraminidase receptor binding variants of human influenza a(h3n2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment? influenza virus neuraminidases with reduced enzymatic activity that avidly bind sialic acid receptors influenza a penetrates host mucus by cleaving sialic acids with neuraminidase mucoproteins in relation to virus action a beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus neuraminidase is important for the initiation of influenza virus infection in human airway epithelium pandemic swine h1n1 influenza viruses with almost undetectable neuraminidase activity are not transmitted via aerosols in ferrets and are inhibited by human mucus but not swine mucus comparative mutational analyses of influenza a viruses rates of spontaneous mutation among rna viruses rapid evolution of rna viruses extreme heterogeneity of influenza virus infection in single cells deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by mxa the inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin accurate measurement of the effects of all amino-acid mutations on influenza hemagglutinin how single mutations affect viral escape from broad and narrow antibodies to h1 influenza hemagglutinin immunogen design for hiv-1 and influenza is it possible to develop a "universal" influenza virus vaccine? toward a universal influenza virus vaccine: potential target antigens and critical aspects for vaccine development hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection structure and function analysis of an antibody recognizing all influenza a subtypes a census of human rna-binding proteins cellular rna binding proteins ns1-bp and hnrnp k regulate influenza a virus rna splicing cellular ddx21 rna helicase inhibits influenza a virus replication but is counteracted by the viral ns1 protein multiple nuclear-replicating viruses require the stress-induced protein zc3h11a for efficient growth dissecting the expression landscape of rna-binding proteins in human cancers rna-binding proteins in cancer: old players and new actors inhibitors of influenza a virus polymerase the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-284156-btb4oodz authors: liu, yiliu; olagnier, david; lin, rongtuan title: host and viral modulation of rig-i-mediated antiviral immunity date: 2017-01-03 journal: front immunol doi: 10.3389/fimmu.2016.00662 sha: doc_id: 284156 cord_uid: btb4oodz innate immunity is the first line of defense against invading pathogens. rapid and efficient detection of pathogen-associated molecular patterns via pattern-recognition receptors is essential for the host to mount defensive and protective responses. retinoic acid-inducible gene-i (rig-i) is critical in triggering antiviral and inflammatory responses for the control of viral replication in response to cytoplasmic virus-specific rna structures. upon viral rna recognition, rig-i recruits the mitochondrial adaptor protein mitochondrial antiviral signaling protein, which leads to a signaling cascade that coordinates the induction of type i interferons (ifns), as well as a large variety of antiviral interferon-stimulated genes. the rig-i activation is tightly regulated via various posttranslational modifications for the prevention of aberrant innate immune signaling. by contrast, viruses have evolved mechanisms of evasion, such as sequestrating viral structures from rig-i detections and targeting receptor or signaling molecules for degradation. these virus–host interactions have broadened our understanding of viral pathogenesis and provided insights into the function of the rig-i pathway. in this review, we summarize the recent advances regarding rig-i pathogen recognition and signaling transduction, cell-intrinsic control of rig-i activation, and the viral antagonism of rig-i signaling. to ensure successful antiviral defenses and to avoid aberrant or dysregulation of host immune signaling, antiviral pathways need to be tightly regulated at each level. in this review, we will summarize the cell-intrinsic regulation of rig-i receptor activity, as well as the viral strategies to subvert the rig-i signaling machinery. the three members of the rlr family: rig-i, mda5 (melanoma differentiation factor 5), and lgp2 (laboratory of genetics and physiology 2) are expressed in most cell and tissue types. they function as cytoplasmic sensors for the recognition of a variety of rna viruses and subsequent activation of downstream signaling to drive type i ifn production and antiviral gene expressions. these three rlr proteins are rna-dependent atpases belonging to the dexd/h-box family of helicases (11) . structurally, rlrs have a similar central helicase core that is comprised of two helicase domains, hel1 and hel2 with an insertion termed hel2i. in addition, they all have a c-terminal domain (ctd). however, only rig-i and mda5 contain two n-terminal caspase activation and recruitment domains (cards) (3) (figure 1a ). among these three, rig-i is the founding member and hence the most intensively studied member of this family. each domain of rig-i plays unique roles during rig-i autorepression and activation. in brief, the ctd and helicase domain are involved in rna ligand binding and atp hydrolysis-involved conformational changes (12) (13) (14) , whereas the rig-i cards facilitate interaction with other downstream card containing molecules (15) . retinoic acid-inducible gene-i has been shown to be involved in the recognition of a variety of rna viruses in the cytoplasm, such as the sendai virus, influenza a and b viruses (iav, ibv), vesicular stomatitis virus, measles virus (mv), newcastle disease virus, ebola virus (ebov), dengue virus (denv), and hepatitis c virus (hcv) (16) (17) (18) (19) . the short double-stranded (ds) rna with a triphosphate (ppp) motif at the 5′-end, as found in these viral genomes, were shown to be a key signature recognized by rig-i (20, 21) . the 5′ppp dsrna of viral nucleocapsids has also been characterized as stimulating rig-i (22) . 5′-diphosphate-bearing rna (5′pprna), either naturally contained in viruses, produced by in vitro transcription, or via chemical synthesis, were all shown to bind to rig-i and were sufficient to activate rig-i (20, 23) . physiologically, the control of in vitro and in vivo infections of reoviruses, which bear the 5′pprna genome, relies on rig-i functionality (24) . it is worth noting that the in vitro-synthesized 5′ppprna sequences also trigger rig-i activation (25) . these agonists have demonstrated their therapeutic potential as broadspectrum antiviral agents and could be optimized as vaccine adjuvant candidates (26) (27) (28) (29) (30) . furthermore, the recognition of several dna viruses, including herpes simplex virus type 1 (hsv-1), epstein-barr virus (ebv), vaccinia virus (vacv), and adenovirus, via the rna polymerase iii were found to be rig-idependent (31, 32) . interestingly, the rig-i-mediated upregulation of sting is required for protection against the hsv-1 by the rig-i agonist, offering new evidence of the overlapping between rig-i signaling and the host response to dna viral infection (33) . notably, viral rna triggered rig-i signaling also mediates the inflammatory response via distinct pathways. the first involves the formation of the rig-i inflammasome through interactions between rig-i, asc, and caspase-1 and the stimulation of il-1β release. the second involves the adaptor proteins card9, bcl-10, mitochondrial antiviral signaling protein (mavs), and the activation of nuclear factor-κb (nf-κb) (34, 35) . upon rna ligand binding, rig-i undergoes a series of conformational changes and posttranslational modifications (ptms) to achieve full activation (further detail below). activated rig-i recruits its downstream adaptor molecule mavs (also known as ips-1, cardif, and visa) through card-card-mediated interactions (36, 37) . the oligomeric rig-i card assembly and the polymeric formation of mavs, together serve as a signaling platform for protein complexes that mediate the bifurcation of signaling into two branches. one branch recruits tumor necrosis factor receptor-associated factors (traf)-2/6 and the receptor-interacting protein 1 to subsequently activate the ikk complex, resulting in nf-κb activation (38) . the other branch signals through traf3 and activates the tank/ikkγ/ikkϵ/tbk1 complex, leading to the phosphorylation and dimerization of interferon regulator factors (irf)-3 and -7 (39, 40) . activated irf3/7 and nf-κb then translocate to the nucleus, together with atf2, c-jun, and the transcription coactivator creb-binding protein/p300, to coordinate the ifn and pro-inflammatory gene expressions (41) . once secreted, ifns bind to specific cell surface receptors and activate the jak-stat pathway. the activated transcription factors stat1, stat2, and irf9 form the interferon-stimulated gene factors (isgf3) complex. isgf3 then translocates to the nucleus and coordinates the transcription of hundreds of isgs including rig-i, thus generating an amplifying loop leading to the accumulation of rig-i during several types of infections (8) (figure 1b ). structural and biochemical studies have demonstrated that the activation of rig-i is a multi-step process and is primarily regulated by conformational changes and ptms. when initially identified as a dsrna sensor, it was hypothesized that rig-i was under negative regulation in physiological conditions. the over expression of the card domain of rig-i alone demonstrated superior signaling activity than full length rig-i in absence of viral pamps (2) . studies by saito et al. showed that the deletion of card was dominant-negative for rig-i signaling. by contrast, the deletion of repressor domain (rd) resulted in constitutive signaling, whereas rd expression alone ablated rig-i signaling actions. together, these findings provided the model of rig-i autoregulation in which the rd is predicted to mask cards for signaling transduction in uninfected cells (42) . the crystal structural analysis further delineated the models of autorepressed and ligand activated states of rig-i, respectively. in a ligand-free state, cards and hel2i interactions hinder dsrna binding and inactivate rig-i (14) . the binding of 5′ppp dsrna to rd leads to a conformational switch of rig-i, which releases the autorepressed cards and exposes the helicase domain for atp binding (14, 43) . atp hydrolysis is essential for rig-i signaling. it enables rig-i to translocate along the dsrna, and further promotes the oligomerization of rig-i cards. these processes assemble rig-i into a filamentous architecture which facilitates the card-card interactions with the mitochondrial mavs, leading to the subsequent signaling transduction for ifn production (44, 45) . importantly, rig-i atpase activity also plays a role in distinguishing self-rna from non-self-rna (46) . it was reported that rig-i atp hydrolysis increases the binding affinity of rig-i and dsrna ligands; whereas the rig-i mutants deficient in atp hydrolysis promotes the interaction of rig-i and self-dsrna and results in unintentional immune signaling (47) . one of the first ptms of rig-i following the initial ligand recognition is performed by the robust ubiquitination machinery (figure 2) . mass spectrometry analysis revealed that trim25, a member of the tripartite motif (trim) protein family possessing e3 ligase activity, induces the covalent lys63-linked ubiquitination of rig-i. mechanistically, the c-terminal spry domain of trim25 interacts with card1 and facilitates the ubiquitination of card2 at k172 (48) . the rig-i-trim25 ubiquitination complex, associates with the adaptor protein 14-3-3ϵ and translocates to mitochondria for mavs binding (49) . mutation figure 2 | regulation of retinoic acid-inducible gene-i (rig-i) activation. (a) in resting cells, rig-i is kept inactivated through the phosphorylation of caspase activation and recruitment domains (cards) and c-terminal domain (ctd) mediated by casein kinase ii and protein kinase c-α/β, respectively. (b) following the binding of 5′ triphosphate (5′ppp) rna and atp hydrolysis, rig-i is dephosphorylated by phosphoprotein phosphatase 1-α/γ and results in a conformational change that opens cards. hdac6-mediated deacetylation of rig-i ctd is critical for rig-i and 5′ppprna binding. the lys63-linked ubiquitination of rig-i mediated by trim25, riplet, oligoadenylate synthetases-like protein, and mex3c at both cards and ctd further activate rig-i and facilitate its tetramerization. (c) interactions between rig-i-trim25 complex and 14-3-3ϵ promote rig-i translocation to mitochondrial mitochondrial antiviral signaling protein (mavs) for downstream signaling, leading to interferon production. interactions between trim25, rig-i, and mavs are further negatively regulated by the lys48-linked ubiquitination, which is meditated by lubac, rnf125, and rnf122. sec14l1 and atg5-atg12 both inhibit the signaling by interrupting rig-i-mavs interactions, whereas sumoylation promotes rig-i-mavs binding. of k172 disrupts the interaction between rig-i and mavs thus abrogating downstream signaling and ifns production (50) . furthermore, a rig-i splice variant which lacks the trim25 interaction domain acts as a feedback inhibitor of rig-i signaling transduction upon viral infections (48) . in addition, riplet (ring-finger protein leading to rig-i activation, also named rnf135 or reul), another e3 ubiquitin ligase, also promotes rig-i ubiquitination. multiple sites within the cards, as well as within the ctd of rig-i, were identified as the crucial ubiquitin anchoring residues (51-53). among which, k63-linked polyubiquitination (pub) at lys788, is demonstrated as being critical for rig-i activation. however, unlike trim25-induced ubiquitination, riplet induced rig-i pub is dispensable for rig-i-rna binding but is essential for releasing card from its autorepressed state. this enhances trim25 functionality as well as promoting the oligomerization of rig-i and the activation of mavs (54) . mex3c (mex-3 rna binding family member c), another recently identified e3 ligase, also mediates lys63-ub at k99 and k169 of card, playing a critical role in rig-i activation (55) . in addition, the oligoadenylate synthetases-like (oasl) protein, although not an e3 ubiquitin ligase itself, contains a dsrna-binding groove and enhances rig-i activation by mimicking the k63-linked pub through its ubiquitin-like (ubl) domain (56, 57) . non-covalent binding of k63-ubiquitin chains to cards also potently activates rig-i (58) . recent structural analysis suggests that covalent and non-covalent binding of ubiquitin synergistically stabilize rig-i tetramerization and enhance polymerization of mavs cards (59) . on the other hand, several deubiquitinating enzymes (dubs) were identified to remove k63-linked pub chains from rig-i, thus dampening rig-i signaling. the tumor suppressor protein cylindromatosis (cyld) removes k63-linked pub chains from rig-i as well as tbk1 and ikkϵ to inhibit the irf3 response, serving as a pathway negative regulator (60) . syndecan-4, a newly identified negative regulator of rig-i, functions through attracting cyld to rig-i complex, thus potentiating the k63-mediated deubiquitination of rig-i (61) . in addition, the ubiquitin-specific protease (usp) family members, such as usp3 and usp21, were also identified as inhibitors of rig-i activation by deubiqutinating rig-i (62, 63) . in contrast to k63-linked ubiquitination, which promotes protein activation, k48-linked ubiquitination triggers proteasomal degradation of its target. for instance, the ring-finger protein 125 (rnf125), together with the ubiquitin e2 ligase ubch5, conjugate k48-linked ubiquitin to rig-i and mavs, targeting them for proteasomal degradation and thereby inhibiting downstream signaling (64) . similarly, rnf122 was recently demonstrated to mediate the proteasomal degradation of rig-i by delivering the k48-linked ubiquitin to rig-i cards (65) . the linear ubiquitin assembly complex (lubac) has been shown to promote k48 pub of trim25, leading to its degradation (66). conversely, the deubiquitinase usp15 antagonizes lubac by removing k48linked ubiquitin from trim25, leading to its stabilization and thereby promoting rig-i-mediated antiviral signaling (67) . in parallel with ubiquitination, phosphorylation has emerged in the past several years as a critical regulator of the rig-i signaling transduction (figure 2) . protein purification and mass spectrometry analysis identified that phosphorylation of thr170 in the cards antagonizes rig-i signaling by inhibiting trim25-mediated lys172 ubiquitination and mavs binding (68) . ser8 phosphorylation of cards also serves as a negative regulator of rig-i (69). in addition, the ctd of rig-i is constitutively phosphorylated at thr770 and ser854/855 by casein kinase ii to promote intermolecular interactions between ctd and cards, thereby maintaining rig-i at an autorepressive state to prevent premature downstream signaling (70) . a recent mass spectrometry analysis revealed that ikk phosphorylates rig-i at ser855, thereby providing a negative feedback regulation of rig-i (71). furthermore, conventional protein kinase c-α (pkc-α) and pkc-β have also been shown to phosphorylate cards, thus suppressing rig-i-trim interaction and subsequent antiviral responses (72) . in fact, rig-i signaling activity is controlled by a dynamic balance between phosphorylation and dephosphorylation. dephosphorylation of rig-i occurs rapidly with the presence of viral rna. a functional sirna screen identified phosphoprotein phosphatase 1-α (pp1α) and pp1γ as essential phosphatases responsible for cards dephosphorylation at ser8 and thr170, leading to rig-i signal activation and viral inhibition (73) . in addition to the ubiquitination and phosphorylation described above, acetylation modulation has recently started to gain more acknowledgment for controlling rig-i activity (figure 2) . mass spectrometry has identified the acetylation of two lysine residues (k858 and k909) in the ctd of rig-i at its inactivate state and are deacetylated during viral infection (74) . the mutation of these two sites restricts rig-i from undergoing the virusinduced interaction with mavs. k858 and k909 acetylation of rig-i has also been shown to control the pamp rna-induced rig-i oligomerization (75) . the cytoplasmic deacetylase hdac6-mediated removal of k909 acetylation has been shown as critical for rig-i binding to dsrna during viral infections (76) . furthermore, hdac6-dependent rig-i deacetylation also regulates rig-i oligomerization upon ligand binding, thus facilitating rig-i activation (75) . rig-i signal transduction is further regulated by additional ptms, regulatory proteins, and other cellular processes (figure 2) . it is worth noting that a number of ubl proteins including sumo, isg15, fat10, and atg8-atg12 are involved in these positive or negative regulatory mechanisms (77) . sumoylation serves as a positive regulator of rig-i by enhancing the rig-i and mavs binding (78) . on the contrary, the hla-f adjacent transcription 10 (fat10), an ubl modifier protein, was shown to negatively regulate rig-i by modulating rig-i solubility through a non-covalent association with cards (79) . in addition, ifn-induced isg15 negatively regulates the rig-i mediated signaling in a feedback-loop control manner (80) . sec14l1 has been observed competing with mavs for rig-i card binding (81) . furthermore, autophagy has been reported to be involved in rig-i modulation through its key regulator, the atg5-atg12 conjugate. atg5-atg12 has been found to suppress rig-i-mavs interaction, thereby inhibiting downstream signaling (82) . recently, deamidation of ctd has been described as a distinct means to induce rig-i activation. for examples, vgat (glutamine amidotransferase), from kshv (kaposi's sarcomaassociated herpesvirus) and γhv68 (murine gamma herpesvirus 68), recruits cellular phosphoribosylformyglycinamide synthase to deamindate and activate rig-i (83, 84). in order to establish infections, viruses have developed sophisticated mechanisms to counteract host immune responses. with regard to rig-i signaling, these include mechanisms such as altering viral genomes and their intermediate transcripts to avoid detection, manipulating the activation and degradation of rig-i and mavs, as well as modulating downstream signaling cascades. studying these antagonistic viral strategies has greatly broadened our understanding of rig-i activation and regulation. since 5′ triphosphate (5′ppp) is an important feature recognized by rig-i, modification of this motif has long been described as one of the major mechanisms for viruses to antagonize rig-i signaling. crimean-congo hemorrhagic fever virus, borna disease virus (bdv), and hantavirus (htnv) remove the 5′ppp group on their genome posttranscriptionally, make rig-i unable to bind to viral rna, and therefore incapable of triggering rig-i activation (85) . mechanistically, htnv uses the "prime and realign" strategy to generate a 5′-terminal monophosphorylate (86, 87) . bdv on the other hand, employs genome trimming to form a 3′-terminal overhang as well as convert 5′ppp to 5′p to avoid detection by rig-i (88) . the arenavirus presents an unpaired 5′ppp-nucleotide overhang to evade recognition by rig-i (89) . the 5′-end of viral rna can also be modified through rna-capping pathways. for example, the genomic rna of polioviruses linked to vpg (viral protein genome-linked) to cap the 5′-end from exposure to rig-i (90). the 5′-end capping with 7-methyl guanosine and methylation of 5′ppp dsrna at the 2′-o position makes viral rna non-distinguishable from the host mrnas, and therefore does not stimulate rig-i (91, 92) . by contrast, some viruses encode viral proteins to prevent rna recognition. the ebov utilizes its vp35 protein to sequester viral rna (18) . the crystal structural analysis indicates that the vp35 interferon inhibitory domain competes with rig-i for dsrna binding by forming an "end-cap" complex with dsrna, resulting in substantially diminished activation of rig-i (93) . similarly, the marburg virus vp35 spirals around the dsrna backbone and end-caps the dsrna to escape from rig-i detection (94, 95) . the iav non-structural protein 1 (ns1) possesses dsrna-binding properties to shield viral rna from rig-i (96). iav has also been shown to antagonize rig-i activation via its viral polymerase subunit pb2. pb2 position 627k in the mammalian strain increases pb2-nucleocapids binding affinity, thus inhibiting rig-i interaction with the nucleoprotein-encapsidated 5′ppp rna (22, 97) . in addition to altering and concealing their genome to prevent rna binding, viruses also re-localize viral rna to specific cellular compartments, such as mitochondria, endoplasmic reticulum (er), and golgi, to avoid cytosolic surveillance by rig-i. for instance, the denv conceals dsrna in the intracellular membrane as an escape strategy (98) . er is an important organelle for viral entry, replication, and assembly. the severe acute respiratory syndrome (sars) coronavirus (sars-cov) has been shown to induce a modified er to hide its replicating rna from detection (99) . these viral antagonism strategies highlight the importance of cellular organelle localization in viral-host interactions during innate antiviral responses. as reviewed above, ubiquitination represents one critical ptm mechanism of rig-i activation and, not surprisingly, is an attractive target for viral manipulation (figure 3a) . viruses have evolved ways to inhibit k63-linked ubiquitination of rig-i by interacting with the e3 ligases trim25 and riplet. for instance, iav ns1 from various strains has been shown to suppress trim25-mediated rig-i cards ubiquitination. among all the trim25 binding amino acids identified in ns1, r38/k41 and e96/e97 were described as critical in interfering with the coil-coiled domain of trim25. these interactions resulted in an inhibition of trim25 multimerization and therefore blocked the rig-i cards ubiquitination (100) . intriguingly, ns1-trim25 binding is found to be preserved in human and avian, but lost in mouse, indicating a species-specific manner of inhibition. this study further demonstrates that the ns1 suppression of rig-i ubiquitination in mouse is riplet-dependent (101) . conversely, phosphorylation of ns1 at thr49 was recently identified as impairing the ns1-trim25 interaction, thereby suppressing its antagonistic activity of rig-i signaling (102) . phosphorylation of another site on ns1, thr80, has also been reported to disrupt ns1 binding affinity with rig-i (103) . similar to iav, the ibv non-structural ns protein (ns1-b) has recently been described as inhibiting rig-i ubiquitination, which involves trim25-ns1 c-terminal effector domain interaction and the rig-i/trim25/ ns1-b complex formation (104) . by contrast, the protease ns3-4a of hcv functions differently, rather than inhibiting trim25, it is thought to target the e3 ligase riplet. ns3-4a directly disrupts riplet, abolishes riplet-mediated rig-i ubiquitination, and further reduces the interaction between trim25 and rig-i (54). on the other hand, some viruses encode enzymes that directly deubiquitinate rig-i. for instance, kshv encoded deubiquitinase orf64 cleaves lys63-ubiquination chains on cards, blocks cards interaction between rig-i and mavs, thereby downregulating rig-i signaling (105) . other viruses including arterivirus, nairovirus, sars-cov, and foot-and-mouth disease virus (fmdv) have also been reported to downregulate rig-i ubiquitination through their viral encoded dubs (106, 107) . few viruses have been shown to manipulate rig-i regulation with regards to targeting the phosphorylation or dephosphorylation process of rig-i. nevertheless, it was reported that mv efficiently escapes antiviral response via suppressing rig-i dephosphorylation in dendritic cells (dcs). in this study, the growth arrest and dna damage protein (gadd34) was shown to form complexes with pp1 to facilitate rig-i activation. the mv infection induced dc-sign signaling results in an inhibition of gadd34-pp1 phosphatases activity and thereby impairs rig-i activation (108). another distinct strategy used by viruses to antagonize rig-i signaling is the direct cleavage or degradation of the receptor and multiple members of the signaling cascade ( figure 3a) . rig-i has been reported in some studies to be cleaved by the proteinase 3c pro during infections with picornavirus, coxsackievirus b3 (cvb3), and enterovirus 71 (ev71) (109, 110) . the encephalomyocarditis virus directs both caspase-and proteasome-dependent degradation of rig-i (111) . intriguingly, the ns1-ns2 degradasome of the respiratory syncytial virus (rsv) has been shown to mediate the proteasomal degradation of rig-i (112) . mitochondrial antiviral signaling protein is also a well-studied molecule which is often targeted by many types of viral-induced cleavage. for example, the hepatitis a virus (hav) cleaves mavs for proteolysis by its protease 3c pro (113) . both cvb3 proteinase 2a pro and 3c pro trigger mavs cleavage at different sites during infection, and the cleavage of mavs by ev71 is accomplished via its 2a pro activity (110, 114) . in addition, serine protease ns3-4a of hcv cleaves mavs, removing it from the mitochondria, thereby inhibiting downstream signaling (36, 115) . in a parallel fashion, many viruses mediate cellular proteolytic degradation of mavs to attenuate rig-i antiviral responses. hepatitis b virus viral protein hbx triggers the proteasome-mediated degradation of mavs through lys136 ubiquitination (116) . another study reported that the hav cysteine protease abc targets mavs for proteolysis at mitochondrial membrane (113) . additionally, viral modulation of cellular organelles such as mitochondria also affects rig-i-mavs signaling. the pb1-f2 of iav, for instance, has been described as decreasing the mitochondrial membrane potential, resulting in the acceleration of mitochondrial fragmentation, thereby inhibiting rig-i-mavs signaling (117) (118) (119) . it is important to note that the proper localization of rig-i and mavs is a prerequisite for effective signaling transduction. mavs resides on the mitochondrial membrane, peroxisomes, and mitochondria-associated membranes for antiviral signaling. in fact, a rig-i translocon has been identified to direct rig-i redistribution from cytosol to membranes during viral infection (49) . studies have shown that several viruses encode proteins to disrupt the proper localization of rig-i or mavs as a novel mechanism of regulation, such as ns3 of denv (113) , nucleoprotein of rsv (120), and non-structural proteins of thrombocytopenia syndrome virus (sftsv) (121) . to ensure successful rig-i signaling transduction, the kinase activities of tbk1 and ikkϵ are tightly controlled via various regulatory mechanisms and are common targets of viruses ( figure 3b) . for example, both the leader proteinase (l pro ) of fmdv (122) and the non-structural protein 3 (ns3) of the mouse hepatitis virus a59 (123) inhibit ubiquitination of tbk1. dengue virus serotype4 non-structural proteins, ns2a and ns4b, as well as the flips proteins encoded by the molluscum contagiosum virus (mcv), all reduce tbk1 phosphorylation, thereby preventing its activation (124, 125) . several viruses have been shown to prevent the formation of functional tbk1-containing complexes. the k7 protein of the vacv prevents tbk1/ikkϵ complex-induced irf activation by targeting host dead box protein 3 (ddx3) (126) . two other viruses, the ny-1 htnv and sars-cov, disrupt the tbk1-traf3 and tank-tbk1/ikkϵ complex, respectively (127, 128) . moreover, sftsv has been shown to irreversibly re-localize tbk1 and ikk from mitochondria and sequester the tbk1/ikkϵ/irf3 complex via the formation of inclusion bodies, causing signaling cascade termination (129) . viral regulation of the transcription factors, irfs and nf-κb, further serve as points of control in rig-i signaling ( figure 3b) . one of the best studied examples is the inhibition of irf3 activity by the iav ns1 protein (130) . besides this, the hsv-1, rabies virus, sars-cov, as well as several paramyxoviruses have been demonstrated to interfere with the phosphorylation state of irf3, thereby blocking ifn induction (131) (132) (133) (134) . the ebv conjugates sumo to irf7 at lysine 452 to decrease irf7 transcriptional activity (135) . the rotavirus ns1, targets both irf3 and irf7 for degradation to prevent irfs from undergoing dimerization (136) . viruses have also developed various means to suppress the irf3 dna binding ability. herpes simplex virus, thogoto virus, and kshv, all developed strategies to downregulate irf3 transcriptional activity by either disrupting irf3 binding complex formations or competing binding regions on the ifnb promoter (137) (138) (139) . viral strategies in inhibiting cytoplasmic or transcriptional activities of nf-κb have been extensively studied during the vacv infection. studies reported that multiple proteins encoded by vacv and hsv-1 suppress nf-κb activation (140) (141) (142) (143) . viruses have also developed multiple inhibitory mechanisms to counteract the ifn stimulation of isgs by targeting stat1 and/or stat2 ( figure 3b ). for example, the langat virus was shown to inhibit the phosphorylation of both stat1 and stat2 (144) . varicella viruses and the japanese encephalitis virus, both block the jak/stat1 pathway through multiple mechanisms including inhibiting stat proteins phosphorylation and nucleotranslocation (145, 146) . the non-structural protein ns5 of several flaviviruses, have been shown to target stat proteins via distinct mechanisms. for example, mnv ns5 inhibits stat1 phosphorylation, whereas denv ns5 interacts with ubr4 to promote stat2 degradation (147, 148) . by contrast, the zika virus ns5 induced proteasomal degradation of stat2 was recently identified as ubr4 independent (149) . furthermore, other viruses, such as hcv (150) , rsv (151) , and paramyxovirus (152) , also demonstrate negative regulation of the jak-stat pathway. studies from the past decade have well established rig-i as one of the principal prrs for the recognition of cytoplasmic viral rna, as well as defining its critical role in the induction of ifns during viral infections. our understanding of the rig-i-mediated antiviral response has been greatly expanded with the key discoveries made regarding the molecular mechanism of rig-i regulation, such as ubiquitination, phosphorylation, and acetylation. meanwhile, investigating viral strategies to manipulate rig-i responses not only allow us to understand the viral pathogenesis, but also significantly contributed to our knowledge of how rig-i is activated and regulated. these new insights into the viral-mediated rig-i regulations are important for vaccine and drug development aiming to suppress infectious diseases and enhance immune responses. yl wrote the manuscript. rl and do revised and approved the manuscript. the authors would like to thank alexandre sze for his critical reading and editing of the manuscript. this research was supported by grant from canadian institutes of health research (mop130401) to rl; do was supported by a peter quinlan mcgill postdoctoral fellowship. the authors would like to acknowledge all the colleagues in the field and apologies to those whose important contributions could not be included in the review due to space constraints. the figures of the review were illustrated using the servier medical art library, http://www. servier.com/powerpoint-image-bank. the role of pattern-recognition receptors in innate immunity: update on toll-like receptors the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses immune signaling by rig-i-like receptors tlr signaling pathways regulation of the antimicrobial response by nlr proteins cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway ifi16 is an innate immune sensor for intracellular dna mechanisms of type-i-and type-ii-interferon-mediated signalling viral evasion and subversion of patternrecognition receptor signalling viral evasion of intracellular dna and rna sensing pattern recognition receptors and inflammation structural insights into rna recognition by rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (mavs) adapter recognition of viral nucleic acids in innate immunity rna recognition and signal transduction by rig-i-like receptors ebola virus vp35 protein binds double-stranded rna and inhibits alpha/beta interferon production induced by rig-i signaling rig-i, mda5 and tlr3 synergistically play an important role in restriction of dengue virus infection 5′-triphosphate rna is the ligand for rig-i recognition of 5′ triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus incoming rna virus nucleocapsids containing a 5′-triphosphorylated genome activate rig-i and antiviral signaling rig-imediated antiviral responses to single-stranded rna bearing 5′-phosphates antiviral immunity via rig-i-mediated recognition of rna bearing 5′-diphosphates systems analysis of a rig-i agonist inducing broad spectrum inhibition of virus infectivity inhibition of dengue and chikungunya virus infections by rig-i-mediated type i interferon-independent stimulation of the innate antiviral response enhanced influenza virus-like particle vaccination with a structurally optimized rig-i agonist as adjuvant defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced dengue virus infection sequence-specific modifications enhance the broad-spectrum antiviral response activated by rig-i agonists cutting edge: the rig-i ligand 3prna potently improves ctl cross-priming and facilitates antiviral vaccination early innate recognition of herpes simplex virus in human primary macrophages is mediated via the mda5/mavs-dependent and mda5/mavs/ rna polymerase iii-independent pathways rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway rig-i mediated sting up-regulation restricts hsv-1 infection recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 regulation and function of nf-kappab transcription factors in the immune system a functional c-terminal traf3-binding site in mavs participates in positive and negative regulation of the ifn antiviral response the nemo adaptor bridges the nuclear factor-kappab and interferon regulatory factor signaling pathways direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 the c-terminal regulatory domain is the rna 5′-triphosphate sensor of rig-i atpase-driven oligomerization of rig-i on rna allows optimal activation of type-i interferon rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner rig-i atpase activity and discrimination of self-rna versus non-self-rna correction: atp hydrolysis by the viral rna sensor rig-i prevents unintentional recognition of self-rna roles of rig-i n-terminal tandem card and splice variant in trim25-mediated antiviral signal transduction the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i a distinct role of ripletmediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity antiviral activity of human oasl protein is mediated by enhancing signaling of the rig-i rna sensor structural and functional analysis reveals that human oasl binds dsrna to enhance rig-i signaling reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity structural basis for ubiquitin-mediated antiviral signal activation by rig-i the tumour suppressor cyld is a negative regulator of rig-i-mediated antiviral response syndecan-4 negatively regulates antiviral signalling by mediating rig-i deubiquitination via cyld usp3 inhibits type i interferon signaling by deubiquitinating rig-i-like receptors usp21 negatively regulates antiviral response by acting as a rig-i deubiquitinase negative regulation of the rig-i signaling by the ubiquitin ligase rnf125 rnf122 suppresses antiviral type i interferon production by targeting rig-i cards to mediate rig-i degradation linear ubiquitin assembly complex negatively regulates rig-i-and trim25-mediated type i interferon induction the ubiquitin-specific protease usp15 promotes rig-i-mediated antiviral signaling by deubiquitylating trim25 phosphorylationmediated negative regulation of rig-i antiviral activity negative role of rig-i serine 8 phosphorylation in the regulation of interferon-beta production phosphorylation of rig-i by casein kinase ii inhibits its antiviral response ikk negatively regulates rig-i via direct phosphorylation conventional protein kinase c-alpha (pkc-alpha) and pkc-beta negatively regulate rig-i antiviral signal transduction dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling lysine acetylation targets protein complexes and co-regulates major cellular functions regulation of retinoic acid inducible gene-i (rig-i) activation by the histone deacetylase hdac6 regulates cellular viral rna sensing by deacetylation of rig-i ubiquitin-like proteins sumoylation of rig-i positively regulates the type i interferon signaling ubiquitin-like modifier fat10 attenuates rig-i mediated antiviral signaling by segregating activated rig-i from its signaling platform negative feedback regulation of rig-i-mediated antiviral signaling by interferon-induced isg15 conjugation negative regulation of rig-i-mediated innate antiviral signaling by sec14l1 the atg5 atg12 conjugate associates with innate antiviral immune responses viral pseudo-enzymes activate rig-i via deamidation to evade cytokine production emerging roles of protein deamidation in innate immune signaling processing of genome 5′ termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction the 5′ ends of hantaan virus (bunyaviridae) rnas suggest a prime-and-realign mechanism for the initiation of rna synthesis old world hantaviruses do not produce detectable amounts of dsrna in infected cells and the 5′ termini of their genomic rnas are monophosphorylated genome trimming: a unique strategy for replication control employed by borna disease virus unpaired 5′ ppp-nucleotides, as found in arenavirus double-stranded rna panhandles, are not recognized by rig-i a protein covalently linked to poliovirus genome rna conventional and unconventional mechanisms for capping viral mrna structural basis for m7g recognition and 2′-o-methyl discrimination in capped rnas by the innate immune receptor rig-i structural basis for dsrna recognition and interferon antagonism by ebola vp35 marburg virus vp35 can both fully coat the backbone and cap the ends of dsrna for interferon antagonism structural basis for marburg virus vp35-mediated immune evasion mechanisms a recombinant influenza a virus expressing an rna-binding-defective ns1 protein induces high levels of beta interferon and is attenuated in mice influenza virus adaptation pb2-627k modulates nucleocapsid inhibition by the pathogen sensor rig-i the dengue virus conceals double-stranded rna in the intracellular membrane to escape from an interferon response sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein phosphorylation of influenza a virus ns1 protein at threonine 49 suppresses its interferon antagonistic activity threonine 80 phosphorylation of non-structural protein 1 regulates the replication of influenza a virus by reducing the binding affinity with rig-i robust lys63-linked ubiquitination of rig-i promotes cytokine eruption in early influenza b virus infection inhibition of rig-i-mediated signaling by kaposi's sarcoma-associated herpesvirusencoded deubiquitinase orf64 regulation of rig-i-like receptor signaling by host and viral proteins arterivirus and nairovirus ovarian tumor domain-containing deubiquitinases target activated rig-i to control innate immune signaling measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp1 phosphatases rig-i is cleaved during picornavirus infection enterovirus 2apro targets mda5 and mavs in infected cells the viral rna recognition sensor rig-i is degraded during encephalomyocarditis virus (emcv) infection viral degradasome hijacks mitochondria to suppress innate immunity disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor the coxsackievirus b 3c protease cleaves mavs and trif to attenuate host type i interferon and apoptotic signaling hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity the hepatitis b virus x protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein influenza virus protein pb1-f2 inhibits the induction of type i interferon by binding to mavs and decreasing mitochondrial membrane potential the influenza virus protein pb1-f2 inhibits the induction of type i interferon at the level of the mavs adaptor protein influenza a virus protein pb1-f2 translocates into mitochondria via tom40 channels and impairs innate immunity human respiratory syncytial virus nucleoprotein and inclusion bodies antagonize the innate immune response mediated by mda5 and mavs hijacking of rig-i signaling proteins into virus-induced cytoplasmic structures correlates with the inhibition of type i interferon responses the leader proteinase of foot-and-mouth disease virus negatively regulates the type i interferon pathway by acting as a viral deubiquitinase plp2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type i interferon production dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk1/irf3 phosphorylation: dengue virus serotype 1 ns4a is a unique interferon-regulating virulence determinant inhibition of interferon gene activation by death-effector domain-containing proteins from the molluscum contagiosum virus viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation the ny-1 hantavirus gn cytoplasmic tail coprecipitates traf3 and inhibits cellular interferon responses by disrupting tbk1-traf3 complex formation sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex evasion of antiviral immunity through sequestering of tbk1/ikkepsilon/irf3 into viral inclusion bodies activation of interferon regulatory factor 3 is inhibited by the influenza a virus ns1 protein inhibition of interferon regulatory factor 3 activation by paramyxovirus v protein the sars coronavirus papain like protease can inhibit irf3 at a post activation step that requires deubiquitination activity genetic dissection of interferon-antagonistic functions of rabies virus phosphoprotein: inhibition of interferon regulatory factor 3 activation is important for pathogenicity herpes simplex virus 1 serine/threonine kinase us3 hyperphosphorylates irf3 and inhibits beta interferon production epstein-barr virus latent membrane protein 1 regulates the function of interferon regulatory factor 7 by inducing its sumoylation rotavirus nsp1 mediates degradation of interferon regulatory factors through targeting of the dimerization domain thogoto virus ml protein suppresses irf3 function binding of kaposi's sarcoma-associated herpesvirus k-bzip to interferon-responsive factor 3 elements modulates antiviral gene expression recruitment of activated irf-3 and cbp/p300 to herpes simplex virus icp0 nuclear foci: potential role in blocking ifn-beta induction the vaccinia virus k1 ankyrin repeat protein inhibits nf-kb activation by preventing rela acetylation vaccinia virus protein c4 inhibits nf-kappab activation and promotes virus virulence herpes simplex virus 1-encoded tegument protein vp16 abrogates the production of beta interferon (ifn) by inhibiting nf-kappab activation and blocking ifn regulatory factor 3 to recruit its coactivator cbp herpes simplex virus 1 protein kinase us3 hyperphosphorylates p65/rela and dampens nf-kappab activation inhibition of interferon-stimulated jak-stat signaling by a tick-borne flavivirus and identification of ns5 as an interferon antagonist blocking of interferon-induced jak-stat signaling by japanese encephalitis virus ns5 through a protein tyrosine phosphatase-mediated mechanism varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms the ns5 protein of the virulent west nile virus ny99 strain is a potent antagonist of type i interferon-mediated jak-stat signaling dengue virus co-opts ubr4 to degrade stat2 and antagonize type i interferon signaling zika virus targets human stat2 to inhibit type i interferon signaling hepatitis c virus targets the interferon-alpha jak/stat pathway by promoting proteasomal degradation in immune cells and hepatocytes respiratory syncytial virus ns1 protein degrades stat2 by using the elongin-cullin e3 ligase paramyxovirus disruption of interferon signal transduction: status report key: cord-256582-x2grfhov authors: sung, pei-shan; hsieh, shie-liang title: clec2 and clec5a: pathogenic host factors in acute viral infections date: 2019-12-06 journal: front immunol doi: 10.3389/fimmu.2019.02867 sha: doc_id: 256582 cord_uid: x2grfhov the protective roles of endosomal toll-like receptors (tlrs) and cytosolic nucleic acid sensors are well elucidated, but the pathogenic host factors during viral infections remain unclear. spleen tyrosine kinase (syk)-coupled c-type lectins (clecs) clec2 and clec5a are highly expressed on platelets and myeloid cells, respectively. clec2 has been shown to recognize snake venom aggretin and the endogenous ligand podoplanin and acts as a critical regulator in the development and immunothrombosis. although clec2 has been reported to interact with type i immunodeficiency virus (hiv-1), its role in viral infections is still unclear. clec5a binds to fucose and mannose moieties of dengue virus membrane glycans, as well as to n-acetylglucosamine (glcnac)/n-acetylmuramic acid (murnac) disaccharides that form the backbone of l. monocytogenes peptidoglycans. recently, we demonstrated that both clec2 and clec5a are critical in microbe-induced “neutrophil extracellular trap” (net) formation and proinflammatory cytokine production. moreover, activation of clec2 by dengue virus (dv) and h5n1 influenza virus (iav) induces the release of extracellular vesicles (evs), which further enhance netosis and proinflammatory cytokine production via clec5a and toll-like receptor 2 (tlr2). these findings not only illustrate the immunomodulatory effects of evs during platelet-leukocyte interactions, but also demonstrate the critical roles of clec2 and clec5a in acute viral infections. the global outburst of severe acute respiratory syndrome coronavirus (sars-cov) infection in 2003 inspired our exploration of how a viral infection can activate a massive over-production of cytokines by the host immune system-a phenomenon known as a "cytokine storm"; this results in a severe inflammatory reaction and increases systemic vascular permeability within 2-5 days following viral exposure. the common features of sars-cov and other acute viral infections are the early onset of inflammatory reactions with elevated local or systemic vascular permeability, before the adaptive immune system is fully activated. these observations suggest that innate immune responses contribute significantly to the pathogenesis of acute viral infections. the identification of endosomal tlrs (tlr3, tlr7, tlr8, tlr9) and cytosolic sensors led to the discovery that their activation by viral nucleic acids induces the production of interferons and proinflammatory cytokines. these intracellular nucleic acid receptors/sensors have been defined as "protective host factors" as they are critical for host defense against viral infections. however, the identity and contribution of "pathogenic host factors" to virus-induced severe inflammatory reactions and lethality, and how different viruses cause distinct clinical symptoms, has remained unclear. in contrast to the relatively conserved nature of nucleic acids (dna and rna), the proteins and glycans of viral envelopes are various have distinct structures; it is enveloped viruses, such as flavivirus, alphavirus, togavirus, coronavirus, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, and filovirus that are primarily responsible for severe inflammatory reactions following infection. therefore, we were interested to determine whether viruses activate membrane receptors on myeloid cells induce cytokine storms, and how these receptors contribute to complex cell-cell interactions during acute viral infections. in this review article, we illustrate the critical roles of c-type lectin receptors in innate immunity and discuss the potential of targeting these cell surface receptors in the treatment of acute viral infections. the common feature of acute viral infections is the rapid onset of inflammatory reactions after exposure to the virus. clinical symptoms range from inapparent and mild fever to severe inflammation and increased endothelial permeability in major organs. severe acute infections in human typically occur in response to enveloped viruses, such as dengue virus (flaviviridae), chikungunya virus (togaviridae), sars-cov (coronaviridae), influenza virus (orthomyxoviridae), measles virus (paramyxoviridae), rabies virus (rhabdoviridae), hanta virus (bunyaviridae), and ebola virus and marburg virus (filoviridae). this implies that membrane components may contribute to the severe inflammatory responses. acute viral infections begin with host exposure to virus and clinical symptoms become apparent gradually as the virus starts to replicate and innate immune responses are activated. abundant proinflammatory cytokines production leads to the characteristic symptoms in acute viral infections, including fever, aches, and pain. when host innate immunity fails to contain virus replication and remove infected cells, high fever and massive cytokine production occur in the host, leading to severe local and systemic changes in vascular permeability, functional failure of affected organs and shock syndromes. it is well-known most of pro-inflammatory cytokines are released from macrophages and severe acute infections are usually associated with the activation of macrophages by enveloped viruses such as dengue virus (1, 2), h5n1 viruses (2) (3) (4) , and ebola viruses (5) . in addition, activation of neutrophils leads to the formation of neutrophil extracellular traps (nets), which may further aggravate virus-induced inflammatory reactions. furthermore, acute viral infections frequently cause thrombocytopenia, and platelet-leukocyte interactions not only regulate inflammatory reactions, but also contribute to the pathogenesis of vascular injury, thrombosis, autoimmune reactions (6) (7) (8) . however, the molecular mechanisms underlying platelet-mediated immunomodulation are still unclear. the human immune system has evolved to provide protection against a diversity of microorganisms, including viral infections. ones of the most important factors involved in suppressing the development of a viral infection is the production of interferons via the activation of intracellular nucleic acid sensors. the endosomal tlrs (such as tlr3, 7, 8, and 9) recognize diverse endogenous danger signals as well as nucleic acid components released from uncoated virions. activation of tlrs results in the production of type i interferons and proinflammatory cytokines, chemokines (including tnf-α, il-6, il-8, ip10, and others) thereby initiating inflammatory responses. in addition, the intracellular rna sensors, such as mda-5 (melanoma differentiation-associated protein 5) and rig-i (retinoic acidinducible gene i) as well as the dna sensors sting (stimulator of interferon genes) and aim2 (absent in melanoma 2) that recognize conserved nucleic structures mediate the production of interferons and proinflammatory cytokines when activated. tlrs are central to the control of innate immune responses through the recognition of pathogen-associated molecular patterns (pamps) and endogenous damage-associated molecular patterns (damps) (9) . therefore, tlrs are not only critical for host defense against pathogens but also contribute to the pathogenesis of autoimmunity (10, 11) . several tlr agonists and antagonists are regarded as promising therapeutic agents for the treatment of sepsis, asthma, vaccine adjuvants, and autoimmunity (9) . the cell surface tlrs (tlr2, tlr4, and tlr5) are activated by diverse pamps and damps, whereas the endosomal tlrs (tlr3, tlr7, tlr8, and tlr9) recognize pathogen-derived nucleic acids and, in the context of therapeutic/experimental approaches, synthetic nucleic acids or nucleic acid mimetics. even though tlrs can bind ligand directly, recent studies have shown that high affinity and stable interactions with ligand also require tlr-associated proteins (i.e., co-receptors such as cd14, lbp) (11) . activation of all tlrs (except tlr3) initiates signaling via myd88-dependent and trif-dependent pathways. it has been shown that activated myd88 triggers two signaling pathways: (1) the traf3-tbk1-ikkε-irf3/irf7 pathway to induce type i interferon (ifn-α and ifn-β) expression; and (2) the traf6-tak1-ikk-nfκb pathway to induce proinflammatory cytokine expression. in contrast, activated trif primarily triggers the traf3-tbk1-ikkε-irf3/irf7 pathway to upregulate type i interferon production. in addition to endosomal tlrs, cells are equipped with cytosolic sensors that detect invading viruses via recognition of viral dna and rna. viral rna is recognized by members of tolllike receptors (tlr3, tlr7/8) and rig-i-like receptors (rig-i, mda5, and lgp2 (laboratory of genetics and physiology 2). in addition, viral dna stimulate ifn production via tlr9 and intracellular dna sensors such as sting (stimulator of ifn genes), mpys (methionine-proline-tyrosineserine), mita (mediator of irf3 activation), or eris (er ifn stimulator) (12, 13) . signaling pathways downstream rna and dna sensors induce the production of restriction factors (such as type i and type iii interferons) that prevent viral replication and give rise cell-intrinsic antiviral immunity (14-16). the superfamily of c-type lectin-like domain (ctld)containing carbohydrate-binding proteins comprises ∼150 members that can be classified into 17 groups in humans (17) . while most ctld family receptors do not contain cytoplasmic domains capable of transducing signals, several are syk-coupled receptors have been reported to play critical roles in host immunity against fungal and non-microbial infections (18) . there is ample evidence to demonstrate the critical roles of c-type lectins in the pathogenesis of dv infection (1, (19) (20) (21) (22) (23) (24) . macrophages, dendritic cells, and liver sinusoid cells are the major targets of dv in humans, where the c-type lectin receptors l-sign/cd299 (expressed in liver sinusoid cells), dc-sign/cd209 and mannose receptor/cd206 (expressed in macrophages/dendritic cells) have been shown to be crucial for dv entry into these cell types. dengue is a mosquitoborne virus and it is interesting to note that dv generated in mosquito cells infects human cells via dc-sign, while dv from human dendritic cells infects human cells via l-sign (1, (19) (20) (21) (22) (23) (24) . these observations led to the hypothesis that distinct glycans associated with dv derived from human or insect cell origins may contribute the differential usage of dc-sign and l-sign in target cell infection. despite the involvement of these c-type lectin receptors in the pathogenesis of viral infections, they do not contain intracellular motifs to enable the initiation of signaling cascades (25) , nor do they transduce signals via associated adaptor proteins as observed in t cell receptors, bcell receptors, or syk-coupled receptors. as well as membraneassociated lectins, the soluble c-type lectin mannose binding lectin (mbl) not only binds dv generated from both human and mosquito cells, but also activates the complement system to neutralize dv infection (26) . all the syk-coupled c-type lectin receptors (clrs) are type ii transmembrane proteins which belong to group ii or group v of the ctld superfamily. group ii includes four syk-coupled clrs (clec4b/dcar2, clec4c/bdca-2, clec4e/mincle, and clec6a/dectin-2), whilst group v (also known as the nk receptor-like lectin family) includes clec2, clec5a/mdl-1, clec7a/dectin-1, and clec9a/cd370 (27) . among these sykcoupled c-type lectins, activation of clec5a by flaviviruses and influenza virus induces the production of inflammatory cytokines and netosis, while these viruses also activate clec2 in platelets to elease extracellular vesicles (evs), which further enhance inflammatory cytokine release and netosis via clec5a and tlr2 in macrophages and neutrophils. clec5a, also known as myeloid dap12-associating lectin-1 (mdl-1), is abundantly expressed in myeloid lineages, including neutrophil, monocyte, macrophage, osteoclast, microglia, and dendritic cells. while clec5a does not contain a cytoplasmic motif, activation of clec5a recruits the dap12 adapter protein, thereby triggering downstream signaling via syk. structural analysis shows that human clec5a is an n-linked homodimeric glycoprotein expressed on cell surface (28) . the ligand for clec5a was unknown prior to the identification of its interaction with dengue virus (1) . we showed that when clec5a is activated by binding to the dengue virion this leads to dap12 phosphorylation and induction of proinflammatory cytokines via syk-mediated pathways. clec5a is responsible for dvinduced hemorrhagic fever (dhf) and dengue shock syndrome (dss), which represent the most severe responses to dv infection and are characterized by plasma leakage due to increased vascular permeability. blockade of the dv-clec5a interaction suppresses the secretion of proinflammatory cytokines, but without affecting interferon-α release. furthermore, blockade of dv-clec5a interaction by anti-clec5a monoclonal antibodies (mabs) efficiently alleviating plasma leakage and systemic hemorrhaging, thus protect mice from dv-induced shock and lethality in mice lack responsiveness to ifn-α and β (signal transducer and activator of transcription 1 (stat1)deficient mice). thus, blockade of clec5a-mediated signaling in dv infected cells provides a promising strategy for attenuating systemic vascular leakage and increasing the survival rate of patients suffering from dhf/dss; this approach might also be applicable to other virus-induced inflammatory diseases (1). however, even though blockade of clec5a protects mice from lethal doses of dv, the protective effect is ∼50% at best. this suggests the presence of yet-discovered pathway in dv-induced lethality, which is the focus of ongoing research. we have shown that dv infection induces abundant il-1β and il-18 production by inflammatory macrophages and also mediates pyroptosis in these cells as a consequence of upregulating pro-il-1β, pro-il-18 and nlrp3 and enabling caspase-1 activation; blockade of clec5a inhibits dv-induced nlrp3 inflammasome activation and cell death (29) . we found that osteoclasts are highly susceptible to dv infection, which induces the production of cytokines and infectious virions at levels similar to macrophages and nuclear translocation of the transcription factor nfatc1, via clec5a. the transient inflammatory response that occurs in bone tissue during dv infection is associated with elevated osteolytic activity and release c-telopeptide of type i collagen (ctx-1) from bone. moreover, clec5a −/− stat1 −/− mice are resistant to dvinduced osteolysis, and anti-clec5a mab inhibits dv-induced osteolytic activity and ctx-1 releases in clec5a ++ stat1 −/− mice. these observations indicate that dv osteoclast and increase osteolytic activity via clec5a. the identification of osteoclasts as a novel target for dv provides a mechanism for some of the clinical symptoms experienced by dengue patients; i.e., as a consequence of transient upregulation of osteolysis (30) . moreover, we demonstrated that japanese encephalitis virus (jev) binds clec5a directly to trigger the phosphorylation of dap12 and the release of proinflammatory cytokines and chemokines in macrophages. even though blockade of clec5a cannot inhibit jev infection to neurons and astrocytes, jev-induced cell death and proinflammatory cytokines in microglia-neurons-astrocyte mixed culture was dramatically inhibited by anti-clec5a mab. this effect is achieved by blockade of jev-clec5a interactions in microglia, thereby attenuating proinflammatory cytokines and toxic substances released from microglia. furthermore, peripheral administration of anti-clec5a mab not only reduces jev-induced neuronal inflammation and lethality, but also restore blood-brain barrier (bbb) integrity after jev infection. this observation suggests that anti-clec5a mab can penetrate bbb and attenuates jevinduced neuronal inflammation in brain, thereby restore bbb integrity and reduces neuronal toxicity and lethality. in contrast to conventional immunosuppressants, anti-clec5a mabmediated immunomodulation does not inhibit host immunity to jev, as all surviving mice produce high titer of jev neutralization ab (both igm and igg) and develop protective cellular immunity against jev infection. these observations indicate that clec5a play a critical role in jev-induced pathological changes, and blockade of jev-clec5a interactions by anti-clec5a mabs is a promising strategy to brain damage and neuronal sequalae after jev infection (31) . in addition to flaviviruses, we further find that clec5a interacts with the hemagglutinin protein of influenza viruses (h1n1, h5n1, h7n9), and blockade of influenza virus-clec5a interactions by anti-clec5a antibodies reduces proinflammatory cytokine production without attenuating influenza virus replication in human macrophages. furthermore, reduced levels of tnf-α and ip-10 were found in clec5a −/− bmdms (bone marrow-derived macrophages) than those in wild type cells. upon lethal challenges with a recombinant h5n1 influenza virus, lower levels of proinflammatory cytokines and less pulmonary cell infiltration with higher survival rates are noted in clec5a −/− mice, despite comparable viral loads are noted in both wt and clec5a −/− mice. these results indicate that influenza virus activates clec5a to enhance inflammatory reactions and cause severe tissue damage, and combination of current anti-viral drugs with anti-clec5a mab will provide better effects to reduce tissue damage and lethality (3). clec2 is highly expressed by platelets and megakaryocytes and low level expression of this syk-coupled clr has also been reported in mouse dendritic cells (32) and neutrophils (33) . human clec2 is a 229 amino acid type ii transmembrane protein containing a putative n-linked glycosylation site in the extracellular domain. in contrast to clec5a, clec2 contains a single yxxl motif (hemitam) in its intracellular domain, and dimerization of clec2 upon ligand engagement, activates platelets via syk and slp-76 (25, 34, 35) . the o-linked glycoprotein podoplanin has been identified as the endogenous ligand of clec2. podoplanin is a mucin-type transmembrane protein expressed in type i alveolar cells, kidney podocytes, and lymphatic endothelial cells (36) . activation of clec2 by podoplanin during embryonic development is required for blood/lymphatic vessel separation (37, 38) , but although clec2 is involved in thrombus stabilization, its absence does not significantly increase bleeding tendency (39) . clec2 also interacts with aggretin (rhodocytin) (40), a snake venom protein isolated from calloselasma rhodostoma (41) that induces platelet activation and aggregation via its binding to clec2 (40) . in addition to protein ligands, clec2 also binds to fucoidans (42) , which are sulfated polysaccharides mainly comprised of fucose, but also containing other monosaccharides and uronic acid (43) . clec2 have been shown to capture human immunodeficiency virus (hiv) via dc-sign and clec-2, thereby facilitate viral dissemination in infected patients (44) . moreover, clec2 is responsible for immunothrombosis in the context of bacterial infections (45, 46) . it has been reported that the absence of clec2 increases clinical severity in a cecal ligation and puncture (clp) model of sepsis following injection of bacterial lipopolysaccharides (47) , and deletion of clec2 in this model exacerbates cytokine storm and inhibits inflammatory macrophage recruitment to the infected peritoneum, resulting in increased bacterial load and organ injury (47) . deletion of clec2 also enhances the severity of brain inflammation in the mouse experimental autoimmune encephalomyelitis (eae) model, where there is evidence that the podoplanin/clec2 axis promotes resolution of inflammatory reactions in autoimmunity (48, 49) . recently, clec2 was shown to be a novel pattern recognition receptor for dv, where dv infection activates platelets to express cd62p, cd63 and to release extracellular vesicles (evs), including microvesicles (mvs) and exosomes (exos) (50) . we have shown that dv binds to clec2 on platelets, promoting the release of evs, including exos (dv-exos) and mvs (dv-mvs). while exos and mvs from resting platelets do not have any activity, dv-exos and dv-mvs are potent "endogenous danger signals" which trigger the activation of clec5a and tlr2, respectively, to promote netosis and production of proinflammatory cytokines in neutrophils and macrophages. while blockade of clec5a offers ∼30% protection rate, simultaneous blockade of clec5a and tlr2 further increase mice survival rate up to 90%. these observations indicate that clec5a/tlr2 is not critical dv-induced pathogenesis, but also plays important roles in platelet-leukocyte interactions via recognizing platelets-derived exos and mvs. thus, targeting clec5a/tlr2 have the potential to underpin novel strategies for treating acute viral infections. it has become clear that pathogens carry multiple pamps and activate immune cells via multiple receptors. for example, dv initiates inflammatory responses through activation of both clec5a and tlr7 associated pathways, while listeria monocytogenes and staphylococcus aureus activate nalp3 (nacht, lrr and pyd domains-containing protein), nlr family nlrc4 (card domain-containing protein 4) and aim2 (absent in melanoma 2) inflammasomes and proinflammatory cytokine release via clec5a and tlr2 (51) . clec2 has been shown to form ligand-dependent multimers with other platelet receptors to activate inflammatory signaling pathways (52) . viral glycans contain multiple terminal sugars, including mannose, fucose, sialic acids with or without sulfation; therefore, it is not surprising that multiple lectin receptors on host cells colocalize during engagement with these pamps. it has been demonstrated that dv interacts with clec5a, dc-sign (dendritic dell-specific intercellular adhesion molecule-3-grabbing non-integrin), dc-signr (1), and mannose receptor (mr) (24) . although dv binds with much lower affinity to clec5a than to dc-sign or dc-signr, only clec5a has been clearly shown to mediate downstream signaling pathways after engagement with dv. dv-induced activation of clec5a is dependent on dc-sign and mr (53) and imaging analysis has revealed that engagement of dv with myeloid cells triggers colocalization of clec5a and mr/dc-sign to form a hetero-multivalent complex (53) . the lectin heterocomplex would facilitate the formation of multivalence interactions between viral glycans and ctype lectins with distinct glycan-binding affinity to enable signaling via clec5a. even though the interaction between dv and clec2 is weak (54) , dv also binds platelets via dc-sign (55) . thus, dv may also trigger the formation of dv-clec2-dc-sign complex to enable signaling via clec2 (figure 1) . figure 1 | heterocomplexes of c-type lectins in myeloid cells and platelets. dengue virus (dv) and influenza virus (h5n1) are captured by the high affinity receptors dc-sign and mannose receptor (mr). the formation of heterocomplexes enables syk-mediated signaling via low affinity clec5a to activate the nalp3 inflammasome and induce the formation of carma1/bcl10/malt1, thereby upregulating proinflammatory cytokine production (right). dv and h5n1 are captured by the high affinity receptor dc-sign and receptor multimerization leads to activation of the low affinity receptor clec2 to stimulate production of extracellular vesicles (evs). aggretin, a high affinity ligand for clec2, induces ev release and platelet aggregation independently of dc-sign (left). in addition to the formation of heterocomplexes between c-type lectins, we have shown that engagement of l monocytogenes with macrophages and neutrophils induces the formation of clec5a/tlr2 heterocomplexes. while tlr2 binds lipoteichoic acid from the l. monocytogenes cell wall, clec5a binds to glcnac-murnac disaccharides within the backbone of bacterial wall teichoic acids (51) . coactivation of clec5a and tlr2 by l. monocytogenes stimulates the production of inflammasomes and induces net formation, proinflammatory cytokine expression and γδ cd4 + th17 cells differentiation (51) . in contrast to viral infection, clec5a-deficient mice are highly susceptible to infections with bacteria including l. monocytogenes and s. aureus (51) . whilst the host immune system is beneficial in promoting the clearance of many pathogens it can also have detrimental effects in some contexts (figure 2) . one of the most cited examples is the high susceptibility to bacterial infection after acute viral infections, for example with influenza virus (56) . potential explanations for this are the association of net formation with lung injury (57) , and the central role of inflammasome activation in viral pathology (58) ; blockade of clec5a/tlr2 attenuates net formation and inflammasome activation and is thus beneficial to the host during acute viral infection (50) . moreover, excessive nets cause damages of epithelium and vascular endothelium during bacterial infections, promotes vascular occlusion and tumor cell metastasis, and enhances autoantibody production (59) . because clec5a is also responsible for cona-induced shock syndrome (60) and synovial inflammation in collagen-induced arthritis (61), thus bloackade of clec5a/tlr2 may reduce bacteria-induced systemic permeability change, inhibit tumor metastasis, and attenuate tissue damages during aseptic inflammation. following our identification of clec5a as the pathogenic host factor in flaviviruses, we further found that clec2 and tlr2 are pathogenic receptors in dv, jev, and h5n1 infections. these viruses are captured by high affinity receptors (dc-sign and mr) to induce inflammatory cytokine release via activation of clec5a and clec2. we have also demonstrated the complex interactions between platelets and leukocytes during viral infection. these studies have demonstrated the molecular mechanisms underlying virus-induced cytokine storms and lethality and revealed a new direction for the development of treatments for acute viral infections. moreover, excessive nets contribute to the pathogenesis of autoimmune diseases (62) , where the risk of developing autoimmune diseases is increased in patients post dv infection (63) . thus, blockade of clec5a and tlr2 using a bi-specific anti-clec5a/tlr2 mab has the potential to reduce the risk of autoimmunity post-dv infection, and have therapeutic effects in autoimmune diseases by limiting excessive net formation and persistent inflammasome activation. there has been a tendency to investigate host responses to pathogen from the perspective of single pamp-receptor interactions with a focus on endosomal tlrs and cytosolic sensors that recognize highly conserved dna and rna structures. however, the complex structures of viruses contain a diversity of pamps that stimulate multiple receptors on host immune cells and modulate innate immune responses to individual viruses. furthermore, viruses frequently infect multiple cell linages, including both immune and nonimmune cells, and induce cell-cell interactions during acute viral infections. our previous studies demonstrated virusmediated activation of platelets to enhance net formation, proinflammatory cytokine release, and inflammasome activation via membrane receptors that include clec5a. this supported the idea that host cells not only recognize conserved ribonucleic acid structures to stimulate production of interferons, but also recognize viral membrane components and extracellular vesicles to further promote multiple innate immune pathways. the ability of dv-evs to enhance inflammatory reactions and netosis suggest that activated evs can be regarded as novel "endogenous danger signals" or "damage-associated molecular patterns" which activate innate immunity via clec5a and tlr2. in this regards, clec5a/tlr2 heterocomplex may play important roles in the pathogenesis of aseptic inflammations. this argument is further supported by the observation that clec5a + cells are responsible for collageninduced autoimmune arthritis (61) and cona-induced lethal shock syndrome (60) . it would be interesting to test whether ev-clec5a/tlr2 interactions contribute to the pathogenesis of aseptic inflammation and autoimmune diseases in the future. in addition to mediating acute infections, some viruses interact with innate immune receptors to establish chronic infections. in this context innate immune receptors act as "pathogenic host factors" that transduce inhibitory signals after binding virus. human hepatitis b virus (hbv) frequently causes chronic and persistent infections following initial inflammatory responses. hbv is an enveloped dna virus that infects hepatocytes and causes persistent infection due to the presence of covalently closed circular (ccc)dna, which becomes inserted into the host genome in hepatocytes. the hbv envelope contains large, middle, and small hbv surface antigens (hbsags); the large hbsag is present on virions, known as dane particles, that contain the viral genome and are able to infect and replicate after entering hepatocytes, but this is not the case for the middle and small hbsags. interestingly, the middle and small hbsags outnumber the large hbsag and it has been speculated that these un-infectious hbsags may suppress host immunity and inhibit the production of anti-hbsag ab. identification of "inhibitory" innate immunity receptor(s) for hbv may provide an answer the un-resolved questions surrounding chronic persistent hbv infection and further our understanding of the roles of viral membrane components in the complex pathogenesis of viral infections. clec5a is critical for dengue-virus-induced lethal disease distinct regulation of dengue virus-induced inflammasome activation in human macrophage subsets clec5a-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza virus pathogenicity in vivo induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? ebola virus: the role of macrophages and dendritic cells in the pathogenesis of ebola hemorrhagic fever platelet-neutrophilinteractions: linking hemostasis and inflammation thrombosis as an intravascular effector of innate immunity the role of platelets in the recruitment of leukocytes during vascular disease toll-like receptors as potential therapeutic targets for multiple diseases the critical role of toll-like receptors-from microbial recognition to autoimmunity: a comprehensive review toll-like receptor co-receptors as master regulators of the immune response induction of type i ifns by intracellular dna-sensing pathways sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity protect this house: cytosolic sensing of viruses sensing and responding to cytosolic viruses invasions: an orchestra of kaleidoscopic ubiquitinations cytosolic sensing of viruses chapter 31: c-type lectins c-type lectins in immunity and homeostasis dc-sign (cd209) mediates dengue virus infection of human dendritic cells dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals lectin switching during dengue virus infection dc-sign (cd209) promoter−336 a/g polymorphism is associated with dengue hemorrhagic fever and correlated to dc-sign expression and immune augmentation transmission-blocking antibodies against mosquito c-type lectins for dengue prevention the mannose receptor mediates dengue virus infection of macrophages the c-type lectin receptors clec-2 and dectin-1, but not dc-sign, signal via a novel yxxl-dependent signaling cascade complement-mediated neutralization of dengue virus requires mannose-binding lectin crystallization and x-ray diffraction analysis of human clec5a (mdl-1), a dengue virus receptor clec5a is critical for dengue virus-induced inflammasome activation in human macrophages clec5a is critical for dengue virus-induced osteoclast activation and bone homeostasis clec5a regulates japanese encephalitis virus-induced neuroinflammation and lethality the expression of mouse clec-2 on leucocyte subsets varies according to their anatomical location and inflammatory state clec-2 is a phagocytic activation receptor expressed on murine peripheral blood neutrophils the platelet receptor clec-2 is active as a dimer clec-2 activates syk through dimerization podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by clec-2 essential in vivo roles of the c-type lectin receptor clec-2: embryonic/neonatal lethality of clec-2-deficient mice by blood/lymphatic misconnections and impaired thrombus formation of clec-2-deficient platelets clec-2 and syk in the megakaryocytic/platelet lineage are essential for development novel platelet activation receptor clec-2: from discovery to prospects a novel syk-dependent mechanism of platelet activation by the c-type lectin receptor clec-2 aggretin, a novel platelet-aggregation inducer from snake (calloselasma rhodostoma) venom, activates phospholipase c by acting as a glycoprotein ia/iia agonist fucoidan is a novel platelet agonist for the c-type lectin-like receptor 2 (clec-2) fucoidan structure and activity in relation to anti-cancer mechanisms dc-sign and clec-2 mediate human immunodeficiency virus type 1 capture by platelets thrombomodulation via clec-2 targeting inflammation drives thrombosis after salmonella infection via clec-2 on platelets the podoplanin-clec-2 axis inhibits inflammation in sepsis podoplanin is a negative regulator of th17 inflammation pdgf upregulates clec-2 to induce t regulatory cells extracellular vesicles from clec2-activated platelets enhance dengue virus-induced lethality via clec5a/tlr2 robust, sensitive and facile method for detection of f-, cn-and ac-anions platelet receptors activated via mulitmerization: glycoprotein vi, gpib-ix-v, and clec-2 dengue virus infection is through a cooperative interaction between a mannose receptor and clec5a on macrophage as a multivalent hetero-complex structural flexibility of the macrophage dengue virus receptor clec5a: implications for ligand binding and signaling dengue virus binding and replication by platelets influenza virusinduced glucocorticoids compromise innate host defense against a secondary bacterial infection high level of neutrophil extracellular traps correlates with poor prognosis of severe influenza a infection delayed oseltamivir plus sirolimus treatment attenuates h1n1 virus-induced severe lung injury correlated with repressed nlrp3 inflammasome activation and inflammatory cell infiltration neutrophil extracellular traps in immunity and disease activation of mdl-1 (clec5a) on immature myeloid cells triggers lethal shock in mice myeloid dap12-associating lectin (mdl)-1 regulates synovial inflammation and bone erosion associated with autoimmune arthritis neutrophil extracellular traps (nets) in autoimmune diseases: a comprehensive review increased risk of autoimmune diseases in dengue patients: a population-based cohort study the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 sung and hsieh. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-259131-36udb7uc authors: hunegnaw, ruth; mushtaq, zuena; enyindah-asonye, gospel; hoang, tanya; robert-guroff, marjorie title: alveolar macrophage dysfunction and increased pd-1 expression during chronic siv infection of rhesus macaques date: 2019-07-03 journal: front immunol doi: 10.3389/fimmu.2019.01537 sha: doc_id: 259131 cord_uid: 36udb7uc hiv infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. alveolar macrophages (ams) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following hiv infection. here, using the siv rhesus macaque model, we document the effect of siv infection on the phenotypic and functional properties of ams. following infection with siv(mac251), ams in bronchoalveolar lavage (bal) sampled over 2to 20-weeks post-infection (wpi) were compared to those in bal samples from naïve macaques. am expression of proinflammatory cytokines tnf-α, il-6, il-1β, and chemokine rantes drastically increased 2-wpi compared to ams of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. phagocytic activity of ams 2-and 4-wpi was elevated compared to ams of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). by 20-wpi the ability of ams from chronically infected animals to perform siv-specific antibody-dependent phagocytosis (adp) was also diminished (p = 0.028). acute siv infection was associated with increased fcγriii expression which subsequently declined with disease progression. frequency of fcγriii(+) ams showed a strong trend toward correlation with siv-specific adp, and at 2-wpi fcγriii expression negatively correlated with viral load (r = −0.6819; p = 0.0013), suggesting a contribution to viremia control. importantly, pd-1 was found to be expressed on ams and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on t-cells, pd-1 expression on ams may also be associated with disease progression. further, ams predominantly expressed pd-l2, which remained consistent over the course of infection. pd-1 blockade enhanced siv-specific adp by ams from chronic infection indicating that the pd-1/pd-l2 pathway may modulate functional activity of ams at that stage. these findings provide new insight into the dynamics of siv infection leading to am dysfunction and alteration of pulmonary innate immunity. our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in hiv-infected individuals. hiv infected individuals have been shown to be pre-disposed to pulmonary infections even while receiving anti-retroviral therapy. alveolar macrophages (ams) play a critical role in lung innate immunity, but contradictory results have been reported regarding their functionality following hiv infection. here, using the siv rhesus macaque model, we document the effect of siv infection on the phenotypic and functional properties of ams. following infection with siv mac251 , ams in bronchoalveolar lavage (bal) sampled over 2-to 20-weeks post-infection (wpi) were compared to those in bal samples from naïve macaques. am expression of proinflammatory cytokines tnf-α, il-6, il-1β, and chemokine rantes drastically increased 2-wpi compared to ams of naïve macaques (p < 0.0001 for all), but dropped significantly with progression to chronic infection. phagocytic activity of ams 2-and 4-wpi was elevated compared to ams of naive animals (p = 0.0005, p = 0.0004, respectively) but significantly decreased by 12-wpi (p = 0.0022, p = 0.0019, respectively). by 20-wpi the ability of ams from chronically infected animals to perform siv-specific antibody-dependent phagocytosis (adp) was also diminished (p = 0.028). acute siv infection was associated with increased fcγriii expression which subsequently declined with disease progression. frequency of fcγriii + ams showed a strong trend toward correlation with siv-specific adp, and at 2-wpi fcγriii expression negatively correlated with viral load (r = −0.6819; p = 0.0013), suggesting a contribution to viremia control. importantly, pd-1 was found to be expressed on ams and showed a strong trend toward correlation with plasma viral load (r = 0.8266; p = 0.058), indicating that similar to over-expression on t-cells, pd-1 expression on ams may also be associated with disease progression. further, ams predominantly expressed pd-l2, which remained consistent over the course of infection. pd-1 blockade enhanced siv-specific adp by ams from chronic infection indicating that the pd-1/pd-l2 pathway may modulate functional activity of ams at that stage. these findings provide new insight into the dynamics of siv infection leading to am dysfunction and alteration of pulmonary innate immunity. our results suggest new pathways to exploit in developing therapies targeting pulmonary disease susceptibility in hiv-infected individuals. keywords: alveolar macrophage, siv, rhesus macaque, antibody-dependent phagocytosis, pd-1, fcγriii, bronchoalveolar lavage introduction macrophages are found in tissues of infected individuals and play a continuous role in pathogenesis. although earlier studies reported that macrophages were the major cell type initially infected by hiv (1), more recent studies have shown that cd4+ t cells are preferentially targeted by hiv/siv at the site of transmission (2, 3). however, macrophages still serve as targets for the virus in vivo (4) . they can sustain viral replication, disseminate virus, and serve as a viral reservoir post-infection (5) (6) (7) . cells of the macrophage/monocyte lineage vary greatly in phenotype, longevity, and in phagocytic, immunoregulatory, and secretory properties (8) (9) (10) (11) . macrophages are categorized as classically (m1) or alternatively (m2) activated based on surface markers and functional role (12, 13) . m1 macrophages mediate inflammatory responses against pathogens while m2 macrophages have anti-inflammatory properties, promoting tissue repair and remodeling (14) . alveolar macrophages (am) in the lung uniquely express both m1 and m2 phenotypic markers, indicating ability to quickly respond to pathogens but also prevent immune activation in response to harmless antigens that enter the alveolar lumen (15) . am express cd4 and chemokine receptors making them vulnerable to hiv infection (16) . however, macrophages may be poorly susceptible to hiv induced cytopathic effects. minimal consequences of hiv infection on the macrophage transcriptome were observed (17) ; in contrast expression of hiv nef and gp120 envelope induced macrophage activation (18, 19) . indirect activation can also occur by exposure of uninfected macrophages to viral gene products or cytokines from other infected cells (20) . am are important lung phagocytes (21) , yet am obtained from hiv-infected individuals have shown contradictory results regarding the impact of viral infection on phagocytosis. some studies have shown impaired phagocytosis of opportunistic pathogens by am (22) (23) (24) (25) (26) ; others have reported no change in am phagocytic activity during infection (27, 28) . such variations in outcome may result from differences in length of infection or reliance on the use of monocyte derived macrophages (mdm) and infected cell lines which may not ideally represent clinical situations. macrophages can utilize fcγ receptors to internalize antibody-opsonized virions or infected cells, potentially leading to antibody-mediated clearance of infectious material (29) . antibody-dependent phagocytosis (adp) by am contributes to protection against viral infections such as influenza (30) , west nile (31), adenovirus (32) , and sars coronavirus (33) . however, adp-mediated protection by macrophages against hiv infection has not been observed. here we investigated the dynamics of siv-related changes in am activity and function by sampling bronchoalveolar lavage (bal) from siv infected rhesus macaques during acute and chronic infection. the am response to siv infection consisted of phenotypic changes and alterations in proinflammatory responses, ability to respond to gp120 antigen, and phagocytic activity. fcγriiib expression on am was linked to siv-specific adp and viral control during acute infection. novel results showed increased expression of programmed cell-death-1 (pd-1) on am from chronically infected macaques and positive correlations between pd-1 expressing ams and siv viremia. we believe this is the first report of pd-1 expression on ams of siv infected macaques. our results suggest associations between pd-1 expression, macrophage dysfunction, and lack of viremia control. forty-nine female indian rhesus macaques used in this study were housed in the nci animal facility, bethesda, md, under protocol vb-020. the nci facility is accredited by the association for assessment and accreditation of laboratory animal care international, and its animal care and use committee approved all animal experiments prior to study initiation. standard practices followed recommendations made in the guide for the care and use of laboratory animals of the united states, national institutes of health, and the weatherall report. thirty macaques were used as naïve controls and 19 macaques were challenged weekly intravaginally using a repeated low-dose of siv mac251 (800 tcid 50 ). infection was confirmed for 14 of the macaques with plasma viral loads of ≥50 siv rna copies/ml as assessed by droplet digital pcr (chung et al., manuscript in preparation). the remaining five macaques were infected intrarectally with a single high dose of siv mac251 (4000 tcid 50 ). out of the 19 infected macaques, 9 were euthanized after 2 wpi and the remaining 10 infected macaques were monitored longitudinally for up to 20 wpi. bal samples were obtained using standard techniques (34) . briefly, rhesus macaques were anesthetized, and an endotracheal tube was inserted through which sterile saline solution (10 ml/kg) was instilled. suction was applied to recover the instilled fluid and the lung lavage was collected in sterile conical tubes. cells were pelleted by centrifuging at 1600 rpm for 10 min at 4 • c and washed in 25 ml cold pbs. centrifugation was repeated, and cells were resuspended for counting. cells showed ∼90% viability as determined by trypan blue staining. bal cells were enriched for ams by depleting epcam + , cd2 + , and cd20 + cells. the following reagents were used: rabbit polyclonal anti-epcam (n3c3) (genetex, irvine, ca); cd2 and cd20 microbeads for non-human primate (miltenyi biotec, auburn, ca). depletion was performed using macs cell separation technology according to the manufacturer's instructions (miltenyi biotec, auburn, ca). enriched ams were stimulated for 6 h in 500 µl r-10 media (rpmi + 10% fbs + 5% pen-strep + 5% anti-anti) containing purified native hiv-1 envelope gp120 (advanced bioscience laboratories, inc., rockville, md) at a concentration of 200 nm or lps (thermofisher scientific, waltham, ma) at 500 ng/ml concentration. for experiments assessing cytokine production by pd-1 + ams, cells were stimulated with 10 µg/ml lps in the presence of golgistop (1 µl, bd biosciences) for 18 h prior to intracellular staining. adp activity was measured as previously described (35) . siv mac251 gp120 was biotinylated with a biotin-xx microscale protein labeling kit (life technologies, grand island, ny) and incubated with a 10-fold dilution of 1 µg avidin coated sky blue fluorescent beads (0.8 µm diameter; spherotech, lake forest, il) overnight at 4 • c. enriched ams were plated in a u-bottom 96 well plate at 40,000 cells/well and undiluted bal-f was added. to determine phagocytic activity of am independent of siv specific antibody, bal-f from naïve macaques was used. for siv-specific adp, autologous bal-f from infected macaques was used. the bead-gp120 mixture was brought to a final 50-fold dilution in r-10 media and 50 µl was added to the cells and incubated for 3 h at 37 • c. for pd-1 blocking experiments, bal samples collected from macaques at 20 wpi were enriched for ams as described above and incubated with either 10 µg/ml ultraleaf purified anti-human pd-1 antibody (eh12.2h7, biolegend) or 10 µg/ml migg1 isotype control (sigma-aldrich). cells incubated with naïve bal-f were used for normalization. in all cases, after 3 h of incubation, 70 µl of 2% paraformaldehyde was added for fixation. fluorescent bead uptake was assessed using a bd biosciences lsrii flow cytometer. bead uptake specifically by am was made possible by focusing on cells that were autofluorescent in the fitc channel. the phagocytic score of each sample was calculated by multiplying the frequency of bead-positive cells by the degree of phagocytosis measured as mean fluorescence intensity (mfi) and dividing by 10 4 . values were normalized to background values (cells and beads with either pbs or naïve bal-f) by dividing the phagocytic score of the test sample by the phagocytic score of the background samples. mouse anti-non-human primate or anti-human fluorochromeconjugated monoclonal abs (except where specified otherwise) known to cross-react with rhesus macaque antigens were used in this study. the following antibodies were used for surface staining: bv711 anti-cd4 (l200), bv786 anti-cd45 (d058-1283), buv395 anti-cd3 (sp34-2), buv496 anti-cd16 (3g8), cells were then washed with bd permwash, and incubated with intracellular staining antibodies for 25 min at rt. finally, cells were washed with pbs and staining data were acquired using the 5 laser bd facsymphony (bd biosciences, san jose, ca). fluorescence minus one (fmo) and isotype controls were used to confirm the phenotype and cytokine expressing population of ams ( figure s1 ). data analyses were performed with flowjo (version 10.5, treestar, inc ashland, or) software. antibodies in bal-f were measured as follows: wells of greiner high-binding ½ area 96-well plates were coated overnight at 4 • c with 100 ng/well of siv mac251 gp120 (for determining gp120specific igg) or 50 ng/well of anti-rhesus igg (alpha diagnostics) (for determining total igg) in sodium bicarbonate buffer (ph 9.6) (sigma-aldrich, st. louis, mo). wells were blocked with 200 µl of 1% bsa diluent/blocking solution (kpl) in distilled water for 2 h at rt. bal-f (50 µl) was added and incubated for 1 h at 37 • c. env-specific igg derived from purified serum igg obtained from siv mac251 -infected macaques and quantified as previously described (36) was used to generate a standard curve for env-specific igg. purified rhesus igg (non-human primate reagent resource) was used to generate a standard curve for total igg. plates were washed 5 times with 1x wash solution (kpl). horseradish peroxidase-labeled goat anti-monkey igg (50 µl at a 1:10,000 dilution; alpha diagnostics) was added, and plates were incubated for 1 h at 37 • c. after washing as described above, 50 µl of 3,3 ′ ,5,5 ′ -tetramethylbenzidine (tmb) peroxidase substrate (kpl) was added for 10-20 min at rt. color development was stopped with 50 µl 1 m phosphoric acid (sigma), and plates were read at 450 nm by using a biotek plate reader. siv gp120-specific antibody levels were expressed as ng gp120-specific igg/µg total igg. statistical analysis was performed using one-way anova or 2way anova with the tukey multiple comparisons test as indicated in the figure legends. the wilcoxon matched-pairs signed rank test was also used where indicated. correlation analyses were performed by non-parametric spearman correlations. statistics were generated using graphpad prism. rhesus macaques were infected with siv mac251 and bal was collected at 2, 4, 8, 12, and 20-wpi. nineteen macaques were sampled at 2-wpi and 10 for the subsequent time points. bal samples were also obtained from 30 naïve macaques. ams from bal were characterized by flow cytometry as hla-dr hi , cd11b int , and cd163 + cd206 + (37) (figure 1a) . the mean frequency of ams was >80% of leukocytes found in the lavage samples, consistent with other reports on bal specimens from naive rhesus macaques (37) . no significant change in the frequency of ams was observed over the 20 weeks of infection ( figure 1b) . expression of cd11c and cd14 has been shown to identify am phenotypes with changes in expression occurring during the fibrotic stage of lung disease (38) here, no significant changes in am expression of cd11c or cd14 over the time period studied were observed (figures 1c,d) . we also did not observe changes in the percentage of ams expressing the costimulatory molecules, cd80 and cd86, indicating that any inflammatory responses recorded may not be associated with the expansion of these cell subsets (figures 1e,f) . macrophages secrete proinflammatory cytokines and chemokines in response to hiv infection (39, 40) . however, a clear dynamic of the cytokine and chemokine response by macrophages over the course of hiv/siv infection has not been shown. here we characterized cytokine levels in am collected from siv infected rhesus macaques between 2 and 20-wpi and compared them to levels in ams from naïve macaques. a strong proinflammatory response was seen during the acute phase of infection. expression of tnf-α, il-6, and il-1β in ams was significantly increased by 2-wpi (p < 0.0001 for all; figures 2a-c) . by 4-wpi expression levels were significantly lower. cytokine expression remained low until the 20-wpi time point except for il-6 expression, which increased significantly by 12-wpi compared to 4-wpi ( figure 2b) . the il-6 expression level at 20-wpi was not significantly higher than levels seen in naïve populations. similarly, a higher level of the chemokine rantes was found in ams from macaques at 2-wpi ( figure 2e) . expression decreased to levels comparable to naïve samples by 4-wpi and remained low until the 20-wpi time point (figure 2e ). mip-1α expression did not change over the course of infection ( figure 2d ). overall, we observed a transient increase in the proinflammatory cytokine responses, which decreased to levels comparable to those of naïve macaques during the chronic phase of infection. to assess whether ams were leaning toward an anti-inflammatory role during chronic infection, we evaluated the intracellular expression of il-10 by ams ( figure 2f ). ams play an important role in maintaining an anti-inflammatory environment in the lung (21, 41) . indeed, il-10 expressing ams were detected in naïve animals and the frequency of il-10+ cells was significantly higher compared to that of acutely infected animals ( figure 2f) . interestingly, the percentage of il-10 expressing ams further decreased significantly as infection progressed into the chronic stage ( figure 2f) . thus, siv infection was associated with a significant spike in the am proinflammatory response by 2-wpi, which waned in chronic infection. in addition, the low proinflammatory cytokine response in chronic infection was not associated with an increase in il-10-expressing ams. to investigate am activation, bal cells obtained from naïve and acute and chronically infected macaques at weeks 2, 4, 8, 12, and 20 wpi, were incubated with native gp120 from r5 tropic siv or lps for 6 h, and intracellular expression of mip-1β and il-6 was assessed (figures 3a,b) . both gp120 and lps induced comparable levels of mip-1β and il-6 in ams from naive and acutely-infected animals (figures 3a,b) . however, in chronically infected macaques gp120 did not induce intracellular il-6 or mip-1β expression in ams (figures 3a,b) , and lps did not stimulate expression of either factor to the level induced in naïve animals suggesting a dysfunctional state of the ams (figures 3c,d) . to assess whether ams contribute to clearance of siv, we evaluated their phagocytic activity and ability to perform adp over the course of siv infection using bal samples from 5 naïve animals and 5 siv-infected macaques sampled between 2 and 20-wpi. while the bal samples contained a high percentage of ams (figure 1b) , the cells were further enriched for ams using macs by depleting cd2, cd20, and epcam positive cells ( figure 4a) . to assess phagocytic capacity independent of siv antibody, enriched cells were incubated with siv mac251 gp120-coated fluorescent beads and bal-f from naïve macaques prior to quantification of bead uptake. a significant increase in phagocytic activity was detected during the acute phase, 2 and 4-wpi (figure 4b) . at 8-wpi, phagocytosis decreased and was comparable to naïve cells. activity further decreased significantly at the 12 and 20-wpi time points compared to the acute and naïve time points (figure 4b ). prior to evaluating adp by ams, we assessed siv gp120specific igg in bal-f ( figure 4c) . specific igg was detected at 8-wpi with levels maintained or continuing to rise until 20-wpi. to determine siv-specific adp, ams from naïve macaques or sampled over the course of siv infection were incubated with antigen-coated beads along with autologous bal-f. phagocytosis scores were normalized against the score obtained using naïve bal-f to highlight phagocytic activity associated with the presence of siv-specific antibody ( figure 4d) . significantly higher phagocytic scores were detected at 12-wpi compared to those at naïve and 4-wpi time points, indicating that ams could perform siv-specific adp ( figure 4d) . to further assess adp by ams in chronic infection, phagocytic scores at chronic time points (figure 4d ) were normalized against siv-specific antibody levels ( figure 4c) . results showed that in the presence of comparable antibody levels, ams mediated adp activity at 12-wpi. however, this activity was significantly compromised by 20 week pi ( figure 4e) . thus, am are capable of gp120specific adp for a limited time period during chronic infection. this diminished functional capacity of ams in the chronic stage further highlights the dysfunction observed upon lps and gp120 stimulation (figure 3) . three classes of fcγr are constitutively expressed on ams with an active role in protecting against pathogens in the airway: high-affinity fcγri (cd64) and low-affinity fcγrii (cd32) and fcγriii (cd16) (42) . fcγriii occurs in two isoforms: fcγriiia (expressed mainly on nk cells and macrophages) and fcγriiib (expressed on neutrophils) (43) (44) (45) . infected mdms have shown compromised expression of the γ signaling chain of fcγriiia, possibly leading to altered phagocytic activity (46) . monocytes from chronically infected individuals have also shown reduced expression of fcγriiia and diminished phagocytic activity (47) . however, thus far no correlations have been found between adp and hiv/siv viral load, cd4 count or fcγr expression. given the unique features of ams compared to other tissue macrophages and monocytes, and the importance of phagocytic function in the lung, we investigated whether there was altered fcγr expression on ams over the course of siv infection and any association with adp. bal from naive macaques contained over 80% of fcγri + and fcγrii + ams (figures 5b,c) . both the frequencies and mfi of fcγrii and fcγri remained high and unchanged over the course of infection (figures 5b,c,e,f) . in contrast, the mean frequency of fcγriii + cells was 30% in naïve macaques. at 2-wpi there was a significant increase in fcγriii + am frequency but levels significantly decreased by 8-wpi (figure 5a) . the low frequency of fcγriii + ams was maintained until 20-wpi. a similar increase in am fcγriii mfi was not observed in the acute phase of infection compared to naïve animals ( figure 5d ). however, expression levels were significantly reduced at the 8 and 20-wpi time points compared to levels at 2-wpi ( figure 5d ) in concert with the decreased frequencies observed. the siv-specific adp observed at 12-wpi ( figure 4e ) exhibited a strong trend toward positive correlation with the percentage of fcγriii + ams at the same time point ( figure 5g) . further, the fcγriii mfi at 2-wpi ( figure 5d ) negatively correlated significantly with acute plasma viral load ( figure 5h) . these data suggest that fcγriii plays an important role in siv-specific adp and that its expression is associated with early viral load control. to characterize the dysfunction observed in ams from chronically infected macaques, we looked for parallels with t cell exhaustion. since the initial description of t cell exhaustion in chronic viral infections (48, 49) , common phenotypic and functional properties have been attributed to a dysfunctional state regardless of the type of pathogen. high and prolonged expression of inhibitory receptors are a key feature of t cell exhaustion in chronic viral infections (50) . in particular, overexpression of the pd-1 receptor on cd8 + t-cells plays a major role in t cell dysfunction associated with hiv infection (51) (52) (53) . here, we identified pd-1 + ams in rhesus macaque bal and assessed changes in its expression on ams from 6 macaques over the course of siv infection (figure 6a) . when the frequency of pd-1 + ams was assessed in naive macaques and compared to frequencies in siv infected animals, five out of six siv + macaques showed an increase in frequency (figure 6b) . the pd-1 + am frequency at 20-wpi was significantly different from both naïve and 2-wpi time points: p = 0.0419 and p = 0.0294, respectively. ams at 20-wpi also displayed a significant increase in pd-1 mfi signifying increased expression of the receptor ( figure 6c) . furthermore, the percentage of pd-1 + ams correlated positively with viral load at 20-wpi ( figure 6d) . we also observed a strong trend toward correlation between pd-1 mfi and viral load at 20-wpi (figure 6e) , indicating an association between pd-1 expression in ams and siv disease progression. pd-1 has two well-known ligands via which inhibitory effects have been reported to occur: programmed death -ligand 1 (pd-l1) and programmed death ligand 2 (pd-l2). while pd-l1 has been shown to be expressed broadly in both hematopoeitic and non-hematopoeitic cells, a more restricted expression pattern has been documented for pd-l2, namely antigen presenting cells (apc) (54) . in order to identify a potential role for the pd-1 pathway on am function, we looked for expression of these ligands on ams after siv infection. ams from naïve macaques predominantly expressed high levels of pd-l2 (figure 7b) . this expression decreased modestly, but not significantly, after siv infection and remained consistent thereafter. on the other hand, we observed minimal expression (∼10 fold lower than that of pd-l2) of pd-l1 on ams from naïve macaques ( figure 7a) . further, pd-l1 expression significantly decreased over the course of siv infection. these results indicate that am dysfunction is more likely associated with increased pd-1 expression rather than changes in the expression of its ligands and may occur through the pd-1/pd-l2 pathway. to identify whether pd-1 blockade can rescue am phagocytosis, we examined the phagocytic activity of ams from 7 chronically infected macaques in the presence of pd-1 blocking antibody or mouse igg as control. interestingly, we found higher phagocytic scores in the pd-1 blockade group in 6 out of the 7 animals (p = 0.0312), indicating that pd-1 expression is likely mechanistically linked to impairment of phagocytic activity (figure 7c) . we further assessed cytokine expression of pd-1 expressing ams. we stimulated ams from chronically infected macaques with 10 µg/ml lps and compared cytokine expression between pd-1 + and pd-1 − populations. although not significant, we found lower expression of tnf-α on pd-1 expressing cells compared to the pd-1 − population ( figure 7d) . comparisons with additional cytokines and chemokines (il-6, il-1β and rantes) are not reported here as expression of these cytokines could not be detected in the ams from the infected macaques that were tested. taken together, these data strongly suggest that pd-1 expression on ams during siv infection may be a marker of dysfunction. our results show that in the siv macaque model, the pro-inflammatory response of ams to siv infection peaks in the acute phase of infection and is diminished in the chronic phase as observed following stimulation with lps or gp120. we report that ams from siv infected macaques can perform adp early in infection, but this activity diminishes in the chronic phase. in novel findings, we report an elevated percentage of pd-1 + ams in chronically infected macaques, which positively correlated with viral load. during the same period diminished inflammatory responses and adp were observed. the percentage of ams stayed consistent over the course of siv infection (figure 1b) . viral infections can lead to activation of macrophages and subsequent release of factors that recruit additional cells of the innate and adaptive immune system (55, 56) . the lack of detection of macrophage recruitment to the lung may have resulted from a concurrent lymphocyte recruitment, making the relative number of macrophages appear consistent. indeed, a higher percentage of lymphocytes in bal of hivinfected patients has been reported (57) . here, estimating actual am numbers by multiplying cell count with percentage of ams did not result in significant differences ( figure s2) . including absolute counting beads as part of the initial analysis might have provided more definitive answers. variations in surface marker expression of ams over 20 weeks of siv infection were also not seen, except for changes in fcγriii expression (figures 1, 5) . phenotypic comparisons of ams from the bal of hiv-infected and uninfected individuals also have not shown differences (57) . proinflammatory cytokines il-6 and il-1β, tnf-α, and chemokine rantes was induced in ams during acute infection (figures 2a-e) . the rapid secretion of cytokines and chemokines was expected as the innate immune system initiates lymphocyte recruitment to establish adaptive immunity prior to viral spread. although the hiv/siv infection rate of ams may be low, macrophages respond to exposure to viral particles or virus-derived gene products such as nef, tat, and the gp120 envelope (18, 58) . following the acute phase, the elevated cytokine and chemokine responses by ams were not sustained despite exposure to viral products and likely lps from the gut (59) . further, stimulation with lps or native gp120 ex vivo showed an impaired il-6 and mip-1β response of ams from chronically infected macaques (figure 3) , indicating a potentially compromised inflammatory response against lung pathogens. this novel observation is consistent with data showing inhibition of tnf-α release by macrophages in response to toll-like receptor (tlr)-4 stimulation during hiv infection, even though the expression level of tlr-4 remained unchanged (60) . desensitization of tlr ligands on ams as a result of viral respiratory infections has also been reported (61) . the β-chemokines mip-1α, mip-1β, and rantes can bind to the hiv-1 coreceptor ccr5 indicating a potential protective effect against entry of r5-tropic viruses (62) (63) (64) (65) . the pattern of chemokine expression here shows that any protective effect of β-chemokines expressed by ams is likely limited to the acute phase of infection (figures 2, 3) . the lowering of cytokine and chemokine responses after the acute phase of infection was not associated with increased il-10 expression (figure 2f ). in fact, ams of naïve macaques expressed the highest level of intracellular il-10 ( figure 2f) . some studies have shown no induction of il-10 secretion or mrna in macrophage-tropic hiv-1 infected mdm compared to uninfected cells (66) . others have shown increased il-10 secretion and mrna levels for in vitro hiv-infected pbmc, monocytes and mdms [reviewed in (67) ]. differences in virus tropism or target cells could account for the variations observed. increased levels of il-10 have been detected in the bal-f of hiv infected patients (68) , however the il-10 in these bal samples could have originated from other lymphocytes that upregulate il-10 during hiv infection (69). in sum, our data indicate that siv disease progression is not associated with increased am il-10 expression, suggesting that ams are not switching to a more regulatory phenotype. ams are essential for lung microbial clearance. despite reports that am function is not affected by hiv-1 infection (27, 28) , recent studies have shown that phagocytic activity can be impaired (26, 70) . as viral load and activation status of macrophages can vary over the course of infection, here we tracked the phagocytic activity of ams following siv infection, providing new insights into the dynamics of am function. ams readily internalized antigen coated beads during acute infection, but this ability markedly decreased in chronically infected animals ( figure 4b ). the initial phagocytic activity was non-specific, as no gp120-specific antibody was present, and was likely due to the rise in proinflammatory responses during acute infection (figures 2a-e) . adp was observed in the chronic stage of infection when gp120specific antibody levels increased in bal-f (figures 4c,d) . however, a continued rise in gp120 antibody levels was not associated with increased adp. in fact, decreased adp activity was seen later in chronic infection. therefore, even though the pattern of chemokine expressing ams over the course of siv infection (figures 2a-e) suggested that frequencies returned to naive levels during the chronic phase of infection, the diminished functional activity observed suggested am dysfunction. fcγriii expression was elevated during acute siv infection but decreased as infection progressed (figures 5a,d) . these data are partly in contrast to data from hiv-infected mdms that showed either elevated or no change in fcγr expression [reviewed in (6) ]. the fcγriii isoform expressed in macrophages (fcγriiia) consists of a ligand bindingα-chain associated with disulfide-linked γ-chains (71, 72) . the γ signaling subunit of fcγrs has been shown to be downregulated as a consequence of hiv infection, resulting in aberrant downstream signaling required for phagocytosis (46, 73) . the fcγriii antibody used here recognizes the ligand binding fcγriii α-chain (74); thus the loss of fcγriii expression cannot be explained by downregulation of the γ-chain alone. our data suggest that in spite of an initial increase in the frequency of fcγriii + ams during acute infection (figure 5a) , prolonged hiv infection may lead to diminished expression of fcγriii. this outcome may not have been observed previously as in vitro mdm infection experiments mainly mimic acute stages of infection. nevertheless, although the frequency of fcγriii + ams at 12-wpi had dwindled to <30% of the level observed in naïve animals, a correlative trend between the frequency of fcγriii + ams and adp was observed ( figure 5g) indicating the importance of this receptor in adp activity mediated by ams. furthermore, fcγriii mfi negatively correlated with acute viremia suggesting an important role for fcγriii-mediated phagocytosis in initial viral load control ( figure 5h) . the pd-1/pd-l pathway plays a key role in negative regulation of adaptive immunity in hiv and other viral infections [reviewed in (75) ], but few studies have explored the role of pd-1 in innate immunity, particularly by macrophages. pd-1 expression on t cells following immune activation and its role in t-cell exhaustion when highly expressed during hiv-1 and other chronic viral infections have been described (52, 76). regarding macrophages, pd-1 expression has been linked to diminished ability to clear microbial invasion in septic mice (77) , inhibition of phagocytic activity and tumor immunity (13) , and inability to perform phagocytosis and intracellular killing in patients with tuberculosis (78) . unlike on t cells, pd-1 expression on macrophages in these studies described a single positive population. here, we also identified a single pd-1 + population of ams derived from chronically infected macaques (figures 6c,d) . the macaques were clear of lung infections at the time of bal collection, suggesting pd-1 expression was only associated with siv infection. until now, no direct evidence has implicated pd-1 in am dysfunction. our data show a direct correlation between pd-1 and siv viremia and suggest that in keeping with correlations described with t cells (53) , disease progression can also be associated with pd-1 expression in ams ( figure 6e) . while pd-1 ligands were found to be expressed on ams from naïve macaques, pd-l1 expression levels were low and subsequently declined after siv infection ( figure 7a ). this result was unexpected and in contrast to a study on mdms that showed up-regulation of pd-l1 and pd-l2 after exposure to inactivated hiv virions (79) . however, the alveolar environment is indeed unique and it is unsurprising that am responses to siv infection differ from in vitro exposure of mdms to virions. rodriguez-garcia et al. further indicated differential regulation of pd-l1 and pd-l2 by il-10, whereby presence of il-10 increased pd-l1 expression and its blockade increased pd-l2 expression (79) . analysis of il-10 expression by ams in our study showed a gradual decrease after siv infection and may be one explanation as to why we observed decreased pd-l1 expression ( figure 2c) . pd-l2 expression, however, remained high and did not increase with decreased il-10 expression. future analysis of the factors found in bal-f could provide further insight into the dynamics of pd-l expression on ams. pd-1 blockade experiments have shown enhancement of siv/hiv-specific responses, proliferative ability and cytokine production by exhausted pd-1 high t cells (80) (81) (82) . in keeping with this, blockade of the pd-1/pd-l pathway has been reported to ameliorate phagocytic function in macrophages found in the tumor microenvironment and in active tuberculosis (78, 83) . here, we also found that blockade of pd-1 could significantly improve phagocytic activity further highlighting pd-1 as a factor playing a role in dysfunction of ams ( figure 7c ). in addition, although not significant potentially due to small sample size, we found lower abundance of tnf-α expressing pd-1 + ams compared to the pd-1 − population during chronic infection ( figure 7d) . these data suggested that the presence of pd-1 on ams is likely a factor in any inhibitory role exerted on ams through the pd-1/pd-l pathway. in summary, our longitudinal investigation has provided important new information about the consequences of siv infection on ams, and in novel findings, propose a role for pd-1, a well-recognized inhibitor of adaptive immune responses, on innate immunity against siv infection. all datasets generated for this study are included in the manuscript and/or the supplementary files. forty-nine female indian rhesus macaques used in this study were housed in the nci animal facility, bethesda, md, under protocol vb-020. the nci facility is accredited by the association for assessment and accreditation of laboratory animal care international, and its animal care and use committee approved all animal experiments prior to study initiation. standard practices followed recommendations made in the guide for the care and use of laboratory animals of the united states, national institutes of health, and the weatherall report. therapeutic strategies towards hiv-1 infection in macrophages early events in sexual transmission of hiv and siv and opportunities for interventions th17 cells are preferentially infected very early after vaginal transmission of siv in macaques hiv-1 infection, response to treatment and establishment of viral latency in a novel humanized t cell-only mouse (tom) model macrophages: a crucial reservoir for human immunodeficiency virus in the body the role of monocytes and macrophages in the pathogenesis of hiv-1 infection macrophages sustain hiv replication in vivo independently of t cells bone marrow derived elements and resident microglia in brain inflammation the prolonged lifespan of alveolar macrophages monocytes and macrophages regulate immunity through dynamic networks of survival and cell death the biology of macrophages: i. general principles and properties m-1/m-2 macrophages and the th1/th2 paradigm alternative activation of macrophages the chemokine system in diverse forms of macrophage activation and polarization human alveolar macrophages predominately express combined classical m1 and m2 surface markers in steady state surface cd4 is critical to in vitro hiv infection of human alveolar macrophages hiv-1 infection of macrophages is dependent on evasion of innate immune cellular activation macrophage activation and human immunodeficiency virus infection: hiv replication directs macrophages towards a pro-inflammatory phenotype while previous activation modulates macrophage susceptibility to infection and viral production cell biology of hiv-1 infection of macrophages lymphocytic alveolitis, bronchoalveolar lavage viral load, and outcome in human immunodeficiency virus infection the role of alveolar macrophages in regulation of lung inflammation the effect of hiv infection on phagocytosis and killing of staphylococcus aureus by human pulmonary alveolar macrophages quantitative differences in phagocytosis and degradation of pneumocystis by alveolar macrophages in aids and non-hiv patients in vivo reduced binding and phagocytosis of pneumocystis carinii by alveolar macrophages from persons infected with hiv-1 correlates with mannose receptor downregulation small alveolar macrophages are infected preferentially by hiv and exhibit impaired phagocytic function. small alveolar macrophages are infected preferentially by hiv and exhibit impaired phagocytic function hiv gp120 in the lungs of antiretroviral therapy-treated individuals impairs alveolar macrophage responses to pneumococci opsonic phagocytosis of streptococcus pneumoniae by alveolar macrophages is not impaired in human immunodeficiency virus-infected malawian adults hiv-1 infection does not impair human alveolar macrophage phagocytic function unless combined with cigarette smoking fcgamma receptors as regulators of immune responses alveolar macrophages are critical for broadly-reactive antibodymediated protection against influenza a virus in mice antibody recognition of cell surface-associated ns1 triggers fc-γ receptor-mediated phagocytosis and clearance of west nile virus-infected cells internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection phagocytic cells contribute to the antibody-mediated elimination of pulmonary-infected sars coronavirus recovery of alveolar macrophages from rhesus and cynomolgus macaques by lung lavage a robust, high-throughput assay to determine the phagocytic activity of clinical antibody samples nasal dna-mva siv vaccination provides more significant protection from progression to aids than a similar intramuscular vaccination in vivo characterization of alveolar and interstitial lung macrophages in rhesus macaques: implications for understanding lung disease in humans flow cytometric analysis of macrophages and dendritic cell subsets in the mouse lung monocyte/macrophage-derived cc chemokines and their modulation by hiv-1 and cytokines: a complex network of interactions influencing viral replication and aids pathogenesis macrophage polarization at the crossroad between hiv-1 infection and cancer development interleukin-10 secretion by alveolar macrophages and monocytes in sarcoidosis quantitative assessment of fc receptor expression and function during in vitro differentiation of human monocytes to macrophages natural killer cell and granulocyte fc gamma receptor iii (cd16) differ in membrane anchor and signal transduction borne von dem ae. the fc-receptor iii of cultured human monocytes. structural similarity with fcriii of natural killer cells and role in the extracellular lysis of sensitized erythrocytes fcγriii (cd16) on human macrophages is a functional product of the fcγriii-2 gene hiv-1 down-modulates γ signaling chain of fcγr in human macrophages: a possible mechanism for inhibition of phagocytosis decreased fc receptor expression on innate immune cells is associated with impaired antibody-mediated cellular phagocytic activity in chronically hiv-1 infected individuals induction and exhaustion of lymphocytic choriomeningitis virusspecific cytotoxic t lymphocytes visualized using soluble tetrameric major histocompatibility complex class i-peptide complexes viral immune evasion due to persistence of activated t cells without effector function redefining chronic viral infection pd-1 is a regulator of virus-specific cd8+ t cell survival in hiv infection upregulation of pd-1 expression on hiv-specific cd8+ t cells leads to reversible immune dysfunction pd-1 expression on hiv-specific t cells is associated with t-cell exhaustion and disease progression the function of programmed cell death 1 and its ligands in regulating autoimmunity and infection alveolar macrophages are a major determinant of early responses to viral lung infection but do not influence subsequent disease development the role of macrophages in the development and progression of aids-related non-hodgkin lymphoma the alveolar microenvironment of patients infected with human immunodeficiency virus does not modify alveolar macrophage interactions with streptococcus pneumoniae induction of rapid and extensive betachemokine synthesis in macrophages by human immunodeficiency virus type 1 and gp120, independently of their coreceptor phenotype microbial translocation is a cause of systemic immune activation in chronic hiv infection hiv impairs tnf-α release in response to toll-like receptor 4 stimulation in human macrophages in vitro sustained desensitization to bacterial toll-like receptor ligands after resolutionof respiratory influenza infection interaction of chemokine receptor ccr5 with its ligands: multiple domains for hiv-1 gp120 binding and a single domain for chemokine binding molecular cloning and functional characterization of a novel human cc chemokine receptor (ccr5) for rantes, mip-1β, and mip-1α ld78β, a nonallelic variant of human mip-1α (ld78α), has enhanced receptor interactions and potent hiv suppressive activity c-c chemokine receptor type five (ccr5): an emerging target for the control of hiv infection lack of interleukin 10 expression in monocyte-derived macrophages in response to in vitro infection by hiv type 1 isolates cytokines and hiv-1: interactions and clinical implications tumour necrosis factor (tnf) and tnf-related molecules in hiv-1+ individuals: relationship with in vitro thl/th2-type response hiv-1 decreases nrf2/are activity and phagocytic function in alveolar macrophages a macrophage fcγ receptor and the mast cell receptor for ige share an identical subunit mechanisms for regulating expression of membrane isoforms of fc gamma riii (cd16) the mechanism underlying defective fcγ receptor-mediated phagocytosis by hiv-1-infected human monocyte-derived macrophages the binding epitopes of human cd16 (fc gamma riii) monoclonal antibodies. implications for ligand binding t cell exhaustion pd-1 expression in acute hepatitis c virus (hcv) infection is associated with hcv-specific cd8 exhaustion pd-1 expression by macrophages plays a pathologic role in altering microbial clearance and the innate inflammatory response to sepsis pd-1/pd-l pathway inhibits m.tb-specific cd4+ t-cell functions and phagocytosis of macrophages in active tuberculosis expression of pd-l1 and pd-l2 on human macrophages is upregulated by hiv-1 and differentially modulated by il-10 enhancing siv-specific immunity in vivo by pd-1 blockade pd-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination responsiveness of hiv-specific cd4 t cells to pd-1 blockade pd-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.01537/full#supplementary-material key: cord-001674-tp4o7fxx authors: oliveira, cláudia c.; van hall, thorbald title: alternative antigen processing for mhc class i: multiple roads lead to rome date: 2015-06-05 journal: front immunol doi: 10.3389/fimmu.2015.00298 sha: doc_id: 1674 cord_uid: tp4o7fxx the well described conventional antigen-processing pathway is accountable for most peptides that end up in mhc class i molecules at the cell surface. these peptides experienced liberation by the proteasome and transport by the peptide transporter tap. however, there are multiple roads that lead to rome, illustrated by the increasing number of alternative processing pathways that have been reported during last years. interestingly, tap-deficient individuals do not succumb to viral infections, suggesting that cd8 t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tap-independent peptides have been identified in the grooves of different mhc class i alleles. some of these peptides are not displayed by normal tap-positive cells and we therefore called them teipp, for “t-cell epitopes associated with impaired peptide processing.” teipps are hidden self-antigens, are derived from normal housekeeping proteins, and are processed via unconventional processing pathways. per definition, teipps are presented via tap-independent pathways, but recent data suggest that part of this repertoire still depend on proteasome and metalloprotease activity. an exception is the c-terminal peptide of the endoplasmic reticulum (er)-membrane-spanning ceramide synthase trh4 that is surprisingly liberated by the signal peptide peptidase (spp), the proteolytic enzyme involved in cleaving leader sequences. the intramembrane cleaving spp is thereby an important contributor of tap-independent peptides. its family members, like the alzheimer’s related presenilins, might contribute as well, according to our preliminary data. finally, alternative peptide routing is an emerging field and includes processes like the unfolded protein response, the er-associated degradation, and autophagy-associated vesicular pathways. these data convince us that there is a world to be discovered in the field of unconventional antigen processing. defective ribosomal product (drip) proteins generates small peptides. potentially, a multitude of proteolytic systems may generate antigenic peptides, but the proteasome is responsible for the liberation of majority of them. inhibition of proteasome activity strongly decreased the pool of mhc class i-binding peptides (1) . proteasomal cleavage typically creates a peptide's c-terminus compatible with mhc class i binding, and peptides are typically extended at their n-terminus (2, 3) . peptides generated in the cytosol are translocated into the endoplasmic reticulum (er) by the tap1/tap2 peptide transporter, where they have access to the peptide loading complex (plc), which is located within the er. tap is a heterodimeric member of the atp-binding cassette (abc) family of transporters, and peptide binding induces atp hydrolysis and transport across the er membrane. once in contact with the plc, the er-amino peptidase eraap (also known as erap1) trims n-extended peptides to a length appropriate for mhc class i binding (4) (5) (6) . chaperone tapasin promotes the formation of stable mhc class i/peptide complexes and acts as an editor. additionally, calnexin facilitates the early folding of mhc class i heavy chains, whereas calreticulin and erp57 are involved in peptide loading (7) (8) (9) . this pathway is also known as the proteasome-tap pathway and is considered as the conventional processing route because it is the mainstream pathway operating in cells under normal conditions (10) (11) (12) . however, cells are equipped with alternative routes leading to liberation and loading of peptides into mhc class i molecules. these routes are independent of one or more molecules from the conventional pathway such as the proteasome, tapasin, or tap. this has become apparent from studies on cells with deficiencies in the conventional processing pathway. in this review, we will discuss what is known to date regarding alternative enzymes and routes to peptide loading compartments of endogenously generated peptides that feed the direct mhc class i pathway, especially important in cases of failure of the conventional route. we have not included interesting literature on cross-presentation pathways for mhc class i peptides. more than 10 years ago, several papers made the important discovery that a large oligopeptidase, called tripeptidyl peptidase ii (tppii), participates in endoproteolytic activity in the cytosol and partially compensates for a deficient proteasomal activity (13) (14) (15) (16) . increased tppii activity even allowed for cell survival in lethal conditions of proteasome inhibition (14, 16) . in these conditions, tppii activity also partially restored peptide presentation in mhc class i molecules and it was speculated that it could account for the generation of some epitopes independently or in cooperation with the proteasome (16) . in fact, a paper from seifert et al. showed that tppii was involved in the generation of an epitope from the human immunodeficiency virus (hiv) protein negative factor (nef) (17) . after that, an increasing number of proteolytic enzymes have been implicated in the generation of peptide-epitopes independently of the proteasome. insulindegrading enzyme (ide) generates an epitope from the human melanoma antigen mage-a3 (18) . thimet oligopeptidase (top) and nardilysin are required for the generation of three other clinically relevant ctl epitopes: the tumor-antigen prame, an epitope from epstein-barr virus (ebv) protein ebna3c, and an epitope from the melanoma protein mart-1 (15) . these enzymes are part of an array of cytosolic endo-and exo-proteases that complement proteasomal activity and degrade proteasome products ultimately into amino acids. importantly, the process of peptide liberation from the protein context is inevitably coupled to the destruction pathway and all proteases mentioned above also destroy some antigenic peptides (19) (20) (21) (22) . peptides that are "rescued" from total destruction are transported by tap into the er and can potentially bind mhc class i molecules. in eukaryotic cells, secretory and membrane proteins contain a signal sequence essential for protein targeting to the er, the entrance for the secretory pathway (23, 24) . these signal sequences are typically composed of three domains: a hydrophobic core (h region) of 6-15 amino acids, a polar c-terminal end (c region) with small uncharged amino acids, and a polar nterminal region (n region) with a positive net charge (25). after insertion into the protein-conduction channel, signal peptides are usually cleaved from the preprotein by signal peptidase (sp) (26). thereafter, signal peptides, which are small domains and trapped in the er membrane, can undergo intramembrane proteolysis by cleavage within their transmembrane region by the presenilintype aspartic protease signal peptide peptidase (spp) (25, 27). peptide ligands suitable for mhc class i binding are thought to be generated after the intramembrane proteolysis by spp that promotes the release of signal peptide fragments from the er membrane (28, 29). the spp-cleaved fragments in the vicinity of the cytosol can get access to the cytosol again and be further processed by the proteasome and transported by tap into the er. most hla class i molecules donate their leader sequences for binding to the non-classical hla-e, and the cleavage of these signal sequences is mediated by sp and spp (30-33). these leader peptides are even the most dominant source of peptides for hla-e. proper surface expression of hla-e prevents cytotoxic action of natural killer cells that continuously sense the presence of peptide/hla-e complexes through their cd94/nkg2 receptors (34-36). the absence of these complexes at cell surface results in failure of interaction with cd94/nkg2a receptors, which activate nk cells for killing their targets. pioneering studies by peter cresswell and victor engelhard in 1992 revealed that most peptides presented at the surface of tap-deficient cells were derived from signal sequences, specific protein regions at the n-terminus of proteins (37, 38). indeed, the parts of the leader peptide within the er membrane that are closest to the er lumen are released there and can get access to mhc class i grooves independent of proteasomes or tap (figure 1) . after the cleavage of the transmembrane sequence by spp, the peptide fragments are released and can associate with mhc class i/β 2 m nascent molecules. this intramembrane proteolysis by spp is thought to be important for the clearance of the er membrane by removing small protein remnants anchored at figure 1 | classical and alternative pathways for mhc class i presentation. cells with deficiencies in components of the mhc class i processing pathway, such as tap, can present a repertoire of peptides derived from alternative processing pathways. different "housekeeping" cell functions such as signal peptide cleavage, protein maturation in the golgi, and protein/organelle disposal via autophagy can provide peptide ligands for mhc class i loading. the membrane rendering them susceptible for subsequent degradation. the intramembrane cleavage by spp is favored by spp topology that conceals the catalytic center within the plane of the membrane. the two aspartic residues required for the proteolytic activity of spp are located within conserved (y/f)d and g(l/i/f)gd amino acid motifs in two adjacent transmembrane domains (39, 40). regarding a cleavage motif for spp, no consensus cleavage site has been described. however, spp demonstrates a strong preference for substrates with helix-destabilizing residues in their transmembrane domain (41-43). amino acids like asparagine, serine, and cysteine disturb a perfect alfa-helical conformation of the transmembrane domain and are therefore referred to as "helix-bending" or "helix-breaking" residues. signal peptides have been shown to contain amino acids with "helixbreaking" capacity within their h region, which critically influence their proteolytic processing by spp (42, (44) (45) (46) . the disturbance of the α-helix caused by these residues is thought to facilitate intramembrane proteolysis. this and other issues would be clarified with the atomic resolution of this protease but this is still lacking due to its technical difficulties. we can have an approximation of that by looking at the crystal structure of presenilin/spp homologs recently published (47) (48) (49) (50) . jr1 is an spp homolog from the archaeon methanoculleus marisnigri that harbors nine transmembrane helices, similar to what is predicted for spp, with tmd 6 and tmd 7 containing the yd and gxgd motif, respectively. the two catalytic aspartate residues are located close to each other and approximately 8 å into the lipid membrane. proteolytic activity occurs in the presence of water molecules that gain access to the catalytic aspartates through a large cavity between two terminal domains (48) . the three-dimensional structure of a human presenilin comprised into the γ-secretase complex has also been described (51) . for the near future, we can expect more information on the catalytic activity of this family of proteases, including spp, which is definitely an important contributor of peptides for mhc class i presentation. our recent data revealed an additional role for intramembrane proteolysis by spp. regardless its name, spp also appeared to liberate a c-terminal peptide, independent of proteasome activity. the processing of c-terminal regions of a type ii protein inserted in the er membrane leads to the presentation of peptides independently of proteasome and tap (52, 53) . the cterminal region from the ceramide synthase trh4, which is a multiple membrane-spanning protein in the er, contains a 9mer peptide-epitope that is located at the very c-terminal end of the protein and protrudes into the lumen of the er (53-56). the trh4 protein has a housekeeping function and is ubiquitously expressed. inhibition of spp activity blocked the generation of the trh4 peptide. experiments with mutant forms of the trh4 protein indicated that the intramembrane cleavage by spp occurs at the direct upstream region of the t cell epitope within the lipid bilayer (52) . we speculated that spp activity in the er membrane is sufficient to liberate the minimal c-terminal 9-mer peptide and release of this peptide into the er lumen. other proteolytic enzymes, such as amino-peptidases, were dispensable. further carboxy-terminal processing was not needed since the epitope is located at the very c-terminal end of the protein. direct release of the liberated peptide into the er lumen is very likely, due to the type ii transmembrane orientation of the trh4 protein tail (nterminus to c-terminus orientation). the exact peptide loading mechanism of the trh4 membrane peptide, however, remains to be determined. what triggers the cleavage of trh4 by spp is not known yet. in addition to its liberation function of small transmembrane substrates, spp has been shown to associate with misfolded membrane proteins in complexes where spp is represented as a monomer, dimer, or multimer (57) . it was suggested that such high molecular weight complexes act as chaperones to dispose of membrane aggregates (57, 58) . the role of spp in this degradation machinery might be to liberate such aggregates from the er membrane. these discoveries were based on the viral us2 and us11 proteins, which successfully labels mhc class i molecules in the er for retrograde transport to push this protein back to the cytosol for degradation by proteasomes (59, 60) . interestingly, the spp family member, presenilin 2, seems to be associated with this membrane proteolysis as well (61) . since spp and presenilin 2 have opposing preference for type i and type ii transmembrane orientations, such a "degradome" machinery might be responsible to clear the er membranes. this type of machinery is called the erassociated degradation (erad) pathway. erad is an er quality control system, which monitors the integrity of nascent or erresident proteins and targets incorrectly folded or misassembled proteins to degradation. the disposal of misfolded mhc class i heavy chains also occurs through the erad pathway in the absence of viral interference (62) . clearly, viruses take advantage of these existing pathways and hijack them in order to evade immune recognition by cytotoxic t cells (63, 64) . another role for spp in erad has come from a recent paper describing the cleavage of the unfolded protein response (upr) regulator xbp1u by spp (65) . xbp1u is a type ii membrane protein and undergoes intramembrane cleavage within a conserved type ii tm domain while integrated in a complex containing spp, the protease derlin-1, and the e3 ligase trc8, which prime spp for xbp1u cleavage. an ectodomain shedding of spp substrates prior to spp cleavage is thought to be required and normally performed by sp in signal sequences but such cleavage was unnecessary in the case of xbp1u, similarly to what we have described for trh4 (52, 65) . in general, the upr induces a strong downregulation of mhc class i molecules at the cell surface (66, 67) . thus, spp activity seems to have a direct impact on mhc class i peptide presentation by cleavage of leader sequences from nascent proteins and the liberation of some c-termini for mhc class i loading. a more indirect role that impacts on mhc class i presentation has been revealed and occurs through an erad pathway. this is also supported by a recent study using a systemslevel strategy reporting the involvement of spp in the network that coordinates the erad response (68). the group of yewdell showed more than a decade ago that peptides located at the c-terminus of er-targeted proteins are very efficiently generated and presented on mhc class i (69) (70) (71) (72) . the location of the peptide was essential to be at the very end of the c-terminus of the protein, not requiring c-terminal trimming, in line with the fact that there is poor carboxypeptidase activity in the er (73) . they described the presentation of tap-independent peptides from one er-resident protein, jaw1, and proteins in the secretory pathway, like ovalbumin and cd23. in each case, the peptides were efficiently liberated from the very c-terminus by the activity of yet unidentified endo-proteases to be generated as class i ligands. based on this pathway of peptide liberation, the authors provided the term "c-end rule" to highlight the capacity of er-resident proteases to liberate class i ligands from the cterminal ends of er-targeted proteins. the liberation of peptides without the intervention of the proteasome can also occur in the trans-golgi network (74, 75) . the main proteolytic enzyme involved was shown to be furin, a known protease of the trans-golgi network normally required for the maturation of secreted proteins (e.g., growth factors and neurotransmitters) by cleaving at precise stretches of three to four basic residues (76) . furin is part of a family of proprotein convertases that comprises nine members (77) . three members (pc5/6, pace4, and pc7) including furin are widely expressed and together they take part in a variety of processes occurring in the trans-golgi network, cell surface, or endosomes. this leads to the activation or inactivation of receptors, ligands, enzymes, viral glycoproteins, or growth factors (78) . furin also has important functions during development by processing substrates like bone morphogenetic protein 10 (bmp10), a member of the tgf-β superfamily that plays a critical role in heart development (79) . furin processes a wide variety of precursor proteins after the c-terminal arginine residue in the preferred consensus motif -arg-x-arg/lys-arg↓-x-(x is any amino acid and "↓" indicates the cleavage position) (80) . initially, this pathway was studied with the use of a model peptide at the cterminus of the secreted hepatitis hbe protein. furin-processed antigens targeted to the secretory route were presented by mhc class i at the cell surface and could elicit functional cd8 t-cell responses in vivo in a tap-independent fashion (75, 81) . the ctermini of secretory or er-localized proteins thus appear to be processed for presentation to cd8 t cells (figure 1 ). the generation of mhc class i ligands described above defines several alternative ways to generate peptide ligands without the intervention of the proteasome. these represent unusual pathways for peptide generation. now, we will discuss a different constraint in the conventional antigen presentation pathway related to the blockade of peptide entrance in the er due to tap deficiency. in human beings, tap-deficiency syndrome has been described in several independent families and results from mutations in either one of the subunits of the peptide transporter, tap1 and tap2 (82, 83) . interestingly, these tap-deficient individuals do not succumb to viral infections, suggesting that cd8 t cell immunity is sufficiently supported by alternative tap-independent processing pathways. to date, a diversity of viral and endogenous tapindependent peptides have been identified in the grooves of different mch class i alleles. importantly, these tap-deficient patients harbor a polyclonal cd8 t-cell repertoire that is capable of recognizing peptides from the ebv virus, like protein lmp2, presented on tap-deficient cells (84) . the tap-independent processing pathway is capable of generating enough mhc class i/peptide complexes in order to keep immunosurveillance and control of viral infections. studies with tap1-knockout mice have shown that surface expression levels of mhc class i are indeed lower, but the remaining complexes do induce a broad and polyclonal repertoire of cd8 t-cells (85) (86) (87) . the tcr usage was shown to be very comparable to that of wild-type mice and tap1-knockout mice were capable of mounting anti-viral cd8 t cell responses. together, these data show that, although crippled, the mhc class i-presented peptide repertoire in the absence of tap is sufficient to support cd8 t cell immunity. interestingly, peptides emerging from alternative tapindependent routes appeared to be immunogenic. following immunizations in mice with tap-deficient tumor cells, specific cd8 t-cells were induced that recognize tap-deficient cells, but not normal cells (56, 88) . these t-cell epitopes seemed to be selectively presented by tap-deficient cells but not under normal conditions. this alternative peptide repertoire emerges due to processing defects and therefore these peptides were named "t cell epitopes associated with impaired peptide processing" (teipp). the molecular identification of some teipp peptides revealed that they can be diverse in length (from 9-mer to 18-mer), amino acid composition, and mhc class i binding, as some are presented by classical mhc class i molecules and others by the non-classical mhc molecule hla-e and the mouse homolog qa-1 b ( table 1 ) (35, [89] [90] [91] [92] . they are derived from normal housekeeping proteins with ubiquitous expression, but are surprisingly not loaded on mhc class i in cells with an intact antigen-processing machinery. they constitute normal self-peptides (non-mutated, not pathogen-or tumor-specific) and can be regarded as real neo-antigens. the immunogenicity of teipp peptides exists because they are not presented by normal cells including the thymus. during thymic development, t cells are subjected to two subsequent processes called positive and negative selection (93, 94) . negative selection is necessary for the maintenance of self-tolerance as it induces the deletion or inactivation of potentially autoreactive thymocytes (95) . we recently demonstrated that teipp-specific cd8 t-cells indeed do not undergo negative selection and are thereby available for therapeutic exploitation. since the peptides recognized by teipp-specific ctl are derived from housekeeping proteins, we tried to understand why teipps are not presented by processing intact cells. collectively, our data show that teipp peptides are actually produced within processing proficient cells, but somehow are not or not sufficiently presented by their surface mhc class i molecules. taking the trh4-derived teipp peptide as a model, we have analyzed the expression of the trh4 gene in several epithelial populations isolated from wild-type and tap1-ko mice (53) . this analysis revealed the same level of rna transcripts between the normal and knockout populations, suggesting comparable protein levels in both cell types. the liberation of the trh4 peptide is performed by spp, which is active in tap-positive as well as tap-negative targets (52) . overexpression of the trh4 gene in tap-positive cells leads to surface presentation in mhc class i (53), still in a proteasome-independent way. moreover, proteasome inhibition in tap-positive cells results in presentation of the endogenous trh4 peptide (56) , indicating that, indeed, this teipp peptide is generated in all cells but loses competition with the overwhelming amount of tap-imported peptides in tappositive cells. some alternative peptides, like the ones derived from ebv proteins were shown to be presented on tap-positive cells to comparable extent, although using alternative pathways. moreover, our data suggest that the limited quantity of the trh4 peptide-epitope in the er is the main cause of selective presentation in tap-deficient cells. interestingly, gradual increase of overexpression correlated with the degree of recognition by the teipp-specific ctl clone, implying that tap transport actually constitutes a strong barrier for teipp peptides. the study of human teipp antigens corroborates these findings. one antigenic peptide is encoded by the human calca gene and derives from the signal sequence of preprocalcitonin (ppct) protein. this peptide is liberated in the er lumen by sequential cleavage with sp and spp, independently from proteasomes ( table 1 ) (96) . the presentation of the ppct peptide to specific ctl was found in human lung and medullary thyroid carcinomas that had very low expression of tap. presentation of the ppct peptide occurred also in normal non-transformed cells, such as dendritic cells (dcs), after knockdown of tap. overexpression of the calca gene in dcs and tap-positive tumor cells resulted in recognition by the specific ctl clone (97) . identification of additional human teipp antigens at the molecular level will enable cd8 t cell targeting of otherwise ctl-resistance tap-negative tumor variants (98, 99) . together, these findings support the model of peptide competition in the er as a factor that prevents presentation of peptides from alternative sources, and shape a picture of alternative processing pathways that emerge upon defects in the conventional one (figure 1 ). the precise loading mechanism of tap-independent signal peptides into mhc class i molecules is not known, since processing by sp and spp is thought to take place outside of the plc. this sophisticated machinery for optimizing ligand length and quality and facilitating peptide loading onto nascent mhc class i molecules greatly facilitates peptide loading by physical bridging transporters to chaperones for loading and also "edits" the repertoire of bound peptides to maximize their affinity (7) . the plc molecule tapasin tethers mhc class i molecules to the peptide transporter acting together with the chaperone calreticulin and the oxidoreductase erp57 (100). tapasin can sense the quality of peptide bound by mhc class i complexes, and allows successive rounds of peptide binding until a certain affinity threshold is met. trimming of incoming peptides by eraap can be necessary for obtaining a good peptide length before selection by a defined mhc class i allele. a recent paper states that mhc class i molecules initially bind a variety of peptides, including some lowaffinity or n-terminally extended ones but then quickly dissociate from the molecule followed by the selection of the best fitting candidates (101) . however, tap-independent leader peptides do not arrive in the er via tap and thus lack these editing and optimizing chaperones. in the absence of tap transporters, the loading of peptides into mhc class i occurs without a fully functional plc at hand. in that respect, inefficient loading of leader peptides in tap-positive cells can be expected, whereas in cells devoid of tap, this alternative er entrance mechanism allows emergence of these peptides. so here, the plc floats in the er membrane and is dispatched from the entry site of peptides. indeed, differential mass spectrometry analysis showed an enhanced presentation of leader peptides in cells lacking the peptide transporter (102) . after all, mhc class i molecules get loaded with the available repertoire: from conventional or unconventional sources. but how do these "untapped" peptides find their way to empty mhc class i molecules at all? it has been shown that loading of peptides in mhc class i can occur with the minimal components of tapasin-erp57-mhc class i complexes (103), but we have to assume that the chances of a peptide to find the plc machinery in the er are scarce. on the other hand, tap-deficient cells harbor more peptide-receptive class i molecules compared to normal cells (104) . these peptides might actually be actively chaperoned toward these open grooves. chaperones with high peptide binding capacity in the er are heat shock proteins (e.g., hsp96) and pdi, an isomerase that efficiently binds free peptide (105) . it is possible that tap-independent peptides are captured by these molecules and chaperoned to mhc class i. recent studies suggest that the tapbpr molecule ("tapasin-like") binds those mhc class i proteins that are not bound to tapasin in the plc, so we might hypothesize that this pool of peptide-receptive grooves may function to load leader peptides (106) . however, this still needs to be investigated. other ways to access peptide-receptive mhc has been proposed and are related to the intracellular traffic of hydrophobic peptides. studies with several epitopes from the lmp2 protein of the ebv showed that peptides, which possess a high hydrophobicity index, were presented in a tap-independent manner (107) . the proposed mechanism describes the generation of these peptides outside the er, since proteasome activity is needed for presentation, and subsequently free diffusion across the er membrane possible due to their high hydrophobicity. these peptides might transgress membranes spontaneously or via alternative membrane transporters. a recent study by tey et al. showed that the processing of a peptide antigen from the human cytomegalovirus (hcmv) latency associated protein, pul138, occurs entirely in the vesicular pathway and is mediated by autophagy (108) . also other examples of autophagy enhanced mhc class i presentation of viral antigens were reported (109, 110) . during autophagy, large portions of cytoplasmic content, including proteins and organelles, are encapsulated in double membrane vesicles called autophagosomes (111) (112) (113) (114) . the autophagosomal membrane has been proposed to originate from the er (115, 116) and peptide-receptive mhc class i molecules might be present in autophagosomes, allowing for loading of peptides in these vesicular compartments (117) . in addition, recirculating mhc class i molecules from the cell membrane end up in endosomes and can have contact with autophagosomes before returning to the endocytic network. transit for membrane-associated proteins between autophagosomes and endosomes has been observed by live cell imaging (118) . moreover, peptides generated by the proteasome seem to get access to the endocytic vesicular pathway as well. we recently found at least one example for this in our teipp repertoire proportions and combinations. the mhc class i peptide repertoire can therefore be compared to a painter's palette where the different "colors" (peptides) are mixed and used to create a colorful and complex "picture." that is generated by the proteasome but is tap-independently presented ( table 1 ) (119) . the presentation of this peptide was surprisingly enhanced by blockade of the proton pump in endosomes, indicating that this antigen most likely crosses the vesicular pathway (119) . finally, mass spectrometry analysis of the tapindependent peptide repertoire pointed at proteins located in the vesicular compartment as an important source (120) (121) (122) . though these alternative processing and loading compartments have not fully been unraveled, these data strongly support the notion that the vesicular pathway and autophagy contributes to antigen presentation by mhc class i molecules. on summarizing, we can conclude that, even though the conventional proteasome-tap pathway represents the major source of the mhc class i peptide repertoire, alternative processing pathways clearly complement the total pool. this multitude of mhc class i processing pathways can be compared to a colorful palette where the painter combines the different colors that will end up in different proportions and combinations in the final picture (figure 2) . in situations of viral infections, cellular transformation, or other initiators of stress, the contribution of these alternative pathways might gain importance. the spp and maybe their family members are convincing examples of alternative proteolytic systems that feed the alternative routes of antigen presentation. the precise molecular identification of alternative loading mechanisms unto peptide-receptive mhc class i molecules, whether it be in the er or in autophagosomes, still needs indepth investigation. in that respect, peptides can walk on multiple different paths before ending up in the grooves of mhc class i and therefore illustrate the old expression that multiple roads lead to rome. the protease inhibitor, n-acetyl--leucyl--leucyl-leucyl--norleucinal, decreases the pool of major histocompatibility complex class i-binding peptides and inhibits peptide trimming in the endoplasmic reticulum 26s proteasomes and immunoproteasomes produce mainly n-extended versions of an antigenic peptide the sizes of peptides generated from protein by mammalian 26 and 20 s proteasomes. implications for understanding the degradative mechanism and antigen presentation an ifngamma-induced aminopeptidase in the er, erap1, trims precursors to mhc class i-presented peptides eraap customizes peptides for mhc class i molecules in the endoplasmic reticulum the er aminopeptidase erap1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues mechanisms of mhc class i-restricted antigen processing and cross-presentation recent advances in antigen processing and presentation proteasome and peptidase function in mhc-class-i-mediated antigen presentation towards a systems understanding of mhc class i and mhc class ii antigen presentation proteases in mhc class i presentation and cross-presentation the known unknowns of antigen processing and presentation a giant protease with potential to substitute for some functions of the proteasome a proteolytic system that compensates for loss of proteasome function antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic t cell epitopes integration of the ubiquitin-proteasome pathway with a cytosolic oligopeptidase activity an essential role for tripeptidyl peptidase in the generation of an mhc class i epitope production of an antigenic peptide by insulin-degrading enzyme a major role for tppii in trimming proteasomal degradation products for mhc class i antigen presentation pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases the efficiency of human cytomegalovirus pp65(495-503) cd8+ t cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases the cytosolic endopeptidase, thimet oligopeptidase, destroys antigenic peptides and limits the extent of mhc class i antigen presentation the translocon: a dynamic gateway at the er membrane approaching the mechanism of protein transport across the er membrane requirements for signal peptide peptidasecatalyzed intramembrane proteolysis on the mechanism of spp-catalysed intramembrane proteolysis; conformational control of peptide bond hydrolysis in the plane of the membrane structural analysis of hepatitis c virus core-e1 signal peptide and requirements for cleavage of the genotype 3a signal sequence by signal peptide peptidase maturation of hepatitis c virus core protein by signal peptide peptidase is required for virus production signal peptide peptidase cleavage of gb virus b core protein is required for productive infection in vivo the crystal structure of gxgd membrane protease flak structure of a presenilin family intramembrane aspartate protease crystal structure of bacillus subtilis signal peptide peptidase a structure of signal peptide peptidase a with c-termini bound in the active sites: insights into specificity, self-processing, and regulation three-dimensional structure of human gamma-secretase new role of signal peptide peptidase to liberate c-terminal peptides for mhc class i presentation peptide transporter tap mediates between competing antigen sources generating distinct surface mhc class i peptide repertoires mammalian ceramide synthases two mammalian longevity assurance gene (lag1) family members, trh1 and trh4, regulate dihydroceramide synthesis using different fatty acyl-coa donors selective cytotoxic t-lymphocyte targeting of tumor immune escape variants signal peptide peptidase (spp) assembles with substrates and misfolded membrane proteins into distinct oligomeric complexes signal peptide peptidase is required for dislocation from the endoplasmic reticulum a high-coverage shrna screen identifies tmem129 as an e3 ligase involved in er-associated protein degradation the human cytomegalovirus us11 gene product dislocates mhc class i heavy chains from the endoplasmic reticulum to the cytosol interactome analyses of mature gamma-secretase complexes reveal distinct molecular environments of presenilin (ps) paralogs and preferential binding of signal peptide peptidase to ps2 er quality control in the biogenesis of mhc class i molecules a membrane protein required for dislocation of misfolded proteins from the er the hcmv gene products us2 and us11 target mhc class i molecules for degradation in the cytosol signal peptide peptidase functions in erad to cleave the unfolded protein response regulator xbp1u the unfolded protein response (upr)-activated transcription factor x-box-binding protein 1 (xbp1) induces microrna-346 expression that targets the human antigen peptide transporter 1 (tap1) mrna and governs immune regulatory genes stimulation of an unfolded protein response impairs mhc class i expression defining human erad networks through an integrative mapping strategy two novel routes of transporter associated with antigen processing (tap)-independent major histocompatibility complex class i antigen processing promiscuous liberation of mhc-class i-binding peptides from the c termini of membrane and soluble proteins in the secretory pathway trimming of antigenic peptides in an early secretory compartment tap-independent delivery of antigenic peptides to the endoplasmic reticulum: therapeutic potential and insights into tap-dependent antigen processing peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by mhc class i major histocompatibility complex class i viral antigen processing in the secretory pathway defined by the trans-golgi network protease furin furin-processed antigens targeted to the secretory route elicit functional tap1-/-cd8+ t lymphocytes in vivo furin at the cutting edge: from protein traffic to embryogenesis and disease the multifaceted proprotein convertases: their unique, redundant, complementary, and opposite functions the activation and physiological functions of the proprotein convertases furin is the major processing enzyme of the cardiacspecific growth factor bone morphogenetic protein 10 proteolytic activation of the spike protein at a novel rrrr/s motif is implicated in furin-dependent entry, syncytium formation, and infectivity of coronavirus infectious bronchitis virus in cultured cells postendoplasmic reticulum rescue of unstable mhc class i requires proprotein convertase pc7 description of hla class i-and cd8-deficient patients: insights into the function of cytotoxic t lymphocytes and nk cells in host defense clinical and immunological aspects of hla class i deficiency human peptide transporter deficiency: importance of hla-b in the presentation of tap-independent ebv antigens positive selection of self-and alloreactive cd8+ t cells in tap-1 mutant mice tap1-deficient mice select a cd8+ t cell repertoire that displays both diversity and peptide specificity tap1 mutant mice are deficient in antigen presentation, surface class i molecules, and cd4-8+ t cells generation of cd8+ t cells specific for transporter associated with antigen processing deficient cells alternative peptide repertoire of hla-e reveals a binding motif that is strikingly similar to hla-a2 cd8+ t cell responses against tap-inhibited cells are readily detected in the human population the nonpolymorphic mhc qa-1b mediates cd8+ t cell surveillance of antigen-processing defects nonclassical mhc class ib-restricted cytotoxic t cells monitor antigen processing in the endoplasmic reticulum antigen presentation in the thymus for positive selection and central tolerance induction selection of the t cell repertoire the thymic medulla: a unique microenvironment for intercellular self-antigen transfer preprocalcitonin signal peptide generates a cytotoxic t lymphocyte-defined tumor epitope processed by a proteasome-independent pathway different expression levels of the tap peptide transporter lead to recognition of different antigenic peptides by tumor-specific ctl strategies to counteract mhc-i defects in tumors a novel category of antigens enabling ctl immunity to tumor escape variants: cinderella antigens tapasin-the keystone of the loading complex optimizing peptide binding by mhc class i molecules in the endoplasmic reticulum the first step of peptide selection in antigen presentation by mhc class i molecules features of tap-independent mhc class i ligands revealed by quantitative mass spectrometry selective loading of high-affinity peptides onto major histocompatibility complex class i molecules by the tapasin-erp57 heterodimer peptide-receptive class i major histocompatibility complex molecules on tap-deficient and wild-type cells and their roles in the processing of exogenous antigens tap-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp96, protein disulfide isomerase and calreticulin the binding of tapbpr and tapasin to mhc class i is mutually exclusive processing of a multiple membrane spanning epstein-barr virus protein for cd8(+) t cell recognition reveals a proteasome-dependent, transporter associated with antigen processing-independent pathway autophagy mediates transporter associated with antigen processing-independent presentation of viral epitopes through mhc class i pathway alternative pathways for mhc class i presentation: a new function for autophagy autophagy enhances the presentation of endogenous viral antigens on mhc class i molecules during hsv-1 infection autophagy and adaptive immunity when autophagy meets viruses: a double-edged sword with functions in defense and offense unveiling the roles of autophagy in innate and adaptive immunity endoplasmic reticulum and golgi complex: contributions to, and turnover by eating the endoplasmic reticulum: quality control by autophagy generation of mhc class i ligands in the secretory and vesicular pathways the itinerary of autophagosomes: from peripheral formation to kiss-and-run fusion with lysosomes dominant contribution of the proteasome and metalloproteinases to tapindependent mhc-i peptide repertoire allele-dependent processing pathways generate the endogenous human leukocyte antigen (hla) class i peptide repertoire in transporters associated with antigen processing (tap)-deficient cells diversity of natural self-derived ligands presented by different hla class i molecules in transporter antigen processing-deficient cells a transporter associated with antigen-processing independent vacuolar pathway for the mhc class i-mediated presentation of endogenous transmembrane proteins financial support was received from the portuguese foundation for science and technology (grant sfrh/bd/33539/2008 to co). gamblin artists colors (portland, or, usa) is acknowledged for the free use of figure 2 . the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-252568-b8sbvy0g authors: marques neto, lázaro moreira; kipnis, andré; junqueira-kipnis, ana paula title: role of metallic nanoparticles in vaccinology: implications for infectious disease vaccine development date: 2017-03-08 journal: front immunol doi: 10.3389/fimmu.2017.00239 sha: doc_id: 252568 cord_uid: b8sbvy0g subunit vaccines are safer but less immunogenic than live-attenuated vaccines or whole cell inactivated vaccines. adjuvants are used to enhance and modulate antigen (ag) immunogenicity, aiming to induce a protective and long-lasting immune response. several molecules and formulations have been studied for their adjuvanticity, but only seven have been approved to formulate human vaccines. metallic nanoparticles (menps), particularly those containing gold and iron oxides, are widely used in medicine for diagnosis and therapy and have been used as carriers for drugs and vaccines. however, little is known about the immune response elicited by menps or about their importance in the development of new vaccines. there is evidence that these particles display adjuvant characteristics, promoting cell recruitment, antigen-presenting cell activation, cytokine production, and inducing a humoral immune response. this review focuses on the characteristics of menps that could facilitate the induction of a cellular immune response, particularly t-helper 1 and t-helper 17, and their potential functions as adjuvants for subunit vaccines. (tlr4 agonists) are immunomodulatory molecules, capable of generating a th1 response (5) . there is a demand for safe adjuvants capable of inducing efficient cellular immunity, especially th1 and th17, to be used against tuberculosis, leishmaniasis, malaria, and other diseases caused by intracellular microorganisms (1, 6) . the majority of molecules with this type of adjuvanticity (th1 driven) are related toward the response of danger receptors to trigger inflammation, thus safety and tolerance could be major barriers that prevent their use in human vaccines (7) . however, comparing alum and cpg/dna adjuvants in human trials, only common adverse effects, including local site reaction, flu-like symptoms and headache were observed when cpg/dna was used (8) . also, verstraeten et al. (9) , analyzing more than 30,000 individuals, who received vaccine-containing as01, observed that only common side effects occurred. nanoparticles (nps) are classically described as structures smaller than 100 nm and can be classified, based on their composition, as polymeric, inorganic, liposomes, immunostimulating complexes, virus-like particles, emulsions, or self-assembled proteins (10) . they are made of different materials and differ in size, shape, and surface properties; interactions with biological systems, therefore, are varied, with several applications in modern medicine. in vaccinology, they are classically thought to have delivery and deposit properties. however, many nps have been shown to stimulate immune responses, including cell recruitment, activation of antigen (ag)-presenting cells (apcs), and induction of cytokine and chemokine release. the development of nanostructures and nanoadjuvants may therefore offer alternatives to currently used adjuvants once studies establish ways for them to elicit innate immune response and support the development of adaptive immune response in the context of vaccine formulations (10) . metallic nanoparticles (menps) are relatively non-biodegradable, have rigid structures, and possess simple synthesis methodology. many have been studied for their immunological properties (11) . however, there are still gaps in understanding the immune response generated by nps, especially menps. few studies have compared nps of different types and there is no standardization among published methodologies, which hampers comparisons of immunostimulatory characteristics. several important characteristics, therefore, have not been well studied. for example, how chemical and physical properties (including material composition, size, shape, surface charge, and hydrophobicity) impact vaccine immune response (5) . this review focuses on the use of menps in formulations against infectious diseases, aiming to assess progress of their use in vaccinology and their possible applications as adjuvant. the immune response generated by menp-formulated vaccines table 1 summarizes the articles that report the use of menps as part of vaccine formulations against infectious diseases and the immune responses they elicited. a range of immune responses is required to fight a diverse group of microorganisms. the type of protective immune response can be simplistically divided based on the type of microorganism: extracellular bacteria and toxin, intracellular bacteria, viruses, fungi, and protozoa. among the vaccines targeting extracellular bacteria and toxin, two were formulated with lipopolysaccharide (lps) in glycopeptide ag. the use of glycoantigen and lps can trigger an intense response through tlr4 and b cell receptor activation; the presence of gold nps (aunps) may have minimal influence on this response. however, in the work of gregory et al. (12) and torres et al. (13) , the use of aunps in the formulation generated a different response, improving anti-lps immunoglobulin g (igg) response, decreasing bacterial burden, generating a more efficient humoral response, and improving animal survival, showing that aunps may influence immune response and protection. using protein ag, barhate et al. (22) formulated a vaccine using aunps and toxoid ag and demonstrated that their formulation could induce a mucosal and systemic igg and iga response. when co-administered with asparagus racemosus extract, a botanically derived adjuvant, the response was further enhanced (22) . dakterzada et al. (24) developed a vaccine against pseudomonas aeruginosa using the flagellin subunit and aunps that elicited an igg response comparable to that induced by freund adjuvant. flagellin is a tlr5 agonist but the recognition and signaling is structure dependent. this study, however, used only the 1-161aa from flagellin and its ability to activated tlr5 could not be maintained (24) . gregory et al. (12) used an f1 yersinia pestis ag conjugated to aunps that induced an ab response with higher igg2a associated with higher levels of interferon gamma (ifnγ), suggesting activation of th1 cells. among the studies that used menps in vaccine formulation, only one targeted intracellular bacteria (listeria monocytogenes). the protective immune response against intracellular bacterial infections requires th1 activation and, therefore, apcs activation and ag presentation through major histocompatibility complex ii (mhc ii). to generate a th1 response, an aunp and listeria ag formulation were used in different strategies. although the authors tested direct vaccination, when dendritic cells (dc), in vitro loaded with aunp plus listeria ag, were adoptively transferred to a naïve animal, they induced th1, cd8+, and natural killer (nk) cells that provided better protection against l. monocytogenes than the traditional vaccine approach (23) . in evaluating vaccines developed with menps against viral infections, niikura et al. (20) (21) also evaluated the addition of cytosine and guanine linked by phosphodiester unmethylated (cpg/ dna) and found that it improved ab levels and animals' survival rates. another important feature of studies by niikura et al. (20) and chen et al. (15) was the use of various np sizes and the demonstration that all different np shapes were capable of inducing a humoral response. the levels of ab were size dependent, but the results were inconsistent: the first study found that a 40 nm sphere was the most efficient ab inducer and the second study found that the 8 nm and 12 nm spheres performed best. a special case of the use of menps was the use of nickelfunctionalized nanolipoprotein particles (ninlps) by yan et al. (28) and wadhwa et al. (27) in combination with hiv ag. ninlps are nanometer-sized nanolipoprotein particles with nickel incorporation into their surface in order to induce polyhistidine tagged proteins adsorption (29) . they demonstrated that specific igg (igg1 and igg2a) levels were greater than those obtained when alum was used in the formulation. fischer et al. (26) used truncated wnv envelope protein ag and found that a single dose vaccination induced a superior anti-wnv igg response and improved protection against a wnv challenge (26) . these responses were associated with nickel functionalization, described as a hapten, and triggered responses through activation of human tlr4 and intracellular transduction signals through myeloid differentiation primary response (myd-88), nuclear factor-κb (nf-κb), and mitogenactivated protein kinase (mapk), inducing pro-inflammatory responses [tumor necrosis factor (tnf)-α and interleukin (il)-8] (30, 31) . for protozoan infections, parween et al. (16) , using plasmodium falciparum merozoite surface protein subunit and aunps, evaluated the humoral immune response (igg1, igg2a, igg2b, and igg3) and found an intense igg1 response compared with the alum formulation (16). kaba et al. (14) , using p. berghei circumsporozoite protein and aunps, generated long-lasting protective immunity with th that produced il-2 and mixed high avidity igg1/igg2a (th2/th1) (14) . in other studies, these authors replaced ag with p. falciparum circumsporozoite protein; vaccination was shown to induce protective cytotoxic (cd8+) cells, high avidity ab titers, and specific effector memory, central memory, and long-term central memory cd8+ t cells in draining lymph nodes, spleen, and liver (18) . this response was shown to be generated by dc cross-presentation, which had delayed fusion and interaction of endosomes with lysosomes caused by the aunp formulation (19) . finally, pfmsp was used with dextrancoated iron oxide nps (ionps) and was capable of inducing a humoral response in two animal models (mouse and monkey). this response was also shown to inhibit parasite growth by 55-100% (25). most studies evaluated immunogenicity through measurement of the humoral immune response. according to their findings, the use of nps was efficient in inducing an ab-based response. based on heavy chain structure, there are five types of ab, each with a different role: igg, igm, iga, igd, and ige. igg and iga can be subdivided as igg1, igg2, igg3, igg4, iga1, and iga2 based on additional small differences in their heavy chain. with regard to vaccination, humoral immunity is especially important in responding to infection by extracellular pathogens, toxins, protozoa, and viruses. its importance is associated with the biological activities of immunoglobulins, including microorganism opsonization and phagocytosis; complement activation (32); toxins and microorganism neutralization (33) ; and mast cells and basophil activation (32, 34) . in addition, immunoglobulins can help target cytotoxicity against infected cells (ab-dependent cell cytotoxicity of cd8 t cells and nk). in some cases, however, the pathogens have the ability to evade the humoral system or can even use immunoglobulins as a way to facilitate cell invasion, as in the cases of mycobacterium tuberculosis and leishmania spp. (35, 36) . the studies described above clearly show that menps (gold, iron, and nickel) can be used for vaccine development. different menps were used in conjunction with several ag for distinct microorganisms and showed the ability to generate humoral and cytotoxic responses. although the generation of igg2a and ifn-γ shown in some studies are indicators of th1 responses using menps as adjuvant, further research is needed to specifically assess the role of different menp vaccines in th1 induction. to understand the possible uses of menps as platforms for vaccines against infectious diseases, analysis is needed of the impact of different physicochemical characteristics of nps on the innate immune response (figure 1) . several strategies have included menps as vaccine platforms, involving menps of different materials (including gold, iron oxide, and nickel); shapes (including spheres, cubes, rods, and disks); sizes (from 2 nm to over 200 nm); and types of coating [e.g., citrate, chitosan, dextran, or cetyltrimethylammonium bromide/4-styrenesulfonic acid-comaleic acid (ctab/pss-ma)]. the material from which an np is made has a direct influence on the functions of apcs; gold nps (aunps) have been most commonly used in vaccinology ( table 2) . the most recent studies involving aunps demonstrate the effects of gold sodium thiomalate on macrophage function, showing lysosomal enzyme inhibition and reducing phagocytosis (37) . similar effects were seen in macrophages of several origins, which, when stimulated with aunps, showed diminished bactericidal activity against staphylococcus aureus (38) and low or absent cytokine production il-6, il-10, and tnf-α (39, 40) . moreover, when splenocytes were stimulated with lps, the addition of aunp reduced il-17 and tnf-α release (40) . some of these results raise the concern on the use of aunps as adjuvants, since these immunomodulatory properties can act inhibiting the generation of th1. however, the response to aunps is also correlated with other physicochemical characteristics that will be discussed below, which may be tailored to improve immunostimulatory or immunomodulatory capacity. iron oxide nanoparticles have also been used as adjuvants. iron is an important ion in the homeostasis of all cells and in generating immune responses to several microorganisms. the effect of ionps phagocytosis have been explored in several studies, for example, m2 macrophages after exposure to ionps induced reactive oxygen species (ros), but after 24 h induced il-10 production (41) . the use of ionps in balb/c mice demonstrated the immunomodulatory capacity of this np by diminishing splenocyte cytokine production (il-4 and ifn-γ) (42) as well as suppressing the response to pancreatic ag in diabetic mice (43) . sindrilaru et al. (44) , however, showed that macrophages, under iron overloaded conditions, became unrestrained m1 (with an incomplete switch to m2 macrophages) and produced more tnf-α, which impaired wound healing and had an important role in the immunopathology of chronic venous leg ulcers. consequently, ionp response seems to have direct correlation with time and dose, once iron overload seems to be a requisite to developed pro-inflammatory response and this aspect must be evaluated to avoid the inhibition of the desired immune response. other critical characteristics are the shape and size of nps, which have a direct impact on vaccine efficiency, ag load capacity, and interaction with cells (phagocytes and apcs). these characteristics have been studied in different nps; shah et al. (45) published a review focusing on the impact of size for alum, oil-in-water, emulsion, polymeric particles, and liposome adjuvanticity, but did not evaluated menps. in the studies reviewed here, np sizes range from 2 nm nanospheres to 270 nm nanocapsules. two authors have evaluated the impact of size and shape for menps ( table 2) : chen et al. (15) evaluated differences in immune response based on aunp sizes (ranging from 2 to 50 nm nanospheres) and found that 8 and 12 nm were the most drained np (15); niikura et al. (20) went further and, using four different shapes of np (20 nm sphere, 40 nm sphere, cube, and rod), showed that ab responses and tnf-α were directly correlated with the specific np surface area (the ratio of the total surface area per single np volume). furthermore, 40 nm spheres appear to be the most efficient in generating immune responses (il-6 and il-12) and granulocyte macrophage colony-stimulating factor production. surface charge and hydrophobicity are additional important np characteristics for immune response induction and are directly influenced by np functionalization (chemical modification of nps surface by adding or replacing functional groups) and coating (ag) (46) . most studies used citrate-coated nps, but dextran and ctab/pss-ma have also been used; all three result in negatively charged (anionic) particles. only one np, revised here, used positive charged (cationic) functionalization [(22); table 2 ]. the higher hydrophobicity of aunp was shown to activate the innate immune system (tnf-α secretion) (47) . although the surface charge of other non-metallic nps has been studied (48) , to our knowledge the studies using menps did not address the other characteristics associated with immune response induction. for non-metallic nps, it appears that a positive charge signified a greater ability to induce immune responses than a negative charge. interestingly, negatively charged non-metallic nps were associated with ag-specific tolerance (48) . further studies are needed to investigate whether or not the charge imputed by np coating influences the immune response. though the size and shape of menps had little to no impact on the innate response elicited, coating modifications may improve the capacity of these molecules to influence immune responses. finally, it is important to note that the majority of adjuvant characteristics were evaluated using non-metallic nps. t-helper 1 cells are associated with immunity against intracellular pathogens and the secretion of ifn-γ, which, in turn, is essential for the activation of mononuclear phagocytes, including macrophages, resulting in enhanced phagocytic activity (49). th17 cells (il-17a and il-17f producer cells) are associated mainly with stimulation and chemotaxis of neutrophils to the site of inflammation. however, their function goes beyond this and includes the targeting of various cells types, including nonlymphoid cells and the stimulation of cytokine, chemokine, and prostaglandin production. another characteristic of these cells is their memory effector subset, which is maintained in mucosal tissues for extended periods. this subset has high plasticity and is able to transform into th1 or th2 phenotypes depending on the cytokine milieu at mucosal sites. this diversity of function and actuation make th17 cells very important in defense against several microorganisms, mainly those acquired through mucosal routes (49, 50) . t-helper 1 and th17 cells have their own distinct sets of functions and differentiation factors. both cell types require t cell receptor downstream activation by ag presentation cells through mhc ii and co-stimulatory molecules (6) . consequently, cytokine release during ag presentation is correlated with the type of adaptive immune response generated. while th1 differentiation requires stimulation by il-12, th17 generation requires transforming growth factor-β and il-6. however, this generation is influenced by other factors and how menp are involved in the possible induction of th1 or th17 will be discussed below. in this review, only one study investigated the development of the direct th1 (type 1 t helper cell) and th17 response. using a listeria ag, rodriguez-del rio et al. (23) showed that in contrast to advax™ adjuvant alone, a combination of 25 nm aunps and advax™ was capable of inducing the highest th1 response. pusic et al. (25) immunized mice with ionps covered with rmsp1, a p. falciparum merozoite ag, and showed that after immunization (intramuscular, subcutaneous, or intraperitoneal), production of il-4 was greater than that of ifn-γ, suggesting a predominant th2 response (although the cellular immune response was not directly evaluated). the first major determinant in generating th1 and th17 populations is the route of vaccine administration, which dictates the cell dynamic and initial response to the vaccine. for example, mohanan et al. (51) , in a cross-sectional study using a liposome plus ag (ova) vaccine formulation, compared intradermal (high igg1; intermediate igg2; and ifn-γ), intralymphatic (high igg1, igg2, and ifn-γ), intramuscular (high igg1; intermediate igg2 and ifn-γ), and subcutaneous (high igg1; low igg2 and ifn-γ) routes of administration (51) . the predominant th1 response to administration through the intradermal route was most likely due to the cooperation between langerhans cells, the primary innate immune response cells and keratinocytes that may also be stimulated by the formulation. these elicited the production of cytokines and chemokines that helped in the activation of other apcs (52) . the early phase of vaccination is characterized by recruitment of neutrophils and monocytes to the site of inoculation. both cell types can also act as apcs, delivering ag-specific and co-stimulatory signals to t cells. their collaborative endeavors have been found to modulate (positively or negatively) the activity of different effector t cell subsets (53, 54) . neutrophils are the first cell lineage to migrate to inflammation sites and, when stimulated, they produce cytokines and chemokines that will attract and activate other cell types. for example, neutrophils were shown to be an important inducer of th1 and th17 cells (55) , but their role in cytokine secretion is much broader (56) . moreover, signals may elicit different function in neutrophils and therefore, influence the quality of t cell responses. for example, aunps have been described as capable of inducing neutrophil extracellular traps, which act as damage-associated molecular patterns and stimulate immune system through dna receptors such as tlr9 (57) . upon stimulation by nps (tio2-titanium dioxide-and alum), duffin et al. (58) demonstrated neutrophil influx to the lungs and also induced production of il-18. silver nps were also shown to be capable of interacting with neutrophils, inducing apoptosis of these cells, and inducing caspase-1\ caspase-4 partially dependent il-1β secretion (59) . in another study, cobalt and nickel nps were shown to induce higher nitric oxide, tnf-α, and cxcl2 chemokine production, by human peripheral blood neutrophils, than titanium nps (tio2np) (60) . nonetheless, tio2nps also induced polymorphonuclear cell activation through phosphorylation of several proteins, including p38 mapk and extracellular signal-regulated kinases-1/2 (erk-1/2), which were associated with increased neutrophil life-span and production of several cytokines and chemokines (61) . classically, apcs, macrophages, and dcs act at the site of vaccine inoculation by sensing foreign agents, through tlrs and other receptors, and triggering inflammation. apcs play a key role in the initiation, maintenance, and selectivity of inflammation, through their three major functions: endocytosis, ag presentation, and production of various cytokines and growth factors (1) . the main family of pattern recognition receptors in microbial recognition is the tlrs, part of the family of transmembrane proteins, which affect the transcription of genes involved in inflammatory and immune response-enhancing cellular activities such as phagocytosis, endocytosis, cytotoxic functions, and cytokine production (62, 63) . the adjuvants most frequently used for the induction of th1 and th17 responses are tlr agonists, such as as04, cpg/ dna, and others. menps seems to have capacity to induce the expression of toll-like receptors, such as tio2nps and zirconium oxide nps that have been described to enhance tlr3, tlr7, and tlr10 expression in macrophages (64) and tlr2 and tlr4 in mouse liver cells (65) . zinc oxide nps (plus ova) generated an inflammatory response in balb/c mice and also improve tlr-2, -4 and -6 expression, followed by activation of src family kinases (66) . consequently, tio2nps and ionps were shown to induce dc upregulation of co-stimulatory molecules (mhc ii, cd80) (25, 67, 68) , which can also be related to tlr stimuli pathways. however, none of these works demonstrate the direct interaction of nps with tlr (using knock-out mice, agonists, or antagonist molecules) thus, this interaction must be further studied. the next step in the generation of adaptive responses is the tailoring of cytokine secretion by apcs at immunological synapses, which will guide the development of the response. several nps have been reported to trigger cytokine and chemokine production, which may be used as biomarkers for immunotoxicity (69). among those described, tio2nps were used in mimetic systems composed of blood vein endothelial component (including pbmc) and was reported to trigger pro-inflammatory cytokines (il-6, ifn-γ, and tnfα) (67); zinc oxide nps were shown to be preferentially associated with monocytes and, when used in pbmc, induced ifn-γ, tnf-α, and il-12 cytokine production (70); aunp-stimulated bone marrow-derived dc produced il-6, tnf-α, and ifn-γ (20) ; and ionps were shown to induce the activation of apcs with an increase of il-6, tnf-α, ifn-γ, and il-12, as well as chemokines. the response generated by ionps, however, was weaker than that generated by the positive control lps which may be beneficial in controlling possible side effects (25) . the generation of a cellular response associated with protection against intracellular pathogens is the ultimate goal of vaccination. however, the direct effects of nps on cellular responses have been evaluated in only a few studies. tio2nps were shown to activate and induce proliferation of naïve cd4+ t cells and to generate a pronounced th1 response with ifn-γ and tnf-α production, associated with pro-inflammatory cytokine production (il-6, il-1a, il-1b) and dc maturation (cd86+ and cd83+ expressions increase). schanen et al. (71) hypothesized that the oxidative capacity of an np could impact the response and trigger pro-inflammatory (oxidant capacity) or anti-inflammatory (antioxidant capacity) responses. this oxidant effect could control ros generation and thus control downstream pro-inflammatory effects while antioxidants prevent the initiation of the innate immunity in lps-stimulated macrophages (71) . this study was, however, conducted with mitogens (non-specific stimuli) and not with vaccine stimuli, but nevertheless serves as a warning about the direct action of nps, not only on the innate immune system but specifically on t cells. there is enough evidence to suggest that menps are not only particulate formulations but also immunostimulatory molecules with several studies demonstrating their capacity to generate humoral and cytotoxic responses. menps clearly have figure 2 | metallic nanoparticles adjuvanticity and its prediction capacity to generate t-helper 1 (th1) and th17 responses. to generate a cellular immune response, the np must be able to be recognized by the host innate immune response and stimulate a sequence of events that will lead to the release of a specific milieu of cytokines and better antigen presentation (bottom arrow). in the top arrow is the immune response elicited by metallic nanoparticles to aid th1 and th17 generation. nf-κb, nuclear factor kappa b; ccl, chemokine ligand; cxcl, chemokine (c-x-c motif) ligand; gm-csf, granulocyte macrophage colonystimulating factor; ifn, interferon; il, interleukin; m-csf, macrophage colony-stimulating factor; myd, myeloid differentiation factor; tcr, t cell receptor; th, t-helper cell; tlr, toll-like receptor; tnf, tumor necrosis factor. immunostimulatory capacity and can induce several reactions in all phases of vaccine development. these capabilities correlated with np physicochemical characteristics such as size, charge, and hydrophobicity, but there are several gaps in our understanding of their mechanism of actions and how they may lead to adjuvanticity, immunomodulation, or tolerance to the ag formulated with nps. there are also evidence of menp being capable of help in the generation of th1 and th17; figure 2 presents an overview of the generation of these cells subsets and the possible role of menp in this induction. ln designed the review and wrote the first draft. aj-k edited the first draft and critically reviewed the manuscript. ak edited the first draft and critically reviewed the manuscript. all authors read and approved the final version of the manuscript and agreed to submission. this work is part of ln phd thesis at biotechnology and biodiversity graduate program from capes. this work was funded by fapeg (grant number 20131026700-1143) and cnpq (grant number: 405198/2015-9). ln received a phd fellow from capes, and apjk (#303675/2015-2) and ak (#307186-2013-0) received a productivity research fellow from cnpq. key roles of adjuvants in modern vaccines novel adjuvant formulations for delivery of anti-tuberculosis vaccine candidates landmark green light for mosquirix malaria vaccine adjuvant system as01: helping to overcome the challenges of modern vaccines vaccine adjuvants: from 1920 to 2015 and beyond. vaccines (basel th1 and th17 cells: adversaries and collaborators different human vaccine adjuvants promote distinct antigen-independent immunological signatures tailored to different pathogens cpg 7909, an immunostimulatory tlr9 agonist oligodeoxynucleotide, as adjuvant to engerix-b hbv vaccine in healthy adults: a double-blind phase i/ii study analysis of adverse events of potential autoimmune aetiology in a large integrated safety database of as04 adjuvanted vaccines nanoparticle vaccines nanoparticles in medicine: current challenges facing inorganic nanoparticle toxicity assessments and standardizations a gold nanoparticle-linked glycoconjugate vaccine against burkholderia mallei protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine a nonadjuvanted polypeptide nanoparticle vaccine confers long-lasting protection against rodent malaria assessment of gold nanoparticles as a size-dependent vaccine carrier for enhancing the antibody response against synthetic foot-and-mouth disease virus peptide induction of humoral immune response against pfmsp-1(19) and pvmsp-1(19) using gold nanoparticles along with alum immunostimulatory effect of gold nanoparticles conjugated with transmissible gastroenteritis virus protective antibody and cd8+ t-cell responses to the plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine mechanisms of protective immune responses induced by the plasmodium falciparum circumsporozoite protein-based, self-assembling protein nanoparticle vaccine gold nanoparticles as a vaccine platform: influence of size and shape on immunological responses in vitro and in vivo gold nanoparticle-m2e conjugate coformulated with cpg induces protective immunity against influenza a virus enhanced mucosal immune responses against tetanus toxoid using novel delivery system comprised of chitosan-functionalized gold nanoparticles and botanical adjuvant: characterization, immunogenicity, and stability assessment a gold glyco-nanoparticle carrying a listeriolysin o peptide and formulated with advax delta inulin adjuvant induces robust t-cell protection against listeria infection induction of humoral immune response against pseudomonas aeruginosa flagellin(1-161) using gold nanoparticles as an adjuvant iron oxide nanoparticles as a clinically acceptable delivery platform for a recombinant blood-stage human malaria vaccine conjugation to nickel-chelating nanolipoprotein particles increases the potency and efficacy of subunit vaccines to prevent west nile encephalitis lipid nanocapsule as vaccine carriers for his-tagged proteins: evaluation of antigen-specific immune responses to hiv i his-gag p41 and systemic inflammatory responses lipid nanoparticles with accessible nickel as a vaccine delivery system for single and multiple his-tagged hiv antigens immobilization of his-tagged proteins on nickel-chelating nanolipoprotein particles crucial role for human toll-like receptor 4 in the development of contact allergy to nickel nickel allergies: paying the toll for innate immunity structure and function of immunoglobulins the function of immunoglobulin a in immunity regulation of mast-cell and basophil function and survival by ige a macrophage invasion mechanism of pathogenic mycobacteria immune adherence-mediated opsonophagocytosis: the mechanism of leishmania infection effect of in vivo administration of gold sodium thiomalate on rat macrophage function effects of gold compounds on function of phagocytic cells. comparative inhibition of activated polymorphonuclear leukocytes and monocytes from rheumatoid arthritis and control subjects effect of silica and gold nanoparticles on macrophage proliferation, activation markers, cytokine production, and phagocytosis in vitro selective inhibitory effects of 50-nm gold nanoparticles on mouse macrophage and spleen cells superparamagnetic iron oxide nanoparticle uptake alters m2 macrophage phenotype, iron metabolism, migration and invasion a single exposure to iron oxide nanoparticles attenuates antigen-specific antibody production and t-cell reactivity in ovalbumin-sensitized balb/c mice reversal of autoimmunity by boosting memory-like autoregulatory t cells an unrestrained proinflammatory m1 macrophage population induced by iron impairs wound healing in humans and mice the impact of size on particulate vaccine adjuvants surface functionalization of nanoparticles for nanomedicine nanoparticle hydrophobicity dictates immune response nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells different subsets of t cells, memory, effector functions, and car-t immunotherapy th17 cells in autoimmune and infectious diseases administration routes affect the quality of immune responses: a cross-sectional evaluation of particulate antigen-delivery systems enhanced antigen-specific antibody production following polyplex-based dna vaccination via the intradermal route in mice enhancement of adaptive immunity by the human vaccine adjuvant as01 depends on activated dendritic cells control of adaptive immunity by the innate immune system mouse neutrophils are professional antigen-presenting cells programmed to instruct th1 and th17 t-cell differentiation neutrophil-derived cytokines: facts beyond expression complexity of danger: the diverse nature of damage-associated molecular patterns proinflammogenic effects of low-toxicity and metal nanoparticles in vivo and in vitro: highlighting the role of particle surface area and surface reactivity silver nanoparticles rapidly induce atypical human neutrophil cell death by a process involving inflammatory caspases and reactive oxygen species and induce neutrophil extracellular traps release upon cell adhesion cytokine and no release from peripheral blood neutrophils after exposure to metal nanoparticles: in vitro and ex vivo studies activation of human neutrophils by titanium dioxide (tio2) nanoparticles innate immunity and vaccines role of fused mycobacterium tuberculosis immunogens and adjuvants in modern tuberculosis vaccines innate defence functions of macrophages can be biased by nano-sized ceramic and metallic particles signaling pathway of inflammatory responses in the mouse liver caused by tio2 nanoparticles zno nanoparticles induced adjuvant effect via toll-like receptors and src signaling in balb/c mice exposure to titanium dioxide nanomaterials provokes inflammation of an in vitro human immune construct activation of the inflammasome by amorphous silica and tio2 nanoparticles in murine dendritic cells cytokines as biomarkers of nanoparticle immunotoxicity the influences of cell type and zno nanoparticle size on immune cell cytotoxicity and cytokine induction immunomodulation and t helper th(1)/th(2) response polarization by ceo(2) and tio(2) nanoparticles the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-298604-tn8hc6jv authors: khawaja, akif a.; chong, deborah l. w.; sahota, jagdeep; mikolasch, theresia a.; pericleous, charis; ripoll, vera m.; booth, helen l.; khan, saif; rodriguez-justo, manuel; giles, ian p.; porter, joanna c. title: identification of a novel hif-1α-α(m)β(2) integrin-net axis in fibrotic interstitial lung disease date: 2020-10-15 journal: front immunol doi: 10.3389/fimmu.2020.02190 sha: doc_id: 298604 cord_uid: tn8hc6jv neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ild) particularly in the most severe form, idiopathic pulmonary fibrosis (ipf), although the underlying mechanisms remain unclear. neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. hypoxia has an important role in inflammation and may also contribute to pulmonary disease. we aimed to determine how neutrophil activation occurs in ild and the relative importance of hypoxia. using lung biopsies and bronchoalveolar lavage (bal) fluid from ild patients we investigated the extent of hypoxia and neutrophil activation in ild lungs. then we used ex vivo neutrophils isolated from healthy volunteers and bal from patients with ild and non-ild controls to further investigate aberrant neutrophil activation in hypoxia and ild. we demonstrate for the first time using intracellular staining, hif-1α stabilization in neutrophils and endothelial cells in ild lung biopsies. hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (pma)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (net) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). hypoxia also increased neutrophil expression of the α(m) (+3.1-fold, p < 0.001) and α(x) (+1.6-fold, p < 0.01) integrin subunits. interestingly, net formation was induced by α(m)β(2) integrin activation and prevented by cation chelation. finally, we observed net-like structures in ipf lung sections and in the bal from ild patients, and quantification showed increased cell-free dna content (+5.5-fold, p < 0.01) and mpo-citrullinated histone h3 complexes (+21.9-fold, p < 0.01) in bal from ild patients compared to non-ild controls. in conclusion, hif-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β(2) integrins. our results identify a novel hif-1αα(m)β(2) integrin axis in net formation for future exploration in therapeutic approaches to fibrotic ild. neutrophilic inflammation correlates with mortality in fibrotic interstitial lung disease (ild) particularly in the most severe form, idiopathic pulmonary fibrosis (ipf), although the underlying mechanisms remain unclear. neutrophil function is modulated by numerous factors, including integrin activation, inflammatory cytokines and hypoxia. hypoxia has an important role in inflammation and may also contribute to pulmonary disease. we aimed to determine how neutrophil activation occurs in ild and the relative importance of hypoxia. using lung biopsies and bronchoalveolar lavage (bal) fluid from ild patients we investigated the extent of hypoxia and neutrophil activation in ild lungs. then we used ex vivo neutrophils isolated from healthy volunteers and bal from patients with ild and non-ild controls to further investigate aberrant neutrophil activation in hypoxia and ild. we demonstrate for the first time using intracellular staining, hif-1α stabilization in neutrophils and endothelial cells in ild lung biopsies. hypoxia enhanced both spontaneous (+1.31-fold, p < 0.05) and phorbol 12-myristate 13-acetate (pma)-induced (+1.65-fold, p < 0.001) neutrophil extracellular trap (net) release, neutrophil adhesion (+8.8-fold, <0.05), and trans-endothelial migration (+1.9-fold, p < 0.05). hypoxia also increased neutrophil expression of the α m (+3.1-fold, p < 0.001) and α x (+1.6-fold, p < 0.01) integrin subunits. interestingly, net formation was induced by α m β 2 integrin activation and prevented by cation chelation. finally, we observed net-like structures in ipf lung sections and in the bal from ild patients, and quantification showed increased cell-free dna content (+5.5-fold, p < 0.01) and mpo-citrullinated histone h3 complexes (+21.9-fold, p < 0.01) in bal from ild patients compared to non-ild controls. in conclusion, hif-1α upregulation may augment neutrophil recruitment and activation within the lung interstitium through activation of β 2 integrins. our results identify a novel hif-1αα m β 2 integrin axis in net formation for future exploration in therapeutic approaches to fibrotic ild. the interstitial lung diseases (ild) are a group of diffuse parenchymal lung disorders that can result in pulmonary fibrosis (pf) (1) . despite recent advances in diagnostics and therapeutics, ild is still associated with substantial morbidity and mortality (2) . neutrophil activation may be important in ild, particularly the most severe fibrotic form, idiopathic pf (ipf). the pathogenesis of ipf is unknown but is thought to involve a "frustrated repair" response to repetitive epithelial injury, with associations to genes and proteins linked to epithelial function, integrity and repair. progressive epithelial damage, and abnormal wound repair leads to extensive scar formation and correlates, clinically, with worsening hypoxia. increasing desaturation during exercise (3) or sleep (4) is a significant predictor of mortality. further evidence from animal models suggests that hypoxia may actually contribute to a vicious cycle of disease progression (5) . this evidence has led to the view that hypoxia itself may contribute to worsening of pf but the mechanistic pathway is unknown. hypoxia, a state in which oxygen supply is inadequate for tissue demands, modulates gene expression via transcriptions factors called hypoxia inducible factors (hif). there are 3 members of the hif family, hif-1α, hif-2α, and hif-3α, which bind conserved dna sequences or hypoxia response elements (hre). although it seems plausible that the ipf lung is hypoxic much of the evidence is indirect. levels of lactic acid, a metabolite generated in response to hypoxia, are high in ipf lung tissue supporting the concept of a hypoxic microenvironment (6) and hif-1α and -2α have been shown, ex vivo, to be expressed in lung biopsies from patients with ipf, in some but not all reports (7, 8) . additional genomic studies in ipf patients show upregulation of hypoxia-related gene signatures, including tgfβ (9), the key fibrotic cytokine in pf, and of the hif-1α pathways (8, 10) . the contribution of neutrophils to ild has also been relatively less studied compared to other inflammatory and fibrotic diseases. early studies began to explore the potential role of neutrophils in ipf (11) (12) (13) (14) , however research focus has since shifted to other cell types. the number of neutrophils in the bronchoalveolar lavage (bal) fluid has been shown to predict both disease severity in ipf (15) and the development of pf in patients with hypersensitivity pneumonitis (16) . in addition, neutrophil extracellular traps (nets) have been shown to indirectly drive pf by stimulation of collagen production from fibroblasts in vitro (17) , and net release has been associated with pf in older mice in vivo (18) with loss of peptidyl arginine deiminase (pad)-4, a key neutrophil enzyme for net formation, being protective (18) . neutrophils are also associated with disease severity in acute lung injury and acute respiratory distress syndrome (ards) (19, 20) however, their precise contribution remains uncertain (21) . neutrophil depletion can ameliorate disease features in mouse models of ards (22) and a reduction in neutrophil infiltration (23) , or knock-down of neutrophil elastase (ne) attenuates fibrosis in bleomycin-induced mouse models of pf (24) . taken together, these studies implicate a contributory role of neutrophils to fibrotic ild. neutrophil survival is a tightly regulated process. prolonged survival can delay resolution of inflammation and can cause damage to surrounding cells and tissues; however, if apoptosis is premature, antimicrobial function can be compromised (25) . hypoxia drives neutrophil survival via hif-1α-dependent nf-κb activation (26) . in addition, hif-2α has also been shown to be important in regulating neutrophil function (27) . few reports address the effects of hypoxia upon net formation. inhibition of hif-1α can reduce net release (28) , whilst pharmacological stabilization of hif-1α increases phagocyte bactericidal activity (29) and net release (30) , implicating a role for down-stream targets of hif-1α in leukocyte function. given the importance of hypoxia and hif signaling in neutrophil function and the emerging role of neutrophils as key drivers of ild, we sought evidence for hypoxia and nets in the lungs of patients with ild and the functional effects of low oxygen levels upon ex vivo neutrophil function and activation. fiber-optic bronchoscopy with bal was performed in line with the american thoracic society guidelines (31) . bal was frozen for later analysis. none of the patients undergoing bronchoscopy had any infections at the time of procedure. bal was obtained from 11 patients with fibrotic ild and seven non-ild controls undergoing diagnostic bronchoscopies. demographics, clinical history and treatments at the time of sample collection are listed in table 1 . within the ild cohort: 4 (36%) had ipf, 3 (27%) had nonspecific interstitial pneumonia, 3 (27%) had chronic hypersensitivity pneumonitis (hp) and 1 (10%) had unclassifiable ild. our non-ild control group underwent diagnostic bronchoscopy due to: 5 (71%) investigation of haemoptysis, 1 (14.5%) right middle lobe collapse and 1 (14.5%) previous tracheal schwannoma patients undergoing yearly bronchial surveillance. only the ild group had lung function tests, as part of standard patient care. none of the patients recruited were taking anti-fibrotic drugs at the time of bronchoscopy. differential cell counts obtained from bal from patients are listed in supplementary table 1 . lung biopsies were collected as part of routine clinical care. ethical approval was given by the uk national research ethics committee (13/lo/0900). ihc was performed using the automated bond-max system (leica biosystems ltd., newcastle) with 4 µm ffpe sections. hif-1α (clone ep1215y, abcam, 1:600 dilution), myeloperoxidase (mpo) (polyclonal, dako, 1:300 dilution) or ne (clone np57, dako, 1:100 dilution) was incubated in epitope retrieval solution 2 for 20 min and stained using the 30, 20, 20 protocol. test antibodies were controlled clinical details were recorded for all subjects at the time of bronchoalveolar lavage. our ild cohort had a mean age of 69 ± 5.9 years and consisted of four patients with ipf, three patients with fibrotic nsip, three patients with chronic hp and one patient with unclassifiable ild. our non-ild control group had a mean age of 51 ± 10.1 years and consisted of five patients with haemoptysis, one patient with rml collapse and one patient with tracheal schwannoma. lung function was only obtained for the ild cohort as part of standard care. fvc, forced vital capacity; hp, hypersensitivity pneumonitis; ild, interstitial lung disease; ipf, idiopathic pulmonary fibrosis; nsip, nonspecific interstitial pneumonia; rml, right middle lobe; tlco, transfer factor for carbon monoxide. for using species-and isotype-matched control antibodies. slides were scanned on a nanozoomer digital slide scanner and images analyzed using ndp viewer software (hamamatsu corportation). a "blinded" reviewer analyzed five randomly selected areas from each subject. representative images were chosen from those selected. neutrophils were isolated as previously described (32) . in brief, neutrophils were isolated by percoll plus density centrifugation from sodium citrate anticoagulated blood obtained by informed consent from healthy volunteers. neutrophils were diluted to 2 × 10 6 neutrophils/ml in phenol-free rpmi (thermo scientific, uk) supplemented with 10% heat-inactivated fbs (thermo scientific, uk) and 2 mm l-gluatamine (lonza, uk). to induce hypoxia, neutrophils were cultured under 1% oxygen in a coy oxygen control glove box (coy laboratory products inc., usa) in a temperature controlled and humidified incubator. human umbilical cord vein endothelial cells (huvec) (lonza, switzerland) were cultured in endothelial growth media 2 (lonza, switzerland) supplemented with 10% fbs (thermo scientific, uk) and 2 mm l-glutamine (lonza, switzerland) and used at passage 5. for endothelial activation, huvec were treated with 10 ng/ml tnf-α (r&d systems, uk) for 24 h prior to experimentation. to induce hypoxia, huvec were cultured under 1% oxygen in a coy oxygen control glove box (coy laboratory products inc., usa) in a temperature controlled and humidified incubator. nets were quantified using the quanti-it tm picogreen r dsdna kit (invitrogen, uk) and using a capture elisa. streptavidin-coated plates (fisher scientific, uk) were coated with an anti-mpo capture antibody (abcam, uk) overnight at 4 • c and blocked with 0.5% bovine serum albumin for 1 h at 37 • c. neutrophil supernatants were incubated for 2 h at 37 • c. further 1 h incubations were performed with an anticitrullinated histone h3 detection antibody (abcam, uk) and hrp-conjugated secondary antibody (dako, uk). sureblue tmb microwell peroxidase substrate (kpl, uk) was then added and incubated in the dark at 37 • c for 20 min and then stopped by the addition of tmb stop solution (kpl, uk). absorbance was read at 450 nm using a tecan genios spectra fluor plate reader (tecan uk ltd., uk). nets were stained for immunofluorescence microscopy as described (32) using methodology modified from (33) . in brief, 5 × 10 5 neutrophils were added to fibrinogen-coated coverslips, stimulated for 4 h with 40 nm pma, 0.5 mm mncl 2 or varying concentrations of leukadherin-1 (la-1; sigma, uk), and fixed with 4% pfa. coverslips were blocked and sequentially incubated with an anti-histone h3 antibody (abcam, uk) and alexa fluor r 488-conjugated goat anti-rabbit igg secondary antibody (life technologies, uk). coverslips were washed, mounted, and sealed using with prolong tm gold antifade mountant with dapi (invitrogen, uk). slides were visualized using a zeiss axio imager.a1 inverted fluorescence microscope (zeiss, germany) and images analyzed using image j. lung sections were stained using a modified protocol based on published reports (34, 35) . five micrometer sections from paraffin-embedded lung biopsies from control or ipf patients were dewaxed prior to heat-induced epitope retrieval with tris-edta buffer, ph 9.0. sections were blocked with fc block (bd biosciences, uk) before incubation with a blocking buffer (5% goat serum/2.5% bsa/pbs/0.1% tween-20) for 1 h. slides were then washed and incubated with anti-citrullinated histone h3 (abcam, uk) and anti-mpo (r&d systems, uk) antibodies diluted in 0.5x blocking buffer overnight at 4 • c. anti-rabbit alexa fluor r 647-conjugated and anti-mouse alexa fluor r 555conjugated secondary antibodies (invitrogen, uk) and dapi (sigma, uk) diluted in 0.5x blocking buffer were then added for 30 min. stained sections were washed, mounted, sealed and visualized using an olympus inverted fluorescence confocal microscope and analyzed using fluoviewer software (olympus). bal fluid was filtered using a 40 µm cell sieve. bal cells were pelleted, counted and 1 × 10 5 viable cells were used to produce cytospin slides (thermo shandon cytospin 3, thermo scientific). cytospin slides were fixed in 4% pfa, washed, and blocked overnight in blocking solution (10% goat serum/1% bsa/2 mm edta/hbss/0.1% tween-20). slides were then washed and incubated with anti-histone h2a.x antibody (abcam, uk) for 1 h before washing. anti-rabbit alexa fluor r 488-conjugated secondary antibody (invitrogen, uk) and dapi (sigma, uk) were then diluted in blocking buffer for 1 h. stained slides were then washed, mounted, sealed and visualized using an olympus inverted fluorescence confocal microscope and analyzed using fluoviewer software (olympus). cell surface expression of neutrophil integrins was evaluated by flow cytometry. following isolation and culture under either normoxia or hypoxia, neutrophils were washed and resuspended in a sodium hepes buffer (20 mm hepes, 140 mm nacl, 2 mg/ml glucose, 0.3% bsa). cells were then stained using integrin subunit specific antibodies or appropriate isotype control for 30 min at room temperature. stained cells were then washed twice, fixed in 2% pfa and assessed using a facs verse (bd biosciences, uk). data was analyzed using flowjo (treestar inc., uk). huvec were cultured in 96-well black tissue culture plates (thermo scientific, uk). twenty-four hours prior to experimentation, huvec were subjected to normoxia or hypoxia in the absence or presence of 10 ng/ml tnf-α. neutrophil adhesion in response to 20 nm pma or 100 ng/ml lipopolysaccharide (lps) were measured as previously described (32) . briefly, neutrophils were cultured under normoxia or hypoxia for 1 h, labeled with 2 ′ ,7 ′ -bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresceinacetoxymethyl ester (life technologies, uk) and then added to wells under normoxia or hypoxia. fluorescence was measured using a tecan genios spectra fluor plate reader (tecan uk ltd., uk). adhesion was calculated by comparing the fluorescence of washed wells to initial fluorescence. trans-endothelial migration assays were performed as previously described (32) . in brief, huvec were grown on transwell inserts (millipore, uk). twenty-four hours prior to experimentation, huvec were cultured under normoxia or hypoxia in the absence or presence of 10 ng/ml tnf-α. neutrophils were cultured under normoxia or hypoxia for 1 h and then labeled with celltracker (invitrogen, uk). 1 × 10 6 neutrophils were added to the upper chamber of transwells and allowed to migrate in the absence or presence of 150 ng/ml il-8 in the lower chamber for 90 min. percent transmigration was calculated by comparing the number of cells in the lower chamber and the number of neutrophils added to the upper chamber. cell lysates (10 µg protein) were resolved by electrophoresis and transferred to a polyvinylidene fluoride membrane (ge healthcare, uk). membranes were blocked for 1 h in 5% skimmed milk/tbs/0.1% tween-20 and incubated with primary antibodies (1:1,000 dilution) overnight at 4 • c. membranes were then washed, incubated with hrp-conjugated secondary antibodies, and visualized using the luminata western hrp substrate system (millipore, ireland). data were evaluated using graphpad prism. data were tested for normality using a kolmogorov-smirnov test. in experimental data sets only comparing two groups, a mann-whitney test was performed or a wilcoxon matched pairs test. in data sets with two variables, data were assessed by two-way anova with a dunnet's or sidak's multiple comparison test. correlations were determined by two-tailed pearson correlation coefficients. a p value below 0.05 was considered significant. given reports of localized hypoxia in pulmonary disease (36) , biopsies from four patients with fibrotic ild, performed to determine a clinical diagnosis of etiology, were examined for evidence of hypoxia. in this representative patient, diagnosed with ipf, hif-1α staining demonstrated positive staining in the endothelium and polymorphonuclear cells, with very little staining in the fibrotic interstitium and overlying epithelium and no staining in control sections (figures 1a,b) . as aberrant net formation has been implicated in several immunopathologies, we also stained lung sections for mpo and ne (figures 1c,d) , highlighting the presence of neutrophils within the pulmonary vasculature. taken together, this staining pattern suggests that tissue-specific hypoxia and neutrophil recruitment may be a feature of the ild lung. these findings led us to examine the effects of hypoxia upon neutrophil function. pharmacological hif-1α stabilization has been reported to enhance bacterial killing and net release (28) (29) (30) , however, these studies were performed using atmospheric oxygen levels. we therefore assessed for any alteration in function, described below, of healthy neutrophils under normoxia (21% oxygen) and hypoxia (1% oxygen). first, we verified hypoxia by examining neutrophil cell lysates for the presence of hif-1α and hif-2α. we observed rapid stabilization of hif-1α under hypoxia, with delayed hif-2α stabilization (figure 2a) . having demonstrated induction of hypoxia, we assessed neutrophil supernatants for mpo-citrullinated histone h3 complexes, which are specific for nets. hypoxic neutrophils displayed greater levels of both spontaneous (+1.31-fold, p < 0.05) and pma-induced (+1.65-fold, p < 0.001) net release ( figure 2b) . as reactive oxygen species generation is thought to drive net formation (37, 38) , we also examined hydrogen peroxidase (h 2 o 2 ) production. rates of h 2 o 2 generation however, were comparable between oxygen states for both unstimulated (1. having found an effect on net release, we next examined integrin activation and neutrophil adhesion, which are also implicated in net induction (39) (40) (41) . we measured neutrophil adhesion to primary human endothelial cells in the absence or presence of pma (a general integrin activator) or lps (to mimic infectious stimuli), stimuli that induce nets via distinct pathways (42) . hypoxia increased both unstimulated (23.6 ± 4.0% vs. 2.7 ± 1.6%, p < 0.05) and lps-stimulated (35.7 ± 4.8% vs. 11.3 ± 1.4%, p < 0.05) adhesion to resting endothelium, whilst pma-stimulated adhesion, which was already high, was unaffected (figures 3a-c) . we then looked at adhesion to endothelium pretreated with tnf-α, to mimic an inflammatory event. whilst unstimulated neutrophil adhesion to tnf-α activated endothelial cells was not altered by hypoxia, there was a 3.22-and 2.11-fold increase in pma-(21.2 ± 6.3% vs. 68.1 ± 8.4%. p < 0.05) and lps-stimulated (23.2 ± 2.8% vs. 49.0 ± 2.3%, p < 0.05) adhesion, respectively (figures 3d-f) . next, we evaluated neutrophil trans-endothelial migration in the absence and presence of il-8, which has been shown to induce neutrophil migration. basal transmigration (in the absence of il-8) across both resting and tnf-α activated endothelium was unaffected by hypoxia. in contrast, in the presence of il-8, hypoxia enhanced neutrophil trans-endothelial migration across both resting and tnf-α activated endothelium (p < 0.05) (figures 3g,h) . hypoxia increases expression of neutrophil β 2 integrins, but not β 1 integrins given the role of integrins in leukocyte extravasation and reports documenting reduced nets following integrin blockade (39), we assessed surface integrin expression. whilst α l expression was unaffected (figure 4a) , significantly higher levels of α m (+3.1-fold, p < 0.001) and α x (+1.6-fold, p < 0.01) were observed under hypoxia (figures 4b,c) . there were no significant differences in β 2 expression (figure 4d) . hypoxia did not have an effect upon α 1 , α 4 , α 5 , or β 1 integrin subunit expression (figures 4e-h) . given reports of reduced net formation following integrin inhibition and our data showing increased α m β 2 and, to a lesser extent, α x β 2 integrin expression, we tested whether integrin engagement induced the release of nets, using an established model in which neutrophils adhere to fibrinogen. although there is some base-line adhesion (5.5%), stimulation with pma increases binding to 83.3% that can be blocked with specific α m β 2 inhibition (supplementary figure 1) . as expected, no nets were observed in unstimulated neutrophils, whilst pma stimulation induced the externalization of histone h3 to form net-like structures (figures 5a,b) . cation chelation through the use of edta, which abolishes integrinmediated adhesion, suppressed pma-induced net formation ( figure 5c) . finally, global integrin activation by means of manganese chloride treatment, induced some histone h3 externalization that could also be suppressed with edta treatment (figures 5d,e) . to confirm the role of α m β 2 , neutrophils were stimulated with la-1, a compound that specifically activates the α m β 2 integrin (43). la-1 stimulation showed a dose dependent effect upon neutrophil adhesion figure 3 | hypoxia enhances neutrophil adhesion and trans-endothelial migration. bcecf-am labeled neutrophil adhesion to endothelial monolayers over 30 min was assessed under normoxia (21% oxygen) and hypoxia (1% oxygen). we first examined neutrophil adhesion to resting huvec using (a) unstimulated neutrophils, (b) neutrophils stimulated with 20 nm pma and (c) cells stimulated with 100 ng/ml lps. next, we examined the effects of hypoxia upon neutrophil adhesion to activated huvec, which had been stimulated with 10 ng/ml tnf-α for 24 h prior to experimentation. we evaluated (d) unstimulated neutrophil adhesion, (e) neutrophil adhesion in response to 20 nm pma and (f) neutrophil adhesion in response to 100 ng/ml lps. finally, the effects of hypoxia upon trans-endothelial migration of celltracker tm green labeled neutrophils over 90 min was evaluated. data are presented as the mean ± sem from four independent experiments and analyzed by a wilcoxon matched pairs test. (g) neutrophil transmigration across resting endothelial monolayers was measured under both normoxia and hypoxia in the absence or presence of 150 ng/ml il-8. (h) huvec were stimulated with 10 ng/ml tnf-α for 24 h. neutrophil trans-endothelial migration was subsequently measured in the absence or presence of 150 ng/ml il-8 under normoxic or hypoxic conditions. data are presented as the mean ± sem from three independent experiments and analyzed by two-way anova with a dunnett's multiple comparison test. * = p < 0.05. hif, hypoxia-inducible factor; pma, phorbol 12-myristate 13-acetate. ( figure 6a) . whilst low concentrations of la-1 failed to induce the formation of net-like structures (figures 6b,c) , high levels of la-1 produced dna-histone structures similar to pma-stimulated cells (figures 6d-f) . these results confirmed that α m β 2 integrin activation can induce nets in human neutrophils. having found evidence of hypoxia within the ild lung and shown that hypoxia augments neutrophil activation, we evaluated lung tissue sections for evidence of nets. in non-ild control lung sections, we observed normal lung architecture with the absence of cellular infiltrates and lung tissue remodeling at both low and high magnification (figures 7a,b) . in contrast, we noted cellular infiltration in ipf lungs accompanied with the presence of mpo and histone citrullination (figures 7c,d) . co-localization of extracellular dna, mpo and citrullinated histones is suggestive of net formation within the ild lung. finally, we examined bal for evidence of neutrophil activation. we generated slides with bal and stained with dapi and citrullinated histone h2a to identify the presence of neutrophils initiating the production of nets. control bal neutrophils displayed punctate dapi staining with the absence of citrullinated histones (figures 8a,b) . in contrast, we observed the presence of citrullinated histones and more diffuse dna staining in bal polymorphonuclear cells obtained from patients with ild, indicative of neutrophils forming net-like structures (figures 8c,d) . we then obtained bal from 11 ild patients (ild-bal) and seven non-ild controls (control bal) and quantified levels of cell-free dna. we found ild-bal had 5.5-fold greater cell-free dna content compared to control bal (p < 0.01) (figure 8e) . cell-free dna content positively correlated with neutrophil counts (% of total cells) isolated from ild-bal (p = 0.0075) (figure 8f) , but not in control bal (figure 8g) . to verify that these were nets, we also examined bal for the presence of mpocitrullinated histone h3 complexes. similar to total cell-free dna, we observed significantly greater values in ild-bal (+21.9-fold, p < 0.01), indicating greater levels of nets ( figure 8h ). neutrophil dysfunction and aberrant activation have been implicated in the pathology of numerous diseases including autoimmune rheumatic diseases (44) (45) (46) (47) (48) and cancer (49) (50) (51) (52) . more recently the release of nets, essential for robust immune defense against pathogens, has also been linked to increased immunopathology in patients with covid-19, a disease characterized by neutrophilic inflammation and endothelial activation (53, 54) . the precise mechanism in which neutrophils contribute to ild pathogenesis is unknown. early work from the 1980s began to explore whether neutrophils might contribute to ipf pathology (11) (12) (13) (14) , however this avenue of research lost momentum. since then, reports have associated increased neutrophil migration and activation with severe pulmonary disease both in animal models (22, 55) and man (16, 19, 20) . we report, for the first time, that neutrophils and endothelial cells in ild lung biopsies display hif-1α expression and provide evidence of the extracellular release of nuclear dna, citrullinated histones and mpo, indicative of net formation in the ild lung. given the profound effects hypoxia exerts upon neutrophil survival and function (26, 27) , these findings led us to investigate whether hypoxia affects neutrophil extravasation and activation, thus contributing to ild pathology. integrins are adhesive molecules that enable leukocytes to interact with their external environment. similar to a previous report (56), we found increased β 2 integrin expression in neutrophils under hypoxia, but specifically found increased α m and, to a lesser extent, α x integrin subunit expression. interestingly, the α m β 2 and α x β 2 integrins also function as complement receptors, which may be relevant to ild pathology given that increased levels of complement c3a and c5a and roles for their receptors have been reported in ipf (57, 58) . moreover, studies using the bleomycin-induced mouse model of ipf highlight roles for both c3 and c5 in pulmonary fibrosis (59, 60) . upregulation of β 1 integrins has been described under hypoxia (61) , however, there are no reports assessing expression in neutrophils. a lack of effect may be explained by the relatively low β 1 integrin expression in human neutrophils. taken together, the evidence indicates that neutrophils predominately engage via β 2 integrins, a mechanism which is enhanced under hypoxia. early studies demonstrated that hypoxia enhances neutrophil adhesion to endothelial cells (62) , epithelial cells (63) , and trans-epithelial migration (64) . in support of these findings, our results show altered function of healthy neutrophils with increased neutrophil adhesion and trans-endothelial migration under hypoxia. in addition, we report that hypoxia enhances net formation. given that the gold standard markers or methods for the induction and detection of nets have not been established (65), we used several different techniques to confirm the release of nets by cultured neutrophils: the co-localization of nuclear dna and histone h3 complexes by immunostaining; figure 8 | bal isolated from ild patients contain more nets and net-releasing neutrophils. bal from ild patients and non-ild controls was examined for evidence of neutrophil activation. we first performed confocal microscopy to identify nets as defined by nuclear dna (dapi, blue staining) and citrullinated histone expression (citrullinated histone h2a, green staining). (a,b) non-ild control bal cells displayed punctate nuclear staining and lack of citrullinated histones, whilst (c,d) bal cells obtained from patients with ild showed degrees of dna externalization from polymorphonuclear cells, along with the presence of citrullinated histones indicative of neutrophils undergoing net release (white arrowheads). representative images from two non-ild or ild donors are shown and the size is denoted by the scale bar. (e) bal fluid was then assessed for the presence of nets by quant-it tm picogreen ® dsdna assay (thermo fisher, uk), which found that bal fluid from ild patients (n = 11) has significantly more cell free dna compared to non-ild controls (n = 8). from differential cell counts, we found that (f) cell free dna positively correlated with the proportion of bal neutrophils in patients with ild, (g) but not in non-ild controls. (h) finally, we also tested bal fluid using an optimized capture elisa detecting mpo-citrullianted histone h3 complexes, demonstrating that bal fluid from ild patients (n = 11) has significantly more nets compared to non-ild controls (n = 8). data were analyzed by either a mann-whitney test or two-tailed pearson correlation coefficients. ** = p < 0.01. bal, bronchoalveolar lavage; ild, interstitial lung disease. confocal imaging of dna, citrullinated histones and mpo; quantification of cell-free dna; and the detection of neutrophilderived proteins (mpo) and citrullinated histone h3 complexes. our observations complement studies in the literature showing that pharmacological hif-1α stabilization enhances net release and inhibition of hif-1α reduces nets and bactericidal activity (28, 29) . whilst hif-1α stabilization has been shown to promote net production (28) , the opposing effect of hypoxia upon net formation has also been described. in contrast to our findings, branitzki-heinemann et al. found hypoxia reduced levels of both spontaneous and pma-induced net release (66) . whilst the definitions of hypoxia and normoxia were identical (1% oxygen vs. 21% oxygen), key methodological differences may explain the contrasting conclusions. branitzki-heinemann et al. isolated neutrophils using gradient centrifugation with polymorphprep, whilst this study used percoll plus. whilst a minor difference, comparative analysis of isolation procedures found reduced cd15 and cd66b expression in neutrophils isolated with polymorphprep (67), which may have further implications on ex vivo function. both reports used pma to initiate net formation, however at different concentrations. in this study, neutrophils were stimulated with 40 nm pma for 4 h, whilst the earlier branitzki-heinemann study treated cells with 25 nm pma for 3 h. it is possible that stimulating neutrophils with a higher concentration of a potent pkc activator may account for the differing response to hypoxia. finally, when quantifying nets, branitzki-heinemann et al. seeded neutrophils on coverslips coated with poly-l-lysine whilst neutrophils in this study were exposed to either nunclontreated or fibrinogen-coated surfaces. this difference may result in varying degrees of α m β 2 engagement and alter neutrophil responses. our data suggest that α m β 2 engagement may induce net formation, which is supported by recent work demonstrating α m β 2 triggering net release (68) . moreover, several studies have shown that α m β 2 blockade reduced net release (39, 40, 69, 70) , which indirectly supports our work. our findings that α m β 2 interaction with ligand may regulate net formation builds on earlier findings, which showed that pma stimulation led to high levels (>80%) of chromatin decondensation (a prelude to net formation) and was not affected by substrate (41) . in contrast, lps stimulation led to lower basal levels of decondensation (∼20%) and levels increased with matrix stiffening and increased cell spreading on β 2 and β 1 integrin ligands. this effect was inhibited by pi3k inhibition suggesting a dependence on integrin outside-in signaling. the impact of matrix stiffness is highly relevant in fibrotic ild as lung fibrosis changes tissue elasticity. there are some methodological differences between this work and that of erpenbeck and colleagues. in particular, our neutrophils were rested in hypoxia or normoxia overnight before pma stimulation. our background net levels were lower in response to pma rather than the dramatic increase from <5 to >80% previously reported. it is possible that when the level of nets is lower, integrin activation related to matrix stiffness plays more of a role regardless of the stimulus. this finding further emphasizes the importance of a complete description of the experimental system (65) . this finding may have particular relevance not only to ild but also to other fibrotic lung diseases. the finding that hypoxia drive net formation may also be of relevance to non-fibrotic pathologies including covid-19, a disease characterized by severe hypoxia, net release and hyperinflammation (53, 71) . further work could build on the level of hypoxia required to produce these effects. in our study we used 1% oxygen, however, normal oxygen levels can range from 5.0 to 13.2% in the circulation and 0.5 to 2.7% in tissues (72, 73) . further experiments could determine whether lesser degrees of hypoxia, seen in clinical practice, also enhance neutrophil adhesion, trans-endothelial migration and net formation. these studies could determine the relative importance of hif transcription factors to net formation and release, through the use of previously identified small molecule inhibitors (74) (75) (76) , and better understand the relationship between neutrophil activation and hypoxia. we observed tissue-specific hif-1α expression in ild lung tissue, mainly restricted to pulmonary endothelial cells and neutrophils, with only minimal upregulation in areas of epithelium and fibrosis, which may hold pathological relevance. previously, markers of hypoxia have been variably reported in the epithelium of patients with ipf. several authors have found hif-2α and ca-ix within the ipf fibrotic reticulum and hif-1α in the overlying epithelium with ihc (7), [albeit sometimes in a single patient (8) ]. hif-1α is more readily found in the mouse bleomycin model of pf raising the question of differences between the two species and insults (77) . whilst epithelium-specific hif-1α deletion has no effect upon radiation-induced enteritis, mice with endothelium-specific hif-1α deletions present with reduced intestinal damage (78) . hif-1α is known to contribute to the pathology of pulmonary hypertension (79, 80) , with some work specifically interrogating endothelial hif signaling (81) . neutrophilic inflammation has also been associated with pulmonary hypertension (82) , and believed to drive angiogenesis via net release (83) . therefore, the pulmonary pathology in ild patients may in part be attributed to endothelial and neutrophil hif-1α expression enhancing neutrophil recruitment and net formation within the lung. in this paradigm, enhanced nets would initiate angiogenic signals and drive lung pathology. interestingly, the model of neo-angiogenesis underlying ild pathology has attracted interest, and the powerful angiogenic inhibitor, nintedanib, shown to have therapeutic benefits in a range of fibrotic ilds (84) and endothelial reactivity with angiogenesis is also noted in covid-19. our findings of hypoxia driving net formation complements the increasing evidence that nets may play a role in many acute and chronic lung diseases (85) , including ild by stimulation of fibroblasts (17) . in pf, we propose that elevated net release may cause epithelial cell damage, dysfunction and death, drive innate and adaptive immune cells activation, and promote a pro-fibrotic environment that ultimately facilitates the progression of pulmonary fibrosis. for our experiments, we used neutrophils isolated from peripheral blood donated from healthy volunteers. further work could examine cells isolated from patients with ild, isolated from either peripheral blood or bal, to determine whether hypoxia has similar or an enhanced effect within this patient population. in addition, it would also be interesting to examine neutrophil function using autologous human serum from patients with ild to provide further insight under more physiological conditions in vitro. hypoxia is also thought to have differential integrinindependent effects upon net formation (66) . further experiments exploring the effects of hypoxia upon neutrophil activation by examining neutrophil responses to a wider range of stimuli, such as bacterial/fungal antigens, ionomycin or monosodium urate crystals, which also activate β 2 integrins (86), may therefore provide further insight into the relationship between neutrophil function, integrin activation and hypoxia. interestingly, the recent consensus article written by opinion leaders to present prevailing concepts and state of the science in net-related research and elaborate on open questions and areas of dispute does not specify which stimulus should be used to induce net formation (65) . instead, this consensus article suggests that researchers should specify in detail culture conditions, including base medium, use of serum, absence of platelets and surface constitution of the cell culture plate, as well as stimulus and source/preparation of inducer used. our analysis of net formation ultimately focuses on the end stages culminating in net release into cell supernatants. further work could explore the effects of hypoxia upon the preceding stages such as cell spreading and chromatin decondensation/nuclear swelling (41, 87) , to better understand how hypoxia affects the entire net formation process. in conclusion, we report that the ild lung contains molecular features of hypoxia, mainly localized to neutrophil and endothelial cells, which may contribute to disease pathology. hypoxia enhanced neutrophil β 2 integrin expression, which translated to augmented adhesion and migration across endothelial cells, and net release. our findings are further supported by demonstration of nets within the human fibrotic lung seen through imaging of ipf lung sections and bal cells, as well as detecting cell-free dna and mpo/citrullinated histone complexes in bal obtained from patients with ild. taken together, our work begins to elucidate a potential role of hypoxia in driving neutrophil recruitment and activation within the airspace to promote a pro-fibrotic environment. these findings offer a rationale for future translational medicine exploration of a novel neutrophil hif-1α-integrin axis as a potential therapeutic target in fibrotic ild. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by uk & national research & ethics committee. the patients/participants provided their written informed consent to participate in this study. ak designed and performed experiments, analyzed the data, and contributed to writing the manuscript. dc, js, and hb obtained and analyzed bal samples and performed microscopy. tm performed lung cyrobiopsies that were used for lung staining. sk and mr-j performed and analyzed ihc images. cp and vr contributed to experimental design and analysis. ig and jp conceived the study, designed the experiments, were involved in data analysis, and contributed to the writing of the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by a ucl impact studentship, the rosetrees trust (m136) and breathing matters, and undertaken at uclh/ucl who received a proportion of funding from the department of health's nihr biomedical research centres funding scheme. jp was funded by an mrc new investigator research grant (mr/k004158/1). open access publication fees were from ucl. we would like to acknowledge professor margaret ashcroft and dr. luke thomas (university of cambridge) for advice and input on hif, dr. andrew smith and professor tony segal (university college london) for their help with the neutrophil h 2 o 2 generation assay, professor nancy hogg (the francis crick institute) for reagents and helpful discussions, professor mikala egeblad and dr. jean albrengues (cold spring harbor laboratory, usa) for their help with staining lung tissue sections, and dr. guiseppe ercoli (university college london) who helped with confocal imaging. this manuscript has been released as a pre-print at biorxiv (88) . interstitial lung disease update in interstitial lung disease 2016 physical activity and exertional desaturation are associated with mortality in idiopathic pulmonary fibrosis sleep oxygen desaturation predicts survival in idiopathic pulmonary fibrosis galectin-1 inhibition attenuates profibrotic signaling in hypoxia-induced pulmonary fibrosis lactic acid is elevated in idiopathic pulmonary fibrosis and induces myofibroblast differentiation via ph-dependent activation of transforming growth factor-beta mir-210 promotes ipf fibroblast proliferation in response to hypoxia integrated genomics reveals convergent transcriptomic networks underlying chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis profibrotic up-regulation of glucose transporter 1 by tgf-beta involves activation of mek and mammalian target of rapamycin complex 2 pathways a system biology study of balf from patients affected by idiopathic pulmonary fibrosis (ipf) and healthy controls mechanisms of neutrophil accumulation in the lungs of patients with idiopathic pulmonary fibrosis role of proteolytic and oxidative products of the neutrophil in determining the specificity of the pulmonary lesion in fibrotic and destructive lung disease familial idiopathic pulmonary fibrosis. evidence of lung inflammation in unaffected family members pharmacologic suppression of the neutrophil component of the alveolitis in idiopathic pulmonary fibrosis peripheral blood proteins predict mortality in idiopathic pulmonary fibrosis increase of lung neutrophils in hypersensitivity pneumonitis is associated with lung fibrosis neutrophil extracellular traps promote differentiation and function of fibroblasts peptidylarginine deiminase 4 promotes age-related organ fibrosis g-csf and il-8 but not gm-csf correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome neutrophils and acute lung injury the mercurial nature of neutrophils: still an enigma in ards? neutrophils as early immunologic effectors in hemorrhage-or endotoxemia-induced acute lung injury doxycycline attenuated pulmonary fibrosis induced by bleomycin in mice mice lacking neutrophil elastase are resistant to bleomycin-induced pulmonary fibrosis cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis hypoxia-induced neutrophil survival is mediated by hif-1alpha-dependent nf-kappab activity hypoxia-inducible factor 2alpha regulates key neutrophil functions in humans, mice, and zebrafish mammalian target of rapamycin regulates neutrophil extracellular trap formation via induction of hypoxia-inducible factor 1 alpha pharmacologic augmentation of hypoxia-inducible factor-1alpha with mimosine boosts the bactericidal capacity of phagocytes iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (nets) in human blood-derived neutrophils an official american thoracic society clinical practice guideline: the clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease autoimmune rheumatic disease igg has differential effects upon neutrophil integrin activation that is modulated by the endothelium neutrophil extracellular traps kill bacteria cancer cells induce metastasis-supporting neutrophil extracellular dna traps immunofluorescence labelling of human and murine neutrophil extracellular traps in paraffin-embedded tissue hypoxia and tissue destruction in pulmonary tb rapid killing of human neutrophils by the potent activator phorbol 12-myristate 13-acetate (pma) accompanied by changes different from typical apoptosis or necrosis novel cell death program leads to neutrophil extracellular traps beta2 integrin mediates hantavirus-induced release of neutrophil extracellular traps synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation effect of adhesion and substrate elasticity on neutrophil extracellular trap formation jnk activation turns on lps-and gram-negative bacteriainduced nadph oxidase-dependent suicidal small molecule-mediated activation of the integrin cd11b/cd18 reduces inflammatory disease leukotriene b4 plays a critical role in the progression of collagen-induced arthritis essential role of neutrophils in the initiation and progression of a murine model of rheumatoid arthritis visualization and in situ analysis of leukocyte trafficking into the ankle joint in a systemic murine model of rheumatoid arthritis a distinct subset of proinflammatory neutrophils isolated from patients with systemic lupus erythematosus induces vascular damage and synthesizes type i ifns low-density granulocytes: a distinct class of neutrophils in systemic autoimmunity tumor-recruited neutrophils and neutrophil timp-free mmp-9 regulate coordinately the levels of tumor angiogenesis and efficiency of malignant cell intravasation a cxcl1 paracrine network links cancer chemoresistance and metastasis tumor-associated neutrophils as a new prognostic factor in cancer: a systematic review and meta-analysis the role of neutrophils in cancer targeting potential drivers of covid-19: neutrophil extracellular traps pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 acid aspirationinduced lung injury in rabbits is mediated by interleukin-8-dependent mechanisms leukocyte adhesion during hypoxia is mediated by hif-1-dependent induction of beta2 integrin gene expression crosstalk between tgf-beta1 and complement activation augments epithelial injury in pulmonary fibrosis contribution of the anaphylatoxin receptors, c3ar and c5ar, to the pathogenesis of pulmonary fibrosis the relationship between complement c3 expression and the muc5b genotype in pulmonary fibrosis opposing regulatory roles of complement factor 5 in the development of bleomycin-induced pulmonary fibrosis selective induction of integrin beta1 by hypoxia-inducible factor: implications for wound healing adhesion of flowing neutrophils to cultured endothelial cells after hypoxia and reoxygenation in vitro hypoxia mediates increased neutrophil and macrophage adhesiveness to alveolar epithelial cells epithelial exposure to hypoxia modulates neutrophil transepithelial migration to net or not to net:current opinions and state of the science regarding the formation of neutrophil extracellular traps formation of neutrophil extracellular traps under low oxygen level impact of human granulocyte and monocyte isolation procedures on functional studies mac-1 triggers neutrophil dna extracellular trap formation to aspergillus fumigatus independently of pad4 histone citrullination regulation of extracellular chromatin release from neutrophils release of neutrophil extracellular traps by neutrophils stimulated with antiphospholipid antibodies: a newly identified mechanism of thrombosis in the antiphospholipid syndrome neutrophil extracellular traps in covid-19 differential effects of physiologically relevant hypoxic conditions on t lymphocyte development and effector functions comparison of tumor and normal tissue oxygen tension measurements using oxylite or microelectrodes in rodents identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway identification of novel small molecule inhibitors of hypoxia-inducible factor-1 that differentially block hypoxia-inducible factor-1 activity and hypoxiainducible factor-1alpha induction in response to hypoxic stress and growth factors echinomycin, a small-molecule inhibitor of hypoxia-inducible factor-1 dna-binding activity comparative expression profiling in pulmonary fibrosis suggests a role of hypoxia-inducible factor-1alpha in disease pathogenesis hif-1alpha deletion in the endothelium, but not in the epithelium, protects from radiation-induced enteritis regulation of hypoxia-induced pulmonary hypertension by vascular smooth muscle hypoxia-inducible factor-1alpha klf5 mediates vascular remodeling via hif-1alpha in hypoxic pulmonary hypertension endothelial hif signaling regulates pulmonary fibrosis-associated pulmonary hypertension exploratory analysis of the neutrophil to lymphocyte ratio in patients with pulmonary arterial hypertension neutrophil extracellular traps promote angiogenesis: evidence from vascular pathology in pulmonary hypertension potential of nintedanib in treatment of progressive fibrosing interstitial lung diseases the emerging role of neutrophil extracellular traps in respiratory disease crystal-induced neutrophil activation vi. involvment of fcgammariiib (cd16) and cd11b in response to inflammatory microcrystals chromatin swelling drives neutrophil extracellular trap release identification of a novel hif-1α-αmβ2 integrin-netosis axis in fibrotic interstitial lung disease. biorxiv conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.02190/full#supplementary-material key: cord-271419-v6dfel3l authors: adachi, shun; koma, takaaki; doi, naoya; nomaguchi, masako; adachi, akio title: commentary: origin and evolution of pathogenic coronaviruses date: 2020-04-21 journal: front immunol doi: 10.3389/fimmu.2020.00811 sha: doc_id: 271419 cord_uid: v6dfel3l nan one of major study concerns in virology is viral adaptive evolution due to its highly replicable and mutable nature in changeable environments. some viruses with this property are severely pathogenic for animals and also for humans. virologists thus must prepare the ground for clinical applications of their findings obtained by structural and functional analyses on viruses. among viruses, some coronaviruses (covs) are notorious for causing the severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). markedly, the very now covid-19 outbreak is brought about by a new coronavirus designated sars-cov-2 (1-3). in this commentary, we focus on the titled review article and mainly introduce evolutionary aspects of the coronaviruses. the said article has successfully predicted today's covid-19 outbreak by pointing out that novel pathogenic variants will readily emerge from very diversified severe acute respiratory syndrome-related coronaviruses (sarsr-covs) of the bat origin through their close coexistence and high genetic recombination ability (figure 1) . therefore, it is very appropriate and timely to introduce this excellent review article here in the general commentary article. as basal knowledge for coronaviruses, they form an envelope structure at the outer surface of virions and their morphology is spherical with 100-160 nm in diameter. their genome is positivesense (+) single-stranded (ss) rna and 27-32 kb in size. coronaviruses have extra accessory genes in addition to those for viral structural proteins. following entry into cells via the specific interaction of viral envelope glycoprotein spike (s) and cellular receptor, coronaviruses replicate in the cytoplasm as the other ssrna (+) viruses. for alpha-and beta-coronaviruses, they are originated in highly metabolized mammals, such as bats and rodents. after being spilled over to alpacas, cows, civets, camels, or pigs, they can also infect humans and frequently cause sars and mers. gamma-and delta-coronaviruses mainly infect birds, but they sometimes infect mammals. molecular phylogenetic trees well support this idea of classifications. among several genes on the genome of interest, an evolutionary biologist tends to focus on a functionally conserved but sequentially diverged gene. this is because the role for the critical gene is conserved in various species, while it is rapidly evolving, indicating that a diverging force acts on the gene, e.g., by co-evolution such as symbiosis, evolutionary arms race, or others. of the cov genes, this review has centered on structural gene s and extra genes orf3/orf8 that encode s and accessory proteins, respectively. importantly, the most frequently observed hotspots for recombination are within s gene and upstream region of orf8 gene. orf8 of sars-cov is assumed to be acquired from sarsr-cov by recombination, and is positively selected (4). s protein contains the receptorbinding domain (rbd) critical for infection, and orf3/orf8 proteins function viral species-specifically, e.g., by prescribing the virulence (orf8), anti-interferon activity (orf3/orf8) or others. because orf3/orf8 are different between sars-cov and sars-cov-2, they might contribute to the difference in their virulence (5) . additionally, s and orf3 genes are positively selected in civet sarsr-covs (4). since rna viruses are easy to mutate and coronaviruses have high potentials for recombination, we can easily see the track of mutations and evolutions of the viruses, especially for sars-cov and mers-cov. rna recombination by rna-dependent rna polymerase with a low fidelity is widely observed and is supposed to shape current viruses by rearranging their genomes or disseminating functional modules (6) . for coronaviruses such as mouse hepatitis virus (mhv), secondary structures of rna genome are responsible for highly dynamic spike structures (7) . these might be an evolutionary cradle from "rna world, " still function in the living organisms nowadays. the non-processive replicase-driven template switching mechanism proposed among coronaviruses (8) thus is a suitable model for the evolution of rnabased replicating system. among many viruses, coronaviruses (recombination frequency for the total genome in vivo is 25%) and picornaviruses are champions of the rna recombination frequency (8) . let us move to the evolution of coronaviruses (figure 1 ). in detail for each gene of the viruses noticed, orf8 is wellknown for viral evolution and the accompanied increase of virulence observed during the late onset of sars. for the rbd in s, sars-cov utilizes ace2 as a cellular receptor for infection, and mers-cov utilizes dpp4 as the receptor. the receptor recognition is important for infection process for the viruses. different use of the receptors among the coronaviruses is due to their sequences/structures of rbd. nonetheless, because of the common nature of rbd (9) , it can be a promising target for development of novel antiviral compounds and antibody therapies for these viruses. however, it is also true in some cases, viruses went beyond the arms race and ingeniously evolved to counteract the clinical strategy. for example, the strategy is applicable for sars-cov wiv1 strain but not for shc014 and hku3. hku3 is remarkable for its truncated form of rbd. for mers-cov, cells expressing suboptimal bat species-derived variant of dpp4 force the viruses to accumulate mutations in the viral spike during passages, resulting in enhanced viral entry merely with two amino acid mutations (10) . the type of adaptation phenomena in virus evolution is testable either in clinical medicine or in vitro evolution system. thus, to consider the origin of new pathogens and the prevention of their transmission to humans, and control of the viruses, not only studies on sars-cov, mers-cov, and sars-cov-2, but also those on their relatives sarsr-covs and mersr-covs are recommendable for bats tracked for the ecology and evolution. we better understand the interaction networks among viruses of the evolution and diversification by their detailed comparison (figure 1) . the review mentions yunnan sarsr-covs might be the origins of the sars-covs, as symbionts, while domestication activity for mammals affects acquisition of pathogenicity to humans [refer also to banerjee et al. (11) ]. both fieldworks and experimental biology are required to understand the viruses concomitantly with predicting or preventing potential outbreaks. sa and aa conceived the idea. sa wrote the manuscript. tk, nd, mn, and aa reviewed the manuscript. tk depicted figure 1. all authors approved its submission genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding genome composition and divergence of the novel coronavirus (2019-ncov) originating in china a novel coronavirus from patients with pneumonia in china severe acute respiratory syndrome (sars) coronavirus orf8 protein is acquired from sars-related coronavirus from greater horseshoe bats through recombination sars-cov-2 and covid-19: the most important research questions new insights into the mechanisms of rna recombination generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted rna secondary structure rna recombination in animal and plant viruses functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses adaptive evolution of mers-cov to species variation in dpp4 bats and coronaviruses we thank ms. fumie nishina (kansai medical university, osaka, japan) and ms. kazuko yoshida (tokushima university, tokushima, japan) for editorial assistance. key: cord-303017-4zx94rm6 authors: barbieri, antonio; robinson, nirmal; palma, giuseppe; maurea, nicola; desiderio, vincenzo; botti, gerardo title: can beta-2-adrenergic pathway be a new target to combat sars-cov-2 hyperinflammatory syndrome?—lessons learned from cancer date: 2020-09-30 journal: front immunol doi: 10.3389/fimmu.2020.588724 sha: doc_id: 303017 cord_uid: 4zx94rm6 sars-cov-2 infection is a new threat to global public health in the 21(st) century (2020), which has now rapidly spread around the globe causing severe pneumonia often linked to acute respiratory distress syndrome (ards) and hyperinflammatory syndrome. sars-cov-2 is highly contagious through saliva droplets. the structural analysis suggests that the virus enters human cells through the ligation of the spike protein to angiotensin-converting enzyme 2 (ace(2)). the progression of covid-19 has been divided into three main stages: stage i—viral response, stage ii—pulmonary phase, and stage iii—hyperinflammation phase. once the patients enter stage iii, it will likely need ventilation and it becomes difficult to manage. thus, it will be of paramount importance to find therapies to prevent or slow down the progression of the disease toward stage iii. the key event leading to hyperinflammation seems to be the activation of th-17 immunity response and cytokine storm. b(2)-adrenergic receptors (b(2)ars) are expressed on airways and on all the immune cells such as macrophages, dendritic cells, b and t lymphocytes. blocking (b(2)ar) has been proven, also in clinical settings, to reduce th-17 response and negatively modulate inflammatory cytokines including il-6 while increasing ifnγ. non-selective beta-blockers are currently used to treat several diseases and have been proven to reduce stress-induced inflammation and reduce anxiety. for these reasons, we speculate that targeting b(2)ar in the early phase of covid-19 might be beneficial to prevent hyperinflammation. coronaviruses are a variable group of enveloped, positive-sense, single-stranded rna viruses (1) . they cause several diseases involving respiratory, enteric, hepatic, and neurological systems with high severity among humans and animals (1, 2) . in the last two decades, the entire world witnessed two novel types of coronavirus, severe acute respiratory syndrome cov (sars-cov) and middle east respiratory syndrome cov (mers-cov), causing severe human diseases (3, 4) . in december 2019 an outbreak of pneumonia of unknown cause occurred in wuhan, hubei province, china that rapidly spread around the globe, reaching to pandemic dimensions, and it was later named as sars-cov-2 or covid19 (5, 6) . the main symptoms of covid-19 include fever, fatigue, and cough, which are similar to that of sars-cov and mers-cov infected cases. sars-cov-2 is closely related to other coronaviruses (88% identity) which likely originated from bats. sars-cov-2 enters the human cells through ace 2 receptors after ligation of spike protein. these receptors are highly expressed in the lung but are also found in other organs such as the heart, kidney, endothelium, and intestine (7) (8) (9) . notably, ace 2 is highly expressed on the luminal surface of intestinal epithelial cells, functioning as a co-receptor for nutrient uptake, in particular for amino acid resorption from food (10) . for this reason, the intestine might also be a putative entry site for sars-cov-2 and that the infection might have been initiated by eating food from wild animals. to date the virus has infected hundreds of thousands of people, and about half of the hospitalized patients show clinical signs of comorbidities such as hypertension (23.7-30%), diabetes mellitus (16.2%), coronary heart diseases (5.8%), and cerebrovascular disease (2.3%). also, a high rate of mortality has been reached when one or more comorbidities coexist. covid-19 infection associated symptoms, including acute respiratory distress syndrome (ards) and septic shock seem to be associated with hyperinflammation and cytokine storm as in patients' serum the levels of several cytokines are elevated (11) . in particular il-6, il-1b, il-10, tnf, gm-csf, ip-10 (ifninduced protein 10), il-17,mcp-3, and il-1ra are mainly involved both in mild and severe forms of disease and altered circulating leukocyte subset and cytokine secretion (12) . as many of these cytokines are involved in the th17 type response, wu and yang suggested that targeting the th17 pathway may counteract the covid-19 symptoms (13). this hypothesis relies on different pieces of evidence: il-6, tnfa, and il-1b promote th17 response and are associated with inflammatory symptoms including fever, and the two latter are also associated with vascular permeability and leakage; il-17 has a broad inflammatory effect and together with gm-csf is involved in inflammatory and autoimmune disease; covid-19 patients have a significantly increased number of ccr6+ th17 cells (4) ; elevated th17 and il-17 related pathways are increased in sars-cov, mers-cov, and h1n1 influenza virus patients (14) (15) (16) ; in mers-cov patients, il-17 and low ifng are associated with worse prognosis (14) . targeting beta-2adrenergic pathway was shown to reduce inflammatory cytokine and th17 response in different settings such as cancer and autoimmune diseases. for this reason we believe that beta-2adrenergic pathway should be more deeply investigated as a possible target to reduce inflammation-related symptoms of sars-cov2. b-adrenergic receptors are g-protein coupled transmembrane proteins. there are three subtypes of b-adrenergic receptors (b1-ars, b2-ars, and b3-ars) that mediate a wide range of physiological responses to catecholamines epinephrine and norepinephrine, and thus play an important role in regulating cardiovascular responses in health and disease. b-adrenoceptors regulate many aspects of airway function, including airway smooth muscle tone, mast cell mediator release, and plasma exudation. b1-adrenergic receptors are located mainly in the heart and in the kidneys (17, 18) . b2-adrenergic receptors are located in the lungs, gastrointestinal tract, liver, uterus, vascular smooth muscle, and skeletal muscle (17, 18) . b3-adrenergic receptors are located in fat cells. more than 90% of all breceptors in the human lung are located in the alveoli where the b2-subtype predominates (70%) (19) . however, b1and b2subtypes also coexist and are distributed uniformly in the alveolar walls. b2-adrenergic receptors are expressed by all the cells of the immune system, including t and b lymphocytes, dendritic cells (dcs) and macrophages (20) . the specific role of adrenergic signaling in regulating immune responses and inflammation is still under debate. however, there are many pieces of evidence supporting a proinflammatory action and promotion of th17 response. manni et al. showed that triggering b2-adrenergic signal stimulates murine dc to secrete il-6 and promote a th17 response (21) . in dc, the b2adrenergic signal also reduces il-12 and ifng production but promotes il-17. b2-ar signaling plays a pivotal role in macrophage activation and proinflammatory cytokine production. similarly, in other immune cells, the overall effect of b2-adrenergic stimulation is an exacerbation of inflammation, promotion of b cell antibody production, and stimulation of dc and macrophages to secrete proinflammatory cytokines. very interestingly, chiarella et al. showed that b2-ar on alveolar macrophages is responsible for il-6 secretion and promotion of inflammation and prothrombotic state in a murine model of particulate-matter-induced thrombosis. administration of b2-ar agonist induces mitochondria-dependent reactive oxygen species (ros) generation which results in camp response element-binding protein (creb) that results in il-6 activation. huang et al. investigated the effect of lymphocyte-derived catecholamines on the differentiation and function of t helper (th) cells, suggesting that this shifted the th1/th2 balance in the direction of greater th2 polarization (22, 23) . panina-bordignon et al. also showed that beta2-adrenergic signaling inhibits the production of il-12, thereby promoting th2 differentiation and inhibiting th1 development associated with antitumor immunity. catecholamines are also known to impact the immune response via down-regulation of ifn-g production (24) . functionally, ifn-g can exert direct antiviral effects on infected cells as well as neighboring cells (25, 26) . it can also activate local immune cells, like tissue-resident dendritic cells, macrophages, and nk cells, to augment antiviral functions (24, (27) (28) (29) . furthermore, ifn-g can also control the antiviral state by modulating the differentiation and maturation of t cells and b cells (30, 31) . khalili et al. showed that propranolol, a nonselective beta-blocker, synergized with an hsp-70-rich tumor lysate vaccine to increase ifn-g production in a murine model of fibrosarcoma (32) . treated animals showed lower rates of tumor growth (p < 0.01) and increased levels of ctl activity (p < 0.05). beta-blockers are a class of medications that are prevalently used to manage abnormal heart rhythms, hypertension and to protect the heart from recurring myocardial infarction. there are two main categories of beta-blockers: non-selective and selective. the first group includes older molecules such as propranolol, nandol, timol, etc., which have different degrees of affinity for b2-ar and b1-ar; second-generation drugs that are more selective such as metoprolol and acebutolol, etc. are specific for b1-ar. after the introduction of cardioselective beta-blockers, the non-selective are not used frequently to treat heart conditions as the selective ones have fewer side effects. however, they are still in use to treat several other conditions such as long qt syndrome, aortic dilation in marfan syndrome, liver cirrhosis to reduce portal hypertension and bleeding esophageal varices. propranolol, a non-selective beta-adrenergic blocker, has been extensively used for over 50 years in the treatment of many cardiovascular problems such as ischemic heart diseases, arrhythmias, and heart malfunction. recently, several clinical trials are supporting its benefit in a number of conditions, including cancer, hemorrhage, sepsis, and hypermetabolic syndrome associated with severe burns, akathisia associated with alzheimer's disease or psychosis, aggression associated with brain injury or disease, and anxiety (33) (34) (35) . two different reports on cancer patients show that propranolol treatment reduces inflammatory cytokines including il-6 and tnfa, inflammation-related transcription factors such as nfkb and stat3 and reduces the activation of treg lymphocytes (36, 37) . they also showed the potential benefit of propranolol on cancer recurrence (cr) and overall survival (os) (36, 37) . in these trials on breast cancer patients, propranolol treatment wellpreserved the anticancer immunological profiles of peripheral blood mononuclear cells, reduced emt and prometastatic and proinflammatory transcription factors in tumor samples. in addition, shaashua and colleagues (38) have suggested a positive synergistic effect of anti-inflammatory and betablockers, when these drugs are co-administered. beta-adrenergic receptor antagonists are also known to have effects on platelet aggregation, and a meta-analysis published in 2014 showed that they decreased platelet aggregation by 13% (95% ci = 8-17%, standardized mean difference = −0.54, 95% ci = −0.85 to −0.24, p < 0.0001) (39) . in particular non-selective lipophilic beta-blockers (including propranolol) decreased platelet aggregation more than that of the selective nonlipophilic beta-blockers. in addition, nonselective beta-blockers have been proved effective on the acute prothrombotic response to psychosocial stress and elevated plasma levels of factor viii:c in patients with deep vein thrombosis (40, 41) . we had previously reported that propranolol reduces the effects of hypothalamic-pituitary-adrenal (hpa) axis stimulation induced by chronic stress. in particular, in a melanoma mouse model, propranolol treatment a week prior to the induction of stress delayed tumor growth. furthermore, propranolol-treated mice showed lower levels of vegf and enos with respect to untreated mice proving that it reduces the production of proangiogenic factors involved in tumor growth (42) . our reported findings also show that propranolol exerts antimetastatic effects on du145 prostate cell lines xenograft in mice. it decreased metastatic foci in the inguinal lymph nodes, significantly reducing mmp2 and mmp9 in tumor samples. moreover, a norepinephrine-dependent increase in epithelial to mesenchymal transition (emt) can be rescued by propranolol treatment (43) . our preliminary data (figure 1) in mouse lung colonization model of melanoma show a significative reduction of il-6 and il-17a levels and an increase of ifn-g in mice treated with selective b2-ar inhibitor ici 118,551 (ici). moreover, our data show an increase of il-12 as shown in icitreated mice with respect to the controls. the patients with sars-cov-2 at the late stages of the disease suffer from many abnormalities, which are the result of immune system imbalance and malfunction which can lead to proinflammatory reactions and immunopathological conditions, presented by lethal inflammation in the lungs and vascular leakage (44, 45) . several pieces of evidence support the pivotal role of il-6 in driving these phenomena. indeed, clinical evidence from china and italy in different hospitals (46) showed that the il-r6 blocker tocilizumab, normally used for the treatment of rheumatoid arthritis can effectively improve clinical conditions of patients. the b2-ar blockade can reduce il-6 and other inflammatory cytokines in the patient's serum contributing to rebalancing the immune system. in 1980, kaplan et al. suggested a possible role of propranolol in the treatment of rheumatoid arthritis (47) . moreover, the stress-induced inflammation, which likely occurs in patients diagnosed with covid-19, could worsen the clinical symptoms; non-selective beta-blockers could prevent this phenomenon and at the same time reduce anxiety in those patients. furthermore, the pericyte injury due to virus infection may result in capillary endothelial cell dysfunction, inducing microvascular dysfunction (48) . indeed, tang et al. proved that anticoagulant treatment is associated with decreased mortality in severe covid-19 patients with coagulopathy (49) . beta-blocker might counteract this by reducing prothrombotic response, vascular tone, and vegf secretion (50) (51) (52) . notably, some authors demonstrated that propranolol inhibits choroidal neovascularization (cnv) in vivo and b2-ar blockade reduces vascular endothelial growth factor (vegf) expression in mouse retinal pigment epithelium and choroidal endothelial cells in culture (53) . since beta-blockers may cause bronchospasm, in some cases, their use is not recommended in asthma or chronic obstructive pulmonary disease (copd). however, a recent study on a large number of patients in denmark showed that in patients taking beta-blockers (including non-selective) the risk of hospitalization for copd is reduced (54) . similarly, in asthma, a recent systematic revision of the literature suggests that the escalating figure 2 | summary of possible benefits of b2-ar targeting in patients with sars-cov-2. explanation step by step of different phases of sars-cov-2 pathogenesis and possible tool offered by beta-blockers to prevent and counteract uncontrolled cytokine storm and thrombus formation in late phase iii that lead to the death of patient. figure 1 | proinflammatory cytokines in mice untreated (control) or treated with beta-2-inhibitor (ici115,881). 12 cytokine multiplex assay was performed through the quantification in serum of il-1a, il-1b, il-2, il-4, il-6, il-10, il-12, il17-a, ifn-g, tnf-a, g-csf, gm-csf, in a lung colonization model of mice bearing murine melanoma cell line b16f10. error bars depict means ± sd. one-way anova and bonferroni post-hoc analysis were used to examine the significance of differences among groups (graph pad prism 8.0). a probability value with p < 0.05 was considered to be statistically significant. barbieri dose of beta-blockers is well tolerated and could be beneficial for airway inflammation and hyperresponsiveness (55) . the patients positive for the sars-cov-2 did not describe any worsening of the symptoms. however, more accurate data are necessary to establish the safety of adrenergic targeting in these patients. many supporting pieces of evidence show that the major symptoms of covid-19 infection are associated with hyperinflammation over-activation of th17 response. indeed, treatments known to reduce th17 response such as tocilizumab and ruxolitinib have been already used to treat covid-19 patients in phases ii and iii. b2-ar signals have been described to have a central role in rheumatoid arthritis in promoting inflammation and th17 response. non-selective beta-blockers have been used in clinical settings to reduce inflammation and th17 response. in addition, the non-selective beta-blocker propranolol blocks at1 induced expression of il-6, nfkb, tnf, ifn-g, vegf, and metalloprotease engagement. we believe that b2-ar signals might be a valuable target for new strategies aiming to block or slow down the transition from the early phases (i and iia) of covid-19 to phase iii by reducing the activation of th17 response and inflammatory cytokine release and prevent venous thromboembolism ( figure 2 ). for these reasons we would like to point out the importance to perform further preclinical and clinical studies to explore this opportunity. the authors acknowledge that the data presented in this study must be deposited and made publicly available in an acceptable repository prior to publication. frontiers cannot accept a manuscript that does not adhere to our open data policies. ab and vd have thought and wrote the manuscript. nr critically revised the manuscript. gp revised immunological aspects and made corrections. nm made important contribution on beta-blockers. gb critically revised the manuscript and supervised the study. all authors contributed to the article and approved the submitted version. this work was funded by an "ricerca corrente" grant from the italian ministry of health. coronaviruses-drug discovery and therapeutic options the emerging novel middle east respiratory syndrome coronavirus: the "knowns" and "unknowns severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease a novel coronavirus from patients with pneumonia in china multiple organ infection and the pathogenesis of sars organ distribution of severe acute respiratory syndrome (sars) associated coronavirus (sars-cov) in sars patients: implications for pathogenesis and virus transmission pathways tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19 coronavirus disease 2019 (covid-19): what we know cytokine storm and leukocyte changes in mild versus severe sars-cov-2 infection: review of 3939 covid-19 patients in china and emerging pathogenesis and therapy concepts th17 responses in cytokine storm of covid-19: an emerging target of jak2 inhibitor fedratinib distinct immune response in two mers-cov-infected patients: can we go from bench to bedside cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus il-17 response mediates acute lung injury induced by the 2009 pandemic influenza a (h1n1) virus molecular mechanisms of b2-adrenergic receptor function, response, and regulation beta-adrenergic receptors, from their discovery and characterization through their manipulation to beneficial clinical application autoradiographic visualization of betaadrenoceptor subtypes in human lung bidirectional role of b2-adrenergic receptor in autoimmune diseases b2-adrenergic agonists bias tlr-2 and nod2 activated dendritic cells towards inducing an il-17 immune response regulation of differentiation and function of helper t cells by lymphocyte-derived catecholamines via a 1 -and b 2 -adrenoceptors lymphocyte-derived catecholamines induce a shift of th1/th2 balance toward th2 polarization stress-induced alterations in interferon production and class ii histocompatibility antigen expression nondegradative role of atg5-atg12/atg16l1 autophagy protein complex in antiviral activity of interferon gamma type i and type ii interferons inhibit the translation of murine norovirus proteins identification of interferon-g, as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity enhancement of mouse natural killer cell activity by type ii interferon gammainterferon is one of several direct b cell-maturing lymphokines distinct requirements for ifns and stat1 in nk cell function ilc1 confer early host protection at initial sites of viral infection long acting propranolol and hsp-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in balb/c mice propranolol attenuates hemorrhage and accelerates wound healing in severely burned adults propranolol for disruptive behaviors in nursing home residents with probable or possible alzheimer disease: a placebo-controlled study propranolol treatment of assaultive patients with organic brain disease: a double-blind crossover, placebo-controlled study perioperative inhibition of b-adrenergic and cox2 signaling in a clinical trial in breast cancer patients improves tumor ki-67 expression, serum cytokine levels, and pbmcs transcriptome propranolol attenuates surgical stress-induced elevation of the regulatory t cell response in patients undergoing radical mastectomy perioperative cox-2 and b-adrenergic blockade improves metastatic biomarkers in breast cancer patients in a phase-ii randomized trial effect of b-blockers on platelet aggregation: a systematic review and meta-analysis the effects of aspirin and nonselective beta blockade on the acute prothrombotic response to psychosocial stress in apparently healthy subjects beta-receptor blockade decreases elevated plasma levels of factor viii: c in patients with deep vein thrombosis role of endothelial nitric oxide synthase (enos) in chronic stress-promoted tumour growth the stress hormone norepinephrine increases migration of prostate cancer cells in vitro and in vivo dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice anti-il6r role in treatment of covid-19-related ards propranolol and the treatment of rheumatoid arthritis the ace2 expression in human heart indicates new potential mechanism of heart injury among patients infected with sars-cov-2 anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy b-adrenergic receptors (b-ar) regulate vegf and il-6 production by divergent pathways in high b-ar-expressing breast cancer cell lines norepinephrine and adenosine-5'-triphosphate synergize in inducing il-6 production by human dermal microvascular endothelial cells norepinephrine upregulates vegf, il-8, and il-6 expression in human melanoma tumor cell lines: implications for stress-related enhancement of tumor progression b2-adrenergic receptor antagonism attenuates cnv through inhibition of vegf and il-6 expression therapy and risk of chronic obstructive pulmonary disease -a danish nationwide study of 1·3 million individuals beta-blockers: friend or foe in asthma? we thank dr. sandra mazzoli for paper formatting, figure and test suggestions and dr. vincenzo quagliariello for elisa test on cytokines. key: cord-273277-4ewvwg4o authors: rudd, christopher e. title: gsk-3 inhibition as a therapeutic approach against sars cov2: dual benefit of inhibiting viral replication while potentiating the immune response date: 2020-06-26 journal: front immunol doi: 10.3389/fimmu.2020.01638 sha: doc_id: 273277 cord_uid: 4ewvwg4o the sars-cov2 (covid-19) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. a key question is whether it is possible to make rational predictions of new therapies based on the presently available scientific and medical information. in this regard, i have noticed an omission in the present analysis in the literature related to the exploitation of glycogen synthase kinase 3 (gsk-3) as a therapeutic approach. this is based on two key observations, that gsk-3 inhibitors can simultaneously block sars viral replication, while boosting cd8+ adaptive t-cell and innate natural killer (nk) responses. firstly, it is already clear that gsk-3 phosphorylation of sars cov1 n protein on key serine residues is needed for viral replication such that small molecule inhibitors (smis) of gsk-3 can inhibit viral replication. in comparing protein sequences, i show here that the key sites in the n protein of sars cov1 n for replication are conserved in sars cov2. this strongly suggests that gsk-3 smis will also inhibit sars cov2 replication. secondly, we and others have previously documented that gsk-3 smis markedly enhance cd8+ cytolytic t-cell (ctl) and nk cell anti-viral effector functions leading to a reduction in both acute and chronic viral infections in mice. my hypothesis is that the repurposing of low-cost inhibitors of gsk-3 such as lithium will limit sars-cov2 infections by both reducing viral replication and potentiating the immune response against the virus. to date, there has been no mention of this dual connection between gsk-3 and sars cov2 in the literature. to my knowledge, no other drugs exist with the potential to simultaneously target both viral replication and immune response against sars cov2. the sars-cov2 (covid19) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. a key question is whether it is possible to make rational predictions of new therapies based on the presently available scientific and medical information. in this regard, i have noticed an omission in the present analysis in the literature related to the exploitation of glycogen synthase kinase 3 (gsk-3) as a therapeutic approach. this is based on two key observations, that gsk-3 inhibitors can simultaneously block sars viral replication, while boosting cd8+ adaptive t-cell and innate natural killer (nk) responses. firstly, it is already clear that gsk-3 phosphorylation of sars cov1 n protein on key serine residues is needed for viral replication such that small molecule inhibitors (smis) of gsk-3 can inhibit viral replication. in comparing protein sequences, i show here that the key sites in the n protein of sars cov1 n for replication are conserved in sars cov2. this strongly suggests that gsk-3 smis will also inhibit sars cov2 replication. secondly, we and others have previously documented that gsk-3 smis markedly enhance cd8+ cytolytic t-cell (ctl) and nk cell anti-viral effector functions leading to a reduction in both acute and chronic viral infections in mice. my hypothesis is that the repurposing of low-cost inhibitors of gsk-3 such as lithium will limit sars-cov2 infections by both reducing viral replication and potentiating the immune response against the virus. to date, there has been no mention of this dual connection between gsk-3 and sars cov2 in the literature. to my knowledge, no other drugs exist with the potential to simultaneously target both viral replication and immune response against sars cov2. (tocilizumab), inhibitors of the co-receptor serine protease tmprss2 (such as camostat mesylate), soluble receptor ace2 as a decoy, amongst others (2). a key objective will be to inhibit sars cov2 transcription while potently boosting the immune response against the viruses and limiting the cytokine release syndrome (crs) associated with severe disease. crs is mediated predominately by t-cells and inflammatory myeloid lineage cells, the latter pathogenically licensed primarily by cd4+ tcells. in this light, the potential for the use of inhibitors of the serine/threonine kinase glycogen synthase kinase 3 (gsk-3) has passed somewhat unnoticed. the hypothesis underlying this article is that gsk-3 inhibitors will both inhibit sars-cov2 replication and potentiate cd8+ t-cell responses for enhanced viral clearance. it should therefore be considered as a new therapeutic approach. the sars-cov2 has four structural proteins, known as the s (spike), e (envelope), m (membrane), and n (nucleocapsid) proteins. the n protein holds the rna genome and is needed for transcription of the viral genome, while the s, e, and m proteins together create the viral envelope (3). the sars cov1 n protein is phosphorylated by gsk-3, an event that is needed for viral replication (4-6). the inhibition of gsk-3 with small molecule inhibitors (smis) prevents sars cov1 n1 phosphorylation, and in the process, limits sars cov1 viral replication (4, 5, 7). as seen in figure 1a , in comparing sequences, it is clear that the sars cov2 n protein sequence has the same conserved key serine residues (serine 189 and 207) as found in the sars cov1 n protein. most residues surrounding these key phosphorylation sites are identical between sars cov1 and sars cov2. it therefore seems likely that gsk-3 phosphorylation of the serine residues in sars cov2 will occur as in related sars cov1. gsk-3 inactivators also inhibit the coronovirus protease (m pro ) (or 3c-like protease) (8) that cleaves the sars-cov-2 encoded polyproteins (pp1a and pp1ab) needed for viral replication and transcription (9) . from these two angles, it is a reasonable hypothesis that the inhibitors of gsk-3 that inhibit sars cov1 replication will inhibit sars cov2 replication. to date, this has not been interrogated, but could be rapidly tested in in vitro assays using vero6 or 293t cells. secondly, studies from my lab and others have shown that gsk-3 negatively regulates t-cell proliferation and function (10) (11) (12) . gsk-3 is most active in resting t-cells, keeping cells in a quiescent state. this function is unlike of other kinases such as p56 lck which initiate the activation of t-cells (13) . as a consequence, the inhibition of gsk-3 with the small molecule inhibitors (smis) such as sb415286 markedly enhance adaptive cd8+ cytolytic cell (ctl) function (11, (14) (15) (16) (figure 1b) . this potentiating effect is due in part to the upregulation in expression of the transcription factor t-bet (tbx21) (11), a central regulator of th1 differentiation (17) . t-bet, in turn, inhibits the transcription and expression of inhibitory receptors pd-1 and lag3, while promoting the expression of cytolytic effector molecules in cd8+ t-cells, granzyme b, perforin and interferongamma (16, 18) . further, and salient to this hypothesis, gsk-3 smis help resolve viral infections in mice, acute infection by the murine gamma-herpesvirus 68, and chronic infection with the lymphocytic choriomeningitis clone 13 (lcmv) (11) . the effects of gsk-3 were preferentially seen in cd8 ctls, and to a lesser extent, cd4+ t-cells, the latter contributing to the crs seen in the more severe clinical manifestations of covid-19. gsk-3 inhibitors have also been found to induce the suppressive cytokine interleukin 10 (il-10) in cd4+ t-cells which might dampen crs in severe disease (19) . il-10 limits the immune response and prevents tissue damage in infection and autoimmune disease (20) . lastly, gsk-3 inhibition drives the maturation and function of natural killer (nk) cells (21) . natural killer (nk) cells are effector cells of the innate immune system and also important in the control of viral infections (22) . the inhibition of the gsk-3 pathway, therefore, plays central roles in promoting both the adaptive and innate immune responses against viruses. the evidence presented here strongly suggests for the first time that gsk-3 inhibitors could constitute an effective therapy in restraining the progression of sars cov-2 infections. to my knowledge, no other drugs exist which might simultaneously target both sars cov-2 viral replication, and the immune response against the virus. it would be a novel and affordable therapeutic approach with the potential dual targeting both the virus and immune system in the treatment of covid-19 patients. in the immediate term, lithium chloride could be administered to patients on a compassionate basis given that citrate, orotate, and carbonate salt formations of the drug are in wide clinical use for the treatment of bipolar disorders. various side effects such as nausea have been reported, although these can be minimized by gradually increasing doses to the desired strength. renal side effects seen in some patients can also be ameliorated with proper drug monitoring (23) . relative to the life-saving potential of gsk-3 inhibition in the treatment of covid-19, the side effects are a minor consideration. with time, more specific gsk-3 reagents such as sb415286 could be tested in clinical trials. both atp competitive and potentially more selective allosteric non-competitive inhibitors could be used. the inhibitor tdzd-8 has been tested in preclinical models of cancer (24) , while another inhibitor tideglusib has been in phase ii clinical trials for alzheimer's disease and progressive supranuclear palsy where it is welltolerated (25, 26) . others recently noted the potential of gsk-3 inhibitors given effects on other viral infections, but failed to note the homologous regulatory phosphorylation sites in sars cov2 and cov1 (27) . it is these known and previously unknown target effects which i argue are key to the potential success of gsk-3 inhibition in the treatment of covid-19. all risks should be understood prior to administration of any treatments and precautions taken under the close supervision of a physician. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. the sars-cov-2 (covid-19) pandemic and uncertainties in developing a vaccine have created an urgent need for new therapeutic approaches. in this opinion or hypothesis article, i propose the exploitation of glycogen synthase kinase 3 (gsk-3) as a therapeutic approach. this is based on two key observations, that gsk-3 inhibitors can simultaneously block sars viral replication, while boosting cd8+ adaptive t-cell and innate natural killer (nk) responses. my hypothesis is that the repurposing of low-cost inhibitors of gsk-3 such as lithium chloride will limit sars-cov-2 infections by both limiting viral replication and potentiating the immune response against the virus. the author confirms being the sole contributor of this work and has approved it for publication. sars-cov-2 vaccines: status report news feature: to counter the pandemic, clinicians bank on repurposed drugs analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational syndrome coronavirus nucleocapsid protein and viral replication identification of phosphorylation sites in the nucleocapsid protein (n protein) of sars-coronavirus inhibition of cell proliferation by sars-cov infection in vero e6 cells structure of m(pro) from sars-cov-2 and discovery of its inhibitors the crystal structures of severe acute respiratory syndrome virus main protease and its complex with an inhibitor glycogen synthase kinase 3: a kinase for all pathways? glycogen synthase kinase 3 inactivation drives t-bet-mediated downregulation of co-receptor pd-1 to enhance cd8(+) cytolytic t cell responses negative regulation of t cell proliferation and interleukin 2 production by the serine threonine kinase gsk-3 the cd4 receptor is complexed in detergent lysates to a protein-tyrosine kinase (pp58) from human t lymphocytes two strings in one bow: pd-1 negatively regulates via co-receptor cd28 on t cells small-molecule inhibition of pd-1 transcription is an effective alternative to antibody blockade in cancer therapy glycogen synthase kinase 3 inactivation compensates for the lack of cd28 in the priming of cd8(+) cytotoxic t-cells: implications for anti-pd-1 immunotherapy. front immunol t-bet in disease small molecule inhibition of gsk-3 specifically inhibits the transcription of inhibitory co-receptor lag-3 for enhanced anti-tumor immunity glycogen synthase kinase-3 controls il-10 expression in cd4(+) effector t-cell subsets through epigenetic modification of the il-10 promoter the regulation of il-10 production by immune cells gsk3 inhibition drives maturation of nk cells and enhances their antitumor activity nk cells in host responses to viral infections what we need to know about the effect of lithium on the kidney rapid and selective death of leukemia stem and progenitor cells induced by the compound 4-benzyl, 2-methyl, 1,2,4-thiadiazolidine, 3,5 dione (tdzd-8) treatment of alzheimer's disease with the gsk-3 inhibitor tideglusib: a pilot study gsk-3 inhibitors: preclinical focus on cns international group for the study of lithium treated, lithium's antiviral effects: a potential drug for covid-19 disease? the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 rudd. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-291941-9a4tt4f7 authors: barber-axthelm, isaac m.; kent, stephen j.; juno, jennifer a. title: understanding the role of mucosal-associated invariant t-cells in non-human primate models of hiv infection date: 2020-08-18 journal: front immunol doi: 10.3389/fimmu.2020.02038 sha: doc_id: 291941 cord_uid: 9a4tt4f7 chronic hiv infection causes systemic immune activation and dysregulation, resulting in the impairment of most t-cell subsets including mait cells. multiple human cohort studies demonstrate mait cells are selectively depleted in the peripheral blood and lymphoid tissues during hiv infection, with incomplete restoration during suppressive antiretroviral therapy. because mait cells play an important role in mucosal defense against a wide array of pathogens, fully reconstituting the mait cell compartment in art-treated populations could improve immunity against co-infections. non-human primates (nhps) are a valuable, well-described animal model for hiv infection in humans. nhps also maintain mait cell frequencies more comparable to humans, compared to other common animal models, and provide a unique opportunity to study mait cells in the circulation and mucosal tissues in a longitudinal manner. only recently, however, have nhp mait cells been thoroughly characterized using macaque-specific mr1 tetramer reagents. here we review the similarities and differences between mait cells in humans and nhps as well as the impact of siv/shiv infection on mait cells and the potential implications for future research. ]. while their role in cancer development and progression has not been fully elucidated, mait cells and specific populations of major histocompatibility complex class i-related (mr1)-restricted t-cells have been shown to infiltrate the tumor microenvironment and are able to lyse cancer cells in vitro and in vivo, indicating a potential role in cancer immunotherapy (2); reviewed in lukasik et al. (3) . in contrast to conventional t-cells which recognize antigens through mhc-mediated antigen presentation, mait cells recognize antigens by mr1-mediated presentation of vitamin b metabolites predominately through a vα7.2 semi-invariant t-cell receptor (tcr) (4, 5) . mait cells are also capable of being activated via a cytokine-mediated, antigen independent pathway, indicating they have properties of both innate and adaptive immune cells (6) . these features of mait cells highlight their importance in rapid immune responses to a wide array of pathogens. while the ability of mait cells to mount an immune response against bacterial pathogens is well established, their role in viral infection is not as clear. viral replication cycles do not involve vitamin b synthesis pathways, which are necessary for tcr-dependent mait cell activation. multiple studies have shown increased mait cell activation in patients with active viral infections, including dengue (7, 8) , influenza (7) , chronic hepatitis b (9) , hepatitis c (7) , and severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (10) . similar findings have also been made in mice following influenza infection (11), as well as in vitro with hepatitis c (7), influenza (12) , and zika virus (8) . virus-induced mait cell activation is mediated through tcr-independent pathways, as shown for influenza (7, 12) , dengue (7) , hepatitis c (7), or zika virus (8) exposure in vitro. this stands in contrast to mait cell stimulation with paraformaldehyde fixed e. coli, which is blocked by treatment with an anti-mr1 blocking antibody. beyond mait cell activation, data on mait cell contributions to viral immunity is limited. supernatant from activated mait cells suppresses hepatitis c virus replication in a dose-dependent manner (7) . mr1 −/− mice, which are deficient in mait cells, have increased morbidity and mortality following influenza challenge; compared to wild type controls (11). increased morbidity and mortality is ameliorated with adoptive transfer of murine pulmonary mait cells prior to viral challenge. adoptive transfer of pulmonary mait cells also reduced morbidity and mortality in rag −/− il-2rγ −/− mice following influenza challenge (11). taken together, these studies indicate that mait cells are capable of suppressing viral replication and providing clinical improvements during viral infections against certain viruses. additional studies are needed to better characterize the impact mait cells have, both beneficial and deleterious, on viral infections, as well as the breadth of viral infections that mait cells are able to mount an immune response against. a better understanding of mait cell function will require continued evaluation of human mait cell dynamics during various disease states, along with in vivo models that best represent the relevant pathologic processes. the mr1 gene, the primary receptor for mait activation through antigen presentation, is highly conserved among mammalian species, but is absent in non-mammalian species (13) . additionally, there have been 3 separate losses of functional mr1 among mammals, including in lagamorpha (rabbits), and in carnivora (dogs, cats, and ferrets) (14) . mice carry a functional mr1 gene but have a relatively low abundance of mait cells in the peripheral blood (median: 0.1%) necessitating the generation of transgenic mice expressing an invariant mvα19-jα33 tcrα to increase mait cell frequencies (15, 16) , or the boosting of tissue mait cell frequencies by administration of antigen and tlr agonists (17) . in contrast, non-human primates (nhps) express a functional mr1 gene and maintain mait cells at frequencies more comparable to humans, providing a superior in vivo model to study mait cell immunological dynamics. herein, we discuss the current state of mait cell characterization in nhps [which has focused on rhesus macaques (rm), pigtail macaques (ptm), and mauritian cynomolgus macaques (mcm)] and the changes in mait cell populations that occur during simian immunodeficiency virus (siv) and simian-human immunodeficiency virus (shiv) infection, which are the critical animal models for hiv infection. human mait cells were originally identified as vα7.2 + cd161 + cells among the bulk t-cell population [reviewed in garner et al. (18) ]. recently, the development of antigen loaded mr1 tetramers has allowed for a more refined identification of mait cells by flow cytometry (19, 20) . similar approaches have been utilized to phenotype macaque mait cells, via identification of vα7.2 + and/or mr1-5-op-ru + t-cells (21) (22) (23) (24) (25) (26) (27) (28) (29) . one important consideration for determining tetramer reactivity in macaque mait cells is the utilization of species specific mr1 tetramers. two studies have identified incomplete cross reactivity of human mr1 tetramers with macaque mait cells (23, 25) . identification of these cells is improved with the use of macaque specific mr1 tetramers. furthermore, the inclusion or exclusion of vα7.2 expression in the definition of a mait cell should be carefully considered. there is growing evidence of a unique vα7.2 − mr1 tetramer + t-cell population in humans (30, 31) , which has also been identified in the peripheral blood of ptms, rms, and mcms (23, 25, 28) . additional work is needed to characterize these cells and to compare their phenotypic and functional properties to their human counterparts. human mait cells are predominately cd8αα + or cd4 − cd8 − , with a minor population of cd4-expressing cells (19) . in contrast, nhp mait cells are almost uniformly cd8α + , with 3 studies noting an absence of cd4 − cd8 − mait cells in nhp (23, 25, 28) . one additional study identified peripheral blood mait cells as predominately cd8α + (36.3%) or cd8 − cd4 − (44.9%) in rms, with minor populations of cd8 + cd4 + (2.9%) and cd4 + (15.8%) mait cells (29) . mait cells were identified based on reactivity to nhp-specific mr1 tetramers without concurrent expression of vα7.2, which may partially explain the presence of cd8 − cd4 − , cd8 + cd4 +, cd4 + mait cells that were not observed in other studies. it is presently unknown if nhp cd8α + mait cells express a homodimeric (cd8αα + ) or heterodimeric (cd8αβ + ) receptor. the cause for this absence of cd4 − cd8 − mait cells in the majority of nhp studies is unknown, and additional studies are needed to characterize this variation from human peripheral mait cells. human cd8 + and cd4 − cd8 − mait cells have been shown to have distinct phenotypic and functional profiles (32) . cd8 + mait cells express higher levels of cytotoxic and coactivating markers compared to cd4 − cd8 − mait cells, and produce higher levels of ifnγ and tnfα following stimulation. cd4 − cd8 − mait cell can be derived in vitro from cd8 + mait cells following tcr-dependent activation. potential causes for the relative paucity of cd8 + mait cells in captive nhps include species-specific variation in mait cell development or differentiation between humans and nhps, or environmental factors related to husbandry practices which drives the altered frequencies in nhp peripheral cd8 + and cd4 − cd8 − mait cells. a lack of cd4 − cd8 − mait cells in nhps may also impact the immune response to certain disease states, and should be considered when utilizing nhps as a model for humans. while nhps predominately lack cd4 − cd8 − mait cells, we caution against pre-gating on cd8 + t cells prior to identifying mr1 tetramer + cells in nhps, as this could hinder observing shifts in mait cell cd4 and cd8 co-receptor usage during disease states or experimental interventions. cd161, a c-type lectin like-receptor, is almost ubiquitously expressed by human mait cells in the peripheral blood and has classically been used as a co-staining marker to gate on vα7.2 + or mr1 tetramer + cells (15, 33, 34) this marker is associated with innate-like, tcr-independent responses to il-12 + il-18 stimulation (35, 36) . three studies have utilized cd161 expression to identify mait cells in nhps (22, 26, 27) . however, two studies have noted poor cross-reactivity of antihuman cd161 antibodies against rm and ptm cd161 antigen (23, 25) . additionally, cd161 downregulation has been identified in mait cells from chronic hiv infected individuals (33, 37, 38) . as a result, we strongly caution against the use of cd161 to define the total mait cell population in nhps; rather, mr1 tetramer in combination with tcr vα7.2 can distinguish major mr1restricted t-cell populations, which can then be phenotyped for additional surface markers. plzf (zbtb16) is most consistently expressed on mait cells in nhps (21, 23-25, 27, 28) , as well as in humans (34, 39) , and is a marker to consider when identifying the bulk mait cell population. additional markers that are expressed on a relatively high frequency of mait cells and may be considered for mait cell identification are ccr6 and il-18rα, which have been shown to be expressed on mait cells from multiple nhp species (21, 25, 27, 28) . mait cells constitute approximately 2.6% (range: 0.1-9.2%) of the total t-cell population in human peripheral blood ( table 1 ) (40) . by comparison, mait cell frequencies in the peripheral blood range from 0.3 to 4.8% in rms (21, 27) , 0.026-1.28% in ptm (25) , and 0.6-17% of cd3 + cd8 + t-cells in mcms (28) . in general, nhps maintain peripheral mait cell frequencies at 1-2% of the bulk cd3 t-cell population. while mait cell frequencies are lower than what is observed in humans, it is substantially higher than baseline mait cell frequencies in mice (16) , and is sufficient to evaluate changes in peripheral mait cell frequencies and phenotype with different experimental interventions. similar to mait cell frequencies in the blood, nhps mait cell frequencies in tissues tend to be lower compared to the frequencies in humans, but higher compared to murine mait cell frequencies. both humans and nhps maintain relatively 1% of the bulk t-cell population, or 16% (range: 9-28%) of the bulk cd3 + cd8 + t-cell population, in the lungs of rms (22, 27) . additionally, mait cell frequencies are highly variable in rm bronchoalveolar lavage fluid (bal), comprising 0.5-14.7% of the bulk cd3 + t-cell population (21, 23, 24, 26) , or approximately 1-17% of the bulk cd3 + cd8 + t-cell population (29) . similar findings have also been made in mcms, with mait cell frequencies ranging from approximately 12-40% of the bulk cd3 + cd8 + t-cell population in the bal (28) . mait cells are consistently maintained at low frequencies in secondary lymphoid organs (lymph nodes and spleen) compared to the peripheral blood, in both humans and nhps. this is attributed to the relative lack of ccr7 and cd62l expression, both required for lymphoid tissue homing, on peripheral mait cells [reviewed in kurioka et al. (41) ]. additionally, it has been postulated that mait cells in the lymph represent a distinct population that recirculate from the tissues, separate from circulating mait cells in the blood (20) . while similar patterns of tissue recirculation in nhps still need to be investigated, differential distribution of mait cell phenotypes between lymphoid and extralymphoid tissues has been described in rms. mait cells within secondary lymphoid tissues are predominately ccr7 + cd28 + (central memory), while mait cells in extralymphoid tissues are predominately ccr7 − cd28 + (transitional memory) or ccr7 − cd28 − (effector memory) (23). species specific variations in t-cell functional activity are important to consider in the context of different animal models. stimulation of peripheral blood mononuclear cells (pbmcs) from ptm with the riboflavin metabolite-based antigen 5-op-ru results in a dose-dependent upregulation of cd69 and selective proliferation of mr1 tetramer + vα7.2 + mait cells ( table 2 ) (25) , demonstrating that human and macaque mait cells are similarly activated by antigen (figure 1) . surprisingly, 5-op-ru stimulated ptm mait cells produced relatively low amounts of tnfα and ifnγ (although ifnγ secretion could be augmented with il-12 and il-18 supplementation to the cultures). in contrast to the ptm data, stimulation of human peripheral mait cells with 5-a-ru resulted in significant production of tnfα and ifnγ (43) . in contrast to humans and nhps, murine mait cells predominately produce il-17 following stimulation with 5-op-ru (16) , highlighting the potential for species specific differences in mait cells functional activity, and the value of the nhps in being able to better model the human peripheral mait cell cytokine profile. the use of fixed e. coli to stimulate human mait cells is now a standard assay, and activates mait cells through both mr1dependent and -independent mechanisms (6, 44) . similarly, lps or e. coli stimulation resulted in significant ifnγ and tnfα production in rm and mcm mait cells (23, 28) , as well as cd107a upregulation in mcm mait cells (28) . in contrast, mcm mait cell stimulation with mycobacterium smegmatis resulted in no significant increase in ifnγ and tnfα production, or cd107a upregulation. following mitogenic stimulation with pma/ionomycin, both ptm and rm mait cells readily produce ifnγ, tnfα, and il-17 (21, 25, 27) . variations in the amount of il-17 production have been reported, with ptm mait cells producing relatively blood mait cells during acute shiv infection (25) . similarly, there was no change in cd69 expression on the effector memory (cd28 − cd95 + ) mait cell population of mcms following siv challenge (28) . however, a transient but significant increase in cd69, cd39, t-bet, and rorγt was observed in the central memory (cd28 + cd95 + ) mait cell population of mcms at 2-3 weeks post-siv challenge (28) . siv/shiv infection is associated with a transient increase in peripheral blood α4β7 + mait cells in ptms at 3-4 weeks post infection (25) . this increase correlated with peak viral load, as well as increased mait cell frequencies with rectal mucosa. one hypothesis is that early viral replication and increased antigen availability in the lymph node may drive α4β7 expression on mait cells. peripheral blood mait cells upregulated α4β7 when cocultured with mesenteric lymph node cells and 5-op-ru, demonstrating that circulating mait cells can upregulate α4β7 when stimulated with antigen in the context of mesenteric lymph node cells (25) . these findings are similar to what has been observed with conventional t-cells in humans and mice, where α4β7 induction is mediated by retinoic acidproducing dendritic cells from gut-associated lymphoid tissues and mesenteric lymph nodes (46) (47) (48) (49) (50) . similarly, α4β7 expression is significantly increased on peripheral mait cells from chronic shiv-infected rms, compared to naïve controls (27) . in humans, β7 integrin expression is increased in hiv + individuals, suggesting this relationship may be conserved between humans and nhp models [reviewed in saeidi et al. (51) ]. this may also partially explain the mechanism for the transient increase in mait cell frequencies observed in the rectal mucosa of hiv + individuals during initial infection (46) . alternatively, increased α4β7 + mait cells in the peripheral blood following siv/shiv infection may be due to expansion and subsequent trafficking of mait cells into the blood from mucosal tissues sites. these changes correlate with peak viremia, suggesting that early viral replication may drive α4β7 induction and increase mucosal tissue trafficking during acute infection. this is potentially driven by direct viral replication, or immune dysregulation with microbial translocation in the gastrointestinal tract. the lack of mait cell depletion during acute infection is consistent with what has been observed in humans during initial hiv infection and emphasizes the importance of the nhp-siv/shiv model to study mait cell dynamics during peracute infection. chronic hiv-1 infection in humans is associated with persistent mait cell depletion in the peripheral circulation [(33, 37) , reviewed in saeidi et al. and juno et al. (51, 52) ]. in contrast, chronic siv/shiv infection has inconsistent effects on peripheral mait cell frequencies in nhps. one study reported a reduced frequency and absolute number of peripheral mait cells in rms during chronic siv infection (21) . a reduction in ccr6 + mait cells was also noted during chronic siv infection, which was hypothesized to reflect impaired tissue trafficking capabilities of the remaining mait cells in the periphery. in contrast, a second study noted increased peripheral mait cell frequencies in rms during chronic shiv infection, which correlated with plasma viral load (27) . despite the overall increase in frequency, mait cell frequencies in the peripheral blood were inversely correlated with plasma viral load, and directly correlated with peripheral cd4 + t-cell frequencies. human mait cells are not preferentially infected by hiv (53) , suggesting that the observed correlation may reflect reduced mait cell frequencies due to reduced cd4 t-cell frequencies, which is secondary to virus-mediated cd4 t-cell depletion. other potential causes for the observed correlation includes increased mait cell consumption in response to higher amounts of virus mediated gastrointestinal inflammation and bacterial translocation. a third study identified no significant difference in peripheral mait cell frequencies during chronic shiv infection in ptms (25) . increased ki-67 expression in the peripheral mait cell populations was identified in 2 studies, despite the variability in mait cell frequency (21, 27) . similar variability has also been observed in mait cell frequencies in tissues during chronic siv/shiv infection in nhps. two separate studies have reported increased and decreased mait cell frequencies in the bal fluid of chronic-shiv infected ptms and chronic-siv infected rms, respectively (21, 25) . additionally, one study identified decreased mait cell frequencies in the inguinal lymph nodes, with concurrent maintenance of mait cells in the mesenteric lymph nodes of chronic shiv-infected ptms (25) . in contrast, a separate study identified a reduction in mait cell frequencies in the mesenteric lymph nodes of chronic siv-infected rms (21) . however, mait cell frequencies are consistently maintained in the liver, spleen, and gastrointestinal tract of chronic siv/shiv-infected macaques (21, 25) . chronic hiv infection is associated with altered expression of a variety of cytokine and chemokine receptors on peripheral mait cells [reviewed in saeidi et al. (51) ]. similar alterations have also been observed in chronic siv/shiv infected nhps. chronic shiv-infection is associated with reduced tbet and eomes expression of peripheral mait cells in ptms, while ccr5 and cxcr3 expression levels are unchanged (25) . in contrast to humans where ccr6 and hla-dr expression is decreased and immune effector functions of peripheral mait cells are significantly impaired during chronic hiv infection. this is marked by reduced cytokine production and cd69 upregulation following e. coli stimulation, which is only partially restored with combination antiretroviral therapy [(37), reviewed in saeidi et al. (51) ]. similar findings were also reported in one study evaluating chronic shiv-infected macaques, with reduced tnfα and il-17 production capacity following mitogenic stimulation (27) . however, two studies looking at chronic siv and shiv infected mcms and ptms, respectively, report no alterations in cytokine stimulation capacity (25, 28) . additionally, chronic shiv-infection in ptms is associated with increased cd69 expression on peripheral mait cells (25) . siv/shiv infection does not consistently alter mait cells' ability to produce granzyme, with 2 studies noting no significant changes in the frequency of granzyme b + mait cells in the peripheral blood during siv or shiv infection (27, 29) . these findings are in contrast to what is observed in hiv + individuals, where peripheral granzyme b + mait cells are increased in frequency compared to uninfected control samples at resting state (54) and have reduced perforin and granzyme expression following e. coli stimulation (54, 55) . in one of the nhp studies, perforin + and perforin + granzyme b + mait cell frequencies were elevated following shiv challenge (27) . the cause for the variability in mait cell frequencies, receptor expression, and effector functions observed in these studies is unknown. the authors hypothesize these may reflect species differences between rms and ptms, and/or differences in viral pathogenesis between different siv/shiv strains. additional sources of variation may be due to inter-study differences in gating strategies to identify mait cells, including the usage of cd161 expression and cd8 + pre-gating. there is also evidence of significant variability in mait cell frequencies within the peripheral circulation and different tissue compartments, both between different species and individual animals. this emphasizes the need for longitudinal studies, in order to evaluate changes in mait cell trends following different experimental manipulations, as opposed to cross sectional studies. it will also be important to consider standards for identifying nhp mait cells in future studies, including the usage of the vα7.2 tcr and species specific mr1 tetramer reactivity, due to evidence of limited cross-reactivity with human mr1 tetramers. one important consideration for hiv + individuals is the risk of co-infection with secondary opportunistic bacterial and fungal pathogens, for which hiv-mediated dysregulation of mait cells may be a predisposing factor. while data on the impact of mait cell responses on fungal infections is currently lacking, mait cells have been shown to become activated in response to candida spp. [reviewed in wong et al. (56) ] and aspergillus spp. (57) . mycobacterium tuberculosis (mtb) is one of the most common opportunistic bacterial infection among hiv + individuals. individuals with active or latent mtb infections tend to have lower peripheral mait cell frequencies, compared to uninfected individuals (58) . additionally, mait cells from hiv/mtb co-infected individuals tended to express lower levels of ccr6 compared to hiv infected individuals and healthy controls, though statistically significant differences were only seen between the hiv/mtb co-infected individuals and the healthy controls. this suggests that hiv-mediated mait cell dysregulation may predispose individuals to opportunistic infection with mtb. in rms, vaccination with bacillus calmette-guérin, a vaccine historically used to prevent tuberculosis, resulted in significantly increased expression of ki-67 and granzyme b in mait cells in the peripheral blood, the vaccine sites, and the draining lymph nodes, compared to pre-vaccination frequencies (23) . while there was evidence of significant mait cell activation following vaccination, no significant changes in mait cell frequencies in the blood or tissue sites were observed. increased peripheral mait cell ki-67 expression, without an increased in peripheral blood mait cell frequencies, was also observed in rms following intrabronchial mtb infection (23) . no significant difference in mait cell frequencies in the peripheral blood or tissue has been reported with siv/mtb co-infection, similar to what has been observed in humans (26, 28, 58) . however, a weak negative correlation between mait cell frequency and bacterial burden was observed in siv/mtb co-infected mcms, which was not maintained in nhps mono-infected with mtb. there was also a significant reduction in tnfα producing capacity of mait cells from the peripheral blood and mtb granulomas of siv/mtb co-infected mcms, compared to mtb mono-infected controls (28) . the results reflect the potential impact of mait cells on the susceptibility of mtb in hiv + individuals, in an nhp animal model. there is growing evidence that mait cells are capable of being activated in response to viral pathogens, despite the lack of vitamin b metabolites that are necessary for tcrdependent mait cell activation. additionally, there is evidence of altered mait cell frequencies, phenotypes, and functional activity during both acute and chronic hiv infection. however, the underlying pathophysiology for why these changes occur is not well understood. having a better understanding of this interaction may provide key insights into developing vaccines and/or therapies for hiv infection and related co-morbidities. the nhp animal model provides a valuable platform to study these changes, and understand the underlying mechanisms for why they occur. while siv/shiv infection in nhps does not reliably recapitulate all aspects of the mait cell changes that occur during hiv infection, notably the lack of mait cell depletion, it does model many aspects of hiv infection well. this includes an initial increase in mait cell frequency with upregulation of α4β7 during acute viral replication, as well as evidence of impaired mait cell function in siv/mtb co-infected nhps. future studies should focus on characterizing the perturbations in the mait cell populations within different tissue compartments during siv/shiv infection; with a special focus on variables that may alter these perturbations, such as macaque species, age, and gender, as well as strain, dose, and route of administration of the virus. by better understanding how these factors impact mait cell dynamics during siv/shiv infection, we will ideally be able to refine the nhp model to better recapitulate the mait cell changes that occur during hiv infection in humans. additionally, the robust response that nhp mait cells generate to both antigen and cytokines, including cytokine independent cell activation, which is comparable to humans, may be advantageous for studying mait cell dynamics with other pathogens. ib-a wrote the initial draft of the manuscript. ib-a, sk, and jj revised the manuscript. all authors contributed to the article and approved the submitted version. we acknowledge funding through the australian national health and medical research council (nhmrc app1149990). the biology and functional importance of mait cells mait cells come to the rescue in cancer immunotherapy? mr1 presents microbial vitamin b metabolites to mait cells t-cell activation by transitory neo-antigens derived from distinct microbial pathways mr1-independent activation of human mucosal-associated invariant t cells by mycobacteria mait cells are activated during human viral infections mait cells are activated in acute dengue virus infection and after in vitro zika virus infection mucosal-associated invariant t (mait) cells are more activated in chronic hepatitis b, but not depleted in blood: reversal by antiviral therapy mait cells contribute to protection against lethal influenza infection in vivo human mucosal-associated invariant t cells contribute to antiviral influenza immunity via il-18-dependent activation exceptionally high conservation of the mhc class i-related gene, mr1, among mammals restricting nonclassical mhc genes coevolve with trav genes used by innate-like t cells in mammals stepwise development of mait cells in mouse and human identification of phenotypically and functionally heterogeneous mouse mucosal-associated invariant t cells using mr1 tetramers mucosalassociated invariant t-cell activation and accumulation after in vivo infection depends on microbial riboflavin synthesis and co-stimulatory signals insights into mucosal-associated invariant t cell biology from studies of invariant natural killer t cells antigen-loaded mr1 tetramers define t cell receptor heterogeneity in mucosal-associated invariant t cells human mait cells exit peripheral tissues and recirculate via lymph in steady state conditions mucosa-associated invariant t cells are systemically depleted in simian immunodeficiency virus-infected rhesus macaques enhanced th1/th17 functions of cd161+ cd8+ t cells in mucosal tissues of rhesus macaques mr1-restricted mucosal-associated invariant t (mait) cells respond to mycobacterial vaccination and infection in nonhuman primates limited pulmonary mucosal-associated invariant t cell accumulation and activation during mycobacterium tuberculosis infection in rhesus macaques cells upregulate α4β7 in response to acute simian immunodeficiency virus/simian hiv infection but are resistant to peripheral depletion in pigtail macaques mucosal-activated invariant t cells do not exhibit significant lung recruitment and proliferation profiles in macaques in response to infection with mycobacterium tuberculosis cdc1551 functional mait cells are associated with reduced simian-human immunodeficiency virus infection mait cells are functionally impaired in a mauritian cynomolgus macaque model of siv and mtb co-infection mucosal-associated invariant t (mait) cells provide b-cell help in vaccinated and subsequently siv-infected human trav1-2-negative mr1-restricted t cells detect s. pyogenes and alternatives to mait riboflavin-based antigens diverse mr1-restricted t cells in mice and humans the cd4-cd8-mait cell subpopulation is a functionally distinct subset developmentally related to the main cd8+ mait cell pool mait cells are depleted early but retain functional cytokine expression in hiv infection mait cells are reduced in frequency and functionally impaired in human t lymphotropic virus type 1 infection: potential clinical implications cd161 ++ cd8 + t cells, including the mait cell subset, are specifically activated by il-12+il-18 in a tcr-independent manner cd161 defines a transcriptional and functional phenotype across distinct human t cell lineages activation, exhaustion, and persistent decline of the antimicrobial mr1-restricted mait-cell population in chronic hiv-1 infection cd161+ mait cells are severely reduced in peripheral blood and lymph nodes of hiv-infected individuals independently of disease progression the transcription factor plzf directs the effector program of the nkt cell lineage human blood mait cell subsets defined using mr1 tetramers mait cells: new guardians of the liver trav1-2+ cd8+ t-cells including oligoconal expansions of mait cells are enriched in the airways in human tuberculosis tcr-or cytokine-activated cd8+ mucosal-associated invariant t cells are rapid polyfunctional effectors that can coordinate immune responses human mait-cell responses to escherichia coli: activation, cytokine production, proliferation, and cytotoxicity human mait cells are xenobiotic-resistant, tissue-targeted, cd161hi il-17-secreting t cells dynamic mait cell response with progressively enhanced innateness during acute hiv-1 infection selective imprinting of gut-homing t cells by peyer's patch dendritic cells retinoic acid imprints gut-homing specificity on t cells retinoic acid receptor signaling levels and antigen dose regulate gut homing receptor expression on cd8+ t cells speciesspecific differences in the expression and regulation of α4β7 integrin in various nonhuman primates functional role of mucosal−associated invariant t cells in hiv infection perturbation of mucosalassociated invariant t cells and inkt cells in hiv infection early and nonreversible decrease of cd161++/mait cells in hiv infection arming of mait cell cytolytic antimicrobial activity is induced by il-7 and defective in hiv-1 infection sustained ifn-i stimulation impairs mait cell responses to bacteria by inducing il-10 during chronic hiv-1 infection the role of mucosal−associated invariant t cells in infectious diseases human mait cells are rapidly activated by aspergillus spp. in an apc−dependent manner low levels of peripheral cd161++cd8+ mucosal associated invariant t (mait) cells are found in hiv and hiv/tb co-infection human mait cell activation in vitro mucosalassociated invariant t cells are depleted and exhibit altered chemokine receptor expression and elevated granulocyte macrophage-colony stimulating factor production during end-stage renal disease mait cells reside in the female genital mucosa and are biased towards il-17 and il-22 production in response to bacterial stimulation key: cord-274557-2071770h authors: späth, peter j.; granata, guido; la marra, fabiola; kuijpers, taco w.; quinti, isabella title: on the dark side of therapies with immunoglobulin concentrates: the adverse events date: 2015-02-05 journal: front immunol doi: 10.3389/fimmu.2015.00011 sha: doc_id: 274557 cord_uid: 2071770h therapy by human immunoglobulin g (igg) concentrates is a success story ongoing for decades with an ever increasing demand for this plasma product. the success of igg concentrates on a clinical level is documented by the slowly increasing number of registered indication and the more rapid increase of the off-label uses, a topic dealt with in another contribution to this special issue of frontiers in immunology. a part of the success is the adverse event (ae) profile of igg concentrates which is, even at life-long need for therapy, excellent. transmission of pathogens in the last decade could be entirely controlled through the antecedent introduction by authorities of a regulatory network and installing quality standards by the plasma fractionation industry. the cornerstone of the regulatory network is current good manufacturing practice. non-infectious aes occur rarely and mainly are mild to moderate. however, in recent times, the increase in frequency of hemolytic and thrombotic aes raised worrying questions on the possible background for these aes. below, we review elements of non-infectious aes, and particularly focus on hemolysis and thrombosis. we discuss how the introduction of plasma fractionation by ion-exchange chromatography and polishing by immunoaffinity chromatographic steps might alter repertoire of specificities and influence ae profiles and efficacy of igg concentrates. i.e., cold-ethanol or ion-exchange chromatography, contaminants, route of application, i.e., intra muscular (imig), intravenous (ivig), or subcutaneous (scig), the rate of increase of the exogenous igg in the circulation of the recipient over time and, last but not least an eventually existing risk factor from patients' side ( figure 1 ) as well as incorrect handling of the concentrate are factors having a role in inducing non-infectious aes related to administration of igg concentrates ( table 1) . igg concentrates represent a defined part of the adaptive immune system, are isolated from pooled human plasma of at least 1000 donors, which contribute to the repertoire diversity in the final product. therapies with igg concentrates manufactured according to regulators requirements are acknowledged to be safe in general. this does not exclude the occurrence of aes which in their majority are rare and clinically mild to moderate. below, we like to give a few insights into various aspects and possible mechanisms of aes. table 1 . contemplating on the active ingredient of the concentrates, the administration of the igg molecules inevitably results in the interactions of the exogenous igg with the various parts of the immune system of the recipient and vice versa. this interaction in its principle generates an inflammatory condition. a key parameter deciding on the intensity of such a condition is the rate of increase over time of exogenous igg in the circulation of the recipient. insert to figure (shadowed): ∆igg in the circulation over ∆time is dependent on the combination of infusion rate (ir) and the strength of the solution applied. main figure: the mode of application, i.e., intravenous or subcutaneous, are additional factors being decisive for kinetics and the area under the curve of generation of pro-inflammatory mediators and for passing the individual threshold (short-dashed line) for a clinically noticeable ae. the threshold in turn depends on eventually exiting risk factors form the patient side. although the area under the curve might be the same for low (long-dashed line) and high (solid line) irs, at high ir, the system might not be able to cope with the extent of reactions. at low ir (long-dashed line), all the events might remain below the threshold of clinically observable ae. subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent than with ivig. in contrast, local mild to moderate aes are more frequent with scig. virus inactivation and virus elimination methods by validating an already performed step of the fractionation process or by introduction of dedicated steps (figure 3) . a hallmark of virus elimination introduced in the late 90s in berne by the team of christoph kempf is the large-scale virus filtration technique (formerly also termed "nanofiltration") (8) . meanwhile, virus filtration became a versatile tool to eliminate a variety of pathogens, the suspected agent of variant creutzfeldt-jakob disease included. thanks to the tightly implemented regulatory framework, pathogen safety of plasma products is at a level never reached before. this is well supported by the fact of reports missing in the last decade of transmission by igg concentrates of emerging viruses (sars coronavirus, west nile virus, mers coronavirus, and others), zoonotic pathogens, or the agent of variant creutzfeldt-jakob disease (vcjd). furthermore, the development of specific mass screening techniques might help to eradicate in any blood product the transmission of vcjd in the future (9) . the human immune system is in charge of controlling invading organisms and mediates homeostasis. the immunoglobulin pools in mammals (igm, igg, and iga) to its smaller part provide defense and to the larger part homeostasis. human igg has a role in both. efficient host defense is supported by "immune antibodies." these have undergone somatic hypermutations and have in their vast majority narrow specificities and high affinities. antibodies, generated in absence of external stimuli are termed "natural antibodies" (nabs). they occasionally recognize self structures. in general, the v(d)j genes of nabs are in germ-line configuration or have undergone a few somatic hypermutations only. furthermore, they have broad specificity, are of low affinity (with exceptions) and high avidity (10) . these nabs can participate in primary host defense, i.e., at a time point when an immunologic reaction has not provided the specific antibodies, react, e.g., with repetitive structures which can be found on bacteria or viruses (11) . the self-reactive nabs, which we like to term physiologic autoantibodies, comprises various populations of antibodies such as (i) those able to interact through their complementary variable regions (v-regions) with the v-regions of circulating and membrane-bound (bcr) immunoglobulins and the t cell receptor (tcr) β-chain variable region, providing a peripheral immune network (v-connected network) (10); (ii) the nabs reacting in a non-idiotypic manner with the hinge region of immunoglobulins (12, 13) ; (iii) populations of nabs showing a wide variety of specificities toward growth factors, cytokines, or anaphylatoxin (10); (iii) and the population reacting with the soluble or membrane-bound forms of cell surface molecules having immunological importance, the last described being the fc receptors cd16 and cd32 (14) . antibodies reacting with docking structures for viruses or bacteria can have additional first-line defense potential (15) . these populations of nabs were described having a peripheral immune network homeostatic and anti-inflammatory function (16, 17) . although the primordial humoral proteins comprising the complement and lectin-like proteins in the plasma play a definite role, another population of self-reactive nabs reacting with, e.g., epitopes conserved over the evolution apparently has tissue homeostatic function and might support the efficient removal of roughly 10 12 altered/senescent cells of the body per day (for references see below). the signal for research on nabs in ivig was the description of igg autoantibody-mediated immune thrombocytopenia (itp) being corrected by infusion of a polyclonal, polyspecific igg concentrate (18, 19) . this research has expanded ever since. the populations of immune antibodies and nabs in igg concentrates upon infusion/injection inevitably react with occasional pathogens, toxins, or superantigens and concomitantly infusion/injection also results in recognition of a wide array of tissue antigens and v-regions of the recipient's immune system. reactions with tissue antigens and v-regions are conveyed by the self-reactive antibodies of the many donors in the igg concentrate. vice versa, the recipient's immune system reacts with the infused igg. a bewildering wide range of possible reactions can occur which primarily are dependent on the immune status of the recipient at the time of therapy and to a smaller part on the igg concentrate(s). the therapeutic effect achieved depends on the disease treated, and can depend on the concentration reached locally, i.e., can have agonistic or antagonistic effects (17, (20) (21) (22) . in summary, it is our opinion that igg concentrates always provide more or less the same "bouquet" of igg specificities (similarity); however, it is the recipient's actual immune condition which decides from which igg specificities the patient's derailed immune system is profiting (diversity). parameters of igg-mediated aes are: (i) the content in the product of biologically highly active likely beneficial ingredients that have to be kept under control (e.g., content of "dimers" devoid of remarkable complement activation in vivo; see below), and the content in unwanted active ingredients that have to be discarded during manufacturing (alloantibodies); (ii) impurities such as iga (anaphylactoid reaction); (iii) activated coagulation and contact activation factors (thromboembolic events) and; (iv) excipients such as sucrose (osmotic nephrosis). below, we like to add and contemplate on how fully native igg molecules not harmed by the manufacturing process might add to aes. the above mentioned inevitable interaction of the exogenous igg with the immune system of the recipient and vice versa in its principle might evoke an inflammatory condition. the sum of the potentially beneficial reactions might overshoot and lead to aes (figure 1) . the principle of induction of mild inflammatory www.frontiersin.org figure 2 | staying within a regulatory network is mandatory for remaining at the sunny side of the moon. handling of blood/plasma products to obtain therapeutic goods has to be performed within a regulatory framework. manufacturing of stable blood product has to adhere strictly to current good manufacturing practice (cgmp). application of cgmp starts from the moment of collecting whole blood and isolation of plasma thereof (r = recovered plasma) or the machine-supported collection of plasma (s = source plasma, apheresis plasma). application of cgmp ends at delivery of blood/plasma products to health care professionals. not following cgmp can lead to withdrawal of a plasma product from the market from one to the other day. conditions upon each infusion of a well-tolerated ivig was confirmed when several dozen normogammaglobulinemic volunteers in all cases except one, showed a more or less moderate inflammatory reaction as indicated by the increase of tumor necrosis factor alpha (tnfα) at 2.5 h post initiation of infusion. the only person in the cohort not showing a measurable tnfα increase was a woman caring at home for her brother with full blown aids (22) . subcutaneously applied igg concentrate reaches the circulation slowly and systemic aes are less frequent compared to ivig but they are not absent (23-28) (a case of unintentional i.v. application of scig is not considered). in contrast, local mild to moderate aes are more frequent with scig (29) . in summary, the intensity of the resulting aes is depending on the immune status of the recipient, the infusion rate (ir), e.g., how rapidly the active ingredients (the various igg specificities), the impurities, and the excipients reach the circulation of the recipient. thus, the i.v. application has the highest chance for the occurrence of aes. in the early days of ivig therapy, complement-mediated "anaphylactoid" (i.e., immediate) and "phlogistic" (i.e., inflammatory) aes were distinguished (30, 31) . the complement-mediated aes were considered to be caused by aggregates in the product ("spontaneous complement activation" or anti-complementary activity or aca) or by in vivo formation of immune complexes (ics, patient's condition related; e.g., subclinical infections or the unnoticed presence of anti-iga antibodies) and therefore only igg concentrates with low or absent aca is accepted by authorities for human use. below, we present one instructive case of each type of reaction. the first reports of rapid onset aes concerned either the application of complement-activating fractions in an igg concentrate or the in vivo formation of complement-activating ics (2) (3) (4) . a very rare but potentially fatal condition is the formation of iga/anti-iga complexes in patients being initiated on replacement therapy and having serum igg antibodies against infused iga not recognized before the start of the ivig infusion (32) . prerequisite for the presence of anti-iga antibodies is the most common primary immunoglobulin defect, i.e., selective iga deficiency (sigad) or igad associated with diminution of other immunoglobulin classes. igad is defined by serum levels of <0.05 or <0.07 g/l (depending on laboratories). a marked diminution of serum iga consistent with igad in various ethnic groups is estimated being 1:155 to 1:18,550 (33) . the mean frequency in caucasians is approximately 1:700 (34) . up to 40% of patients with igad have been reported having anti-iga antibodies in the serum with titers ranging between 1:4 and 1:262,144. in approximately 10% of patients with common variable immunodeficiency (cvid), and occasionally in patients with other primary immunodeficiency diseases, measurable anti-iga can be detected (35, 36). these antibodies are predominantly of the igg class, but anti-iga antibodies of other immunoglobulin classes have been described as well (37, 38) . the reason for their emergence remains unknown. taken the above numbers, the infusion of human-derived products containing iga resulting in severe anaphylactoid type aes should be considerable. this is not the case (39) . questions about the clinical relevance of above numbers emerge as soon as blood banks (i) estimate the theoretical risk of iga anaphylactic reactions (32); (ii) assess the relation of severe igad with the presence or absence of anti-iga antibodies (40) ; (iii) screen donors for very low iga levels in order to become able to provide blood and plasma-derived products free of iga and find a considerably lower frequency than expected (41) . alternatively, the test systems may not reliably detect anti-iga antibodies being as yet insensitive and inaccurate or -at least -do not correspond to the clinically relevant fraction of antibodies. this comes to mind when a more close look to "anti-iga" gives "unexpected" results, including "anti-iga" in blood donors with normal serum iga level or "anti-iga" that cannot be neutralized with purified iga (42); or when blood products containing proven anti-iga do not elicit severe aes (43) . among patients on replacement therapy, those with cvid may rarely develop severe immediate aes (32) . the discrepancy between anti-iga positive patients and frequency of aes raises the question about the nature of the many reported anti-iga antibodies and also raises the question about the immunologic condition which allows the formation of anaphylactoid anti-iga antibodies. there might be some logic in supposing that anaphylactoid anti-iga cannot evolve at iga levels otherwise fulfilling the definition of igad. such a condition would constantly generate ics which in turn could activate complement, react with immune cells, and be deposited in lung and kidney. indeed, horn et al. found anti-iga frontiers in immunology | primary immunodeficiencies antibodies in cvid patients missing iga + b cells and presenting with iga levels <0.0009 g/l, a level which is more than 50-to 70fold lower than the threshold for igad (44) . however, a possibility for an iga-mediated anaphylactoid reaction at measurable iga serum levels might exist. serum iga contains approximately 85% subclass 1 of iga (iga1) and only 15% subclass 2 of iga (iga2). selective deficiency of iga2 and -although evidence is lackingthe presence of a highly specific anti-iga2 antibody theoretically could elicit a severe ae. the kinetics of anti-iga after infusion of blood products have been studied in a few cases. in these patients, a fall in anti-iga titers has been noticed followed by an increase during subsequent weeks or months. this suggests that at appropriate proportions, iga of the infused material and anti-iga present in the patients' serum combine with each other to form ics. in turn, ics activate complement that are bound and eliminated by macrophages most likely leading to cytokine release. the increase in anti-iga titers over time indicates that the infused iga-containing product has a booster effect (36, 37, 45) . such boosting effect together with the presence of anti-iga before the application of an igg concentrate can be taken as the ultimate confirmation of a supposed iga/anti-iga reaction. figure 4 depicts a well-documented case of iga/anti-iga reaction in a patient who progressed from sigad to cvid. the events during the first 12 h at occasion of the first infusion of ivig were as follows (shadowed area in figure 4 ): 2 min after the start of the infusion, having received eight drops of an igg solution (iga < 1.2 g/l; 3% solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped. after approximately 1 h, the reaction has weaned, and 2 h later the patient felt well again, and the infusion of total 6 g igg could be continued without further complications. although the patient fairly assured having never received any blood or plasma product in the past, the follow-up of her anti-iga titers from before infusion to 1 year later confirmed a true anaphylactoid reaction mediated by anti-iga, as the anti-iga became undetectable immediately after the infusion and showed a boosting phenomenon during the following months. true anaphylactoid reaction was further confirmed by follow-up of total complement hemolytic activity (ch50) on the day of infusion. interestingly enough, the ch50 value reached its nadir at the end of the infusion when the patient had no complains. although a single case only, the events during the first infusion call for the following remarks: (i) severe aes most likely occur at concomitant complement and cell activation with cytokine release; (ii) infusion of minute to low amounts of ivig hours before the main infusion can "anergize" cells and stop release of pro-inflammatory www.frontiersin.org figure 4 | an individual's slithering into the dark. a female patient has been suffering from recurrent airway infections since adolescence, occasionally complicated by pneumonia. at age 34, selective iga deficiency was diagnosed. ten years later, she was hospitalized with pneumonia. within these 10 years, her serum igg had dropped from 7 to 0.87 g/l (long-dashed line) and iga was undetectable. the diagnosis was corrected into cvid, and ivig replacement therapy was initiated (shadowed area). two minutes after the start of the infusion of a 3% igg solution (iga < 1.2 g/l; 3% solution), she experienced a flush, back pain, rigors, difficulty in breathing, and hypotension. the infusion was immediately stopped and later continued without further complications (see text). the confirmation of a true anaphylactoid reaction due to anti-iga in the serum of the patient was achieved by follow-up of anti-iga (solid line) and ch50 (short-dashed line). cytokines; (iii) "anergized" cells loose reactivity toward ongoing formation of ics and complement activation products. a non-complement-mediated anaphylactoid reaction was ascribed to the unforeseen release of elastase and other proinflammatory substances from neutrophils activated by the formation of in vivo iga/anti-iga complexes. complement activation or mast cell-dependent release of vasoactive substances was excluded as pathogenic mechanisms. although the iga/anti-iga complexes usually do not cause clinically relevant neutrophil degranulation within the circulation, the presence of a rare genotype encoding a novel gain-of-function igg receptor on neutrophils may provoke premature degranulation by these complexes. this phenomenon was only relevant in hypogammaglobulinemic patients in the presence of in vivo iga/anti-iga complexes (46). the low prevalence of this genotype combined with an igad or cvid may add how to explain the rarity of serious anaphylactoid reactions in newly ivig-treated patients. authors share the opinion of janne björkander who at occasion of a discussion panel "dilemmas in diagnosis and management of antibody deficiencies: ask the experts" held at occasion of the 58th annual meeting of the american academy of allergy, asthma & immunology (aaaai), new york city, march 1-6, 2002 came to the following conclusion: a clinician has to be aware of the risk, particularly at occasion of first infusions, but otherwise iga is not a major concern (from tape record). in the early days of ig-therapy, the nature of the "phlogistic" aes was obscure. however, it was already known that an ae can be prevented or its evolution halted when the patient receives a low dose of ivig first or the infusion is stopped early and is continued several hours later. hours later the infusion can be (re)started at high rates without further problems ( figure 5) . one of the authors had a particular opportunity to get an insight into what a "phlogistic reaction" might be. at the occasion of a voluntary infusion of an investigational liquid ivig, he encountered a severe flu-like ae of more than 12 h duration. before injection, the investigational liquid preparation had passed all release criteria for human use, including spontaneous complement activation assessed by aca and was free of prekallikrein activator (pka). in those days, assays for cytokines in biological samples just began to become available and were included into the parameters assessed in the study. infusion was stopped after 1 h because of a drop of pulse rate and heavy discomfort provoking the laconic comment by the proband's technician who was taking samples:"you look green." the infusion was continued after another 90 min when the heart rate had almost normalized. the infusion could be completed within an additional 3.25 h (a total of 0.4 g/kg b.w.) without further aggravation of malaise. the leukocyte count transiently had dropped to a nadir of 40% at 2 h followed by a leukocytosis peak at 8 h. complement activation, as assessed by generation of c3a/c3a[desarg] and the formation of the terminal complement complex c5b-9, apparently did not occur: the c3a/c3a[desarg] value reached a maximum of 260 ng/ml (norm: <200 ng/ml) at 7 h while the c5b-9 value never moved outside the normal range. instead, a sequence of rapid transient massive increases of pro-inflammatory cytokines was observed: (i) tnfα started to increase 30 min post initiation of infusion from a value of 20 pg/ml to a peak value at 2 h which was above the calibration range of the test kit of 1500 pg/ml; (ii) interleukin 8 (il-8) increase started after 1 h from 29 pg/ml and peaked at 2.5 h with 4400 pg/ml post initiation of infusion; (iii) interleukin 6 (il-6) secretion started after 1 h with an undetectable level and peaked at 4 h with 345 pg/ml. all pro-inflammatory cytokines fell sharply while the second part of the infusion was still ongoing. the day after infusion, the pro-inflammatory cytokine profiles were back to normal and the flu-like syndrome was gone. in contrast, interleukin 1 receptor antagonist (il-1ra) values started increasing at 1.5 h (300 pg/ml), peaked at 4 h (>32,000 pg/ml), and decreased slowly to reach a value of 10,500 pg/ml 24 h after initiation of the infusion. soluble tnf receptor p75 level started at 2.25 ng/ml, reached a peak with 16.5 ng/ml at the same time as il-1ra, and 24 h after initiation of infusion was still at 10.5 ng/ml. thus, in this normogammaglobulinemic subject similar cytokine profiles and leukocyte number changes were observed as reported for hypogammagobulinemia under replacement therapy (47, 48) . a series of further experiments with investigational and marketed ivigs was performed. all igg concentrates were analyzed for their molecular weight (mw) distribution. the most remarkable differences emerged in the mw range of dimers while the presence of minute amounts of higher oligomers could not be excluded with certainty. below, we will use the term "dimers" for that fraction of igg with higher mw. subsequent findings indicated that levels of "dimers" >12% were responsible for complement-independent cell activation and cytokine release. the tnfα peaks assessed at 2.5 h post initiation of infusions correlated with "dimer" content of the ivigs and mirrored a clinical score of aes (49) (50) (51) . frontiers in immunology | primary immunodeficiencies a few years before a complement-independent induction of a hypotensive factor by igg di-and oligomers was reported in animal experiments (52) . a key role for macrophages in the generation of the hypotensive lipid factor was identified as plateletactivating factor, being induced by dimers and polymers (53, 54) . several years later, the dimer-mediated aes in animal experiments were confirmed (55, 56) . yet, at the same time, the dimer content of ivig apparently correlated with the clinical efficacy in a murine itp model (55, 57) . variables such as "ir," "genetic background," "endogenous immunoglobulin levels," or "proportional fraction of polymers versus dimers" may impact on the balance between the phlogiston (being cytokines, active lipid substances, or a combination of factors) and the therapeutic efficacy (blocking igg receptors on liver/spleen macrophages to prevent clearance of "opsonized" material such as platelets in itp). as of today, reports on release of cytokines in humans in association with aes or tolerability toward dimers remain scarce and to the best of our knowledge studies in humans of causative factors/fraction in an igg concentrate has not been adequately addressed (47, 48, (58) (59) (60) (61) . in igg preparations, various forms of dimers might be present: formed through covalent binding (62) by denaturation, hydrophobic interactions of the fc-parts, and by idiotype/anti-idiotype interactions, as part of the v-connected network of peripheral immune homeostasis (63) . for a commercially viable fractionation process, pooling of donated plasma is mandatory in order to obtain a volume of starting material large enough to cover ever increasing costs for documentation, in-process, and batchrelease testing as it is required by cgmp. pooling also intends smoothing the batch-to-batch differences in antibody titers, a goal apparently difficult to achieve to levels as theory might imply (64) . consequences of pooling are on the one hand the enrichment of public/common immune antibodies while diluting out individual specificities; on the other hand, the antibodies of the immune network of an individual donor are exposed to those of many other donors. the more individuals contribute to the pool, the more complex the possible "immune-network" interactions among iga, igg, and igm will become. the subsequent fractionation process has far-reaching effects on immunoglobulins from a given pool: only trace amounts of iga and igm are retained in the final product, i.e., igg is deprived of its counterparts of the v-connected immune network. the igg molecules of the homeostatic network "naked" at their v-regions can interact with each other at random combining site-interactions of single donor-derived monomeric igg (65), otherwise not existing in vivo. this interaction is largely reversible. with increasing numbers of donors included into the pool, the immune network recognition among the "naked" igg molecules of the v-connected network becomes more and more complex, and the dimer and lower oligomer content in the resulting igg concentrate increases (66) (67) (68) . in lyophilized igg concentrates, the dimer formation is "frozen" at a low level while in liquid preparations an equilibrium between monomers and dimers is achieved over time reaching a dimer content of 12% or more if not hampered by stabilizers. specificities, as far as they have been addressed, in the dimer fraction considerably differ from the monomeric fraction (69) (70) (71) (72) (73) . in conclusion, the immunomodulatory efficacy of igg concentrates in part depends on the capacity and extent to form "dimer" fractions devoid of remarkable complement activation in vivo. the "art" of manufacturing a liquid igg concentrate is not to eliminate the monomeric igg having potential for "dimer" formation but to inhibit extensive"dimerization."in summary,aes might be associated with the induction of pro-inflammatory cytokines in absence of measurable complement activation in vivo where all regulatory mechanisms and removal processes of a body are at disposition. at reasonable irs in the open system of the human body, clinically relevant systemic complement activation apparently needs oligomers formed of three or more igg molecules. there are multiple reports of ig-induced hemolytic anemia (ha) in patients receiving high doses of ivig (60, 74-110) (table 2; figure 6 ; www.adrreports.eu). by spontaneous reporting, risk factors recognized for ig-induced hemolysis include beside high doses (more than 100 g ivig over 2-4 days), female gender and histo-blood group type a, b, or ab of recipients. www.frontiersin.org a significant proportion of patients receiving ivig develop a positive direct antiglobulin test (dat) detectable after 24 h for up to 10 days after the ivig infusion (109, 110) . however, it should be underlined that the dat positivity due to the factors mentioned above (111, 112) is not sufficient per se to diagnose hemolysis and dat positivity does not necessarily imply the presence of active hemolysis. dat-positive mild hemolytic reactions can be easily missed and the true incidence of such reactions is difficult to document without careful clinical and laboratory follow-up. in the majority of reports on ha, intravascular red blood cell (rbc) destruction via complement activation or extravascular rbc sequestration and removal by the reticulo-endothelial system was proposed to result from igg alloantibodies with specificity for rbc antigens a, b, d, or c. hemolytic anemia induced by high-dose ivig has an average incidence of 5.8% (85) . low-dose igg replacement therapy is considered universally as safe, and only few cases of hemolysis following low-dose ivig or scig administration have been described (80, 95, 110) . a baseline wbc and rbc count prior to ivig initiation and a close clinical and laboratory follow-up was suggested as a useful tool for early diagnosis and treatment. a possible work up might be to check hemoglobin (hb) level prior and 48-78 h after ig infusion. in case of a drop of hb, the presence of dat, an increase in unconjugated bilirubin, lactate dehydrogenase (ldh), and reduced haptoglobin level, followed by a rise in reticulocyte count should be assessed (figure 7) . we systematically reviewed case reports related to ivig-induced hemolysis from 1987 to 2014 and identified 29 articles containing reports of 109 patients. baseline characteristics of the patients are shown in table 2 . when available, blood group, dat, hb drop, and outcome are indicated. all reports showed positive dat, except for a case of yin et al. (87) ; in this case, dat was performed 10 days after ivig administration and the dat negativity might have been due to a rapid removal of sensitized rbcs. in the majority of patients, the outcome was positive: 106 out of 109 patients recovered with or without packed rbc transfusions; three patients died after ha, with the hemolytic episode representing a precipitating factor of a severe underlying condition. elution experiments were performed and the search for blood group antibodies revealed anti-a and anti-b specificity in the majority of cases; anti-d specificity was assessed in four reports, often associated with other specificities (95, 106, 110) . a search for other specificities such as anti-band 3 or anti-gal was not performed. only one report detected anti-c specificity in three patients; in one of them associated with anti-d irregular antibodies (110) . although studies were restricted to blood group antibodies, this finding demonstrated that polyvalent igg preparations might contain clinically significant non-blood group antibodies, which are not part of the lot-release criteria in that their titration is not yet required by the european pharmacopeia. antibodies in ha, such as anti-c, may have unexpected hemolytic consequences (113) (114) (115) (116) (117) . beside passive transfer of alloantibodies, igg administration also has been demonstrated to lead to unspecific enhanced erythrocyte sequestration, in particular, in patients with underlying inflammatory disorders (109, 118) . in 2009, the canadian ivig hemolysis pharmacovigilance group elaborated criteria to define an "ivig-induced hemolysis" (83) . they included a reduction of hb levels ≥1 g within 10 days after ig administration, with frontiers in immunology | primary immunodeficiencies appearance of a positive dat and, at least, two of the following criteria: increase in the reticulocyte count, elevation of ldh and unconjugated bilirubin serum levels, low haptoglobin, hemoglobinuria, hemoglobinemia, presence of significant spherocytosis, in the absence of alternative causes of anemia. the passive transfer of igg alloantibodies through igg concentrates is difficult to explain as polyvalent igg is prepared from plasma of thousands of donors. since immunization to rbc alloantigens can occur because of past transfusions or pregnancy, the hypothetical numbers of alloimmunized plasma donors should be rather low. recently, other mechanisms underlying alloimmunization related to molecular mimicry have been demonstrated (119) . the mechanism of high-dose ivig-induced ha is complex and it might vary from patient to patient. ivig cause hemolysis due to: (i) diseaseassociated pre-coating of rbcs; (ii) igg with hemolysis triggered by passive transfer of igg binding to blood group antigens; (iii) transfer of high levels of alloantibodies to rbc pre-coated at a low level only; or (iv) transfer of clinically tolerable levels of isoagglutinins plus transfer of additional rbc-reacting physiological autoantibodies. indeed, hemolytic reactions could not be related exclusively to transfer of alloantibodies. hence, antibodies other than histo-blood group alloantibodies (pre-)coated to rbcs might contribute to hemolysis in igg recipients need to be identified. in addition, hemolytic episodes may possibly be precipitated by some sort of complexed/denatured igg that co-purify with other igg in the product (76, 109, 118, 120) . recently, a two hit mechanism for ivig-induced hemolysis has been proposed: the passive transfer of alloantibodies through ivig representing the first hit and the underlying inflammatory state representing the second hit (121) . nowadays all commercial ig products have to undergo anti-a and anti-b testing and regulatory requirement ask for respective igg antibody titers of ≤1:64 at 5% solution strength (w/v) (103, 104) . nevertheless, hemolysis might occur even in recipients of igg products that meet these specifications (76) . consequently, it has been suggested that igg recipients should be monitored for clinical signs and symptoms of hemolysis (122) . with the detection of the immunomodulatory potential of igg concentrates, their clinical use has continuously increased (123) . to cover the need, at a first glance, an increase of the volume of plasma fractionated seems to be the most convenient option. however, this might economically not be viable because fractionation of plasma products is interconnected (124) and before increasing output of one product (e.g., ivig), the market absorbance of the other products as well (e.g., albumin) must be ascertained. on a longer-run, a more viable option is to improve recovery. considering recovery, the cold-ethanol fractionation apparently has reached its limits. as of today, four manufactures have invested into a"modern"fractioning technique on the basis of ion-exchange chromatography. ion-exchange chromatography allows elevated recovery at high purity. as of today, five ivigs, one scig, and one anti-d concentrate are fractionated by ion-exchange chromatography. pharmacovigilance has shown that all chromatographically fractionated ivig and scig, more or less prominently, show a tendency for elevated frequencies of hemolytic aes. anti-a and anti-b alloantibody titers are now lot-release criteria (see above) as they constitute the major risk parameter for hemolytic reactions mediated by igg concentrates. to overcome the threat of end up on the dark side of the moon, two manufacturers have taken measures to reduce anti-a and anti-b titers in their igg products. one measure chosen was adsorption of the alloantibodies by affinity chromatography (125) . reported reduction in both alloantibodies was significant and levels were similar to those in cold-ethanol fractionated immunoglobulins (126) . the other measure chosen was reduction in anti-a using an automated indirect agglutination test for donor screening and exclusion of high-titer donations (approximately 5.1%) from plasma pooling and fractionation (127) . this measure reduced anti-a in the igg concentrate by one titer step. to ensure staying on the safe and sunny side, the manufacturer has announced the introduction of an alloanti-a and alloanti-b immune-affinity chromatography step into the manufacturing process (128) . preliminary results indicate depletion in anti-a and anti-b by >80% in investigational lots. subsequently, we want to discuss possible consequences of (extensive) removal of antibodies reacting with histo-blood group antigens a and b. three facts have initiated our thinking about possible consequences of removal of histo-blood group a and b reacting www.frontiersin.org antibodies from igg concentrates. (i) in collaboration with hans u. lutz, formerly biochemistry eth zurich, we have observed the non-intended removal of natural anti-c3 autoantibodies regulating complement activation by large-scale immune-affinity adsorption of iga from an igg concentrate (129) . anti-c3 antibodies belong to the family of "nabs" and have a particular role in homeostasis: they control activation of complement, among others, in the frame of nab-mediated opsono-phagocytosis of altered or senescent cells, including rbcs (130) (131) (132) . thus, the intention to target one particular antibody by affinity chromatography might reduce that antibody specificity but at the same time affect other specificities as well. (ii) it should be kept in mind that the blood groups a and b are in fact "histo-blood group" antigens, i.e., they are also found on white blood cells, t lymphocytes, and proteins and also can be found in soluble form (133) . alloantibodies reacting with histo-blood group antigens a and b thus have much broader tissue recognition than rbcs only. in addition, alloanti-a and alloanti-b belong to the population of nabs recognizing non-self and most likely participate in primary host defense (134) . (iii) in contrast to cold-ethanol fractionation, where low titers of alloanti-a and alloanti-b are achieved on basis of their isoelectric points (ieps), the (extensive) immune-affinity removal might affect a much wider iep range, thereby removing broadly reacting antibodies and impairing some desirable functions of the igg concentrate. thus, the struggle for staying on the sunny side of the moon might have consequences for the antibody repertoire in an igg concentrate. antibodies reacting with terminal di-, tri-, and tetrasaccharides belong to the large family of human anti-glycan nabs. histo-blood group a and b epitopes in terminal position are tetrasaccharides. alloantibodies to these tetra-saccharides are found in the plasma of healthy individuals depending on the blood group they have. a considerable body of research into the nature of these nabs has been performed so far, all using for isolation the corresponding terminal di-or tri-saccharides (135) (136) (137) . recently, the repertoire and epitope specificity of such immunoglobulins was addressed in depth by including the tetra-saccharide as well (138, 139) . it proved that serum of healthy individuals contain respectable amounts of di-or tri-saccharide-reacting nabs. these nabs proved to be pseudo-anti-a and pseudo-anti-b nabs as they are not reacting with the tetra-saccharide of histo-blood groups a and b. in contrast, alloanti-a and -b antibodies able to react with tetra-saccharides are reacting with the corresponding terminal di-and tri-saccharides. reasoning about the biological role of these "high-titer and population conservative" anti-di-and anti-tri-saccharide nabs and the consequence of their potential removal by immunoaffinity is outlined below. a population of the anti-glycan nabs are the anti-αgal nabs which recognize galα1-3gal and galα1-3(fucα1-2)gal epitopes. anti-αgal nabs have been described being xenoreactive, recognizing bacterial galα1-3gal (140) and having tissue homeostatic function. the daily removal of altered/senescent cells of the body is~10 12 . removal is mainly mediated by apoptosis (no inflammation, no necrosis). rbcs, when they do not encounter a pathological condition, over their life span of 100-120 days remain intact although they shrink, do not undergo apoptosis but progressively become senescent, mainly due to cumulative oxidative stress. removal of intact rbcs with a daily turnover of~2 × 10 11 , corresponding to~20 g cell mass, is effectuated by increased exposure of otherwise cryptic structures such as spectrin, band 3, or αgal epitopes. these exposed structures are recognized by low affinity, high avidity, c3-bearing nabs, which promote the efficient removal of intact senescent rbcs (130, 141, 142) . immunoaffinity adsorption by tri-saccharides columns of di-and tri-saccharide reacting nabs from igg concentrates can eliminate anti-histoblood a and b alloantibodies while it also eliminates αgal and this might have a janus effect. the face directed to the sun tells that adsorbing αgal nabs reacting with altered and senescent self on rbc might prevent an increase in the igg load of rbcs over the threshold level of relevant hemolysis in individuals at risk. the face directed to the dark indicate that adsorption of tissue homeostatic antibodies might deprive an igg concentrate of potentially beneficial antibodies. although they are nabs, tri-saccharide reacting antibodies can be induced by feeding bacteria bearing the corresponding carbohydrate epitopes (134) . these inducible nabs are considered to participate in primary host defense. other antibodies possibly involved in primary host defense are the anti-αgal nabs. they show a broad specificity and can react with a number of related αgal-terminated oligosaccharides, including those on bacteria (143) . thus, the immunoadsorption of di-and trisaccharide reacting nabs might diminish the potential of an igg concentrate to mediate primary host defense. therefore, when choosing affinity resins for immunoadsorption, there might be some aspects worth to consider. in summary, the principles of avoiding co-fractionation through cold-ethanol fractionation (144) versus immune-affinity removal of histo-blood group alloantibodies can have an impact on the presence of homeostatic and first-line defense antibodies. according to present knowledge, only resins coated with the corresponding tetra-saccharides can ascertain the selective removal of histo-blood group alloantibodies presumably involved in ha. resins coated with the corresponding di-and tri-saccharides also remove blood group alloantibodies, however not selectively. such resins in addition might remove a broad range of nabs present in igg concentrates at relative high amounts. in the literature, the use of tri-saccharide-coated resin was reported (145) (146) (147) . we have found no information available in the public domain indicating which type of resin is/will be used for reduction of the histo-blood group alloantibodies in large-scale fractionation of igg. furthermore, we suggest that the effect of reduction of anti-a and ant-b reacting antibodies by immune-affinity on the antibody repertoire of igg concentrates can only be assessed by, e.g., using pathogens/commensals, which share the saccharide epitopes, that have been used to coat the affinity resins or alternatively by exposing senescent rbcs stripped off the iggs coated in vivo. finally, techniques are required, which allow detection of low affinity, high avidity nabs. ivig administration-related aes, including thrombosis, have been extensively described (148) . thrombotic aes are severe aes and patients with risk factors require a special care. reported average incidence of ivig-induced thrombosis ranges from 3 to 13% (149) . recognized risk factors for ivig-induced thrombosis frontiers in immunology | primary immunodeficiencies include male gender; age >60; diabetes; renal insufficiency, dyslipidemia; hypertension; immobility; coronary disease; pre-existing vascular disease, family history of early thromboembolic disease; atrial fibrillation, high-dose and high-speed ivig infusions. ivig-induced thrombosis is reported both as venous events such as thrombosis stroke, pulmonary embolism (pe), deep venous thrombosis (dvt), and arterial ischemia events such as myocardial infarct and stroke. the mechanisms leading to ivigassociated thrombosis are still not completely clear; three main mechanisms have been proposed, emphasizing the role of an increased blood viscosity causing a hypercoagulable state (150) , the role of anticardiolipin antibodies passively transferred through ivig (151) , and the role of factor xia or other biologically highly active factors passively transferred via igg concentrates, such as pka. avoiding activated coagulation factors in igg concentrates starts with appropriate anticoagulation of donated blood/plasma, i.e., careful mixing of anticoagulant and sample over the whole donation process. alterations in an established manufacturing process neglecting appropriate controls can also lead to increased risk of transmission of activated coagulation factors. high mw proteins passively transferred by ivig are probably contributing to this phenomenon (152) . in patients with other risk factors, such as vascular disease, the increase in blood viscosity can precipitate thromboembolic events. as elderly individuals are prone for such aes, we like to point to the possibility of elevated altered/senescent self-reacting with infused homeostatic nabs being a possible factor facilitating thrombotic events as well. a relationship between ivig administration and cerebral vasospasm has also been suggested by sztajzel et al. (153) ; blood viscosity is a determinant for oxygen delivery to the tissues, and changes in viscosity can lead to a reduction in cerebral or myocardial perfusion. we systematically reviewed case reports related to iviginduced thrombosis from 1986 to 2014 (figure 8) . literature search identified 35 articles containing reports concerning 65 patients (6, 24, 149, . when data were available, diagnosis, risk factors, the number of ivig infusion prior to thrombosis event, and outcome have been indicated. baseline characteristics of the patients are shown in table 3 . high-dose ivig induced thromboembolic events in 59 patients at low to medium ivig doses. marie et al. (163) observed that the frequency and type of arterial events was inversely related to the time elapsed from ivig infusion; almost 50% (23 versus 21 reports) of arterial ischemic events occurred within 12 h following ivig, while about 75% of venous thrombosis occurred after more than 24 h. no correlation between number of infusions and occurrence of ae was observed. the main risk factors observed in this review were hypertension (19 cases, 33% of prevalence), previous vascular disease (18%), and dyslipidemia (17%). average mortality for thrombotic events was 10% (arterial ischemia 9% versus venous thrombosis 11%, pe representing the main venous fatal event). predicting iviginduced thrombosis is difficult. risk factors should be assessed for each patient including instrumental exams when needed. doppler ultrasound can be useful as early diagnostic tool for thrombosis or to detect the presence of abnormal blood flow especially after prolonged immobility. ivig should be administered at low ir to reduce the risk. the administration of antiplatelet or anticoagulant prophylaxis was suggested in patients with several risk factors (162) . however, thrombotic events have been reported even after several previous uncomplicated courses of treatment. www.frontiersin.org (2), arrhythmia (1) first (3), several (2) arterial (3), venous (2) 14 days recovery (4), death (1) al-riyami et al. (1) second (1), third (2), several (5) arterial (6), venous (1) 1 h (2), 2 days (3), 2 weeks (2) recovery (5) stamboulis et al. (1) several (4) arterial (4) 2 h (3), 2 days (1) death (3) clinical and immunological characteristics of patients described in case reports. numbers in parenthesis indicate the number of patients with the given condition. in such cases, patients should be examined for signs and symptoms of thrombosis during each courses of ivig. immunoglobulin g concentrates are widely acknowledged to offer a safe, high-dose, long-term therapy option for a variety of diseases. aes occur rarely and mainly are mild to moderate. deviations from this rule of thumb are addressed by authorities and the plasma fractionation industry to achieve corrections. above, we have reviewed two types of ae which have shown elevated frequency in the near past. we tried to give some insights which might help in reducing frequencies of aes bed side. authors are deeply grateful to hans-hartmut peter, freiburg, germany, for his careful reading of the manuscript, the valuable comments, and the correction of english. epidemic hepatitis b caused by commercial human immunoglobulin intravenous administration of human γ-globulin adverse reactions following administration of human gamma-globulin igg antibodies to iga in two patients with hypogammaglobulinemia treated with commercial gammaglobulin contact-activated factors: contaminants of immunoglobulin preparations with coagulant and vasoactive properties fatal thrombotic events during treatment of autoimmune thrombocytopenia with intravenous immunoglobulin in elderly patients effect of high-dose intravenous immunoglobulin therapy on blood rheology pathogen safety of immunoglobulin preparations population screening for variant creutzfeldt-jakob disease using a novel blood test: diagnostic accuracy and feasibility study intravenous immunoglobulin: exploiting the potential of natural antibodies inherent specificities in natural antibodies: a key to immune defense against pathogen invasion the natural human igg anti-f(ab')2 antibody recognizes a conformational igg1 hinge epitope stimulation of complement amplification by f(ab') 2 -containing immune complexes and naturally occurring anti-hinge antibodies -possible role in systemic inflammation natural autoantibodies to fcγ receptors in intravenous immunoglobulins multi-faceted role of naturally occurring autoantibodies in fighting pathogens immunomodulation of autoimmune and inflammatory diseases with intravenous immune globulin intravenous immunoglobulin: an update on the clinical use and mechanisms of action demonstration of a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura high-dose intravenous gammaglobulin for idiopathic thrombocytopenic purpura in childhood concurrent presence of agonistic and antagonistic anti-cd95 autoantibodies in intravenous ig preparations a differential concentration-dependent effect of ivig on neutrophil functions: relevance for anti-microbial and anti-inflammatory mechanisms verträglichkeit und virussicherheit von intravenösen immunglobulinen occurrence of hemolytic reactions (hrs) on the same day as immune globulin (ig) product administrations during cerebral venous and sinus thrombosis associated with subcutaneous immunoglobulin injection and oral contraceptive use multiple immunoglobulin intolerance without antibody's anti-immunoglobulin a: a case report common variable immunodeficiency: a patient with anaphylaxis to intravenous and subcutaneous immunoglobulin prospective study on cvid patients with adverse reactions to intravenous or subcutaneous igg administration severe adverse reaction to subcutaneous immunoglobulin therapy in a patient with common variable immunodeficiency evaluation of the relationship between injection site reaction rate and scig doses in patients with primary immunodeficiencies immunthemotherapy: a guide to immunoglobulin prophylaxis and therapy prophylactic infusions with an unmodified intravenous immunoglobullin product causing few side-effects in patients with antibody deficiency syndromes the role of anti-iga antibodies in causing adverse reactions to gamma globulin infusion in immunodeficient patients: a comprehensive review of the literature prevalence of immunoglobulin a deficiency (igad) in shanghai blood donors and efforts to establish a rare blood bank of igad in shanghai hammarström l, persson maa, smith cie. anti-iga in selective iga deficiency -in vitro effects and ig subclass pattern of human anti-iga immunoglobulin prophylaxis in patients with antibody deficiency syndromes and anti-iga antibodies follow-up of anti-iga antibodies in primary immunodeficient patients treated with gamma-globulin anaphylactic reactions after gamma globulin administration in patients with hypogammaglobulinemia: detection of ige antibodies to iga management of immunoglobulin a deficiency: lessons from haemovigilance screening of canadian blood services donors for severe immunoglobulin a deficiency large scale detection of iga deficient blood donors unexpected reactions of the anti-iga antibody particle gel immunoassay allergic transfusion reactions from blood components donated by iga-deficient donors with and without anti-iga: a comparative retrospective study anti-iga antibodies in common variable immunodeficiency (cvid): diagnostic workup and therapeutic strategy induction of unresponsiveness against iga in iga-deficient patients on subcutaneous immunoglobulin infusion therapy release of cytokines, soluble cytokine receptors, and interleukin-1 receptor antagonist after intravenous immunoglobulin administration in vivo neutropenia as a complication of intravenous immunoglobulin (ivig) therapy in children with immune thrombocytopenic purpura: common and non-alarming igg dimers in liquid intravenous immunoglobulin preparations laboratory parameters measured during infusion of immunoglobulin preparations for intravenous use and related tolerability welltolerated liquid intravenous immunoglobulin g preparations (ivgg) have a low immunoglobulin g dimer (igg-dimer) content an animal model for the detection of hypotensive side effects of immunoglobulin preparations key role of macrophages in hypotensive side effects of immunoglobulin preparations. studies in an animal model vasoactive side effects of intravenous immunoglobulin preparations in a rat model and their treatment with recombinant platelet-activating factor acetylhydrolase intravenous immunoglobulin preparations induce mild activation of neutrophils in vivo via triggering of macrophages -studies in a rat model hypotension with intravenous immunoglobulin therapy: importance of ph and dimer formation therapeutic efficacy of intravenous immunoglobulin preparations depends on the immunoglobulin g dimers: studies in experimental immune thrombocytopenia tumor necrosis factor and intravenous gammaglobulins in common variable immunodeficiency ivig adverse reactions: potential role of cytokines and vasoactive substances acute hemolysis after intravenous immunoglobulin amid host factors of abo-mismatched bone marrow transplantation, inflammation, and activated mononuclear phagocytes intravenous immunoglobulin induces interferon-γ and interleukin-6 in vivo human igg2 can form covalent dimers variable regionconnected, dimeric fraction of intravenous immunoglobulin enriched in natural autoantibodies ivig -mechanisms of action igg dimers in multidonor-derived immunoglobulins: aspects of generation and function immunoglobulin g dimer: an idiotype-anti-idiotype complex a view of the human idiotypic repertoire -electron microscopic and immunologic analyses of spontaneous idiotype-anti-idiotype dimers in pooled human igg polyreactive antibodies in multidonorderived immunoglobulin g: theory and conclusions drawn from experiments comparative analysis of antigen specificities in the monomeric and dimeric fractions of intravenous immunoglobulin monomerization of dimeric igg of intravenous immunoglobulin (ivig) increases the antibody reactivity against intracellular antigens antibodies in the dimer fraction of ivig have the capacity to bind beta amyloid an analysis of anti-fas and anti-siglec-9 autoantibodies in monomeric and dimeric fractions of ivig dimeric ivig contains natural anti-siglec-9 autoantibodies and their antiidiotypes intravenous immunoglobulininduced hemolytic anemia in a patient with juvenile dermatomyositis intravenous immunoglobulin-induced haemolysis: a case report and review of the literature massive intravascular haemolysis after high dose intravenous immunoglobulin therapy maternal hemolysis after intravenous immunoglobulin treatment in fetal and neonatal alloimmune thrombocytopenia hemolytic anemia following intravenous immunoglobulin therapy in patients treated for kawasaki disease: a report of 4 cases ivig -a hemolytic culprit haemolysis after treatment with intravenous immunoglobulin due to anti-a hemoglobinuria and acute kidney injury requiring hemodialysis following intravenous immunoglobulin infusion a pediatric case series of acute hemolysis after administration of intravenous immunoglobulin acute hemolysis after high-dose intravenous immunoglobulin therapy in highly hla sensitized patients hemolytic transfusion reactions after administration of intravenous immune (gamma) globulin: a case series analysis hemolytic anemia following intravenous immunoglobulin administration acute hemolysis in a patient with cytomegalovirus pneumonitis treated with intravenous immunoglobulin (ivig) a prospective study of the immediate and delayed adverse events following intravenous immunoglobulin infusions adverse effect of polyvalent immunoglobulin in the treatment of guillain-barré syndrome hemolysis after administration of high-dose immunoglobulin in a patient with myocarditis haemolytic anaemia associated with high dose intravenous immunoglobulin therapy in a child with guillain-barré syndrome insisting on intravenous polyvalent immunoglobulin therapy in polymyositis in spite of the occurrence of sever hemolytic anemia -poursuite du traitement d'une polymyosite par les immunoglobulines intraveineuses polyvalentes malgré la survenue d'une anémie hémolytique sévère severe hemolytic anemia following high-dose intravenous immunoglobulin administration in a patient with kawasaki disease hemolytic anemia associated with intravenous immunoglobulin hemolysis during immunoglobulin therapy -hemolisis durante el tratamiento con inmunoglobulinas hemolytic anemia following highdose intravenous immunoglobulin administration acute hemolysis due to passively transfused high-titer anti-b causing spontaneous in vitro agglutination hemolysis after high-dose intravenous ig immune hemolysis, disseminated intravascular coagulation, and serum sickness after large doses of ivig for kawasaki disease acute intravascular haemolysis associated with high dose immunoglobulin after bone marrow transplantation for acute myelogenous leukemia hemolysis after intravenous immune globulin therapy: relation to igg subclasses of red cell antibody autoimmune hemolytic anemia in kawasaki disease: a case report haemolysis induced by intravenously-administered immunoglobulin massive intravascular hemolysis associated with intravenous immunoglobulin in bone marrow transplant recipients hemolytic anemia following intravenous gamma globulin administration hemolysis following intravenous immune globulin therapy transient haemoglobin drop following high dose intravenous immunoglobulin in vivo administration of intravenous immunoglobulin (ivig) can lead to enhanced erythrocyte sequestration hemolysis in patients with antibody deficiencies on immunoglobulin replacement treatment batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti-rh d activity hematologic toxicities associated with intravenous immunoglobulin therapy anti-a and anti-b activity in batches of different intravenous immunoglobulin products determined using a direct haemagglutination method international collaborative study to evaluate candidate reference reagents to standardize haemagglutination testing for anti-a and anti-b in normal intravenous immunoglobulin products european directorate for the quality of medicines and healthcare. anti-a and anti-b haemagglutinins european directorate for the quality of medicines and healthcare. test for anti-d antibodies in human immunoglobulin immune complex-like moieties in immunoglobulin for intravenous use (i.v.ig) bind complement and enhance phagocytosis of human erythrocytes regulation of primary alloantibody response through antecedent exposure to a microbial tcell epitope a follow-up study of 49 adult patients with idiopathic thrombocytopenic purpura treated with high-dose immunoglobulins and anti-d immunoglobulins hemolysis upon intravenous immunoglobulin transfusion report of the fda meeting on strategies to address hemolytic complications of immune globulin infusions kreuth ig working group. european consensus proposal for immunoglobulin therapies the art of balanced production in vitro and in vivo properties differ among liquid intravenous immunoglobulin preparations patient safety through an ivig mastered manufacturing process. posters isoagglutinin reduction in human immunoglobulin products by donor screening igg product development isoagglutinin reduction measures. oral presentation naturally occurring antibodies/autoantibodies in polyclonal immunoglobulin concentrates. lutz, h. u. naturally occurring antibodies (nabs) naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes high doses of immunoglobulin g attenuate immune aggregate-mediated complement activation by enhancing physiologic cleavage of c3b in c3bn-igg complexes intravenously applied igg stimulates complement attenuation in a complementdependent autoimmune disease at the amplifying c3 convertase level histoblood group antigens as allo-and autoantigens blood group isoantibody stimulation in man by feeding blood group-active bacteria normal human serum contains natural antibodies reactive with autologous ab0 blood group antigens a unique natural human igg antibody with anti-alpha-galactosyl specificity normal human serum contains high levels of anti-galα1-4glcnac antibodies natural anti-a and anti-b of the ab0 system: allo-and autoantibodies have different epitope specificity repertoire of human natural anti-glycan immunoglobulins. do we have autoantibodies? interaction between human natural anti-alpha-galactosyl immunoglobulin g and bacteria of the human flora innate immune and non-immune mediators of erythrocyte clearance comparison of serum anti-band 3 and anti-gal antibody binding to density-separated human red blood cells specificity of human antibodies against galα1-3gal carbohydrate epitope and distinction from natural antibodies reacting with galα1-2gal or galα1-4 gal anti-a and anti-b haemagglutinin trend analysis during manufacturing process of ivig. presentation at workshop "strategies to address hemolytic complications of immune globulin infusions in vitro evaluation of the efficacy and biocompatibility of new, synthetic ab0 immunoabsorbents high molecular weight blood group a trisaccharide-polyacrylamide glycoconjugates as synthetic blood group a antigens for anti-a antibody removal devices specific antibody filter (saf) binding capacity enhancement to remove anti-a antibodies intravenous immunoglobulin: adverse effects and safe administration thromboembolic complications of intravenous immunoglobulin therapy in patients with neuropathy: a two-year study high-dose intravenous immunoglobulin and serum viscosity: risk of precipitating thromboembolic events antired blood cell antibodies, free light chains, and antiphospholipid antibodies in intravenous immunoglobulin preparations soluble hla class i and class ii concentrations in commercial immunoglobulin preparations high-dose intravenous immunoglobulin treatment and cerebral vasospasm: a possible mechanism of ischemic encephalopathy? high dose intravenous immunoglobulin may be complicated by myocardial infarction fatal case of bilateral internal jugular vein thrombosis following ivig infusion in an adolescent girl treated for itp cerebral sinus thrombosis following iv immunoglobulin therapy of immune thrombocytopenia purpura graft rupture after high-dose intravenous immunoglobulin therapy in a renal transplant patient cerebral sinus thrombosis in a patient with humoral immunodeficiency on intravenous immunoglobulin therapy: a case report a case of deep vein thrombosis and pulmonary thromboembolism after intravenous immunoglobulin therapy acute stroke with high-dose intravenous immune globulin venous and arterial thrombosis following administration of intravenous immunoglobulins intravenous immunoglobulin-associated vena cava thrombosis intravenous immunoglobulin-associated arterial and venous thrombosis; report of a series and review of the literature thromboembolic events as an emerging adverse effect during high-dose intravenous immunoglobulin therapy in elderly patients: a case report and discussion of the relevant literature diffuse venous thromboemboli associated with ivig therapy in the treatment of streptococcal toxic shock syndrome: case report and review deep vein thrombosis after intravenous immunoglobulins associated with methylprednisolone deep venous thrombosis after high-dose intravenous immunoglobulin in the treatment of pemphigus vulgaris thromboembolic complications of intravenous immunoglobulin treatment acute myocardial infarction following intravenous immunoglobulin therapy for chronic inflammatory demyelinating polyneuropathy in association with a monoclonal immunoglobulin g paraprotein stroke and deep venous thrombosis complicating intravenous immunoglobulin infusions thrombosis complicating high dose intravenous immunoglobulin: report of three cases and review of the literature acute thromboembolic events associated with intravenous immunoglobulin infusion in antibody-deficient patients transverse sinus thrombosis and ivig treatment: a case report and discussion of risk-benefit assessment for immunoglobulin treatment thrombotic complications after intravenous immunoglobulin therapy in two patients pulmonary embolism after intravenous immunoglobulin adverse effects of intravenous immunoglobulin therapy in 56 patients with autoimmune diseases acute myocardial infarction associated with high dose intravenous immunoglobulin infusion for autoimmune disorders. a study of four cases deep venous thrombosis of the arm after intravenous immunoglobulin infusion: case report and literature review of intravenous immunoglobulin-related thrombotic complications cerebral infarction complicating intravenous immunoglobulin therapy in a patient with miller fisher syndrome central retinal vein occlusion complicating treatment with intravenous immunoglobulin acute myocardial infarction during treatment with intravenous immunoglobulin for idiopathic thrombocytopenic purpura (itp) myocardial infarction as a complication of immunoglobulin therapy iatrogenic central retinal vein occlusion and hyperviscosity associated with high-dose intravenous immunoglobulin administration the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-278081-tk7vn1v1 authors: brooks, wesley h. title: viral impact in autoimmune diseases: expanding the “x chromosome–nucleolus nexus” hypothesis date: 2017-11-28 journal: front immunol doi: 10.3389/fimmu.2017.01657 sha: doc_id: 278081 cord_uid: tk7vn1v1 viruses are suspected of significant roles in autoimmune diseases but the mechanisms are unclear. we get some insight by considering demands a virus places on host cells. viruses not only require production of their own proteins, rna and/or dna, but also production of additional cellular machinery, such as ribosomes, to handle the increased demands. since the nucleolus is a major site of rna processing and ribonucleoprotein assembly, nucleoli are targeted by viruses, directly when viral rna and proteins enter the nucleolus and indirectly when viruses induce increased expression of cellular polyamine genes. polyamines are at high levels in nucleoli to assist in rna folding. the size and activity of nucleoli increase directly with increases in polyamines. nucleolar expansion due to abnormal increases in polyamines could disrupt nearby chromatin, such as the inactive x chromosome, leading to expression of previously sequestered dna. sudden expression of a large concentration of alu elements from the disrupted inactive x can compete with rna transcripts containing intronic alu sequences that normally maintain nucleolar structural integrity. such disruption of nucleolar activity can lead to misfolded rnas, misassembled ribonucleoprotein complexes, and fragmentation of the nucleolus. many autoantigens in lupus are, at least transiently, components of the nucleolus. considering these effects of viruses, the “x chromosome–nucleolus nexus” hypothesis, which proposed disruption of the inactive x by the nucleolus during stress, is now expanded here to propose subsequent disruption of the nucleolus by previously sequestered alu elements, which can fragment the nucleolus, leading to generation of autoantigens. previously, we presented the "x chromosome-nucleolus nexus" hypothesis (1) (2) (3) . in the hypothesis it was proposed that enlargement of the nucleolus in response to cellular stress could disrupt neighboring chromatin, such as the inactive x chromosome. as a result, sequestered alleles (e.g., polyamine genes on the inactive x), elements (e.g., alu elements), and viruses could be opened for transcription. this could lead to eventual creation of autoantigens due to overexpression of genes and elements from both the previously active chromatin and the, now, reactivated chromatin. here is presented new details to the hypothesis, explaining how the disrupted chromatin can lead to subsequent disruption of the nucleolus, even nucleolar fragmentation, which results in ineffective nucleolar functioning, misfolded rnas, misassembled or incompletely assembled ribonucleoprotein (rnp) complexes, and stabilization of nucleolar components in autoantigenic conformations. many of the major autoantigens in autoimmune diseases like systemic lupus erythematosus (sle) are, at least transiently, components of the nucleolus (e.g., splicosome subunits). among the factors that could cause extraordinary cellular stress, viruses are highly suspected of causing such disruption in autoimmune diseases. exposomics is the study of all environmental factors which a person may encounter during their lifetime, even including prenatal exposure. these environmental factors in the exposome can include components of the diet, gut microbiota, chemicals, air pollutants, heavy metals, and infectious agents. these factors can cause cellular stress and can have a cumulative effect in cells through accumulation of genetic damage and/or disruption of epigenetic control, especially in genetically predisposed individuals, that establishes the conditions for eventual manifestation and progression of an autoimmune disease. within the exposome is the infectome which is the collection of pathogens that may contribute to an individual's onset and progression of an autoimmune disease (4) . this can be complicated by the latency of some pathogens and the synergistic effects of multiple pathogens. however, unless one is looking for pathogen antigens, the specific pathogen and its effect on the immune system may be masked by a larger response to more abundant autoantigens some of which the pathogen's antigens may mimic. in addition, it has been difficult to prove these associations since, in the case of viruses, many viruses can establish latent infections but the autoimmune disease may not manifest itself until several years after the initial infection, thus clouding the true extent of their association. for example, a mononucleosis infection, a.k.a. the "kissing disease, " which involves the epstein-barr virus (ebv), increases the risk for subsequent appearance of multiple sclerosis (ms) but manifestation of the ms might not occur until as long as 30 years after the mononucleosis episode (5) . add to this the fact that in the interim the individual has had other infections caused by other pathogens that complicate the situation, potentially triggering the actual autoimmune disease for which an initial ebv infection set the stage. a subsequent infection with another virus could allow activation of latent viruses giving a combined stressful impact on the cell. for example, a primary infection by cytomegalovirus can lead to reactivation of latent ebv which provokes an immune response (6) . the induction of one latent virus by another virus shows the potential complexity underlying autoimmune diseases. the general population has had exposure to many of the viruses associated with autoimmune diseases but for most individuals there is no autoimmune disease development, suggesting that genetic susceptibility is also important as well as possible epigenetic and environmental factors. as an example, most adults have had exposure to ebv but few develop an autoimmune disease, suggesting other factors are involved rather than just ebv. a genetic possibility for these differing responses may be based on different hla types, for example, entry of ebv into a host cell via binding of the ebv's gp42 glycoprotein to human cd21 and lymphocytic antigen type hla-dr. other hla sub-types may have different expression levels or have different affinity for the gp42 and not be as compliant for ebv entry. in addition, the extracellular portion of the ebv bzlf2 protein can suppress antigen presentation by binding hla-dr delaying detection of the ebv (7) . table 1 lists many of the viruses that have shown associations with autoimmune diseases. we should note that there is variety in the route of transmission and entry among these viruses: (1) respiratory and oral secretions (saliva, sputum, nasal mucus) (e.g., ebv, parvovirus); (2) gut (e.g., enteroviruses); (3) insect vector transmission (e.g., mosquitos for zika, west nile); (4) sexual interactions (e.g., hpv, hiv); and (5) transfusions (e.g., hiv). the tissue types in which viral sequestration occurs may vary, such as ebv behind the blood-brain barrier associated with ms or possible ebv in the synovium associated with ra. we should also note that there are both rna and dna viruses listed in table 1 and most of these viruses can persist in a latent state in the host. appearance of viral antigens does not necessarily mean that the virus is the cause of the autoimmune disease episode since it may only be the result of stress from an autoimmune disease episode that leads to subsequent activation of a hidden virus. for most of these associations (table 1) , it remains to be determined if the virus is the causative agent, one of several combined contributing agents, or simply appearing as a result of impaired host cell suppression of the virus. for example, the measles virus is suspected of involvement in ms due to the appearance of antibodies to measles virus antigens in cerebrospinal fluid of ms patients (36) . whether the ms is a direct result of the measles virus or the appearance of measles antigens is a consequence of the ms or is simply coincidental is not known. there may, in fact, be another virus or another environmental agent that has disrupted the suppression of the latent measles virus. as it is, the situation is even more perplexing since the introduction of measles vaccination for the general population has not caused a significant change in the occurrence rate of ms (37) . other viruses with an infrequent association with an autoimmune response can have reemergence in other forms, such as the varicella zoster virus which causes chicken pox and which can reemerge as shingles and is associated with ms (38) . and we should bear in mind that human endogenous retroviruses are suspected of involvement in serious diseases, including autoimmune diseases (25, 39) . among the viruses listed in table 1 , ebv has received the most attention as a virus with links to autoimmune diseases (15, 40) . an association with ebv infection has been observed in sle (41, 42) , ms (43) , ra, and sjögren's syndrome (sjs) (44) and prior ebv infection, as indicated by sero-positivity for ebv antigens, is observed in 94.2% of controls and 99.5% of ms patients (45) . in addition, ebv (human gammaherpesvirus 4, hhv-4) is representative of several herpes viruses that have shown associations with autoimmune diseases. therefore, based on current knowledge, ebv is most useful for describing possible viral involvement in autoimmune diseases in general. one way in which a viral infection could provoke an autoimmune reaction is by disrupting the host cell's epigenetic control during a particularly strong cellular stress response to the viral activity leading to expression of previously sequestered gene alleles. expression from those newly opened sites could lead to imbalance in the protein and rna products. the female predominance of many autoimmune diseases suggests that the x chromosome and possibly disruption of the inactive x chromosome, a major epigenetic structure in the cell, could be of significance in such a scenario of viral disruption of epigenetic control (1, 3) . one point of concern is fragile sites, which are particularly susceptible to viral insertion and dna breaks. fragile sites can be hundreds of thousands, even millions of base pairs in length. the x chromosome has four major fragile sites (fraxa at xq28; fraxb at xp22; fraxc at xq22; and fraxd at xq27) (1, 46) . reactivation of part or all of the inactive x chromosome could open these fragile sites for expression of hidden viruses in the fragile sites, adding to the cellular stress. once a virus becomes active in a cell, one of its prime targets in taking over the cell is the nucleolus. the virus is dependent on the host cell's machinery, including ribosomes and transfer rnas (trna), in order for viral proteins to be synthesized. and viral rnas need to be properly folded and assembled into ribonucleoprotein complexes (rnps). rna and rnp processing and assembly are major functions of the nucleolus. the virus puts additional demands on the nucleolar functions beyond the host cell's needs and, since the virus does not code for ribosomes and such, the virus needs to induce increased nucleolar capacity and activity. localization of viral rna and proteins to the nucleolus takes advantage of nuclear and nucleolar localization signals (nols) and chaperones. in the case of viral rna transcripts, rna pol iii transcribed rnas bind ssb/la and ssa/ro which assist in nucleolar entry and processing along with any required refolding. for viral proteins, nuclear localization signals (nls), which are sequences of basic amino acids, and nols are used but, since these signals are frequently closely positioned, it has been difficult to decipher the nols from the more recognizable nls (47) . some progress has been made in determining nols, such as for the adeno-associated virus serotype 2 assembly activating protein (48) , and for nucleolar retention signals, such as found in coronavirus nucleocapsid proteins (49) . the nucleolus-structure, function, and dynamics the nucleolus [reviewed in ref. (50) (51) (52) (53) (54) ] is a prominent feature in the nucleus, appearing as a vacant area when imaging nuclear dna content. there is, in fact, some dna in the nucleolus. as far as active dna in the nucleolus, nucleoli associate primarily with repetitive copies of ribosomal dna (rdna) genes expressing ribosomal rnas from nucleolar organizing regions (nors) which are located on the acrocentric autosomes 13, 14, 15, 21, and 22. other dna sequences may be involved at the periphery of the nucleolus transiently, such as in repair of breaks in dna near the rdna genes. a role in dna repair in general is emerging for the nucleolus since many proteins involved in dna repair have associations with the nucleolus (55) . further, centromeric dna is associated with nucleoli as part of the nucleolar regulation of the cell cycle (56) . the nucleolus does not have a membrane defining its structure but nucleoli are typically surrounded by a shell of heterochromatin established in chromosomes containing nucleolar-associated chromatin domains (nads) (52) . this then serves to define the boundaries of the nucleolus. the nads contain satellite dna, mostly from centromeric and pericentromeric regions of chromosomes. the nads also contain gene poor and silent chromatin. in addition, the inactive x chromosome (a.k.a. the barr body), a heterochromatic body found in most human female cells, is found in close proximity to nucleoli in one-third of cells throughout the cell cycle and 90% of cells in s phase suggesting a putative role for the nucleolus in maintaining x chromosome inactivation (57) . the heterochromatin-nucleolar associations are facilitated, in general, by insulator proteins, ctcf (ccctc-binding factor) and nucleophosmin and additionally, in the case of the inactive x chromosome, by x inactivation specific transcript rna. ebv latency can be controlled by ctcf bound in the promoter region of the epstein-barr virus nuclear antigen 2 (ebna-2) gene (58) . disruption of chromatin-nucleolar interactions could lead to changes in ebv latency when ctcf interactions with inserted ebv genes are disrupted. in addition, another very important point to keep in mind is that nucleolin bound to rna polymerase ii (rna pol ii) transcripts that contain intronic alu elements appear to have a critical role in maintaining the integrity of the nucleolus (59) . when caudron-herger and colleagues added rna pol iii transcribed alu element sequences, even as short as 20 nucleotides, there was fragmentation of nucleoli into small nucleolar-like units that were very inefficient in carrying out nucleolar functions of rna and rnp processing and assembly (figure 1) . the authors proposed that the nucleolar fragmentation was attributable to dicer-facilitated degradation of hybridized rna pol iii alu sequences with rna pol ii intronic alu sequences. the work of caudron-herger and colleagues demonstrates a close connection between nucleolar integrity and the complexes of nucleolin with rna pol ii transcripts containing intronic alu sequences. another possibility for nucleolar disruption by rna pol iii alu transcripts that caudron-herger and colleagues did not mention is possible competition for nucleolin between the rna pol ii intronic alu sequences and a sudden abundance of rna pol iii alu transcripts. we believe this could have a major role in generation of autoantigens as we will explain below. normally nucleoli contain three discernable sub-regions: the fibrillary centers (fcs), the dense fibrillary centers (dfcs), and the granular components (gcs). the fcs are the sites of rdna transcription by rna polymerase i (rna pol i) to generate the initial ribosomal rna transcript, the pre-rrna. only 50% of the ~400 rdna repeats in the human diploid genome are transcriptionally active (60) . processing of the pre-rrna occurs primarily in the dfcs assisted by small nucleolar rnas (snornas). assembly of the final ribosomal subunits occurs in the gc, which has a high concentration of proteins needed to complete the rnps (61). other rnas and rnps processed and assembled in the nucleolus include: the signal recognition particle (srp) which controls translation and localization of extracellular proteins by transporting them to the endoplasmic reticulum (er) for eventual extracellular release; trnas which require extensive folding; small nuclear ribonucleoprotein complexes involved in splicing of messenger rnas (mrnas); and centromere components. therefore, the nucleolus is involved directly or indirectly in many cellular functions, such as regulation of mitosis; cell-cycle progression; cell proliferation; mrna processing via splicing; translation; protein localization; and various forms of stress response. the nucleolar proteome contains over 4,500 proteins according to the nucleolar proteome database, nopdb3.0 (62). about 30% of these proteins are involved in ribosome biogenesis. since the demands on nucleolar output can change rapidly, the nucleolar proteome is very dynamic. in addition, the size of the nucleolus can change dramatically depending on the needs. increased nucleolar size correlates directly with increases in polyamine synthesis (63) . the polyamines, spermidine and spermine, are involved in many cellular functions but their highest concentrations are found in the nucleoli where the polyamines assist in folding of rna transcripts and assembly of rnps. the polyamines have a unique combination of length (spermidine ~11 å spermine ~14 å), flexibility (all single bonds) and high cationic charge at physiological ph (spermidine +3; spermine +4) which makes them ideal counter ions to assist in folding the negatively charged rna transcripts in the nucleolus. nucleoli are very dynamic structures in the cell and cell cycle. there can be more than one nucleolus in the nucleus and, figure 1 | nucleolar integrity from rna pol ii intronic alu sequences. (a) caudron-herger and colleagues reported that the nucleolus has a high content of rna pol ii transcripts with intronic alu sequences (red) and these transcripts associate with nucleolin to maintain nucleolar integrity (59) . (since the actual localization in the nucleolus and structure of the nucleolin-rna complexes are not known, they are shown simply as part of the nucleolar perimeter.) (b) addition of rna fragments from rna pol iii alu sequences, even as short as 20 bases, leads to loss of nucleolar integrity which caudron-herger and colleagues attribute to dicer degradation of hybridized alu sequences. this leads to fragmentation of the nucleolus into subunits that are substantially less efficient in nucleolar functions of rna folding and ribonucleoprotein assembly. combined, they can occupy up to 25% of the nucleus. they can expand rapidly, facilitated by increased polyamines, in response to cellular stress since the cell may need to have more ribosomes and trnas to synthesize new proteins to recover from the stress. however, we should remember that the nucleolus surrounds itself with heterochromatin so there is the possibility of displacement or disruption of neighboring heterochromatin due to the nucleolar dynamics (1) . with regards to the cell cycle, nucleoli disappear in mitosis and reappear in telophase and early g1 forming around nors with the rdna genes and pre-existing rrna and ribosomal complexes (64) . in addition, cdk1 cyclin kinases have a key role in controlling nucleoli during cell cycling, and centromere complexes are generated in the nucleolus giving further importance to nucleoli in cell cycling. once in the host cell, the virus can be sequestered into the host chromatin or it can initiate viral replication. in the case of ebv, the multi-functional epstein-barr nuclear antigen 1 (ebna-1) protein from the ebv genome can assist in the spread and attachment of viral dna to metaphase chromosomes (65) . ebna-1 can also disrupt the host cell's usp7-assisted stabilization of p53/ tp53, thereby preventing the host cell from entering apoptosis, setting the stage for continual viral replication (65) . however, viruses do need host cell machinery produced in the nucleolus, such as ribosomes, to facilitate viral replication. therefore, the virus will attempt to increase nucleolar activity and turn on viral gene expression. the ebv genome has a snorna, called v-snorna1, which is found in the nucleolus in infected cells (66) . the v-snorna1 appears to be involved in activation of the viral dna polymerase. another ebv early gene is epstein-barr nuclear antigen 2 (ebna-2) which is a transactivator of viral and host genes. ebna-2 can associate with rna polymerase ii promoters to induce increased transcription and this includes the myc gene (67, 68) . myc induces increased rna polymerase iii (rna pol iii) activity which creates viral rna transcripts and many of the rna transcripts for nucleolar assembled complexes (69) . and myc induces increased transcription by rna pol i to create rrna transcripts (70) . the myc interactome consists of approximately 15% of genes throughout the host genome (71, 72) . included among these are genes involved in polyamine synthesis: ornithine decarboxylase (odc); spermidine synthase (sds); and spermine synthase (sms) (73) (74) (75) . and so the virus induces polyamine synthesis which is directly associated with an increase in the size and activity of the nucleolus (63, 76) . the cationic polyamines are ubiquitous and have many important interactions throughout the cell and local extracellular environment (e.g., spermine in neural synapses). the highest levels of polyamines are found in the nucleolus where the polyamines play a critical role in rna folding by neutralizing anionic charges in the rna sufficiently for intra-strand rna-rna interactions to form. polyamine availability directly correlates with increased rna expression and processing (77) . and the polyamines can assist in rnp complex assembly. mostly these are transient interactions of the polyamines but, in some cases, (a) the inactive x chromosome (xi) is typically located at the nuclear periphery next to the nuclear membrane and is associated with a nucleolus in 90% of cells in s phase and one-third of cells throughout the cell cycle except during mitosis when nucleoli disappear (57) . this places one of the most inactive structures in the cell, the inactive x, next to one of the most active, multi-functional, and dynamic structures, the nucleolus. (b) the nucleolus can rapidly expand during cellular stress, such as viral activation. trapped between the nucleolus and the nuclear membrane, the xi could be disrupted by the expanding nucleolus. the polyamines remain as part of the final rna complex, such as in trnas (1). since viral genes need to be expressed and viral rnas need to be folded and some viral proteins localize to the nucleolus for folding and assembly into the final virion, it is understandable why a virus would want to increase the host cell's polyamine content to increase nucleolar capacity and activity (78) . however, the relation between viral activation and subsequent rna synthesis is more complex and can vary among viral types. for example, poliovirus inhibits rna pol i activity by inducing sl1 cleavage and ubf posttranslational modification (79) , whereas hepatitis c virus stimulates rna pol i activity which is involved in transcription of the rdna genes (60) . viruses can influence the nucleolar proteome leading to abnormal redistribution of nucleolar components to the nucleus, cytoplasm, and even cell surface (80) . for example, viruses induce cell surface exposure of ssb/la which normally facilitates termination of rna pol iii transcription in the nucleus and then, along with ssa/ro, acts as a chaperone for the rna transcript as it is processed in the nucleolus (81) . viruses can also disrupt the cell-cycle related kinases in the nucleolus, suppressing normal cell cycling and, thereby, hijacking the nucleolus to focus on viral rna and protein synthesis and assembly of viral rnp complexes (78) . the usual effect of cellular stress, such as viral activity, on the nucleolus is to cause enlargement of the nucleolus but on some occasions the nucleolus can actually decrease in size. inhibition of rna pol i by poliovirus, as mentioned above, could be such a situation since a drop in rrna transcripts would inhibit the major nucleolar function of ribosome synthesis. the "x chromosome-nucleolus nexus" hypothesis: disruption of the inactive x the stress that viral activity can put on the nucleolus can lead to extensive enlargement of the nucleolus. this could potentially disrupt the epigenetic silencing in heterochromatin neighboring the nucleolus. the inactive x chromosome, a.k.a. the barr body, would be especially vulnerable, as we proposed in the original version of the "x chromosome-nucleolus nexus" hypothesis (1), since the barr body is frequently found in close proximity to a nucleolus (57) and against the nuclear membrane (82), as depicted in figure 2a . sandwiched between the nucleolus and the nuclear membrane, the barr body would not be able to avoid exposure and disruption due to the nucleolin and nucleophosmin that are involved in chromatin remodeling from an expanding nucleolus (83, 84) . in addition, exposure of the chromatin to the high content of polyamines in the nucleolus could add to the disruption of chromatin since the cationic polyamines can compete with histones for dna binding. moreover, the polyamines have the potential to stabilize alternate dna conformations, such as z-dna, which is targeted by autoantibodies in some cases of sle and ra. negative supercoiling stress is stored in nucleosomes as the double-stranded right-hand coiled b-dna makes a left-handed supercoil over the surface of the nucleosome's histones. displacement of the histones during chromatin remodeling could release the negative supercoiling stress, allowing it to flux through the dna and potentially flipping into left-hand coiled z-dna which is also a form of negative supercoiling storage. z-dna appears only transiently in chromatin since most dna is wrapped up as b-dna in nucleosomes. z-dna is not flexible enough to bend around histones so it is excluded from the 145 bp bound to the surface of the histones. since nucleosomes in human chromatin occur every 200 bp on average, there is normally little opportunity for z-dna to form and persist. exposure to high levels of polyamines from the nucleolus concomitant with disruption of nucleosomes could increase the likelihood of z-dna persistence when there is a shift of negative supercoiling storage from nucleosomes to z-dna (85) . in a similar manner, dna cruciforms are formed from negative supercoiling stress but their occurrence is also suppressed by positioned nucleosomes. the alu elements, of which there are more than one million throughout the human genome, contain sequences capable of cruciform formation (1). since there are approximately 15 million nucleosomes associated with the human genomic dna, there is ample stored negative supercoiling stress. this potential for rapid dynamic changes in chromatin, including disruption of higher order "stacked" nucleosomes, alternate dna conformations, displacement of bound proteins, and dna strand separation, is perhaps figure 3 | establishment of the inactive x chromosome (xi). early in embryogenesis one of the two x chromosomes in female cells is inactivated by persistent expression of the x inactivation specific transcript rna (xist) from the x inactivation center (xic). xist rna does not code for protein but remains in the nucleus and binds contiguous chromatin (i.e., the xi, a.k.a. the barr body), recruiting epigenetic silencing effectors (e.g., dna methyltransferases). approximately 95% of genes from the long arm (xq) and 65% of genes from the short arm (xp) are silenced. silenced genes shown as dark blue, while genes that escape inactivation are shown as light blue [based on ref. (89) ]. the result is the barr body which appears as a dense heterochromatic structure near the nuclear membrane. the bulk of the heterochromatic core contains xq genes with some xp genes, and the euchromatic-like surface layer has primarily xp genes that are: actively expressed; potentiated for expression; or silenced but adjacent to expressed genes. particularly interesting in the xp is the pseudo-autosomal region 1 (par1) which has an abundance of alu elements (46) . in addition, xp22 contains a "hot" line-1 sequence that can code for a fully functional reverse transcriptase. xp22 also contains a fragile site (fraxb). fragile sites are preferential locations for viral insertions. and xp22 on the xi contains epigenetically silenced genes for spermine synthase (sms) and spermidine/ spermine n1 acetyltransferase (sat1). overexpression of sms and/or sat1 that could occur with disruption of epigenetic silencing on the xi can impact cellular methylation and polyamine types and levels. this could also impact polyamine activity in the nucleoli. under-appreciated aspects of epigenetics and could come into play when the nucleolus encroaches on surrounding chromatin. normally males have only one x chromosome whereas females have two x chromosomes. most genes on the x are not sex-related so females only need one active x chromosome. therefore, early in embryogenesis, each female cell inactivates one x chromosome, either the maternally derived or the paternally derived x, and each daughter cell will inherit that inactivation pattern. the process of x chromosome inactivation [reviewed in ref. (86) ], which results in the heterochromatic barr body, begins from the x inactivation center at xq13 of the x chromosome's long arm (xq) (figure 3) . approximately 95% of genes on the xq and 65% of genes on the short arm (xp) are inactive and form the heterochromatic core of the barr body (87) . the surface of the barr body would be more characteristic of euchromatin with genes that escape inactivation and some inactive genes that are surrounded by active genes or genes potentiated for activity. especially interesting are genes on the xp from xp22 to the xp telomere, including the pseudo-autosomal region 1 (par1). these would be at the surface of the barr body and more readily disrupted by an expanding nucleolus under stress. sms and spermidine/spermine n1 acetyltransferase (sat1) are involved in polyamine synthesis and recycling, respectively, and normally sms and sat1, located at xp22.1, are inactive on the barr body (87) . however, disruption of the barr body by enlargement of the nucleolus, as shown in figure 2b , could lead to reactivation of sms and sat1. this would result in a rapid increase in polyamine synthesis and recycling beyond what was already induced by the host cell and the viral activity. there would be an increase in acetylated polyamines by sat1 that could interfere with rna folding and, through oxidation, generate putrescine which, in turn, could allosterically increase s-adenosylmethionine (sam) decarboxylase activation reducing sam needed for dna and protein methylation (72) . excess free polyamines can be conjugated to proteins by transglutaminases and acetylated polyamines, and the conjugated polyamines and putrescine can be oxidized to toxic acrolein. there is a close relationship between the intensity of sjs and the appearance of acrolein-conjugated proteins (88) . the net effect of expression of sms and sat1 from the disrupted barr body is dysregulation of polyamine levels. add to this the fact that sat1 can undergo super induction, meaning there could be a several 100-fold increase in polyamine acetylation. in the nucleolus, there could be an increase in polyamines and now acetylated polyamines that interfere with normal rna folding and rnp assembly. once sat1 becomes active from both x chromosomes from nucleolar disruption of the barr body, going forward there could be a drop in polyamines during subsequent stress events as super induction of sat1 acetylates polyamines. it may follow that the nucleolus can no longer expand sufficiently to adapt to new stresses and the nucleolus can no longer work efficiently in proper folding and assembly of rnas and rnps, leading to creation of autoantigens. in other words, an initial stress-induced polyamine-driven expansion of the nucleolus could disrupt the barr body leading to rna pol iii alu transcript-driven fragmentation of the nucleolus. subsequently, with reactivation of sat1 from the barr body, it may reduce polyamines and reduce the ability of the nucleolus to function normally in folding and assembly of rnas and rnps and fail to react effectively to future stressful events since polyamines will be rapidly acetylated as they are synthesized. this scenario could result in ongoing generation of abnormal, potentially autoantigenic rnps due to the compromised nucleolus that lacks sufficient polyamines. another problem that could arise is reverse transcription. most line-1 elements have mutated sufficiently so that they no longer code for functional reverse transcriptases. a few, including one in xp22, can still produce functional reverse transcriptases but are suppressed by positioned nucleosomes (1) . reverse transcription of alu elements could be particularly consequential. alu elements are rich in g-c base pairs; therefore, reverse transcribed alu dna would require significant methylation since hypomethylated dna would be interpreted as exogenous. line-1 reverse transcriptases preferentially reverse transcribe line-1 rna at a rate of 1,000× and alu rna at a rate of 300× in comparison to other rnas (90) . the cell could quickly become inundated with hypomethylated alu dna. li and steinman reported a high content of alu dna (up to 55%) in the free dna in sera of lupus patients whereas alu elements only account for 10.8% of the human genome (91) . those authors suggested that reverse transcription could be a possible cause. we proposed that fully functional line-1 elements activated from disruption of the x chromosome could be involved in such reverse transcription. expanding the "x chromosome-nucleolus nexus" hypothesis: disruption of the nucleolus the earlier version of the "x chromosome-nucleolus nexus" hypothesis suggested that there could be consequences from expression of previously sequestered alu elements, particularly from the par1 of the x chromosome short arm where there is an exceptionally high content of alu elements (1). we can now add detail to the hypothesis regarding what consequences could arise from rna pol iii expression of these alu elements. there are over 1,000,000 alu elements spread throughout the human genome but most are suppressed by a positioned nucleosome. displacement of the nucleosome could open the alu element's internal rna pol iii transcription start site. rna pol iii can be quite prolific since it does not require energy (atp), does not require extensive assembly of transcription factors, can initiate from the intragenic promoter in alu elements, and can rapidly reinitiate to generate multiple transcripts. in addition, since alu elements average only 300 bp and the intragenic promoter requires only about 70 bp for transcription factor binding, displacement of only one nucleosome would be all the opening needed. the abundance of rna pol iii typically found near the nucleolus, particularly the perinucleolar compartment, could rapidly generate thousands of alu rnas if there were a disruptive event, such as encroachment of the nucleolus into the barr body. especially vulnerable is the dense cluster of alu elements in the par1 region near the surface of the barr body. whereas alu elements comprise 10.8% of the human genome, they are at only 8% in the x chromosome. however, alu elements account for 28.8% of the par1 region and 19% of the adjacent s5 region (46) . since par1 has approximately 2.5 million base pairs, there are estimated to be more than 2,500 alu elements in par1 that could potentially flood the nucleus and nucleolus with alu rna transcripts ( figure 4a ). contrast this with the approximately 200 active ribosomal rna genes in the nucleolus. the alu rnas could interfere with assembly of the srp which contains an alu domain that binds srp 9/14 heterodimers (72) . free alu rna could compete in binding the srp 9/14 leaving incomplete srps that cannot halt ribosomal activity in the cytoplasm when needed during synthesis of extracellular targeted proteins. this could lead to improper modifications (e.g., transglutamination) and localization of proteins. and, opening of the alu elements, which have extensive intra-strand matching sequences, could also facilitate formation of cruciforms in the dna which could be stabilized by polyamines. these cruciforms could be interpreted as autoantigens by the immune system. perhaps, the greatest danger from expression of alu elements from the disrupted barr body is deduced from the work of caudron-herger and colleagues mentioned previously (59) . an abundance of rna pol iii alu transcripts from the disrupted barr body could compete with or lead to degradation of the rna pol ii intronic alu rna that, along with nucleolin and nucleophosmin, provides structural integrity for the nucleolus. this would lead to fragmentation of the nucleolus into inefficient subunits (figure 4b) . these nucleolar-derived subunits could have abnormal levels of polyamines and acetylated polyamines that cannot properly fold rna and assemble rnps. in fact, the needed components for assembly of an rnp like the ribosome may be unequally distributed among the nucleolar fragments preventing complete assembly. and there could be viral proteins and rnas competing to join rnp assemblies. the rnps and partial assemblies could be stabilized in abnormal conformations and associations by the polyamines and become autoantigenic when released from the cell. such extracellular exposure could occur by blebbing and microparticle release as the cell enters apoptosis, netosis or other forms of termination (92) . with a loss of integrity of the nucleolus and expression of viral components, some nucleolar material could be displayed on the cell surface, such as the la protein (81) . in addition, nucleolar fragmentation could lead to loss of nucleolar control of cell cycling, such as assembly of centromeres. another problem that could arise is involvement of cyclic gmp-amp synthase (cgas) which detects cytosolic dna. this includes detection of micronuclei that contain dna from a disrupted nucleus or from dna damage (93) . fragmentation of the nucleolus, as described above, could potentially generate such micronuclei, especially when centromere assembly and functioning are disrupted or when there are lagging chromosomes during mitotic segregation of chromosomes. the appearance of hypomethylated reverse transcribed alu dna in the cytosol, possibly originating from x-linked line-1 reverse transcription of par1 alu element rna, could also trigger the cgas-sting pathway. formation of cyclic gmp-amp (cgamp) can trigger activation of the stimulator of interferon genes (sting) protein which induces transcription of interferon β (ifnβ) as part of the innate immune response in antiviral, antibacterial, and anticancer activity and is suspected of involvement in autoinflammatory and autoimmune diseases (94) . therefore, the original "x chromosome-nucleolus nexus" hypothesis, which explained how the nucleolus could disrupt the inactive x chromosome, can now be expanded to include disruption of the nucleolus by x-linked alu rna transcripts that lead to nucleolar fragmentation and generation of autoantigenic material. the "x chromosome-nucleolus nexus" hypothesis in relation to other diseases the "x chromosome-nucleolus nexus" hypothesis was developed primarily with sle in mind but it could be involved in many autoimmune diseases. the mechanism could have differing effects due to the cell types and locations involved. for example, ra and sle can have some of the same autoantigens targeted, figure 4 | autoantigens generated by disruption of the nucleolus. (a) the original version of the "inactive x chromosome and nucleolus nexus" hypothesis proposed that there is disruption of the inactive x by the nucleolus due to an extraordinary expansion of the nucleolus under stress (figure 2b) . this disruption could open previously sequestered dna, especially alu sequences and genes in the short arm of the xi that are located in the euchromatic-like surface layer of the xi (1). now, based on the work by caudron-herger and colleagues (59), we can propose additions to the hypothesis, that x-linked alu transcripts generated by the abundant rna pol iii near the nucleolus can disrupt the nucleolin-rna pol ii intronic alu complexes, either by dicer degradation or by direct competition between the rna pol iii alu transcripts and the intronic alu sequences. (b) the subsequent fragmentation of the nucleolus could result in nucleolar fragments that contain conformationally abnormal autoantigenic structures due to improperly folded rnas or improperly assembled ribonucleoprotein complexes (rnps). for example, in some nucleolar fragments there may be insufficient quantities of ribosomal components (either rnas or proteins) and, therefore, complete functional ribosomes cannot be formed. there may also be incorporation of viral rna and/or viral proteins into the rnps. also, overexpression of x-linked spermine synthase and/or spermidine/spermine n1 acetyltransferase could result in abnormal types and levels of polyamines in the nucleolus and nucleolar fragments. for example, there may be putrescine, acetylated polyamines, and/or nuclear aggregates of polyamines in the nucleolus that interfere with rna folding. normally one would expect only spermine and spermidine to be present in large quantities. extracellular release (by apoptosis, necrosis, netosis) of these abnormal nucleolar products could provoke an autoimmune reaction that later targets the more abundant normal products due to epitope spreading. such as z-dna, but ra is primarily behind the synovial membrane reducing full exposure of antigenic and autoantigenic material to the immune system. however, continual attraction of neutrophils to the same confined inflammation site in ra where the neutrophils undergo netosis in an ineffective attempt at clearing abnormal material would produce chronic local exposure of cells and extracellular material to the neutrophil's active peptidyl arginine deiminases (pads) producing high levels of citrullinated proteins (e.g., collagen) that eventually provokes the adaptive immune system into producing autoantibodies targeting the modified collagen as a major autoantigen in ra (85) . in a similar manner, ms is confined behind the bloodbrain barrier reducing access of autoantigenic material to the immune system but, again, neutrophils continually attracted to an ms lesion could be releasing pads that citrullinate myelin, eventually triggering autoantibodies to citrullinated myelin basic protein which is a major autoantigen in ms. sle is a systemic disease suggesting that the immune system can more readily react to the broad array of abnormal material seen in sle and generate autoantibodies. compared to ra or ms, the autoantigens, autoantibodies and complexes of the two in sle have easier access to the circulatory system allowing the reaction to spread to and deposit in different organs rather than being confined behind a membrane barrier. sjs can be a primary disease or it can be secondary to sle, ra or ms. this suggests that there could be similar mechanisms occurring in all four of these disorders. involvement of polyamines, possibly due to loss of epigenetic control of x-linked polyamine genes, is suspected in sjs since the appearance of acrolein conjugated proteins is related to the intensity of sjs and acrolein is an oxidation product of polyamines (88) . the hypothesis may even play a role in alzheimer's disease (alz) since there is a female bias in the disease and there are autoantibodies involved in alz (95) . in addition, polyamine levels are altered in alz (96, 97) along with increased acrolein (98) ; sam levels are greatly decreased (99) ; polyamines are involved in plaque formation (100) and nucleolar poly (adp-ribose) polymerase 1 (parp1) is decreased in alz (101) . cellular stress that leads to disruption of the inactive x chromosome and/or the nucleolus could play a role in the altered polyamine activity, decreases in sam, decreases in parp1, and appearance of acrolein. the hypothesis as a whole or in parts (part 1: inactive x disruption and/or part 2: nucleolar disruption) could have a role in some cancers. the inactive x chromosome is often missing in tumor cells from breast and ovarian cancers (102) . the inactive x may have reactivated from a decrease in methylation (possibly due to over activity of polyamine synthesis and recycling) or the inactive x was lost due to improper segregation of chromosomes to daughter cells (possibly due to centromere assembly in the nucleolus). viruses could be involved in the loss of x inactivation since viruses increase polyamine levels in order to increase nucleolar activity for their benefit, as exemplified by ebv induction of odc, sds, and sms via increased myc activity. the subsequent reduction in sam due to polyamine synthesis would make it difficult for the cell to maintain chromatin methylation required for epigenetic silencing in the x chromosome and other chromosomes leading to disruption of control of oncogenes and tumor suppressor genes and there is the possibility of opening previously sequestered viruses that then try to take control of the nucleoli. the inactive x has the greatest demands for methylation but it is the last chromosome to be replicated and repackaged in late s phase or even early g2 when sam levels would have already been impacted by methylation of other chromosomes. reactivation or loss of the inactive x chromosome could explain some of the cases of triple-negative breast cancer in which there is no overexpression of her2, estrogen, or progesterone receptors (103) . so this epigenetic scenario of barr body disruption could explain some of the enigmatic cases of cancers. also, keep in mind that viral disruption of nucleoli could interfere with nucleolar involvement in dna repair, nucleolar assembly of centromeres, and alter nucleolar control of cell-cycle kinases (78) . fragmenting of nucleoli as centromeres are being formed could lead to abnormal distribution of chromosomes resulting in daughter cells of differing karyotypes, such as a parent (46,xx) cell generating daughter cells of (45,x0) and (47, xxx) . disruption of the nucleolus in tumor cells could also lead to appearance of autoantigens. autoantigens can arise in cancers but they differ from those normally seen in autoimmune diseases such as sle. the differences could arise from: the cell type involved (proliferating versus mature, differentiated); the nucleolar activity and content at the time of disruption; and the rapidity of the disruption (acute versus gradual accumulation). we can consider that nucleoli in proliferating cells would be heavily involved in cell cycling, such as generating centromere components, and so many of the autoantigens that arise would be expected to be related to cell cycling and suppression of apoptosis (104) . autoimmune diseases, such as sle, give rise to autoantigens that are components more routinely found in abundance in nucleoli, such as ribosomal or splicosomal components. there is a slightly higher risk of cancers among autoimmune patients but the risk varies with regards to the type of cancer (105) . therapeutics taken by the patient targeting the autoimmune disease could contribute to cancer development. hematological, thyroid, lung, and vulva cancers show an increased risk with non-hodgkin's lymphoma showing a 3× to 4× greater risk in lupus patients, while breast, endometrial, and ovarian cancers show a lower risk. for now there is no direct connection between viruses and the "x chromosome-nucleolus nexus" hypothesis to the increased risk of cancers among autoimmune disease patients but we can consider the induction by viruses of increased polyamine levels and the possible reactivation of x-linked polyamine genes as means by which competition for the cellular methyl donor, sam, could reduce dna methylation and open oncogenes for overexpression in proliferation competent cells. increased nucleolar size due to increased polyamines could add to the disruption of neighboring epigenetically silenced chromatin to expose alleles for expression. the focus of this discussion has been on viruses and the "x chromosome-nucleolus nexus" hypothesis since we now understand the effects a virus can have on the nucleolus and how it is to the benefit of the virus to influence the nucleolar activity. and ebv has been used as the primary example of viral involvement in autoimmune diseases since it is one of the viruses most suspected of having such a role and we can connect ebv actions (e.g., increased myc activity) to increases in polyamines that could directly impact the nucleolus and trigger the hypothesized mechanism. other factors besides viruses, such as bacteria or chemicals, can contribute to autoimmune diseases but the means is less clear and may not closely follow the mechanism proposed. bacteria, for example, produce putrescine and spermidine without the extensive controls on polyamine synthesis seen in eukaryotes but the bacteria can produce their own machinery and are not as dependent as viruses on the cell's nucleolus. in addition, disruption of heterochromatin, such as the inactive x chromosome, can open latent viruses. the x chromosome has four major fragile sites and fragile sites are frequently the locations chosen for viral insertion. the particulars of the fragile site and its vulnerability to viral insertion may add to the genetic susceptibility of an individual. the previous version of the "x chromosome-nucleolus nexus" hypothesis, or simply the "nucleolus" hypothesis, explained how an overly stressed nucleolus could disrupt neighboring heterochromatin, especially the barr body. as a result, there could be detrimental increases in synthesis and recycling of polyamines that could impact cellular methylation and potentially stabilize autoantigenic complexes of nucleolar and chromatin components. the point was made that many autoantigens in sle are, at least transiently, components of the nucleolus but the means by which such nucleolar components could become autoantigenic was not presented. now, with this work, the hypothesis has been extended to include disruption of the nucleolus as an additional step. a disrupted barr body could generate an abundance of polyamines and alu rna from x-linked genes and elements that further stress and damage the nucleolus, making it very inefficient in its functions, even fragmenting it and possibly leading to cell death. and there may be overexpression of sat1 that hampers the nucleolus in subsequent stress events since polyamines may be converted to a predominance of acetylated polyamines that are less effective at or even detrimental to proper nucleolar folding and assembly of rnas and rnps. meanwhile during nucleolar disruption autoantigens may be created, stabilized and released extracellularly. further work is needed to understand how the various autoantigens provoke the autoimmune response. is it, for example, a conformational alteration of the ribosomal subunits stabilized by polyamines, or incomplete assembly of an rnp? or could it be "guilt by association" such as ssa/ro bound to misfolded rnas stabilized with polyamines that prevent proper refolding? there may be incorporation of viral components in the rnps that make it autoantigenic. or could it be abnormal localization and/or modification of proteins that are misdirected due to alu rna interference with srp assembly (72) . epitope spreading from the autoantigenic complex to the normal endogenous protein would seem to have a role in the autoimmune response with the greater abundance of the endogenous protein then providing more of the provocation than the original autoantigenic complex. and the fragmentation of nucleoli, as described here, could lead to extracellular signaling and extracellular exposure of autoantigens. testing of the hypothesis can use powerful approaches, such as computational molecular dynamics and single cell analysis, that have now reached sophistication that allow us to explore the interactions of nucleolar components as they are normally processed and the possibilities of how abnormalities could occur. for example, what is the effect of acetylated polyamines if they were to compete with spermidine and spermine in the nucleolar folding and assembly of rnps? what are the interactions and resulting structures of intronic alu rna with nucleolin? and what is the distribution and composition of rnas and proteins in nucleolar fragments compared to intact nucleoli? and, perhaps most important, what does this new hypothesis present as far as therapeutic targets? certainly suppressing viral activity, myc activity, polyamine synthesis, and polyamine recycling are important targets but also newer areas, such as the cgas-sting pathway are promising targets too. the importance of alu elements implied by this hypothesis calls into question the use of mouse models of autoimmune diseases since mice do not have the extensive amount of alu elements seen in humans (certainly not a cluster of 28.8% seen in the par1 of the human x). in addition, the mouse x chromosome is telocentric (just one long arm) whereas the human x is submetacentric (a long arm and a short arm). the mouse x inactivation would be relatively consistent since it does not negotiate a centromere. x inactivation researchers complain that it is difficult to study partial x reactivation in mice due to the consistency of inactivation along the mouse x. this makes the murine x more robust under stress, at least with regards to this hypothesis (1). we have previously discussed the short arm of the x chromosome (xp) and especially the portion from xp21.2 to the terminus as having a major role in sle, particularly with regards to possible reactivation of the inactive x chromosome (1-3, 72, 82, 106, 107) . this section includes: a "hot" line-1 (codes for a fully functional reverse transcriptase); the polyamine genes spermidine/spermine n1 acetyltransferase and sms; and the par1 region with an abundance of alu elements. other groups are just now coming to the conclusion that the xp arm has a major role in autoimmune diseases (108) although they have not mentioned the alu elements, line-1 and polyamine genes we have mentioned and they have not made the connection to the nucleolus. it is hoped that autoimmune disease researchers will consider the "x chromosome-nucleolus nexus" hypothesis since it is the most comprehensive explanation yet for autoimmune diseases. it does involve areas with which those researchers may not be familiar, such as polyamines, x inactivation, epigenetics, and the nucleolus making it a rather complex scenario but autoimmune diseases are very complex phenomena. the author confirms being the sole contributor of this work and approved it for publication. the author has no funding other than as a research assistant professor in the chemistry department at the university of south florida, tampa, fl usa. epigenetics and autoimmune diseases: the x chromosome-nucleolus nexus a review of autoimmune disease hypotheses with introduction of the "nucleolus" hypothesis the 'nucleolus' hypothesis of autoimmune diseases and its implications tracing environmental markers of autoimmunity: introducing the infectome multiple sclerosis after iinfectious mononucleosis immunoreactivation of epstein-barr virus due to cytomegalovirus primary infection the extracellular domain of the epstein-barr virus bzlf2 protein binds the hla-dr beta chain and inhibits antigen presentation coxsackievirus b1 is associated with induction of β-cell frontiers in immunology | www autoimmunity that portends type 1 diabetes human cytomegalovirus and autoimmune disease autoimmune hepatitis: a comprehensive review the mosaic of environment involvement in autoimmunity: the abrogation of viral latency by stress, a non-infectious environmental agent, is an intrinsic prerequisite prelude before viruses can rank as infectious environmental agents that trigger autoimmune diseases correlation between systemic lupus erythematosus and cytomegalovirus infection detected by different methods autoimmunity in dengue pathogenesis expression of innate immunity genes and damage of primary human pancreatic islets by epidemic strains of echovirus: implication for post-virus islet autoimmunity systematic review and meta-analysis of the sero-epidemiological association between epstein-barr virus and systemic lupus erythematosus the immunology of epstein-barr virus-induced disease hepatitis b virus (hbv) and autoimmune disease hepatitis c virus, autoimmunity and rheumatic disease herpes simplex virus encephalitis is a trigger of brain autoimmunity molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection autoimmunity and dysmetabolism of human acquired immunodeficiency syndrome aids virus infection and autoimmunity: a perspective of the clinical, immunological, and molecular origins of the autoallergic pathologies associated with hiv disease the enemy within: endogenous retroelements and autoimmune disease human endogenous retrovirus group e and its involvement in diseases on the relationship between human papilloma virus vaccine and autoimmune diseases human parvovirus b19, autoimmunity and systemic lupus erythematosus human parvovirus b19 infection and autoimmunity possible role of human herpesvirus 6 as a trigger of autoimmune disease association of human herpes virus 6 (hhv-6) with multiple sclerosis: increased igm response to hhv-6 early antigen and detection of serum hhv-6 dna virus infection, antiviral immunity, and autoimmunity increased prevalence of varicella zoster virus dna in cerebrospinal fluid from patients with multiple sclerosis west nile virus infection and myasthenia gravis zika virus and neurologic autoimmunity: the putative role of gangliosides classification and characterization of human endogenous retroviruses; mosaic forms are common measles virus antibodies in serum and cerebrospinal fluid in patients with multiple sclerosis and other neurological disorders, with special reference to measles antibody synthesis within the central nervous system multiple sclerosis incidence in the era of measles-mumps-rubella mass vaccinations varicella-zoster virus in cerebrospinal fluid at relapses of multiple sclerosis viruses and endogenous retroviruses in multiple sclerosis: from correlation to causation epstein-barr virus in autoimmune diseases epstein-barr virus and systemic lupus erythematosus strong viral associations with sle among filipinos the role of ebv in ms pathogenesis potential role of epstein-barr virus in sjogren's syndrome and rheumatoid arthritis an updated meta-analysis of risk of multiple sclerosis following infectious mononucleosis the dna sequence of the human x chromosome nuclear entry of dna viruses identification and characterization of nuclear and nucleolar localization signals in the adeno-associated virus serotype 2 assembly-activating protein delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein new insights into nucleolar structure and function. f1000prime rep the nucleolus: structure/function relationship in rna metabolism nucleolus and nuclear periphery: velcro for heterochromatin proteins of the nucleolus: an introduction the nucleolus -guardian of cellular homeostasis and genome integrity crosstalk between the nucleolus and the dna damage response nucleolar dna: the host and guests perinucleolar targeting of the inactive x during s phase: evidence for a role in the maintenance of silencing regulation of epstein-barr virus latency type by the chromatin boundary factor ctcf alu element-containing rnas maintain nucleolar structure and function the nucleolus under stress the multifunctional nucleolus nopdb: nucleolar proteome database-2008 update role of polyamine in the regulation of rna synthesis in uterine nucleoli assembly and disassembly of the nucleolus during the cell cycle structure of the p53 binding domain of hausp/usp7 bound to epstein-barr nuclear antigen 1 implications for ebv-mediated immortalization expression and processing of a small nucleolar rna from the epstein-barr virus genome the proto-oncogene c-myc is a direct target of gene of epstein-barr virus nuclear antigen 2 epstein-barr virus induces cellular transcription factors to allow active expression of eber genes by rna polymerase iii direct activation of rna polymerase iii transcription by c-myc c-myc binds to human ribosomal dna and stimulates transcription of rrna genes by rna polymerase i an overview of myc and its interactome autoimmune diseases and polyamines the ornithine decarboxylase gene is a transcriptional target of c-myc c-myc targets genes involved in cell growth, apoptosis, and metabolism targeting ornithine decarboxylase in myc-induced lymphomagenesis prevents tumor formation ultrastructural changes in vitro of rat liver nucleoli in response to polyamines relationship between polyamine accumulation and rna biosynthesis and content during the cell cycle rna viruses: hijacking the dynamic nucleolus modifications of both selectivity factor and upstream binding factor contribute to poliovirus-mediated inhibition of rna polymerase i transcription viruses and the nucleolus: the fatal attraction virus infection induces redistribution and membrane localization of the nuclear antigen la (ss-b): a possible mechanism for autoimmunity x chromosome inactivation and autoimmunity nucleolin: the most abundant multifunctional phosphoprotein of nucleolus nucleophosmin: from structure and function to disease development increased polyamines alter chromatin and stabilize autoantigens in autoimmune diseases noncoding rnas and epigenetic mechanisms during x-chromosome inactivation x-inactivation profile reveals extensive variability in x-linked gene expression in females intense correlation between protein-conjugated acrolein and primary sjogren's syndrome recent insights into the regulation of x-chromosome inactivation line-mediated retrotransposition of marked alu sequences plasma dna in systemic lupus erythematosus post-translational modifications, subcellular relocation and release in apoptotic microparticles: apoptosis turns nuclear proteins into autoantigens cgas surveillance of micronuclei links genome instability to innate immunity sting: infection, inflammation and cancer autoantibodies in patients with alzheimer's disease: pathogenetic role and potential use as biomarkers of disease progression brain polyamine levels are altered in alzheimer's disease polyamines in frontal cortex of patients with downs syndrome and frontiers in immunology | www alzheimer disease acrolein is increased in alzheimer's disease brain and is toxic to primary hippocampal cultures brain s-adenosylmethionine levels are severely decreased in alzheimer's disease endogenous polyamines reduce the toxicity of soluble aβ peptide aggregates associated with alzheier's disease nucleolar parp1 expression is decreased in alzheimer's disease: consequences for epigenetic regulation of rdna and cognition the disappearing barr body in breast and ovarian cancers triple-negative breast cancer epigenetic connections between autoimmune disorders and haematological malignancies cancer risk in systemic lupus: an updated international multi-centre cohort study systemic lupus erythematosus and related autoimmune diseases are antigen-driven epigenetic diseases autoimmune disorders result from loss of epigenetic control following chromosome damage rare x chromosome abnormalities in systemic lupus erythematosus and sjögren's syndrome key: cord-288496-7rrh2gg6 authors: stryhn, anette; kongsgaard, michael; rasmussen, michael; harndahl, mikkel nors; østerbye, thomas; bassi, maria rosaria; thybo, søren; gabriel, mette; hansen, morten bagge; nielsen, morten; christensen, jan pravsgaard; randrup thomsen, allan; buus, soren title: a systematic, unbiased mapping of cd8(+) and cd4(+) t cell epitopes in yellow fever vaccinees date: 2020-08-31 journal: front immunol doi: 10.3389/fimmu.2020.01836 sha: doc_id: 288496 cord_uid: 7rrh2gg6 examining cd8(+) and cd4(+) t cell responses after primary yellow fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 cd8(+) and 50 cd4(+) t cell epitopes, many inducing strong and prevalent (i.e., immunodominant) t cell responses. restricted by 40 and 14 hla-class i and ii allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. the broad coverage of epitopes and hla overcame the otherwise confounding effects of hla diversity and non-hla background providing the first evidence of t cell immunodomination in humans. also, double-staining of cd4(+) t cells with tetramers representing the same hla-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many cd4(+) t cell responses. we suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of cd4(+) t cell responses. our t cell epitope discovery approach uses a combination of (1) overlapping peptides representing the entire yellow fever virus proteome to search for peptides containing cd4(+) and/or cd8(+) t cell epitopes, (2) predictors of peptide-hla binding to suggest epitopes and their restricting hla allotypes, (3) generation of peptide-hla tetramers to identify t cell epitopes, and (4) analysis of ex vivo t cell responses to validate the same. this approach is systematic, exhaustive, and can be done in any individual of any hla haplotype. it is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both cd4(+) and cd8(+) t cell epitopes. it is efficient and, importantly, reduces the false discovery rate. the unbiased nature of the t cell epitope discovery approach presented here should support the refinement of future peptide-hla class i and ii predictors and tetramer technologies, which eventually should cover all hla class i and ii isotypes. we believe that future investigations of emerging pathogens (e.g., sars-cov-2) should include population-wide t cell epitope discovery using blood samples from patients, convalescents and/or long-term survivors, who might all hold important information on t cell epitopes and responses. examining cd8 + and cd4 + t cell responses after primary yellow fever vaccination in a cohort of 210 volunteers, we have identified and tetramer-validated 92 cd8 + and 50 cd4 + t cell epitopes, many inducing strong and prevalent (i.e., immunodominant) t cell responses. restricted by 40 and 14 hla-class i and ii allotypes, respectively, these responses have wide population coverage and might be of considerable academic, diagnostic and therapeutic interest. the broad coverage of epitopes and hla overcame the otherwise confounding effects of hla diversity and non-hla background providing the first evidence of t cell immunodomination in humans. also, double-staining of cd4 + t cells with tetramers representing the same hla-binding core, albeit with different flanking regions, demonstrated an extensive diversification of the specificities of many cd4 + t cell responses. we suggest that this could reduce the risk of pathogen escape, and that multi-tetramer staining is required to reveal the true magnitude and diversity of cd4 + t cell responses. our t cell epitope discovery approach uses a combination of (1) overlapping peptides representing the entire yellow fever virus proteome to search for peptides containing cd4 + and/or cd8 + t cell epitopes, (2) predictors of peptide-hla binding to suggest epitopes and their restricting hla allotypes, (3) generation of peptide-hla tetramers to identify t cell epitopes, and (4) analysis of ex vivo t cell responses to validate the same. this approach is systematic, exhaustive, and can be done in any individual of any hla haplotype. it is all-inclusive in the sense that it includes all protein antigens and peptide epitopes, and encompasses both cd4 + and cd8 + t cell epitopes. it is efficient and, importantly, reduces the false discovery rate. the unbiased nature of the t cell epitope discovery approach presented here should support the refinement of future peptide-hla class i and ii predictors and tetramer technologies, which eventually should cover all hla class i and ii isotypes. we believe that future investigations of emerging the immune system attempts to protect its host against invading pathogens; yet, it can also cause serious pathology. the ability to discriminate between foreign and self is key to exerting immune protection without inflicting immune pathology. immune recognition is therefore of immense interest and efficient methods to identify and validate immune epitopes are a high priority. in this context, t cells, which effectively orchestrate the overall immune response, are of particular interest. t cells are specific for compound ligands consisting of peptides, generated intracellularly by proteolytic degradation of protein antigens, which are presented in the context of major histocompatibility complex (mhc) [or human leucocyte antigens (hla)] molecules on the surface of antigen presenting cells (apc) (1) . the interaction between peptide and hla is specific; the resulting hla-mediated t cell epitope selection process being greatly diversified by the polygenic and polymorphic nature of the hla. this significantly affects the peptide-binding specificity of the set of hla molecules that are available to any given host; something that effectively individualizes our immune responses. although other events are also involved in antigen processing and presentation, the single most selective event is that of peptide-hla binding. it is estimated that ca. 0.5% of all possible peptide-hla combinations are of a sufficiently high affinity that they potentially, but not necessarily, could be immunogenic (2) . major efforts have been devoted to understand, quantitate and preferably predict peptide-hla binding as a means to identify t cell epitopes. proposed in 1999, the "human mhc project" aims at mapping all human mhc (or hla) specificities (3, 4) . established in 2004, the "immune epitope database" (iedb) has become an authoritative repository of hla binding peptides and t cell epitopes, and of methods to predict these (5) . the recent breakthrough in cancer immunotherapy has reinforced the interest in fast and efficient methods to identify t cell epitopes with special emphasis on identifying immunogenic neoepitopes for personalized cancer immunotherapy. thus, several recent international research efforts, such as the "human immunopeptidome project and consortium, " "tumor neoantigen selection alliance" and others, have focused on t cell epitope discovery. employing recent advances in mass spectrometry to perform large-scale identification of peptides eluted of hla molecules, these efforts promise to identify natural ligands thereby capturing information on both antigen processing and hla binding (6) . over the past decades, substantial progress has been made on predicting peptide-hla interactions, particularly for hla class i (hla-i), which restricts cd8 + cytotoxic t cells (ctl's), and to a lesser degree on predictions for hla class ii (hla-ii), which restricts cd4 + helper t cells (th) (7) (8) (9) (10) (11) (12) (13) (14) (15) . state-of-theart predictors such as netmhcpan, an artificial neural network method based on a large collection of experimental peptide-hla-i binding data, can successfully identify 96.5% of cd8 + t cell epitopes, while rejecting 98.5% of non-epitopes (16) . however, considering that only 1 of 2,000 (2) to 8,000 (17) random peptides is a t cell immunogen in the context of a given hla molecule, even a rejection rate as high as 98.5% translates into a high false discovery rate (fdr) (8, 10, 11, 18) . this is a general problem of current peptide-hla binding predictors (10, 11) , and it is particularly problematic when trying to develop a neoepitope-specific, personalized cancer immunotherapy where timely delivery of a few unique cancer neoepitopes is of paramount importance; something that potentially could be achieved with even better predictors (8, (19) (20) (21) . yellow fever virus (yfv) is a mosquito-borne flavivirus (i.e., a ssrna virus) (22, 23) . it remains an important human pathogen despite the existence of an effective live attenuated vaccine (24) . particularly relevant to this study, previous analyses of the cd8 + t cell response against a limited number of epitopes have revealed that vaccination with this live vaccine represents an excellent model for studying the host response to a viral infection (25, 26) . the main advantages are that the precise time and the exact identity of the immune challenge are both known [note that the vaccine strain used here is known to be stable (27) ]; issues that otherwise might complicate the interpretation of immune responses observed in patients that are naturally infected with a variable pathogen. here, we have generated a comprehensive, population-wide t cell epitope discovery approach with a much-reduced fdr, and used it to identify and validate immunodominant cd8 + and cd4 + t cell epitopes in a cohort of 210 hla-typed, primary yfv vaccinees. this involves using a "forward (or direct) immunology" approach, where you start with a specific t cell response of interest and then search for the epitope(s) being recognized (28, 29) , to perform an initial identification of t cell stimulatory peptides. subsequently, a "reverse immunology" approach, where you start by predicting possible t cell epitopes and then search for a t cell response of the corresponding specificity (30, 31) was used, to perform a final identification and validation of the underlying specific t cell epitopes and their hla restriction elements. from here on, this approach is denoted as a "hybrid forward-reverse immunology" (hfri) approach. briefly, in the "forward immunology" step, pbmc's obtained 2-3 weeks after primary yfv vaccination were ex vivo stimulated with an overlapping peptide library representing the entire 3,411 amino acid yfv proteome and tested by an ifnγ-specific intracellular cytokine secretion (ics) assay thereby identifying cd8 + and cd4 + t cell stimulatory yfv-derived peptides. in the subsequent "reverse immunology" step, predictors were used to select appropriate peptide-hla combinations for the generation of peptide-hla tetramers, which then were used to identify and validate the underlying t cell epitopes and their hla restriction elements. applying this hfri approach to t cell epitope discovery in 50 yfv vaccinees, we identified and tetramervalidated 92 cd8 + and 50 cd4 + t cell epitopes covering 40 hla-i and 14 hla-ii allotypes, respectively (note that he tetramer-validation step could not be performed exhaustively for the cd4 + t cell epitope discovery process and that the true number of cd4 + t cell epitopes probably was many times larger than the 50 validated cd4 + t cell epitopes reported here). with a cohort of 210 yfv vaccinees, the prevalence of responses against the cd8 + t cell epitopes could be examined. about a third (31%) of these epitopes were recognized in >90% of the individuals expressing the hla-i in question. by this token, they could be considered strongly immunodominant. we conclude that t cell epitope discovery using this hfri approach is highly efficient, in particular when examining larger populations responding to the same pathogen (e.g., an infectious pathogen e.g., sars, ebola, zika, sars-cov-2). furthermore, we suggest that the hfri approach is unbiased and that the resulting t cell epitopes should serve as a valuable benchmark for future improvements of predictive algorithms of immunogenicity. primary vaccination with the attenuated yfv vaccine, 17d-204, is known to trigger a prompt and vigorous cellular immune reaction (25, 26) . here, 210 vaccinees were recruited, and peripheral blood mononuclear cells (pbmc) were prepared from 50-to 200-ml blood samples obtained before and ca. 2 weeks after primary vaccination, respectively (26) . the typical yield from the latter was ca. 450 million pbmc. all vaccinees were hla typed at high-resolution (i.e., 4 digit) including all nine classical, polymorphic hla loci (i.e., hla-a, b, c, drb1, drb3/4/5, dqa1, dqb1, dpa1, and dpb1) (26) . the 17d-204 vaccine encodes a single polyprotein precursor of 3,411 amino acids (aa), which is processed into 15 proteins. the full genome (genbank accession# x15062) and proteome (swiss-prot accession# p03314) sequences of the 17d-204 have been determined (32) . a library of 850 overlapping 15 mer peptides overlapping by 11 aa, spanning the entire yfv precursor protein (essentially the yfv proteome), was generated. additionally, 50 peptides representing potentially aberrant yfv translation products were selected. of the resulting 900 peptides, synthesis failed for 30 peptides (3%) leaving 870 peptides for analysis. since testing each of these peptides individually would exhaust the available pbmc's, the peptides were tested in pools. initially, the peptides were organized into a single 30×30 matrix from which 30 "column pools" and 30 "row pools" were generated leading to a total of 60 pools each containing ca. 30 different peptides. each peptide would be present in two pools: one column and one row pool (supplementary figure s1a) . the intersections of stimulatory column and row pools should ideally identify which peptide might be immunogenic and therefore should be further investigated on an individual basis. this 30×30 matrix strategy was initially tested using an exvivo ifnγ elispot assay as readout. after the first 94 primary vaccinated donors had been recruited, the average number of positive column/row intersections was found to be 418 (range 26-870) ( figure 1a) suggesting that the hit rate from pools containing 30 peptides was too high, at least in the setting of this acute viral response, to be effective in eliminating nonstimulatory peptides from further consideration. to reduce the hit rate per peptide pool, the peptides were re-organized into four smaller matrices, three 15×15 matrices and one 14×15. for each matrix, 14 to 15 column pools and 15 row pools were generated leading to a total of 119 pools, which each contained 14 to 15 different peptides (supplementary figure s1b) . to further reduce the number of relevant intersections, the ifnγ elispot assay was replaced by an ifnγ intracellular cytokine staining (ics) assay, which can discriminate between cd8 + and cd4 + t cells and therefore eliminate intersections with mismatched cd8 + and cd4 + t cell responses. furthermore, to increase the number of t cells available for the ics assay, pbmc were expanded in four separate in vitro cultures containing a pool of ca. 225 peptides corresponding to each of the four matrices, respectively (the potential bias introduced by this in vitro culture is discussed in supplementary results and discussion). after 8 days, each matrix-expanded pbmc culture was tested against the appropriate row and column pools using ifnγ ics as readout. for comparison, 36 donors, which had already been analyzed using the 30×30-matrix, elispot-based screening strategy, were re-screened using the 4×(15×15)-matrix, icsbased screening strategy (figures 1b-d) . the aggregated cd4 + and cd8 + t cell responses were calculated for ics responses (denoted "all t cells" in figure 1 ) and compared to those from elispot responses. the total number of intersections needing deconvolution was significantly lower for the 4×(15×15 ics strategy [average intersections 253 (range 20-589)] than for the 30×30 elispot strategy [average 418 (range 26-870), (p < 0.0001, n = 36, mann-whitney u-test), figures 1a,b] . the 253 intersections, which on average were detected by the ics-based screening strategy, could further be broken down into an average of 80 (range 2-374) ( figure 1c ) and 197 (range 7-514) ( figure 1d ) intersections representing cd8 + and cd4 + t cell responses, respectively (30 of these intersections were shared). the peptides corresponding to these intersections were subsequently tested individually to identify which of the intersections truly represented cd8 + and/or cd4 + t responses. figure 1 | comparing the number of peptide-containing peptides identified by the two different approaches. t cells obtained ex vivo from primary yfv vaccinees were stimulated with matrix-derived pools of yfv peptides and responses were read by ifnγ-specific elispot or ics. the peptides were distributed into matrixes, column and row pools of peptides were generated, and used to test t cell stimulation. intersections of stimulatory column and row pools putatively identified single stimulatory peptides for further analysis (supplementary figure s3) . (a) peptides were distributed into one 30×30 matrix generating 30 + 30 = 60 pools, which were used to stimulate t cell responses in 94 donors using an ifnγ-specific elispot assay as readout of all (i.e., cd4 + and cd8 + ) t cell responses [average 418 positive intersections (range ]. (b,c) peptides were distributed into four ca. 15×15 matrices generating 4x(ca. 15 + 15) ca. 120 pools, which were used to stimulate t cell responses in 36 donors using an ifnγ-specific ics assay as readout of (b) all t cell responses [average 253 intersections (range 20-589)], (c) cd8 + t cell responses [average 80 intersections (range 2-374), and (d] cd4 + t cell responses average 197 intersections (range 7-514). the symbols representing the 36 donors that were examined by both elispot and ics have been shaded. mann whitney u test was used to determine the significance of the difference between the indicated groups (***p < 0.0001). the complete screening and validation procedure is illustrated using donor yf1067. the blood sample for donor yf1067 was collected at day 16 post vaccination, which is within the time span of optimal post vaccination yfv responses (25, 26) . it gave a relatively high yield of 700 × 10 6 pbmc's for the subsequent epitope discovery effort. donor yf1067 was initially analyzed by the 30×30-matrix, ifnγ elispot-based screening strategy where 690 positive intersections were identified. a total (i.e., cumulative) yfv-specific response of 8000 sfu were obtained suggesting a t cell response of considerable breath and magnitude. re-analyzing this donor using the four-matrix, ics-based screening strategy, the number of intersections for figure 2 | t cell epitope screening strategy. (a) identification of stimulatory 15mer peptides: pbmc's from yfv vaccinated donors were divided into four cultures and in vitro stimulated with peptide sublibraries corresponding to each of the four 15×15 peptide matrices. after 8 days, each sublibrary expanded pbmc culture was tested by ics against the matrix-specific row and column peptide pools. subsequently, individual peptides representing stimulatory matrix intersections were analyzed to identify single t cell stimulatory 15-mer peptides. (b) identification of t cell epitopes and their hla restriction elements: cd4 + t cell epitope deconvolution: single cd4 + t cell stimulatory 15mer peptides were tested for binding to the donor's hla-dr molecules using a biochemical hla class ii binding assay, positive interactions were used to generate peptide-hla class ii tetramers, and these tetramers were used to stain expanded t cells, and the resulting epitopes were eventually validated by ex vivo elispot analysis. cd8 + t cell epitope deconvolution: single cd8 + t cell stimulatory 15mer peptides were submitted to the netmhcpan 2.4 predictor together with the donor's hla class i haplotype to identify optimal epitopes and their hla-restriction elements. these optimal epitopes were subsequently synthesized and validated by ex vivo peptide-hla class i tetramer staining. follow-up analysis could be reduced to 253; 78 representing cd8 + t cell responses and 218 representing cd4 + t cell responses (43 of the intersections contained both cd4 + and cd8 + t cell responses). peptides corresponding to the 253 intersections were tested individually by ics; this identified 27 and 31 cd8 + and cd4 + t cell stimulatory 15mer peptides, respectively (for a detailed listing of these peptides, see tables 1,2 below). the next steps aimed at identifying the underlying cd8 + and cd4 + t cell epitope(s) and their hla restriction element(s), preferably by generating the corresponding tetramer(s), and validate the epitope(s). for a general outlined of this epitope discovery scheme, see figure 2 . identification and validation of cd8 + t cell epitopes exemplified by donor yf1067 the sequences of the 27 15mer peptides, which stimulated cd8 + t cell responses in donor yf1067, were submitted, along with the donor's hla-i allotypes (in casu hla-a * 02:01, -a * 32:01, -b * 07:02, -b * 40:01, -c * 03:04 and -c * 07:02), to our webserver netmhcpan (version 2.4 at http://www.cbs.dtu.dk/ services/netmhcpan-2.4 was available at the time of this analysis). in silico, this predictor considered all 26 submer peptides of 8-11mer length, which could possibly be generated from a 15mer peptide, and predicted their binding to all six hla-i allotypes of donor yf1067, a total of 6 × 26 = 156 submer-hla-i combinations per 15mer peptide, and returned a ranked list across all six hla-i allotypes of the most likely epitope(s) and their hla-i restriction element(s). for all 27 cd8 + t cell stimulatory peptides, this amounted to predicting the binding affinities of 27 × 156 = 4,212 submer-hla-i combinations. for each 15mer peptide, submers representing the top one to three predicted affinities were synthesized and the stabilities of the corresponding peptide-hla-i interactions were measured experimentally ( table 1) . fluorochrome-labeled tetramers corresponding to the most stable peptide-hla-i interactions were generated and used to label relevant cd8 + t cells. when available, surplus t cells from the initial expansion cultures were used as a first line of identification of cd8 + t cell epitopes and their restriction elements, however, ex vivo tests were always used for the final cd8 + t cell epitope validation, and for enumerating and characterizing epitope-specific cd8 + t cells. the matrix-identified cd8 + t cell stimulatory 15mer peptides and the corresponding tetramer-validated optimal the 27 cd8+ t cell stimulatory 15mer peptides are given including their sequences and ics stimulation values. the epitopes eventually identified are given in red. as shown, many epitopes were found in consecutive 15mer peptides. these 15mer peptides were submitted along with the donors hla-i haplotype to netmhcpan 2.4, which returned suggested epitopes and restriction elements. in most cases, the epitope was found as a top ranking prediction. the stabilities of the suggested peptide-hla-i complexes were measured and the corresponding tetramers generated. finally, these tetramers were used to validate the cd8+ t cell epitopes and to enumerate the responding cd8+ t cells ex vivo. cd8 + t cell epitopes and their restriction elements are listed ( table 1) . for each of the 27 cd8 + t cell stimulatory 15mer peptides identified in donor yf1067, one or more cd8 + t cell epitopes and their hla-i restriction elements were identified. some of the epitopes were present in two consecutive overlapping 15mer peptides and should therefore only be counted as epitopes once. with this in mind, 19 unique cd8 + t cell epitopes were recognized by donor yf1067 (7 epitopes restricted by hla-a * 02:01, 2 by hla-a * 32:01, 4 by hla-b * 07:02, 6 by hla-b * 40:01, and none by hla-c * 03:04 or -c * 07:02) ( table 1) . cd8 + t cell specific for the 19 unique yfv-derived epitopes were readily detectable and enumerable ex vivo during the acute primary response of donor yf1067. the frequencies of total, as well as activated, tetramer-positive cd8 + t cells were determined ( table 1) . the most frequently and immunodominant epitope of all, the hla-a * 02:01-restricted ns4b 214−222 epitope, was recognized by 4.6% of cd8 + t cells in donor yf1067. the frequencies of cd8 t cells recognizing each of the other 18 epitopes ranged from 0.03 to 0.9%; the total frequency of cd8 + t cells recognizing the 19 yfv epitopes was ca. 8%. the yfv vaccine induced a measurable increase in the overall frequency of activated cd8 + t cells (i.e., cd38 + hla-dr + cd8 + t cells) (26) . in donor yf1067, the yfv vaccine induced an increase in activated cd8 + t cell from 0.6% preto 7% post-vaccination. notably, the hla-a * 02:01-restricted ns4b 214−222 -epitope was recognized by 41% of the activated cd8 + t cells in donor yf1067. the frequencies of activated cd8 + t cells recognizing each of the other 18 epitopes ranged from 0.1 to 4.1%. in total, the 19 identified yfv-specific cd8 + t cell epitopes accounted for the majority (ca. 60%) of activated cd8 + t cells observed during the acute response following primary yfv vaccination. the cd8 + t cell epitope discovery strategy described above for donor yf1067 was extended to 50 randomly selected donors, who were sampled at day 12-21 after vaccination i.e., at the peak of a primary anti-yf vaccine response (25, 26) . cd8 + t cell responses specific for 120 different peptide hla-i combinations were identified and validated by ex vivo tetramer staining (for an overview, see figure 3 , and for details, see supplementary table si) . this represented 92 different cd8 + t cell epitopes restricted by 40 different hla-i molecules; 68, 20 and 4 epitopes were restricted by 1, 2, and 3 different hla-i molecules, respectively. the hla-a, -b and -c allotypes covered by the 50 donors were, respectively 19, 30, and 20 of which the majority, 15, 27, and 16, were available to us for tetramer validation. thirteen of the 15 different hla-a allotypes tested served as restriction elements of 38 different cd8 + t cell peptide epitopes leading to the presentation of 44 immunogenic peptide-hla-a combinations; 26 of the 27 different hla-b allotypes tested served as restriction elements of 56 different epitopes leading to the presentation of 74 immunogenic peptide-hla-b combinations; whereas only one of the 16 different hla-c allotypes tested served as restriction elements of 2 different epitopes leading to the presentation of 2 immunogenic peptide-hla-c combinations. the average number of cd8 + t cell epitopes identified per hla-a and -b allotype, 3.4 and 2.8, respectively, were not significantly different [p > 50%, fishers exact test, two-tailed (graphpad)]. to the best of our knowledge, 84 of the 92 yf-specific cd8 + t cell epitopes, and 110 of the 120 epitope-hla-i combinations reported here and in previous publications (26, 33) , were first identified as a result of this hfri project. for the previously reported epitopes or epitope-hla-i combinations, minor adjustments of the already available information could be made: some had not been tetramer validated before, and others were also found to be restricted by other, albeit closely related, hla-i allotypes than those previously reported. in a few cases, tetramers representing the exact epitope-hla-i combinations previously reported failed to label cd8 + t cells in our donors despite expressing the appropriate hla-i allotype (for details see supplementary table si) . our in-house peptide repository included 533 yfv-derived peptides from previous hla mapping efforts (34) . using the contemporary netmhcpan2.4 at %rank cut-off of 0.5% to select putative binders from this repository, we generated 90 additional peptide-hla-i tetramers (i.e., tetramers that had not already been prepared in the course of the present hfri approach). we included these tetramers in the immunodominance analysis described below. nine additional peptide-hla-i combinations, which had not been observed previously, were identified; four representing previously identified epitope presented by an alternative hla-i restriction element, and five representing new yfv-specific cd8 + t cell epitopes (supplementary table sii) . thus, the total number of cd8 + t cell epitopes discovered and tetramer validated here was 97 of which 92 (or 95%) were identified by the hfri approach. we systematically extended the analysis of ex vivo responses to additional donors expressing relevant hla-i restriction elements and evaluated them in terms of prevalence (the frequency of responders in donors with the hla-i restriction element in question) and response magnitude (the average ex vivo frequency of tetramer positive, activated cd8 + t cells of the responding donors) supplementary tables si, sii. to allow for a reasonable assessment of prevalence, the final analysis included epitopes restricted by hla-i molecules represented by at least 5 donors, who had donated blood samples 12-21 days after vaccination. this involved a total of 98 peptide-hla combination representing 81 epitopes presented by 24 hla-i allotypes (figure 4) . immunodominance was frequently observed. from an epitope point of view, 25 (or 31%) of the 81 epitopes had a prevalence of ≥90% and a median magnitude >0.03%, and 50 (or 62%) had a prevalence of ≥50% and a median magnitude >0.02%. from an hla point of view, 16 (or 67%) of the 24 hla-i molecules presented at least one epitope with ≥90% prevalence, and all 24 hla-i molecules presented at least one epitope with at least 50% prevalence. in terms of hla-i coverage and immunodominance, the vast majority of our cohort, 97, 79, and 43%, carried at least one, two or three hla-i allotypes, respectively, which presented at least one epitope with ≥90% prevalence. a selection of 10 immunodominant epitopes representing the most frequent hla-a and -b allotypes would cover 95% of the caucasian population. the "all cd8" bar indicates the positions of each cd8 + t cell stimulatory 15mer peptide relative to the yfv polyprotein. each 15mer is shown as a frame that horizontally indicates the starting and ending positions of the 15mer peptide, vertically indicates the number of donors, of the 50 donors tested, who responded to the peptide, and the color-coded is according to the polyprotein coloring scheme (to enhance the visualization of overlapping peptide sequences, this coloring is translucent). the lower hla-i allotype-designated bars indicate the tetramer-validated epitopes and their hla-i restriction elements (e.g., a*01:01 is shorthand for hla-a*01:01). again, the frame horizontally indicates the starting and ending positions of the epitopes; however, for visual clarity, all frames have the same vertical dimension. the details of each epitop (epitope sequence, cd8+ t cell stimulation, ex vivo tetramer staining frequency, and response prevalence is given in supplementary table si) . figure 4 | prevalence and magnitude of cd8 + t cell responses. to determine the prevalence and the median magnitude of the cd8 + t cell responses toward the epitopes discovered by the hfri approach in 50 donors were extended to additional donors expressing the relevant hla-i restriction elements. only donors sampled 12-21 days after vaccination were included. the prevalence (gray columns) and the median magnitude of the responses (black diamond) were determined for each epitope-hla combination. only epitope-hla combinations analyzed in 5 or more donors were included. the epitopes are organized according to restriction elements. the top figure shows the hla-a restriction elements; the bottom figure shows the hla-b and -c restriction elements. it has been suggested that immunodominant epitopes can curtail responses to other epitopes (reviewed in 35). the hla-a * 02:01 restricted, yfv ns4b 214−222 -epitope may represent a unique opportunity to address this in an outbred human population: it represents an exquisitely dominant cd8 + t cell response as all 93 hla-a * 02:01-positive donors examined here responded to this epitope and an average of 29% of all activated cd8 + t cells from ex vivo blood samples obtained 2-3 weeks after yfv vaccination were specific for this epitope. it has recently been suggested that this massive response can be explained by the invariant cdr1α loop of trav12-2 taking part in the recognition of this epitope (35) . in donors, who had donated blood samples at the peak of the response (12-21 days after vaccination), we examined whether the presence of hla-a * 02:01, -a * 01:01, or -a * 03:01, could be correlated to the strength of cd8 + t cell responses restricted by other restriction elements, in casu all available hla-b allotypes. we included 142 donors, which, respectively, could be split into 71 and 71 hla-a * 02:01 positives and negatives, 39 and 103 hla-a * 01:01 positives and negatives, or 30 and 112 hla-a * 03:01 positives and negatives; and used tetramers to examine the ex vivo frequencies of up to 43 different hla-b-restricted responses. in the presence or absence of each of the three hla-a restriction elements, the average frequencies of each of the hla-b-restricted responses were determined leading to the generation of up to 43 matched-pairs per hla-a. the frequencies, or magnitude, of the hla-b-restricted responses were significantly reduced in the presence vs. absence of hla-a * 02:01 (median reduction of 0.0432%, p < 0.0001). in contrast, in the presence vs. absence of hla-a * 01:01, which have lesser immunodominant cd8 + t cell responses, there was a smaller and not significant reduction (median reduction of 0.0165%, p = 0.1353); in the presence vs. absence of hla-a * 03:01, which have even fewer immunodominant cd8 + t cell responses, there was a very small and non-significant increase (median increase of 0.0041%, p = 0.51) (wilcoxon signed-rank test, figure 5 ). we suggest that this may be the first demonstration of immunodomination in an outbred human population. the length of the 97 discovered cd8 + t cell epitopes ranged from 8 to 11mers with a predominance of 9mers (67 (69%) 9mers, 18 (19%) 10mers, 5 (5%) 11mers, and 7 (7%) 8mers) (supplementary figure s2) . this matches well with available data for peptides eluted of hla-a and -b molecules (33) . some of the epitopes were size variants of the same peptide sequence. in six cases, such size variants were presented by the same hla-i restriction element (four cases involving two size variants each and two cases involving three size variants each, supplementary table si). we reasoned that cd8 + t cell recognition of these identically restricted size variants could either involve cross-recognition of shared epitope structure(s) by the same tcr(s), or involve recognition of genuinely different epitope structures by different tcr(s). to evaluate this, tetramers of the different epitope size variants and the relevant hla restriction elements were produced with unique fluorochrome labels and used to determine whether the epitope size variants were recognized by the same or different t cell populations. in some cases, distinctly defined and shared subpopulations were observed (figures 6a,d,i,j) indicating that one or more unique shared epitope structures were presented and recognized; in other cases, we observed shared subpopulations merging with populations that were single-stained with one of the lengthvariant tetramers (figures 6c,g,h,i) ; and finally, in some cases, no shared subpopulations were observed suggesting that the corresponding length-variants were presented and recognized as being distinctly different (figures 6b,e,f) . accommodating length-variants by extending the peptide-binding groove or by one or more aa's bulging out of the groove (34) could affect the presented epitopes dramatically, whereas accommodating length-variants by protruding out of the n-or c-terminal ends of the groove could leave the non-protruding end of the epitope unaltered. elucidating the structural basis of these various recognition modes is beyond the scope of this paper. one of the more frequent hla allotypes, hla-b * 07:02, offered an opportunity to compare the hfri approach with a strictly reverse immunology approach. theoretically, a total of 13,610 peptides of 8-11mer size could be generated from the yfv proteome. netmhcpan 2.4 predicted 54 of these as being strong hla-b * 07:02 binders at a %rank of <0.5%. we selected 40 of those for further examination (supplementary table siii) . with one exception, all of these predicted binders supported hla-b * 07:02 tetramer generation, which subsequently were used to examine ex vivo obtained pbmc's from at least 16 hla-b * 07:02 + donors. apart from the epitopes that had already been described (supplementary tables si, sii) , no additional hla-b * 07:02-restricted cd8 + t cell epitopes were identified. thus, a final count can be made: combining the hfri and a strictly reverse immunology approach, a total of 10 unique hla-b * 07:02-restricted cd8 + t cell epitopes were found; the hfri strategy identified nine of these, whereas the reverse immunology strategy identified eight; seven (70%) of these epitopes were shared. assuming that the number of true positive hla-b * 07:02 epitopes is ten, then both strategies were sensitive (correctly identifying 80-90% of the 10 epitopes) and at the same time very specific (correctly rejecting 99.6% of the 13,600 non-epitopes); the hfri approach being slightly more sensitive and specific than the reverse immunology approach. the major performance difference between the two strategies arose from the lower false discovery rate (fdr) where the hfri screening strategy required 13 peptides to identify nine of the ten epitopes found (a fdr of 17%; albeit some of these apparently false positive peptides were eventually identified as epitopes restricted by other hla-i restricting elements expressed by the donors suggesting that the true false discovery rate of the hfri approach was even smaller), whereas the reverse immunology approach required 54 peptides to identify eight of the ten epitopes suggesting a false discovery rate of 85%. in conclusion, the present hfri approach ranks epitope at the very top of the list of candidates while decimating the false discovery rate (further comparisons of hfri vs. reverse immunology is described in the discussion and detailed in supplementary results and discussion). the unbiased nature of our cohort of 120 different hfriidentified peptide hla-i combinations covering 40 hla-i restriction elements provided an opportunity to evaluate the performance and discriminatory power of various prediction methods such as the authoritative netmhcpan [both the contemporary version 2.4 (36) and the most recent version 4.0 (37) trained on both eluted ligands (el) and peptide binding affinity (ba)], and the recent mhcflurry (38) (trained either only on ba data or on both el and ba data) (38) and mixmhcpred (trained only on el data) (39) . in addition to these peptide-hla-i affinity predictors, we also included a stability predictor, netmhcstabpan 1.0 (40) . for each of these methods, predictions scores of all 13,610 peptides of length 8-11 aa that could be generated from the 3,411 aa yfv proteome were predicted for the relevant hlas (using %rank scores allowing comparisons across hla allotypes and predictors as read-outs). subsequently, a receiver operating characteristics (roc) analysis was performed and the area under the curve (auc) was determined. a non-discriminatory predictor has an auc of 0.5, whereas a perfectly discriminating predictor has an auc of 1.0. applied to this unbiased and validated set of epitopes, all of these predictors gave highly discriminatory auc's of 0.98743 to 0.99797 (supplementary figure s3) . these impressive auc's are heavily influenced by the many nonimmunogenic peptides being correctly rejected; however, this may still leave considerable room for false positive discovery rates (fdr). in this case, a more fdr-averse way to visualize the performance is to use the frank score, which is the number of false positive predictions (fp) relative to the total number of peptides (n) that can be generated from the source protein (i.e., frank = fp/n). a frank score of 0 indicates a "perfect prediction" where a true epitope receives the highest prediction value of all peptides within the source protein and avoids any false positive predictions, whereas a frank score of 0.5 indicates a random prediction where half of the predictions are false positives. frank values were calculated for each epitope-hla pair and predictor (supplementary figure s2) . the best predictors were netmhcpan 4.0 el and mixmhcpred, which, respectively, scored 21 and 20 "perfect" predictions, obtained an average frank score of 0.001875 and 0.003809, and a median frank score of 0.000405 and 0.000588, respectively. the median, being a more "outlier-resistant" measure, would, respectively, indicate that the netmhcpan 4.0 el and mixmhcpred methods would place 6 and 8 false-positive non-epitopes ahead of each epitope, corresponding to a false discovery rate of 85 and 89%. these numbers should be appreciated in the context of a random predictor, which would yield a fdr of 99%, and a perfect predictor which would yield an fdr of ca. 50% [assuming that only 50% of hla-presented peptides are immunogenic (2)]. in line with earlier work (41) , comparing the predictive power of the various predictors in terms of the frank values, netmhcpan 4.0 el was found to significantly outperform all other predictors (p < 0.02 in all cases, wilcoxon matched-pairs signed rank test) (supplementary figure s2) . identification and validation of cd4 + t cell epitopes (exemplified by donor yf1067) in donor yf1067, the ics-based screening analysis identified 31 15mer peptides as stimulating cd4 + t cell responses. at face value, these 15mer peptide sequences qualified as cd4 + t cell epitopes (the iedb epitope curation manual 2.0 defines a cd4 + t cell epitope of 15 residues or less in length as an "exact epitope"). to identify the underlying hla class ii restriction elements, the binding of each of the 31 15mer peptides to each of the hla-dr molecules of donor yf1067 (in casu hla-drb1 * 13:02, -drb1 * 15:01, -drb3 * 03:01 and -drb5 * 01:01) was tested in a biochemical binding affinity assay (42) . nine (28%), eleven (34%), four (13%) and four (13%) of the epitopes bound with an affinity better than 50 nm to one, two, three and four of the donor's hla-dr molecules, respectively ( table 2) , whereas three (9%) bound to none of them. secondly, we generated tetramers for 50 of the (9×1 + 11×2 + 4×3 + 4×4) = 59 strongly interacting peptide-hla combinations and used these to label in vitro expanded cd4 + t cells from donor yf1067. twenty-two of the 50 tetramers successfully identified cd4 + t cell epitopes and their hla-dr-restriction elements (figure 7) . the final validation and enumeration of specific cd4 + t cell was performed by an ex vivo ifnγ elispot analysis ( table 2) . in one case, the same epitope was presented by two different hla-drb allotypes and should therefore only be counted as epitope once. thus, 21 of the 31 different hla-dr-restricted cd4 + t cell epitopes observed in donor yf1067 were identified at the tetramer level; the remaining eleven epitopes were not resolved. the latter could potentially be explained as being restricted by hla-dq or dp molecules; something that could not be readily addressed by our tetramer capabilities at the time; albeit, in one case, we successfully generated a ns4b 233−247 -dpa1 * 01:03-dpb1 * 04:01 tetramer and identified an hla-dprestricted epitope. although 19 of the 32 cd4 + t cell stimulatory peptides bound to more than one of the four hla-dr allotypes of donor yf1067, there was only one epitope that exploited more than one of the available hla-dr allotypes as restriction element: the ns5 551−565 epitope, which was recognized by cd4 + t cells in the context of both hla-drb1 * 15:01 and hla-drb5 * 01:01. that this was not a case of tcr cross-recognition was shown by double staining with the two tetramers showing two distinctly different cd4 + t cell populations recognizing the ns5 551−565 -epitope presented by either hla-drb1 * 15:01 or hla-drb5 * 01:01 (see section recognizing the same cd4+ t cell epitope presented by two to three different hla-dr allotypes below). thus, in donor yf1067, a total of 31 cd4 + t cell epitopes were identified; 22 of these could be hla-dr or -dp tetramer validated. the cd4 + t cell epitope discovery strategy was extended to the same 50 donors used for cd8 + t cell epitope discovery. a total of 192 cd4 + t cell stimulatory 15mer epitopes were identified (for an overview, see figure 8 "all cd4", and for details, see supplementary table siv) . some of these epitopes were frequently recognized. thus, the single most recognized cd4 + t cell epitope, enve 44−58 , was recognized in 22 (71%) of 31 tetramer-tested donors tested, and another 12 epitopes were recognized in 10-16 (32-52%) of 31 donors. however, most of the 192 epitopes were much less frequently recognized; in fact, 76 of the peptides were recognized in only one (3%) of the 31 donors. we suggest that the strongest and most immunodominant cd4 + t cell epitopes have been found. an important objective was to identify and validate the hla-dr restriction element(s) used to present these epitopes (for an overview, see figure 8 , and for details, see supplementary table sv) . we have evaluated the restriction elements for 74 of the 192 epitopes. for each epitope, the most likely hla-dr restricting element was selected based on its affinity to one or more of the hla-dr allotypes available to the donor. guidance was also obtained from which hla-dr allotypes were shared amongst the epitope-responding donors. in some cases, more than one strong binding hla-dr allotype and/or more than one shared hla-dr allotype were found highlighting that multiple hla-dr allotypes would have to be considered as potential restriction elements. in total, 152 peptide-hla-dr tetramers were generated and used to validate the cd4 + t cell epitopes. of these, 64 the 31 cd4 + t cell stimulatory 15mer peptides are given including their sequences and ics stimulation values. overlaps between two consecutive peptides are given in red. the binding affinity of the 15mer peptides to the four hla-drb1 allotypes of donor yf1067 were measured. tetramers corresponding to the strongest binders were generated. the resulting tetramers were used to stain and analyze expanded cd4 + t cells by flow cytometry gating on cd3 + cd4 + t cells. in 21 cases, staining with a hla-dr tetramer was demonstrated ( figure 7) . note, that no hla-dr-restricted cd4 + t cell responses were found for the ns4b (233-247) epitope, yafvgvmynlwkmkt. eventually, it was found to be a dpa1 * 01:03-dpb1 * 04:01 binder, the corresponding tetramer was generated, and cd4 + t cell staining could be demonstrated (figure 7) . finally, an ex vivo elispot assay was performed to validate the cd4+ t cell epitopes. tetramers were tested positive for cd4 + t cell staining in one or more donors. this covered 50 cd4 + t cell peptide epitopes restricted by 13 different hla-dr molecules (some epitopes were presented by more than one hla-dr allotype) and one hla-dp molecule. for 17 of the 50 epitopes, the hla-dr molecules available to us for tetramer generation did only partially cover the hla-dr molecules observed in one or more of the responding donors. as an example, the most frequently recognized epitope, enve 44−58 , was found in 23 donors (supplementary tables siv, sv) . using appropriate tetramers, two restriction elements, hla-drb1 * 03:01 and -drb3 * 03:01, were identified, however, four of the enve 44−58 responding donors expressed neither the drb1 * 03:01 nor the drb3 * 03:01. this suggested that one or more additional, not yet identified, restriction element(s) existed for this epitope; something that could apply to more of the 17 epitopes. for each peptide, a biochemical hla class ii binding assay was used to identify which of donor yf1067's hla-ii molecules could bind the peptide and therefore could serve as restriction elements. productively interacting peptide-hla-ii combinations were used to design and generate peptide-hla class ii tetramers. the resulting tetramers were used to stain and analyze expanded cd4 + t cells by flow cytometry gating on cd3 + t cells. note, that the selective hla class ii tetramer staining of cd4 + , not cd8 + , t cells is a demonstration of the specificity of the tetramer staining. the identities of the epitopes and their restricting hla-ii elements are indicated. some of the 15mer peptides could stimulate cd4 + t cell responses restricted by two or three different hla-dr restriction elements (supplementary table sv) . no donor happened to possess three appropriate hla-dr molecules, but some did possess two and could generate appropriate cd4 + t cell response restricted by both of these restriction elements. in these cases, staining cd4 + t cells with two uniquely labeled tetramers, representing either of the two restriction elements, allowed us to address whether the same epitope presented by two different restriction elements were recognized by the same, or by distinctly different, cd4 + t cells. (figures 9a-e) . the remaining three epitopes were presented by the closely related hla-dr allotypes (hla-drb1 * 13:01 and -drb1 * 13:02 (one amino acid difference, a v86g, a part of the peptide binding site interacting with p1 of the core sequence), ns3 57−71 , ns5 59−73 , and ns1 111−125 , showed various degrees of cross-recognition. the ns3 57−71 peptide presented by hla-drb1 * 13:01 and -drb1 * 13:02 is mostly recognized by separate t cell populations; only a small population recognized the peptide presented by both molecules (figure 9f ). for peptides, ns5 59−73 and ns1 111−125 about half of the t cells recognizing the peptides presented by hla-drb1 * 13:01 cross-recognized the peptides presented by hla-drb1 * 13:02, with none or a very small t cell population recognizing the peptides presented only by hla-drb1 * 13:02 (figures 9g,h) . we speculate that a peptide presented by two restricting hla-dr molecules with only a few polymorphic amino acid differences may be cross-recognized by some, but not necessarily all, cd4 + t cells of appropriate specificity, whereas, presentation by two restricting hla-dr molecules with many polymorphic amino acid differences are more likely to be recognized as being distinctly different. in 16 cases, two consecutive overlapping 15mer peptides stimulated cd4 + t cell responses restricted by the same hla-dr restriction element. if the two peptides of such an overlapping 15mer peptide pair were presented through two different core regions, one for each, then the two neighboring epitopes should be perceived as being distinctly different and should be recognized by two disparate cd4 + t cell populations. alternatively, if the two peptides were presented through the exact same core region, then the two neighboring epitopes could potentially be perceived as being identical and be recognized by the same cd4 + t cell populations. to examine this, cd4 + t cells were double-stained with hla-dr tetramers, which had been prepared with each of the overlapping peptides of a 15mer pair and labeled with a unique fluorochrome. we analyzed 10 such pairs and found a wide variety of staining patterns. in no case did two peptides of an overlapping pair engage two distinctly different cd4 + t cell populations; rather, in all cases observed, the two peptides engaged at least some shared cd4 + t cell populations suggesting usage of shared core regions. in most cases, a plethora of shared, yet subtly different, cd4 + t cell populations were observed (figure 10) . by way of examples, tetramers representing the overlapping hla-drb1 * 01:01-restricted 15mer peptides, capc 49−63 and capc 53−67 , revealed multiple distinct cd4 + t cell subpopulations, which recognized one, the other, or both tetramers at various efficiencies ( figure 10a) ; whereas tetramers representing the overlapping hla-drb1 * 01:01-restricted 15mer peptides, ns5 471−485 and ns5 475−489 , revealed almost exclusively cd4 + t cell subpopulations recognizing both tetramers, albeit clearly comprising multiple distinct subpopulations ( figure 10b) . we argue that this phenomenon increases and diversifies cd4 + t cell responses. apart from two small proteins, the 20 aa er anchor and the 164 aa prm proteins, all yfv proteins contained both cd8 + and cd4 + t cell epitopes. on average, the frequencies of cd8 + and cd4 + t cell epitopes were ca. 3 and 6 per 100 aa, respectively (table 3) . notably, the cd8 t cell epitopes, which have been tetramer mapped exhaustively, exhibited stretches of overlapping epitopes restricted by several different hla class i molecules: twelve stretches encompassing two epitopes, five stretches encompassing three epitopes, and three larger hot-spots areas encompassing four to six epitopes, many of which were presented by several different hla molecules. thus, the frequently recognized enve 200−240 sequence comprised six peptide epitopes and ten hla-restriction elements giving a total of twelve epitope-hla combinations (figure 11) . these three hot-spot regions accounted for about 15 of the 97 (15%) cd8 + t cell epitopes identified, and encompassed 15 hla-i restriction elements covering ca. 77% of the caucasian race. although the yf protein was generated as one long precursor polyprotein, no epitopes were found in any of the overlaps between the different processed proteins. no epitopes were found in any of the peptides representing products of alternative translation initiation codons. in order to understand the complexity of the human t-cell response to a circulating pathogen, and its potential impact on population dynamics of both pathogen and host, knowing a wide range of epitopes relevant for t-cell/pathogen interplay is essential. however, identifying the exact epitope sequence and the exact hla allotype involved in t cell recognition of a specific pathogen is a demanding challenge. over the years, a plethora of methods have been used to identify t cell epitopes. there are two major and principally different approaches of t cell epitope discovery. the "forward immunology" approach (28, 29) uses specific t cell responses as a starting point to search for the underlying t cell epitope and its mhc restriction, whereas the "reverse immunology" approach (30, 31) uses predictions (e.g., of peptide-mhc interactions) to suggest possible t cell epitopes and then screen them for their ability to stimulate specific t cell responses [reviewed in (9, 43, the frequencies of epitopes per 100 aa are also given. ]. the experimental procedures involved in both of these epitope discovery modes tend to involve slow, low throughput, cumbersome and expensive processes [e.g., expression cloning of antigen libraries and/or hla genes (28, [45] [46] [47] [48] , synthesis of peptide libraries etc.]. in contrast, the bioinformatics component of a reverse immunology approach offers a process that is fast, of high capacity and throughput, yet very easy and inexpensive; a process, which is well-suited to support systematic analyses of genomic and proteomic information (3, 4, 9, 30) . it is not surprising that reverse immunology has become the preferred approach to t cell discovery. the need for high speed and capacity is of obvious importance in emerging infectious diseases (including bioterrorism), and even more so in personalized cancer immunotherapy where fast and high-throughput methods are essential for the selection of relevant and safe cancer neoepitopes in real time. current peptide-mhc predictors are highly sensitive and specific [96.5 and 98.5%, respectively (16) ]. however, despite continued improvements of these predictors, the false discovery rate (fdr) is very high (8, 10, 18) ; something that compromises the successful inclusion of one, or preferably more, t cell epitopes in cancer immunotherapy even if these encompass up to 10-20 predicted epitopes (20, 21) . reducing the fdr while maintaining the sensitivity will be needed if reverse immunology in the future should fully support neoantigen discovery and secure timely, personalized immunotherapy of cancer (19) . indeed, most of the larger cd8 + and cd4 + t cell epitope submissions to the iedb have been identified by "reverse immunology." thus, sette and coworkers used "reverse immunology" to identify dengue virus-specific t cell epitopes and have, as of july 2019, contributed with the single largest submissions of cd8 + and cd4 + t cell epitopes (iedb reference id 1027503, 1031475, and 1031301). in contrast, the "forward immunology" approach has fallen relatively into disuse. an innovative approach pioneered by koelle and co-workers, which has resulted in larger iedb submissions of cd8 + and cd4 + t cell epitopes (e.g., iedb reference id 1021375), have used a "forward" component where co-transfecting panels of apc with cdna encoding antigen and hla class i or ii, each apc representing a single antigen and a single hla restriction element, were used to interrogate cd4 + and cd4 + t cell responses of virus infected donors (48) . the "forward" component of this approach identified intact immunogenic protein antigens and their restriction element(s); however, for the epitope discovery part of this work, the entire antigen was subjected to a "reverse" component predicting the epitope(s) and its hla restriction element(s). another innovative approach, tetramer guided epitope mapping (tgem), pioneered by kwok and james, which has resulted in large cd4 + t cell epitope submissions [iedb references id 1026930 (49) , 1013360, 1016040, and 1020783], have also used a "forward" component. longer overlapping peptides representing entire antigens were offered to single hla class ii molecules and the resulting peptide-hla class ii complexes were multimerized and the ensuing tetramers used to interrogate cd4 + t cell responses of appropriate donors. using shorter overlapping peptides suitable as cd8 + t cell epitopes, maeurer and coworkers established a tetramer-based approach for cd8 + t cell epitope discovery, which also resulted in larger iedb submissions [iedb id 1026840 (50) ]. this latter approach would obviously be very peptide intensive if every relevant peptide was to be tested in that way (e.g., the yellow fever proteome would require 13610 peptides to represent all possible 8-11mer peptides). here, we have generated a "hybrid forward-reverse immunology" (hfri) approach capable of doing concurrent cd8 + and cd4 + t cell epitope discovery and demonstrated that it can perform large-scale epitope discovery and at the same time decimate the false positive discovery rate. for the initial "forward immunology" screen, we used an overlapping peptide library of 850 15mer peptides overlapping by 11 aa, which represented the entire 3,411 aa yellow fever virus proteome, to stimulate pbmc's obtained ex vivo from primary yellow fever virus vaccinees at the peak of the resulting t cell response. since 15mer peptides are further processed during in vitro ics and/or elispot assays, this peptide library represented all possible yfv-specific cd4 + and cd8 + t cell epitopes of up to 12 aa in length; some, but not all, epitopes of 13-15 aa in length; and no epitopes of a length longer than 15 aa. distributing this peptide library into 4 matrices, the initial screening effort could be reduced to testing ca. 120 peptide pools for their ability to stimulate t cell responses preferably by ics analysis. the matrix design subsequently allowed us to home in on the individual t cell stimulatory peptides. following the "forward immunology" screening, a "reverse immunology" approach was applied to all the 15mer peptides containing cd8 + t cell epitopes. in silico, the affinities of all possible 8-11mer peptides that could be generated from the 15mer were predicted in the context of up to 6 different hla-a, -b and -c allotypes per individual. this reduced the number of potential peptide-hla-a, -b or -c combinations from 156 per stimulatory 15mer peptide to typically one to three combinations. the most likely peptide binders were synthesized and used to generate appropriate peptide-hla-i tetramer(s), which subsequently were used to validate cd8 + t cell epitope(s). for the vast majority of t cell stimulatory 15mer peptides, at least one epitope was identified per 15mer peptide. once the stimulatory 15mer peptides had been identified, predicting the exact epitope and its restriction element was a highly efficient process; typically, the epitopes ranked first, second or third amongst the many potential epitope-hla combinations. as a cost-saving measure, if the predictions clearly discriminated between the candidates, a consecutive process was applied whereby the top peptide(s) were synthesized and tested before any next tier peptides were synthesized and tested. this hfri approach was extended to 50 primary yfv vaccinees, where it identified and tetramer-validated 92 cd8 + t cell epitopes (predominantly of size 9-10mer, range 8-11mer) covering 40 hla-i allotypes (representing a total of 120 peptide-hla-i combinations). before this work, the iedb had registered ten yfv-specific cd8 + t cell epitopes as being "exact epitopes" (i.e., length from 7 to 11 aa) and restricted by an hla allotype defined at high (4-digit) resolution; however, none of them were tetramer validated. four of the ten already registered yfv-specific, cd8 + t cell epitopes were included in the 92 epitopes identified here. thus, the present approach identified and validated 92 -4 = 88 new, or ca. 90% of all currently known, yfv-specific, cd8 + t cell epitopes. the total number of exact cd8 + t cell epitopes with high resolution hla-i restriction, which are currently registered in the iedb is 2,612 of which 1,101 have been tetramer validated (extracted from the iedb, july 2019). thus, this study accounts for >8% of these tetramer-validated human cd8 + t cell epitopes. to evaluate the prevalence of the different yfv-specific cd8 + t cell immune responses, the tetramer analysis was extended to additional vaccinees with the appropriate hla-i allotypes. many epitopes were frequently observed (i.e., were highly prevalent) in vaccinees with the appropriate hla allotype. thus; 25 (ca. 31%) and 50 (ca. 62%) of 81 cd8 + t cell epitopes were observed in ≥90 and ≥50%, respectively, of vaccinees with the appropriate hla-i allele. conversely, 18 (ca. 75%) of 24 hla-i allotypes presented at least one cd8 + t cell epitope with a prevalence of ≥90%. thus, the hfri approach identified a cohort of immunodominant yellow fever-derived peptides, which could be of broad diagnostic and therapeutic interest. large-scale t cell epitope discovery could also address more fundamental issues in immunobiology. pertinent examples of phenomena that are poorly understood include the closely related immunodominance (that the immune response is focused on just a few of the many available determinants expressed by a pathogen) and immunodomination (that the immune response of one specificity can suppress the response of another specificity). not surprising, these phenomena are closely related to antigen processing and presentation including mhc and t cell repertoire (51) . the vast majority of experimental data on immunodominance and immunodomination emanates from studies involving inbreed mice. few studies in humans address immunodominance [e.g., (52) ]; to the best of our knowledge none involve immunodomination. the latter is particularly difficult to address in an outbreed system like the human where the extremely diverse hla creates context dependent effects that confounds attempts to address immunodomination. assuming that the context-dependent effects hla could even out in larger donor cohorts, we exploited the size of our study to ask whether the presence of hla-a * 02:01, which restricts a strongly immunodominant, ns4b 214−222 -specific t cell response, would correlate with a reduction of responses restricted by other hla allotypes. indeed, under these conditions, we could demonstrate such a correlation in the presence of hla-a * 02:01, but not in the presence of hla-a * 01:01 or -a * 03:01. note that the hla-a * 01:01 or -a * 03:01 allotypes themselves featured a hierarchy of immunodominant t cell responses i.e., they are valid hla restricting elements. this may be the first demonstration of primary anti-virus responses being subjected to immunodomination in humans. a further analysis of the mechanism of behind these phenomena is beyond the scope of this paper. hla-c restricted, cd8 + t cell epitopes were scarcely represented (<3%) in the iedb; something that potentially could be explained by hla-c being insufficiently investigated. a priori, we expected that the unbiased nature of our approach would reveal several hla-c restricted cd8 + t cell epitopes, however, we only found one case of a strong and highly prevalent cd8 + t cell response, which could not be explained by any of the hla-a or -b allotypes available to the responding donors. instead, a strongly predicted binding to a shared hla-c allotype amongst the responding donors suggested an hla-c * 06:02 restricted response. eventually, two hla-c * 06:02restricted epitope length variants; ns3 207−213 (trrflpqil) and ns3 208−213 (rrflpqil), were tetramer validated. these were the only hla-c restricted cd8 + t cell epitope identified; all other identified cd8 + t cell epitopes were validated as being either hla-a or -b restricted. hla-c is less polymorphic and is known to be expressed at a lower level than hla-a and -b (53) (54) (55) (56) ; something that has been correlated with reduced cytotoxic t lymphocyte responses (57, 58) . in the case of the hla-c * 06:02-restricted ns3 207−213 (trrflpqil) epitope identified here, any reduced expression level of hla-c * 06:02 might have been compensated by the very strong predicted binding affinity for ns3 207−213 . although weaker hla-c-restricted cd8 + t cell responses may have been missed, we would argue that it is unlikely that we have missed strong and prevalent hla-c restricted cd8 + t cell epitopes. thus, we suggest that the paucity of strong hla-c restricted cd8 + t cell responses, at least in an acute viral infection like yellow fever virus, is not due to hla-c having been neglected in the scientific literature, but rather reflects a true biological phenomenon. notwithstanding, future cd8 + t cell discovery efforts should include hla-c, in particular if one or more hla-c restricted epitopes can be suggested in a situation where there are no obvious hla-a or -b restricted candidates. concurrent with cd8 + t cell discovery, the "forward-reverse immunology" approach also allowed hla-ii-restricted cd4 + t cell epitope discovery. the initial matrix-driven "forward" analysis of 50 donors identified 192 cd4 + t cell stimulatory yfv-derived 15mer peptides. this suggests that cd4 + t cell epitopes are more numerous than cd8 + t cell epitopes, perhaps as much as 2-3 times greater. if generalizable, this would have important implications for cd4 + t cell immunity since, everything else being equal, it would be more difficult for a microorganism to escape many cd4 + t cell epitopes than fewer cd8 + t cell epitopes. addressing the number of immunogenic open reading frames, other have also hinted at a greater preponderance of cd4 + than cd8 + t cell epitopes (59, 60) ; to the best of our knowledge, ours is the first proteome-wide study that have made this observation at the epitope level. the identification of the restricting hla class ii element(s) is a serious challenge in part due to different hla-ii allotypes having overlapping peptide binding repertoires (61) . in fact, this problem is so manifest that sette et al. have developed a panel of 46 different single hla-ii transfected cell lines to identify hla-ii restriction elements (62) . it would be ideal if hla-ii restrictions could be identified by predictors and then validated by tetramer analysis. unfortunately, the contemporary cd4 + t cell epitope discovery tools were immature (e.g., the early netmhciipan predictors were relatively inefficient and focused solely on the hla-dr isotypes), and access to peptide-mhc class ii tetramers was very limited. moreover, ex vivo frequencies of tetramer-positive cd4 + t cells tend to be <0.01%, which make them difficult to detect. thus, our cd4 + t cell epitope discovery process was not exhaustive; however, as cd4 + t cell discovery tools mature, we believe that the efficiency of cd4 + t cell epitope discovery eventually should approach that of cd8 + t cell epitope discovery. here, using a panel of recombinant hla-dr molecules, we measured the binding affinity of the overlapping 15mer peptides to the most common hla-dr allotypes. for each stimulatory 15mer peptide, this suggested which of the donor's hla-dr molecules should be used to generate peptide-hla-dr tetramers for validation of cd4 + t cell epitopes. this "brute force" approach was extended to 31 donors, where we tetramervalidated 50 cd4 + t cell epitopes covering 13 different hla-dr allotypes (and one hla-dp allotype). as of july 2019, the iedb has registered a total of 1,915 yfv-specific cd4 + t cell epitopes as being "exact cd4 + t cell epitopes" (i.e., length 15 aa, or less) and restricted by an hla-ii defined at high (i.e., 4-digit) resolution; 368 of which have been tetramer-validated. thus, the tetramer-validated yfv-specific cd4 + t cell epitopes reported here represents a significant increase in the number of tetramer-validated cd4 + t cell epitopes. it should be noted that james and coworkers have identified and tetramer-validated 94 different yfv-specific cd4 + t cell epitopes [iedb reference id 1026930 (49) ] that are 17 aa long and therefore fall just outside the definition of an exact cd4 + t cell epitope. a detailed examination of cd4 + t cell responses revealed a phenomenon that could have profound biological and practical implications for cd4 + t cell recognition. in many cases, two consecutive overlapping 15mer peptides stimulated cd4 + t cell responses, which were restricted by the same hla-dr restriction element. when the responding cd4 + t cells were double-stained with hla-dr tetramers, which had been prepared with each of the overlapping peptides of a 15mer pair and labeled with a unique fluorochrome, we observed a plethora of different, yet partially shared, cd4 + t cell specificities. situations where overlapping peptides are presented must occur regularly in vivo since experiments sequencing natural peptides eluted of hla-ii molecules frequently find large series of staggered peptides surrounding each core region (63) . exploiting this wealth of closely related peptides to engage a large number of different cd4 + t cell specificities recognizing the same core region in slightly different ways [something that actually was noted years ago (64) ], may represent a biologically significant diversification mechanism of cd4 + t cell responses reducing the risk of pathogen escape and increasing the chances of recognizing a given target. this phenomenon is also important for how cd4 + t cell responses should be analyzed. a single peptide-hla-ii tetramer is likely to engage a range of t cells of various avidities for the tetramer; something that might explain why single hla-ii tetramers often appear to label a poorly defined, non-base line separated, mono-specific cd4 + t cell population of low frequency. if examined with two "overlapping" tetramer, this could in reality turn out to be a heterogeneous collection of better defined and separated cd4 + t cell populations of a higher accumulated frequency. from a practical perspective, this implies that double staining involving two overlapping peptide-hla-ii tetramers will be needed to faithfully enumerate and monitor any cd4 + t cell response. from a technical perspective, this will increase the observed frequencies of specific cd4 + t cells for a given specificity (i.e., increase the sensitivity of the analysis), and it will increase the resolution of the flow cytometric analysis since it may separate various positively staining subpopulations and provide a better discrimination against negatively staining cd4 + t cells. the t cell epitope discovery approach described here has several advantages. in the forward immunology component, an overlapping peptide library is used to search for peptides containing cd4 + or cd8 + t cell epitopes. in the subsequent reverse immunology component, pan-specific predictors are used to identify the underlying epitope and its hla restriction element. these steps can be done in any (obviously outbred) individual of any hla haplotype using simple and standardized conditions. this reduces the number and combinations of peptides and hla allotypes that should be considered for peptide-hla tetramer generation and used in the final validation of the discovered t cell epitopes. as shown here, this approach is efficient and, not surprisingly, it reduces the false discovery rate. as peptide-hla class i and ii predictors and tetramer technologies mature, this approach will eventually be able to cover all frequently found hla class i and ii iso-and allotypes. this approach is systematic, all-inclusive, complete, and global in the sense that it includes all protein antigens and peptide epitopes, encompasses both cd4 + and cd8 + t cell epitopes, and can be extended to all individuals of all populations. this approach could be extended to other attenuated live virus vaccines (e.g., those targeting measles, mumps, rubella, polio, and hpv). compared to a strictly reverse approach, significant disadvantages of the hfri approach include the time and cost associated with establishing a complete overlapping peptide library as well as using a cellular readout as an initial selection step. therefore, this will probably not be justified if the aim is to identify epitopes in an urgent effort involving one donor (e.g., for cancer immunotherapy purposes); rather, it would be appropriate if the aim is to examine a large panel of donors in order to get population-wide data including immunodominance, candidates for diagnostics and vaccine development for infectious disease purposes (examples include a range of emerging and re-emerging infectious diseases like hiv, sars, mers, chikungunya, dengue, west nile, zika, ebola and sars-cov-2). for all of the above examples, the proteomes are small enough that their entire proteomes could be addressed by an overlapping peptide strategy using the number of pbmc's that could be obtained from a donor. addressing larger pathogen proteomes (e.g., herpes virus, bacteria or parasites; or smaller highly variable virus like hiv and dengue) in their entirety would require either a selection process downsizing the source target protein antigens, or the development of novel miniaturized, yet high-capacity, technologies. one could envision that future investigations of emerging diseases would include population-wide t cell epitope discovery efforts using blood samples from patients, convalescents and/or longterm survivors, which all possess important information on t cell epitopes and responses. similarly, one could envision that approval and registration of new vaccines could include population-wide analysis of t cell epitopes and responses. one could also envision that population-wide information on t cell epitopes and immunodominance could be used to design subunit vaccines. another important advantage of the forward-reverse approach presented here is the unbiased nature of the t cell epitope discovery process. whereas, current data-driven bioinformatics peptide-mhc predictors are quite accurate, the need for even better predictors stresses not only the need for high-quality training data, but also the need for high-quality validation data. in this context, there is an inherent problem in most epitope discovery efforts being dependent on peptide-mhc predictors since this effectively means that current t cell epitope discovery submissions tend to be biased by current predictors; something that might compromise the validation and benchmarking of predictors. having reasoned that our forward-reverse approach captures about 90% of the true t cell epitopes, we would like to propose that the resulting data is largely unbiased and should serve as an appropriate benchmark [others have reached similar conclusions (47)]. as an example, we used the cd8 + t cell epitopes identified here to benchmark current predictors. all current predictors were quite efficient and accurate. the newer predictors, some of which included immunopeptidomics and therefore may also reflect antigen processing, were better than the older predictors [as also noted by others (41) ]. however, these improvements are incremental and even the newest predictors were afflicted by high fdr's. taken together, this could be interpreted as a need for a change in how we predict t cell epitopes that is more fundamental than merely acquiring more peptide-mhc affinity and/or stability data e.g., by including t cell receptor specificities and repertoire propensities. a source of unbiased t cell epitope data would be instrumental in improving predicting tools. in conclusion, for smaller proteomes, it is possible to design a limited set of overlapping peptides spanning the entire proteome and use these to reveal the vast majority, if not all, specific cd4 + and cd8 + t cell responses concurrently; use predictors to identify the underlying combination of peptide epitopes and mhc restriction elements; and finally use this information to construct suitable peptide-mhc multimers and validate the t cell epitopes discovered. performing this in cohorts of patients or vaccinees allows for a systematic, global and cost-efficient analysis of cd4 + and cd8 + t cell epitopes, and evaluation of their immunodominance. as previously described (26) , this study was approved by the danish national committee on health research ethics (protocol # h-1-2009-095), and the collection of data and cells was approved by the danish data protection agency (permission 2008-41-2732). all volunteers gave written informed consent prior to participation. based on previous yfv vaccination history and their international card of vaccination, healthy volunteers, who for traveling purposes were about to receive a primary yfv vaccination, were recruited. the attenuated yfv vaccine, 17d-204 (sanofi pasteur; marketed as stamaril in more than 70 countries globally and as yf-vax in the usa) was administered intramuscularly. about 42% of the volunteers received an yfv vaccination only, whereas the remaining 58% received additional vaccines, typically killed, inactivated or subunit vaccines; in no case was the yfv vaccine co-administered with another live attenuated vaccine. as previously described (26) , blood samples were obtained just prior to and after the yfv vaccination (typically day 10-20 post vaccination, range 9-41 days). peripheral blood mononuclear cells (pbmc) were isolated by density gradient centrifugation using ficoll-paque tm plus (ge healthcare europe, brøndby, denmark). they were either examined directly ex vivo or cryopreserved in 10% dmso and 90% fcs at −150 • c for later in vitro analysis. chromosomal dna was prepared from the pbmc's and sequence-based typing (sbt) was used to perform highresolution (i.e., 4 digit) hla-typing (genome diagnostics, utrecht, the netherlands). all loci encoding classical hla molecules were typed i.e., the three class i ioci, hla-a, -b, -c and the six class ii loci, hla-drb1, -drb3/4/5, -dqa1, -dqb1, -dpa1, and -dpb1. the pbmcs were analyzed ex vivo for the t cell markers, cd3, cd4, and cd8, and the extracellular t cell activation markers, cd38 and hla-dr as previously described (26) . briefly, pbmcs were incubated with fluorochrome-conjugated anti-cd3, -cd4, -cd8, -cd38, and -hla-dr antibodies for 30 min at room temperature, washed, fixed with 1% formaldehyde, and analyzed by flow cytometry (lsr-ii, bd biosciences) using diva software. all antibodies were obtained from biolegend (san diego, ca, usa). all peptides were synthesized by standard 9fluorenylmethyloxycarbonyl (fmoc) chemistry and purified by reversed-phase high-performance liquid chromatography (purity at least 80%, usually >95%), mass spectrometry validated and lyophilized (schafer-n, copenhagen, denmark). an overlapping peptide library systematically covering the entire proteome of the vaccine strain, yf-17d-204 (uniprot# p03314), was synthesized. this encompassed the entire yf precursor protein of 3411 aa, which could be represented by 850 peptides, each 15 aa long and overlapping its neighboring peptides by 11 aa. in addition, 50 peptides, which were predicted to be binders to hla-a or -b supertype representatives by our netmhcpan predictor, were selected from putative products of alternative translation initiation codons (65) . one hundred and seven (107, or 11.9%) of the total of 900 selected peptides were difficult to synthesize and/or purify; many of which had long stretches of hydrophobic aa. adding one or two lysine to their n-or c-termini allowed the successful synthesis and purification of 77 of these "difficult to synthesize" peptides leaving 30 peptides that could not be synthesized and/or purified. thus, 870 (97%) of the selected peptides could be included in this epitope screening effort. these peptides were initially organized in a 30×30 matrix, and eventually in four 15×15 matrices (supplementary figure s1 ). fresh or thawed pbmcs were tested using an interferon-γ (ifnγ) specific eli spot assay as previously described (66) . briefly, 2-3 × 10 5 cells/well were plated in an eli spot plate (mahas4510, merck millipore, usa) and in vitro cultured for 18-24 h in media supplemented with or without peptide at 0.5 µm [or, as positive control, with 1 µg/ml staphylococcal enterotoxin b (seb, sigma aldrich, st. louis, usa)]. an ap conjugate substrate kit (bio-rad) was used for visualization of spots. eli spots were counted using a ctl immunospot series 5 uv analyzer. immunospot 5.0.9 software (c.t.l., shaker heights, usa) was used for analysis. wells with spotforming units spu > 2 times the background wells were considered positive. pbmcs were incubated overnight (37 • c, 5% co 2 ) in x-vivo 15 media (fisher scientific) supplemented with 5% ab serum (invitrogen) and a mixture of relevant peptides at a concentration of 0.5 µm of each peptide. the cells were harvested and washed, and subsequently plated in 24-well plates at a concentration of 5 × 10 6 /ml supplemented with 50 u/ml il-2 for expansion. fresh media and il-2 were supplemented every second day until the cells were harvested at day 8, and il-15 (15 ng/ml) was added the last 4 days. in vitro cultured pbmc's were harvested, washed, and aliquoted at 2-4 × 10 5 cells/well in a round bottom 96 well-plate. the in vitro cultured pbmc's were stimulated for 4 h at 37 • c, 5% co 2 with, for the matrix analysis, each of the 15 column and 15 row mixes (1 µm/peptide), and for the epitope identification with a single peptide (0.8 µm). after 1 h of stimulation 1 µg/ml brefeldin a (bd biosciences) was added. the cells were subsequently permeabilized (becton dickinson permeabilizing solution 2) and stained with anti-cd3-pacific blue, anti-cd4-percp, anti-cd8-apc, anti-cd69-pe, and anti-ifnγ-fitc, according to the "fastimmune" protocol (becton dickinson). the cells were subsequently analyzed by flow cytometry using a lsrii (bd biosciences). the gating strategy is illustrated in supplementary figure s4 . staining of more than 0.8% of cd8 + or cd4 + t cells was considered positive. hla class i tetramers were produced as previously described (67) . briefly, recombinant, biotinylated hla class i heavy chain, human β 2 -microglobulin and peptide were incubated in 50 mm tris-maleate ph 6. hla-dra and hla-drb chains were produced as previously described (42) . for tetramer production, hla-dra and hla-drb chains were mixed in a 1:1.5 ratio and incubated in 3 µm peptides in pbs (ph 7.4) with 20% glycerol and 0.1% pluronic f68 for 96 h at 18 • c. the resulting monomers were buffer changed into pbs with 5% glycerol and concentrated on 10kd vivaspin (satorius) and quantitated by luminescent oxygen channeling immunoassay (loci)-driven assay (42) . the resulting monomers were tetramerized by addition of fluorochrome labeled streptavidin (streptavidin-phycoerythrin (sa-pe) or streptavidin-allophycocyanin (sa-apc); both from biolegend) sequentially over 60 min at a 1:4 molar ratio of streptavidin to monomer. for t cell analysis, pellets of 4 × 10 5 cells obtained from in vitro peptide stimulated cell cultures, were re-suspended in a 40 µl tetramer diluted in media to a final concentration of ca. 30 nm, and incubated for 1 h at 37 • c, followed by 30 min incubation with fluorochrome conjugated anti-cd3, -cd8, -cd38, and -hla-dr antibodies. the cells were analyzed by flowcytometry (fortessa or lsr-ii, bd biosciences) using diva software. for each donor, all 15mer peptides eliciting a cd8 + t cell response were submitted to our bioinformatics predictor, netmhcpan (36) , which returned a prioritized list of predicted optimal epitopes, which could bind to any of the up to six hla-a, -b, or-c molecules of the donor in question. for each donor, all 15mer peptides eliciting a cd4 + t cell response were submitted to our bioinformatics predictor, netmhciipan (68) , which returned a prioritized list of predicted epitopes including a predicted core-region, which could bind to any of the up to four hla-drb1, or-drb3/4/5 molecules of the donor in question. the stability of peptide-hla class i complexes was measured using dissociation of 125 i radiolabelled β 2 m in a scintillation proximity assay (spa) as previously described (69) . briefly, recombinant, biotinylated hla class i heavy chains were diluted into a refolding buffer containing the test peptide and trace amounts of 125 i radiolabeled β 2 m, and allowed to refold at 18 • c for 24 h in a streptavidin-coated scintillation microplate (flashplate plus, perkin elmer, boston, ma). dissociation was initiated by adding excess unlabeled β 2 m and placing the microplate in a scintillation counter (topcount nxt, packard) adjusted to 37 • c. reading the microplate continuously for 24 h allowed determination of the dissociation of radiolabeled β 2 m. peptide-hla-ii binding affinities were determined using a previously described luminescent oxygen channeling immunoassay (loci)-driven assay (42) . briefly, denatured and purified recombinant hla-ii alpha and beta chains were diluted into a refolding buffer (tris-maleate buffer, ph 6.6) with graded concentrations of the test peptide, and incubated for 48 h at 18 • c to allow for equilibrium to be reached. the peptide concentration leading to half-saturation (ed 50 ) was determined as previously described (42) . under the limited receptor concentrations used here, the ed 50 reflects the affinity of the interaction. graphpad prism 8 was used for statistical analyses (unpaired and paired mann-whitney-wilcoxon tests, unpaired and paired t-tests, fishers exact test, and roc analysis). all datasets presented in this study are included in the article/supplementary material. towards a systems understanding of mhc class i and mhc class ii antigen presentation immunodominance in major histocompatibility complex class i-restricted t lymphocyte responses description and prediction of peptide-mhc binding: the human mhc project identifying cytotoxic t cell epitopes from genomic and proteomic information: the human mhc project the immune epitope database and analysis resource program 2003-2018: reflections and outlook a case for a human immuno-peptidome project consortium computational tools for the identification and interpretation of sequence motifs in immunopeptidomes editorial: the problem with neoantigen prediction identification of t-cell epitopes for cancer immunotherapy bassani-sternberg: 'hotspots' of antigen presentation revealed by human leukocyte antigen ligandomics for neoantigen prioritization best practices for bioinformatic characterization of neoantigens for clinical utility an automated benchmarking platform for mhc class ii binding prediction methods robust prediction of hla class ii epitopes by deep motif deconvolution of immunopeptidomes mhc peptidome deconvolution for accurate mhc binding motif characterization and improved t-cell epitope predictions footprints of antigen processing boost mhc class ii natural ligand predictions netmhcpan-3.0; improved prediction of binding to mhc class i molecules integrating information from multiple receptor and peptide length datasets a quantitative analysis of the variables affecting the repertoire of t cell specificities recognized after vaccinia virus infection why 99% may not be as good as you think it is: limitations of screening for rare diseases predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing personalized rna mutanome vaccines mobilize poly-specific therapeutic immunity against cancer an immunogenic personal neoantigen vaccine for patients with melanoma yellow fever vaccine yellow fever yellow fever expert: yellow fever in africa: estimating the burden of disease and impact of mass vaccination from outbreak and serological data human effector and memory cd8+ t cell responses to smallpox and yellow fever vaccines adaptive immune responses to booster vaccination against yellow fever virus are much reduced compared to those after primary vaccination. sci rep high stability of yellow fever 17d-204 vaccine: a 12-year restrospective analysis of large-scale production a nonapeptide encoded by human gene mage-1 is recognized on hla-a1 by cytolytic t lymphocytes directed against tumor antigen mz2-e linking genomics to immunotherapy by reverse immunology-'immunomics' in the new millennium discovery of t cell epitopes implementing hla-peptidomics into a reverse immunology approach nucleotide sequence comparison of the genome of two 17d-204 yellow fever vaccines abacavir-reactive memory t cells are present in drug naive individuals human leukocyte antigen (hla) class i restricted epitope discovery in yellow fewer and dengue viruses: importance of hla binding strength fuertes marraco: t cell receptor alpha variable 12-2 bias in the immunodominant response to yellow fever virus netmhcpan, a method for quantitative predictions of peptide binding to any hla-a and -b locus protein of known sequence netmhcpan-4.0: improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data mhcflurry: open-source class i mhc binding affinity prediction deciphering hla-i motifs across hla peptidomes improves neoantigen predictions and identifies allostery regulating hla specificity pan-specific prediction of peptide-mhc class i complex stability, a correlate of t cell immunogenicity a comprehensive review and performance evaluation of bioinformatics tools for hla class i peptide-binding prediction functional recombinant mhc class ii molecules and high-throughput peptide-binding assays strategies for the identification of t cell-recognized tumor antigens in hematological malignancies for improved graft-versus-tumor responses after allogeneic blood and marrow transplantation human tumor antigens recognized by t lymphocytes tumor antigens recognized by t lymphocytes diversity in the acute cd8 t cell response to vaccinia virus in humans analysis of cd8(+) t cell response during the ebola epidemic in west africa extensive cd4 and cd8 t cell cross-reactivity between alphaherpesviruses yellow fever vaccination elicits broad functional cd4+ t cell responses that recognize structural and nonstructural proteins a broad profile of co-dominant epitopes shapes the peripheral mycobacterium tuberculosis specific cd8+ t-cell immune response in south african patients with active tuberculosis immunodominance and immunodomination: critical factors in developing effective cd8+ t-cell-based cancer vaccines cd8+ tcr bias and immunodominance in hiv-1 infection the killer immunoglobulin-like receptor gene cluster: tuning the genome for defense molecular structure of human histocompatibility antigens: the hla-c series distinctive polymorphism at the hla-c locus: implications for the expression of hla-c relative expression levels of the hla class-i proteins in normal and hivinfected cells international histocompatibility working group in hematopoietic cell: hla-c expression levels define permissible mismatches in hematopoietic cell transplantation influence of hla-c expression level on hiv control broadly targeted human cytomegalovirus-specific cd4+ and cd8+ t cells dominate the memory compartments of exposed subjects cd4 t-cell memory responses to viral infections of humans show pronounced immunodominance independent of duration or viral persistence functional classification of class ii human leukocyte antigen (hla) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes a strategy to determine hla class ii restriction broadly covering the dr, dp, and dq allelic variants most commonly expressed in the general population different binding motifs of the celiac disease-associated hla molecules dq2.5, dq2.2, and dq7.5 revealed by relative quantitative proteomics of endogenous peptide repertoires structural characteristics of an antigen required for its interaction with ia and recognition by t cells major histocompatibility class i molecules can present cryptic translation products to t-cells ctl epitopes for influenza a including the h5n1 bird flu; genome-, pathogen-, hla-wide screening one-pot, mix-and-read peptide-mhc tetramers quantitative predictions of peptide binding to any hla-dr molecule of known sequence: netmhciipan real-time, high-throughput measurements of peptide-mhc-i dissociation using a scintillation proximity assay the authors thank doctors and nurses at the two recruiting centers and the blood bank of the university hospital, and members of the buus laboratory for expert technical assistance. the studies involving human participants were reviewed and approved by danish national committee on health research ethics (protocol # h-1-2009-095). the patients/participants provided their written informed consent to participate in this study. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.01836/full#supplementary-material key: cord-267166-ecmayzr6 authors: savarin, carine; dutta, ranjan; bergmann, cornelia c. title: distinct gene profiles of bone marrow-derived macrophages and microglia during neurotropic coronavirus-induced demyelination date: 2018-06-11 journal: front immunol doi: 10.3389/fimmu.2018.01325 sha: doc_id: 267166 cord_uid: ecmayzr6 multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns) characterized by demyelination and axonal loss. demyelinating lesions are associated with infiltrating t lymphocytes, bone marrow-derived macrophages (bmdm), and activated resident microglia. tissue damage is thought to be mediated by t cell produced cytokines and chemokines, which activate microglia and/or bmdm to both strip myelin and produce toxic factors, ultimately damaging axons and promoting disability. however, the relative contributions of bmdm and microglia to demyelinating pathology are unclear, as their identification in ms tissue is difficult due to similar morphology and indistinguishable surface markers when activated. the cd4 t cell-induced autoimmune murine model of ms, experimental autoimmune encephalitis (eae), in which bmdm are essential for demyelination, has revealed pathogenic and repair-promoting phenotypes associated with bmdm and microglia, respectively. using a murine model of demyelination induced by a gliatropic coronavirus, in which bmdm are redundant for demyelination, we herein characterize gene expression profiles of bmdm versus microglia associated with demyelination. while gene expression in cns infiltrating bmdm was upregulated early following infection and subsequently sustained, microglia expressed a more dynamic gene profile with extensive mrna upregulation coinciding with peak demyelination after viral control. this delayed microglia response comprised a highly pro-inflammatory and phagocytic profile. furthermore, while bmdm exhibited a mixed phenotype of m1 and m2 markers, microglia repressed the vast majority of m2-markers. overall, these data support a pro-inflammatory and pathogenic role of microglia temporally remote from viral control, whereas bmdm retained their gene expression profile independent of the changing environment. as demyelination is caused by multifactorial insults, our results highlight the plasticity of microglia in responding to distinct inflammatory settings, which may be relevant for ms pathogenesis. introduction multiple sclerosis (ms) is a chronic inflammatory disease of the central nervous system (cns), characterized by demyelination and axonal damage. active demyelinating lesions are characterized by cd8 t cells, cd4 t cells expressing both th1 and th17 cytokines, bone marrow-derived macrophages (bmdm) and activated cns resident microglia (1, 2) . myeloid cells activated by t cell effector functions are thought to participate in tissue damage by removing or "stripping" myelin (3) , and secreting toxic factors, such as reactive oxygen species, nitric oxide and the pro-inflammatory cytokines, tumor necrosis factor (tnf), and il-1β (4, 5) . activated microglia also secrete chemokines, which recruit innate and adaptive immune cells into the parenchyma, further amplifying the destructive inflammatory response (5) . however, both bmdm and microglia effector functions are highly heterogeneous depending on the environment and may not only contribute to disease progression but also to resolution (6, 7) . for example, by removing apoptotic cells and debris, their phagocytic activity favors tissue repair and is essential for disease resolution (3) . in addition, both cell populations secrete anti-inflammatory cytokines, such as il-10 and tgf-β, as well as trophic factors, which provide an environment that promotes tissue repair and neuronal protection (8) . the heterogeneity of the inflammatory response associated with ms lesions at the cellular and functional levels, thus makes it difficult to establish detrimental versus disease resolving functions of bmdm and microglia in ms pathogenesis. in addition to the inherent limitations associated with sampling cns tissues for longitudinal studies, the individual role of bmdm versus microglia as pathological mediators remains ambiguous due to morphological similarities and lack of reagents uniquely identifying each population. however, increasing evidence from animal models supports the concept that microglia and bmdm comprise two effector populations with distinct origins (derived from progenitors in the embryonic yolk sac and circulating monocytes respectively) and functions during ms and other neuroinflammatory disorders (9) . a variety of murine models, including autoimmune-and viral-induced demyelination, have been developed to study pathogenic features of ms (10) . the most common is the experimental autoimmune encephalomyelitis (eae), an autoreactive cd4 t cell-induced autoimmune demyelination characterized by infiltration of myelin-specific th1 and th17 cells, bmdm and microglial activation (11, 12) . pathogenesis during eae is associated with temporally distinct microglial activation and bmdm cns infiltration. early microglia activation is insufficient to trigger clinical disease, whereas delayed cns recruitment of bmdm directly correlates with disease progression. importantly, depletion of bmdm but not microglia inhibits eae (13, 14) . similarly, mice deficient in ccl2 (ccl2 −/− ), a chemokine essential for inflammatory monocyte recruitment into the cns (15) , are resistant to eae (16) . in support of detrimental bmdm functions, a combined histological and gene profiling study showed that demyelination is mediated by bmdm associated with nodes of ranvier, whereas debris clearance is achieved by microglia (17) . altogether, studies in the eae model demonstrate that bmdm recruitment into the cns is essential for the process of myelin loss and clinical manifestation. inflammatory demyelination is also induced following infection with two natural viral mouse pathogens, theiler's murine encephalomyelitis virus (tmev) and members of the neurotropic mouse hepatitis viruses (mhv). tmev infection induces an autoimmune disease in which bmdm are essential for both viral persistence and demyelination (18, 19) . however, the function of bmdm as a main reservoir of active viral replication during chronic tmev infection, limits efforts to assess their role in demyelination independent of virus load (20) . in contrast, infection with the non-lethal glia tropic mhv strain designated jhmv predominantly targets oligodendrocytes (olg) and to a lesser extent microglia and astrocytes. viral replication peaks at day 5 post infection (p.i.), but infectious virus is reduced below detection by day 14 p.i. acute infection initiates rapid cns recruitment of predominantly bmdm, but also neutrophils and nk cells, followed by infiltration of both cd8 and cd4 t cells, as observed in active ms lesions. the t cell response, which is essential to reduce viral replication, is highly th1 polarized with no evidence of il-17 or gm-csf production (21) (22) (23) . importantly, t cell-mediated virus control coincides with initiation of demyelination, which peaks between days 14-21 p.i. after infectious virus is cleared (24, 25) . although olg tropism is a requirement for demyelination, immunodeficient mice demonstrated that infection of olg in the absence of adaptive immunity is insufficient to cause demyelination. however, transfer of either virus-specific cd4 or cd8 t cells into virus infected immunodeficient mice leads to demyelination (26, 27) . furthermore, ifn-γ dependent control of infectious virus within olg and no evidence for olg apoptosis, suggested that direct t cell-mediated cytolysis of olg does not play a major role in myelin loss (28) . this implicates t cell activated bmdm and microglia as the most probable mediators of myelin destruction. moreover, both myeloid populations are abundant in lesions and occasionally associated with damaged axons (29) . however, in contrast to eae, genetic or chemical depletion of monocytes during jhmv infection does not alter disease severity, virus replication or myelin loss (30, 31) , suggesting that bmdm are dispensable for jhmv-induced demyelination. this study takes advantage of the distinct tissue environments established during eae and jhmv infection to characterize temporal alterations in gene expression profiles of bmdm versus microglia in a th1 biased demyelination model. to date, we are not aware of any reports evaluating the signature profile of microglia associated with pathogenic functions during demyelination. the results reveal that cns infiltrating bmdm rapidly establish a characteristic profile including m1 and m2 markers, which prevails throughout infection as the population declines. by contrast, gene expression in microglia is only prominently altered remote from viral control concomitant with demyelination; distinct from bmdm, the gene expression pattern is skewed to a highly pro-inflammatory and phagocytic profile. the results overall highlight the plasticity of microglia responses in distinct inflammatory settings, which may be relevant for ms pathogenesis at distinct stages of disease. wild-type (wt) c57bl/6 mice were purchased from the national cancer institute (frederick, md, usa). homozygous ccl2 deficient (ccl2 −/− ) mice were originally obtained from b. j. rollins (dana-farber cancer institute, boston, ma, usa). cx3cr1 gfp/gfp (b6.129p-cx3cr1 tm1litt /j) and ccr2 rfp/rfp (b6.129 (cg)-ccr2 tm2.1ifc /j) mice were purchased from the jackson laboratory (bar harbor, me, usa) and crossed to generate cx3cr1 gfp/+ ccr2 rfp/+ mice. transgenic mice were bred and maintained at the biological research institute under sterile conditions. all procedures were preformed in compliance with the cleveland clinic institutional animal care and use committee approved protocols. the glia tropic jhmv neutralizing monoclonal antibody (mab)derived 2.2v-1 variant was used for all infections (32) . mice of both sexes between 6 and 7 weeks of age were infected in the left hemisphere with 1,000 pfu of jhmv diluted in endotoxin-free dulbecco's phosphate-buffered saline (pbs) in a final volume of 30 µl. mice were monitored daily for clinical disease severity according to the following scale: 0, healthy; 1, hunched back and ruffled fur; 2, partial hind limb paralysis or inability to maintain the upright position; 3, complete hind limb paralysis; 4, moribund or dead. for analytical flow cytometry, anesthetized mice were perfused with ice-cold pbs, and resected brains and spinal cords homogenized using a ten-broeck tissue grinder as described (33) . tissue homogenates were adjusted to 30% percoll (pharmacia, uppsala, sweden) and underlaid with 1 ml 70% percoll prior to centrifugation at 850 g for 30 min at 4°c. cns mononuclear cell were recovered from the 30/70% interface, washed and resuspended in facs buffer (pbs + 1% bovine serum albumin). cells were blocked with anti-mouse cd16/cd32 (clone 2.4g2) mab for 15 min on ice prior to staining. staining was performed for 30 min on ice using fluorescein isothiocyanate, phycoerythrin, peridin chlorophyll protein complex (percp), or allophycocyanin (apc) conjugated mab (all from bd biosciences except where indicated) specific for cd45 (clone ly-5), cd11b (clone m1/70), f4/80 (serotec, raleigh, nc, usa) and major histocompatibility complex (mhc) class ii (clone 2g9). cells were then washed twice in facs buffer prior to analysis using a bd accuri flow cytometer (bd biosciences) and flowjo software (tree star inc., ashland, or, usa). for cell purification, spinal cords from pbs-perfused cx3cr1 gfp/+ ccr2 rfp/+ mice were finely minced with a razor blade. minced tissues were enzymatically digested in rpmi 1640 medium containing 10% fetal calf serum, 0.5% collagenase d (100 mg/ml) roche, basel, switzerland and 1% dnase i (1 mg/ml) (sigma aldrich, st. louis, mo, usa) for 40 min at 37°c. collagenase was then inactivated by addition of 1% 0.1 m edta for 5 min at 37°c prior centrifugation at 400 g for 7 min at 4°c. spinal cord-derived cells from seven mice were pooled and isolated using percoll gradients as described above and then stained with cd11b-percp and cd45-apc for 30 min on ice. spinal cord-derived bmdm (cd45 hi cd1 1b + ccr2 rfp+ ) and microglia (cd45 low cd11b + cx3cr1 gfp+ ) were purified using a facsaria cell sorter (bd biosciences) and resuspended in trizol. yields from 7-pooled mice ranged between 5.4-20 × 10 5 cells for bmdm and 0.5-1.2 × 10 5 cells for microglia depending on the time p.i. microglia from naïve mice were used to assess baseline expression, whereas circulating monocytes were used as controls for cns infiltrated bmdm after infection. monocytes were isolated from blood treated with gey's solution to lyse red blood cells prior to staining and cell sorting. gene expression profiling using ncounter analysis rna was prepared by extraction with trizol reagent (invitrogen, carlsbad, ca, usa) and direct-zol rna mini prep (zymo research, irvine, ca, usa) according to the manufacturer's instructions. gene expression profiles were analyzed using the ncounter mouse myeloid innate immune panel comprising 754 targets representing all major myeloid cell types and generated according to the manufacturer's protocol (nanostring technologies, seattle, wa, usa). the nanostring ncounter system directly captures and counts individual mrna transcripts using a multiplexed measurement system thereby omitting cdna based amplification (34) . analysis was performed using nsolver analysis software v3.0 and ingenuity pathway analysis (qiagen, hilden, germany). venn diagrams from individual gene lists and protein-protein interaction networks were constructed using genespring (agilent, inc.) and string software (http://www. string-db.org). to confirm validity of nanostring ncounter analysis, a small set of selected genes were analyzed by real-time pcr ( figure s1 in supplementary material). following rna extraction as described above, first-strand cdna was synthesized using reverse transcriptase (invitrogen) with oligo-dt and random primers (promega, madison, wi, usa) as described (35) . gene expression analysis was performed using a 7500 fast real-time pcr system (applied biosystems, foster city, ca, usa), sybr green master mix (applied biosystems) and the following primers: gapdh, 5′-catggccttccgtgttccta-3′ (forward) and 5′-atgcctgcttcaccaccttct-3′ (reverse); il15, 5′-tg aggctggcattcatgtctt-3′ (forward) and 5′-tccagtt ggcctctgttttagg-3′ (reverse); il1rn 5′-agatagacatg gtgcctattgacctt-3′ (forward) and 5′-catctccagac ttggcacaaga-3′ (reverse) and arg1 5′-tgggtggatgct cacactga-3′ (forward) and 5′-caggttgcccatgcaga tt-3′ (reverse). transcripts levels were normalized to the housekeeping gene gapdh and converted to a linearized value using the following formula: 2 (c t gapdh-c t gene) × 1,000, where ct represents the threshold cycle value. following pbs perfusion, spinal cords were fixed in 10% neutral buffered formalin, embedded in paraffin and sections stained with luxol fast blue as described to visualize demyelination (36) . for analysis of iba1 + cells spinal cords from ice-cold pbs-perfused mice were quickly embedded in oct and kept at −80°c until 10 µm sections were prepared. sections were fixed with paraformaldehyde for 20 min, treated with blocking solution for 30 min and then stained with rabbit anti-iba1 mab (wako, osaka, japan) overnight at 4°c. goat anti-rabbit secondary ab (invitrogen) was added for 1 h at room temperature and sections mounted with vectashield mounting medium (vector laboratories, burlingame, ca, usa). sections were analyzed using a leica tcs confocal microscope. to better characterize reactivity of microglia and infiltrating bmdm following jhmv infection, we initially monitored cns infiltration of bmdm, as well as upregulation of mhc class ii as an activation marker on both cns bmdm and microglia by flow cytometry. bmdm with a typical cd45 hi cd11b + f4/80 + phenotype comprised the majority of inflammatory leukocytes as early as day 3 p.i. and then progressively decreased as virus replication is controlled by t cells. at the onset of demyelination at day 10 p.i., the bmdm population stabilized at ~10% of the infiltrating leukocytes ( figure 1a ). bmdm initially infiltrated as mhc class ii lo expressing cells, but the vast majority upregulated mhc class ii by day 7 p.i. mhc class ii expression on microglia was sparse at days 3 and 5 p.i., but rapidly increased by day 7 p.i. and then gradually declined by day 14 p.i. (figure 1a) . these kinetics supported that microglia and bmdm activation peaks delayed relative to peak bmdm accumulation and coincides with peak t cell ifn-γ production (36, 37) . enhanced activation of microglia at day 7 p.i., compared to earlier times p.i., was also supported by progression of morphological changes, evidenced by enlarged cell bodies and retracted and thickened processes ( figure 1b) . the decline of bmdm, but an ongoing activation phenotype of microglia at the time of evident demyelination implicated microglia as mediators of tissue damage during jhmv encephalomyelitis. biochemical depletion of peripheral monocytes indeed supported that bmdm are not essential to tissue destruction in jhmv-infected mice (30) . data from our own laboratory further demonstrated that the chemokine ccl2 is essential for bmdm accumulation within the cns (31) . the absence of ccl2 resulted in an ~80% reduction of bmdm at all time points, including day 14 p.i. (31) and ( figure 1c ) when demyelination is prominently evident in wt mice. nevertheless, microglia activation, as monitored by mhc class ii expression, was independent of ccl2 ( figure 1d ). most importantly, the absence of ccl2-dependent bmdm within the cns did not alter demyelination ( figure 1e ). similar myelin loss at day 21 p.i. comparing wt and ccl2 −/− infected mice supported the concept that microglia mediate demyelination during jhmv infection. we next evaluated effector functions of bmdm versus microglia associated with jhmv-induced demyelination by comparing gene expression profiles using ncounter analysis of mrna isolated from purified bmdm and microglia of infected cx3cr1 gfp/+ ccr2 rfp/+ mice. characteristic expression of cx3cr1 gfp and ccr2 rfp on cd45 high cd11b + bmdm (population #1) and cd45 low cd11b + microglia (population #2) is shown in figure 2 throughout days 5-14 p.i. microglia were characterized by high expression of cx3cr1 and undetectable ccr2 expression (figures 2b,c) similar to other inflammatory models (17, 38) . in contrast, cns infiltrating bmdm expressed ccr2 and low levels of cx3cr1 compared to microglia ( figure 2b ). co-expression of ccr2 and cx3cr1 was maintained on bmdm at all time points p.i. and no cx3cr1 − cells were detectable ( figure 2c ). as both microglia and infiltrating bmdm retained their phenotype throughout infection, cd45 low cd11b + cx3cr1 gfphi ccr2 − and cd45 hi cd11b + cx3cr1 gfplow ccr2 + populations were isolated by facs from spinal cords at days 5, 7, 10, and 14 p.i. for subsequent mrna expression analysis. age-matched naïve animals were used to isolate microglia and blood circulating monocytes as precursors of cns-infiltrating bmdm. gene expression profiles for all purified populations were obtained using ncounter analysis and the innate myeloid immune panel. the respective naïve populations were used to assess signature gene expression profiles under homeostatic conditions (figure 3) . figure 3a shows the top 50 highly expressed genes within each population relative to three ncounter platform housekeeping genes, namely g6pdx, polr1b, and tbp, selected for three high, medium, and low expression, respectively, in this part of analysis platform. figure 3b lists the top 50 enriched genes specific for microglia compared to monocytes, or monocytes versus microglia, respectively. among the top 50 genes highly expressed in microglia, 15 were also specific and included genes of the complement cascade (c1qa, c1qb, c1qc, and c3ar1) and trem2, encoding a cell surface receptor involved in phagocytic functions and known to be expressed by microglia (39) . other genes, such as adamts1, f11r or hpgds, found within the top 50 enriched genes expressed by microglia ( figure 3b ) were also previously described as microglia specific (17, 40) . cx3cr1 mrna encoding the fractalkine receptor and used as a marker for microglia (41) , was also among the top 50 highly expressed genes ( figure 3a) , but not unique, consistent with the cx3cr1 lo phenotype on circulating monocytes. similarly, ccr2 expression characteristic of monocytes was confirmed by ccr2 mrna as the second in place of the top 50 expressed genes specific for circulating monocytes (figure 3b) . other specific signature genes of monocytes are related to motility and migration/tissue invasion, e.g., s100a4, s100a8, s100a9, fn1, sema4, mmp8, and to a lesser extent mhc class ii antigen presentation, e.g., h2-ab1, cd74, and fas. microglia and circulating monocytes also shared 17 highly expressed genes, including genes characteristic of the myeloid lineage such as csf1r, a gene coding for a cell surface receptor essential for hematopoietic figure 4a ). interestingly however, three genes among the common and highly expressed genes were regulated differently. ctss mrna, encoding for cathepsin s, a lysosomal cysteine proteinase participating in the mhc class ii molecule antigen presentation pathway as well as nociception (42, 43) , was specifically upregulated within bmdm, with highest levels observed at days 10 and 14 p.i. (figure 4a) , when demyelination increases. by contrast, microglia transiently downregulated ctss mrna at day 7 p.i. opposite regulation was also observed for dusp1 mrna (figure 4a ). dusp1 mrna encodes the dual specificity protein phosphatase 1, an enzyme involved in the cellular stress response and a negative regulator of cell proliferation (44) . while dusp1 mrna levels were vastly upregulated early following bmdm accumulation, but declined by day 7 p.i. and thereafter, levels were rapidly downregulated within microglia ( figure 4b) . finally, tyrobp mrna encoding for the trem2 adaptor dap12 and known to regulate microglia phagocytic functions (39) , was downregulated in bmdm throughout infection, but specifically upregulated within microglia at days 10 and 14 p.i. (figure 4b ). this expression pattern on microglia correlated with the onset of myelin loss and supported trem2 signaling specifically by microglia in response to tissue damage. to determine whether apparently differential functions of bmdm and microglia associated with jhmv-induced demyelination are reflected in distinct gene profiles, we monitored overall up-and downregulation of gene expression relative to basal levels in each population. analysis times were chosen to correlate with innate responses (d5 p.i.), peak t cell effector function (d7 p.i.), resolution of infection and initiation of demyelination (d10 p.i.), and finally viral clearance and overt demyelination (d14 p.i.). at day 5 p.i. a higher number of genes were differentially regulated within infiltrating bmdm compared to microglia (231 versus 76; figure 5a ). moreover, almost 80% of the genes showing altered expression in early infiltrated bmdm were increased compared to basal levels, while only 54% were increased in microglia; the remaining differentially expressed mrnas were decreased ( figure 5b) . the overall number of differentially expressed genes slightly declined in bmdm by day 7 p.i., when t cells exert maximal effector function (37) , and remained fairly constant throughout day 14 p.i. (figure 5a) . furthermore, the relative decline in the proportion of upregulated mrnas coincided with an increased proportion of downregulated genes, reaching a roughly equal distribution at days 10-14 p.i., when virus is largely controlled (figure 5b ). in contrast, microglia altered their gene expression pattern extensively at day 7 p.i. (97 genes, figure 5a ) with 67% of differentially regulated genes showing increases ( figure 5b ). by day 10 p.i., overall altered gene expression remained stable relative to day 7 p.i., with equal proportions showing increases and decreases. however, as myelin loss progresses by day 14 p.i., differentially regulated genes increased again in numbers, with the proportion of upregulated mrnas reaching 95% (figure 5a ). altogether these data show unique regulation of gene profiles in bmdm compared to microglia throughout the course of infection. while most changes were evident in bmdm following initial cns accumulation, microglia revealed most pronounced changes at the time of myelin loss. we further analyzed differential gene expression across time points focusing on upregulated genes using venn diagrams to reveal the relative proportion of genes that were commonly increased at all time points (figure 5c ). of the 183 upregulated genes in bmdm at day 5 p.i., 62 were unique to day 5. on the other hand, 77 genes (representing 42% of all upregulated genes) remained highly expressed at all other time points (figure 5c ). of note, not a single gene transcript was specifically upregulated at day 7 p.i., and only three overlapped with sustained upregulation at days 10 and 14 p.i. similarly, only 4 gene transcripts were specifically elevated at day 10 p.i., 7 were unique to both days 10 and 14, and only one was unique to day 14 p.i. these results indicate that the gene expression profile characterizing bmdm is established early following infection, with sparse unique alterations as bmdm decline during infection. in stark contrast, only 3 of 41 gene transcripts upregulated in microglia at day 5 p.i. were unique to day 5, and no gene transcript was commonly upregulated across all time points analyzed. furthermore, distinct from bmdm, 59 gene transcripts were figure 3 | gene expression characterizing microglia and circulating monocytes under homeostatic conditions. spinal cord-derived microglia (cd45 low cd11b + cx3cr1 gfp+ ) and circulating blood monocytes (cd11b + ccr2 rfp+ ) were purified from naïve cx3cr1 gfp/+ ccr2 rfp/+ mice by facs and rna subjected to ncounter analysis using the myeloid cell probe panel. panel (a) depicts the top 50 highly expressed genes and (b) the enriched genes uniquely characterizing each population. in (a) * highlights genes that are both highly expressed and enriched in each population, while # highlights genes highly expressed and common to both microglia and circulating monocytes. uniquely upregulated by day 7 p.i., with none common to day 10 p.i., and only six overlapping with those upregulated at day 14 p.i. although no gene transcripts were upregulated uniquely at day 10 p.i., 22 overlapped with those upregulated at day 14 p.i. a further 76 genes, comprising 53% of all upregulated genes at day 14 p.i., were specifically expressed at elevated levels at day 14 p.i. coinciding with overt myelin loss ( figure 5c) . these profiles reveal a dynamic range of responses and extensive plasticity of gene expression profiles in microglia throughout jhmv infection ( figure 5c ). we next used a protein-protein network connection constructed based on differential gene expression to specifically examine upregulation of gene transcripts within bmdm and microglia correlating with demyelination at day 14 p.i. for comparison, we also analyzed the network connection at day 5 p.i., when expression profiles were most prominently altered in bmdm, but more modestly in microglia. this comparative analysis was chosen to provide clues about specific functions and involvement of microglia relative to bmdm in tissue destruction (figures 6 and 7) . our initial focus was on temporally altered networks in bmdm (figure 6) . at day 5 p.i., early infiltrated bmdm expressed a wide array of chemokines regulating cns infiltration of both innate (cxcl2, ccl2, ccl4, ccl5, ccl7, and ccl12) and adaptive (cxcl9, cxcl10) immune cells (figure 6a) . a large cluster of molecules regulating the innate immune response, essential to limit early viral replication (45, 46) , was also expressed by bmdm (figure 6a) . these include pathogen recognition receptors such as tlrs (tlr1-4 and tlr9) and molecules linked to the tnf pathway (tnf, traf6, etc.). finally, bmdm expressed molecules involved in antigen presentation, including tap1 and tap2, as well as t cell activating co-stimulatory molecules (cd80 and cd86) or il-12, which induce th1 differentiation (figure 6a) . these results indicate that early infiltrating bmdm orchestrate the acute innate immune response crucial for limiting cns viral spread, as well as initiating the adaptive immune response by recruiting and activating t cells. at day 14 p.i., correlating with peak demyelination, a more restrained number of mrna transcripts were upregulated in bmdm (figure 6b) . the cluster of chemokines mobilizing immune cells was sustained ( figure 6b) . in contrast, the molecular network extended from tnf was more limited at d14 p.i. compared to d5 p.i. (figures 6a,b) . molecules regulating primarily the cd4 t cell response were expressed at day 14 p.i. and comprised gene transcripts involved in antigen presentation including mhc class ii (h2-ab1) and co-stimulatory molecules such as cd80 and cd86 (figure 6b) . interestingly, among the more restricted number of gene transcripts upregulated at day 14 p.i. in bmdm, several were transcripts encoding m2 molecules, which included arg1, il1rn and tgm2 (figure 6b) . in contrast to the vast number of genes upregulated early following infection in bmdm, a significantly lower number of upregulated genes characterized microglia at day 5 p.i. (figure 7a) . transcripts for chemokines regulating migration of both innate and adaptive immune cells, such as ccl2, ccl3, ccl4, ccl12, cxcl10, and cxcl16, were expressed by microglia at day 5 p.i. (figure 7a) . transcripts for tnf and other inflammatory cytokines generally associated with the innate responses, e.g., il1a, il1b, and il18 were also upregulated early in microglia ( figure 7a) . another extended network of tnf comprised psmb8 and psmb9, subunits of the immunoproteasome, essential for antigen presentation by mhc class i molecules. by day 14 p.i., the number of upregulated transcripts extensively increased in microglia (figure 7b) . the most clustered network comprised proteins like ccl5, cxcl9, cxcl10, cxcl13, cxcl16, and cxcr4, all chemokines and chemokine receptors regulating migration and arrest of adaptive immune cells within the cns during inflammation (47) . this chemokine cluster was linked to tnf and inflammatory cytokines previously detected at d5 p.i., e.g., il1a, il1b, and il18. another extended network of tnf comprised psmb8 and psm9, also present at d5 p.i., ctnnb1 encoding b-catenin, a cellular adhesion molecule, and cdkn1a, a cyclin inhibitor. other upregulated gene transcripts associated with class i antigen presentation, e.g., tap1 and tap2, were also linked through a network associated with complement component genes (c3, c3ar1, c4b, c1qa, c1qb, c1qc), which are highly expressed within microglia under homeostatic conditions (figure 3) . similarly, tyrobp and trem2, which formed phagocytic synapses (48), are both highly expressed in microglia during myelin loss. finally, a wide variety of upregulated gene transcripts are associated with mhc class ii antigen presentation and modulation of t cell function. this includes h2-ab1, encoding for the mhc class ii molecules and h2-dm, encoding for a second accessory protein, which facilitates peptide loading. similarly, genes associated with the invariant chain of mhc class ii were increasingly expressed within microglia, such as cd74 and ctss (cathepsin s, which cleaves invariant chain thereby promoting loading on mhc class ii). in addition, genes encoding for modulators of the cd4 t cell response, such as itgax (cd11c), cd86 and cd83 were also expressed by microglia ( figure 7b) . overall, the upregulated networks are related to complement activation, enhanced class i and class 2 antigen processing and presentation (potentially related to ifn-γ responses) as well as migration and phagocytic activity. however, there does not appear to be a bias toward phagocytic receptors over other components activated by pro-inflammatory mediators. pathogenic versus protective functions of myeloid cells following activation have also been correlated to expression of key molecules defined as m1 versus m2 markers. while the strict classification of myeloid cells into the m1 or m2 category has been tempered based on a more dynamic and mixed phenotype during inflammatory responses (49, 50) , the m1 and m2 markers remain helpful to gage overall effector functions. among the 89 analyzed gene transcripts in the nanostring myeloid panel related to m1/m2 polarization (51 m1 and 38 m2 genes), between 50 and 59% were upregulated across the course of jhmv infection with no difference comparing m1 versus m2 genes ( figure 8a) . among the total upregulated m1 markers, about 50% were commonly increased within both infiltrating bmdm and microglia; representative genes were il12b and il15ra (figures 8a,b) . however, while high levels of il12b mrna were observed in both bmdm and microglia at d5 p.i., expression was only sustained in microglia at day 7 p.i. and thereafter (figure 8b ). by contrast, il15ra was increased in both bmdm and to a lesser extent in microglia at d5 p.i., but was decreased in both populations at later time points p.i. (figure 8b ). in addition, between 35 and 45% of m1 markers were specifically expressed by infiltrating bmdm during the course of jhmv infection (figure 8a) , including cd86, atf3, ifng, ptgs2, ccr7, cxcl11, and cxcl12 transcripts ( figure 8b and data not shown). however, only 5 m1 related gene transcripts were specifically upregulated in microglia at the time of demyelination, including ccl5, fas, cxcl13, tnfsf10, and psmb9. (figures 8a,b and data not shown) . importantly, the most prominent difference between microglia and bmdm was noted in m2 marker regulation. among the 50-58% m2 gene transcripts upregulated following jhmv infection, only a small proportion (21-32%) was expressed by microglia ( figure 8a) . fn1 was the only m2 marker specifically expressed by microglia at the time of demyelination ( figure 8a) . although il1rn transcript expression was elevated in both bmdm and microglia, the increase was at best modest in microglia ( figure 8c) . the majority (86-95%) of m2 markers upregulated during jhmv infection were rather expressed by infiltrating bmdm, including arg1, erg2, il-10, and ccl22 (figures 8a,c and data not shown). increased transcript levels were most pronounces at days 5 and 7 p.i., but dropped off thereafter. altogether, these data showed that while infiltrating bmdm express a mixed phenotype of m1 and m2 markers during jhmv infection, microglia expressed primarily pro-inflammatory genes while not expressing m2 markers. microglia and infiltrating macrophages are major components of ms active lesions (51) . their effector functions are highly heterogeneous as evidenced by both pathogenic and protective functions during the course of ms (7) . they can promote tissue damage by releasing toxic and pro-inflammatory molecules, mediate demyelinated axons through phagocytosis as well as propagate inflammation by recruiting and activating adaptive immune cells. on the other hand, both populations also display protective functions by clearing myelin debris, which facilitates remyelination, as well as releasing trophic and anti-inflammatory factors, which promote tissue repair. while it remains a challenge to distinguish infiltrating macrophages from microglia in ms lesions due to morphological and phenotypic similarities, they are disparate effector cells based on animal ms models (17, 52) . questions relating to the pathogenicity of infiltrating macrophages and/or microglia in ms remain unanswered. can both populations display protective functions? do they display dynamic functions throughout the evolution of ms lesions? deciphering the respective roles of macrophages versus microglia in facilitating tissue damage and/or repair is essential to our understanding of ms pathogenesis and development of effective therapeutic strategies. in the murine eae model of ms, infiltrating bmdm are essential in mediating demyelination (53) . gene expression profiles demonstrated that bmdm are indeed highly phagocytic and inflammatory at disease onset, while microglia display a repressed phenotype (17) . by contrast, during jhmv-induced demyelination, recruited bmdm are dispensable for the demyelinating process (30) . distinct from eae, where microglia activation precedes cns infiltration of bmdm (52) , jhmv infection elicits early bmdm infiltration, prior to microglia activation. these distinct kinetics of bmdm recruitment relative to microglia activation thus appear to correlate with the apparently opposing roles of microglia as demyelinating populations. these data further suggest that early responses set the stage or imprint subsequent effector functions of bmdm and microglia. using a similar approach with nanostring analysis as in eae, the present study used gene expression profiling to characterize both bmdm and microglia myeloid functions at various times post jhmv infection. analysis of overall gene expression patterns revealed that the most extensive changes in bmdm were evident early after infection, while microglia showed a more dynamic profile throughout the course of viral encephalomyelitis. importantly, the most drastic gene upregulation in microglia was observed coincident with demyelination, at which time peak viral load and t cell effector function have substantially subsided (54) . our data contrast with eae (17) , where bmdm upregulated far more genes compared to microglia at disease onset, supporting opposing functions of bmdm and microglia in mediating demyelination in these two models. further, while bmdm exhibited a mixed expression profile of both pro-and anti-inflammatory markers, microglia expressed a highly pro-inflammatory profile and repressed most of the m2 markers across the entire time course of jhmv encephalomyelitis. analysis of protein-interacting networks within genes upregulated in microglia at the time of myelin loss revealed several key functions linked to promoting tissue damage. genes associated with complement activation were notably increased, although they were already highly expressed by microglia under homeostatic conditions. complement activation as a pathogenic component in ms has been reported following detection of deposits of the activated products of the complement component c3 in ms lesions (55) . the classical complement pathway has also been shown to mediate olg death thus promoting demyelination (56) . microglia phagocytic activity may also initiate tissue damage by directly removing myelin from axons, especially at the node of ranvier (17) . genes associated with trem2/dap12 signaling were also highly expressed by microglia at time of demyelination. trem2 modulates phagocytic capacity of myeloid cells via dap12 signaling (57) and is expressed on myelin-loaded myeloid cells in ms lesions (58) , supporting a role in ms pathogenesis. similar to jhmv infection, trem2 is predominantly expressed by microglia during eae and cuprizone-induced demyelination (59) (60) (61) . however, trem2-modulated phagocytic functions are essential for removal of myelin debris and remyelination implicating repair-promoting functions of microglia in the specific tissue environments defining these two demyelination models (17, 62) . preferential trem2 expression within microglia compared to bmdm following jhmv infection support a more pathogenic role of trem-2 in jhmv-induced demyelination, potentially by promoting myelin stripping after recognition of glycolipids and phospholipids exposed on damaged myelin. in this context, it is critical to note that jhmv infection is associated with extensive transient production of ifn-γ and its inducible genes, i.e., inos, and cxcr3 ligands, which may drive a more phagocytic pathway in microglia in efforts to remove damaged proteins and lipids (54) . further investigation is required to define inflammatory conditions under which trem-2 modulated or other phagocytic pathways promote tissue damage or repair and whether these are transient and reversible. microglia may also induce demyelination by secreting toxic factors including inflammatory cytokines that are highly expressed by microglia at time of demyelination, including tnf. tnf can induce olg death (63, 64) and is expressed in active ms lesions, as well as elevated tnf in serum and cerebral spinal fluid correlates with enhanced ms pathology (65, 66) . finally, microglia functions during jhmv infection were also associated with promoting adaptive immune response. an extensive network of chemokines and chemokines receptors relating to the recruitment and arrest of t and b cells within the inflamed cns were highly expressed by microglia. similarly, several genes associated with antigen processing and presentation by mhc class i and ii molecules were upregulated within microglia, suggesting that microglia promote t cell reactivation upon cns entry. however, microglia explanted during eae, tmev as well as jhmv infections failed to support myelin-specific cd4 t cell responses ex vivo, despite detection of internalized myelin (67) (68) (69) . a potential deficit in antigen processing was supported by the ability of exogenous peptide to overcome the inability of microglia to prime myelinspecific cd4 t cells (69) . nevertheless, this notion is opposed by our microglia profiling showing upregulation of genes involved in protein degradation and class ii peptide loading, e.g. cd74 (invariant chain), h2-dma (peptide loading), ctss (cathepsin s), which cleaves invariant chain. the apparent inability of microglia to elicit cd4 t cell effector function ex vivo thus remains intriguing. our present study reveals new insights into the plasticity of microglia in adapting to inflammation and expressing pathogenic functions associated with demyelination, characteristics which have previously been ascribed to bmdm (17) . moreover, altered bmdm expression profiles coincided with their early infiltration into the cns and remained largely similar throughout infection. while altered microglia gene expression coincided with the time of early, yet robust demyelination, it remains to be determined whether these changes are sustained at later time points during jhmv persistence, when clinical disease improves and remyelination occurs. it will be of specific interest to assess whether the microglia pro-inflammatory phenotype evolves to an anti-inflammatory, repair-promoting phenotype, as evidenced by the plasticity of myeloid cells in cns autoimmunity (70) . furthermore, our study emphasizes that the distinct tissue environments during eae and jhmv infection drive opposite effector functions of microglia versus infiltrating macrophages. the interplay between t cells, infiltrating macrophages and microglia, as well as astrocytes drives ms pathogenesis, yet mechanisms ultimately leading to loss of repair remain unclear. taking advantage of demyelinating models characterized by distinct inflammatory factors such as both th1 and th17 cells in eae (11) , strong th1 polarized responses during jhmv infection, distinct kinetics of bmdm recruitment versus glia activation promises to reveal essential new insights into the interplay of microglia and bmdm functions in debris clearance versus active myelin stripping and ongoing axonal damage. longitudinal studies will aid in developing efficient future therapies to combat ms pathogenesis. all procedures were performed in compliance with protocols approved by the cleveland clinic institutional animal care and use committee. author contributions cs designed and performed experiments, collected and interpreted the data, and wrote the manuscript. rd analyzed data and edited manuscript. cb designed the research, provided materials, interpreted the data, and wrote the manuscript. all authors approved the final manuscript. the authors would like to thank natasha towne for her exceptional technical assistance, as well as jennifer powers for performing facs purification. this work was supported by the national institutes of health grant ns091183 and national ms society research grant nmss rg4450. rd is supported by the national institutes of health grant ns096148. the supplementary material for this article can be found online at https://www.frontiersin.org/articles/10.3389/fimmu.2018.01325/ full#supplementary-material. figure s1 | validation of nanostring ncounter gene expression analysis by real-time pcr. to validate nanostring ncounter data, expression of il-15, il1rn, and arg1 was analyzed by q-pcr in naïve circulating monocytes and microglia, as well as in bone marrow-derived macrophage (bmdm) and microglia isolated from the spinal cord of jhmv-infected mice at days 5, 7, 10, and 14 p.i. levels of mrna expression are normalized to gapdh mrna levels. pathogenesis of tissue injury in ms lesions multiple sclerosis -the plaque and its pathogenesis janus-faced microglia: beneficial and detrimental consequences of microglial phagocytosis microglia: active sensor and versatile effector cells in the normal and pathologic brain role of microglia in cns autoimmunity microglial physiology: unique stimuli, specialized responses the benefits and detriments of macrophages/microglia in models of multiple sclerosis microglia as neuroprotective, immunocompetent cells of the cns non-identical twins -microglia and monocyte-derived macrophages in acute injury and autoimmune inflammation modeling the heterogeneity of multiple sclerosis in animals communication between pathogenic t cells and myeloid cells in neuroinflammatory disease using eae to better understand principles of immune function and autoimmune pathology the effects of macrophage depletion on the clinical and pathologic expression of experimental allergic encephalomyelitis suppression of experimental allergic encephalomyelitis in lewis rats after elimination of macrophages abnormalities in monocyte recruitment and cytokine expression in monocyte chemoattractant protein 1-deficient mice absence of monocyte chemoattractant protein 1 in mice leads to decreased local macrophage recruitment and antigen-specific t helper cell type 1 immune response in experimental autoimmune encephalomyelitis differential roles of microglia and monocytes in the inflamed central nervous system ccr2 regulates development of theiler's murine encephalomyelitis virus-induced demyelinating disease neuropathogenesis of theiler's murine encephalomyelitis virus infection, an animal model for multiple sclerosis anti-ccl2 treatment inhibits theiler's murine encephalomyelitis virus-induced demyelinating disease cd8 t cell mediated immunity to neurotropic mhv infection interleukin-12 (il-12), but not il-23, deficiency ameliorates viral encephalitis without affecting viral control cd4 t cells promote cd8 t cell immunity at the priming and effector site during viral encephalitis cd4 and cd8 t cells have redundant but not identical roles in virus-induced demyelination interleukin-10 is a critical regulator of white matter lesion containment following viral induced demyelination macrophage infiltration, but not apoptosis, is correlated with immune-mediated demyelination following murine infection with a neurotropic coronavirus perforin and gamma interferon-mediated control of coronavirus central frontiers in immunology | www nervous system infection by cd8 t cells in the absence of cd4 t cells ifn-gamma is required for viral clearance from central nervous system oligodendroglia maturation and localization of macrophages and microglia during infection with a neurotropic murine coronavirus depletion of blood-borne macrophages does not reduce demyelination in mice infected with a neurotropic coronavirus monocytes regulate t cell migration through the glia limitans during acute viral encephalitis pathogenicity of antigenic variants of murine coronavirus jhm selected with monoclonal antibodies inverted immunodominance and impaired cytolytic function of cd8+ t cells during viral persistence in the central nervous system direct multiplexed measurement of gene expression with color-coded probe pairs factors supporting intrathecal humoral responses following viral encephalomyelitis memory cd4+ t-cell-mediated protection from lethal coronavirus encephalomyelitis cd4 t cells contribute to virus control and pathology following central nervous system infection with neurotropic mouse hepatitis virus the fractalkine receptor but not ccr2 is present on microglia from embryonic development throughout adulthood trem2, microglia, and neurodegenerative diseases identification of a unique tgf-beta-dependent molecular and functional signature in microglia role for neuronally derived fractalkine in mediating interactions between neurons and cx3cr1-expressing microglia cathepsin s controls mhc class ii-mediated antigen presentation by epithelial cells in vivo cathepsin s causes inflammatory pain via biased agonism of par2 and trpv4 dusp1 is controlled by p53 during the cellular response to oxidative stress type i interferons are essential in controlling neurotropic coronavirus infection irrespective of functional cd8 t cells alpha/beta interferon (ifn-alpha/beta) signaling in astrocytes mediates protection against viral encephalomyelitis and regulates ifn-gamma-dependent responses chemokines and chemokine receptors in inflammation of the cns deficiency of tyrobp, an adapter protein for trem2 and cr3 receptors, is neuroprotective in a mouse model of early alzheimer's pathology the m1 and m2 paradigm of macrophage activation: time for reassessment a polarizing question: do m1 and m2 microglia exist heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination infiltrating monocytes trigger eae progression, but do not contribute to the resident microglia pool ccr2+ly-6chi monocytes are crucial for the effector phase of autoimmunity in the central nervous system coronavirus infection of the central nervous system: host-virus stand-off complement in multiple sclerosis: its role in disease and potential as a biomarker myelin-specific multiple sclerosis antibodies cause complement-dependent oligodendrocyte loss and demyelination the triggering receptor expressed on myeloid cells 2: a molecular link of neuroinflammation and neurodegenerative diseases identification of soluble trem-2 in the cerebrospinal fluid and its association with multiple sclerosis and cns inflammation blockade of trem-2 exacerbates experimental autoimmune encephalomyelitis trem2 regulates microglial cell activation in response to demyelination in vivo trem2 sustains microglial expansion during aging and response to demyelination glial response during cuprizone-induced de-and remyelination in the cns: lessons learned oligodendrocyte apoptosis and primary demyelination induced by local tnf/p55tnf receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy tumor necrosis factor-alpha mediates oligodendrocyte death and delayed retinal ganglion cell loss in a mouse model of glaucoma identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions multiple sclerosis: prospective analysis of tnf-alpha and 55 kda tnf receptor in csf and serum in correlation with clinical and mri activity dendritic cells permit immune invasion of the cns in an animal model of multiple sclerosis epitope spreading initiates in the cns in two mouse models of multiple sclerosis self-reactive cd4(+) t cells activated during viral-induced demyelination do not prevent clinical recovery myeloid cell plasticity in the evolution of central nervous system autoimmunity the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-275779-ocbygkyb authors: ye, zi-wei; yuan, shuofeng; poon, kwok-man; wen, lei; yang, dong; sun, zehua; li, cun; hu, meng; shuai, huiping; zhou, jie; zhang, mei-yun; zheng, bo-jian; chu, hin; yuen, kwok-yung title: antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin head region of pandemic h1n1 influenza virus play detrimental roles in h1n1-infected mice date: 2017-03-21 journal: front immunol doi: 10.3389/fimmu.2017.00317 sha: doc_id: 275779 cord_uid: ocbygkyb engaging the antibody-dependent cell-mediated cytotoxicity (adcc) for killing of virus-infected cells and secretion of antiviral cytokines and chemokines was incorporated as one of the important features in the design of universal influenza vaccines. however, investigation of the adcc epitopes on the highly immunogenic influenza hemagglutinin (ha) head region has been rarely reported. in this study, we determined the adcc and antiviral activities of two putative adcc epitopes, designated e1 and e2, on the ha head of a pandemic h1n1 influenza virus in vitro and in a lethal mouse model. our data demonstrated that sera from the e1-vaccinated mice could induce high adcc activities. importantly, the induction of adcc response modestly decreased viral load in the lungs of h1n1-infected mice. however, the elevated adcc significantly increased mouse alveolar damage and mortality than that of the pbs-vaccinated group (p < 0.0001). the phenotype was potentially due to an exaggerated inflammatory cell infiltration triggered by adcc, as an upregulated release of cytotoxic granules (perforin) was observed in the lung tissue of e1-vaccinated mice after h1n1 influenza virus challenge. overall, our data suggested that adcc elicited by certain domains of ha head region might have a detrimental rather than protective effect during influenza virus infection. thus, future design of universal influenza vaccine shall strike a balance between the induction of protective immunity and potential side effects of adcc. introduction influenza viruses, as one of the major zoonotic pathogens, have continuously caused global health concerns due to their high propensity for unpredictable genetic mutation in major surface antigens, hemagglutinin (ha), and neuramindase. antivirals and vaccines are vital in combating the threat of influenza epidemics and pandemics. however, the increasing usage of licensed antivirals has resulted in the global emergence of amantadine-and/or oseltamivir(1) . on the other hand, seasonal influenza vaccines have to be updated annually due to the continuous antigenic drift and shift (2) . otherwise, the mismatch between vaccinated formulations and that of natural selection would considerably limit the effectiveness of influenza vaccines. neutralizing antibodies have traditionally been thought to provide protection against influenza viruses. nevertheless, the effectiveness induced by such vaccines is limited by the emergence of mutant viruses that are resistant to antibodymediated neutralization (3) . in this regard, the quest for universal influenza vaccines has fueled the interest in broadly reactive antibodies in addition to direct virus neutralizations. antibody-dependent cell-mediated cytotoxicity (adcc) uses effector arms of both innate and adaptive immune responses to eliminate target cells. natural killer (nk) cells, upon triggered by specific adcc antibodies, mediate the clearance of virusinfected cells (4) . the nk cell cd16 receptor recognizes the fc portion of igg1 antibodies that in turn bind to antigens on virus-infected cells (5) . this interaction induces degranulation of nk cells to release perforin/granzymes as well as secretion of antiviral cytokines such as interferon-γ (ifn-γ) and tumor necrosis factor-α (tnf-α) (5) . since adcc could invoke protective immune response against infections from a broad array of viruses, the adcc antibody response was incorporated as one of the most important features of potential universal vaccine candidates by the world health organization. notably, multiple components of influenza viruses are known to induce adcc, including the viral surface proteins such as ha (6) and m2 ectodomain (7, 8) , as well as the internal proteins including np and m1 (9) . the glycoprotein ha consists of two functional domains, the immunodominant globular head domain and the more conserved stalk domain (10) . conventionally, neutralizing antibody response to influenza virus is dominated by antibodies that target the head region, which block the virus receptor-binding site. compared with the bulky globular ha head, the ha stem region is far less immunogenic, and antibodies directed to this domain occur at a relatively low frequency. however, a rare class of neutralizing antibodies that target a conserved site in the ha stem was reported recently, which might shed new light for the development of universal influenza vaccines (6) . we have previously identified two putative adcc epitopes that mapped to the ha head of a pandemic h1n1 influenza virus (11) . both epitopes, designated e1 and e2, revealed by epitope mapping of convalescent-phase plasma igg antibodies from six h1n1-infected human subjects, are dominant and highly conserved (11) . in this study, we further dissected the function of the two adcc epitopes in vitro and in a lethal mouse model. surprisingly, our results demonstrated that the adcc response elicited by the e1 epitope triggered a detrimental rather than protective effect against influenza virus infection. while the antiviral efficacy provided by the stalk-specific adcc antibodies has been confirmed (12) , our data raised concerns on the side effect of certain ha head epitopes in devising a universal influenza vaccine. in this regard, our study suggested that a delicate balance between protective immunity and over induction of adcc should be maintained, which should be an important consideration in evaluating vaccine safety. the la4 cell line, which was derived from mouse lung adenoma, was maintained in dmem/f-12 medium (gibco) supplemented with 20% heat-inactivated fetal bovine serum (fbs), 50 u/ml penicillin, and 50 μg/ml streptomycin (p/s). peripheral blood mononuclear cells (pbmcs) were prepared by ficoll-paque separation (13) of heparinized whole blood obtained from healthy balb/c mice (6-8 weeks old). to prepare the adcc target cells, la4 cells were transfected with an ha expression plasmid that based on the cdna fragment of influenza virus strain a/hong kong/415742/2009(h1n1)pdm09. specifically, the full-length ha fragment was cloned into a mammalian expression vector peak10 plasmid containing a mouse igg1 fc gene (ch2 + ch3) (14) . the pandemic h1n1 influenza virus strain a/hong kong/415742/2009(h1n1)pdm09 was used for in vitro virus infection; while its mouse-adapted version, a/hong kong/415742md/2009 (h1n1)pdm09 was propagated in embryonated hens' eggs and utilized for in vivo experiment (15) . the viruses were stored in −80°c in aliquot and titrated by standard plaque assay. all experiments with live viruses were conducted using biosafety level 2 facilities as described previously (16) . balb/c female mice, 6-8 weeks old, were kept in biosafety level 2 housing and given access to standard pellet feed and water ad libitum. all experimental protocols followed the standard operating procedures of the biosafety level 2 animal facilities and were approved by the animal ethics committee in the university of hong kong (17) . vaccinations were carried out to immunize the mice with e1 or e2 or ha epitopes by dna priming and protein boost. pbs was used as a negative control. the specified vaccination scheme was listed in table 1 . to prepare the dna plasmids, either of the e1 or e2 fragment (11) was cloned into the mammalian expression vector peak10 as described for the ha plasmid construction. priming of the mice by electroporation. to prepare protein for vaccination, recombinant ha, e1, and e2 fusion proteins were expressed in freestyle 293ft™ transient expression system (invitrogen) and purified by protein a affinity (ge healthcare). subsequently, proteins were dialyzed and concentrated with vivaspin 20 centrifugal concentrator (ge healthcare), followed by protein boosting through intramuscular route. each mouse received 25 μg protein at each protein boosting. sera were obtained at day 68 postimmununization before virus challenge. antibody titers raised against e1, e2, and ha in mouse sera were evaluated by elisa as previously described with some modifications (18) . mouse sera collected from the pbs-treated group were taken as a background control. immunized mice (15 mice/group) were inoculated with five 50% lethal dose (ld50) of mouse-adapted pandemic h1n1 influenza virus by intranasal route, i.e., 1,000 pfu/mouse. animal survival and body weight were monitored daily for 14 days after virus challenge. a body weight loss of 30% was set as the humane endpoint. three mice per group were randomly selected and euthanized on day 3 and 5 post-challenge, respectively. mouse lungs were collected from the euthanized mice. half of the lung tissues were harvested for virus titration by rt-qpcr methods (19) , while the other half were immediately fixed in 10% of pbs buffered formaldehyde for histopathological analyses as described previously (20) . slides were examined in a blinded manner and scored with a semiquantitative system according to the relative degree of inflammation and tissue damage (21) (22) (23) (24) . inflammation was scored as follows: 0, no inflammation; 1, perivascular cuff of inflammatory cells; 2, mild inflammation (extending throughout 25% of the lung); 3, moderate inflammation (25-50% of the lung); 4, severe inflammation involving over one half of the lung. antibody-dependent cell-mediated cytotoxicity activity, reflected by the rate of cell death, was measured by a flow cytometry-based assay that described previously with some modifications (11) . generally, the pkh-67 dye (sigma) was utilized to label the target cells, i.e., ha-transfected la-4 cells; while 7-aminoactinomycin d (7-aad; invitrogen) was used to stain the dead cells that mediated by adcc activity. experimentally, pkh-67-labeled target cells and unlabeled effector cells (i.e., pbmcs) were prepared in rpmi 1640 medium (gibco) containing 10% fbs and 1% p/s with a cell density of 10 6 and 10 8 cells/ml, respectively. subsequently, 50 μl of target cells were dispensed into a round-bottom 96-well plate, followed by addition of 1 μl of mouse serum. mouse serum concentration in each group was normalized before addition according to their titers determined by elisa. one hour after incubation, 50 μl of effector cells were added to incubate with the target cells. three hours later, another 1 μl of 7-aad was added to each well before performing the flow cytometry. cell death was determined with a facsaria iii flow cytometer using the bd facs software (bd biosciences). percent cell death was calculated by software analysis of four identifiable cell populations: live effector cells (no dye), dead effector cells (7-aad positive), live target cells (pkh-67 positive), and dead target cells (pkh-67 and 7-aad double positive). assay controls used to define cell populations included target cells alone (background cell death) and target cells with 1 μl triton x-100 added (maximum cell death). percent adcc was calculated as [(percent experimental lysis − percent spontaneous lysis)/(percent maximum lysis − percent spontaneous lysis)] × 100%, in which percent spontaneous lysis refers to the percent lysis of infected cells with effectors but in the absence of serum, while percent maximum lysis refers to the percent lysis of infected cells with effectors in the presence of 1% triton x-100. experiments were performed in triplicate and repeated twice for confirmation. immunostaining was performed as previously described to visualize perforin expression in mouse lung tissues (25) . rat anti-mouse perforin (abcam ab16074) and goat anti-rat alexa 594 were used as primary and secondary antibodies, respectively. images were acquired with a carl zeiss lsm 780 system. table s1 in supplementary material. statistical comparison was performed by student's t-test using graphpad prism 6. differences were considered statistically significant when p < 0.05. results adcc responses are enhanced by the sera of e1-vaccinated mice in our previous study, we mapped two putative adcc epitopes, e1 and e2, on the ha head region. by depleting e1 and/or e2 from clinical plasma igg antibodies, the adcc activity dropped significantly, which suggested that the two epitopes played potential roles in eliciting adcc (11) . in this study, we sought to confirm the function of these putative epitopes in the induction of adcc activity using a mouse model. immunization of mice gave raise to igg titers against the e1, e2, or ha protein, as quantified by elisa (figure 1) . a prime/boost immunization strategy was adopted, and mice that immunized with pbs or ha were included as a negative or a positive control, respectively ( table 1 ). our results indicated that serum samples from mice vaccinated with e1 ( figure 1a ) or e2 ( figure 1b) both exhibited strong dilution-dependent antibody responses, reaching a titer of more than 1:5000. additionally, using ha as the coating antigen for elisa, we demonstrated that e1 and e2 sera could bind fulllength ha at a comparable efficiency ( figure s1 in supplementary material). taken together, our data suggested that the vaccination was successful and the resultant serum samples could be utilized for further investigations. next, adcc activities in serum samples from e1-, e2-, or ha-immunized mice were evaluated by flow cytometry-based adcc assays. to this end, the ha-transfected la4 cells were labeled with the cell-membrane marker pkh67 and utilized as target cells for adcc-specific antibody binding. subsequently, the vaccinated mouse serum was added to bridge the interaction between target cells and pbmc effector cells (figure 2) . the presence of adcc-mediating antibody was determined by analyzing the cytotoxicity rate within the cell mixture that contained the target cells, serum, and effector cells, in which the dead target cell population was revealed by the cell death marker, 7aad (figure 3) . as shown in figures 3a-g , sera from the e1-vaccinated mice consistently triggered the highest 7aad + rate among all evaluated groups, suggesting that a higher percentage of cell lysis was induced in the e1 group in comparison to the other groups. the percentage of cytotoxicity was normalized using the formula reported by srivastava et al., with spontaneous and maximum cell cytotoxicity rate taken into consideration (11) . quantitatively, sera from the e1-vaccinated mice elicited a significantly increased (p < 0.05) adcc activity in comparison with the pbs-vaccinated group (figure 3h) . of note, though sera from the e2-vaccinated mice triggered a subtle increase in adcc activity, the difference was not statistically significant ( figure 3h) . intriguingly, albeit ha contained the e1 epitope, sera from the ha-vaccinated group did not induce a significantly elevated cytotoxicity response in comparison to that of the pbs control group (figure 3h ). since e1 was capable of inducing adcc activities, we hypothesized that e1-vaccinated mice could potentially be protected by the elicited adcc activity after influenza virus challenge. to this end, we inoculated the vaccinated mice with pandemic h1n1 influenza virus in a lethal mouse model ( figure 4a) . as shown in figure 4b , mice in the ha-vaccinated group, as a positive control, demonstrated a substantial reduction of viral load on both day 3 and day 5 post-inoculation in comparison to the pbs-vaccinated group. importantly, we detected an approximately one log decrease of viral load in the mouse lungs of the e1-vaccinated group in comparison to that of the pbs-vaccinated group on day 5 post-inoculation, while no significant difference could be observed between the two groups on day 3 post-virus challenge (figure 4b ). in addition, the viral load in the lungs of the e1-vaccinated mice was notably lower on day 5 when compared with that of day 3, suggesting that the adcc effect was triggered between day 3 and 5 postinoculation ( figure 4b) . figure 2 | schematic diagram of the antibody-dependent cell-mediated cytotoxicity (adcc) assay. adcc activity was determined by a flow cytometrybased assay using two fluorescent dyes. pkh-67, a membrane-labeling dye, was used to specifically identify the ha-transfected target cells. 7aad was excluded by viable cells but could penetrate the cell membrane of dead or dying cells and intercalate into double-stranded dna. briefly, 50 μl of pkh-67-labeled target cells (10 6 cells/ml) was dispensed into a round-bottom 96-well plate, followed by addition of e1/e2/ha/pbs sera and effector/pbmc cells. following a 3-h incubation, 7-aad was added. cell death was determined on a facsaria iii flow cytometer using bd facs diva software (bd biosciences). percent cell death was analyzed by the flowjo software. in parallel, we measured the survival rate and body weight changes of the mice. as shown in figure 4c , all mice from the ha-vaccinated group survived the course of infection while all mice received pbs-treatment died, indicating that the virus challenge was successful. unexpectedly, our results demonstrated that the mice in the e1-vaccinated group succumbed to influenza virus infection at a significantly earlier time (p < 0.0001) postinoculation when compared with that of the pbs-vaccinated control group (figure 4c ). in line with the survival rate, mice from the e1-vaccinated group suffered from an apparently accelerated weight lost starting on day 3 post-inoculation in comparison to mice from the pbs-and e2-vaccinated groups, although the difference did not reach statistical significance ( figure 4d) . next, we carried out histopathological examinations on the lung sections of the virus-infected mice. using uninfected mouse lung tissues as a control (figures 5i,j) , our observation showed that on both day 3 and day 5, mice from the ha-vaccinated group (figures 5g,h) exhibited ameliorated alveolar morphology changes when compared with those from the e1 (figures 5c,d) , e2 (figures 5e,f) , and pbs (figures 5a,b) groups. importantly, the lung pathological scores of mice from the ha-vaccinated group on both day 3 and day 5 were significantly lower than those of the pbs-treated mice (figures 5k,l) ; while mice from the e1-vaccinated group suffered from a significantly more dramatic interstitial inflammatory infiltration than that of the pbs-treated mice on day 5 ( figure 5l ). this result indicated that the detrimental lung damage of e1-vaccinated mice, possibly triggered by adcc, might account for the reduced viral load in lungs as well as the earlier drop in survival. to address whether the severe lung damage in the e1-vaccinated group could be attributed to the adcc effect, we performed immunofluorescence staining to visualize the expression level of perforin among different mouse groups. perforin is released by activated nk cells and is known as a marker of adcc activation (28) . as quantitated in figure 6m , the e1-vaccinated mice (figures 6d-f) demonstrated the highest perforin expression level in the lung sections amongst the other three groups on day 5 post infection (figures 6a-c,g-l) . however, the mrna expression level of perforin was not significantly different across all evaluated groups ( figure 6n) . binding of fc receptor (fcr) on effector cells triggers the secretion of cytotoxic granules as well as antiviral cytokines and chemokines, such as ifn-γ and tnf-α (4). to investigate if elevated expression of proinflammatory cytokines might contribute to the lung damage, we measured the mrna expression level of representative cytokines including tnf-α (figure 7a) , il-1β (figure 7b) , and ifn-γ (figure 7c ) in the mouse lungs samples. our results showed that the gene expression of all three proinflammatory cytokines were significantly enhanced in the e1-vaccinated group when compared with those of the pbstreated group (figure 7) , which were in line with the perforin protein expression pattern in figure 6 . together, our data suggested that the e1 epitope from the ha head domain mediated unfavorable adcc that resulted in a more severe lung damage and exacerbated mouse mortality despite a successful control of the h1n1 influenza viral load. antibody-dependent cell-mediated cytotoxicity, as a bridge of the innate and adaptive immunity, plays important roles in humoral and cellular immune response (4, 9) . since adcc antibodies are known to recognize a wide range of viral proteins that lead to the lysis of virus-infected cells, a better understanding on the adcc mechanism during influenza virus infections will facilitate the development of universal influenza vaccines with broader protections (4, 9, 29) . the conserved viral proteins or domains, such as m2 extracellular domain and ha stem domain, have been widely studied as potential targets of domain-based universal influenza vaccines. jegerlehner and colleagues have demonstrated that the protective role of m2 adcc-mediating antibodies was dependent on fcr (7, 8) . dilillo et al. provided further support that the influenza-specific adcc antibody, though elicited by the ha stem, also required fcrs interaction for protection against lethal influenza infection (6) . collectively, both studies highlighted the functional importance of fcr during adcc-mediated virus clearance. on the other side, unexpected cases have been reported that young adults without prior exposure to the 1968 h3n2 virus produced robust adcc-mediating antibody response upon infection of this virus strain. some individuals even possessed cross-reactive adcc-mediating antibodies toward avian h5n1 and h7n7 strains in the absence of prior exposure (30) . however, the mechanism of such antibody responses remains unclear to date. in this study, we investigated the adcc effect of the two putative ha head epitopes in vitro and in vivo. our data demonstrated that e1-induced adcc activity against h1n1 influenza virus infection in vitro (figure 3) . unexpectedly, although e1 vaccination decreased the viral load in h1n1-infected mice (figure 4b) , it induced exacerbated lung damage ( figure 5 ) and a higher level of nk activity (figure 6 ) that accelerated mouse death ( figure 4c ). nk cells, which offer the first line of defense against virus infection, have been widely considered to be beneficial to the host during viral infections. however, a recent report by zhou et al. revealed that adoptive transfer of nk cells from influenza virus-infected lungs, but not uninfected lung, resulted in a more rapid weight loss and increased mortality of virus-infected mice (31) . this finding was in line with our observation that e1-induced adcc exhibited deleterious impact to promote mortality during influenza virus infection. most healthy donors have a persistently low level of crossreactive adcc-mediating antibodies, while these cross-reactive antibodies are found in individuals in the absence of detectable neutralization (4, 9) . in our previous study, both e1 and e2 epitopes were identified as putative regions that could induce adcc activity. the depletion of such antibodies in human plasma significantly decreased the adcc effect. however, for certain samples, it appeared that more diluted plasma exhibited higher adcc activity than less diluted plasma, and the use of igg antibodies at a low concentration led to a higher adcc activity than the use of igg antibodies at a high concentration (11) . to date, there is no conclusive study on the correlation between antibody concentration and adcc activity, neither was the optimal concentration of adcc antibodies that could protect against virus infection elucidated. in this context, we demonstrated here that an overwhelming production of adcc antibodies in the absence of neutralization might not play a protective role against influenza virus infection. indeed, multiple factors such as saturation of antibodies or interference from non-adcc antibodies may contribute to the induction of adcc (4, 11) . in this case, the threshold level of protective adcc-mediating antibodies should be investigated in further studies. various adcc assays that mainly differ in the choice of effector cells and measurement of adcc activity have been reported (4, 9) . for example, some studies used ha-transfected or virus-infected a549 cells as target cells, which were susceptible results are presented as bar charts with mean values ± sd. differences between groups were compared using the t test. *p < 0.05 and **p < 0.01 as compared to the pbs-immunized group. survival rate (c) and body weight (d) of the mice (9 mice per group) vaccinated with pbs (red), e1 (blue), e2 (green), and ha (purple) were monitored for 14 days. the body weight values are shown as means ± sd for the mice that were alive at each time point (***p < 0.0001). to nk cell-mediated adcc after incubating with the sera from healthy donors or clinical blood samples (6, 32, 33) . in our case, we isolated pbmcs from healthy mice as effector cells and measured the death rate of target cells in the presence of vaccinated mouse sera (figure 2) . at the same time, utilization of flow cytometry for quantitation of cell cytotoxicity provided an efficient and precise way to assess the adcc responses (figure 3) . importantly, the h1n1-infected la4 cells showed a low background of cell death in the absence of antibodies (figure 3c) , which suggested la4 as an ideal cell line for the mouse-specific adcc assay. collectively, the established in vitro adcc assay, together with the balc/c mouse model, might be applied for the evaluation of other influenza-specific adcc epitopes. experimental mouse models are an invaluable scientific resource that allow comprehensive investigation of key biological questions in vivo and provide an essential platform in the study of many human diseases. it has been widely acknowledged that the mouse and human antibody repertoire share a general similarity (34) (35) (36) . however, differences in germline antibody repertoire exist between species, and the number of mature naïve b cells from mice is smaller than that from humans (37) . both variations may contribute to the dissimilarities in the antibodies elicited by the e1-containing fragments in humans compared to those in mice. due to the diversity of the b cell antigen receptor repertoire between the mouse and human model, the antibodies bind to the same fragments in distinct host models might potentially have slightly different epitopes. alternatively, there may be fundamental differences between murine and human in terms of the regulation in nk cell cytotoxic granule secretion (38) . surprisingly, distinct expression patterns of perforin were detected between protein ( figure 6m ) and mrna (figure 6n ) levels. the discrepancy might be explained by a previous finding that resting murine nk cells are "pre-armed" with high amounts of perforin mrna, which can be rapidly translated into protein upon activation in vitro and in vivo (38). this mechanism of murine nk cells facilitates a better control of perforin expression, allowing a rapid production of effector proteins without the need of de novo gene transcription (38). upon activation, adcc effector cells produce various cytokines such as tnf-α, il-1β, and ifn-γ. further, cytokines may represent one of the arming pathways that stimulate the translation of perforin (38). notably, human nk cell is minimally cytotoxic at rest, expresses little perforin protein, and becomes potently cytotoxic only after cytokine activation (39, 40) . our result showed that tnfα, il-1β, and ifn-γ were significantly elevated in e1-vaccinated group when compared with those of the pbs-treated group (figure 7) . in this regard, the upregulation of these cytokines may activate the cytotoxic perforin to cause the detrimental damage in mouse lungs. in our previous study, e1 and e2 epitopes were located on the ha head ( figure s2 in supplementary material), both of which are conserved in h1n1 strains from 2009 (2009_h1n1) (11) . to date, however, the role of ha head during influenza virus infection and adcc activation has not been fully delineated. in a recent study, dilillo and colleagues reported that neutralizing antibodies targeting the ha stem but not the ha head were capable of conferring influenza-specific adcc (6) . they proposed that only the anti-stem antibodies could bind in a correct conformation to ligate fcrs, which was based on the observation that a strain-specific anti-ha head antibody (py102) was unable to mediate fcγr binding and to protect mice. on the other hand, ha head-induced adcc activities were reported by a number of other groups (41) (42) (43) , which was in agreement with our findings. the discrepancy between these observations might be due to the sequential and structural variations among subtypes/strains of influenza virus used. interestingly, although both e1 and e2 epitopes are located on the ha head, serum from the ha-vaccinated group did not trigger a significantly elevated cytotoxicity response (figure 3h) , implying that additional regulators exist to control the adcc activity. in summary, we provided in vitro and in vivo evidence to verify the effect of the ha head epitope e1-mediated adcc. importantly, our data suggested that e1-mediated adcc alone caused detrimental effect during influenza virus infection, which raised concerns on using this conserved non-neutralizing region of the ha head in future designs of a universal influenza vaccine. in fact, the "headless ha" has been recommended in several vaccine designs that aimed to make use of adcc antibodies (44) (45) (46) (47) . our data provided further evidence in support of this "headless ha" vaccine design strategy. emerging influenza antiviral resistance threats influenza vaccine -outmaneuvering antigenic shift and drift variable influenza vaccine effectiveness by subtype: a systematic review and meta-analysis of test-negative design studies influenza-specific antibody-dependent cellular cytotoxicity: toward a universal influenza vaccine activation of nk cells by adcc responses during early hiv infection broadly neutralizing hemagglutinin stalk-specific antibodies require fcγr interactions for protection against influenza virus in vivo influenza a vaccine based on the extracellular domain of m2: weak protection mediated via antibody-dependent nk cell activity a human anti-m2 antibody mediates antibody-dependent cell-mediated cytotoxicity (adcc) and cytokine secretion by resting and cytokine-preactivated natural killer (nk) cells what lies beneath: antibody dependent natural killer cell activation by antibodies to internal influenza virus proteins enabling the 'host jump': structural determinants of receptor-binding specificity in influenza a viruses identification of dominant antibody-dependent cell-mediated cytotoxicity epitopes on the hemagglutinin antigen of pandemic h1n1 influenza virus epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza a virus middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways crossreactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody that recognizes a novel conformational epitope on gp41 and lacks reactivity against self-antigens d225g mutation in hemagglutinin of pandemic influenza h1n1 (2009) virus enhances virulence in mice delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza a/h5n1 virus a novel small-molecule compound disrupts influenza a virus pb2 cap-binding and inhibits viral replication identification of a novel small-molecule compound targeting the influenza a virus polymerase pb1-pb2 interface cross-protection of influenza a virus infection by a dna aptamer targeting the pa endonuclease domain peptide-mediated interference of pb2-eif4g1 interaction inhibits influenza a viruses' replication in vitro and in vivo severity of pneumonia due to new h1n1 influenza virus in ferrets is intermediate between that due to seasonal h1n1 virus and highly pathogenic avian influenza h5n1 virus age-related sensitivity and pathological differences in infections by 2009 pandemic influenza a (h1n1) virus critical role of il-17ra in immunopathology of influenza infection a critical role of il-17 in modulating the b-cell response during h5n1 influenza virus infection carcinoembryonic antigen-related cell adhesion molecule 5 is an important surface attachment factor that facilitates entry of middle east respiratory syndrome coronavirus active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis productive replication of middle east respiratory syndrome coronavirus in monocyte-derived dendritic cells modulates innate immune response nk cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy universal flu vaccine": can nk cell-mediated adcc tip the scales? cross-reactive influenza-specific antibody-dependent cellular cytotoxicity in intravenous immunoglobulin as a potential therapeutic against emerging influenza viruses nk cells exacerbate the pathology of influenza virus infection in mice protection against h5n1 influenza virus induced by matrix-m adjuvanted seasonal virosomal vaccine in mice requires both antibodies and t cells human seasonal influenza a viruses induce h7n9-cross-reactive antibodydependent cellular cytotoxicity (adcc) antibodies that are directed towards the nucleoprotein similarity and divergence in the development and expression of the mouse and human antibody repertoires of mice and not men: differences between mouse and human immunology transitional b cells: how well are the checkpoints for specificity understood? differences in mouse and human nonmemory b cell pools acquisition of murine nk cell cytotoxicity requires the translation of a pre-existing pool of granzyme b and perforin mrnas the biology of human natural killer-cell subsets differential expression of human granzymes a, b, and k in natural killer cells and during cd8+ t cell differentiation in peripheral blood influenza virus a(h1n1)2009 antibody-dependent cellular cytotoxicity in young children prior to the h1n1 pandemic antibody-dependent cell-mediated cytotoxicity to hemagglutinin of influenza a viruses after influenza vaccination in humans cross-reactive influenza-specific antibody-dependent cellular cytotoxicity antibodies in the absence of neutralizing antibodies the quest for a universal flu vaccine: headless ha 2.0 preparation of influenza virus subviral particles lacking the ha1 subunit of hemagglutinin: unmasking of cross-reactive ha2 determinants vaccination with soluble headless hemagglutinin protects mice from challenge with divergent influenza viruses stalking influenza by vaccination with pre-fusion headless ha ministem key: cord-268483-joiajgs4 authors: shah, vibhuti kumar; firmal, priyanka; alam, aftab; ganguly, dipyaman; chattopadhyay, samit title: overview of immune response during sars-cov-2 infection: lessons from the past date: 2020-08-07 journal: front immunol doi: 10.3389/fimmu.2020.01949 sha: doc_id: 268483 cord_uid: joiajgs4 after the 1918 flu pandemic, the world is again facing a similar situation. however, the advancement in medical science has made it possible to identify that the novel infectious agent is from the coronavirus family. rapid genome sequencing by various groups helped in identifying the structure and function of the virus, its immunogenicity in diverse populations, and potential preventive measures. coronavirus attacks the respiratory system, causing pneumonia and lymphopenia in infected individuals. viral components like spike and nucleocapsid proteins trigger an immune response in the host to eliminate the virus. these viral antigens can be either recognized by the b cells or presented by mhc complexes to the t cells, resulting in antibody production, increased cytokine secretion, and cytolytic activity in the acute phase of infection. genetic polymorphism in mhc enables it to present some of the t cell epitopes very well over the other mhc alleles. the association of mhc alleles and its downregulated expression has been correlated with disease severity against influenza and coronaviruses. studies have reported that infected individuals can, after recovery, induce strong protective responses by generating a memory t-cell pool against sars-cov and mers-cov. these memory t cells were not persistent in the long term and, upon reactivation, caused local damage due to cross-reactivity. so far, the reports suggest that sars-cov-2, which is highly contagious, shows related symptoms in three different stages and develops an exhaustive t-cell pool at higher loads of viral infection. as there are no specific treatments available for this novel coronavirus, numerous small molecular drugs that are being used for the treatment of diseases like sars, mers, hiv, ebola, malaria, and tuberculosis are being given to covid-19 patients, and clinical trials for many such drugs have already begun. a classical immunotherapy of convalescent plasma transfusion from recovered patients has also been initiated for the neutralization of viremia in terminally ill covid-19 patients. due to the limitations of plasma transfusion, researchers are now focusing on developing neutralizing antibodies against virus particles along with immuno-modulation of cytokines like il-6, type i interferons (ifns), and tnf-α that could help in combating the infection. this review highlights the similarities of the coronaviruses that caused sars and mers to the novel sars-cov-2 in relation to their pathogenicity and immunogenicity and also focuses on various treatment strategies that could be employed for curing covid-19. the whole world is currently confronting a crisis situation that first appeared in late december 2019 as merely a few cases of pneumonia in wuhan, china. the patients were exhibiting common symptoms like fever, dry cough, sore throat, breathlessness, and fatigue. sample swabs from the oral cavity and anal region were collected along with the blood and bronchoalveolar lavage fluid (balf) from all seven of the patients, irrespective of their age and gender, which were then sent to the wuhan institute of virology for further examination. as the outbreak initiated at the seafood market with the onset of winter, similar to that of the previous severe acute respiratory syndrome (sars) infection, the scientists first screened the samples using pan-cov qpcr primers. surprisingly, five samples were reported positive for coronavirus. thorough investigation employing next-generation sequencing and phylogenetic analysis led to the identification of the causative agent of this respiratory disease, a novel coronavirus (2019-ncov) (1) . as more cases started to appear around the world, on february 11, 2020, the world health organization assigned a name, corona virus disease 2019 or covid-19, to the disease and declared it a pandemic on march 11, 2020. the virus was renamed from 2019-ncov to sars-cov-2 by the international committee on taxonomy of viruses on the basis of its genetic similarity to a previously known coronavirus, severe acute respiratory syndrome coronavirus (sars-cov) (2) . transmission of sars-cov-2 occurs when a healthy individual inhales or comes into contact with respiratory droplets from an infected person. the average incubation period before patients exhibit disease symptoms ranges from 2 to 14 days (3) . before the spread of covid-19, sars emerged as an epidemic in 2003, followed by middle east respiratory syndrome (mers) in 2012, both caused by a novel coronavirus of zoonotic origin and assigned to the genus betacoronavirus (4) . the worldwide outbreak of sars-cov-2 has put life on hold, having a major impact on the world's economy, and has claimed ∼436,167 lives globally as of june 15, 2020 (5, 6) . unlike previous episodes of coronavirus spread, where it took months to identify the cause of infection and perform genome sequencing (7) , advancement in science and technology made it possible to identify the causative organism swiftly. within a few weeks of the outbreak, different laboratories across the world had sequenced the whole viral genome and had also provided structural and functional insights into the essential proteins required by the virus for its survival. these immediate scientific inputs helped with developing diagnostic kits and defining treatment strategies for effective prognosis and prevention (8) (9) (10) . in this review, we are emphasizing the immunological aspect of sars-cov-2 pathogenesis by taking into consideration the previous experimental and clinical knowledge obtained from the coronaviruses that were responsible for causing sars and mers. this approach will assist in utilizing immunotherapies, repurposing the previously approved antiviral drugs, and developing therapeutic vaccines specific to novel coronavirus more effectively. initial genome sequencing and phylogenetic analysis of novel coronavirus sars-cov-2 has shown that it is genetically similar to previously known coronavirus sars-cov and hence is placed under the family coronaviridae. coronavirus contains positivesense single-stranded rna (+ve ssrna) as its genetic material, which can be about 30 kb in length and is mostly protected by an outer fatty layer of an envelope that also helps the virus to evade host immune response and assists its entry inside the host cell (11, 12) . the subfamily coronavirinae is further subdivided into four genera, namely alpha-, beta-, gamma-, and delta-coronavirus (α-cov, β-cov, γ-cov, and δ-cov). viruses having the potential to infect humans are placed under the genus α-cov and β-cov (sars-cov & mers-cov), whereas viruses of γ-cov and δ-cov genera are mostly known to infect avians and pigs (13) . the novel coronavirus, sars-cov-2 falls under the genus β-cov, as it shares 88% sequence identity with sars-cov-like coronaviruses (derived from bat) but is only 79% identical to sars-cov and 50% identical to mers-cov (3) . thus, it can be deduced by its genome identity that the immediate host of this virus could be a bat, which then transmitted it to some unknown intermediate host that acted as a source for the transmission of the virus to humans. like those of sars-cov and mers-cov, the sars-cov-2 genome comprises of 12 open reading frames (orfs) in number. at the 5 ′ end of the viral genome, overlapping orfs 1a and 1b are present that encode the rna polymerase and other nonstructural proteins of the virus and occupy approximately twothirds of the genome. genes encoding structural proteins such as spike (s), membrane (m), envelope (e), and nucleocapsid (n), are present in the remaining one-third of its genome spanning from the 5 ′ to the 3 ′ terminal, along with several genes encoding non-structural proteins (nsps) and accessory proteins scattered in between, as shown in figure 1 . despite being in the same serogroup, there is a slight difference in the nucleotide number, sequence, gene order, and expression method among previously known coronaviruses and the novel sars-cov-2 (1, 14, 15) . recent reports highlight that a few amino acid substitutions have occurred in the novel coronavirus genes encoding the s protein, nsp2, nsp3, and receptor-binding domain (rbd). these mutations in the nsp2 & nsp3 are also believed to impart the enhanced infection abilities of the novel coronavirus (16, 17) . rna viruses are prone to acquiring genetic mutations that eventually help them to escape the host immune system and develop drug resistance. researchers have also found minor mutations in sars-cov-2 genotype in different covid-19 patients (18) . one such hotspot of mutation in the sars-cov-2 genome is the rna-dependent rna polymerase gene. on analyzing 220 sequences across the globe, eight repetitive novel point mutations were observed. viral genetic sequences accessed from europe exhibited five mutation hotspots, whereas the remaining three point mutations were solely present in the sequences from north america. these unique mutations suggest that the viral strains are continuously evolving across the globe figure 1 | schematic representation of the coronavirus structure and genomic comparison of coronaviruses. (a) representation of coronavirus showing different components of the particle, which is 100-160 nm in diameter. the single-stranded rna (ssrna) genome, covered with the envelope and membrane proteins, gains access into the host cell and hijacks the replication machinery. (b) the ssrna of sars-cov-2 is about 30 kb and has similarities with the genomes of sars-cov and mers-cov. translation of this ssrna results in the formation of two polyproteins, namely pp1a and pp1ab, that are further sliced to generate numerous non-structural proteins (nsps). the remaining orfs encode for various structural and accessory proteins that help in assembly of the viral particle and evading immune response. and that the strains from europe, north america, and asia might have co-existed the whole time (19) . another similar report analyzed 7,666 global viral genomic sequences and found 198 unique mutation sites on sars-cov-2 genome that encodes nsps and s protein, suggesting that the virus is trying to adapt to its new host (20) . as numerous drugs are currently being designed to target the proteins that are essential for the survival of the virus, rapid genetic mutation occurring in these proteins might not prove to be a potential candidate for drug design. therefore, the invariable region of the virus could be a better target to avoid drug failures. interestingly, sars-cov-2, similar to sars-cov, exploits the angiotensin-converting enzyme 2 (ace2) receptor to gain access inside human cells, whereas mers-cov binds specifically to dipeptidyl peptidase 4 (dpp4) receptor (21, 22) . binding of the virus particle to the specific receptor on the host cell plays a key role in governing its pathogenicity. functional evaluation was carried out to reveal the potential receptors for different betacoronaviruses (β-cov) including sars-cov-2, and it was found out that the entry of the virus particle was enhanced in human cells expressing ace2 receptor instead of dpp4 or aminopeptidase n (apn) in the case of the novel coronavirus (23) . recent structural insights provided by cryo-em studies of s protein in prefusion conformation highlighted that the binding efficiency of ace2 and s protein of sars-cov-2 is 10-20 times greater than for the previously known sars-cov (24, 25) . the latest reports suggest that the trimeric s protein of sars-cov-2 is sliced by transmembrane protease serine 2 (tmprss2), similar to sars-cov (26, 27) . hence, profound knowledge of the potential receptors to which the virus particle can bind and its associated proteases will help us in designing specific antiviral drugs and neutralizing antibodies and will lead us to foresee whether particular coronaviruses of zoonotic origin could be able to adapt and infect humans. all coronaviruses initiate entry inside the target cell by engaging the host receptor with the s glycoprotein present on their surface so as to gain entry inside the target cell. the region of s protein containing the rbd is present on the s1 subunit. in a few coronaviruses, rbd is present at the n-terminus region of s1, whereas in sars-cov, it is situated at the c-terminus region (28, 29) . the fusogenic activity of virus-cell membrane is governed by two tandem domains, heptad repeats (hr1,2) that are present on the s2 region of s protein (30, 31) . initially, it was believed that sars-cov enters the target cell merely by virtue of cell membrane integration of virus particle and host cell membrane (32) . later, it was discovered that an essential proteolytic cleavage event takes place in the s protein at the s2 position of sars-cov that results in membrane fusion and facilitates virus entry inside the cell (33) . once the coronavirus is inside the host cell via membrane fusion, it releases its +ve ssrna genome into the cytoplasmic compartment, where the translation of orf-1a and orf-1b begins resulting in the formation of two large polyproteins (pp1a and pp1ab). three functional proteases then cleave the polyproteins into 16 non-structural proteins (nsp1-16), which eventually create the viral rna polymerase and other accessory proteins for virus assembly (34) (35) (36) . an uninterrupted replication-transcription event results in the formation of various nested sets of subgenomic (sg) mrnas that eventually translate into numerous structural and accessory proteins (37) . the e glycoproteins after synthesis are incorporated into the rough endoplasmic reticulum or golgi membrane. the +ve ssrna combines with capsid protein to form the nucleocapsid, followed by budding of assembled virus particles in the er-golgi intermediate compartment (ergic) (38) . lastly, the virus particle-loaded vesicles are fused with the cell membrane for effective shedding of the virus (4). these new virions are now accessible to infect the neighboring healthy cells and are also released into the surrounding environment via respiratory droplets that are highly contagious and hence potentially spread the disease to healthy individuals. the path followed by sars-cov-2 to reach the lungs is via the naso-oral cavity. once the virus is inhaled, it enters the epithelial cells of the nasal cavity by engagement of ace2 receptor with the viral rbd and initiates its replication (27, 39, 40) . this initial asymptomatic phase lasts for about 1-2 days, during which the virus multiplies in the upper respiratory tract, where no major hindrance is caused by the innate immune cells. within 2-14 days of initial encounter, the common symptoms of covid-19 start to appear, which are similar to those of sars and mers, i.e., fever, dry cough, pharyngitis, shortness of breath, joint pain, and tiredness. numerous problems arise during this phase of the disease, including nosocomial and fomite transmission of infection, which enhances the chances of community spread (41) . soon, the virus begins to move toward the lower respiratory tract via airways, and this triggers a strong innate immune response. patients at this stage start exhibiting enhanced pro-inflammatory response that leads to viral sepsis accompanied by other complications, including pulmonary edema, acute respiratory distress syndrome (ards), different organ failures, and death in the worst scenarios (42) . the infected individuals rarely show the intestinal symptoms like diarrhea that were evident in other coronavirus infections. patients are recommended to be quarantined to prevent community spread of this pandemic virus (43) . the severity of covid-19 has been found to be greater in aged individuals and in people with a health history, such as those immune-compromised by hiv infection or by chemotherapy for cancer. diabetic and asthma patients, along with individuals with hypertension, obesity, or heart, kidney, or liver disorders, are also at higher risk if they acquire the disease (44) . autopsy reports of individuals who died due to sars show multi-organ dysfunction, with the highest viral titers in the lungs and immune cells in circulation, thus damaging the pulmonary and immune system (45, 46) . as opposed to adults, only a very small population of children has been infected with sars-cov-2. in one study, the symptoms displayed by children above 15 years were found to be milder as compared to those of younger children, who showed severe symptoms but with rare deaths and better prognosis (47) . the study speculated two major possibilities related to covid-19 severity in children among different age groups. one of these rests on the finding that ace2 activity is higher in children aged 4-13 years; after this age, it starts to decline until adolescence. this could be one of the reasons why lung fibrosis is observed mainly in younger children. secondly, differential cd4 + and cd8 + t cell populations have been seen in children as compared to adults (48, 49) . a large number of clinical and epidemiological criteria were defined to assess probable pediatric cases of covid-19 (50) . a preliminary report from a cross-sectional study of children admitted to us and canadian pediatric intensive care units (picus) during march 14-april 3, 2020, revealed that the 48 children were admitted in the usa whereas no covid-19 cases were reported in canadian picus. the study revealed that there are fewer covid-19 cases in children as compared to adults and that there is a median picu time of 5 days (51). a recent preprint from paris reports that 11 children (age 3.7-16.6) were admitted experiencing symptoms similar to kawasaki disease (kd) along with gastrointestinal issues and elevated inflammatory markers. further investigation suggested that they were also sars-cov-2-positive, speculating that this could be the reason for kd shock syndrome (52) . similar cases have been observed in new york, where four otherwise healthy sars-cov-2-positive children started displaying symptoms similar to kd and toxic shock syndrome, thereby needing intensive care (53) . therefore, medical practitioners should be prepared to tackle such sudden post-infection complications to avoid the associated risks. once the virus gains access inside the target cell, the host immune system recognizes the whole virus or its surface epitopes, eliciting the innate or adaptive immune response (figure 2 ). pathogen recognition receptors (prrs) present on immune cells, mainly toll-like receptors 3, 7, and 8, are the first to identify the virus, which leads to enhanced interferon (ifn) production. the function of host innate immune cells is impaired during sars-cov and mers-cov infection by their non-structural proteins, which affects the overall cytokine production (54) (55) (56) . humoral response against sars-cov-2 has been found to be similar to that against other coronavirus infections, involving the characteristic igg and igm production. at the onset of sars-cov infection, b cells elicit an early response against the n protein, while antibodies against s protein could be detected after 4-8 days from the appearance of initial symptoms (57, 58) . although n protein is smaller than s protein, it is highly immunogenic, and the absence of glycosylation sites on it results in n-specific neutralizing antibody production at an early stage of acute infection (59) . sars-cov-specific iga, igg, and igm antibodies were detected after the onset of symptoms at different time points in infected patients. a persistent level of igg was detected for a longer period, whereas igm levels started to decline after 3 months (60, 61) . in an observational case study of 16 sars-cov-2 patients, anti-s-rbd igg was detected in all of the subjects, whereas anti-n igg and anti-s-rbd igm were detected in 15 patients and anti-n igm in 14 patients (62) . an elisa-based time kinetics study to detect the covid-19 specific humoral immune response showed that the patients produced igm and igg antibodies that did not cross-react with other human coronaviruses except sars-cov. igm and iga antibodies were detected 5 days after the onset of initial symptoms, whereas igg was detected after 14 days (63) . another kinetic study of viral shedding and antibody detection was published in a preprint and reported the presence of higher igg and igm antibody titers in severe patients. they also observed that weak responders for igg antibody had higher viral clearance than strong responders. this observation suggests that robust antibody response leads to disease severity while feeble response is associated with the elimination of virus (64) . a case study on pediatric patients reports that 5 out of 6 children showed a protective humoral response, with neutralizing igg and igm antibodies targeting the n and s-rbd proteins of sars-cov-2 (65) . these studies propose that igm-based elisa can be used for early diagnosis of patients along with qpcr techniques to improve the sensitivity and specificity of the technique. in addition to neutralizing antibodies, which are defensive and useful, there are numerous non-neutralizing antibodies in the system that aid the infection of immune cells and apcs. previously existing sars-cov antibodies may promote the viral infection in fcr-expressing cells (66) . this ace2-independent pathway of viral entry does not result in viral replication; rather, viral shedding by macrophages enhances inflammation and tissue injury by myeloid cell activation. this mechanism of viral entry through non-neutralizing antibody that results in aberrant activation of immune cells is called ade (antibody-dependent enhancement) (66, 67) . ade has been observed in a number of viral infections, including sars and mers. in the case of sars, anti-s antibodies were observed to be involved in ade to gain entry into fcr-expressing cells (68) , while in mers, a neutralizing mab (mersmab1) targeting rbd aided in mers pseudo-virus entry via the dpp4 pathway (69) . although there is no clear evidence regarding ade in sars-cov-2 infection, it is still necessary to consider all of the odds in the pursuit of developing vaccines and treatment regimens involving antibodies (70) . during viral infection, t cells also recognize the viral antigens presented by mhc class i [mhc; human leukocyte antigen (hla) in humans], which in turn promotes the cytokine release and cytotoxic activity of cd8 + t cells (71) . but in some other cases, mhc class ii is also found to present sars-cov peptides to cd4 + t cells. due to the genetic polymorphism of hla, some haplotypes, like hla-b * 07, hla-b * 46, hla-drb1 * 12 (72) , and hla-cw * 08 (73) , are found to be more susceptible to coronavirus infection, whereas the hla-drb1 * 03, hla-a * 02, and hla-cw * 15 haplotypes are protected from sars-cov infection (74) . similarly, hla-drb1 * 11 and hla-dqb1 * 02 were found to be vulnerable to mers-cov infection (75) . additionally, mhc expression is also found to be reduced during the infection due to epigenetic modifications of downstream molecules (76, 77) . so far, hla association is not very wellidentified for sars-cov-2 infection, and this could be crucial for the prevention and treatment of covid-19. however, in a recent report, blood plasma from covid-19 patients was able to block the expression of hla-dr on cd14 + monocytes, which was restored effectively on inhibiting il-6, suggesting that decreased hla-dr expression in sars-cov-2 patients is due to the buildup of hyper-inflammatory conditions (78) . decrease in mhc expression is also evident in cancer cells, which is a mechanism by which they evade the immune response by epigenetically modifying calnexin promoter. but infection with influenza virus in these cancer cells results in enhanced mhc-i presentation due to the increased expression of chromatin remodeling proteins, which stabilizes p53 expression and hence augments the immune surveillance of cancer cells (79) . therefore, molecules that can upregulate chromatin regulators and increase the mhc-i expression could potentially be used for covid-19. most of the t-cell epitopes presented by mhc complex are derived from structural proteins such as the s and n proteins of the coronavirus in both humans and animal models, while the nsps have regulatory effects on the signaling cascade (80, 81) . t cells can be stimulated by 14 epitopes, most of which are observed to be located on orf3 and the s protein in sars patients (61) . in a large cohort study during sars-cov infection, s protein was the only immuno-dominant epitope for cd8 + t-cell activation (61) , whereas, in mers, cd8 + response was against the s and n proteins along with some of the m/e epitopes (82) . these t-cell epitopes have been tested in animal models by assessing the lung pathology and t-cell response upon infection in balb/c and c57bl/6 mice (80, 83). the sequence of sars-cov-2 being more similar to sars-cov than to mers-cov, with no mutation in 19 epitopes, provides a prospective subunit vaccine for stimulating a strong t-cell response in covid 19 patients (84) . in a recent study, samples from 20 convalescing covid-19 patients were analyzed to check the development of adaptive immune response during infection. the results highlighted that helper t cells were eliciting a robust immune response against s, m, and n protein. the effect of adaptive immune response on humoral immunity was also compared, where a strong cd4 + t-cell response against sars-cov-2 eventually resulted in an increase in anti-s-rbdspecific igg and iga antibody titer. along with cd4 + t cells, immunogenic epitopes on s, m, and n proteins were also able to activate cd8 + t cells. however, such t-cell response was not specific to recovered patients only but was also present in 40-60% of the individuals who were not exposed to sars-cov-2. further analysis showed that they had pre-existing cross-reactive cd4 + t cells, which might have been generated in response to some previous coronavirus infection. hence, these t-cells could impart protective immunity in such individuals against sars-cov-2 to some extent (85) . these epitopes could be a promising factor in developing immunotherapy by small molecules that can increase the presentation of viral epitopes. a rapid and coordinated immune response during viral infection leads to enhanced secretion of various cytokines, which acts as a defense mechanism against the virus. numerous reports suggest that individuals affected with sars-cov or mers-cov have dysregulated cytokine production from both innate and adaptive immune cells. in the case of sars, infected hematopoietic cell, monocyte-macrophages, and other immune cells trigger enhanced secretion of pro-inflammatory cytokines like tnf-α, il-6, and ifn-α/-γ, with reduced anti-inflammatory cytokines (86) (87) (88) . similarly, mers-cov infection leads to delayed but increased production of ifn-α and pro-inflammatory cytokines like il-6, il-8, and il-1β (89) (90) (91) . such elevated levels of cytokines were associated with multi-organ dysfunctional syndrome (mods) and ards due to the accumulation of numerous immune cells like macrophages, neutrophils, and dendritic cells in the lungs causing alveolar damage and edema (56, 92, 93) . similarly, in covid-19 patients, secretion of cytokines and chemokines, which attract the immune cells to the lungs, was increased, hence causing ards, which is fatal to critically ill individuals (94, 95) . signature cytokines in severely ill covid-19 patients were consistent with those in sars and mers, i.e., enhanced expression of il-6, tnf-α, macrophage inflammatory protein 1-α (mip-1α), mcp3, gm-csf, il-2, and ip-10 along with elevated chemokines (ip-10, ccl2/mcp1, cxcl1, cxcl5) were also detected in sars-cov-2 infection (96) (97) (98) (99) . in children, the increased inflammatory markers include il-6, il-1, and c-reactive protein along with procalcitonin in serum (52) . in a case study, a 14-year-old child with cytokine storm was treated with anakinra (il-1 receptor antagonist) in order to stabilize the respiratory illness and other clinical symptoms (100) . transcriptomic analysis of pbmc and balf showed that a number of immune regulators were upregulated, particularly cxcl10, with respect to balf. this study also reported that several apoptotic genes and p53 signaling molecules were upregulated, suggesting a possible reason for lymphopenia in these patients (101) . therapeutic measures to control such cytokines involve neutralizing antibodies or small molecular drugs that can stop the signaling cascade for cytokine production. the most potent antiviral machinery acquired by immune cells is the secretion of interferons that act as secondary messengers stimulating the neighboring cells. most innate immune cells are efficient in producing ifns that are involved in obstructing cell proliferation, apoptosis, and immunomodulation (54, 102) . as an escape mechanism, sars-cov or mers-cov uses several ways to overcome the host immune response, one of which is by severe leukopenia and lymphopenia (103) (104) (105) . after gaining entry to the cell, these viruses encode different proteins that interact with downstream signaling molecules of tlrs and the jak-stat pathway. mers-cov encoded matrix protein, accessory proteins from orf 4a, 4b, and 5, which directly inhibits the ifn promoter and nuclear localization of irf3 (106) . plpro, encoded by sars-cov and mers-cov, prevents the dissociation of nf-κb from iκbα, whereas nonstructural proteins of sars-cov, i.e., plpro and orf3b, inhibit irf3 phosphorylation and hence its translocation to the nucleus (4, 107, 108). these viral accessory proteins also inhibit the jak-stat pathway, resulting in inhibition of genes by isre promoters (109) (110) (111) (figure 3) . a new investigation revealed that sars-cov-2 infection leads to an overall decrease in the transcription of antiviral genes because of the lower production of type i and iii interferons with sufficient isg expression, along with elevated chemokine secretion. results obtained from in-vivo and ex-vivo covid-19 experiments were in tune with the in-vitro findings. therefore, a decrease in the innate antiviral response, along with hyper-inflammation, could be one of the causes of covid-19 severity (112) . in addition to reduction in t cells, sars-cov-2 infection also enhances the exhaustion of effector t cells, decreasing the immune response against the virus (94, 113) . exhaustion and loss in function of effector t cells is the result of increased expression of inhibitory receptors like pd-1, tim-3, and tigit on its surface as a result of cytokines like il-6, il-10, and tnf-α or by decreasing the regulatory t-cell population (114, 115) . following viral/antigen clearance, most of the effector t cell undergoes apoptosis in the contraction phase. subsequently, a pool of memory t cells are generated that are programmed to fight against re-infection. cd4 + memory t cells, upon restimulation, trigger b cells and other immune cells by cytokine production, while cytotoxic memory t cells help in destroying the infected cells during subsequent infection (116, 117). case studies in recovered sars patients showed that both cd4 + and cd8 + memory t cells were efficient in eliciting immune response from 3 months to 6 years without the presence of any antigens (118) . in a case study of 23 recovered sars-cov patients, the patients showed very low frequencies of memory b cells, while memory t cells elicited a response against the s protein in 60% of recovered individuals (119) . considering the memory t-cell subset, n-specific helper t cells had more of central memory markers (cd45ra − , ccr7 + , cd62l − ) while the cd8 + t cell population had the effector memory (cd45ra + , ccr7 − , cd62l − ) phenotype in a steady-state manner (120) . the study suggests that an effective vaccine or t cell epitopes could be used to target a particular population for rapid viral clearance. in recent reports, covid-19 subjects have shown reduced regulatory t cell populations and memory t cells, which may aggravate the inflammatory response leading to cytokine storm and hence enhance the tissue damage and organ failure (114) . in a mouse model, the use of cd4 + memory t cells as a vaccine by the intranasal, but not the subcutaneous, route imparted a protective response against the human coronavirus. the infused cd4 + memory t cell, upon re-stimulation, produces ifn-γ and recruits cd8 + t cells for rapid clearance in response to sars-s366 peptide (121) . recently, a human ace-2-expressing mouse model has been developed by crispr/cas9 technology that recapitulates the human symptoms upon infection with sars-cov-2 through the intra-nasal route. this tool will be beneficial for evaluating the efficacy of vaccines for covid-19 and also to study its transmission and pathogenesis (122) . just like sars and mers, there are no specific clinically approved drugs available for covid-19 as of june 15, 2020 (123) . currently, the treatment regime focuses mainly on providing intensive care in order to alleviate the symptoms and discomfort associated with covid-19. conservative fluid therapy accompanied by broad-spectrum antibiotics are also given to the patients as a protective measure to avoid opportunistic bacterial infections. however, ventilator support for respiration is provided to the patient under extreme conditions (124) . numerous fda-approved antiviral drugs, vaccines, and immunotherapies that are already being used to treat other diseases have also been considered as a possible approach for treating covid-19 ( table 1) . but this approach may reduce the availability of these drugs and vaccines for the intended diseases and for the patients with the greatest need. the molecular, structural, and functional relationships of lopinavir viral protease involved in immature, noninfectious hiv virus particle, and inhibits plpro or 3clpro in sars-cov-2. favilavir viral rna polymerase purine analog blocking viral rna synthesis. remdesivir (141) ribavirin guanosine nucleoside binds to nucleoside binding pocket of the enzyme. (133, 140, 142) galidesivir adenosine analog, effective against ebola, zika, and other rna viruses. chloroquine/hydroxychloroquine heme polymerase and ace2 increases endosomal ph and terminal glycosylation of ace2, inhibiting sars-cov-2 entry. (144, 145) nitazoxanide glutathione-s-transferase alters ph and inhibits viral maturation. reported against tb, helminthic, and protozoan infection. umifenovir/arbidol n/a interacts with aromatic residues of viral glycoproteins. is being trialed for prophylactic action against covid-19. sars-cov-2 with sars-cov might define the use of existing anti-viral drugs against covid-19 (147, 148) , considering the total time it takes to perform clinical trials and get fda approval for the use of novel drugs and vaccines. the increasing knowledge of the genetic, immunological, and molecular mechanisms behind its enhanced pathogenicity might help in developing specific treatment approaches for covid-19 in the future. considering the studies on the molecular mechanism of coronavirus infection (147) , several antiviral drugs could be repurposed for the treatment of covid-19. remdesivir is a nucleotide analog that acts as an antiviral agent for a wide variety of viruses and has been tested widely against previous epidemics of coronavirus infections in both in-vitro and in-vivo models (138, (149) (150) (151) . this adenosine analog gets incorporated into the newly synthesized viral rna, which inhibits the addition of further nucleotides by viral rnadependent rna polymerase and hence terminates the ongoing transcription. administration of intravenous remdesivir was found to be effective in treating the first known patient of covid-19 in the usa (152) . a randomized double-blinded clinical trial on 1,059 adult hospitalized covid-19 patients was sponsored by the national institute of allergy and infectious diseases, usa, to further test the potency of intravenously administered remdesivir. the preliminary outcomes of the trial reported that remdesivir treatment decreased the median recovery time in the treatment group (11 days) as compared to the placebo group (15 days). the mortality rate was also less in the treatment group (7.1%) in contrast to the placebo group (11.9%) (153) . numerous clinical studies, similar to this, are required so as to validate the proposed drugs for covid-19. favipiravir, ribavirin, and galidesivir are also potential nucleoside analogs that might be useful against novel coronavirus infection (154) . the combinatorial therapy approach of using remdesivir along with chloroquine, a well-known anti-malarial drug, has also been tested in vitro so as to study its effectiveness against sars-cov-2 (141, 155) . it has been reported that chloroquine immuno-modulates the host microenvironment and also interferes with the replication of the virus and its interaction with the receptor (156, 157) . in a randomized clinical trial (nct04308668) involving 821 asymptomatic individuals across the us and canada who had come into close contact with potential covid-19 patients, the individuals were given either hydroxychloroquine or placebo as a prophylactic measure. the results revealed that hydroxychloroquine treatment had the same effect as did the placebo group. the usage of hydroxychloroquine resulted in minor side effects (40.1%) as compared to the placebo treatment (6.8%). however, no cardiovascular disorder or treatment-related major complications were observed (158) . based on the putative function of hydroxychloroquine on the endosomal acidification, whereby it is presumed to hinder viral uncapping, it can be observed that it has a great potential for prophylaxis, not to prevent infection but to reduce effective viral load in patients and thus lead to milder disease. numerous clinical trials to further explore the usage of hydroxychloroquine in different combinations are in the pipeline and will finally provide a better understanding of the efficacy of this drug for covid-19. a few anti-hiv drugs, such as lopinavir/ritonavir in combination with interferon beta (ifn-β), have been tested in vivo for treating coronavirus infections (sars-cov, mers-cov) and have also been used in the case of covid-19 (138, 139, 159) . various complementary therapies could also be employed as a preventive measure against viral infections. many essential proteases, such as chymotrypsin (3c-like protease) and plpro, which are required by coronavirus for completing the replication process, can also be targeted using drugs. cinanserin, flavonoids, and some small molecules are known to inhibit 3clpro, whereas diarylheptanoids are used to inhibit plpro (160) (161) (162) . in a recent study, 16 potential anti-hcov drugs were identified through a systems biology-based approach, such as melatonin, mercaptopurine, sirolimus, dactiomycin, and toremifene, which are to be tested further for their potency (163) . in the absence of any dependable vaccine or drugs with tested efficacy and when the pandemic onslaught is ongoing, a worthy therapeutic approach is passive immunization using purified antibodies. the source of such antibodies could be the sera of convalescing individuals, mabs, or genetically modified antibodies from an animal host, which can efficiently neutralize the virus. this is an age-old practice, with pioneering work having been done by the nobel laureate, emil behring, who applied this approach for diphtheria, and has been used whenever there are sudden outbreaks of viral diseases like sars, mers, h1n1, h5n1, ebola, and many others (61, 164, 165) . as opposed to active vaccination, plasma therapy is the only means to provide immediate immunity for viral clearance, as in the case of sars-cov-2. as in other epidemic diseases, convalescent sera are currently being employed for covid-19 in a number of countries (166, 167) . although a randomized controlled trial is yet to be reported, limited studies in 10 patients have been documented with no remission of severe respiratory afflictions on receiving neutralizing antibodies from 39 convalesced donors with antibody titers of 1:160, along with drugs and oxygen support (168) . a report from hong kong suggested that this therapy had poor outcome in sars patients, with a number of limitations in their study (169) . as with transfusion of any blood products, precautionary screening of infectious agent is warranted in plasma transfusion. recently, the fda in the usa has approved trials of convalescent plasma therapy in covid-19 under specific guidelines; plasma donation is advised 3 weeks after a patient becomes virus-negative on pcr. the major challenge in this therapy is obtaining donors with similar blood antigens with a high antibody titer of sars-cov-2 (170) . another potential adverse effect of this approach is ade of infection, which is common in so many other viruses. but, to date, the incidence of ade has not been reported in the case of sars-cov-2. another major point of contention is the selection of patients for this therapeutic approach. in most clinical trials, patients with severe diseases are being recruited, while the presumed mechanism of action of convalescent plasma, based on its content of virus-neutralizing antibodies, rather points to plausible favorable outcomes in earlier phases of the disease because in the later, more severe phases, the hyperimmune response, rather than the viral load, becomes the more critical pathology. finally, there are no available data on the heterogeneity of response to convalescent plasma transfusion, which may further illustrate the importance of careful evidencebased patient selection, as heterogeneity of response may result from both virus and host-intrinsic factors which are, to date, not revealed. researchers around the world are working hard to develop a potential vaccine candidate so as to stop the deadly pandemic caused by sars-cov-2. however, vaccine development is not an easy task, as a number of successful clinical trials are required before approval for patients. different approaches are being utilized for designing a specific vaccine targeting either the structural proteins or viral replication process, which eventually results in the inhibition of viral growth and its further transmission. the common strategies involve the use of live attenuated vaccine (lav), inactivated virus, subunit vaccines, monoclonal antibody vaccine, virus vectors, protein vaccines, and dna/rna-based vaccines (171) (172) (173) (174) . there are numerous subunit vaccines targeting all or a part of s protein that have already been tested for sars and mers in animal models (175) and could be potential candidates for testing against sars-cov-2. a recent pilot study with a purified inactivated sars-cov-2 virus vaccine displayed very promising outcomes in different animal models. the neutralizing antibodies generated after vaccination were able to effectively target 10 different strains of sars-cov-2 without developing any ade of infection (176) . various randomized controlled trials (nct04327206, nct04328441) are also underway to evaluate the effectiveness of the bcg vaccine against sars-cov-2 for healthcare professionals. an adenovirus vector-based vaccine candidate, chadox1 (presently azd1222), developed by oxford university (licensed to astrazeneca) for use against sars-cov-2 has been reported to activate both the humoral and cell-mediated immune response when tested in rhesus monkey (177) . the phase i clinical trial to confirm its potency is also in progress (nct04324606). another group has followed a similar approach by using a recombinant adenovirus type 5 (ad5-ncov) vectorbased vaccine for covid-19. the full report from the phase i clinical trial (nct04313127) of ad5-ncov shows that it is very effective in generating both humoral and rapid t-cell response post immunization. the group is now ready for the next clinical trial phase to further strengthen the effectiveness of the ad5-ncov vaccine (178) . it should be noted that there are potential risks associated with the usage of live attenuated viruses, for example, complications resulting in lung damage by infiltrating eosinophils, as seen in in vivo models (179, 180) . however, eosinophil immunopathology due to sars-cov vaccine could be reduced by using tlr4 agonist as an adjuvant (181) . viral neutralizing antibodies specifically targeting various regions of s, i.e., s1-rbd, s1-ntd, or the s2 region, and blocking the interaction of virus with the receptor are well-known for sars and mers (182) . these neutralizing antibodies could prove to be the best and potential candidate for cross-neutralization of sars-cov-2. despite being structurally related, some of the sars-cov neutralizing monoclonal antibodies failed to interact with the s-protein of sars-cov-2, which could be attributable to the substantial differences in their rbd (183) . a recent study reported the presence of high titres of neutralizing anti-s-rbd igg antibodies, but no antibodies were detected against the n protein in recovered covid-19 patients, suggesting that anti-s igg persists longer than does anti-n igg. along with the humoral immune response, they also observed an s protein-specific t cell-population producing ifn-γ, which further contributes to conferring protective immunity against sars-cov-2 infection (184). recently, a monoclonal antibody (47d11) has been identified from 51 sars-spike hybridomas that targets the conserved s-rbd region (residue 338-506) and therefore can very effectively neutralize sars-cov-2 along with sars-cov (185). on similar lines, a group has isolated a single-domain antibody from a phage display library targeting the s-rbd region of sars-cov-2. the fully humanized singledomain antibody was able to neutralize the virus by interacting with a cryptic epitope in s protein (186) . these mab and singledomain antibodies could be used to treat as well as to design quick diagnostic kits for covid-19. the new technology of the microneedle array (mna) has been employed for delivering sars-cov-2 s1 subunit vaccine, which could be really helpful in the treatment of the emerging covid-19 outbreak (187) . the transfer of s1 subunit by mna elicited a strong virus specific-antibody response in sars-cov-2 (187) . a novel encapsulated mrna vaccine candidate developed by modernatx, inc. that encodes full length s protein of sars-cov-2, is also under clinical trial (nct04283461). there is an urgent need to develop more such specific vaccines that could neutralize the novel coronavirus effectively (188) . the host innate immune system encounters upcoming infections, and this results in elevated production of various cytokines and type i interferons (ifns). in the case of prolonged infection, hyperactivation of the immune system may also result in the development of a pro-inflammatory microenvironment, leading to adverse outcomes and even death. the induction of numerous lymphokines, such as il-6, il-1β, tnf-α, and ccl2, that are pro-inflammatory in nature has also been observed in the case of covid-19 (189) (190) (191) . a previous study in a mers animal model showed that treatment with recombinant type-1 ifn (rifn) decreased the viral rna level in lungs with a decrease in ifn-stimulating gene expression. early treatment with rifn resulted in a dampening of cytokine and chemokine release that lowered the migration of neutrophils and other cells in lung (91) . an allogenic mesenchymal stem cell-based (remestemcel-l) therapy developed by mesoblast, which has been previously used for inflammatory conditions and graft vs. host disease in children and adults, is now being assessed for covid-19 (192) (193) (194) . in this therapy, bone marrow-derived mscs from the donor are grown in vitro and are then transfused to the recipient patients. upon infusion, these cells exhibit antiinflammatory activity by reducing pro-inflammatory cytokine production via the recruitment of anti-inflammatory cells in the affected tissue (195) . currently, a randomized placebo-controlled trial (nct04371393) with 300 patients is ongoing for treating ards caused by covid-19. treatment with rifn, inhibitors of the pro-inflammatory pathway, cytokine inhibitors such as tocilizumab, lenzilumab, and many others are still to be used in combination with other drugs for treating covid-19. so far, there is not much evidence from clinical trials of such inhibitors with which to predict the outcome of these anticytokine therapies. considering the current situation of more than 8 million people being infected, with ∼436,167 deaths as of june 15, 2020, there is an urgent need to control the sars-cov-2 pandemic. the fatality rate of sars-cov-2 in lower than those of other coronaviruses that caused catastrophes in the past, but the higher infectivity rate makes it worse. raising awareness of this contagious virus is one of the many ways by which its spread can be prevented. the governing authorities concerned in every country have approved guidelines and taken necessary action to quarantine infected people and break the chain of community spread. antibodies, vaccines, and drugs developed for previously emerged coronaviruses could potentially be used for treating sars-cov-2. the combination of various neutralizing antibodies against s protein could enhance the effectiveness of viral clearance. among various antivirals and other small molecules that are fda approved, chloroquine/hydroxychloroquine has shown better positive outcome in covid-19 patients. in clinical trials, some of the combinational antiviral drugs like lopinavir + ritonavir and blockers like angiotensin receptor blocker that were thought to be effective, have failed in curing the disease (139, 196) . cytokine storm being one of the symptoms of infected individuals, anticytokine therapy for tnf and il-6 should be attempted to determine the efficacy of these antibodies in the treatment of sars-cov-2 infection. clinical trial chictr2000029765 with tocilizumab, a monoclonal humanized antibody against il-6 receptor, has shown some efficacy, but this still needs to be tested in a larger cohort. with the increasing number of deaths, there is an immense need to accelerate the development of rapid and sensitive diagnostic kits and to commence clinical trials of the readily available and safe drugs to reduce the rising infections and covid-19-related deaths so as to bring life back on track. vs and pf contributed equally in writing the review. conception of idea was done by sc, vs, and pf. manuscript writing and editing was done by all the authors. we are thankful to csir-indian institute of chemical biology, jadavpur, kolkata and national centre for cell science (nccs), pune for providing infrastructure facilities. we would also like to acknowledge department of biotechnology-systems medicine cluster (dbt-symec) grant and j c bose fellowship-serb (science and engineering research board) to sc, for the financial support. a pneumonia outbreak associated with a new coronavirus of probable bat origin genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding air, surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient sars and mers : recent insights into emerging coronaviruses estimates of the severity of coronavirus disease 2019 : a model-based analysis united nation, department of economic and social affairs (2020) the genome sequence of the sars-associated coronavirus a new coronavirus associated with human respiratory disease in china crystal structure of sars-cov-2 main protease provides a basis for design of improved α-ketoamide inhibitors structural basis for the recognition of the sars-cov-2 by full-length human ace2 a novel coronavirus from patients with pneumonia in china coronavirus envelope protein: current knowledge human coronaviruses: a review of virus-host interactions origin and evolution of pathogenic coronaviruses from sars to mers, thrusting coronaviruses into the spotlight genome composition and divergence of the novel coronavirus (2019-ncov) originating in china covid-2019: the role of the nsp2 and nsp3 in its pathogenesis origin and evolution of the 2019 novel coronavirus emerging sars-cov-2 mutation hot spots include a novel rna-dependent-rna polymerase variant emergence of genomic diversity and recurrent mutations in sars-cov-2 angiotensinconverting enzyme 2 is a functional receptor for the sars coronavirus wenhui dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses cryo-em structure of the sars coronavirus spike glycoprotein in complex with its host cell receptor ace2 cryo-em structure of the 2019-ncov spike in the prefusion conformation host cell proteases: critical determinants of coronavirus tropism and pathogenesis sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor localization of neutralizing epitopes and receptor-binding site in murine coronavirus spike protein viral shedding patterns of coronavirus in patients with probable severe acute respiratory syndrome current advancements and potential strategies in the development of mers-cov vaccines fusion mechanism of 2019-ncov and fusion inhibitors targeting hr1 domain in spike protein characterization of severe acute respiratory syndrome-associated coronavirus (sars-cov) spike glycoprotein-mediated viral entry activation of the sars coronavirus spike protein via sequential proteolytic cleavage at two distinct sites characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus rna-dependent rna polymerase coronavirus transcription: a perspective host factors in coronavirus replication identification of novel subgenomic rnas and noncanonical transcription initiation signals of severe acute respiratory syndrome coronavirus the c-terminal domain of the mers coronavirus m protein contains a trans-golgi network localization signal structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes available online at infection control: severe acute respiratory syndrome coronavirus 2 (sars-cov-2). (2020) immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic people who are at higher risk for severe illness | coronavirus | covid-19 severe acute respiratory syndrome-associated coronavirus in lung tissue multiple organ infection and the pathogenesis of sars sars-cov-2 infection in children vulnerability of children with covid-19 infection and ace2 profiles in lungs possible causes for decreased susceptibility of children to coronavirus diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus characteristics and outcomes of children with coronavirus disease 2019 (covid-19) infection admitted to us and canadian pediatric intensive care units outbreak of kawasaki disease in children during covid-19 pandemic: a prospective observational features of covid-19 post-infectious cytokine release syndrome in children presenting to the emergency department links between innate and adaptive immunity via type i interferon interferon and cytokine responses to sarscoronavirus infection sars-cov regulates immune function-related gene expression in human monocytic cells profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers early detection of antibodies against various structural proteins of the sarsassociated coronavirus in sars patients serological assays for emerging coronaviruses : challenges and pitfalls profile of specific antibodies to the sars-associated coronavirus t cell responses to whole sars coronavirus in humans temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2 : an observational cohort study profiling early humoral response to diagnose novel coronavirus disease (covid-19) viral kinetics and antibody responses in patients with covid-19 protective humoral immunity in sars-cov-2 infected pediatric patients the potential danger of suboptimal antibody responses in covid-19 medical countermeasures analysis of 2019-ncov and vaccine risks for antibody-dependent enhancement (ade) antibody-dependent enhancement of sars coronavirus infection and its role in the pathogenesis of sars molecular mechanism for antibody-dependent enhancement of coronavirus entry avoiding pitfalls in the pursuit of a covid-19 vaccine novel immunodominant peptide presentation strategy: a featured hla-a * 2402-restricted cytotoxic t-lymphocyte epitope stabilized by intrachain hydrogen bonds from severe acute respiratory syndrome coronavirus nucleocapsid protein association of human leukocyte antigen class ii alleles with severe acute respiratory syndrome in the vietnamese population epidemiological and genetic correlates of severe acute respiratory syndrome coronavirus infection in the hospital with the highest nosocomial infection rate in taiwan in 2003 humanleukocyte antigen class i cw 1502 and class ii dr 0301 genotypes are associated with resistance to severe acute respiratory syndrome (sars) infection association of human leukocyte antigen class ii alleles with severe middle east respiratory syndrome-coronavirus infection cell host response to infection with novel human coronavirus emc predicts potential antivirals and important differences with sars coronavirus mers-cov and h5n1 influenza virus antagonize antigen presentation by altering the epigenetic landscape complex immune dysregulation in covid-19 patients with severe respiratory failure smar1 favors immunosurveillance of cancer cells by modulating calnexin and mhc i expression t cell responses are required for protection from clinical disease and for virus clearance in severe acute respiratory syndrome coronavirus-infected mice priming with sars cov s dna and boosting with sars cov s epitopes specific for cd4+ and cd8+ t cells promote cellular immune responses recovery from the middle east respiratory syndrome is associated with antibody and t cell responses sars coronavirus nucleocapsid immunodominant t-cell epitope cluster is common to both exogenous recombinant and endogenous dna-encoded immunogens preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals human immunopathogenesis of severe acute respiratory syndrome (sars) plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome temporal changes in cytokine/chemokine profiles and pulmonary involvement in severe acute respiratory syndrome delayed induction of proinflammatory cytokines and suppression of innate antiviral response by the novel middle east respiratory syndrome coronavirus: implications for pathogenesis and treatment active replication of middle east respiratory syndrome coronavirus and aberrant induction of inflammatory cytokines and chemokines in human macrophages: implications for pathogenesis ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes clinicopathologic, immunohistochemical, and ultrastructural findings of a fatal case of middle east respiratory syndrome coronavirus infection in the united arab emirates elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) pathological findings of covid-19 associated with acute respiratory distress syndrome covid-19 : consider cytokine storm syndromes and immunosuppression pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients anti-inflammation treatment of severe coronavirus disease 2019 (covid-19): from the perspective of clinical immunologists from china the cytokine storm in covid-19: an overview of the involvement of the chemokine/chemokine-receptor system novel paediatric presentation of covid-19 with ards and cytokine storm syndrome without respiratory symptoms transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients the interferon in tlr signaling: more than just antiviral haematological manifestations in patients with severe acute respiratory syndrome:retrospective analysis significant changes of peripheral t lymphocyte subsets in patients with severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia the structural and accessory proteins m, orf 4a, orf 4b, and orf 5 of middle east respiratory syndrome coronavirus (mers-cov) are potent interferon antagonists severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of regulation of irf-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus molecular determinants for subcellular localization of the severe acute respiratory syndrome coronavirus open reading frame 3b protein severe acute respiratory syndrome coronavirus open reading frame (orf) 3b, orf 6, and nucleocapsid proteins function as interferon antagonists pathogenic influenza viruses and coronaviruses utilize similar and contrasting approaches to control interferon-stimulated gene responses imbalanced host response to sars-cov-2 drives development of covid-19 elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients dysregulation of immune response in patients with covid-19 in wuhan, china lees jr, farber dl. generation, persistence and plasticity of cd4 t-cell memories characterization of sars-cov-specific memory t cells from recovered individuals 4 years after infection lack of peripheral memory b cell responses in recovered patients with severe acute respiratory syndrome: a six-year follow-up study longlived memory t lymphocyte responses against sars coronavirus nucleocapsid protein in sars-recovered patients airway memory cd4+ t cells mediate protective immunity against emerging respiratory coronaviruses a mouse model of sars-cov-2 infection and pathogenesis coronaviruses-drug discovery and therapeutic options a rapid advice guideline for the diagnosis and treatment of 2019 novel coronavirus (2019-ncov) infected pneumonia (standard version) potent cross-reactive neutralization of sars coronavirus isolates by human monoclonal antibodies potent neutralization of mers-cov by human neutralizing monoclonal antibodies to the viral spike glycoprotein the cytokine release syndrome (crs) of severe covid-19 and interleukin-6 receptor (il-6r) antagonist tocilizumab may be the key to reduce the mortality preventing covid-19-induced pneumonia with anticytokine therapy fda approves emergency ind use of humanigen's lenzilumab for compassionate use in covid-19 patients altasciences completes phase i study on gimsilumab for ards in covid-19 type 1 interferons as a potential treatment against covid-19 covid-19 and emerging viral infections: the case for interferon lambda learning from the past: possible urgent prevention and treatment options for severe acute respiratory infections caused by 2019-ncov research and development on therapeutic agents and vaccines for covid-19 and related human coronavirus diseases small molecule inhibitors of the sars-cov nsp15 endoribonuclease an orally bioavailable broad-spectrum antiviral inhibits sars-cov-2 in human airway epithelial cell cultures and multiple coronaviruses in mice baricitinib as potential treatment for 2019-ncov acute respiratory disease comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a trial of lopinavirritonavir in adults hospitalized with severe covid-19 old weapon for new enemy: drug repurposing for treatment of newly emerging viral diseases remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro ribavirin and interferon alfa-2a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study protection against filovirus diseases by a novel broad-spectrum nucleoside analogue bcx4430 new insights on the antiviral effects of chloroquine against coronavirus: what to expect for covid-19? mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology the efficacy of lopinavir plus ritonavir and arbidol against novel coronavirus infection molecular mechanisms of severe acute respiratory syndrome (sars) receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus gs-5734 and its parent nucleoside analog inhibit filo-, pneumo-, and paramyxoviruses. sci rep prophylactic and therapeutic remdesivir (gs-5734) treatment in the rhesus macaque model of mers-cov infection gs-5734) protects african green monkeys from nipah virus challenge first case of 2019 novel coronavirus in the united states remdesivir for the treatment of covid-19 -preliminary report new nucleoside analogues for the treatment of hemorrhagic fever virus infections chloroquine analogs as antimalarial candidates with potent in vitro and in vivo activity chloroquine is a potent inhibitor of sars coronavirus infection and spread effects of chloroquine on viral infections: an old drug against today's diseases? a randomized trial of hydroxychloroquine as postexposure prophylaxis for covid-19 treatment of middle east respiratory syndrome with a combination of lopinavir-ritonavir and interferon-β1b (miracle trial): study protocol for a randomized controlled trial cinanserin is an inhibitor of the 3c-like proteinase of severe acute respiratory syndrome coronavirus and strongly reduces virus replication in vitro inhibition of sars-cov 3cl protease by flavonoids diarylheptanoids from alnus japonica inhibit papain-like protease of severe acute respiratory syndrome coronavirus network-based drug repurposing for novel coronavirus 2019-ncov/sars-cov-2 feasibility of using convalescent plasma immunotherapy for mers-cov infection, saudi arabia treatment with convalescent plasma for influenza a (h5n1) infection the convalescent sera option for containing covid-19 breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 effectiveness of convalescent plasma therapy in severe covid-19 patients use of convalescent plasma therapy in sars patients in hong kong available online at sars vaccines: where are we? neutralizing antibody against severe acute respiratory syndrome (sars)-coronavirus spike is highly effective for the protection of mice in the murine sars model sars corona virus peptides recognized by antibodies in the sera of convalescent cases severe acute respiratory syndrome (sars) coronavirus: application of monoclonal antibodies and development of an effective vaccine subunit vaccines against emerging pathogenic human coronaviruses development of an inactivated vaccine candidate for sars-cov-2 chadox1 ncov-19 vaccination prevents sars-cov-2 pneumonia in rhesus macaques safety, tolerability, and immunogenicity of a recombinant adenovirus type-5 vectored covid-19 vaccine: a dose-escalation, open-label, non-randomised, first-in-human trial a double-inactivated severe acute respiratory syndrome coronavirus vaccine provides incomplete protection in mice and induces increased eosinophilic proinflammatory pulmonary response upon challenge immunization with sars coronavirus vaccines leads to pulmonary immunopathology on challenge with the sars virus effects of toll-like receptor stimulation on eosinophilic infiltration in lungs of balb/c mice immunized with uv-inactivated severe acute respiratory syndrome-related coronavirus vaccine neutralizing antibodies against sars-cov-2 and other human coronaviruses potent binding of 2019 novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals a human monoclonal antibody blocking sars-cov-2 infection identification of human single-domain antibodies against sars-cov-2 microneedle array delivered recombinant coronavirus vaccines: immunogenicity and rapid translational development sars-cov-2 vaccines: status report induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies mast cells contribute to coronavirus-induced inflammation: new anti-inflammatory strategy clinical features of patients infected with 2019 novel coronavirus in wuhan a phase 3, single-arm, prospective study of remestemcel-l, ex vivo culture-expanded adult human mesenchymal stromal cells for the treatment of pediatric patients who failed to respond to steroid treatment for acute graft-versus-host disease remestemcel-l: human mesenchymal stem cells as an emerging therapy for crohn's disease transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia mesoblast to evaluate anti-inflammatory cell therapy remestemcel-l for treatment of covid-19 lung disease are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? key: cord-278839-uu2wlpmp authors: alberca, ricardo wesley; pereira, nátalli zanete; oliveira, luanda mara da silva; gozzi-silva, sarah cristina; sato, maria notomi title: pregnancy, viral infection, and covid-19 date: 2020-07-07 journal: front immunol doi: 10.3389/fimmu.2020.01672 sha: doc_id: 278839 cord_uid: uu2wlpmp pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov-2) can induce the coronavirus diseases-2019 (covid-19) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid-19-positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid-19 during pregnancy, to establish a correlation and possible implications of covid-19 during pregnancy and neonatal's health. pregnancy comprises a unique immunological condition, to allow fetal development and to protect the host from pathogenic infections. viral infections during pregnancy can disrupt immunological tolerance and may generate deleterious effects on the fetus. despite these possible links between pregnancy and infection-induced morbidity, it is unclear how pregnancy interferes with maternal response to some viral pathogens. in this context, the novel coronavirus (sars-cov-2) can induce the coronavirus diseases-2019 (covid19) in pregnant women. the potential risk of vertical transmission is unclear, babies born from covid-19-positive mothers seems to have no serious clinical symptoms, the possible mechanisms are discussed, which highlights that checking the children's outcome and more research is warranted. in this review, we investigate the reports concerning viral infections and covid-19 during pregnancy, to establish a correlation and possible implications of covid-19 during pregnancy and neonatal's health. keywords: covid-19, sars-cov-2, pregnancy, neonatal, immunology pregnancy pregnancy comprises a unique immunological condition, to protect the fetus from maternal rejection, allowing adequate fetal development and protection against microorganisms (1, 2) . the maternal immune system is challenged by paternal alloantigens expressed both by the fetus and the placenta. however, through a complex range of cells and molecules, the mother does not develop a classic response to this allograft (3) . during pregnancy, fetal microquimerism occurs, where fetal cells, such as nucleated erythrocytes, trophoblastic cells, and leukocytes (3), cross the placental barrier and expose the mother to fetal alloantigens. these cells can remain in the bloodstream and maternal tissues many years after delivery (4, 5) . in comparison to the post-partum period, pregnancy increases monocytes, granulocytes, pdcs, mdcs in the blood, peaking during 2 trimesters. simultaneously, during pregnancy occurs a reduction in cd3, cd4, and cd8 t cells in comparison with post-partum. b cells are decreased during the third trimesters. nk cells cd56 dim are reduced in the second and third trimester of pregnancy in comparison with the first trimester and post-partum period. during the second and the third trimesters, nk and cd4 t cells present a reduction in the production of ifn-γ, tnf, il-6 cells, compared with post-partum (6) but the variability and contradictory reports are noted (7) . maternal monocytes do not show differences in absolute numbers, however, they show some phenotypic changes including an increase in the expression of adhesion molecules (cd11a, b; cd54), and the high-affinity igg receptor, fcγr-i (cd64) (8) . the absolute number of nk cells in maternal blood increases in the first trimester of pregnancy (9) . like lymphocytes, b cells are decreased during pregnancy and remain lower until 1 month after delivery. in vitro, b cells of pregnant women were less responsive, with suppression of lymphopoiesis and exclusion of autoreactive b cells (10). despite this, vaccine response during pregnancy remains effective (11, 12) . from the 13th week of gestation, maternal peripheral blood monocytes also undergo phenotypic and functional changes. there is an increase in the ability to produce cytokines il-1β and il-12 and a reduction in the potential for tnf-α secretion (13) . the placenta is a transient chimeric organ that develops from the uterine wall and can express different receptors and dynamically delivered microvesicles through pregnancy (14) . this organ mediates hormonal, nutritional, and oxygen support to the fetus while modulating maternal's immune response (15) . the placental maternal face is formed from decidual cells, with the presence of wide range of immune cells, including uterine natural killer (unk), dendritic cells (dcs), and regulatory t cells (tregs). the fetal face consists of the placental villus, which contains fetal blood vessels surrounded by fibroblasts and placental villous macrophages of fetal origin, hofbauer cells (16, 17) . treg cells are crucial for proper gestational development and are numerically elevated during pregnancy, in peripheral, deciduous and umbilical cord blood (18) . paternal hla-c is a crucial molecule that can elicit allogeneic immune responses by maternal cell and aid in the development of maternal-fetal tolerance (19) , also t reg may regulate cd4 + and cd8 + t lymphocyte activation through the expression of il-10 and tgfβ (20) . another striking feature of the maternal-fetal interface is the accumulation of nk cells, which comprise up to 70% of deciduous leukocytes in early pregnancy (21) . these cells are important for the regulation of cytokines production, especially il-10, and act in the production of angiogenic factors, chemokines, controlling the invasion of trophoblasts and availability of adequate maternal blood at the implantation site (17, 21, 22) . during pregnancy, hormonal variations can modulate immune responses, generating a reduction in the number of dcs and monocytes, and a decrease in the activation of macrophages, t, and b cells (23) . to better establish the tolerogenic milieu, estrogen induces efficiently foxp3 t regs cells (24) (25) (26) . changes in hormonal levels and immune system function generated by pregnancy may increase women's vulnerability to infections. pregnant women show higher mortality rates and complications associated with viral infections compared to the general population (27, 28) . for example, varicella disease in children is mild, but primary infections during pregnancy can progress to varicella pneumonia and death (29) . in 2009, during the h1n1 flu pandemic, an increased ratio of female to male cases was verified, in which pregnant women developed more complications, as severe acute respiratory syndrome, and higher mortality compared to the general population (30, 31) . similarly, in 1918 the pandemic spanish flu, among 1,350 reported cases of influenza in pregnant women, 27% died as a result of the infection (32) . in 1957, with the h5n1 pandemic, 50% of influenza deaths in women of reproductive age in minnesota occurred in pregnant women (33) . although influenza viruses are restricted to maternal lungs, inflammatory cytokines can lead to fetal complications mainly preterm birth and fetus miscarriage (34, 35) . in the ebola epidemic in 1995, 46% of infected women (out of a total of 177) were pregnant (36) . some evidence suggests that during pregnancy there is a greater risk of developing serious illnesses, spontaneous abortion, hemorrhage, and death when infected with the ebola virus (37) . additionally, infection by the lassa virus in pregnant women shows high levels of placental replication, and the risk of maternal-fetal mortality increases with the duration of pregnancy (38, 39) . viruses can gain access to the decidua and placenta by ascending from the lower reproductive tract or via hematogenous transmission, viral tropism for the decidua and placenta is then dependent on viral entry receptor expression in these tissues as well as on the maternal immune response to the virus (16) . a range of viral infections in pregnancy are associated with specific placental findings, including lymphoplasmacytic villitis with associated enlargement of villi and intravillous hemosiderin deposition in the setting of maternal cytomegalovirus infection (40) , as well as rare reports of intervillositis in the setting of zika virus (41) and dengue virus (42), among others. although there is little knowledge about placental findings associated with the common coronaviruses, ng et al. reported placental pathology in seven women with sars infection in hong kong (43) . in three placentas delivered in the acute stage of sars, demonstrated increased perivillous or subchorionic fibrin, while in two women who had recovered from third-trimester infection by the time of delivery, there were large zones of avascular villi, with one of the two additionally demonstrating a large villous infarct; both contained increased nucleated red blood cells in the fetal circulation. none of the seven placentas examined had any acute or chronic inflammatory processes (43) . the covid-19 pandemic is still in its early stages, with preliminary case series of infection in pregnant women available. a study of three placentas delivered from pregnant women with sars-cov-2 infection, infected in their third trimester with emergency cesarean section, describe various degrees of fibrin deposition. the fibrin deposition occurred inside and around the villi with local syncytial nodule increases in all three placentas, multiple villous infarcts in one placenta, and a chorangioma in another case. all samples from three placentas were negative for the nucleic acid of sars-cov-2 (44) . another study with 16 placentas from patients with sars-cov-2 were examined and the most significant finding is an increase in the rate of features of maternal vascular malperfusion (mvm), most prominently decidual arteriopathy including atherosis, fibrinoid necrosis, and mural hypertrophy of membrane arterioles (45). maternal hypertensive disorders, including gestational hypertension and preeclampsia, are the major risk factors for mvm (46) , although only 1 of the patients was hypertensive in this study. notwithstanding, sars-cov-2 is a virus that is expected to induce inflammation, it is relevant that neither acute inflammatory pathology (aip) nor chronic inflammatory pathology (cip) were increased in covid-19 patients relative to the controls. however, none of the covid-19 patients in this study were severely ill or undergoing a cytokine storm and it may be possible that cip could be induced in those cases of severe systemic inflammation (45). there few knowledges about miscarriage in women with covid-19, one case was a pregnant woman with symptomatic coronavirus disease who experienced a second-trimester miscarriage. a stillborn infant was delivered vaginally and swabs from the axillae, mouth, meconium, and fetal blood obtained within minutes of birth tested negative for sars-cov-2 and bacterial infection. the fetal autopsy showed no malformations, and fetal lung, liver, and thymus biopsies were negative for sars-cov-2. furthermore, amniotic fluid and vaginal swabs sampled during labor tested negative for sars-cov-2 and bacterial infection. placental histology demonstrated mixed inflammatory infiltrates composed of neutrophils and monocytes in the subchorial space and unspecific increased intervillous fibrin deposition (47) . during the worldwide sars-cov-1 (severe acute respiratory syndrome coronavirus-1) epidemic in 2003, a notable increase in mortality and morbidity was documented in pregnant patients (48) . agreeing with previous observations that the risk of viral pneumonia is significantly higher among pregnant women compared to the rest of the population (49) . in 2012, infection with the middle east respiratory syndrome (mers-cov) coronavirus in saudi arabia after the isolation of a male patient who died of severe pneumonia (50, 51) . data on the effects of mers-cov on pregnancy are limited, whereas there is a description of stillbirth at 5 months of gestation (52) . between 2012 and 2016, the ministry of health of saudi arabia reported the occurrence of 1,308 cases of mers-cov infection, five of which were pregnant (53) . despite the few descriptions, the immunological changes in pregnancy may alter the susceptibility to mers-cov and the severity of the clinical disease (51) . in a mice model of herpes virus infection, even in the absence of herpes virus placental passage, there was a marked increase in the levels of pro-inflammatory cytokines, including ifn-γ and tnf-α, as well as changes in fetal development (30) . this scenario may result from the placenta's pro-inflammatory response generated by the infection, or it may be due to other physiological changes in the mother or placenta related to the infectious process (54) . placental cells, predominantly trophoblasts, express tlr (toll-like receptors) and this expression varies according to the gestational age and the differentiation stage of these cells. viral infections can disturb the fine immune regulation at the maternal-fetal interface and lead to fetal damage, even without the virus reaching it directly (55) . for example, tlr-3 expressed by trophoblasts in the first trimester of pregnancy (56) , mediates rapid antiviral response (57) , and induces the production of cytokines, type i interferon (ifn) and type iii ifn (58) . tlr7 is also expressed in trophoblasts, which induces the synthesis of anti-viral cytokines and plays a role in preventing intrauterine transmission of hbv (59) . however, these inflammatory responses can be associated with complications in pregnancy, such as pre-eclampsia and/or intrauterine growth deficit (1) . in general, cytokines and ifns are important mediators in a healthy pregnancy, due to their role in the regulation of cell function, proliferation, and gene expression. however, when dysregulated, they have the potential to interrupt fetal and placental development pathways (60). the world health organization (who) estimates that about 2.5 million children died within the first month of life in 2018. every day ∼7,000 newborns die, amounting to 47% of all child mortality under the age of 5 years (61) . the majority of all neonatal's deaths are due to preterm birth, intrapartum-related complications (birth asphyxia or lack of breathing at birth), infections and birth defects. regarding the highest incidence of infection observed in early-life, it is generally attributed to an immature immune system during the transitional post-natal period (62) . innate immune cells are composed of specialized cells, such as granulocytes (e.g., neutrophil), monocytes, macrophages, dcs and innate lymphocytes. around 5 weeks gestation, neutrophils are present in human fetal liver parenchyma (63) , when compared to the adult response, neonatal neutrophils have qualitative and quantitative impairments in the response under stress conditions, including reduced chemotaxis, respiratory burst, and extracellular traps formation (64) . the cytokine profile produced by antigen-presenting cells (apcs) monocyte/macrophage and dcs in newborn differs from those produced by adults. typically, apcs from neonates produce less pro-inflammatory cytokines like il-1β, tnf-α, il-12p70, and type i ifn upon stimulation on tlrs (65) . otherwise, it produces great amounts of th17-promoting cytokines (il-6 and il-23) when compared with adult cells (66) . following, the importance of anti-inflammatory response in early life is highlighted through the great amount of il-10 produced by newborn monocyte/conventional dc (cdc) compared to adults (67) . the pattern of innate cytokine response can be attributed to two mechanisms: (i) high mononuclear cell levels of intracellular cyclic adenosine monophosphate (camp), a secondary messenger that suppresses th1 but enhances th2 and anti-inflammatory cytokine production (68) and (ii) altered dna binding capacity of transcription factors, such as irf3 to the promoter regions of cytokine genes secondary to age-specific chromatin (69) . curiously, neonates' dcs activation with clr agonist dectin or macrophage-inducible c-type lectin (mincle), simultaneously with tlr7/8 potently drives caspase-1 and nf-kb activation and th1-supporting cytokine production (including il-12p70), overcoming the age-specific epigenetic barrier in early life for irf3 function and leading to a th-1 phenotype (70, 71) . on 14 weeks of gestation, mature fetal αβ t lymphocytes can be detected. during the second and third trimesters of gestation, the repertoire of fetal t cell receptors diversifies (72) . generally, neonates have a limited th1 profile response to some vaccines and pathogens, agreeing with a lower capacity of cd4 t cells to produce ifn-γ and of apcs to produce th1-skewing cytokines (73) . although there are some situations where the responsiveness of the th1 profile is efficient, for example, neonates and infants develop adult-like th1 responses to bcg or pertussis vaccines, and a fetus can develop th1 responses in congenital cmv infection (74) (75) (76) . recent studies suggested that the early life immune system could present advantages for the elicitation of broadly neutralizing antibodies (bnabs), a response highly desired for an hiv vaccine. in fact, hiv-infected children develop bnabs responses earlier and more frequently than infected adults (77) . congenital and perinatally acquired viral infections do occur and may lead to major disabilities in infancy and childhood, the main causes can be attributed to pathogens like toxoplasma gondii, rubella virus, cytomegalovirus (cmv), herpes viruses, syphilis, and zika virus (78) . while congenital rubella virus syndrome is no longer seen in countries with compulsory immunization against this virus, an outbreak of zika virus (zikv) recently occurred in brazil resulting in the zikv syndrome, with brain lesions comparable to, but more severe than congenital cmv infection (79) . neonates display an immature immune response, the first exposition to an environmental stimulus can shape the lung's immune response (80) . furthermore, there is a predominant type 2 immune response in the lungs (81), these characteristics make infants susceptible to respiratory viral infections, a common cause of infant's death (82) . rsv is an important cause of lower respiratory tract illness in infants globally and is responsible for one-third of deaths due to lower respiratory tract infections in children <1 year of age (83) . pregnant women are considered at high risk for severe influenza disease, for this reason, influenza vaccination has been recommended for pregnant women and introduced into immunization programs (84) . influenza vaccination is safe and protective on preterm birth (ptb) and low birth weight (lbw) (85) . one of the benefits of maternal immunization has also been shown to extend to neonates through the transfer of maternal antibodies, providing passive immunization against the influenza virus (86). on the severe 2009 pandemic h1n1 influenza illness, some studies suggested an association between severe h1n1 disease, preterm birth, and fetal death; however, these limited data do not permit firm conclusions (35) . sars-cov-1 infected ∼100 pregnant women during the pandemic (87), causing a high lethality and miscarriage rate (88) , but no neonatal infection has been reported (88) . in 2017, cynthia maxwell postulated possible intensive care and procedures to properly manage maternal and neonatal sars-cov-1 infections (89) . vertical transmission of mers has not been documented. in a case report by alserehi et al., a mother was diagnosed with mers, treated and a cesarean section was performed to deliver a healthy preterm baby with 32 weeks of gestation (52) . hon et al. described 14 children with mers, that presented persistent fever and cough, after treatment no fatal case was reported. all children in this report obtained the infection via adult-to-children transmission, and no children-tochildren transmission was reported (90) . iqbal et al. reported a case of spontaneous vaginal delivery in covid-19-positive pregnant, with no signs of neonatal infection up to 7-days post-partum (91) . nevertheless, it is important to highlight contact precautions were made in this report to prevent post-partum transmission. in late 2019, a respiratory infectious disease began to be investigated in wuhan, china (92) . at first, contagion occurred through contact with some infected animals but, soon there were the first reports of human-to-human transmission (93), the virus was identified as belonging to the coronaviridae family and was designated sars-cov-2 (severe acute respiratory syndrome coronavirus-2) (94). like other members from this viral family, mers and sars-cov-1, the new coronavirus causes a respiratory disease, named covid-19 (coronavirus disease−2019) (95) . although very similar, sars-cov-1 and sars-cov-2 impacted the world differently. sars-cov-1 emerged in 2002 and killed almost 800 people in 26 countries (96) and, even without a vaccine, it was taken preventive actions as patient isolation. the new coronavirus has killed more than 480,000 people in just 6 months and has spread to 5 continent (97). sars-cov-2 shares genetic similarities between sars-cov-1 and mers, 79 and 50%, respectively (98) . sars-cov-2 is an enveloped single-stranded rna virus and has a genome of ∼30,000 nucleotides that encode structural and accessory proteins-the largest known viral rna genome (99) . sars-cov-1 and sars-cov-2 enter the host's cells via the ace2 receptor (angiotensin-converting enzyme 2) (100). in the lung, the most affected organ among those infected, the main target is the type 2 alveolar cell (101) . the ace2 receptor is also expressed in cells from kidneys, esophagus, heart (102) . moreover, a small percentage of monocytes and macrophages express the ace2 receptor (94, 99) . thus, there may be another alternative receptor or infectious pathway, such as antibody-dependent enhancement (ade). however, unlike other coronaviruses, limited to respiratory disorders, sars-cov-2 caused multiple organ failure. furthermore, this receptor is more expressed in the elderly, which associated with immunosenescence and other comorbidities common among the elderly may justify the high lethality rate in this age group (103) . the viral load peaks occur during the first week of infection and then gradually decrease over the next few days. in addition, the viral load is correlated with the patient's age. igg and igm antibodies start to increase 10 days after disease and most patients are seroconverted in the first 20 days (104) . moreover, in vitro assays, has shown that the serum from sars-cov-2-infected patients were able to neutralize the virus (101) . thereby, the humoral response can be another antiviral strategy via plasma transfer (105) . in sars-cov-1 and mers, as a viral escape mechanism, the virus can suppress ifn type i response, either by cytosolic sensors of ubiquitination, inhibiting nuclear factors translocation or decreasing stat1 phosphorylation (106) . neutrophils, c-reactive protein and several cytokines (as il-6, tnf, il-10) are increased in covid-19, and this elevation is correlated with disease severity and death (97) . in serious illness, the same protein levels were detected and inflammatory cytokines increase is correlated with t cd4 + and t cd8 + lymphocytes decrease and lower ifnγ production. b-lymphocytes do not appear to be affected by the disease, regardless of severity (92, 103, 107) . these characteristics observed in patients indicate that a covid-19 can be mediated by an intense inflammatory process that follows the disease severity. as with sars-cov-1 and mers, this increase in cytokine levels-known as a cytokine storm-can be involved with the pathogenesis of the disease (92). to defend itself against an aggressive agent (such as infection, trauma, acute inflammation, among others) the body produces an exaggerated response to localize and then eliminate the damage. this response is known as the systemic inflammatory response syndrome (sirs) or, if the source infection sepsis (108) , this process leads to the release of acute-phase proteins and endocrine, hematological and immunological changes, among them, the cytokine storm can lead to tissue damage and even death (109) . cytokine storm is produced, mainly, by highly activated macrophages and can cause lung damage and start viral sepsis (110) . this inflammation leads to other complications, such as acute respiratory distress syndrome (ards) and respiratory and cardiac failure (48, 111) . studies in mice infected with sars-cov-1, also demonstrate the cytokine storm dampening adaptive immunity (112) . other factors may also influence the susceptibility for covid-19 infected persons, and some gene polymorphisms, well-documented for other viral infections (113) . at the moment no vaccine or specific treatments are available for disease control of the sars-cov-2. in pregnancy, pneumonia infections may trigger an increased mortality risk to the mother and fetus (114) , which can also lead to complications as preterm birth and small for gestational age (115) . placental syncytiotrophoblast cells express the ace2 receptor and this receptor is highly expressed in the first months of pregnancy. associated with placental immaturity, the early ace2 expression can make the first trimester the most likely period for sars-cov-2-infection (14) . a serine protease, tmprss2, is also required for viral entry (100, 116) and there is still no consensus about placenta expression. some studies report low, but present, mrna expression in human placentas (117) , others describe that expression is not detectable (118) . the association of tmprss2 and ace2 expression, in the first months of pregnancy, would make this phase more susceptible to sars-cov-2-infection. blood tests in pregnant women revealed regular covid-19 markers, such as lymphopenia, neutrophilia, and elevated c-reactive protein level in pregnant women (119, 120) . some reports also verified an increase in alt, ast, and d-dimer (120) (121) (122) ). an important report verified that 3 mothers developed anemia and dyspnea, which could potentially be a risk factor during c-section labor (123) . chen and collaborators, verified alteration in calcium and albumin levels in the blood of pregnant women with sars-cov-2 infection (124) , which could potentially increase the severity in covid-19 (125) . furthermore, in a recent report involving maternal death in consequence to covid-19, 2 cases reported a low number of platelets, which is associated with an increase in mortality by covid-19 (126, 127) . it is still under investigation the effects of sars-cov-2infection in the maternal-fetal context ( table 1) . some reports describe that symptomatic infected-mothers did not transmit the virus during pregnancy. in a case report of seven cases, showed that three babies were tested to sars-cov-2 and only 1 was positive 36 h post-partum (138) . on the other hand, another report shows increase in inflammatory cytokines and virus-specific igm levels in newborns, from infected-mothers, 2 h after birth (120) , and in another report, newborns presented virus-specific igm and igg, but no sars-cov-2-infection ( table 1 ) (128) . this lead to the possibility of the activation of the maternal immune system by sars-cov-2 may have some implication of the offspring's health and immune system development. although the number of pregnant women with covid-19 studies is limited, there is no conclusive report of vertical transmission ( table 1 ) (129, 139) . a recent case report, was described two cases of rashes and one with facial ulcerations (123) . another important factor, besides the immune activation, the maternal usage of antiviral drugs can also permanently affect the offspring's immune response (140) , as there is no current standard protocol of treatment regarding the usage of antibiotics or antivirals ( table 1 ) (115) . only a fraction of patients infected with sars-cov-2 develops severe respiratory disorders, it is unknown whether the pregnant could be more susceptible to pulmonary diseases. covid-19 can progress to a severe lung inflammation that can progress to life-threatening illness at the severe stage (141) . this inflammatory process is associated with high plasma levels of cytokines, as cytokines storm, including il-2, il-7, il-10, g-csf, ip-10, mcp-1, mip-1a, and tnfα (92) . this might play an important role in pregnancy as il-2 has been implicated to be upregulated in pre-eclampsia (142) and miscarriage (143) and il-7/il-7r signaling pathway in fetal miscarriage (144) , due to the upregulation in the ratio of th17/treg cells (145) . another relevant aspect is the possible implication of polymorphisms in covid-19 diseases, as is well-documented for other viral infections (114) . also, cytokines polymorphisms, such as tnf-α 308g/a (rs1800629) polymorphism is associated with recurrent miscarriage (146) . in fact, tnf-α and tnf-α receptor play an important role in the development of the fetus, being present in the ovary, endometrium, placenta, and fetus, and in the amniotic fluid in different concentration (147) . this increase in tnf-α during pregnancy may implicate in different health outcomes depending on the gestational period (148) , leading to tissue necrosis in the placenta and hypoxia (149) . interestingly, an acute increase of this cytokine during pregnancy in animals may cause abortion (7) . moreover, alteration in the health status of the mother during pregnancy can have long-term effects on the offspring's health (150) . inflammatory processes during pregnancy can also impact women's health, as the increase in tnf-a during pregnancy can also lead to impaired insulin sensitivity (151) and gestational diabetes mellitus (152) . in animal models, inflammation during pregnancy has been shown to alterations in the behavior (153, 154) fetal brain development (155) (156) (157) , metabolic disturbance (158, 159) , and shape offspring's immune response to antigens and infections (160, 161) . the physiological response, as stress and the control of temperature, during the infection may present a long-term effect in pregnant women with covid-19. the increase in stressrelated hormones can also affect the offspring's immune system (162) and fever during pregnancy increase the chances of neural disorders in the children (163) . moreover, an increase in anti-inflammatory cytokine il-10 in covid-19 mothers is probably a regulatory mechanism crucial to regulate the inflammation (164) and pregnancy maintenance (165). even though no vertical transmission for covid-19 has been reported until now, several reports of early-life infections have been described with very low death rates (98, 119) . reports with recommendations to the treatment of pregnant women with covid-19 (166) and for neonates with covid-19 have been published (167, 168) . another possible route for sars-cov-2 is oral transmission by fecal samples (169) , and via breastfeeding from a sars-cov-2 infected mother. regarding breastfeeding, a small study found no evidence of covid-19 in breast milk, of six patients (139) . however, the primary concern is whether an infected mother can transmit the virus through respiratory droplets during breastfeeding. other viruses in the past have also caused concern in pregnant women. the zika virus has been linked to several cases of microcephaly in newborns during an epidemic in 2015 in brazil (170) . the infection had a high point in the first trimester of pregnancy, where there were more favorable conditions for its entry and replication in placental cells. in the case of sars-cov-2, it has not yet possible due to the time of infection occurs in the world, to observe the consequences of infection in the firsttrimester pregnancy. taking into account the early pregnancy, the placental tissue immaturity together with the up-regulation of ace2 expression in placental cells, perhaps the more susceptible period for sars-cov-2 infection is around the first trimester of pregnancy. it is important to highlight that after the 2009 influenza pandemic there have been reports of reduced cytokine response to bacterial infections. this leads to the hypothesis that covid-19 can lead to impairments of the immune response to other pathogens and vaccines in the future. future investigations are needed to identify the possible implications of sars-cov-2/covid-19 in pregnancy, the possible infection of the placenta in the first trimester of pregnancy and implications of the cytokine storm to the neonatal's health. ra and ms: write, conception, and review. np, lo, and sg-s: write and review. all authors contributed to the article and approved the submitted version. ra holds a post-doctorate fellowship from fapesp (19/02679-7) and lo also holds a post-doctorate fellowship from fapesp (19/07976-0). sg-s holds a master degree fellowship from fapesp (19/22448-0). the immune system in pregnancy: a unique complexity immune mechanisms at the maternal-fetal interface: perspectives and challenges self-recognition and the role of fetal microchimerism male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum chimerism occurs in thyroid, lung, skin and lymph nodes of women with sons characterizing the pregnancy immune phenotype: results of the viral immunity and pregnancy (vip) study how immune mechanisms are affected by pregnancy the transforming growth factor-ss superfamily cytokine macrophage inhibitory cytokine-1 is present in high concentrations in the serum of pregnant women maternal b lymphocytes specific for paternal histocompatibility antigens are partially deleted during pregnancy influenza immunization in pregnancy-antibody responses in mothers and infants neonatal outcomes after antenatal influenza immunization during the 2009 h1n1 influenza pandemic: impact on preterm birth, birth weight, and small for gestational age birth monocytes are progressively activated in the circulation of pregnant women the expression and localization of the human placental prorenin/renin-angiotensin system throughout pregnancy: roles in trophoblast invasion and angiogenesis? regulation of placental development and its impact on fetal growth-new insights from mouse models risks associated with viral infections during pregnancy hla-g: at the interface of maternal-fetal tolerance membrane progesterone receptors in human regulatory t cells: a reality in pregnancy the dual role of hla-c in tolerance and immunity at the maternal-fetal interface new paradigm in the role of regulatory t cells during pregnancy single-cell reconstruction of the early maternal-fetal interface in humans endocrine factors modulating immune responses in pregnancy enhanced foxp3 expression and treg cell function in pregnant and estrogen-treated mice induction of regulatory t cells by physiological level estrogen cutting edge: estrogen drives expansion of the cd4 + cd25 + regulatory t cell compartment viral infections during pregnancy the association between viral infections, maternal and fetal mortality/morbidity intrauterine infection with varicellazoster virus after maternal varicella preparing for influenza after 2009 h1n1: special considerations for pregnant women and newborns pandemic 2009 influenza a(h1n1) virus illness among pregnant women in the united states influenza occurring in pregnant women: a statistical study of thirteen hundred and fifty cases deaths from asian influenza associated with pregnancy the influence of pregnancy on systemic immunity maternal influenza and birth outcomes: systematic review of comparative studies ebola hemorrhagic fever and pregnancy what obstetrician-gynecologists should know about ebola: a perspective from the centers for disease control and prevention a prospective study of maternal and fetal outcome in acute lassa fever infection during pregnancy lassa fever in pregnancy: report of 2 cases seen at the university college hospital demethylation profile of the tnf-α promoter gene is associated with high expression of this cytokine in dengue virus patients pathology of congenital zika syndrome in brazil: a case series sickle-cell erythrocytes in the placentas of dengue-infected women the placentas of patients with severe acute respiratory syndrome: a pathophysiological evaluation pregnancy with new coronavirus infection: clinical characteristics and placental pathological analysis of three cases placental histopathological lesions in correlation with neonatal outcome in preeclampsia with and without severe features second-trimester miscarriage in a pregnant woman with sars-cov-2 infection a casecontrolled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome pneumonia in pregnancy sars and mers: recent insights into emerging coronaviruses potential maternal and infant outcomes from (wuhan) coronavirus 2019-ncov infecting pregnant women: lessons from sars, mers, and other human coronavirus infections impact of middle east respiratory syndrome coronavirus (mers-cov) on pregnancy and perinatal outcome hospital outbreak of middle east respiratory syndrome coronavirus viral infection of the placenta leads to fetal inflammation and sensitization to bacterial products predisposing to preterm labor toll-like receptors and pregnancy: trophoblast as modulators of the immune response a role for tlrs in the regulation of immune cell migration by first trimester trophoblast cells recognition of doublestranded rna and activation of nf-kappab by toll-like receptor 3 type iii interferons produced by human placental trophoblasts confer protection against zika virus infection maternal-derived hepatitis b virus e antigen alters macrophage function in offspring to drive viral persistence after vertical transmission protecting the newborn and young infant from infectious diseases: lessons from immune ontogeny ontogeny of myeloid cells neutrophil production and function in newborn infants innate cellular immune responses in newborns innate immunity of the newborn: basic mechanisms and clinical correlates regulatory activity of autocrine il-10 on dendritic cell functions the adenosine system selectively inhibits tlr-mediated tnfalpha production in the human newborn age-related gene expression differences in monocytes from human neonates, young adults, older adults dectin-1 activation unlocks il12a expression and reveals the th1 potency of neonatal dendritic cells age-specific adjuvant synergy: dual tlr7/8 and mincle activation of human newborn dendritic cells enables th1 polarization timely and spatially regulated maturation of b and t cell repertoire during human fetal development unbalanced neonatal cd4(+) t-cell immunity newborns develop a th1-type immune response to mycobacterium bovis bacillus calmette-guérin vaccination bordetella pertussis infection in 2-monthold infants promotes type 1 t cell responses functional exhaustion limits cd4 + and cd8 + t-cell responses to congenital cytomegalovirus infection immunological mechanisms of inducing hiv immunity in infants treatment of perinatal viral infections to improve neurologic outcomes viral infections and the neonatal brain age-specific incidence of influenza a responds to change in virus subtype dominance pulmonary susceptibility of neonates to respiratory syncytial virus infection: a problem of innate immunity? respiratory viruses and sudden infant death evaluation of maternal mortality under-reporting in the heights of chiapas using the ramos and modified ramos strategies maternal immunisation: what have been the gains? where are the gaps? what does the future hold? the safety of inactivated influenza vaccines in pregnancy for birth outcomes: a systematic review influenza vaccination of pregnant women and protection of their infants consensus document on the epidemiology of severe acute respiratory syndrome (sars) pregnancy and perinatal outcomes of women with severe acute respiratory syndrome 225-management guidelines for obstetric patients and neonates born to mothers with suspected or probable severe acute respiratory syndrome (sars) clinical presentations and outcome of severe acute respiratory syndrome in children an uncomplicated delivery in a patient with covid-19 in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating personto-person transmission: a study of a family cluster a novel coronavirus from patients with pneumonia in china coronaviruses as the cause of respiratory infections world health organization. coronavirus disease (covid-2019) situation reports 158 coronavirus disease (covid-19) and neonate: what neonatologist need to know genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection clinical and immunologic features in severe and moderate coronavirus disease 2019 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study convalescent plasma as a potential therapy for covid-19 interaction of sars and mers coronaviruses with the antiviral interferon response the pathogenesis of spinal cord involvement in dengue virus infection definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis the systemic inflammatory response syndrome the landscape of lung bronchoalveolar immune cells in covid-19 revealed by single-cell rna sequencing mers-cov infection in humans is associated with a proinflammatory th1 and th17 cytokine profile dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice host single nucleotide polymorphisms modulating influenza a virus disease in humans pneumonia in pregnancy pneumonia and pregnancy outcomes: a nationwide population-based study tmprss2 isoform 1 activates respiratory viruses and is expressed in viral target cells expression of transmembrane serine protease tmprss2 in mouse and human tissues prostatelocalized and androgen-regulated expression of the membrane-bound serine protease tmprss2 a case report of neonatal covid-19 infection in china possible vertical transmission of sars-cov-2 from an infected mother to her newborn clinical analysis of 10 neonates born to mothers with 2019-ncov pneumonia neonatal early-onset infection with sars-cov-2 in 33 neonates born to mothers with covid-19 in wuhan, china infants born to mothers with a new coronavirus (covid-19). front pediatr clinical analysis of pregnant women with 2019 novel coronavirus pneumonia mortality from covid-19 increases with unsaturated fat, and may be reduced by early calcium and albumin supplementation association between platelet parameters and mortality in coronavirus disease 2019: retrospective cohort study thrombocytopenia is associated with severe coronavirus disease 2019 (covid-19) infections: a meta-analysis antibodies in infants born to mothers with covid-19 pneumonia lack of vertical transmission of severe acute respiratory syndrome coronavirus 2, china first case of neonatal infection due to covid 19 in spain covid-19 vaginal delivery-a case report analysis of the pregnancy outcomes in pregnant women with covid-19 in hubei province coronavirus disease 2019 (covid-19) during pregnancy: a case series covid-19 in a 26-week preterm neonate covid-19 infection in first trimester of pregnancy marked by a liver cytolysis: a case report evidence of mother-to-newborn infection with covid-19 maternal death due to covid-19 clinical features and obstetric and neonatal outcomes of pregnant patients with covid-19 in wuhan, china: a retrospective, single-centre, descriptive study clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records testicular effects following in utero exposure to the antivirals acyclovir and ganciclovir in rats pathological findings of covid-19 associated with acute respiratory distress syndrome interleukin-2 receptor serum concentrations in normal pregnancy and pre-eclampsia the involvement of inflammatory cytokines in the pathogenesis of recurrent miscarriage abnormal il-2 receptor levels in non-pregnant women with a history of recurrent miscarriage il-7/il-7r signaling pathway might play a role in recurrent pregnancy losses by increasing inflammatory th17 cells and decreasing treg cells association of tumor necrosis factoralpha 308g/a polymorphism with recurrent miscarriages in women human tumour necrosis factor: physiological and pathological roles in placenta and endometrium tumor necrosis factor-α and pregnancy complications: a prospective study placental tnf-α signaling in illness-induced complications of pregnancy programming of fetal insulin resistance in pregnancies with maternal obesity by er stress and inflammation tnf-alpha is a predictor of insulin resistance in human pregnancy maternal circulating concentrations of tumor necrosis factor-alpha, leptin, and adiponectin in gestational diabetes mellitus: a systematic review and meta-analysis prenatal immune challenge affects growth, behavior, and brain dopamine in offspring in utero exposure to virus infections and the risk of developing anorexia nervosa maternal infection: window on neuroimmune interactions in fetal brain development and mental illness prenatal exposure to maternal infection alters cytokine expression in the placenta, amniotic fluid, fetal brain prenatal lps-exposurea neurodevelopmental rat model of schizophrenia-differentially affects cognitive functions, myelination and parvalbumin expression in male and female offspring prenatal lipopolysaccharide exposure promotes dyslipidemia in the male offspring rats prenatal viral exposure followed by adult stress produces glucose intolerance in a mouse model prenatal initiation of endotoxin airway exposure prevents subsequent allergeninduced sensitization and airway inflammation in mice thrown off balance: the effect of antenatal inflammation on the developing lung and immune system maternal stress during pregnancy increases neonatal allergy susceptibility: role of glucocorticoids hertz-picciotto i. is maternal influenza or fever during pregnancy associated with autism or developmental delays? results from the charge (childhood autism risks from genetics and environment) study effect of anti-tnf-α on the development of offspring and pregnancy loss during pregnancy in rats sars-cov-2 infection during pregnancy. information and proposal of management care emergency plan for inter-hospital transfer of newborns with sars-cov-2 infection chinese expert consensus on the perinatal and neonatal management for the prevention and control of the 2019 novel coronavirus infection detectable sars-cov-2 viral rna in feces of three children during recovery period of covid-19 pneumonia zika virus in the americas: early epidemiological and genetic findings the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 alberca, pereira, oliveira, gozzi-silva and sato. this is an openaccess article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-284409-xiyeceib authors: prabakaran, ponraj; chen, weizao; dimitrov, dimiter s. title: the antibody germline/maturation hypothesis, elicitation of broadly neutralizing antibodies against hiv-1 and cord blood igm repertoires date: 2014-08-28 journal: front immunol doi: 10.3389/fimmu.2014.00398 sha: doc_id: 284409 cord_uid: xiyeceib we have previously observed that all known potent broadly neutralizing antibodies (bnabs) against hiv-1 are highly divergent from their putative germline predecessors in contrast to bnabs against viruses causing acute infections such as henipaviruses and sars cov, which are much less divergent from their germline counterparts. consequently, we have hypothesized that germline antibodies may not bind to the hiv-1 envelope glycoprotein (env) because they are so different compared to the highly somatically mutated hiv-1-specific bnabs. we have further hypothesized that the immunogenicity of highly conserved epitopes on the hiv-1 envelope glycoproteins (envs) may be reduced or eliminated by their very weak or absent interactions with germline antibodies and immune responses leading to the elicitation of bnabs may not be initiated and/or sustained. even if such responses are initiated, the maturation pathways are so extraordinarily complex that prolonged periods of time may be required for elicitation of bnabs with defined unique sequences. we provided the initial evidence supporting this antibody germline/maturation hypothesis, which prompted a number of studies to design vaccine immunogens that could bind putative germline predecessors of known bnabs and to explore complex b cell lineages. however, guiding the immune system through the exceptionally complex antibody maturation pathways to elicit known bnabs remains a major challenge. here, we discuss studies exploring the antibody germline/maturation hypothesis as related to elicitation of bnabs against hiv-1 and present our recent data demonstrating the existence of germline-like precursors of vrc01 antibodies in a human cord blood igm library. elicitation of broadly neutralizing antibodies (bnabs) targeting the hiv-1 envelope glycoproteins (envs), the key to an effective hiv-1 vaccine, remains elusive. previous studies have demonstrated several properties of the hiv-1 envs that could limit their ability to elicit bnabs. these include protection of the conserved structures by variable loops (1) (2) (3) , remarkable genetic diversity (4), a glycan shield (5) , steric occlusion (6) , and conformational masking (7) . until 6 years ago, only a handful of bnabs, including b12, 2g12, 2f5, and 4e10, were known. although the structural and functional studies of those bnabs revealed some important neutralization epitopes (8) , such bnabs have not been successfully elicited by any vaccination approach. in 2007, we first noted that in hiv-1 specific bnabs the number of amino acid mutations from their closest corresponding germline sequences was significantly higher than that of bnabs against the sars cov coronavirus, and hendra and nipah viruses, which cause self-limiting acute infections (9) . using a large nonimmune igm library, we identified several hiv-1 env specific antibodies and found that they had fewer somatic mutations than the hiv-1 bnabs, as well as limited neutralizing activity (9) . these findings indicated that elicitation of hiv-1 bnabs would require far more extensive maturation processes than those needed to generate the bnabs against the sars cov and henipaviruses. so, we have suggested that the difficulty of eliciting these bnabs may be due, at least in part, to the complex and prolonged maturation pathways required for the development of bnabs against the hiv-1, which can take long time (10) . we thus speculated that this may represent another significant challenge in the development of effective hiv-1 vaccines. we quantified the number of mutations in human monoclonal antibodies (mabs) that we selected from phage libraries generated from an hiv-1-infected patient with a known time of infection (10) . we calculated the number of amino acid mutations per heavy chain v gene, and defined it as antibody somatic mutational diversification (asmd). we compared the extent and dynamics of the asmd between hiv-1-specific mabs and a panel of sars covspecific mabs. our experiments based on the asmd predicted that elicitation of hiv-1-specific bnabs would take at least 3 years. an illustrative mathematical model using the asmd rate based on www.frontiersin.org an exponential time dependent function suggested that a much longer time would be needed for the required maturation, unless somatic diversification had already been initiated from an intermediate antibody. thus, all these initial studies corroborated our hypothesis that the infrequent occurrence or absence of bnabs in hiv-1-infected patients could be due, at least in part, to the lack or limited availability of b cell receptors that rapidly mature into bnabs. therefore, we suggested that appropriate immunization protocols of long duration need to be developed using the knowledge gained from the exploration of antibody maturation pathways in humans (10) . from the striking observation that all known potent hiv-1 bnabs are highly divergent from their putative germline predecessors in contrast to bnabs against henipaviruses and sars cov coronavirus, we hypothesized that, since the germline antibodies are so different compared to the highly somatically mutated hiv-1 bnabs, they may not bind to the env. this led us to the hypothesis that the immunogenicity of the highly conserved epitopes on the hiv-1 native envelope glycoproteins (envs) is reduced or eliminated by their very weak or absent interactions with germline antibodies, which could not initiate and/or sustain immune responses leading to elicitation of bnabs: even if immune responses are initiated and sustained, the maturation pathways are so complex that help and long times may be needed for their elicitation. to test our antibody germline/maturation hypothesis, we designed germline-like antibodies corresponding to the known bnabs b12, 2g12, 2f5, x5, m44, and m46 (the latter three antibodies were discovered in our laboratory and possess hiv-1 cross-reactivity with moderate neutralizing activities) and evaluated them for binding to envs (11) . we found that while germline-like x5, m44, and m46 bound to envs with relatively high affinity, the germline-like precursors of b12, 2g12, and 2f5 failed to bind envs in an elisa assay although their corresponding mature bnabs bound strongly. these results provided initial evidence that the env structures containing conserved epitopes might not initiate humoral responses due to limited or absent binding to the germline precursors of bnabs. these germline precursors may also be of limited availability as recently reported (12) . following that initial study, we expanded our investigation to different variants of the two different antibodies (b12 and x5) including their closest germline counterparts and several germline-like intermediates (13) . the experiments showed that b12 intermediate antibodies neutralized only some hiv-1 isolates with relatively weak potency. in contrast, intermediates of x5 neutralized a subset of the tested hiv-1 isolates with efficiencies comparable to those of the matured x5. these results helped explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of cd4-induced (cd4i) antibodies in hiv-1-infected patients (x5 is a cd4i antibody) as well as the maturation pathway of x5. in the case of b12, germlinelike intermediates along the maturation pathway were shown to not only bind some envs but also human self antigens, suggesting that antigens other than the envs could help guide the immune system through the b12 maturation pathway. therefore, we proposed a conceptually new vaccination approach, in which it is critical to identify primary immunogens that bind to the germline antibodies that are predecessors of bnabs. if needed, these immunogens should be combined with secondary immunogens that recognize intermediate and/or matured antibodies to guide the immune system through the prolonged, complex maturation pathways (14) . in this respect, we envisioned that the knowledge of human antibodyomes would become indispensable to elucidate the origin, diversity, and maturation pathways of bnabs and discover germline-like intermediates of bnabs that could provide a basis for the design of novel hiv-1 vaccine immunogens (14, 15) . in recent years, several groups have reported a number of new bnabs that were identified from multiple hiv-1 infected individuals using designed novel antigen baits and advanced technologies implemented in isolating human mabs and high-throughput sequencing (16) . particularly, haynes, kwong, stamatatos, scheif et al. have dealt with a large amount of data delineating structural, genetic determinants, and maturation pathways of different bnabs. these studies not only confirmed our previous findings that the envs fail to engage germline versions of bnabs but also suggested possible holes in b cell repertoires and demonstrated the implications of our antibody germline/maturation hypothesis for finding germline-like precursors, intermediates as well as for designing immunogens that could potentially bind to such bnab intermediates. in this report, we discuss the recent advancements in hiv-1 vaccine research in the context of the antibody germline/maturation hypothesis, and highlight critical factors to be considered when exploring germline-like precursors and intermediates of bnabs. we also report for the first time using 454 sequencing data analysis of a human cord blood igm library to identify putative germline precursors of the heavy and light chains of vrc01-like antibodies. these naturally occurring cord blood-derived vrc01-like heavy and light chains may be useful as putative templates for designing novel vaccine immunogens that can lead to the elicitation of vrc01-like antibodies and for understanding the maturation pathways of this bnab. still there are major challenges to be overcome. new empirical and semiempirical approaches could be successful; recently, new paradigms were discussed that could better fit our increased knowledge of hiv immunopathology and which could possibly be more helpful in guiding future vaccine research than did past unsuccessful approaches (17) . dna isolation, amplification, and 454 sequencing of the human cord blood igm library were previously described in detail (18, 19) . for quality control, we trimmed the 454 sequence reads and retained only sequences with lengths of more than 300 nucleotides, covering the entire antibody variable domains consisting of all three complementarity determining regions (cdrs) along with framework regions (frs). we used imgt/highv-quest for immunogenetic and statistical analyses (20) . the output results from the imgt/highv-quest analysis were stored in a local postgresql database, and structured query language (sql) was used to retrieve the data for further analysis. statistical calculations were carried out using jmp10® statistical software (sas institute, cary, nc, usa). antibody sequences from ighv1-2 and igk3-11 lineages were retrieved from our local antibodyome database consisting of immunogenetic data derived from 454 sequencing of the human cord blood igm library using sql statements. amino acid sequence identities between each of the selected lineage sequences from the 454 sequence data and pertinent germline sequences were calculated based on the pairwise alignment using local blast as implemented in bioedit v7.0.9 (21) . phylogenetic analysis was carried out using the archaeopteryx software (22) . our earlier observation of the extensive maturation of hiv-1 bnabs in contrast to those against some viruses causing acute infections led to the antibody germline/maturation hypothesis (9-11, 13, 14) . according to this hypothesis, it is critical to identify immunogens that would bind to germline and/or intermediate antibodies of bnabs, as well as the exploration of antibodyomes could be useful for identifying such immunogens (14) . figure 1 describes the timeline involving some of the key developments in current hiv-1 vaccine research focused on antibody germline-like intermediates and maturation pathways of bnabs. major research efforts in this direction were spearheaded by deep sequencing and structural biology studies of vrc01-like and other cd4-binding site (cd4b) antibodies from hiv-1-infected individuals. these studies delineated possible maturation pathways of such antibodies with high levels of somatic mutations and convergence in antibody recognition (23, 24) . both studies revealed that the putative germline precursors of these antibodies had weak or no apparent affinity for env, and acquisition of a large number of somatic mutations were needed for the breadth and potency of these antibodies. these studies also explored antibody diversity and found many intermediates of similar lineages of the heavy chain genes from the two ighv families vh1-2 and vh1-46 that paired with different light chain genes. thus, analysis of the vrc01-related antibodyome from hiv-1 infected patients revealed b cell maturation pathways that may help guide the vaccine-induced elicitation of such antibodies. however, if we could find germline-like intermediates of such bnabs from a naïve antibody repertoire, then www.frontiersin.org potential vaccine immunogens developed based on those templates would stimulate an adequate b cell immune response in healthy humans. to this end, we identified vrc01-like intermediate antibodies from a naïve antibody library of human cord blood, which is presented later in the text. we previously analyzed the igm repertoires of healthy individuals and identified several intermediates of b12 from the vh1-3 gene family (15) . sequence analysis of 28,925 unique sequences from the igm repertoires revealed a cdrh3 with a length (20 amino acids) and sequence similar (50%) to that of the b12 cdrh3, but the v gene associated with that cdrh3 was found to be hv4-b (15) . this finding indicates that long cdrh3s may not be a limiting factor for the development of bnabs (25) although long cdrh3 motifs with certain amino acid preferences and/or associations with particular heavy or light chain families favoring polyreactivity may not be undermined. stamatatos and coworkers have conducted experiments screening a large panel of recombinant envs for binding to the germline predecessors of b12, nih45-46, and 3bnc60 to test how env immunogens interact with the predicted germline versions of known bnabs (26) . they found that the mature bnabs reacted with diverse envs but the corresponding germline antibodies did not. they examined in detail the germline b12 and its chimeric forms -either the germline heavy chain paired with the mature light chain and vice versa -to test whether they could interact with any of the recombinant envs derived from clade a, b, and c viruses. among all the recombinant envs tested, at least one env (qh0692) was found to bind a b12 chimera with a mature heavy chain. however, this chimera failed to mediate calcium mobilization, indicating no bcr activation via bcr-antigen engagement. in other studies, they found that the elimination of certain conserved glycosylation sites on envs led to the binding of germline versions of vrc01 and nih45-46 and bcr activation (27) but that the modified envs did not interact with pg9 and 447-52d germlines (28) . haynes and coworkers have succeeded in finding envs capable of engaging the germline versions of a cd4bs bnab, ch103, while studying the co-evolution of the antibody in an hiv-1 infected patient (29) . they found that ch103 is less mutated than most other cd4bs bnabs, and importantly that the unmutated common ancestor of the ch103 lineage avidly bound the transmitted/founder hiv-1 envs. this finding suggests that early founder envs could bind optimally to the germline and intermediate versions of ch103, and therefore, are promising vaccine immunogens, representing an important step forward in hiv-1 vaccine development. similarly, the maturation pathway of the potent v1v2-directed hiv-neutralizing antibody, cap256-vrc26, has been described, in which a germline-like intermediate with a 35-amino acid residue long cdrh3 was shown to bind and neutralize the superinfecting virus weakly, but did not bind or neutralize heterologous viruses (30) . these results suggest that the cap256-vrc26 lineage could be initiated by using a rare superinfecting-virus-like v1v2 env. in another successful effort in identifying an env that could engage the germline versions of bnabs, scheif and coworkers devised a computation-guided approach combined with in vitro screening to engineer a gp120 outer domain. the designed protein not only bound to multiple vrc01-class bnabs and their germline precursors but also activated b cells expressing diverse intermediates of the bnabs (31) . therefore, priming with the protein and subsequent boosting with more native immunogens could help induce early somatic mutations and the ultimate elicitation of vrc01-class bnabs. interestingly, nussenzweig and coworkers' study showed that somatic mutations of the frs and insertions of some bnabs are required for their broad and potent hiv-1 neutralizing activity (32) . based on structural information, they made different germline versions of vrc01, nih45-46, 12a21, and 3bnc117, and found that mutations in frs were also essential for binding, breadth, and potency of most bnabs. this suggested that certain framework mutations could be critical and should be preserved for designing the intermediates of such bnabs. several other studies mining the hiv-1 infected donors' antibodyomes (33-35) revealed putative intermediates of bnabs. many of them with lower levels of somatic hyper mutations could bind to selective envs; for example, intermediates of pgt121-134 were able to preferentially bind native envs relative to monomeric gp120 (36) . we also identified 2f5-like antibodies (m66 and m66.6) with much fewer mutations than 2f5 and suggested their use as a model system for elicitation of such antibodies (37, 38) . all these newly discovered bnabs raise the hopes for effective hiv-1 vaccine development as they reveal characteristic features of bnabs that could help us understand the immunological basis critical for their production and also serve as templates for rational vaccine design. therefore, the focus has been dramatically shifted to explore and overcome the immunological hurdles associated with the elicitation of bnabs, namely, extensive somatic mutations of bnabs. major challenges remain in identification of intermediates with a minimal number of mutations, and appropriate env immunogens that would bind such intermediates and activate bcrs, which can lead to the maturation of the intermediate antibodies to bnabs. recently, new paradigms that better fit our increased knowledge of hiv immunopathology and which may be more helpful in guiding future vaccine research than did past unsuccessful approaches were discussed (17) . we previously characterized the human cord blood cell-derived igm antibodies using 454 sequencing to study gene diversity and somatic mutations (19) . naïve germline antibody repertoires, particularly from babies, may be quite unique for understanding the b cell maturation pathways, as they can also mount an immune response against hiv-1 as recently found (39) . our earlier gene usage analysis of the cord blood igm repertoire showed the biased ighv gene usages (19) as similar to adult igm repertoires (40) . however, we already noted that the ighv1-2 gene usage was significantly higher in the cord blood igm repertoire, i.e., an overall contribution of 20% as compared to 8% in adult igm repertoires. this suggested that the cord blood igm repertoire may be advantageous for the exploration of the ighv1-2*02 lineages when studying germline precursors and intermediates of vrc01 heavy chain. a total of 5,624 heavy chain and 1,096 light chain sequences of ighv1-2 and igkv3-11 lineages, respectively, were used to frontiers in immunology | hiv and aids select the top 10 sequences as closest intermediates for vrc01 in each heavy and light chain categories by using local blast searching. we performed phylogenetic analysis of the selected sequences to identify genetic relationships among vrc01-like intermediates of heavy (figure 2a) and light (figure 2b ) chains. we found two of the antibody heavy chains, hwav6 and jhedt, which were 100% identical to the ighv1-2*02 germline sequence. remarkably, their cdrh3 sequences had the same length (14 amino acids) as that of the vrc01 heavy chain. for these 10 heavy chain sequences, the cdrh3 lengths ranged from 8 to 16 amino acids with sequence variations at the junctions. one of the germline sequences, jhedt, had a point mutation at cys100tyr (kabat numbering) of cdrh3 that exactly mimicked the residue tyr100 of cdrh3 in vrc01. the residue tyr100 at cdrh3 of vrc01 is most likely contributed by the ighd3-16*02 germline with a point mutation cys100tyr. the other heavy chain sequence i76at, which was the closest to vrc01 heavy chain, also had the same mutation at cys100tyr. one of the other germline sequences, hwav6, had trp100b (kabat numbering) of cdrh3 that exactly mimicked the residue trp100b of cdrh3 in vrc01. intriguingly, the trp100b residue is a junctional amino acid of the cdrh3 in germline hwav6, and it exactly replicates the trp100b junctional residue of cdrh3 in vrc01. this suggests a possible maturation mechanism involved in the vrc01-like intermediates where junctional amino acids could determine the maturation pathway far preceding the somatic hypermutation required for affinity maturation (41) . most of the closest ighv genes, 8 out of 10 shown in the figure 2a , have at least one mutation in the v region, and www.frontiersin.org two sequences, g2w0t and gd60c, have two mutations at each of the cdrh1. the pre-existing amino acid mutations found in the v region and cdrh3 sequence information may inform the design of heavy chain germline-like precursors and intermediates, and help naturally reconstruct the b cell clonal lineages in the maturation pathways of vrc01. light chain recognition of envs by vrc01 and vrc01-related antibodies has been studied in detail using structural and 454 sequencing data (33) . the vrc01 light chain commonly uses the igkv1-33 lineage and has a characteristic five amino acid long cdrl3 and a distinctive two amino acid deletion in cdr l1. therefore, we selected the igkv1-33 lineage sequences with five amino acid length cdrl3s, but no sequences were found with a two amino acid deletion in cdr l1 ( figure 2b ). all of them had either framework or cdr mutations or both. four of them had a point mutation at cdrl1 and seven of them had a point mutation at cdrl3. the structural basis for germline gene usage of vrc01-related antibodies targeting the cd4bs has been previously described (42) , which revealed a set of signature features for these antibodies that were verified by mutagenesis. these signature features explained the origin of the igvh1-2 gene and antibody resistance for some env sequences. we found that characteristic residues including the trp100b of heavy chains were conserved while light chains did not have any characteristic residues as reported previously (42) . however, other pre-existing amino acid mutations in light chains could have implications for the vrc01-related intermediates with a characteristic cdrl3 of five amino acid length. we analyzed the amino acid length distributions of cdrh3 and cdrl3 sequences that were of vrc01 origins, namely, ighv1-2 and igkv3-11 for heavy and light chains, respectively, as derived from the human cord blood igm library (figure 3) . the cdrh3 lengths ranged from 4 to 27 amino acids, indicating high cdr3 length diversity (figure 3a ). vrc01 has a cdrh3 length of 14 amino acids, which is shorter than those of most other anti-hiv-1 antibodies (25) . the lcdr3 lengths ranged from 4 to 14 amino acids ( figure 3b) . the cdrl3 of vrc01 has a characteristic length of five amino acids with a mature genetic signature (33) . analysis of the human cord blood igm repertoire showed only a fraction of such light chains with a shorter length of five amino acids ( figure 3b) respectively. these plots show that there are position specific variations in the cdrh1 and cdrh2 regions of ighv1-2 genes. these could indicate possible ighv1-2 specific pre-existing amino acid mutations in cdrh1 and cdrh2, as observed in several naïve antibody heavy chain sequences, which could inform the design of germline precursors and intermediates of vrc01-like antibodies. we previously observed that the v-d-j rearrangement patterns occurred at different frequencies with 1,430 v-d-j combinations in a human cord blood igm repertoire (19) . figure 4a shows the v-d-j diversity associated with ighv1-2 gene sequences using a bubble plot for comparison with different d and j genes. the vrc01 heavy chain uses ighd3-16 and ighj2 genes to recombine with ighv1-2. however, other vrc01-related antibodies exhibit a skewed usage of ighj genes although at least three different ighj genes (ighj1, ighj2, and ighj4) are involved (23) . as the human cord blood igm library has a large functional v-d-j diversity, it can be used to identify potential vrc01-like heavy chain germline precursors and intermediates. in jawed vertebrates the expressed heavy chains may use any of the six ighd reading frames (rfs); however, rf1 is thought to be the preferred one as it mostly encodes tyrosine and glycine. the remaining five rfs encode either hydrophobic or charged amino acids, but the use of inverted rf1, rf2, and rf3 are discouraged. preferential usage of ighd rfs has been long implicated in b cell development and antigen-specific antibody production (43) (44) (45) , and selected based upon its amino acid content (46) . genetic control of igdh rf preference over the regulation of repertoire development has been recognized (47) . here, we have analyzed the productive ighd rf usages in a human cord blood igm library. frequency distribution of rfs is plotted using a pie chart as depicted in figure 4b . we noted that there were not any highly restricted usages of the ighd rfs although some preferential usages depending on the ighd genes were found. this clearly indicates that ighd rfs diversity could add more diverse amino acid contents leading to enormous cdrh3 diversity. it may also be possible that intermediates with different rf choices play a critical role in selecting certain maturation pathways efficiently. the antibody germline/maturation hypothesis led to a paradigm shift in the design of immunogens for bnab elicitation, as well as the realization of the importance of the complexity of the bnab maturation pathways, and exploration of human antibodyomes (14) . in fact, human antibodyome exploration is also promising for other fields of science and medicine (14, 48) . this antibodyome approach is now a major direction of research in the hiv-1 vaccine field (16, 49) . an important goal is to precisely identify naturally occurring germline-like precursors and intermediates of bnabs that could help designing novel immunogens, which could activate the corresponding bcrs and drive the immune system to produce bnabs within a short period of time. we presented an approach using a human cord blood igm library to identify putative germline precursors and intermediates of vrc01-like heavy and light chains, which could be useful in reconstructing the b cell clonal lineages in the maturation pathways of vrc01-related bnabs. this method has the potential to help in the identification of naturally occurring germline-like precursors and intermediates of any known bnab and in the development immunogens based on hiv-1 envs (50) and peptides (51) , as well as non-hiv-1 molecules (12) . however, major challenges remain and new paradigms that better fit our increased knowledge of hiv immunopathology could possibly be more helpful in guiding future vaccine research than did past unsuccessful approaches (17) . we thank the laboratory of molecular technology of saic-frederick, inc., for providing roche 454 sequencing service. we thank tina ju for critically reading the manuscript. this research was supported by the intramural research program of the nih, national cancer institute, center for cancer research, the intramural aids targeted antiviral program (iatap) of the nih and by federal funds from the nih, national cancer institute, under contract nos. no1-co-12400 and hhsn261200800001e. the www.frontiersin.org the antigenic structure of the hiv gp120 envelope glycoprotein crystal structure of a soluble cleaved hiv-1 envelope trimer cryo-em structure of a fully glycosylated soluble cleaved hiv-1 envelope trimer viral sequence diversity: challenges for aids vaccine designs antibody neutralization and escape by hiv-1 examination of the contributions of size and avidity to the neutralization mechanisms of the anti-hiv antibodies b12 and 4e10 hiv-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites hiv vaccine design and the neutralizing antibody problem extensive maturation of cross-reactive hiv-1 neutralizing antibodies but not of neutralizing antibodies against the sars cov, nipah and hendra viruses all known cross reactive hiv-1 neutralizing antibodies are highly divergent from germline and their elicitation may require prolonged periods of time germline-like predecessors of broadly neutralizing antibodies lack measurable binding to hiv-1 envelope glycoproteins: implications for evasion of immune responses and design of vaccine immunogens identification of non-hiv immunogens that bind to germline b12 predecessors and elicit cross-reactive neutralizing hiv-1 antibodies maturation pathways of cross-reactive hiv-1 neutralizing antibodies therapeutic antibodies, vaccines and antibodyomes origin, diversity, and maturation of human antiviral antibodies analyzed by high-throughput sequencing hiv-1 neutralizing antibodies: understanding nature's pathways paradigm changes and the future of hiv vaccine research: a summary of a workshop held in baltimore on 20 construction of a large naive human phage-displayed fab library through one-step cloning expressed antibody repertoires in human cord blood cells: 454 sequencing and imgt/highv-quest analysis of germline gene usage, junctional diversity, and somatic mutations imgt((r)) tools for the nucleotide analysis of immunoglobulin (ig) and t cell receptor (tr) bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt phyloxml: xml for evolutionary biology and comparative genomics focused evolution of hiv-1 neutralizing antibodies revealed by structures and deep sequencing sequence and structural convergence of broad and potent hiv antibodies that mimic cd4 binding immunologic basis for long hcdr3s in broadly neutralizing antibodies against hiv-1 recombinant hiv envelope proteins fail to engage germline versions of anti-cd4bs bnabs engineering hiv envelope protein to activate germline b cell receptors of broadly neutralizing anti-cd4 binding site antibodies diverse recombinant hiv-1 envs fail to activate b cells expressing the germline b cell receptors of the broadly neutralizing anti-hiv-1 antibodies pg9 and 447-52d co-evolution of a broadly neutralizing hiv-1 antibody and founder virus developmental pathway for potent v1v2-directed hiv-neutralizing antibodies rational hiv immunogen design to target specific germline b cell receptors somatic mutations of the immunoglobulin framework are generally required for broad and potent hiv-1 neutralization multidonor analysis reveals structural elements, genetic determinants, and maturation pathway for hiv-1 neutralization by vrc01-class antibodies mining the antibodyome for hiv-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains de novo identification of vrc01 class hiv-1-neutralizing antibodies by next-generation sequencing of b-cell transcripts the effects of somatic hypermutation on neutralization and binding in the pgt121 family of broadly neutralizing hiv antibodies cross-reactive hiv-1-neutralizing human monoclonal antibodies identified from a patient with 2f5-like antibodies structural basis for hiv-1 neutralization by 2f5-like antibodies m66 and m66.6 early development of broadly neutralizing antibodies in hiv-1-infected infants precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire junctional amino acids determine the maturation pathway of an antibody structural basis for germ-line gene usage of a potent class of antibodies targeting the cd4-binding site of hiv-1 gp120 forced usage of positively charged amino acids in immunoglobulin cdr-h3 impairs b cell development and antibody production preferential use of dh reading frame 2 alters b cell development and antigen-specific antibody production b cell development regulated by gene rearrangement: arrest of maturation by membrane-bound d mu protein and selection of dh element reading frames the restricted dh gene reading frame usage in the expressed human antibody repertoire is selected based upon its amino acid content regulation of repertoire development through genetic control of dh reading frame preference the promise and challenge of high-throughput sequencing of the antibody repertoire b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study immunogen design for hiv-1 and influenza recognition of synthetic glycopeptides by hiv-1 broadly neutralizing antibodies and their unmutated ancestors content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the guest associate editor marc h. v. van regenmortel declares that, despite having collaborated with the author dimiter s. dimitrov, the review process was handled objectively and no conflict of interest exists. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-269222-g2ibmo75 authors: valenti, piera; rosa, luigi; capobianco, daniela; lepanto, maria stefania; schiavi, elisa; cutone, antimo; paesano, rosalba; mastromarino, paola title: role of lactobacilli and lactoferrin in the mucosal cervicovaginal defense date: 2018-03-01 journal: front immunol doi: 10.3389/fimmu.2018.00376 sha: doc_id: 269222 cord_uid: g2ibmo75 the innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal ph, modulation of immunity, and production of bioactive compounds. among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by neisseria gonorrheae, chlamydia trachomatis, and trichomonas vaginalis infections promoting both innate and adaptive immune responses. in vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. the innate defense system of the female mucosal genital tract involves a close and complex interaction among the healthy vaginal microbiota, different cells, and various proteins that protect the host from pathogens. vaginal lactobacilli and lactoferrin represent two essential actors in the vaginal environment. lactobacilli represent the dominant bacterial species able to prevent facultative and obligate anaerobes outnumber in vaginal microbiota maintaining healthy microbial homeostasis. several mechanisms underlie the protection exerted by lactobacilli: competition for nutrients and tissue adherence, reduction of the vaginal ph, modulation of immunity, and production of bioactive compounds. among bioactive factors of cervicovaginal mucosa, lactoferrin, an iron-binding cationic glycoprotein, is a multifunctional glycoprotein with antibacterial, antifungal, antiviral, and antiparasitic activities, recently emerging as an important modulator of inflammation. lactobacilli and lactoferrin are largely under the influence of female hormones and of paracrine production of various cytokines. lactoferrin is strongly increased in lower genital tract mucosal fluid of women affected by neisseria gonorrheae, chlamydia trachomatis, and trichomonas vaginalis infections promoting both innate and adaptive immune responses. in vaginal dysbiosis characterized by low amounts of vaginal lactobacilli and increased levels of endogenous anaerobic bacteria, the increase in lactoferrin could act as an immune modulator assuming the role normally played by the healthy microbiota in vaginal mucosa. then lactoferrin and lactobacilli may be considered as biomarkers of altered microbial homeostasis at vaginal level. considering the shortage of effective treatments to counteract recurrent and/or antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lactoferrin could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. keywords: lactobacilli, lactoferrin, cervicovaginal defense, vaginal homeostasis, inflammation introduction the basic structures of the female reproductive system are ovaries, fallopian tubes, uterus, cervix, and vagina. ovaries are responsible for the production of the ovum and secrete both estrogen and progesterone. when an ovum is developing in an ovary, it is encapsulated in a sac known as an ovarian follicle. on maturity of the ovum, the follicle and the ovary's wall rupture, allowing the ovum to escape and enter the fallopian tube to reach uterus. the uterus is a muscular organ useful to accept a fertilized ovum, which becomes implanted into the endometrium. the cervix is the neck of the uterus, which protrudes through the upper anterior vaginal wall. the vagina is a fibromuscular canal that connects the upper part of female genital tract to the outside of the body and represents the portal of entry of pathogenic microorganisms. the epithelial mucosa of the lower genital tract is extensively colonized by commensal microorganisms, while the tissues of the upper genital tract are generally considered to be sterile (1) . however, bacterial colonization of the upper genital tract of healthy asymptomatic women remains a somehow controversial issue (1) . the vaginal tract is colonized by microorganisms, recognized as the vaginal microbiota (vm). these microorganisms, in addition to a complex synergism among secretion's proteins and peptides, epithelial, and immune cells, perform a pivotal role in the defense of female genital tract against infectious and inflammatory processes. in the state of mucosal health, the various components are in balance. the rupture of mucosal homeostasis determined by the alteration of one of the various actors often results in an increased host susceptibility to infections. healthy vm is dominated by lactobacillus spp., but other microorganisms can be present at lesser extent (gardnerella, prevotella, streptococcus, ureaplasma, peptostreptococcus, staphylococcus, corynebacterium, clostridium, mycoplasma, enterococcus, bacteroides, escherichia, bifidobac terium, veillonella, and candida) (2) (3) (4) (5) (6) . over 20 species of lactobacillus have been detected in the vagina. however, in the majority of women, the healthy vaginal microflora contains one or two lactobacillus species among lactobacillus crispatus, lactobacillus gasseri, lactobacillus jense nii, and lactobacillus iners (7, 8) . currently, the role of l. iners in vaginal health is still unclear (9) . indeed, l. iners has been recently detected in both dysbiotic and healthy women, and its presence and amount are inversely correlated with l. crispatus (8, 10, 11) . lactobacilli are involved in maintaining the healthy vaginal environment by counteracting overgrowth of other resident microorganisms (12) . lactobacilli can also colonize the human cervix. in different studies, a range of 29-52% of women resulted colonized by lactobacilli in the cervix, and within a single subject, usually the same lactobacillus strains colonized both the cervix and the vaginal tract (13) (14) (15) . lactobacilli exert their protective effects by several mechanisms: (i) microbial competition for the nutrients and for adherence to the vaginal epithelium; (ii) reduction of the vaginal ph by the production of organic acids, especially lactic acid, through the degradation of glycogen released by vaginal cells thus exerting selective antimicrobial activity against non-resident microbiota; (iii) production of antimicrobial substances, such as bacteriocins and hydrogen peroxide (h2o2) able to suppress the growth of several microorganisms; and (iv) modulation of the local immune system (16) . homeostasis of vaginal environment results from complex interactions and synergies among the host and different microorganisms that colonize the vaginal mucosa, and the maintenance of high numbers of resident lactobacilli is an effective hallmark of woman's health and a wellorganized protection against pathogens causing sexually tranmitted infections (stis). abnormal vm involving a strong reduction or disappearance of lactobacilli characterizes a pathologic condition known as bacterial vaginosis (bv) that afflicts fertile, premenopausal, and pregnant women with an incidence rate ranging from 20 to 50% (17) . bv is a polymicrobial clinical syndrome resulting from the replacement of the normal lactobacillus spp. with high number of anaerobic bacteria such as gardnerella vaginalis, prevotella spp., mobiluncus spp., ureaplasma, mycoplasma, and other fastidious or not culturable anaerobes (5, 18) . in bv, the overgrowing anaerobes produce compounds such as polyamines and other molecules capable of inducing the release of proinflammatory cytokines such as il-1β, il-6, and il-8 (19, 20) . bv represents an independent risk factor for severe reproductive tract sequelae associated with pelvic inflammatory disease and tubal factor infertility (21, 22) . changes in the vm have been also associated with obstetrical complications such as late miscarriage and premature birth (23), thus exerting a profound impact also on the health of newborns. moreover, women with lactobacillus poor flora show an increased susceptibility to sexually transmitted pathogens. several studies indicate that abnormal vm lacking lactobacilli is associated with the acquisition of infections by neisseria gonorrhoeae, chlamydia trachomatis, and trichomonas vaginalis (24) (25) (26) (27) (28) . furthermore, the alterations in vm are associated with increased risk of acquiring viral sexually transmitted diseases (stds). indeed, longitudinal and cross-sectional studies demonstrated the association between altered vm and the increased prevalence/incidence of many viral stis such as human immunodeficiency virus (hiv), herpes simplex virus (hsv), human papillomavirus, and cytomegalovirus infection (29) . in addition to lactobacilli, the cervicovaginal fluid (cvf) exerts a significant microbicidal activity against gram-positive and gram-negative bacteria, fungi, and certain viruses as well as an anti-inflammatory activity through several peptides and proteins, all characterized by common cationic features (30) . the main antimicrobial peptides and proteins present in the cvf are shown in table 1 (31) (32) (33) (34) (35) (36) (37) (38) (39) (40) (41) (42) (43) (44) (45) (46) . these peptides and proteins act through different mechanisms: (i) microbial lysis; (ii) depletion of environmental nutrients essential for microbial growth; (iii) competitive binding to host cells; (iv) degradation of negatively charged microbial surface components; (v) interference with host cell signaling pathways; and (vi) modulation of inflammation and other functions involved in host defense (34, 47) . the bactericidal activity of many of these compounds is strictly associated with their cationic features. the concentration of some of these molecules in cvf is lower than that required for in vitro inhibition of pathogens; however, it is known that several antimicrobials display synergistic effects. indeed, human β defensin 2 and cathelicidin antimicrobial peptide ll-37 (48) , secretory leukocyte protease inhibitor (slpi) and lysozyme (49) , and lactoferrin (lf) and lysozyme (49) display synergistic effects that potentially increase innate immune protection in the female reproductive tract (50) . many of these antimicrobial/immunomodulatory compounds appear to be under hormonal control (31) . in cvf α and β defensins, slpi and lysozyme levels are high during the proliferative phase, greatly decrease at mid-cycle/ovulation, and increase again during the late secretory phase. lactoferrin, belonging to transferrin family, is a multifunctional glycoprotein of about 690 amino acids and a mw of (44, 51, 52) . lf, abundantly found in most biological fluids of mammals, is synthesized by exocrine glands, many mucosal epithelial cells and released by neutrophils during inflammation. the highest level of the human lf (hlf) is found in colostrum (7 mg/ml), while decreases in mature milk (1.5-4.0 mg/ml). in the tears, hlf is detected at low concentration (about 2.0 mg/ml), while in saliva, small intestine, earwax, vaginal fluid, amniotic fluid, upper airway fluid, seminal plasma, and the cervical mucus at very low levels (<0.1 mg/ml) (53) . in particular, the concentration of hlf in human vaginal fluid corresponds to 1-3 µg/ml, while it is extremely high (100 µg/ml) in the cervical mucus plug (30, 54, 55) . of note, a total number of 10 6 neutrophils release 15 µg of hlf in sites of inflammation and infection (53) . in human body fluids, the concentration of free available iron must not overcome 10 -18 m to avoid microbial multiplication and to hinder the precipitation of insoluble ferric hydroxides as well as the formation of reactive oxygen species. hlf, by its iron-binding ability, guarantees that free available iron does not exceed 10 -18 m. hlf and bovine milk derivative lf (blf) possess high homology of sequence. from three-dimensional structure, lf is folded into two homologous lobes, each structured in two domains (n1 and n2, c1 and c2). one fe (iii) ion is chelated by each lobe. when lf is completely iron saturated, its conformation appears in a closed state, more resistant to proteolytic enzymes than the unsaturated open form (51) . the low iron availability (10 -18 m), hindering microbial growth, is a signal of health and wellness, while iron concentration higher than 10 -18 m favors not only microbial replication but also the biofilm formation (56, 57) and persistence (44) . interestingly, wiesner and vilcinskas have reported that proteins and peptides of mucosal secretions possess several functions (58) . accordingly, hlf and blf are multifunctional glycoproteins effective against bacteria, mycetes, viruses, and parasites, possessing also anti-inflammatory and immunomodulatory properties (44) . in particular, blf, available in large quantities and recognized by food and drug administration (fda, usa) as a safe substance, is the main lf used in in vitro studies (44) as well as in clinical trials (52, (59) (60) (61) (62) and in mice (63) . the level of vaginal hlf and others antimicrobial peptides change in response to microbial infections. it has been demonstrated that hlf and defensin levels increase in genital secretion of women with c. trachomatis, n. gonorrhoeae, t. vaginalis, or candida spp. infection and bv in comparison to healthy condition (64, 65) . lactobacilli the innate defense system of the female mucosal genital tract involves a complex interaction among the healthy vaginal flora, immune cells, and several proteins that defend the host from pathogens. it is broadly suggested that the crucial role of vaginal lactobacilli is to protect female genital tract through the production of lactic acid responsible for low vaginal ph that inhibits sexually transmitted pathogens. lactic acid is in equilibrium with lactate anion. the former is the predominant form in healthy vaginal conditions and low ph (<4.5), thus exerting antimicrobial activity against pathogens. lactate anion predominates at higher ph (>4.5) conditions in women with dysbiosis (66) . in vitro experiments demonstrated that at physiological concentrations (55-111 mm) of lactic acid and at ph 4.5, bv-associated bacteria such as g. vaginalis and atopobium vaginae were inactivated without effects on typical vaginal species of lactobacilli (67) . furthermore, inactivation of bv-associated species was dependent on lactic acid itself rather than ph, since a ph value of 4.5 determined by other acids was significantly less microbicidal. recent in vitro studies, however, demonstrated that c. trachomatis multiplication is inhibited by different strains of vaginal lactobacilli, independently from ph alterations (68) . c. trachomatis, an obligate intracellular pathogen, responsible for the most common bacterial std worldwide, causes acute and chronic infections. unlike acute infections, which can be cured with oral or topical administration of antibiotics, chronic infections are difficult to eradicate and need prolonged therapies, thus increasing the risk of developing antibiotic resistance (69) . therefore, novel alternative therapies are needed. the difficulty in finding new agents against c. trachomatis infection resides in the complex life cycle of this peculiar pathogen. in fact, c. trachomatis has a unique biphasic developmental cycle, alternating between the extracellular infectious elementary bodies (ebs), metabolically inactive, and the intracellular non-infectious reticular bodies (rbs), which are metabolically active. it has been recently demonstrated that vaginal lactobacilli inhibit ebs adhesion to epithelial cells as well as the intracellular rbs replication (68) . the effect on the early phases of infection was related both to co-aggregation between lactobacilli and c. trachomatis and to competition for epithelial cell adhesion. the inhibition of chlamydial infection by lactobacilli was strain and dose dependent, suggesting that the strains and the amount of lactobacilli in the vagina are responsible for the protection from chlamydial infection. lactobacilli have been demonstrated to protect lower female genital tract also from n. gonorrhoeae infection, the second most common bacterial sti. the interaction between bacteria and host cells determines the success of the pathogen mucosal colonization or its elimination through the continuous fluid flow. in this respect, vielfort et al. (15) showed that lactobacilli compete with n. gonorrhoeae for adhesion to human cervical cells. it has been also demonstrated that l. jensenii atcc 25258 could reduce both adhesion and invasion of n. gonorrhoeae, whereas l. gasseri atcc 33323 could displace adherent gonococci from the cell surface (70) . the protection exerted by healthy vm toward viral infections can be ascribed to a direct virucidal effect or to the maintenance of natural defense factors present in the vaginal milieu. some mechanisms have been suggested by results obtained from both in vitro experiments and clinical observations in infected women (29) . lactobacillus metabolites possessing antimicrobial activity may be directly protective against viral infections. hydrogen peroxide (h2o2) produced by lactobacilli plays an important role as a natural microbicide within the vaginal ecosystem due to its toxic activity against a number of microorganims and viruses, including hiv-1 (71) and hsv-2 (72) . it has been observed that a range of 70-95% of lactobacilli present in the vaginal flora of healthy women produce h2o2. this percentage drops to 5% in women affected by vaginal infections (73) . the physiological acid vaginal ph value (≤4.5) determined by lactobacilli inactivates hiv (74) and hsv-2 (75) . in addition, hsv-2 is inactivated by lactic acid concentrations leading to ph values similar to the ones detected in the healthy human vagina (72) . several compounds released from lactobacilli can impair the efficiency of target cells in supporting viral replication. a nonprotein cell wall component extracted from a vaginal strain of lactobacillus brevis strongly reduces hsv-2 replication in cell culture (76) , whereas acid lactobacillus metabolic products decrease activation of t lymphocytes, with a consequent lower lymphocyte susceptibility to hiv-1 infection (77) . a healthy vm contributes to the maintenance of the natural defense mechanisms from invading pathogens. the gel layer coat of the vaginal and cervical mucosa represents a physical barrier that hinders viral binding to cell membrane receptors, thus protecting women from viral infections. indeed, in vitro studies demonstrated that hsv could be trapped into the viscous cervical mucus (78) . bv-related microorganisms are able to produce higher levels of mucin-degrading enzymes, such as mucinase and sialidase, in comparison to lactobacilli-dominated healthy vaginal flora (79) (80) (81) . therefore, an increased degradation of the protective mucus layer may promote binding of hsv-2 and other viruses to the underlying epithelial cell receptors. further studies have demonstrated that vaginal lactobacilli are able to inhibit the first steps of hsv-2 infection in cell culture (72, 76) . the antiviral activity exerted by the presence of lactobacilli during hsv-2 binding to the cell membrane is strain dependent and appears directly related to the adhesion capacity of lactobacillus strains (82) . in conclusion, several mechanisms may be involved in the antimicrobial effect of vaginal lactobacilli: interference with microorganisms in the process of adhesion or entry into host cells, production of metabolites with a direct antimicrobial effect, production of compounds able to inhibit obligate intracellular pathogen replication, and contribution to the maintenance of natural defense factors present in the vaginal milieu. as already discussed, hlf is one of the most important defense proteins of cvf. in fact, endogenous hlf has been found increased in the genital fluid of women affected by neisseria gonorrheae, c. trachomatis, and t. vaginalis infections and/or vaginal dysbiosis (64) . in this respect, hlf released by neutrophils recruited in situ could represent a marker of non-healthy conditions and one of the mediators involved in counteracting the inflammatory mileu. the first function of hlf, recognized in vitro, was the bacteriostatic activity depending on its ability to sequester iron necessary for bacterial survival and growth (83) . hlf and blf establish a battle for iron acquisition with pathogens, capable to counteract these iron-binding proteins by synthesizing siderophores, small high affinity iron-chelating molecules, or through iron acquisition from other sources (65) . as a matter of fact, g. vaginalis, lacking of siderophores, acquires iron by the lysis of erythrocytes, using hemoglobin as iron source. this is consistent with the observation that g. vaginalis level increases during menses (6) . moreover, independently from iron-binding ability, blf exerts several antibacterial activities: (i) bacterial lysis through its binding to lipopolysaccharide (lps); (ii) inhibition of bacterial adhesion to the epithelial cells; and (iii) inhibition of the entry into host cells by facultative or obligate intracellular bacteria through competitive binding to host cells and/or to microbial surface components [(44) and references therein (46, 84) ]. of note, facultative intracellular pathogens require intracellular nutrients, including iron, for replication in mammalian cells, and obligate intracellular c. trachomatis is no exception (85) . a preparation of blf, iron saturated at 20% to consent further iron chelation, was utilized in in vitro model to check its antichlamydial activity (84) . similar to that observed using vaginal lactobacilli, the incubation of cell monolayers with blf before the infection or at the moment of the infection significantly inhibited the adhesion and entry of c. trachomatis into epithelial cells. therefore, the inhibition of c. trachomatis infectivity by blf was dependent on its interaction with the cell surface and especially with glycosaminoglycans and heparan sulfate proteoglycans (86) , which are potential receptors for c. trachomatis adhesion (87) . conversely, the preincubation of blf with c. trachomatis ebs did not influence its infectivity, supporting the idea that the specific interaction between blf and epithelial host cells could be the sole mechanism responsible for the inhibition of c. trachomatis invasion (84) . recently, the inhibition of il-6 and il-8 synthesis by blf has been demonstrated in in vitro model, mimicking the in vivo chlamydial infection. blf, added to infected cells 3 h postinfection, produced a significant decrease of il-6 and il-8 without any effect on the number of intracellular chlamydia. similarly, il-6 levels were reduced when blf was added 3 h postinfection to epithelial monolayers infected with other facultative intracellular bacteria or to lps-stimulated macrophages (88) (89) (90) . the anti-chlamydial activity of blf related to its anti-inflammatory function has been shown in vivo. in a pilot study, 7 of 176 pregnant women showing cervical specimens positive for c. trachomatis were treated with the intravaginal administration of blf (100 mg) every 8 h for 30 days. interestingly, after 1 month, six women resulted negative for c. trachomatis and showed significant decreased il-6 levels in their cvf (84) . similar to what observed in in vitro model, intravaginal administration of blf seems to act by protecting mucosal host cells against the adhesion and entry of chlamydial ebs, which are released extracellularly after redifferentiation of rbs to ebs. the decrease of il-6 levels could be a marker for the inhibition of c. trachomatis ebs infection of host cells due to the presence of blf. in other words, blf protects host cells and prevents the early phase of infection by ebs. unlike lactobacilli (68), blf does not affect the replication of rbs (84) . the potential influence of exogenous blf on microbial communities populating vagina has been recently investigated. vaginal blf administration to 60 women with bv has been shown to be able to modify vm composition. in fact, the treatment induced a reduction of bv-associated gardnerella, prevotella, and lachnospira genera as well as an increase of lactobacillus species (91) . these data suggest the therapeutic potential of blf in counteracting female genital tract diseases. indeed, it would be relevant to unveil the molecular mechanisms as well as the immunological changes accompanying blf effects on microbiota. furthermore, blf antiviral activity, verified against both enveloped and naked viruses, is exerted in the early stage of infection, thus inhibiting viral binding and entry into the host cell. this activity is mainly due to blf binding to heparan sulfate glycosaminoglycan cell receptors or viral particles or both (45) . similar to viral particles, the inhibition of plasmodium endocytosis is attributed to the interaction between blf and both cell surface heparan sulfate and lipoprotein receptor-related protein (92) (93) (94) (95) . in this respect, blf represents the most relevant protein symbolizing a brick in the wall of natural non-immune defenses of human mucosal fluids against microbial infections (44) . it is well known that lactobacilli are endowed with healthpromoting and immunomodulatory properties. along with bifidobacteria, they have been proposed as candidates for prevention and/or treatment of allergy, colitis, infections, and other inflammatory conditions (96) . some lactobacillus strains have been also proposed as vaginal microbicide candidates against sti (e.g., n. gonorrhoeae, candida albicans, and hiv). besides mechanisms related to the bacterium itself (e.g., enhancement of epithelial barrier function and competition with pathogens), the capability to redirect the immune response underlies many of the beneficial effects of lactobacilli. in vitro data demonstrate that l. crispatus (97) (98) (99) and l. jensenii (100) act not only through colonization of epithelial cells but also influencing the cytokine secretion pattern. in particular, upon recognition through tolllike receptor (tlr) 2/6 and 2/4, nf-κb signaling is activated without induction of pro-inflammatory mediators (il-1β, il-1α, and tnf-α). furthermore, secretion of cytokines as il-8 is inhibited, while production of il-10, il-6, and defensins can be induced ensuring homeostasis of immune responses. although innate immunity is the first level to be influenced by the probiotic interaction with mucosal epithelium, other cells (e.g., dendritic cells) can be shaped by lactobacilli to skew adaptive responses. an example is represented by l. crispatus sj-3c-us strain, which was shown to confer anti-inflammatory properties to dendritic cells by inducing upregulation of il-10 production and induction of regulatory t cells (101). as mentioned above, a key metabolite produced by lactobacilli is lactic acid. besides its antimicrobial properties, many of the immune modulation mechanisms exerted by lactobacilli can be ascribed to this compound. in particular, lactic acid has been shown to induce an anti-inflammatory response from vaginal and cervical epithelial cells by inhibiting il-6, il-8, rantes, and tnf-α secretion stimulated by tlr agonists used to mimic pathogen-associated molecular patterns (pamps) from microbes (102) . given that il-6, il-8, and tnf-α are known to promote replication of hiv through activation of nf-κb transcription in hiv target cells, lactic acid produced by lactobacilli in the vaginal environment could be relevant in the context of viral infection acquisition. furthermore, the anti-inflammatory cytokine il-1ra was induced by lactic acid treatment of cervicovaginal epithelial cells. all these anti-inflammatory effects were mediated by both l-and d-lactic acid isomers and by the protonated form which predominates in healthy conditions with low values of vaginal ph (102, 103) . very few data about the immunological changes associated with benefits induced by lactobacilli administration are available in the vaginal tract in vivo in both physiological and pathological conditions. lactobacillus salivarius crl 1328 and l. gasseri crl 1263 have been proposed as good candidates to keep a balanced microbiota and immune surveillance. indeed, in a murine model set up to evaluate the benefits of lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells, lactobacilli inoculation did not modify the amounts of granulocytes and macrophages in vaginal washings (104) . in humans, it has been shown that administration of probiotic lactobacillus vaginal tablets produces a significant reduction in the levels of vaginal il-1β and il-6 cytokines demonstrating the capacity of lactobacilli to modulate the production of inflammatory cytokines in both women with bv and women with healthy vaginal flora (105, 106) . a link between oral probiotic administration and vm/immune markers has been recently demonstrated by vitali et al. on pregnant women (107) . the authors investigated the effects of dietary supplementation with vsl#3 probiotic mixture containing eight species of lactobacillus, bifidobacterium, and streptococcus on the vm during late pregnancy. interestingly, no changes in the bacterial counts of the most represented populations were revealed upon probiotic administration. however, the probiotic mixture was able to change the composition of less abundant vaginal microorganisms by avoiding the reduction of bifidobacterium and the increase of atopobium recorded in the last trimester of pregnancy in control healthy women. significant modifications of the local immune system were also associated with the consumption of the probiotic showing anti-inflammatory effects. in particular, vaginal levels of il-4 and il-10 were maintained in balance compared to the reduction observed in control group. furthermore, vaginal levels of eotaxin, a pro-inflammatory chemokine, were reduced upon probiotic dietary supplementation. similar to lactobacilli, also blf exhibits effects on the host immune system, ranging from inhibition of inflammation to promotion of both innate and adaptive immune responses (108) . innate immunity is shaped by endogenous hlf through its interaction with pamps and/or pattern recognition receptors (prrs) expressed by host cells. in vitro studies demonstrated that carbohydrate chains of hlf make it able to interact directly with tlr4 resulting in moderate activation of tlr4 associated pathways (109) . lps is a typical pamp, which is bound by hlf, resulting in the inhibition of cell activation and inflammatory responses (110, 111) . it has been proposed that in vivo hlf inhibits lps-stimulated tlr4 signaling and depresses endotoxemia (112) . in particular, upon lps binding, hlf acts in reducing tnf-α, il-1, and il-6 production by immune cells (macrophages, neutrophils, and lymphocytes), as well as il-8 release by endothelial cells (113) . it has been demonstrated that levels of hlf are increased in inflammatory diseases such as rheumatoid arthritis (114) , severe acute respiratory syndrome (115) , inflammatory bowel disease (116) , and as mentioned above, some std and bv (64) . these observations suggest that hlf could be used as a clinical marker of inflammatory conditions. besides prrs and pamps binding, hlf displaces proteases from heparin in mast cells thus playing antiallergic effects (117) . furthermore, hlf competes with il-8 through the binding to proteoglycans on endothelium, thus interfering with neutrophils recruitment to the site of inflammation (118) . hlf has been shown to bind also dna in the neutrophil extracellular traps (nets), and this capability plays a key role in the context of netosis, which is the nets production by neutrophils. hlf adheres to the dna structures released due to chromatin decondensation and spreading exerting its antimicrobial properties (119) . in vitro experiments showed that recombinant hlf is able to induce maturation of antigen-presenting cells such as dendritic cells, thus suggesting that it can represent a link in shaping adaptive immunity (120) . depending on the external stimulus (pathogens, allergen, tumor antigens, etc.) and host immune status, hlf can modulate il-4, il-2, il-12, or ifn-γ levels, thus providing different outcomes: strong th1 polarization (infections, tumor), reduction of excessive th1 responses, and correction of th1/th2 balance (allergy, autoimmunity). furthermore, lf is able to support proliferation of t cell precursors and their differentiation. besides its role in cellular-mediated immunity, hlf influences activation of b cells, thus playing a role also in humoral responses (113) . the effects of exogenous lf on immune responses have been evaluated in different in vitro systems. different epithelial cell monolayers infected with various facultative or obligate intracellular pathogens produce pro-inflammatory cytokines and the addition of blf significantly decreased il-1β, il-6, il-8, and nf-kb levels (84, 88, 121, 122) . the results obtained in different in vitro models and in various clinical trials confirm the blf ability in downregulating pro-inflammatory cytokine synthesis. it has been demonstrated that exogenous blf localizes into cell nucleus, thus acting as transcriptional factor and inhibiting pro-inflammatory cytokines (52, (122) (123) (124) . although the mechanisms by which blf exerts its anti-inflammatory activity are still under debate, recently, the blf ability to decrease the high levels of il-6 in cvf seems strictly related to its capacity to restore iron homeostasis disorders (52, 84) . therefore, lf is not only a key element in the host defense system (44, 125, 126) but also a pivotal component that is able to regulate the inflammatory response and iron homeostasis (52, (60) (61) (62) . recently, blf is emerging as an attractive molecule for treating inflammation by ranging pro-inflammatory macrophagic phenotypes m1 to regulatory/ anti-inflammatory m2 phenotypes (90). women life: estrogens, lactobacilli, and lf lactobacilli defenses of female mucosal genital tract are largely under the influence of hormones and paracrine production of various cytokines. vaginal environment undergoes overtime shifts in the representation and abundance of microbial key species that are influenced by factors that may include age, hormonal fluctuations, sexual activity, use of medication, and hygiene (12) . the vagina is lined by stratified not keratinizing squamous epithelium, which is variable in thickness and structure depending on life stages. the vaginal epithelium consists of three cell layers: superficial, intermediate, and basal capable of storing glycogen under the influence of estrogen. in the pre-pubertal and the postmenopausal women, the epithelium is thin and characterized by a basal layer of cells and several layers of parabasal cells. this thin atrophic epithelium is susceptible to infection and frequently shows degenerative and inflammatory changes. vaginal epithelium reflects the hormonal changes of the menstrual cycle with increased mitosis of the basal layers. under the influence of estrogen in the proliferative phase of the cycle, the whole epithelium thickens and is multilayered. during the secretory phase of the cycle, the intermediate layers become thick and the cells stuffed with glycogen. therefore, the glycogen content of the vaginal epithelium co-variates with estrogen levels. breakdown of glycogen by resident healthy vm produces an acid ph in the vagina, which deters infections. indeed, reproductive-age women carry lactobacillus species (predominant lactic acid bacteria) and genera of streptococcus and atopobium, which conserved the ecological function of lactate production in the vaginal microbiome (127) . it is well demonstrated that fluctuations in the vm occur not only based on intercourse and infections but also during menstrual cycle. in general, high levels of estradiol may favor a lactobacillidominant environment, especially l. crispatus, l. gasseri, and/or l. jensenii (128) , which can be underrepresented in low estrogen conditions such as the beginning of a menstrual cycle or in postmenopausal women. in one study, l. crispatus appears to decline during menses, while g. vaginalis increases along with l. iners and subsequently the concentration of both species decreases after menses (6) . in other studies, l. crispatus has also been reported to decline 100-fold during menses, while the numbers of l. iners strongly increase (10, 11, 129) . recently, cultivation-independent methods have highlighted the complexity and temporal variability of the vm (8, 130) . in particular, gajer et al. described temporal changes in the composition of vm in reproductive-age women within a 16-week period (129) . in general, the highest variability of microbiota community was associated with menses. for example, a subject can show a community dominated by l. crispatus, which could be replaced by l. iners during menses or by streptococcus spp. in a different subject. the same community could then revert to a community dominated by l. crispatus at the end of menses. moreover, the results also showed how some bacterial communities changed greatly over short time periods, while others were more stable. hormone levels were combined with diversity data showing that an increase in estradiol and progesterone corresponded to decreased microbial variability. vaginal metabolome data added information about community function, which was maintained despite changes in its composition. indeed, shifts in community composition involved only changes in the relative dominance of a little number of different bacteria that are able to produce lactic acid (129) . interestingly, a recent study performed to investigate timing and sequence of changes that occur in the vaginal and vulvar microbiota during puberty showed that vm of perimenarcheal girls resembles those of reproductive-age women (131) . in fact, l. crispatus, l. iners, l. gasseri, l. jensenii, and, in some subjects, streptococcus spp. were dominant in the microbiota of girls before the onset of menarche in the early to middle stages of puberty. further studies should be performed to increase knowledge about the link between estrogen, vaginal glycogen levels, lactic acid bacteria abundance, and vaginal ph. other important fluctuations in the vaginal microbiome are recorded during pregnancy. aagaard et al. showed that microbiome was enriched in l. iners, l. crispatus, l. jensenii, and l. johnsonii (132) . the increase in lactobacilli may be due to the increase in estrogen levels that occurs during pregnancy although further investigations are needed to better understand the relationship between specific species of lactobacillus and estrogen levels. however, another study shows that l. crispatus and l. iners dominate the vaginal flora as pregnancy progresses and maternal age seems to be important for the dominance of l. crispatus or l. iners, with l. iners being dominant in older gravidae (133) . as mentioned above, menopause usually represents a phase in which lactobacilli levels are low, but it seems that their implication is even more pronounced involving other features. actually, inverse correlation has been found between lactobacillus levels and vaginal dryness, a common condition of postmenopausal period, which was shown to be associated with changes in vaginal epithelial cell integrity and inflammation (134) . besides the endogenous hormonal fluctuations, clinical evidences demonstrate that the use of hormonal contraceptives is also able to induce changes in vm, thus influencing the susceptibility to sti and bv. therefore, sti and bv (which in turn predisposes to sti) depend, in part, on modification of vaginal bacterial communities induced by some contraception methods. clinical trials based on nugent score endpoint and questionnaires have revealed a reduced bv rate in women who use estrogen-containing contraceptives (135, 136) . the effects of progestin-containing contraceptives such as depot medroxyprogesterone acetate (dmpa) and levonorgestrel are less clear. in a systematic review of 36 eligible studies, authors have shown that combined oral contraceptives (cocs; combination of an estrogen and a progestin) and dmpa reduce bv by a range of 10-20 and 18-30%, respectively (137) . accordingly, in a recent retrospective study on 682 fertile women, based on 16s rrna sequencing, authors found that women using coc or dmpa showed a reduced colonization by bv-associated bacteria compared to women using condoms. in the same study, women using progestinbased therapies have a significantly higher abundance of taxa associated with a dysbiotic vm. on the other hand, coc users have a lactobacilli-dominated vm and a higher proportion of h2o2-producing lactobacillus species (l. crispatus, l. gasseri, and l. jensenii) correlating with vaginal health compared to progestin-containing contraceptives (138) differences in the results reported in other studies, which do not reveal any changes in vaginal bacterial communities associated with progestin use, could be due to the different study population characteristics (e.g., ethnicity of subjects) and/or the design of the study itself such as the specific bv status examination (139, 140) . in women with bv, vm is not the only level that hormonal contraception acts on. in fact, changes in genital tract immunity by effect of hormonal contraceptives have been shown in terms of both suppression and activation of responses. in a study on 81 women, cervical secretions contained lower levels of pro-inflammatory molecules (tnf, ifn-γ, and gm-csf) in subjects with bv using hormonal contraceptives compared to those not using them (141) . on the other hand, an increase of inflammatory cytokines (mip-1α, mip-1β, il-6, il-8, ip-10, and rantes) has been associated with hormonal contraception in a different cross-sectional analysis including 376 african women (142) . conflicting data existing in these studies on immunomodulatory potential of hormonal contraception in female genital tract are complicated by their variability in terms of in vitro versus in vivo models used and sample tested (plasma or blood versus cervical fluid). however, the investigation on these features may represent a key point in understanding the association between hormonal contraception and hiv acquisition. in fact, women using progestin dmpa, but not those who use coc have been found to be at significantly increased risk of hiv infection compared to women not using hormonal contraception (143) . in a more recent case-control selection of specimens from a large, prospective, clinical study, the same authors have investigated on innate immunity mediators in cervical samples collected from 199 women at their visit before hiv seroconversion and matched visits from 633 women remaining hiv uninfected. higher levels of pro-inflammatory markers such as rantes have been found in hiv seroconversion and in dmpa users, suggesting a possible role of this cytokine in the association between the contraceptive and hiv risk acquisition (144) . the possible explanation could be that the upregulation of rantes, observed in women using dmpa and women with bv, may simultaneously block the ccr5 cellular hiv receptor but also facilitate transmission through recruiment of target cells. also other sti can be influenced by hormonal contraception, such as candidiasis, which has been found increased in women using coc but not dmpa (137) . taken together, these observations suggest that altered immune responses by effect of hormonal contraception may predispose to infections from pathogens. in cases of a pre-existing condition of dysbiosis or specific cervicovaginal infection, suppression or activation of immunity by pathogens could increase the risk of infections by cumulative action with exogenous hormones. similar to the estrogen-induced changes in the species and number of lactobacilli, the hlf concentration fluctuates accordingly to circulating estrogen levels (145) (146) (147) . in addition to the hlf levels produced by uterine epithelium and released in cvf, the synthesis of iga and igg is also modulated by estrogen and progesterone, thus exerting an immune protection against sexually transmitted pathogens (148) . figure 1 shows a comparison among estrogen, progesterone, and lf levels during the proliferative, ovulatory, and secretory phase. the hlf levels increase in line with estrogen production, reaching the highest levels during ovulatory phase, while they are inversely correlated with progesterone levels. as a matter of fact, secretory phase accordingly to the increase of progesterone shows the lowest levels of hlf. at the end of the secretory phase, hlf returns to increase in parallel to the progesterone decrease (145) (146) (147) (148) (149) . obviously, the use of oral contraceptives by decreasing estrogen synthesis suppresses the production of hlf as well as immunoglobulins for the duration of hormone exposure, thus possibly increasing the susceptibility of women to infections (148) . in rats, pretreatment with progesterone prior to exposure to chlamydia thracomatis infection induced a persistent infection (150) . furthermore, the rats treated with progesterone were also found to be more vulnerable to chlamydial intrauterine infection, whereas the treatment with estradiol, the major female estrogen sex hormone, reduced the susceptibility to infection (151) . the sex steroid hormones are also important mediators of inflammation (152) and may influence resistance or susceptibility to parasitic infections (153) . sex steroid hormones are also involved in viral infections. it has been proposed that hiv-1 utilizes a window of vulnerability during the menstrual cycle. this crucial period overlaps with the mid-cycle when innate and adaptive immune responses are suppressed by estrogens and/or progesterone to facilitate reproductive processes. hiv-1 presumably exploits this time frame, during which antiviral factors are suppressed, to establish and propagate infection in the female mucosal genital tract (31, 148, 154) . in menopausal women, the low concentration of hlf in the secretions, related to the low levels of sexual hormones (155) , may lead to recurrent infections. it is important to underline that during menses the decrease of this important natural defense glycoprotein is balanced by the presence of neutrophils that, through the synthesis of granules, can restore, at least partially, hlf concentration and its antimicrobial activity when the epithelial barrier is disrupted. the recruitment of neutrophils occurs through high levels of inflammatory biomarkers as pro-inflammatory cytokine as il-8, il-6 or c-reactive protein (119) . as matter of fact, during menstruation, as well as in aging and menopause, the decrease of estrogens is related to the increase of inflammatory processes (156) . interestingly, hlf expression in endometrium suggests that, during the gestational period, hlf produced by uterine epithelium and neutrophils and released in cvf is controlled by sex steroid hormones (149) . it has been reported that high levels of progesterone parallel low levels of estrogens in normal pregnancy, while this ratio is inverted in pregnant women with the preterm delivery threat (157) . determining the existence of a regulatory circuit linking hlf synthesis and sex steroid hormone fluctuations may unravel novel mechanisms leading to preterm birth. figure 2 | lactobacillus spp. and lactoferrin interplay on infection and inflammation in female genital tract. a schematic representation of lactobacillus spp. and lactoferrin balance: a multitasking strategy to protect against pathogen challenge and maintain immune homeostasis. (a) healthy genital tract; (b) vaginosis: high levels of pro-inflammatory cytokines, decrease of lactobacilli and increase of gram-negative anaerobes, and increase of lactoferrin concentration released by neutrophils; (c) decrease of pro-inflammatory cytokines by lactoferrin and restoration of healthy microbiota. lactobacillus spp. and lf are pivotal components of first-line defense in the female mucosal genital tract involved in protection against a multitude of microbial infections and the most effective natural mechanism to dampen inflammatory processes. to inhibit cervicovaginal infections, an ideal drug should inhibit: • microbial growth; • microbial adhesion and entry into host cells; • microbial intracellular replication; and • infection of new host cells by microbes extracellularly released from the infected cells. in the vaginal environment of women of childbearing age, the inhibition of bacterial multiplication through the synthesis of antibacterial substances by lactobacilli or by competition between microbes and lf for iron acquisition represents an effective natural defense mechanism. both lactobacilli and lf can inhibit the adhesion and consequently the microbial entry inside the cells through an interaction with the cell surface components potential receptors for pathogens. lactobacilli and lf appear complementary since lactobacilli inhibit microbial intracellular replication and together with lf hinder the infection of still healthy cells by microbes extracellularly released. this close cooperation is also exerted through their anti-inflammatory function. in this scenario, the mucosal environment represents a good model of mutualism and reciprocity against the injury by microbes. a schematic representation of lactobacillus spp. and lf balance in protecting against pathogens and maintaining immune homeostasis in the vaginal tract is shown in figure 2 . considering the shortage of effective treatments to counteract antibiotic-resistant bacterial infections, the intravaginal administration of lactobacilli and lf could be a novel efficient therapeutic strategy and a valuable tool to restore mucosal immune homeostasis. author contributions pm, rp, and pv conceived the topic concept and wrote and revised the final manuscript; lr, ac, ml, dc, and es provided figures and contributed to manuscript preparation and editing. all authors read and approved the final version. acknowledgments this work was granted by sapienza university of rome funds to pv. microbiota of the upper and lower genital tract emerging role of lactobacilli in the control and maintenance of the vaginal bacterial microflora understanding the bacterial flora of the female genital tract characterization of vaginal flora and bacterial vaginosis in women who have sex with women the human vaginal bacterial biota and bacterial vaginosis temporal variability of human vaginal bacteria and relationship with bacterial vaginosis the vaginal microbiome: new information about genital tract flora using molecular based techniques vaginal microbiome of reproductive-age women lactobacillus iners: friend or foe? longitudinal study of the dynamics of vaginal microflora during two consecutive menstrual cycles longitudinal qpcr study of the dynamics of l. crispatus, l. iners, a. vaginae, (sialidase positive) g. vaginalis, and p. bivia in the vagina lactobacilli in the female genital tract in relation to other genital microbes and vaginal ph cervical bacterial flora in infertile and pregnant women microbial flora of the vagina and cervix adherence of clinically isolated lactobacilli to human cervical cells in competition with neisseria gonorrhoeae defense factors of vaginal lactobacilli prevalence of bacterial vaginosis: 2001-2004 national health and nutrition examination survey data the microbiology of bacterial vaginosis local and systemic cytokine levels in relation to changes in vaginal flora cytokines, pregnancy, and bacterial vaginosis: comparison of levels of cervical cytokines in pregnant and non-pregnant women with bacterial vaginosis bacterial vaginosis as a risk factor for upper genital tract infection rates of bacterial vaginosis in women undergoing in vitro fertilisation for different types of infertility bacterial vaginosis as a risk factor for preterm delivery: a meta-analysis vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition bacterial vaginosis is a strong predictor of neisseria gonorrhoeae and chlamydia trachomatis infection bacterial vaginosis (bv) and the risk of incident gonococcal or chlamydial genital infection in a predominantly black population bacterial vaginosis, race, and sexually transmitted infections: does race modify the association? bacterial vaginosis assessed by gram stain and diminished colonization resistance to incident gonococcal, chlamydial, and trichomonal genital infection vaginal microbiota and viral sexually transmitted diseases antimicrobial components of vaginal fluid innate immunity in the human female reproductive tract: endocrine regulation of endogenous antimicrobial protection against hiv and other sexually transmitted infections antibacterial activity of antileukoprotease antibacterial activity and specificity of the six human α defensins antiviral mechanisms of human defensins gene expression, immunolocalization, and secretion of human defensin-5 in human female reproductive tract human β-defensins the antibacterial and antifungal properties of trappin-2 (pre-elafin) do not depend on its protease inhibitory function antiviral activity of trappin-2 and elafin in vitro and in vivo against genital herpes calprotectin -a pleiotropic molecule in acute and chronic inflammation metal chelation and inhibition of bacterial growth in tissue abscesses cathelicidins: family of antimicrobial peptides. a review protective roles of the skin against infection: implication of naturally occurring human antimicrobial agents β-defensins, cathelicidin ll-37 and lysozyme female genital tract secretions inhibit herpes simplex virus infection: correlation with soluble mucosal immune mediators and impact of hormonal contraception lactoferrin: an important host defence against microbial and viral attack antiviral properties of lactoferrin -a natural immunity molecule lactoferrin: a natural glycoprotein involved in iron and inflammatory homeostasis the role of cationic polypeptides in modulating hiv-1 infection of the cervicovaginal mucosa endogenous antimicrobial peptides and skin infections in atopic dermatitis synergistic and additive killing by antimicrobial factors found in human airway surface liquid host antimicrobial defence peptides in human disease molecular structure, binding properties and dynamics of lactoferrin body iron delocalization: the serious drawback in iron disorders in both developing and developed countries lactoferrin: an alternative view of its role in human biological fluids innate host defense of human vaginal and cervical mucosae antimicrobial factors in the cervical mucus plug a component of innate immunity prevents bacterial biofilm development iron availability influences aggregation, biofilm, adhesion and invasion of pseudomonas aeruginosa and burkholderia cenocepacia antimicrobial peptides: the ancient arm of the human immune system oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women the influence of lactoferrin, orally administered, on systemic iron homeostasis in pregnant women suffering of iron deficiency and iron deficiency anaemia lactoferrin efficacy versus ferrous sulfate in curing iron disorders in pregnant and non pregnant women safety and efficacy of lactoferrin versus ferrous sulphate in curing iron deficiency and iron deficiency anaemia in hereditary thrombophilia pregnant women: an interventional study aerosolized bovine lactoferrin reduces neutrophils and pro-inflammatory cytokines in mouse models of pseudomonas aeruginosa lung infections multiplex immunoassay of lower genital tract mucosal fluid from women attending an urban std clinic shows broadly increased il1ß and lactoferrin siderophores in iron metabolism: from mechanism to therapy potential antimicrobial and immune modulatory effects of lactic acid and short chain fatty acids produced by vaginal microbiota associated with eubiosis and bacterial vaginosis in vaginal fluid, bacteria associated with bacterial vaginosis can be suppressed with lactic acid but not hydrogen peroxide effects of vaginal lactobacilli in chlamydia trachomatis infection treatment of chlamydial infections: 2014 update inhibition of neisseria gonorrhoeae epithelial cell interactions by vaginal lactobacillus species viricidal effect of lactobacillus acidophilus on human immunodeficiency virus type 1: possible role in heterosexual transmission inhibition of herpes simplex virus type 2 by vaginal lactobacilli prevalence of hydrogen peroxide-producing lactobacillus species in normal women and women with bacterial vaginosis disinfection and inactivation of the human lymphotropic virus type iii/lymphadenopathy-associated virus acidform inactivates herpes simplex virus and prevents genital herpes in a mouse model: optimal candidate for microbicide combinations antiviral activity of lactobacillus brevis towards herpes simplex virus type 2: role of cell wall associated components human vaginal leukocytes and the effects of vaginal fluid lymphocyte and macrophage defense functions diffusion of macromolecules and virus-like particles in human cervical mucus glycosidase and proteinase activity of anaerobic gram negative bacteria isolated from women with bacterial vaginosis a novel bacterial mucinase, glycosulfatase, is associated with bacterial vaginosis among pregnant women with bacterial vaginosis, the hydrolytic enzymes sialidase and prolidase are positively associated with interleukin-1β technological and biological evaluation of tablets containing different strains of lactobacilli for vaginal administration iron-binding proteins in milk and resistance to escherichia coli infections in infants effect of bovine lactoferrin on chlamydia trachomatis infection and inflammation response of chlamydia trachomatis serovar e to iron restriction vitro and evidence for iron-regulated chlamydial proteins characterization of the glycosaminoglycan-binding region of lactoferrin the chlamydia trachomatis ctad1 invasin exploits the human integrin 1 receptor for host cell entry lactoferrin differently modulates the inflammatory response in epithelial models mimicking human inflammatory and infectious diseases lactoferrin prevents lps-induced decrease of the iron exporter ferroportin in human monocytes/macrophages lactoferrin efficiently counteracts the inflammation-induced changes of the iron homeostasis system in macrophages bacterial biota of women with bacterial vaginosis treated with lactoferrin: an open prospective randomized trial plasmodium falciparum inhibition in vitro with lactoferrin, desferrithiocin, and desferricrocin iron chelators as therapeutic agents against pneumocystis carinii dual interaction of the malaria circumsporozoite protein with the low density lipoprotein receptor-related protein (lrp) and heparan sulfate proteoglycans reduction of the infectivity of toxoplasma gondii and eimeria stiedai sporozoites by treatment with bovine lactoferricin challenges in translational research on probiotic lactobacilli: from in vitro assays to clinical trials lactobacillus crispatus modulates epithelial cell defense against candida albicans through toll-like receptors 2 and 4, interleukin 8 and human β-defensins 2 and 3 probiotic properties of lactobacillus crispatus 2,029: homeostatic interaction with cervicovaginal epithelial cells and antagonistic activity to genitourinary pathogens lactobacillus crispatus mediates anti-inflammatory cytokine interleukin-10 induction in response to chlamydia trachomatis infection in vitro homeostatic properties of lactobacillus jensenii engineered as a live vaginal anti-hiv microbicide lactobacillus crispatus strain sj-3c-us induces human dendritic cells (dcs) maturation and confers an anti-inflammatory phenotype to dcs vaginal lactic acid elicits an anti-inflammatory response from human cervicovaginal epithelial cells and inhibits production of pro-inflammatory mediators associated with hiv acquisition the role of lactic acid production by probiotic lactobacillus species in vaginal health effectiveness of vaginal tablets containing lactobacilli versus ph tablets on vaginal health and inflammatory cytokines: a randomized, double-blind study biological control of vaginosis to improve reproductive health dietary supplementation with probiotics during late pregnancy: outcome on vaginal microbiota and cytokine secretion bovine lactoferrin counteracts toll-like receptor mediated activation signals in antigen presenting cells human lactoferrin activates nf-kappab through the toll-like receptor 4 pathway while it interferes with the lipopolysaccharide stimulated tlr4 signaling lactoferrin is a lipid a-binding protein lactoferrin binds cpg-containing oligonucleotides and inhibits their immunostimulatory effects on human b cells human milk components modulate toll-like receptor-mediated inflammation lactoferrin, a key molecule in immune and inflammatory processes increased levels of lactoferrin in synovial fluid but not in serum from patients with rheumatoid arthritis expression profile of immune response genes in patients with severe acute respiratory syndrome update of fecal markers of inflammation in inflammatory bowel disease the inhibition of mast cell activation by neutrophil lactoferrin: uptake by mast cells and interaction with tryptase, chymase and cathepsin g lactoferrin inhibits the lipopolysaccharide-induced expression and proteoglycan-binding ability of interleukin-8 in human endothelial cells neutrophil extracellular traps and its implications in inflammation: an overview recombinant human lactoferrin induces human and mouse dendritic cell maturation via toll-like receptors 2 and 4 lactoferrin downregulates pro-inflammatory cytokines upexpressed in intestinal epithelial cells infected with invasive or noninvasive escherichia coli strains lactoferrin decreases inflammatory response by cystic fibrosis bronchial cells invaded with burkholderia cenocepacia iron-modulated biofilm cellular internalization of lactoferrin in intestinal epithelial cells the n1 domain of human lactoferrin is required for internalization by caco-2 cells and targeting to the nucleus lactoferrin: a modulator of immune and inflammatory responses multifunctional roles of lactoferrin: a critical overview differences in the composition of vaginal microbial communities found in healthy caucasian and black women changes in the predominant human lactobacillus flora during in vitro fertilisation temporal dynamics of the human vaginal microbiota daily temporal dynamics of vaginal microbiota before, during and after episodes of bacterial vaginosis vaginal microbiota of adolescent girls prior to the onset of menarche resemble those of reproductive-age women a metagenomic approach to characterization of the vaginal microbiome signature in pregnancy pregnancy's stronghold on the vaginal microbiome vaginal microbiome and epithelial gene array in post-menopausal women with moderate to severe dryness prevalent and incident bacterial vaginosis are associated with sexual and contraceptive behaviours in young australian women recurrence of bacterial vaginosis is significantly associated with posttreatment sexual activities and hormonal contraceptive use hormonal contraception decreases bacterial vaginosis but oral contraception may increase candidiasis: implications for hiv transmission changes in vaginal community state types reflect major shifts in the microbiome cervicovaginal bacteria are a major modulator of host inflammatory responses in the female genital tract association between injectable progestin-only contraceptives and hiv acquisition and hiv target cell frequency in the female genital tract in south african women: a prospective cohort study hormonal contraceptive use modulates the local inflammatory response to bacterial vaginosis injectable progestin-only contraception is associated with increased levels of proinflammatory cytokines in the female genital tract hormonal contraception and hiv acquisition: reanalysis using marginal structural modeling cervical inflammation and immunity associated with hormonal contraception, pregnancy, and hiv-1 seroconversion estrogen regulation of lactoferrin expression in human endometrium lactoferrin gene expression and regulation: an overview lactoferrin: the path from protein to gene regulation of mucosal immunity in the female reproductive tract: the role of sex hormones in immune protection against sexually transmitted pathogens differential expression and estrogen response of lactoferrin gene in the female reproductive tract of mouse, rat, and hamster chlamydia trachomatis infection in the female reproductive tract of the rat: influence of progesterone on infectivity and immune response antigen-presenting cells in the female reproductive tract: influence of estradiol on antigen presentation by vaginal cells the complex role of estrogens in inflammation immunoendocrine mechanisms associated with resistance or susceptibility to parasitic diseases during pregnancy a new strategy to understand how hiv infects women: identification of a window of vulnerability during the menstrual cycle bone health in menopausal women: a role for general practitioners. clin cases miner bone metab molecular mechanisms of aging-associated inflammation dydrogesterone supplementation in women with threatened preterm delivery -the impact on cytokine profile, hormone profile, andprogesterone-induced blocking factor key: cord-313117-0qur0isb authors: gardinassi, luiz g.; souza, camila o. s.; sales-campos, helioswilton; fonseca, simone g. title: immune and metabolic signatures of covid-19 revealed by transcriptomics data reuse date: 2020-06-26 journal: front immunol doi: 10.3389/fimmu.2020.01636 sha: doc_id: 313117 cord_uid: 0qur0isb the current pandemic of coronavirus disease 19 (covid-19) has affected millions of individuals and caused thousands of deaths worldwide. the pathophysiology of the disease is complex and mostly unknown. therefore, identifying the molecular mechanisms that promote progression of the disease is critical to overcome this pandemic. to address such issues, recent studies have reported transcriptomic profiles of cells, tissues and fluids from covid-19 patients that mainly demonstrated activation of humoral immunity, dysregulated type i and iii interferon expression, intense innate immune responses and inflammatory signaling. here, we provide novel perspectives on the pathophysiology of covid-19 using robust functional approaches to analyze public transcriptome datasets. in addition, we compared the transcriptional signature of covid-19 patients with individuals infected with sars-cov-1 and influenza a (iav) viruses. we identified a core transcriptional signature induced by the respiratory viruses in peripheral leukocytes, whereas the absence of significant type i interferon/antiviral responses characterized sars-cov-2 infection. we also identified the higher expression of genes involved in metabolic pathways including heme biosynthesis, oxidative phosphorylation and tryptophan metabolism. a btm-driven meta-analysis of bronchoalveolar lavage fluid (balf) from covid-19 patients showed significant enrichment for neutrophils and chemokines, which were also significant in data from lung tissue of one deceased covid-19 patient. importantly, our results indicate higher expression of genes related to oxidative phosphorylation both in peripheral mononuclear leukocytes and balf, suggesting a critical role for mitochondrial activity during sars-cov-2 infection. collectively, these data point for immunopathological features and targets that can be therapeutically exploited to control covid-19. the current pandemic of coronavirus disease has affected millions of individuals and caused thousands of deaths worldwide. the pathophysiology of the disease is complex and mostly unknown. therefore, identifying the molecular mechanisms that promote progression of the disease is critical to overcome this pandemic. to address such issues, recent studies have reported transcriptomic profiles of cells, tissues and fluids from covid-19 patients that mainly demonstrated activation of humoral immunity, dysregulated type i and iii interferon expression, intense innate immune responses and inflammatory signaling. here, we provide novel perspectives on the pathophysiology of covid-19 using robust functional approaches to analyze public transcriptome datasets. in addition, we compared the transcriptional signature of covid-19 patients with individuals infected with sars-cov-1 and influenza a (iav) viruses. we identified a core transcriptional signature induced by the respiratory viruses in peripheral leukocytes, whereas the absence of significant type i interferon/antiviral responses characterized sars-cov-2 infection. we also identified the higher expression of genes involved in metabolic pathways including heme biosynthesis, oxidative phosphorylation and tryptophan metabolism. a btm-driven meta-analysis of bronchoalveolar lavage fluid (balf) from covid-19 patients showed significant enrichment for neutrophils and chemokines, which were also significant in data from lung tissue of one deceased covid-19 patient. importantly, our results indicate higher expression of genes related to oxidative phosphorylation both in peripheral mononuclear leukocytes and balf, suggesting a critical role for mitochondrial activity during sars-cov-2 infection. collectively, these data point for immunopathological features and targets that can be therapeutically exploited to control covid-19. keywords: covid-19, transcriptomics, inflammation, metabolism, sars-cov-2, sars-cov, influenza, oxidative phosphorylation introduction the outbreak of coronavirus disease 19 (covid-19) , first recognized in wuhan, china, rapidly became a pandemic of major impact not only on global public health but also on economy and social well-being (1). sars-cov-2 infection results in clinical outcomes ranging from asymptomatic status to severe disease and ultimately, death (2) . understanding of the molecular mechanisms underlying the pathology of covid-19 is required to design effective therapies and safe vaccines. in this context, current investigations have been devoted to biochemical characterization and cellular phenotyping in patients to development of animal models of covid-19 (3). transcriptomics of peripheral blood cells has been a powerful tool to characterize human immune responses to diverse pathogens, including respiratory viruses (4-6). gene expression profiling by different analytical platforms and sample types revealed that covid-19 patients exhibit: (i) activation of humoral immunity, hypercytokinemia, apoptosis (7) , and dynamic toll like receptor (tlr) signaling (8) in peripheral leukocytes; (ii) induction of interferon stimulated genes (isgs), chemokines and inflammation in the lower respiratory tract (7, 9, 10) . of importance, the results and interpretation of these data were based on single-gene-level analyses, in which significance of quantitative changes of each gene are calculated separately and they are latter submitted to pathway enrichment analysis. however, the statistical power and sensitivity to identify pathways, or gene modules (computational gene networks), associated with disease phenotypes can be enhanced by the use of non-parametric rank-based tests such as the robust positional framework gene set enrichment analysis (gsea) (11) . moreover, interpretation of transcriptional changes during covid-19 has been primarily evaluated using canonical pathways that do not often reflect human responses. therefore, we propose alternative strategies to analyze and interpret transcriptomics data, which provide novel insights into immune and metabolic responses during covid-19. datasets used in this study included public transcriptomes available at the genome sequence archive (gsa) or human gsa in national genomics data center, beijing institute of genomics (big), chinese academy of sciences for rna-seq data related to sars-cov-2 infection (cra002390 and hra000143); gene expression omnibus (geo) for rna-seq data related to sars-cov-2 infection (gse147507) and microarray data related to sars-cov-1 infection (gse1739) or influenza a virus (iav) infection (gse34205, gse6269, gse29366, gse38900, gse20346, gse52428, gse40012, gse68310, gse61754, gse90732); and arrayexpress for nanostring ncounter data related to sars-cov-2 infection (e-mtab-8871). deseq2-normalized counts were used for the rna-seq dataset cra002390 (7), while raw read counts for the rna-seq datasets gse147507 (9) or hra000143 (10) were treated and normalized to log 2 counts per million with edger package for r (12) . normalized data was acquired for nanostring ncounter e-mtab-8871 (8) . normalized microarray datasets were acquired with omicc platform (13) . detailed information about the datasets used in this study are described in table 1 . data were analyzed with the positional framework gene set enrichment analysis (gsea) (11), using pre-ranked mode, 1,000 permutations and weighted enrichment statistics. the blood transcriptional modules (btms) (24) and metabolic pathways annotated in the kyoto encyclopedia of genes and genomes (kegg) database (25) were used as gene sets. to construct the network of btms from peripheral blood mononuclear cell (pbmc) transcriptomes, genes were preranked by the wald test statistics score calculated with deseq2 package comparing each gene in covid-19 patients and healthy controls, as described (7) . btms detected with a false discovery rate (fdr) adjusted p < 0.001 were then linked by the number of genes shared between two gene modules. to perform the btm-driven meta-analysis between respiratory viruses, gene lists from each dataset were preranked by log 2 fold change of experimental samples over healthy controls. gene modules significantly associated with at least 50% of the datasets were selected by a nominal p < 0.001 for pbmcs and whole blood. the datasets were not merged at the single-gene-level. each dataset was composed by a different number of genes and samples, and different types of samples ( table 1 ). the output of the gsea provides a normalized enrichment score (nes) for each btm associated with each dataset. the nes was then compared between datasets selected at the determined cut-off (p < 0.001). to enforce confidence in the enrichments, we also retained only the btms that were associated with at least 50% of the datasets, independently of infection, sample type and regulation. metabolic pathways from kegg database were selected by a fdr adjusted p < 0.05 for pbmcs from covid-19 patients. for balf datasets (cra002390 and hra000143), genes were also pre-ranked by log 2 fold change of experimental samples over healthy controls and used as input in pre-ranked gsea. btms and kegg metabolic pathways were selected by relaxed significance (nominal p < 0.05) and consistent up-or downregulation in both datasets. for lung biopsies (gse147507), one sample from covid-19 patients shows a distinct read count profile and was considered an outlier as described (26) . the remaining sample was used to perform single sample gsea, in which genes were pre-ranked by log 2 fold change of the experimental sample over healthy controls. networks were visualized and generated with cytoscape v3.7.2 (27) . heat maps were generated with the package gplots for r and hierarchical clustering with the package amap for r, using euclidian distance metric and ward linkage. the bubble plots were generated with the package ggplot2 for r. graphpad prisma v. 8 was used to perform t-tests on nanostring ncounter data and generate bar plots. to evaluate the robustness of our approach, validate previous findings and obtain novel perspectives into immune responses to sars-cov-2 infection, we constructed a modular transcriptional network of pbmcs from covid-19 patients. genes were pre-ranked by the wald test statistics score calculated with deseq2 package [7[, and used as input in pre-ranked gsea. we interpreted the dynamics in gene expression of covid-19 patients using the alternative tool to conventional pathways, the btms, which were particularly devised to evaluate human immune responses (24) . to ensure maximal confidence, we applied a conservative statistical cutoff (fdr adjusted p < 0.001) to select significant btms ( figure 1a) . the transcriptional network captured several cellular characteristics of sars-cov-2 infection in peripheral blood, including t and nk cell ( figure 1d ) cytopenia (28) , and upregulation of cell cycle or genes associated with plasma cells and immunoglobulins (7) . in addition, our approach also detected increased signals of monocytes (figure 1b) , dendritic cells ( figure 1c ) and of the mitochondrial respiratory electron transport chain in sars-cov-2 infection (figure 1a) , suggesting a critical role of metabolic pathways for the immune response of covid-19 patients. to gather further insights on host responses to sars-cov-2 infection, the modular transcriptional signature of covid-19 patients was compared to that of individuals infected with sars-cov-1 or iav. for this, we analyzed 11 additional public transcriptome datasets, spanning over 600 samples from human pbmcs or whole blood. gene lists from each dataset were preranked by the log 2 fold changes relative to healthy controls and used as input in pre-ranked gsea. the statistical cutoff was established at nominal p < 0.001, whereas only bmts present in at least 50% of datasets are shown (figure 2a) . independently of the cohort, technology to quantify gene expression (rnaseq or microarray) and type of sample (pbmcs or whole blood), we observed a core transcriptional response that is comparable between infections caused by sars-cov-2, sars-cov-1, and iav. this core response includes modules of cell cycle and proliferation, monocytes and dendritic cells. indeed, the module m67 (dendritic cells) was upregulated in almost all datasets. of interest, sars-cov-1 and iav infections also induced significant reduction of peripheral t lymphocytes and nk cells. datasets from iav infection induced activation of type i interferon/antiviral responses or rig-1 like receptor signaling, while only sars-cov-1 induced significant association to one module, antiviral ifn signature. data from a different cohort of patients and analytical platform also demonstrated that several genes involved in type i interferon/antiviral responses were not significantly altered in whole blood of covid-19 patients (figure 2b) . we also evaluated btms that were uniquely associated to the transcriptomes from covid-19 patients, which showed enrichment in immune-related modules and heme biosynthesis (figure 2c) . data indicates an upregulation of heme biosynthesis in pbmcs from covid-19 patients ( figure 2d ). because immune responses are tightly connected to metabolic programs (4, 29-31), we explored metabolic pathway enrichment with the kegg database. in addition to porphyrin metabolism, which shares significant proportion of genes with btm m222 (heme biosynthesis ii), our analysis confirmed the upregulation of glycolysis and gluconeogenesis (7), and detected other pathways such as tricarboxylic acid (tca) cycle, oxidative phosphorylation, tryptophan metabolism, glycan degradation, nucleotide metabolism and galactose metabolism (figure 2e ). because the lung is the primary site of infection and failure of this organ is a severe complication of sars-cov-2 infection, we also evaluated immune and metabolic signatures in the lower respiratory tract of covid-19 patients. for that, we performed a btm-driven meta-analysis of transcriptomes from samples of bronchioalveolar lavage fluid (balf) (7) . using a relaxed statistical cutoff (nominal p < 0.05), there were nine significant btms and three kegg metabolic pathways that were consistently up or downregulated among both datasets ( figure 3a) . btms reflect upregulated networks of chemokines and neutrophils, as well as reduced expression of genes related to dendritic cells, monocytes, and t cell activation. we also found consistent upregulation of the modules related to chemokines ( figure 3b ) and neutrophils ( figure 3c ) in lung tissue data from one covid-19 patient. few metabolic pathways were consistently regulated between the balf datasets, including the upregulation of oxidative phosphorylation and downregulation of fructose and mannose metabolism and other glycan degradation ( figure 3a) . none of these metabolic pathways were significantly enriched on the sample of lung tissue. here, we used a robust modular transcriptomics approach that captured significant changes of cellular patterns in peripheral blood of covid-19 patients, including t lymphopenia and reduced numbers of nk cells (28) . several hypothesis have been formulated to explain the lymphopenia during covid-19, including t cell infection by sars-cov-2 (32), or t cell exhaustion (33) . in addition, we identified upregulated expression of chemokines and neutrophils in the lung tissue and balf of covid-19 patients that support an immunopathological role for these granulocytes (34) . these data are in line with findings by zhou et al. (10) , which also suggest higher proportion of neutrophils, activated dendritic cells and activated mast cells via cell deconvolution of balf transcriptomes. interestingly, our data suggest increased proportion of monocytes and dendritic cells in the circulation, but not in the balf. using single-cell rna-seq, some studies demonstrated that dendritic cells are indeed reduced in the balf (35) and there are significant phenotypical alterations of monocytes from covid-19 patients compared to healthy controls (36) . we demonstrated that compared to sars-cov-1 or iav, sars-cov-2 infection fails to induce significant type i interferon responses in pbmcs (figure 2a) or whole blood (figure 2b) , which corroborates the low concentrations of type i interferon in the circulation of covid-19 patients (9, 37, 38) . these findings contrast with induction of isg expression in both lung tissue (9) and balf (10) of covid-19 patients, while recent studies indicate that type i and iii interferons negatively affect the lung epithelium during viral infections (39, 40) . the transcriptional response of peripheral leukocytes reflects the systemic adaptations to the inflammatory environment imposed by sars-cov-2 infection, whereas type i interferon signaling in peripheral leukocytes might affect immunity in other organs such as the kidneys (41) . importantly, recent data suggest an improvement of patients with uncomplicated covid-19 treated with interferon-alpha2b (42) . we expect that several factors will contribute to differences in transcriptional profiles of larger cohorts of covid-19 patients, especially those bearing comorbidities associated with severe disease. higher expression of angiotensin-converting enzyme 2 (ace2) has been suggested as a potential mechanism of susceptibility of individuals with comorbidities associated with covid-19 (43) . however, severe disease and death also occur after infection of otherwise healthy individuals, indicating that a series of mechanisms account for the severity of covid-19. upregulated expression of genes that coordinate heme biosynthesis has been described in sepsis secondary to pneumonia and suggest a protective mechanism against oxidative stress (44) . hypoxia also modulates the expression of genes coding for proteins that coordinate heme biosynthesis (45) . we hypothesize that excessive heme accumulation could amplify pro-inflammatory cytokine production (46, 47) or cause intravascular coagulation (48) and promote pathology during covid-19. strikingly, we observed the modulation of several metabolic pathways in pbmcs and balf, while oxidative phosphorylation was the only significant metabolic pathway overlapping in both compartments. this suggests a critical role for mitochondrial activity during covid-19. many metabolites composing the pathways identified in the current study have been quantified via metabolomics of plasma or serum from covid-19 patients (49, 50) . mass spectrometry measurements revealed the modulation of pathways such as tca cycle and fructose and mannose metabolism (50) , tryptophan metabolism, glycolysis and gluconeogenesis and others (49) . metabolomics analysis of human pbmcs infected with iav showed activation of tryptophan metabolism and glycolysis, whereas glucose consumption via hexosamine biosynthesis underlies the cytokine storm promoted by iav infection (51) and could also affect covid-19. taken together, this study demonstrates unappreciated inflammatory networks and metabolic pathways that are associated with covid-19. publicly available datasets were analyzed in this study. this data can be found here: genome sequence archive (gsa) or human gsa in national genomics data center, beijing institute of genomics (big), chinese academy of sciences for rna-seq data related to sars-cov-2 infection (cra002390 and hra000143); gene expression omnibus (geo) for rna-seq data related to sars-cov-2 infection (gse147507) and microarray data related to sars-cov-1 infection (gse1739) or influenza a virus (iav) infection (gse34205, gse6269, gse29366, gse38900, gse20346, gse52428, gse40012, gse68310, gse61754, gse90732); and arrayexpress for nanostring ncounter data related to sars-cov-2 infection (e-mtab-8871)/b. lg: selected the data, performed data analysis, interpreted the results, and wrote the manuscript. cs, hs-c, and sf: interpreted the results and critically reviewed the manuscript. all authors contributed to the article and approved the submitted version. pandemic fear" and covid-19: mental health burden and strategies clinical characteristics of 3,062 covid-19 patients: a meta-analysis infection and rapid transmission of sars-cov-2 in ferrets integrative metabolomics and transcriptomics signatures of clinical tolerance to plasmodium vivax reveal activation of innate cell immunity and t cell signaling blood transcriptional profiling reveals immunological signatures of distinct states of infection of humans with leishmania infantum transcriptomics in human challenge models. front immunol transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients a dynamic immune response shapes covid-19 progression imbalanced host response to sars-cov-2 drives development of covid-19 heightened innate immune responses in the respiratory tract of covid-19 patients gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles edger: a bioconductor package for differential expression analysis of digital gene expression data expanding the immunology toolbox: embracing public-data reuse and crowdsourcing expression profile of immune response genes in patients with severe acute respiratory syndrome plasticity and virus specificity of the airway epithelial cell immune response during respiratory virus infection gene expression patterns in blood leukocytes discriminate patients with acute infections aberrant cell cycle and apoptotic changes characterise severe influenza a infection-a metaanalysis of genomic signatures in circulating leukocytes a distinct influenza infection signature in the blood transcriptome of patients with severe community-acquired pneumonia whole blood gene expression profiles to assess pathogenesis and disease severity in infants with respiratory syncytial virus infection a host transcriptional signature for presymptomatic detection of infection in humans exposed to influenza h1n1 or h3n2 transcriptomic profiling facilitates classification of response to influenza challenge host transcriptional response to influenza and other acute respiratory viral infections-a prospective cohort study development of an objective gene expression panel as an alternative to selfreported symptom scores in human influenza challenge trials molecular signatures of antibody responses derived from a systems biology study of five human vaccines kegg: kyoto encyclopedia of genes and genomes plasmin cascade mediates thrombolytic events in sars-cov-2 infection via complement and plateletactivating systems cytoscape: a software environment for integrated models of biomolecular interaction networks complex immune dysregulation in covid-19 patients with severe respiratory failure metabolic phenotypes of response to vaccination in humans mtor regulates metabolic adaptation of apcs in the lung and controls the outcome of allergic inflammation antibiotics-driven gut microbiome perturbation alters immunity to vaccines in humans sars-cov-2 infects t lymphocytes through its spike protein-mediated membrane fusion reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) targeting potential drivers of covid-19: neutrophil extracellular traps single-cell landscape of bronchoalveolar immune cells in patients with covid-19 a single-cell atlas of the peripheral immune response in patients with severe covid-19 impaired type i interferon activity and exacerbated inflammatory responses in severe covid-19 patients. medrxiv type i ifn immunoprofiling in covid-19 patients type iii interferons disrupt the lung epithelial barrier upon viral recognition type i and iii interferons disrupt lung epithelial repair during recovery from viral infection multiorgan and renal tropism of sars-cov-2 interferon-alpha2b treatment for covid-19 ace2 expression is increased in the lungs of patients with comorbidities associated with severe covid-19 genetic signature related to heme-hemoglobin metabolism pathway in sepsis secondary to pneumonia hypoxia decreases the expression of the two enzymes responsible for producing linear and cyclic tetrapyrroles in the heme biosynthetic pathway characterization of heme as activator of toll-like receptor 4 hemolysis-induced lethality involves inflammasome activation by heme excess of heme induces tissue factordependent activation of coagulation in mice proteomic and metabolomic characterization of covid-19 patient sera plasma metabolomic and lipidomic alterations associated with covid-19 o-glcnac transferase promotes influenza a virus-induced cytokine storm by targeting interferon regulatory factorâ��5 the authors thank the administrative and technical support provided by the iptsp -ufg. we thank all authors that contributed to the generation of the datasets used in the study. we also thank dr. key: cord-263433-oldy0gta authors: barriocanal, marina; carnero, elena; segura, victor; fortes, puri title: long non-coding rna bst2/bispr is induced by ifn and regulates the expression of the antiviral factor tetherin date: 2015-01-09 journal: front immunol doi: 10.3389/fimmu.2014.00655 sha: doc_id: 263433 cord_uid: oldy0gta many long non-coding rnas (lncrnas) are expressed in cells but only a few have been well characterized. in these cases, lncrnas have been shown to be key regulators of several cellular processes. therefore, there is a great need to understand the function of more lncrnas and their regulation in response to stimuli. interferon (ifn) is a key molecule in the cellular antiviral response. ifn binding to its receptor activates transcription of several ifn-stimulated genes (isgs) that function as potent antivirals. in addition, several isgs are positive or negative regulators of the ifn pathway. this is essential to ensure a strong antiviral response and a later return of the cell to homeostasis. as the isgs described to date are coding genes, we sought to determine whether ifn also regulates the expression of long non-coding isgs. to this aim, we used rna sequencing to analyze the transcriptome of control and huh7 cells treated with ifnα2. the results show that ifn-treatment regulates the expression of several unknown non-coding transcripts. we have validated two lncrnas upregulated after treatment with different doses of type i ifnα2 in different cells or with type iii ifnλ. these lncrnas were also induced by influenza and vesicular stomatitis virus mutants unable to block the ifn response, but not by several wild-type lytic viruses tested. these lncrna genes were named lncisg15 and lncbst2 as they are located close to isgs isg15 and bst2, respectively. interestingly, inhibition experiments showed that lncbst2 is a positive regulator of bst2. therefore lncbst2 has been renamed bispr, from bst2 ifn-stimulated positive regulator. our results may have therapeutic implications as lncbst2/bispr, but also lncisg15 and their coding neighbors, are increased in cells infected with hepatitis c virus and in the liver of infected patients. these results allow us to hypothesize that several lncrnas could be activated by ifn to control the potency of the antiviral ifn response. the interferon (ifn)-mediated innate immune response provides a potent defense against pathogens (1) . upon invasion, pathogenassociated molecular patterns (pamps) are detected by specific receptors in the cells. these can be located on the surface of the cell, as in the case of toll-like receptors (tlrs), or intracellularly, as in the case of the retinoic acid-inducible gene i (rig-i). pamp recognition triggers a series of signaling cascades that lead to the production and secretion of type i ifn. type i ifn (ifnα, ifnβ, and others) binds to ifn receptors present on the surface of all cell types and activates janus-activated kinase/signal transducer and activator of transcription (jak/stat) signaling. this gives rise to the nuclear translocation of the stat1/stat2/irf9 (ifn regulatory factor 9) complex that binds ifn-stimulated response elements (isre) in the promoters of ifn-stimulated genes (isgs) and activates their transcription. a similar response is induced by type iii ifn (ifnλ) upon binding to its receptor (2, 3) . in contrast, type ii ifn or ifnγ, produced by cells of the immune system, binds to the widely expressed ifnγ receptor (4, 5) leading to nuclear translocation of stat1 homodimers, which bind to gamma-activated sequences (gas) in the promoter of immunoregulatory genes. ifn-stimulated genes are antiviral factors, positive regulators of the ifn pathway (stat1 and 2 and irf1) or negative regulators that help ifn-induced cells to return to cellular homeostasis (socs and ups18) (6) (7) (8) (9) (10) (11) . among antiviral genes, there are factors that function to increase cell sensitivity to pamps (oas and pkr) or true antiviral effectors that block viral entry (mx, ifitm, and trim), virus replication, translation and stability (ifit, oas, pkr, and isg15), or viral release (viperin and tetherin/bst2) (8) . while most ifn-induced factors known to date are proteins, ifn also activates the expression of several micrornas that contribute to the antiviral state or to the control of ifn response (12) . few studies have been performed to address whether ifn could also regulate expression of long non-coding rnas (lncr-nas) (13) (14) (15) . in recent years, viral infection has been reported to be able to induce the expression of cellular lncrnas. this has been shown for infection with enterovirus, influenza, hiv, hepatitis b and c viruses, and the sars coronavirus (13, (16) (17) (18) (19) (20) (21) (22) (23) (carnero et al., in preparation) . the lncrna signature found after infection www.frontiersin.org should be a mixture of transcripts induced by the virus and transcripts that respond to the cellular antiviral pathways activated by the infection. in fact, activation of tlrs by pamps induces the expression of several lncrnas. tlr2 signaling leads to the activation of lncrna-cox2, which regulates the expression of genes related to the immune system (24) . activation of tlr3 results in increased neat1, which increases the expression of genes such as il8 (19) . tlr4 controls il1b-erna and il1b-rbt46 lncr-nas whose downregulation diminishes il1b and accumulation of lps-induced rnas (25) . likewise, the lps-induced inflammatory response is controlled by lnc-il7r (26) . innate activation also induces linc1992/thril, which controls tnfα and other genes involved in the immune response (27) . in turn, tnfα induces lethe, a pseudogene that responds to nfκb and reduces inflammation by inhibiting nfκb dna binding activity (28) . lncrna responses are also critical for the functionality of dendritic cells, cd4+ and cd8+ t-cells (29) (30) (31) (32) . thus, nest lncrna controls ifnγ locus in cd8+ t-cells leading to decreased salmonella enterica pathogenesis (33, 34) . these studies illustrate the interest in identifying novel lncr-nas and elucidating their function and regulation. lncrnas are thought to be at least as numerous as protein-coding genes, but only a few are well characterized (35) (36) (37) (38) . lncrnas are transcripts similar to mrnas but with poor coding potential. they are more cell type-specific, less expressed, and less well conserved than mrnas (29, 39) . interestingly, lncrnas are cell regulators that can function in cis, co-transcriptionally, or in trans. some control the expression of coding genes located in the same genomic region. therefore, the genomic location of lncrnas can provide hints as to their functionality. they can be sense or antisense (when overlapping with one or more exons of another transcript in the same or in the opposite strand, respectively); intronic (when derived from an intron of another transcript); divergent or bidirectional (when they share a promoter with another transcript in the opposite strand and therefore are co-regulated); or intergenic (when they are independent, located in between two other genes). several mechanisms are involved in the regulation of neighboring or antisense genes by lncrnas. these include transcriptional activation or interference, recruitment of chromatin modifiers and remodelers, regulation of imprinting, editing, splicing or translation, and stability (40) (41) (42) (43) (44) . to address the issue of whether ifn could also regulate expression of lncrnas, which may play key roles in the antiviral response, we analyzed the transcriptome of cells treated or not with ifnα2 by rna sequencing (rnaseq). in this analysis, we identified two lncrnas upregulated in response to ifn in different cell lines. interestingly, these lncrnas are expressed from positions in the genome divergent from the well-characterized isgs isg15 and bst2. therefore, we have called them lncisg15 and lncbst2. these lncrnas and their coding counterparts are also induced in cells infected with mutants of influenza or vesicular stomatitis viruses (vsv) that fail to block the ifn response. surprisingly, they are also induced in culture cells infected with hepatitis c virus (hcv) and in the liver of patients with hcv infections. finally, according to hugo regulation, we have renamed lncbst2 bispr, from bst2 ifn-stimulated positive regulator, as we show that inhibition of lncbst2 expression by rnai leads to decreased levels of bst2 mrna, providing a new layer of regulation of the ifn response. the huh7 cell line, derived from a human hepatocarcinoma, was provided by dr. chisari's lab (scripps research institute, la jolla, ca, usa). a549 cells, from human non-small cell lung carcinoma, were kindly provided by estanislao nistal (cima, university of navarra, spain). human liver samples with or without hcv infection were obtained from the biobank of the university of navarra under approval from the ethics and scientific committees. liver tissue sections were snap frozen and stored at −80°c. the clinical data from hcv-infected subjects are shown in table s1 in supplementary material. cells were grown in dulbecco's modified eagle medium (dmem) supplemented with 10% fetal bovine serum (fbs) and 1% penicillin-streptavidin and maintained at 37°c in a 5% co 2 atmosphere. twenty-four hours before treatment with ifn, huh7 and a549 cells were seeded in six-well plates. then, 0, 5, 50, 250, 1000, or 10000 units/ml of ifnα2 (sicor biotech, lithuania) were used in a final volume of 2 ml. huh7 cells were also treated with 250 ng/ml of il28b/ifn-λ3 (r&d systems) in a final volume of 2 ml. for treatment with ruxolitinib (selleckchem), cells were seeded out 24 h before and treated with 0.8 µm ruxolitinib in a final volume of 2 ml. one hour after treatment media were discarded and replaced by media containing 100 units/ml ifnα. cells were harvested for rna extraction at the indicated times post-treatment. sirnas targeting lncbst2/bispr were designed using iscore designer and rna scales (45, 46) and purchased from dharmacon. the lncbst2/bispr sirnas targeted the sequence gacuagugugagcaacaaa. for cell transfection with sir-nas, lipofectamine 2000 reagent (invitrogen) was used according to manufacturer's instructions. cells were seeded 24 h before transfection. for each well of a six-well plate, 80 pmoles sirna were used. the sirna was mixed with 50 µl optimem. furthermore, 6 µl lipofectamine were mixed with 250 µl optimem media and incubated for 5 min. then, lipofectamine and sirna solutions were mixed and incubated for 20-60 min at room temperature. after incubation, half of the volume of the cell media was discarded and 300, 150, or 75 µl of the lipofectamine mixture were added to each well of 6, 12, or 24-well plates, respectively. six hours post-transfection the media from the cells was discarded and substituted with dmem media enriched with 10% fbs and antibiotics. hepatitis c virus jfh-1 was obtained from an initial viral stock from the genotype 2a jfh-1 plasmid (pjfh-1) previously described by wakita et al. (47) . the virus was amplified as described (15) . influenza virus strain a/pr8/34 wt (pr8), a mutant lacking ns1 (∆ns1), vsv-gfp, and the mutant m51r frontiers in immunology | molecular innate immunity were kindly provided by estanislao nistal (cima, university of navarra, spain) (48) (49) (50) , semliki forest virus (sfv) was a gift from cristian smerdou (cima, university of navarra, spain), and adenovirus serotype 5 (ad5) was amplified as previously described (48) . vsv-egfp titration was performed in quadruplicates on a549 cells. the supernatant from infected cells was collected and 1:10 serial dilutions were performed. cells were seeded 24 h before infection in 96-well plates and infected with 50 µl of each dilution. twenty-four hours after infection, gfp expression was visualized by microscopy and used to determine the titer. cells were infected with hcv at a multiplicity of infection (moi) of 0.3, with vsv at a moi of 5 and with a moi of 10 of influenza a, ∆ns1, ad5, and sfv. in the case of the lytic viruses, we used a moi of 5 or 10 as this causes cytopathic effects at 24 h (for vsv, influenza and sfv) or 48 h (for ad) in huh7 or a549 cells. after infection, the virus was removed and fresh medium was added to the cells. cells were harvested for rna extraction at the indicated times post-infection. two million huh7 cells were incubated in 100 µl of cytoplasmic buffer (50 mm tris hcl ph7.4, 1 mm edta, and 1% np40) for 5 min at 4°c. then, cells were centrifuged for 5 min at 3000 g and the supernatant was used to isolate cytoplasmic rna. the pellet was washed with cytoplasmic buffer and centrifuged as before. the supernatant was discarded and the pellet was used to isolate the nuclear rna. rna from nuclear and cytoplasmic fractions was isolated with maxwell 16 research system (promega). human tissue was homogenized using the ultra-turrax dispersing machine (t25 basic ika-werke) (51) . total rna from the tissue was extracted in 1 ml trizol (sigma-aldrich) and recommendations of the supplier were followed (52) . dnase (fermentas) treatment was performed to eliminate dna from the samples before rt-pcr reactions. rna was extracted from cells with the maxwell 16 research system from promega following the manufacturer's recommendations. rna concentration was measured using nanodrop 1000 spectrophotometer. the quality of the rna was analyzed by bioanalyzer (agilent technologies). reverse transcription (rt) was performed as described (53) . the reaction was performed in the c1000 touch thermal cycler from bio-rad. the samples were incubated at 37°c for 60 min, then at 95°c for 60 s and then immediately cooled to 4°c. qpcr was performed in the cfx96 real-time system from bio-rad as described (54) . the results were analyzed with bio-rad cfxmanager software. gapdh levels were evaluated in all the cases as a reference. only the samples with similar gapdh amplification were analyzed further. the primers used are listed in table s2 in supplementary material and were designed with the primer3 program 1 . rna of excellent quality, as determined by bioanalyzer (agilent technologies) was treated with the ribo-zero rrna removal kit 1 http://frodo.wi.mit.edu (epicenter) to deplete from ribosomal rna. library preparation with truseq rna sample preparation kit (illumina) and sequencing was performed at the embl genomics core facility (genecore) in an illumina hiseq 2000. sequences were paired-end, 150 bases long, and strand specific. rnaseq data are available at the ncbi gene expression omnibus (geo) data repository 2 . rna sequencing data analysis was performed using the following workflow: (1) the quality of the samples was verified using fastqc software; (2) the preprocessing of reads was performed by elimination of contaminant adapter substrings with scythe and by quality-based trimming using sickle; (3) the alignment of reads to the human genome (hg19) was performed using the tophat2 mapper (55); (4) transcript assembly and quantification using fpkm of genes and transcripts was carried out with cufflinks 2 (56); (5) the annotation of the gene locus obtained was performed using cuffmerge with gencode v16 as reference; and (6) differential expression analysis was performed using cuffdiff 2 (56) . genes were selected as differentially expressed using a p-value threshold of 0.01. further analysis and graphical representations were performed using an r/bioconductor (57) . reads from all the differentially expressed sequences were visualized in the integrative genomics viewer (igv) 3 (58, 59) and the sequences were compared to the ensembl and encode databases and searched for in the genome browser from ucsc 4 for more information (60, 61) . candidates were divided into coding, non-coding (according to ucsc classification), or non-assigned, when the transcription of the sequence had not been annotated in the databases. functional enrichment analysis of gene ontology (go) categories was carried out using a standard hypergeometric test (62) . biological knowledge extraction was complemented through the use of ingenuity pathway analysis (ingenuity systems) 5 , with a database that includes manually curated and fully traceable data derived from literature sources. open reading frame finder (ncbi) was used to evaluate the length of all probable open reading frames (orfs) in lncisg15 and lncbst2/bispr. coding potential was assayed with the coding potential assessment tool (cpat) (63, 64) and by searching the lncipedia database (65) for the presence of our candidates in the pride archive (66) or in lists of transcripts associated with ribosomes (67, 68) . phylogenetic codon substitution frequencies (phylocsf) were also used to predict the coding potential of lncisg15 and lncbst2/bispr (69). statistical analysis of the rnaseq data has been already described. remaining analysis was performed using graph-path. statistical significance of infected versus non-infected samples was calculated using a two-tailed non-parametric mann-whitney t -test or with a two-tailed students t -test when the samples followed a normal distribution according to the shapiro-wilk test. welch's correction was applied for samples with heterogeneous variance. for correlation studies, a two-tailed non-parametric spearman analysis was used. p values lower than 0.05 were deemed as significant. to identify lncrnas that respond to ifn, we treated huh7 cells with 10000 units/ml of ifnα2 for 3 days. these conditions serve to induce the expression of well-known isgs such as gbp1, irf1, bst2, oas, or isg15 (15) . in addition, this treatment induces an antiviral effect, as hcv-infected huh7 cells treated with 10000 units/ml of ifnα2, show decreased levels of viral proteins and viral genomes compared to untreated infected cells (data not shown). finally, the rna isolated from huh7 cells treated with 10000 units/ml of ifnα2 for 3 days was used to hybridize an agilent array. analysis of the array showed that well-characterized isgs such as mx1, stat1, irf9, isg15, bst2, and several members of the gbp, oas, and ifi families were upregulated with a very high statistical significance (b > 7) (15) . ingenuity analysis of the data showed that ifn signaling was the pathway with the highest enrichment followed by other antiviral responses. the microarrays were used to identify lncrnas regulated by ifnα (15) . however, an array will only evaluate the expression levels of the transcripts that hybridize to probes spotted in the array. in the case of the lncrnas, the array used only addresses the expression of 7419 regions described as long intergenic noncoding rnas (lincrnas). however, it has been estimated that there could be as many lncrna genes as coding genes, and some authors consider that the number of lncrnas could be as high as 200000 (37, 38) . therefore, to achieve a more complete identification of lncrnas that respond to ifn, we analyzed the transcriptome by rnaseq. rna isolated from control cells or huh7 cells treated with ifn as described, was sequenced after ribodepletion. around 130 million reads were obtained per sample. analysis was performed using a bioinformatic workflow that includes tophat2 and cufflinks 2 as described in the methods section. the analysis showed that, among the genes upregulated in response to ifn, there were several isgs such as mx1, isg15, bst2, or members of the ifi and oas families ( figure 1a and table s3 in supplementary material). ingenuity analysis showed that ifn signaling is a top canonical pathway (p = 3.3 × 10 −3 ), the top upstream regulator is ifnα2 (p = 1.9 × 10 −8 ), and cell signaling and infectious and inflammatory diseases are among the main functions. the expression of~1000 coding genes was altered by ifn (table s3 in supplementary material). the rnaseq analysis also showed that the expression levels of many regions that do not correspond to coding genes were also significantly modified in response to ifn ( figure 1b) . out of the 890 putative non-coding genes whose expression was significantly altered, half were upregulated (table s3 in supplementary material). all candidates where visualized using igv ( figure s1 in supplementary material) (58, 59) . we also paid special attention to altered sequences located close to well-known isgs and to genomic regions that were highly expressed and deregulated in response to ifn. eight candidates that fulfill at least one of these two criteria were chosen for further validation ( table 1 and figure s1 in supplementary material). to validate the eight candidates chosen, we treated huh7 cells with different doses of ifnα2. rna was isolated from the cells at 6, 24, 48, or 72 h post-treatment and the expression levels of the candidates were evaluated by quantitative rt-pcr (qrt-pcr) (table 1; figure 2 ). all the candidates were induced after ifn-treatment from 2 to more than 1000-fold. however, many of the candidates were detected at very low cycles in the pcr amplification. a closer examination of their sequences indicated that they contained repetitive sequences or sequences similar to mitochondrial or ribosomal rnas that could have led to an erroneous alignment of the rnaseq reads to the human genome. we believe that, even when the oligonucleotides used for amplification were specific, a partial homology to other sequences could allow cross-amplification and thus increased possibilities of misleading results. these candidates were not studied further. we focused on two lncrnas with no repetitive sequences whose expression was highly upregulated in response to ifn (table 1; figure 2 ). interestingly, database analysis showed that they are expressed from positions in the genome located close to isg15 and bst2, both of which are well-characterized isgs. this may have functional relevance as some lncrnas have been described to regulate the expression of neighboring genes. therefore, we originally named these lncrnas after their neighbor, lncisg15 and lncbst2. later, lncbst2 was renamed bispr to follow hugo regulations. when we evaluated the expression of these lncrnas and their neighboring transcripts, we observed that both were strongly upregulated at early times in response to ifn (figure 2a) . furthermore, they responded to ifnα2 doses as low as 5 units/ml. these are similar levels to those found in the sera of some hcv patients (70) . the induction was also observed at late times post-ifn-treatment. to evaluate further the robustness of the effect www.frontiersin.org of ifn on these lncrnas, we tested whether they also respond to ifnλ, a type iii ifn. huh7 cells treated with ifnλ for 6, 12, 24, 48, or 72 h also showed increased levels of lncisg15, lncbst2/bispr, and their neighbors ( figure 2b) . in this case, all the transcripts showed a higher upregulation at later times post-ifnλ treatment. viruses activate the ifn response by several mechanisms. therefore, they have evolved to block ifn production and the activation of the ifn pathway. the molecular mechanisms involved in this ifn blockade have been characterized for many viruses. thus, for instance, ns1 protein from influenza virus and matrix protein from vsv are key factors in controlling ifn in infected cells (48) (49) (50) 71) . we sought to check whether lncisg15 and lncbst2/bispr were induced by the physiological ifn induced by an influenza virus that lacks ns1. therefore, we evaluated the expression of these lncrnas in cells infected with an influenza wild-type virus or a ns1 mutant. we also included cells infected with other rna viruses such as sfv and hcv or dna viruses such as adenovirus. all these viruses have developed mechanisms to block the cellular antiviral response and, with the exception of hcv, lead to a lytic infection. different times post-infection were evaluated. the last point was collected when the cytopathic effect was apparent. this occurred at 24 h post-infection in the case of influenza and sfv or 48 h post-infection, in the case of adenovirus. hcv-infected cells were collected at 48 and 72 h postinfection. the results showed that at later times post-infection with the influenza virus lacking ns1, there was increased expression of lncisg15, lncbst2/bispr, and their neighboring coding transcripts (figure 3a) . this increase was not observed in cells infected with wild-type influenza virus, or with other wildtype lytic viruses, suggesting that the induction may be mediated by ifn. most lncrnas are tissue-specific. to determine whether lncisg15 and lncbst2/bispr respond to infection only in huh7 cells or whether this effect is specific for influenza viruses, we infected alveolar epithelial a549 cells with vsv-gfp wild-type virus or with a m51r matrix mutant that fails to control ifn. we chose a549, because lung cells serve as the primary site for productive infection of vsv and many respiratory viruses (72) . infection with the wild-type virus did not increase the expression of lncbst2/bispr or bst2 (data not shown). however, a549 cells infected with the vsv mutant m51r for 4, 6, or 10 h did show increased levels of lncisg15, lncbst2/bispr, isg15, bst2, and other isgs such as gbp1 ( figure 3b) . surprisingly, infection with hcv also increased the expression of lncisg15, lncbst2/bispr, and other isgs, including isg15, bst2, and irf1 ( figure 3a and data not shown). to determine whether these genes were also upregulated in infected patients, we used liver samples from hcv-negative and hcv-positive donors. after quantification of the rna levels, we observed a significant increase in lncisg15, lncbst2/bispr, isg15, and bst2 in hcvinfected patients compared to controls (figure 4a ). with the number of patients evaluated, a significant correlation was not found between expression levels and infection with a particular genotype of hcv, presence of hcv-induced hepatocellular carcinoma (hcc), liver cirrhosis, or with a particular cirrhosis stage. therefore, there were no significantly different levels of these transcripts in hcv-infected livers without hcc compared with the peritumoral tissue of hcv-infected livers with hcc. although most of the samples belong to patients that are still alive, no significant correlation was observed between the levels of the evaluated transcripts and survival post-diagnosis. finally, we performed correlation studies to analyze whether in the patients, the expression level of lncisg15 or lncbst2/bispr correlates significantly with the expression level of their neighboring coding genes. the results show a highly significant positive correlation between lncisg15 and isg15 or lncbst2/bispr and bst2 (figure 4b ). the experiments performed so far suggest that a general correlation could exist between the expression of lncisg15 and isg15 or lncbst2/bispr and bst2. each lncrna and its neighboring coding gene have similar induction patterns in response to ifn or to viral infection (compare their levels in figures 2 and 3) . furthermore, the levels of each coding/non-coding pair correlate significantly in patient samples (figure 4b) . to analyze this in more detail, we performed correlation studies of the coding/noncoding pairs in all the samples evaluated in figures 2 and 3 . the results show that the correlation of each pair was highly significant ( figure s2 in supplementary material). this suggests that they could be co-regulated, and therefore, they could share similar functions. however, expression of lncisg15 and lncbst2/bispr also correlated significantly with the expression of other isgs such as oas, gbp1, or irf1 (data not shown). to obtain more information on the relationship between the coding/non-coding pairs, we searched several databases. lncisg15 and lncbst2/bispr genes are in head-to-head orientation with their coding neighbors ( figure s3 in supplementary material) and they could share the same promoter. this is based on the following facts: (i) the distance between the two genes is <1000 bp, a cut-off for bidirectional promoters (73, 74) ; (ii) there is a single dnase hypersensitivity region located between the genes, and (iii) polymerase ii (pol ii) chipseq analysis of k562 cells shows a single peak covering the h3k27ac region between both genes. interestingly, the peaks observed for pol ii chipseq are increased at 30 min or 6 h post-treatment with ifnα or ifnγ. finally, the promoter regions contain conserved isre sites and binding sequences for irf1, irf2, and irf7. to discriminate whether lncisg15 and lncbst2/bispr are induced directly by the jak/stat signaling pathway or by a secondary wave of the ifn response, we evaluated the expression of these lncrnas and their coding neighboring genes in huh7 or a549 cells incubated or not with the jak/stat inhibitor ruxolitinib. expression of gbp1, a bona fide isg, was also evaluated as a positive control (figure 5) . the results show that the levels of gbp1, bst2, and lncbst2/bispr are significantly reduced liver samples from hcv-negative and hcv-positive donors were used to quantify the levels of lncisg15, lncbst2/bispr, isg15, bst2, and gapdh mrnas. statistical significance was calculated using a two-tailed non-parametric mann-whitney t -test for lncbst2/bispr, isg15, and lncisg15 and with a two-tailed students t -test with welch's correction for bst2, which follows a normal distribution according to the shapiro-wilk test. (b) expression levels observed for lncisg15, lncbst2/bispr in patient samples were compared to the expression levels of their coding neighbors isg15 and bst2, respectively. a correlation analysis was performed and statistical significance was calculated using a two-tailed non-parametric spearman analysis. www.frontiersin.org in the presence of ruxolitinib, indicating that their expression is stat-dependent. levels of bst2 and lncbst2/bispr were also reduced in cells treated with sirnas targeting stat1 or by inhibition of irf1, a transcription factor that acts downstream of ifn (data not shown). this indicates that bst2 and lncbst2/bispr respond to stats but also to other transcription factors induced by ifn. these results agree with the possibility that bst2 and lncbst2/bispr share a bidirectional promoter. in contrast, the effect of the jak/stat pathway on isg15 and lncisg15 expression was less robust. treatment with ruxolitinib decreased the expression of isg15 and lncisg15, but in the latter, this effect was only observed in a549 cells. no effect on isg15 or lncisg15 expression was observed with a milder inhibition of stat1 or inhibition of irf1 using rna interference (data not shown). thus, although isg15 is induced very rapidly after ifntreatment, we do not observe a strong regulation of isg15 or lncisg15 by the stat pathway under the conditions used. in fact, it has been reported that a major regulator of isg15 is irf3, a transcription factor activated in response to pamps, but also a downstream effector of the ifn response (75) . we evaluated the coding capacity of lncisg15 and lncbst2/bispr bioinformatically. orf finder (ncbi) was used to determine all possible orfs in these lncrnas ( figure s4 in supplementary material). the analysis shows that all putative orfs are shorter than 50 amino acids. only two orfs could be translated according to their poor susceptibility to nonsense mediated decay. however, these orfs have non-consensus kozak sequences at the initiation codon and therefore a poor coding capacity. then, we evaluated the coding potential of lncisg15 and lncbst2/bispr with the cpat (63, 64) ( figure 6a) . cpat uses a model built with orf size and coverage together with codon (ficket score) and hexamer (hexamer score) usage bias. according to this program, lncisg15 and lncbst2/bispr are non-coding as they have a coding probability of 0.001 and 0.064, respectively, much lower than 0.364, used as a threshold with the highest sensitivity and specificity to differentiate between coding and non-coding transcripts in humans figure 6 | lncisg15, lncbst2/bispr have poor coding potential and accumulate preferentially in the nucleus. (a) bioinformatic analysis of the coding potential of lncisg15 and lncbst2/bispr. results obtained from cpat and lncipedia. two transcripts have been evaluated for lncbst2/bispr. lncisg15 and lncbst2/bispr have a coding probability and a coding label of "non-coding rnas" according to these analyses. "lists" indicated the number of times that these transcripts have been found in the pride archive or in lists containing ribosome-associated rnas published by lee or bazzini. see the text for other details. (b) subcellular localization of lncisg15 and lncbst2/bispr. huh7 cells were mock-treated or treated with 10000 units/ml of ifnα2 and divided into nuclear and cytoplasmic fractions. rna was isolated from each fraction and used to evaluate the expression levels of lncisg15 and lncbst2/bispr by qrt-pcr. malat1, gapdh, and isg15 mrna was also quantified and used as a reference to calculate the relative levels of each transcript and as a control to evaluate the subcellular fractionation. the ratio of cytoplasmic to nuclear levels is shown. the experiment was performed three times and each value shows the average of three replicas from a representative experiment. error bars indicate standard deviations. (64) . lncisg15 and lncbst2/bispr were also described as noncoding in lncipedia (65) . this lncrna database shows that these lncrnas are not found in the pride archive, a database for proteomic data, or in lists of transcripts associated with ribosomes in ribosome profiling experiments (66) (67) (68) . further, lncisg15 and lncbst2/bispr were also described as non-coding by the analysis of phylocsf, which uses multiple alignments to calculate the frontiers in immunology | molecular innate immunity phylogenetic conservation score and determines whether a multispecies nucleotide sequence alignment is likely to represent a protein-coding region (69) . finally, we evaluated the subcellular localization of lncisg15 and lncbst2/bispr in huh7 cells mock-treated or treated with 10000 units/ml of ifnα. rna was isolated from nuclear or cytoplasmic fractions and quantified by qrt-pcr. we found that the coding gapdh or isg15 mrnas accumulate preferentially in the cytoplasm while the nuclear rnas malat1 or u6 are preferentially nuclear (figure 6b data not shown) . similarly, lncisg15 and lncbst2/bispr, compared to mrnas, accumulate preferentially in the nucleus. this result, together with the bioinformatic analyses, strongly suggests that lncisg15 and lncbst2/bispr are non-coding rnas. to address the role of lncbst2/bispr, we used rna interference. huh7 cells treated or not with ifn, were transfected with sir-nas targeting lncbst2/bispr and rna expression was evaluated by qrt-pcr. the results show that expression of lncbst2/bispr was decreased compared to cells transfected with control sirnas ( figure 7a) . surprisingly, inhibition of lncbst2/bispr also led to decreased levels of bst2 mrna. expression of lncisg15, isg15, gbp1 or expression of genes located in the genome close to bst2 or lncbst2/bispr, such as gtpbp3 or mvb12a, was not affected (figure 7a and data not shown). to determine whether this was a general phenomenon, we transfected the sirnas targeting lncbst2/bispr into a549 cells infected or not with the vsv m51r mutant or treated with ifn. similarly to what has been observed in huh7 cells, the sirna that targets lncbst2/bispr leads to decreased levels of lncbst2/bispr and bst2 mrna while the levels of isg15 mrna are not significantly affected (figure 7b) . similar results were observed with a different sirna targeting lncbst2/bispr. rna sequencing analysis of human cells treated with ifnα2 and controls has allowed the identification of lncrnas induced in response to ifn (figure 1) . analysis of the rnaseq data shows that several of the upregulated genes are well-known coding isgs such as isg15 or oas ( figure 1a , table s3 in supplementary material). ingenuity analysis confirms the enrichment of genes involved in the ifn response among the regulated factors. we have used rnas from similar ifn-treated and control cells to hybridize expression arrays (15) . comparison of the datasets obtained in the analysis of the array and the rnaseq shows that only 13 coding genes were identified in both studies, including oas, isg15, mx1, and some members of the ifi family. generally, overlap between microarray and rnaseq analysis is not high (76) . furthermore, the overlapping decreases with sequencing depth and when low fold-changes or low abundance genes are analyzed. (77) . this is because sequencing of low transcript abundances is characterized by high variance, which impedes their identification in rnaseq analysis. we believe that this may explain the poor correlation found between the array and the rnaseq datasets. in fact, we have determined by qrt-pcr that some ifnrelated low abundance transcripts are detected only in the array analysis. these are early responders to ifn, which increased only marginally 3 days after ifn-treatment, when the analysis was performed. therefore, we believe that some lncrnas induced early post-ifn-treatment may have not been identified in our analysis. interestingly, in the process of writing this manuscript, a paper www.frontiersin.org was accepted describing the identification of ifn-induced lncr-nas by rnaseq in samples treated with ifn for short times (14) . we believe that this study will be complementary to our work. together, the datasets should contain lncrnas regulated at early and later times post-ifn-treatment. similarly, the lack of correlation between the microarray and rnaseq datasets also indicates that they can complement each other. we have identified two lncrnas whose expression is highly upregulated in response to different doses of ifnα (table 1; figure 2a ) or ifnλ (figure 2b) . our results show that induction of these lncrnas by ifnα seems faster than that observed for ifnλ. we cannot rule out the possibility that a fast response to ifnλ may also be observed when higher doses are used. encode analysis of polymerase ii binding to the promoters of these lncr-nas also shows that they may be induced by treatment with ifnα and ifnγ ( figure s3 in supplementary material). these lncrnas have been named lncisg15 and lncbst2/bispr after their neighboring genes, which play a key role in the antiviral ifn response. our molecular and bioinformatic analyses strongly suggest that lncisg15 and lncbst2/bispr are indeed lncrnas, as they accumulate preferentially in the nucleus of ifn-treated or untreated cells ( figure 6b ) and they have poor coding potential ( figure 6a and figure s4 in supplementary material). in general, the upregulation of lncisg15 and lncbst2/bispr mimics that of their coding counterparts (figures 2-4) . in fact, analysis performed with all the expression data obtained in our studies, shows a highly significant correlation between lncisg15 and isg15 and between lncbst2/bispr and bst2. significant correlations also exist between these lncrnas and other ifn-induced genes such as oas, gbp1, or irf1 ( figure s2 in supplementary material and data not shown). this may reflect the fact that all these genes are induced by ifn with a similar kinetics. in the case of the lncrnas and their coding counterparts, correlation of the expression may result from their transcriptional co-regulation. experimental and bioinformatic analyses indicate that bst2 and lncbst2/bispr are bona fide isgs strongly induced by the jak/stat pathway in response to ifn (figure 5 and figure s3 in supplementary material). furthermore, expression of bst2, isg15, and their neighboring non-coding genes is induced by downstream effectors of the ifn response. these studies allow us to suggest that lncisg15/isg15 and lncbst2/bispr/bst2 may share bidirectional promoters. other ifn-induced gene pairs may also be co-regulated by bidirectional promoters as these are enriched in stat1 binding (78) . bidirectional promoters often couple genes involved in the same process, allowing for coordinated temporal and environmental responses (73, (78) (79) (80) (81) (82) . non-coding rnas generated from bidirectional promoters may have functional roles that affect the bidirectional promoter, the neighboring protein-coding gene, or more distal genes (83) . these effects could lead to activation or repression of the expression and could be mediated by either the transcription process itself or by the produced ncrna transcript (84) . in this study, we show that post-transcriptional inhibition of lncbst2/bispr leads to reduced levels of bst2 mrna (figure 7) . therefore, lncbst2/bispr should increase transcription or stability of its coding neighboring gene. our results demonstrate that this regulation seems specific for bst2, as lncbst2/bispr downregulation does not affect the expression of genes located nearby, which has been described for compact genomes (85) . moreover, inhibition of lncbst2/bispr does not affect expression levels of other ifn-related genes such as isg15 or gbp1 (figure 7 and data not shown). we anticipate that inhibition of lncbst2/bispr, and therefore of bst2, could impact the antiviral effects of ifn. bst2 is also named tetherin, as it inhibits viral budding by using anchors that trap virions on the cell membrane (86) (87) (88) . several enveloped viruses have been shown to be susceptible to the action of bst2/tetherin and have evolved to develop evasion strategies (87) . interestingly, hiv, influenza, hcv, and vsv are among the susceptible viruses and could be used to test the antiviral role of lncbst2/bispr (49, (89) (90) (91) (92) (93) (94) (95) . in fact, we show that lncbst2/bispr and bst2 are induced after infection with hcv or influenza and vsv mutant viruses that activate the ifn response (figure 3) . upregulation of lncbst2/bispr, lncisg15, and their coding neighbors was also observed in patients infected with hcv compared to controls (figure 4) . similarly, a significant upregulation of lncbst2/bispr and isg15 was also detected in human tlymphocytes infected with hiv compared to controls (data not shown). a non-significant increase in bst2 and lncisg15 was also observed in these samples. this leads to the possibility that interference with these factors could have therapeutic relevance. it is unclear why cells infected by these viruses, which employ several viral proteins to block the ifn pathway, show activation of these ifn response genes (96) . in the case of hcv, it has been previously shown that patients with chronic hcv infections express isgs, including high levels of isg15 (97) (98) (99) . in fact, hcv has evolved to use some isgs for viral replication (100, 101) . this is the case for isg15. isg15 is an ubiquitin-like protein that attaches to its targets in a process called isgylation (102, 103) . protein isgylation may result in increased or decreased functionality depending on the target (104) . isg15 preferentially conjugates newly synthesized proteins affecting more strongly viral proteins or cellular proteins translated into ifn-induced cells (105) . viruses such as influenza, hiv, or vsv are susceptible to the action of isg15 (103, 106) . in the case of hcv, a pro-hcv role for isg15 has been reported (105, 107) . isg15 has been shown to negatively regulate rig-i and thus to inhibit the signaling process leading to ifn induction that affects hcv replication (108) . furthermore, isg15 expression in the liver of chronically infected patients is considered a negative predictive biomarker of the ability of the patients to respond to ifn therapy (97) (98) (99) (figure 4) . in our study, we cannot address whether lncisg15, bst2, or lncbst2/bispr are markers for the susceptibility of hcv patients to respond to ifn-treatment, as the hcv patients that we have studied are non-responders to ifn. we believe that, similar to lncbst2/bispr, lncisg15 could affect the expression of isg15 or other genes. this lncrnamediated control has also been described for a lncrna located close to the isg viperin, which has been shown to regulate the levels of many ifn-inducible genes (14, 109) . further experiments will be required to address the role of lncisg15 and to decipher the molecular mechanisms that allow the control exerted by lncbst2/bispr on bst2. we believe that these studies may be important to better understand the ifn response and its pro or antiviral functions on hcv and other viruses. frontiers in immunology | molecular innate immunity regulation of type i interferon responses dynamic expression profiling of type i and type iii interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression interferons alpha and lambda inhibit hepatitis c virus replication with distinct signal transduction and gene regulation kinetics interferon gamma: a master regulator of atherosclerosis the two faces of interferon-gamma in cancer complex modulation of cell typespecific signaling in response to type i interferons synergistic activation of inflammatory cytokine genes by interferon-gammainduced chromatin remodeling and toll-like receptor signaling interferon-stimulated genes: a complex web of host defenses socs proteins, cytokine signalling and immune regulation alpha interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp18/ubp43 fine tuning type i interferon responses interferons and micrornas annotation of long non-coding rnas expressed in collaborative cross founder mice in response to respiratory virus infection reveals a new class of interferon-stimulated transcripts negative regulation of the interferon response by an interferon-induced long non-coding rna type interferon regulates the expression of long noncoding rnas lncrna expression signatures in response to enterovirus 71 infection evidence for a crucial role of a host non-coding rna in influenza a virus replication unique signatures of long noncoding rna expression in response to virus infection and altered innate immune signaling long noncoding rna neat1-dependent sfpq relocation from promoter region to paraspeckle mediates il8 expression upon immune stimuli hepatitis b virus x protein (hbx)-related long noncoding rna (lncrna) down-regulated expression by hbx (dreh) inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin long noncoding rna high expression in hepatocellular carcinoma facilitates tumor growth through enhancer of zeste homolog 2 in humans elevation of highly up-regulated in liver cancer (hulc) by hepatitis b virus x protein promotes hepatoma cell proliferation via down-regulating p18 neat1 long noncoding rna and paraspeckle bodies modulate hiv-1 posttranscriptional expression a long noncoding rna mediates both activation and repression of immune response genes long noncoding rnas and enhancer rnas regulate the lipopolysaccharide-induced inflammatory response in human monocytes the human long noncoding rna lnc-il7r regulates the inflammatory response the long noncoding rna thril regulates tnfalpha expression through its interaction with hnrnpl a mammalian pseudogene lncrna at the interface of inflammation and antiinflammatory therapeutics chromatin signature reveals over a thousand highly conserved large non-coding rnas in mammals genome-wide identification of long noncoding rnas in cd8+ t cells role of micrornas and long-non-coding rnas in cd4(+) t-cell differentiation the stat3-binding long noncoding rna lnc-dc controls human dendritic cell differentiation the nest long ncrna controls microbial susceptibility and epigenetic activation of the interferon-gamma locus cutting edge: influence of tmevpg1, a long intergenic noncoding rna, on the expression of ifng by th1 cells an integrated encyclopedia of dna elements in the human genome the rise of regulatory rna human cancer long non-coding rna transcriptomes specific expression of long noncoding rnas in the mouse brain landscape of transcription in human cells molecular interplay of the noncoding rna anril and methylated histone h3 lysine 27 by polycomb cbx7 in transcriptional silencing of ink4a control of alternative splicing through sirna-mediated transcriptional gene silencing expression of a noncoding rna is elevated in alzheimer's disease and drives rapid feed-forward regulation of beta-secretase epigenetic silencing of tumour suppressor gene p15 by its antisense rna long noncoding rnas: fresh perspectives into the rna world thermodynamic instability of sirna duplex is a prerequisite for dependable prediction of sirna activities comparison of approaches for rational sirna design leading to a new efficient and transparent method production of infectious hepatitis c virus in tissue culture from a cloned viral genome adenovirus va rna-derived mirnas target cellular genes involved in cell growth, gene expression and dna repair induction and evasion of type i interferon responses by influenza viruses the vesicular stomatitis virus matrix protein inhibits transcription from the human beta interferon promoter effect of adenovirus-mediated rna interference on endogenous micrornas in a mouse model of multidrug resistance protein 2 gene silencing combination of rna interference and u1 inhibition leads to increased inhibition of gene expression increased in vivo inhibition of gene expression by combining rna interference and u1 inhibition aav vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers tophat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions transcript assembly and quantification by rna-seq reveals unannotated transcripts and isoform switching during cell differentiation integrative genomics viewer (igv): high-performance genomics data visualization and exploration integrative genomics viewer the ucsc genome browser database: 2014 update gencode: the reference human genome annotation for the encode project data analysis tools for dna microarrays cpc: assess the proteincoding potential of transcripts using sequence features and support vector machine cpat: coding-potential assessment tool using an alignment-free logistic regression model lncipedia: a database for annotated human lncrna transcript sequences and structures the proteomics identifications (pride) database and associated tools: status in 2013 global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution identification of small orfs in vertebrates using ribosome footprinting and evolutionary conservation revisiting the protein-coding gene catalog of drosophila melanogaster using 12 fly genomes quantification of different human alpha interferon subtypes and pegylated interferon activities by measuring mxa promoter activation influenza a virus lacking the ns1 gene replicates in interferon-deficient systems inhibition of vesicular stomatitis virus infection in epithelial cells by alpha interferon-induced soluble secreted proteins an abundance of bidirectional promoters in the human genome complex loci in human and mouse genomes identification of a member of the interferon regulatory factor family that binds to the interferonstimulated response element and activates expression of interferon-induced genes comparison of microarrays and rna-seq for gene expression analyses of dose-response experiments large scale comparison of gene expression levels by microarrays and rnaseq using tcga data transcription factor binding and modified histones in human bidirectional promoters systematic analysis of headto-head gene organization: evolutionary conservation and potential biological relevance bidirectional gene organization: a common architectural feature of the human genome expression of the murine ranbp1 and htf9-c genes is regulated from a shared bidirectional promoter during cell cycle progression divergent transcription is associated with promoters of transcriptional regulators functional consequences of bidirectional promoters induced ncr-nas allosterically modify rna-binding proteins in cis to inhibit transcription antisense expression increases gene expression variability and locus interdependency tetherin inhibits hiv-1 release by directly tethering virions to cells bst-2/tetherin: structural biology, viral antagonism, and immunobiology of a potent host antiviral factor intrinsic cellular defenses against human immunodeficiency viruses bst-2 expression in human hepatocytes is inducible by all three types of interferons and restricts production of hepatitis c virus mechanism of hiv-1 virion entrapment by tetherin bst2/tetherin inhibits hepatitis c virus production in human hepatoma cells vpu binds directly to tetherin and displaces it from nascent virions influenza virus partially counteracts restriction imposed by tetherin/bst-2 tetherin inhibits retrovirus release and is antagonized by hiv-1 vpu interferoninduced cell membrane proteins, ifitm3 and tetherin, inhibit vesicular stomatitis virus infection via distinct mechanisms regulation of hepatic innate immunity by hepatitis c virus interferon type i gene expression in chronic hepatitis c interferon signaling and treatment outcome in chronic hepatitis c intrahepatic gene expression during chronic hepatitis c virus infection in chimpanzees hepatitis c virus controls interferon production through pkr activation hepatitis c virus blocks interferon effector function by inducing protein kinase r phosphorylation interferon-induced isg15 pathway: an ongoing virus-host battle the antiviral activities of isg15 positive regulation of interferon regulatory factor 3 activation by herc5 via isg15 modification the interferon stimulated gene 15 functions as a proviral factor for the hepatitis c virus and as a regulator of the ifn response ifn-stimulated gene 15 functions as a critical antiviral molecule against influenza, herpes, and sindbis viruses isg15, a ubiquitin-like interferon-stimulated gene, promotes hepatitis c virus production in vitro: implications for chronic infection and response to treatment negative feedback regulation of rig-imediated antiviral signaling by interferon-induced isg15 conjugation the antiviral protein viperin inhibits hepatitis c virus replication via interaction with nonstructural protein 5a regulation of interferon-stimulated gene bst2 by a lncrna transcribed from a shared bidirectional promoter barriocanal contributed to the writing of the manuscript; victor segura was in charge of all the bioinformatic analyses; and puri fortes conceived the project and the required experiments, provided the budget, interpreted the data, and wrote the manuscript. we thank nerea razquin and celia prior for excellent technical assistance, paul miller for editorial work, estanis nistal, cristian smerdou, and rafael aldabe for influenza and vsv viruses, sfv, and hcv, respectively. we also thank pablo gastaminza for the huh7 cells sensitive to hcv infection used in all the experiments, esther larrea for ifnα2 and primers for isgs, elizabeth guruceaga for the initial studies on rnaseq data and ruben hernandez for helpful comments on the manuscript. we would like to thank patients for the generous donation of samples and virginia villar and the biobank of the university of navarra for their mediation. this work was supported by grants from ministerio de ciencia e innovacion bio2009/09295, and saf2012-40003, feder funding, funds from the "ute project cima" and by the project rnareg [csd2009-00080], funded by the ministry of science and innovation under the program consolider ingenio 2010. a manuscript from dr. valadkhan's laboratory has been accepted for publication that describes similar results (110) . the supplementary material for this article can be found online at http://www.frontiersin.org/journal/10.3389/fimmu.2014.00655/ abstract the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. the review editor saba valadkhan declares that, despite having collaborated on a publication in the last 2 years with authors puri fortes, marina barriocanal, and elena carnero, the review process was handled objectively. key: cord-305936-tdswzj7r authors: freitas, andré ricardo ribas; donalisio, maria rita title: excess of mortality in adults and elderly and circulation of subtypes of influenza virus in southern brazil date: 2018-01-08 journal: front immunol doi: 10.3389/fimmu.2017.01903 sha: doc_id: 305936 cord_uid: tdswzj7r purpose: in the elderly population, the influenza infection and its clinical complications are important causes of hospitalization and death, particularly, in longer-lived age. the objective of this study is to analyze the impact of influenza virus circulation on mortality in the elderly and adults, in years with different predominant virus strains. methods: we performed a time trend study to evaluated excess of mortality for pneumonia and influenza, respiratory disease, and all-causes in southern region of brazil, from 2002 to 2015. after considering other models, we opted for serfling regression. excess of death rates per 100,000 inhabitants were analyzed in specific age groups (24–59, 60–69, 70–79, ≥80 years) and by year of occurrence. mortality information were taken from brazilian mortality information system and etiological data were accessed in sentinel virological surveillance database, getting the weekly positivity of the immunofluorescence tests for influenza a (h1n1, h3n2), and b. results: in southern brazil, there is an evident seasonal pattern of all death outcomes among different age groups in the dry and cold season (april–september). the highest excess mortality rates occurs among older, particularly in years of circulation of influenza ah3n2, especially among people ≥80 years, in 2003 and 2007—years of great severity of influenza activity. after 2009, with the introduction of the pandemic influenza ah1n1, we observed a lower impact on the mortality of the elderly compared to <60 years. discussion: a cross reactivity antibody response from past exposure probably provided protection against disease in the elderly. despite not controlling for comorbidities, climate, and vaccination, for the >70 years, ratio of respiratory diseases excess mortality rates between ah1n1 (2009) and severe year of h3n2 (2007) shows protection in the pandemic year and great vulnerability during ah3n2 virus predominance. conclusion: the reduced immune response to infection, and to vaccination, and presence of comorbidities recommend a special attention to this age group in brazil. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases are some of the preventive and public health measures. introduction human influenza viruses can cause diseases through many direct and indirect pathological effects. consequences are destruction of infected cells, release of cytokines leading to fever, malaise, damage to respiratory epithelium and pulmonary parenchyma, and pneumonia. it includes secondary bacterial infections because of tissue damage and exacerbation of preexisting comorbidities such as cardiovascular and renal diseases, diabetes, or chronic lung disease (1) (2) (3) . the rates of hospitalization and mortality associated with influenza are higher among patients with chronic diseases, children under 1 year and after 65 years of age (4, 5) . with the aging population in recent decades, the raw number of hospitalizations and deaths related to pneumonia and influenza tends to increase (4) , this phenomenon has been observed also in brazil (6, 7) . however, the impact and severity of influenza virus circulation depend in part, on the strain that predominates in the season each year. due to the lack of laboratory confirmation, influenza-associated morbidity and mortality are often classified as pneumonia, other respiratory diseases, or other causes. given the difficulty of directly measuring influenza morbidity and mortality, time series models are used to elucidate disease patterns in various age groups. trends are usually determined by means of statistical inference, based on seasonal coincidence of the occurrence of certain diseases or death and laboratory confirmation of the viral circulation (4, 8) . different approaches, with and without the quantification of the proportion of viral isolates, can produce average estimates of excess deaths associated with the circulation of certain viral variants (9) (10) (11) . viral surveillance data, hospitalization, or death indicators are particularly useful for the study of influenza in the tropics, as seasonality may be less evident (11) (12) (13) . serfling regression has been used to analyze excess of mortality related with respiratory virus circulation (7, (14) (15) (16) . despite some limitations (17) , the inclusion of sinusoidal terms in weekly regression may reduce spurious correlation between influenza occurrence and death (18, 19) . it is particularly useful when no other covariables are available, and with small samples of viral sentinel surveillance data (18) . poisson regression and the generalized linear model (glm) can produce more specific estimates and support adjustments for variables (temperature, humidity, comorbidities, other circulations of viruses), although they require a more robust and consistent virological surveillance and cannot be used for pandemics (4) . in brazil, surveillance for influenza syndromes was implemented in 2000, monitoring the occurrence of respiratory viruses (influenza a and b, parainfluenza 1, 2, and 3, respiratory syncytial virus, adenovirus). the brazilian ministry of health provides vaccination coverage annually since 1999 for seniors and some risk groups, with vaccine coverage of the elderly population at around 80% in southern brazil, the region with the highest coverage of the country. despite the adequate coverage, protective titers after vaccination (hi ≥ 0) are consistently lower with poorer cell mediate and antibody responses in the elderly comparing to adults (20) . considering the vulnerability of the elderly to influenza virus infection, and the lack of studies on its repercussion in brazil, the objective of this study was to analyze the impact of different strains of influenza a virus circulation. we analyzed particularly the most predominant variants (ah1n1 and ah3n2) on excess of mortality in the adults and elderly of different age groups in a region with marked seasonality of respiratory diseases in brazil. this is a time trend study to evaluated excess of mortality from 2002 to 2015 in southern region of brazil (states of paraná, santa catarina e rio grande do sul), total area is 576,774,31 km 2 , population is 27,386,891 inhabitants with subtropical climate (köppen-geiger classification cfa). we choose these states for analysis because of the consistent seasonal pattern of influenza, as well as the availability and quality of etiological data from the virological surveillance system in that region. for the mortality rates of specific age groups (24-59, 60-69, 70-79, and ≥80 years) and death causes, we took data from brazilian mortality information system. causes are classified according to international causes of death icd-10 revision, pneumonia, and influenza (icd j 10 to j18.9), respiratory diseases (icd j00 to j99), and all-cause (excluding external causes of mortality). we obtained population of each year and age group from instituto brasileiro de geografia e estatística-ibge from the census-2010, and population estimates for the following years. etiologic information of flu-like syndrome was accessed in database of the national sentinel virological surveillance system. it has data from 128 sentinel units distributed in all regions of the country-north (21 units), northeast (26 units), southeast (34 units), south (38 units), and central west (9 units). surveillance is performed through the systematic collection of weekly samples of nasopharyngeal secretions from patients who present flu-like syndrome. reference laboratories process samples by using indirect immunofluorescence (iif), with tests for influenza a and b, parainfluenza 1, 2, and 3, respiratory syncytial virus, and adenovirus. a portion of the samples is submitted to polymerase chain reaction tests to identify the virus genotype. we calculated the laboratory positivity indicator using weekly positive results of iif divided by the total of weekly valid tests, i.e., excluding the results within inadequate samples (not enough biological material, improper storage, incorrect material in the sample) or inconclusive results (no valid results). influenza vaccination coverage (%) of southern region from 2002 to 2015 was obtained from brazilian national program of immunization data base (datasus). the criteria used to define the period of increase of influenza activity was when the positivity of the samples tested exceeded twice the annual mean of the weekly positivity of samples processed by surveillance, during two consecutive weeks. in the year 2009, we consider the period officially recognized by the brazilian ministry of health as epidemic by the influenza ah1n1pmd2009 strain, due to irregularity of the sample collection by the sentinel surveillance system at the end of epidemic. we calculated the weekly mortality rates by age group using the number of deaths per group of causes divided by the estimated population in the middle of the year multiplied by 100,000. we constructed a serfling cyclical regression model (14) for weekly data applied to each age group and causes of death (pneumonia and influenza, respiratory diseases, and all causes), as seen in others studies (7, 15) , to estimate baseline of predicted deaths in the absence of influenza epidemics. to fit regression, we used period of 13 years (from 2002 to 2015), excluding the weeks of epidemics periods. a cyclical linear regression was adjusted with the equation: where y is the mortality rate, β is the coefficients of regression, t is time in weeks, and t 2 and t 3 are variables for adjusting the secular trend of the disease. we used of sine and cosine for adjust of annual and semiannual periodic components. after adjusting a linear regression and define the expected mortality rate, we delimited 95% upper confidence limit of the baseline as the reference threshold in the absence of influenza epidemics. we calculated the excess of deaths as the observed mortality minus the expected mortality in the periods when mortality was above 95% of the confidence interval during epidemics periods. we also present ratios of excess mortality rates among years of predominant circulation of influenza strains ah3n2 (mean and years of severity), ah1n1 pre-pandemic, and ah1n1 postpandemic for each age group. for data compilation, we used microsoft office excel 2007, and for statistical analysis, spss for windows, version 24.0. results table 1 shows the proportion of positivity of the iif nasopharyngeal samples and the annual prevalence of strains of influenza in the period. before 2009, the year of entry of the pandemic strain ah1n1pmd 2009, there was a predominance of influenza ah3n2 in the years 2003 to 2007. after 2009, there is alternation of strains in the southern brazil. annual elderly vaccination coverage in southern region is high and homogeneous, around 80%, and even higher in the recent years. there is an evident seasonal pattern of deaths from pneumonia and influenza, respiratory diseases, and all-causes among the elderly in different age groups in the dry, cold months (april-september) in southern region (figure 1) . we note a progressive increase in the rates of excess deaths (of all outcomes) with increasing age, especially among those older than 70 years. in the pre-pandemic years with dominance of the ah1n1 strain, the excess of mortality rates associated with influenza were relatively low, compared to years of prevalence of ah3n2 strain ( table 2) . among those over 80 years, the ratio of excess mortality rates between 2009 and the years with dominium of h3 strains was less than one. this ratio suggests that this age group was spared in the 2009 pandemic. however, in years of predominance of strain h3, excess of mortality rate of all causes in this group were 449.6 per 100,000 (corresponding to 1,598 obits), 5, and 8.2 times greater than the same rate in years of circulation of h1n1 in pre-and post-pandemic period, respectively. among adults (24-59 years), we observe a large excess of deaths rates during the 2009 pandemic (953 obits), which correspond to 7.1 excess deaths from all causes, and 99 excess mortality from respiratory diseases associated with viral infection in every 100,000 individuals of the age group. the ratio between excess mortality rates due to pneumonia/influenza in the pandemic year (2009) and the mean rate of the period was 12 times higher among the youngest ( table 2) . rates of excess mortality by pneumonia and influenza and respiratory diseases are lower than all causes in all age groups, but particularly high in older than 80 years ( table 2) . the results highlight the great vulnerability of elderly to influenza ah3n2, especially among older than 70 years in severe years of influenza activity, like 2003 and 2007. the study also shows the lower impact of influenza ah1n1pdm 2009 in this age group compared to younger. risk of dying among the elderly in years of circulating ah3n2 influenza has been reported in several parts of the world (9, 10, 21, 22) ; however, in brazil, there are no recent estimates available. few studies analyze the circulation and impact of influenza in tropical and subtropical regions (6, 7, (9) (10) (11) . influenza b virus is also associated with severe disease (23); however, this variant did not circulate with intensity during the study years in brazil. although the elderly are the most vulnerable group to viral respiratory infections, we found relative small excess of deaths in years of circulating ah1n1 pre pandemic (2002 and 2008) . study comparing excess deaths from respiratory diseases in the elderly in latin america shows stable rates (mean of 89.4 per 100,000 inhabitants) in southern brazil between 1998 and 2008 (prepandemic flu a-h1n1), although higher in brazil than in other countries (24) . in the usa and in european countries, influenza seasons dominated by subtype ah3n2 are typically associated with mortality two to three times higher than in seasons with predominance of ah1n1 (prior to pandemic strain 2009) and of influenza b viruses (9, 10, 19, 25) . when all causes of death are studied, the overall mortality associated with influenza among elderly exceeds that observed in younger age group. it should be considered that all causes mortality is a non-specific measure and a distant outcome of influenza infection. however, it is difficult to determine which group of causes of death could better characterize the influenza burden in mortality. by choosing only the respiratory causes, we may underestimate clinical complications of pulmonary viral infection (e.g., cardiovascular). therefore, in this study, we analyzed all causes, respiratory, and pneumonia and influenza deaths. the unfavorable evolution of infection in the elderly is possibly due to the prevalence of comorbidities, deficiencies in defense mechanisms, and poor antibody response to vaccination, as cellmediated and humoral responses limit severity of disease (26) . patients with chronic diseases are more susceptible to infection due to decline of the immune function through inflammatory mechanisms, hindering the mucosal barrier, and the adaptive and innate immunological defense mechanisms (27) . the immune response to infection in the elderly tend to be delayed and weak, with prolonged inflammatory responses, which involves different types of host reaction, mainly to clearance virus. the exacerbations of these mechanisms may induce immunemediated pathology causing tissue damage (28) . cytokine high serum levels of il-6, tnf-a, ifn-g and sil-2r, chemokines ip-10, mcp-1, and monokine induced by ifn-g (mig), are associated with severe clinical cases and lung damage (29) . immunological abnormalities in people with diabetes, chronic respiratory diseases, cardiopathy, or other chronic diseases have increased risk of severe infection and bad prognosis (19) . for example, there is the consistent association of influenza infection with cardiovascular mortality, particularly acute myocardial infarction (30) . in part, it is attributed to altering endothelial function due to an acute inflammatory and procoagulant stimulus during viral infection (31, 32) . clinical complications of diabetes triggered by influenza infection cause impairment of leukocyte function and increase post-infection colonization rates resulting in poor prognosis in the elderly (33, 34) . in young people and adults, in 2009, the emerging influenza ah1n1 strain had a notable impact on the mortality of people up to 59 years in various parts of the world, including brazil (7, 25, 35, 36) . excess mortality of individuals aged 24-59 years in the state of são paulo, brazil was identified during the pandemic ah1n1 virus (7) . pregnant women adults with metabolic conditions, including obesity, chronic respiratory disease, and other chronic diseases were significantly associated with severe acute respiratory syndrome and the lethality in brazil (37) . our study showed a 41.5-fold higher rate of mortality from pneumonia and influenza in adults (24-59 years) in the pandemic year ah1n1 than the average of years with predominance of ah3n2 circulation in southern region. in addition to the clinical severity and the large portion of the affected population, pandemics affect age groups in different ways (38) . while only 10% of deaths from seasonal influenza occur among those under 65 years of age, in the pandemics of 1918, 1957-1958, and 1968 , this proportion was 95, 40, and 50%, respectively (39) . therefore, pandemics tend to affect a larger proportion of young people than seasonal influenza. in this study, higher rates of death due to pneumonia, influenza, respiratory, and all causes were observed among those aged 24-59 years in 2009. one explanation for the higher mortality observed among the youngest is that they would be more prone to the situation known as "cytokine storm, " i.e., a dysfunctional overproduction of cytokines that would lead to diffuse damage to the respiratory tract with severe and potentially lethal systemic repercussions (40) . viral replication and production of inflammatory mediators seem to be involved in the pathogenesis of infection with influenza a h1n1pmd2009, hindering the clearance of virus in lung tissue and leading to pathologic lesions (41) . another explanation for the lower mortality in the elderly is that they were exposed previously to antigens of the pandemic virus. hancock et al. (42) suggested a cross-reactive antibody response to 2009 pandemic ah1n1. similarities between ah1n1 antigen from 2009 and 1918 were detected. this last virus strain has not circulated since 1958 (39) , when the ah1n1 strain was displaced by ah2n2 (asian flu). at that time, ah1n1viral circulation occurred mainly in children, the current elderly of 2009. the emergence of the ah3n2 strain in the pandemic year 1968 (hong kong flu) affected several age groups. this new strain resulted from a large genetic mutation (shift) recombining virus material of the circulating ah2n2 with the avian h3, of asian origin, resulting in the new variant ah3n2 (38) . in 2002-2003, under selective pressure an antigenic small mutation (drift), resulted in a/fujian/411/02(h3n2) a strains emerged after a "jump" in genes evolution of hemagglutinin and neuraminidase proteins of virus surface (43, 44) . the circulation of the fujian strain had a great impact on the mortality from pneumonia in several parts of the world in 2003-2004 and 2004-2005 (22) and in brazil (45) . in 2007, a new drift resulted in influenza ah3n2 detected in south brazil (46) also affecting hospitalizations and deaths in various parts of the world (47) . we observed high rates of excess mortality in the elderly, in the years of 2003 and 2007. limitations of this study refer mainly to the ecological analysis of pooled data. we did not analyze individual information regarding comorbidities and history of vaccination that could be important confounders influencing mortality (17) . we just had the overall annual vaccination coverage which were in general, around 80% in the period. estimates of the number of deaths (all causes, respiratory, and pneumonia-influenza) supposedly related to influenza may be inaccurate in inferring the impact of respiratory viruses. correlations in time series studies may produce spurious associations, especially between all causes of death and influenza infection, due to the distance between cause and outcome, and to multiple components of the obits. serfling addresses part of this limitation by introducing sinusoidal terms in equation, since non-influenza mortality is not expected to coincide exactly with sinusoidal pattern (14, 19) . moreover, excess mortality of pneumonia, respiratory diseases, and all causes can be considered as an alert to surveillance of viral respiratory diseases, such as a sentinel indicator to be investigated (4, 48) . although all causes mortality is a non-specific indicator, it does not underestimate the complications of chronic diseases associated with influenza (4). despite the influenza component in all causes mortality is small, the indicator can be considered an indirect measure, a warning, useful in epidemiological monitoring. another limitation is the lack of robust etiologic data from virological surveillance in the years 2002-2012, which could lead to imprecision in the analyses; however, the data on the predominance strains in the southern region are reliable, and influenced the composition of the vaccine of each season. considering the option for the analysis model, serfling linear regression may produce different estimates when compared with other models (poisson, arima, and glm) (9, 10); poisson and arima models produce higher mortality estimates than serfling, and serfling higher than glm, especially among the elderly (16, 17, 21) . we chose serfling model because we do not have robust virological surveillance data, before 2013, and the study period includes a pandemic year (4) . besides, in this study, we did not analyze climatic variables (minimum temperatures and relative air humidity) that could also interfere with viral transmission and increase the impact of the disease, particularly in the elderly. in conclusion, probably previous exposures to influenza ah1n1 in the past influenced the mortality of brazilian elderly in 2009, despite the vulnerability of this age group to clinical complications. for the >70 years, we observe higher excess mortality rates (of all outcomes) in severe year of ah3n2 circulation (2003, 2007) . it is also worth noting that vaccination has been associated with the prevention of death particularly at age 65 (49) . therefore, the high elderly vaccination cover in southern brazil may have attenuated excess of mortality estimated, although the immune response is limited among those. more attention should be given to the circulation of influenza ah3n2 in subtropical regions in brazil. the reduced immune response to infection and to vaccination, and associated comorbidities recommend a special attention to this age group. besides medical assistance, the timeliness of vaccine campaigns, its composition, and etiological surveillance of respiratory diseases in the region are some of the preventive and public health measures. both authors made contributions to the conception of the work, acquisition, analysis, interpretation of data, and writing the manuscript. center for disease control and prevention. prevention and control of influenza: recommendations of the advisory committee on immunization practices (acip) influenza and the winter increase in mortality in the united states, 1959-1999 impact of respiratory virus infections on persons with chronic underlying conditions epidemiology of seasonal influenza: use of surveillance data and statistical models to estimate the burden of disease influenza-related hospitalizations among children in hong kong trends in mortality from respiratory diseases among the elderly and the influenza vaccine intervention mortality associated with influenza in tropics, state of sao paulo, brazil, from 2002 to 2011: the pre-pandemic, pandemic, and post-pandemic periods influenza-related deaths -available methods for estimating numbers and detecting patterns for seasonal and pandemic influenza in europe excess mortality associated with influenza epidemics in portugal time series methods for obtaining excess mortality attributable to influenza epidemics influenza associated mortality in the subtropics and tropics: results from three asian cities influenza in tropical regions seasonality of influenza in brazil: a traveling wave from the amazon to the subtropics methods for current statistical analysis of excess pneumonia-influenza deaths impact of influenza vaccination on seasonal mortality in the us elderly population influenza-related mortality in spain is influenza-like illness a useful concept and an appropriate test of influenza vaccine effectiveness? estimates of us influenza-associated deaths made using four different methods mortality associated with influenza and respiratory syncytial virus in the united states role of humoral and cell-mediated immunity in protection from influenza disease after immunization of healthy elderly mortality burden of the 2009 a/h1n1 influenza pandemic in france: comparison to seasonal influenza and the a/h3n2 pandemic the global circulation of seasonal influenza a (h3n2) viruses comparing clinical characteristics between hospitalized adults with laboratory-confirmed influenza a and b virus infection trends in mortality from respiratory disease in latin america since 1998 and the impact of the 2009 influenza pandemic estimated global mortality associated with the first 12 months of 2009 pandemic influenza a h1n1 virus circulation: a modelling study t cell mediated immunity to influenza: mechanisms of viral control t-cell immunity to influenza in older adults: a pathophysiological framework for development of more effective vaccines a question of self-preservation: immunopathology in influenza virus infection fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia influenza as a trigger for acute myocardial infarction or death from cardiovascular disease: a systematic review acute respiratory tract infections: a potential trigger for the acute coronary syndrome influenza and atherosclerosis: vaccination for cardiovascular disease prevention multiple immunological abnormalities in patients with type 1 (insulin dependent) diabetes mellitus use of influenza and pneumococcal vaccines in people with diabetes mortality attributable to influenza in england and wales prior to, during and after the 2009 pandemic risk factors for severe outcomes following 2009 influenza a (h1n1) infection: a global pooled analysis pandemic h1n1 influenza in brazil: analysis of the first 34,506 notified cases of influenza-like illness with severe acute respiratory infection (sari) pandemic versus epidemic influenza mortality: a pattern of changing age distribution epidemiology of influenza and its control aberrant innate immune response in lethal infection of macaques with the 1918 influenza virus cytokine and chemokine profiles in lung tissues from fatal cases of 2009 pandemic influenza a (h1n1): role of the host immune response in pathogenesis crossreactive antibody responses to the 2009 pandemic h1n1 influenza virus the evolution of human influenza a viruses from 1999 to 2006: a complete genome study molecular evolution of human influenza a/h3n2 virus in asia and europe from 2001 to molecular characterization of influenza viruses collected from young children in uberlandia, brazil-from virus influenza detectados no estado do rio grande do sul durante center for disease control and prevention. influenza activity -united states and worldwide, 2007-08 season the impact of influenza epidemics on mortality: introducing a severity index deaths averted by influenza vaccination in the us during the seasons 2005/06 through 2013/14 authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-259748-x7dq1sy4 authors: wan, dongshan; jiang, wei; hao, junwei title: research advances in how the cgas-sting pathway controls the cellular inflammatory response date: 2020-04-28 journal: front immunol doi: 10.3389/fimmu.2020.00615 sha: doc_id: 259748 cord_uid: x7dq1sy4 double-stranded dna (dsdna) sensor cyclic-gmp-amp synthase (cgas) along with the downstream stimulator of interferon genes (sting) acting as essential immune-surveillance mediators have become hot topics of research. the intrinsic function of the cgas-sting pathway facilitates type-i interferon (ifn) inflammatory signaling responses and other cellular processes such as autophagy, cell survival, senescence. cgas-sting pathway interplays with other innate immune pathways, by which it participates in regulating infection, inflammatory disease, and cancer. the therapeutic approaches targeting this pathway show promise for future translation into clinical applications. here, we present a review of the important previous works and recent advances regarding the cgas-sting pathway, and provide a comprehensive understanding of the modulatory pattern of the cgas-sting pathway under multifarious pathologic states. pattern-recognition receptors (prrs) serve as innate cellular sensors of danger signals, such as pathogen-associated molecular patterns (pamps) or danger-associated molecular patterns (damps), and yield cellular-stress response. dna molecules are vital genetic components within cells, which are compartmentalized restrictively into specific regions. the occasionally misplaced dna is degraded rapidly by scavenger cells and extracellular or intracellular ribonucleases. aberrant accumulation of dna is relevant to tissue damage (1) . in 2008, several research teams discovered a new protein on the endoplasmic reticulum (er) which can be activated by immune-stimulatory dna (isd) and initiate type-i interferon (ifn) responses, which was named "stimulator of interferon genes" (sting, also known as mita, eris) (2) (3) (4) . sting does not bind to dna directly, and bacteria-derived cyclic di-guanylate monophosphate (c-dgmp) or cyclic di-adenosine monophosphate (c-damp) were confirmed to be ligands for sting (5, 6) . subsequently, it was found that some dna sensors can facilitate sting activation, such as interferon gamma inducible protein 16 (ifi16) (7) . however, sting activation could not be fully explained by the upstream factors/ligands that had been found. it was postulated that an unknown upstream regulator might be responsible for sting activation. in 2013, wu and sun found that cyclic guanosine monophosphate-adenosine monophosphate (cgamp) was a novel secondary messenger serving as a ligand of sting (8) . beside it, they purified a new protein named "cyclic-gmp-amp synthase" (cgas) that had cytosolic dna-sensing ability and can synthesize cgamp (8) . also, they found that the cgas-cgamp-sting pathway was indispensable for host anti-viral immunity (9) . their work filled in the gaps missing from upstream of sting. stimulator of interferon genes or cyclic-gmp-amp synthase is expressed widely in a broad spectrum of cells including immune, non-immune, cancer cells (10) . mounting evidence has demonstrated that the cgas-sting pathway is important for mediating cellular immune sensing, and shows particular responses pattern to the isd distinguished from other nucleotide-sensing pathways. it is also regulated delicately by several molecules or feedback loops to maintain cellular homeostasis. nevertheless, cgas-cgamp-sting pathway itself has distinctive or even opposing effects under different conditions. in this review, we cover the roles of cgas-sting pathway in cellular type-i ifn immune response, and several cellular processes including autophagy, survival and senescence. we also summarize the literature on intrinsic cellular mechanisms modulating cgas-sting pathway as well as its cross-regulations with other dna-sensing pathways. moreover, the inflammationmodulation capacities of this pathway in infectious disease, inflammation and cancers have been elucidated too, and a pervasive pattern of this pathway has been described, which could provide a plausible explanation of the contradictory findings of studies. finally, current or prospective therapeutic strategies targeting the pathway, and issues that need to be addressed in the future, are discussed. cyclic-gmp-amp synthase belongs to the structurally conserved cgas/dncv-like nucleotidyltransferase (cd-ntases) superfamily. the latter is expressed universally in prokaryotes and eukaryotes, and can use purines or pyrimidines selectively as substrates for the production of linear or cyclic di-or even tri-nucleotide compounds, which act as secondary intracellular messengers (11) . cyclic-gmp-amp synthase is distributed mainly in the cytosol (also nucleus in some specific conditions) (8) . generally speaking, cgas is activated upon the recognition of b-type double-stranded dna (dsdna) without sequence-specificity but not a-type dsdna or rna (12, 13) . hybrid dna:rna or stemlike single-stranded dna (ssdna) are also low-affinity ligands for cgas (14, 15) . after binding with ligands, cgas undergoes an allosteric structural change, and subsequently catalyzes its substrates guanosine triphosphate (gtp) and adenosine triphosphate (atp) to produce a mixed phosphodiester-linked cyclic dinucleotide: g(2 -5 )pa (3 -5 ) p cgamp (abbreviated as 2 ,3 -cgamp or cgamp) (16) . cgas also catalyze the synthesis of linear dinucleotides such as amp-2 -atp, gmp-2 -gtp, and amp-2 -gtp as intermediate products (17) . there are two major dsdna-binding sites on opposite sides of the catalytic pocket: a and b site. site a is the primary contact surface for dsdna, whereas site b is complementary, binding another dsdna. it allows for cgas to the formation of a 2:2 cgas:dsdna complex structure directed into two orientations with dsdna at least 20 bp (18) (19) (20) (figure 1a) . increased numbers of back-to-back dimers of cgas hold the two dsdna molecules together and permit successive recruitment of cgas which, consequently, forms a 2n:2 cgas:dsdna higherordered "ladder-like" oligomerization, with cgas arrayed "head to head/tail to tail" (19, 21) . the dna-binding protein hu, mitochondrial transcription factor a (tfam), or bacterial high mobility group box 1 protein (hmgb1) can bend the dsdna into a u-shaped structure and, thus, facilitate binding of cgas dimers to the same strand as it travels in opposite directions (21) (figure 1b) . human cgas, unlike mouse cgas, is prone to formation of this ladder-like network with long dsdna, because of the human-specific residues k187 and l195. these two dsdna-interfacing residues of site a loosen the interaction of dsdna with cgas, leading to dsdna curving and allowing more convenient binding for the next adjacent cgas (20, 21) ( figure 1c) . finally, accumulated cgas-dsdna complexes can go through a liquid-phase separation and condense into gellike droplets as a reaction unit ( figure 1d) . this conformation requires a sufficiently long dsdna strand to form multivalent interaction positions, also requires the function of the n-terminal tail of cgas and a recently discovered dsdna-binding site in the catalytic domain of cgas (site c) (22, 23) . meanwhile, the n-terminal tail of cgas mediates cgas localization onto the membrane by binding to phosphatidylinositol 4,5bisphosphate (pi (4, 5) p2) and prevents liberation of cgas and oligomerization, but can release cgas during cell stress (24). the structure of cgas determines long strand dsdna (>500-1,000 bp) could potentially stimulate the enzyme activity and cgamp production of cgas (25). the ability of human cgas to discriminate long dsdna strands from shorter dsdna may contribute to the specific sensing and recognition of the "danger dna" of pathogens, necrotic cells or cancer cells rather than irrelevant shorter dsdna, thereby enhancing the immunity against them specifically. double-stranded dna is restricted into the nucleus or mitochondria and is rarely present in the cytoplasm. extrinsic dsdna from pathogens such as viruses, bacteria, transcellular vesicles or rupture of dying cells can be internalized into the cytosol in several diverse ways (26-28). these extrinsic dsdna sources are engulfed by endosome through phagocytosis and digested immediately by dnaseii when fusing with lysosomes (29, 30). however, some escaping mechanisms under certain conditions could help protect them from being degraded. for example, antimicrobial peptide ll37 could efficiently transports self-dna from endosome into cytosol of monocytes (28). cell oxidative stress can lead to phagosomal acidification delay and probably release endosome context including dsdna owing to increased membrane permeability (27, 31). the intrinsic self-dsdna can also be segregated inaccurately and released into the cytosol (32, 33). for example, genomic dna (gdna) injury as a result of genotoxic stress and dna self-instability or replication errors leads to double-strand breaks (dsbs) and can be repaired by several ways (34). impaired mediators of dna-damage repair response mediators, such as ataxia telangiectasia mutated (atm)-rad3, poly adpribose polymerase (parp) and breast cancer1/2 (brca1/2) multiple cgas molecules can bind two double-stranded dnas (dsdna) to form a 2n:2 cgas:dsdna higher-ordered "ladder-like" oligomerization. mitochondrial transcription factor a (tfam) can bend the dsdna into a u-shaped structure and promote polymerization. (c) cgas can recognize b-type dsdna. in humans, the cgas dna-interfacing residue of site a loosens the interaction of dsdna to curve dsdna away for more convenient binding with next adjacent cgas. cgas can catalyze gtp and atp to synthesize cyclic guanosine monophosphate-adenosine monophosphate (cgamp). the n-terminal tail binds to the cell membrane, associating with phosphatidylinositol 4,5-bisphosphate [pi (4,5)p2]. (d) accumulation of cgas-dna complex goes through a liquid-phase separation and condenses into gel-like droplets. are associated with persisting dsbs and accumulation of cytosolic dna (35-37). extra-nuclear micronuclei formation during mitosis is a source of cytosolic dsdna caused by dsbs (32, 38). followed by homologous recombination repair of collapsed replication forks, dna cleavage by methyl methanesulphonate (mms) and ultraviolet-sensitive 81 (mus81) also lead to cytosolic dsdna presenting (39). furthermore, manually cre/loxp recombination technology can induce dsdna damage during dna cleavage, which results in the accumulation of cytoplasmic dsdna (40). in normal cellular mitotic processes, chromosomal dna can be exposed to the cytoplasm, while it is hard to bind and trigger cgas (41). in addition, mitochondrial dna (mtdna) is also a considerable ligand of cgas and can be released into the cytosol under mitochondrial stress or dysfunction of proteins which participates in maintaining mitochondrial operations (33, 42, 43) (figure 2a) . cells have several types of nucleases to restrict cytosolic dna to avoid cgas activation. for example, three-prime repair exonuclease 1 (trex1 also known as dnaseiii) is a cytosolic dna exonuclease which removes unprotected dsdna from the cytosol (44) . rnaseh2 locates to the nucleus and specifically degrades the rna in rna:dna hybrids participating in dna replication (45) . dnaseii is a lysosomal dnase which degrades undigested dna in endosomes or autophagosomes to prevent their entry into the cytoplasm (30). sam domain and hd domain-containing protein 1 (samhd1) is characterized as a dntpase and restricts reverse transcription of the rna virus (46, 47) . samhd1 can also stimulate the exonuclease (but not the endonuclease) activity of mre11 to degrade nascent cdns (including cgamp) might be exported by some ways. (c) inflammatory signaling mediated by the cgas-sting pathway. after sensing dna, cgas produces cgamp and extracellular cdns, promoting stimulator of interferon genes (sting) to undergo dimerization. sting can exit from the endoplasmic reticulum (er), and be translocated from the er to the er-golgi vesicle, and arrives at the golgi. sting and tank binding kinase 1 (tbk1) can be oligomerized and cluster at the golgi. the sting-tbk1/iκb kinase (ikk) signalosome forms a scaffold to phosphorylate interferon regulatory factor 3 (irf3) and inhibitor of nf-κbα (iκbα). then, dimerized irf3 and the activated canonical nf-κb p50/p65 complex can be translocated into the nucleus as transcription factors to promote transcription of type-i ifn. (d) autophagy initiation and degradation. sting activation on er triggers er stress and mechanistic target of rapamycin complex1 (mtorc1) dysfunction. er stress and mtorc1 dysfunction can stimulate the unc-51 like autophagy activating kinase (ulk1) complex and beclin1-phosphatidylinositol 3-kinase catalytic subunit type 3 (pi3kc3) complex. autophagy-related protein 9 (atg9) and light chain 3 (lc3) are associated with genesis and elongation of the autophagosome. after autophagy initiation, cgas-sting is ubiquitinated and binds with p62. then, they are packaged into autophagosomes and terminally sorted to lysosomes (bold arrows represent main signaling pathways, thinner arrows represent regulatory signaling pathways, and dashed arrows represent bypass or suspicious pathways). ssdna, and start dna-repair responses at stalled replication forks (48) . depletion of samhd1 leads to the cleaving of nascent ssdna by the activity of mre11 endonuclease and cytosolic translocation of gdna (48) . deficiency of any of these nucleases can lead to accumulation of self-dna in the cytoplasm, thereby activating the cgas-sting pathway against dna molecules (30) (figure 2a ). the production of asymmetrically linked 2 ,3 -cgamp catalyzed by cgas has the highest affinity for sting to promotes sting dimerization (49, 50) . cgamp as a second messenger can be also transferred among cells in several ways to pass danger signaling of frontiers in immunology | www.frontiersin.org cytosolic dna. intercellular gap junction consists of two docking hexamer channels formed by different connexins, which allows many small molecules, including cgamp, to pass bi-directionally through cells. and intercellular transfer of cgamp through gap junction is largely dependent on connexin 43 (51) (52) (53) . additionally, cgamp can be packaged into virons and pre-notify newly infected cells (54, 55) . cell fusion is a distinct manner for intracellular transmission of the human immunodeficiency virus (hiv); cgamp also enter membrane-fused bystander cells in this way (56) . extracellular vesicles such as exosomes can contain cgamp along with viral dna, host gdna or mtdna, and mediate cells communication (57, 58) . there were no evidences that cgamp could be pumped out to extracellular space by a channel/transporter. however, it was found that slc19a1 can transmit cyclic dinucleotides (cdns) into cell plasma (59, 60) . notably, ectonucleotide pyrophosphatase/phosphodiesterase family member 1 (enpp-1) can degrade extracellular cgamp (61) (figure 2b ). besides triggering sting, these exogenous cgamp can directly bind to cgas and prompt its activation as well (62) . after binding to cgamp, the "lid" region of the sting dimer undergoes a conformational change that converts sting from an inactive "open" formation to an active "closed" formation. following that, the sting dimer translocates from the er to perinuclear er-golgi intermediate compartment (ergic) vesicles, finally arriving at the golgi to form punctuate structures with downstream molecules (2, 63) . er-retention of sting caused by mutations results in reduced ifn signaling (64, 65) . the translocon-associated protein β (trapβ) recruited by inactive rhomboid protein 2 (irhom2) initially forms the trap translocon complex that mediates sting exit from the er (2, 66). they both assist cytoplasmic coat protein complex-ii (copii) to drive er-vesicle formation and carry the sting complex to the golgi (67, 68) . trafficking sting can bind directly to and be phosphorylated by tank binding kinase 1 (tbk1) dimer or iκb kinase (ikk) complex (3, 69, 70) . the c-terminal tail (ctt) of sting is a linear unfolded segment, which determines the optimization of combination specificity. sting ctt in mammals tends to bind tbk1, whereas in fish it tends to activate nuclear factor-kappab (nf-κb) signal (71) . the sting phosphorylation site ser366 in the ctt cannot reach the kinase-domain active site of its directly bound tbk1, instead can reach the kinase-domain active site of the next adjacent tbk1 binding with another sting and be phosphorylated, while tbk1 phosphorylate each other (72, 73) . hence, sting and tbk1 can aggregate on the golgi to form the sting signalosome. clustering sting undergoes palmitoylation and full activation (74) . it is also possible for sting-ikk to cluster and form the sting signalosome in this manner. the sting-tbk1/ikk signalosome produces a scaffold to phosphorylate interferon regulatory factor 3 (irf3) or inhibitor of nf-κbα (iκbα). activated irf3 undergo dimerization (70) . the activation of iκbα leads to its polyubiquitination and degradation by the proteosome, thereby eliminating its inhibition of nf-κb. there is also evidence suggesting that nf-κb activation might not require sting trafficking from the er (75) . then, the dimerized irf3 or activated nf-κb p50/p65 (p65 is also known as rela) complex are translocated into the nucleus as transcription factors and bind to the promoter of type-i ifn to aid the transcription of type-i ifn (2, 3, 70) . meanwhile, activation of nf-κb p52/relb can prevent recruitment of p65 and inhibit the p50/p65 signal (76) (figure 2c ). expressed type-i ifn can propagate among cells in paracrine or autocrine manners. the binding of ifnα/β with its receptor triggers janus kinase (jak) and signal transducer and activator of transcription (stat) pathways, then induce transcription of type-i ifn-stimulated genes (isgs), which have ifn-sensitive response elements (isres) in their 5 -untranslated regions (utrs) (77) . irf3 can also bind partially to several isres alone (78) . herein, the expression of some isgs including interferon-induced protein with tetratricopeptide repeats (ifit) and pro-inflammatory cytokines such as tumor necrosis factor α (tnfα), interleukin (il)-6, c-x-c motif chemokine ligand 10 (cxcl10) and c-c motif chemokine ligand 5 (ccl5) is increased remarkably by the cgas-sting pathway (79) . furthermore, cgas and sting are both isgs, suggesting a positive feedback loop in spreading of the ifn signal (80, 81) . stimulator of interferon genes activation on the er also triggers an er stress response with an "unfolded protein response (upr) motif " on the c-terminus of sting, which leads to and er stress-mediated autophagy (82, 83) . sting-tbk1 activation and er stress also induce mechanistic target of rapamycin complex 1 (mtorc1) dysfunction (84) . er stress or reduced mtorc1 signaling activates unc-51-likeautophagy activating kinase (ulk1) complex and the beclin-1-class iii phosphatylinositol 3-kinase (pi3kc3 also known as vps34) complex, which promotes initiation of the classical autophagy path (85) . cgas can also interact directly with the autophagy protein beclin-1-pi3kc3 complex and trigger autophagy (86) . furthermore, cgas-dsdna polymer can form a liquid-phase condensate (as mentioned above), which could theoretically be an initiator of autophagy (87) . after autophagy initiation, autophagy-related protein 9 (atg9) undertakes the genesis of the autophagosome along with light chain3 (lc3) undergoing lipidation, thereby resulting in elongation of the autophagosome (88) . lc3 can also be recruited directly by ergic-loading sting and bypass the classical autophagy pathway (68, 89) . cgas-sting-mediated autophagy can spread to the whole cell and help the elimination of intracellular microorganisms, subcellular organelles or misfolded proteins, as well as the er itself that loads the sting signalosome (90-92) ( figure 2d ). cgas-sting-mediated autophagy is also indispensable for removing cytosolic dna and inflammatory signaling factors to restrict the inflammatory response raised by the pathway itself (93) . excessive signaling of the autophagy cascade can lead to irreversible apoptosis termed "autophagic cell death" (94) . consequently, oligomerized cgas or sting undergoes ubiquitination and is packaged into autophagosomes with the help of p62, to be terminally sorted into lysosomes (79, 83, 95, 96) . cgas or sting is digested immediately in the autophagolysosome after transient activation of downstream signaling (68, 79, 83, 89) . autophagy functions as a negative feedback loop which ensures transient cgas-sting signaling and avoids consistent over-reaction of the pathway. thus, impairment of autophagy may give rise to destructive inflammatory diseases (31). we cataloged factors in the literature that could potentially up-or down-regulate expression of cgas/cgamp/sting in pre-translational or post-translational stages (tables 1, 2) . the regulatory mechanisms of tbk1, irf, and nf-κb in signaling pathways associated with expression of type-i ifn are outside the scope of this review. pyhin family member absent in melanoma 2 (aim2) is a cytoplasmic dsdna sensor. it can recruit apoptosis-associated speck-like protein containing a card (asc) by its pyhin domain and form the aim2 inflammasome. the inflammasome activates caspase-1, which activates il-1 and trigger pyroptosis (97) . the aim2 pathway could counteract the cgas-sting pathway (98) . first, cgas is a target for caspase-1 cleavage (99) . second, gasdermin d activated by caspase-1 can lead to potassium ion (k + ) efflux which inhibits cgas (100) . conversely, the cgas-sting pathway can trigger the aim2 inflammasome or nlr family pyrin domain containing 3 (nlrp3) by several means, and the process lags behind canonical ifn signaling (96, 101) . in this way, the inhibitory nucleic-acid sensor nlr family card domain containing 3 (nlrc3) can counteract sting by binding and occupying it, but viral dna as a possible nlrc3 ligand can reverse its occupation of sting (102) (figure 3a) . another pyhin family member, ifi16, is a dna sensor located in the nucleus. ifi16 can bind to viral dna sequences or damaged chromatin dna and be translocated to the cytoplasm to recruit sting cooperatively with tnf receptor associated factor 6 (traf6) and p53 (103, 104) . several studies have shown that ifi16 (which can stimulate the phosphorylation and recruitment of sting and tbk1) is required for the full response of sting (105, 106) ( figure 3b) . conversely, cgas can partially enter the nucleus and interact with ifi16 to promote its stability (107) . therefore, it is inferred that during viral infection, ifi16 can facilitate recognition of decapsidated viral dna in the nucleus, while cgas in the cytoplasm engages with viral gene transcription products (104, 108) . however, sting signaling can trigger ifi16 degradation by tripartite motif-containing 21 (trim21) ubiquitination (109) . tlr is also an important prr for multiple pamps (110) . tir domain-containing adaptor-inducing ifnβ (trif) is downstream of several subtypes of tlrs (including tlr3). trif may be responsible for interacting with sting and helping the dimerization of sting (111) . during viral infection, the tlr9-myeloid differentiation primary response 88 (myd88)-irf3/7 pathway is necessary for mouse monocytes recruitment to lymph nodes, whereas the sting pathway is necessary for local production of type-i ifn (112) . however, sting signaling can induce suppressor of cytokine signaling1 (socs1) expression, which can negatively regulate myd88 activity (113) (figure 3c ). oxidized mtdna can be released into the cytoplasm during cell stress elicited by hypoxia, viral infection and mitochondrial damage, etc.; oxidized mtdna is resistant to degradation by the cytosolic nuclease trex1 (114) . in addition, mtdna accompanied with tfam (a mtdna-binding protein that can bend mtdna) is also a reasonable target for recognition by cgas (21, 33) . however, during regulated cell death (as represented by apoptosis), it undergoes mtdna release but has certain mechanisms to ensure a minimal cgas-stingmediated immune response. mitochondrial outer membrane permeabilization (momp) activation, which is executed by bcl-2-associated x protein (bax) and bcl-2 antagonist or killer (bak), is a highly controlled conserved process in regulated cell figure 3 | interaction of the cgas-sting pathway with other dna-sensing pathways and its role in cell survival. (a) absent in melanoma2 (aim2) pathway and pyroptosis and necroptosis. aim2 can be triggered by cgamp and form an inflammasome, consequently triggering interleukin (il)-1 production and pyroptosis. stimulator of interferon genes (sting) trafficking to the lysosome ruptures the lysosome membrane, resulting in k + efflux and activation of the nlrp3 inflammasome, leading to pyroptosis. cyclic-gmp-amp synthase (cgas) and interferon regulatory factor 3 (irf3) can be a target for caspase-1 cleaving. gasdermin d can lead to k + efflux and inhibition of cgas. (b) interferon gamma inducible protein 16 (ifi16). ifi16 can be transported to the cytoplasm to help to recruit sting and tank binding kinase 1 (tbk1). ifi16 as a pyhin family protein may form the inflammasome only in theory. (c) toll-like receptor (tlr) pathway. tir domain-containing adaptor-inducing ifnβ (trif) may be responsible for helping the dimerization of sting. sting signaling can induce suppressor of cytokine signaling 1 (socs1) expression, which negatively regulates myd88 activity. (d) apoptosis. mitochondrial outer membrane permeabilization (momp) formed by bax/bak induced by mitochondrial stress can release oxidized mitochondrial dna (mtdna) and cytochrome c into the cytosol. oxidized mtdna is a suitable ligand for cgas recognition and is resistant to dnaseiii (trex1) degradation. cytochrome c binds to apoptotic protease-activating factor 1 (apaf1) and initiates the formation of an apoptosome cooperatively with caspase-9 to activate caspase-3, which can induce apoptosis. caspase-3 can cleave cgas. death. bak and bax activated by apoptosis signals cooperatively form a pore-like conformation on the mitochondrial outer membrane, leading to a permeability change of outer and also inner membranes (115, 116) . consequently, the mitochondrial matrix, including cytochrome c and oxidized mtdna-tfam, is released into the cytoplasm (115, 117) . cytochrome c binds to apoptotic protease-activating factor 1 (apaf1) and initiates the formation of the apoptosome cooperatively with caspase-9, which further triggers the intrinsic apoptosis program (117) . in vivo and in vitro studies have shown that an absence of caspase-9 is associated with greater release of type-i ifn (43, 117) . this occurs because caspase-9 and its downstream caspase-3 can cleave cgas and irf3 to restrain deleterious inflammation (118) (figure 3d) . the cgas-sting pathway can also initiate programmed cell death. activation of sting enhances phosphorylation and activation of receptor interacting serine/threonine kinase 3 (rip3) and mixed lineage kinase domain-like pseudokinase (mlkl). proapoptotic bcl2 binding component 3 (puma), a member of bh3-only family, is subsequently activated in a rip3/mlkl-dependent manner, which promotes leakage of mtdna by momp (119, 120) . activated irf3 can bind directly to bax to form irf3/bax complex and induce apoptosis (47) . excessive cgas-sting-mediated autophagy signaling can cause "autophagic cell death" and prevent malignant transformation of cells through dna damage (94, 121) . sting trafficking to the lysosome can broaden permeabilization of the lysosome membrane, thereby rupturing the lysosome and releasing its contents, resulting in "lysosomal cell death (lcd)". lcd further triggers k + efflux and nlrp3 activation, ultimately resulting in pyroptosis (96, 101) (figure 3d) . moreover, stimulating sting-dependent type-i ifn and tnfα signals simultaneously can lead to necroptosis of tumor cells (122, 123) . cell senescence is recognized as a permanent arrest of the cell cycle, and is common in aging, immunity, ontogenesis and infectious defense (124) . it lacks a specific biomarker but can be identified by the expression of several antiproliferative molecules (representatively rb-p16 andp53-p21 pathway) (125) . during senescence, changes in the nuclear structure and loss of the nuclear lamina protein disrupt the integrity of the nuclear envelope, leading ultimately to dna damage and cytoplasmic chromatin fragments (126) . cellular senescence can be accelerated by accumulation of cytoplasmic chromatin in turn (127) . these senescent cells produce the senescence-associated secretory phenotype (sasp), which shapes an inflammatory microenvironment (128) . the cgas-sting pathway has been reported to be involved in the recognition of cytoplasmic chromatin fragments from senescence-related dna damage, and mediate the expression of sasp genes (129) (130) (131) (132) . along with these actions, the expression of trex1 and dnaseii is inhibited by dna damage through the inhibition of e2f/dp (a potential transcription factor of trex1 and dnaseii) (130) . for hematopoietic stem cells (hscs), dna damage can promote excessive secretion of type-i ifn in the hsc niche and activate p53 pathway, both of which can lead to long-term senescence and exhaustion of hscs (133, 134) . hscs expressing a circular rna named "cia-cgas" in the nucleus, however, are protected from this exhaustion as a result of cia-cgas having stronger affinity than that of self-dna, which prevents it from being sensed (135) . it implied a novel target to manipulate the immune environment in bone marrow and help for finding treatment approaches for hematopoiesis-based diseases, such as aplastic anemia. utilizing cellular senescence to restrain tumor growth is discussed below. cgas-sting signaling has an essential role in defense against a broad spectrum of intracellular dna and rna viruses (9, 26, 50) . hiv is a typical rna retrovirus: there is neither dsdna in its genome, nor production of nucleic acids (50) . nevertheless, cgas can detect the presence of hiv. rna:dna hybrids synthesized during reverse transcription that can be sensed by cgas explain (at least in part) this phenomenon (14) . cgas may be triggered by endogenous dna broken and released during hiv infection as well theoretically. however, some studies found the new mechanisms. the early reverse-transcription production of hiv-1 can flank short stem loops with paired base, which lead to the production of y-type dna containing unpaired guanosines that can activate cgas well (15) . moreover, nucleolus protein non-pou domain-containing octamer-binding protein (nono), as a sensor of capsid components of hiv, can help cgas to be translocated to the nucleus and assist cgas to sense hiv dna accompanied by polyglutamine-binding protein 1 (pqbp1) (136, 137) . the assistance proffered by nono in assisting cgas to sense dna is also associated with its role in constructing a ribonuclear complex with dna-dependent protein kinase (dna-pk) subunits around hexamethylene bisacetamide-inducible protein1 (hexim1), termed as "hexim1-dna-pk -paraspeckle components-ribonuclear protein complex (hdp-rnp), " which also has a role in repair of dna damage and transduction of genotoxic signals (138) . this complex is also required to accompany cgas-pqbp1 in sensing dna virus, such as kaposi's sarcoma-associated herpes virus (139) . in addition, during virus infection, sting activation can lead to global suppression of translation in cells, which restricts viral replication (140) . compared with hiv-1, hiv-2 is less infective because it can infect dendritic cells (dcs) and elicit an anti-virus immune response. as a result, hiv-2 can cross-protect against hiv-1 (141). this phenomenon has been attributed to the fact that hiv-2 (instead of hiv-1) can encode protein vpx, which overcomes the samhd1 restriction of dntp in dcs (46, 142) . hiv-1 can infect dcs via vpx presentation, nevertheless, hiv-1 still cannot be fully sensed and induce an efficient immune response owing to certain escape mechanisms. whether it is hiv-1 or hiv-2, a completely robust ifn response is required at pre-and post-integration sensing stages (143) . cgas in dcs can detect reverse-transcribed cdna of hiv-2 before and after integration, whereas hiv-1 sensing is after genome integration owing to its capsid protection (144, 145) . it was suggested that during initial infection by hiv-1, nucleotides are recruited into the intact capsid through the hexamer pores on the hiv-1 capsid. therefore, the capsid-coated hiv-1 virus prevents the encapsidated reverse-transcription production from being sensed by the cytosolic nucleic-acid sensors (146) . hiv-1 capsids can be ubiquitinated and then degraded by the host e3 ubiquitin ligase function of trim5, which leads to detection of viral dna, meanwhile hiv-1 could use some host protein like cyclophilins to evade the sensing (147, 148) (figure 2a) . similarly, other viruses also have evasion mechanisms to escape cgas-sting pathway surveillance (table 3) . therefore, identifying and preventing such viral-evasion factors could be a viable means to design novel anti-viral drugs. cgas-sting pathway is responsible to protect against intracellular or extracellular bacterial infection (especially hsv, herpes simplex virus; cmv, cytomegalovirus; irhom2, inactive rhomboid protein 2; trapβ, translocon-associated protein β; kshv, kaposi's sarcoma-associated herpes virus; hdp-rnp, hexim1-dna-pk-paraspecklecomponents-ribonuclear protein complex; lana, latency-associated nuclear antigen; virf1, viral interferon regulatory factor 1; plpro, papain-like protease; lc3, light chain3; mtor, mechanistic target of rapamycin; traf3, tnf receptor associated factor 3; hcv, hepatitis c virus; hpv, human papilloma virus; hbv, hepatitis b virus; hiv, human immunodeficiency virus; nf-κb, nuclear factor-κ b. intracellular infections). cdns (e.g., c-dgmp, c-damp, and cgamp) produced by bacteria are essential for the regulation of bacterial function, such as biofilm formation, colonization, and reproduction (149, 150) . as ligands for sting, cdns can bind directly to and activate sting independently of cgas, which contributes to several immune responses from bacteria (151) . usually, bacteria can enter or be engulfed by the cell through the endophagosome and be sequestered from the cytosolic sense receptor. some bacteria, such as mycobacterium tuberculosis (mtb), can survive in vacuoles, resulting in an insufficient cellular immune response to defend against it (152) . in contrast, the esx-1 secretion system of the mycobacterium can translocate the phagosomal vacuolar matrix including bacterial genome molecules into the cytoplasm and trigger the cgas-sensing pathway (153) . for other bacteria, such as legionella pneumophila or and brucella abortus, the host guanylate binding proteins (gbps) facilitate rupture of phagosome vacuoles and are indispensable for controlling their infection (154, 155) . autophagy signaling mediated by cgas/sting is also involved in microorganism clearance mentioned above (90, 91) . bacteria have evolved strategies to confront this pathway too. bacterial phosphodiesterase cdnp produced by mtb or group-b streptococci can degrade cdns (156, 157) . cpsa (a type of mtb lytr-cpsa-psr domain-containing protein) can prevent autophagy responses for eliminating pathogens (90) . chlamydia trachomatis inclusion membrane proteins can maintain the stability of the inclusion membrane and avoid inclusion lysis (leading to pathogen antigens leaking out and being detected by the host cell) (158, 159) . yersinia outer protein j (yopj) deubiquitinates sting and impedes the formation of the sting signalosome (160) . the cgas-sting pathway activation even impedes the elimination of listeria monocytogenes because bacterial dna can be packaged into evs and transferred into t cells, where it induces apoptosis of t cells (161, 162) . several protozoans, such as toxoplasma gondii and malaria parasites, have an intracellular period in their lifecycle. t. gondii could engage cgas-sting exclusively (163) . however, irf3 activation inducing isg expression promotes t. gondii development independently of ifn expression (163) . p. falciparum can target erythrocytes, lacking a nucleus and unable produce ifn, but infected erythrocytes can secrete evs containing parasitic gdna to monocytes and trigger cgas (164) . the immune system is regulated by a complicated network. disorder of immune signaling can elicit non-infectious inflammatory or autoimmune diseases. excessive, uncontrolled production of type-i ifn can lead to a spectrum of inflammation diseases termed "type-i interferonopathies, " which have some common manifestations (165) . cgas-sting is the one of main sources of type-i ifn, acts as a cellular immune-sensing signaling axis, and is involved in type-i interferonopathies. stimulator of interferon genes -associated vasculopathy with onset in infancy (savi) is a typical sting-related hereditary inflammatory type-i interferonopathy, and is manifested by interstitial lung disease, dermatomyositis and arthritis. its pathology is featured by leukocytoclastic vasculitis and microthrombotic angiopathy of small dermal vessels (166, 167) and patients can also suffer from lymphopenia (166) (167) (168) (169) . the etiology of savi is a gain of function (gof) mutant in sting which leads to constitutive sting activation without cdns stimulation (166) . currently, several mutant amino acids residues have been found in or close to the dimerization domain (v155m, n154s, g166e, v147l, and v147m) (64, 166, 168, 170) , as well as r284g, r284s, r281q, and c206y in the cgamp-binding domain (171) . other types of type-i interferonopathies, such as systemic lupus erythematosus (sle) and aicardi-goutières syndrome (ags), have relationships with defective clearance of cytosolic nucleic acids caused by congenital dysfunction of trex1, rnaseh2, and samhd1. sle is a heterogeneous autoimmune disease which has prominent type-i and also -ii ifn signatures (172) . ags comprises some systemic autoimmune syndromes overlapping with sle, and can be classified as a "lupuslike disease" (173) . additionally, ags also causes severe developmental neurological disorders, including cerebral calcifications, encephalopathy and cerebral atrophy. systemic lupus erythematosus is a representative model for elucidating the mechanism of type-i interferonopathies. in sle, the level of self-dna which is packaged into apoptosis-derived membrane vesicles along with the level of anti-dsdna antibody is increased in the serum of patients (174) . a study revealed increasing levels of isgs (including cgas/sting) as well as the cgamp-detected ratio in peripheral-blood mononuclear cells of sle patients (175) . as innate immune cells, dcs have essential roles in antigen presentation, cytokine secretion, and priming the adaptive response of immune cells (176) . plasmacytoid dcs (pdcs) can internalize and recognize self-dna and they are the main source of type-i ifn in serum during sle (177) . ifnα/β is essential for complete function of immature pdcs (178) . ifnα/β and il-1 can induce mitochondrial oxidative stress in dcs and decrease atp production, which blocks proton-pump function and increases ph of lysosomes. this process inhibits mitochondrial degradation and blocks mtdna clearance, which engages the cgas-sting pathway (31, 179). moreover, monocytes may sense mtdna through cgas-sting and differentiate into dcs (31, 180). neutrophil extracellular traps (nets) are complexes released by neutrophils exposed to stimuli or autoantibody immune complexes. nets comprise extracellularly released chromatin, myeloperoxidase enzymes, and also oxidized mtdna. in lupus-like diseases, nets can be induced by ifnα/β and may play a major part in priming pdcs (181, 182) . all mechanisms stated above contribute to a more aggravated type-iifn response and exacerbate disease. a similar phenomenon can be observed in savi, ataxia telangiectasia (at) and artemis deficiency (183) . however, compared with savi, dcs in sle can prime t-cell maturation significantly and increasing secretion of pro-inflammatory cytokines, such as il-6 and tnfα can also lead to activation of adaptive immunity ( figure 4a) . the cgas-sting pathway can mediate systemic inflammation as well as autoimmune activation. however, it is also involved in the local inflammation of multiple tissues. with regard to ischemic myocardial infarction (mi), cardiac macrophages can sense dying ruptured cells and lead to fatal post-mi cardiac inflammation, which is reversed by ablation of cgas/sting/irf3 (184, 185) . in a non-alcoholic steatohepatitis (nash) model induced by a methionine-and choline-deficient or high-fat diet, lipotoxicity can cause mitochondrial damage and up-regulate sting/irf3 expression in hepatocytes, which in turn promotes lipid accumulation and inhibits glycogen synthesis. all above bring out hepatic inflammation and hepatocytes apoptosis (186) . in this model, mice with deficiency of sting presents alleviated insulin resistance and lower levels of low-density lipoprotein in serum, and also decreased hepatic inflammation and fibrosis/steatosis, in which hepatic macrophages/kupffer cells may take a big part (187, 188) . lipotoxicity can induce p62 to be phosphorylated through the cgas-sting-tbk1 pathway, which causes aggravated protein inclusions in hepatocytes and it indicates that p62 could be a biomarker for nash prognosis (189) . mtdna-dependent inflammation induced by lipotoxicity also occurs in adipose tissue and endothelial cells of blood vessels, which contributes to tissue inflammation, insulin resistance, and cardiovascular diseases (42, 190) . in traumatic brain injury (tbi), local injury initiates breakdown of the blood-brain barrier and global neuroinflammation (191) . sting expression is up-regulated in tbi and can lead to increased expression of pro-inflammatory cytokines and enlargement of secondary injury (192) . reduced autophagy-associated protein expression induced by sting may contribute to the dysfunction of autophagy and dampen the elimination of necrotic tissue, thereby intensifying inflammation (192) . during silicosis, silica can yield cytosolic dsdna release and engage cgas-sting, which activates dcs and macrophages to cause severe lung inflammation. it also leads to death of epithelial cells through the nlrp3 pathway and pulmonary fibrosis (193) . similarly, mtdna release in renal tubule cells has been found to be associated with acute kidney injury by cytotoxic drugs and chronic renal fibrosis (194, 195) . neurodegenerative diseases are correlated with local inflammation (196) . in the central nervous system (cns), microglia is considered to be the main source of cgas-sting-dependent ifn expression (197) . in neurodegenerative diseases, levels of the marker of microglia activation-cluster of differentiation 68 (cd68), and pro-inflammatory cytokines are increased (198) . a significant feature of parkinson's disease (pd) is the neuronal loss of cerebral nuclei (especially dopaminergic neurons in the substantianigra). serine/threonine-protein kinase pink1 and e3 ubiquitin-protein ligase parkin are ubiquitinrelated factors that take part in removing damaged mitochondria by autophagy, and their dysfunction lead to the early onset of pd (199) . parkin −/− and pink1 −/− mice, following exhaustive exercise, show inflammation and loss of dopaminergic neurons, which can be rescued by loss of sting (200) . similarly, at is a genetic disease caused by missense mutation of a dna-repair protein: atm. at patients usually show neurodegenerative defects (especially ataxia) complicated with telangiectasia on their eyes or body, deficiency of adaptive immune cells, and predisposition to cancer (201) . nevertheless, up-regulation of expression of type-i ifn can also be found in at patients and mice, causing them to be prone to autoimmune diseases (36, 183, 202) . this syndrome is related to p53-mediated senescence but also the chronic inflammation mediated by the cgas-sting pathway which engages cytosolic uncombined broken gdna caused by atm dysfunction (127) . in addition, accumulation of cellular mtdna occurs in age-related macular degeneration characterized by retinal pigmented epithelium (rpe) degeneration. this can trigger chronic inflammation by cgas-sting pathway, in which nlrp3 inflammasomes, inflammatory/apoptotic caspases are also involved (203, 204) . with regard to other diseases in which adaptive immune cells prime, cgas-sting has a different role. multiple sclerosis (ms) is a local inflammatory disease of cns. ms is characterized by over-reactive microglia, infiltration of self-reactive t cells, demyelization of nerve fibers and hyperplasia of gliocytes. autoantibodies against proteins expressed in immune-privileged regions of cns also contribute to its pathogenesis (205) . in ms, ifnα/β can attenuate disease severity effectively. this implies a protective role for type-i ifn in cns, which is considered to counteract the pro-inflammatory ifnγ (206) . using experimental autoimmune encephalitis (eae) as a ms model, sting was found to be indispensable for amelioration of type-i ifnmediated neuroinflammation, and it could be induced by a conventional anti-viral drug ganciclovir (207) . ultraviolet (uv) radiation is a factor inversely related to the morbidity of ms (208) . it was found that uv-b irradiation can recruit inflammatory monocytes and produce type-i ifn in a sting-dependent manner (209) . all above indicate that cgas-sting-ifnα/β pathway may have a beneficial effect on some cns inflammatory diseases such as ms. a possible reason for the observed effect above is due to indoleamine 2,3-dioxygenase (ido), which can catabolize tryptophan (trp) oxidatively. trp withdrawal or trpoxidative catabolites can interact with general control non-derepressible 2 (gcn2) and mtor, of which both can control cellular aminoacid metabolism and suppress t helper 1 (t h 1) cells immunity (210) . ifnα/β is a potent ido inducer to suppress proliferation of cd4 + t h 1 cells and promote differentiation of foxp3 + regulatory t (t reg ) cells, which are believed to suppress cnsspecific autoimmunity (210, 211) . in addition, dna released from dying cells can be internalized directly by t cells and sensed by cgas-sting pathway, which leads to enhancement of the t h 2 transcription factor gata3 but suppression of the t h 1 transcription factor t-bet. consequently, this process polarizes naive t cells toward t h 2 differentiation (212). studies mentioned above may (at least in part) explain why the cgas-sting signal is a negative regulator of ms. the inhibitory role of cgas-sting in inflammation is also attributed to its apoptosis-triggering role. in some subtypes of savi and mouse models, apoptosis of blood-vessel endothelial cells or bronchial epithelial cells and leucopenia can be observed (especially t-cell lymphopenia) (166, 169, 170) . when the sting signal is stimulated, apoptosis occurs more frequently in normal or cancerous t cells (119) . also, bone-marrow chimeras and gene-knockout studies have shown that t cells defect in savi are not associated with type-i ifn signaling or cgas (213, 214) . localization of sting at the golgi can cause delay of t-cell mitosis and reduced proliferation independently of irf3 and tbk1 (215) . furthermore, a "upr motif " on the c-terminus of sting can cause er stress and upr, resulting in ca 2+ overloading and t-cell death (82) . a controversial view is that b cells express sting variously and may undergo apoptosis through this way (166, 216, 217) . however, simultaneous signaling by sting and the b-cell antigen receptor can promote b-cell activation and antibody production independently of type-i ifn (217) (figure 4b) . as for some diseases with inflammatory responses involved, the acute phase of pancreatitis causes dying acinar cells to produce free dsdna, which activates cgas-sting signaling in macrophages, and exacerbates inflammation severity (218) . however, in the chronic phase of pancreatitis, cgas-sting activation decreases pancreatic inflammation, which may be mediated by limiting t h 17 response (219). for gut mucosal immunity, transient stimulation of sting could strengthen the function of antigen-presenting cells (apcs) and promote t h 1 and t h 17 immune responses against microbes (220) . chronic sting signaling, however, elicits an il-10 response to control the inflammation and avoids inflammatory enterocolitis such as bowel disease (221) . sting knockout mice present reduced numbers of goblet cells, a decreased ratio of commensal versus harmful bacteria and compromised t reg cells in the gut, making it prone to enterocolitis (222) . chromosomal instability (cin) is an intrinsic feature of cancer, and results spontaneously from errors in chromosome segregation during the mitosis of cancer cells. cin can also be induced manually by radiotherapy or chemotherapy, which causes dsbs. it results in micronuclei formation outside the nucleus, of which rupture brings out irrepressible accumulation of cytosolic self-dna and engages cgas (32, 38, 223). however, normal mitotic processes involve exposure of chromosomal dna to the cytoplasm, but this cannot initiate a substantial inflammatory reaction or apoptosis because nucleosomes can suppress dsdna-cgas binding in a competitive manner (41). an appropriate immune response against tumors via a type-i ifn plays an indispensable part in limiting tumors and prolonging host survival (224) . it was found sting-deficient mice are prone to developing several types of cancer and have poor survival under a tumor burden, whereas stimulation of sting can elicit robust immunity to tumors (225) (226) (227) . a mechanism is many cancer cells expressing cgas can recognize cytosolic dna and produce cgamp to stimulate secretion of type-i ifn through sting (228, 229) . excessive expression of trex1 in cancer cells, which can be induced by radiotherapy, attenuates this progression (228) . cgas-sting can also promote senescence of cancer cells through the p53-p21 pathway (129) . cgas-sting-mediated autophagy contributes to autophagic cell death if mitotic crisis occurs to avoid transformation of cancer cells (121) . melanoma cells can also transfer cgamp produced by them to proximal non-cancerous host cells through gap-junction channels and activate sting in these cells, which contributes to the recruitment of tumor-infiltrating immune cells such as natural killer (nk) cells (51, 230) . expression of the nk cellspecific ligand nkg2d retinoic acid early transcript 1 (rae1) on cancer cells is highly up-regulated by sting once nk cells permeate into tumor tissue (231) . the activation of sting in the endothelium within the tumor microenvironment (tme) could contribute to the remodeling of tumor vasculatures, and may have positive effects on tumor regression (232) . dendritic cells are the main source of type-i ifn in several types of tmes and are dependent on sting signaling (229) . more preferentially than macrophages, infiltrating dcs take up tumor-derived dna or cgamp from dying cell fragments by phagocytosis (27, 29, 129, 226, 233) . moreover, cancer cells can package dna into exosomes and transfer dna to dcs (234) . produced cgamp by cancer cells can also be transferred to dcs through forming gap junction (53) . by activating cgas-sting signal in dcs, cd8α + subtype dcs secret chemokines such as ccl5 and cxcl10 and crossprime infiltrating anti-tumor cd8 + t cells (29, 226, (235) (236) (237) . in contrast, numbers of immune-suppressing cells such as t reg cells, myeloid-derived suppressor monocytes and m2 macrophages have been reported to be decreased (225, 238, 239) . expression of il-15/il-15rα complex is up-regulated in myeloid cells with the help of sting/type-i ifn and promotes tumor regression (240) . tumor cells can evade intrinsic cellular surveillance in different ways. in various cancer cell lines, cgas, sting, tbk1, and irf3 are mutated frequently and their decreased expressions are also related to the high level of methylation (241) . sting expression has been shown to be suppressed by the alternative lengthening of telomeres (alt) pathway, which is responsible for prolonging the telomere length and maintaining the proliferation of tumor cells (242) . a hypoxic environment in tumor cells can lead to accumulation of lactic acid and is associated with the inhibition of tumor-conditional dcs and reduced expression of ifn signaling molecules (243) . in breast cancer, functional up-regulation of expression of human epidermal growth factor receptor 2 (her2), a ligand-independent receptor tyrosine kinase (rtk), can arrest the expression of rac-alpha serine/threonineprotein kinase (akt1) (a key factor in the mtor pathway), which is reported to inhibit the activation of cgas and tbk1 (244, 245) . patients with lung adenocarcinoma have a low probability of survival if they have reduced expression of cgas (132) . thus, expression of the cgas-sting and dna-damage marker histone γh2ax in tumor cells could be considered as independent prognostic factors to predict therapy response and clinical outcome, and could be superior to that of traditional markers like immunogenic cell death and t cells number (246) . however, some scholars have arrived at opposite conclusions. when dsbs occur in cancer cells, cgas can be relocated to the nucleus and obstruct the formation of the parp1-timeless complex, thereby inhibiting homologous recombination repair and maintaining cin, which potentiates tumor evolution (35, 223) . it has also been reported that cgas recognizing cin activates non-canonical nf-κb signaling and potentiates cellular metastasis programs (247) . furthermore, sting −/− mice are resistant to skin carcinogenesis in a 7,12-dimethylbenz(a)anthracene (dmba)-treatment model. it has been demonstrated that when dmba-induced nuclear dna leaks into the cytoplasm, sting can induce chronic inflammatory stimulation that contributes to cancer development (248) . during brain metastasis, cgamp transferred to bystander cells (e.g., astrocytes) can also produce ifnα and tnfα in the tme but, in this context, it will support tumor development and chemoresistance (249) . coordinating with myeloid cells penetrating into the tumor, myeloidderived suppressor cells can also be recruited through the c-c chemokine receptor type 2 (ccr2) (250) . another study found that microparticles yielded by tumor cells can turn macrophages into the m2 type through cgas-sting-tbk1, contrary to previous findings (251) . immune-system interactions with tumor cells are complicated. the effect of cgas-sting on cancer is dependent on the type of tumor, host immune state, activated cell types, therapeutic intervention, and the magnitude of cgas-sting activation. like inflammation generated by cgas-sting, a time-dependent inflammatory anti-tumor response mediated by cgas-sting may be present. temporary activation of cgas-sting in innate immune cells could enhance the anti-tumor effect, whereas sustained activation of cgas-sting might induce immune tolerance of the tumor. more investigations are necessary to ascertain the exact role of cgas-sting in oncology, and elucidate the specific advantages and adverse effects of targeting the cgas-sting pathway in cancer therapy ( figure 5 ). considering the pivotal role of the cgas-sting pathway in infection, inflammation and cancer, positive modulation of the pathway signaling is a promising way to enhance the immune state and restrict microorganisms or heterogeneous cells, whereas negative modulation can control aberrant inflammation. radiotherapy or chemotherapy drugs such as cisplatin or cyclophosphamide can induce dsbs and micronuclei, then trigger the cgas-sting pathway to enhance tumor immunogenicity (252) (253) (254) . in addition, parp inhibitors such as olaparib have promising effects on cancer cells lacking brca2 because of their cooperative dna-repair functions (253) . although cgas activation is inhibited by nucleosomes, taxol can induce mitotic cell-cycle arrest and sustain divided chromatin in the cytosol to activate the cgas-sting pathway slowly, and accumulation of signaling could stimulate apoptosis of cancer cells (41). inhibitors of topoisomerase 1 or 2 used conventionally as chemotherapy drugs trigger minor damage to dna and accumulation of cytosolic dna, which can engage cgas and enhance the anti-tumor or anti-infection responses of cells (255) (256) (257) . cgas-sting is essential on anti-tumor immune checkpoints therapies. for example, blockade of cd47-signal regulatory protein α (sirpα) signaling on dcs can activate nadph oxidase 2 (nox2) and increase the ph in phagosomes along with incomplete degradation of mtdna, which can trigger cgas-sting (129) . sting deficiency in mice abrogates the anti-tumor effect of cd47 blockade (258) . a similar phenomenon also can be seen in anti-programmed cell death-1 (pd-1) therapy (259) . there is greater infiltration of ifnγ + cells and cd8 + t cells and pd-1/pd-1 ligand 1 (pd-l1) expression in tme treated by sting-ligand derivatives (260) . therefore, in several types of tumors, combined administration of a sting agonist and immune-checkpoint antibody could elicit a more curative outcome compared with one therapy alone (238, 261) . viruses can infect cells lacking cgas-sting more effectively, and have higher oncolytic activity compared with virus therapy alone. hence, the use of oncolytic viruses such as talimogene laherparepvec is beneficial for treating tumors with low expression of cgas/sting. sting expression can be regarded as a prognostic measurement for such therapy (262) . some artificial analog molecules of cdns, such as 5,6dimethylxanthenone-4-acetic acid (dmxaa) and 10-carboxymethyl-9 (10h) acridone (cma) can bind the cdn pocket of mouse-specific sting dimers and promote conformational transition of sting from inactive "open" to an active "closed" state (263, 264) . dmxaa showed convincing efficacy in restraining tumors in mice (265) . however, dmxaa is restricted in activating mouse-specific sting but not humanspecific sting, which could be an explanation for the failure of dmxaa in treating 1299 non-small-cell lung cancer patients in a phase-iii clinical trial (nct00662597) (266) . nonetheless, with three substitutions (g230i, q266i, and s162a), human sting can also be induced by dmxaa to undergo conformational transition (264) . another new compound, amidobenzimidazole, has been found to be an agonist of sting without lid closure and has potential therapeutic value (267) . cyclic dinucleotides and their derivates that can stimulate sting directly are candidate adjuvants to restrain tumors. intratumoral administration of c-damp, c-dgmp, or cgamp analogs alone or combined with other adjuvants or tumor antigens have shown anti-tumor effects (259, 268) ; phase-i or ii clinical trials (nct02675439, nct03172936, and nct03937141) of dithio-(rp,rp)-c-damp (known as adu-s100) are ongoing (261) . to avoid the degradation and ensure maximal phagocyte internalization of cdns, endosomolytic nanoparticles have been designed to package and deliver cdns. for example, ph-sensitive nanoparticles (e.g., stingnanoparticles) can release their contents if located in acidic endosomal environments (269) . for treatment of type-i interferonopathies, lessons can be taken from the treatment of canonical autoimmune disease such as sle, but there are several differences. for example, it was found that corticosteroid pulse therapy, γ-immunoglobulins, disease-modifying anti-rheumatic drugs, anti-cd20, and some immunosuppressants (e.g., methotrexate) have limited efficacy against savi (166, 171) . jak inhibitors such as ruxolitinib, tofacitinib and baricitinib that reduce type-i ifn downstream signaling have shown therapeutic value against savi, but further verification of their efficacy is needed (270) . moreover, novel immune therapies, such as anti-ifnα and anti-ifnar immunoglobin, are in clinical trials for sle. these could also be tested against savi in the future (165) . pharmaceutically screening has revealed that some antimalaria drugs, such as suramin, have an inhibitory effect on cgas by blockade of interaction between dna and cgas (271) . in addition, novel small molecules such as ru320521 or g150 can occupy the enzymatic pocket of species-specific cgas to abrogate cgamp synthesis (272, 273) . recently, a study found that aspirin can acetylate cgas at three lysine residues and block cgas activity (274) . with regard to sting, the cyclopeptide astin c can block irf3 recruitment onto the sting signalosome (275) . the molecule h-151 can block the palmitoylation of human-sting (276) . all of these agents are potential candidates for alleviating type-i interferonopathies. the cgas-sting pathway is primarily responsible for the modulation of immune response in cells when facing cytosolic dsdna challenge. moreover, it is complicatedly cross-regulated by other cellular processes or cellular signaling networks. the exact fundamental mechanism of the pathway in cells and the effect on the whole organism in specific states is not completely clear and requires further investigation. in conclusion, the cgas-sting pathway has dichotomous roles in the immune system. in general, cgas-sting-type-i ifn signaling can promote the innate immune response in myeloid cells but alleviate the adaptive immune response exerted by t cells and b cells. cgas shows high expression in apcs such as macrophages and dcs, but sting is expressed in most cells (10) . cgas-sting signaling corresponding to cytoplasmic dsdna in apcs can boost innate and adaptive immunity transiently. in this situation, the dna challenge signal is limited to only macrophages and dcs. their pro-inflammatory and antigenpresenting functions to adaptive immune cells are promoted in the short-term. if the signal spreads to other bystander cells, such as t cells, b cells, local resident cells by means of cgamp transfer, or just aberrant sting activation by gof, it causes apoptosis in bystander cells or adaptive immune cells and immune tolerance in the long-term. therefore, it is reasonable to conclude that the intrinsic function of the cgas-sting pathway is essential for the innate immune system responses of the host immediately after pathogen invasion or abnormal cell appearing. once the challenge persists, the cgas-sting pathway controls the adaptive immune system to avoid chronic, detrimental inflammatory reactions or autoimmune diseases. the inflammatory response exists universally in almost all physiologic and pathologic progressions. cgas-sting is pivotal in modulating cellular inflammation, so it is promising to extend our conception of the cgas-sting pathway onto more diseases with inflammatory responses involved, especially cns-based diseases such as stroke, in which the inflammatory reaction exists but was recognized less. moreover, for targeting the cgas-sting pathway for therapeutic purposes, drugs should be optimized to augment the desirable effect and prevent its unwanted effects. for example, to eliminate tumor cells or infectious agents, agonists of cgas-sting would be a rational option if designed to target apcs exclusively but not t cells or b cells. in this scheme, the antitumor immune response is enhanced while avoiding apoptosis of adaptive immune cells and infiltration of immune suppressor cells. also, most research on the cgas-sting pathway has focused on its ifn-expressing role but overlooked autophagy and cell-death roles, which are also main downstream signaling of the pathway. therefore, some drugs, such as emricasan, are potential apoptosis inhibitors that may have a complementary effect on ameliorating apoptosis of blood-vessel endothelial cells or bronchial epithelial cells, and lymphopenia, in savi. until now, studies of the cgas-sting pathway have been done mainly in the laboratory but it has large space to be explored in clinical or translational fields. additionally more prrs and cellular immune-surveillance pathways may remain to be discovered to piece together the molecular puzzles of the cell. dw drafted the manuscript and drew the figures. wj and jh conceived and revised the review. frontiers in immunology | www.frontiersin.org recognition of endogenous nucleic acids by the innate immune system sting is an endoplasmic reticulum adaptor that facilitates innate immune signalling the adaptor protein mita links virus-sensing receptors to irf3 transcription factor activation an endoplasmic reticulum ifn stimulator, activates innate immune signaling through dimerization c-di-amp secreted by intracellular listeria monocytogenes activates a host type i interferon response sting is a direct innate immune sensor of cyclic di-gmp ifi16 is an innate immune sensor for intracellular dna cyclic gmp-amp synthase is a cytosolic dna sensor that activates the type i interferon pathway pivotal roles of cgas-cgamp signaling in antiviral defense and immune adjuvant effects an expression atlas of human primary cells: inference of gene function from coexpression networks bacterial cgas-like enzymes synthesize diverse nucleotide signals structural mechanism of cytosolic dna sensing by cgas structure of human cgas reveals a conserved family of second-messenger enzymes in innate immunity cytosolic rna:dna hybrids activate the cgas-sting axis sequence-specific activation of the dna sensor cgas by y-form dna structures as found in primary hiv-1 cdna cgas produces a 2 -5 -linked cyclic dinucleotide second messenger that activates sting the catalytic mechanism of cyclic gmp-amp synthase (cgas) and implications for innate immunity and inhibition the cytosolic dna sensor cgas forms an oligomeric complex with dna and undergoes switch-like conformational changes in the activation loop cyclic gmp-amp synthase is activated by double-stranded dna-induced oligomerization structure of the human cgas-dna complex reveals enhanced control of immune surveillance cgas senses long and hmgb/tfam-bound u-turn dna by forming protein-dna ladders dna-induced liquid phase condensation of cgas activates innate immune signaling human cgas catalytic domain has an additional dna-binding interface that apoptotic caspases suppress mtdna-induced sting-mediated type i ifn production obsessive-compulsive disorder is associated with broad impairments in executive function: a meta-analysis ribonuclease h2 mutations induce a cgas/sting-dependent innate immune response restriction by samhd1 limits cgas/sting-dependent innate and adaptive immune responses to hiv-1 host restriction factor samhd1 limits human t cell leukemia virus type 1 infection of monocytes via sting-mediated apoptosis samhd1 acts at stalled replication forks to prevent interferon induction cyclic gmp-amp containing mixed phosphodiester linkages is an endogenous high-affinity ligand for sting cyclic gmp-amp synthase is an innate immune sensor of hiv and other retroviruses cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cgamp connexin-dependent transfer of cgamp to phagocytes modulates antiviral responses cancer-cell-intrinsic cgas expression mediates tumor immunogenicity viruses transfer the antiviral second messenger cgamp between cells transmission of innate immune signaling by packaging of cgamp in viral particles cgas-mediated innate immunity spreads intercellularly through hiv-1 envinduced membrane fusion sites priming of dendritic cells by dna-containing extracellular vesicles from activated t cells through antigen-driven contacts cells infected with herpes simplex virus 1 export to uninfected cells exosomes containing sting, viral mrnas, and micrornas slc19a1 is an importer of the immunotransmitter cgamp slc19a1 transports immunoreactive cyclic dinucleotides hydrolysis of 2 3 -cgamp by enpp1 and design of nonhydrolyzable analogs cgas facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity sting regulates intracellular dna-mediated, type i interferon-dependent innate immunity activation by translocation from the er is associated with infection and autoinflammatory disease atg9a controls dsdna-driven dynamic translocation of sting and the innate immune response irhom2 is essential for innate immunity to dna viruses by mediating trafficking and stability of the adaptor sting tmed2 potentiates cellular ifn responses to dna viruses by reinforcing mita dimerization and facilitating its trafficking autophagy induction via sting trafficking is a primordial function of the cgas pathway phosphorylation of innate immune adaptor proteins mavs, sting, and trif induces irf3 activation sting specifies irf3 phosphorylation by tbk1 in the cytosolic dna signaling pathway modular architecture of the sting c-terminal tail allows interferon and nf-kappab signaling adaptation structural basis of sting binding with and phosphorylation by tbk1 a conserved plplrt/sd motif of sting mediates the recruitment and activation of tbk1 activation of sting requires palmitoylation at the golgi ubiquitination of sting at lysine 224 controls irf3 activation non-canonical nf-kappab antagonizes sting sensor-mediated dna sensing in radiotherapy immunomodulatory functions of type i interferons dna-binding landscape of irf3, irf5 and irf7 dimers: implications for dimer-specific gene regulation attenuation of cgas-sting signaling is mediated by a p62/sqstm1-dependent autophagy pathway activated by tbk1 positive feedback regulation of type i ifn production by the ifn-inducible dna sensor cgas positive feedback regulation of type i interferon by the interferon-stimulated gene sting sting-mediated disruption of calcium homeostasis chronically activates er stress and primes t cell death sting senses microbial viability to orchestrate stress-mediated autophagy of the endoplasmic reticulum chronic innate immune activation of tbk1 suppresses mtorc1 activity and dysregulates cellular metabolism mtor signaling in growth, metabolism, and disease crosstalk between the cgas dna sensor and beclin-1 autophagy protein shapes innate antimicrobial immune responses a brief history of autophagy from cell biology to physiology and disease autophagy in the renewal, differentiation and homeostasis of immune cells sting directly activates autophagy to tune the innate immune response mycobacterium tuberculosis is protected from nadph oxidase and lc3-associated phagocytosis by the lcp protein cpsa extracellular m. tuberculosis dna targets bacteria for autophagy by activating the host dna-sensing pathway upr, autophagy, and mitochondria crosstalk underlies the er stress response dnase2a deficiency uncovers lysosomal clearance of damaged nuclear dna via autophagy self-consumption: the interplay of autophagy and apoptosis trafficking-mediated sting degradation requires sorting to acidified endolysosomes and can be targeted to enhance anti-tumor response the dna inflammasome in human myeloid cells is initiated by a sting-cell death program upstream of nlrp3 aim2 recognizes cytosolic dsdna and forms a caspase-1-activating inflammasome with asc antagonism of the sting pathway via activation of the aim2 inflammasome by intracellular dna inflammasome activation triggers caspase-1-mediated cleavage of cgas to regulate responses to dna virus infection gasdermin d restrains type i interferon response to cytosolic dna by disrupting ionic homeostasis a noncanonical function of cgamp in inflammasome priming and activation viral dna binding to nlrc3, an inhibitory nucleic acid sensor, unleashes sting, a cyclic dinucleotide receptor that activates type i interferon non-canonical activation of the dna sensing adaptor sting by atm and ifi16 mediates nf-kappab signaling after nuclear dna damage nuclear ifi16 induction of irf-3 signaling during herpesviral infection and degradation of ifi16 by the viral icp0 protein ifi16 is required for dna sensing in human macrophages by promoting production and function of cgamp ifi16 and cgas cooperate in the activation of sting during dna sensing in human keratinocytes cgas-mediated stabilization of ifi16 promotes innate signaling during herpes simplex virus infection viral dna sensors ifi16 and cyclic gmp-amp synthase possess distinct functions in regulating viral gene expression, immune defenses, and apoptotic responses during herpesvirus infection sting-mediated ifi16 degradation negatively controls type i interferon production assembly and localization of toll-like receptor signalling complexes sting requires the adaptor trif to trigger innate immune responses to microbial infection sequential activation of two pathogen-sensing pathways required for type i interferon expression and resistance to an acute dna virus infection cross-regulation of two type i interferon signaling pathways in plasmacytoid dendritic cells controls anti-malaria immunity and host mortality oxidative damage of dna confers resistance to cytosolic nuclease trex1 degradation and potentiates sting-dependent immune sensing mitochondria and cell death: outer membrane permeabilization and beyond mitochondrial inner membrane permeabilisation enables mtdna release during apoptosis apoptotic caspases prevent the induction of type i interferons by mitochondrial dna apoptotic caspases suppress type i interferon production via the cleavage of cgas, mavs, and irf3 signalling strength determines proapoptotic functions of sting puma amplifies necroptosis signaling by activating cytosolic dna sensors autophagic cell death restricts chromosomal instability during replicative crisis induction of necroptotic cell death by viral activation of the rig-i or sting pathway tnfalpha and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide sting agonists in nonimmunogenic tumors cellular senescence: aging p16ink4a induces an age-dependent decline in islet regenerative potential autophagy mediates degradation of nuclear lamina extranuclear dna accumulates in aged cells and contributes to senescence and inflammation senescenceassociated secretory phenotypes reveal cell-nonautonomous functions of oncogenic ras and the p53 tumor suppressor cytoplasmic chromatin triggers inflammation in senescence and cancer downregulation of cytoplasmic dnases is implicated in cytoplasmic dna accumulation and sasp in senescent cells innate immune sensing of cytosolic chromatin fragments through cgas promotes senescence cgas is essential for cellular senescence dna-damage-induced type i interferon promotes senescence and inhibits stem cell function bacterial c-di-gmp affects hematopoietic stem/progenitors and their niches through sting a circular rna protects dormant hematopoietic stem cells from dna sensor cgas-mediated exhaustion pqbp1 is a proximal sensor of the cgas-dependent innate response to hiv-1 nono detects the nuclear hiv capsid to promote cgas-mediated innate immune activation p53 induces formation of neat1 lncrna-containing paraspeckles that modulate replication stress response and chemosensitivity hexim1 and neat1 long non-coding rna form a multi-subunit complex that regulates dna-mediated innate immune response stingdependent translation inhibition restricts rna virus replication inhibition of hiv-1 disease progression by contemporaneous hiv-2 infection samhd1 is the dendritic-and myeloid-cell-specific hiv-1 restriction factor counteracted by vpx reshaping of the dendritic cell chromatin landscape and interferon pathways during hiv infection the capsids of hiv-1 and hiv-2 determine immune detection of the viral cdna by the innate sensor cgas in dendritic cells hiv-1 activation of innate immunity depends strongly on the intracellular level of trex1 and sensing of incomplete reverse transcription products hiv-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated dna synthesis restriction of hiv-1 and other retroviruses by trim5 hiv-1 evades innate immune recognition through specific cofactor recruitment cyclic di-gmp: second messenger extraordinaire cyclic gmp-amp signalling protects bacteria against viral infection a bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis tuberculosis pathogenesis and immunity mycobacterium tuberculosis differentially activates cgas-and inflammasome-dependent intracellular immune responses through esx-1 brucella abortus triggers a cgas-independent sting pathway to induce host protection that involves guanylate-binding proteins and inflammasome activation beneficial effects of yogasanas and pranayama in limiting the cognitive decline in type 2 diabetes group b streptococcus degrades cyclic-di-amp to modulate stingdependent type i interferon production inhibition of innate immune cytosolic surveillance by an m. tuberculosis phosphodiesterase absence of specific chlamydia trachomatis inclusion membrane proteins triggers premature inclusion membrane lysis and host cell death the chlamydia trachomatis inclusion membrane protein cpos counteracts sting-mediated cellular surveillance and suicide programs yersinia yopj negatively regulates irf3-mediated antibacterial response through disruption of sting-mediated cytosolic dna signaling intracellular bacteria engage a sting-tbk1-mvb12b pathway to enable paracrine cgas-sting signalling sting-dependent type i ifn production inhibits cell-mediated immunity to listeria monocytogenes induction of interferon-stimulated genes by irf3 promotes replication of toxoplasma gondii malaria parasite dna-harbouring vesicles activate cytosolic immune sensors type i interferon in rheumatic diseases activated sting in a vascular and pulmonary syndrome inherited sting-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations stimulator of interferon genes-associated vasculopathy with onset in infancy: a mimic of childhood granulomatosis with polyangiitis severe combined immunodeficiency in stimulator of interferon genes (sting) v154m/wild-type mice sting-associated vasculopathy develops independently of irf3 in mice disease-associated mutations identify a novel region in human sting necessary for the control of type i interferon signaling personalized immunomonitoring uncovers molecular networks that stratify lupus patients aicardi-goutieres syndrome and the type i interferonopathies apoptosisderived membrane vesicles drive the cgas-sting pathway and enhance type i ifn production in systemic lupus erythematosus expression of cyclic gmp-amp synthase in patients with systemic lupus erythematosus. arthritis rheumatol the role of dendritic cells in autoimmunity human plasmacytoid dentritic cells elicit a type i interferon response by sensing dna via the cgas-sting signaling pathway interferon priming is essential for human cd34+ cell-derived plasmacytoid dendritic cell maturation and function interleukin-1beta induces mtdna release to activate innate immune signaling via cgas-sting induction of dendritic cell differentiation by ifn-alpha in systemic lupus erythematosus neutrophil extracellular traps enriched in oxidized mitochondrial dna are interferogenic and contribute to lupus-like disease netting neutrophils are major inducers of type i ifn production in pediatric systemic lupus erythematosus type i ifnrelated netosis in ataxia telangiectasia and artemis deficiency cytosolic dna sensing promotes macrophage transformation and governs myocardial ischemic injury irf3 and type i interferons fuel a fatal response to myocardial infarction activation of the sting-irf3 pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease expression of sting is increased in liver tissues from patients with nafld and promotes macrophage-mediated hepatic inflammation and fibrosis in mice sting-mediated inflammation in kupffer cells contributes to progression of nonalcoholic steatohepatitis lipotoxicity induces hepatic protein inclusions through tank binding kinase 1-mediated p62/sequestosome 1 phosphorylation sting-irf3 triggers endothelial inflammation in response to free fatty acid-induced mitochondrial damage in diet-induced obesity priming the inflammatory pump of the cns after traumatic brain injury stingmediated type-i interferons contribute to the neuroinflammatory process and detrimental effects following traumatic brain injury sting-dependent sensing of self-dna drives silica-induced lung inflammation mitochondrial damage and activation of the sting pathway lead to renal inflammation and fibrosis mitochondrial damage causes inflammation via cgas-sting signaling in acute kidney injury how neuroinflammation contributes to neurodegeneration sensing of hsv-1 by the cgas-sting pathway in microglia orchestrates antiviral defence in the cns chronic neurodegeneration induces type i interferon synthesis via sting, shaping microglial phenotype and accelerating disease progression the roles of pink1, parkin, and mitochondrial fidelity in parkinson's disease parkin and pink1 mitigate sting-induced inflammation atm and the molecular pathogenesis of ataxia telangiectasia autoimmune phenomena in ataxia telangiectasia mitochondrial dna has a pro-inflammatory role in amd cgas drives noncanonical-inflammasome activation in age-related macular degeneration multiple sclerosis type i/ii interferon balance in the regulation of brain physiology and pathology activation of the sting-dependent type i interferon response reduces microglial reactivity and neuroinflammation modulation of the immune system by uv radiation: more than just the effects of vitamin d? ultraviolet b irradiation causes stimulator of interferon genes-dependent production of protective type i interferon in mouse skin by recruited inflammatory monocytes. arthritis rheumatol engineering dna nanoparticles as immunomodulatory reagents that activate regulatory t cells activation of the sting adaptor attenuates experimental autoimmune encephalitis nucleic acid sensing by t cells initiates th2 cell differentiation sting-associated lung disease in mice relies on t cells but not type i interferon hierarchy of clinical manifestations in savi n153s and v154m mouse models intrinsic antiproliferative activity of the innate sensor sting in t lymphocytes agonist-mediated activation of sting induces apoptosis in malignant b cells b cell-intrinsic sting signaling triggers cell activation, synergizes with b cell receptor signals, and promotes antibody responses sting signaling promotes inflammation in experimental acute pancreatitis habtezion a. sting signalling protects against chronic pancreatitis by modulating th17 response stingactivating adjuvants elicit a th17 immune response and protect against mycobacterium tuberculosis infection sting-dependent signaling underlies il-10 controlled inflammatory colitis the cytosolic sensor sting is required for intestinal homeostasis and control of inflammation the multifaceted role of chromosomal instability in cancer and its microenvironment immunogenic cell death in cancer and infectious disease sting contributes to antiglioma immunity via triggering type i ifn signals in the tumor microenvironment sting-dependent cytosolic dna sensing mediates innate immune recognition of immunogenic tumors suppression of sting associated with lkb1 loss in kras-driven lung cancer dna exonuclease trex1 regulates radiotherapy-induced tumour immunogenicity growing tumors induce a local sting dependent type i ifn response in dendritic cells tumorderived cgamp triggers a sting-mediated interferon response in nontumor cells to activate the nk cell response rae1 ligands for the nkg2d receptor are regulated by sting-dependent dna sensor pathways in lymphoma sting activation reprograms tumor vasculatures and synergizes with vegfr2 blockade dendritic cells but not macrophages sense tumor mitochondrial dna for cross-priming through signal regulatory protein alpha signaling exosomes shuttle trex1-sensitive ifn-stimulatory dsdna from irradiated cancer cells to dcs host type i ifn signals are required for antitumor cd8+ t cell responses through cd8{alpha}+ dendritic cells sting-dependent cytosolic dna sensing promotes radiation-induced type i interferondependent antitumor immunity in immunogenic tumors targeting dna damage response promotes antitumor immunity through sting-mediated t-cell activation in small cell lung cancer intratumoral sting activation with t-cell checkpoint modulation generates systemic antitumor immunity sting signaling remodels the tumor microenvironment by antagonizing myeloid-derived suppressor cell expansion il-15 is a component of the inflammatory milieu in the tumor microenvironment promoting antitumor responses suppression of sting signaling through epigenetic silencing and missense mutation impedes dna damage mediated cytokine production extrachromosomal telomere repeat dna is linked to alt development via cgas-sting dna sensing pathway downregulation of membrane trafficking proteins and lactate conditioning determine loss of dendritic cell function in lung cancer her2 recruits akt1 to disrupt sting signalling and suppress antiviral defence and antitumour immunity akt kinase-mediated checkpoint of cgas dna sensing pathway dna damage predicts prognosis and treatment response in colorectal liver metastases superior to immunogenic cell death and t cells chromosomal instability drives metastasis through a cytosolic dna response inflammation-driven carcinogenesis is mediated through sting carcinomaastrocyte gap junctions promote brain metastasis by cgamp transfer host stingdependent mdsc mobilization drives extrinsic radiation resistance tumor cellderived microparticles polarize m2 tumor-associated macrophages for tumor progression parp inhibition enhances tumor cell-intrinsic immunity in ercc1-deficient non-small cell lung cancer parp inhibitor efficacy depends on cd8(+) t-cell recruitment via intratumoral sting pathway activation in brca-deficient models of triple-negative breast cancer radiotherapy combined with novel sting-targeting oligonucleotides results in regression of established tumors topoisomerase 1 inhibition promotes cyclic gmp-amp synthasedependent antiviral responses topoisomerase ii inhibitors induce dna damage-dependent interferon responses circumventing ebola virus immune evasion cgas/sting axis mediates a topoisomerase ii inhibitor-induced tumor immunogenicity cd47 blockade triggers t cell-mediated destruction of immunogenic tumors sting activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity a sting agonist given with ox40 receptor and pd-l1 modulators primes immunity and reduces tumor growth in tolerized mice magnitude of therapeutic sting activation determines cd8(+) t cellmediated anti-tumor immunity recurrent loss of sting signaling in melanoma correlates with susceptibility to viral oncolysis species-specific detection of the antiviral small-molecule compound cma by sting bindingpocket and lid-region substitutions render human sting sensitive to the species-specific drug dmxaa the sting agonist dmxaa triggers a cooperation between t lymphocytes and myeloid cells that leads to tumor regression randomized phase iii placebo-controlled trial of carboplatin and paclitaxel with or without the vascular disrupting agent vadimezan (asa404) in advanced non-small-cell lung cancer design of amidobenzimidazole sting receptor agonists with systemic activity sting ligand c-di-gmp improves cancer vaccination against metastatic breast cancer endosomolytic polymersomes increase the activity of cyclic dinucleotide sting agonists to enhance cancer immunotherapy efficacy of the janus kinase 1 /2 inhibitor ruxolitinib in the treatment of vasculopathy associated with tmem173-activating mutations in 3 children suramin potently inhibits cgamp synthase, cgas, in thp1 cells to modulate ifnbeta levels small molecule inhibition of cgas reduces interferon expression in primary macrophages from autoimmune mice development of human cgas-specific small-molecule inhibitors for repression of dsdnatriggered interferon expression the cyclopeptide astin c specifically inhibits the innate immune cdn sensor sting targeting sting with covalent small-molecule inhibitors trim56-mediated monoubiquitination of cgas for cytosolic dna sensing the ubiquitin ligase trim56 regulates innate immune responses to intracellular doublestranded dna the e3 ubiquitin ligase rnf185 facilitates the cgas-mediated innate immune response rnf26 temporally regulates virus-triggered type i interferon induction by two distinct mechanisms the e3 ubiquitin ligase amfr and insig1 bridge the activation of tbk1 kinase by modifying the adaptor sting trim14 inhibits cgas degradation mediated by selective autophagy receptor p62 to promote innate immune responses p38 inhibition provides anti-dna virus immunity by regulation of usp21 phosphorylation and sting activation usp18 recruits usp20 to promote innate antiviral response through deubiquitinating sting/mita the deubiquitinase cyld is a specific checkpoint of the sting antiviral signaling pathway sumoylation promotes the stability of the dna sensor cgas and the adaptor sting to regulate the kinetics of response to dna virus senp7 potentiates cgas activation by relieving sumo-mediated inhibition of cytosolic dna sensing g3bp1 promotes dna binding and activation of cgas zcchc3 is a cosensor of cgas for dsdna recognition in innate immune response manganese increases the sensitivity of the cgas-sting pathway for double-stranded dna and is required for the host defense against dna viruses tmem203 is a binding partner and regulator of sting-mediated inflammatory signaling in macrophages the erassociated protein zdhhc1 is a positive regulator of dna virus-triggered, mita/sting-dependent innate immune signaling association of abnormal elevations in ifit3 with overactive cyclic gmp-amp synthase/stimulator of interferon genes signaling in human systemic lupus erythematosus monocytes s6k-sting interaction regulates cytosolic dna-mediated activation of the transcription factor irf3 glycogen synthase kinase 3beta regulates irf3 transcription factor-mediated antiviral response via activation of the kinase tbk1 trim9 short isoform preferentially promotes dna and rna virus-induced production of type i interferon by recruiting gsk3beta to tbk1 lsm14a plays a critical role in antiviral immune responses by regulating mita level in a cell-specific manner trim29 promotes dna virus infections by inhibiting innate immune response trim30alpha is a negative-feedback regulator of the intracellular dna and dna virustriggered response by targeting sting the ubiquitin ligase rnf5 regulates antiviral responses by mediating degradation of the adaptor protein mita usp13 negatively regulates antiviral responses by deubiquitinating sting oligoadenylatesynthetase-family protein oasl inhibits activity of the dna sensor cgas during dna virus infection to limit interferon production interactome and proteome dynamics uncover immune modulatory associations of the pathogen sensing factor cgas sensing of bacterial cyclic dinucleotides by the oxidoreductase recon promotes nf-kappab activation and shapes a proinflammatory antibacterial state the ca(2+) sensor stim1 regulates the type i interferon response by retaining the signaling adaptor sting at the endoplasmic reticulum nitro-fatty acids are formed in response to virus infection and are potent inhibitors of sting palmitoylation and signaling nlrc3, a member of the nlr family of proteins, is a negative regulator of innate immune signaling induced by the dna sensor sting ppm1a regulates antiviral signaling by antagonizing tbk1-mediated sting phosphorylation and aggregation ptpn1/2-mediated dephosphorylation of mita/sting promotes its 20s proteasomal degradation and attenuates innate antiviral response nlrx1 sequesters sting to negatively regulate the interferon response, thereby facilitating the replication of hiv-1 and dna viruses nlrx1 promotes immediate irf1-directed antiviral responses by limiting dsrnaactivated translational inhibition mediated by pkr mitigating sox2-potentiated immune escape of head and neck squamous cell carcinoma with a sting-inducing nanosatellite vaccine mediates hypoxia-induced immunosuppression by repressing cgas nrf2 negatively regulates sting indicating a link between antiviral sensing and metabolic reprogramming kdm5 histone demethylases repress immune response via suppression of sting herpes simplex virus 1 abrogates the cgas/sting-mediated cytosolic dna-sensing pathway via its virion host shutoff protein, ul41 herpes simplex virus 1 tegument protein vp22 abrogates cgas/sting-mediated antiviral innate immunity species-specific deamidation of cgas by herpes simplex virus ul37 protein facilitates viral replication evasion of the sting dna-sensing pathway by vp11/12 of herpes simplex virus 1 herpes simplex virus 1 gamma134.5 protein inhibits sting activation that restricts viral replication human cytomegalovirus protein ul31 inhibits dna sensing of cgas to mediate immune evasion type i interferon production by inactivating the dna sensor cgas without affecting sting essential role of hcmv deubiquitinase in promoting oncogenesis by targeting anti-viral innate immune signaling pathways human cytomegalovirus tegument protein ul82 inhibits sting-mediated signaling to evade antiviral immunity the herpesviral antagonist m152 reveals differential activation of sting-dependent irf and nf-kappab signaling and sting's dual role during mcmv infection human cytomegalovirus-encoded us9 targets mavs and sting signaling to evade type i interferon immune responses inhibition of cgas dna sensing by a herpesvirus virion protein cytoplasmic isoforms of kaposi sarcoma herpesvirus lana recruit and antagonize the innate immune dna sensor cgas modulation of the cgas-sting dna sensing pathway by gammaherpesviruses sars coronavirus papain-like protease inhibits the type i interferon signaling pathway through interaction with the sting-traf3-tbk1 complex poxviruses evade cytosolic sensing through disruption of an mtorc1-mtorc2 regulatory circuit viral and metazoan poxins are cgamp-specific nucleases that restrict cgas-sting signalling zika virus elicits inflammation to evade antiviral response by cleaving cgas via ns1-caspase-1 axis denv inhibits type i ifn production in infected cells by cleaving human sting dengue virus ns2b protein targets cgas for degradation and prevents mitochondrial dna sensing during infection dna tumor virus oncogenes antagonize the cgas-sting dna-sensing pathway influenza a virus targets a cgas-independent sting pathway that controls enveloped rna viruses hepatitis c virus ns4b blocks the interaction of sting and tbk1 to evade host innate immunity hepatitis c virus ns4b protein targets sting and abrogates rig-i-mediated type i interferon-dependent innate immunity hepatitis b virus polymerase disrupts k63-linked ubiquitination of sting to block innate cytosolic dnasensing pathways hiv-2/siv vpx targets a novel functional domain of sting to selectively inhibit cgas-stingmediated nf-kappab signalling we thank k. wood from barrow neurological institute for discussions and editing. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 wan, jiang and hao. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-312074-0nqmjdek authors: zhong, jixin; rajagopalan, sanjay title: dipeptidyl peptidase-4 regulation of sdf-1/cxcr4 axis: implications for cardiovascular disease date: 2015-09-25 journal: front immunol doi: 10.3389/fimmu.2015.00477 sha: doc_id: 312074 cord_uid: 0nqmjdek dipeptidyl peptidase-4 (dpp4) is a ubiquitously expressed protease that regulates diverse number of physiological functions. as a dipeptidase, it exerts its catalytic effects on proteins/peptides with proline, alanine, or serine in the penultimate (p1) amino acid residue from the amino terminus. the evidence to date supports an important effect of dpp4 in catalytic cleavage of incretin peptides and this perhaps represents the main mechanism by which dpp4 inhibition improves glycemic control. dpp4 also plays an important role in the degradation of multiple chemokines of which stromal cell-derived factor-1 (sdf-1, also known as cxcl12) is perhaps an increasingly recognized target, given its importance in processes, such as hematopoiesis, angiogenesis, and stem cell homing. in the current review, we will summarize the importance of dpp4-mediated enzymatic processing of cytokines/chemokines with an emphasis on sdf-1 and resultant implications for cardiovascular physiology and disease. identified as a new dipeptide naphthylamidase in 1966 (1) and subsequently found to be identical to t cell activation antigen cd26, ada-binding protein, mouse thymocyte-activating molecule, and rat liver membrane glycoprotein gp110 (2) . dpp4 consists of a short n-terminal intracellular domain (6 residues), a 22-residue-long transmembrane α-helix domain (23 amino acids), and a large c-terminal extracellular domain. the c-terminal extracellular domain is responsible for its catalytic activity and binding to a number of ligands, such as ada and matrix proteins (3) . the catalytic activity of dpp4 depends on its dimerization and glycosylation of specific residues (2) . dpp4 can also assemble into tetramers on the cell surface, which may involve the linkage of dimers located on the surface of two different cells, enabling it to function as a cell-cell communication molecule. in addition to its membrane-bound form, dpp4 also circulates as a soluble form in the plasma, which lacks the cytoplasmic and transmembrane domain with preserved catalytic activity. soluble dpp4 (sdpp4) is a homodimer with a molecular weight range of 210-290 kda (4), but can form higher molecular weight assemblies migrating as 900-kda complexes (5) . whether sdpp4 is cleaved from the membrane or is secreted is unclear. for instance, studies investigating viral liver infection suggested that sdpp4 is shed from membrane-bound dpp4 (6) . sdpp4 has, however, also been detected in the lumen of secretory granules in pancreatic α cells and in the exocytic secretory lysosomes of natural killer cells (7, 8) . sdpp4 is commonly elevated in many disorders, such as solid tumors, reactive airways disease, hepatitis c, type 2 diabetes, and obesity (6, 9, 10) . tissue distribution and cell specific expression and phenotype of dpp4 −/− mice dipeptidyl peptidase-4 is widely distributed throughout the body ( table 1) , with particularly high expression on the apical surface of endothelial and differentiated epithelial cells. bone marrow cells, brush border of the small intestine, proximal tubular cells, and glomerular cells in the kidney express high levels of dpp4 as well (11) . dpp4 is present on endothelial cells and fibroblasts throughout the body. among hematopoietic cells, dpp4 is expressed at the highest level on t cells with lower levels in monocytes and dendritic cells (12) . dpp4 expression increases potential function serves as an adipokine mediating obesity-induced metabolic syndrome (9) adipose tissue macrophage and dendritic cells enhances t cell inflammation and obesity-induced insulin resistance (12) t cells promotes t cell activation by providing co-stimulatory signaling (17, 18) endothelial cells regulates endothelial function and vascular tone (19, 20) epithelial cells expressed in the epithelial cells in the kidney, lung, and gi tract. mediates mers-cov infection in the lung (21), kidney fibrosis (22) , diabetic nephropathy (23), intestinal growth (24) hepatocytes involved in lipogenesis (25) and liver damage (26) as monocytes differentiate into antigen-presenting cells as well as during t cell activation (12, 13) . dpp4 is expressed at high levels in kidney, spleen, lung, pancreas, and prostate (14) . mice lacking the gene encoding dpp4 are refractory to the development of obesity and hyperinsulinemia and demonstrate improved post-prandial glucose control (15, 16) . mice deleted for dpp4 are fertile and appear healthy. only slight decrease of body weight in dpp4 −/− mice was observed compared to wild types. they have normal fasting blood glucose level, but shows reduced glycemic excursion after an oral glucose challenge (16) . increased intact insulinotropic form of glp-1 and circulating insulin were seen in dpp4 −/− mice after oral glucose stimulation (16) . pair feeding and indirect calorimetry studies indicate that reduced food intake and increased energy expenditure accounted for the resistance to high fat diet-induced obesity in the dpp4 −/− mice. ablation/ deletion of dpp4 is associated with improved metabolic control with improved insulin sensitivity, reduced pancreatic islet hypertrophy, and protection against streptozotocin-induced loss of β cell mass and hyperglycemia (15) . pharmacological inhibition of dpp4 enzymatic activity improves glucose tolerance in wildtype but not in dpp4 −/− mice. interestingly, dpp4 inhibitor's also improve glucose tolerance in glp1r −/− mice, indicating that dpp4 contributes to blood glucose regulation by controlling the activity of glp-1 as well as additional substrates (16) . dipeptidyl peptidase-4 has been shown to be able to cleave a number of chemokines and cytokines, including sdf-1, granulocyte-macrophage colony-stimulating factor (gm-csf), granulocyte colony-stimulating factor (g-csf), interleukin-3 (il-3), erythropoietin (epo), regulated on activation normal t-cell expressed and presumably secreted (rantes, also known as ccl5), macrophage-derived chemokine (mdc, also known as ccl22), eotaxin (also known as ccl11), monokine induced by ifn-γ (mig, also known as cxcl9), ifn-γ-induced protein-10 (ip-10, also known as cxcl10), and interferon-inducible t-cell α chemoattractant (itac, also known as cxcl11) ( table 2 ). the regulation of cytokine levels through catalytic cleavage may influence their levels in tissue domains. the differential contribution of dpp4 in the regulation of each of these cytokines is obviously dependent on the levels of expression of dpp4, which may vary depending on the tissues, the cells predominantly expressing the cytokine of interest and the disease context. additionally, circulating or cell free dpp4 may also contribute to catalytic activity. dpp4-mediated truncation of rantes abolishes the chemotactic activity to monocytes but not to t cells (27) . eotaxin (ccl11) is an eosinophil chemotactic protein and has been shown to be involved in allergic responses. dpp4 truncation of eotaxin inactivates its chemotactic activity for eosinophils. dpp4-truncated eotaxin shows impaired binding and signaling through ccr3. in addition, truncated eotaxin suppresses calcium signaling and chemotaxis of intact eotaxin (28) . dpp4 inhibition by either genetic deletion or pharmacological inhibition, enhances eotaxin-induced mobilization of eosinophils into the blood and recruitment into the injury/injection site (29) . mdc (ccl22) is a chemoattractant for monocytes, dendritic cells, nk cells, and chronically activated t lymphocytes (30, 31) . dpp4-truncated mdc displays preserved chemotactic activity toward monocytes, but less potency toward lymphocytes and dendritic cells (32) . cxcr3 interacts with mig (cxcl9), ip10 (cxcl10), and itac (cxcl11), all of which are targets of dpp4. cleavage of those chemokines by dpp4 attenuates their chemotactic activity, with the cleaved products serving as endogenous antagonists for cxcr3 binding (33) . dipeptidyl peptidase-4 is also involved in the inactivation of multiple colony-stimulating factors (csfs) and thus regulates hematopoietic stem cells (hscs) and hematopoietic progenitor cell (hpc) function. the proliferative action of gm-csf, g-csf, il-3, and erythropoietin (epo) on hpcs is enhanced in dpp4 knockout mice or by pretreatment with a dpp4 inhibitor (35) . catalytic inhibition of dpp4 or dpp4 deficiency promotes the engraftment of hscs and hpcs after bone marrow transplantation in mice (35) . dpp4-truncated csfs may suppress the activity of their respective full-length csf via antagonism (35) . stromal cell-derived factor-1 is an 8-kda peptide that is encoded by cxcl12 (36) . it is a chemoattractant for t lymphocytes, bone marrow stem cells [such as hsc, endothelial progenitor cell (epc), and mesenchymal stem cells (mscs)], endogenous cardiac stem cells (cscs), and adipose-derived regenerative cells (37) (38) (39) . there are several isoforms of sdf-1 (sdf-1α-ζ), resulting from alternative splicing of its mrna (40) . among these isoforms, sdf-1α is the best described. sdf-1α is expressed in many tissues, including bone marrow, heart, liver, kidney, thymus, spleen, skeletal muscle, and brain (36, (40) (41) (42) (43) . in the cardiovascular system, sdf-1α is expressed in stromal cells, endothelial cells, and cardiomyocytes (44, 45) . sdf-1 is typically inactivated by exopeptidases, such as dpp4, matrix metalloproteinase (mmp)-2, and -9 (34) . unlike cleavage of sdf-1 by dpp4 at position 2-3, mmps cleave sdf-1 at position 4-5, leading to the loss of its binding activity to cxcr4 (46) . the relative contribution of each of these peptidases in regulation of sdf-1 levels is unclear. cxcr4 is an alpha-chemokine receptor specific for sdf-1 and belongs to a family of g-protein-coupled receptors. cxcr4 is expressed on a range of progenitor cells (including hematopoietic, endothelial, and cscs) and thus is important for cell migration and organ development during embryogenesis (39, 40, 47) . mice deficient for either cxcr4 or sdf-1 display abnormal b-lymphocyte, hepatic, and cardiac (ventricular septal defects) development, and die in utero (48) (49) (50) . loss-of-function cxcr4 mutations in humans also causes impaired neutrophil mobilization and b-cell lymphopenia (51) . in addition to cxcr4, cxcr7 has also been suggested to be an important receptor for sdf-1 (52, 53) . however, the relative contribution and interactions of cxcr4 and cxcr7 is not fully elucidated. the involvement of cxcr7 in cardiovascular disease, if any, is also not yet known (39) . dpp4 may also play a more general role in regulating csf activity and stem cell homing (35) . it was previously believed that disruption of the interaction between cxcr4 receptor expressed by hematopoietic progenitors and sdf-1 expressed by bone marrow stromal cells is sufficient to detach anchored progenitors from their bone marrow niches, leading to their rapid mobilization to the peripheral blood. amd3100 (also termed plerixafor) inhibits sdf-1-mediated migration in vitro by blocking the chemokine binding to its major receptor cxcr4 (54) . amd 3100 mobilizes immature progenitor cells from the bone marrow into the blood and has been approved for clinical mobilization in lymphoma and multiple myeloma patients undergoing autologous transplantation. when combined with g-csf, amd3100 synergistically augments mobilization of progenitor cells, with increased in vitro migration to sdf-1 gradients and facilitates repopulation of transplanted non-obese diabetic/severe combined immunodeficient mice (55) . amd 3100 has recently been shown to directly induce sdf-1 release from cxcr4 + human bone marrow osteoblasts and endothelial cells, with sdf-1 release from these cells into the circulation, representing a pivotal mechanism essential for steady-state egress and rapid mobilization of hpcs (56). the sdf-1/cxcr4 axis has been shown to be critical in tissue repair in multiple organ systems, including the eye, heart, kidney, liver, brain, and skin. specific to the heart, the sdf-1/cxcr4 axis has been shown to be essential for cardiogenesis (57, 58) . sdf-1 is now well known as a key regulator of stem cell migration to sites of tissue injury (44, 59) . sdf-1 was first identified by askari et al. as a key regulator of stem cell migration to ischemic cardiac tissue (44) . cd34 + stem cells express the sdf-1 receptor cxcr4 at high levels (37, 60) . during myocardial infarction, sdf-1 levels are elevated 1 h after infarction and return to baseline at day 7 and further reduced to a low level thereafter (44) . overexpression of sdf-1 in ischemic cardiomyopathy by either engineered cellbased or plasmid-based approach improved cardiac function in rats via enhancing stem cell homing and promoting revascularization of the infarct area (61, 62) . therefore, the ability to express sdf-1 locally is believed to enhance the vasculogenic potential of adult cardiac progenitor cells (63) . however, the enhancement of endogenous stem cell-based repair appears to be blunted due to the short half-life of sdf-1 at the time of acute myocardial infarction owing to its degradation by proteases (44) . as a major enzyme mediating the degradation of sdf-1, dpp4 may represent a potential target for improving stem cell homing with stem cell-based therapy. preservation of sdf-1 by dpp4 inhibition has been shown to promote stem cell repopulation and homing to ischemic tissues. dpp4 inhibitors diprotin a or val-pyr, enhance chemotaxis of hscs and hpcs and greatly increase homing and engrafting capacity of hscs (64, 65) . pretreatment of hsc with dpp4 inhibitor diprotin a, enhanced their repopulation ability in lethally irradiated mice (66) . enhancement of engraftment of human cd34 + cord blood cells with dpp4 inhibition has also been observed in xenogeneic mouse recipients (nod/scid or nod/scid/beta 2 null ) (67, 68) . pretreating either donor cells in vitro or recipients in vivo is able to enhance the engraftment of stem cells (66, 69) . in a lung transplantation model, systemic dpp4 inhibition by vildagliptin increases sdf-1 levels in plasma, spleen, and lung, accompanied by a significant increase of stem cells in the lung grafts. dpp4 inhibitor-treated mice also shows less alveolar edema compared with untreated recipients (70) . liebler showed that dpp4 inhibition enhances sdf-1/cxcr4 axis and increased the retention of human bone marrow-derived cells in the injured lungs of immune deficient mice by 30% (71) . in addition to sdf-1, dpp4 inhibition also enhances bone marrow engraftment by preserving g-csf and gm-csf. both g-csf and gm-csf are substrates for dpp4, with inhibition of dpp4 promotes bone marrow engraftment not only through sdf-1 but also csf-dependent mechanisms (35) . g-csf and gm-csf in turn may also increase the expression of dpp4 on cd34 + cells, which results in their decreased responsiveness to sdf-1 (72). angiogenesis and vasculogenesis are an immensely complex process that requires the coordinated action of a multitude of cells, transcription factors, and cytokines working in concert in a precisely choreographed manner. it is widely believed that these processes can be recapitulated in the adult through the participation of a progenitor cell population of which epcs are perhaps the best described and widely believed to be important building blocks for the assembly of functional vasculature in adults. while the origins of epcs are still controversial, what is clear is that these cells have the capacity to differentiate into mature endothelial cells (73) . implantation of ex vivo-expanded epc has been shown to improve neovascularization of injured tissues in animal models (74) (75) (76) . sdf-1 plays a pivotal role in the trafficking and homing of epcs to ischemic tissues (77) (78) (79) . sdf-1 levels increase in plasma and ischemic tissue shortly after ischemic injury, in response to hypoxia which upregulates hif-1α (79) . hif-1α upregulates sdf-1, by binding to the promoter of sdf-1 and initiating its transcription (80) . ex vivo priming with sdf-1, enhances the proangiogenic potential of epc as evidenced by improved blood flow recovery when transplanted into a nude mouse model of hind-limb ischemia (81) . disease states, such as diabetes associated with upregulation of dpp4, may represent prototypical conditions associated with defective homing and integration of epc's owing to rapid degradation of sdf-1 (82) . kanki et al. reported that sdf-1 could be cleaved by dpp-4 in both plasma and ischemic heart tissue (34) . shih demonstrated an improvement in epc number and endothelial nitric oxide synthetase (enos) expression after dpp4 inhibition by mk-0626 (83) . transient engineered cell-based or plasmid-based overexpression of sdf-1 in ischemic cardiomyopathy has been shown to improve cardiac function in animal models (62) . in a study that compared the effects of sdf-1 overexpressed on mscs alone or mesenchymal stem cells engineered to overexpress sdf-1 (msc-sdf) on cardiac function in lewis rats after acute myocardial infarction, tail vein infusion of msc and msc-sdf-1, 1 day after acute myocardial infarction, led to improved cardiac function by echocardiography by 70.7 and 238.8%, respectively, compared with saline controls. the beneficial effects of msc-sdf transplantation were suggested to be mediated through preservation rather than regeneration of cardiac myocytes within the infarct area (84) . cardiac progenitor cell and cxcr4 expression on cardiac myocytes are required for further local trophic effects of msc (85) . the mechanism of action of sdf-1 overexpression in myocardial infarction and heart failure are likely multifactorial, including both systemic and direct trophic effects. an important effect of sdf-1 is its effect on the recruitment of cscs to the infarct and infarct border zone (59) . delivery of mscs engineered to overexpress sdf-1 at the time of acute myocardial infarction has been shown to lead to improvement in cardiac function (61) . the myocardial repair initiated by endogenous stem cell appears blunted because of the natural short-term expression of sdf-1 at the time of acute myocardial infarction. in light of these effects in regulation of sdf-1, dpp4 inhibition has been suggested to be of potential benefit in cardiovascular diseases, such as myocardial infarction and peripheral artrerial disease. in combination with g-csf, dpp4 inhibition augments myocardial regeneration and improves cardiac function after myocardial infarction in mice (86, 87) . in combination with cxcr4 overexpression, diprotin a treatment has shown to improve myocardial function and repair of infarcted myocardium (88) . a bioengineered protease-resistant form of sdf-1 has shown greater potency in promoting blood flow recovery after hind-limb ischemia (89) and improving cardiac function as well as capillary density in the infarcted heart (34) . dual injection of g-csf and sitagliptin resulted in the mobilization of progenitor cells and relieved the symptom of end-stage heart failure in a 19-month-old boy (90) . protease-resistant forms of sdf-1 display an enhanced potency in improving blood flow in experimental peripheral artery disease and myocardial infarction (34, 89) . it has been shown that parathyroid hormone treatment after myocardial infarction improves dpp4 in chemotaxis and cardiovascular disease frontiers in immunology | www.frontiersin.org survival and myocardial function with potential involvement of enhanced homing of bone marrow-derived stem cells. huber et al. demonstrated that parathyroid hormone serves as a dpp4 inhibitor and increases cardiac sdf-1 level, which in turn enhances cxcr4 + bone marrow-derived stem cell homing to ischemic heart and attenuates ischemic cardiomyopathy after infarction (91) . haverslag showed sdf-1 preservation by dpp4 inhibitor increases monocyte extravasation and thus accelerating perfusion recovery without detrimental side effects on plaque stability in atherosclerosis-prone apoe −/− mice (92) . figure 1 depicts modulation of sdf-1 levels in the myocardium by dpp4 inhibition and enhancement of myocardial angiogenesis by dpp4 levels. in a porcine model of hf, delivery of a plasmid sdf-1 with an endomyocardial injection catheter demonstrated safety at doses up to 100 mg while improving cardiac function and vasculogenesis up to 90 days post-injection at doses of 7.5 and 30 mg (59) . in a phase i dose escalation study with 12 months follow-up in ischemic cardiomyopathy, 17 subjects in new york heart association class iii heart failure, with an ejection fraction ≤40% on stable medical therapy, were enrolled to receive 5, 15, or 30 mg of plasmid sdf-1 via endomyocardial injection. the primary end points for safety and efficacy were at 1 and 4 months, respectively. the primary safety end point was a major adverse cardiac event while efficacy end points were changes in quality of life, new york heart association (nyha) class, 6-min walk distance, single photon emission computed tomography, n-terminal pro-brain natruretic peptide, and echocardiography at 4 and 12 months. the primary safety end point was met. at 4 months, all of the cohorts demonstrated improvements in 6-min walk distance, quality of life, and nyha class (93) . stromal cell-derived factor-1 plasmid treatment for patients with heart failure (stop-hf) was a phase ii, double-blind, randomized, placebo-controlled trial to evaluate safety and efficacy of a single treatment of plasmid sdf-1 delivered via endomyocardial injection to patients with ischemic heart failure. the primary endpoint was a composite of change in 6 min walking distance and minnesota living from heart failure questionnaire from baseline to 4 months follow-up. the primary endpoint was not met (p = 0.89). for the patients treated with psdf-1, there was a trend toward an improvement in left ventricular ejection fraction at 12 months (placebo vs. 15 vs. 30 mg δlvef: −2 vs. −0.5 vs. 1.5%, p = 0.20). patients in the first tertile of ef (<26%) that received 30 mg of psdf-1 demonstrated a 7% increase in ef compared with a 4% decrease in placebo (δlvef = 11%, p = 0.01) at 12 months (94) . although the reasons for the overall failure are currently unclear, the differential benefit in those with advanced left ventricle dysfunction raises the possibility of differential mechanisms that would be operational in more advanced patients. these include the possible overexpression of cxcr4 in cardiac myocytes in the infarct border leading to a negative inotropic state (95) . the transient overexpression of sdf-1 in ischemic cardiomyopathy has been shown to lead to long-term down-regulation of cardiac myocyte cxcr4 expression, re-recruiting the contractile function of the border zone (61) . patients with greater left ventricle dysfunction are likely to have a greater volume of myocardial tissue under stress; therefore, a greater demonstrable response to sdf-1 overexpression. another important reason could be that the upregulation of dpp4 in the border zone of the infarct or around ischemic areas may have resulted in rapid degradation of sdf-1 limiting the efficacy of such an approach. since both the phase i and phase ii studies were performed in the absence of dpp4 inhibition, it could be speculated that the results may have been different if the trials had been performed either in the presence of a dpp4 inhibitor. figure 1 | dipeptidyl peptidase-4 inhibition in modulation of sdf-1 and myocardial angiogenesis: expression of dpp4 increases in myocardial infarction. suppression of dpp4 enzymatic activities by pharmacological inhibitors preserves sdf-1, which results in an enhanced homing of cxcr4 + progenitor cells from bone marrow to infarcted tissues. cxcr4, chemokine (c-x-c motif) receptor 4; dpp4i, dpp4 inhibitor; mi, myocardial infarction; sdf-1, stromal-derived factor-1. dpp4 in chemotaxis and cardiovascular disease frontiers in immunology | www.frontiersin.org due to the importance of sdf-1/cxcr4 axis in the stem cell and progenitor cell survival and function, understanding this axis and molecules that modulate their production and action will be of utility for the treatment of cardiovascular disease. there are a number of clinically approved drugs, including dpp4 inhibitors and parathyroid hormone, which have the ability to enhance sdf-1/cxcr4 responsiveness and may improve the outcome of cardiovascular diseases. several recent large scale clinical trials have indicated that unlike most other oral anti-diabetic drugs that promote cardiovascular disease, dpp4 inhibitors are safe from cardiovascular standpoint despite lack of evidence showing beneficial effect (96) (97) (98) . to what extent sdf-1/cxcr4 axis contributes to this effect requires further investigation. this work was supported by grants from aha (15sdg25700381 and 13post17210033) and mid-atlantic nutrition obesity research center (norc pilot & feasibility program) to dr. zhong. a new dipeptide naphthylamidase hydrolyzing glycyl-prolyl-beta-naphthylamide pharmacology, physiology, and mechanisms of action of dipeptidyl peptidase-4 inhibitors dpp4 in cardiometabolic disease: recent insights from the laboratory and clinical trials of dpp4 inhibition serum high molecular weight dipeptidyl peptidase iv (cd26) is similar to a novel antigen dppt-l released from activated t cells a prediction of dpp iv/cd26 domain structure from a physico-chemical investigation of dipeptidyl peptidase iv (cd26) from human seminal plasma similar increased serum dipeptidyl peptidase iv activity in chronic hepatitis c and other viral infections specific localization of membrane dipeptidase and dipeptidyl peptidase iv in secretion granules of two different pancreatic islet cells natural killer cell cytotoxicity: how do they pull the trigger? dipeptidyl peptidase 4 is a novel adipokine potentially linking obesity to the metabolic syndrome altered dipeptidyl peptidase-4 activity during the progression of hyperinsulinemic obesity and islet atrophy in spontaneously late-stage type 2 diabetic rats organ distribution of aminopeptidase a and dipeptidyl peptidase iv in normal mice a potential role for dendritic cell/macrophage-expressing dpp4 in obesity-induced visceral inflammation an emerging role of dipeptidyl peptidase 4 (dpp4) beyond glucose control: potential implications in cardiovascular disease dipeptidyl peptidase iv and related enzymes in cell biology and liver disorders mice lacking dipeptidyl peptidase iv are protected against obesity and insulin resistance enhanced insulin secretion and improved glucose tolerance in mice lacking cd26 the structure and function of cd26 in the t-cell immune response role of cd26/dipeptidyl peptidase iv in human t cell activation and function long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis acute dpp-4 inhibition modulates vascular tone through glp-1 independent pathways molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 linagliptinmediated dpp-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen diabetic nephropathy amelioration by a low-dose sitagliptin in an animal model of type 2 diabetes (zucker diabetic fatty rat) stimulation of intestinal growth and function with dpp4 inhibition in a mouse short bowel syndrome model glucagon-like peptide-1 reduces hepatic lipogenesis via activation of amp-activated protein kinase serum dipeptidyl peptidase-4 activity in insulin resistant patients with non-alcoholic fatty liver disease: a novel liver disease biomarker cd26/dipeptidyl peptidase iv differentially regulates the chemotaxis of t cells and monocytes toward rantes: possible mechanism for the switch from innate to acquired immune response cd26/dipeptidyl-peptidase iv down-regulates the eosinophil chemotactic potency, but not the anti-hiv activity of human eotaxin by affecting its interaction with cc chemokine receptor 3 inhibition of cd26/dipeptidyl peptidase iv enhances ccl11/eotaxin-mediated recruitment of eosinophils in vivo human macrophage-derived chemokine (mdc), a novel chemoattractant for monocytes, monocyte-derived dendritic cells, and natural killer cells stcp-1 (mdc) cc chemokine acts specifically on chronically activated th2 lymphocytes and is produced by monocytes on stimulation with th2 cytokines il-4 and il-13 truncation of macrophage-derived chemokine by cd26/dipeptidyl-peptidase iv beyond its predicted cleavage site affects chemotactic activity and cc chemokine receptor 4 interaction amino-terminal truncation of cxcr3 agonists impairs receptor signaling and lymphocyte chemotaxis, while preserving antiangiogenic properties stromal cell-derived factor-1 retention and cardioprotection for ischemic myocardium dipeptidylpeptidase 4 negatively regulates colony-stimulating factor activity and stress hematopoiesis stromal derived factor 1α: a chemokine that delivers a two-pronged defence of the myocardium the chemokine sdf-1 is a chemoattractant for human cd34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of cd34+ progenitors to peripheral blood implantation of adipose-derived regenerative cells enhances ischemia-induced angiogenesis role of the sdf-1-cxcr4 axis in stem cellbased therapies for ischemic cardiomyopathy sdf-1α as a therapeutic stem cell homing factor in myocardial infarction trafficking of normal stem cells and metastasis of cancer stem cells involve similar mechanisms: pivotal role of the sdf-1-cxcr4 axis molecular cloning and characterization of a murine pre-b-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin the pleiotropic effects of the sdf-1-cxcr4 axis in organogenesis, regeneration and tumorigenesis effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy structural localization and expression of cxcl12 and cxcr4 in rat heart and isolated cardiac myocytes matrix metalloproteinase activity inactivates the cxc chemokine stromal cell-derived factor-1 defects of b-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the cxc chemokine pbsf/sdf-1 the chemokine receptor cxcr4 is essential for vascularization of the gastrointestinal tract function of the chemokine receptor cxcr4 in haematopoiesis and in cerebellar development mutations in the chemokine receptor gene cxcr4 are associated with whim syndrome, a combined immunodeficiency disease cxcr7: a new sdf-1-binding receptor in contrast to normal cd34+ progenitors is functional and is expressed at higher level in human malignant hematopoietic cells the role of sdf-1-cxcr4/ cxcr7 axis in the therapeutic effects of hypoxia-preconditioned mesenchymal stem cells for renal ischemia/reperfusion injury rapid mobilization of functional donor hematopoietic cells without g-csf using amd3100, an antagonist of the cxcr4/sdf-1 interaction human progenitor cells rapidly mobilized by amd3100 repopulate nod/scid mice with increased frequency in comparison to cells from the same donor mobilized by granulocyte colony stimulating factor rapid mobilization of hematopoietic progenitors by amd3100 and catecholamines is mediated by cxcr4-dependent sdf-1 release from bone marrow stromal cells sdf-1-enhanced cardiogenesis requires cxcr4 induction in pluripotent stem cells cardiogenic induction of pluripotent stem cells streamlined through a conserved sdf-1/vegf/ bmp2 integrated network sdf-1 in myocardial repair the chemokine sdf-1 activates the integrins lfa-1, vla-4, and vla-5 on immature human cd34(+) cells: role in transendothelial/stromal migration and engraftment of nod/scid mice mechanical and electrical effects of cell-based gene therapy for ischemic cardiomyopathy are independent plasmid-based transient human stromal cell-derived factor-1 gene transfer improves cardiac function in chronic heart failure heterogeneity in sdf-1 expression defines the vasculogenic potential of adult cardiac progenitor cells modulation of hematopoietic stem cell homing and engraftment by cd26 cell surface peptidase cd26/ dipeptidylpeptidase iv regulates cxcl12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood cd34+ progenitor cells amd3100 and cd26 modulate mobilization, engraftment, and survival of hematopoietic stem and progenitor cells mediated by the sdf-1/cxcl12-cxcr4 axis inhibition of cd26 in human cord blood cd34+ cells enhances their engraftment of nonobese diabetic/severe combined immunodeficiency mice cd26 inhibition on cd34+ or lineage-human umbilical cord blood donor hematopoietic stem cells/hematopoietic progenitor cells improves long-term engraftment into nod/scid/beta2null immunodeficient mice diprotin a infusion into nonobese diabetic/severe combined immunodeficiency mice markedly enhances engraftment of human mobilized cd34+ peripheral blood cells cd26/dpp-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemiareperfusion injury in mouse lung transplantation retention of human bone marrow-derived cells in murine lungs following bleomycin-induced lung injury upregulation of cd26 peptidase downregulates the functional chemotactic response of cd34+cd38-human cord blood hematopoietic cells isolation of putative progenitor endothelial cells for angiogenesis sphingosine-1-phosphate stimulates the functional capacity of progenitor cells by activation of the cxcr4-dependent signaling pathway via the s1p3 receptor psgl-1-mediated activation of ephb4 increases the proangiogenic potential of endothelial progenitor cells stromal cell-derived factor-1 effects on ex vivo expanded endothelial progenitor cell recruitment for ischemic neovascularization a novel mechanism for endothelial progenitor cells homing: the sdf-1/cxcr4-rac pathway may regulate endothelial progenitor cells homing through cellular polarization statin and stromal cell-derived factor-1 additively promote angiogenesis by enhancement of progenitor cells incorporation into new vessels sdf-1 involvement in endothelial phenotype and ischemia-induced recruitment of bone marrow progenitor cells comp-ang1 stimulates hif-1α-mediated sdf-1 overexpression and recovers ischemic injury through bm-derived progenitor cell recruitment ex vivo priming of endothelial progenitor cells with sdf-1 before transplantation could increase their proangiogenic potential nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes mk-0626, a dipeptidyl peptidase-4 inhibitor, improves neovascularization by increasing both the number of circulating endothelial progenitor cells and endothelial nitric oxide synthetase expression sdf-1 expression by mesenchymal stem cells results in trophic support of cardiac myocytes after myocardial infarction myocardial cxcr4 expression is required for mesenchymal stem cell mediated repair following acute myocardial infarction synergy between cd26/dpp-iv inhibition and g-csf improves cardiac function after acute myocardial infarction granulocyte colony-stimulating factor treatment plus dipeptidylpeptidase-iv inhibition augments myocardial regeneration in mice expressing cyclin d2 in adult cardiomyocytes genetically manipulated progenitor cell sheet with diprotin a improves myocardial function and repair of infarcted hearts proteaseresistant stromal cell-derived factor-1 for the treatment of experimental peripheral artery disease first treatment of a child suffering from severe ischemic cardiomyopathy with g-csf and sitagliptin parathyroid hormone is a dpp-iv inhibitor and increases sdf-1-driven homing of cxcr4(+) stem cells into the ischaemic heart cd26 inhibition enhances perfusion recovery in apoe-/-mice an open-label dose escalation study to evaluate the safety of administration of nonviral stromal cell-derived factor-1 plasmid to treat symptomatic ischemic heart failure changes in ventricular remodelling and clinical status during the year following a single administration of stromal cell-derived factor-1 non-viral gene therapy in chronic ischaemic heart failure patients: the stop-hf randomized phase ii trial cxcr4 modulates contractility in adult cardiac myocytes saxagliptin and cardiovascular outcomes in patients with type 2 diabetes mellitus alogliptin after acute coronary syndrome in patients with type 2 diabetes effect of sitagliptin on cardiovascular outcomes in type 2 diabetes key: cord-304623-j9ay4jkf authors: entrican, gary; lunney, joan k.; wattegedera, sean r.; mwangi, william; hope, jayne c.; hammond, john a. title: the veterinary immunological toolbox: past, present, and future date: 2020-07-28 journal: front immunol doi: 10.3389/fimmu.2020.01651 sha: doc_id: 304623 cord_uid: j9ay4jkf it is well-recognized that research capability in veterinary species is restricted by a lack of immunological reagents relative to the extensive toolboxes for small rodent biomedical model species and humans. this creates a barrier to the strategic development of disease control solutions for livestock, companion animals and wildlife that not only affects animal health but can affect human health by increasing the risk of transmission of zoonotic pathogens. there have been a number of projects aimed at reducing the capability gaps in the veterinary immunological toolbox, the majority of these focusing on livestock species. various approaches have been taken to veterinary immunological reagent development across the globe and technological advances in molecular biology and protein biochemistry have accelerated toolbox development. while short-term funding initiatives can address specific gaps in capability, they do not account for long-term sustainability of reagents and databases that requires a different funding model. we review the past, present and future of the veterinary immunological toolbox with specific reference to recent developments discussed at the international union of immunological societies (iuis) veterinary immunology committee (vic) immune toolkit workshop at the 12th international veterinary immunology symposium (ivis) in seattle, usa, 16–19 august 2019. the future availability of these reagents is critical to research for improving animal health, responses to infectious pathogens and vaccine design as well as for important analyses of zoonotic pathogens and the animal /human interface for one health initiatives. the development of novel tools and technologies has been fundamental to the advancement of basic and applied immunology across species. the rate of progress of immunological reagent development for veterinary species has been much slower than that for humans and small rodent biomedical model species, and has impacted research capability in those species (1) . historically, however, innovations in surgical procedures in veterinary species have resulted in major stepchanges in our understanding of the ontogeny, compartmentalization and function of the immune system. for example, bursectomy in chickens shed new light on mechanisms of b cell development and immunoglobulin production (2) , in utero thymectomy of lambs revealed the importance of the thymus for lymphocyte development (3) and lymphatic cannulation of sheep revealed that lymphocyte subsets differ between blood, afferent and efferent lymph (4) . these ground-breaking experiments were feasible, in part, due to the size of the species under investigation, particularly for the technique of lymphatic cannulation due to the diameter of lymphatic vessels in ruminants (5) . however, this momentum in veterinary immunological studies was not maintained; the vast majority of technological innovations and discoveries in immunology in the past 50 years have been made in mice. the development of congenic mice, differing at a single histocompatibility locus, was a fundamental technological innovation in immunology that led to mice being the primary species of choice for research. that pioneering work of george snell and the later capability of genetically manipulating congenic mice has allowed immunologists to ascribe functions to genes, molecules and cells with high precision (6) . the development of monoclonal antibody (mab) technology using congenic mice subsequently created almost boundless opportunities for research in basic and translational immunology (7) . the availability of mabs that could phenotype cells and detect cytokines by elisa underpinned the discovery of two distinct cd4 +ve t-cell subsets in congenic mice (8) . the subsequent th1/th2 paradigm provided a fundamental framework for investigating immune activation and regulation that has expanded far beyond those original two subsets. current capability now extends to multi-parametric analyses such as simultaneous fourteen-color flow cytometry that can identify 89 functionally-relevant cd4 +ve t-cell subsets in human blood (9) . mass cytometry (cytof) methods using panels of well over 40 conjugated antibodies are now allowing for even deeper analysis of single cell expression, offering new insights into cellular subsets and their differentiation (10, 11) . such technologies cannot usually be applied directly to different species since molecular differences in immunological orthologs result in low cross-reactivity of reagents across species (12) as affirmed by a recent comparison of reactivity of immune protein reagents for other species with swine orthologs (13) . thus, reagent development needs to be evaluated on a caseby-case basis. gaps in capability for veterinary species are often prioritized based on the extensive mouse and human immunological toolboxes. the expansion of the toolboxes has revealed substantial differences in the ways that humans, mice and veterinary species respond to disease and highlighted to need for studying different species in their own right (14, 15) . there have been coordinated efforts to evaluate species crossreactivity of anti-human cd antigen mabs through the animal homologs section of the human leukocyte differentiation antigen (hlda) workshops: for horses (16) , dogs (17) , pigs (18) , and ruminants (19) . in an effort to generate greater international co-ordination for immune reagent characterization activities, the international union of immunological societies (iuis) veterinary immunology committee (vic) supported a toolkit workshop (20) . it is almost 10 years since the last published review of the veterinary immunology toolbox from the iuis vic toolkit workshop at the 9th ivis in tokyo, japan (1) . here, we review progress over the past decade by reporting on the iuis vic toolkit workshop at the 12th ivis in seattle, usa in 2019 and take a forward look to the future of the veterinary immunology toolbox. the success of the hlda workshops was based on good co-ordination, high-quality work and collective effort by the veterinary immunology community, as well as results from past species-specific cd workshops supported by iuis vic. common standards were applied to the distribution and evaluation of anti-human cd reagents being assessed in different laboratories and the collective generated data being reviewed centrally. the outcome was an evidence-based assessment for the activity of species cross-reactive mabs, with affirmation that only a limited number of mab directed against human cd antigens actually cross-react with other animal species (21) . these results instilled confidence in the performance of those reagents and promoted their uptake by the research community and industry, including companies that market and sell veterinary immunological reagents. although the hlda workshops were primarily focused on evaluation of species cross-reactive antibodies, they played an important role in informing of capability gaps and therefore the prioritization of reagents for future development. a major step-change in the way veterinary immunological reagent development was supported came with the inception of a uk immunological toolbox funded by the biotechnology and biological sciences research council (bbsrc) and the scottish executive environment and rural affairs department (seerad) in 2003. this was unique as it united several laboratories within a single project to take a collective multispecies approach to immunological reagent development. this was followed by the veterinary immune reagent network (virn) funded by united states department of agriculture (usda)/national institute of food and agriculture (nifa) in the us in 2005. both projects included the creation of databases listing available veterinary immunological reagents, which will be discussed later. they also expanded the emphasis from mab anti-cd antigens to expression of immune proteins (cytokines and chemokines) and protein reactive mabs. the us project included direct collaboration with commercial partners to express these immune proteins. the us and uk projects worked together under a memorandum of understanding (mou) to avoid duplication of effort. this mou was created in the absence of a mechanism for joint international funding by the respective national agencies. the structure, priorities and achievements of these projects has been published previously (1) . a key output from these initiatives was an increased recognition of the importance of coordinated, complementary approaches to reagent prioritization and development. their success has also been reflected by continued support for reagent development initiatives by funders seeking to build on the significant benefits from their original investments, with the assertion that long-term sustainability is essential. the funding for veterinary immunology reagent development has changed over the past 10 years, moving from the multispecies models of the uk immunological toolbox and us virn, to single-species projects. with the exception of ruminants, there is very little species cross-reactivity of veterinary reagents, highlighting that the genes involved in immune responses are amongst the most rapidly evolving in vertebrate genomes (22, 23) . however, this does not diminish their potential as disease models. bbsrc and usda/nifa have supported reagent development projects for ruminants, swine, horses, aquaculture species and poultry in the past 10 years (box 1). a barrier to formal international collaboration was lifted in 2013 when usda/nifa and bbsrc launched a pilot call to support animal disease research of strategic importance to both the us and uk which included the development of veterinary immunological reagents for agriculturally-relevant animal species. the swine toolkit was a landmark first transatlantic veterinary immunology reagent project funded under this initiative in 2015 (box 1). although we have focused here on projects funded specifically to develop reagents and supporting technologies, this is not intended to ignore the veterinary immunological reagent development that is conducted within disease-driven projects, networks and within strategic programmes of government research institutes across the globe. the challenge is in capturing the outputs of these diverse activities. the websites of commercial reagent suppliers and peer-reviewed publications are sources of validated information on reagent activity. however, they do not capture everything, a particular gap being the paucity of "negative" data when reagents are found to be non-functional or where repeated attempts fail to generate specific antibodies. these are very valuable data as they can potentially prevent the duplication of wasted effort. the solution lies in community engagement for the sharing of knowledge on reagent availability and performance. workshops such as those hosted by iuis vic toolkit are a focal point for international information exchange, but they do not have the facility to capture, store and disseminate information at a detailed level. it has been recognized for many years that a major unmet need in veterinary immunology is the lack of centralized, non-commercial, searchable reagent databases (20) . the original uk immunological toolbox (2003) (2004) (2005) (2006) (2007) (2008) (2009) and the us virn (2005-2015) both created lists of reagents but the databases were not sustainable beyond the term of funding. this is not surprising as curation is timeconsuming, requiring expert knowledge of immunology and information technology input to create web-based interfaces. this also highlighted the problem of sustainability when there is reliance on short-term funding for reagent development projects. finding solutions to these problems has been the focus of several recent workshops as discussed below. one exception to this has been the usda agricultural research service (ars) supported porcine translational research database (ptrd, http://tinyurl. com/hxxq3ur) (15) . the current landscape of the veterinary immunology toolbox has been shaped by new funding approaches to facilitate reagent development while also addressing the complex issues of database construction, collection and validation of data, and sustainability of the database and biobanks of the reagents listed therein. this report summarizes the outcomes of several international workshops where these various elements have been considered. before summarizing those outcomes, it is worth reviewing the scope of the toolbox in terms of species coverage and knowledge of immunological capability within those species. in the broadest sense, the concept of a veterinary immunological toolbox encompasses a broad range of livestock, companion animal, biomedical model and wildlife species. there has been progress in reagent development across all of those species in the past 10 years which has been presented at various meetings and workshops. we have identified a number of published articles where reagent availability for different species have been reviewed. for the purposes of the toolbox, livestock species can largely be regarded as belonging to one of four major groupings, namely swine (24, 25) , ruminants (22, 26) , poultry (27) (28) (29) , and aquaculture (30, 31) . companion animals include horses (32, 33) , cats (34) , and dogs (35) . as previously discussed, mice are the most common small-animal biomedical model for human (12) . however, rabbits (36) and ferrets (37) are also popular small-animal biomedical models for human disease. there is interest in expanding the immunological toolboxes for wildlife species, for example buffalo (38) and badgers (39) due to their potential to act as reservoirs for economically-important livestock diseases. there is also interest in developing immunological reagents for marine mammals such as dolphins (40) . in addition, although camelid species are not often regarded as a major target host species for disease studies, they have come to the fore with heightened awareness of mers-cov and the potential to reduce zoonotic transmission by investigating vaccine-induced responses in camels (41) . importantly, camels make a unique technological contribution to the immunological toolbox via the production of nanobodies (42, 43) . to date, the concept of the veterinary immunological toolbox has largely (but not exclusively) focused on reagent development for livestock species due to their strategic relevance for funders with a stake in livestock health, food safety and global food security. in the period between the last published review of the iuis vic toolkit workshop at the 9th ivis in tokyo (1) and the iuis vic toolkit workshop at the 12th ivis in seattle, there have been several key meetings whose outcomes are directly relevant to the current status and future directions of the toolbox and merit discussion here. the first was at the 10th ivis in milan, italy in 2013 when bbsrc and the global strategic alliances for the coordination of research on the major infectious diseases of animals and zoonoses (star-idaz) supported a vaccinology https://www.pirbright.ac.uk/news/2019/11/bill-melinda-gates-foundation-funds-development-pirbright%e2%80%99s-livestock-antibody-hub. to study cattle, pig and poultry antibody responses at high resolution to expand the understanding of protective immunity in those species and that can also be used as models for a range of human infectious diseases. usda/nifa: cattle (2019-2022): https://portal.nifa.usda.gov/web/crisprojectpages/1016686-immune-reagents-for-ruminants-with-primary-focus-on-bovine-specific-reagents.html. to develop, and make commercially available, mab reagents needed to elucidate cattle immune mechanisms by focusing on cd antigens, cytokines, and chemokines and relevant assays. https://portal.nifa.usda.gov/web/crisprojectpages/0221344-us-veterinary-immune-reagent-network.html. to clone, express, develop mab reagents specific for ruminants, swine, poultry, equine and aquaculture species, sharing methods across species. work with commercial partner to market expressed proteins for use by veterinary immunology community. workshop. the lack of immunological tools and reagents was recognized as a major barrier to progress. this can be seen in the subsequent bbsrc veterinary vaccinology strategy (https:// bbsrc.ukri.org/about/reviews/scientific-areas/1506-veterinaryvaccinology-strategy/) and the creation of the bbsrc uk veterinary vaccinology network (vvn). in 2017, bbsrc vvn hosted a workshop to discuss the toolbox initiatives in the uk and us with specific relevance to the aims and objectives of the newly-formed global challenges research fund (gcrf) international veterinary vaccinology network (ivvn). a full report is available on the bbsrc vvn website (http://www.vetvaccnet.ac.uk/publications/veterinaryimmunology-toolbox-meeting-uk-veterinary-vaccinologynetwork). at this workshop, the pirbright institute and the roslin institute at the university of edinburgh announced plans for a new uk immunological toolbox project. the combined project would be underpinned by core institute funding from the bbsrc, with additional support from the bbsrc gcrf tools and resources (https://www.immunologicaltoolbox.co. uk/about/funders). this project is addressing major gaps in capability and sustainability. the first of these is the creation of a publicly accessible, searchable database of veterinary immunological reagents to be accessed via a dedicated website. a follow up meeting was held at the vvn conference in stirling in early 2018 (https://www.vetvaccnet.ac.uk/news/2018/01/ uk-veterinary-vaccinology-network-conference-2018-report) to discuss in more detail the focus of the website and new reagent development. it was agreed by the community that a key driver for the website would be the facility for researchers to submit information on reagent performance and request reagent production where gaps exist. it was discussed that the primary focus of new reagent development should be around t cell and b cell subsets to help dissect in more detail pathogen and vaccine responses. as well as new reagent development the toolbox aims to exploit new technologies to translate current hybridoma stocks into gene blocks via sequencing and create a recombinant antibody pipeline, express recombinant proteins (including cytokines and chemokines), build multiplex platforms and develop high-throughput screening systems for new antibodies. these sequences act as the template from which the constant region can be switched between different species while maintaining target specificity. a toolkit workshop was held at the 6th european veterinary immunology workshop (eviw) conference in utrecht, netherlands in 2018. although this conference was organized under the auspices of the european veterinary immunology group (evig), as opposed to iuis vic, the iuis vic toolkit committee took a leading role in the organization of the toolkit workshop. notably, the toolkit workshop was structured to reflect four newly-formed major livestock groupings (swine, ruminants, poultry, aquaculture) of iuis vic toolkit which were announced for the first time at this meeting. the leaders of the species groups represented their respective areas at the workshop. they are listed on the iuis vic webpage and can be contacted by members of the community who are seeking information or looking to engage in reagent development for each of those areas (https://iuis.org/committees/vic/). the workshop covered the major projects in europe and the us on reagent development, including a presentation on the plans for the new uk immunological toolbox. in the panel discussion, there was broad international support for the approaches being taken within the new toolbox project and recognition of the complementary work being supported by usda/nifa in all of the target species (box 1). this meeting cemented the requirement for community engagement in the website to provide and maximize information exchange about the availability and performance of reagents and the focus on the generation of novel antibodies and methods to distinguish t and b cell subsets. this particular area will be advanced by the development of a new livestock antibody hub centered at the pirbright institute which aims to improve both animal and human health globally by translating research outcomes in livestock diseases (box 1). a core aim of this antibody hub is to develop tools, techniques and reagents for livestock research that bring the research capability to the same level as that for humans and mice. the iuis vic toolkit workshop at the 12th ivis in seattle was the forum for the international launch of the pirbright/roslin uk immunological toolbox website and the associated database (http://www.immunologicaltoolbox.co.uk). this database was built around the original information collated during the 2003-2009 bbsrc seerad-funded uk immunological toolbox and is therefore skewed toward three of the four major livestock groupings (swine, ruminants and poultry). however, aquaculture species, companion animals and now major animal pathogens are also included, and as the community engages the amount of information will increase. the main aim of the website is to collate reagent information and act as a centralized source to increase information exchange but is not the only source for any particular species. for example, the usda porcine translational research database (http://tinyurl.com/hxxq3ur) is considered a very wide ranging and valuable community resource and cannot be duplicated but information is shared with the uk immunological toolbox via mutual awareness and direct communication. the uk immunological toolbox database contains data on reagents that are held in research laboratories, and also those available commercially, which immediately raises questions on the quality and reproducibility of reagents from different sources. the standardized production, evaluation and storage of commercially-available reagents would be expected to reduce batch-to-batch variation, whereas the same reagent produced and stored in different research laboratories is likely to have more variability due to the different conditions. when reagents are listed on the uk immunological toolbox website there will be information on their specificity and performance, preferably supported by peer-review publication wherever possible. there is also a facility for registered users to provide feedback on performance to add to the available information. such information will be checked before posting against the user's identification. it was emphasized that such a database can be as complete and useful as the community wants it to be. the website and database will be curated centrally, but the community has to take collective ownership by submitting reagents and information on their performance. it is pleasing to see that this is already happening. the toolbox website also serves as a reference point for non-veterinary immunologists looking to expand their choice of biomedical models and facilitate comparative immunology research (44) . finally, several new opportunities were identified during the open discussion at the iuis vic toolkit workshop in seattle. these included the unique opportunity to salvage and store "orphan" mabs via the sequencing technology within the uk immunological toolbox. the preservation of sequences does not incur the high costs associated with maintaining hybridoma cells in liquid nitrogen. in addition, the sharing of sequences circumvents many of the logistical and financial issues involved in the shipment of live cells, particularly across international borders. as we enter the third decade of the 21st century, the "one health" agenda has never been more important. the development of solutions for controlling infectious diseases in livestock, companion animals and wildlife not only has direct benefits for the target species but can reduce disease transmission across species, including zoonotic transmission, thereby reducing the wider global disease burden (45) . close contact between different animal species and between animals and humans is a risk for zoonotic disease, which can be difficult to manage in low and middle income countries (lmics) (46) . given the importance of livestock to lmics, the veterinary immunological toolbox provides economic and health benefits by underpinning animal vaccine development. the quality of toolbox reagents and associated information in the uk immunological toolbox database are paramount. evidence-based validation and standardization of new technologies is essential to generate confidence in performance and encourage uptake by the community. there remain major capability gaps in multi-analyte protein technologies for veterinary species. the development of such technologies is technically challenging, but entirely feasible with the appropriate resources and effort. the key to success is in working together. the single-tube technology that simultaneously identifies 89 functionally-relevant cd4 +ve t-cell subsets in human blood was developed and validated through the collective efforts of the multiple partners in the euroflow and periscope consortia (9) . multiparametric technologies are extremely powerful; one way of expanding the flexibility of the relatively limited range of antibodies in veterinary species is the ability to efficiently conjugate small amounts of antibodies with different labels for defining immune correlates. the identification and quantification of immunological correlates of protection are aspirational goals for the development of safe and effective vaccines (47, 48) . however, with the exception of anti-virus neutralizing antibodies, immunological correlates of protection tend to be multifactorial rather than singular, particularly in the case of cell-mediated protective immunity requiring not only cell subset identification but appropriate cytokine co-expression. the solution to identifying such correlates lies in the application of a range of multi-plex technologies that all detect multiple analytes at the genetic, protein, and cellular level, so called "systems vaccinology" (49) . we are also moving into an era of high dependency on computational infrastructure as the data generated by such complex studies require specialized programmes for full analysis. hence, collective approaches are becoming increasingly important if we are to maximize our potential to develop and adopt complex technologies in the future. the importance of genomic information and alternate expression systems such as pichia pastoris, insect and mammalian cells has meant wider availability of species-specific immune proteins. the veterinary immunology community has a long history of working together for collective good, such as the hlda workshops, international cd workshops, toolkit committees, collaborative funding initiatives and the immunological toolbox. in doing so we need to maintain a global perspective and consider technologies that create solutions for animal diseases across borders. one example is the antibody sequencing technologies of the new uk immunological toolbox. in addition to the advantages described earlier, this technology offers particular cost-effective and sustainability benefits for the transfer and storage of reagents to lmics where veterinary immunology research is being conducted. in parallel to sequencing, expressing and engineering mabs, companies and research groups all over the world are adapting single b cell sequencing technologies to a range of host species. these technologies often rely to some extent on existing reagents to identify b cells (including antigen specific b cells) but are generally very adaptable to any given species and synergise well with existing mouse recombinant antibody expression methods. these methods are providing a completely new route to identifying antibodies against specific epitopes on pathogens as well as other foreign immunizing antigens. these antibodies can be used as reagents, including mapping complex epitope landscapes to inform structural vaccinology approaches to increase efficacy, and may also be used as therapeutics. antibodies are now a primary therapeutic goal of many companies for a range of human diseases. cats and dogs are not only a profitable target market for immunotherapeutics, they provide value data on in vivo mab function (50) . although the cost of such treatments is currently prohibitive for food producing species, large animal models and speciesspecific reagents can have a very important role in testing manufacture, delivery and efficacy of mabs as part of the one health approach. the impact of veterinary immunology research will ultimately be measured by the development, or contribution to the development, of disease-control solutions including diagnostic tests, vaccines and genetic-based strategies. the range of vaccine-delivery platforms is rapidly expanding, including improved adjuvants, vector-based delivery systems and genetic vaccination with dna and rna. although viralvectored vaccines are successfully deployed in humans and companion animals (51), public safety concerns remain regarding their use food animals (52) . the immunological toolbox can be applied to safety and efficacy studies in livestock, thereby informing on the benefit-risk ratio that would be impossible to do at the same scale in humans or primates. animal genetics can provide insights into responses to infection and vaccination which can be translated into livestock breeding programmes (53, 54) . breeding programmes require several generations to observe population effects and conclusive proof for the effect of a specific genotype on immune status requires functional evidence, hence reliance on the toolbox. new gene-editing technologies such as crispr now allow very targeted approaches to livestock production (55) . this is the future of livestock farming and the immunological toolbox not only has a role to play in the identification of genes to be targeted, but it will also be important for defining subsequent immune function, including potential off-target effects. genome editing is also creating the opportunities to engineer species to act as better models for human diseases alongside or in addition to genetically defined and tailored breeds, such as scid pigs and mhc homozygous pigs (56, 57) . for example, pigs are emerging as a very powerful model to predict human influenza vaccine responses but to achieve the maximum benefit of such models a complete toolkit is required (58) . gene editing is already providing pig organs for future human xenotransplantation, a biomedical application that has helped drive reagent development in pigs (59, 60) . the veterinary immunological toolbox is very broad in its scope and has evolved from multiple efforts across the globe. in the broadest sense, the toolbox incorporates livestock, companion animals, wildlife and biomedical animal species. each is important in its own right, but all are collectively important for the one health agenda and for controlling existing and emerging diseases that infect different animal populations and have zoonotic potential. as human populations expand, there is a need to protect food security without compromising food safety. disease prevention and control results in improvements in animal health and welfare, which not only has economic and ethical benefits but can also address concerns for climate change by making food production more efficient. basic immunology underpins these approaches, from vaccine design to understanding the effects of gene editing. the immunological toolbox website and associated searchable database provides a new focal point for information and knowledge exchange for the veterinary immunology community. the key to future success is global collective working facilitated by networks such as national immunological societies, eviw, ivvn, american association of veterinary immunologists (aavi), and iuis vic toolkit committee. veterinary immunology committee toolkit workshop 2010: progress and plans effect of early bursectomy on germinal centre and immunoglobulin production in chickens the growth and development of lambs thymectomized in utero lymphocyte subsets show marked differences in their distribution between blood and the afferent and efferent lymph of peripheral lymph nodes a road less travelled: large animal models in immunological research a methods paper that led to much more continuous cultures of fused cells secreting antibody of predefined specificity two types of murine helper t cell clone. i definition according to profiles of lymphokine activities and secreted proteins age distribution of multiple functionally relevant subsets of cd4+ t cells in human blood using a standardized and validated 14-color euroflow immune monitoring tube use of mass cytometry to profile human t cell exhaustion immune monitoring using mass cytometry and related high-dimensional imaging approaches advanced model systems and tools for basic and translational human immunology porcine cytokines, chemokines and growth factors: 2019 update genomic responses in mouse models poorly mimic human inflammatory diseases an in-depth comparison of the porcine, murine and human inflammasomes; lessons from the porcine genome and transcriptome report of the first international workshop on equine leucocyte antigens monoclonal antibodies that define canine homologues of human cd antigens: summary of the first international canine leukocyte antigen workshop (claw) summary of the first round analyses of the second international swine cd workshop cross-reactivity with bovine cells of monoclonal antibodies submitted to the 6th international workshop on human leukocyte differentiation antigens a current perspective on availability of tools, resources and networks for veterinary immunology summary of the animal homologue section of hlda8 exploiting ovine immunology to improve the relevance of biomedical models the porcine translational research database: a manually curated, genomics and proteomics-based research resource expressed gene sequence and bioactivity of the ifngamma-response chemokine cxcl11 of swine and cattle development of new immune reagents for swine health, vaccine and disease studies contributions of farm animals to immunology the long view: a bright past, a brighter future? forty years of chicken immunology pre-and post-genome marek's disease in chickens: a review with focus on immunology development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes perspective on the development and validation of ab reagents to fish immune proteins for the correct assessment of immune function standardized imgt r nomenclature of salmonidae igh genes, the paradigm of atlantic salmon and rainbow trout: from genomics to repertoires the development of equine immunity: current knowledge on immunology in the young horse transcriptome analysis of immune genes in peripheral blood mononuclear cells of young foals and adult horses cats are not small dogs: is there an immunological explanation for why cats are less affected by arthropod-borne disease than dogs? parasites vect immunotherapy for dogs: running behind humans the wide utility of rabbits as models of human diseases moving forward: recent developments for the ferret biomedical research model characterization of leukocyte subsets in buffalo (bubalus bubalis) with cross-reactive monoclonal antibodies specific for bovine mhc class i and class ii molecules and leukocyte differentiation molecules immunological responses of european badgers (meles meles) to infection with mycobacterium bovis identification of monoclonal antibodies cross-reactive with bottlenose dolphin orthologues of the major histocompatibility complex and leukocyte differentiation molecules humoral immunogenicity and efficacy of a single dose of chadox1 mers vaccine candidate in dromedary camels a toolbox of nanobodies developed and validated for use as intrabodies and nanoscale immunolabels in mammalian brain neurons the therapeutic potential of nanobodies the uk veterinary immunological toolbox website: promoting vaccine research by facilitating communication and removing reagent barriers quantitative outcomes of a one health approach to study global health challenges global patterns of zoonotic disease in mammals a threshold method for immunological correlates of protection erratum to: a threshold method for immunological correlates of protection systems vaccinology and big data in the vaccine development chain anti-nerve growth factor monoclonal antibodies for the control of pain in dogs and cats multivalent and multipathogen viral vector vaccines working with gm vaccines: engaging the public genes controlling vaccine responses and disease resistance to respiratory viral pathogens in cattle host genetics of response to porcine reproductive and respiratory syndrome in nursery pigs on-farm livestock genome editing using cutting edge reproductive technologies the major histocompatibility complex homozygous inbred babraham pig as a resource for veterinary and translational medicine cd3epsilon(+) cells in pigs with severe combined immunodeficiency due to defects in artemis t and b cell immune responses to influenza viruses in pigs the role of genetically engineered pigs in xenotransplantation research tolerance in xenotransplantation all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. our collective thanks to star-idaz, bbsrc vvn, gcrf ivvn, and aavi for supporting workshops and iuis for supporting the 12th ivis/iuis vic immune toolkit workshop. key: cord-295416-y3lvcjqd authors: eichinger, katherine m.; kosanovich, jessica l.; gidwani, sonal v.; zomback, aaron; lipp, madeline a.; perkins, timothy n.; oury, tim d.; petrovsky, nikolai; marshall, christopher p.; yondola, mark a.; empey, kerry m. title: prefusion rsv f immunization elicits th2-mediated lung pathology in mice when formulated with a th2 (but not a th1/th2-balanced) adjuvant despite complete viral protection date: 2020-07-29 journal: front immunol doi: 10.3389/fimmu.2020.01673 sha: doc_id: 295416 cord_uid: y3lvcjqd respiratory syncytial virus (rsv) remains the most common cause of lower respiratory tract infections in children worldwide. development of a vaccine has been hindered by the risk of developing enhanced respiratory disease (erd) upon natural exposure to the virus. generation of higher quality neutralizing antibodies with stabilized pre-fusion f protein antigens has been proposed as a strategy to prevent erd. we sought to test whether there was evidence of erd in naïve balb/c mice immunized with an unadjuvanted, stabilized pre-fusion f protein, and challenged with rsv line 19. we further sought to determine the extent to which formulation with a th2-biased (alum) or a more th1/th2-balanced (advax-sm) adjuvant influenced cellular responses and lung pathology. when exposed to rsv, mice immunized with pre-fusion f protein alone (pref) exhibited increased airway eosinophilia and mucus accumulation. this was further exacerbated by formulation of pref with alum (aluminum hydroxide). conversely, formulation of pref with a th1/th2-balanced adjuvant, advax-sm, not only suppressed rsv viral replication, but also inhibited airway eosinophilia and mucus accumulation. this was associated with lower numbers of lung innate lymphocyte cells (ilc2s) and cd4+ t cells producing il-5+ or il-13+ and increased ifnγ+ cd4+ and cd8+ t cells, in addition to rsv f-specific cd8+ t cells. these data suggest that in the absence of preimmunity, stabilized pref antigens may still be associated with aberrant th2 responses that induce lung pathology in response to rsv infection, and can be prevented by formulation with more th1/th2-balanced adjuvants that enhance cd4+ and cd8+ ifnγ+ t cell responses. this may support the use of stabilized pref antigens with th1/th2-balanced adjuvants like, advax-sm, as safer alternatives to alum in rsv vaccine candidates. respiratory syncytial virus (rsv) is the most common cause of lower respiratory tract infections (lrti) in children worldwide with nearly every child infected by 2 years of age (1) (2) (3) . in children >5 years of age, rsv causes an estimated 33 million acute lrti annually, with over 3 million episodes requiring hospitalization (4) . in 2017, the global cost estimate for inpatient and outpatient rsv lrti management in young children (< 5 years of age) was ∼4.8 billion euros (equivalent to ∼$5.2 billion usd) (5) . in addition to young children, rsv is a common cause of severe respiratory disease in the elderly and those who are immunocompromised (6) (7) (8) . given that severe rsv disease affects ages spanning infancy to geriatrics, it is clear that natural rsv infection does not induce long-lasting immunity and individuals are re-infected throughout their lives (9, 10) . thus, rsv immunization has the potential to boost rsv immunity and alleviate the morbidity associated with repeated rsv infections across all age groups. however, despite the immense economic and healthcare burden posed by rsv infection, there is currently no licensed rsv vaccine. the discovery and stabilization of the prefusion conformation of the rsv f protein (pref) re-ignited hopes for an rsv vaccine due to its ability to elicit potent neutralizing antibodies (11) . a number of studies have demonstrated the protective potential of high levels of rsv neutralizing antibody and as such, boosting serum neutralizing antibody levels has been an important objective of rsv vaccine research (12, 13) . the incorporation of adjuvants into vaccine formulations can enhance the vaccine's effect as well as reduce antigen concentrations and the number of immunizations required for a protective effect (14) . differential stimulation of various pattern recognition receptors can further shift the immune response toward vaccine antigens to promote th1-or th2-type immune responses. based on the known th2-bias associated with early rsv infections, it is imperative to understand the extent to which preventative rsv vaccine adjuvants shift the th1/th2 balance. in use since the 1930's, aluminum salts have long been recognized for their ability to enhance immunogenicity and boost antibody production (15) . alum adjuvant was used in the formalin-inactivated rsv (fi-rsv) trials of the 1960's that produced enhanced respiratory disease (erd) requiring hospitalization upon natural rsv exposure in 80% of vaccinees (16) . subsequent investigations into the cause of fi-rsv-induced erd have suggested that alum exacerbated the t helper type 2 (th2) pathology associated with erd (17) . other studies have disputed alum's role and have instead suggested that formalin inactivation of rsv resulted in poor neutralizing antibody development and immune complex deposition (18, 19) . reports have also demonstrated that formalin-inactivation of rsv resulted in post-fusion f (postf) protein being the predominant protein presented on the surface of the virion (20) . the implication of this data is that pref is more representative of live rsv and therefore, may be less likely than postf subunit vaccines to induce pathology. moreover, in a naïve cotton rat model, both pref and postf immunization elicited protective rsv immunity without inducing alveolitis when paired with the th1-skewing toll-like receptor 4 agonist (tlr4), glucopyranosyl lipid a (gla) (21) . these results suggest that more th1-biased adjuvants may provide a safe alternative to alum in models of pref immunization. importantly, th1skewing adjuvants, including tlr9 agonists, have recently been fda-approved for use in other vaccine systems, like hepatitis b (22) . advax-sm is an adjuvant comprised of delta inulin polysaccharide formulated with the tlr9 agonist, cpg oligodeoxynucleotides (cpg odn). the advax-sm adjuvant system has demonstrated greater th1-skewing properties when formulated with live rsv immunization (23) and ameliorated th2-related airway eosinophilia in a model of immunization against severe acute respiratory syndrome (sars)-associated coronavirus (24) . in the immunization studies presented here, we conducted a detailed evaluation of the protective capacity of rsv pref antigen alone or combined with the th1/th2-balanced adjuvant, advax-sm (pref/advax-sm), or the th2-skewing adjuvant, alum (pref/alum/aluminum hydroxide) in naïve balb/c mice. these studies evaluated immunogenicity, efficacy, innate and adaptive immune responses, and safety at acute and convalescent time points following rsv challenge. despite pref/alum immunization generating higher neutralizing antibody titers, both pref/advax-sm and pref/alum had undetectable viral replication, while pref alone lacked the immunogenicity to fully protect from rsv infection in naïve animals. pref/advax-sm induced more balanced th1/th2 immunity characterized by the generation of neutralizing antibody, a mean pref-specific igg2a/igg1 ratio >2, rsv f-specific and cytotoxic cd8+ t cells, and th1 cd4+ t cells. importantly, pref/advax-sm immunization protected from increased inflammation and mucus production, even when compared to pbs controls. in contrast, pref alone and pref/alum generated robust th2 immunity evidenced by pref-specific igg2a/igg1 ratios <1 and increased il-5+ and il-13+ cd4+ t cells. interestingly, despite undetectable viral replication at 4dpi, pref/alum immunization induced large populations of type 2 innate lymphoid cells (ilc2) in the lung producing the th2-type cytokines, il-5 and il-13. combined, the th2 immunity of pref/alum was consistent with enhanced inflammation featuring airway eosinophils and increased mucus production. overall, our observations have significant implications for the rsv vaccine field demonstrating that higher neutralizing antibody titers, while protective, are not implicitly tied to rsv pref vaccine safety and more th1/th2balanced adjuvants may generate protective responses while eliciting a desirable safety profile in naïve mice. animal studies were carried out in accordance with the university of pittsburgh's iacuc guidelines for the use and care of laboratory animals. seven to eight week old balb/cj female mice were purchased from the jackson laboratory (bar harbor, me). female mice were immunized via intramuscular (i.m.) injection with 50 mcl of vehicle (naïve and pbs), stabilized rsv prefusion protein (pref; 10 mcg; calder biosciences) alone, or formulated with advax-sm tm (pref/advax-sm; 1 mg/mouse; vaxine pty ltd, bedford park, australia) or alum (pref/alum; 2 mg/ml). specifically, alhydrogel adjuvant 2%, an aluminum hydroxide wet gel suspension from invivogen, was used in these studies. advax-sm is composed of microparticles of polyfructofuranosyl-d -glucose (delta inulin) combined with cpg55.2-odn (5 ′ atcgactctcgagcgttctc-3 ′ ), which was synthesized by genedesign (osaka, japan). the immunized mice were boosted 3 weeks later with their respective vaccine formulations. at 9 weeks post-prime, mice were challenged intranasally (i.n) with rsv l19 (5 × 10 5 pfu/gm) and culled at 4 or 8 days post-infection (dpi) using 100% isoflurane and cervical dislocation. rsv l19 was propagated and viral titers quantified as previously described (25) . all animal studies were approved by the university of pittsburgh institutional animal care and use committee; protocol #20047209. bronchoalveolar lavage (bal) was collected through intratracheal instillation of hbss + edta. bal samples were centrifuged and the soluble fraction stored at −80 • c for cytokine analysis and the cellular fraction analyzed via flow cytometry. cytokine concentrations were determined using the bio-plex pro tm mouse cytokine 23-plex assay (biorad, ca), per manufacturer's protocol. the right lung was harvested and enzyme-digested into a single cell suspension for flow cytometry, as described previously (26, 27) . where indicated, bal cells and lung homogenate were stimulated ex-vivo for intracellular cytokine detection. briefly, live cells from bal and lung homogenate were enumerated using a hemacytometer and trypan blue. for intracellular cytokine staining of lung homogenate or bal, 1 million cells in duplicate (singular for bal) were plated in a cd3-coated (5 mcg/ml, biolegend) 96-well flat-bottomed tissue culture plate in 200 mcl of 10% rpmi supplemented with cd28 (2 mcg/ml) and incubated at 37 • c overnight. after overnight stimulation with cd3/cd28, lung homogenate underwent a secondary stimulation with pma (1:1,000), ionomycin (1:1,000), and brefeldin a (1:1,000) for 2 h prior to t cell surface and intracellular flow staining. to obtain intracellular ilc2 cytokine staining, lung homogenate (3 million cells) was plated in 24-well tissue culture plates and stimulated with pma (30 ng/ml), ionomycin (500 ng/ml), and brefeldin a (1:1,000) in 10% rpmi at 37 • c for 3 h prior to surface and intracellular flow staining. bal cells were surface stained with combinations of the following (clone): molecular probes live/dead fixable blue, cd16/32 (2.4g2), siglec-f (e50-2440), f4/80 (t45-2342), cd11b (m1/70), ly6g (1a8), cd4 (gk1.5), cd8α (53-6.7), cd44 (im7) (bd biosciences, ca), cd11c (n418), cd19 (6d5), and tcr β (h57-597) (biolegend, ca). lung homogenate was surface stained with combinations of the following antibodies: cd16/32 (2.4g2), lineage cocktail, cd45 (30-f11), st2 (dih9), il-7rα (a7r32), cd19 (6d5), tcr β (h57-597) (biolegend), cd4 (gk1.5), and cd8α (53-6.7) (bd bioscience). following surface staining, cells were fixed and permeabilized for intracellular staining with bd cytofix/cytoperm tm solution kit (bd biosciences) according to the manufacturer's protocol. intracellular cytokines were stained with a combination of the following: cd206 (c068c2), il-5 (trfk5), ifnγ (xmg1.2), granzyme b (qa16a02) (biolegend), and il-13 (ebio13a) (thermofisher scientific, ma). where indicated and prior to surface staining, bal samples were incubated with rsv a strain f-protein 85−93 mhc i pentamer (h-2kd kyknavtel; proimmune, fl) to identify rsv f 85−93 -specific cd8 + t cells. samples were run on a bd lsrfortessa managed by the united flow core of the university of pittsburgh. data was analyzed using flowjo v10 software (flowjo, llc, or). cell populations were defined as follows: eosinophils (siglec f+/ f4/80+/ cd206lo/-/ cd11b+); neutrophils (siglec f-/ cd11bhi/ ly6g+/ cd11c-/lo); monocytes (siglec f-/ f4/80+/ cd11c+/ cd11b+); ilc2s (lin-/ cd45+/ st2+/ il-7rα+); t cells (± cd19-/ tcr β +/ cd4+ or cd8+). a lps-treated negative control was used to set the gate for rsv a strain f-protein 85−93 mhc i pentamer+ cd8+ t cells. pre-challenge serum was collected via submandibular bleed 2-3 days prior to rsv challenge and separated using gel-z serum separator tubes (sarstedt, germany). serum was stored at −80 • c until heat inactivation (56 • c for 30 min) and neutralizing antibody titers were performed. serial dilutions of heat inactivated serum (50 mcl in phenol-free mem supplemented with 5% fbs and pen/strep, invitrogen) were incubated for 2 h in a 37 • c co 2 incubator in a 96well plate format with 100 pfu/well line 19 rsv-renilla luciferase virus (provided by martin moore) in 50 mcl phenol free mem medium as above. after 2 h, hep-2 cells were trypsinized and a total of 2.5 × 10 4 cells were added per well in 25 mcl of phenol free mem with fbs and antibiotics as above. cells were incubated for a total of 64-66 h at 37 • c, 5% co 2 and luciferase readout was then obtained using the renilla-glo luciferase kit (promega) according to the manufacturer's instructions. luciferase activity (luminescence) was measured using a novostar plate reader after a 15 min incubation at 25 • c. all plates were run in duplicate and averaged. co-star 96-well, high binding elisa plates were coated with rsv pref at a concentration of 5 mcg/ml overnight at 4 • c. each plate included standards of either mouse igg1 or igg2a (invitrogen) at 10 and 2 mcg/ml in a 2-fold dilution series for intra-plate quantification of signal on uncoated wells. plates were then washed with pbs, and blocked for 1 h at 37 • c with 1% bsa in pbs. heat-inactivated serum samples were diluted 1:500 in 1% bsa in pbs for the first well, and then 3-fold serially diluted a total of 3 times. serum was incubated on the plates for 1 h at 25 • c, followed by three washes with pbs 0.05% tween-20, and secondary antibody incubation with anti-igg1 or anti-igg2a (isotype specific, bd pharmingen), respectively at a 1:10,000 dilution for 30 min at 25 • c in 1% bsa. 1-step tmb (thermo scientific) was used to develop the plates and the reaction was quenched by the addition of 4 n h 2 so 4 . plates were read at 450 nm in a novostar plate reader. data analysis was performed in excel and data points were interpolated from the linear region of the standards on each individual plate. samples were run in duplicate and the data presented represents the average values from both runs. left lungs were gravity filled with 10% formalin at 4 and 8 days post rsv challenge, as previously described (28) . the mcgowan institute for regenerative medicine (university of pittsburgh, pa) stained and processed the preserved lungs. periodic acid-schiff (pas) stained lungs were assessed by two pathologists blinded to treatment groups to quantify airway mucus production, according to previously published methods (26) . briefly, a score of 0-4 was given to all airways (average 50) with the following scale: 0 = no pas+ cells; 1 = 1-25% pas+ cells; 2 = 26-50% pas+ cells; 3 = 51-75% pas+ cells; 4 = 76-100% pas+ cells. scores were averaged and the total percentage of pas+ airways were graphed along with a more detailed breakdown of the proportion of each severity score (0-4) (# of airways of individual severity scores/total airways scored). standard hematoxylin and eosin (h&e) staining was performed on lung sections and scored by two pathologists (oury and perkins) blinded to treatment groups, according to previously described methods (29) . in short, each field (average 28 fields) in the lung was observed with a light microscope (x200 magnification) and scoring was based on the percentage of lung tissue affected according to the following scale: 0 = no inflammation, 1 = up to 25%, 2 = 25-50%, 3 = 50-75%, and 4 = 75-100%. scores were averaged and reported as a proportion of the sum of scores divided by the total number of fields counted. statistics were performed with graphpad prism 8 software (graphpad software, la jolla, ca). results in the figures are displayed as the mean ± sem. neutralizing antibody data was analyzed by nonlinear regression to obtain ic 50 values, which were compared between immunization groups using anova with a tukey's post-test, with pbs serving as the control. statistical significance was determined between each immunization cohort using anova with tukey's multiple comparison test between all groups (note: naive animals were not included in analysis but are graphed for reference). comparisons of the inflammatory and % pas+ scores within immunization groups over time were made using t-tests corrected for multiple comparisons with the holm-sidak method (α = 0.05). p-values <0.05 were considered significant. data is representative of 2 separate experiments. the th2-biased humoral immunity of pref/alum and the greater th1-skewed humoral immunity of pref/advax-sm protect against rsv replication the objective of this study was to determine the capacity of rsv pre-fusion (pref) vaccines formulated with different adjuvants to generate neutralizing antibody, prevent virus replication, and protect from pulmonary pathology following rsv challenge. to that end, 8 week old balb/cj mice were immunized twice with pbs (vehicle control), pref alone, or pref formulated with the th1/th2-balanced adjuvant, advax-sm (pref/advax-sm), or the t helper type 2 (th2)-skewing adjuvant, aluminum hydroxide (pref/alum). six weeks after their final immunization, sera were collected from mice in each group prior to viral challenge with rsv line 19; a control group of pbs-immunized mice received vehicle only (naïve) ( figure 1a) . a comparison of pref-specific igg2a and igg1 in prechallenge sera provided initial evidence supporting the roles of advax-sm as a more th1-biased adjuvant and alum as a th2polarizing adjuvant; production of igg2a and igg1 subclasses are reflective of their respective th1 and th2 biased immune responses in mice (30) . pref/advax-sm immunization produced higher titers of igg2a than all immunization groups tested ( figure 1b ) and greater igg1 compared to pbs ( figure 1c ). the mean igg2a/igg1 ratio of pref/advax-sm mice was > 2 and significantly higher than pref or pref/alum animals, indicating a more th1-skewed humoral response ( figure 1d ). in contrast, pref/alum immunization produced negligible titers of igg2a ( figure 1b) and instead elicited increased titers of igg1 ( figure 1c) , with a resulting igg2a/igg1 ratio < 1 ( figure 1d ). similar to pref/alum, pref alone elicited an igg2a/igg1 ratio < 1, indicating th2-dominant antibody responses in both groups. to assess differences in protective antibody responses between immunization groups, neutralizing antibody titers were measured in pre-challenge sera and rsv lung titers were quantified at 4 days post-infection (dpi). mice immunized with pref/alum generated greater neutralizing antibody titers than all other immunization groups ( figure 1e) . despite lower neutralizing antibody titers in mice immunized with pref/advax-sm, as compared to pref/alum, both immunization groups had undetectable rsv in their lungs at 4 dpi ( figure 1f ). in contrast, pref alone generated measurable neutralizing antibody titers in 50% (n = 4/8) of the animals and only 38% (n = 3/8) had undetectable rsv lung titers ( figure 1f ). all immunized mice had lower viral lung titers when compared to pbs controls following rsv challenge, however, only mice figure 1 | the th2-biased humoral immunity of pref/alum and the th1 skewed-humoral immunity of pref/advax-sm protect from rsv challenge. at 7-8 weeks of age, female adult balb/cj mice were immunized with phosphate buffered saline vehicle control (naïve & pbs), prefusion rsv f protein alone (pref), or pref formulated with advax-sm (pref/advax-sm) or alum (pref/alum) as depicted in (a). six weeks post-boost, pre-challenge serum was collected and mice were subsequently intranasally challenged with vehicle (naïve) or 5 × 10 5 pfu/gram of rsv line 19. animals were culled for sample collection at 4 or 8 days post infection (dpi) (a). pre-challenge serum was analyzed for pref-specific igg2a (b), igg1 (c), igg2a to igg1 ratios (d) and neutralizing antibody titers (e). at 4 dpi, left lungs were harvested and virus quantified using standard h&e plaque assays (f). data are represented as mean ± sem (n = 6-8 mice per group);*p < 0.05, **p < 0.01, and ****p < 0.0001. due to a lack of pre-existing pref-specific antibody, pbs was not included in the analysis in (d). immunized with pref/alum or pref/advax-sm achieved full viral protection at 4 dpi. to evaluate potential immunopathology in mice that received th2-skewing immunizations (pref alone and pref/alum) as compared to the greater th1-biased regimen (pref/advax-sm), left lungs were harvested from immunized mice at 4 dpi and stained with h&e to evaluate inflammation. representative images (10x) taken from animals in each immunization group demonstrated increased inflammation in all immunization groups relative to pbs controls, with pronounced perivascular inflammation seen in pref-and pref/alum-immunized animals (figures 2a-e) . inflammatory scores increased at 4 dpi in all groups that received pref-formulated immunizations but only pref alone and pref/alum elicited significantly greater inflammation compared to pbs (figure 2f) . to determine the contribution of innate cellular responses to the enhanced figure 2 | pref and pref/alum immunization elicited enhanced pulmonary inflammation characterized by robust airway eosinophil recruitment. naive mice were immunized and challenged with rsv as described in figure 1 . at 4 dpi, left lungs were formalin filled, paraffin embedded and sectioned for staining with h&e. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). two blinded, independent pathologists scored all slides as described in the methods. scores between the two investigators were averaged and data is represented as mean ± sem (n = 3 mice) (f). at 4 dpi, bal was collected and eosinophils (g), neutrophils (h), and monocytes (i) were identified via flow cytometry. data are represented as mean ± sem (n = 6-8 mice per group);*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. yellow arrows highlight perivascular inflammation and blue arrows highlight peribronchial inflammation. inflammation observed in pref-and pref/alum-immunized groups, bronchoalveolar lavage (bal) was collected at 4 dpi and analyzed via flow cytometry (gating strategy for discriminating innate immune cells is shown in supplementary figure 1) . immunization with pref alone and pref/alum elicited a dramatic recruitment of eosinophils to the airways following rsv challenge, whereas eosinophil populations remained at baseline in pref/advax-sm-immunized mice ( figure 2g ). neutrophils were increased in the bal of pref-and pref/advax-smimmunized mice compared to pbs controls ( figure 2h ) and total monocytes were greater in pref-immunized animals as compared to all other groups tested (figure 2i) . taken together these results show that increased airway eosinophils paralleled increases in inflammation scores in mice immunized with pref alone and pref/alum, whereas neutrophils and monocytes were more pronounced in the unadjuvanted pref group. human rsv disease is characterized by both inflammation and extensive mucus production. thus, to determine if the enhanced inflammation seen in pref-and pref/alum-immunized groups was also associated with enhanced mucus production, left lungs were harvested at 4 dpi and periodic acid-schiff (pas) stained to analyze mucus metaplasia. representative images (10x) were taken from a sample within each immunization group to visualize the extent of mucus production (figures 3a-e) . a majority of airways from naïve ( figure 3a) , pbs (figure 3b) , and pref/advax-sm immunization groups ( figure 3d ) had little to no pas+ staining, while pref alone ( figure 3c ) and pref/alum ( figure 3e ) groups had extensive pas+ staining. consistent with the representative lung sections, pref-and pref/alumimmunization groups had higher percentages of pas+ airways compared to all other groups tested ( figure 3f) . to discriminate the degree of airway mucus production, the level of severity was reported for each group, whereby no pas+ staining in the airway yielded a score of "0, " the frequency of airways with 1-25% pas+ staining received a score of "1, " 26-50% pas+ yielded a score of "2, " 51-75% pas+ airways received a score of "3, " and 76-100% pas+ staining received a score of "4." four days after diluent (figure 3g ) or rsv challenge, pbs ( figure 3h ) and pref/advax-sm ( figure 3j ) immunization groups had the largest percentage of unaffected airways and a small proportion of airways with mild pas+ staining (1 score). in contrast, pref-( figure 3i ) and pref/alum-immunized mice ( figure 3k ) had smaller percentages of unaffected airways and greater frequency of airways with higher severity scores (2-4 scores). pref/advax-sm immunization produced th1-dominant immunity with increased rsv f-specific cd8+ t cells to delineate the relationship between distinct th cell subsets and associated pulmonary inflammation and mucus production, figure 3 | increased mucus production parallels enhanced inflammation in mice immunized with pref alone or pref/alum. mice were immunized and challenged with rsv as described in figure 1 . at 4 dpi, lungs were formalin filled, paraffin embedded and sectioned for staining with pas. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). to quantify the extent of pas staining, lungs were scored as previously described in the methods. scores were averaged and the total percentage of pas+ airways were graphed (n = 3) (f); **p < 0.01 and ***p < 0.001. a more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score (0-4) divided by the total number of airways, according to the methods (g-k). th1-and th2-type cytokines were quantified from bal. ifnγ, the canonical th1 cytokine, was highest in pref/advax-sm mice at 4 dpi (figure 4a) , whereas levels of the th2-associated cytokines, il-5 ( figure 4b ) and il-13 ( figure 4c) , were similar between pref-advax-sm-immunized mice and control groups (naïve and pbs). conversely, pref-and pref/alum-immunized mice had increased concentrations of il-5 and il-13 with no appreciable increase in ifnγ levels in these groups. to identify cellular sources contributing to the th1associated cytokine bias of pref/advax-sm and the th2associated cytokine bias of pref and pref/alum immunization, ilc2 and cd4+ t cell populations were analyzed from lung homogenate at 4 dpi via flow cytometry (gating strategy to discriminate t cell populations and ilc2s are shown in supplementary figures 2, 3 , respectively). consistent with their th2 cytokine profiles, pref/alum immunization elicited greater ilc2 populations ( figure 4d ) following rsv challenge compared to all other groups. though ilc2s trended higher in pref alone immunization than control and pref/advax-sm groups, the difference was not significant. moreover, pref/alum-immunized mice had increased il5+ ( figure 4e ) and il-13+ ( figure 4f ) ilc2 populations as compared to pref/advax-sm-immunized mice and pbs controls; once again, increased trends in the pref alone group did not achieve significance. in conjunction with increased ilc2s, pref/alumimmunized mice had marked increases in il-5-producing cd4+ t cells in lung homogenate ( figure 4h ). pref-immunized mice had similar trends in increased il-5+ cd4+ t cells but were not significantly different compared to pbs controls. in contrast, pref/advax-sm-immunized mice had the largest population of ifnγ+ cd4+ t cells (figure 4g ) with distinctly increased ifnγ+:il-5+ cd4+ t cell ratios (figure 4i ) as compared to pref-and pref/alum-immunized mice. a similar increase in the ratio of ifnγ+:il-5+ cd4+ t cells was observed in the pbs group. lastly, due to the importance of cd8+ t cells in clearing rsv, general cd8+ t cell populations and rsv f 85−93 -specific cd8+ t cells were compared in mice from each immunization group. mice immunized with pref/advax-sm had increased total cd8+ t cells as compared to pref-immunized mice and pbs controls ( figure 4j) . moreover, only pref/advax-smimmunized mice had increased rsv f 85−93 -specific cd8+ t cells, which were greater than all other groups tested ( figure 4k) . collectively, these results identified ilc2s and cd4+ t cells as figure 4 | pref/advax-sm immunization produced th1-dominant immunity with increased rsv f-specific cd8+ t cells. at 4 dpi, first wash samples were harvested from the airways for quantification of ifnγ (a), il-5 (b), and il-13 (c) by luminex. right lungs were harvested and homogenized for quantification of ilc2s (d) and ilc2 intracellular cytokine staining of il-5 (e) and il-13 (f) by flow cytometry. intracellular production of ifnγ (g) and il-5 (h) by cd4+ t cells from lung homogenate were quantified by flow cytometry and the ratio of ifnγ/il-5-producing cd4+ t cells was calculated (i). total cd8+ t cells (j) and rsv f 85−93 -specific cd8+ t cells (k) were measured from bal. data are represented as mean ± sem (n = 6-8 mice per group);*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. frontiers in immunology | www.frontiersin.org cellular sources of th2-type cytokines associated with pref-and pref/alum-immunized mice following rsv challenge. in stark contrast, pref/advax-sm immunization elicited an increase in ifnγ-producing cd4+ t cells and rsv f 85−93 -specific cd8+ t cells in response to rsv exposure that likely contributed to viral protection. to determine if immunization with pref, pref/alum, or pref/advax-sm expedites disease resolution, innate inflammatory cells and lung inflammation were examined later at 8 days post-rsv challenge. representative images (10x) were taken from h&e stained lung sections from each immunization group (figures 5a-e) . each rsv-challenged group displayed enhanced inflammation that was predominately perivascular in nature with non-significant reductions in inflammation in the immunization groups compared to the pbs group ( figure 5f ). similar to the 4-day time point, pref/alum mice maintained greater airway eosinophil populations ( figure 5g ) as compared to pref/advax-sm-immunized mice and pbs controls. neutrophils were elevated in pbs controls and the pref/alum group compared to pref/advax-sm-immunized mice, although no significant difference was observed in the pbs group (figure 5h) . pref-immunized and pbs control groups had the greatest number of monocytes in the bal when compared to pref/advax-sm-immunized mice ( figure 5i) . overall, populations of innate inflammatory cells had largely resolved in the bal of pref/advax-sm-immunized mice by 8 dpi. in pbs controls, increases in neutrophils and monocytes likely contributed to the dramatic increase in inflammation between 4 and 8 dpi, whereas inflammatory scores for the groups immunized with pref remained largely unchanged (figure 5j) . these results suggest that inflammation and recruitment of inflammatory cells dramatically increased in pbs controls, innate inflammatory cells largely resolved in the pref/advax-sm group, and pref/alum immunization failed to resolve airway eosinophilia by 8 days post-rsv challenge. to determine if mucus production resolved or worsened in immunized mice over the course of infection, left lungs were collected and pas-stained at 8 dpi. representative images (10x) were taken from samples in each group (figure 6a-e) . readily discernable mucus was observed in pbs ( figure 6b ) and pref/alum ( figure 6e ) lung sections, with a lower frequency of pas+ staining seen in mice immunized with pref-alone ( figure 6c ). quantitative analysis of the percentage of pas+ figure 5 | inflammation worsens over time in unimmunized compared to immunized groups. at 8 dpi, left lungs were formalin fixed and stained with h&e to assess inflammation. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). two blinded, independent pathologists scored all slides and scores were averaged. data is represented as mean ± sem (n = 3-4 mice) (f). bal was collected at 8 dpi for quantification of eosinophils (g), neutrophils (h), and monocytes (i) via flow cytometry. data are represented as mean ± sem (n = 6-8 mice). inflammatory scores from 4 and 8 dpi were compared for each immunization groups over time (j). data are represented as mean ± sem (n = 3-4 mice); *p < 0.05 and **p < 0.01. airways revealed that pbs controls and pref/alum-immunized groups had the highest proportion of pas+ airways (figure 6k) . pref/advax-sm immunized mice maintained a low percentage of pas+ airways through 8 dpi. a more detailed analysis revealed that >50% of airways were pas+ in pbs-( figure 6g ) and pref/alum-immunized mice ( figure 6j ) and these groups had the largest proportions of airways that received higher severity scores (scores of 2-4) relative to all other groups (figure 6f-j) . at 8 dpi, the majority of airways in the prefimmunization group were unaffected and when pas+ was observed, it was generally mild (score of 1; figure 6i ). mice immunized with pref/advax-sm (figure 6j ) had the largest percentage of pas-free airways, with a smaller proportion of airways receiving higher severity scoring (scores of [2] [3] [4] . similar to what was seen with inflammation over time, pbs controls demonstrated a distinct enhancement of mucus production between 4 and 8 dpi (figure 6l) , while pref/alum immunization elicited a high degree of pas+ staining throughout the rsv time course. in contrast, by 8 dpi, pref-immunized mice resolved some of the high proportion of pas+ airways observed at 4 dpi, while mice immunized with pref/advax-sm maintained low levels of mucus production. together, these data suggest that the high degree of mucus produced by 8 dpi in pbs controls is mitigated more effectively in mice immunized with pref/advax-sm as opposed to pref/ alum immunization. based on the changes in lung pathology observed between immunization groups at 8 dpi, we asked whether there was an associated change in th phenotypes among immunization figure 6 | mucus production persisted in the lungs of pref/alum-immunized mice at 8 dpi and remained low in mice immunized with pref/advax-sm. at 8 dpi, left lungs were formalin fixed and stained with pas to assess mucus production. each panel represents an individual mouse from the indicated group (scale bar 100 µm) (a-e). a more detailed breakdown of scores for each cohort is provided, calculated as a proportion i.e., number of airways of each severity score (0-4) divided by the total number of airways, according to the methods (f-j). to quantify the extent of pas staining, lungs were scored as previously described in the methods. scores were averaged and the total percentage of pas+ airways were graphed. total percentage pas+ airways from 8 dpi are shown in (k) (n = 3-4) and are further compared over time between 4 and 8 dpi in each group in (l); **p < 0.01. (figure 7a) , while mice immunized with pref alone and pref/alum had larger populations of il-5+ cd4+ t cells ( figure 7b) . additionally, il-13+ cd4+ t cell populations in pref/alum immunized mice were significantly higher than pref/advax-sm animals ( figure 7c ). examination of cd8+ t cell populations revealed greater granzyme b+ ( figure 7d) and ifnγ+ cd8+ t cells + (figure 7e ) in the airways of pref/advax-sm immunized mice and pbs controls, with pbs mice having significantly more ifnγ+ cd8+ t cells than the other immunization groups tested. in addition to cellular analysis in the bal, t cells and ilc2s were measured in lung homogenate to address potential differences in cell localization. in accordance with their role as early immune responders, ilc2 populations ( figure 7f ) had contracted by 8 dpi and no differences were detected between immunization groups. moreover, populations of il-5+-( figure 7g ) and il-13+-producing ilc2s ( figure 7h) were similar between all groups. finally, th phenotypes were examined at 8 dpi in lung homogenate. pref/advax-sm immunized mice had the highest number of ifnγ+ cd4+ t cells (figure 7i ) compared to other groups, while il-5+ cd4+ t cells ( figure 7j) were greatest in pref-vaccinated mice. consistent with the data at 4 dpi, the cd4+ ifnγ+:il-5+ ratio ( figure 7k ) was higher in pref/advax-sm immunized mice as compared to all other groups tested. this demonstrates the persistent th1-dominant immune response elicited by pref/advax-sm immunization. here, we explored the ability of a rsv pref subunit proteinbased immunization to protect naïve balb/c mice from rsv infection and pulmonary pathology when formulated with either a th2-or more th1/th2-balanced adjuvant. alum has been in use in human vaccines since the early 20th century and is well recognized for its th2-skewing and antibody boosting properties. as expected, pref/alum immunization produced th2-biased immunity with high titers of pref-specific igg1 and neutralizing antibody, which were associated with undetectable viral replication following rsv challenge. while pref alone lacked the immunogenicity to offer complete rsv protection, likely due to low neutralizing antibody production, prefimmunized mice generated th2-type immune responses similar to mice immunized with pref/alum. on the other hand, advax-sm is a delta-inulin polysaccharide adjuvant formulated with cpg55.2 odn, a tlr9 agonist. the combination of delta inulin and tlr9 agonism has been reported to generate a th1/th2balanced adjuvant response in models of sars-associated coronavirus, japanese encephalitis virus, and west nile virus (24, 31, 32) . in models of rsv immunization, tlr9 agonists formulated with formalin-inactivated rsv have increased immunogenicity while ameliorating pulmonary pathology and reducing airway hyperresponsiveness normally exacerbated by fi-rsv immunization (33, 34) . in one report, an intramuscular immunization of live rsv formulated with advax-sm protected from rsv infection with increased neutralizing antibody titers and greater rsv-specific igg2a/igg1, but had similar lung inflammation as unadjuvanted control mice, suggesting the th2 polarization of the live rsv vaccine may have overcome the greater th1 bias of the cpg oligonucleotide (23) . these studies support the idea that tlr9 agonists may be combined with stabilized pref proteins to induce a more th1-biased response with an acceptable safety profile and highlight the importance of evaluating adjuvant safety as well as efficacy when developing vaccine strategies. in our study, mice immunized with pref/advax-sm elicited a greater th1-type response, while still generating high levels of pref-specific igg2a and igg1 antibody. despite producing lower neutralizing antibody titers than pref/alum immunized, pref/advax-sm mice were resistant to rsv infection. moreover, the discreet th profiles produced by pref/alum and pref/advax-sm generated distinct differences in pulmonary pathology. the th2 profile of pref/alum-immunized mice was associated with enhanced pulmonary inflammation and mucus production at 4 dpi and continued to have the greatest proportion of pas+ airways as late as 8 dpi. the immunopathology associated with pref/alum immunization occurred in spite of high neutralizing antibody titers and undetectable viral replication. previous studies of fi-rsv-associated erd have demonstrated that poor avidity and affinity maturation resulted in non-protective antibody development and th2-associated immunopathology (19) . furthermore, it has been suggested that alum did not alter the erd profile induced by fi-rsv immunization (18) . however, in our study, alum adjuvant exacerbated the pathology induced by immunization with pref alone despite protection from rsv infection. in contrast, the th1-type responses generated by immunization with pref/advax-sm elicited less overall inflammation with low levels of mucus production. importantly, pref/advax-sm immunization protected mice from the worsening inflammation and mucus production that occurred between 4 and 8 dpi in pbs controls. taken together, these data indicate that rsv pref immunization formulated with th1-skewing adjuvants, like advax-sm, provide protection against rsv infection in naïve balb/c mice as compared to alum by inhibiting viral replication without eliciting enhanced pulmonary pathology. though ige was not measured in these studies, alum, as opposed to cpg-odn, adjuvanted antigens have been associated with an increased production of ige (35) . future studies will be needed to address the role of ige following immunization with rsv pref adjuvanted with alum. neutralizing antibodies have been shown to reduce the severity of rsv disease (12, 13) . moreover, the risk of reinfection is inversely correlated to the level of serum neutralizing antibodies (36) . as such, boosting serum neutralizing antibody titers has been an important objective of rsv vaccination, especially since the discovery and subsequent stabilization of the pre-fusion conformation of rsv f protein (11) . rsvneutralizing activity in human sera is primarily derived from pref-specific antibodies (37, 38) . thus, the use of stabilized pref as a vaccine antigen has sparked new hope that high neutralizing antibody titers can be produced to provide long-term protection. pre-clinical studies in young and aged mice have demonstrated the neutralizing antibody boosting-potential of rsv pref when formulated with both th2-and th1/th2-skewing adjuvants (39) . in agreement with our data, pref immunization alone has been shown to lack sufficient immunogenicity to increase neutralizing antibody titers above a protective threshold (39) . our study expanded these findings by directly challenging pref-immunized mice and demonstrated incomplete protection and enhanced lung pathology following rsv infection in association with little to no measurable neutralizing antibody production (i.e., only 50% of pref-immunized mice produced any measurable neutralizing antibody). earlier work found that higher titers of neutralizing antibody were associated with rsv pref immunization formulated with th1/th2-balanced adjuvants (39) . in contrast, our results show that the th2-dominant immune response of pref/alum immunization produced higher neutralizing antibody titers as compared to the th1-biased immune response elicited by pref/advax-sm immunization. importantly, however, both pref/alum and pref/advax-sm immunization fully protected mice from rsv infection with a complete absence of detectable viral replication in the lungs at 4 dpi. additional studies will be required to determine if lower neutralizing antibody production, as seen in pref/advax-sm, affects the durability of protection against rsv challenge. despite evidence of the correlation between neutralizing antibody in the serum and protection from severe rsv disease (12, 13, 36, 37) , other data suggests that high serum levels of neutralizing antibody provide insufficient protection from rsvdisease or reinfection in some individuals (40) (41) (42) . therefore, rsv vaccines that rely on neutralizing antibody as the sole correlate of protection against rsv disease may not provide universal or long-lasting immunity. numerous studies have demonstrated the importance of t cells in clearing rsv in primary rsv infection (43) (44) (45) . in mouse models of primary infection, cd8+ t cells are critical for viral control and both the number of cd8+ t cells and their production of ifnγ are crucial for effective clearance (46) (47) (48) . despite conferring protection against rsv replication, immunopathology has been linked to ifnγ-producing cd8+t cells during primary infection (47) and in memory responses resulting from immunization (49) . in contrast, our data shows that immunization with pref/advax-sm protects against rsv infection and elicits rsv f-specific and ifnγ-producing cd8+ t cells without inducing additional pulmonary pathology. alternatively, by 8 dpi, a robust ifnγ+ cd8+t cell response was induced in pbs-immunized control mice that was further associated with increased inflammation. these divergent outcomes in lung pathology associated with ifnγ+ cd8+t cells may be explained by the presence of pre-existing neutralizing antibody in pref/advax-sm as compared to the lack of pre-existing neutralizing antibody in pbs controls. another model of rsv vaccination has demonstrated that pre-existing neutralizing antibody can protect mice from severe immunopathology caused by potent memory ifnγ-producing cd8+t cells (50) . therefore, rsv vaccination approaches that combine pref with th1/th2-balanced adjuvants may prove to be efficacious and safe through the combination of increased neutralizing and rsv-specific antibody production and memory ifnγ+ cd8+t cell responses. like cd8+ t cells, research has demonstrated the importance of cd4+ t cells in the control of rsv infection and their role in inducing pathology (43) (44) (45) . the 1960's vaccine trials of formalin-inactivated rsv formulated with alum (fi-rsv) demonstrated the sobering potential for vaccine-associated erd. upon natural rsv exposure, 80% of vaccinees developed erd, requiring hospitalization and two children died (16) . investigations into the causes of erd using multiple animal models have revealed th2 cd4+ t cells to be closely linked to the increased inflammation, mucus, and airway hyperresponsiveness observed in erd (51) (52) (53) (54) (55) . in our study, both pref alone and pref/alum immunization elicited th2 cd4+ t cell profiles associated with enhanced inflammation, airway eosinophils, and extensive mucus production. interestingly, despite their similar th2 phenotypes, pref-immunized mice resolved airway neutrophils, eosinophils, and mucus more rapidly than pref/alum immunized mice. this resolution occurred in pref-immunized mice despite their poor neutralizing antibody production and detectable rsv replication, which was likely the cause of increased inflammatory innate cell recruitment as compared to pref/alum immunization. whereas, mice immunized with pref/alum had high titers of neutralizing antibody and undetectable rsv in the lung, yet persistent airway mucus and inflammation. in contrast to data showing the ability of pre-existing antibody to temper the immunopathology induced by ifnγ+ cd8+t cells (50) , our data suggests that pre-existing neutralizing antibody alone may not modulate the immunopathology associated with th2 cd4+ t cells in a similar manner. in addition to increases in th2 cd4+ t cells, pref-but especially pref/alum-immunization was associated with large populations of ilc2s producing il-5 and il-13 in the lung. ilc2s are the dominant innate lymphoid cell in the lung and are well recognized for their ability to produce large amounts of type 2 cytokines upon stimulation (56) . in primary rsv, ilc2 expansion and activation result from damage to airway epithelial cells via rsv infection and subsequent release of il-33 (57) and/or thymic stromal lymphopoietin (tslp) (58). however, in pref/alum immunized animals, ilc2s increased and became activated in spite of effective rsv neutralization and undetectable virus in the lung at 4 dpi. our data suggests that an alternative mechanism may contribute to the induction of ilc2 responses in pref/alum immunized mice. a murine model of nippostrongylus brasiliensis demonstrated that crosstalk between ilc2s and antigen-specific th2 cd4+ t cells occurs, promoting ilc2 proliferation and il-13 production (59). this ilc2-cd4+ t cell crosstalk was shown to be mutually beneficial to both cell types' maintenance, expansion, and cytokine production. future studies are necessary to understand how pref/alum immunization contributes to ilc2 expansion and activation. however, this is the first study to suggest a relationship between ilc2 activation and th2-associated immunopathology following pref/alum immunization in naïve mice. in conclusion, our studies demonstrated that pref/advax-sm and pref/alum immunizations provided equivalent protection from rsv infection but elicited dramatically different outcomes regarding lung pathology. the th2 immunity induced by pref/alum immunization was associated with increased inflammation and abundant mucus production that persisted through 8 dpi. in addition to il-5-and il-13-producing cd4+ t cells, pref/alum immunization was also associated with increased il-5-and il-13-producing ilc2 populations. this previously unrecognized relationship between pref/aluminduced th2 immunity and ilc2 activation in the absence of rsv replication may have important implications for rsv vaccine development. it suggests that despite eliciting high titers of neutralizing antibody, the extensive th2 immune bias generated by pref/alum may still lead to overt pathology in naïve individuals. it has been suggested that rsv exposure prior to immunization may mitigate the overwhelming th2 immunity and lung pathology observed here with pref/alum, however, considering the important safety concern, further studies are needed to confirm this hypothesis. in contrast to the th2-skewed immunity of pref/alum, the combination of neutralizing antibody production, th1 immunity, and cytotoxic ifnγproducing cd8+t cells induced by pref/advax-sm provided vigorous protection from rsv replication without inducing pathology. taken together, these data suggest that rsv pref protein subunit vaccinations formulated with th1/th2-balanced adjuvants may provide complete rsv protection with a desirable safety profile. future studies will be needed to elucidate safety and protection of preimmune mice when immunized with pref alone or when formulated with a th1-vs th2-biased adjuvant. the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by the university of pittsburgh institutional animal care and use committee. kei, jk, and kem contributed to the overall study design, execution, and interpretation of data and writing of the paper. sg and az contribute through development of novel assays for generation of data. ml contributed to data interpretation, figure development, and writing. my contributed to data generation, study design, and analysis. tp and to contributed to the analysis and interpretation of data. cm and np contributed to study design. all authors contributed to the article and approved the submitted version. viral and host factors in human respiratory syncytial virus pathogenesis respiratory syncytial virus-associated hospitalizations among children less than 24 months of age global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study cost of respiratory syncytial virus-associated acute lower respiratory infection management in young children at the regional and global level: a systematic review and meta-analysis respiratory syncytial virus infection in elderly and high-risk adults viral respiratory infections in the institutionalized elderly: clinical and epidemiologic findings respiratory syncytial viral infection in children with compromised immune function serological array-in-well multiplex assay reveals a high rate of respiratory virus infections and reinfections in young children. msphere respiratory syncytial virus infection in adults structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus risk of respiratory syncytial virus infection for infants from low-income families in relationship to age, sex, ethnic group, and maternal antibody level correlates of immunity to respiratory syncytial virus (rsv) associated-hospitalization: establishment of minimum protective threshold levels of serum neutralizing antibodies an overview of novel adjuvants designed for improving vaccine efficacy vaccine adjuvants: understanding the structure and mechanism of adjuvanticity respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine alum adjuvant enhances protection against respiratory syncytial virus but exacerbates pulmonary inflammation by modulating multiple innate and adaptive immune cells a role for immune complexes in enhanced respiratory syncytial virus disease lack of antibody affinity maturation due to poor toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease pre-fusion f is absent on the surface of formalin-inactivated respiratory syncytial virus immunization with low doses of recombinant postfusion or prefusion respiratory syncytial virus f primes for vaccine-enhanced disease in the cotton rat model independently of the presence of a th1-biasing (gla-se) or th2-biasing (alum) adjuvant recommendations of the advisory committee on immunization practices for use of a hepatitis b vaccine with a novel adjuvant delta inulin-derived adjuvants that elicit th1 phenotype following vaccination reduces respiratory syncytial virus lung titers without a reduction in lung immunopathology severe acute respiratory syndrome-associated coronavirus vaccines formulated with delta inulin adjuvants provide enhanced protection while ameliorating lung eosinophilic immunopathology primary respiratory syncytial virus infection in mice stimulation of immature lung macrophages with intranasal interferon gamma in a novel neonatal mouse model of respiratory syncytial virus infection localization of the t-cell response to rsv infection is altered in infant mice alveolar macrophages support interferon gamma-mediated viral clearance in rsv-infected neonatal mice rage-dependent vcam-1 expression in the lung endothelium mediates il-33-induced allergic airway inflammation regulation of antibody isotype secretion by subsets of antigen-specific helper t cells je-advax vaccine protection against japanese encephalitis virus mediated by memory b cells in the absence of cd8(+) t cells and pre-exposure neutralizing antibody an inactivated cell culture japanese encephalitis vaccine (je-advax) formulated with delta inulin adjuvant provides robust heterologous protection against west nile encephalitis via cross-protective memory b cells and neutralizing antibody tlr9 agonist, but not tlr7/8, functions as an adjuvant to diminish fi-rsv vaccine-enhanced disease, while either agonist used as therapy during primary rsv infection increases disease severity cpg in combination with an inhibitor of notch signaling suppresses formalin-inactivated respiratory syncytial virus-enhanced airway hyperresponsiveness and inflammation by inhibiting th17 memory responses and promoting tissue-resident memory cells in lungs dendritic cells expressing myd88 molecule are necessary and sufficient for cpg-mediated inhibition of ige production in vivo risk of primary infection and reinfection with respiratory syncytial virus prefusion f, postfusion f, g antibodies, and disease severity in infants and young children with acute respiratory syncytial virus infection prefusion f-specific antibodies determine the magnitude of rsv neutralizing activity in human sera adjuvants and the vaccine response to the ds-cav1-stabilized fusion glycoprotein of respiratory syncytial virus impaired antibody-mediated protection and defective iga b-cell memory in experimental infection of adults with respiratory syncytial virus immunity to and frequency of reinfection with respiratory syncytial virus infection and disease with respect to age, immunologic status, race and sex distinct types of lung disease caused by functional subsets of antiviral t cells cd4+ t cells clear virus but augment disease in mice infected with respiratory syncytial virus. comparison with the effects of cd8+ t cells role of t lymphocyte subsets in the pathogenesis of primary infection and rechallenge with respiratory syncytial virus in mice cytotoxic t cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus virus clearance and immunopathology by cd8(+) t cells during infection with respiratory syncytial virus are mediated by ifn-gamma pulmonary t cells induced by respiratory syncytial virus are functional and can make an important contribution to long-lived protective immunity memory cd8 t cells mediate severe immunopathology following respiratory syncytial virus infection pre-existing neutralizing antibodies prevent cd8 t cell-mediated immunopathology following respiratory syncytial virus infection secreted respiratory syncytial virus g glycoprotein induces interleukin-5 (il-5), il-13, and eosinophilia by an il-4-independent mechanism a human respiratory syncytial virus (rsv) primate model of enhanced pulmonary pathology induced with a formalin-inactivated rsv vaccine but not a recombinant fg subunit vaccine rsv vaccine-enhanced disease is orchestrated by the combined actions of distinct cd4 t cell subsets vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with lot 100 or a newly prepared reference vaccine respiratory synctial virus infection in balb/c mice previously immunized with formalin-inactivated virus induces enhanced pulmonary inflammatory response with a predominant th2-like cytokine pattern group 2 innate lymphoid cells in pulmonary immunity and tissue homeostasis for clinical pharmaceutical sciences, university of pittsburgh school of pharmacy. this work benefitted from special bd lsrfortessatm funded by nih 1s10od011925-01 (pi: borghesi). np (vaxine pty ltd., bedford park, australia) generously provided advax-sm but did not otherwise provide financial assistance for this project. development of advax-sm adjuvant was supported by national institutes of health contracts hhsn272201400053c, hhsn272200800039c and ai061142. this publication's contents are solely the responsibility of the authors and do not necessarily represent the official views of the national institutes of health, national institute of allergy and infectious diseases. we would like to pay special thanks to dr. kevin legge for his thoughtful and constructive feedback during the writing of this manuscript. conflict of interest: np is affiliated with vaxine pty ltd., which hold commercial interests in advax adjuvants cm and my are affiliated with calder biosciences, which holds commercial interests in the stabilized pref protein. the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 eichinger, kosanovich, gidwani, zomback, lipp, perkins, oury, petrovsky, marshall, yondola and empey. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-277529-z2r14w2k authors: stella, alessandro; lamkanfi, mohamed; portincasa, piero title: familial mediterranean fever and covid-19: friends or foes? date: 2020-09-18 journal: front immunol doi: 10.3389/fimmu.2020.574593 sha: doc_id: 277529 cord_uid: z2r14w2k familial mediterranean fever (fmf) and covid-19 show a remarkable overlap of clinical symptoms and similar laboratory findings. both are characterized by fever, abdominal/chest pain, elevation of c-reactive protein, and leukocytosis. in addition, colchicine and il-1 inhibitors treatments that are effective in controlling inflammation in fmf patients have recently been proposed for off-label use in covid-19 patients. thus, fmf may resemble a milder recapitulation of the cytokine storm that is a hallmark of covid-19 patients progressing to severe disease. we analyzed the sequence of the mefv-encoded pyrin protein – whose mutations cause fmfin mammals, bats and pangolin. intriguingly, although pyrin is extremely conserved in species that are considered either a reservoir or intermediate hosts for sars-cov-2, some of the most common fmf-causing variants in humans are present as wildtype residues in these species. we propose that in humans, pyrin may have evolved to fight highly pathogenic infections. the world health organization reported a novel coronavirus on december 30, 2019 as the cause of a cluster of pneumonia cases in the city of wuhan in the hubei province of china. since then, the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has infected nearly 30 million individuals worldwide causing more than 900,000 deaths during the past 6 months of the covid-19 pandemic. among european countries, italy has been severely hit by covid-19 (1) . since the initial outbreak, a huge body of clinical and scientific information has been accumulated on covid-19, a multifaceted disease hitting not only lungs, but also other organs, with different defined stages (2, 3) . in most cases, sars-cov-2 enters the human body through inhaled droplets and aerosols. although contact with contaminated surfaces has been hypothesized as a second possible infection route, the importance of this alternative mode of infection has not been assessed systematically (4, 5) . upon infection, sars-cov-2 enters its target cells via: (a) binding of its spike protein (s) to angiotensin converting enzyme 2 (ace2); (b) activation through proteolysis of the viral s protein catalyzed by the cellular transmembrane protease serine 2 (tmprss2); and (c) fusion of sars-cov-2 virus with the host cell membrane. a wide variation in allele frequencies at ace2 expressions single nucleotide polymorphisms (esnps) loci can partly explain the differences in covid-19 prevalence across different countries (6) . also, differential expression of ace2 occurs in several human cancers and chronic diseases, possibly influencing covid-19 susceptibility and severity (7) . the involvement of ace2 and tmprss2 in viral cell entry has been exploited to plan experimental therapies based on using protease inhibitors such as camostat mesylate or nafamostat to block sars-cov-2 entry into cells (8, 9) . once inside the host cells, the sars-cov-2 positive single stranded rna (ssrna) genome begins replication and cytoplasmic accumulation. the ssrna or its double stranded intermediate (dsrna) are recognized by the innate immune nucleic acid sensing systems whose activation exerts a first antiviral response through the production of type 1 interferons and the secretion of pro-inflammatory cytokines. the immunomodulation at this early stage of infection might determine the growth of the viral load, with most infected individuals still being asymptomatic or paucisymptomatic, while almost 15% of sars-cov-2 positive patients develop fever, coughing, occasionally ageusia and anosmia and even gastrointestinal symptoms (3), with or without hepatic involvement (10) . patients progressing to the second phase show a strong immunological and hyperinflammatory response that is defined as a "cytokine storm" and which may lead to respiratory worsening and bilateral pneumonitis. in this second stage, covid-19 patients manifest symptoms mimicking those present in patients with auto-inflammatory diseases such as fever, arthralgia, leucopenia and myocarditis (11) (12) (13) . we were intrigued by the remarkable overlap between these clinical manifestations and some of the typical manifestations of familial mediterranean fever (fmf), a largely recessively inherited monogenic inflammasomopathy (autoinflammatory disorder involving the inflammasome) caused by mutations in the mefv gene that is particularly prevalent in the mediterranean basin (14) . while the previously mentioned clinical signs are not specific to fmf and shared with other hereditary recurrent fevers and inflammatory diseases, yet they represent an indication of similarities among fmf pathogenesis and the hyperinflammatory response observed in covid-19 patients. worthy of note, taste alteration has been reported amongst the prodromal manifestations preceding fever attacks in fmf (15, 16) . we should stress that the cytokine storm observed in the inflammatory stage of covid19, has been reported in other autoinflammatory diseases (aids) such as the macrophage activation syndrome (mas) and adult onset still's disease (17) . however, authors have often reported genetic heterogeneity and overlap between these aids and hereditary recurrent fevers (18) (19) (20) . colchicine, a natural alkaloid from colchicum autumnale, has a long history as a drug to treat pain and swelling since ancient egypt. it is nowadays used to treat fmf, gout, behçet syndrome and recurrent non-infective pericarditis. unsurprisingly, it is currently being investigated in several covid-19 therapeutic trials (21) (22) (23) . notably, in colchicine-resistant or colchicineintolerant fmf patients, alternative treatments include biologics that neutralize the pro-inflammatory cytokine interleukin (il)-1β directly (canakinumab) or inhibit il-1β-mediated activation of the il-1 receptor (anakinra and rilonacept) (24) (25) (26) . similarly to colchicine, several ongoing trials are evaluating the use of il-1 pathway inhibitors to treat covid-19 patients (27) (28) (29) (30) . although first results from covid-19 patients treated with these repurposed drugs have been conflicting (31) (32) (33) , the role of pyrin (the inflammasome sensor protein that is encoded by mefv) in modulating severity and outcome of covid-19 is still unknown. the pyrin domain architecture shows intriguing features which may add insights into its possible role in covid-19 disease. pyrin has a n-terminal pyrin domain (pyd) that is frequently found in other innate immune pathogen sensors that mount inflammasome responses such as nlrp1, nlrp3, and aim2. the n-terminal pyd domain engages in homotypic interactions with its pyd counterpart in the adaptor protein apoptosisassociated speck-like protein with a caspase recruitment domain (asc) to assemble asc specks, which recruit the inflammatory protease procaspase-1 inducing its self-cleavage and activation (34) . caspase-1 in turn matures the proinflammatory cytokines il-1β and il-18 and cleaves gasdermin d to trigger a lytic cell death mode termed pyroptosis that promotes secretion of aforementioned cytokines along with danger-associated molecular patterns (damps) such as il-1alpha, hmgb1 and atp (35) . pyroptosis is a double-edged sword with both antiviral and proviral activities during viral infections (36) . in fact, cell death can lead to halting viral replication and infection, frequently at the price of increased inflammation. conversely, dead cells release a large number of viral particles contributing to viral dissemination. the mefv encoded pyrin sensor contains in its central region three domains: a bzip domain (aa 370-412), a b-box domain (aa 370-412) and a coiled-coil domain (cc, aa 420-440). the role of these three domains has not been thoroughly investigated and few fmf-causing variants localize to pyrin's central region (37) . this region may have an autoinhibitory role precluding the pyd domain from interacting with asc and activating pyroptosis (38) . the c-terminal b30.2 (also known as pry/spry) domain is extremely important in fmf pathogenesis since most of the disease-penetrant mefv variants cluster in this region (39) . although this uneven distribution of fmf-associated mefv mutations suggests that the b30.2 domain is crucial in regulating pyrin inflammasome activity, the precise molecular mechanisms by which the b30.2 domain regulates fmf pathogenesis have not been elucidated (40) . likely, fmf-causing mutations in the b30.2 domain may derail intramolecular interactions that keep pyrin in an autoinhibitory state. interestingly, the degree of amino acid conservation along the pyrin protein sequence is rather variable and may offer some insights in the dangerous liaisons between fmf and covid-19. we aligned the mefv-encoded pyrin amino acid sequences from 19 different species including the only pangolin and all bat species sequences available in genbank (figure 1) . bats and pangolins have been considered the reservoir and intermediate host, respectively, of sars-cov-2 before transmission to humans. the alignment of the pyrin sequences presented unique evolutionary features (figure 1 ). in fact, some of the most prevalent fmf-associated mutations in human pyrin were present as wild type in all bat species analyzed and in pangolin (v726a, r761h). this resembles previous findings that some fmf-associated mutations are retrieved as wild type in primates, suggesting evolutionary pressure on pyrin (41) . in contrast, other fmf-associated amino acids residues that are largely prevalent in middle eastern populations (m680i, m694i, m694v) were not observed in bats and pangolin. worthy of note m680i, m694v, m694i, and r761h were all associated with derailed pyrin-induced il-1β secretion in a recently developed blood-based functional test for fmf alleles (42) . it is tempting to speculate that fmf patients carrying v726a and r761h variants-which represents the wild type residues in all bats and pangolin sequences-might modulate better their cytokine response to sars-cov-2 infection. further, in a mouse knock-in model the v726a variant causes a more severe fmf phenotype compared to m694v, m680i, and elevated production of multiple cytokines (43) . given that the m680i and m694v/i alleles in patients provoke a hyperinflammatory response not different from v726a and r761h (at least in fmf), one may hypothesize that they could warrant a comparable attenuation of viral infection. an elaboration on this hypothesis would stem from the increased fmf severity associated to m680i and m694v/i mutations. this raised level of inflammation can either move the balance toward excess inflammation in covid-19 or be causing an even improved immunomodulation in covid-19 compared to v726a, r761h. moreover, historic pandemics and different pathogens may have selected for different mefv variants in their respective host species and populations. thus, a pathogen different from coronaviruses (i.e., yersinia pestis) might have selected the m680i, m694v/i mutations in humans but not in bats. indeed, confirming this hypothesis, and shortly after the submission of this work, genetic and experimental evidences have been reported linking these mefv variants with resistance to yersinia pestis (44, 45) . the e148q and r202q mefv variants, which according to a current consensus are considered neutral polymorphisms, showed a response to colchicine challenge alike normal controls (42) . these two variants presented rather divergent evolutionary features. while the glutamic acid at position 148 was strictly conserved in all species analyzed, the arginine at position 202 was conserved in pangolin and in 7 of 13 bat species, and changed to a glutamine in mouse and rats (figure 2) . therefore, e148q and r202q which appear to be neutral in functional assays seem to be under apparently different evolutionary pressures. the frequency of mefv mutations and polymorphisms shows a great variability in countries where fmf has a high frequency. in fact, the m694v and m694i, are prevalent among turks, non-ashkhenazi jews, and arabs, while the m680i is frequent in the armenian population (46) (47) (48) (49) . the v726a and r761h are generally associated with a milder phenotype, and generally reported in clusters of fmf patients of ashkhenazi jewish origin, and in the western mediterranean area (50) . the e148q variant, whose pathogenicity is still debated, also presents a wide variation in frequency across different populations (51) . this peculiar distribution of the mefv variome led to the hypothesis that the mefv gene has been subjected to a balancing selection and nucleotide variation, particularly in the b30.2 region, is adaptive (52) . thus, the severity of covid-19 disease in fmf patients, once infected, might be influenced, at least partially, depending on specific mefv genotypes which shows country-specific differences. the mefv gene displays other intriguing evolutionary features. in fact, while the n-terminal pyd domain is conserved across all species, the b30.2 domain appears of more recent origin. of note, when considering the entire pyrin amino acid sequence, human pyrin is more closely related to its bats and pangolin homologs than to other mammal species (figure 3) . in contrast, the human nlrp3 protein presented a high level of homology in all analyzed species. it clustered with other mammalian nlrp3 proteins in the phylogenetic tree and was more distantly related to bat sequences (figure 4) . in addition, differently from mefv, the most common human nlrp3 mutations-r260w, d303n, t348m, and l355p-which represent more than 40% of the total mutation burden for this gene (53, 54) , were never present as wild type amino acid residues in all bat species and pangolin ( figure 5) . therefore, considering the pyrin inflammasome a passive bystander in sars-cov-2 infection could lead to overlooking an important innate immune pathway that coordinates the response to pathogens. in fact, inflammasome-driven pyroptosis is one of the results of the cytokine storm which concurs to the high pathogenicity of both sars-cov, and sars-cov-2 (55, 56). recent findings demonstrated that the sars-cov orf3a protein can provoke a cytokine storm by activation of the nlrp3 inflammasome through traf3-dependent ubiquitination of asc (57) . a recent analysis of 2782 sars-cov-2 strains showed that non-synonymous substitutions in the sars-cov-2 orf3a protein may alter virulence and infectivity (58) . additional viral proteins are able to activate the nlrp3 inflammasome. the nlrp3-mediated response to influenza a virus (iav) has been extensively studied and plays a crucial role in protecting the host while helping in clearing the infection. however, if the inflammatotory response is particularly prolonged and excessive it can increase disease burden (59) . the nlrp3 inflammasome plays a critical role in guarding against iav infection and reducing lung damage consequent to infection (59, 60) . other viruses are capable of activating the nlrp3 inflammasome. the viroporin 2b, released upon human rhinovirus infection (hrv), causes proteolytic activation of procaspase-1 and il-1ß secretion in a nlrp3 dependent manner (61) . several other viruses can provoke a sustained response from the nlrp3 inflammasome as extensively reviewed (62) . similarly to the mefv-encoded pyrin, the n-terminal pyd domain of nlrp3 can recruit asc via homotypic interaction with its pyd counterpart on asc to assemble asc filaments. the assembly of asc filaments in macromolecular structures known as asc specks allow the recruitment of procaspase-1 to induce its self-cleavage and activation. caspase-1 in turn cleaves the proinflammatory and the answer is. . .. before the covid-19 pandemic, a recent report (63) demonstrated that bats, when infected with different zoonotic figure 5 | alignment of 14 nlrp3 orthologs from amino acid 235 to amino acid 394 (human gene). the four residues whose mutations are responsible of more than 40% of nlrp3 -associated autoinflammatory diseases are boxed. frontiers in immunology | www.frontiersin.org 6 september 2020 | volume 11 | article 574593 viruses, can sustain high viral loads while presenting a dampened nlrp3-mediated inflammatory response associated with a decrease in both asc speck formation and il-1β secretion. hence, a dysregulation of the nlrp3 inflammasome can also contribute to the utterly complex individual immune response to viral infections (64) . these findings suggest that the modulation of innate immunity rather than an enhanced antiviral defense might shape the different outcome of covid-19 disease. thus, we hypothesize that competition between pyrin and the nlrp3 inflammasomes for asc recruitment may tilt the balance between a cytokine storm and a finely adjusted protective inflammation (figure 6) . fmf, in which pyrin activity and consequent asc oligomerization are increased because of mefv pathogenic variants, may therefore represent a unique opportunity as a disease model to investigate the regulation of the inflammatory response to novel emerging viruses. we presented here the unique evolutionary features of the mefv-encoded pyrin suggesting its putative contribution in shaping the individual risk to develop severe complications consequent to infectious diseases. several factors could have contributed to the rapid spreading of covid-19 pandemic, including access to adequate health care, aging demographic, metabolic dysfunctions, socio-cultural differences. it would be wise not to discard individual genetics, including the mefv gene, from the mix. future investigation on how carriers of different mefv genotypes have responded to coronavirus infections could help in refining existing and novel therapeutics in development for present and future challenges. in conclusion, the answer to the title question might not be blowing in the wind but could hopefully be found in a deeper knowledge of inflammasome regulation in well-known inflammatory diseases. the datasets presented in this study can be found in online repositories. the names of the repository/repositories and accession number(s) can be found in the article/ supplementary material. as designed the study and drafted the manuscript. ml and pp drafted the manuscript. all authors contributed to the article and approved the submitted version. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.574593/full#supplementary-material covid-19, internists and resilience: the north-south italy outbreak tocilizumab: from the rheumatology practice to the fight against covid-19, a virus infection with multiple faces covid−19: focus on the lungs but do not forget the gastrointestinal tract epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan comparative genetic analysis of the novel coronavirus (2019-ncov/sars-cov-2) receptor ace2 in different populations systematic profiling of ace2 expression in diverse physiological and pathological conditions for covid-19/sars-cov-2 identification of nafamostat as a potent inhibitor of middle east respiratory syndrome coronavirus s protein-mediated membrane fusion using the splitprotein-based cell-cell fusion assay sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor hepatic consequences of covid-19 infection. lapping or biting? dysregulated adaptive immune response contributes to severe covid-19 dysregulation of immune response in patients with coronavirus 2019 (covid-19) in wuhan, china cardiovascular disease in patients with autoinflammatory syndromes familial mediterranean fever in the world the prodrome: a prominent yet overlooked pre-attack manifestation of familial mediterranean fever familial mediterranean fever: clinical, molecular and management advancements the immunology of macrophage activation syndrome evidence for genetic overlap between adult onset still's disease and hereditary periodic fever syndromes still's disease mefv gene mutations and their clinical significance in korean patients with adult-onset still's disease the greek study in the effects of colchicine in covid-19 complications prevention (grecco-19 study): rationale and study design severe acute respiratory syndrome coronavirus viroporin 3a activates the nlrp3 inflammasome colchicine-a short history of an ancient drug biologic agents and more for the treatment of familial mediterranean fever. the old, the new, and the rare effect of interleukin-1 antagonists on the quality of life in familial mediterranean fever patients anti-il1 treatment in colchicine-resistant paediatric fmf patients: real life data from the helios registry repositioning parp inhibitors for sars-cov-2 infection (covid-19); a new multi-pronged therapy for ards? il−1 blockade with anakinra in acute leukaemia patients with severe covid−19 pneumonia appears safe and may result in clinical improvement safety and efficacy of early high-dose iv anakinra in severe covid-19 lung disease covid-19: consider cytokine storm syndromes and immunosuppression should we stimulate or suppress immune responses in covid-19? cytokine and anticytokine interventions colchicine may not be effective in covid-19 infection; it may even be harmful? continuous hydroxychloroquine or colchicine therapy does not prevent infection with sars-cov-2: insights from a large healthcare database analysis mechanisms and functions of inflammasomes emerging inflammasome effector mechanisms viruses and the diversity of cell death familial mediterranean fever: breaking all the (genetic) rules pyrin activates the asc pyroptosome in response to engagement by autoinflammatory pstpip1 mutants improvement of mefv gene variants classification to aid treatment decision making in familial mediterranean fever the b30.2 domain of pyrin, the familial mediterranean fever protein, interacts directly with caspase-1 to modulate il-1beta production episodic evolution of pyrin in primates: human mutations recapitulate ancestral amino acid states blood-based test for diagnosis and functional subtyping of familial mediterranean fever gain-offunction pyrin mutations induce nlrp3 protein-independent interleukin-1beta activation and severe autoinflammation in mice ancient familial mediterranean fever mutations in human pyrin and resistance to yersinia pestis the pyrin inflammasome in health and disease lack of clear and univocal genotype-phenotype correlation in familial mediterranean fever patients: a systematic review the population genetics of familial mediterranean fever: a meta-analysis study mefv mutations and their relation to major clinical symptoms of familial mediterranean fever a novel cluster of patients with familial mediterranean fever (fmf) in southern italy genetic epidemiology of familial mediterranean fever through integrative analysis of whole genome and exome sequences from middle east and north africa global epidemiology of familial mediterranean fever mutations using population exome sequences a population genetics study of the familial mediterranean fever gene: evidence of balancing selection under an overdominance regime nlrp3-associated autoinflammatory diseases: phenotypic and molecular characteristics of germline versus somatic mutations phenotypic and genotypic characteristics of cryopyrinassociated periodic syndrome: a series of 136 patients from the eurofever registry lessons learned to date on covid-19 hyperinflammatory syndrome: considerations for interventions to mitigate sars-cov-2 viral infection and detrimental hyperinflammation expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars severe acute respiratory syndrome coronavirus orf3a protein activates the nlrp3 inflammasome by promoting traf3-dependent ubiquitination of asc sars-cov-2 and orf3a: nonsynonymous mutations, functional domains, and viral pathogenesis. msystems the intracellular sensor nlrp3 mediates key innate and healing responses to influenza a virus via the regulation of caspase-1 the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna rhinovirus-induced calcium flux triggers nlrp3 and nlrc5 activation in bronchial cells response of host inflammasomes to viral infection dampened nlrp3-mediated inflammation in bats and implications for a special viral reservoir host severe covid-19: nlrp3 inflammasome dysregulated key: cord-306600-cxz8hf9q authors: matarazzo, laura; casilag, fiordiligie; porte, rémi; wallet, frederic; cayet, delphine; faveeuw, christelle; carnoy, christophe; sirard, jean-claude title: therapeutic synergy between antibiotics and pulmonary toll-like receptor 5 stimulation in antibiotic-sensitive or -resistant pneumonia date: 2019-04-09 journal: front immunol doi: 10.3389/fimmu.2019.00723 sha: doc_id: 306600 cord_uid: cxz8hf9q bacterial infections of the respiratory tract constitute a major cause of death worldwide. given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. in this context, innovative therapeutic strategies are urgently needed. stimulation of innate immune cells in the respiratory tract [via activation of toll-like receptors (tlrs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. previous studies of the tlr5 agonist flagellin in animal models showed that standalone tlr stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic–susceptible pneumococcus. we next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than 100-fold compared to standalone treatments. our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. in conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria. bacterial infections of the respiratory tract constitute a major cause of death worldwide. given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. in this context, innovative therapeutic strategies are urgently needed. stimulation of innate immune cells in the respiratory tract [via activation of toll-like receptors (tlrs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. previous studies of the tlr5 agonist flagellin in animal models showed that standalone tlr stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic-susceptible pneumococcus. we next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than 100-fold compared to standalone treatments. our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. in conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria. keywords: flagellin, toll-like receptor 5, antibiotic, resistance, streptococcus pneumoniae, pneumonia, superinfection introduction pneumonia constitutes a major cause of death, morbidity and health resource use worldwide. the main causative agents identified in adult patients hospitalized for community-acquired pneumonia (cap) are viruses (in 27-30% of cases, the most common being rhinovirus, influenza and coronavirus) and bacteria (14-23% of cases, with a marked predominance of streptococcus pneumoniae infections) (1) (2) (3) . when faced with overt clinical signs of bacterial pneumonia, the standard of care is antibiotic treatment. the combination of a constant rise in antibiotic resistance in recent decades with a decline in the discovery of new drugs has led to an increase in treatment failure and mortality (4) . in 2017, the world health organization's global action plan highlighted the urgent need to control the emergence of antibiotic resistance (5) . given this context, a number of new anti-infectious treatment strategies are being developed. the modulation of innate immunity [by targeting immune receptors, such as toll-like receptors (tlrs)] is a promising approach (6, 7) . indeed, innate immunity is highly conserved in evolution, and this system constitutes the first line of defense against invading pathogens. moreover, innate immunity triggers a broad range of antimicrobial defense mechanisms and immune cells-thereby greatly reducing the risk of resistance in the pathogens. moreover, activation of tlr signaling has been associated with a favorable outcome in infections with antibiotic-resistant bacteria or colonization resistance by such pathogens (8) (9) (10) . these observations support that stimulation and effector activities of innate immunity are not influenced by the antibiotic resistance mechanisms carried by bacteria. flagellin is the main protein component of the bacterial flagellum and is a natural agonist of tlr5; the latter is expressed at the surface of a many different cell types, including mucosal epithelial cells and immune cells such as dendritic cells, macrophages, and lymphocytes (11) . various studies in animal models have highlighted the antimicrobial potency of flagellin against a wide variety of bacterial infections [such as intestinal infections caused by salmonella enterica, enterococcus faecium, clostridium difficile, and escherichia coli (8, (12) (13) (14) , respiratory infections caused by pseudomonas aeruginosa and s. pneumoniae (15, 16) ], and viral and fungal infections (17) (18) (19) . although most studies have demonstrated the protective effect of flagellin administered before or during exposure to a microbial pathogen, the protein's immunostimulatory efficacy in therapeutic context has not been extensively characterized. using a mouse model of s. pneumoniae lung infection, we recently demonstrated that combination treatment with mucosally administered flagellin and an orally or intraperitoneally administered low-dose (i.e., subtherapeutic) antibiotic is more effective than the antibiotic alone (i.e., with a lower bacterial load in the lung, and a lower mortality rate). furthermore, the combination treatment was also effective in a model of post-flu pneumococcal superinfection (20) . the effectiveness of these combination therapies depends on tlr5 signaling as demonstrated using tlr5-deficient animals and tlr5-mutated recombinant flagellin (20) . our studies highlighted that the airway epithelium is the main tlr5-specific signaling compartment (21) (22) (23) . taken as a whole, these observations are the first to highlight the added value of respiratory delivery of flagellin as an immunomodulatory biologic for the adjunct treatment of bacterial pneumonia (i.e., in addition to the standard of care). our working hypothesis was that simultaneous treatment with an antibiotic and intranasal, i.e., respiratory flagellin constitutes a "double hit" against the pathogen. a combination of two drugs may result in independent actions or specific (i.e., additive, synergistic, or antagonistic) effects that define the biological outcome (24) (25) (26) . an interaction between two drugs is considered to be synergistic when the measured effect of the combination treatment exceeds the predicted cumulative value of the two components given separately. synergy increases treatment efficacy, and is expected to limit the emergence of drug resistance. furthermore, synergy allows the physician to decrease the dose level or the frequency of dosing, which thereby dampens adverse drug reactions and may even enable the rehabilitation of neglected drugs. conversely, an antagonistic combination treatment has a smaller effect than the predicted cumulative value of the two components given separately. most studies of potentially synergistic antimicrobial agents are performed in in vitro systems such as bacterial cultures, using checkerboard assays and increasing doses of each drug (25, 27) . unlike antibiotics that directly affect the bacteria, immunomodulatory biologic activity requires sentinel cells for detection, downstream signaling and thus the production of antimicrobial effectors and the recruitment and/or activation of innate immune cells. at present, there are no comprehensive in vitro models of this complicated physiological system. in the present study, we quantified the nature and magnitude of the interactions between antibiotics and intranasal instillation of flagellin with regard to antibacterial effectiveness in a murine model of s. pneumoniae respiratory infections. furthermore, we wanted to assess the efficacy of this novel therapeutic strategy against infection with antibiotic-resistant bacteria, which represents major public health issues today. to this aim, we investigated the combination's effect on antibiotic-resistant s. pneumoniae in a relevant model of post-flu pneumococcal pneumonia, and characterized the immune response induced by the flagellin-mediated protection. serotype 1 s. pneumoniae (sp1; clinical isolate e1586) was obtained from the national reference laboratory-ministry of health, uruguay (15) . serotype 3 s. pneumoniae (sp3; strain 104491) was provided by the institut pasteur (paris, france); it is a multidrug-resistant clinical isolate from a human bronchial secretion, and is resistant to amoxicillin (amx), cefotaxime, doxycycline, erythromycin, chloramphenicol, streptomycin, and cotrimoxazole (sxt). working stocks were prepared as described previously (15, 28) . briefly, fresh colonies grown on bloodagar plates were incubated in todd hewitt yeast broth (thyb) (sigma-aldrich, saint-louis, mo) at 37 • c until the od 600nm reached 0.7-0.9 units. cultures were stored at −80 • c in thyb + glycerol 12% (vol./vol.) for up to 3 months. for infection, working stocks were thawed and washed with sterile dulbecco's phosphate-buffered saline (pbs, gibco, grand island, ny) and diluted to the appropriate concentration. the number of bacteria (as colony forming units [cfus] ) was confirmed by plating serial dilutions onto 5% sheep blood agar plates. female balb/cj mice, female swiss mice, and male c57bl/6j mice (6-8 weeks old) (janvier laboratories, saint berthevin, france, or envigo, huntingdon, uk) were maintained in individually ventilated cages and handled in a vertical laminar flow cabinet (class ii a2, esco, hatboro, pa). all experiments complied with institutional regulations and ethical guidelines (c59-350009, institut pasteur de lille; protocol 2015121722429127). prior to intranasal infection, the mice were anesthetized via the intraperitoneal injection of 1.25 mg (50 mg/kg) ketamine plus 0.25 mg (10 mg/kg) xylazine in 250 µl of pbs. for primary infections with sp1, 2-4 × 10 6 cfu were inoculated intranasally in 30 µl pbs, as described previously (20) . the influenza infection model was developed in our laboratory on the c57bl/6j mice (29, 30) . the sp3 pneumococcal superinfection model was therefore performed in these animals. briefly, mice were first infected intranasally with 30 µl pbs containing 50 plaque-forming units (pfus) of the pathogenic, murine-adapted h3n2 influenza a virus strain scotland/20/74, as described previously (30, 31) . seven days later, animals were infected intranasally with 10 3 cfu of sp3 in 30 µl pbs. for the determination of bacterial counts in lung and spleen, mice were sacrificed at selected times via the intraperitoneal injection of 5.47 mg of sodium pentobarbital in 100 µl pbs. tissues were collected and homogenized with an ultraturrax homogenizer (ika-werke, staufen, germany), and viable counts were determined by plating serial dilutions onto blood agar plates and incubating them at 37 • c for 12-24 h. the recombinant flagellin flic 174−400 came from s. enterica serovar typhimurium flic and was produced with an histidine tag, as described previously (20, 32) . the protein flic 174−400 was certified to be immunologically active in reporter cells and in mouse assays, and the residual lipopolysaccharide concentration was determined to be <20 pg per µg of flagellin (20) . the treatments' effects on s. pneumoniae lung infection were quantified as the percentage bacterial growth (% growth ), corresponding to the ratio of the mean bacterial load in the lungs of infected, treated mice to the load in infected, nontreated (control) mice. for example, the effect of treatment a was calculated as follows: % growth[a] = (mean cfu [a] /mean cfu [control] ) × 100. the predicted additive effect (or predicted % growth ) of a combination treatment was calculated as described previously (33) . briefly, the predicted % growth of a treatment combining compounds a and b is the product of the experimentally defined % growth values for each standalone treatment (predicted% growth[a+b] = % growth[a] × % growth [b] ). if the experimental % growth for the combination treatment is lower or higher than the predicted % growth , then the two drugs are synergistic or antagonistic, respectively. when the experimental and predicted % growth values are identical, the two drugs' effects are additive. total lung rna was extracted with the nucleospin rna plus kit (macherey-nagel, duren, germany) and reverse-transcribed with the high-capacity cdna archive kit (applied biosystems, foster city, ca). the cdna was amplified using sybr greenbased real-time pcr on a quantstudio 12k pcr system (applied biosystems). relative mrna levels (2 − ct ) were determined by comparing first the pcr cycle thresholds (c q ) for the gene of interest and the reference genes actb and b2m ( c q ), and then the c q values for infected mice treated with the amx+flagellin combination treatment and with amx alone (control group) ( cq). all the primers used in the study (listed in table 1 ) were validated for efficacy. bronchoalveolar lavage (bal) fluid samples were obtained after intratracheal injection of 3 × 1 ml of pbs supplemented with 5% fetal calf serum (fcs). lungs were perfused with pbs, excised and finely minced then digested in a solution of rpmi 1640 medium (gibco) containing 1 mg/ml collagenase viii (sigma-aldrich) and 80 µg/ml dnase i (sigma-aldrich) for 20 min at 37 • c. after washes, red blood cells were removed using a lysis solution (pharmlyse, bd bioscience). lung cell homogenates were then suspended in a 20% percoll gradient and centrifuged at 2,000 rpm without brake at room temperature for 10 min. the cell pellets were washed with pbs supplemented with 2% fcs and cells were filtrated before antibody labeling. bal and lung cells were stained with anti-cd45-allophycocyanin-cyanine 7 (clone 30f11), anti-cd11b-brilliant violet 785 (clone m1.70), anti-siglecf-alexafluor 647 (clone e50-2440), anti-ly6c-peridinin chlorophyll protein-cyanine 5.5 (clone hk1.4), anti-ly6gphycoerythrin (clone 1a8), anti-cd11c-phycoerythrin-cyanine 7 (clone hl3), and ccr2-brillant violet 421 (clone sa203g11) antibodies. dead cells were excluded from the analysis using propidium iodide. the antibodies were purchased from bd biosciences (san jose, ca) or biolegend (san diego, ca). data were collected on a bd lsr fortessa and analyzed with the bd facsdiva software. concentration of ccl20, cxcl1, cxcl2, il-6, il-1β, and tnf was determined in bal fluids and lung homogenates by enzymelinked immunosorbent assay (elisa kit from ebioscience, r&d systems or becton dickinson). bal fluids were obtained by intratracheal injection of 2 × 1 ml pbs supplemented with protease inhibitors (roche). lungs were perfused with pbs and collected in t-per reagent (pierce) supplemented with protease inhibitors and debris were eliminated by centrifugation. all samples were stored at −20 • c. the results were described as the mean ± standard error of the mean (sem) or the median (range), as indicated. intergroup differences were analyzed using the mann-whitney test and the log rank test. all analyses were performed with prism software (version 5.0, graphpad software, la jolla, ca). the threshold for statistical significance was set to p < 0.05. in earlier research, we had shown that the intranasal administration of a combination of flagellin flic 174−400 and low-dose antibiotics improved the therapeutic outcome of lung infection with the antibiotic-susceptible sp1 [minimum inhibitory concentration (mic) amx = 0.016 µg/ml] (20) . given the difficulty of performing in vitro checkerboard assays with immunomodulators, we therefore sought to evaluate the nature of antibiotic-flagellin interactions in vivo. we first defined the dose of flagellin that promoted saturating immune responses in sp1-infected mice (figure 1) . intranasally administered flagellin was associated with the production of various innate immunity-related components, including chemokines (cxcl1, cxcl2, and ccl20), inflammatory cytokines (il-1β and il-6), and antimicrobial peptides (s100a9), along with the recruitment of neutrophils to the airways (15, 16, 20, 21, 23, 28) . mice were treated simultaneously with oral amx (0.2 mg/kg) and intranasal flagellin flic 174−400 (at doses of 0.4 µg to 1 mg/kg, i.e., 1 ng to 25 µg per animal). immune responses were analyzed by monitoring the lung transcription of inflammatory genes associated with tlr5 signaling and by comparing mrna levels to animals that received amx alone. the results showed that doses from 1 to 25 µg per animal saturated the upregulation of transcriptional response for cxcl1, cxcl2, ccl20, il1b, and il6 genes. ultimately, the dose of 2.5 µg of flic 174−400 was selected as a saturating immunostimulatory dose in the context of pneumococcal infection and lung inflammation. the next set of experiments was designed to characterize the therapeutic interaction between intranasal flagellin flic 174−400 and oral amx. mice were infected with sp1 and treated 12 h later with either a single intranasal instillation of flagellin (2.5 µg), a single intragastric administration of suboptimal amx doses of 5 µg (0.2 mg/kg) or 40 µg (1.6 mg/kg) or the combination treatment. to define the treatments' efficacy, lung bacterial counts were measured at 12 h post-treatment. the results showed that flagellin alone had mostly no antibacterial effect, whereas 5 and 40 µg doses of amx alone were, respectively, associated with 5-and 7-fold smaller bacterial loads, relative to untreated mice (figure 2a) . the combination treatment (amx + flic 174−400 ) induced a 10-fold relative decrease in bacterial counts for 5 µg of amx and a 82-fold relative decrease for 40 µg of amx-showing that amx-flagellin combination treatment is more effective than the corresponding dose of amx or flagellin as monotherapy (figure 2a) . the nature of the interactions between flagellin and antibiotics was further analyzed by comparing the bacterial growth upon treatment. the % growth values for the combination treatment (8.4% for flagellin + amx 5 µg and 1.2% for flagellin + amx 40 µg) were much lower than the corresponding predicted % growth values for additive effects, calculated as % growth[amx] × % growth[flagellin] (19.2% for flagellin + amx 5 µg and 12.3% for flagellin + amx 40 µg) (figure 2b) . this experiment indicated strong synergy between the two compounds. similar experiments were carried out with the combination of the antibiotic sxt and flagellin (figures 2c,d) . the antibiotic sxt was administered intraperitoneally at doses of 1 and 4 mg (40 and 160 mg/kg, respectively). flagellin (2.5 µg) significantly improved the therapeutic outcome of sxt treatment, as evidenced by cfu counts in the mice's lungs 12 h after administration of the treatments ( figure 2c) . the experimental % growth values for the combination treatment were lower than the corresponding predicted % growth values (14 vs. 23.5% for sxt 1 mg, and 0.88 vs. 7.3% for sxt 4 mg)-reflecting a synergy between flagellin and sxt ( figure 2d ). taken as a whole, these results show that antibiotics + flagellin had a strong synergistic effect on pneumococcal lung infection in mice. furthermore, the synergy seems to be independent of the type of antibiotic, since it was observed with a compound that inhibits bacterial cell wall (amx) and a pair of compounds that inhibits folic acid synthesis (sxt). next, we looked at whether the combination treatment's effect on an antibiotic-sensitive s. pneumoniae strain was also exerted on antibiotic-resistant bacteria. to this end, a mouse model of infection with a sp3 strain that is resistant to a wide range of antibiotics including amx (mic amx = 2 µg/ml, i.e., 125-fold higher than for sp1) was developed. we found that the sp3 strain failed to induce a lethal infection and other signs of disease (weight loss) in naïve mice-even at high doses of challenge (10 6 or 10 7 bacteria per animal) (figures 3a,b) . given that the influenza virus infection increases susceptibility to bacterial infections even after it has been eliminated (34-37), sp3 infection was assessed in mice that had already been exposed to the virus. briefly, mice were infected first with an intranasal, sublethal dose of h3n2 virus (50 pfu) and then infected 7 days later with 10 3 cfu of sp3. this bacterial superinfection induced significant weight loss and was 100% lethal (figures 3a,b) . the bacterial counts increased gradually over time, and reached 10 7 cfu per lung 24 h post-infection ( figure 3c) . sp3 was also detected in the spleen-indicating a translocation and systemic dissemination of the bacteria-from 24 h post-infection onwards (figure 3c ). in conclusion, the antibiotic-resistant sp3 strain induced effective pneumonia when animals had been previously exposed to experimental flu. in order to test the efficacy of an antibiotic+flagellin combination treatment in the post-influenza sp3 superinfection model, mice were treated with amx alone, flagellin flic 174−400 alone, or a combination of both compounds 12 h after the bacterial infection ( figure 4a ). due to the high level of amx resistance, the doses of antibiotic used were 100 µg (4 mg/kg) and 350 µg (14 mg/kg). using this regimen, the serum concentration levels of amx in naïve animals were expected to be close to 1 × mic and 4 × mic, respectively (professor charlotte kloft, personal communication). flagellin treatment alone decreased bacterial counts in the lungs by 5.6-fold, whereas amx treatments decreased bacterial counts by 3.7-fold (for a dose of 100 µg) and 74.6-fold (for a dose of 350 µg). when amx was combined with flagellin, bacterial counts were of 5,526-and 5,485-fold lower for the 100 and 350 µg doses of antibiotic, respectively. these results show a significant therapeutic advantage for the combination treatment, relative to standalone amx or flagellin treatments ( figure 4b) . we also determined cfu counts in the spleen; both amx and amx + flagellin treatments (either with 100 or 350 µg of the antibiotic) were able to prevent systemic dissemination of the infection (data not shown). comparison of % growth for the observed effect of the combination treatment vs. predicted additive effect (0.7 vs. 8.9% for amx 100 µg, and 0.02 vs. 0.9% for amx 350 µg) demonstrated the synergy of the combination in the context of superinfection and antibiotic resistance ( figure 4c) . after two administrations of treatments 12 and 36 h after sp3 superinfection, the flagellin + amx combination was found to significantly improve the survival of mice, relative to standalone treatments ( figure 4d) . these data strongly suggest that flagellin + amx have synergistic therapeutic effects to control the antibiotic-resistant pneumococcal infections in relevant pathophysiological contexts. since infection by influenza virus induces major changes in lung integrity and immune cell populations, we investigated the immunomodulatory impact of flagellin on post-flu respiratory infections by the antibiotic-resistant sp3 strain. to this end, c57bl/6 mice were infected with influenza a virus at day 0 and then challenged with antibiotic-resistant sp3 at day 7. treatments with oral amx (100 µg) combined or not with intranasal flagellin (2.5 µg) were administered 12 h after sp3 infection. lungs were collected 2 h post-treatment for transcriptional analysis using rt-qpcr assays, as described in figure 1 . we observed that despite the superinfection, flagellin still enhanced the transcription of cxcl1, cxcl2, ccl20, il1b, il6, and s100a9 genes, i.e., surrogate markers of tlr5mediated lung stimulation ( figure 5a) . we next quantified the cytokine/chemokine production after 6 h of treatment both in the bal fluids and lung protein extracts. delivery of flagellin in the lung of amx-treated pneumococcal superinfection significantly increased levels of ccl20, cxcl1, cxcl2, and tumor-necrosis factor (tnf) both in the lungs ( figure 5b ) and in the bal fluids ( figure 5c ) in amx+flagellin-treated mice compared with amx+pbs-treated mice. we also observed increased il-6 production in both compartments although it was not statistically significant. production of il-1β (or pro-il-1β) was detected only in the lung tissue and was increased in flagellin-treated mice. finally, we used flow cytometry to evaluate immune cell populations in bal fluids and lung tissue collected 12 h posttreatment. the analysis showed that the neutrophil counts were higher in mice having receiving the combination treatment (i.e., tlr5 stimulation and amx) than in mice having receiving amx alone both in the lung tissues ( figure 5d ) and the bal fluids ( figure 5e) . interestingly, the innate response to combination treatment was also detectable in blood since the production of the inflammatory mediators were significantly augmented at 2 h (for il-6, ccl20, cxcl1, and cxcl2) and 6 h (ccl20 and cxcl1) compared to amx alone treatment (supplementary figure 1) . the blood cytokine production then diminished to an undetectable or very low level at 12 h. thus, these observations showed that the mucosal delivery of flagellin does not induce sustained systemic inflammation. overall, the innate immune response to flagellin was effectively stimulated in the context of the influenza immunological imprinting, the superinfection challenge, and the antibiotic treatment. our present results demonstrated the synergistic efficacy of a combination of an antibiotic (amx or sxt) and the local administration of the immunomodulatory biologic flagellin against respiratory infections caused by s. pneumoniae. of note, the efficacy of combined antibiotic + flagellin treatment, previously demonstrated in inbred balb/c and c57bl/6 mice by porte et al. (20) , was here extended to outbred swiss mice, showing genetic background independence of the protection. remarkably, flagellin was able to trigger lung innate immune responses in the context of inflammation (i.e., airways damaged by bacterial pneumonia and flu). immunostimulation in the lung was a dose-dependent process that was saturating by microgram-per-animal levels of flagellin. the synergy appeared to be independent of the antibiotic dose level and the antibiotic's target, since amx acts on the bacterial cell wall and sxt inhibits dna synthesis. the present study is also the first to have demonstrated that stimulating innate immunity can treat severe pneumonia induced by antibiotic-resistant pathogenic bacteria; this may open up new avenues for the treatment of pneumonia in the context of growing antimicrobial resistance. it has been demonstrated that intranasal administration of flagellin activates tlr5-dependent local innate responses with broad-spectrum antibacterial activity (11, 15, 16, 20, 21, 23) . the pulmonary response includes the production of various antimicrobial peptides (i.e., cathelicidin antimicrobial peptide and the β-defensins), cytokines (tnf, il-1β, and il-6), and chemokines (i.e., ccl20, cxcl1, cxcl2, cxcl5, and cxcl8). this cytokine and chemokine production is in line with the observed recruitment of phagocytes (and especially neutrophils) in the lung following the intranasal administration of flagellin to naïve mice (15, 23, 38) . flagellin intranasal administration specifically triggers tlr5-mediated transcription in the lungs from 2 to 30 h after a pneumococcus infection or from 7 to 14 days after an influenza infection (20) . here, we demonstrated that the lung innate immune signature induced by intranasal instillation of flagellin is still effective in a highly inflammatory context with associated lung damage (pneumococcal postinfluenza superinfection), and is not influenced by antibiotic treatment (figure 5) . interestingly, earlier reports indicated that influenza infections promote the partial but sustained desensitization of tlr-mediated lung innate responses and a reduction in tlr expression (39) . our observations demonstrate that, in the physiopathological context of superinfection, flagellin is still able to trigger sufficient levels of innate defense and exert synergy with antibiotics (figure 4) . airway epithelial cells have been identified as an important component for detection of flagellin and tlr5 signaling at homeostasis (21, 22) . these sentinel cells not only sense danger signals introduced in the conducting airways but also produce factors to directly impair the colonization and growth of pathogens or indirectly mobilize phagocytic and immune cells to clear infection. more generally, airway epithelium tlr signaling represent a key driving force in antibacterial defense (40). recently, anas et al. demonstrated an essential contribution of epithelial signaling in the respiratory tract in response to flagellin in the context of infection with pseudomonas aeruginosa (41) . our data showed that several antimicrobial peptides (s100a9), cytokines (il-1β and tnf), and chemokines (ccl20, cxcl1, and cxcl2) that were associated to epithelial responses are also upregulated after the administration of the combination treatment in the post-flu superinfection model, suggesting that the epithelium is also an important flagellin-specific driving force in the lung damaged by viral and bacterial infections. targeting epithelium is a serious benefit for immunostimulation since it allows reducing the dose and bypassing systemic adverse effects. our data contribute to highlight the therapeutic potential of the association of two drugs with distinct modes of action: an antibiotic with a direct effect on bacteria, and a tlr5specific stimulator of innate immunity with indirect antibacterial activity mobilizing both multiple phagocytic host cells and various antimicrobial factors such as antibacterial peptides, and chemokines and cytokines that mobilize and activate immune cells. besides pathogen killing, the multitargeting of innate immunity by flagellin could impact on bacterial fitness and thereby increase susceptibility to the antibiotic. the innate immune response induced by tlr5 signaling may also modify the distribution of antibiotic in lung tissues while the antibiotic, by damaging the pathogen, could also enhance the immune signaling. in addition, the pharmacokinetics of the antibiotic and the immunostimulator, i.e., a short-term dose-dependent effect for the antibiotic, and an immediate and long-lasting impact of the immunostimulator due to cell mobilization, are likely complementary. finally, flagellin, by modulating innate immunity in the respiratory tract, has been shown to enhance the mucosal and systemic adaptive immunity (22, 23) . such property may be of interest to elicit anti-pathogen immune memory and prevent recurrent/relapse infections. as an opportunistic bacterium, s. pneumoniae frequently colonizes the upper respiratory tract and thus represents lungs were collected 2 h after treatment, and homogenized. after rna extraction, expression levels of selected genes were then analyzed using rt-qpcr assays. the relative expression level for each gene is expressed against that of the reference genes actb and b2m and the reference condition amx+pbs (arbitrarily set to a value of 1). the data represent the mean ± sem. lungs (b) and bal fluids (c) were collected 6 h after treatment and cytokine (continued) figure 5 | and chemokine levels were measured by elisa. data from amx+flagellin-treated and amx+pbs-treated mice were compared in a mann-whitney test and are represented as individual values and mean. lungs (d) and bals (e) were collected 12 h after treatment. lungs and bal cell suspensions were stained using a mixture of antibodies specific for surface markers before flow cytometry analysis. neutrophils were defined as cd45 + cd11b + ly6g + cells after exclusion of dead cells and alveolar macrophages (cd45 + siglecf + cd11c + cells) from the analysis. numbers of neutrophils in the lung parenchyma (d) and bal fluids (e) are shown for individual animal and the line represents the mean. data from amx+flagellin group were compared to those of amx+pbs group in a mann-whitney test. statistical significance is indicated as follows: *p < 0.05, and **p < 0.01. the prime cause of bacterial-associated cap (42) . however, other microorganisms can cause cap and healthcare-associated pneumonia; they include gram-positive bacteria such as staphylococcus aureus, gram-negative bacteria like p. aeruginosa, klebsiella pneumoniae, haemophilus influenzae, mycoplasma (m. pneumoniae) and intracellular bacteria (legionella pneumophila) (1). the diagnosis and treatment of cap is complicated by the broad variety of causative agents, and the progression of antibacterial resistance. in this context, immunomodulators such as flagellin are of great interest because they activate a large number of antimicrobial immune mechanisms. indeed, flagellin has already demonstrated its ability to protect against various pathogens including gram-negative and gram-positive bacteria (8, (12) (13) (14) (15) (16) 20) . furthermore, our present results showed that the therapeutic synergy between antibiotic and intranasal flagellin is independent of the antibiotic's mechanism of actionsuggesting that flagellin can potentially be combined with various antibiotics for a wide range of clinical situations. the synergistic effects of the combined therapy have been determined to be independent of capsule antigenicity (serotype 1 or 3) of pneumococcus, suggesting that the general innate immune protecting mechanisms triggered by flagellin could potentially be effective against a large variety of serotypes. given the progression of antibiotic resistance, a model of infection by antibiotic-resistant bacteria would constitute an important tool for developing alternative anti-infectious approaches. we first attempted to develop such a model in immunocompetent animals. the multidrug-resistant clinical isolate of pneumococcus sp3 was unable to induce a lethal infection, even at high doses. acquisition of antibiotic resistance is often associated with a loss of bacterial fitness (43) , which might explain the sp3's very low virulence in naïve mice. it is now becoming clear that many cases of bacterial pneumonia result from co-infections or consecutive infections (especially influenza virus infections) (37) . as shown by figures 3d,e, 4 , influenza virus infection creates a favorable environment for colonization and invasion by the low-virulence antibioticresistant pneumococcus sp3 strain. our data demonstrated that the flagellin+amx combination treatment effectively reduces the bacterial burden caused by the sp3 strain in the lung, and improves the survival rate among treated mice. our proof-of-concept findings may be transposable to the clinic for patients with co-infections and superinfections, which are relevant physiopathological causes of hospitalization and complicated pneumonia. antibiotics constitute the current standard of care for bacterial pneumonia, and the growing threat of antibiotic resistance is a major public health concern. when defining the dosing regiments of antibiotics used to treat a patient, the physician must take account of the antibiotic' pharmacokinetic and pharmacodynamic characteristics. the relationship between in vivo exposure to the drug and in vitro susceptibility of the bacteria conditions not only the treatment's clinical outcome (i.e., clearance of the infection) but also adverse effects or drug toxicity (44) . thus, the maximum dose of antibiotic that can be administered to a patient may not be enough to totally clear highly resistant bacteria. our data suggest that the antibacterial efficacy of these antibiotic dose levels can be synergistically enhanced by the effect of flagellin on lung innate immunity. taken as a whole, the present results suggest that the selective boosting of innate lung immunity by flagellin improves the therapeutic outcome of antibiotic treatment. in humans, this approach might be a useful generic alternative to the treatment of bacterial pneumonia, thereby reducing the antibiotic dose and regimen as well as the emergence of antibiotic resistance. moreover, such strategy promotes multitarget inhibition through multiple innate immune effectors that should be more resistant to the development of resistance and may restore some antibacterial activity to antibiotic in the context of antibiotic resistance. characterization of flagellin's contribution to the lung antibacterial defenses at the molecular and cellular level and the protein's synergy with antibiotics is likely to open up new avenues for the immunotherapy of respiratory tract infections. all experiments complied with institutional regulations and ethical guidelines (c59-350009, institut pasteur de lille; protocol apafis #5164 -2015121722429127). the protocols were validated by the ethical committee for animal experiments (comitéd'éthique en expérimentation animale-nord-pas-de-calais ceea 75). lm performed all animal, rt-qpcr, and flow cytometry experiments. fc, rp, dc, and cf provided lm with technical assistance. fw analyzed the bacterial species and antibiotic resistance. lm, cc, and j-cs designed experiments and wrote the manuscript. j-cs and cc supervised the experimental work as a whole. the study was funded by inserm, institut pasteur de lille, université de lille, inserm-transfert (grant: copoc innatebiotic r12041es), and the era-net joint programming initiative on antimicrobial resistance and the agence nationale de la recherche (grant anr-15-jamr-0001-01). lm is a fellow of the innovation pharmaceutique et recherche program. aetiology of lower respiratory tract infection in adults in primary care: a prospective study in 11 european countries community-acquired pneumonia requiring hospitalization among u.s. adults dynamics of lung defense in pneumonia: resistance, resilience, and remodeling overcoming the current deadlock in antibiotic research global action plan on antimicrobial resistance targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases modulating immunity as a therapy for bacterial infections bacterial flagellin stimulates toll-like receptor 5-dependent defense against vancomycin-resistant enterococcus infection vancomycinresistant enterococci exploit antibiotic-induced innate immune deficits tlr-7 activation enhances il-22-mediated colonization resistance against vancomycin-resistant enterococcus compartmentalized antimicrobial defenses in response to flagellin flagellin treatment protects against chemicals, bacteria, viruses, and radiation toll-like receptor 5 stimulation protects mice from acute clostridium difficile colitis cutting edge: tlr5-/-mice are more susceptible to escherichia coli urinary tract infection mucosal administration of flagellin protects mice from streptococcus pneumoniae lung infection flagellin stimulates protective lung mucosal immunity: role of cathelicidin-related antimicrobial peptide viral infection. prevention and cure of rotavirus infection via tlr5/nlrc4-mediated production of il-22 and il-18 recombinant tlr5 agonist cblb502 promotes nk cell-mediated anti-cmv immunity in mice flagellin-induced expression of cxcl10 mediates direct fungal killing and recruitment of nk cells to the cornea in response to candida albicans infection a toll-like receptor 5 agonist improves the efficacy of antibiotics in treatment of primary and influenza virus-associated pneumococcal mouse infections radioresistant cells expressing tlr5 control the respiratory epithelium's innate immune responses to flagellin airway structural cells regulate tlr5-mediated mucosal adjuvant activity indirect toll-like receptor 5-mediated activation of conventional dendritic cells promotes the mucosal adjuvant activity of flagellin in the respiratory tract analysis of drug combinations: current methodological landscape assessment and modelling of antibacterial combination regimens when does 2 plus 2 equal 5? a review of antimicrobial synergy testing comparison of techniques for measurement of in vitro antibiotic synergism activation of type 3 innate lymphoid cells and interleukin 22 secretion in the lungs during streptococcus pneumoniae infection interleukin-22 is produced by invariant natural killer t lymphocytes during influenza a virus infection: potential role in protection against lung epithelial damages potential role of invariant nkt cells in the control of pulmonary inflammation and cd8+ t cell response during acute influenza a virus h3n2 pneumonia invariant nkt cells modulate the suppressive activity of il-10-secreting neutrophils differentiated with serum amyloid a deletion of flagellin's hypervariable region abrogates antibody-mediated neutralization and systemic activation of tlr5-dependent immunity synergy testing of fda-approved drugs identifies potent drug combinations against trypanosoma cruzi both influenza-induced neutrophil dysfunction and neutrophil-independent mechanisms contribute to increased susceptibility to a secondary streptococcus pneumoniae infection influenza a inhibits th17-mediated host defense against bacterial pneumonia in mice influenza and bacterial superinfection: illuminating the immunologic mechanisms of disease the co-pathogenesis of influenza viruses with bacteria in the lung mucosal administration of flagellin induces innate immunity in the mouse lung flagellin promotes myeloid differentiation factor 88-dependent development of th2-type response lung epithelial cells: therapeutically inducible effectors of antimicrobial defense lung epithelial myd88 drives early pulmonary clearance of pseudomonas aeruginosa by a flagellin dependent mechanism understanding the pneumococcus: transmission and evolution antibiotic resistance and its cost: is it possible to reverse resistance? clinical applications of population pharmacokinetic models of antibiotics: challenges and perspectives we thank dr. aneesh vijayan for technical assistance in the production and quality control of recombinant proteins. we also thank the bicel flow cytometry core facility and the animal facility at the institut pasteur de lille for technical assistance. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.00723/full#supplementary-material key: cord-313227-6zwkfzab authors: scala, stefania; pacelli, roberto title: fighting the host reaction to sars-cov-2 in critically ill patients: the possible contribution of off-label drugs date: 2020-05-27 journal: front immunol doi: 10.3389/fimmu.2020.01201 sha: doc_id: 313227 cord_uid: 6zwkfzab the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the etiologic agent of the 2019 coronavirus disease (covid19). the majority of infected people presents flu like symptoms and among them 15–20% develops a severe interstitial pneumonitis (ip) that may eventually evolve in acute respiratory distress syndrome (ards). ip is caused by the viral glycoprotein spike (s) binding to the angiotensin converting enzyme 2 (ace2) expressed on the surface of alveolar pneumocytes. the virus is recognized by the “pattern recognition receptors” (prr) of the immune cells that release cytokines activating more immune cells that produce a large number of pro-inflammatory cytokines, tissue factors and vasoactive peptides. affected patients might develop the “cytokine storm syndrome,” a fulminant and fatal hypercytokinaemia with multiorgan failure. in patients infected by sars-cov-2 increase in t-helper 2 (th2) cytokines (il-4 and il10) are reported in addition to the t-helper 1 (th1) cytokines (il1b, ifnγ, ip10, and mcp1) previously detected in other coronavirus infections. cytokines and other molecules involved in immune response and inflammation are conceivable therapeutic targets for ip and ards, improving symptoms and decreasing intensive care unit admissions. to this aim off label drugs may be used taking into consideration the window timing for immunosuppressive drugs in virus infected patients. some off label therapeutic options and preclinical evidence drugs are herein considered. in a report on more than 70 thousands patients of the chinese province of hubei, the majority of infected symptomatic people presented flu like symptoms (mainly fever and cough), with 15-20% of patients developing a severe interstitial pneumonitis (ip) that could evolve in acute respiratory distress syndrome (ards). the case fatality rate in the whole population resulted 2.3% (8 and 15%, for patients older than 70 and 80, respectively). in critical patients 49% of case fatality rate was registered (4) . ip is caused by the attack of the virus against the alveolar pneumocytes (aps) through the binding of the viral glycoprotein (spike, s) to the angiotensin converting enzyme 2 (ace2) expressed on the surface of the aps (5) . the virus enters in the host target cells through receptor-mediated endocytosis and quickly replicates; virus release in the extracellular space occurs through either budding or cell death. in the extracellular space the virus is recognized by the prr of immune cells (6) . this process contributes to the virus elimination through an amplification cascade in which the immune cells produce a large number of pro-inflammatory cytokines, tissue factors, and vasoactive peptides. these molecules reach the blood vessel wall causing a burst of nitric oxide, damages to the blood vessels and to the coagulation system (7) . among the most involved cells, macrophages play an important role in acute lung injury, which identify pathogen-associated molecular patterns (pamp) and trigger innate immunity (8, 9) . macrophages secrete a large number of inflammatory mediators and cytokines, such as tumor necrosis factor-alpha (tnf-α), interleukin-1beta (il-1β), interleukin-6 (il-6), inducible nitric oxide synthase (inos), and macrophage migration inhibitory factor (mif). tnf-α can directly damage cells of the pulmonary vascular endothelium, increasing capillary endothelial permeability, causing pulmonary edema, predicted by il-6 level (10). progression to acute respiratory distress syndrome (ards) is based on the acute onset of lung inflammation, determined by monocyte/macrophage polarization and function. during active infection, inflammatory monocytes/macrophages (imms), and resident macrophages undergo marked phenotypic and functional changes, from m1 proinflammatory (classically activated) to m2 inflammatory-resolving macrophages, with a dynamic continuum through discrete categories. during acute infection, monocytes/macrophages often display a phenotype of classically activated macrophages that mediate antiviral host defenses but also promote lung injury by producing nitric oxide (no), reactive oxygen species (ros), il-1, il-6, and il-8 and tnf-α. simultaneously, some macrophages may become m2 macrophages alternatively activated, exerting anti-inflammatory function and regulating wound healing by producing matrix metalloproteinases (mmps), growth factors, and anti-inflammatory cytokines, particularly tgf-β. pro-inflammatory macrophages diminish at the removal of stimulus (11) (12) (13) . evidence of a cytokine storm has been found in severe pneumonitis linked to coronavirus infection (14) . previously, in patients with sars, il1b, il6, il12, ifnγ, ip10, and mcp1 were found to be increased (15) . in patients with mers, ifnγ, tnfα, il15, and il17 were shown to participate in the severity of the pneumonitis (16) , and an elevated inflammatory innate immune response has been shown in the lower respiratory tract. although those cytokines were elevated, down-regulation of genes encoding inflammatory th1 and th2 molecules was noted (17) . interestingly, in patients infected by sars-cov-2, there is an increase in il1β, ifnγ, ip10, and mcp1, probably leading to activated t-helper-1 (th1) cell responses, and increased production of t-helper-2 (th2) immunosuppressive cytokines, such as il4 and il10 (18) . in particular, a significant increase in il2, il7, il10, g-csf, ip10, mcp1/ccl2, mip1a, and tnfα was noted in patients requiring admission to the intensive care unit (icu) compared to patients with a milder disease. as the infiltrate of monocytes, neutrophils, lymphocytes, and macrophages are the cellular actors of the inflammatory response (14) , chemokine ligands and receptors play an important role in driving immune cell migration and homing (19) . these cytokines may explain the observation of reduced levels of circulating lymphocytes. peripheral blood examinations on admission in the majority of patients with covid-19 displayed lymphopenia, elevated infection-related biomarkers (i.e., procalcitonin, erythrocyte sedimentation rate, serum ferritin, and c-reactive protein) (20) and several elevated inflammatory cytokines (i.e., tumor necrosis factor (tnf)-α, interleukin (il)-2r and il-6). patients with more severe cases had higher leukocyte and neutrophil count, lower lymphocyte count and higher neutrophil-to-lymphocyte ratio (nlr) (21) . lymphocyte subsets showed that the total number of b cells, t cells and nk cells was significantly decreased in patients with covid-19, more significantly so in severe cases. in particular, t cells (t helper, t suppressor, and tregs cells) were mostly affected by sars-cov-2. in addition, recent evidence in sars-cov infection suggests that seroconversion may also play a role in lung injury. a detrimental role of early appearance of antispike (s)-igg was demonstrated during sars-cov infection in a macaque model (22) . despite markedly reducing virus titers, anti-s-igg caused lung injury during the early stages of infection, impairing the wound-healing macrophage response and tgf-β production, while promoting pro inflammatory cytokine il-8, mcp1 production, and inflammatory macrophage accumulation (22) . interestingly, in sars patients who died in hong kong during the 2002 outbreak, the anti-spike (s) glycoprotein neutralizing antibodies appeared significantly before and reached a higher titer than in patients surviving (23) . consistently, preexisting serum antibodies, derived by exposition to influenza seasonal strains, may recognize but fail to neutralize, the new pandemic strain and were found to associate with worse clinical severity during the 2009 influenza pandemic (24, 25) . the inflammatory status together with pulmonary edema and respiratory failure define the clinical picture of the ards associated with covid-19 (26) . the most compelling emergency that the health system faces in this epidemic is the shortage of critical care units. the saturation of intensive care units (icu) precludes the rescue of patients who might be saved, increases covid-19 lethality rate and worsens the prognosis for other pathological conditions requiring icu admission. the severe ip or ards of the covid-19 requires ventilator support and can kill infected people averaging in 2 weeks from the appearance of the first symptoms (27, 28) . therapy in use for hiv and other viral disease have been empirically administered without much benefit (29) , while promising experimental antiviral drugs such as remdesivir and chloroquine, an old antimalarial drug with in vitro activity on the viral infection, are currently in clinical trials (30, 31) . in the absence of specific validated approaches, and waiting for a vaccine, a clinical empirical rational management is needed. another reasonable approach would be drugs targeting the host immune-inflammatory reaction. methylprednisolone, although somewhat controversial, seems to be overall useful in these patients (32) , while dexamethasone has been shown to be useful in patients with ards of different etiologies (32, 33) . cytokines and the other molecules involved in the immune response regulation and inflammation are conceivable targets to improve ip and ards lung injury. to this aim off label drugs may be used considering the timing for immunosuppressive drugs in virus infected patients. unfortunately, the time window is not univocally defined and data may derive from clinical studies. several therapeutic options that could be rapidly translated to clinical trials are available. some of them are listed below. tocilizumab is an anti-il6 receptor antibody (roactemra, roche) approved to treat moderate to severe rheumatoid arthritis (ra). tocilizumab has been used to counteract the side effects of immune checkpoint inhibitors and car-t therapy in cancer bearing patients (34) and, recently, to antagonize the host reaction in patients affected by ards linked to covid 19 (35) . at today covid-19 national management guidelines of chinese health authorities include the use of tocilizumab for severe pneumonia. a preliminary report on 21 critical cases of covid-19 suggests efficacy of the treatment with faster recovery and lower risk of death for treated patients, while no toxicity was associated with the reported administration schedule (one or maximum two doses) (36) . timing of administration seems to be crucial as tocilizumab may be more efficient if administered earlier than actual use (37) . anakinra is an interleukin-1 receptor antagonist (il-1ra) previously evaluated in clinical trials for ra patients. il-1beta/il-1alpha are two stimulating cytokines of monocyte-macrophage cells acting upstream of the inflammatory signaling pathway induced by inflammasome, thus anakinra should block the cytokine storm. in a small open-label study, anakinra has been tested as agent preventive of mechanic ventilation in 9 patients hospitalized for moderate-severe covid-19. amelioration of oxygen flow and blood inflammation markers was described without significant toxicity (38) . in clinically moderate and severe covid-19 patients preliminary evidence reported high levels of three cytokines, cxcl10, ccl7 and il-1, rather than il-6, (39). in chronic use anakinra could determine reaction at the site of injection and infection as the main side effects (40) . emapalumab is a fully human igg1 monoclonal antibody that binds free and receptor-bound interferon-γ. emapalumab is approved by the us fda for the treatment of haemophagocytic (hlh) (41) a rare disorder characterized by pathologic immune activation and hyperinflammation that eventually damage multiple organs. a prospective study has shown a good safety profile of emapalumab in pediatric and adolescent patients affected by hlh, with the infection susceptibility being the major threat (42) . blocking ifn γ activity could counteract the host immune hyper-reaction to sars-cov-2. mycophenolic acid has been used as immunosuppressant agent in pemphigus as a corticosteroid-sparing agent and in kidney transplant patients to avoid rejection. it inhibits inositol monophosphate dehydrogenase, that causes depletion of guanosine and deoxyguanosine nucleotide pools impairing the activity of b and t lymphocytes. the drug has also been demonstrated to inhibit mrna expression of pro-inflammatory cytokines tnf-α, il-6, and il-1β4 (43) . mycophenolic acid has been shown to have activity in vitro against zika virus replication (44) and coronavirus through a non-competitive inhibition of mers-cov papain-like protease (45). urinary infections, diarrhea, and leukopenia are the side effects more often described (46) . anti-tnfα agents used in autoimmune diseases, such as ra and ulcerative colitis, in principle, may have a role in treating severe respiratory syndrome of covid-19. infliximab is a monoclonal antibody targeting tnf alpha while etanercept is a receptor fusion protein (human igg1-fc plus soluble p75 tnf alpha extracellular domain). tnf-α is a proinflammatory cytokine produced by macrophages, lymphoid cells, endothelial cells, cardiac myocytes, adipose tissue, and brain cells such as microglia and astrocytes. its receptors are widely expressed and tnf-α plays a key role in immunological defense processes such as inducing fever, inhibiting viral replication during infections, and leading to a permanent growth arrest in cancer (47, 48) . toxicity profile includes augmented risk of infections (49) . proteasomal system regulates different cell functions, among which nuclear factor kb (nf-kb) key transcription factor for innate and adaptive immunity (50) . bortezomib inhibits proteasome and it is used in the treatment of myeloma and mantle cell lymphoma. it has been shown to have antiviral activity against herpes virus, targeting viral entry, replication, and assembly (51) . another proteasome inhibitor, vr23, possess powerful antiinflammatory activity reducing il-6 in synovial cells from ra patients, and improving lps-induced acute lung injury by decreasing neutrophil migration, tnf-α secretion, and tissue inflammation in a mice model (52) . the doselimiting toxicity of proteasome inhibitors is the peripheral neuropathy (53) a clinically relevant complication, which negatively impacts the quality of life of multiple myeloma survivors (54) . pandemic viruses decrease type i interferon (ifn) abundance (24) . in humans 17 different types of poly-adenosine 5 ′diphosphate (adp)-ribose polymerase (parp) are recognized. parps transfer adp ribose from nicotinamide adenine dinucleotide (nad+) to targeted proteins achieving a post translational modification called adp-ribosylation, generally in response to stress conditions such as dna damage, heat shock and viral attack (55) . parp11 is an adp ribosyl-transferase that inhibits interferon type i (ifn-i) antiviral activity. ifn-i is a key component of the immune response against viral pathogens that induces the expression of several genes (interferon stimulated genes -isgs) with diverse antiviral properties (56) . parp11 inhibitor, rucaparib has been shown to restore the activity of ifn-i against different viruses in a murine model (57) . there is evidence that zikv infection triggers type i ifn production by host cells, zikv is sensitive to the antiviral activity of ifn and ifn i seems crucial also in sars-cov-2 infection (58, 59) . parp inhibitors are used in subgroup of patients with breast or ovarian cancer. toxicity is mainly hematological (60) . peroxisome proliferator-activated receptor gamma (pparγagonists, rosiglitazone and pioglitazone, are drugs in clinical use for diabetes (61) . insulin resistance amplifies inflammation, associated with an increase in c-reactive protein, il-6, and tnf-α (62) and produces a pro-coagulant state with increased fibrinogen and plasminogen activator inhibitor, (pai-1) (63). pioglitazone, in clinical studies on diabetic patients, was able to reduce the plasma level of different inflammatory factors among which cpr, il-1, il-6, and tnf-α (64). thus, it is of great interest that pioglitazone can produce an anti-inflammatory effect also on lung inflammation and fibrosis (65) . considered the excellent tolerability, pparγ agonists may be tested for amelioration of virus induced lung injuries. plerixafor is a cxcr4 antagonist used for stem cell mobilization in patients undergoing autologous stem cell transplantation. cxcr4-mediated inflammatory responses is based on the efficient chemotaxis function of inflammatory cells, such as neutrophils, lymphocytes, and monocytes (66) . in murine models of acute lung insufficiency cxcr4 expression was significantly increased in macrophages sorted from bronchoalveolar lavage fluid and receptor downregulation reduced il-6 and tnf-α. administration of amd3100 significantly attenuated the influx of inflammatory cells to the airway and reduced the levels of il-4, il-5, and il-13 in an murine asthmatic model either in the lavage fluid and lung homogenate through attenuation of the th17 (67), cell population. no adverse events have been described for a single injection of plerixafor (68). fingolimod, a sphingosine-1-phosphate (s1p) receptor agonist is approved for the treatment of multiple sclerosis (ms). s1p is mainly expressed in vascular endothelial cells and lymphocytes in lung tissue. s1p1 agonists (cym-5442 and rp-002) have been reported to protect mice from death caused by severe influenza infection, attenuating cytokine production and inhibiting infiltration of innate immune cells. in a mouse model of 2009 h1n1 pandemic influenza, the s1p1 receptor agonist significantly inhibited synthesis of il-1α, il-1β, il-6, il-10, mcp-1, tnf-α, and gm-csf, and reduced deaths from lethal infections by more than 80%. in addition the combination of oseltamivir can reduce mouse mortality by 96% (69) . recently a multiple sclerosis (ms) patient in treatment with fingolimod that was diagnosed with covid-19 reported a favorable outcome (70) . as reported, the toxicity profile even for long term use, is reassuring (71) . in conclusion, while specific antiviral therapies are in rapid development (remdesivir, chloroquine, vaccine), controlling the powerful inflammatory response causing severe ip or ards is a reasonable approach. agents that are available now to improve the lung injuries due to the host reactions and reduce the lethality of the disease are badly needed, and some are already in clinical studies. drugs targeting multiple cyto/chemokines involved in sars-cov-2 ip are available for trial or for off-label use, but close attention is needed to the schedule of administration, considered the immunosuppressive action of these drugs. to this aim rapid identification of prognostic factors in the peripheral immune profile may support therapeutic approach. careful clinical studies are warranted. ss and rp both equally contributed in designing and realizing the manuscript. the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china emerging coronaviruses: genome structure, replication, and pathogenesis severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention angiotensinconverting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target covid-19: consider cytokine storm syndromes and immunosuppression cytokine storm and sepsis disease pathogenesis diverse macrophage populations mediate acute lung inflammation and resolution lipoxin a4 receptor agonist bml-111 induces autophagy in alveolar macrophages and protects from acute lung injury by activating mapk signaling effect of tlr4/myd88 signaling pathway on sepsis-associated acute respiratory distress syndrome in rats, via regulation of macrophage activation and inflammatory response protective and pathogenic functions of macrophage subsets exploring the full spectrum of macrophage activation macrophages in tissue repair, regeneration, and fibrosis pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome mers-cov infection in humans is associated with a pro-inflammatory th1 and th17 cytokine profile mers-cov infection is associated with downregulation of genes encoding th1 and th2 cytokines/chemokines and elevated inflammatory innate immune response in the lower respiratory tract clinical features of patients infected with 2019 novel coronavirus in wuhan inflammatory chemokines direct and restrict leukocyte migration within live tissues as glycan-bound gradients clinical and biochemical indexes from 2019-ncov infected patients linked to viral loads and lung injury antispike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection antibody responses against sars coronavirus are correlated with disease outcome of infected individuals severe pandemic 2009 h1n1 influenza disease due to pathogenic immune complexes high titer and avidity of nonneutralizing antibodies against influenza vaccine antigen are associated with severe influenza acute respiratory distress syndrome clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study recent advances in the understanding and management of ards. f1000res a trial of lopinavirritonavir in adults hospitalized with severe covid-19 remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of covid-19 associated pneumonia in clinical studies risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial successful treatment of cytokine release syndrome with il-6 blockade in a patient transitioning from immune-checkpoint to mek/braf inhibition: a case report and review of literature advances in the research of cytokine storm mechanism induced by corona virus disease effective treatment of severe covid-19 patients with tocilizumab tocilizumab, an anti-il6 receptor antibody, to treat covid-19-related respiratory failure: a case report targeting the inflammatory cascade with anakinra in moderate to severe covid-19 pneumonia: case series novel coronavirus (covid-19) with il-6 inhibitor: are we already that far? anakinra for the treatment of rheumatoid arthritis: a safety evaluation emapalumab: first global approval emapalumab in children with primary hemophagocytic lymphohistiocytosis occurrence of sars-cov-2 during mycophenolate mofetil treatment for pemphigus mycophenolic acid, an immunomodulator, has potent and broad-spectrum in vitro antiviral activity against pandemic, seasonal and avian influenza viruses affecting humans a single-centre retrospective study of the safety and efficacy of mycophenolate mofetil in children and adolescents with nephrotic syndrome t-helper-1-cell cytokines drive cancer into senescence regulation of proliferation, survival and apoptosis by members of the tnf superfamily safety and efficacy of tumor necrosis factor antagonists in older patients with ulcerative colitis: patient-level pooled analysis of data from randomized trials nf-kappab and the link between inflammation and cancer early steps in herpes simplex virus infection blocked by a proteasome inhibitor the vr23 antitumor compound also shows strong anti-inflammatory effects in a human rheumatoid arthritis cell model and acute lung inflammation in mice a practical review of proteasome pharmacology neurological monitoring reduces the incidence of bortezomib-induced peripheral neuropathy in multiple myeloma patients regulation of poly(adp-ribose) polymerase 1 functions by post-translational modifications interferon-stimulated genes as enhancers of antiviral innate immune signaling adp-ribosyltransferase parp11 modulates the interferon antiviral response by mono-adpribosylating the ubiquitin e3 ligase beta-trcp antiviral activities of type i interferons to sars-cov-2 infection parp12 suppresses zika virus infection through parp-dependent degradation of ns1 and ns3 viral proteins exploring and comparing adverse events between parp inhibitors an overview on medicinal perspective of thiazolidine-2,4-dione: a remarkable scaffold in the treatment of type 2 diabetes tnf-alpha and inflammatory cytokines and risk of type 2 diabetes: a systematic review and meta-analysis selective insulin resistance and the development of cardiovascular diseases in diabetes: the 2015 edwin bierman award lecture can pioglitazone be potentially useful therapeutically in treating patients with covid-19? med hypotheses pioglitazone, a peroxisome proliferator-activated receptor gamma ligand, suppresses bleomycin-induced acute lung injury and fibrosis cxcr4 knockdown prevents inflammatory cytokine expression in macrophages by suppressing activation of mapk and nf-kappab signaling pathways cxcr4 inhibitor attenuates ovalbumin-induced airway inflammation and hyperresponsiveness by inhibiting th17 and tc17 cell immune response plerixafor enables safe, rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection covid-19 infection in a patient with multiple sclerosis treated with fingolimod benefitrisk profile of sphingosine-1-phosphate receptor modulators in relapsing and secondary progressive multiple sclerosis the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 scala and pacelli. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-333670-qv1orlv5 authors: mutti, luciano; pentimalli, francesca; baglio, giovanni; maiorano, patrizia; saladino, rita emilena; correale, pierpaolo; giordano, antonio title: coronavirus disease (covid-19): what are we learning in a country with high mortality rate? date: 2020-05-28 journal: front immunol doi: 10.3389/fimmu.2020.01208 sha: doc_id: 333670 cord_uid: qv1orlv5 nan covid-19 has been declared a pandemic by the who (1) . following the outbreak of the disease in china, italy was the first european country to be heavily struck (2, 3) . initially, three covid-19 cases were reported in early february, which were all related to individuals who had traveled to china; then, on the 20th, a young man who had not traveled abroad presented with severe sars-cov-2-induced pneumonia in lombardy, a region in the north of the country (2) . over the next 2 weeks, many patients in the surrounding areas were diagnosed with covid-19, which was often severe, and another cluster was identified in the nearby region of veneto (2) . there then followed an exponential increase in cases, mostly in the north, although the disease spread throughout the whole country, leading to the hypothesis that the virus had been circulating since january (2, 4) . at that point, italy reached incidence and mortality rates that were amongst the highest in the world (2) (3) (4) . many factors explain differences from other countries, including different application of detection tests, a larger elderly population, and different prevention policies and capacity to provide intensive care (2) . while it is paramount to conceive preventive strategies and apply more effective early treatments, it is also crucial to understand the biological mechanisms underlying these fatal outcomes. in italy, the possibility of performing autopsies or post-mortem diagnostic studies on suspect, probable, or confirmed covid-19 cases has been intensively debated (5, 6) ; however, postmortem pathological analysis of covid-19 patients in china has shown findings consistent with acute respiratory distress syndrome (ards) (7-9) (figure 1 ). at present, the exact nature of the acute lung injury trigger is not yet fully clarified; however, it could be ignited by t cells overreacting to virus-specific epitopes, thus recruiting multiple cytokineactivated inflammatory cell lineages (10) (11) (12) . other possibilities that deserve further experimental evidence include an exaggerated antibody-mediated response with complement activation and/or fcγ1 receptor-mediated leukocyte engagement and/or a hypothetical cytopathic effect of the virus (13, 14) . the latter could explain the recently described microvascular damage leading to disseminated intravascular coagulation (manifested as thrombosis, thrombocytopenia, and gangrene of extremities), anti-phospholipid syndrome, and mimicry of vasculitis, which are described in both chinese cohorts (15) and italian patients (16) (17) (18) . in our experience, ≈18% of patients develop interstitial pneumonia, and a subset of these (≈5%) develop ards that, especially when so serious as to require invasive ventilation, is mostly fatal. the risk of ards rises with age, and almost all deaths regard patients with pre-existing chronic conditions (19, 20) . pre-admission hypertension, in particular, has been reported as a key mortality risk factor (19) . the risk of death further rises where there is a lack of ventilators or ventilation is refused, as described in xu et al. (9) . moreover, an increasing number of clinical reports describe a biphasic behavior: a first phase where covid-19-infected patients are completely asymptomatic, which lasts on average seven days, and a second phase where the patients present mild to moderate flu-like symptoms, anosmia, ageusia, and blind conjunctivitis, which may last 10-15 days (21, 22) . a minority of patients who are unable to achieve complete virus coverage develop severe cardio-respiratory symptoms with radiological signs of pneumonitis, ards, and then multiorgan failure (23) . the last phase occurs, on average, 15-30 days after infection. in the latter case, patients may test negative for covid genome research standard molecular tests. altogether, these clinical findings, as well as the available pathology studies, support the hypothesis of an inappropriate immunerelated inflammatory response to covid19 epitopes and consequent auto-antigen release and t-cell cross-presentation in the damaged alveolar tissue. consistently, recent results indicate that a systemic immune dysregulation that triggers auto-sustaining inflammatory lung damage, causing fatal respiratory-failure and consequent multiorgan-failure, is the main virus-related-death cause in patients who develop sars-cov-2 (10). the culprit is the cytokine storm unleashed in this context by the infection and already described in cancer patients treated with cart or immunotherapy, including the "old" treatments with interleukins (il2 and il12, in particular) and the newest anti-ctla-4 and or anti-pd-1/pdl1 immunecheckpoint inhibitors. a greater risk of pneumonitis has already been recorded in chinese patients bearing a highfrequency of specific class-i and ii hla alleles associated with poor virus clearance and development of immune-related pneumonitis and other inflammatory-related autoimmune diseases (24) . this viral-load-independent different response to the infection might depend on a genetic predisposition causing extreme and often lethal inflammatory reactions. given the inefficacy of steroids (9), understanding the molecular features underlying such threatening immune-related events provides a strong rationale for using biological drugs for the early treatment of symptomatic patients, aimed at hampering the effects of the most relevant cytokines able to trigger an antibody response and acute inflammatory reaction, such as il6 and il1α. to this purpose, abs against the il6 receptor, or drugs able to disrupt its downstream signals, can inhibit its function on specific inflammatory cell subsets. these agents have so far been promising in the clinical setting for curbing the inflammatory response to control the severe immune-related adverse events related to cart-therapy and immune-checkpoint blockade and autoimmune diseases, including juvenile rheumatoid arthritis, psoriatic arthritis, and ulcerative colitis, all related to particular hla class i and ii alleles, some of which, like class i b * 27 and b * 35, might sustain both mitochondrial stress and cross-reactivity with several pathogens (25) . therefore, while antiviral drugs help to contain viral replication, moabs to il6 in the early phase of respiratory involvement could control the risk of a fatal virus-inducedcytokine storm. a great effort should be made to recognize lung involvement as, at least theoretically, the earlier the treatment, the better the outcome will be, with il6 inhibitors being able to "nip in the bud" the inflammatory cascade and prevent the fatal permanent damage to the alveolar pneumocytes. on this basis, il6 inhibitors are currently being tested in china and italy in patients with respiratory failure, and other il6 inhibitors are also being considered. iatrogenic cues might also contribute to exacerbating the acute inflammatory lung injury triggered by the virus. most hospitalized patients in fact received oxygen either through intubation or mechanical or non-invasive ventilation (20) ; however, oxygenation in ards patients with acute lung inflammation has been previously shown to interfere with the anti-inflammatory response induced locally by hypoxia through the activation of the adenosine a2a receptor (26) . similarly, in covid-19, patients, oxygen therapy could worsen lung injury by weakening such anti-inflammatory pathways. consistently with this hypothesis, in a cohort of 5,700 patients hospitalized with covid-19 in the new york city area, mortality reached 88.1% for those requiring mechanical ventilation (27) . in lombardy, the intensive care unit mortality was 26%, and indeed, a large proportion of admitted patients required mechanical ventilation (20) . these data support the possible use of adenosine agonists in patients presenting with ards (figure 1) . identifying infected patients at higher risk of poor prognosis even without evident risk factors could represent an important step forward. in this direction, zhou et al. reported some predictive biomarkers of the severity of the infection (23). nguyen and colleagues, in a preprint article, analyzed the sars-cov-2 proteome and identified a range of hla alleles potentially able to present (or not) viral epitopes. they suggest that individuals bearing hla-b * 46 (which has the fewest predicted binding peptides for sars-cov-2) may be particularly vulnerable to covid-19, whereas individuals bearing hla-b * 15 (which has the greatest predicted capacity to present sars-cov-2 peptides) could exhibit cross-protective t-cell based immunity. the authors highlight that a thorough understanding of how hla variation correlates with covid-19 onset and outcome could help identify high-risk subjects (28) . indeed, we have preliminary evidence that the prevalence of specific hla class i alleles across italian regions/provinces correlates with increased covid-19 incidence (correale p., mutti l., submitted for publication). if confirmed in wide case-control studies, the identification of hla alleles that are more permissive to viral infection would provide the first genetic explanation for the wide differences in covid-19 incidence rates among italian regions and also among nearby provinces with similar environmental factors. figure 1 | host response and possible outcomes of sars-cov-2 infection. viral infection seems to occur mainly upon sars-cov2 engagement of angiotensin i converting enzyme 2 (ace2), which acts as a functional receptor for the spike glycoprotein of the coronavirus. the hla genetic system acts as a key player in determining the anti-viral immune response. in particular, the ability of hla to trigger an adequate cytotoxic t-lymphocyte (ctl) response will result in viral clearance and host healing, along with the development of the igm, iga, and igg humoral response. conversely, an inadequate hla asset will result in an inefficient ctl response and, consequently, incomplete viral clearance. in this context, various factors underlie increased covid-19 severity, including an exaggerated ab response, complement activation, leukocyte-mediated antibody-dependent cell-mediated cytotoxicity (adcc), and t-cell-mediated inflammation, as discussed in the text. without a protective immune response, the virus is able to migrate, propagating into other ace2-expressing tissues, while the damaged lung cells induce high inflammation, triggering the cytokine storm that represents the main cause of the acute respiratory distress syndrome (ards) and subsequent multiorgan failure. incomplete viral clearance can also lead to virus hiding in sanctuary sites and patient relapse with symptoms arising in new districts. in the purple boxes, different therapeutic approaches aimed at targeting either the virus or endogenous host players are represented. overall, understanding the role of pro-inflammatory cytokines certainly unravels a new battleground against the lethal clinical effect of codiv-19 infection; this, along with the identification of a high-risk autoimmune profile, including the genotyping of class i and ii hla, which have a key role in shaping the anti-viral immune response and th1/th2 lymphocyte subset response (figure 1) , and immune-profiling, could also help to prevent these dangerous evolutions of the disease (29) . in particular, the isolation of genetically at-risk individuals, including healthcare workers, will inform future vaccination campaign priorities and clinical management strategies. the finding of healed patients retesting positive after an apparent complete virus clearance is a matter of intense debate in italy and worldwide. assuming that testing was reliable, various hypotheses are being considered, including viral mutation, although variation among sequences seems very low at present (30) . a preprint study in rhesus macaques argues against a risk of re-infection (31) . host inability to develop immunological memory with subsequent longterm protection is also being evaluated. interestingly, another preprint study identified specific sars-cov-2 neutralizing antibodies (nabs) in the plasma of patients who had recovered from infection and recorded that 30% of patients failed to develop high titers of nabs after covid-19 infection (32) . another possibility is that newborn sars-cov-2 might hide in sanctuary sites, such as the ncs and/or testis, which are protected from both antiviral drugs and proficient immuno-effectors; this hypothesis is supported by the recent description of viral detection in the cerebrospinal fluid but not in the nasopharyngeal swab in a case report (33) . overall, these distinct biological patterns of response to the virus should be taken into account for the design of new preventive and therapeutic strategies. lm, pc, and ag conceived the study. fp and gb evaluated current data. rs and pm studied hla involvement. pc conceived and fp sketched the figure. all authors contributed to manuscript writing and agreed with content. this work was supported by the sbarro health research organization (www.shro.org) and the commonwealth of pennsylvania. available online at case-fatality rate and characteristics of patients dying in relation to covid-19 in italy covid-19) situation report -57 coronavirus disease 2019 (covid-19) in italy the autopsy debate during the covid-19 emergency: the italian experience covid-19 deaths: are we sure it is pneumonia? please, autopsy, autopsy, autopsy! a new coronavirus associated with human respiratory disease in china clinical features of patients infected with 2019 novel coronavirus in wuhan pathological findings of covid-19 associated with acute respiratory distress syndrome molecular immune pathogenesis and diagnosis of covid-19 immunoinformatics-aided identification of t cell and b cell epitopes in the surface glycoprotein of 2019-ncov covid-19 infection and rheumatoid arthritis: faraway, so close monitoring kinetics reveals critical parameters of iga-dependent granulocyte-mediated anti-tumor cell cytotoxicity identification of coronavirus isolated from a patient in korea with covid-19. osong public the use of antiinflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the experience of clinical immunologists from china is covid evolution due to occurrence of pulmonary vascular thrombosis? covid-19 pulmonary involvement: is really an interstitial pneumonia? pulmonary embolism or pulmonary thrombosis in covid-19? is the recommendation to use high-dose heparin for thromboprophylaxis justified? characteristics, treatment, outcomes and cause of death of invasively ventilated patients with covid-19 ards in baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region objective evaluation of anosmia and ageusia in covid-19 patients: a single-center experience on 72 cases conjunctivitis and covid-19: a meta-analysis clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study werner syndrome: clinical features, pathogenesis and potential therapeutic interventions major histocompatibility complex genomics and human disease oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area human leukocyte antigen susceptibility map for sars-cov-2 could pd-1/pdl1 immune checkpoints be linked to hla signature? the establishment of reference sequence for sars-cov-2 and variation analysis lack of reinfection in rhesus macaques infected with sars-cov-2. biorxiv neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications. medrxiv a first case of meningitis/encephalitis associated with sars-coronavirus-2 we are grateful to barbara colombo for the figure graphics. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 mutti, pentimalli, baglio, maiorano, saladino, correale and giordano. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-316702-dj2fo8sn authors: vignesh, ramachandran; shankar, esaki m.; velu, vijayakumar; thyagarajan, sadras panchatcharam title: is herd immunity against sars-cov-2 a silver lining? date: 2020-09-30 journal: front immunol doi: 10.3389/fimmu.2020.586781 sha: doc_id: 316702 cord_uid: dj2fo8sn nan the emergence of a novel coronavirus severe acute respiratory syndrome coronavirus-2 (sars-cov-2) in late 2019 and its wide global spread has led to millions of infections and substantial morbidity and mortality (1) . coronavirus disease 2019 (covid-19) caused by sars-cov-2 infection can range from mild self-limiting disease to acute respiratory distress syndrome and death (2) . with the who having reported 31,174,627 confirmed cases and 962,613 deaths globally as of 22nd september 2020, the covid-19 pandemic seems to show almost poor indication of abating (3) . while the global scientific community is racing against time to strategize combating possibilities, with several vaccine trials, drug discoveries and validations underway, we still need a practical and sustainable solution to face the ongoing threat to global public health. the terminology "herd immunity", coined by topley and wilson in 1923, later formed the basis for vaccines, applications and vaccination programs, especially against certain viral infections (4). the concept of herd immunity refers to the indirect protection from infection conferred on susceptible individuals when a sufficiently large proportion of individuals immune to the infection exist in a population. herd immunity concept is generally applicable to those infections that are transmitted directly from person to person and for those humans serving as the reservoir of infection. history has shown that a significant drop in the number of cases and even eradication is rendered by vaccines, and herd immunity is achieved against infectious diseases like small pox, polio, measles, rubella, diphtheria, pertussis and mumps (4-7). the concept of herd immunity appears to be highly critical in the context of disease elimination programs because the said eradication of an infectious agent becomes possible in spite of the absence of an effectual vaccine. interestingly, natural herd immunity is a classical concept that has been successfully accomplished in instances like the 1918 h1n1 influenza pandemic wherein no vaccine was available (8) . going by this old school modus operandi, in the absence of a vaccine, building natural herd immunity against sars-cov-2 is theoretically considered feasible. however, letting an existing super infectious condition to run amok in the pretext of building up effective herd immunity might not be a smart strategy to end the pandemic. it requires judicious analyses to avoid the colossal burden it could inflict on the healthcare system and the surge in mortalities and associated complications. in a detailed classical analysis, fox et al. has listed down the following four conditions for effective herd immunity to ensue (i) the infectious agent must exist and be restricted to a single host, (ii) the transmission must occur primarily through direct contact, (iii) the infection must induce a robust and life-long immunity, and (iv) the cumulative or herd immunity is amplified if the population possesses a random mixing pattern (9) . applying the aforementioned conditions to herd immunity against sars-cov-2, though the infectious agent has been identified and believed to be zoonotic, we are yet to place a finger on the intermediate host (10) . secondly, the transmission, of course, occurs through direct person-to-person contact (11) . however, regarding the third condition, we have a paucity of data on the immune response elicited by sars-cov-2 in humans, till date (12) and the questions remains about the long-lasting immunity for the exposed individuals. finally, though the entire human population is susceptible to covid-19, the mixing of the pattern varies that is dependent on several societal factors and practices such as universal lockdown, mass quarantine, isolation, social distancing and public health preventive measures, particularly for those at risk (12) . table 1 listed the various infectious agents and their corresponding r 0 values and herd immunity thresholds. while earlier studies have estimated the basic reproductive number (r 0 ) of sars-cov-2 to be in the range of 2 to 3, recent estimates place the r 0 at 5.7 (13, 15) . this indicates the highly infective nature of the virus, meaning that on an average each infected individual can give rise to about 5.7 newer infections and subsequently spread the agent on an exponential scale. assuming an r 0 estimate of 5.7, using the mathematical formula 1-1/r 0 , the herd immunity threshold for covid-19 would be~82.5%, meaning that the incidence of infection will begin to decline once the proportion of individuals with acquired immunity to sars-cov-2 in the population exceeds 82.5%. however, it remains to be noted that the estimate of the proposed threshold is only theoretical with the assumptions of a homogenous population and presence of uniform sterilizing immunity in the recovered patients. mathematical modeling studies have shown that disease-induced herd immunity threshold would be substantially lower than the values calculated by conventional formula due to the population heterogeneity (16, 17) . furthermore, there are several instances of "super spreading events" reported from various countries, wherein a single patient goes on to infect far more number of people than an average patient does, based on estimated r 0 value (18) . several factors such as increased viral load, asymptomatic individuals, immune suppression and extensive social interactions have been implicated in these "super-spreading events" (19) . studies point towards these "super-spreaders" as the reason for the heterogeneous propagation of sars-cov-2 across geographical locations and since these present with a relatively higher r 0 value, they could potentially impact the dynamics of spread and lower the herd immunity threshold (20, 21) . the whole concept of herd immunity against sars-cov-2 hinges on the assumption that an infection would generate sufficient, effective and protective long-lasting immunity. however, data are scarce if the acquired immunity developed by humans is sterilizing enough and whether it would stay long enough. earlier studies on survivors of sars-cov-1 and mers-cov infections have shown that the antibodies against sars-cov-1 persisted for nearly two years and antibodies to mers-cov lasted for almost 3 years (22, 23) . besides, studies have also demonstrated evidence of seroconversion within 14 to 19 days of disease onset among sars-cov-2 patients with covid-19 (24, 25) . a study has reported the interesting finding of t cell reactivity to sars-cov-2 in about 50% of specimens collected between 2015 and 2018 before the viral emergence (26) . this could reflect the circulating t cell memory to the seasonal coronaviruses-229e, oc43, nl63, and hku1. a study also suggests that sars-cov-2-reactive antibody responses have been detected in unexposed individuals who are igg seropositive for oc43 and nl63 and this cross-immune reactivity mainly targets viral 1ab polyprotein and s proteins, these viral antigens have high sequence similarity with low pathogenic human coronaviruses and sars-cov-2 (27, 28) . in addition, a study has also shown that these antibodies were particularly prevalent in children and adolescents (29) . it is also important to note that sera from uninfected individuals exhibited variability in their reactivity, they are reactive with sars-cov-2 s and nucleoprotein but not with the s1 subunit or the receptor binding domain (rbd) of spike protein. since many studies from different geographical locations are documenting preexisting immunity to sars-cov-2, it will be important to define specificities of these t and b cell immune response carefully to assess their association with covid-19 disease severity. this preexisting cross-reactive t and b cell immunity to sars-cov-2 may have wide implications as this could explain differential clinical outcomes in covid-19 patients, disease severity, vaccine development, and important in accessing herd immunity for sars-cov-2 viral infection/covid-19 disease. studies characterizing the sars-cov-2-specific t cell responses suggest that there is marked activation of t cells in acute covid-19 patients (30) (31) (32) . several studies have provided strong evidence for the importance of sars-cov-2 specific ctls, and t helper cells in mild and moderate patients compared to severe covid-19 disease (27, 28, (31) (32) (33) . the effector memory cd8 t cell population is decreased in the severe patients compared to the recovered patients (34) (35) (36) . similarly, the t cells in severe patients compared to convalescent patients express higher levels of exhaustion markers pd-1 and cd39 on the memory cd8 t cells with the signature pertaining to hyperimmune activation (hladr + cd38 + cd8 + ) with lower cd8 t cell response. in line with cd8 t cells, the effector memory cd4 t cell frequencies are also elevated in the covid-19 patients (34) (35) (36) . interestingly, the recovered patients seem to have higher levels of activated circulating t follicular helper (tfh) population, indicative of recent antigen encounter and emigration from germinal center. similar to the pd-1 levels of cd8 t cells, the pd-1 levels were increased in the cd4 t cells. as a result of the decrease in the t cells, non-t cell frequencies that includes, macrophages, monocytes, neutrophils were observed to be elevated in severe covid-19 patients (30, 34) . similar to t cells the b cell subpopulations are altered in the covid-19 disease and it has been shown that the frequency of plasmablast is highly elevated (30% of the circulating b cells) in the covid-19 patients (34) . these blood plasmablast frequencies are shown to correlate with activated circulating tfh cells (34, 37) . the proliferating b cells are markedly elevated in the covid-19 patients with activated phenotype, suggestive of altered b cell response during covid-19 disease. several studies have provided strong evidence for the importance of sars-cov-2specific neutralizing antibodies in association with less disease severity in covid-19 patients (38, 39) . sars-cov-2 specific antibodies are found in convalescent plasma and most recovered individuals have rbd-specific antibodies with potent antiviral activity (40, 41) , importantly severe and critical patients are currently being treated with convalescent plasma therapy in many parts of the world (42, 43) . overall, these data suggest that vaccines or therapeutic strategies that induce functional anti-viral sars-cov-2 specific t and b cell responses are important to curtail sars-cov-2 infection in vivo. there remain many unknowns and several other research questions unanswered. the degree of protection afforded by the antibodies against sars-cov-2 among the infected is still ambiguous, and strong evidence-based findings are awaited to know if the host responses generated would be of strong sterilizing or weak and waning immune responses. earlier studies on the coronavirus 229e, responsible for common cold have shown that the titers of antibodies produced were not sufficient to prevent reinfection in all the individuals studied (44) . interestingly, similar pattern exists in sars-cov-2 infection and where there is a rapid decay of sars-cov-2 of antibodies in persons with mild symptoms and recovered patients (45, 46) . similarly, as a recent study demonstrated the evidence of reinfection of sars-cov-2 by phylogenetically distinct sars-cov-2 re-infection, this may also suggest that the immunity generated during primary sars-cov-2 infection may be short lived and may not protect if reinfection happens by the second distinct virus (47) . these results also suggest that sars-cov-2 may continue to circulate among human population despite herd immunity with natural infection or with vaccination. however, a recent study on monkeys has indicated the development of protective immunity against reexposure to sars-cov-2 (homologous) (48) . hence, massive longitudinal monitoring of sars-cov-2 seroprevalence remains the need of the hour to determine the percentage of the population already infected and if reaching a herd immunity threshold is even feasible. it also remains logical to consider the viral factors such as the possibility of mutations and emergence of new strains of a virus, which can go on to make herd immunity futile (49) . given the unavailability of a vaccine against sars-cov-2, it is prudent to foresee the consequences of achieving the herd immunity threshold in epidemiological and immunological viewpoints. a recent population-based sero-epidemiological study from spain involving 61075 participants has revealed 5% seroprevalence of sars-cov-2, thereby highlighting that the majority of the spanish population remains seronegative (50) . figure 1 represents this scenario of a population with only 5% seroprevalence of sars-cov-2 antibodies and the importance of achieving herd immunity threshold. similarly, seroprevalence among the community in los angeles county has been reported as 4.34%, about 3.3% in japan, and 2.73% in hong kong (51) (52) (53) . based on a serological survey conducted by the indian council for medical research (icmr) in may -june, 2020 across 83 districts in india involving over 26,400 participants, the seroprevalence was found to be only 0.73% (54) . the lower rates of seroprevalence of sars-cov-2 antibodies reported from various geographical locations point in the direction that we are still a long way ahead of achieving herd immunity. according to a modeling study, assuming a scenario of achieving uniform herd immunity threshold of 67% and with an infection fatality rate of 0.6% for sars-cov-2, the estimate of the absolute number of deaths worldwide would easily exceed a whopping 30 million (14) . since these estimates are based on various assumptions and the real-life scenario could throw us curve balls of multiple factors influencing the outcomes, the fatality rate could even be higher. a recent modelling study has estimated that about one in five individuals worldwide would be at increased risk of severe covid-19, upon infection with sars-cov-2, owing to the underlying conditions. the study projects an alarming figure of about 349 million people requiring hospital admission and substantial mortality rates (55) . furthermore, in the case of highly populated and resourcestrapped countries, the increase in the number of infected cases could overwhelm the healthcare facilities and could lead to a shortage of essential medical services exacerbating further complications and deaths. thus, weighing on the immunological and epidemiological consequences, achieving natural herd immunity at the cost of severe morbidities and mortalities worldwide, in the absence of an effective and safe vaccine appears farfetched and seemingly an impracticable solution. with so many vaccines being tested in various phases of clinical trials, the availability of an effective vaccine seems to be the only way forward in this war against covid-19. echoing dr. anthony fauci's statements, even when a vaccine with a modest efficacy is made available in near future, anti-science and anti-vaccine campaigns need to be counteracted and cooperation of the general public is needed to achieve an efficient and successful herd immunity against covid-19 (56). rv, es, vv, and st led the writing of this opinion article. all authors contributed to the article and approved the submitted version. the views, opinions, assumptions, or any other information set out in this article are solely those of the authors and should not be attributed to anyone. the authors salute all the health care workers who are at the front lines of the covid-19 pandemic, helping patients and their families. frontiers in immunology | www.frontiersin.org september 2020 | volume 11 | article 586781 an interactive web-based dashboard to track covid-19 in real time clinical features of patients infected with 2019 novel coronavirus in wuhan who. coronavirus disease (covid-19) dashboard. available at: https://covid19. who.int/?gclid=cjwkcajw_qb3braveiwavwq6vvsmd-becu3ak qlye88rcmzf-muzkht9fj70phh5wl5hb3fcqzsmhrocvoiqavd_bwe the spread of bacterial infection. the problem of herd-immunity herd immunity: history, theory, practice herd immunity": a rough guide vaccination and herd immunity: what more do we know? dynamics of population immunity due to the herd effect in the covid-19 pandemic herd immunity: basic concept and relevance to public health immunization practices a review of coronavirus disease-2019 (covid-19) transmission routes of 2019-ncov and controls in dental practice perspectives on therapeutic neutralizing antibodies against the novel coronavirus sars-cov-2 early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia herd immunity: understanding covid-19 high contagiousness and rapid spread of severe acute respiratory syndrome coronavirus 2 a mathematical model reveals the influence of population heterogeneity on herd immunity to sars-cov-2 herd immunity thresholds for sars-cov-2 estimated from unfolding epidemics super-spreading events and contribution to transmission of mers, sars, and covid-19 identifying and interrupting superspreading eventsimplications for control of severe acute respiratory syndrome coronavirus 2 do superspreaders generate new superspreaders? a hypothesis to explain the propagation pattern of covid-19 heterogeneity in sir epidemics modeling: superspreaders. medrxiv (2020) longitudinal profile of antibodies against sars-coronavirus in sars patients and their clinical significance persistence of antibodies against middle east respiratory syndrome coronavirus severe acute respiratory syndrome coronavirus 2-specific antibody responses in coronavirus disease antibody responses to sars-cov-2 in patients with covid-19 targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals pre-existing immunity to sars-cov-2: the knowns and unknowns selective and cross-reactive sars-cov-2 t cell epitopes in unexposed humans pre-existing and de novo humoral immunity to sars-cov-2 in humans immunologic perturbations in severe covid-19/sars-cov-2 infection breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 systems-level immunomonitoring from acute to recovery phase of severe covid-19 longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients deep immune profiling of covid-19 patients reveals distinct immunotypes with therapeutic implications reappearance of effector t cells is associated with recovery from covid-19 sars-cov-2-specific t cell immunity in cases of covid-19 and sars, and uninfected controls humoral and circulating follicular helper t cell responses in recovered patients with covid-19 neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications. medrxiv (2020) 2020.03.30 rapid generation of neutralizing antibody responses in covid-19 patients convergent antibody responses to sars-cov-2 in convalescent individuals potent neutralizing antibodies against multiple epitopes on sars-cov-2 spike convalescent plasma as a potential therapy for covid-19 effect of convalescent plasma therapy on time to clinical improvement in patients with severe and life-threatening covid-19: a the time course of the immune response to experimental coronavirus infection of man rapid decay of anti-sars-cov-2 antibodies in persons with mild covid-19 longitudinal evaluation and decline of antibody responses in sars-cov-2 infection covid-19 reinfection by a phylogenetically distinct sars-coronavirus-2 strain confirmed by whole genome sequencing sars-cov-2 infection protects against rechallenge in rhesus macaques covid-19: herd immunity and convalescent plasma transfer therapy prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study seroprevalence of sars-cov-2-specific antibodies among adults estimation of seroprevalence of novel coronavirus disease (covid-19) using preserved serum at an outpatient setting in kobe, japan: a cross-sectional study seroprevalence of sars-cov-2 in hong kong and in residents evacuated from hubei province, china: a multicohort study prevalence of sars-cov-2 infection in india: findings from the national serosurvey global, regional, and national estimates of the population at increased risk of severe covid-19 due to underlying health conditions in 2020: a modelling study fauci says covid-19 vaccine may not get us to herd immunity if too many people refuse to get it the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright â© 2020 vignesh, shankar, velu and thyagarajan. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-318418-uqxzds6g authors: inatomi, takio; amatatsu, masaaki; romero-pérez, gustavo a.; inoue, ryo; tsukahara, takamitsu title: dietary probiotic compound improves reproductive performance of porcine epidemic diarrhea virus-infected sows reared in a japanese commercial swine farm under vaccine control condition date: 2017-12-22 journal: front immunol doi: 10.3389/fimmu.2017.01877 sha: doc_id: 318418 cord_uid: uqxzds6g lactogenic immunity transferred to piglets after inoculation of a live vaccine to pregnant sows was proved limited to control porcine epidemic diarrhea (ped). hence, here we evaluated the efficacy of administration of a probiotic compound containing bacillus mesentericus, clostridium butyricum, and enterococcus faecalis together with a commercial live-attenuated ped vaccine (nisseiken ped live vaccine, nisseiken, tokyo, japan) to improve the health and reproductive performance of ped-infected sows. twenty pregnant sows in a ped-positive farm were equally divided into probiotics-administered (vp) and control (vc) sow groups. a commercial live-attenuated vaccine was injected as per the manufacturer’s instruction. the probiotic compound (15 g/day) was orally administered to vp from 6 weeks pre-parturition to 7 days post-parturition (ppd7). vp had a significantly higher body weight at ppd7 than vc (191 vs 186 kg; p < 0.05). at day 3 post-parturition (ppd3) (4.18 vs 3.63 kg/day) and ppd7 (5.14 vs 4.34 kg/day), milk produced by vp was significantly (p < 0.05) greater than that by vc. total immunoglobulin (ig)a and igg concentrations at day 0 were significantly (p < 0.05) higher in whey of vp (1.9 and 6.6 g/dl, respectively) than in that of vc (1.7 and 6.1 g/dl, respectively). however, total igg concentration in whey of vp and vc at ppd3 and ppd7 did not differ. antibody titer was significantly higher at day 0 in serum of vp than it was that of vc (60 vs 37 in geometric mean; p < 0.05). likewise, the antibody titer in whey of vp and vc was found to be similar at day 0 (416 vs 208 in geometric mean; p = 0.13). consequently, vp had fewer days between weaning and return to estrus than did vc (7 vs 10 days; p < 0.05). moreover, piglets of vp had a significantly (p < 0.05) higher litter weight at birth (9,252 g/litter) and a lower mortality (12%) during suckling than those of vc (8,686 g/litter and 28%, respectively). in summary, probiotic-supplemented, ped-vaccinated sows were healthier, transferred ped-specific antibodies via colostrum to piglets, had greater litter weight at birth, and reduced mortality during suckling. introduction porcine epidemic diarrhea (ped) is an enteric disease that causes severe economic losses to the pig industry worldwide (1) . ped was first recognized in the uk and quickly spread to other european countries (2, 3) . in the following years, ped was detected in many asian countries, including japan (4), where it re-emerged in 2013 and caused approximately 1,000 outbreaks in 39 of 47 prefectures (3) . intriguingly, the ped strains emerged in asia are quite distinct from those previously reported (5) , as they cause more deleterious effects on all pigs regardless of age (6) . porcine epidemic diarrhea virus is an enveloped, singlestranded rna virus belonging to the group 1 of the genus coronavirus (7) . ped is characterized by watery diarrhea, dehydration, vomiting, anorexia, and reduced appetite in pigs of all ages. for newborn piglets, mortality caused by ped is close to 100% (8, 9) , whereas for suckling piglets mortality can be as high as 80% (10) . in addition, ped can seriously diminish the production performance of surviving animals (11) . interestingly, the deleterious effects of ped on reproductive performance of gilts and sows depend on the pregnancy stage during which they contract the disease (12) . for example, sows infected with ped during the first 30 days of pregnancy have decreased farrowing rates, increased abortion rates, and more mummified fetus per litter, whereas females infected with ped during 91-120 days of pregnancy have more stillbirth piglets per litter (12) . although depressed milk secretion has been previously reported in pedinfected sows (9) , the extent of the effect of this viral infection on lactating sows, including passive immunity or the health status and survival rate of suckling piglets, is yet to be fully investigated. in recent years, vaccines have been developed in an attempt to eradicate ped (13) (14) (15) (16) . in theory, pre-parturition vaccination of sows putatively permits enteric sensitization to antigens, after which immunoglobulin (ig)a immune cells are transferred to the mammary gland and secrete antibodies (17) . as a result, passive immunity against ped is putatively conferred by the sow to suckling piglets via colostrum and milk (17, 18) . in fact, however, lactogenic immunity by live vaccines has been proven only somewhat efficacious in providing protection to piglets against ped (19) . for example, ped infections have recently re-emerged in japan (3), even though, following infection outbreaks, live vaccines were administered to pigs in this country. this lack of effectivity is likely due to either strains from live vaccines producing more infectious mutations in the wild (20) or vaccine strain sequences having limited compatibility with those of wild types (21) . it is, therefore, imperative to find antiviral agents that act as adjuvant to existing vaccines and help increase their effectivity. recently, a probiotic bacteria fused with ped virus core neutralizing epitope antigen was developed to use an anti-ped vaccine (22) . in mice, oral administration was the effective strategy for this vaccine, targeting mainly mucosal dendritic cells in the intestine and stimulating ped virus specific immunity. in contrast, efficacy of this oral vaccine for pigs is still obscure, because no validation study for this species has been conducted yet. administration of probiotics such as lactic acid bacteria (lab) has been recognized as a viable alternative to antiviral medication for treating viral infections. for example, viable lactobacillus acidophilus and lactobacillus reuteri have been shown to protect experimental models against viral strains such as human rotavirus (hrv) by improving total intestinal iga-releasing cell immune responses, as well as total serum igm, and intestinal igm and igg titers (23) . similarly, colonization of the gut of neonatal gnotobiotic pigs with probiotic strain such as lactobacillus rhamnosus strain gg and bifidobacterium animalis ssp. lactis bb-12 resulted in significantly lower fecal scores and reduced hrv shedding concentrations but increased intestinal iga hrv antibody concentrations and hrv-specific iga antibody-releasing cell numbers in infected animals (24) . likewise, in our premises we demonstrated that a cell preparation of enterococcus faecalis strain ec-12, which is a heat-killed lab, protected weaning piglets from rotavirus infection (25) and stimulated luminal iga secretion in young calves (26) and chicks (27) . recently, sirichokchatchawan et al. (28) demonstrated that live lab could reduce ped infectivity in vitro. these data highlight the potential that probiotics and immunogenics may have to enhance lactogenic immunity and the efficacy of live vaccines administered to farm animals. in a separate study, we demonstrated that a probiotic compound containing bacillus mesentericus, clostridium butyricum, and e. faecalis prevented colibacillosis in weaning piglets (29) . we also reported that twofold concentration of total iga was detected in the ileum in piglets when this probiotic compound was administered. finally, we also reported in a different study that this threeprobiotic strain compound induced twofold concentration of total iga in the ileum of chicks affected by coccidian infection (30) . the aim of the present study was to evaluate the efficacy of the aforementioned probiotic compound mixed with peptide-zinc to improve the health and reproductive performance of pedinfected lactating sows when administered along with a ped vaccine injection in japan. probiotic product bio-three pz (toa pharmaceutical co. ltd., tokyo, japan) containing a mixture of bacterial strains b. mesentericus to-a (1 × 10 6 cfu/g), c. butyricum to-a (1 × 10 6 cfu/g), and e. faecalis t-110 (1 × 10 8 cfu/g) in a peptide-zinc compound (10 mg/g) was used in this study. the present work was carried out in a commercial swine farm in kyushu region of japan. the farm operates a farrow-to-finish business and has approximately a stock of 900 sows (landrace x large white). for the present work, all sows were impregnated by duroc boars. experiments were carried out between april and may 2014. sows ate commercial feed during gestation (shuton-b; minami nihon kumiai siryo, kagoshima, japan) or lactation (shuton-lactation; minami nihon kumiai siryo) period. the diet was free from intestinal microbiota modifiers, such as antimicrobials and probiotics. a preliminary enteropathogen survey of the farm showed that none of the following diseases were detected in sows and suckling piglets: rotavirus, transmissible gastroenteritis virus, clostridium 3.20 ± 0.04 3.62 ± 0.04 <0.001 perfringens, enterotoxigenic escherichia coli, salmonella sp., brachyspira hyodysenteriae, lawsonia intracellularis, or classical swine fever virus. detection of other pathogens such as porcine reproductive and respiratory virus and porcine circovirus type 2 was positive, but not active during this study. the infection rate of these two viruses in the farm was constant at about 40%, according to the survey of 10 randomly selected sows conducted every 4 months. infection outbreaks of ped virus occurred in december 2013 in the region where the farm was located. as a consequence, an infection screening was conducted at the farm by an independent livestock hygiene center. the screening results showed that all sows used in the present study were indeed naturally infected with ped virus. the present experiment was approved by the ethical committee at inatomi animal hospital (approval number 09121018). the experiment was carried out between may 2014 and june 2014. twenty pregnant sows were equally divided and allocated to either a probiotic-administered group (vp) or a control group (vc) with a similar mean parity (= 3.1). six weeks before parturition to 1 week after parturition, 15 g/day of the probiotic compound was orally administered to sows in the vp group, whereas 15 g/ day of a standard, probiotic-free diet was given to sows in the vc group. a commercial live-attenuated vaccine (nisseiken ped live vaccine, nisseiken, tokyo, japan) containing ped virus strain p-5v (seed) 1 was injected to all sows (10 4.5 tcid50/ head) 6 and 2 weeks before parturition. each sow fostered nine neonates throughout the experiment. some piglets died during the experiment. clinical signs included watery yellowish diarrhea and dehydration. an autopsy showed that their small intestine was thin and flaccid. a qualified clinical veterinarian (dr. takio inatomi) diagnosed that piglets died from ped infection. blood was collected from the jugular vein of sows 14 days prior to parturition and 0 and 7 days post-parturition. milk secretion was determined by a general method described in the standard methods of evaluation of reproductive performance compiled by the japan's pork producers association. 2 daily volume of milk secretion of all sows was measured at days 3 and 7 postparturition. a portion of milk was collected at days 0, 3, and 7 post-parturition. in addition, the total body weight of neonates was repeatedly measured immediately prior to and after daily suckling. when piglets of the experimental sows died during lactation, healthy and similar-age foster piglets were introduced from other sows so that all piglets ingested a similar amount of maternal milk. protein and fat percentages in milk at days 0 and 7 post-parturition were determined by a milk analyzer (milkoscan™ ft1; foss, eden prairie, mn, usa). 1 http://www.jp-nisseiken.co.jp/en/products/pdf/pig/ped_en.pdf. 2 http://www.jppa.biz. whey was collected from milk after centrifugation (13,000 × g, 30 min, 4°c). serum was collected from blood after centrifugation (1,200 × g, 20 min; room temperature). total iga or g concentrations were measured by a commercial elisa kit (porcine iga or igg elisa quantitation set; bethyl, montogomery, tx, usa). the determination method was as previously described elsewhere (31) . a portion of whey was sent to the nansatu livestock hygiene center (kagoshima, japan), and neutralized antibody titer against ped in whey and serum was determined by a general method as previously described elsewhere (10, 32) . either the student's or the welch's t-test was applied to analyze differences between mean values in all parameters. values are shown as mean ± se. differences between mean values were considered significant at p < 0.05 and a tendency to be significant at p < 0.1 in all statistical analyses. all calculations were made using statcel3 (oms, tokyo, japan) as add-in application for microsoft excel ® (microsoft, seattle, wa, usa). the mean feed intake of sows is shown in table 1 and probiotic compound had a significantly higher (p < 0.001) feed intake before (2.85 kg) and after parturition (3.62 kg) compared with sows vaccinated against ped virus but receiving no dietary probiotic supplementation (2.69 and 3.20 kg, respectively). in comparison with that of vc sows, the mean body weight of vp sows showed only a tendency (p < 0.1) to be greater as they approached parturition (191 vs 196 kg, respectively), but it was significantly higher (p < 0.05) by day 7 post-parturition (186 vs 191 kg, respectively) (figure 2 ). following parturition, milk produced by vp sows was found to be significantly greater (p < 0.05) than that by vc sows at days 3 (4.18 vs 3.63 kg/day, respectively) and 7 after parturition (5.14 vs 4.34 kg/day, respectively) ( figure 3) . the protein percentage in milk of vp sows was significantly greater (p < 0.05) at parturition day (11.1%) than that measured in milk of vc sows (10.1%) (figure 4) . however, when it was measured again at day 7 postparturition, no difference in protein percentage was detected between milk samples of vp and vc sows (4.9 vs 4.6%). when looking at the lactogenic immunity parameters, total iga concentration at day 0 (parturition day) was significantly (p < 0.05) higher in whey of vp sows (1.90 g/dl) than in that of vc sows (1.72 g/dl), but afterward, total iga concentration only showed a tendency (p < 0.1) to increase in whey of vp but not vc sows, when it was measured at days 3 (1.82 vs 1.75 g/dl, respectively) and 7 post-parturition (1.11 vs 1.04 g/dl, respectively) (figure 5a) . similarly, the concentration of total igg was significantly (p < 0.05) greater in whey of vp sows (6.58 g/dl) at day 0 when compared with that of vc sows (6.10 g/dl). however, unlike iga, the concentration of igg was the same in whey of both vp and vc sows, when it was measured at days 3 (2.12 vs 2.04 g/dl, respectively) and 7 post-parturition (0.36 vs 0.35 g/dl, respectively) ( figure 5b ). in addition, while the antibody titer was found significantly (p < 0.05) higher at day 0 in serum of vp sows (59.7 in geographic mean) when compared with that of vc sows (36.8) , it was similar in serum of sows in both experimental groups 14 days prior to parturition (78.8 vs 59.7, respectively) and 7 days post-parturition (36.8 vs 27.9, respectively) ( figure 5c) . likewise, the antibody titer was found to be similar (415.9 vs 207.9 at day 0; 168.9 vs 84.4 at day 7) in whey of both vp and vc sows ( figure 5d ). reproductive performance the combined administration of probiotics and vaccine had a positive effect on reproductive performance. indeed, the days between weaning and return to estrus for most vp sows were fewer (7 days) (p < 0.05) than those for vc sows (10 days) ( figure 6) . moreover, piglets farrowed by vp sows had a significantly (p < 0.05) higher weight (9,252 g/litter) ( figure 7a ) and a lower (p < 0.05) mortality percentage (12%) than those farrowed by vc sows (8,686 g/litter and 28%, respectively) ( figure 7b ). porcine epidemic diarrhea-vaccinated sows that were also supplemented with probiotics improved their feed intake, gained reasonable bodyweight during the period prior to parturition, and had only marginal weight loss by day 7 post-parturition than did sows that were vaccinated but did not receive probiotics supplementation (figures 1 and 2) . moreover, those sows receiving both probiotic supplementation and vaccine administration produced more milk than did sows that were only vaccinated (figure 3) . previously, alexopoulos et al. (33) showed that supplementing feed with a probiotic compound containing bacillus licheniformis and bacillus subtilis spores not only increased the body weight of sows but also helped minimize their weight loss during the suckling period. likewise, kritas et al. (34) reported that supplementing with b. subtilis c-3102 resulted in higher feed consumption and better body condition of sows. thus, it is very likely that in the present study there were similar effects, in which probiotics indirectly promoted weight gain in sows by competing with pathogens in the gut and stimulating the immune system of the host and hence making the animal more resistant to infections (35) . it is also possible that probiotics fought back the viral infection directly and decreased the viral load in sows and eventually in piglets as sirichokchatchawan et al. (28) recently suggested when discussing the potency of probiotic lab to reduce ped infectivity in vitro. in addition, the peptide-zinc mixture added to the probiotic compound may have also contributed to enhance the immune response. indeed, zinc has been previously shown to help reduce diarrhea incidence and increase body weight in pigs by stimulating the immune response against viral infection (36) . ultimately, healthier sows in the vp group consumed more feed, utilized better feed nutrients, and consequently gained more weight and produced more milk (figures 3 and 4) . in commercial swine farms, pregnant sows are inoculated with vaccines to trigger an immunoprophylactic reaction known as lactogenic immunity (15) . lactogenic immunity protects piglets against infections via the suckling of colostrum and milk of vaccinated sows (17) . nonetheless, in the present work, probiotics supplementation significantly improved the concentration of total iga and igg in the milk of sows than did ped vaccination alone, possibly resulting from stimulation of gut immunity by bacteria in the administered probiotic compound (figures 5a,b) . the sole administration of ped vaccine to sows has been shown to increase ped-specific igg levels in serum (15) . interestingly, by administering the ped vaccine along with probiotics supplementation, we were able to trigger an even higher serum ped-specific antibody titer than did ped vaccination alone, very likely due to a greater stimulation of the immune system of sows. then, as our results show, these antibodies were mobilized to fight back ped infection before and after parturition (figure 5c) , which consequently also helped to stimulate ped-specific antibodies production in whey ( figure 5d) . finally, once piglets started to suckle, passive immunity against ped was putatively transferred from sows via colostrum and milk (17, 18) , possibly inducing early maturation of gut immunity of piglets that lessened the infection, which in turn heightened the effect of vaccination (37) . this ultimately resulted in the farrowing of piglets with a greater weight and a lower mortality percentage by sows receiving both probiotics and the ped vaccine (figure 7) . viral infections can cause serious biological disturbances in sows including a higher recurrence of estrus (12) , as well as abnormal tissue development such as ovarian cysts formation (38) . these disorders can lead to low fertility (12) . in addition, it is believed that a decrease in appetite and lower efficiency of nutrient utilization during the course of an infection can cause a lower body weight of sows which in turn cause a longer period before sows can return to estrus (39) . nonetheless, it has been previously shown that b. subtilis c-3102 supplementation helped reduce the number of days between weaning and return to estrus (34) . furthermore, a probiotic compound containing the same bacteria used in this study improved the percentage of sows returning to estrus (29) . in agreement with this, in the present work ped vaccination and probiotic administration to ped-infected sows considerably lowered the number of days of recurrence of estrus than did ped vaccination alone ( figure 6 ). as previously mentioned, it is very likely that an enhanced microbiota permitted sows to eat more and utilized nutrients more efficiently, thus preventing malnutrition from causing a longer period between weaning and return to estrus (39) . olanratmanee et al. (12) found that ped infection caused a reduction in the body weight of piglets at birth. in the present study, piglets farrowed by sows vaccinated against ped and supplemented with probiotics had a greater mean litter weight at birth and a lower mortality percentage than did those farrowed by sows receiving ped vaccination but no probiotic supplementation (figures 7a,b) . however, unlike our study, kritas et al. (34) found that supplementation of a single probiotic strain (b. subtilis c-3102) to sows infected with pathogenic e. coli did not alter the body weight at birth of piglets. this apparent discrepancy may be due to in the present study and unlike that by kritas et al. a probiotic mixture was given to sows. indeed, it has been demonstrated both in vivo and in vitro that multistrain probiotic compounds are more effective at inhibiting pathogens than single-strain probiotics and that some strain mixtures are more effective than others (40, 41) . in conclusion, we demonstrated that supplementation of a mixture of probiotics and a peptide-zinc compound enhanced the immune system of ped virus-infected sows. in addition, we also showed that probiotics were able to act as adjuvant to commercial live ped vaccines administered to pregnant sows. this experiment was approved by the ethical committee of inatomi animal hospital in japan. takio inatomi belongs to inatomi animal hospital informed consent to animal owners. improvement of ped symptom by probiotic administration frontiers in immunology | www.frontiersin.org december 2017 | volume 8 | article 1877 author contributions ti designed and performed this experiment and contributed to the discussion and interpretation of data. ma supplied the experimental materials and contributed to the discussion and interpretation of data. gr-p wrote the manuscript. ri contributed to the discussion and interpretation of data and wrote the manuscript. tt designed this experiment, contributed to the discussion and interpretation of data, and wrote the manuscript. all authors have read and approved the final manuscript. references epidemiological factors associated to spread of porcine epidemic diarrhea in japan porcine epidemic diarrhea virus: an emerging and re-emerging epizootic swine virus molecular characterization of pig epidemic diarrhoea viruses isolated in japan from an outbreak of swine diarrhea of a newtype associated with coronavirus-like particles in japan comparative genome analysis and molecular epidemiology of the reemerging porcine epidemic diarrhea virus strains isolated in korea new variants of porcine epidemic diarrhea virus, china vet virol porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis an immunohistochemical investigation of porcine epidemic diarrhoea isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages impact of porcine epidemic diarrhea on performance of growing pigs impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen derivation of attenuated porcine epidemic diarrhea virus (pedv) as vaccine candidate oral efficacy of vero cell attenuated porcine epidemic diarrhea virus dr13 strain antibody response of pregnant sows to porcine epidemic diarrhea virus live vaccine and maternally-derived antibodies of the piglets porcine epidemic diarrhoea virus: a comprehensive review of molecular epidemiology, diagnosis, and vaccines probiotics and colostrum/milk differentially affect neonatal humoral immune responses to oral rotavirus vaccine evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study molecular epidemiology of porcine epidemic diarrhea virus in china molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus (pedv) field isolates in korea oral delivery of probiotics expressing dendritic cell-targeting peptide fused with porcine epidemic diarrhea virus coe antigen: a promising vaccine strategy against pedv influence of probiotic lactobacilli colonization on neonatal b cell responses in a gnotobiotic pig model of human rotavirus infection and disease lactobacilli and bifidobacteria enhance mucosal b cell responses and differentially modulate systemic antibody responses to an oral human rotavirus vaccine in a neonatal gnotobiotic pig disease model the effect of enterococcus faecalis cell preparation (ec-12) against the diarrhea in the nursing and weaning piglets under the clinical condition a cell preparation of enterococcus faecalis strain ec-12 stimulates the luminal immunoglobulin a secretion in juvenile calves cell preparation of enterococcus faecalis strain ec-12 prevents vancomycin-resistant enterococci colonization in the cecum of newly hatched chicks protective effects of cell-free supernatant and live lactic acid bacteria isolated from thai pigs against a pandemic strain of porcine epidemic diarrhea virus dietary administration of probiotics to sows and/or their neonates improves the reproductive performance, incidence of post-weaning diarrhea and histopathological parameters in the intestine of weaned piglets morphometric and histopathological evaluation of a probiotic and its synergism with vaccination against coccidiosis in broilers the evaluation of secretion volume and immunoglobulin a and g concentrations in sow colostrum from anterior to posterior teats isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate field evaluation of the efficacy of a probiotic containing bacillus licheniformis and bacillus subtilis spores, on the health status and performance of frontiers in immunology | www sows and their litters reproductive performance of sows was improved by administration of a sporing bacillary probiotic (bacillus subtilis c-3102) evaluation of probiotics as a substitute for antibiotics in a large pig nursery highdose dietary zinc oxide mitigates infection with transmissible gastroenteritis virus in piglets strategies for design and application of enteric viral vaccines pathological evaluation of reproductive system of porcine reproductive and respiratory syndrome virus-vaccinated and nonvaccinated anestrus sows and gilts body weight loss during lactation and its influence on weaning-to-service interval and ovulation rate in landrace and yorkshire sows in the tropical environment of thailand in vitro evaluation of single-and multi-strain probiotics: inter-species inhibition between probiotic strains, and inhibition of pathogens health benefits of probiotics: are mixtures more effective than single strains? key: cord-272491-a84pahdr authors: maron-gutierrez, tatiana; rocco, patricia r. m. title: cell-free therapies: novel approaches for covid-19 date: 2020-09-18 journal: front immunol doi: 10.3389/fimmu.2020.583017 sha: doc_id: 272491 cord_uid: a84pahdr nan first described in late 2019, coronavirus disease (covid)-19, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), rapidly escalated into a global pandemic with a high case fatality rate. covid-19 patients with acute respiratory failure exhibit some distinctive pathological characteristics, which to some extent resemble the acute respiratory distress syndrome (ards), sars, and middle east respiratory syndrome (mers); these include hypoxemia, diffuse alveolar damage with cellular exudates, extensive pulmonary inflammation, lung edema, and hyaline membrane formation (1) . in addition to respiratory failure, these patients present with a dysfunctional systemic host response that affects multiple organs, including the central nervous, cardiovascular, renal, and gastrointestinal systems (2) (3) (4) , as well as a wide range of coagulation disturbances, such as thrombocytopenia, sustained systemic clotting activation, massive thrombin and fibrin formation, and disseminated intravascular coagulation (5) . few therapies are effective for covid-19 patients, and to date there are still no vaccines available. there are vaccine candidates in development and ongoing clinical trials, but they are expected to be available, at best, in early 2021 (6) . thus, novel effective and safe therapies are urgently required to treat covid-19 patients (7) . in this context, several clinical trials have begun pursuing new therapies as well as repurposing existing ones, largely through drug repositioning, including of antiviral, antimalarial, and anti-inflammatory agents. these therapies aim to target entry of the virus into host cells, multiplication of the viral genetic material, and/or the immune response and inflammatory process (6) . although some therapies shows promising results, they raise some concerns, such as limited cohort sizes, non-randomized trials, lack of considerations for gender, comorbidities, concurrent treatments, and route of drug delivery, among others (6) . early in the covid-19 pandemic, corticosteroids were not recommended because of the adverse effects previously observed in influenza, sars-cov, and mers-cov infections (8) . nevertheless, a recent controlled, open-label trial of dexamethasone for up to 10 days resulted in lower 28-day mortality in mechanically ventilated patients (9) . mesenchymal stromal cell (msc)-based treatment has been proposed as a suitable therapeutic approach for covid-19 (10) . as mscs have the potential to interact directly with immune cells, their transplantation may improve outcomes in covid-19 patients through modulation of the immune response, mitigation of the inflammatory cascade, and promotion of tissue repair and regeneration (11, 12) . in addition, cd147, the second entry receptor for sars-cov-2, can be expressed by tissue-specific stem cells (13) . together with the loss of airway epithelial cells by viral infection and replication, the additional loss of regenerating stem cells may be responsible for diminished cellular and lung regeneration (13) . cell-based therapies have been quite extensively studied for potential applicability in covid-19, especially given the short time since the onset of the pandemic (14, 15) ; however, due to the risk of macro-and microthrombosis, cell-free therapies may be more appealing. cell-free therapies might decrease injury to different organs, such as lung, heart, kidney, liver, and brain, as well as reduce thrombus formation and endothelial inflammation (figure 1 ). cell-free therapies, such as the msc secretome (obtained as conditioned medium) and extracellular vesicles (evs) from mscs, have been studied in ards (16) and multiple organ dysfunction syndrome (mods) (17, 18) for their antiinflammatory and anti-fibrogenic effects, as well as their epithelial and endothelial regenerative properties. however, figure 1 | potential effects of pharmaceutical preparations of msc secretome on different organs involved in covid-19. the msc secretome, in the form of conditioned medium containing extracellular vesicles (evs) and mitochondria, could be transformed into a stable product for the treatment of patients with covid-19. secretome-based therapies might mitigate cardiac, kidney, liver, nervous system, and lung injury; decrease macro-and micro-thrombus formation and endothelial inflammation; and repair lung epithelial and endothelial cells. many researchers and international societies, including the international society for extracellular vesicles (isev) and the international society for cellular and gene therapies (isct), have expressed concern regarding the use of evs-whether derived from mscs or from other cell sources-in the treatment of covid-19 (19) . clinical trials are encouraged; however, the use of evs for any purpose in covid-19 is not endorsed by isev and isct until proper regulation of manufacturing, quality control protocols, and clinical trial design are in place, in order to avoid the stem-cell industry trying to sell unregulated msc treatments (19) . in this context, the implementation of computer-controlled bioreactors (20) and the development of standard operating procedures (sops) for obtaining a good manufacturing practice (gmp)-grade msc secretome and its components are necessary for clinical applications (21) . these must be reproducible, scalable, and well-controlled to limit heterogeneity and enhance predictability in the composition and function of secretome-derived products (22) . further studies are still needed to better understand the best route of cell-free therapy delivery, dose, and timing of administration (23) . moreover, important factors should be taken into consideration such as the culture medium, cell and tissue source, donor variability, and culture conditions (cell priming with hypoxia, biochemical or mechanical stimuli, three-dimensional spheroid culture, among others), as well as the timing and method of msc-secretome harvesting (20, 22) . in short, cell-free therapies could be a more suitable treatment for covid-19 than mscs, but additional investigations are required (2). the msc secretome is a complex mixture of soluble components (growth factors and cytokines), a vesicular portion that comprises evs, and cell organelles (e.g., mitochondria) (24) (25) (26) . considering that sars-cov-2 infection is being associated with an increased inflammatory process (27), we believe that msc secretome products might help reverse covid-19related immune dysregulation, due to their anti-inflammatory, immunomodulatory, and regenerative effects (14) . the msc secretome has properties similar to those of its parent mscs (28) . moreover, the secretome is generally considered safer than parent cells, since it (1) lacks the potential for endogenous tumor formation, as it cannot self-replicate; (2) can be classified as non-immunogenic, due to the limited number of antigenic components; and (3) may lead to less formation of emboli when injected intravenously (14, 29) . as recently reported elsewhere, the msc secretome (in the form of conditioned medium) can be stored more easily than mscs (14) , which is an important consideration given the lack of adequate facilities in developing countries. transforming msc-secretome components into a freeze-dried, stable powder product which can be reconstituted for intravenous injection or inhalation might be a suitable approach for the treatment of patients with covid-19 (figure 1 ) (17) . mitochondria are intracellular organelles that play a vital role in cellular homeostasis and enable stress adaptation (30) . most cellular energy generation takes place in the mitochondria (31), and excessive mitochondrial dysfunction leading to defects in energy flow leads to unsustainable maintenance of life and adaptation to stress (30) . one of the main mechanisms associated with the pathophysiology of sepsis is mitochondrial dysfunction (32, 33) . in 2012, the first evidence that mscs restore alveolar bioenergetics through cx43-dependent alveolar attachment and mitochondrial transfer was observed in experimental ards (25) . in 2015, phinney et al. observed mitochondrial transfer from mscs to macrophages in response to oxidative stress (34) . recently, court et al. investigated the effect of mscmediated transfer of mitochondria on lymphoid cells. they observed mitochondria-labeled mscs mainly in cd4+t cells, paving the way for exploration of organelle-based therapies in immune diseases (26) . interestingly, mscs are not the only cell type able to transfer mitochondria. lipopolysaccharide (lps)stimulated monocytes release free and microvesicle-associated mitochondria as part of their secretome (35) . these studies demonstrate the complexity of cell-to-cell communication by identifying mitochondria as a source for target cells to restore their bioenergetics, enable immunomodulatory effects, and suppress inflammation. clinical trials failed to show efficacy of immunomodulatory therapies in sepsis (36, 37) . since bacterial sepsis shares some similarities with covid-19, we may consider a new route for therapeutic intervention focused on mitochondrial cell transfer. another option is therapy with engineered evs containing mitochondria. the immunomodulatory and regenerative potential of mscs may be independent of direct cellular cross-talk (21, 38) . mscs act through a paracrine mechanism based primarily on evs, which interact with neighboring target cells or can reach distant organs (39) . distinctions between the subtypes of evs were previously based on subcellular origin, with exosomes being of endosomal origin and microvesicles derived from the cell membrane. however, given the historically contradictory definitions and inaccurate expectations of biogenesis associated with these terms, in 2018, isev recommended the use of new terms for ev subtypes that refer to their physical characteristics, such as size (small and medium/large evs) or density; their biochemical composition (cd63+/cd81+-evs, annexin a5stained evs, etc.); or their cell of origin (msc evs, podocyte evs, etc.) (40) . ev biochemistry varies according to composition and cell source (41) . evs can carry membrane and cytosolic proteins, transcription factors, dna, coding and non-coding rnas and various signal transduction molecules (5, 21, 38, 42) , acting on both physiological and pathological events, e.g., modulating the inflammatory response (2, 14) . evs also carry different cytokines and growth factors, such as interleukin (il)-6 and il-10, transforming growth factor (tgf)-β, and hepatocyte growth factor (hgf) (43) . in addition, evs contain matrix-remodeling enzymes, such as matrix metalloproteinases (mmps), heparanases, hyaluronidases, and tissue inhibitors of metalloproteinases (timps); ev-mediated proteolytic activities have also been described, which might modulate the remodeling process and contribute to tissue repair (39) . in this context, our group demonstrated that mscs increased mmp-8 expression and decreased timp-1 expression in an experimental model of ards, suggesting an effect on the extracellular matrix (44) . rather than suppressing immune responses, evs appear to act as true modulators, inducing regulatory responses and tolerance in order to restore homeostasis (45) . administration of evs has proven safe and effective in preclinical studies of lung injury and sepsis models (16, 18, 19, (46) (47) (48) . in preclinical studies, ev therapy ameliorated acute lung injury (49, 50) and was equally or even more effective than mscs in mitigating lung inflammation and pathological damage (41, 51) . evs have also been shown to attenuate e. coli and influenza infections (47, 48, 52) , including a mixed swine (h3n2, h1n1) and avian (h9n5, h7n2) influenzainduced lung injury model (48) . the beneficial effects of evs have further been observed in an ischemic stroke model (53, 54) . since covid-19 may be associated with damage to other organs in addition to the lungs and has been associated with ischemic stroke, evs could be a particularly promising therapy in this context (19) . however, only one prospective study has evaluated the effects of evs (specifically, exosomes from bone marrow-derived mscs) in covid-19 patients (55) . even though the inflammatory response was reduced significantly, and no adverse events were observed, this study encountered limitations regarding ev characterization and biological properties, the actual dose of ev administered, and how the injection of evs was monitored. at the time of writing, there are three ongoing clinical trials of msc-derived evs for covid-19 treatment, to be administered intravenously (chictr2000030484) or by inhalation (nct04276987, chictr2000030261); however, recruitment has not yet begun. it is important to consider both the source of mscs from which evs are derived, which can be obtained from different tissues and donors, and their preparation. depending on these factors, mscs and their evs can have different therapeutic properties. compared to bone marrow-derived mscs, for instance, adipose tissue-derived mscs express more tissue factor (an important initiator of coagulation in sepsis) and reduce hemocompatibility, which has been shown to vary according to donor and culture handling conditions (56, 57) . therefore, evs obtained from adipose tissue-derived mscs may have greater thrombogenic activity than those from bone marrowderived mscs (58, 59) , and thus should not be considered for use in covid-19 patients (5, 19) . furthermore, the therapeutic effects of evs are known to vary according to their preparation method, even when obtained from the same msc source (53) . moreover, differences in donor parameters, including age, have been associated with significant variations in cytokine content, thus resulting in different effects on injury mitigation (60, 61) . in addition to their natural cargo, evs can be loaded with biochemical compounds or genetically engineered to target infected cells, thus providing additional perspective for covid-19 treatment beyond msc-derived evs, including ev-based drug delivery, inhibition of ev biogenesis and uptake, and evbased vaccines (41) . the latter might be a particularly promising cell-free approach to covid-19 treatment. exosome vaccines may contain membrane-anchored ectodomains of sars-cov-2 components on their surface, facilitating cross-linking of the bcell receptor (2) . exosome-based vaccines containing the spike (s) proteins of sars-cov-2, one of the structural proteins that mediate viral entry into the host cells (62) , could induce high levels of neutralizing antibodies (63) . in addition, the cargo of ev-based vaccines can be modified to include proteins and mirnas to help modulate the immune response (62) . however, further research is needed to assess the safety and clinical pharmacology of ev-based therapies in order to provide guidance for manufacturing, storage, dosing, and administration (19) before these potential treatments can be made more accessible worldwide (14, 17) . in the specific setting of covid-19, administration of msc-evs may have several advantages over mscs: (i) there is no risk of emboli formation in the injured microcirculation; (ii) no risk of mutagenicity or oncogenicity is observed; (iii) nebulized delivery can be used (despite several controversies regarding this route of administration); and (iv) tolerance of longer storage periods allow for later therapeutic use, reducing the stringency of storage and transportation requirements. in short, cell-free therapies should be considered a promising alternative for covid-19 treatment. clinical trials of ev-based therapies for covid-19 should clearly describe the dose, route of administration, characteristics of the administered evs, timing of administration, any monitoring performed during administration, and detailed primary and secondary outcomes. all authors conceived the concept of this article and wrote the manuscript. mesenchymal stem cells as a potential therapy for covid-19 regenerative medicine in covid-19 treatment : real opportunities and range of promises the involvement of the central nervous system in patients with covid-19 multiple organ dysfunction in sars-cov-2 : mods-cov-2 rationale for the clinical use of adipose-derived mesenchymal stem cells for covid-19 patients can stem cells beat covid-19: advancing stem cells and extracellular vesicles toward mainstream medicine for lung injuries associated with sars-cov-2 infections emerging therapies for covid-19 pneumonia clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury dexamethasone in hospitalized patients with covid-19 -preliminary report mesenchymal stem cell therapy for covid-19: present or future transplantation of ace2-mesenchymal stem cells improves the outcome of patients with covid-19 pneumonia cytokine storm and leukocyte changes in mild versus severe sars-cov-2 infection: review of 3939 covid-19 patients in china and emerging pathogenesis and therapy concepts cd147 as a target for covid-19 treatment: suggested effects of azithromycin and stem cell engagement current status of cell-based therapies for respiratory virus infections: applicability to covid-19 emerging trends in covid-19 treatment: learning from inflammatory conditions associated with cellular therapies extracellular vesicles : a new frontier for research in acute respiratory mesenchymal stromal cell secretome for severe covid-19 infections: premises for the therapeutic use current understanding of the therapeutic benefits of mesenchymal stem cells in acute respiratory distress syndrome international society for extracellular vesicles and international society for cell and gene therapy statement on extracellular vesicles from mesenchymal stromal cells and other cells: considerations for potential therapeutic agents to suppress coronavirus disease-19 modulation of the mesenchymal stem cell secretome using computer-controlled bioreactors: impact on neuronal cell proliferation, survival and differentiation non-coding rnas in mesenchymal stem cell-derived extracellular vesicles: deciphering regulatory roles in stem cell potency, inflammatory resolve, and tissue regeneration bioprocessing of mesenchymal stem cells and their derivatives: toward cell-free therapeutics exosomes derived from bone marrow mesenchymal stem cells as treatment for severe covid-19 cell-based therapies for the acute respiratory distress syndrome mitochondrial transfer from bone-marrow-derived stromal cells to pulmonary alveoli protects against acute lung injury mitochondrial transfer from mscs to t cells induces treg differentiation and restricts inflammatory response clinical features of patients infected with 2019 novel coronavirus in wuhan molecular mechanisms responsible for therapeutic potential of mesenchymal stem cell-derived secretome respiratory syncytial virus-infected mesenchymal stem cells regulate immunity via interferon beta and indoleamine-2,3-dioxygenase an energetic view of stress: focus on mitochondria mitochondria as key components of the stress response the third international consensus definitions for sepsis and septic shock (sepsis-3) neuroanatomy of sepsis-associated encephalopathy mesenchymal stem cells use extracellular vesicles to outsource mitophagy and shuttle micrornas mitochondria are a subset of extracellular vesicles released by activated monocytes and induce type i ifn and tnf responses in endothelial cells is a "cytokine storm" relevant to covid-19? the role of mitochondrial dysfunction in sepsis-induced multiorgan failure nexus between extracellular vesicles, immunomodulation and tissue remodeling: for good or for bad? ann transl med extracellular vesicles and matrix remodeling enzymes: the emerging roles in extracellular matrix remodeling, progression of diseases and tissue repair minimal information for studies of extracellular vesicles 2018 (misev2018): a position statement of the international society for extracellular vesicles and update of the misev2014 guidelines the role of extracellular vesicles in covid-19 virus infection current knowledge and future perspectives on mesenchymal stem cell-derived exosomes as a new therapeutic agent functional proteins of mesenchymal stem cell-derived extracellular vesicles effects of mesenchymal stem cell therapy on the time course of pulmonary remodeling depend on the etiology of lung injury in mice identification of the right cell sources for the production of therapeutically active extracellular vesicles in ischemic stroke human mesenchymal stem cell microvesicles for treatment of escherichia coli endotoxin-induced acute lung injury in mice therapeutic effects of human mesenchymal stem cell-derived microvesicles in severe pneumonia in mice mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model mesenchymal stem cells-derived extracellular vesicles in acute respiratory distress syndrome: a review of current literature and potential future treatment options exosomes derived from endothelial progenitor cells ameliorate acute lung injury by transferring mir-126 extracellular vesicles derived from mesenchymal stromal cells: a therapeutic option in respiratory diseases? mesenchymal stem cell-derived extracellular vesicles decrease lung injury in mice mesenchymal stromal cell-derived small extracellular vesicles induce ischemic neuroprotection by modulating leukocytes and specifically neutrophils extracellular vesicles improve post-stroke neuroregeneration and prevent postischemic immunosuppression exosomes derived from bone marrow mesenchymal stem cells as treatment for severe covid-19 clinical cellular therapeutics accelerate clot formation are therapeutic human mesenchymal stromal cells compatible with human blood? stem cells the effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles effect of mscs and msc-derived extracellular vesicles on human blood coagulation differential effects of extracellular vesicles from aging and young mesenchymal stem cells in acute lung injury msc-derived exosomes: a novel tool to treat therapy-refractory graft-versushost disease detection of antibodies against sars-cov in serum from sars-infected donors with elisa and western blot exosomal vaccines containing the s protein of the sars coronavirus induce high levels of neutralizing antibodies the authors thank filippe vasconcellos, b.a., for his assistance in editing the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 maron-gutierrez and rocco. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-295302-vwrxentv authors: shivarov, velizar; petrov, peter k.; pashov, anastas d. title: potential sars-cov-2 preimmune igm epitopes date: 2020-04-30 journal: front immunol doi: 10.3389/fimmu.2020.00932 sha: doc_id: 295302 cord_uid: vwrxentv while studying the human public igm igome as represented by a library of 224,087 linear mimotopes, three exact matches to peptides in the proteins of sars-cov-2 were found: two in the open reading frame 1ab and one in the spike protein. joining the efforts to fast track sars-cov-2 vaccine development, here we describe briefly these potential epitopes in comparison to mimotopes representing peptides of sars-cov, hcov 229e and oc43. the covid19 pandemic has put to test the capacity of vaccinology to produce as fast as possible relevant vaccines. a number of recent reports predict possible sars-cov-2 epitopes for vaccine development but there are no reports on experimentally defined b cell epitopes (1) (2) (3) (4) (5) . the closest to identification of actual epitopes is the finding of pentapeptide sequences from the viral proteome in other known epitopes form iedb (5) . a library of 224,087 mimotopes corresponding to the human public igm repertoire as represented in a plasma pool from 10,000 healthy donors was recently designed (6) . the mimotopes were selected from a commercial 7 amino acid random peptide phage display library (ph.d. 7, new england biolabs). conceptually, this mimotope library reflects at a certain level of detail, the repertoire of igm specificities in the plasma focusing on the recurring ones. the latter can be just natural antibodies or they may represent the product of fast extrafollicularly expanding igm clones that may serve as precursors of highly specific, somatically mutated, class-switched b cells. the preimmune repertoire has to be quasi-complete to provide for rapid expansion of clones reactive with any newly encountered antigen. the same may not be true for our library although, due to the polyspecific binding, most of the available public repertoire may be partially represented in it (6) . here we report that the igm mimotope library contains heptapeptides identical to peptides in the proteome of sars-cov-2. one of them may serve as a potentially neutralizing epitope on the spike protein. the design and the properties of the mimotope database has been published elsewhere (6) . the available sequences of the genomes of sars-cov (nc_004718.3), sars-cov-2 (asm985889v3), hcov229e (nc_002645.1), and hcovoc43 (ay391777.1) were split into consecutive overlapping heptamers shifted by one residue and the resultant sequence sets were compared to the sequences in the database of natural mimotopes. only exact matches were considered. the homologous sequences in the non-redundant databases of the human proteome and viridae (taxid:10239) were blast searched using the ncbi blastp suite (https://blast.ncbi.nlm.nih. gov/blast.cgi?page=proteins). as part of an ongoing analysis, the natural mimotope database was represented as a graph by connecting the sequences having at least 5 exact matches (i.e., of maximal hamming distance 2). the graph had one giant component containing approximately 90% of the sequences which was further considered as the graph of interest. for the present study, the degrees of the vertices representing the natural sars-cov-2 epitopes, all of which belonged to the giant component, were used as the number of adjacent mimotopes parameter. for a set of words of length l based on an alphabet of l symbols, the theoretical average number of neighbors n at hamming distance d was calculated using the following formula for the number of neighbors: l d for the present study, l = 20, l = 7, and d < 3. for the first layer of neighbors n1 = 133 and for the second n2 = 7581. under the hypothesis that the database is a random sample from the set of heptamer peptides, the probability of the occurrence of each neighbor is: and the expected mean number of distinct neighbors at d < 3 was calculated as p.(n(1,7)+n(2,7)) ≈1.33. the value of p was used subsequently also in a binomial test to calculate the probabilities of finding equal or higher number of adjacent mimotopes ( table 1) . the structure of the spike of sars-cov-2 was recently published [6vsb.pdb (2) ]. it was used to visualize the molecular context of the spike epitope found. the visualization of the structure and the calculation of the relative solvent exposed surface were done using ucsf chimera, developed by the resource for biocomputing, visualization, and informatics at the university of california, san francisco, with support from nih p41-gm103311. to demonstrate linear b cell epitope prediction uncertainty, we have reanalyzed data from he et al. (7) on patients' sera reactivity to sars-cov peptides comparing them to bepipred (http://tools.iedb.org/bcell/help/#bepipred2) scores of the same sequences. a simple comparison for exact matches to peptides from the sars-cov-2 proteome yielded 3 heptapeptides-two in the open reading frame 1ab ( 3518 aqtgiav 3524 and 5198 tkgphef 5204 ) and one in the spike protein ( 108 ttldskt 114 ). the expect value (e) is a parameter that describes the number of hits one can "expect" to see by chance when searching a database of a particular size. essentially, the e value describes the random background noise (https://blast.ncbi.nlm.nih.gov/ blast.cgi?cmd=web&page_type=blastdocs&doc_type= faq#expect). the e value of search results with so short sequences is very high and the mere number of sequences is not statistically significant. yet, this does not refute the fact that 3 heptapeptides which are operationally defined mimotopes of human preimmune antibodies, are part of the viral proteome and, thus, represent (parts of) possible epitopes. on the other hand, the mimotopes in the database sometimes form non-random clusters of homologous sequences much like the mimotopes selected by a single monoclonal antibody. each one among 224,087 randomly selected heptamers should have on the average 1.33 homologous sequences in that same database that differ from it by up to 2 mismatches. as seen from table 1 , all sars sequences but not those from trivial hcov were members of clusters significantly greater than random (binomial test, p < 0.05, false discovery rate adjusted). this is an indication that the presence of these sequences is non-random and they represent clusters of mimotopes representing well-represented individual (poly)specificities. an important prerequisite for the functionality of these epitopes is their degree of exposure to the solvent. the recently published structure of the spike (s protein) of sars-cov-2 (2) shows that 108 ttldskt 114 forms a loop exposed to the solvent (figure 1a) . the relative solvent exposed surface greatly exceeds the threshold of 5% for participating in contacts ( figure 1b) . this loop is adjacent to the loop representing the epitope of the neutralizing antibody lca60 on the sars-cov spike (8, 9) . presumably, it is similarly exposed further in the open conformation of the spike domains. the adjacent n-glycosylation sites are n165 and n234. dependent on the size of the carbohydrate sidechains, they may partially occlude the epitope. the closest sequences in the human proteome are 540 tltldskt 547 of the prostate-specific transglutaminase (tgm4) and 462 ttldski 468 of mucin-16 [also known as ovarian tumor marker ca125, q8wxi7.3, (10) ]. both are on tumor associated antigens (10, 11) . while tgm4 is an intracellular antigen, mucin-16 is highly accessible on cell surfaces and in a soluble form. the mucin sequence 462 ttldski 468 is t/s biased, represents part of the highly o-glycosylated n-terminal part of mucin-16 and is predicted to be o-glycosylated itself. normally, such mucin protein core epitopes are occluded by glycosylation and thus, cryptic with respect to immune tolerance. yet, monoclonals to similar epitopes turned out to bind specifically to tumor expressed mucins (12) (13) (14) (15) which are aberrantly/hypo glycosylated. the sequences 108 ttldskt 114 has several exact matches in viruses outside the family coronaviridae in hypothetical proteins (7)]. the two predicted natural epitopes are overlaid in red. there is antibody reactivity in patients' sera to these epitopes although one of them has bepipred score far below the threshold of 0.5. of various phages. at least one of them infects l. plantarum which is a common species in the gut microbiome. it is not surprising that the public igm repertoire has clones potentially capable of binding to non-conserved regions of novel viruses. similarly, the igm igome contained sequences found also in sars-cov, although the epidemic was too restricted to be reflected in the antibody repertoires of the donors (table 1) . furthermore, no signs of persistent antibody titers after exposure were observed. the representation of clones reactive with the trivial human coronaviruses 229e and oc43 was rather narrower than that of the unknown strains. some of the epitopes were conserved between sars-cov and sars-cov-2 (aqtgiav and tkgphef) but they were found in non-structural proteins and are hardly targets for neutralizing antibodies ( table 1) . on the other hand, all potential epitopes found could play a role in targeting the viral proteins to specific b1 cells which produce the bulk of natural igm. the latter are known to be excellent antigen presenting cells able to prime cd4 + t cells, and initiate th1 immune responses (16) (17) (18) in antigen specific manner much like activated specific b2 cells (17). it has been shown that b1 cells secreted igm is a non-redundant and essential arm of the humoral responses to influenza in mice (19) . this implies that natural antibody epitopes might be essential components of subunit vaccines even though they may not represent typical dominant epitopes. the role of overlapping t and b cell epitopes is not clear except when the b cell receptor has a high enough affinity for the epitope to protect it during processing (20) , but it is interesting that one of the sars-cov natural epitopes ( 922 ttstalg 928 ) is also part of a cd4 t cell epitope in the context of hla-dr b1 * 04:01 (21) . using the iedb preferred method the epitope 108 ttldskt 114 is predicted to overlap a potential class ii epitope in the context of hla-drb1 * 07:01, while two other potential epitopes just up-and downstream overlap it partially (in the context of hla-dpa1 * 02:01/dpb1 * 01:01 and hla-drb1 * 04:01, hla-drb1 * 04:05 and hla-drb1 * 13:02, respectively). in this respect, maybe a more useful epitope would be the continuous sequence 99niirgwifgttldsktqsllivnnatnv126. the current thinking separates the repertoire of natural and induced antibodies (22) . the preimmune igm mimotopes we describe could represent also epitopes of naïve b cell clones which may have undergone extrafollicular expansion poised to initiate also follicular immune responses. as to the capacity of these epitopes to induce fully mature antibody response, it is interesting to note that the two preimmune igm epitopes found for the spike of sars-cov (922ttstalg928 and 389vkgddvr395) are proven antibody targets in approximately one fourth of the sars patients (7) . thus, our mimotope library has the capacity to identify potential true precursor epitopes and not only natural antibody epitopes. furthermore, a recent report indicates the importance of igm antibodies in the control of the diseases in mild cases of covid19 (23) . thus, it is quite possible that the sars-cov-2 spike epitope ttldskt is bound by b cells that will contribute to the induced immune response. none of the in silico predicted epitopes (1-5) overlaps with 108 ttldskt 114 which is also specific to sars-cov-2. the correlation between the actual reactivities in sars-cov patients' sera and the bepipred score ( figure 1c) confirms the low power of linear b cell epitope predicting algorithms, and underlies the necessity to base the proposals of new epitopes as much as possible on actual binding data. these considerations make the novel sars-cov-2 epitopes valid targets in the search for a vaccine for covid-19. the whole paradigm followed here focuses exclusively on the relatively rare linear epitopes. a lot more information about conformational epitopes may be hidden in the natural mimotope database but the approaches for sorting out clusters of mimotopes defining a conformational epitope are still being developed. the proposed actual preimmune igm epitopes of sars-cov-2 can be instrumental both as parts of subunit vaccines or in the design of nanoparticlebased vaccines but also in the development of therapeutic monoclonal antibodies. the datasets analyzed and the scripts for this study can be found in the github repository (https://github.com/ansts/ sars-cov-2). vs and ap: conceptualizing, manuscript preparation, and data analysis. pp: data analysis. novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov structure, function and antigenicity of the sars-cov-2 spike glycoprotein candidate targets for immune responses to 2019-novel coronavirus (ncov): sequence homology-and bioinformatic-based predictions preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies epitopes for a 2019-ncov vaccine diagnostic profiling of the human public igm repertoire with scalable mimotope libraries identification of immunodominant sites on the spike protein of severe acute respiratory syndrome (sars) coronavirus: implication for developing sars diagnostics and vaccines cryo-em structure of the 2019-ncov spike in the prefusion conformation unexpected receptor functional mimicry elucidates activation of coronavirus fusion muc16 (ca125): tumor biomarker to cancer therapy, a work in progress overexpression of transglutaminase 4 and prostate cancer progression: a potential predictor of less favourable outcomes effect of modification of carbohydrate side chains on the reactivity of antibodies with core-protein epitopes of the muc1 gene product development and characterization of breast cancer reactive monoclonal antibodies directed to the core protein of the human milk mucin epitope mapping of anti-muc1 mucin protein core monoclonal antibodies epitopes of muc1 tandem repeats in cancer as revealed by antibody crystallography: toward glycopeptide signatureguided therapy an overview of b-1 cells as antigenpresenting cells b cells are the dominant antigen-presenting cells that activate naive cd4+ t cells upon immunization with a virus-derived nanoparticle antigen b-1 and b-2 cell-derived immunoglobulin m antibodies are nonredundant components of the protective response to influenza virus infection modulation of antigen processing by bound antibodies can boost or suppress class ii major histocompatibility complex presentation of different t cell determinants searching immunodominant epitopes prior to epidemic: hla class ii-restricted sars-cov spike protein epitopes in unexposed individuals inherent specificities in natural antibodies: a key to immune defense against pathogen invasion breadth of concomitant immune responses prior to patient recovery: a case report of non-severe covid-19 the authors wish to thank prof. angel galabov for critical reading of the manuscript. key: cord-256998-or73in8m authors: nguyen, khue g.; vrabel, maura r.; mantooth, siena m.; hopkins, jared j.; wagner, ethan s.; gabaldon, taylor a.; zaharoff, david a. title: localized interleukin-12 for cancer immunotherapy date: 2020-10-15 journal: front immunol doi: 10.3389/fimmu.2020.575597 sha: doc_id: 256998 cord_uid: or73in8m interleukin-12 (il-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid-1990's showed that systemic delivery of il-12 incurred dose-limiting toxicities. nevertheless, il-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il-12-based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered. interleukin-12 (il-12) is a potent, pro-inflammatory type 1 cytokine that has long been studied as a potential immunotherapy for cancer. unfortunately, il-12's remarkable antitumor efficacy in preclinical models has yet to be replicated in humans. early clinical trials in the mid-1990's showed that systemic delivery of il-12 incurred dose-limiting toxicities. nevertheless, il-12's pleiotropic activity, i.e., its ability to engage multiple effector mechanisms and reverse tumor-induced immunosuppression, continues to entice cancer researchers. the development of strategies which maximize il-12 delivery to the tumor microenvironment while minimizing systemic exposure are of increasing interest. diverse il-12 delivery systems, from immunocytokine fusions to polymeric nanoparticles, have demonstrated robust antitumor immunity with reduced adverse events in preclinical studies. several localized il-12 delivery approaches have recently reached the clinical stage with several more at the precipice of translation. taken together, localized delivery systems are supporting an il-12 renaissance which may finally allow this potent cytokine to fulfill its considerable clinical potential. this review begins with a brief historical account of cytokine monotherapies and describes how il-12 went from promising new cure to ostracized black sheep following multiple on-study deaths. the bulk of this comprehensive review focuses on developments in diverse localized delivery strategies for il-12-based cancer immunotherapies. advantages and limitations of different delivery technologies are highlighted. finally, perspectives on how il-12-based immunotherapies may be utilized for widespread clinical application in the very near future are offered. keywords: interleukin-12 (il-12), cancer immunotherapy, cancer vaccine, cytokine delivery system, localized delivery, intratumoral administration overview of il-12-based immunotherapies a brief history of cytokine and il-12 immunotherapies since the discovery of an "endogenous pyrogen, " now known as il-1, in 1953, scientists have anticipated the use of exogenous cytokines to manipulate a patient's immune system in an effort to control malignant neoplasms (1) . early obstacles to cytokine-based immunotherapy centered on difficulties achieving reproducible manufacture of a sufficient and pure supply of cytokines for clinical trials. in the early 1980s, recombinant dna technology and advances in the biochemical characterization of proteins combined to overcome this hurdle. finally, in 1986, ifn-α broke through as the first cytokine to win fda approval as a single agent cytokine therapy for cancer (2) . since then, hundreds, if not thousands, of studies have evaluated more than 40 cytokines against a range of preclinical tumor models. a number of promising cytokines, including gm-csf, il-1, tnfα, ifn-γ, and il-12, subsequently entered clinical trials as single agents but failed to provide clinical benefit. currently, only 2 of 40+ identified cytokines are approved as single agent immunotherapies for a limited number of indications ( table 1) . another fda approved cancer immunotherapy, talimogene laherparepvec (t-vec; imlygic tm ) is an oncolytic herpes simplex virus that uses gm-csf expression as an immune enhancer (7) . perhaps the greatest disappointment in cytokine immunotherapy development thus far is il-12. il-12 is a potent, pro-inflammatory cytokine produced by antigen presenting cells typically in response to microbial pathogens. it is comprised of two subunits, p35 and p40, that are linked by three disulfide bridges to form a p70 heterodimer (7) (8) (9) (10) . il-12 is chiefly responsible for the induction and enhancement of cell-mediated immunity. among its diverse functions, il-12 has been shown to: (i) induce t h 1 cell differentiation; (ii) increase activation and cytotoxic capacities of t and nk cells; and (iii) inhibit or reprogram immunosuppressive cells, such as tumor associated macrophages (tams) and myeloid-derived suppressor cells (mdscs) (11) (12) (13) (14) (15) (16) . il-12 also induces the production of large amounts of ifnγ which itself is cytostatic/cytotoxic (17, 18) , anti-angiogenic (19, 20) and can upregulate mhc i and ii expression on tumor cells for enhanced recognition and lysis (21) . not surprisingly then, il-12 has demonstrated remarkable antitumor effects against a range of malignancies in preclinical studies (22) (23) (24) (25) . these effects are largely dependent on cd8 + t cells, nk cells, and nk t cells (23, 26, 27) . in clinical studies, il-12 has been evaluated as an experimental treatment for numerous malignancies (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) . unfortunately, the efficacy of il-12 at tolerated doses has been minimal (29, 30, 33) . atkins and colleagues were the first to employ il-12 immunotherapy in a clinical trial (28) . this phase i study enrolled 40 patients, including 20 with renal cancer and 12 with melanoma, to investigate intravenous administration of recombinant hil-12 (rhil-12). one melanoma patient experienced a transient complete response and one renal cancer patient had a partial response (28) . subcutaneous rhil-12 was employed in a separate pilot study that enrolled 10 advanced melanoma patients (29) . in this study, a fixed dose of rhil-12 (0.5 µg/kg) was given to patients on days 1, 8, and 15 for two sequential cycles of 28 days. no partial or complete responses were reported. minor tumor shrinkages involving some subcutaneous metastases and hepatic metastases were observed (29) . in yet another early melanoma study, the administration of il-12 was found to induce a striking peripheral burst of hla-restricted ctl precursors directed to autologous tumors and to multiple immunogenic tumor-associated antigens (39) . significantly, the infiltration of cd8 + t cells with an effector-memory phenotype was identified in posttreatment metastatic lesions, but not in pretreatment metastatic lesions of three patients (39) . il-12 has also been shown to induce productive antitumor responses against cutaneous t cell lymphoma variants (38) , aids-related kaposi sarcoma (37) , and non-hodgkin's lymphoma (38) . although il-12 has demonstrated robust antitumor activity in preclinical studies and potent immune-stimulating potential in humans, systemic administrations of il-12 have been shown to be exceedingly toxic. in one phase ii trial, a maximal dose of 0.5 µg/kg per day resulted in severe side effects in 12 out of 17 enrolled patients and the deaths of two patients (40) . interestingly, the same dose of 0.5 µg/kg il-12 per day was found to be well-tolerated in patients that were enrolled in a previous phase i study. a change in dosing schedule accounted for the differences in toxicity between the phase i and phase ii trials. in the phase i trial, a single tester dose of il-12 was administered 1 week before a multiple-dose regimen. the tester dose was found to blunt the toxicity induced by subsequent doses (41) . overall, severe toxicities in early clinical trials, including 2 on-study deaths (42) due to frequent systemic injections of il-12, together with disappointing clinical responses in large phase 2 studies (43, 44) , dampened enthusiasm for il-12-based immunotherapy. the disappointing antitumor responses in clinical trials raised the possibility that il-12 is simply less active in humans. however, the severe toxicities outlined above indicate that il-12 has potent biological activity in humans. another possibility for the limited clinical efficacy is insufficient delivery of il-12 to the tumor microenvironment in humans. il-12, like most cytokines, functions locally through paracrine and autocrine mechanisms. the ideal targets of il-12 immunotherapy are not lymphocytes in circulation, but rather immune cells within the tumor and nearby lymph nodes, including activated but exhausted t cells, nk cells, tams, and mdscs. therefore, maximizing the amount of il-12 that reaches the tumor seems critical for a robust antitumor response. we and others have noted that il-12 immunotherapeutics would be more effective and less toxic if delivered and maintained in the tumor through the use of novel delivery technologies. there are five benefits of local, persistent il-12 delivery. the first is enhanced spatiotemporal distribution of il-12 compared to systemic delivery. the failure of il-12-based immunotherapies to achieve widespread clinical success may be at least partially attributed to the inability of a tolerated dose of systemically administered il-12 to reach therapeutic concentrations within human tumors. in mice, implanted or induced tumors are disproportionately large. for example, a 1.5-g tumor (∼1.25 cm 3 ) comprises 6% of the body weight of a 25-g mouse. a comparably sized tumor in a 70 kg (154 lbs) human would weigh 4.2 kg (9.2 lbs). furthermore, rapidly growing murine tumors are highly vascularized relative to their human counterparts (45, 46) . taken together, a significantly larger fraction of a systemically administered il-12 dose can be expected to reach the tumor in a mouse compared to a human. localized delivery strategies, on the other hand, are capable of enhancing il-12 concentrations in the tumor microenvironment by one or more orders of magnitude (47) (48) (49) (50) . a second benefit of localized il-12 delivery is the ability to generate systemic antitumor immunity from a locally initiated intravenous injection (4, 5) talimogene laherparepvec (t-vec) unresectable advanced melanoma intratumoral injection (6) * subcutaneous and intramuscular injections are local deliveries. however, they result in systemic cytokine distribution and are utilized in the clinic as systemic therapies. immune response. as cancer metastasizes and becomes a "systemic" disease, conventional wisdom has suggested that metastases must be treated with systemic therapies such as i.v. administered chemotherapies or immune checkpoint inhibitors. however, systemic delivery increases the frequency of adverse events through off-target interactions. for instance, systemic il-12 therapy has the potential to cause activation and/or differentiation of all circulating t cells whereas activation/differentiation of only tumor-specific or tumor antigen-experienced t cells is preferred. fortunately, a growing mountain of evidence demonstrates that localized il-12 can generate systemic, adaptive immunological memory capable of controlling anestic tumors, inhibiting metastases, and preventing tumor recurrences (51) (52) (53) . in particular, local administration of il-12 has been shown to activate or reactivate tumor infiltrating cd8 + t cells, improve antigen presenting machinery and subsequently cause the expansion of tumor-specific cd8 + effector t cells. this often leads to enhanced infiltration of contralateral untreated tumors (54) . research from our own lab demonstrated that local/intravesical administration of il-12 eliminated untreated flank tumors only when a primary orthotopic bladder tumor was treated. these data indicated that t cells must be educated with antigens from a primary tumor in order to find and eliminate abscopal tumors. similarly, intratumoral injections of il-12 neoadjuvant to resection have been shown to inhibit metastases by multiple groups in a t cell, nk cell, or ifn-γ dependent manner (52, 55) . taken together, the convincing evidence demonstrating that localized il-12 can induce abscopal immunity renders systemic il-12 delivery unnecessary even for the treatment of metastatic disease. third, as mentioned above, il-12 is a pleiotropic cytokine with context dependent consequences. when il-12 is administered systemically, it induces rapid increases in pro-inflammatory cytokines, such as ifn-γ, tnf-α, and il-6 (56). this "cytokine storm" combined with rapid decreases in peripheral blood lymphocytes, monocytes, and neutrophils can be lethal (42) . however, when controlled locally, pleiotropic cytokines have the potential to engage multiple antitumor effector mechanisms. for instance, il-12 increases the activation and cytolytic capacity of cd8 + t cells and nk cells and induces the production of ifn-γ. ifn-γ, in turn, may kill tumor cells directly, inhibit angiogenesis (57) (58) (59) (60) , and stimulate nk cells, ctls (61, 62) , and macrophages (63) while upregulating mhc i and ii molecules (64) on the surfaces of tumor cells. fourth, high levels of locally administered il-12 can reverse tumor-supporting immunosuppression. the immunosuppressive tumor microenvironment is a major hindrance to the clinical efficacy of all cancer immunotherapies. in fact, the cancer vaccine literature teaches that the majority of patients in clinical studies are able to mount significant antigen-specific t cell responses, yet few patients experience clinical benefit (65) (66) (67) . similarly, the extraordinary activity of car t cells against hematologic malignancies becomes less than ordinary against solid tumors. many solid tumors lack the chemokines and inflammation necessary to recruit cytotoxic t cells (68) . moreover, dense tumor stroma prevents t cell penetration while immunosuppressive factors released by tumor cells, suppressor t cells and tams can cause t cell anergy. regarding the latter, many tumors bathe in a cocktail of immunosuppressive factors such as tgfβ, il-10, ido, and larginase. fortunately, high intratumoral concentrations of il-12 can cause apoptosis and elimination of cd4 + cd25 + foxp3 + suppressor t cells in tumors (69) . in addition, the tumor suppressive phenotype of tams can be converted to a cytotoxic, antitumor phenotype in the presence of localized il-12 (11) . finally, il-12 has been shown to modulate and alter the suppressive activities of tumor-associated mdscs (12) . lastly, and perhaps most importantly, activation of t cells in the presence of il-12 can not only enhance ctl function, but also reduce negative regulatory mechanisms such as pd-1/pd-l1 signaling and autocrine ifnγ-induced apoptosis. this "protective" effect has been observed mostly in the cellular immunotherapy literature. standard protocols for ex vivo expansion of tumor infiltrating lymphocytes for adoptive cell therapy (act) traditionally used high dose il-2 to facilitate t cell proliferation (70) . the inclusion of il-12 in conditioning/expansion media has been explored recently because it had been shown previously to result in optimal t cell priming (71) . indeed, adoptive transfer of tumor-specific cd8 + t cells primed ex vivo in the presence of il-12 resulted in enhanced antitumor responses (72, 73) , increased persistence of infused t cells (73, 74) , as well as increased expression of il-2rα (cd25], icos, ox40, granzyme b, and ifnγ (73) . importantly, cytotoxic t lymphocytes (ctls) stimulated with il-12 were more effective in controlling tumors following adoptive transfer than ctls stimulated with ifnα (75) . il-12-stimulated t cells expressed lower levels of pd-1 and higher levels of ifnγ and il-2 compared to ifnα-stimulated t cells (75) . il-12 conditioning caused downregulation of ifnγr2 with a concomitant decrease in susceptibility to ifnγ-induced apoptosis of tumor-infiltrating cd8 + t cells (74, 76) . il-12 delivery strategies can be divided into three general approaches. the first involves fusion of a targeting moiety to il-12 in order to facilitate accumulation in a tumor following a systemic injection. the most common of class of fusion molecules are immunocytokines, which involve linking a tumor binding antibody fragment to a cytokine. the second approach involves delivery of genetic material encoding il-12 directly to the tumor or a tissue of interest. this category can be further divided based on the type or method of gene delivery. plasmids, mrna, viruses, and transduced cells are all capable of expressing and delivering il-12 after a local injection. the third major approach involves controlled release of recombinant il-12 protein from a sustained delivery system. here, the cytokine delivery system is injected or implanted directly in a tumor or tissue of interest. the remainder of this section will present and discuss the most relevant preclinical and clinical data pertaining to each il-12 delivery strategy. a summary of current clinical trials utilizing localized il-12 delivery is presented in table 2 . as mentioned above, immunocytokines are part or whole cytokines that have been engineered to contain antibody fragments or other targeting moieties. these "targeted" cytokines are administered systemically but are expected to accumulate within tumors at higher levels compared to non-targeted cytokines. various tumor-related features have been targeted by immunocytokines including: (1) tumor antigens which are overexpressed or uniquely expressed by tumor cells; (2) cryptic extracellular matrix epitopes found only in tumors; and (3) neovasculature markers as tumors require angiogenesis for growth. developments in immunocytokines are discussed below. the pan-carcinoma antigen, epithelial cell adhesion molecule (epcam), is highly expressed by cancer cells of epithelial origin such as colon, prostate, breast, and lung carcinomas. huks-il-12 is an immunocytokine of il-12 fused to the fc fragment of a humanized antibody that recognizes epcam. in a murine prostate cancer model, huks-il-12 was found to suppress experimental metastases in scid mice reconstituted with activated human t and nk cells lymphocytes (78) . another immunocytokine, hu14.18-il-12, which is irrelevant in this system due to its targeting of ganglioside gd2, was found to be somewhat less effective than huks-il-12, although differences in antitumor activity were not statistically significant (78) . dual immunocytokines in which both il-2 and il-12 were fused to the huks1/4 antibody fragment were found to eliminate epcam-expressing llc flank tumors following intratumoral (i.t.) injection (79) . interestingly, a mixture of huks-il-2 and huks-il-12 was less effective than the dual immunocytokine if delivered i.t., but exhibited similar in antitumor activity if administered i.v. (79) . the epidermal growth factor receptor her2/neu is overexpressed in roughly a third of breast and ovarian cancers, with high expression correlating with poor prognosis. trastuzumab, a monoclonal antibody targeting her2, has been approved for the treatment of certain breast cancers for more than 20 years. a mouse single chain il-12 fused to an anti-her2/neu igg3 (mscil-12.her2.igg3) retarded the growth of ct26-her2/neu tumors in immunocompetent mice (80) . a direct comparison demonstrated that mscil-12.her2.igg3 and free il-12 induced similar activities against ct26-her2/neu tumors (81) . follow up studies revealed that mscil-12.her2.igg3 also displayed robust antitumor activity against mc38/her2/neu and d2f2/e2 tumors (82, 83) . a more recent study revealed that disruption of the heparin binding domain in the mscil-12.her2.igg3 immunocytokine, reduced il-12 bioactivity (84) . this result was consistent with recent studies showing that heparin and heparan sulfate bind to and enhance the activity of il-12 (85) (86) (87) (88) (89) . while eliminating heparin binding reduces il-12 activity, we speculate that this reduction could be counterbalanced by an enhancement in tumor targeting as the il-12 immunocytokine may no longer bind to ubiquitous sulfated glycosaminoglycans in non-targeted tissues. mesothelin is a differentiation antigen that is highly expressed in a number of human cancers including mesotheliomas, pancreatic and lung adenocarcinomas, and ovarian and breast carcinomas. to direct il-12 to mesothelin expressing cancer cells, a scfv, called ss1, that specifically binds to mesothelin was fused to the p35 subunit of a single-chain il-12 (90) . human peritoneal mesotheliomas established in nude mice were significantly inhibited by i.p. injections of il12-ss1 (90) . that these studies were successful in nude mice seems to imply a prominent role for nk cells in this model. ca166-9 is a cancer antigen that is expressed in about half of human ovarian cancers (91) . a scfv of the 6b11 monoclonal antibody that binds to ca166-9 was fused to mil-12 (92) . systemically (i.v.) administered 6b11scfv-mil-12 was found to inhibit the growth of subcutaneously implanted id8 ovarian tumors more effectively than non-targeted mil-12 (92) . cd30 is expressed by activated lymphocytes and thus serves as a useful target for several types of lymphoma. a cd30-targeted il-12 fusion protein was developed for cd30 + hodgkin's lymphoma therapy (93) . the immunocytokine was found to induce activation of t and nk cells and secretion of proinflammatory cytokines resulting in enhanced cytotoxicity of cd30 + mc38 cells. interestingly, a cd30-targeted il12-il2 fusion protein outperformed targeted il-2 or il-12 alone in all in vitro measures. the dual cytokine construct induced regression of cd30 + but not cd30-mc38 tumors in vivo (93) . whether the dual cytokine fusion protein was better than the single cytokine constructs is not known as the latter were not evaluated in vivo. many solid tumors overexpress extracellular matrix (ecm) which serves as transport barrier to the penetration of therapeutics and immune cells. in ecm-rich solid tumors, it may frontiers in immunology | www.frontiersin.org targeting ecm proteins instead of cancer cells, therefore, is a promising strategy to encourage immunocytokine accumulation in tumors. there are two immunocytokines, hubc1-il12 and il-12-l19, that have been developed to target the splice variant extra domain b (ed-b) of fibronectin, which is highly expressed in tumor tissues but undetectable in normal adult tissues with the exception of endometrium (94) . bc-1 is a monoclonal antibody that recognizes the ed-b isoform, thus a hubc1-il12 immunocytokine has been constructed from two molecules of il-12 fused to each of the igg heavy chains of humanized bc-1. systemic administration of hubc1-il12 was found to eliminate experimental pc3 metastases and suppress the growth of multiple human tumor lines in immunocompromised mice more effectively than il-12 alone (95) . a phase i trial evaluated the safety of weekly infusions of as1409 (hubc1-il12) in 13 renal carcinoma and malignant melanoma patients (96) . the maximum tolerated dose (mtd) was found to be 15 µg/kg. in contrast, the mtd of twice weekly i.v. il-12 was previously found to be 0.5 µg/kg (97) . dose limiting toxicities, including fever, fatigue, and elevated transaminase levels, were consistent with known toxicities of il-12 (96) . the second ed-b targeted immunocytokine, il-12-l19, is comprised of the ed-b-binding l19 scfv and il-12 (98) . l19-targeted cytokines have been shown to selectively accumulate in tumors following i.v. administration (99, 100) . intravenous administration of il-12-l19 every 48 h was found to control the growth of primary c51 colon adenocarcinomas, f9 teratocarcinomas as well as experimental pulmonary c51 metastasis (101) . biodistribution studies confirmed that a greater percentage of the injected dose of il-12-l19 was found in tumors as compared to an il-12-fusion negative control. il-12-l19 also demonstrated synergistic antitumor activity when combined with l19-tnfα (102) . most recently, il-12 was fused to the collagen-binding proteoglycan lumican and mouse serum albumin (msa), to create il12-msa-lumican (103) . lumican binds to collagen types i and iv, components of the thick fibrotic capsule surrounding tumors and perivascular basement membrane, respectively. in mice bearing established subcutaneous flank b16f10 tumors, treatment with il12-msa-lumican resulted in prolonged tumor control and longer survival. significant weight loss was observed following il12-msa compared to il12-msa-lumican treatment, indicating that collagen targeting may reduce systemic toxicities of il-12. finally, the combined treatment of lumican-msa-il2 and il12-msa-lumican potentiated anti-pd-1 increasing survival in multiple models and completely protecting cured mice from live tumor rechallenge (103) . another collagen-binding immunocytokine comprised of the a3 cbd of von willebrand factor fused to both subunits of il-12 was also recently developed (104) . systemic (i.v.) administration of this cbd-il12 was found to accumulate in emt6 mammary carcinomas at significantly higher levels and induce higher rates of complete tumor regression against 1-week old b16f10 and emt6 tumors compared to il-12 (104) . inclusion of the cbd resulted in a 5-6-fold decrease in plasma half-life despite the larger size of cbd-il12. the distributions of cbd-il12 and il-12 in normal tissues following i.v. injection were surprisingly similar although typical sites of collagen targeted drugs, e.g., bone and skin, were not examined. most importantly, although elevated liver enzymes were observed, levels following cbd-il12 at an impressive dose of 50µg/mouse were similar to 10µg/mouse of il-12 (104) . in general, the key advantage of systemically administered immunocytokines is their ability to preferentially accumulate within a site of disease, e.g., a tumor. however, immunocytokines retain complete cytokine activity in circulation which allows them to interact with circulating lymphocytes and induce similar cytokine-induced toxicities as parental cytokines (105) . one clever strategy has been developed to reduce adverse effects associated with systemic il-12 by separating the targeted delivery of the p35 and p40 subunits. this split-immunocytokine approach involves first delivering a bivalent p35-based antibody fusion protein (f8-p35s-f8). f8 binds to the alternatively spliced extra domain a (ed-a) domain which is present on the subendothelial extracellular matrix of tumor neovasculature (106) . after allowing time for binding and clearance of unbound f8-p35s-f8, a subsequent administration of p40, which has no activity by itself, interacts with p35 to recover il-12 activity. quantitative biodistribution investigation in f9 teratocarcinomas bearing mice showed that both targeted subunits accumulated in the tumor (106) . furthermore, the recombined subunits displayed robust il-12 activity in terms of ifnγ production and stat4 phosphorylation (106) . tumor necrosis is a common feature of most advanced solid tumors. approaches to target dna strands that become uniquely exposed in necrotic foci are under investigation. the monoclonal antibody, chtnt-3, recognizes single-stranded dna (107) . a fusion between the variable heavy chain of chtnt-3 and hil-12 forms the necrosis-targeting immunocytokine, chtnt-3/hil-12 (94) . chtnt-3/hil-12 was retained in a subcutaneous tumor after i.v. injection and resulted in a significant inhibition of du145 prostate tumors in human pbl-engrafted scid mice (94) . another necrosis-targeting il-12, capitalizes on the specificity of the nhs76 antibody for ssdna and dsdna (108, 109) . nhs-il12 is comprised of the full length nhs76 antibody fused to 2 single-chain il-12 molecules. systemic administration of a murine analog, nhs-muil12, has been shown to delay the growth of mc38-cea+ colorectal carcinomas in cea.tg mice (108) . furthermore, tumorbearing mice treated with nhs-muil12 developed cd8 + t cell responses against an endogenous tumor antigen, p15e. in vivo imaging studies have shown that nhs-muil12 accumulated in flank tumors following a s.c. injection (108) . subcutaneous administrations of nhs-muil12 were also recently shown to provide significant reductions in orthotopic mb49luc bladder tumors (110) . tumor control was associated with a noticeable reduction in markers of immunosuppression, e.g., mdscs, macrophages and tumor-associated tgf-β (110) . the combination of nhs-muil12 with avelumab, an anti-pd-l1 antibody, resulted in improved control of both mc38 and mb49 flank tumors with higher frequencies of cd8 + t cells and enhanced t cell activation compared to either agent alone (111) . against orthotopic emt-6 mammary tumors (∼100 mm 3 ) the combination of nhs-muil12 and avelumab induced complete regression in 7 of 8 mice (112) . the same treatment was shown to delay, but not completely regress, the growth of 350-400 mm 3 established emt-6 tumors. importantly, nhs-muil12 plus avelumab was shown to induce protective immunity as all cured mice resist an emt-6 tumor challenge but not a 4t1 challenge. furthermore, treatments enhanced cytotoxic nk and cd8 + tcell proliferation, t-bet expression, plasma cytokine levels, and innate and adaptive immune genes (112) . combining nhs-il12 with fcil-7 or il-2mab602 resulted in improved antitumor immunity, increased survival, and longterm remission in sarcoma-bearing mice (113) . fcil-7 is a fusion of interleukin-7 and an fc fragment while il-2mab602 is a fusion of il-2 and a monoclonal antibody against il-2, mab602. separately, the combination of nhs-il12 with local tumor irradiation was shown to increase treatment efficacy (114, 115) . in preparation for first-in-human clinical trials, a comparative oncology study in client-owned dogs with melanoma revealed that s.c. injections of nhs-il-12 induced transient increases in serum ifnγ and il-10. two of 7 dogs in a dose escalation cohort experienced a partial response while 5 of 7 dogs had increased levels of tumor-infiltrating cd8 + t cells (116) . nhs-il12 is currently in phase i clinical studies either as a monotherapy (nct01417546) or in combination with avelumab (nct02994953). in the former study, nhs-il12 induced transient lymphopenia and elevated liver transaminases, but was otherwise well-tolerated with a mtd of 16.8 µg/kg (117) . no objective tumor responses were observed, however, 5 of 59 patients experienced stable disease. immune assays revealed that nhs-il12 treatment increased nk cell frequencies and broadened the tcr diversity of tumor-infiltrating t cells (117) . systemically administered immunocytokines can significantly reduce but are unlikely to completely avoid il-12-related toxicities. as mentioned above, in circulation, immunocytokines will interact with immune cells and induce signaling outside of the tumor. in addition, all targeting moieties are susceptible to non-specific binding and distribution in normal, untargeted tissues. for example, radiolabeled nhs76 has been found in all major tissues in mice for 2-3 days after i.v. administration (109) . furthermore, substantial amounts of il-12-l19 were found in the livers of treated animals, likely leading to hepatotoxicity (101) . on-target/off-tissue specific binding may create additional concerns. in the case of nhs-targeting moieties, cancer patients have high levels of circulating cell-free dna that is shed from tumors (108). it is not clear how circulating dna impacts nhs targeting. in the case of neovasculature targeting moieties, angiogenesis is a normal process of wound healing and promotes collateral circulation for atherosclerotic blood vessels. disrupting non-cancerous angiogenesis could induce hypertension and cardiac ischemia which are among the adverse events associated with anti-angiogenic agents, such as bevacizumab. moreover, the potential immunogenicity of a nonendogenous immunocytokine is another factor that may limit therapeutic potential. as non-native proteins, immunocytokines could contain immunogenic epitopes against which an immune response, likely an antibody response, could be raised. antiimmunocytokine antibodies could induce pharmacological abrogation, therapeutic alteration, or hypersensitivity reactions (118) . because of the potential for anti-immunocytokine antibodies, novel immunocytokines should be engineered to minimize the presence of immunogenic epitopes. overall, although immunocytokines remain capable of inducing il-12-related adverse events, the use of targeting moieties may improve biodistribution enough to expand the therapeutic window of il-12-based immunotherapies. intratumoral (i.t.) injections of dna and rna encoding il-12 have the potential to localize and sustain the production of il-12 in the tumor microenvironment. nucleic acids are much easier to produce, purify and manipulate than recombinant cytokines. however, mammalian host cells are not easy to transfect and typically require chemical, physical, or electrical assistance to achieve reasonable transfection rates. this section will highlight progress in nucleic acid-based approaches both preclinically and clinically. around the same time that recombinant il-12 was failing in clinical trials, a limited number of preclinical and clinical studies explored i.t. injection of plasmid dna encoding il-12 (pil-12) as a potentially less toxic approach. preclinically, i.t. pil-12 inhibited but did not eliminate b16 melanomas (119) . in this study, il-12 was not detected in the serum following i.t. injection. in another study involving gray horses with metastatic melanoma, i.t. pil-12 resulted in detectable levels of pil-12 in the serum for up to 36 h (120). however, it is not clear if systemic dissemination of pil-12 resulted in significant systemic increases in serum il-12 or ifnγ as these were not measured (120) . in a phase i/ii trial of intralesional injections with pil-12, 3 of 9 and 8 of 9 patients with stage iv malignant melanoma experienced clinical and local responses, respectively (121) . in a phase i/ib study, i.t. pil-12 was found to reduce the size of treated lesions by at least 30% in 5 of 12 malignant melanomas and renal cell carcinomas (122) . pil-12 injections were welltolerated as no patient in either study experienced a significant treatment-related adverse event. despite successful safety studies, the use of naked pil-12 for cancer immunotherapy has not progressed, mostly likely due to poor transfection efficiency. the application of pulsed, high electric fields to facilitate cellular uptake and expression of genes has been a part of the molecular biologist's toolbox for decades. intratumoral injection of pil-12 immediately followed by electroporation, referred to here as pil-12+ep, has been explored in several murine tumor models (123) (124) (125) (126) (127) (128) (129) (130) . as expected, the benefit of adding electroporation was immediately apparent as one early study showed pil-12 alone had no effect on b16f10 tumor growth while pil-12+ep significantly inhibited tumors and extended survival (123) . importantly, the increase in antitumor efficacy was not associated with an increase in systemic il-12 levels (123) . among the more notable responses in other early preclinical studies, nearly half of mice bearing established b16f10 melanomas experienced complete tumor regression following 2 weekly treatments with pil-12+ep (124) . in a follow up study, pil-12+ep induced tumor regression in up to 80% of mice, whereas i.t. injections of pil-12 alone delayed but could not eliminate b16f10 primary tumors (125) . cured mice displayed protective immunity as 20 of 21 rejected a b16f10 challenge (125) . in the sccvii squamous cell carcinoma (scc) model, complete regressions were observed in 40% of mice following pil-12+ep (127). furthermore, 3 of 6 cured mice resisted a tumor challenge containing five times the original dose of tumor cells (127, 128) . against bjmc3879 murine mammary adenocarcinomas, ct26 murine colon adenocarcinomas and renca renal cell carcinomas, pil-12+ep significantly suppressed, but did not eliminate implanted tumors (129, 131) . against murine sa-1 fibrosarcomas, pil-12+ep suppressed tumor growth and induced complete regression in 90% of treated mice with 11 of 18 becoming resistant to tumor rechallenge (132) . in this study, il-12 and ifnγ were detected in the serum of treated mice, however, no side effects were observed (132) . abscopal responses have been documented in several studies. against bilateral sa-1 tumors, pil-12+ep treatment consistently eliminated primary, treated tumors, while slowing the growth of secondary, untreated tumors (132) . similarly, pil-12+ep treatment of mh134 hepatocellular carcinomas, inhibited both treated and untreated tumors while preventing spontaneous pulmonary metastases (133) . a recent study using bilateral b16 tumors demonstrated that an optimized pil-12+ep protocol (134) was capable of regressing treated lesions while inhibiting the growth of contralateral untreated tumors (54) . injecting pil-12 directly into tumors is important as multiple studies have confirmed that i.t. pil-12+ep treatments were significantly more effective than either peritumoral or intramuscular (i.m.) routes (124, 125, 128, 132) . the i.m. route also resulted in significantly more il-12 and ifn-γ in the serum (124, 128) . in terms of mechanism, multiple studies agreed that pil-12+ep treatment was associated with increased t cell infiltration, increased ifnγ expression and decreased angiogenesis (124, 127, 128, 131, 133, 135) . these findings are consistent with known antitumor mechanisms of il-12. more recent mechanistic studies have focused on changes in immune cell phenotype and function. for example, b16f10 tumor regression following pil-12+ep was mediated via the perforin/granzyme lytic pathway while antigen-specific cd8 + t cell responses were directed against tyrosinase-related protein epitope trp2 [180] [181] [182] [183] [184] [185] [186] [187] [188] (136) . in another study, pil-12+ep-induced elimination of b16f10 tumors was associated with increased tumor infiltration and polarization of macrophages toward an m1 phenotype (137) . another group found that pil-12+epinduced antitumor responses against b16f10 tumors were correlated with a reduction in pd-1 expression on cd4 + and cd8 + t cells (138) . yet, another group found that the treatment of bilateral b16f10 tumors induced a unique population of cd8 + effector t cells with low pd-1 expression in both untreated tumors and systemically (54) . this finding suggested that a subset of cd8 + effectors generated by pil-12+ep may be protected or "armored" against checkpoint-mediated exhaustion (54) . a unique feature of pil-12+ep immunotherapy is the potential to manipulate electric field parameters to enhance transfection, and therefore, efficacy. an exploration of electric field parameters demonstrated that pil-12+ep-induced cures ranged from 65 to 80% in b16f10 tumor-bearing mice depending on pulsing conditions (138) . about half of these cured mice resisted a b16f10 rechallenge (138) . by further enhancing electric field intensity and/or pulse length, it is possible to directly kill tumor cells and release tumor antigens via irreversible electroporation. in one recent study, partial-irreversible electropermeabilization (pire) administered after peritumoral electrotransfection with pil-12 caused complete regression of about 40-50% of treated b16f10 tumors (139) . two of 4 cured mice completely resisted tumor rechallenge while the remaining two experienced delayed tumor growth from the rechallenge. this pil-12 plus pire approach was found to delay, but not eliminate distant, untreated tumors in about half of the mice (139) . there have been several attempts to enhance antitumor activity through the incorporation of additional cytokineencoding plasmids. of note, two studies have demonstrated that ep using a combination of il-12 and il-18 plasmids outperformed il-12 alone in terms of antitumor activity (140, 141) . the rationale to combine these two cytokines is wellsupported given that il-12 and il-18 synergize to enhance th1 responses and ifn-γ production. however, adverse events are also enhanced as systemic co-administration of recombinant il-12 and il-18 proteins leads to lethal toxicity in mice (142) . in one study, addition of pil-18 to pil-12 increased serum il-12 and ifn-γ levels for at least 6 days after ep although no inflammation was observed in liver, lung, and intestine samples (140) . in a second study, pil-18 did not increase il-12-induced serum ifn-γ, but intratumoral ifn-γ was significantly higher (141) . several notable canine clinical studies have explored pil-12+ep in dogs with naturally occurring tumors. in one such study, pil-12+ep resulted in a 13-83% reduction in mast cell tumor volume (143) . treated nodules displayed increases in leukocytic inflammation and decreases in the number of malignant mast cells (143) . in beagles with canine transmissible venereal tumors (ctvts), pil-12+ep induced complete regression of all treated lesions (144) . contralateral untreated tumors were also significantly inhibited. serum il-12 levels peaked 7 days after treatment; however, relevant blood chemistries, i.e., liver and kidney enzymes, as well as cell counts were not different from those of control dogs (144) . another study investigated pil-12+ep for the treatment of canine oral malignant melanoma (omm). there were no differences in the percentages of helper cd4 + and cd8 + cells before and after treatment, while t reg frequencies declined from 1.2 to 0.3%. one month post treatment, the objective response rate was 67% (6/9) but by the end of the observation period, all but one of the dogs developed progressive disease (145) . a more recent study in 9 dogs with a range of spontaneous cancers, demonstrated that pil-12+ep induced immunostimulatory and anti-angiogenic effects (146) . administration of three pil-12+ep treatments every other day caused significant systemic toxicities, including anemia and thrombocytopenia. after switching to a weekly schedule, treatments were well-tolerated, however, all treated tumors continued to progress (146) . several human clinical trials, mainly against advanced melanoma, have investigated the safety and efficacy of pil-12+ep. in a phase i study, 24 patients with stage iii or iv melanoma received i.t. pil-12+ep (six 100 µs, 1,300v/cm pulses) on days 1, 5, and 8 during a single 39-day cycle. fifty three percent of patients experienced a systemic response, defined as either stable disease or regression of untreated lesions, following pil-12+ep (147) . most notably, 2 of 19 patients showed complete regression of all metastases. no grade 3 or higher adverse events were observed and neither il-12 nor ifnγ was detectable in serum samples. in a follow up phase ii study, 29 patients with in-transit or m1a melanoma were treated with up to four 12-week cycles of pil-12+ep as described above (148) . intermediate results revealed an objective response rate of 33% with 11% complete responses. the treatment was found to increase nk cell levels both intratumorally and systemically (148) . no grade 3/4 drug-related adverse events were noted. a subsequent analysis of clinical samples revealed that responses were associated with increased intratumoral infiltration of cd3 + t cells (148) . in addition, t cell receptor beta chain (tcrβ) sequencing revealed a focusing of the tcr repertoire following treatment. however, there were no differences in t cell clonality between responders and non-responders to pil-12+ep (148) . a second study from the same trial, nct01502293, found a complete response rate of up to 17.9% and a best overall response rate of 35.7% in patients with stage iii/iv melanoma (149) . nearly half of these patients experienced regression of at least one anenestic lesion. an analysis of transcripts in melanoma biopsies found increases in t cell trafficking, immune activation, and antigen presentation (149) . genes associated with adaptive resistance, e.g., pd-l1, tgfβ, and trail, were also increased (149) . recent results from a safety study in 3 patients with locoregional merkel cell carcinoma (mcc) and 12 patients with metastatic mcc received 1 or up to 4 cycles of pil-12+ep, respectively (150) . the overall response rate in the metastatic mcc cohort was 25% (3/12). of 10 patients with measurable untreated lesions, 3 experienced abscopal regressions. in addition, 2 patients experienced clinical responses lasting 16 and 55+ months, respectively. two of the locoregional mcc patients, all of whom were treated with definitive surgery after pil-12+ep, were recurrence-free at 44+ and 75+ months, respectively (150) . serum il-12 levels were not measured, but treatments were well-tolerated, and no serious adverse events were observed. lipoplexes, polyplexes, and lipopolyplexes are complexes of lipids, polymers, and lipids plus polymers, respectively, with dna. these complexes are under investigation to enhance the delivery and transfection efficiency of plasmids encoding genes of interest, including pil-12. numerous studies have explored a range of different materials to create novel pil-12 complexes (151) (152) (153) (154) (155) . studies demonstrating antitumor efficacy following local or targeted delivery of pil-12 complexes are highlighted below. polyethyleneimine (pei), a highly cationic polymer that readily complexes with negatively charged dna, has been widely used to enhance gene delivery. pei protects dna from degradation in vivo, encourages interaction with negatively charged cell membranes, and enhances release from lysosomes by acting as proton sponge (156) . pei:il-12 complexes were shown to transfect lung tissue following delivery via nebulization (157) . this approach led to production of il-12 in the lungs which was not detectable in the plasma of treated mice (157, 158) . weekly or twice weekly administration of aerosolized pei:il-12 was found to suppress or eliminate experimental pulmonary metastases of saos-2 human osteosarcomas in athymic nude mice (157) . recent attempts to enhance uptake and il-12 production have focused on modification of pei with tetraiodothyroacetic acid (tetrac) which binds the α v β 3 integrin receptor that is overexpressed in some tumors (159) or diethylene triamine penta-acetic acid (dpta) which can reduce the surface charge of pei:il-12 complexes (160, 161) . as of this writing, no in vivo data using either modification have been published. polyvinylpyrridilone (pvp) is another cationic polymer that readily complexes with dna. twice weekly i.t. injections of pil-12/pvp complexes were found to eliminate 30 and 50% of 8-10 mm 3 renca and ct26 tumors, respectively (162) . most mice that were cured of a primary tumor rejected a subsequent rechallenge (162) . in a follow-up study, pil-12/pvp was found to be more effective than pifnα/pvp in controlling preclinical tumors, while the combination of pil-12/pvp and pifnα/pvp synergized to eliminate 100% of renca and 50% of ct26 tumors (163) . in both studies, cd8 + t cells but not cd4 + t cells were identified as primary effectors. complexation with poly-a-(4-aminobutyl)-l-glycolic acid (paga), a biodegradable polyester, enhanced transfection efficiency of pil-12 and expression of il-12 in vitro and in vivo (164, 165) . however, t cell infiltration of injected ct26 colon adenocarcinomas and antitumor activities following repeated injections of paga/pil-12 and naked pil-12 were similar (164) . encapsulation of pil-12 in nanoparticles comprised of poly-(d,l-lactic-co-glycolic acid) (plga) and 1,2-dioleoyl-3-(trimethylammonium) propane (dotap) demonstrated complete regression of up to 75% of established heterotopic bnl hepatocarcinomas following a single i.t. injection (166) . importantly, treatment with encapsulated pil-12 was more effective than treatment with nanoparticles with pil-12 adsorbed to the surface (166) . long term expression of inflammatory cytokines could be a concern as both il-12 and ifnγ were detected in the serum for up to 30 days after treatment (166) . a similar, so-called dmp nanoparticle, comprised of dotap and methoxy-poly(ethylene glycol)-poly(lactide) (mpeg-pla), has been developed to facilitate gene delivery (167) . complexation with pil-12 resulted in inhibition of ct26 tumors with no signs of systemic toxicity, as determined by appearance, body weight, fecal output, and urinary excretion (167) . a slightly different dmp nanoparticle that uses polycaprolactone (pcl) instead of pla, inhibited the growth of intraperitoneal c26 colon carcinomas and ll/2 lewis lung carcinomas (168) . in addition to delaying tumor growth, dmp/il-12 particles resulted in high il-12 gene expression and t cell infiltration although the treatment regimen consisted of 7 daily or every other day treatments (168) . plasmids complexed with mannosylated chitosan (mc) are under development as a method to target mannose receptors on i.t. dcs. chitosan is a linear co-polymer of β-linked dglucosamine and n-acetyl-d-glucosamine. it is primarily derived from the structural polysaccharide, chitin, found in shells of crustaceans. i.t. injection of mc/pil-12 complexes elicited modest growth delay of ct26 tumors, which was associated with increased tumor cell apoptosis and decreased angiogenesis (169) . lipopolymers, which incorporate a lipid tail on a polymer backbone, are also under exploration for non-viral gene delivery. water soluble lipopolymers (wslp) comprised of cholesterol conjugated to pei have been developed to increase cell membrane permeability and reduce pei-mediated toxicity (170) . wslp/p2cmvmil-12 dna complexes inhibited the growth of ct26 tumors and improved survival following a single i.t. injection (170) . however, the antitumor efficacies of wslp/pil-12 complexes and naked pil-12 appeared similar. in a later study, wslp/p2cmvmil-12 complexes injected every 4 days outperformed single injections and multiple injections with naked pil-12 or pei/pil-12 complexes (171) . the vast majority of injected wslp/p2cmvmil-12 was found in the tumor for up to 24 h with small but increasing accumulation in the liver and blood (171) . subsequent studies demonstrated that i.t. wslp/p2cmvmil-12 significantly suppressed the growth of primary and metastatic 4t1, tsa, and emt-6 mammary carcinomas (172, 173) . polytraxane (prx) is a composite molecule made of polyethylene glycol (peg) and cationic cyclodextrin (cd) that self assembles with dna into a spherical particle (174) . a 4-arm configuration, rather than a linear configuration, had a higher accumulation in mc38-luc tumors and lower accumulation in the lungs following systemic delivery. the 4-arm prx/pil-12 also produced higher levels of il-12 and significantly slowed mc38-luc tumor growth after five i.v. injections of the complex starting 10 days after tumor implantation. (174) systemic injections of prx/pil-12 complexes induced moderate lymphopenia, but no elevation of liver enzymes (174) . pil-12 complexed with a polyethyleneglycolpolyethylenimine-cholesterol (ppc) lipopolymer was shown to inhibit 4t1 and sccvii tumors (175) and increase survival in mice with intracranial gl261 gliomas (176) following localized injections. of note, although the i.p. route is frequently used to deliver drugs systemically, i.p. injections of pmil-12/ppc for treatment of id8 ovarian carcinomas resulted in high levels of il-12 and ifn-γ in ascites but low levels in serum (177) . in this study, i.p. pmil-12/ppc was well-tolerated with no significant changes in serum chemistries (177) . in a phase i study, phil-12/ppc was administered i.p. to 13 women with chemo-resistant recurrent ovarian cancer (178) . escalating doses of phil-12/ppc were well-tolerated with no dose-limiting toxicities. five of the 13 treated patients reported a serious adverse event, however, only one was possibly related to the phil-12/ppc (178) . similar to preclinical studies, no detectable increase in serum il-12 was found following phil-12/ppc treatment (178) . a subsequent phase ii study in 20 patients with platinum-resistant recurrent ovarian cancer demonstrated similar safety following weekly i.p. phil-12/ppc, however, with no objective clinical responses observed (179) . a different phase i trial evaluated the safety of phil-12/ppc in ovarian cancer patients when combined with carboplatin and docetaxel chemotherapy (180) . while there were no dose limiting toxicities, grade 3 adverse events included manageable abdominal pain and cytokine release syndrome. two of 12 patients experienced complete response while 4 of 12 experienced a partial response (180) . a more recent phase i trial combined weekly i.p. pil-12/ppc with i.v. pegylated liposomal doxorubicin in 14 patients with persistent or recurrent platinum-resistant ovarian or peritoneal cancers (181) . although increased levels of il-12, ifn-γ, and tnf-α were found in peritoneal fluid, no dose limiting toxicities were observed and a maximum tolerated dose was not reached. the best partial response (28.6%) and stable disease (57.1%) rates were found at the highest dose (36 mg/m 2 ) of pil-12/ppc (181) . pil-12 complexed with a cationic lipid (+/-)-n-(2-hydroxyethyl)-n,ndimethyl-2,3-bis(tetradecyloxy)-1propanaminium bromide/dioleoylphosphatidylethanolamine (dmrie/dope), and injected i.t. was found to inhibit and eliminate ct26 and renca tumors while protecting up to 96 and 100%, respectively, of mice from rechallenge (182) . interestingly, a direct comparison between naked pil-12 and dmrie/dope/pil-12 revealed no difference in antitumor activity (182) . polyphosphazene particles were modified with hydrophobic n,n-diisopropylethylenediamine (dpa) and hydrophilic monomethoxy poly-(ethylene glycol) (mpeg) to create weakly cationic particles to complex with pil-12 (183) . mpeg/pil-12 polymersomes delayed ct26 tumor growth when administered i.v. body weights were unaffected by mpeg/pmil-12 treatments. tumor il-12 levels steadily increased, reaching about 80 pg/g tumor on day 16, while serum il-12 concentration remained on average about 40 pg/ml after day 9 and continued to day 16. the concentration of ifn-γ in the tumor reached a maximum of 350 pg/g tumor on day 9, while serum levels of ifn-γ slightly increased from day 9 to day 16, reaching a concentration of only 4 pg/ml. while mpeg/pmil-12 polymersomes did not affect cd3 + cd4 + cells in the tumor, there was a 2-fold increase of cd3 + cd8 + cells and significant increases of cd3 − nk1.1 + and cd3 + nk1.1 + cells in the tumor. in another study, all-trans-retinoic acid (atra) was incorporated in cationic liposomes and complexed with pil-12 (184) . atra was previously found to increase the expression of tnf receptor 1 and mediate apoptosis of lung cancer cells via tnfα (185, 186) . i.v. injections of atra-cationic liposome/pil-12 reduced lung nodules and extended survival compared to cationic liposome/pil-12 treatment in an experimental pulmonary metastasis model using c26 cells expressing luciferase (184) . concentration of il-12 in the lungs reached a maximum of 12 pg/mg protein at 6 h post injection, while levels of il-12 in the spleen and liver were significantly lower and nearly eliminated by 24 h. interestingly, the incorporation of atra reduced liver enzymes levels and thus hepatic toxicity suggesting a possible anti-inflammatory role (184) . recently, mrna delivery platforms have received tremendous attention, most notably as front running vaccines against sars-cov-2. mrna, like dna, can encode an unlimited number of proteins and polypeptides. although mrna-based platforms are less stable than dna-based platforms, mrna can be protected from digestion through encapsulation in polymeric or lipidbased micro-or nanoparticles. a key advantage of mrna is their ability to directly translate encoded proteins in the cytoplasm. in contrast, dna must first translocate to the nucleus to be transcribed to mrna before translation in the cytoplasm. regarding the use of mrna to deliver il-12 locally, a recent study demonstrated that weekly i.v. delivery of lipid nanoparticles (lnp) loaded with mrna encoding il-12 reduced tumor burden in a myc-driven transgenic mouse model of hepatocellular carcinoma (hcc) (187) . the tumor inhibition and extended survival were attributed to increased infiltration of cd3 + cd4 + cd44 + immune cells and not suppression of myc (188) . similarly, moderna/astrazeneca has developed, medi1191, an lnp formulation with il-12 mrna. medi1191 is currently in phase i clinical trials for intratumoral injection of advanced solid tumors in combination with durvalumab ( table 2 ) (189) . in preclinical studies, a single intratumoral injection of mrna encoding murine il-12 (mil-12) increased ifnγ expression and genes associated with a th1 response in mc38 tumor-bearing mice (190) . when combined with anti-pd-l1, enhanced t cell infiltration and expanded tumor-specific t cell subsets were observed (190) . in another phase i clinical trial, sanofi and biontech are testing sar44100 (bnt131), an mrna platform encoding a cocktail of il-12sc, il-15sushi, ifnα, and gm-csf for intratumoral injection as a monotherapy and in combination with cemiplimab (191) . to our knowledge, no preclinical data with sar44100 have been disclosed. in general, nucleic acid-based il-12 delivery approaches are limited by variable transfection rates as well as unregulated production of gene products. in other words, it is easy to control the amount of pil-12 or il-12 mrna delivered but it is not easy to control the dose of recombinant il-12 that each subject receives. variable transfection rates may be responsible for conflicting reports on whether pil-12+ep does (131-133, 140, 143, 144) or does not (124, 126, 130) produce significant increases in serum il-12 and ifn-γ. fortunately, lethal il-12-related toxicities have not been reported in the any of the preclinical or clinical studies detailed above. nevertheless, the potential for severe il-12-related adverse events caused by continued and/or unregulated production of il-12 remains. strategies to enhance safety and efficacy, either by localizing gene-based il-12 though incorporation of an anchoring or binding domain or by incorporation of an inducible safety switch to turn off il-12 production, will help improve the therapeutic window of promising nucleic-acid-based il-12 delivery technologies. another potential limitation that must be considered, is any type of adverse reaction against components of the delivery vehicle or against the nucleic acid vector itself. regarding the former, foreign delivery components have the potential to induce immune responses which could influence il-12 delivery. most notably, it has been reported that about 7 in 10 humans have circulating anti-peg antibodies (192) . the high prevalence of anti-peg antibodies could limit the efficacy of any peg-based delivery vehicle. regarding the immune responses against il-12 vectors, nucleic acids, particularly dna that is found in the cytoplasm have the potential to stimulate the cgas-sting pathway, which may induce its own inflammatory response. thus, studies utilizing nucleic acid-based delivery must take care to decouple the effects of il-12 from sting activation. lastly, although an exceedingly rare event, dna vectors could become integrated within a cell's genome. depending on the site, such integration could have deleterious or even transforming effects. given that only transient il-12 expression is desirable, strategies capable for preventing integration, like the use of circular instead of linearized plasmids, should be preferred. adenoviruses, herpes simplex viruses, semliki forest viruses, poxviruses, and other viral vectors have been engineered to express biologically active il-12. these engineered viruses injected directly into a tumor are able to infect cancer cells and induce expression of il-12 within the tumor microenvironment. furthermore, many viruses have the unique ability to selectively lyse cancers cells after infection. such oncolytic viruses take advantage of defective cell cycle and interferon signaling pathways that are hallmarks of cancer cells but not normal cells (193) . oncolytic viruses can kill compromised cancer cells in a variety of ways from direct virus-mediated cytotoxicity to indirect destruction of tumor-feeding blood vessels (194) . the following sections discuss the progress and limitations of il-12encoding viral vectors. adenoviruses are the most well-studied among the il-12 expressing vectors (47, (195) (196) (197) (198) (199) (200) (201) . in preclinical studies, i.t. injections of adenoviruses encoding il-12 (ad-il-12) have mediated regressions of murine colorectal carcinomas (202) (203) (204) , breast carcinomas (47, 201, 202) , prostate carcinomas (205, 206) gliomas (207, 208) , bladder carcinomas (209) , fibrosarcomas (202, 210) , laryngeal squamous cell carcinoma (211) , hepatomas (212) and hepatocellular carcinomas (213, 214) medullary thyroid carcinomas (215) , thyroid follicular cancer (216) , and ewing's sarcoma (217) . among the more robust responses, ad-il-12 induced complete regression of subcutaneous neuro-2a neuroblastomas in nearly half of mice receiving a single i.t. injection (218) . mice becoming tumor-free also rejected a subsequent tumor rechallenge (218) . similar results were found against ct26 colon adenocarcinomas with more than three-fourths of mice completely eliminating their tumors and all cured mice rejecting a tumor rechallenge (203) . the antitumor immune response was mediated primarily by cd8 + t cells. impressively, 3 of 7 mice with bilateral tumors experienced complete regression of an untreated tumor (203) . against 6-23 rat medullary thyroid carcinomas, i.t. injection of adtcpmil-12 caused complete regression of more than 60% of treated tumors (215) . all cured rats rejected a tumor rechallenge while separate experiments showed that treatment of a single tumor resulted in inhibition of a distant untreated tumor (215) . while liver infection following i.t. adtcpmil-12 injection was documented, no toxicity was observed (215) . a follow up study found similar antitumor and abscopal responses against rat thyroid follicular cancer (216) . in the pymt-derived transplanted mammary carcinoma model, adenoviral vectors encoding il-12 induced complete regression in 31% and partial regression in 47% of mice (47) . ten of 11 tumor-free mice completely rejected a tumor rechallenge. in contrast with similar studies using ad vectors expressing il-2, no obvious toxic side effects due to admil-12 were noted (47) . in another difficult model, a single i.t. injection of ad.5/3.crgd-mil12p70 resulted in >60% long term survival of mice with intracranial gl261 gliomas (207) . in a murine model of ewing's sarcoma (tc71), twice weekly i.t. injections of ad.mil-12 significantly delayed treated tumors as well as untreated tumors (217) . ad.mil-12 also induced complete regression in all treated mice bearing heterotopic mb49 bladder carcinomas (209) . although body weights were not affected, serum ifn-γ levels due to i.t. ad.mil-12 were maximal from 2 (∼3,500 pg/ml) to 5 days (∼1,500 pg/ml) post injection (209) . in a useful comparison against other cytokines, one study demonstrated that ad-ifn-γ had no greater antitumor activity than an empty ad vector, whereas admil-12 induced complete regressions of p815 mastocytomas in >80% of treated mice (219) . similarly, ad-gm-csf inhibited the growth of frtl-tc rat thyroid tumors, however, adil-12 was found to be much more effective (220) . adil-12 also generated systemic immunity capable of inhibiting the growth of distant tumors (220) . an adenoviral vector expressing a single chain il-12 (scil-12) was developed to enhance bioactivity over the native heterodimeric form (221) . long-term tumor-free survival was observed in up to 90% of rats with established mh-7777a hepatocellular carcinomas following i.t. infections with ad.scil-12 (221) . all tumor-free mice were protected from tumor rechallenge. however, despite i.t. injections, il-12 and ifn-γ were detected in the serum of treated mice (221) . an oncolytic adenovirus expressing single-chain il-12 (ad-dhscil12) was more effective than non-replicating (nononcolytic) adenoviruses expressing il-12 at controlling liver metastases of pancreatic cancer in hamsters (222) . however, il-12 levels in serum and non-tumor tissues were similar (222) . in clinical studies, i.t. ad.il-12 was well-tolerated with no dose-limiting toxicities in a phase i trial in 21 patients with advanced pancreatic, colorectal, or primary liver malignancies (223) . serum ifnγ levels peaked 1 day after ad.il-12 administration and was likely responsible for the 16 grade 3 adverse events observed. one patient had a partial response and 29% of patients experienced stable disease. four of 10 assessable patients experienced increases in tumor infiltrating cd4 + and cd8 + cells. delayed-type hypersensitivity tests with inactivated adenovirus indicated that all patients developed an immune response against the adenovirus (223) . despite robust antitumor immune responses, interest in ad-il-12 waned in the mid-2000s due to the aforementioned lethal toxicities associated with systemic administration of ril-12 and the inability of ad-il-12, even if administered intratumorally, to prevent systemic dissemination of the cytokine. in recent efforts to mitigate systemic il-12 dissemination and associated toxicities, two strategies have been developed. the first involves engineering il-12 to prevent its dissemination. this has been accomplished either by anchoring il-12 to the surface of tumor cells via fusing a transmembrane domain or glycosylphosphatidylinositol (gpi)-anchored signal sequence to the cytokine (224, 225) or by deleting the n-terminal signal peptide and thus preventing il-12 secretion (226) . using the former technology, i.t. injection of an adenoviral vector encoding membrane-anchored il-12 (ad/scil-12-b7tm) eliminated the majority of primary ct26 tumors and suppressed the growth of untreated contralateral tumors (5 out of 5 mice), in which complete regression occurred in 1 out of 5 mice (227). importantly, negligible il-12 was found in the circulation of mice treated with ad/scil-12-b7tm. the second technology deletes the signal peptide from the p35 subunit of il-12 to prohibit il-12 secretion (226) . newly designed oncolytic adenoviral vectors with three genes deleted, i.e., triple deletion (td), and encoding either wild-type il-12 (ad-td-il-12) or a non-secreting il-12 (ad-td-nsil-12) were evaluated in syrian hamster models of pancreatic cancer. six i.t. injections of either ad-td-il-12 or ad-td-nsil-12 were found to eliminate subcutaneous hpd1nr tumors in all mice (226) . against peritoneally disseminated shpc6 tumors and orthotopic hap-t1 pancreatic tumors, ad-td-nsil-12 outperformed ad-td-il-12 in terms of overall survival (226) . most importantly, ad-td-nsil-12 resulted in significantly lower serum il-12 levels and reduced systemic inflammatory cytokine expression (226) . the second strategy to mitigate systemic il-12 dissemination involves conditional expression of il-12. the rheoswitch therapeutic system r (rts) is an ecdysone receptor-based gene regulation platform in which a transcription factor becomes activated only in the presence of a synthetic small molecule ligand (228) . an adenoviral vector encoding the rts switch and mil-12 (ad-rts-mil-12) and controlled by the oral activator, veledimex (vdx) was recently shown to extend survival in mice bearing intracranial gl261 gliomas (229) . intratumoral ad-rts-mil-12 plus oral vdx was found to induce il-12 expression in a dose-dependent manner. local il-12 expression correlated with increases in tumor-infiltrating lymphocytes. il-12 and ifnγ were detected in the sera of treated animals, albeit at an order of magnitude lower than levels found in tumors (229) . several clinical studies utilizing the regulatable ad-rts-il-12 platform are underway ( table 2) . recent results from a phase i study in 31 patients undergoing resection of recurrent highgrade glioma demonstrated that vdx induced il-12 expression in a dose-dependent manner (230) . likewise, the frequency and severity of adverse events, including grade 3 cytokine release syndrome, also increased with vdx dose. demonstrating the advantage of the inducible system, all serious adverse events were reversible with vdx discontinuation. interestingly, the use of corticosteroids negatively impacted survival (230) . at the optimal dose, and in the absence of corticosteroids, the median overall survival of 17.8 months was encouraging (230) . while the localized injection of ad-rts-hil-12 in the resected tumor bed induced il-12 expression in a recurrent tumor microenvironment, systemic dissemination of il-12 and its resultant toxicities could not be avoided. herpes simplex viruses (hsvs) are another family of viruses that have been widely explored for localized il-12 delivery. wild-type hsv are cytolytic and thus must be significantly attenuated or rendered replication-incompetent to avoid systemic infection. injection of replication-incompetent hsv-il-12 into established hepatomas prior to partial hepatectomy inhibited the engraftment of an intraportal tumor cell challenge in preclinical studies (231) . importantly, no changes in serum il-12 were detected in treated buffalo rats (231) . in order to capitalize on their lytic potential, hsvs have been engineered, through deletion or mutation of genes responsible for viral replication such that only cancer cells are lysed. not surprisingly, these oncolytic hsvs (ohsvs) have been shown to exhibit greater antitumor potential than their replicationincompetent, parental counterparts. for instance, treatment of established hepatomas with a non-cytokine encoding ohsv exhibited significant antitumor activity which was further increased with an il-12 insert (232). the ohsv-il-12 treatment was also more effective than ohsv at protecting animals from a tumor rechallenge (232) . similar antitumor responses to ohsv and increased efficacy with ohsv-il-12 were observed against flank ct26 and sccvii tumor models (233) (234) (235) . a single i.t. injection was able to delay the growth of established sccvii tumors whereas multiple injections induced complete regressions in 5 of 6 treated mice (235) . in contrast, multiple injections of ohsv-gm-csf cured only half of treated mice (235) . in the ct26 model, ohsv-il-12 inhibited or eliminated injected 5 mm tumors while inhibiting non-injected, contralateral tumors (236, 237) . when low dose mil-12 was added to i.t. ohsv-il-12, both treated and untreated ct26 tumors were eliminated in more than two-thirds of mice (237) . the importance of t cells in this model was established as ohsv-il-12 had no antitumor effect in ct26-bearing nude mice (236, 237) . intraperitoneal administration of ohsv-il-12 also increased survival in misiir-tag mice bearing ovarian carcinomas (238) . treatment was associated with tumor antigen-specific cd8 + t cell infiltration (238) . against flank sarc-043 and sarc-045 sarcomas, ohsv-il-12 extended survival compared to saline injections (239) . although there was no difference is survival between control and il-12 encoding ohsv, the ohsv-il-12 was found to increase tumor-infiltrating effector t cells while decreasing immunosuppressive mdsc and t reg populations (239) . in an interesting comparison of cytokines, ohsv-il-12 was more effective at inhibiting flank prostate tumors, tramp-c2 and pr14-2, than ohsv encoding gm-csf (ohsv-gm-csf) which was no more effective than non-cytokine encoding ohsv (240) . only 1 of 18 mice treated with ohsv-il-12 exhibited an increase in serum il-12 4 days after treatment (240) . ohsv-il-12 also significantly outperformed ohsv-gm-csf in a model of ct26 metastasis (241) . in addition to localizing il-12, i.t. injections may be key in assuring safety as intrasplenic injections of ohsv-il-12 induced concerning increases in il-12 and ifnγ in both serum and liver specimens (241) . frequent and worthwhile targets of ohsv-il-12 immunotherapy are the various forms of brain cancer. in one early study, ohsv-il-12 was found to extend survival and cure approximately one-fourth of mice bearing 5-day-old intracranial neuro-2a neuroblastomas (242) . treatment was associated with an influx of cd4 + and cd8 + t cells as well as macrophages. the inclusion of il-12 was critical, as median survivals of mice treated with the parental, non-cytokine encoding ohsv or saline were similar (242) . likewise, ohsv-il-12 but not non-cytokine ohsv was effective at extending survival against intracerebral mouse 005 glioblastomas (243) . in an intracranial 4c8 glioma model, ohsv-il-12 was significantly more effective than ohsv at extending survival and curing mice (244) . in a model of breast cancer metastasizing to the brain, i.t. ohsv-il-12 modestly extended the survival of mice bearing intracranial sck tumors (245) . intracerebral injections of ohsv-il-12 in owl monkeys resulted in neither histopathological changes in brain tissue nor clinical evidence of toxicity, as assessed by changes in temperature, neurologic performance, feeding or social behavior, or weight (246) . in addition to encoding cytokines, hsvs can be engineered to target cancer-associated antigens. four i.t. injections of a her2-targeted ohsv-il-12 was significantly more effective at inhibiting both day 3 and day 10 tumors than the non-cytokine encoding parental hsv (day 3: 15/22 ohsv-il-12 vs. 7/20 hsv becoming tumor free; day 10: 3/18 ohsv-il-12 vs. 1/12 hsv becoming tumor free) (247) . immune responses to ohsv-il-12 included elevated levels of ifnγ, il-2, granzyme b, tbet, and tnfα as well as th1 polarization and nk activation (247) . this her-2 targeted ohsv-il-12 was also found to induce complete remission in more than one-fourth of treated mice bearing orthotopic high grade gliomas (hgg) expressing her2 (248) . cured mice were protected from hgg rechallenge regardless of her2 expression (248) . this is an important finding given the heterogeneity of her2 expression among and within tumors. the clinical precedence for hsv has been established with talimogene laherparepvec, which encodes for gm-csf and is approved for intratumoral injection in patients with advanced, non-resectable melanoma. a clinical grade preparation of hsv-1 encoding hil-12 (m032) induced no adverse clinical signs after intracerebral injection in non-human primates (249) . a phase 1 clinical trial exploring the safety of m032 in patients with recurrent or progressive glioblastoma multiforme, anaplastic astrocytoma, or gliosarcoma is currently recruiting [nct02062827]. semliki forest virus (sfv) is an alphavirus that was first isolated from mosquitos in uganda and has a broad range of hosts, making it ideal for translation (250) . in addition, modified sfv can produce higher levels of recombinant protein than retroviral vectors, and expresses protein more stably than adenoviral vectors (251) . sfv is also less pathogenic in humans, and induces apoptosis of tumor cells at the end stage of virulence (250) . sfv encoding il-12 (sfv-il-12) was found to extend the survival of mice with established orthotopic 203 gliomas (252) . because sfv infection induces apoptosis, uptake of infected tumor cells by dendritic cells in the presence of il-12 is posited as a potential mechanism of enhanced antitumor activity. in a b16 brain tumor model, the same group demonstrated a prolonged median survival by 5 days when immunizing with sfv-il-12 pulsed dendritic cells, compared to a retroviral vector encoding il-12 (253) . in a woodchuck hcc model, induced by chronic infection with woodchuck hepatitis virus (whv), using a single intratumoral treatment with sfv-enhil-12, which included 10 separate injections at different sites of one tumor, partial tumor regressions of up to 80% occurred in 5 out of 6 animals (254) . although all tumors regrew after treatment, injections of sfv-enhil-12 resulted in a favorable safety profile with only transient reductions in body weight (254) . in a transgenic mouse model of spontaneous hcc, i.t. sfv-il-12 treatments achieved 100% survival for at least 135 days (255) . interestingly, sfv-il-12, which induces transient infection and expression of il-12, was found to be more effective and less toxic than long-term il-12 expression induced by a plasmid encoding il-12 which was delivered hydrodynamically (255) . i.t. sfv-il-12 inhibited tumor growth and extended survival in mice bearing orthotopic 4t1 mammary carcinomas (256) . when administered neoadjuvant to resection and combined with attenuated salmonella (lvr01) as a post-surgery adjuvant, an impressive 90% long-term tumor free survival was achieved (256) . while there is consensus on its benefits, the mechanism of sfv-il-12-induced tumor regression is debated. initial studies performed in a subcutaneous b16 melanoma model did not find a significant decrease in tumor regression when using t cell-deficient nude or nk cell-deficient beige mice. rather, this group suggested that inhibition of angiogenesis mediated by ifnγ production causes massive tumor necrosis (257) . a decade later, the same group further supported this conclusion using inos deficient mice that express high levels of vegf (258) . the effect of il-12 was even more pronounced, with fewer tumor vessels in inos −/− mice than in wild-type mice given the same treatment with sfv-il-12 (258) . however, they did find immunohistochemical evidence that nk cell activation and recruitment is correlated with murine endothelial cell death (258) . more recently, the efficacy of sfv-il-12 was found to be dependent on type i interferons produced by macrophages and dendritic cells (259) . furthermore, the type i interferon receptor, ifnar, is necessary for il-12-dependent cd8 + t cell expansion (259) . different routes of administration were compared using a subcutaneous p815 model and demonstrated that i.t. injection of sfv-il-12 was superior in producing ifnγ compared to either s.c. or i.v. routes (260) . importantly, none of the sfv-il-12 injections resulted in increased serum levels of ifnγ (260) . the combination of sfv-il-12 with monoclonal antibodies or adjuvants is another strategy that has been studied. by administering sfv-il-12 and anti-cd137 to provide costimulation to t cells, survival was improved in both s.c. b16-ova and s.c. tc-1 models with a maximum longterm survival of 75% in both models (261) . in the bilateral b16-ova model, 90% of treated and 22% of untreated tumors experienced complete regression with 10 8 sfv-il-12 viral particles and anti-cd137 (261) . cd8 + t cells were crucial for this tumor regression and sfv-il-12 increased the ratio of cd8 t/t regs compared to anti-cd137 alone. a subsequent paper further demonstrated that sfv-il-12 induces pd-l1 expression on b16-ova cells and therefore combined pd-1/pd-l1 blockade with the viral construct (262) . this combination significantly enhanced survival compared to individual components using b16-ova and mc38 models, with long-term survival >75% (262) . modifications to improve the performance of sfv-il-12 vectors have also been explored. an enhanced (sfv-enhil-12) vector with separate promoters for each subunit of il-12 increased il-12 expression 8-fold over the single promoter construct (251) . one i.t. injection of 10 8 viral particles using either the original or enhanced constructs resulted in >80% longterm tumor free survival of mc38 tumor-bearing mice (251) . lower doses of sfv-enhil-12 induced tumor regression more efficiently than sfv-il-12, although the enhanced vector induced higher levels of serum il-12 (251) . a separate enhanced sfv vector with 10x higher gene expression, called psfv10-e-il12, has also been developed (263) . this vector also included separate promoters for p40 and p35 with an additional capsid enhancer to drive enhanced expression of the recombinant protein. two helper vectors with instructions for structural proteins were cotransfected with the enhanced sfv to produce virus-like particles (vlps). intratumoral injections in subcutaneous k-balb and ct26 tumors caused complete regression and furthermore inhibited primary tumor growth and reduced metastases in a heterotopic 4t1 model (263) . in an effort to limit undesirable virus-mediated cytotoxicity for intracranial applications, sfv vlps expressing il-12 have been developed (264) . low dose (5 × 10 7 vlps) treatment decreased orthotopic rg2 rat glioma volumes by 70% and extended survival by a little more than 20% over vehicle controls. high dose (5 × 10 8 vlps) treatment resulted in enhanced tumor reduction but also caused cns inflammation, necrosis, and treatment-related death (264) . the broad infectivity of sfv-based vectors is a major limitation to their use in cns applications. vaccinia virus (vv), which was used extensively during the eradication of smallpox, has a large capacity for gene insertion, a wide host range and high gene expression efficiency. vv expressing il-12 (vv-il-12) has been found to inhibit, but not eliminate, c6 gliomas in nude mice following i.t. injection (265) . low vv-il-12 doses, 10 2 -10 4 pfu, and high vv-il-12 doses, 10 5 -10 7 pfu, resulted in similar levels of tumor inhibition (265) . all doses above 10 pfu resulted in cytokine-associated toxicities punctuated with a 40% mortality rate in the high dose group (265) . similar tumor inhibition and mortality were found in a subsequent publication which also correlated high plasma levels of ifnγ and tnfα with toxicity (266) . vv-il-12 and il-2 expressing vv (vv-il-2) exhibited similar inhibition of c6 gliomas (265, 266) however, against ae17 mesotheliomas, vv-il-12 cured 80% of mice compared to only 20% of mice treated with vv-il2 (267) . recently, an oncolytic vv-il-12 was shown to increase lymphocytic infiltration and inhibit llc and b16f10 tumors with 14-25% complete responses (268) . antitumor efficacy was enhanced by the addition of an il-7 expressing vv (vv-il7) as well as immune checkpoint inhibitors (268) . the combination of vv-il-12 and vv-il7 did not affect mouse body weight (268) and appears to be better tolerated than previous vv-il-12 treatments which utilized different vv strains (265, 266) . a recombinant canarypox virus (alvac) encoding il-12 has been shown to induce complete regression of ts/a mammary carcinomas after six i.t. injections, whereas a single i.t. injection of alvac-il-12 had no effect on tumor growth (269) . of the mice that were rendered tumor-free, 70% were protected from a contralateral rechallenge. antitumor activity was driven by il-12 as the alvac vector itself displayed very limited tumor inhibition. in a phase i study in 9 patients with unresectable metastatic melanoma, i.t. injections of alvac-il-12 resulted in increased intratumoral il-12 mrna expression in only 4 of 9 patients (270) . three patients experienced modest increases in serum il-12 and ifnγ levels and no dose-limiting toxicities were observed. one patient had a complete response of an injected lesion and an uninjected in-transit metastases (270) . in a similar phase i study performed by the same group, only 1 of 4 evaluable patients exhibited an increase in intratumoral il-12 mrna after alvac-il-12 administration (271) . attenuated measles viruses (mevac) have been used safely as a vaccine to prevent measles in billions of people. mevac is another type of oncolytic virus which makes it an attractive potential cancer therapy. mevac encoding il-12 (mevac fmil-12) achieved complete regression of carcinoembryonic antigen (cea) expressing mc38 tumors in 90% of mice treated i.t. (272) . mev encoding anti-pd-l1 or igg-fc induced complete regressions in 60 and 40% of treated mice, respectively. antitumor activity was cd8 + t cell dependent and correlated with high levels of intratumoral ifnγ. all mice experiencing complete regressions following mevac fmil-12 also complete rejected tumor rechallenge (272) . a follow up study demonstrated that mevac fmil-12 was more effective at controlling tumors than mev encoding il-15 in two murine tumor models (273) . against mc38-cea tumors, mevac fmil-12 eliminated tumors in all treated mice whereas mev-il15 induced complete tumor regressions in 60% of mice (273) . effects appear to be model dependent as treatment of b16 tumors expressing human cd46 with mev-il-12 and mev-il15 only modestly extended survival (273) . vesicular stomatitis virus (vsv) is another potent oncolytic virus under exploration for cancer therapy. vsvs engineered to express il-12 (vsv-il-12) were recently evaluated in an orthotopic floor of mouth model of sccvii murine squamous cell carcinoma (274) . multiple i.t. injections of vsv-il-12 were more effective at eliminating tumors and induced higher longterm survival rates (40 vs. 0%) compared to non-cytokine encoding vsv (274) . moloney murine leukemia virus (momlv) is a positive sense, single-stranded rna (ssrna) retrovirus. a momlv expressing il-12 was engineered to display an anti-erbb2 scfv against the her2 receptor (275) . eight daily i.t. injections of 6-day-old flank mbt2 tumors was found to result in inhibition and complete regression in about one-fifth of mice treated with the her2targeted momlv-il-12 (275) . a hybrid viral vector which pairs the high infectivity of adenoviruses with the high gene expression of sfv has been engineered to specifically infect and express il-12 in hepatocellular carcinoma cells (276) . in buffalo rats bearing orthotopic mca-rh7777, the adv-sfv hybrid vector expressing il-12 induced complete regression in 4 of 12 treated animals. while the non-hybrid sfv-il-12 induced complete regression in 50% of treated rats, treatment was accompanied by elevated liver enzymes indicating hepatotoxicity (276) . in contrast, liver enzyme levels following administration with the tissue-specific, hybrid vector were no different than levels in saline-treated control rats (276) . newcastle disease virus (ndv) is an oncolytic, enveloped, negative-sense, single-stranded rna virus. ndvs have been engineered to express checkpoint inhibitor molecules and checkpoint inhibitor-cytokine conjugates (277) . although the antitumor activity of i.t. ndv expressing il-12 alone was not tested, 5 i.t. injections of ndv-anti-cd28-mil-12 plus i.p. anti-ctla-4 or ndv-antipdl1-mil-12 plus i.p. anti-ctla-4 resulting in complete responses in 77 or 70%, respectively of mice bearing 9 day-old b16f10 flank tumors (277) . the same treatments of one of two bilateral tumors resulted in 10 and 62% complete responses, respectively, of untreated tumors (277) . a major benefit of virus-based il-12 delivery is high transfection rates, particularly when compared to non-viral vectors. however, virus-based delivery is limited in several aspects. first, neutralizing immune responses including anti-viral antibody production (271, 278, 279) and dth responses (223) may preclude repeated injections with the same vector. however, the extent to which anti-viral immunity limits repeated injections is unclear. mice previously exposed to a viral vector demonstrated a 2.4-fold reduction in intratumoral transgene expression. this level of reduction did not affect antitumor activity; however, the potential exists for reduced antitumor immune responses when lower doses of virus are used (280) . on the positive side, viral dissemination and transgene expression in non-targeted liver tissue was reduced by more than 1,000-fold in mice pre-exposed to the viral vector (280) . secondly, viral il-12 delivery requires transfection of host cells, which has been shown to be highly heterogeneous due to inherent susceptibility or previous exposure to related vectors (223) . in an extreme example of susceptibility, a dose of adcmvmil-12 that was found to be lethal in 100% of treated c57bl/6 mice did not result in a single death in the balb/c strain (281) . livers of c57bl/6 mice exhibited much higher levels of adenovirus transduction (281) . the current coronavirus outbreak further illustrates the point that some individuals appear to be more susceptible than others to viral infections. third, although clinical trials have shown that recombinant viruses are tolerated, viral dissemination, and infection in liver cells following i.t. injection were reported at an alarming degree (282) (283) (284) . even when small volumes (10-20 µl) of recombinant adenoviruses were injected i.t., transgene expression was found in the liver, intestine, spleen, kidney, and brain. as much as 34% of the injected adv infected non-targeted normal tissues (284) . similar levels of transgene expression was found in the liver and tumor 3 days after an i.t. injection (280) . fourth, some viral vectors, such as momlv and mev, can insert their genetic information into an infected cell's genome causing undesirable mutations and uncontrolled longterm expression of il-12. the benefits of using such integrating vectors must be weighed against these risks as other viruses, such as adv, hsv, vsv, and ndv, are non-integrating and induce transient expression (285) . lastly, and most relevant to the topic of localizing il-12, even local injections of viruses encoding il-12 are not able to prevent il-12 dissemination (47, 202, 282, 286, 287) . consequently, antitumor responses and il-12-mediated adverse events are strongly correlated with virus dose. similar to plasmidbased il-12 delivery, novel strategies that anchor il-12 to an injection site or prevent its secretion appear best suited to break this correlation and widen the therapeutic window of virusencoding il-12. dendritic cells (288, 289) , fibroblasts (290, 291) , macrophages (292, 293) , tumor cells (294, 295) , and mesenchymal stem cells (mscs) (296) (297) (298) have all been engineered to express il-12. transduced dcs, fibroblasts, tumor cells, and macrophages have been directly injected into tumors, while mscs have been injected systemically for migration to tumor beds (296, 297) . a much larger number of preclinical and clinical studies have used il-12-transduced tumor cells for s.c. immunization. these studies are not covered in this review as il-12 in this context is functioning as a vaccine adjuvant following injection of transduced cells in healthy, non-cancerous s.c. tissue rather than a strategy to localize il-12 to a tumor. dendritic cells and macrophages have been engineered to express a variety of cytokines including il-12. in addition to supplying the tumor microenvironment with il-12, transduction of these antigen presenting cells increases their ability to induce robust antitumor immune responses. following i.t. injection, dcs and macrophages are capable of capturing tumor antigen, migrating to draining lymph nodes, presenting antigen and priming tumorspecific t cell responses. transfection of antigen presenting cells with il-12 helps polarize t cells to a th1 phenotype and facilitate robust tumor-specific ctl responses (288) . in an early preclinical study, bone marrow-derived dendritic cells (bmdcs) expressing il-12 were more effective than dcs expressing il-2 in controlling the growth of b16f10 melanomas and generating tumor-specific ctl activity (299) . il-12-transduced bmdcs were also found to significantly suppress established mca205 fibrosarcomas, b16 melanomas, d122 adenocarcinomas, and 178-2 bma prostate carcinomas following a single i.t. injection (288, 300, 301) . in one study, 36% of treated mca205 tumors were completely rejected. tumor-free mice also rejected a subsequent mca205 rechallenge (288) . the growth of contralateral, non-treated tumors was also suppressed, implying that systemic tumor-specific immunity can be induced following i.t. injection. il-12-transduced bmdcs were substantially more effective than il-12-transduced fibroblasts or non-transduced bmdcs at inducing tumor-specific ctl activity and ifnγ production by draining lymph node cells and splenocytes (288) . enhanced trafficking of dcs to regional lymph nodes provides a major advantage over non-migratory cells such as il-12transduced tumor cells or fibroblasts. dcs transfected with adenovirus to express il-12 (dc-adcmvil-12) were found to eliminate ct26 and mc38 colon adenocarcinomas in 67-84% and 50% of mice, respectively (289, 302) . mice experiencing complete regression exhibited tumor-specific ctl activity and rejected tumor rechallenge. i.t. injections of adcmvil-12-infected fibroblasts or allogeneic dc-adcmvil-12 were not effective (289) . in a similar study, dc-adcmvil-12 reversed immune suppression mediated by tbj neuroblastomas and induced complete tumor regression (303) . dcs engineered to co-express both il-12 and il-18 displayed synergistic antitumor activity that was greater than either dcencoding cytokine alone (304) . all mice experienced complete regression of cms4 tumors while treated metha tumors were substantially reduced (304) . while serum ifn-γ levels reached 400 pg/ml 2 days after dc administration, no toxicities were noted (304) . bmdcs transfected by simian virus 40 (sv40) to express il-12 or il-15 demonstrated complete regression of ct26 colon adenocarcinomas in 40 or 73% of mice, respectively (305) . there was no synergy between il-12 and il-15 when delivered together. interestingly, maturation of dcs with lps prior to i.t. injection impaired antitumor activity, presumably due to reduced antigen loading in the tumor. however, this hypothesis was not evaluated (305) . transfection of bmdcs isolated from cms4 sarcoma-bearing mice with an adenoviral vector expressing il-12 was shown to restore antigen-presenting and allostimulatory functions (306) . direct injections of the rescued bmdcs were shown to eliminate intrahepatic cms4 tumors and induce protective immunity (306) . dcs can also be isolated from mouse spleens prior to transduction with an il-12 encoding vector. a recent study demonstrated that splenic dcs overexpressing il-12 can inhibit the growth of melanomas in mice (307) . il-12-transduced macrophages are less well-studied, however, like dcs, macrophages have antigen presenting capabilities that can be enhanced by il-12. a single i.t. injection of peritoneal exudate-derived macrophages transduced by admil-12 induced suppression of primary and metastatic 178-2 bma orthotopic prostate tumors (293) . serum il-12 levels peaked 3 days after injection and remained elevated for at least 2 weeks. antitumor activity was correlated with increased leukocytic infiltrate and cytolytic activity (293) . the admil-12-transduced macrophages were found to migrate to draining lymph nodes following i.t. injection. similar to some viral constructs, to reduce the potential for toxicity, il-12 expression can be placed under the control of an inducible promoter. one inducible system consists of 2 cassettes including gal4-ecr;vp16-rxr and il-12. first, gal4-ecr and vp16-rxr are expressed under the constitutive ubiquitin c promoter. the heterodimerization of gal4-ecr and vp16-rxr are triggered by addition of small-molecule ligand, e.g., pharmacologically inert diacylhydrazine. gal4-ecr-vp16-rxr heterodimers bind to a synthetic promoter containing gal4 binding site and induce the expression of il-12 (308) . dcs with inducible il-12 demonstrated inhibition of renca and metha tumors following i.t. administration (309) . combining il-12expressing dcs with either il-21-or ifnα expressing dcs did not improve antitumor efficacy (309) . the ability to turn cytokine expression "on" and "off " is an attractive feature which may potentially allow for better control over cytokine delivery. to date, only one clinical study has been published. in a phase i study, autologous dendritic cells transfected to express il-12 were administered i.t. to patients with metastatic gastrointestinal carcinomas (310) . of the 17 treated patients, one experienced a partial response and two experienced stable disease. serum ifnγ, but not il-12, levels were elevated following treatment. four instances of transient grade 3 lymphopenia were observed, but no grade 4 toxicities were observed. in vitro il-12 production was highly variable, most likely due to differences in susceptibility of dendritic cell infection with adenoviruses encoding il-12 (310) . fibroblasts expressing il-12 caused the elimination of 7-day mca207 tumors in 70-100% of mice after a single peritumoral injection (291) . in addition, local il-12-mediated regression was also able to control tumors on the contralateral flank. as has been shown in other systems, i.t. injections were more effective than distant s.c. injections at controlling tumor growth. repeated injections of il-12-engineered fibroblasts were welltolerated, with no abnormalities detected in liver and renal function tests (291) . a similar peritumoral implantation of alginate encapsulated nih3t3 fibroblasts expressing il-12 was found to inhibit ct26 tumor growth (311) . in a phase i study, peritumoral injections of il-12-transduced autologous fibroblasts induced transient tumor reductions in four of nine patients with disseminated cancers (312) . treatment with fibroblasts capable of secreting up to 5,000 ng of il-12 per day was well-tolerated (312) . mesenchymal stem cells (mscs) are multipotent adult stem cells that divide and differentiate in order to repair skeletal tissues, such as bone, cartilage, and muscle. as such, mscs are predisposed to traffic from bone marrow to a wound site based on expression of soluble factors and unique receptors in damaged tissues. tumors can express many of the same factors and receptors which results in mscs trafficking to, and infiltrating tumors following systemic injections (313) . thus, using il-12 transduced mscs as a "trojan horse" to target tumors and deliver il-12 is a promising approach. in one preclinical study, ad.il-12-transduced mscs inhibited the growth of tc71 human ewing sarcomas in mice (314) . one potential concern about il-12-expressing mscs (msc/il-12) is their tropism for some normal tissues. in the above study, msc/il-12 were found in tumor, liver, lung and spleen tissues but not kidney, heart, or brain tissues 10 days after i.v. injection (314) . in a different study, high levels of msc/il-12 were found in the tumor but not in the liver, lung, and spleen 20 days after administration (297) . earlier time points were not presented. il-12 levels were high in the tumor; however, significant serum il-12 levels were found to persist for at least 2 weeks after msc/il-12 administration (297) . these high levels of intratumoral il-12 resulted in the inhibition of 786-0 renal cell carcinomas in nude mice (297) . in a study that illustrates the importance of tropism, mscs that were non-virally transfected with pil-12 significantly inhibited b16f10 lung metastases but not subcutaneous b16f10 tumors following i.v. administration (315) . only when administered i.t., were msc/il-12 cells able to inhibit s.c. b16f10 tumors (315) . despite the tumor-homing potential of mscs, published data suggest that msc/il-12 may be most effective at treating tumors in tissues where mscs have a natural tropism, e.g., lung, liver and bone (315) . several other studies have favored local over systemic administration of msc/il-12 to treat various tumors. intracranial gl26 glioma-bearing mice treated with i.t. ucb-msc-il12m generated from human umbilical cord blood experienced significantly prolonged survival (316) . specifically, 70% of ucb-msc-il12m treated mice survived more than 90 days post tumor implantation whereas all mice treated with mscs expressing gfp succumbed within 65 days (316) . cured mice were completely protected from ipsilateral and contralateral tumor rechallenge (316) . in another study, bone marrow derived mscs were transfected with a lentivirus to express mil-12 (317) . these lenti-mil-12-mscs reduced the volume of h22 and metha ascites and increased survival when administered i.p. 2 and 7 days after tumor inoculation (317) . no pathological abnormalities were noted on biopsies taken from internal organs of lenti-mil-12-msc treated mice (317) . human mscs transduced with a retroviral vector expressing a modified il-12 (mscs/il-12m) inhibited b16f10 melanomas following five i.t. injections over 3 weeks (298) . safety was implied as no il-12 was detected in the serum following treatment. interestingly, the antitumor activities elicited by i.t. injections of mscs/il-12m and il-12 plasmid dna were similar (298) . in a rare comparison of cytokine delivery technologies, i.t. injections of mscs transfected with rad/il-12m inhibited b16f10 primary and metastatic tumors more effectively than i.t. injections of rad/il-12m alone (318) . despite the homing feature tsa mammary carcinoma cells transduced to secrete il-12 were found to cure 10 of 40 mice bearing 1-day established tsa tumors following local injection (294) . when tumors were allowed to establish for seven days prior to treatment, antitumor efficacy dropped, with only 2 of 40 mice experiencing tumor regression. local delivery of 9l gliosarcoma cells engineered to express il-12 (9l-il12) significantly prolonged the survival of rats challenged intracranially with wild-type 9l tumors (295) . similar results were observed whether the 9l-il12 treatment was given at the same time or 5 days later than the tumor inoculation. the authors noted that only mice receiving the delayed treatment were protected from tumor rechallenge (295) . natural killer (nk) cells and chimeric antigen receptor (car) t cells have also been transduced to produce il-12. in one recent study, primary nk cells isolated from c57bl/6 mice and transduced to express il-12 (nk il−12 ) increased mhc class i expression in ova-transfected melanoma cells (b16m05) compared to mock plasmid transduced nk cells (319) . combination treatment studies involving tumor-specific cytotoxic t lymphocytes (ctls), pegylated il-2, and activated nk il−12 revealed that only groups containing activated nk il−12 demonstrated statistically significant prolonged survival over mock constructs, untreated, and ctl alone controls in b16-ova tumor bearing mouse models. in particular, nk il−12 plus il-2 treatment increased survival by 11.5 days, while nk il−12 plus ctl and il-2 prolonged survival for 16.5 days. nevertheless, all mice eventually succumbed to the disease and no stable tumor regressions were reported (319) . car t cells have shown remarkable efficacy against hematologic malignancies but have yet to achieve clinical benefit against solid tumors. engineering car-t cells to express il-12 is under investigation to improve antitumor efficacy against solid tumors. in one study, i.v. infusion of il-12 expressing anti-vegfr2 car t cells induced curative regressions in 40-80% of mice in five different tumor models including 10-12-day-old b16f10 melanomas, mca-205 sarcomas, mc38 colorectal adenocarcinomas, mc17-51 sarcomas or 12-14 days old ct26 colon adenocarcinomas (320) . t cells singularly transduced with either anti-vegfr-2 or il-12 were markedly less effective (320) . because constitutive systemic expression of il-12 can be toxic, inducible il-12 expression strategies have been developed. by using an nfat-responsive promoter, il-12 could be expressed only after tcr engagement (320) (321) (322) . this modification reduced toxicity but required lymphodepletion to achieve durable tumor regressions (320) . the efficacy of car t cell therapy in general is dependent upon lymphodepletion via chemotherapy or radiotherapy. in an attempt to eliminate the need for lymphodepleting preconditioning, cd19 car t cells expressing il-12 (cd19/il-12) have been investigated (323, 324) . indeed, treatment with cd19/il-12 car t cells significantly enhanced the survival of cd19-el4 tumor-bearing mice vs. treatment with cd19 car t cells (75 vs . 0% survival at day 60 after tumor inoculation) (323) . similarly, second generation cd19/il-12 car t cells produced a 25% long-term survival rate in mice with established a20 b cell lymphomas. without the il-12 insert, all mice treated with cd19 car t cells succumbed to disease (324) . 4h11-28z/il-12 car t cells, which are specific for the muc-16 ecto antigen and secrete il-12, controlled skov3 human ovarian carcinomas in scid-beige mice following i.p. infusion 2 or 4 weeks after tumor implantation (325) . specifically, ∼80 or 100% of mice treated 4 or 2 weeks, respectively, after tumor implantation with 4h11-28z/il-12 car t cells survived for 90 days. in contrast, ∼0% and 20% of mice treated 4 or 2 weeks, respectively, after tumor implantation with 4h11-28z car t cells, not containing the il-12 gene, survived for 90 days (325). glypican-3 (gpc3)-specific car t cells with inducible expression of il-12 (gpc3-28z-nfat-il-12 car t) significantly inhibited or eliminated established plc/prf/5 and huh-7 human hccs (326) . the il-12 secreting car t cells resulted in decreased t reg infiltration. serum ifnγ and il-12 levels were elevated although neither pathological changes to critical organs nor significant loss in body weight were observed (326) . it should be noted, however, that these studies were performed in immunocompromised mice which is not a reliable model for immune-related adverse events. from a clinical standpoint, administrations of il-12-transduced cells have been well-tolerated, with patients experiencing only transient symptoms of lymphopenia, fever, and malaise; however, significant, durable clinical responses have been elusive. from a manufacturing perspective, the transduction of allogeneic and xenogeneic cells is easier and more reproducible. however, these cells are rapidly recognized and rejected by the host immune system. thankfully, recent advances in autologous cell transduction due to the emergence of car t cell immunotherapy has eliminated many concerns related to technical feasibility and patient heterogeneity (310) . local administration of recombinant il-12 protein is the most direct and most quantifiable strategy in terms of ensuring the accuracy and reproducibility of a delivered dose. however, recombinant cytokines disseminate rapidly from local injection sites into the systemic circulation (327) . thus, in order to maintain high levels of recombinant il-12 in the tumor microenvironment while minimizing systemic exposure, sustained delivery technologies, capable of locally releasing proteins and cytokines over extended periods of time following direct injection, are under development (328) . the encapsulation of pharmaceuticals in polymeric microspheres for sustained delivery has been explored for several decades (329, 330) . the use of microspheres to deliver cytokines is a more recent trend. synthetic polymers, such as poly-lactic coglycolic acid (plga), poly-caprolactone (pcl), and poly-lactic acid (pla), that have been widely explored for drug delivery, are being adapted to encapsulate and deliver various cytokines including il-12 (55, (331) (332) (333) (334) (335) . human il-12-loaded pcl:pla microspheres, when co-delivered with human peripheral blood lymphocytes (pbls), were shown to prevent engraftment of human tumors in scid mice (333) . peg-il-2 also suppressed tumor engraftment and growth, but not as effectively as il-12-loaded microspheres. although high levels of il-12 and ifn-γ were found in sera after administration of il-12-loaded microspheres, this localized delivery strategy was more effective at preventing tumor engraftment than repeated i.p. injections of il-12 (336) . similarly, a single i.t. injection of il-12-loaded pcl:pla microspheres outperformed bolus injections of il-12 in the inhibition of human head and neck tumor tissue xenografts containing human pbls (337) . il-12-loaded microspheres completely suppressed engraftment in 50% of mice, whereas il-12 alone slowed but could not eliminate tumor growth (337). in the above xenograft studies, antitumor activity was mediated by cd8 + t cells and cd56 + natural killer cells (333, 337) . in yet another study, lung tumor xenografts using non-disrupted human lung tumor tissue were completely suppressed when injected with il-12-loaded pla microspheres 1 week after implantation (338) . mechanistically, quiescent cd4 + effector memory t cells present within the tumor microenvironment were reactivated upon exposure to high local levels of il-12 (339) . the proliferation of reactivated cd4 + cells and their production of ifn-γ facilitated tumor destruction (339) . in murine tumor models, i.t. injections of il-12-loaded pla microspheres eliminated up to 70% of line-1 lung alveolar carcinomas (332) . in contrast, i.t. injections of free il-12 induced a complete response in only one of five treated mice. interestingly, mice experiencing complete tumor regression following microsphere administration were more resistant to tumor rechallenge than mice vaccinated with either live or irradiated line-1 cells co-injected with il-12-loaded microspheres (332) . in other studies, i.t. injections of il-12loaded pla microspheres significantly reduced the incidence and number of spontaneous pulmonary tumor nodules (331, 340) . when administered neoadjuvant to tumor resection, il-12-loaded microspheres inhibited local recurrence, reduced pulmonary metastases, and improved disease-free survival as compared to resection alone (331) . a similar study using more advanced line-1 tumors, which were 1,000-1,300 mm 3 at study onset, demonstrated that neoadjuvant i.t. injections of il-12-loaded microspheres prevented both local recurrence and pulmonary metastases better than surgery alone (341) . the addition of gm-csf-loaded pla microspheres enhanced antitumor activity and survival. however, more cytokines were not always better, as the addition of low dose il-2 to neoadjuvant immunotherapy with il-12-and gm-csf-loaded microspheres abrogated antitumor immunity (341) . a theme common to nearly all il-12-based immunotherapies was that il-12-loaded microspheres induced cd8 + t cell and nk cell activation, including increases in cytolytic function and ifn-γ expression. in-depth effector mechanisms are elegantly described in a series of papers by egilmez and kilinc et al. (69, 340, (342) (343) (344) (345) . in addition to inducing a strong effector response, a potentially more important feature of i.t. injected il-12-loaded microspheres is their potential to reverse immunosuppression in the tumor microenvironment. for example, a single i.t. injection of il-12-loaded microspheres with and without gm-csf-loaded microspheres induced apoptosis and elimination of tumor-infiltrating cd4 + cd25 + foxp3 + t suppressor cells (69, 344) . il-12-loaded microspheres were also shown to reprogram immunosuppressive tumor-infiltrating macrophages (tims) and tumor-associated macrophages (tams) to a proinflammatory, antitumor phenotype (11, 346) . microsphere platforms are well-suited to explore codelivery of multiple cytokines. the combination of il-12-and tnfα-loaded pla microspheres was shown to be more effective than il-12 and gm-csf-loaded microspheres at eliminating mt-901 mammary carcinomas (347) and b16 melanomas (55) . nearly 70 and 40% of mt-901 tumor-bearing mice were rendered tumor-free following i.t. injections with dual cytokine loaded microspheres of il-12/tnfα and il-12/gm-csf, respectively (347) . dual cytokine loaded microspheres of il-12/tnfα also induced increases in tumor-specific cytokine release from effector cells as compared to microspheres containing either cytokine alone (334) or the combination of il-12 and gm-csf (55, 347) . a single i.t. injection of il-12-and il-18-loaded pla microspheres outperformed injections of either cytokine-loaded microsphere alone in terms of delaying the growth of b16 melanomas (348) . unlike previous studies demonstrating the elimination of 70% of line-1 tumors (332) and impressive reductions in metastatic lesions (331, 341) , il-12-loaded microspheres had no impact on either primary b16 tumor growth or pulmonary metastases (348) . interestingly, the triple combination of il-18/il-12/tnfα loaded in microspheres was found to reduce antitumor activity in the 4t1 model (335) . in her-2/neu transgenic mice, which develop spontaneous, multifocal mammary carcinomas, il-12-and gm-csf-loaded microspheres injected intratumorally caused complete regression of primary tumors in up to 40% of mice (349) . unfortunately, responses were temporary, and all tumors recurred. interestingly, chronic immunotherapy with cytokine-loaded microspheres could not control tumor burden due to a progressive decline in the intensity of antitumor t cells and an increase in tumor-infiltrating cd4 + cd25 + foxp3 + t suppressor cells with repeated injections (349, 350) . subsequent studies have shown that administration of cyclophosphamide can block the postimmunotherapy increase in t suppressor cells (351, 352) . recently, intratumoral delivery of il-12 microspheres (il-12 ms) in combination with stereotactic body radiation (sbrt) resulted in remarkable tumor regression in several types of preclinical pancreatic ductal adenocarcinoma mouse models (353). this study showed that intratumoral levels of ifnγ were enhanced following the treatment of sbrt/il-12 ms, which in turn increased cd8 + t cell activation. sbrt/il-12 ms treatments also established systemic tumor immunity that was confirmed by antitumor effects on liver metastases (353). chitosan (cs) is a bioadhesive polysaccharide derived primarily from the exoskeletons of crustaceans. it is nontoxic, biodegradable, and non-immunoreactive (354) with an established safety profile in humans. co-formulation of il-12 in cs (cs/il-12) has been shown to increase the i.t. residence of il-12 from 1-2 days to 1-2 weeks (23) . cs is believed to hinder il-12 diffusion through viscous and electrostatic interactions. the sustained presence of cs/il-12 induced complete regression of 80-100% of mice bearing mc38, panc02, ct26, e0771, and mb49 tumors (23, 355) . administration of cs/il-12 neoadjuvant to 4t1 tumor resection resulted in complete clearance of lung metastases and long-term survival in about two-thirds of mice. in contrast, all mice treated with surgery alone died within 38 days and mice receiving il-12 without cs neoadjuvant to resection had a median survival of 46 days (52). cured mice rejected a subsequent challenge with live 4t1 cells, but not live renca cells illustrating the specificity of the antitumor immune response. in another demonstration of systemic tumorspecific immunity from localized il-12, 70% of mice bearing both flank and orthotopic mb49 bladder tumors were able to clear the flank tumor following intravesical administration of cs/il-12 (53) . in contrast, mice with only a flank tumor, did not benefit from intravesical cs/il-12 in a naïve bladder. these data indicate that intravesical cs/il-12 induced systemic tumor-specific immunity only when il-12 was localized in or near a tumor. in a subsequent study, orthotopic bladder tumor regression following intravesical cs/il-12 immunotherapy was associated with a rapid infiltration of macrophages and granulocytes followed by increases in cd4 + and cd8 + effector t cells along with a reduction of immunosuppressive t regs and mdscs (14) . importantly, local administrations of cs/il-12 did not result in significant toxicity, such as elevated liver enzymes, commonly observed following regular, free il-12 injections (52) . a polysaccharide gel matrix, designated f2 gel, is comprised of 70% deacetylated poly-n-acetyl glucosamine (p-glcnac) coformulated with lactate salt. p-glcnac gels were evaluated as slow-release cytokine depots for gm-csf, il-2, and il-12 (356) (357) (358) (359) (360) . in particular, i.t. injections of il-2 in p-glcnac gel delayed the growth of ova-transfected ae17 malignant mesotheliomas (359) . interestingly, gm-csf and il-12 depots had no impact on mesothelioma growth, although only a single i.t. injection was given. the p-glcnac gel by itself induced a strong, transient inflammatory response which was thought to be due to a foreign body reaction or toxic species that may have been contained within the gel (361, 362) . human il-12 has been encapsulated in multilamellar liposomes for sustained release after i.t. injection (363) . antitumor activity on xenografts of human lung tissues indicated that liposomal encapsulation is a promising approach capable of eliminating tumor cells and inducing lymphocyte infiltration 2 weeks after i.t. injection. liposomal leakage and/or il-12 liposome dissemination could be problematic following delivery of large doses (10 µg/mouse) as significant sustained levels of il-12 and ifn-γ were found in sera of treated mice (363) . capitalizing on the tumor disruption caused by cationic liposomes, il-12-and tlr4 ligand monophosphoryl lipid a (mpl)-loaded cationic liposomes were injected i.t. to treat 4t1 tumors. this combination produced an abscopal effect which arrested growth in both treated and distal tumors but did not result in tumor elimination (364) . to date, sustained release technologies for recombinant il-12 have not been evaluated in clinical studies. while sustained release technologies are designed to maximize il-12 concentrations in the tumor microenvironment, some amount of delivered il-12 can be expected to reach the systemic circulation. however, due to the infrequency of administration for these long-acting platforms, they are unlikely to induce the same lethal toxicities that were observed following the frequent systemic il-12 administrations in early clinical trials (42) . nevertheless, it will be essential to perform rigorous pharmacokinetic and toxicology studies prior to clinical translation of any sustained recombinant il-12 platform. a possible limitation of the microsphere encapsulation technology is the use of organic solvents during the encapsulation process. organic solvents can denature cytokines immediately or adversely affect long term storage. one study indicated that cytokines lose about half of their bioactivity during microencapsulation (365) . loss of bioactivity appears to be cytokine dependent (366) . specific activity of il-12 after encapsulation in pla microspheres was 90%. however, more than 80% of the bioactivity of il-12 was lost when pla/il-12 microspheres were stored for 8 days (366) . in addition, because many commonly used biodegradable polymers are comprise of acidic monomers, polymer degradation often forms highly acidic microenvironments that may inactivate cytokines (188) . a number of techniques are under investigation to either raise ph (367) or stabilize encapsulated cytokines (368) . a common limitation of liposomal carriers is their inherent leakiness, and therefore the choice of lipid and modifications to stabilize the bilayer are critical for sustained release. high temperature phase-transition lipids, such as dspc, increase the retention of cargo and enhance drug circulation time. incorporation of cholesterol into the lipid bilayer increases the rigidity and further decreases leakage of interior cargo (369) . another major limitation of liposomes is opsonization by serum proteins and clearance by phagocytes of the reticuloendothelial system (res). pegylation can reduced res clearance, however, peg can sterically inhibit ligand-cell interaction (370) . if administered i.t., loss of tumor targeting is likely not detrimental. while some localized il-12 immunotherapies have shown limited efficacy in clinical studies, a long-awaited breakthrough leading to widespread application of localized il-12 as a monotherapy does not appear imminent. however, there are a number of near-term opportunities to combine localized il-12 with traditional or experimental cancer management approaches. and in fact, most contemporary experimental therapies for aggressive or advanced cancers involve combination approaches. thus, near future opportunities involving translatable combination approaches are explored in the following sections. the majority of cancer patients with solid tumors undergo tumor resection as part of their treatment. surgery, by itself, is unable to prevent recurrence and/or metastasis which is responsible for 9 out of 10 cancer-related deaths. patients at high risk of recurrence receive adjuvant chemotherapy and/or radiotherapy following resection. while chemotherapy and radiotherapy may help reduce the risk of recurrence, they induce life-altering side effects and fail in a significant fraction of patients. as a result, most metastatic cancers arise from previously treated non-metastatic disease (371) (372) (373) . administration of localized il-12 to the tumor microenvironment prior to surgery has the potential to reduce recurrence rates and/or eliminate occult metastases through the induction of systemic, tumor-specific immunity. as mentioned above, our own investigation demonstrated that neoadjuvant i.t. injections of cs/il-12 prior to resection can improve overall survival from 0 to 65% in a highly metastatic 4t1 breast carcinoma model (52) . cured mice demonstrated tumor-specific immunity via tumor rechallenge, ctl activity, and elispot assays. if proven to be safe, localized il-12 can be administered neoadjuvant to any solid tumor resection, not just in high risk patients. in fact, at the time of surgery, it is likely that a significant fraction of patients already has occult metastases that cannot be detected via radiographic imaging. the median primary breast tumor size for which 100% of patients had micrometastases was found to be only 1.8 cm (374) . while most immune-oncology trials focus on developing therapies for metastatic disease, an approach, such as neoadjuvant localized il-12, capable of preventing metastasis in the first place, may be a more effective strategy to reducing cancer mortality. the combination of il-12-based immunotherapy and chemotherapy ("biochemotherapy") is also under exploration. several clinical studies have shown significant clinical benefit when certain cytotoxic drugs are combined with il-12 (181, 375, 376) . there are three distinct advantages for combining chemotherapy with il-12 immunotherapy. first, chemotherapy provides a debulking benefit with the elimination of some or most of the tumor. second, some chemotherapeutics can deplete immunosuppressive cells. cyclophosphamide has been shown to deplete t regs while 5-fluorouricil and gemcitabine have been shown to deplete mdscs. third, some chemotherapies cause upregulation of t cell-attracting chemokines in the tumor (377) . for example, melanoma-bearing mice treated with temozolomide, cisplatin, or dacarbazine increase expression of ccl5, cxcl9, and cxcl10 (378) . the timing of chemotherapy relative to local il-12 immunotherapy is expected to be critical. unlike the neoadjuvant resection approach mentioned above, chemotherapy should be administered prior to il-12 therapy to avoid elimination of beneficial proliferating immune cells. chemotherapy may synergize with il-12 by reducing tumor burden and creating a source of tumor antigen for in situ vaccination. in fact, certain chemotherapies can induce an immunogenic cell death that is capable of priming an antitumor immune response (379) . to date, several examples demonstrate the promise of chemotherapy plus local il-12 delivery. antitumor efficacy of pil-12/ppc is improved with paclitaxel, cyclophosphamide, or taxol/paraplatin (172, 175, 177) . the antitumor effect of il-12 lipoplexes against intracranial glioma was substantially improved when combined with fda-approved carmustine (bcnu) wafers (176) . finally, a phase-1 clinical trial found that intraperitoneal il-12 gene therapy in combination with intravenous pegylated liposomal doxorubicin resulted in greater clinical benefit relative to the chemotherapeutic alone (57.1% vs. 40-45.3%) in patients (n = 11) with platinum-resistant epithelial ovarian cancer (181) . standard-of-care radiotherapy of inoperable tumors can be administered prior to local il-12 delivery. radiation-induced tumor cell death can supply a source of tumor antigens, while radiation has been shown to upregulate co-stimulatory molecules on the surface of tumor cells which makes them more susceptible to immune-mediated killing (380) . the addition of tumor irradiation after pil-12+ep was found to improve antitumor activity (130) . similarly, radiotherapy improved the antitumor activity of i.t. oncolytic adenoviruses expressing il-12 and gm-csf (381) . it has also been found that radiotherapy can induce infiltration of mscs in the tumor site. through a combination of radiation and il-12-expressing mscs, both primary tumor growth and metastases were reduced in hca-i and hepa 1-6 hepatoma models (382) . in another study, radiation along with an i.t. injection of an il-12-encoding adenoviral vector greatly reduced tumor growth and metastasis compared to either treatment alone in a bnl-p2 hepatocellular carcinoma model, while a near-complete abscopal response occurred in a ct26 colorectal cancer model (383) . an abscopal response was also induced in a fully humanized rhabdomyosarcoma xenograft model. mice with bilateral tumors were treated with a combination of single-tumor radiation and systemic nhs-il12, which resulted in significantly lower tumor burdens than either individual treatment (114) . cryoablation, radiofrequency ablation (rfa), microwave ablation (mwa), embolic microspheres and high intensity focused ultrasound (hifu) are routinely used in the clinic to destroy some operable and many inoperable solid tumors. irreversible electroporation is another ablative technology in clinical and preclinical development (384) (385) (386) (387) . all of these strategies have the potential to induce in situ vaccinations through the release of tumor antigen, although abscopal responses or robust anti-tumor immune responses are rare. among the ablation strategies, cryoablation appears distinct from most other ablation approaches that kill via high temperatures and result in coagulation, enzyme inactivation, and antigen denaturation. cryoablation is thought to mostly preserve tumor antigen structure (388, 389) and has been shown to induce greater levels of tumor antigen uptake by dendritic cells compared to rfa (390) . in addition, cryoablation also induces higher levels of inflammatory cytokines, such as il 1, il 6, and tnfα, compared to rfa and mwa (391, 392) . perhaps most importantly, cryoablation was recently shown to induce expansion of oligoclonal t cells both in tumor tissues and in peripheral blood of kidney cancer patients (393) . the addition of localized il-12 to the above ablation strategies may enhance tumor control as well as antitumor immunity. our recent preclinical data, demonstrated that the combination of cryoablation and cs/il-12 could eliminate multiple tumor types and induce abscopal immunity in a bilateral flank model (394) . ongoing studies are aimed at determining mechanisms of systemic tumor control. immune checkpoint inhibitors are ineffective against noninflamed, "cold" tumors. localized il-12 induces profound intratumoral infiltration that can be expected to synergize with checkpoint inhibitors. indeed, the addition of anti-pd-1 or anti-ctla-4 improved the antitumor activity of l19-il-12 in multiple murine tumor models (395) . similarly, systemic anti-ctla-4 in combination with intratumoral il-12 induced synergistic antitumor responses in gl-26 glioblastoma and sma-560 astrocytoma models (396) . also, as mentioned in a previous section, i.t. injections of vv-il-12 and vv-il7 enhanced the responsiveness of poorly immunogenic tumors to checkpoint inhibition (268) . finally, a recent phase ii study of i.t. pil-12+ep and systemic pembrolizumab in patients with non-infiltrated melanomas resulted in a 41% objective response rate with 36% complete responses (397) . correlative studies demonstrated increased antigen cross-presentation along with enhance t cell infiltration and activity leading to higher than expected clinical responses (397) . il-12 enhances cytotoxic t and nk cell activity while reversing tumor-induced immunosuppression, inhibiting angiogenesis, increasing lymphocyte trafficking and antigen presentation either directly or through induction of ifnγ (398) . based on these diverse mechanisms as well as its profound antitumor activity in numerous preclinical studies, il-12 is arguably one of, if not, the most potent antitumor cytokine evaluated to date. unfortunately, the much-anticipated clinical translation of il-12-based immunotherapies suffered a tremendous setback in the late 1990s/early 2000s due to severe clinical toxicities associated with systemic il-12 injections. through the development of several clever approaches to localize il-12 to the tumor microenvironment while limiting systemic exposure, il-12-based immunotherapies are making a comeback. several clinical studies evaluating localized il-12 have been initiated ( table 2 ) with more on the way. while each delivery strategy has limitations, the approaches reviewed above may retain enough il-12 in the tumor while eliminating the need for frequent systemic injections. by reducing the potential for il-12-mediated toxicities associated with systemic injections, localized il-12 can expand the therapeutic window and finally allow il-12 to fulfill its considerable potential. as exemplified by the current immune-oncology clinical trial landscape, combination approaches appear to be most effective for accelerating clinical impact. several promising preclinical combination approaches with localized il-12 are likely to be translated in the near future. there is unprecedented interest in finding immunomodulators that can enhance lymphocytic infiltration in order to improve the efficacy of checkpoint inhibitors. localized il-12, based on its ability to drive th1 responses, enhance cytolytic activity, and protect t cells from pd-1/pd-l1 exhaustion and ifnγ-induced apoptosis may be an ideal partner for checkpoint inhibitors. if safety concerns are assuaged, localized il-12 could be used in earlier stage cancer patients as a neoadjuvant to resection. for tumors that are inoperable, combining localized il-12 with ablation may help increase local as well as distant tumor control. with the interesting combination approaches, as well as the uptick in il-12-based immunotherapies in clinical trials, there is reason to believe that localized il-12 may play a major role in cancer immunotherapy in the near future. studies on the pathogenesis of fever. ii. characterization of fever-producing substances from polymorphonuclear leukocytes and from the fluid of sterile exudates anticancer cytokines: biology and clinical effects of interferon-alpha2, interleukin (il)-2, il-15, il-21, and il-12 melanoma: therapeutic options with recombinant interferons results of treatment of 255 patients with metastatic renal cell carcinoma who received high-dose recombinant interleukin-2 therapy high-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and (1993) talimogene laherparepvec improves durable response rate in patients with advanced melanoma coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor identification and purification of natural killer cell stimulatory factor (nksf), a cytokine with multiple biologic effects on human lymphocytes cloning of cdna for natural killer cell stimulatory factor, a heterodimeric cytokine with multiple biologic effects on t and natural killer cells cloning and expression of murine il-12 il-12 rapidly alters the functional profile of tumor-associated and tumor-infiltrating macrophages in vitro and in vivo the role of interleukin-12 on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis interleukin-12: a master regulator immunological mechanisms of intravesical chitosan/interleukin-12 immunotherapy against murine bladder cancer. oncoimmunology differential effects of il-12 on tregs and non-treg t cells: roles of ifn-gamma, il-2 and il-2r il-12 triggers a programmatic change in dysfunctional myeloidderived cells within mouse tumors antiproliferative effects of four different cytokines on renal carcinoma cell lines ifn-gamma induces apoptosis in ovarian cancer cells in vivo and in vitro inhibition of angiogenesis in vivo by interleukin 12 inhibition of tumor-induced angiogenesis by the administration of recombinant interferon-gamma followed by a synthetic lipid-a subunit analogue (gla-60) cellular responses to interferon-gamma interleukin-12 in anti-tumor immunity and immunotherapy intratumoral immunotherapy of established solid tumors with chitosan/il-12 effect of interleukin 12 on tumor induction by 3-methylcholanthrene therapeutic effect of interleukin 12 on mouse haemangiosarcomas is not associated with an increased antitumour cytotoxic t-lymphocyte activity the anti-tumor activity of il-12: mechanisms of innate immunity that are model and dose dependent antitumor and antimetastatic activity of interleukin 12 against murine tumors phase i evaluation of intravenous recombinant human interleukin 12 in patients with advanced malignancies pilot study of subcutaneous recombinant human interleukin 12 in metastatic melanoma phase i trial of subcutaneous recombinant human interleukin-12 in patients with advanced renal cell carcinoma interleukin-12 therapy of cutaneous t-cell lymphoma induces lesion regression and cytotoxic t-cell responses phase i study of intraperitoneal recombinant human interleukin 12 in patients with mullerian carcinoma, gastrointestinal primary malignancies, and mesothelioma phase 1 study of the intravesical administration of recombinant human interleukin-12 in patients with recurrent superficial transitional cell carcinoma of the bladder a phase ii trial of interleukin-12 in patients with advanced cervical cancer: clinical and immunologic correlates. eastern cooperative oncology group study e1e96 nk and cd8+ t cell-mediated eradication of poorly immunogenic b16-f10 melanoma by the combined action of il-12 gene therapy and 4-1bb costimulation intratumoral recombinant human interleukin-12 administration in head and neck squamous cell carcinoma patients modifies locoregional lymph node architecture and induces natural killer cell infiltration in the primary tumor activity of subcutaneous interleukin-12 in aids-related kaposi sarcoma phase ii clinical trial of interleukin-12 in patients with relapsed and refractory non-hodgkin's lymphoma and hodgkin's disease peripheral burst of tumor-specific cytotoxic t lymphocytes and infiltration of metastatic lesions by memory cd8+ t cells in melanoma patients receiving interleukin 12 after initial setback, il-12 regaining popularity interleukin 12: still a promising candidate for tumor immunotherapy? effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production evaluation of recombinant human interleukin-12 in patients with recurrent or refractory ovarian cancer: a gynecologic oncology group study randomized multicenter phase ii trial of subcutaneous recombinant human interleukin-12 versus interferon-alpha 2a for patients with advanced renal cell carcinoma selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal patterns of vasculature in mouse models of lung cancer are dependent on location direct intratumoral injection of an adenovirus expressing interleukin-12 induces regression and long-lasting immunity that is associated with highly localized expression of interleukin-12 co-delivery of antigen and il-12 by venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects right time and place for il12: targeted delivery stimulates immune therapy new insights into il-12-mediated tumor suppression gene therapy of cancer based on interleukin 12 neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis intravesical chitosan/interleukin-12 immunotherapy induces tumorspecific systemic immunity against murine bladder cancer characterization of abscopal effects of intratumoral electroporation-mediated il-12 gene therapy neoadjuvant intratumoral cytokine-loaded microspheres are superior to postoperative autologous cellular vaccines in generating systemic anti-tumor immunity repeated administrations of interleukin (il)-12 are associated with persistently elevated plasma levels of il-10 and declining ifn-gamma, tumor necrosis factor-alpha, il-6, and il-8 responses transcriptionally active stat1 is required for the antiproliferative effects of both interferon alpha and interferon gamma ifn-gamma regulates donor cd8 t cell expansion, migration, and leads to apoptosis of cells of a solid tumor ifn-gamma-mediated upmodulation of mhc class i expression activates tumor-specific immune response in a mouse model of prostate cancer ifn-gamma-mediated inhibition of tumor angiogenesis by natural killer t-cell ligand, alpha-galactosylceramide interferon: a cytotoxic t lymphocyte differentiation signal il-2 and ifn-gamma are two necessary lymphokines in the development of cytolytic t cells gamma interferon enhances macrophage transcription of the tumor necrosis factor/cachectin, interleukin 1, and urokinase genes, which are controlled by short-lived repressors interferon and major histocompatibility complex genes: a model to analyse eukaryotic gene regulation? placebo-controlled phase iii trial of patient-specific immunotherapy with mitumprotimut-t and granulocyte-macrophage colony-stimulating factor after rituximab in patients with follicular lymphoma evaluation of ipilimumab in combination with allogeneic pancreatic tumor cells transfected with a gm-csf gene in previously treated pancreatic cancer cancer immunotherapy comes of age cancer immunotherapy strategies based on overcoming barriers within the tumor microenvironment reversing tumor immune suppression with intratumoral il-12: activation of tumor-associated t effector/memory cells, induction of t suppressor apoptosis, and infiltration of cd8+ t effectors use of tumor-infiltrating lymphocytes and interleukin-2 in the immunotherapy of patients with metastatic melanoma. a preliminary report il-12 priming during in vitro antigenic stimulation changes properties of cd8 t cells and increases generation of effector and memory cells synergy of brief activation of cd8 t-cells in the presence of il-12 and adoptive transfer into lymphopenic hosts promotes tumor clearance and anti-tumor memory ex vivo interleukin-12-priming during cd8 + t cell activation dramatically improves adoptive t cell transfer antitumor efficacy in a lymphodepleted host ex vivo conditioning with il-12 protects tumor-infiltrating cd8 + t cells from negative regulation by local ifn-gamma cutting edge: il-12 and type i ifn differentially program cd8 t cells for programmed death 1 re-expression levels and tumor control switching on of the proliferation or apoptosis of activated human t lymphocytes by ifn-gamma is correlated with the differential expression of the alpha-and beta-chains of its receptor clinical trial listed in this table meet the following four criteria: 1) involve a delivery technology; 2) be administered locally, i.e. intratumoral or peritumoral; 3) use il-12 as an effector and 4) demonstrate antitumor activity of il-12. clinical trials listed in this table include: 1) nhs-il12 for solid tumors 14) a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar441000 as monotherapy and in combination with cemiplimab in patients with advanced solid tumors antibody-il-12 fusion proteins are effective in scid mouse models of prostate and colon carcinoma metastases bifunctional cytokine fusion proteins for gene therapy and antibody-targeted treatment of cancer a single-chain il-12 igg3 antibody fusion protein retains antibody specificity and il-12 bioactivity and demonstrates antitumor activity mechanism of antitumor activity of a single-chain interleukin-12 igg3 antibody fusion protein (mscil-12.her2.igg3) cytokines fused to antibodies and their combinations as therapeutic agents against different peritoneal her2/neu expressing tumors long-term immunity elicited by antibody-cytokine fusion proteins protects against sequential challenge with murine mammary and colon malignancies amino acid residues involved in the heparin-binding activity of murine il-12 in the context of an antibody-cytokine fusion protein modulation of interleukin-12 activity in the presence of heparin molecular mechanisms of heparin-induced modulation of human interleukin 12 bioactivity species-specific differences in heparin-induced modulation of il-12 family cytokines efficient production and purification of recombinant human interleukin-12 (il-12) overexpressed in mammalian cells without affinity tag novel immunocytokine il12-ss1 (fv) inhibits mesothelioma tumor growth in nude mice prognostic value and characterization of the ovarian cancer-specific antigen ca166-9 construction, expression, and function of 6b11scfv-mil-12, a fusion protein that attacks human ovarian carcinoma an il12-il2-antibody fusion protein targeting hodgkin's lymphoma cells potentiates activation of nk and t cells for an anti-tumor attack expression of the extra domain b of fibronectin, a marker of angiogenesis, in head and neck tumors hubc1-il12, an immunocytokine which targets edb-containing oncofetal fibronectin in tumors and tumor vasculature, shows potent anti-tumor activity in human tumor models a phase 1 study of as1409, a novel antibody-cytokine fusion protein, in patients with malignant melanoma or renal cell carcinoma phase i trial of twice-weekly intravenous interleukin 12 in patients with metastatic renal cell cancer or malignant melanoma: ability to maintain ifn-gamma induction is associated with clinical response an engineered antibody-interleukin-12 fusion protein with enhanced tumor vascular targeting properties enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix engineered vascular-targeting antibody-interferon-gamma fusion protein for cancer therapy enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature synergistic therapeutic effects of a tumor targeting antibody fragment, fused to interleukin 12 and to tumor necrosis factor alpha anchoring of intratumorally administered cytokines to collagen safely potentiates systemic cancer immunotherapy collagen-binding il-12 enhances tumour inflammation and drives the complete remission of established immunologically cold mouse tumours a dose-escalation and signal-generating study of the immunocytokine l19-il2 in combination with dacarbazine for the therapy of patients with metastatic melanoma targeted reconstitution of cytokine activity upon antigen binding using split cytokine antibody fusion proteins a new chemically modified chimeric tnt-3 monoclonal antibody directed against dna for the radioimmunotherapy of solid tumors the immunocytokine nhs-il12 as a potential cancer therapeutic characterization of a phage display-derived human monoclonal antibody (nhs76) counterpart to chimeric tnt-1 directed against necrotic regions of solid tumors temporal changes within the (bladder) tumor microenvironment that accompany the therapeutic effects of the immunocytokine nhs-il12 enhanced antitumor effects by combining an il-12/anti-dna fusion protein with avelumab, an anti-pd-l1 antibody combination therapy with nhs-muil12 and avelumab (anti-pd-l1) enhances antitumor efficacy in preclinical cancer models cancer-targeted il-12 controls human rhabdomyosarcoma by senescence induction and myogenic differentiation tumortargeted il-12 combined with local irradiation leads to systemic tumor control via abscopal effects in vivo. oncoimmunology enhanced binding of necrosis-targeting immunocytokine nhs-il12 after local tumour irradiation in murine xenograft models defining the pharmacodynamic profile and therapeutic index of nhs-il12 immunocytokine in dogs with malignant melanoma first-in-human phase i trial of a tumor-targeted cytokine (nhs-il12) in subjects with metastatic solid tumors immunogenicity to biotherapeutics -the role of anti-drug immune complexes tumor regression of human and murine melanoma after intratumoral injection of il-12-encoding plasmid dna in mice in vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid dna coding for equine interleukin 12 intratumoral injection of dna encoding human interleukin 12 into patients with metastatic melanoma: clinical efficacy intratumoral injection of il-12 plasmid dna-results of a phase i/ib clinical trial effective tumor therapy with plasmid-encoded cytokines combined with in vivo electroporation il-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous b16.f10 melanoma il-12 gene therapy using an electrically mediated nonviral approach reduces metastatic growth of melanoma evaluation of toxicity following electrically mediated interleukin-12 gene delivery in a b16 mouse melanoma model regression of tumor growth and induction of long-term antitumor memory by interleukin 12 electro-gene therapy administration route-and immune cell activation-dependent tumor eradication by il12 electrotransfer intratumoral delivery of interleukin 12 expression plasmids with in vivo electroporation is effective for colon and renal cancer radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma in vivo electrogene transfer of interleukin-12 inhibits tumor growth and lymph node and lung metastases in mouse mammary carcinomas local and systemic antitumor effect of intratumoral and peritumoral il-12 electrogene therapy on murine sarcoma electroporation-mediated interleukin-12 gene therapy for hepatocellular carcinoma in the mice model improving therapeutic efficacy of il-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters systemic administration of interleukin-12 can restore the antitumor potential of b16 melanoma-draining lymph node cells impaired at a late tumor-bearing state intratumoral electroporation of il-12 cdna eradicates established melanomas by trp2(180-188)-specific cd8+ ctls in a perforin/granzyme-mediated and ifn-gamma-dependent manner: application of trp2(180-188) peptides gene electrotransfer of plasmid-encoding il-12 recruits the m1 macrophages and antigen-presenting cells inducing the eradication of aggressive b16f10 murine melanoma il-12 gene electrotransfer triggers a change in immune response within mouse tumors pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma in vivo electroporation-mediated transfer of interleukin-12 and interleukin-18 genes induces significant antitumor effects against melanoma in mice combination of il-12 and il-18 of electro-gene therapy synergistically inhibits tumor growth coadministration of interleukin-18 and interleukin-12 induces a fatal inflammatory response in mice: critical role of natural killer cell interferongamma production and stat-mediated signal transduction electrogene therapy with interleukin-12 in canine mast cell tumors electroporation-mediated il-12 gene therapy in a transplantable canine cancer model a combination of electrochemotherapy, gene electrotransfer of plasmid encoding canine il-12 and cytoreductive surgery in the treatment of canine oral malignant melanoma intratumoural interleukin 12 gene therapy stimulates the immune system and decreases angiogenesis in dogs with spontaneous cancer phase i trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma intratumoral plasmid il12 electroporation therapy in patients with advanced melanoma induces systemic and intratumoral t-cell responses intratumoral delivery of tavokinogene telseplasmid yields systemic immune responses in metastatic melanoma patients intratumoral delivery of plasmid il12 via electroporation leads to regression of injected and noninjected tumors in merkel cell carcinoma integrin-targeted nanocomplexes for tumour specific delivery and therapy by systemic administration conjugation of poly(amidoamine) dendrimers with various acrylates for improved delivery of plasmid encoding interleukin-12 gene preparation and characterization of gelatin nanoparticles containing pdna encoding il-12 and their expression in ct-26 carcinoma cells preparation and characterization of chitosan/beta-cyclodextrin nanoparticles containing plasmid dna encoding interleukin-12 efficient gene delivery by egf-lipoplexes in vitro and in vivo polyethylenimine: a versatile, multifunctional nonviral vector for nucleic acid delivery aerosol gene therapy with pei: il-12 eradicates osteosarcoma lung metastases eradication of osteosarcoma lung metastases following intranasal interleukin-12 gene therapy using a nonviral polyethylenimine vector tetraiodothyroacetic acid-conjugated polyethylenimine for integrin receptor mediated delivery of the plasmid encoding il-12 gene enhanced delivery of plasmid encoding interleukin-12 gene by diethylene triamine penta-acetic acid (dtpa)-conjugated pei nanoparticles delivery of plasmid encoding interleukin-12 gene into hepatocytes by conjugated polyethylenimine-based nanoparticles intratumoral delivery of il-12 gene by polyvinyl polymeric vector system to murine renal and colon carcinoma results in potent antitumor immunity combination of interleukin 12 and interferon alpha gene therapy induces a synergistic antitumor response against colon and renal cell carcinoma biodegradable polymer-based interleukin-12 gene delivery: role of induced cytokines, tumor infiltrating cells and nitric oxide in anti-tumor activity soluble biodegradable polymer-based cytokine gene delivery for cancer treatment in vivo targeted gene delivery by cationic nanoparticles for treatment of hepatocellular carcinoma modified nanoparticle mediated il-12 immunogene therapy for colon cancer local and systemic delivery of interleukin-12 gene by cationic micelles for cancer immunogene therapy mannosylated chitosan nanoparticle-based cytokine gene therapy suppressed cancer growth in balb/c mice bearing ct-26 carcinoma cells intratumoral delivery of p2cmvmil-12 using water-soluble lipopolymers tumor regression by repeated intratumoral delivery of water soluble lipopolymers/p2cmvmil-12 complexes combination of local, nonviral il12 gene therapy and systemic paclitaxel treatment in a metastatic breast cancer model local, non-viral il-12 gene therapy using a water soluble lipopolymer as carrier system combined with systemic paclitaxel for cancer treatment development of self-assembled multi-arm polyrotaxanes nanocarriers for systemic plasmid delivery in vivo synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer a safety and efficacy study of local delivery of interleukin-12 transgene by ppc polymer in a model of experimental glioma treatment of disseminated ovarian cancer using nonviral interleukin-12 gene therapy delivered intraperitoneally phase-i clinical trial of il-12 plasmid/lipopolymer complexes for the treatment of recurrent ovarian cancer a phase ii trial of intraperitoneal egen-001, an il-12 plasmid formulated with peg-pei-cholesterol lipopolymer in the treatment of persistent or recurrent epithelial ovarian, fallopian tube or primary peritoneal cancer: a gynecologic oncology group study phase i trial of a formulated il-12 plasmid in combination with carboplatin and docetaxel chemotherapy in the treatment of platinum-sensitive recurrent ovarian cancer a phase i trial of intraperitoneal gen-1, an il-12 plasmid formulated with peg-pei-cholesterol lipopolymer, administered with pegylated liposomal doxorubicin in patients with recurrent or persistent epithelial ovarian, fallopian tube or primary peritoneal cancers: an nrg oncology/gynecologic oncology group study intratumoral injection of interleukin-12 plasmid dna, either naked or in complex with cationic lipid, results in similar tumor regression in a murine model cationic polyphosphazene vesicles for cancer immunotherapy by efficient in vivo cytokine il-12 plasmid delivery enhanced growth inhibition of metastatic lung tumors by intravenous injection of atra-cationic liposome/il-12 pdna complexes in mice retinoic acid receptors and cancers all-trans-retinoic acid upregulates tnf receptors and potentiates tnf-induced activation of nuclear factors-kappab, activated protein-1 and apoptosis in human lung cancer cells lipid nanoparticles that deliver il-12 messenger rna suppress tumorigenesis in myc oncogene-driven hepatocellular carcinoma visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (plga) microspheres a study of medi1191 in sequential and concurrent combination with durvalumab in subjects with advanced solid tumors abstract 5017: medi1191, a novel il-12 mrna therapy for intratumoral injection to promote t<sub>h</sub>1 transformation of the patient tumor microenvironment a first-in-human dose escalation and expansion study to evaluate intratumoral administration of sar441000 as monotherapy and in combination with cemiplimab in patients with advanced solid tumors analysis of pre-existing igg and igm antibodies against polyethylene glycol (peg) in the general population viruses as anticancer drugs oncolytic virotherapy adenovirus-mediated interleukin-12 gene therapy for metastatic colon carcinoma construction of a double recombinant adenovirus vector expressing a heterodimeric cytokine: in vitro and in vivo production of biologically active interleukin-12 adenovirus-mediated expression of interleukin-12 induces natural killer cell activity and complements adenovirus-directed gp75 treatment of melanoma lung metastases construction and characterization of a recombinant adenoviral vector expressing human interleukin-12 intranasal therapy with an adenoviral vector containing the murine interleukin-12 gene eradicates osteosarcoma lung metastases antitumor mechanism of intratumoral injection with il-12-expressing adenoviral vector against il-12-unresponsive tumor the antitumor effects of adenoviralmediated, intratumoral delivery of interleukin 23 require endogenous il-12 induction of antitumor immunity by direct intratumoral injection of a recombinant adenovirus vector expressing interleukin-12 regression of colon cancer and induction of antitumor immunity by intratumoral injection of adenovirus expressing interleukin-12 role of nk and t cells in il-12-induced anti-tumor response against hepatic colon carcinoma adenovirusmediated interleukin-12 gene therapy for prostate cancer: suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model attenuation of the glucocorticoid response during ad5il-12 adenovirus vector treatment enhances natural killer cell-mediated killing of mhc class i-negative lncap prostate tumors depletion of myeloid-derived suppressor cells during interleukin-12 immunogene therapy does not confer a survival advantage in experimental malignant glioma in situ adenoviral interleukin 12 gene transfer confers potent and longlasting cytotoxic immunity in glioma eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cdnas for the il-12 heterodimer and its inhibition by the il-12 p40 subunit homodimer a single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with meth-a fibrosarcoma growth suppression of human laryngeal squamous cell carcinoma by adenoviral-mediated interleukin-12 enhanced antitumor effect of combined replicative adenovirus and nonreplicative adenovirus expressing interleukin-12 in an immunocompetent mouse model adenovirus-interleukin-12-mediated tumor regression in a murine hepatocellular carcinoma model is not dependent on cd1-restricted natural killer t cells gene therapy of orthotopic hepatocellular carcinoma in rats using adenovirus coding for interleukin 12 effective gene therapy for medullary thyroid carcinoma using recombinant adenovirus inducing tumor-specific expression of interleukin-12 gene therapy of a rat follicular thyroid carcinoma model with adenoviral vectors transducing murine interleukin-12 intratumor murine interleukin-12 gene therapy suppressed the growth of local and distant ewing's sarcoma neuroblastoma regression and immunity induced by transgenic expression of interleukin-12 high frequency of specific cd8+ t cells in the tumor and blood is associated with efficient local il-12 gene therapy of cancer thyroid cancer immuno-therapy with retroviral and adenoviral vectors expressing granulocyte macrophage colony stimulating factor and interleukin-12 in a rat model low-dose adenoviral immunotherapy of rat hepatocellular carcinoma using single-chain interleukin-12 treatment of pancreatic cancer with an oncolytic adenovirus expressing interleukin-12 in syrian hamsters phase i trial of intratumoral injection of an adenovirus encoding interleukin-12 for advanced digestive tumors membrane-tethered proteins for basic research, imaging, and therapy tumor therapy applying membrane-bound form of cytokines re-designing interleukin-12 to enhance its safety and potential as an anti-tumor immunotherapeutic agent cancer immunotherapy using a membrane-bound interleukin-12 with b7-1 transmembrane and cytoplasmic domains the rheoswitch system for inducible up-and down-regulation of gene expression regulated intratumoral expression of il-12 using a rheoswitch therapeutic system((r)) (rts((r))) gene switch as gene therapy for the treatment of glioma regulatable interleukin-12 gene therapy in patients with recurrent highgrade glioma: results of a phase 1 trial neoadjuvant interleukin-12 immunogene therapy protects against cancer recurrence after liver resection in an animal model neoadjuvant treatment of hepatic malignancy: an oncolytic herpes simplex virus expressing il-12 effectively treats the parent tumor and protects against recurrence-after resection interleukin 12 secretion enhances antitumor efficacy of oncolytic herpes simplex viral therapy for colorectal cancer angiogenesis inhibition by an oncolytic herpes virus expressing interleukin 12 cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma in situ cancer vaccination: an il-12 defective vector/replication-competent herpes simplex virus combination induces local and systemic antitumor activity augmentation of antitumor immune responses by multiple intratumoral inoculations of replicationconditional hsv and interleukin-12 il-12 expressing oncolytic herpes simplex virus promotes anti-tumor activity and immunologic control of metastatic ovarian cancer in mice newly characterized murine undifferentiated sarcoma models sensitive to virotherapy with oncolytic hsv-1 m002 enhanced therapeutic efficacy of il-12, but not gm-csf, expressing oncolytic herpes simplex virus for transgenic mouse derived prostate cancers cytokine-secreting herpes viral mutants effectively treat tumor in a murine metastatic colorectal liver model by oncolytic and t-cell-dependent mechanisms engineered herpes simplex virus expressing il-12 in the treatment of experimental murine brain tumors multifaceted oncolytic virus therapy for glioblastoma in an immunocompetent cancer stem cell model increased efficacy of an interleukin-12-secreting herpes simplex virus in a syngeneic intracranial murine glioma model preclinical evaluation of oncolytic deltagamma(1)34.5 herpes simplex virus expressing interleukin-12 for therapy of breast cancer brain metastases preclinical evaluation of a genetically engineered herpes simplex virus expressing interleukin-12 a fully-virulent retargeted oncolytic hsv armed with il-12 elicits local immunity and vaccine therapy towards distant tumors eradication of glioblastoma by immuno-virotherapy with a retargeted oncolytic hsv in a preclinical model evaluation of the safety and biodistribution of m032, an attenuated herpes simplex virus type 1 expressing hil-12, after intracerebral administration to aotus nonhuman primates the molecular pathogenesis of semliki forest virus: a model virus made useful? semliki forest virus vectors engineered to express higher il-12 levels induce efficient elimination of murine colon adenocarcinomas induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered semliki forest virus to produce interleukin-12 marked enhancement of antitumor immune responses in mouse brain tumor models by genetically modified dendritic cells producing semliki forest virus-mediated interleukin-12 semliki forest virus expressing interleukin-12 induces antiviral and antitumoral responses in woodchucks with chronic viral hepatitis and hepatocellular carcinoma short-term intratumoral interleukin-12 expressed from an alphaviral vector is sufficient to induce an efficient antitumoral response against spontaneous hepatocellular carcinomas neoadjuvant administration of semliki forest virus expressing interleukin-12 combined with attenuated salmonella eradicates breast cancer metastasis and achieves long-term survival in immunocompetent mice transfer of the murine interleukin-12 gene in vivo by a semliki forest virus vector induces b16 tumor regression through inhibition of tumor blood vessel formation monitored by doppler ultrasonography the anti-angiogenic activity of il-12 is increased in inos-/-mice and involves nk cells strict requirement for vector-induced type i interferon in efficacious antitumor responses to virally encoded il12 immunotherapy with recombinant sfv-replicons expressing the p815a tumor antigen or il-12 induces tumor regression immunotherapeutic synergy between anti-cd137 mab and intratumoral administration of a cytopathic semliki forest virus encoding il-12 virotherapy with a semliki forest virus-based vector encoding il12 synergizes with pd-1/pd-l1 blockade regression of mouse tumours and inhibition of metastases following administration of a semliki forest virus vector with enhanced expression of il-12 semliki forest virus-mediated gene therapy of the rg2 rat glioma low-dose vaccinia virus-mediated cytokine gene therapy of glioma evaluation of cytokine toxicity induced by vaccinia virus-mediated il-2 and il-12 antitumour immunotherapy cytokine-armed vaccinia virus infects the mesothelioma tumor microenvironment to overcome immune tolerance and mediate tumor resolution intratumoral expression of il-7 and il-12 using an oncolytic virus increases systemic sensitivity to immune checkpoint blockade canarypox virus-mediated interleukin 12 gene transfer into murine mammary adenocarcinoma induces tumor suppression and long-term antitumoral immunity intratumoral administration of a recombinant canarypox virus expressing interleukin 12 in patients with metastatic melanoma phase i study of the intratumoral administration of recombinant canarypox viruses expressing b7.1 and interleukin 12 in patients with metastatic melanoma oncolytic measles virus encoding interleukin-12 mediates potent antitumor effects through t cell activation. oncoimmunology immunological effects and viral gene expression determine the efficacy of oncolytic measles vaccines encoding il-12 or il-15 agonists interleukin-12 expression enhances vesicular stomatitis virus oncolytic therapy in murine squamous cell carcinoma enhancement of antitumor activity of gammaretrovirus carrying il-12 gene through genetic modification of envelope targeting her2 receptor: a promising strategy for bladder cancer therapy increased efficacy and safety in the treatment of experimental liver cancer with a novel adenovirus-alphavirus hybrid vector engineering newcastle disease virus as an oncolytic vector for intratumoral delivery of immune checkpoint inhibitors and immunocytokines immunotherapy of established tumors in mice by intratumoral injection of an adenovirus vector harboring the human il-2 cdna: induction of cd8 + t-cell immunity and nk activity effect of viral dose on neutralizing antibody response and transgene expression after aav1 vector re-administration in mice pre-existing immunity to adenovirus does not prevent tumor regression following intratumoral administration of a vector expressing il-12 but inhibits virus dissemination genetic heterogeneity in the toxicity to systemic adenoviral gene transfer of interleukin-12 systemic vector leakage and transgene expression by intratumorally injected recombinant adenovirus vectors systemic dissemination of viral vectors during intratumoral injection in vivo cancer gene therapy with a recombinant interleukin-2 adenovirus vector gene therapy leaves a vicious cycle intratumoral delivery of adenovirus-mediated interleukin-12 gene in mice with metastatic cancer in the liver preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for prostate cancer induction of systemic and therapeutic antitumor immunity using intratumoral injection of dendritic cells genetically modified to express interleukin 12 intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas fibroblasts genetically engineered to secrete interleukin 12 can suppress tumor growth and induce antitumor immunity to a murine melanoma in vivo cancer immunotherapy of established tumors with il-12. effective delivery by genetically engineered fibroblasts therapeutic effects of gelatin matrix-embedded il-12 gene-modified macrophages in a mouse model of residual prostate cancer macrophages transduced with an adenoviral vector expressing interleukin 12 suppress tumor growth and metastasis in a preclinical metastatic prostate cancer model antitumor efficacy of adenocarcinoma cells engineered to produce interleukin 12 (il-12) or other cytokines compared with exogenous il-12 paracrine delivery of il-12 against intracranial 9l gliosarcoma in rats expression of interleukin-12 by adipose-derived mesenchymal stem cells for treatment of lung adenocarcinoma. thorac cancer therapeutic potential of human mesenchymal stem cells producing il-12 in a mouse xenograft model of renal cell carcinoma anti-tumor activity of mesenchymal stem cells producing il-12 in a mouse melanoma model enhancement of antitumor immunity against b16 melanoma tumor using genetically modified dendritic cells to produce cytokines route of administration influences the antitumor effects of bone marrowderived dendritic cells engineered to produce interleukin-12 in a metastatic mouse prostate cancer model augmented anti-tumor effect of dendritic cells genetically engineered by interleukin-12 plasmid dna upregulation of natural killer cells functions underlies the efficacy of intratumorally injected dendritic cells engineered to produce interleukin-12 interleukin-12 transduced dendritic cells induce regression of established murine neuroblastoma intratumoral delivery of dendritic cells engineered to secrete both interleukin (il)-12 and il-18 effectively treats local and distant disease in association with broadly reactive tc1-type immunity intratumoral injection of dendritic cells transduced by an sv40-based vector expressing interleukin-15 induces curative immunity mediated by cd8+ t lymphocytes and nk cells injection of il-12 gene-transduced dendritic cells into mouse liver tumor lesions activates both innate and acquired immunity intratumoral injection of dendritic cells overexpressing interleukin12 inhibits melanoma growth conditional interleukin-12 gene therapy promotes safe and effective antitumor immunity therapeutic effect of intratumoral administration of dcs with conditional expression of combination of different cytokines intratumoral injection of dendritic cells engineered to secrete interleukin-12 by recombinant adenovirus in patients with metastatic gastrointestinal carcinomas continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy interleukin 12 gene therapy of cancer by peritumoral injection of transduced autologous fibroblasts: outcome of a phase i study concise review: mesenchymal stem cell tumorhoming: detection methods in disease model systems murine bone marrow-derived mesenchymal stem cells as vehicles for interleukin-12 gene delivery into ewing sarcoma tumors reversal of tumor growth by gene modification of mesenchymal stem cells using spermine-pullulan/dna nanoparticles gene therapy of intracranial glioma using interleukin 12-secreting human umbilical cord blood-derived mesenchymal stem cells mesenchymal stem cells genetically modified by lentivirus-mediated interleukin-12 inhibit malignant ascites in mice the effects of mesenchymal stem cells injected via different routes on modified il-12-mediated antitumor activity adoptive transfer of natural killer cells promotes the anti-tumor efficacy of t cells local delivery of interleukin-12 using t cells targeting vegf receptor-2 eradicates multiple vascularized tumors in mice il-12 release by engineered t cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression improving adoptive t cell therapy by targeting and controlling il-12 expression to the tumor environment tumor-targeted t cells modified to secrete il-12 eradicate systemic tumors without need for prior conditioning cd19 car t cells expressing il-12 eradicate lymphoma in fully lymphoreplete mice through induction of host immunity il-12 secreting tumor-targeted chimeric antigen receptor t cells eradicate ovarian tumors in vivo armored inducible expression of il-12 enhances antitumor activity of glypican-3-targeted chimeric antigen receptor-engineered t cells in hepatocellular carcinoma chitosan solution enhances the immunoadjuvant properties of gm-csf sustained release technology and its application in environmental remediation: a review designing hydrogels for controlled drug delivery recent advances in hydrogel based drug delivery systems for the human body neoadjuvant therapy with interleukin-12-loaded polylactic acid microspheres reduces local recurrence and distant metastases in situ tumor vaccination with interleukin-12-encapsulated biodegradable microspheres: induction of tumor regression and potent antitumor immunity cytokines delivered by biodegradable microspheres promote effective suppression of human tumors by human peripheral blood lymphocytes in the scid-winn model synergistic effect of intratumoral il-12 and tnf-alpha microspheres: systemic antitumor immunity is mediated by both cd8+ ctl and nk cells intratumoral delivery of encapsulated il-12, il-18 and tnf-alpha in a model of metastatic breast cancer antitumor immune response of human peripheral blood lymphocytes coengrafted with tumor into severe combined immunodeficient mice interleukin-12 delivered by biodegradable microspheres promotes the antitumor activity of human peripheral blood lymphocytes in a human head and neck tumor xenograft/scid mouse model human cd4+ t cells present within the microenvironment of human lung tumors are mobilized by the local and sustained release of il-12 to kill tumors in situ by indirect effects of ifn-gamma human cd4+ effector memory t cells persisting in the microenvironment of lung cancer xenografts are activated by local delivery of il-12 to proliferate, produce ifn-gamma, and eradicate tumor cells transient activation of tumor-associated t-effector/memory cells promotes tumor eradication via nk-cell recruitment: minimal role for long-term t-cell immunity in cure of metastatic disease cancer immunotherapy with interleukin 12 and granulocyte-macrophage colony-stimulating factor-encapsulated microspheres: coinduction of innate and adaptive antitumor immunity and cure of disseminated disease dichotomous effects of ifn-gamma on dendritic cell function determine the extent of il-12-driven antitumor t cell immunity central role of tumorassociated cd8+ t effector/memory cells in restoring systemic antitumor immunity activated cd8+ t-effector/memory cells eliminate cd4+ cd25+ foxp3+ tsuppressor cells from tumors via fasl mediated apoptosis central role of ifngamma-indoleamine 2,3-dioxygenase axis in regulation of interleukin-12-mediated antitumor immunity rapid release of cytoplasmic il-15 from tumor-associated macrophages is an initial and critical event in il-12-initiated tumor regression intratumoral il-12 and tnf-alpha-loaded microspheres lead to regression of breast cancer and systemic antitumor immunity generation of a tumor-specific systemic response after intratumoral injection of il-12 and il-18-loaded polylactic acid microspheres il-12 + gm-csf microsphere therapy induces eradication of advanced spontaneous tumors in her-2/neu transgenic mice but fails to achieve long-term cure due to the inability to maintain effector t-cell activity chronic immune therapy induces a progressive increase in intratumoral t suppressor activity and a concurrent loss of tumor-specific cd8+ t effectors in her-2/neu transgenic mice bearing advanced spontaneous tumors chronic chemoimmunotherapy achieves cure of spontaneous murine mammary tumors via persistent blockade of posttherapy counterregulation effect of chitosan properties on immunoreactivity intravesical immunotherapy of superficial bladder cancer with chitosan/interleukin-12 characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-n-acetyl glucosamine-based polymer matrix sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific t-cell response paracrine release of il-12 stimulates ifn-gamma production and dramatically enhances the antigen-specific t cell response after vaccination with a novel peptide-based cancer vaccine intratumoral poly-n-acetyl glucosamine-based polymer matrix provokes a prolonged local inflammatory response that, when combined with il-2, induces regression of malignant mesothelioma in a murine model using poly-n-acetyl glucosamine gel matrix to deliver il-12 with anti-schistosomasis vaccination evaluation of the biocompatibility of a chitosan scaffold in mice biocompatibility evaluation of crosslinked chitosan hydrogels after subcutaneous and intraperitoneal implantation in the rat il-12 delivered intratumorally by multilamellar liposomes reactivates memory t cells in human tumor microenvironments adjuvant cationic liposomes presenting mpl and il-12 induce cell death, suppress tumor growth, and alter the cellular phenotype of tumors in a murine model of breast cancer cytokine immunotherapy of cancer with controlled release biodegradable microspheres in a human tumor xenograft/scid mouse model characterization of cytokine-encapsulated controlled-release microsphere adjuvants stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide) biodegradable microand nanoparticles as long-term delivery vehicles for interferon-alpha challenges in development of targeted liposomal therapeutics advances and challenges of liposome assisted drug delivery surgery for cancer: a trigger for metastases chemotherapy-induced metastasis in breast cancer radiotherapy targeting cancer stem cells "awakens" them to induce tumour relapse and metastasis in oral cancer biological behavior of human breast cancer micrometastases a novel synergistic combination of cyclophosphamide and gene transfer of interleukin-12 eradicates colorectal carcinoma in mice a phase i clinical trial of adoptive t cell therapy using il-12 secreting muc-16(ecto) directed chimeric antigen receptors for recurrent ovarian cancer the next challenge in cancer immunotherapy: controlling t-cell traffic to the tumor chemotherapy induces intratumoral expression of chemokines in cutaneous melanoma, favoring t-cell infiltration and tumor control immunogenic effects of chemotherapy-induced tumor cell death radiation-induced effects and the immune system in cancer a novel combination treatment of armed oncolytic adenovirus expressing il-12 and gm-csf with radiotherapy in murine hepatocarcinoma irradiation-induced localization of il-12-expressing mesenchymal stem cells to enhance the curative effect in murine metastatic hepatoma combination of radiation and interleukin 12 eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment the role of irreversible electroporation (ire) for locally advanced pancreatic cancer: a systematic review of safety and efficacy irreversible electroporation reverses resistance to immune checkpoint blockade in pancreatic cancer systematic review of surgical and percutaneous irreversible electroporation in the treatment of locally advanced pancreatic cancer high-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity immunologic response to cryoablation of breast cancer thermal ablation of tumours: biological mechanisms and advances in therapy synergy between in situ cryoablation and tlr9 stimulation results in a highly effective in vivo dendritic cell vaccine changes in interleukin-1beta and 6 after hepatic microwave tissue ablation compared with radiofrequency, cryotherapy and surgical resections image-guided thermal ablation of tumors increases the plasma level of interleukin-6 and interleukin-10 characterization of the cryoablation-induced immune response in kidney cancer patients abscopal immunity achieved via in situ vaccination using a novel combination of cryoablation and interleukin-12 the antibody-based delivery of interleukin-12 to solid tumors boosts nk and cd8 + t cell activity and synergizes with immune checkpoint inhibitors intratumoral il-12 combined with ctla-4 blockade elicits t cell-mediated glioma rejection phase ii trial of il-12 plasmid transfection and pd-1 blockade in immunologically quiescent melanoma revisiting interleukin-12 as a cancer immunotherapy agent abbreviations a-nk il−12 or activated nk il−12 , activated primary natural killer (nk) cells transduced to express il-12 adcmvil-12 or adcmvmil-12, recombinant defective adenovirus expressing il-12 or mil-12 under control of a cmv promoter adenoviral vector expressing murinedil-12 protein under the control of the hcmv promoter and sv40 polyadenylation sequences; ad-dhscil12, adenovirus with replication dependent on hypoxia-inducible factor (hif) activity expressing single-chain il-12; ad-il-12 or adil-12 or ad.il-12 or admil-12 or ad.mil-12 inducible adenoviral vector engineered to express il-12 under the control of the rheoswitch therapeutic system r (rts) gene switch adtcpmil-12, recombinant replication-defective adenoviral vector expressing murine interleukin-12 (mil-12), driven by a modified calc-i promoter oncolytic triple-deletion (td) adenoviral vector encoding wild-type il-12 oncolytic triple-deletion (td) adenoviral vector encoding non-secreting il-12 antibody-dependent cellular cytotoxicity; ad.scil-12, adenoviral vector expressing single-chain il-12 ad5-ycd/muttksr39rep-hil12, a replication-competent oncolytic adenovirus encoding the murine pro-inflammatory cytokine interleukin-12 (il-12) gene and two suicide fusion genes, a yeast cytosine deaminase (ycd) and a mutant form of herpes simplex virus type 1 thymidine kinase /3.crgd-mil12p70, a replication-deficient double targeted ad5 backbone-based vector carrying a chimeric ad5/3 fiber with integrin-binding rgd motif incorporated in its ad3 knob domain expressing murine il-12p70 adenoviral vector encoding membrane-anchored murine single-chain il-12 with b7-1 transmembrane and cytoplasmic domains atra-cationic liposome/il-12 pdna, pil-12 complexed with cationic liposomes incorporating all-trans-retinoic acid chimeric antigen receptor; cbd, collagen binding domain; cbd-il-12, collagen-binding immunocytokine comprised of a3 cbd of von willebrand factor fused to both subunits of il-12 chtnt-3/huil-12, necrosis-targeting immunocytokine comprised of huil-12 fused to the variable heavy chain of chtnt-3 antibody dendritic cells transfected with adenovirus expressing il-12 under control of the cmv promoter enzyme-linked immune absorbent spot fda, food and drug administration; gm-csf, granulocyte-macrophage colony-stimulating factor glypican-3-specific car t cells with nfat-inducible expression of il-12 herpes simplex viruses (hsv) encoding il-12; hubc1-il12, immunocytokine comprised of two molecules of il-12 fused to each of the igg heavy chains of humanized bc-1 antibody immunocytokine comprised of il-12 fused to the fc fragment of humanized ks antibody; hu-14.18-il-12, immunocytokine comprised of il-12 fused to gd2 targeting hu14 dmp/il-12, pil-12 complexed with dmp cationic micelles 3-dimyristyloxypropyl)-n,n-dimethyl-(2-hydroxyethyl)ammonium bromide/dioleoyl phosphatidylethanolamine (dmrie/dope) cationic lipids; il-12, interleukin-12; il-12m, modified il-12; an n-glycosylation mutant of il-12 at asn220; il12-il2, immunocytokine comprised of il-12 and il-2 fused to anti-cd30 scfv antibody immunocytokine comprised of il-12 fused to l19 scfv antibody; il12-msa, il-12 fused to mouse serum albumin (msa) il-12 fused to mouse serum albumin (msa) and lumican, a collagen-binding proteoglycan immunocytokine comprised of p35 subunit of single-chain il-12 fused to ss1, a mesothelin-binding single-chain variable fragment lenti-mil-12-mscs, bone-marrow derived mscs transfected with lentivirus to express il-12 mc/pmil-12, pmil-12 complexed with mannosylated chitosan (mc) myeloid-derived suppressor cells mevac fmil-12, attenuated measles viruses (mevac) encoding il-12 histocompatibility complex; momlv-mil-12, moloney murine leukemia virus (momlv) expressing il-12; mpeg/pmil-12, polymersomes comprised of pmil-12 complexed with polyphosphazene particles modified with dpa and mpeg; mscs, mesenchymal stem cells; mscil-12.her2.igg3, mouse single-chain il-12 fused mesenchymal stem cells (mscs) expressing il-12; l19-mil12, immunocytokine comprised of murine il-12 fused to l19 scfv antibody; nhs-il12, necrosis-targeting immunocytokine comprised of two single-chain il-12 molecules fused to fulllength nhs76 antibody; nhs-muil12, murine analog of nhs-il12; nk cells a biodegradable polyester; pbl, peripheral blood lymphocytes; pd-1, programmed cell death protein 1; pd-l1, programmed death-ligand 1; pei:il-12, il-12 complexed with polyethyleneimine (pei); pil-12 enhanced sfv vector with 10x higher gene expression of recombinant il-12 protein; rad/il-12m, recombinant adenovirus expressing il-12m; rndv-anti-cd28-mil-12, newcastle disease virus (ndv) expressing immunocytokine comprised of anti-cd28 antibody fused to mil-12; rndvanti-pdl1-mil-12, newcastle disease virus (ndv) expressing immunocytokine comprised of anti-pdl1 antibody fused to mil-12; rsfv/il12, recombinant semliki forest viruses (sfv) encoding il-12; rvsv-il12, recombinant vesicular stomatitis virus (vsv) encoding il-12; rvv-mil-12 severe combined immunodeficient; scil-12, single-chain il-12; sfv-il12, semliki forest viruses encoding il-12 sfv-il-12, semliki forest viruses encoding il-12 tumor associated macrophages; tlr, toll-like receptor; tnf, tumor necrosis factor; ucb-msc-il12m, human umbilical cord blood-derived mesenchymal stem cells (ucb-mscs) expressing il-12m vv-il-12, vaccinia virus (vv) expressing il-12 il-12 expression plasmid complexed with water-soluble lipopolymers car t cells which are specific for the muc-16ecto antigen and secrete il-12; 6b11scfv-mil-12, immunocytokine comprised of mil-12 fused to scfv of 6b11 antibody key: cord-285760-y37ji92k authors: connell, anna r.; connell, jeff; leahy, t. ronan; hassan, jaythoon title: mumps outbreaks in vaccinated populations—is it time to re-assess the clinical efficacy of vaccines? date: 2020-09-18 journal: front immunol doi: 10.3389/fimmu.2020.02089 sha: doc_id: 285760 cord_uid: y37ji92k history illustrates the remarkable public health impact of mass vaccination, by dramatically improving life expectancy and reducing the burden of infectious diseases and co-morbidities worldwide. it has been perceived that if an individual adhered to the mmr vaccine schedule that immunity to mumps virus (muv) would be lifelong. recent mumps outbreaks in individuals who had received two doses of the measles mumps rubella (mmr) vaccine has challenged the efficacy of the mmr vaccine. however, clinical symptoms, complications, viral shedding and transmission associated with mumps infection has been shown to be reduced in vaccinated individuals, demonstrating a benefit of this vaccine. therefore, the question of what constitutes a good mumps vaccine and how its impact is assessed in this modern era remains to be addressed. epidemiology of the individuals most affected by the outbreaks (predominantly young adults) and variance in the circulating muv genotype have been well-described alluding to a collection of influences such as vaccine hesitancy, heterogeneous vaccine uptake, primary, and/or secondary vaccine failures. this review aims to discuss in detail the interplay of factors thought to be contributing to the current mumps outbreaks seen in highly vaccinated populations. in addition, how mumps diagnoses has progressed and impacted the understanding of mumps infection since a mumps vaccine was first developed, the limitations of current laboratory tests in confirming protection in vaccinated individuals and how vaccine effectiveness is quantified are also considered. by highlighting knowledge gaps within this area, this state-of-the-art review proposes a change of perspective regarding the impact of a vaccine in a highly vaccinated population from a clinical, diagnostic and public perspective, highlighting a need for a paradigm shift on what is considered vaccine immunity. muv is an enveloped, non-segmented, negative-sense, single stranded rna virus that varies between a spherical and pleiomorphic shape of ∼200 nm (85-300 nm) (1, 2) . muv is responsible for an acute viral infection, spread by respiratory droplets (via coughs, sneezes) and urine (3, 4) . with an incubation period of 14-25 days, muv replicates in the nasopharynx and regional lymph nodes, with a secondary viremia occurring late in the incubation period (5, 6) . muv can be detected from saliva up to 7 days prior, and as late as 9 days after clinical onset of parotitis (7) . the muv genome of seven genes consists of 15,384 nucleotides, and encodes six structural proteins and at least two non-structural proteins; the nucleocapsid protein (np), v protein (v), phosphoprotein (p), matrix (m) protein, fusion (f) protein, small hydrophobic (sh) protein, hemagglutininneuraminidase (hn) protein, and large (l) protein. the role of the i protein is not known (1, 6, 8) . the sh gene is the most variable region of the muv genome; a 2-4% intravariation and 8-18% inter-variation has been documented (9) . this gene is used in molecular phylogeny for genotyping and to identify transmission patterns in populations (6) . despite being serologically monotypic, 12 muv genotypes (a to l) have been described to date (muv genotypes e and m are omitted, as the muv previously assigned to these groups were later re-assigned) (1, 9, 10) . the geographic distributions of the muv genotypes varies worldwide but can co-circulate and thus drive temporal shifts in their distribution. genotype a was frequently isolated in europe until the 1990's. currently genotypes c, d, e, g, and h are prevalent in europe and the united states of america (usa) whereas genotypes b, f and i are more common in asian countries ( table 1) (10, 18, 86, 87) . since 1946 numerous mumps vaccines have been developed worldwide, varying in efficacy and safety profiles but primarily consisting of an attenuated live muv without an adjuvant (6, (87) (88) (89) . currently in europe and for the majority of the g20 countries who have a mumps vaccine in their immunization schedule (table 1) , the mumps vaccine is included as part of the trivalent measles, mumps rubella (mmr) vaccine, and is primarily administered in two doses (90, 91) . the jeryl lynn (jl) vaccine, derived from the genotype a muv strain was first developed in the usa and has been used extensively in the united kingdom (uk), ireland and usa since it was licensed in 1967 (92) . derived from a single clinical sample, and propagated in a chick embryo cell culture, two viral isolates (jl2 and jl5) are present, differing by ∼414 nucleotides and 87 amino acid changes (93) (94) (95) . the rit 4385 mumps vaccine, developed from the dominant viral component (jl5) in the jl vaccine strain appears to have comparative safety and efficacy (seroconversion) profiles to the jl vaccine strain (87, (96) (97) (98) . however, since no controlled clinical trials of efficacy have been published to compare the two doses of the two vaccines, the clinical significance of this observation is not known. despite the integration of the mmr vaccine into childhood immunization programs, cyclical outbreaks [defined as two or more cases linked by place and time (96) ] of muv have been documented in several highly vaccinated populations such as ireland and the united kingdom (6, (97) (98) (99) (100) (101) (102) (103) . between august 2018-and january 2020, 3,736 mumps cases were notified in ireland, primarily affecting individuals between the ages of 15-24 years. of the 32% of cases that stated vaccination status, 72% had received two doses of the mmr vaccine (104) . an upsurge of mumps cases has also occurred in 47 states of the united states over the last 2 decades, primarily affecting people between 18 and 24 years in close contact/shared settings (105) . in indiana, 76.9% of mumps cases (84.9% of university affiliated and 52% of community cases) had documented evidence of mmr vaccination (106) . this results in a significant resource burden for public health departments to control. several reviews, both observational and systematic have demonstrated the clinical benefit of a mumps vaccine (107, 108) , the pathogenesis and genomic diversity of the muv (10, 107, 108) and the epidemiology surrounding the outbreak (1, 10, 82) . it is not clear why these mumps outbreaks occur, although it has been alluded to be due to a number of interrelated factors, such as sub-optimal vaccine uptake (1, 109, 110) , primary or secondary vaccine failure or failure of the mumps vaccine to protect individuals from infection (vaccine efficacy) (107) (figure 1 ). history depicts the remarkable public health impact of mass vaccination. previously inevitable childhood diseases with potentially debilitating or deadly outcomes have seen their rates plummet worldwide or become successfully eradicated. immunizations of vaccine preventable diseases are estimated to prevent ∼2-3 million deaths per annum and increase life expectancy by ∼29 years (111) . more recently there has been a shift in the public and media perception of vaccines to their safety, which has facilitated outbreaks such as mumps (112) . organized opposition to vaccinations has a long history; public outcry and resistance following the introduction of the smallpox vaccine in the nineteenth century led to the introduction in england of the vaccination act of 1853 (113) . with one in eight children in the usa under the age of 2 currently thought to be unvaccinated due to parental choice, the who now considers vaccine hesitancy as one of the ten threats to global health in 2019 (114) . vaccine hesitancy, defined as a "delay in acceptance or refusal of vaccines despite availability of vaccination services" involves a multitude of social, political, cultural and emotional factors in highly vaccinated, western populations (115, 116) . one of the main issues is the parental concerns regarding the perceived risk of a vaccine to their child (such as timing/schedules of vaccines, associated pain of administration, and potential adverse effects) vs. the disease morbidity and mortality associated with the vaccine preventable disease (117, 118) . the retracted paper published in the lancet in 1999 (56) and "anti-vaccination" opinions on social media have also contributed to the persistent and insistent misinformation (116) , despite vast follow-up epidemiological studies showing no relationship between the mmr vaccine and autism, or differing cognitive development/intelligence (118) (119) (120) . however, the resultant reaction of the public led to the uptake of the first mmr vaccine falling sharply from 1999, with uptake falling to below 75% in 2002 (104, 121) . the age demographic that are experiencing the most cases of mumps in ireland during the current ongoing outbreak would have been scheduled to have received the first mmr vaccine between 1997 and 2003. nevertheless, no deductions can be made, due to the lack of vaccination status information provided with reported cases (104) . frontiers in immunology | www.frontiersin.org heterogeneity of immunization coverage in specific populations or geographic locations of susceptibility is also becoming an important epidemiological issue in maintaining proficient population immunity for mumps (3, 109, 122) . the who recommends a >95% mmr vaccine coverage for herd immunity. maintenance of such coverage is well-demonstrated in finland, where a country-wide 2-dose mmr vaccination program initiated in the 1980's eliminated measles, mumps and rubella within 25 years (123, 124) . recent publications from around the world indicate that the level of mmr vaccine uptake is far lower than what is recommended [reviewed in ramanathan et al. (125) ] (101, (126) (127) (128) (129) . of the g20 nations that implement a mumps vaccine within their vaccination schedule, only 3 countries have maintained vaccine coverage levels of >95% (table 1) . however, poor uptake/incomplete vaccination alone may not be the only issue relating to mumps outbreaks. in the netherlands, mumps outbreaks still occurred with an overall herd immunity threshold of 86-92%, and where 96 and 93% received the first and second mmr at 14 months and 9 years, respectively (125, 130) . the clinical presentation of mumps is pathognomic (bi-lateral parotitis); therefore supporting laboratory diagnosis was rarely employed in the past. as the classical symptoms of mumps are not always typical, there may have been a significant number of individuals in the past who may have been infected but were not identified as such. when mumps vaccination was introduced in 1967, the criteria the vaccine had to meet was the proof that it was clinically effective, i.e., that it reduced the risk of disease in vaccinated individuals in real-world conditions over a set period. such an example was seen the usa; the reported cases (i.e., diagnosis of clinical symptoms) of mumps declined from >100 cases per 100,000 population before 1967 (pre-vaccine era) to 10 cases per 100,000 population in 1977, a reduction of 99% (105, 110, 126, 131) . to note, clinical efficacy was probably based upon the reduction of the "classical bilateral presentation" rather than the milder mumps presentation. therefore, one could argue that the original vaccine efficacy for clinical manifestations was over estimated. currently the laboratory diagnosis of mumps infection in ireland is based upon two approaches: detection of mumps rna by reverse transcriptase pcr (rt-pcr) in a buccal swab containing saliva, throat swab or urine specimen, and serological detection of immunoglobulin m (igm) using a capture assay (132, 133) . both approaches for diagnosis are impacted significantly by the quality and timing of sample collection post-onset of symptoms and also if the subject is mumps naïve or had received mumps containing vaccine (87, 126, 134, 135) . there are challenges in using standard serological laboratory diagnostic methods to reliably confirm mumps re-infection of individuals who had been previously naturally infected or vaccinated (130, 136) . briefly, vaccinated individuals re-infected with muv may only generate a weak or undetectable igm response (133) . although a rise in igg titer may also not occur in vaccinated individuals (87, 137) , numerous studies have documented a rapid, variable increase in mumps-specific igg levels, with neutralization antibody concentrations present up to 10 months post-infection (130, 138, 139) . therefore, reverse transcriptase-polymerase chain reaction (rt-pcr) is recommended (133, 140) , and was formally introduced in 2015 as the principle diagnostic tool in ireland to detect mumps in oral fluids (141) . rt-pcr can identify current mumps infection more effectively in vaccinated individuals than serological techniques alone as it identifies the presence of the muv vs. the immunological response (igg, igm), and has been previously shown to 100% correlate with viral culture results (140, 141) . the case numbers of more recent mumps outbreaks should always be assessed with this question in mind; are the number of mumps cases increasing, or/and are we better at diagnosing an acute infection? the latter seems to be the most probable, as many individuals who are being tested do not present with classical symptoms. in addition to enhanced surveillance of mumps cases, further optimizations of technologies are also occurring; the utilization of next-generation sequencing demonstrated that by editing one 2-fold degenerate nucleotide in the forward primer and three 2-fold degenerate nucleotides in the probe sequence optimized the fluorescence intensity and clinical sensitivity of the real-time rt-pcr when compared to the cdc-developed and who-recommended rt-pcr target [(np) gene] leading to ∼11% increase in clinical sensitivity (i.e., ct values that were ∼3.7 cycles lower) (142) . much is not known about the immunological response to the mumps vaccine strain. however, a number of young adults who were vaccinated as children over the last two decades have demonstrated an increased risk of muv infection with time, which is assumed to be related to a decline of antibodies to sub-protective levels of immunity (40, 101, 125, 128, (143) (144) (145) (146) . primary vaccine failure is defined as the lack of a sufficient initial antibody response to a vaccine in a recipient resulting in a lack of protective immune responses (6, 147) . although this type of vaccine failure may be because of improper storage/handling or administration of the vaccine, impacting its efficacy, it may also be due to the initial immunological response of an individual to the vaccine, which is usually quantified by the presence of antibodies that should be detectable in the weeks following vaccination. primary vaccine failure was attributed to primaryschool outbreaks of both mumps and measles in ireland, which subsequently resulted in reducing the age for the second dose of mmr2 vaccine from 10-14 years in 1999 to 4-5 years of age (6) . with the cyclical outbreaks occurring, it has been proposed that primary vaccine failure could again be a factor. how is a response to a vaccine determined? in pre-licensure studies of the jl and urabe mumps vaccines, high seroconversion and low failure rates were observed in children after the first vaccine dose (>90 and 5.5%, respectively), demonstrating that the vaccine induced an antibody response (148) (149) (150) (151) (152) (153) . a more recent study by ong et al. demonstrated that a ≥2fold increase in mumps antibodies 30-days post-vaccination was considered to be an adequate response of immunity (154) . vaccine effectiveness (i.e., seroconversion post-vaccination) of 2 vaccine doses has only been conducted on the jl strain; 6 studies provided a median vaccine efficacy of 88%. these studies have shown that 2 doses of mmr were more effective (but not statistically significant) than a single mmr dose to combat the incidence of mumps infection (101, 126, 145, 151, 152, 155) . mumps-specific antibodies have been detected 1-2 years postvaccination and without substantial decline for 8 years after mumps vaccination, with the immunogenicity and efficacy of the mmr vaccine showing comparable immunogenicity levels to post-vaccination levels at 3 years (148, 156) . however, most studies of this vaccine (involving either a mumps-specific vaccine or a combined vaccine) only followed-up to 30-56 days postvaccination (157) (158) (159) (160) (161) (162) (163) (164) (165) (166) (167) . despite few follow-up studies estimating post-vaccination antibody titers specific to the vaccine mumps strain, the evidence of seroconversion post-vaccination in a number of studies indicate that primary vaccine failure does not seem to be a significant contributor to the outbreaks that have been recently observed (118, 149, 150, 152, 158, (168) (169) (170) (171) . it has been noted that a small percentage of the population do not seroconvert post-vaccination; <1% who received the mmr vaccine were seronegative 4-9 years after the first dose of mmr (n = 616) (143) . poor immune responses to primary vaccination has been shown to be a good indicator of infection susceptibility (172) . this is in agreement with the correlation of pre-outbreak jl virus neutralization titres and elisa results being significantly lower in individuals who became infected compared to non-infected individuals (173) . further studies of these individuals may provide insights of which immunological process are integral to develop immunity. the current methods used to determine immunity against mumps cannot discriminate between primary and secondary vaccine failure; only the timing of these tests can assess whether an individual ever mounted an immune response postvaccination or whether the response is detectable years postvaccination. primary vaccine failure encompasses the failure to mount an immune response to a dose of a vaccine, secondary vaccine failure refers to a more gradual loss of immunity after a successful initial response that occurs over a number of years post-vaccination (174) . several factors have been proposed to be implicated with secondary vaccine failure, such as waning immunity, a lack of cross-neutralization, and natural boosting. waning immunity is defined as a decline in immunological protection proportional to time since vaccination. potential waning immunity has been documented in the current mumps outbreaks seen in europe and the usa, mostly affecting young adults within highly vaccinated populations attending tertiary education who have received two doses of the mmr vaccine in early childhood (40, 110, 126, 144, 145, (175) (176) (177) (178) (179) (180) (181) . a number of studies from the usa, where a jl vaccine has been used since 1971 have demonstrated waning immunity within the population. the risk of developing clinical mumps was shown to increase by 10-27% for every year post-mmr vaccination (125) , with the rates of mumps infection rising from 1.6 cases per 1,000 in those who received the second dose of the vaccine within 2 years of the outbreak, to 11.3 cases per 1,000 in those who received it over 13 years prior. using a mathematical model with analytical limitations, a recent metaanalysis of six studies estimated that vaccine-derived immune protection to muv wanes about 27 years post-vaccination (182) . kennedy et al. (183) also demonstrated a decrease of ∼20% in mumps neutralizing antibody titers over 10 years. in contrast, other studies appear to contradict, these findings, showing no link between mumps protection and time elapsed following administration of mumps vaccine (138, 148, 149, 184, 185) . lebaron et al. (143) and gothefors et al. (186) demonstrated that 70-99% of individuals still had detectable anti-mumps antibodies ∼10 years after initial vaccination. cohen et al. (101) also demonstrated minimal antibody level decline after two mmr doses 6-7 years after second vaccination. neutralizing antibodies against the jl-5 vaccine strain has also been detected in ∼80% for age groups 2-20 years, 67% for age group 24-26 years; and 77% for age group 50+ years (187) . implementation of a third dose of the mmr vaccine has been shown to be effective as a stop gap measure in limiting disease spread in outbreak settings situations (129) . individuals vaccinated for the third time had a 78% lower risk of contracting mumps, with a decreased attack rate of 6.7 vs. 14.5 cases per 1,000 when compared to those who received a second dose. more than 50% of those who received a third dose of the mmr vaccine showed a 4-fold increase in mumps antibody titers (105, 106, 168, 188) . an increase in mumps igg humoral immunity was also observed post-vaccine administration. however, this immunity boost has been shown to be a transient effect, with mumps antibody titers returning to pre-third dose of mumps-vaccination levels 1 year after vaccination. therefore, as waning immunity is thought to be an important factor facilitating mumps outbreaks, the emphasis placed on the quantity/quality of mumps-specific antibodies may need to be re-assessed. it is yet undetermined if the total loss of detectable antibodies correlates to a loss of clinical protection, as the minimal level of neutralizing antibody required for protection against mumps has not yet been defined (184). antigenic variation and thus reduced cross-neutralization between the vaccine and circulating strains of different muv genotypes have been cited as possible explanations for mumps outbreaks in highly vaccinated populations (125, 184, (189) (190) (191) . recent outbreaks in europe and northern america (including ireland) have shown the circulating muv during the current outbreaks to be genotype g (135, 184, 192, 193) . this muv genotype was first identified in 1996, and has demonstrated intra-genotype diversity of up to 7% (table 1) (6, 134) . the jl vaccine strain (genotype a), differs phylogenetically to the circulating muv (genotype g) (125) . in vitro studies of the genotypic distribution and temporal shift of muv suggest that cross neutralization between wild type and vaccine genotypes may be approximately half the concentration measured against the vaccine strain (130) . pre-infection neutralization titers in mumps positive cases were also significantly lower against genotype g vs. mumps vaccine strain, potentially due to amino acid differences in b-cell epitopes and/or n-linked glycosylation sites on the hn and also within the f protein (194) . santak et al. (195, 196) also demonstrated that conformational changes within the f protein may lead to immunological escape. despite the decline/scarcity of cross-neutralizing antibodies, different mumps vaccines used worldwide have been shown to prevent significant clinical mumps infection during outbreaks (101, 197) . dependent on the strain, a 2-16-fold variation of patient sample titers has been shown to be protective in in vitro plaque reduction neutralizations (149, 151, 198) . although the sera of one of these studies, was collected only 6 weeks after mmr vaccination, a time point that may not signify the concept of waning immunity and antigenic differences, several other groups have shown that the most divergent strains of muv can be neutralized in vitro with only slight variations in titers, supporting the concept that muv is serotypically monotypic (184, 190, 195, 198) . epitopes of the muv that are presented to cd8+ t-cells have been shown to be present in not only the circulating strains of virus but also in a number of vaccine strains (199) . in addition, lewnard et al. (182) also found no evidence that recent mumps outbreaks were due to the emergence of muv strains escaping vaccine-driven immunological pressure. therefore, the limited data does not suggest that antigenic drift of the muv leading to diminished neutralization capacity of the vaccine strain could fully explain the recent outbreaks (125) . further studies into the cross-neutralizing capacity of the mumps vaccine strain administered 15-20 years previously to the current circulating strain of muv in countries where outbreaks are being observed will allow better deductions to be made. it is possible that differences in the neutralization capacity of vaccine-induced antibodies against different muv strains may be more significant when levels of neutralizing antibody are low and become "overwhelmed" when the mumps viral load challenge is high (200) . several prominent mmr/mumps vaccine studies were undertaken at a time when there was still a high prevalence of circulating wild type virus, which enabled sub-clinical boosting to occur in an individual. such natural boosting is illustrated in belarus, where a subpopulation of vaccinated individuals only had a small amount of their overall mumps igg antibody levels specific to the vaccine-strain (201) . neutralization antibodies against iowa-g/usa06 (the circulating wild type virus) were also present in pre-infection plasma of all mumps cases during a recent outbreak in the us (173) . this indicates that the mumps vaccine alone is not solely responsible for the high levels of mumps antibodies (202) , and that longterm antibody persistence or protective efficacy data of the vaccines used may not truly reflect the current circumstance of viral transmission/circulating within a highly vaccinated population (99) . herd immunity increases the chance for natural mumps boosting for an individual is at a minimum, reducing the potential of the frequency of mumps outbreaks (123, 124, 184) . with less opportunity for subclinical boosting (asymptomatic response to the circulating virus), the impact of other elements of waning immunity may play an increasingly critical role in the re-emergence of mumps outbreaks (98, 171) . additionally, as the heterogeneous uptake of vaccines in this modern era is leading to susceptible individuals within the community, future work will need to encompass genotyping of circulating muv to examine how impactful subclinical boosting was on early measures of vaccine efficacy in current populations. why do we consider antibodies to be the best measurement of vaccine efficacy? the evolution of an individual's immune response differs between natural infection and vaccination, in particular the difference in the affinity and specificity of an immunological marker such as antibodies (203) . true correlates of mumps immunity after vaccination have been poorly characterized; to date, there are no reliable correlates of protection from either symptomatic mumps infection (clinical immunity), or individuals previously exposed to muv (204) . therefore, a serological surrogate/ substitute is used (205). mumps vaccine efficacy is quantified by a single measure, igg which may not suffice to evaluate the magnitude of the actual humoral response. borgmann et al. (206) proposed an increase in mumps-specific igg titer in sera as a diagnostic criteria of mumps reinfection (206) . it has been suggested that vaccinated individuals have modified b-cell responses to muv that allow for the rapid generation of igg antibodies and a blunted or absent igm response (207, 208) . in addition, emerging data in simian immunodeficiency virus studies suggests that not all antibody responses are equal, and qualitative features of antibodies may be key to defining protective immune profiles (209) . despite its use, the correlation to mumps-specific igg concentrations and neutralization titers against the jl virus is poor, suggesting that igg concentrations do not adequately represent a sufficient surrogate correlate of protection (194) . this is demonstrated in finland; only 24% of vaccinees had no detectable mumps antibodies after 21 years (123, 124) . data from the european sero-epidemiology network (esen2) project in 2004 reported that mmr immunization uptake in ireland in 2004 was 92% (6), however it was also suggested that only 80-85% of 15-to 24-year-olds in ireland had detectable antibodies to muv by either natural immunity or immunization (210) . in 2011, vaccine coverage of medical students in germany was reported to be 75.1% (211) . in children between the ages of 1-17 years, where 88.8% had been vaccinated with the mmr vaccine at least once, only 76.8% showed prevalence of antibodies (212) . however, 7.8% showed a prevalence of antibodies to measles and rubella in the absence of mumps-specific antibodies. therefore, previous measurement of anti-mumps-specific igg that represented immunity induced by the mumps vaccine appears to be overestimated (99, 213) . antibody levels of other components of the mmr vaccine have seen similar trends. waning rubella antibody titers have been observed, despite the number of acute rubella and congenital rubella syndrome cases not increasing. it has also been shown that college students who received rubella vaccination during childhood and had low/no antibody response were able to mount a secondary response when challenged with rubella indicating that an individual's low antibody levels are not always indicative of susceptibility to infection (214) . measles antibodies can also be detected for up to a decade post-vaccination, with >90% of individuals still measles igg positive at 6-7 years of age (144, 215) . however, as with mumps and rubella, waning measles antibody titers have been observed (143, 216) . despite this, a recent longitudinal study of up to 10 years demonstrates how effective the mmr vaccine has been in preventing diagnosed measles cases during the 1990's/2000's (217) . similarly, three doses of the hepatitis b (hbv) vaccine in a cohort of alaskan natives showed >95% seroconversion in children and young adult post-vaccination and provided long term and durable protection against chronic hbv infection. although no increase of hbv prevalence were observed 51% individuals had low to undetectable antibody levels after 30 years. these observations suggest that an individual's antibody levels do not indicate susceptibility to infection, that either an antibody titer lower than recommended guidelines is still protective, or/and is an ineffective surrogate of protection. this is emphasized in a study by amanna et al.; (218) responses to non-replicating protein antigens (tetanus and diphtheria) were shown to have approximate antibody half-lives of 11-19 years. in comparison, antibodies following wild type infection were shown to have half-lives of 50 years or more which was thought until recently to confer a more prolonged lifelong protection (214, 218, 219) . however, reinfections observed in individuals that were previously naturally infected have demonstrated that the quantitative measurement of antibodies do not indicate sterile immunity (220) . it is also important to stress that seroconversion rates due to immunization/natural infection only reflects a change of antibody status from negative to positive, but not necessarily the intensity of antibody response. in addition, there is no consistency in the timing of sample collected post-vaccination to test vaccine efficacy, and between the serological tests utilized for detecting mumps antibodies. as a result, documented seroconversion rates of the mumps vaccines used vary widely (jl: 74-100%, rit 4385 strain: 88-98%, urabe am 9: 79-100%, rubini: 35-95%). this highlights that the assays used to detect immunity to muv may not always detect an adequate post-vaccination response. only a small number of serological commercial assays such as the detection of hepatitis b surface antibody (anti-hbs) (221) and rubella igg (222) have been designed using who reference material as a standard for quantification. however, even utilizing this reference standard demonstrates significant differences in the determined quantification of either anti-hbs or rubella igg depending on the assays used; although a value for anti-hbs of 10 iu/ml is regarded as protective against significant hbv infection, the detection of this anti-hbs is significantly influenced by which anti-hbs assays is used (223) (224) (225) (226) (227) . therefore, it is possible that the current assays/tests mechanisms utilized to measure mumps antibodies are too insensitive/inappropriate/crude to identify nuances in the immune response which could correlate with immunity against mumps. in addition, variation within neutralization epitopes i.e., the quality of the antibody present could be a more important correlate than quantity (190, 198) . though labor-intensive, neutralizing antibodies are considered to be a better correlate of mumps immunity. antibodies against the haemagglutinin-neuraminidase protein (hn) and nucleoprotein (np) have been shown to neutralize muv, however, repeated attempts to define a titer that provides a protective threshold titer have been inconclusive (203, 228) . in older studies, during field evaluations of the jl vaccine, neutralizing antibody titers of 1:2-1:4 in unvaccinated individuals was considered seropositive and protective from mumps infection (149, 151, 152) . using a more contemporary wild-type isolate (iowa-g/usa06), a 1:8 neutralizing titer cut off was defined between case patients and exposed patients, despite the fact that no cut-off could fully discern between the two groups (173) . however, that these results are dependent on the challenge virus strain used in the assay. rasheed et al. demonstrated a 6fold lower neutralization titer to the g-genotype when compared to the jl vaccine strain in 18-23 year olds (229) . this has also been seen between mumps vaccine strains vs. circulating strains in india and china (47, 197) . despite studies in more highly vaccinated populations demonstrating that hn-inhibiting titers after natural disease were 1:9 compared to 1:5 post-vaccination, neither appeared to prevent reinfection (173, (218) (219) (220) 230) . there is increasing evidence that the mumps-specific antibody response is broader than neutralization alone (112) . avidity testing for virus-specific igg has been proposed (3, 220, 229) . individuals who lack measurable mumps-specific antibody levels may be susceptible to infection but protected from significant illness as they may be protected by cell-mediated immune memory. prolonged t-cell responses are reported after other vaccinations; 14-16 years after a single dose of the rubella vaccine ra27/3, a t-cell proliferative response to neutralizing antibodyinducing peptides suggest t helper and b-cell interactions. this indicates that full vaccine effectiveness could be dependent on mounting both an antibody and cell-mediated immune response (214) . although cell mediated immunity has not been as wellassessed in mumps infection, a lymphoproliferative response was induced in infants vaccinated at 6, 9, or 12 months of age was induced (231) with antigen-specific t-cells reported to appear within 1 month of infection (183) . lymphoproliferative responses to measles and mumps vaccine viruses were shown to persist in two thirds of the population at least 6 years after immunization (232), with t-and b-cell immunity persisting for 10 years post-immunization (202) . low levels of mumps-specific memory b-cells have also been documented suggesting that mumps infection or vaccination may not generate a robust b-cell memory (136, 233) . two principal mechanisms for maintaining long-term humoral immunity have been proposed and reviewed by amanna et al. (218) : associations between memory b-cell levels and antibody may reflect an epiphenomenon in which serum antibody levels and memory b-cells are equally stable but independently maintained. if memory b-cells and plasma cells are independently regulated, then multiple re-exposures to antigens may cause divergence between memory b-cell levels and antibody levels (218) . antigens with the highest rates of boosting through vaccination or latent viral infection coincidentally showed the weakest association between memory b-cell titers and antibody titers (234). although the role and efficacy of t-cell immunity to mumps infection is unclear, there is a possibility that certain muv strains may be capable of escaping vaccine induced t-cell responses, which may not be considered of significance until b-cell waning immunity comes into play (198) . in individuals who did not respond to vaccination (i.e., had a ≤2-fold of mumps antibody titers 30 days post-vaccination), several genes including those implicated in antigen presenting, processing, t-cell response and function showed significantly increased expression, with mhc class ii hla-drb3 and hla-dra, and cd86 induced when compared to responders 1 day post-mmr vaccination. this may indicate that the stimulation of a rapid adaptive immune response limits antigenic presentation and hence prevent the differentiation of memory b-cells to antibody-producing plasma cells (154) . differences in predicted b-cell and t-cell epitopes between jl5 vaccine strain and other vaccine strains may also be implicated in the outbreaks witnessed (235) . although, it has also been shown that natural mumps infection or vaccination do not always induce both cellular and humoral immunity. de wit et al. (199, 236, 237) has shown the presence of th1-type cd4 + t-cells recognizing a muv epitope in a hlr-dr restricted manner. in addition, the response of ifn-γ and tnf producing cd8 + t-cells specific to muv epitopes are lower in vaccinated individuals when compared to individuals who were naturally infected (199, 213, (236) (237) (238) . utilizing current knowledge and new technologies may help define a better surrogate correlate of protection and potentially determine a cut-off between the immunity of a vaccinated individual and a secondary mumps infection. this may potentially move the diagnostic preference from serological tests to more comprehensive functional assays. despite the large resurgence of mumps outbreaks, there is insurmountable evidence highlighting the benefit of the mumps vaccine ( table 2) . routine childhood mmr vaccination has resulted in a dramatic decrease in the incidence of mumps cases, and has shifted the peak age-specific attack rates from a young children (manifesting between 5 and 15 years) to one that affects young adults, in particular those who have close interaction with other young adults (18-24 years) (6, 110) . additionally, clinical manifestations and severity of disease in vaccinated vs. unvaccinated individuals differ (129, 248) . although muv can be clinically asymptomatic in about 15-30% of those who become infected, the vaccine against mumps confers protection in a dose response manner; unvaccinated individuals saw an attack rate of based on the reduction seen upon the introduction of a mumps vaccine, it has been proposed that mmr vaccination also prevents the transmission of the virus. there is limited knowledge regarding the shedding and transmission of muv, but it is thought that close contact and transmission of a certain viral load may induce clinical symptoms (243, 246, 249) . modeling data suggests that infectious muv shedding decreases rapidly after the onset of symptoms, however 8-15% are patients are thought to still be virally shedding 5 days after the onset of symptoms (244) . this could be the reason why the transmission of muv can be exacerbated by close social situations within a heterogeneously vaccinated population. outbreaks generally occur in situations of intense contact such as college dormitories, boarding schools, and youth summer camps (191) , with up to a third reporting some contact with a mumps case (105) . evidence of lower levels of viral replication also suggests a clinical benefit of the vaccine (243, 244) . viral load and presence of the mumps vaccine genome in areas of viral replication was lower in vaccinated individuals vs. unvaccinated individuals (243) . in addition, patients who contracted mumps but had two doses of mmr have been shown to shed less muv in their urine, with fewer experiencing bilateral parotitis or orchitis than unvaccinated individuals (239), this suggests that immunity induced by mmr vaccination limits virus transmission and complications (241, 242) . it should be noted also that individuals who received two doses of mmr, and had a positive correlation between viremia, salivary viral loads and systematic clinical mumps infection may have an increased risk of transmitting virus. these individuals also lacked mature functional responses, with low neutralizing antibody titers and avidity indexes (239) . overall, evidence demonstrates a clinical advantage to receiving a mumps vaccine ( table 2) . currently no global consensus exists for the measurement of mumps antibodies, mumps avidity or neutralizing titers that correlate to vaccine response and protection in healthy individuals. if a biomarker is discovered, it could be utilized as an international diagnostic reference standard to allow global harmonization and evaluation of the relative effectiveness of the different vaccination programs worldwide. such an attempt was conducted by andrews et al. (250) , who reported on the european sero-epidemiology network project which was established to harmonize the seroepidemiology of five vaccine preventable infections including measles, mumps, and rubella in eight european countries. the study concluded that the development of an international standard for mumps would help in the standardization and comparability of mumps antibodies in the different enzyme immunoassays used in laboratories. however, to date, no international reference standard for mumps has been established. in response to infection, the human immune system launches a series of immunological responses with the goal of controlling or eliminating the pathogen. if the pathogen circumvents the frontline defense of the innate immune system, an adaptive immune response specific for the pathogen will become activated to respond, with the intention to generate humoral-and cell-mediated immunity. humoral immunity, represented by antibodies secreted by b-cells are not effective against pathogens that invade host cells. therefore, cell-mediated immunity instructed by the vaccination aims to stimulate the host immunological process and formation of cell-mediated immunological memory via the use of live-attenuated or of inactivated/subunit vaccine components to promote a cell-mediated immune response. extensive knowledge gaps significantly hinder improvements to the mumps vaccine and prospects for mumps eradication and maintaining proficient population immunity (3, 122, 187) . few studies have collected data that examines different aspects of mumps immunity and are limited in their predictive value for future outbreaks (253) . for example, the importance of t and b-cell responses in protective mumps immunity and how memory/plasma cell numbers are homeostatically maintained post-infection or vaccination is relatively unknown (252) . it should be acknowledged that the mechanism of protection of infection may not be the same mechanism of recovery from infection, which may make the identification of a common correlate of protection and recovery difficult (203) . therefore, if a correlate or surrogate correlate is unobtainable to define an individual's protection to mumps, should we re-consider and re-focus efforts on optimizing the vaccine using available historical clinical and trial data? it has been suggested that wild-type infection could confer a "better quality, " broader and prolonged immuno-activation than vaccine-induced immunity. this is reflected in mean neutralizing antibody titers detected post-mumps vaccination, which were over five times lower than those detected following wild type infection. similarly, hemagglutination-inhibiting titers after natural disease were 1:9 compared to 1:5 post-vaccination (214, 218, 219) . the use of a live-attenuated virus vaccine is intended to mimic immunological reactions and responses between the host and wild type virus (254) . the current liveattenuated mmr vaccine is intramuscularly injected, a route that significantly differs from the natural infection mode of transmission. however, emphasized by differing immunological kinetics between immunized and naturally infected individuals when subjected to wild type pathogens, injectable vaccines are considered not to be the best inducer of antigen-specific mucosal immune responses for mucosal pathogens, especially if the mode of administration is not the natural route (the respiratory tract) (255, 256) . improvements on a broader range of antigen delivery systems will improve vaccination strategies and potentially prolong the effect of a vaccination by producing a localized immunological response in the relevant tissues (257, 258) . mucosal vaccines such as intra-nasal vaccination have advantages over traditional injectable vaccines as they can induce an effective, more robust immune response without any physical discomfort and more closely replicate the natural route of infection for mumps (255, 259) . b-cells induced by the mucosal response are also capable of secreting iga class of antibodies in the lumen, where the interaction and neutralization of specific antigens form iga-antigen complexes are easily able to be entrapped in the mucus and eliminated by cilial epithelial cells (259) . activated mucosal lymphocytes can also reach other mucosal sites via the lymphatic system and have the capability to transfer immunity (260) . such an example is the intranasal immunization of inactivated influenza. with a 70-90% similar efficacy between the injectable and intranasal influenza in healthy individuals this intranasal vaccine can elicit the secretion of haemagglutinin and neuraminidase specific iga antibodies in the upper respiratory tract, and corresponding igg antibodies (258) . live, cold adapted attenuated nasal influenza vaccine has been routinely used in russia for over 50 years (261) . other liquid live-attenuated intranasal vaccines are available; "nasovac r " in india, and "flumist r " in the us, uk and new zealand (258, 259, 262) . inactivated vaccines consisting of heat/chemical or liveattenuating monovalent or multivalent pathogens in animals/cell lines were developed to protect against disease causing microorganisms (263) . less emphasis was placed on understanding the mechanisms related to conferring immunological memory; the focus lay on the availability, mass production and administration of the vaccine to introduce herd immunity into populations (264) . currently, the least expensive and time effective method to licensure is the comparison of serologic responses of the new vaccine to an existing licensed vaccine, which can lead to a bias on the development of novel vaccines (222) . this methodology also does not account for the fact that each vaccine developed elicits its own immunological signature and may need to be considered on an individual basis (265) . raymond et al. (266) has suggested that embryonated chicken egg-based vaccines may induce antibodies that are more preferential to egg adapted strains better than wild type virus. amino acid substitutions/differences in key antigenic targets due to the passage of the growing virus within this environment may optimize the growth of the virus, but could lead to differences over time that could affect the immunogenicity or potency of the vaccine (172, 222, 267) . the jl vaccine contains two isolates of the jl strain (jl2 and jl5) and whilst no immunological differences have been documented, jl2 grows to higher titers than jl5 in embryonic eggs and also demonstrates significant sequence variability (94, 268) . zost et al. (269) also demonstrated that an egg selected mutation within a glycosylation site in the 2016-2017 influenza vaccine strain led to the production of poorer neutralizing antibodies to the vaccine strain compared to wild type influenza virus. vaccine rit 4385 strain derived from one of the two distinct virus subtypes of the jl vaccine (jl5) showed comparable seroconversion rates despite inducing a significantly lower geometric mean antibody titer when compared to recipients of the jl vaccine, but does not have any longitudinal trials investigating its efficacy, even though there are populations who are currently receiving it (101, 270) . the significant time gap between pathogen emergence and vaccine licensure, could potentially lead to antigenic drift. there is potential that modern biotechnologies could be utilized to design novel vaccine platforms (251, 271, 272). clinically derived recombinant muv lacking the expression of the immunomodulatory v or sh protein are currently being investigated (273) . in china, a vaccine consisting of the prevalent wildtype virus genotype (f) has recently been produced and is currently undergoing trials (269) . in addition, despite being extremely pleomorphic, utilizing mhc epitopes as potential b-cell and t-cell vaccine candidates are also being investigated (81, 274, 275) . vaccine design has involved the utilization and templating of epitopes that previously induced a b-or t-cell response during natural disease that are considered to be immunogenic enough to induce similar responses if administered in a vaccine. however, the appropriate b-cell and t-cell epitope/peptide candidates to induce a protective immunological response can be difficult to correctly identify and synthesize, as it may differ to the immunodominant epitope and host presentation of that antigen (251, 276). prediction of mhc-peptide binding and cleavage has demonstrated mismatches in both vaccine tcell and b-cell epitopes in vaccinated individuals highlighting small number of distinguishing amino acid changes of the jl5 major strain (235) . the importance of understanding tand b-cell responses and how antigen-specific memory cells numbers are homeostatically maintained post-infection is crucial to understand to ensure successful vaccine development (252, 277) . since the 1990's, significant progress has also been made in developing flexible, amplifiable, scalable, inexpensive, and cold-chain free rna vaccines, such as synthetic mrna molecules encoding only the antigen of interest and selfamplifying rna (sa-rna) (264) . such examples include an experimental mrna vaccine candidate (mrna-1273) which encodes a stable form of the sars-cov-2 spike protein and has been accepted as a trial candidate for clinical trials in healthy male and female individuals (278, 279) . in addition, sa-rna viruses as gene delivery and vaccine vectors have also demonstrated therapeutic efficacy in a number of preclinical studies. in the context of influenza, sa-rna vaccines have shown comparable results of protection at lower doses than mrna vaccines (272, 280, 281) . exponential developments in the "omic" area has enabled further vaccine development and understanding of the immunological response and challenges surrounding this area (282) . systems vaccinology, which includes immunoformatics, dna/rnaseq, microarrays, mass spectrometry proteomics, transcriptomics, and metabolomics have all shown huge potential in elucidating differences in vaccine strains, vaccine growth and individual response in depth and on an epigenetic level allowing the identification of new vaccine antigens with increased speed and sensitivity (235, 263, (283) (284) (285) . adjuvants, a group of biological and chemical compounds could also be considered to enhance and improve the longevity of the immune response of a vaccine such as the mmr. adjuvants have been successful in significantly reducing overall antigen dose in vaccine formulations as well as alter and broaden the host response through epitope spreading and qualitatively shaping the effector function of antibodies through subclass selection (173, 286) . the re-purposing of live-attenuated vaccines as tibv are also being investigated. trained immunity based vaccines (tibv) elicit heterologous protective effects by inducing a broader, lasting priming of innate immune cells, in addition to the intended specific immunological response and memory of conventional vaccines [reviewed in (287) ]. mmr and bcg vaccines have been considered as potential tibv in the context of the current coronavirus disease 2019 (covid-19) pandemic (288) , however further research is needed. the mumps component of a vaccine is an unpurified product whose potency is measured through a biological assay for the substance rather than through evaluation of integrity of physical form (quantitative pcr after cell culture) (289) . a monovalent mumps vaccine lot is used to characterize the performance of the mumps potency assay with international reference standards. degradation products are neither identified nor quantified (290) . currently, the minimum potency of the mumps vaccine used varies between brands used [summarized by su et al. (107) ] (291) . however, this potency measurement differs to other mmr vaccines strains previously used [reviewed in (10) ]. in addition, the maximum required potency is not usually specified. atrasheuskaya et al. (172) demonstrated that the four out of 14 lots of vaccine associated with six cases of viral transmission postvaccination to previously vaccinated contacts were in fact twice as potent as the lots that were not associated with viral transmission post-vaccination (172, 292) . this may impact the use and efficacy of specific vaccines. due to their neurovirulence and increased incidence of aseptic meningitis and mumps cases, the urabe am 9 and rubini mumps vaccine strains were discontinued in many countries (87, 293, 294) . comparing alternative culturing technologies and defining a viral potency range for vaccines could help reduce variability within the mmr vaccine (292) . ensuring the use of a reference sample that had similar replication rate and composition as the virus to be tested will allow accurate determination of the quantity of virus present per lot of vaccine. investigating novel vaccine candidates shown to induce a similar quantity but qualitatively different antibodies will help segregate and reveal potential correlates of protection (209) . incorporating more modern technologies such as microarray technology or antibody pattern/profiling (rather than single antibody measures) to investigate biomarkers of neutralizing antibody response and/or correlates of protective immunity, in addition to incorporating what has been accomplished in finland will allow further understanding of mumps immunity (123, 124, 173, 195, 196, 295) . the efficacy of a vaccine is defined by disease prevention (sterile immunity, establishment of primary infection and shedding of mature virus particle), or complications associated with infection (orchitis, neurological issues etc.) (203) . despite the well-documented success of the global immunization programs demonstrating how vaccines significantly attenuate disease and onward transmission of infection, they are rarely totally efficacious (demonstrated in pre-licensure clinical trials) or effective (determined by practical use) (99, 173, 296) . therefore, does "immunity" refer to sterile immunity or solely to protection from symptomatic infection? what defines an effective vaccine, or what constitutes vaccine failure? does the medical profession and the "pro-vaccine" message contribute to the public skepticism regarding immunization? is it time to shift the medical and public perception paradigm from "protection of infection following vaccination" to "protection from serious clinical mumps manifestation"? the lack of definition leads to misinterpretation by health professionals and media of what is truly occurring. such an example is currently observed with influenza; individuals who have recently being vaccinated against influenza and subsequently become infected with influenza, assume that the vaccine has "failed" even though there is a reduction in symptoms. the current assertion that vaccines "protect against" or "eliminate" the risk of infection may contribute to the misperception about what level of protection a vaccine actually provides (vaccination efficacy) perpetuated by the witnessing of visible clinical disease and outbreaks despite vaccination (116, 297, 298) . therefore, definition and consensus of what is termed a true "vaccine failure" is required to inform both the clinical and public perception of what the function of a vaccine is. deciding what the clinical endpoint of a vaccine is i.e., infection with mild clinical symptoms vs. natural infection/disease with its associated complications and assessing the impact of the vaccine in a heterogeneously vaccinated population will allow a better consensus of what is required. a paradigm shift in what is considered to be a good vaccine i.e. one that provides protection against serious clinical sequalae, in addition to identifying a reliable laboratory marker for this protection is required (203) . by focusing on, and acknowledging that vaccines may not prevent infection but will attenuate the clinical complications/consequences that arise from infection in addition to reducing onward transmission will provide a more realistic view of the benefits of vaccination (297) . immunity is therefore beneficial but does not necessarily mean protection. if we can decide whether the end point of a vaccine is either the prevention of infection or protection against serious sequalae of infection, its efficacy and impact can be determined and will have enormous implications on how vaccine failure can be studied, quantified and interpreted. this teasing out of the immunological response to muv will ultimately provide potential correlates with robust predictive power, suggest directions for further vaccine improvement, and enable the discovery of potential biomarkers to help create a more efficient diagnostic assay that can discern between different infectious diseases and vaccination vs. disease status. the identification and incorporation of a correlate into diagnostic protocols which can be widely accessible may potentially allow global harmonization of criteria defining immunological protection against mumps. the medical and scientific field needs to inform the public more accurately about what a good vaccine consists of, which may result in a more positive attitude toward vaccines. in the majority of individuals, a vaccine can prevent serious clinical sequalae and associated complications following wild type infections, but also significantly reduce onwards transmission in particular to the cohorts who are not vaccinated due to a contraindication to vaccination. this is the positive and realistic view of vaccination which should be presented rather than the current flawed message of "get the vaccine and be protected from infection." the public deserves, and will appreciate, a more accurate and informed message. ac, jc, and jh contributed to the conception and design of the review. ac wrote the first draft of the manuscript. jc, tl, and jh contributed to manuscript revision. all authors have read and approved the submitted version. this work was funded by the national children's research centre, children's health ireland, dublin, ireland with grant number c/18/11 awarded to jh. mumps virus analysis of mumps vaccine failure by means of avidity testing for mumps virus-specific immunoglobulin g infectiousness of communicable diseases in the household (measles, chickenpox, and mumps) hearing loss due to mumps molecular epidemiological evaluation of the recent resurgence in mumps virus infections in ireland isolation of virus during the incubation period of mumps infection function of small hydrophobic proteins of paramyxovirus proposed criteria for classification of new genotypes of mumps virus mumps in the vaccination age: global epidemiology and the situation in germany measles resurgence in argentina: 1997-8 outbreak available online at triple viral/double viral immunization: coverage data of mmr1 and mmr2 national vaccination calender everything you need to know about the new vaccination law in argentina molecular identification of mumps virus genotypes from clinical samples: standardized method of analysis genomic diversity of mumps virus and global distribution of the 12 genotypes world health organization. mumps reported cases (as of austrailian government department of health. 2.7 mumps (last updated mumps laboratory case definitnion austrailian institute of health and welfare parental opinions towards the "no jab, no pay a protracted mumps outbreak in western australia despite high vaccine coverage: a population-based surveillance study immunogenicity and safety of the combined vaccine for measles, mumps, and rubella isolated or combined with the varicella component administered at 3-month intervals: randomised study calendários de vacinação sbim vacina sarampo, caxumba, rubéola e varicela (atenuada) outbreak of aseptic meningitis associated with mass vaccination with a urabe-containing measles-mumps-rubella vaccine: implications for immunization programs outbreak of aseptic meningitis and mumps after mass vaccination with mmr vaccine using the leningrad-zagreb mumps strain detection of a new mumps virus genotype during parotitis epidemic of 2006-2007 in the state of sao paulo, brazil reemergence of mumps in sao paulo, brazil-the urgent need for booster shot campaign to prevent a serious infectious disease guidelines for the prevention and control of mumps outbreaks in canada (archived) guidelines for the prevention and control of mumps outbreaks in canada. mumps-containing vaccine and immunization programs in canada immunization coverage report for school pupils in ontario. 2016-17 school year. toronto, on: queen's printer for ontario investigation and management of a large community mumps outbreak among young adults in toronto guidelines: mumps in canada assessment of onedose mumps-containing vaccine effectiveness on wild-type genotype f mumps viruses circulating in mainland china mumps epidemiology and mumps virus genotypes circulating in mainland china during 2013-2015 importation of mumps virus genotype k to china from vietnam waning immunity against mumps in vaccinated young adults pediatric vaccines: global brands and country availability available online at information sheet observed rate of vaccine reactions. measles, mumps and rubella vaccines routine immunization: regional and country profiles summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for 2017 submitted through the who/unicef joint reporting form on immunization measles elimination efforts and 2008-2011 outbreak progress toward measles elimination in germany immunogenicity of mumps virus vaccine candidates matching circulating genotypes in the united states and china routine immunization: regional and country profiles; germany. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for 2017 submitted through the who/unicef joint reporting form on immunization list of the names, pharmaceutical forms, strengths of the medicinal products, routes of administration, marketing authorisation holders in the member states (annex 1, article 31) mumps and mumps vaccine: a global review live attenuated measles mumps and rubella vaccines: an over view the epidemiology of mumps in italy pediatric sentinel surveillance of vaccinepreventable diseases in italy available online at the law on compulsory vaccination in italy: an update 2 years after the introduction mmr vaccination and autism european centre for disease prevention and control. 2020: annual epidemiological report for 2017; mumps measles in mexico, 1941-2001: interruption of endemic transmission and lessons learned detection of mumps virus genotype h in two previously vaccinated patients from mexico city missed opportunities for measles, mumps, and rubella (mmr) immunization in mesoamerica: potential impact on coverage and days at risk el programa nacional de vacunacion: orgullo de mexico progress toward measles elimination in the russian federation routine immunization: regional and country profiles; russian federation. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for 2017 submitted through the who/unicef joint reporting form on immunization mumps vaccine failure investigation in safety evaluation of mmr vaccine during a primary school campaign in saudi arabia measles in saudi arabia: from control to elimination measles immunization in saudi arabia: the need for change ministry of health saudi arabia. ministries of health and education start a vaccination campaign against measles, mumps and rubella reemergence of mumps genetic characteristics of mumps viruses isolated in korea from 2007 to 2012 increasing mumps incidence rates among children and adolescents in the republic of korea: age-period-cohort analysis compulsory vaccines to be applied to baby after birth in turkey the national vaccination schedule in previously healthy children: the practical recommendations about additional vaccines routine immunization: regional and country profiles; turkey. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for 2017 submitted through the who/unicef joint reporting form on immunization genotyping of mumps virus circulating in turkey in the 2006-2007 winter season risks of convulsion and aseptic meningitis following measles-mumps-rubella vaccination in the united kingdom nhs vaccinations and when to have them routine immunization: regional and country profiles; united kingdom of great britain and northern ireland. summary of routine immunization and vaccine-preventable diseases surveillance data, based primarily on data for 2017 submitted through the who/unicef joint reporting form on immunization proteomics for development of vaccine viral mumps: increasing occurrences in the vaccinated population. oral surg oral med oral pathol oral radiol centre for disease control and prevention. recommended child and adolescent immunization schedule for ages 18 years or younger, united states vaccination coverage for selected vaccines and exemption rates among children in kindergarten -united states, 2017-18 school year vaccination coverage among children aged 19-35 months -united states the molecular epidemiology of mumps virus the immunological basis for immunization series module 16: mumps. world health organization vaccination of human beings against mumps; vaccine administered at the start of an epidemic. i. incidence and severity of mumps in vaccinated and control groups preparation of mumps vaccines and immunization of monkeys against experimental mumps infection european centre for disease prevention and control. mumps: vaccine scheduler available online at: https://vaccineschedule.ecdc.europa.eu/scheduler/bydisease? live attenuated mumps virus vaccine. 1. vaccine development increased mumps incidence in the netherlands: review on the possible role of vaccine strain and genotype molecular differences between two jeryl lynn mumps virus vaccine component strains, jl5 and jl2 the jeryl lynn vaccine strain of mumps virus is a mixture of two distinct isolates health protection surveillance centre. definition of an outbreak estimation of the efficacy of three strains of mumps vaccines during an epidemic of mumps in the geneva canton (switzerland) comparative efficacy of rubini, jeryl-lynn and urabe mumps vaccine in an asian population estimates of mumps seroprevalence may be influenced by antibody specificity and serologic method mumps outbreak in a highly vaccinated student population mumps outbreak, england. emerg infect dis mumps outbreak in the republic of moldova mumps outbreak in jerusalem affecting mainly male adolescents ongoing mumps outbreak among adolescents and young adults mumps outbreak in a highly vaccinated university-affiliated setting before and after a measles-mumps-rubella vaccination campaign-iowa mumps outbreaks at four universities-indiana current status of mumps virus infection: epidemiology, pathogenesis, and vaccine mumps: an update on outbreaks, vaccine efficacy, and genomic diversity a report on the status of vaccination in europe mumps resurgences in the united states: a historical perspective on unexpected elements failure to vaccinate and vaccine failure the burden of disease and the changing task of medicine the politics of prevention: anti-vaccinationism and public health in nineteenth-century england a population-based cohort study of undervaccination in 8 managed care organizations across the united states world health organization. meeting of the strategic advisory group of experts on immunization misinformation lingers in memory: failure of three pro-vaccination strategies delaying vaccination is not a safer choice reactogenicity and immunogenicity of a new live attenuated combined measles, mumps and rubella vaccine in healthy children measles, mumps, rubella vaccination and autism: a nationwide cohort study measles, mumps and rubella (mmr) vaccination has no effect on cognitive development in children-the results of the polish prospective cohort study available online at sustained transmission of mumps in a highly vaccinated population: assessment of primary vaccine failure and waning vaccine-induced immunity rapid effect on endemic measles, mumps, and rubella of nationwide vaccination programme in finland measles, mumps, and rubella in finland: 25 years of a nationwide elimination programme knowledge gaps persist and hinder progress in eliminating mumps recent resurgence of mumps in the united states brief report: update: mumps activity-united states an outbreak of mumps in sweden mumps outbreaks in canada and the united states: time for new thinking on mumps vaccines dynamics of the serologic response in vaccinated and unvaccinated mumps cases during an epidemic challenges in interpretation of diagnostic test results in a mumps outbreak in a highly vaccinated population laboratory-based investigation of suspected mumps cases submitted to the german national reference centre for measles, mumps, and rubella comparison of the sensitivity of laboratory diagnostic methods from a wellcharacterized outbreak of mumps in new york city in 2009 enzyme-linked immunospot assay detection of mumps-specific antibodysecreting b cells as an alternative method of laboratory diagnosis mumps outbreak in a highly vaccinated school population: assessment of secondary vaccine failure using igg avidity measurements seroepidemiology of the recent mumps virus outbreaks in ireland laboratory testing and phylogenetic analysis during a mumps outbreak in ontario \sim:text=rt%2dpcr%20and %20viral%20culture,aid%20in%20diagnosing%20mumps%20infection diagnosis of acute mumps infection during an outbreak in a highly vaccinated population: mumps rna or mumps igm detection? monitoring viral genetic variation as a tool to improve molecular diagnostics for mumps virus persistence of mumps antibodies after 2 doses of measles-mumps-rubella vaccine persistence of measles, mumps, and rubella antibodies in an mmr-vaccinated cohort: a 20-year follow-up mumps vaccine performance among university students during a mumps outbreak emerging mumps infection epidemiology of a mumps outbreak in a highly vaccinated island population and use of a third dose of measles-mumps-rubella vaccine for outbreak control-guam long-term follow-up for immunity after monovalent or combined live measles, mumps, and rubella virus vaccines persistence of antibody in human subjects for 7 to 10 years following administration of combined live attenuated measles, mumps, and rubella virus vaccines studies on live attenuated mumps vaccine. i. comparative field trials with two different live vaccines live attenuated mumps-virus vaccine. iv. protective efficacy as measured in a field evaluation live attenuated mumps-virus vaccine. 3. clinical and serologic aspects in a field evaluation experiences with jeryl lynn strain live attenuated mumps virus vaccine in a pediatric outpatient clinic genomic signature of early t-cell response is associated with lower antibody titer threshold for sterilizing immunity the effectiveness of the mumps component of the mmr vaccine: a case control study measles, mumps, rubella vaccine (priorix; gsk-mmr): a review of its use in the prevention of measles, mumps and rubella immunogenicity and safety of a measles-mumps-rubella vaccine administered as a first dose to children aged 12 to 15 months: a phase iii, randomized, noninferiority, lot-to-lot consistency study a new measles mumps rubella (mmr) vaccine: a randomized comparative trial for assessing the reactogenicity and immunogenicity of three consecutive production lots and comparison with a widely used mmr vaccine in measles primed children immunogenicity and safety of measles-mumps-rubella-varicella (mmrv) vaccine followed by one dose of varicella vaccine in children aged 15 months-2 years or 2-6 years primed with measles-mumps-rubella (mmr) vaccine similar immunogenicity of measles-mumps-rubella (mmr) vaccine administrated at 8 months versus 12 months age in children immune response to the mumps component of the mmr vaccine in the routine of immunisation services in the brazilian national immunisation program immunogenicity and safety of measles-mumpsrubella vaccine delivered by disposable-syringe jet injector in india: a randomized, parallel group, non-inferiority trial safety and immunogenicity of human serum albumin-free mmr vaccine in us children aged 12-15 months immunogenicity and safety of early vaccination with two doses of a combined measles-mumps-rubella-varicella vaccine in healthy indian children from 9 months of age: a phase iii, randomised, non-inferiority trial duration of the immune response to mmr vaccine in children of two age-different groups antibody persistence for 3 years following two doses of tetravalent measlesmumps-rubella-varicella vaccine in healthy children a combined measles, mumps, rubella and varicella vaccine (priorix-tetra): immunogenicity and safety profile centers for disease control and prevention. prevention of measles, rubella, congenital rubella syndrome, and mumps, 2013: summary recommendations of the advisory committee on immunization practices (acip) primary vaccine failure to routine vaccines: why and what to do? evaluation in young children of the urabe am 9 strain of live attenuated mumps vaccine in comparison with the jeryl lynn strain safety and characterization of the immune response engendered by two combined measles, mumps and rubella vaccines horizontal transmission of the leningrad-3 live attenuated mumps vaccine virus mumps antibody levels among students before a mumps outbreak: in search of a correlate of immunity primary vaccine failure after 1 dose of varicella vaccine in healthy children outbreak of mumps in a vaccinated child population: a question of vaccine failure? measles, mumps, and rubella-vaccine use and strategies for elimination of measles, rubella, and congenital rubella syndrome and control of mumps: recommendations of the advisory committee on immunization practices (acip) antibodies to measles, mumps and rubella in uk children 4 years after vaccination with different mmr vaccines vaccine-induced measles virus antibodies after two doses of combined measles, mumps and rubella vaccine: a 12-year follow-up in two cohorts evaluation of cellular immunity to mumps in vaccinated individuals with or without circulating antibodies up to 16 years after their last vaccination longterm persistence of mumps antibody after receipt of 2 measles-mumpsrubella (mmr) vaccinations and antibody response after a third mmr vaccination among a university population immunity to mumps before and after mmr vaccination at 12 years of age in the first generation offered the two-dose immunization programme vaccine waning and mumps reemergence in the united states differential durability of immune responses to measles and mumps following mmr vaccination antibody induced by immunization with the jeryl lynn mumps vaccine strain effectively neutralizes a heterologous wild-type mumps virus associated with a large outbreak mumps outbreaks in a highly vaccinated population: investigation of a neutralization titre against the current circulating wildtype genotype g5 mumps virus immunogenicity and reactogenicity of a new measles, mumps and rubella vaccine when administered as a second dose at 12 y of age sera from different age cohorts in belgium show limited crossneutralization between the mumps vaccine and outbreak strains effectiveness of a third dose of mmr vaccine for mumps outbreak control antigenic relationships between six genotypes of the small hydrophobic protein gene of mumps virus serological and phylogenetic evidence of monotypic immune responses to different mumps virus strains remembering mumps phylogenetic analysis of clinical mumps virus isolates from vaccinated and non-vaccinated patients with mumps during an outbreak rt-pcr based diagnosis and molecular characterisation of mumps viruses derived from clinical specimens collected during the 1996 mumps outbreak in portugal mumps-specific cross-neutralization by mmr vaccine-induced antibodies predicts protection against mumps virus infection antigenic differences between vaccine and circulating wild-type mumps viruses decreases neutralization capacity of vaccine-induced antibodies identification of conformational neutralization sites on the fusion protein of mumps virus cross-neutralization between three mumps viruses & mapping of haemagglutininneuraminidase (hn) epitopes recent mumps outbreaks in vaccinated populations: no evidence of immune escape identification of naturally processed mumps virus epitopes by mass spectrometry: confirmation of multiple cd8+ t-cell responses in mumps patients seroprevalence of mumps in the netherlands: dynamics over a decade with high vaccination coverage and recent outbreaks investigation of mumps vaccine failures in minsk immune responses to mumps vaccine in adults who were vaccinated in childhood correlates of protection induced by vaccination correlations among measles virus-specific antibody, lymphoproliferation and th1/th2 cytokine responses following measles-mumps-rubella-ii (mmr-ii) vaccination mumps virus infection in vaccinated patients can be detected by an increase in specific igg antibodies to high titres: a retrospective study laboratory diagnosis of mumps in a partially immunized population: the nova scotia experience mumps outbreak and laboratory diagnosis antibody fab-fc properties outperform titer in predictive models of siv vaccine-induced protection increase in mumps in ireland in late assessment of mumps virus-specific antibodies by different serological assays: which test correlates best with mumps immunity? seroprevalence of measles-, mumpsand rubella-specific igg antibodies in german children and adolescents and predictors for seronegativity cellular and humoral immunity after vaccination or natural mumps infection rubella specific cell-mediated and humoral immunity following vaccination in college students with low antibody titers measles, mumps, and rubella antibody patterns of persistence and rate of decline following the second dose of the mmr vaccine development and durability of measles antigen-specific lymphoproliferative response after mmr vaccination childhood mmr vaccination and the incidence rate of measles infection: a ten year longitudinal cohort study of american children born in the 1990s duration of humoral immunity to common viral and vaccine antigens mumps outbreaks in vaccinated populations: are available mumps vaccines effective enough to prevent outbreaks? symptomatic mumps virus reinfections complete genome sequence of the who international standard for hepatitis b virus dna the establishment of surrogates and correlates of protection: useful tools for the licensure of effective influenza vaccines? duration of immunogenicity and efficacy of hepatitis b vaccine in a yupik eskimo population a longitudinal hepatitis b vaccine cohort demonstrates long-lasting hepatitis b virus (hbv) cellular immunity despite loss of antibody against hbv surface antigen antibody levels and protection after hepatitis b vaccine: results of a 30-year follow-up study and response to a booster dose protection provided by hepatitis b vaccine in a yupik eskimo population. seven-year results long-term efficacy of active postexposure immunization of infants for prevention of hepatitis b virus infection. united states-people's republic of china study group on hepatitis b mumps virus-specific antibody titers from pre-vaccine era sera: comparison of the plaque reduction neutralization assay and enzyme immunoassays decreased humoral immunity to mumps in young adults immunized with mmr vaccine in childhood mumps virus neutralizing antibodies do not protect against reinfection with a heterologous mumps virus genotype immune responses to measles and mumps vaccination of infants at 6, 9, and 12 months correlates of lymphoproliferative responses to measles, mumps, and rubella (mmr) virus vaccines following mmr-ii vaccination in healthy children detection of mumps virus-specific memory b cells by transfer of peripheral blood mononuclear cells into immune-deficient mice are cases of mumps in vaccinated patients attributable to mismatches in both vaccine t-cell and b-cell epitopes?: an immunoinformatic analysis the human cd4(+) t cell response against mumps virus targets a broadly recognized nucleoprotein epitope mumps infection but not childhood vaccination induces persistent polyfunctional cd8(+) t-cell memory cellular immunity to mumps virus in young adults 21 years after measles-mumps-rubella vaccination severity of mumps disease is related to mmr vaccination status and viral shedding mumps vaccination coverage and vaccine effectiveness in a large outbreak among college students-iowa characteristics of a large mumps outbreak: clinical severity, complications and association with vaccination status of mumps outbreak cases transmission of mumps virus from mumps-vaccinated individuals to close contacts the duration of mumps virus shedding after the onset of symptoms detection of rna of mumps virus during an outbreak in a population with a high level of measles, mumps, and rubella vaccine coverage notes from the field: absence of asymptomatic mumps virus shedding among vaccinated college students during a mumps outbreak-washington guidance for isolation precautions for mumps in the united states: a review of the scientific basis for policy change mumps clinical diagnostic uncertainty two major mumps genotype g variants dominated recent mumps outbreaks in the netherlands the european sero-epidemiology network: standardizing the enzyme immunoassay results for measles, mumps and rubella from vaccines to memory and back systematic review of measles and rubella serology studies how to determine protective immunity in the post-vaccine era challenges in mucosal vaccines for the control of infectious diseases correlates, surrogates, and vaccines aerosolized mmr vaccine: evaluating potential transmission of components to vaccine administrators and contacts of vaccinees vaccines against mucosal infections intranasal immunization with dry powder vaccines mucosal immunity and vaccines live attenuated pandemic influenza vaccine: clinical studies on a/17/california/2009/38 (h1n1) and licensing of the russian-developed technology to who for pandemic influenza preparedness in developing countries live attenuated influenza vaccine (flumist(r); fluenz): a review of its use in the prevention of seasonal influenza in children and adults proteomic contributions to our understanding of vaccine and immune responses advances in mrna vaccines for infectious diseases molecular signatures of antibody responses derived from a systems biology study of five human vaccines influenza immunization elicits antibodies specific for an egg-adapted vaccine strain influenza vaccine failure: failure to protect or failure to understand? sequence diversity of jeryl lynn strain of mumps virus: quantitative mutant analysis for vaccine quality control contemporary h3n2 influenza viruses have a glycosylation site that alters binding of antibodies elicited by egg-adapted vaccine strains stobart cc, moore ml. development of next-generation respiratory virus vaccines through targeted modifications to viral immunomodulatory genes self-amplifying rna vaccines give equivalent protection against influenza to mrna vaccines but at much lower doses immunogenicity of novel mumps vaccine candidates generated by genetic modification discovering protective cd8 t cell epitopes-no single immunologic property predicts it! characterization of peptides bound to the class i mhc molecule hla-a2.1 by mass spectrometry identifying epitopes of hiv-1 that induce protective antibodies contrasting b celland t cell-based protective vaccines preclinical and clinical demonstration of immunogenicity by mrna vaccines against h10n8 and h7n9 influenza viruses safety and immunogenicity study of 2019-ncov vaccine (mrna-1273) for prophylaxis of sars-cov-2 infection (covid-19) kunjin virus replicons: an rnabased, non-cytopathic viral vector system for protein production, vaccine and gene therapy applications rna viruses as tools in gene therapy and vaccine development vaccinomics: current findings, challenges and novel approaches for vaccine development vaccines for the 21st century the role of systems biology approaches in determining molecular signatures for the development of more effective vaccines adjuvants and alternative routes of administration towards the development of the ideal influenza vaccine trained immunity-based vaccines: a new paradigm for the development of broad-spectrum anti-infectious formulations could an unrelated live attenuated vaccine serve as a preventive measure to dampen septic inflammation associated with covid-19 infection? mbio potency estimation of measles, mumps and rubella trivalent vaccines with quantitative pcr infectivity assay evaluation of medicines for human use quality of biotechnological products: viral safety evaluation of biotechnology products from cell lines of human or animal origin a framework for research on vaccine effectiveness the urabe am9 mumps vaccine is a mixture of viruses differing at amino acid 335 of the hemagglutininneuraminidase gene with one form associated with disease studies on live mumps virus vaccine. v. development of a new mumps vaccine "am 9" by plaque cloning highly parallel characterization of igg fc binding interactions the concept of vaccination failure effective messages in vaccine promotion: a randomized trial it's not all about autism: the emerging landscape of anti-vaccination sentiment on facebook key: cord-282081-qaagup4d authors: flicker, sabine; zettl, ines; tillib, sergei v. title: nanobodies—useful tools for allergy treatment? date: 2020-09-30 journal: front immunol doi: 10.3389/fimmu.2020.576255 sha: doc_id: 282081 cord_uid: qaagup4d in the last decade single domain antibodies (nanobodies, v(h)h) qualified through their unique characteristics have emerged as accepted and even advantageous alternative to conventional antibodies and have shown great potential as diagnostic and therapeutic tools. currently nanobodies find their main medical application area in the fields of oncology and neurodegenerative diseases. according to late-breaking information, nanobodies specific for coronavirus spikes have been generated these days to test their suitability as useful therapeutics for future outbreaks. their superior properties such as chemical stability, high affinity to a broad spectrum of epitopes, low immunogenicity, ease of their generation, selection and production proved nanobodies also to be remarkable to investigate their efficacy for passive treatment of type i allergy, an exaggerated immune reaction to foreign antigens with increasing global prevalence. type i allergy, an ige antibody mediated hypersensitivity disease, represents a common health problem affecting almost 30% of the population worldwide (1) . the recognition of allergens by specific ige antibodies that are generated after sensitization is a key event for the initiation of allergic inflammation (2) . allergic patients suffer from a variety of allergic symptoms including rhinoconjunctivitis and asthma (3) but also food allergy and skin inflammation (4) . these clinical manifestations cause a major burden by reducing the quality of life of affected persons (5) . while anti-inflammatory treatment based on pharmacotherapy reduces allergic symptoms and is the most commonly prescribed medication for treatment of allergic patients (6) , only allergenspecific immunotherapy (ait) represents a causative treatment of type i allergy. in fact, ait induces a protective immunity in allergic patients based on the modification of cellular and humoral responses to the disease causing allergen (7) . besides the inhibition of ige binding to their specific allergen, the immune deviation from a th2 to th1 response, and the decreases in numbers of effector cells in target organs, the generation and maintenance of allergen-specific regulatory t and b cells and the involvement of their suppressive cytokines are essential for the induction of allergen tolerance (8) (9) (10) . beyond doubt the improvement of allergic symptoms is further caused by ait-induced igg antibodies found in serum and nasal secretions (8, (11) (12) (13) (14) . for many years ait was conducted with aqueous natural allergen extracts and patients experienced considerable side effects due to the unpredictable composition and poor quality of the injected extracts (1) . recent developments like next-generation forms of ait based on molecular approaches may overcome the limitations of current forms of ait (15, 16) . the last generation of improved vaccines, i.e. peptide carrier vaccines, induces an igg response that targets ige binding sites on allergens. induced igg antibodies effectively block ige binding and are termed blocking antibodies (1, 17) . however, the efficacy of such blocking antibodies was long questioned because it revealed to be cumbersome to isolate reproducible defined, i.e. monoclonal allergen-specific antibodies comprising the capacity to inhibit allergen-induced allergic reactions. a recent proof of concept study re-stimulated the idea to generate monoclonal allergen-specific antibodies and to evaluate their feasibility for allergy treatment. the authors could show that a single subcutaneous injection of a mixture of two human monoclonal allergen-specific igg4 antibodies significantly reduced allergic symptoms in allergic patients (18, 19) . moreover, validated in a pca mouse model, the mixture of these two monoclonal antibodies proved to be more potent in inhibiting mast cell degranulation than igg antibodies purified from patients' sera who underwent successful ait (18) . furthermore, these human monoclonal igg4 antibodies recently completed the phase ii clinical trial in treatment of cat allergic patients (https://clinicaltrials.gov/ct2/show/nct03838731). these results proved for the first time that allergy treatment with monoclonal allergen-specific antibodies is a well-tolerated, rapid, and effective approach to reduce allergic inflammation and rekindled the blocking antibody concept (11, 20, 21) . nevertheless, the generation and identification of blocking conventional human or humanized antibodies is connected with high costs for production, validation and application (22, 23) . therefore, cost-effective alternatives are currently sought. the nanobody technology represents such an alternative implying a significant improvement to the laborious methods to obtain monoclonal blocking conventional antibodies. due to their beneficial properties of small molecules and monoclonal antibodies, nanobodies in general are an attractive agent for development of novel therapeutic strategies (24, 25) . the ease of their generation and production, the single domain organization, their beneficial biochemical properties and their feature to recognize small cavities on the surface of antigens and hence bind to epitopes inaccessible for conventional antibodies (26) have raised the particular interest of allergologists recently. can the nanobody technology provide enhanced opportunity to generate a panel of antigen-binding molecules with various epitope specificities for certain allergens different to conventional antibodies? will these identified allergen-specific nanobodies be more efficient in blocking than conventional igg antibodies due to their pronounced cleft recognition? will it be possible with this technology to find single nanobodies that are able to abrogate ige-mediated allergic inflammation? these questions and our wish to answer these questions attracted our attention. within this review, we focus on the powerful nanobody technology to generate allergen-specific nanobodies and report on their evaluation for prospective application for passive allergy treatment. if allergologists are asked why the search for effective protective allergen-specific monoclonal antibodies is complex and laborious, they will describe this issue by the typical quest for a needle in a haystack. through intense and precise molecular and immunological exploration of available allergen-specific monoclonal antibodies in the past it was proven that epitope specificity and affinity are decisive for their inhibitory potential to block ige binding and thus ige-mediated reactions (21, (27) (28) (29) . the commitment to find and isolate monoclonal antibodies with specificity and high affinity for certain allergens and even more for certain epitopes always started with several fundamental decisions. amongst them the choice for the perfect source to gain dna coding for antibodies and the applied technology to generate allergen-specific antibodies are two of the most critical ones. regarding the dna source both animals, mainly mice, and humans served as blood, spleen, tonsils and even bone marrow donors in the last decades to isolate b cells or plasma cells and thus dna coding for antibodies (30) (31) (32) . for the proof of principle, murine igg antibodies overlapping with human ige binding sites are valuable tools to investigate the effects to inhibit ige epitope recognition on allergens and consequently to contribute to the design of hypoallergenic derivatives suitable for ait (33) . however, the direct therapeutic use of these murine monoclones in humans is limited by the high incidence of harmful immune responses against these administered foreign proteins (34) . to mitigate this limitation numerous murine monoclonal antibodies have been re-engineered by chimerization and humanization technologies. these expensive procedures are justified for fatal diseases like different forms of cancer but were barely applied for allergen-specific murine antibodies so far with a few exceptions (35, 36) . this was one of the main reasons why allergologists in the recent past endeavour to focus on human donors including allergic patients, ait-treated patients and even healthy individuals depending on the research question (28, 37, 38) . various methods were utilized to generate allergen-specific genuine, i.e. native antibodies with the preservation of the natural vh and vl pairing including hybridoma technology, epstein-barr-virus (ebv) transformation, single b cell sorting and cloning and humab mice (transgenic mice that produce fully human antibodies) (18, (39) (40) (41) (42) (43) (44) (45) (46) (47) (48) (49) . in parallel, versatile approaches were developed to generate non-genuine antibodies by random combination of vh and vl chains, i.e., combinatorial fab/scfv libraries or (semi-) synthetic libraries (37, 38, (50) (51) (52) (53) (54) (55) (56) (57) (58) (59) (60) . based on pcr amplification as strong tool to depict large antibody repertoires and phage display to screen these large repertoires, many recombinant allergen-specific antibody fragments (fabs or scfvs) were isolated (37, 38, (50) (51) (52) (53) (54) (55) (56) (58) (59) (60) (61) (62) (63) (64) . all mentioned technologies have definitely contributed to the isolation and evaluation of monoclonal allergen-specific igg, ige antibodies and fragments thereof and furthermore to assess their feasibility for allergy treatment. nevertheless, all mentioned technologies are also reported to have some limitations. while the hybridoma technology and ebv transformation are generally unsuitable for a comprehensive screening of large antibody repertoires because of their inefficient fusion and transformation events, the single b cell sorting was long hampered by inadequate staining technologies to clearly identify allergen-specific antibody producing cells (32, 39) . the main drawback of combinatorial libraries is that they usually rely on random combination and thus most likely unnatural vh and vl antibody pairings. additionally, it turned out independent of the applied technology to be very difficult to isolate monoclonal igg and ige antibodies with a broad epitope spectrum for each allergen. it also revealed that besides several blocking antibodies also many non-blocking or even enhancing antibodies were isolated (44, (63) (64) (65) . while all three types of monoclonal antibodies were unambiguously supportive to study the structural requirements for efficient effector cell activation and hence contribute to elucidate the underlying mechanisms of type i allergy, non-blocking and enhancing antibodies were fully useless for the prospective application as protective antibodies. these insights forced allergologists to look beyond the conventional antibody horizon. about 30 years ago, a group of belgian scientists made an unexpected discovery, which was patented and later presented to the scientific community in the form of the well-known discovery publication in the journal nature in 1993 (66) . they found that a significant amount of non-canonical types of antibodies is naturally present in blood of camelidae in addition to conventional antibodies. this exceptional type of antibody called heavy chain-only antibody (hcab) lacks light chains and consists of a homodimer of shortened (without ch1 domain) heavy chains. the antigen-recognition region in hcabs is formed by only one variable domain (v h h) that is directly linked via a hinge region to the fc-domain (66) . later on, similar non-canonical hcabs were found in some cartilaginous fishes such as sharks and ratfish (67) (68) (69) . the antigen-binding variable domain of these antibodies was named vnar as opposed to v h h in camelids. a recombinant protein version of the v h h-or vnar-domain is usually called "single domain antibody" or "nanobody". the very popular term "nanobody" is the commercial name given by the belgian biopharmaceutical company ablynx, a pioneer in hcab-based therapeutic applications that was acquired by sanofi in 2018. the nanobody generation technology was proven to be a very efficient machinery to generate nanobodies with required properties and offered crucial advantages compared to traditional techniques utilized to produce murine or human conventional antibodies. after the typical initial immunization (of camelids) step, the full repertoire of cdna coding for functional nanobodies can be efficiently cloned from peripheral blood lymphocytes of immunized animals using pcr amplification and then a panel of nanobodies of required specificity can be easily selected using phage (or other type of) display-based methods (66, (70) (71) (72) . in addition, there are different in vitro affinity maturation approaches to improve features of initially selected nanobodies (71, 73, 74) . in some cases, especially if the antigen of interest is toxic, unstable, non-immunogenic or not available in sufficient quantity, other types of libraries (naive, semisynthetic or fully synthetic libraries) can be efficiently used instead of immune libraries for generation of nanobodies (75) (76) (77) (78) (79) . synthetic libraries can be made using special predesigned scaffolds such as humanized scaffolds optimized for intracellular stability (77) or optimized for bacterial expression (80) . non-immune libraries are typically much larger than immune libraries and a ribosome display was suggested for the initial selection round of such large libraries to work with higher concentrations of nanobody variants than in case of phage display (79, 80) . synthetic libraries combined with different selection procedures were successfully used to obtain conformationally selective nanobodies against g protein-coupled receptors (78), sybodies against very challenging targets such as the heterodimeric bacterial abc exportertm287/288 (81) or the intracellular kdel receptor (82) to name a few examples from many others. nanobodies comprise unique features that distinguish them from classical antibodies. nanobodies are the smallest known antibody fragments (4 × 2.5 x 3 nm, 12-15 kda) of natural origin that are able to specifically bind their cognate antigens. due to their often extended cdr3 loop they can form unusual paratopes, i.e. finger-like extensions and thus recognize special native antigenic epitopes (small cavities, concave surfaces, conformational epitopes, active sites of enzymes) that are hidden for conventional antibodies (figure 1) . indeed, nanobodies have proven to be useful tools for modulating the activity of enzymes (26, 83, 84) . it could be therefore speculated that allergen-specific nanobodies that modulate or inhibit the proteolytic activity of certain allergens (e.g., phl p 1, der p 1) might reduce their penetration capacity through mucosal surfaces. furthermore, nanobodies are able to bind small peptides with high affinity (85) (86) (87) (88) (89) . their high affinity, solubility and stability over a wide range of temperatures and ph, ease of producing in bacteria or other expression systems make them convenient molecules for different applications, as well as for all possible engineering modifications e.g., development of complex constructs and conjugates. nanobodybased tools are therefore increasingly used for research, molecular visualization, diagnostics and development of new treatment options for various pathologies, including cancer and other socially significant diseases (71, 72, (90) (91) (92) (93) (94) . so far, only one allergen-specific nanobody is described in the literature. this nanobody is reported to be specific for the major peanut allergen, ara h 3 and was isolated from a synthetic library of humanized nanobodies via phage display (95) . the interaction between ara h 3 and the ara h 3-specific nanobody resulted in a dissociation constant of 400 nm representing medium affinity binding and was further investigated by the structural determination of formed co-crystals (95) . the authors acknowledged that additional work is needed to improve the affinity of the isolated nanobody to make it an attractive tool for the development of biosensors for peanut allergen detection. this finding clarifies that the selection procedure is only one part of the successful discovery of potent ige-blocking nanobodies, thus the evaluation of selected nanobodies is critical as well. nevertheless, we are confident that soon more allergenspecific nanobodies will arise to be studied for their potential to abrogate ige-mediated allergic inflammation. similar to the evaluation of conventional antibodies with the focus to identify effective protective monoclones, generated nanobodies have to be assessed first for their allergen specificity, epitope recognition, cross-reactivity to homologous allergens present in related species, for their affinity to their corresponding allergens and most importantly for their ability to inhibit patients´ige binding to these allergens (figures 2a-c) . after the allergen specificity of isolated nanobodies is confirmed, the proof for cross-reactivity (figure 2a) is of great importance because ige antibodies from allergic patients often displayed cross-reactivity to allergens from other allergen sources (28, 96) . high affinity and slow dissociation of formed nanobody/allergen complexes will be critical prerequisites for allergen-specific nanobodies to be chosen as suitable candidate ( figure 2b) . however, the pivotal characteristics for an allergen-specific nanobody to be attractive for further processing will be the determination of its potential to block patients' ige binding and hence ige-mediated effector cell activation ( figure 2c) . additionally, specific nanobodies have to be tested as well for their cross-protectivity to homologous allergens. all these properties are crucial requirements for allergen-specific nanobodies to be selected for further essential investigations concerning half-life, clearance and safety. nanobodies are considered as proteins of weak immunogenicity due to the shared similarities with variable vh domains of human immunoglobulins (igg3 subclass), and they can be further improved by a humanization approach (97) ( figure 2d) . consequently, no immune response against applied nanobodies was raised in mice or humans that were injected with nanobody-containing constructs (98) (99) (100) . safety of nanobody-based drugs is confirmed by several completed phase 1 and phase 2 clinical trials (101) and recent approval by the us food and drug administration (fda) and the european medicines agency (ema) of the first therapeutic nanobody, caplacizumab, a bivalent nanobody designed for the treatment of thrombotic thrombocytopenic purpura and thrombosis (102) . though advantageous for in vivo imaging, the small size of nanobodies could be seen as a disadvantage for passive treatment of allergy due to a quick renal clearance of nanobodies from blood (approx. 30 min). many different strategies to extend the in vivo half-life of nanobody-based construct have been developed (103) . they include increasing the hydrodynamic radius of a protein by attaching highly flexible and hydrophilic molecules such as polyethylene glycol (peg) and carbohydrates or by genetic fusion with polypeptide chains mimicking the biochemical properties of peg, fusion of v h h to the fc region of igg, fusion or non-covalent binding to albumin (104) ( figure 2d ). nanobodies can also be used as modules to engineer larger molecules with several valencies and/or specificities, such as multivalent (105) (106) (107) (108) , bispecific (105, 109) , and other (110, 111) constructs that may acquire considerably higher specificity, binding efficiency and biological activity (106, 107, 111) . nanobodies were also considered as possible ligands to design new highly specific immunosorbents (112) (113) (114) . different types of nanobody-based tools/approaches can be envisaged to be potentially profitable for an allergy treatment: a) bispecific nanobodies for topical application to capture allergens before they penetrate epithelial mucosa in airways, b) very stable nanobodies to capture food allergen in gastrointestinal tract, c) anti-idiotypic nanobodies mimicking allergenic epitopes as a possible replacement for a complex natural allergen for a new kind of ait vaccine development, d) multivalent nanobody-based constructs for systemical administration to efficiently block allergen interaction with ige on mast or basophil cells, e) efficient immunosorbents to remove ige from the blood by immune apheresis. correspondingly, different administration approaches for nanobody-based constructs can be developed: aerosol or topical applications, oral route or subcutaneous administrations. temporary blocking of allergen-ige interaction (i.e. by topical or systemic administration of specific nanobodies) or a subtraction of ige from the periphery blood (i.e. apheresis) may give a short-term treatment effect. for a long-term treatment effect we could hypothesize the use of anti-idiotypic nanobodies to ige. such nanobodies may represent "internal images" of an allergen and mimick hypoallergenic b cell epitopes. to efficiently induce igg response that targets ige binding sites on allergens, these nanobodies should be fused to a viral coat protein as it was described for next-generation forms of ait (15) . the generation of allergen-specific nanobodies unambiguously represents a reasonable progress in the field of allergy. with their well-documented qualities including their ability to recognize unusual "hidden" epitopes, high affinity binding, solubility, extreme stability and low immunogenicity, nanobodies attracted the interest of allergologists to study their suitability for passive allergy treatment. the chance to find allergen-specific nanobodies with this powerful technology that ideally comprise high affinity and bind to epitopes partly or fully overlap with ige binding sites on allergens is tempting. however, so far no allergen-specific nanobody fulfilling these criteria was reported indicating that it might be rather difficult to raise allergenspecific nanobodies of sufficient affinities. whether the current lack of such nanobodies is owed to some inherent structural or functional properties of nanobodies and/or the camelid immune system or the simple reason that the current research focus in the allergy field is on ait and its improvement has to be resolved. if allergen-specific nanobodies are identified that competitively block allergen binding to ige and thus abrogate ige-mediated allergic inflammation, we assume that they will represent appropriate tools for future allergy treatment. their economic properties, i.e. low production costs encouraged researchers to elaborate antibody engineering of these single-domain antibodies for diverse applications in biotechnology and medicine. this gathered knowledge will facilitate the implementation of modified allergen-specific nanobodies tailored to the needs of allergy treatment. nanobodies can be easily formatted for a particular application e.g., modified as recognition modules in large constructs or as bi-or oligo-specific, bi-or oligovalent derivatives. with the availability of allergen-specific nanobodies or their derivatives with inhibitory potential, it should be possible to examine engineered candidates in proof of concept testings for efficacy and safety in experimental animal models to identify promising nanobody-based drugs for clinically relevant allergens. molecular aspects of allergens and allergy allergy and allergic disease association between asthma, rhinitis, and conjunctivitis multimorbidities with molecular ige sensitization in adults quality of life in children and adolescents with respiratory allergy, assessed with a generic and disease-specific instrument histamine, histamine receptors and antihistamines: new concepts past, presence, and future of allergen immunotherapy vaccines mechanisms of allergen immunotherapy for inhaled allergens and predictive biomarkers mechanisms of immune regulation in allergic diseases: the role of regulatory t and b cells mechanisms of allergen-specific immunotherapy: diverse mechanisms of immune tolerance to allergens renaissance of the blocking antibody concept in type i allergy monitoring allergen immunotherapy effects by microarray nasal allergen-neutralizing igg4 antibodies block ige-mediated responses: novel biomarker of subcutaneous grass pollen immunotherapy allergenspecific nasal igg antibodies induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity next-generation of allergen-specific immunotherapies: molecular approaches recombinant allergens for immunotherapy: state of the art a nonallergenic birch pollen allergy vaccine consisting of hepatitis pres-fused bet v 1 peptides focuses blocking igg toward ige epitopes and shifts immune responses to a tolerogenic and th1 phenotype treating cat allergy with monoclonal igg antibodies that bind allergen and prevent ige engagement a randomized clinical trial of passive immunotherapy with single-dose anti-fel d 1 monoclonal antibodies regn 1908-1909 in cat induced rhinoconjunctivitis: exploratory efficacy endpoints, safety, and pharmacokinetics mechanisms of immunotherapy: igg revisited passive immunization with allergen-specific antibodies pricing of monoclonal antibody therapies: higher if used for cancer? examining trends in cost and clinical benefit of novel anticancer drugs over time nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics the therapeutic potential of nanobodies molecular basis for the preferential cleft recognition by dromedary heavychain antibodies inhibition of allergen-dependent ige activity by antibodies of the same specificity but different class epitope specificity determines cross-protection of a sit-induced igg4 antibody the cloning and expression of human monoclonal antibodies: implications for allergen immunotherapy rapid production of antigen-specific monoclonal antibodies from a variety of animals human b cell memory tracing ige-producing cells in allergic patients a human ige antibody binding site on der p 2 for the design of a recombinant allergen for immunotherapy the safety and side effects of monoclonal antibodies omalizumab, a novel anti-ige therapy in allergic disorders a drug safety review of treating eosinophilic asthma with monoclonal antibodies construction of a combinatorial ige library from an allergic patient. isolation and characterization of human ige fabs with specificity for the major timothy grass pollen allergen, phl p 5 isolation of a high-affinity bet v 1-specific igg-derived scfv from a subject vaccinated with hypoallergenic bet v 1 fragments single b cell antibody technologies continuous cultures of fused cells secreting antibody of predefined specificity human-human hybridomas producing monoclonal antibodies of predefined antigenic specificity human ige mabs define variability in commercial aspergillus extract allergen composition human ige monoclonal antibodies for epitope mapping of der p 2 human igg monoclonal antibodies that modulate the binding of specific ige to birch pollen bet v 1 immunologic characterization of monoclonal antibodies that modulate human ige binding to the major birch pollen allergen bet v 1 allergen specificity of igg4-expressing b cells in patients with grass pollen allergy undergoing immunotherapy ige-expressing memory b cells and plasmablasts are increased in blood of children with asthma, food allergy and atopic dermatitis high-affinity allergen-specific human antibodies cloned from single ige b cell transcriptomes human bcr analysis of single-sorted, putative ige+ memory b cells in food allergy conversion of grass pollen allergen-specific human ige into a protective igg1 antibody isolation of high-affinity human ige and igg antibodies recognising bet v 1 and humicola lanuginosa lipase from combinatorial phage libraries selection of recombinant ige antibodies binding the betalactoglobulin allergen in a conformation-dependent manner hevein-specific recombinant ige antibodies from human single-chain antibody phage display libraries the human ige-encoding transcriptome to assess antibody repertoires and repertoire evolution a common idiotype in ige and its relation to recognition of the grass pollen allergen phl p 2 delineating the specificity of an ige-encoding transcriptome acoustic microfluidic chip technology to facilitate automation of phage display selection highdensity ige recognition of the major grass pollen allergen phl p 1 revealed with single-chain ige antibody fragments obtained by combinatorial cloning human recombinant antibody fragments specific for a rye-grass pollen allergen: characterization and potential applications generation of human monoclonal allergen-specific ige and igg antibodies from synthetic antibody libraries a human monoclonal ige antibody defines a highly allergenic fragment of the major timothy grass pollen allergen, phl p 5: molecular, immunological, and structural characterization of the epitope-containing domain spatial clustering of the ige epitopes on the major timothy grass pollen allergen phl p 1: importance for allergenic activity molecular characterization of bip 1, a monoclonal antibody that modulates ige binding to birch pollen allergen, bet v 1 molecular characterization of human igg monoclonal antibodies specific for the major birch pollen allergen bet v 1. anti-allergen igg can enhance the anaphylactic reaction molecular characterization of a human immunoglobulin g4 antibody specific for the major birch pollen allergen, bet v 1 naturally occurring antibodies devoid of light chains a new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks distinct patterns of igh structure and organization in a divergent lineage of chrondrichthyan fishes isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein loop libraries a general protocol for the generation of nanobodies for structural biology natural single-domain antibodies nanobodies and their potential applications affinity maturation of a v(h)h by mutational hotspot randomization exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar kd of a single-domain antibody with a flat paratope facile generation of heat-stable antiviral and antitoxin single domain antibodies from a semisynthetic llama library immunological applications of single-domain llama recombinant antibodies isolated from a naïve library nali-h1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies yeast surface display platform for rapid discovery of conformationally selective nanobodies generation of synthetic nanobodies against delicate proteins a novel nanobody scaffold optimized for bacterial expression and suitable for the construction of ribosome display libraries the extracellular gate shapes the energy profile of an abc exporter structural basis for ph-dependent retrieval of er proteins from the golgi by the kdel receptor potent enzyme inhibitors derived from dromedary heavy-chain antibodies selection of nanobodies that block the enzymatic and cytotoxic activities of the binary clostridium difficile toxin structure and properties of a complex of alpha-synuclein and a singledomain camelid antibody peptides in headlock-a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy a peptide tag-specific nanobody enables high-quality labeling for dstorm imaging the alfa-tag is a highly versatile tool for nanobodybased bioscience applications a nanobody that recognizes a 14-residue peptide epitope in the e2 ubiquitin-conjugating enzyme ubc6e modulates its activity properties, production, and applications of camelid single-domain antibody fragments nanobodies as therapeutics: big opportunities for small antibodies nanobodies as versatile tools to understand, diagnose, visualize and treat cancer singledomain antibodies and the promise of modular targeting in cancer imaging and treatment prospective applications of single-domain antibodies in biomedicine screening of nanobody specific for peanut major allergen ara h 3 by phage display an ige epitope of bet v 1 and fagales pr10 proteins as defined by a human monoclonal ige general strategy to humanize a camelid single-domain antibody and identification of a universal humanized nanobody scaffold efficient cancer therapy with a nanobody-based conjugate experimental therapy of african trypanosomiasis with a nanobodyconjugated human trypanolytic factor formatted anti-tumor necrosis factor a vhh proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen-induced arthritis camelid and shark single domain antibodies: structural features and therapeutic potential caplacizumab: first global approval single-domain antibodies and their formatting to combat viral infections strategies for extended serum half-life of protein therapeutics the assembly of single domain antibodies into bispecific decavalent molecules llama-derived single domain antibodies to build multivalent, superpotent and broadened neutralizing anti-viral molecules formatted single-domain antibodies can protect mice against infection with influenza virus (h5n2) multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction vhh-based bispecific antibodies targeting cytokine production construction of a pix-modified adenovirus vector able to effectively bind to nanoantibodies for targeting genetic passive immunization with adenoviral vector expressing chimeric nanobody-fc molecules as therapy for genital infection caused by mycoplasma hominis multivalent anchoring and oriented display of single-domain antibodies on cellulose single-domain antibody-based ligands for immunoaffinity separation of recombinant human lactoferrin from the goat lactoferrin of transgenic goat milk a method for the parallel and sequential generation of single-domain antibodies for the proteomic analysis of human blood plasma the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 flicker, zettl and tillib. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-257662-viy65y72 authors: burrack, kristina s.; morrison, thomas e. title: the role of myeloid cell activation and arginine metabolism in the pathogenesis of virus-induced diseases date: 2014-09-08 journal: front immunol doi: 10.3389/fimmu.2014.00428 sha: doc_id: 257662 cord_uid: viy65y72 when an antiviral immune response is generated, a balance must be reached between two opposing pathways: the production of proinflammatory and cytotoxic effectors that drive a robust antiviral immune response to control the infection and regulators that function to limit or blunt an excessive immune response to minimize immune-mediated pathology and repair tissue damage. myeloid cells, including monocytes and macrophages, play an important role in this balance, particularly through the activities of the arginine-hydrolyzing enzymes nitric oxide synthase 2 (nos2; inos) and arginase 1 (arg1). nitric oxide (no) production by inos is an important proinflammatory mediator, whereas arg1-expressing macrophages contribute to the resolution of inflammation and wound repair. in the context of viral infections, expression of these enzymes can result in a variety of outcomes for the host. no has direct antiviral properties against some viruses, whereas during other virus infections no can mediate immunopathology and/or inhibit the antiviral immune response to promote chronic infection. arg1 activity not only has important wound healing functions but can also inhibit the antiviral immune response during some viral infections. thus, depending on the specific virus and the tissue(s) involved, the activity of both of these arginine-hydrolyzing enzymes can either exacerbate or limit the severity of virus-induced disease. in this review, we will discuss a variety of viral infections, including hiv, sars-cov, lcmv, hcv, rsv, and others, where myeloid cells influence the control and clearance of the virus from the host, as well as the severity and resolution of tissue damage, via the activities of inos and/or arg1. clearly, monocyte/macrophage activation and arginine metabolism will continue to be important areas of investigation in the context of viral infections. tissue -resident and monocyte-derived macrophages are innate immune cells that play a key role in normal tissue homeostasis, presentation of foreign and self antigens following infection or injury, pathogen clearance, and resolution of inflammation and wound healing. depending on the microenvironment, macrophages can be programed to adopt a variety of proinflammatory, regulatory, resolving, and immunosuppressive activation phenotypes, particularly in vivo. these activation states exist as a complex continuum of overlapping phenotypes; however, macrophage subsets with distinct functions have been defined (1) . macrophages are considered m1-polarized when stimulated by ifn-γ or toll-like receptor (tlr) ligands, such as lipopolysaccharide (lps), to express inducible nitric oxide synthase (inos; nos2) and produce nitric oxide (no). nos enzymes metabolize l-arginine to citrulline and no. no is a short-lived gaseous messenger with physiological and pathological effects. nanomolar concentrations of no, generated by endothelial nos and neuronal nos, are important for maintaining homeostasis, regulating vasodilation, and for the aggregation, recruitment, and adhesion of platelets to the vascular endothelium. inos generates micromolar levels of no that modulates various pathophysiological processes and is important for killing intracellular pathogens (2) . in contrast, m2-polarized macrophages result following stimulation of cells with a variety of stimuli, including type 2 cytokines such as interleukin (il)-4 or il-13. m2-polarized macrophages express a distinct l-arginine-metabolizing enzyme, arginase 1 (arg1), which hydrolyzes l-arginine to l-ornithine and urea. l-ornithine can be further metabolized to polyamines, which participate in a variety of fundamental cellular functions (e.g., proliferation, cell membrane transport), and l-proline, which is an essential component of collagen. in addition to playing important roles in defense against extracellular parasites and tissue repair, arg1 expression and activity in myeloid cells have emerged as a major regulator of innate and adaptive immune responses (3) . other m2-like suppressive or anti-inflammatory macrophages include myeloid-derived suppressor cells (mdscs) and tumor-associated macrophages (tams). mdscs are considered to be an immature population of myeloid cells, including both monocyte-like (gr-1/ly-6c + ) and neutrophil-like (gr-1/ly-6g + ) populations, associated with tumors or infections that suppress proinflammatory responses (4, 5) . depending on the context, mdscs have been shown to mediate their suppressive activity via no-and/or arg1-dependent mechanisms. importantly, macrophages are not permanently programed, but are considered "plastic" -that is, macrophages have been shown to change activation phenotypes depending on the local environment. although the m1/inos and m2/arg1 division is generally appropriate, arg1 can be induced in m1-like macrophages under certain conditions. thus, due to the spectrum of activation states for macrophages, a framework for macrophage-activation nomenclature was recently suggested (6) . in an attempt to avoid confusion in this review, we focused on the specific effects of the l-arginine metabolizing enzymes inos or arg1 on the pathogenesis of viral infections, noting other activation markers where appropriate. increasing evidence suggests that myeloid cell programing, inos, and arg1 contribute to the pathogenesis of numerous virus infections, suggesting that therapies that target these cells and pathways may be beneficial for the treatment of some virus diseases. in this review, we highlight recent studies of viral infections where myeloid cell polarization -resulting in expression of inos or arg1 -contribute to viral control or the development of chronic virus infection and mediate the resolution of tissue damage or cause immunopathology. no has antimicrobial activity against a number of bacteria, parasites, and fungi (7, 8) . additionally, no has been shown to have direct antiviral effects in vitro and/or in vivo against several viruses, including dna viruses such as herpes simplex virus type-1 (hsv-1), ectromelia virus (ev), and vaccinia virus (vv) (9, 10), as well as some rna viruses such as vesicular stomatitis virus (vsv) (11), japanese encephalitis virus (jev) (12) , dengue virus (denv) (13) , and coxsackievirus ( table 1 ) (14) (15) (16) (17) . there are several advantages of using no as an antiviral agent. for instance, unlike complement and antibody, no can readily pass through cellular membranes into neighboring cells as well as some viruses. additionally, no is likely to act on a variety of both viral and virally exploited cellular targets, inhibiting viral replication as well as limiting the capacity of viruses to develop resistance. lastly, the effect of no is independent of immune recognition of the infected cell, in contrast to that of antiviral lymphocytes, which could be important in virusinfected cells where expression of mhc class i molecules may be downregulated and in some virally infected tissues such as the brain where expression of mhc class i and ii molecules is limited. in initial studies in vitro, inhibition of ev, vv, and hsv-1 replication in mouse raw 264.7 macrophages and in primary mouse macrophages following ifn-γ treatment was shown to be largely dependent on no production (9, 10) . additionally, pharmacologic inhibition of nos or genetic deletion of nos2 resulted in increased viral titers and mortality following ev infection in mice (9, 18) . moreover, no affects several events in the late stages of the life cycle of vv, including viral dna replication, viral protein synthesis, and virion maturation in vitro (32) . these studies provided some of the first evidence that macrophage-produced no has direct antiviral effects. in addition to inhibiting hsv-1 replication in vitro, macrophage-derived no has been shown to have anti-hsv properties in vivo. in a mouse model of hsv-1-mediated corneal disease, inos was highly induced in the trigeminal ganglion (tg) of hsv-1-infected mice, and its expression was markedly reduced in mice depleted of macrophages (22) . depletion of macrophages prior to hsv-1 infection resulted in markedly reduced inos expression and higher viral loads in the tg of infected mice (22, 23) , suggesting that macrophages were the main source of inos expression in the affected tissues following hsv-1 infection and that no had important anti-hsv-1 properties in vivo. consistent with these data, inhibition of nos activity resulted in increased viral loads in the tg (22) . additional studies showed that f4/80 + gr-1 + inflammatory monocytes were recruited to the eye via an ifn-α-driven ccl2 gradient and restricted hsv-1 replication in that tissue via no production (24) . it was further shown that no production by f4/80 + macrophages in the brains of hsv-1-infected mice blocked viral replication in a partially tlr2-and tlr9-dependent mechanism (25) . finally, following footpad inoculation, hsv-1-infected nos2 −/− mice displayed a delayed clearance of virus from the dorsal root ganglia (drg) and exhibited an increase in the frequency of virus reactivation in drg (26) . the reactivity of no and its higher oxides and nitrosothiol products (84) makes it likely that a variety of molecular targets are involved in its antiviral action. it has been shown that no can inhibit ribonucleotide reductase (85, 86) , a rate-limiting enzyme in dna synthesis, and no can lead to the deamination of mammalian and bacterial dna (87, 88) , which may be important antiviral mechanisms. indeed, hsv-1 encodes its own ribonucleotide reductase and although it is not required for hsv-1 replication in vitro, it is necessary under conditions where the intracellular pool of deoxynucleotides is limited (89, 90) . thus, by inactivating this cellular and/or viral enzyme, no may halt virus replication by directly inhibiting viral dna synthesis. in addition to hsv-1, treatment of primary human cells with an no donor following infection with human cytomegalovirus (hcmv), a beta-herpesvirus, resulted in a significant reduction of early and late viral protein expression (28) . consistent with these in vitro data, nos2 −/− mice (129/sv/ev x c57bl/6 f1) exhibited increased viral titers and mortality following infection with murine cmv (mcmv; smith vr194 strain) (29) . nitric oxide has also been shown to have antiviral properties on a chicken herpesvirus, marek's disease virus (mdv), which can cause t cell lymphomas in chickens: addition of no-generating compounds inhibited viral replication in chicken fibroblasts (33) . additionally, the treatment of chickens with an inhibitor of inos increased the level of mdv replication in vivo (34) . further studies demonstrated that no production was limited to chickens that were genetically resistant to tumor development following mdv infection or to chickens that were vaccinated before being inoculated with mdv (35) . thus, no appeared to be produced in both types of resistance to tumor development in marek's disease, either acquired after vaccination or genetic. together, these findings suggest a role of no in the protective immune mechanisms against marek's disease, possibly through its activity on viral replication. finally, studies with hbv, a hepadnavirus associated with acute and chronic hepatitis, demonstrated that hbv replicated to higher levels in the livers of hbv-transgenic nos2 −/− mice than control transgenic mice, and transgenic nos2 −/− mice had increased frontiers in immunology | antigen presenting cell biology liver disease (36) . it was further demonstrated that no production by mononuclear cells, most likely macrophages, in the liver mediated most of the antiviral activity resulting from ifn-γ production by virus-specific t cells (36) , suggesting an antiviral role for macrophage-derived no following hbv infection in mice. in addition to dna viruses, macrophage-derived no also exerts antiviral effects against a number of rna viruses. inhibition of jev, a mosquito-transmitted flavivirus that causes encephalitis in humans, in ifn-γ-activated raw 264.7 macrophages in vitro correlated with no production, and ifn-γ-activated raw 264.7 macrophage-mediated inhibition of jev replication in murine neuroblastoma n18 cells was no-dependent (12) . moreover, inhibition of nos activity led to increased mortality in jev-infected mice (12) . in terms of its mechanism of action, no was found to inhibit jev rna synthesis, viral protein accumulation, and virus release from infected cells in vitro (12) . these data suggest that no may be directly or indirectly inhibiting viral enzymes and/or other www.frontiersin.org cellular components required for viral replication, and this may subsequently block viral protein synthesis. additionally, no may interfere with the release and/or maturation of virions. monocyte/macrophage-derived no may also block replication of denv, another mosquito-transmitted flavivirus. infection with denv resulted in increased levels of no in patients with dengue fever, the classic form of the disease (37) . additionally, inos expression was induced in cd14 + monocytes from a subset of acutely infected individuals (13) . it was further shown that ex vivo infection of human monocytes with denv-1 resulted in increased inos expression, and inhibition of inos activity led to increased denv antigen detection in these cells (13) . moreover, treatment of c6/36 mosquito cells with an no donor resulted in reduced denv-positive cells (13) . these data suggest that denv replication is susceptible to no-mediated inhibition. consistent with this, nos2 −/− mice were shown to be more susceptible to denv infection, resulting in more severe disease and increased lethality in mouse models of denv-2 and denv-3 infection (38, 39) . it was further demonstrated that, following denv infection in vivo, il-12 and il-18 induced ifn-γ production, resulting in inos expression and no production, which contributed to viral control (38, 39) . in addition to monocyte/macrophage-derived no, a recent study demonstrated that platelets isolated from patients with dengue fever had increased l-arginine transport and increased no production compared to platelets from healthy controls (40) . however, no has anti-aggregatory properties, and mendes-ribeiro et al. (40) found that dengue patients exhibited decreased collagen-induced platelet aggregation, consistent with the vascular leak and hemorrhagic manifestations of dengue hemorrhagic fever/dengue shock syndrome (dhf/dss), thus establishing an association between reduced platelet aggregation, enhancement of the l-arginine-no pathway, and dhf/dss (41) . in contrast, getts and colleagues showed that experimentally abrogating no activity during west nile virus (wnv) encephalitis, a related flavivirus, in no-competent mice at a specific, relatively late time point prolonged survival of infected mice, while pharmacological inactivation throughout disease did not (42) . combined, these data suggest that although during denv infection ifn-γ-induced no production has a role in antiviral defense, it is likely that dysregulation of the il-12/18-ifn-γ-no axis leads to immune-mediated damage in certain flavivirus infections. along these lines, it has also been shown that treatment of mice with a nos inhibitor increased mortality rates following sindbis virus (sinv) infection (43) , suggesting a protective role for no during this particular cns infection. however, sinv replication in the brain was unaffected. furthermore, treatment of neuroblastoma cells with no donors had little effect on sinv replication but increased cell viability (43) . these data suggest that no protects mice from fatal sinv-induced encephalitis by a distinct mechanism that does not directly involve the inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication. nitric oxide also plays an antiviral role during cns infection with reovirus. infection of neonatal mice with the prototypic neurotropic reovirus strain (t3a) induced inos expression in brain areas demonstrating reovirus antigen expression and associated virus-induced injury (44) . reovirus also induced inos expression following in vitro infection of primary neuronal and glial cultures. reovirus was shown to infect a subpopulation of microglial cells in vitro (44) , suggesting that direct virus interaction may induce inos in this specialized population of macrophages. treatment of neuronal cultures with an no donor inhibited viral replication whereas a nos inhibitor increased viral growth (44) , suggesting inos has the potential to exert antiviral activity in vivo. finally, coxsackievirus infection has been shown to induce expression of inos in macrophages infiltrating the hearts of infected mice (17) . treatment of wt mice with a nos inhibitor and infection of nos2 −/− mice resulted in more severe coxsackievirus-induced pancreatitis and myocarditis, elevated viral loads in tissues, and decreased survival compared to wt mice following coxsackievirus b3 (cvb3) infection (14, 15, 17) . similarly, nos2 −/− mice infected with coxsackievirus b4 exhibited decreased survival and delayed viral clearance compared to wt mice (16) . these data suggest an antiviral effect of no against coxsackievirus infection. consistent with this, it was demonstrated that no inhibits the 2a and 3c proteinases of cvb3 in vitro (91) . additionally, cvb3-infected outbred mice showed significantly reduced signs of myocarditis after treatment with no donors (91) . despite its protective capacity during some viral infections, no can also contribute to immunopathology. the pathological effects of no are likely due, at least in part, to oxidative damage caused by the interaction of no with oxygen radicals such as the superoxide anion radical o − 2 and hydrogen peroxide (h 2 o 2 ). for example, although addition of an no donor to virusinfected mdck cells reduced influenza a and b viral burden in vitro (45) , treatment of mice with inhaled no (ino) did not decrease the viral load of influenza a (mouse-adapted h1n1 strain)-infected mice; in fact, prophylactic treatment with ino resulted in enhanced weight loss and decreased survival following infection (46) , suggesting a pathogenic role for no. consistent with this, chickens, which show a high level of mortality and associated pathology following avian influenza infection, had higher levels of inos expression in the lungs compared with h5n1 influenza-infected ducks, which show relatively minor symptoms following influenza infection (47) . additionally, akaike and colleagues (48) found evidence of the production of peroxynitrite, which is generated through the reaction of no and o 2 -, in the lungs of influenza a (mouse-adapted h2n2 strain)-infected mice. moreover, inhibition of nos resulted in enhanced survival and decreased pneumonia, but not decreased viral loads, in influenzainfected mice (48, 49) , suggesting that no was contributing to pathogenesis rather than having direct antiviral effects. nos2 −/− mice also survived a lethal dose of influenza a virus (pr/8/34 strain) infection with little histopathologic evidence of pneumonitis; however, in these studies no infectious virus was detected in nos2 −/− mice at day 6 after infection (49) . the enhanced viral control in nos2 −/− mice was shown to require the activity of ifn-γ (51), with nos2 −/− mice also producing increased virusspecific igg2a antibody titers (50) . additionally, genetic deletion frontiers in immunology | antigen presenting cell biology of nos2 or pharmacologic inhibition of nos enhanced survival of mice inoculated with the highly pathogenic (non-mouse-adapted) 1918 influenza virus strain, although mice exhibited similar viral loads to control mice in lung tissue at the peak of viral replication (51) . influenza infection in vitro was shown to induce apoptosis, and a reduction in influenza-mediated apoptosis was noted in cells treated with a nos inhibitor (52) . similarly, fewer apoptotic cells were found in the lungs of influenza-infected nos2 −/− mice, suggesting that no mediates cell death following influenza infection (52) . the cellular source of inos/no following influenza infection in mice was shown to be ccr2 + inflammatory monocytes that accumulate in the lungs: ccr2 −/− mice survived a lethal challenge of influenza infection (pr/8/34 strain) and had significantly reduced accumulation of inos-expressing macrophages in the lung, with no associated increase in viral titers or dissemination (53) . it was also recently shown that a subset of monocyte-derived dendritic cells (dcs), described as tnf-α/inos-producing dcs (tipdcs), accumulate in greater numbers during the course of lethal versus sublethal influenza infections, suggesting a pathogenic role for this subpopulation of myeloid cells (54) . interestingly, though, aldridge et al. (54) found that the tipdcs also stimulated a local, protective cd8 + t cell response in the virusinfected respiratory tract, indicating both protective and pathogenic roles for these cells in influenza infection. it was further shown that partially compromising tipdc recruitment via treatment with pioglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (ppar-γ), was protective against lethal influenza challenge (54). pioglitazone treatment led to a reduction in the levels of ccl2 (mcp-1) and mcp-3 in the bal fluid of influenza-infected mice (54) . pioglitazone has also been shown to reduce the production of a wide range of proinflammatory molecules, including inos (55), providing further evidence for the importance of no production by monocyte-derived cells in the pathogenesis of influenza infection. pharmacologic inhibition of nos using l-nmma also decreased pneumonitis and increased survival following intranasal infection of cba/j mice with hsv-1, despite a 17-fold increase in viral titers in the lung at day 3 after inoculation (58). in contrast, treatment of balb/c mice with a different nos inhibitor [aminoguanidine (ag), administered intranasally] resulted in enhanced pneumonitis, viral titers, and mortality following infection with a different strain of hsv-1 (59) . thus, the precise role of no in hsv-1 pneumonitis remains to be determined. no and other ros/rns were also shown to be pathogenic in the brains of mice with herpes encephalitis: inos was induced in cd11b + resident microglia following intranasal infection with hsv-1, and oxidative and nitrative damage was found in the brains of infected animals (60) . a common neurological complication of hiv infection in the developed world is sensory neuronal injury accompanied by inflammation, which is clinically manifested as disabling pain and gait instability. feline immunodeficiency virus (fiv) infection of cats, which causes similar neuroinflammation together with immunosuppression in cats, resulted in induction of inos and stat-1, which were predominantly produced by macrophages, in drg (64) . additionally, inhibition of nos resulted in reduced nitrotyrosine and prevented neuronal injury in fiv-infected drg cultures in vitro (64) . these data suggest that lentivirus infection contributes to axonal and neuronal injury through a mechanism involving m1 macrophage immune activation mediated by stat-1 and inos activation. in addition to these studies, infection of mice with the retrovirus lp-bm5, which causes profound immunodeficiency, induces cd11b + gr-1 + ly-6c + mdsc-like cells that inhibit both t-and b-cell responses in an inos/no-dependent but arginase-independent fashion (65) . this study identified an important -and only recently appreciated -role for inosexpressing myeloid cell-mediated suppression of b cell responses in retrovirus infection. the oxidative effects of no have also been shown to inhibit immune cells, particularly t cells. this phenomenon has been appreciated for a number of years in the context of tumors (92), where myeloid suppressor cells can inhibit the anti-tumor t cell response via the effects of no in addition to other mechanisms (2, 4) . in a similar manner, it has been shown that no can inhibit the antiviral immune response. mcmv clearance from balb/c mice is predominantly cd8 + t cell-mediated. a recent report showed that mcmv infection in balb/c mice induced cd11b + ly-6c hi inflammatory monocyte recruitment from the bone marrow to infected tissues that was dependent on ccr2 signaling (66) . this recruitment was shown to inhibit antigen-specific cd8 + t cell activation, expansion, and cytotoxic activity via no production, thus facilitating viral persistence (66) . in a similar fashion, no may contribute to a defective immune response following infection of mice with an attenuated neurotropic coronavirus (rj2.2 strain of mouse hepatitis virus). rj2.2infected wt mice exhibited mild acute encephalitis, followed by a non-lethal, chronic demyelinating disease (68) . in marked contrast, rj2.2 infection of mice that transgenically express ccl2 in the brain (ccl2 tg) ineffectively cleared virus and rapidly succumbed to the infection (68). ccl2 tg mice mounted a dysregulated immune response, characterized by increased accumulation of inos-expressing macrophages and microglia as well as regulatory t cells, but decreased arg1 expression (68) . these data suggest that persistent ccl2 overexpression establishes and sustains an immunological milieu that may predispose mice to a defective immune response to a typically minimally virulent virus. arginase activity is important for wound healing and tissue regeneration through the production of polyamines and proline (2) . in the context of some viral infections, arginase activity and m2 macrophage activation have been shown to be beneficial for tissue repair following virus-induced damage. for instance, resolution of severe respiratory syncytial virus (rsv)-induced bronchiolitis in mice is mediated by m2 macrophages that counteract cyclooxygenase (cox)-2-induced lung pathology (19, 20) . arg1 was induced in the lungs of rsv-infected mice, and its induction was shown to be il-4rα-dependent (19) . additionally, wt macrophages adoptively transferred into rsv-infected il-4rα −/− mice restored the www.frontiersin.org m2 phenotype in the lungs and decreased lung pathology (19) . it was further shown that the lipoxogenase pathway was important for m2 macrophage activation and lung resolution following rsv infection (20) . most recently it was demonstrated that treating mice with agents that sustain arg1 expression (e.g., il-4/anti-il-4 immune complexes) limited rsv-induced lung pathology (21) . consistent with a pathogenic role for inos/no following influenza infection (described above), it was recently shown that the presence of airway bacteria polarize alveolar macrophages into a m2 phenotype, thus limiting influenza-mediated lethal lung inflammation. wang and colleagues (27) demonstrated that priming with staphylococcus aureus, which commonly colonizes the upper respiratory mucosa, attenuated influenzamediated lung injury via tlr2 signaling that recruited peripheral ccr2 + cd11b + monocytes into the alveoli (27) . these monocytes polarized alveolar macrophages into a m2 phenotype characterized by high arg1 as well as ym1, fizz1, and il-10 expression (27) . it was further shown that s. aureus-primed m2 alveolar macrophages inhibited inflammatory cell recruitment to the lung, including neutrophils, nk cells, and cd8 t cells (27) . s. aureus-primed m2 alveolar macrophages also expressed higher levels of the inhibitory ligand pd-l1 (27) , suggesting that expression of a combination of anti-inflammatory cytokines and inhibitory ligands could be the mechanisms by which s. aureusprimed m2 alveolar macrophages limit influenza-mediated lung inflammation. as discussed above, coxsackievirus b3 (cvb3) infection causes myocarditis in human beings as well as in male balb/c mice. although female mice do not develop severe myocarditis, both male and female mice have comparable numbers of infiltrating macrophages and viral titers in the heart following cvb3 infection (30) . the macrophages infiltrating the heart in male mice were skewed toward a m1 phenotype characterized by high expression of inos (17) as well as m1-associated cytokines such as ifn-γ and il-12 (30) . additionally, inhibition of nos resulted in increased viral titers and higher mortality in cvb3-infected mice (17), consistent with an antiviral role for no during cvb3 infection (see above). however, in contrast to male mice, the heart-infiltrating macrophages in female mice were skewed toward a m2 phenotype characterized by high expression of arg1 as well as il-4 and il-10 (30). moreover, adoptive transfer of ex vivo-programed m1 macrophages significantly increased myocarditis in both male and female mice. strikingly, transfer of m2-programed macrophages into susceptible male mice alleviated myocardial inflammation by modulating the local cytokine profile from a m1 to m2 phenotype and promoting peripheral regulatory t cell (treg) differentiation (30) . using different variants of cvb3, one that caused myocarditis in c57bl/6 mice and one that did not, it was additionally shown that the myocarditic variant induced a m1 macrophage phenotype (31) . in contrast, the amyocarditic variant induced a m2 macrophage phenotype, which was also associated with the activation of nkt cells that promoted a treg response (31) . the ability of nkt cells to suppress myocarditis was shown by adoptive transfer of purified nkt cells into nkt knockout (jα18 knockout) mice infected with the myocarditic cvb3 variant, which inhibited cardiac inflammation and increased treg response (31) . cardiac virus titers were equivalent in all mouse strains indicating that nkt cells did not participate in control of virus infection (31) . thus, although no appears to have antiviral properties against cvb3, these data indicate an important role for arg1-expressing m2 macrophages in controlling cvb3-induced myocarditis. as a consequence of their co-evolution with their hosts, viruses have developed numerous strategies to evade the host immune system and ensure their own replication and survival. recent studies have identified a new evasion strategy for viruses: exploitation of the host's anti-inflammatory, wound repair response to promote chronic infection. two strains of lcmv -armstrong (arm) and clone 13 (c13)have been studied for decades as models for acute and chronic infections (93) . infection of mice with the arm strain leads to a robust cd8 + t cell response that rapidly clears the virus (94) , whereas infection with c13 results in t cells with impaired functionality, enabling the virus to persist (95) . it was recently demonstrated that c13 infection led to an enhanced and sustained expansion of cells that resembled mdscs (70) . these suppressive myeloid cells inhibited t cell proliferation ex vivo via an inos/no-dependent but arg1-independent mechanism. another study, however, found that arg1-expressing immunoregulatory antigen presenting cells induced during c13 infection suppressed t cell responses (67) . most recently, it was demonstrated that t cell responses were improved -resulting in clearance of the normally chronic c13 infection -when either myeloid cells or t cells lacked il-10 production (96) . overall, these data demonstrate the importance of inos/arg1-expressing myeloid cells in viral persistence. similar to lcmv c13 infection, it was recently demonstrated that infection of mice with the arthritogenic alphaviruses ross river virus (rrv) and chikungunya virus (chikv) resulted in the induction of arg1 in macrophages in the infected and inflamed musculoskeletal tissues (69) . it was further shown that genetic deletion of myeloid cell arg1 resulted in enhanced viral control in inflamed muscle tissue and reduced tissue pathology following rrv infection in mice (69) , suggesting an important role for arg1-expressing macrophages in the persistence of these chronic viruses. infection of mice with theiler's murine encephalomyelitis virus (tmev) results in persistent virus infection in the cns, which contributes to the development of a demyelinating disease that has similarities with multiple sclerosis. bowen and olson (97) showed that cd11b + ly-6c + cells infiltrated the cns following infection and were the dominant cell type during the innate immune response. depletion of the cd11b + ly-6c + cells via administration of an anti-gr-1 ab resulted in reduced development of demyelinating disease and enhanced virus-specific cd4 + and cd8 + t cell responses (97) . additionally, tmev-infected, anti-gr-1 ab-treated mice had decreased myelin-specific cd4 + t cell responses compared to control ab-treated mice during the demyelinating disease at a later time post-infection (97) . although the expression of arg1 was not investigated in this study, tmevinfected mice had elevated expression of il-10 in the brain and spinal cord (97), suggesting a role for this cytokine in the suppression of antiviral t cell responses, potentially through the effects of arg1. interestingly, a role for the modulation of arginine metabolism in viral control versus persistence along with associated disease has recently been demonstrated for the tumor-inducing, chickenspecific herpesvirus mdv. we mentioned above that mdv was vulnerable to the antiviral properties of no, with inos being induced in genetically resistant chickens and in vaccinated chickens (35) . in contrast, mdv induced strong macrophage arginase activity in cell extracts from adherent monocytes from genetically susceptible chickens, but not in chickens that were resistant to marek's disease, either genetically or acquired after vaccination (35) . together, these data suggest that in the case of marek's disease, the state of resistance versus sensitivity to disease was correlated with a reciprocal balance of nos versus arginase activities in macrophages. this phenomenon of arg1-mediated t cell suppression has also been recognized in human viral infections. arg1 mrna and protein levels were elevated in hcv-infected liver cell lines in vitro and in hcv-infected liver samples compared with paired hepatocellular carcinoma samples from the same patients or with uninfected liver tissues (71) . additionally, the number of mdscs in chronic hcv patients correlated with levels of plasma hcv-rna (98). cai et al. (98) also found that mdscs from patients with chronic hcv infection suppressed t cell function via an arg1-dependent mechanism. an additional study found that more pbmcs from chronic hcv patients expressed the phenotypic markers of mdscs than pbmcs from healthy controls, and these cells expressed increased levels of p47 phox , a component of the nadph oxidase complex (99) , suggesting a role for ros in mdsc-mediated suppression. consistent with this, cd33 + mononuclear cells co-cultured with hcv-infected hepatocytes or hcv core protein suppressed t cell proliferation in a ros-dependent manner (99) . overall, these data suggest that multiple mechanisms -including arginine metabolism and ros -may be at play in myeloid cell-mediated suppression of anti-hcv t cell responses. it has been suggested that prolonged immune activation during chronic virus infections, such as hcv and hiv, provides an environment that drives viral replication and disease progression (100, 101) . moreover, immune activation can drive an anti-inflammatory response to limit immunopathology, which can be characterized by the presence of m2-like macrophages. indeed, similar to hcv infection, a role for arginase and m2polarized mdsc-like cells has been identified in the suppression of antiviral t cell responses following hiv infection. individuals with detectable hiv-1 infection showed an increase in the frequency of cd163 + cd16 + cd14 + monocytes, which are thought to be precursors of m2 macrophages, when compared to seronegative or hiv-1-infected persons with undetectable viral loads, and monocyte frequency correlated positively with hiv-1 viremia and negatively with cd4 + t cell counts (in patients with counts <450 cells/µl) (72) . furthermore, qin and colleagues (73) observed elevated levels of mdscs, defined as hla-dr −/low cd11b + cd33 +/high cd14 + cd15 − cells, in the peripheral blood of hiv-1-seropositive subjects compared with healthy controls, and these mdscs suppressed t cell responses in an arg1-dependent manner. moreover, pbmcs from hivseropositive patients exhibited increased levels of arginase activity (73) . cloke and colleagues (74) found that increased arginase activity correlated with lower cd4 + t cell counts, and this association was abrogated following antiretroviral treatment (75) . additionally, exposure of pbmcs to hiv gp120 expanded t cellsuppressive mdscs in vitro (76) . these data point to a direct role for arginase-expressing mdsc-like cells in the suppression of anti-hiv t cell responses. consistent with that, individuals co-infected with hiv and leishmania parasites had increased arginase activity in pbmcs and plasma compared with leishmania-only infected individuals, even though leishmania infection alone results in increased arginase activity (77) . in addition, the parasite load in the spleen was significantly higher in co-infected patients (77) . the arginase-expressing cells were identified as low-density granulocytes (77) . these results suggest that increased arginase might contribute to the poor immune responses and disease outcome characteristic of patients with leishmania and hiv co-infection. hepatitis b virus (hbv) infection is another common chronic viral infection, with estimates as high as 350 million chronically infected humans (102) . bility and colleagues (78) recently developed a humanized mouse model with both a human immune system and human liver cells, named the a2/nsg-hu hsc/hep humanized mouse model, to study the pathogenesis of hbv infection. following hbv infection, the mice developed persistent hbv infection as well as chronic hepatitis and liver fibrosis (78) . the liver disease was associated with a high level of infiltrating human macrophages with a m2-like activation phenotype (78) . similarly, m2-like macrophage accumulation was seen in chronic hbv-infected patients, and m2-like macrophage induction in the liver was associated with accelerated liver fibrosis and necrosis in patients with acute hbv-induced liver failure (78), suggesting a role for m2 macrophages in persistent hbv infection. additionally, patients with acute hbv infection had increased serum levels of arginase, and this serum inhibited ifn-γ production by cd8 + t cells (79) . das et al. (80) also found decreased l-arginine levels in the circulation of chronic hbv patients with marked liver inflammation (>100 alt) and increased arginase activity in liver extracts taken directly ex vivo from patients with chronic hbv compared with those from patients with other types of liver pathology (80) . they further showed that cd8 + t cells from chronic hbv patients, regardless of their antigen specificity, exhibited less il-2 but not ifn-γ or tnf-α production and impaired proliferation following tcr-dependent stimulation, indicating an aberrant antiviral t cell response in chronic hbv infection (80) . in the a2/nsg-hu hsc/hep humanized mouse model, hbv-infected mice had impaired liver t cell responses, and m2 macrophages were associated with t cells in the liver (78) . expression of the tcr signaling molecule cd3ζ was reduced in both peripheral and intrahepatic cd8 + t cells from chronic hbv patients; similarly, cd28 was also downregulated on cd8 + t cells from high viral load hbv patients (80) . downregulation of the cd3ζ molecule has previously been shown to occur in the arginine-depleted tumor microenvironment. consistent with this, in vitro transfection of cd3ζ and cd28 restored il-2 production and supplementation of l-arginine partially restored cd3ζ expression and t cell proliferation (80) . these data suggest a role www.frontiersin.org for arginase activity and arginine depletion in the impairment of anti-hbv t cells functions. in the absence of inkt cells, influenza a (pr/8 strain) infection was shown to induce the expansion of cd11b + gr-1 + mdscs in the lungs of mice, which suppressed influenza-specific t cell and antibody responses through the activity of both arginase and nos, resulting in higher viral titers and increased mortality (81) . adoptive transfer of inkt cells reversed this phenotype; mice had an increased survival rate, reduced viral titers, and increased virusspecific immune responses, suggesting a novel immunomodulatory role for inkt cells during influenza virus infection (81) . moreover, these authors identified that influenza infection in humans induced the expansion of cd11b + myeloid cells with suppressive activity that could be reduced by inkt cell activation or the inhibition of arginase and nos activity. similarly, it was recently shown that highly pathogenic h5n1 and h1n1 influenza virus infection induced the accumulation of cd11b + gr-1 + cells and the expression of arg1 in the lungs (82) , further supporting a role for m2-polarized mdsc-like cells in promoting viral persistence and immunopathology. helminth infection induces the expression of type 2 cytokines and is associated with m2 macrophage activation, as determined by arg1, fizz1, and ym1 expression. indeed, osborne and colleagues (83) found that arg1, fizz1, and ym1 were highly induced in the ileum of mice infected with the helminth trichinella spiralis (ts). interestingly, they further showed that co-infection of mice with ts and murine norovirus (mnv) resulted in decreased frequencies and numbers of mnv-specific cd8 + and cd4 + t cells within the small intestine and spleen as well as decreased polyfunctionality of these t cells, compared to ts-only infected mice (83) . additionally, the defective t cell responses were associated with increased viral loads in the double-infected mice compared to the mono-infected controls (83) , suggesting that ts-elicited m2-activated macrophages inhibited the antiviral t cell response to mnv. lastly, neutralization of ym1, a chitinase-like molecule, in co-infected mice partially restored antiviral immunity and was associated with enhanced control of viral replication (83) . these data point to a new mechanism by which arg1-expressing macrophages inhibit antiviral responses. cumulatively, these data are reminiscent of macrophages found in tumors (e.g., mdscs, tams) that have been shown to suppress anti-tumor t cell responses via a variety of no-and/or arg1-dependent mechanisms (4, 5) . indeed, in a mouse model of human papillomavirus (hpv)-induced cancer, arg1-expressing cd11b + f4/80 + macrophages infiltrated the tumors and inhibited t cell responses, including virus-specific t cells, by suppressing t cell proliferation and promoting a regulatory phenotype (103) . moreover, depletion of the tumor-infiltrating macrophages resulted in reduced tumor growth and increased tumor infiltration by virus-specific cd8 + t cells (103) . thus, increasing evidence points to a direct role for arginase-expressing m2-polarized cells in the suppression of antiviral t cell responses and the persistence of a variety of important pathogenic viruses. in addition to the actions of inos and arg1, mdsc-like cells can employ other mechanisms to promote chronic viral infections, which were recently reviewed by goh and colleagues (104) . in contrast to some parasitic infections where m2 macrophages limit th2 cell-mediated immunopathology, m2-polarized macrophages have been shown to promote immunopathology in some viral infections. for example, it was recently demonstrated that sars-cov infection of mice induced suppressive alveolar macrophages that inhibited the induction of antiviral t cell responses, a phenotype that was reversed by the adoptive transfer of activated bone marrow-derived dcs into mice prior to virus infection (56) . additionally, sars-cov-infected mice lacking hematopoietic stat-1 expression were shown to have greater weight loss and lung pathology, and this was associated with the activation of m2 macrophages (57) . to further test the role of m2 macrophages in enhanced pathogenesis following sars-cov infection, the authors generated stat-1/stat-6 double knockout mice due to the established role for stat-6 in driving m2 macrophage activation in response to il-4/il-13 stimulation. stat-1/stat-6 double knockout mice, which reversed the upregulation of m2 macrophages observed in stat-1-deficient mice, had reduced lung disease and prefibrotic lesions (57) . these data support the notion that m2 macrophages contribute to sars-cov pathogenesis. in another example, mice deficient in the ifn-γr exhibit more severe disease following infection with murine gammaherpesvirus-68 (mhv-68), including interstitial and intra-alveolar fibrosis that is reminiscent of idiopathic pulmonary fibrosis (ipf) in human beings. in this model, alveolar macrophages were recruited to the lungs of mhv-68-infected ifn-γr −/− mice, were associated with areas of fibrosis, and exhibited a m2-polarized phenotype characterized by the expression of fizz1, ym1, and arg1 (61) . additionally, lung tissue from patients with ipf showed increased expression of arg1 in alveolar macrophages compared with normal lung (61) . these results suggest that virus-induced upregulation of arg1 could be mediating lung fibrogenesis. mhv-68 infection in ifn-γr −/− mice also resulted in fibrosis in lymphoid tissues such as the spleen, which is a site of latent mhv-68 infection, and the liver (62, 63) . similar to the lung, mhv-68 infection in the absence of ifn-γr signaling induced a m2 macrophage response in the spleen, characterized by high arg1 expression along with fizz1 and m2/th2 cytokines such as il-13, resulting in fibrotic disease in the spleen (105) . moreover, depletion of t cells prevented mhv-68-mediated fibrosis in ifn-γr −/− mice (62) , suggesting that m2 macrophages were further driving th2 activation to possibly create a m2/th2 cytokine-induced cycle, resulting in the exaggerated pathology. in contrast to ifn-γr −/− mice, inos was induced in the spleen of mhv-68-infected wt mice (105) , indicating an important role for ifn-γ in inducing a m1-associated immune response to control gamma-herpesvirus infection and limiting arg1-mediated immunopathology. macrophages and other myeloid cells have marked phenotypic heterogeneity, as a result of distinct cellular differentiation programs, distribution in tissues, and responsiveness to various endogenous and exogenous stimuli. indeed, macrophages have well-established roles in development, tissue homeostasis, coordinating the adaptive immune response and inflammation, as well as directing tissue resolution and repair following damage -processes that are often modulated via the actions of the arginine-hydrolyzing enzymes nos2 and arg1. we have highlighted a number of viral infections in which these enzymes have a beneficial effect: no has antiviral properties against a variety of viruses, and arginase activity can mediate tissue repair and regeneration following a viral insult (table 1) . however, no production can also result in immunopathology in some virus infections, and the suppressive functions of arg1-expressing macrophages can promote immunopathology. additionally, some viruses have exploited the immune-suppressive properties of inos-and/or arg1-expressing macrophages to evade the immune response, particularly the antiviral t cell response, resulting in chronic viral infections. clearly, inos-and/or arg1-mediated responses are important in many viral infections. thus, there is the potential to develop the means to selectively stimulate or inhibit either m1 or m2 responses to mediate viral clearance or repair tissue damage. due to the overlap in immunosuppressive mechanisms of inos-and/or arg1-expressing suppressor cells, therapeutic strategies under development to limit the immunosuppressive effects of myeloid cells in cancer may be beneficial in treating persistent/chronic virus infections. however, as described above, inos and arg1 activity can be both beneficial and detrimental during certain viral infections. therefore, further research is needed to define the molecular and tissue-specific mechanism(s) by which inos and arg1 influence the clearance of viral pathogens as well as the injury and repair of tissues. in addition, a better understanding of the pathways regulating macrophage polarization (specifically inos and/or arg1 induction and activity), macrophage trafficking, and the precise effects of inos and arg1 activity on other immune cells following different virus infections will inform the development of therapeutics that target critical effector molecules to promote viral control and limit immunopathology. protective and pathogenic functions of macrophage subsets m1 and m2 macrophages: oracles of health and disease arginase: an emerging key player in the mammalian immune system coordinated regulation of myeloid cells by tumours myeloid derived suppressor cells and their role in diseases macrophage activation and polarization: nomenclature and experimental guidelines role of nitric oxide synthesis in macrophage antimicrobial activity role of nitric oxide in parasitic infections inhibition of viral replication by interferon-gamma-induced nitric oxide synthase evidence for antiviral effect of nitric oxide. inhibition of herpes simplex virus type 1 replication inhibition of vesicular stomatitis virus infection by nitric oxide inhibition of japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on rna virus replication inducible nitric oxide synthase (inos) expression in monocytes during acute dengue fever in patients and during in vitro infection the role of inducible nitric oxide synthase in the host response to coxsackievirus myocarditis inducible nitric oxide synthase protection against coxsackievirus pancreatitis sarvetnick n. a critical role for inducible nitric oxide synthase in host survival following coxsackievirus b4 infection nitric oxide inhibits viral replication in murine myocarditis identification of nitric oxide synthase 2 as an innate resistance locus against ectromelia virus infection control of rsv-induced lung injury by alternatively activated macrophages is il-4r alpha-, tlr4-, and ifn-beta-dependent role of the lipoxygenase pathway in rsv-induced alternatively activated macrophages leading to resolution of lung pathology agents that increase aam differentiation blunt rsv-mediated lung pathology macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system il-4 and il-10 antagonize il-12-mediated protection against acute vaccinia virus infection with a limited role of ifn-gamma and nitric oxide synthetase 2 ifn-alpha-driven ccl2 production recruits inflammatory monocytes to infection site in mice defense against hsv-1 in a murine model is mediated by inos and orchestrated by the activation of tlr2 and tlr9 in trigeminal ganglia mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced th1 cell responses bacterial colonization dampens influenza-mediated acute lung injury via induction of m2 alveolar macrophages role of ifn-gamma-induced indoleamine 2,3 dioxygenase and inducible nitric oxide synthase in the replication of human cytomegalovirus in retinal pigment epithelial cells role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus differential macrophage polarization in male and female balb/c mice infected with coxsackievirus b3 defines susceptibility to viral myocarditis cross-regulation of t regulatory-cell response after coxsackievirus b3 infection by nkt and gammadelta t cells in the mouse gamma interferon-induced, nitric oxidemediated inhibition of vaccinia virus replication nitric oxide inhibits marek's disease virus replication but is not the single decisive factor in interferongamma-mediated viral inhibition inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of marek's disease virus resistance and susceptibility to marek's disease: nitric oxide synthase/arginase activity balance nitric oxide inhibits hepatitis b virus replication in the livers of transgenic mice short report: increased level of serum nitric oxide in patients with dengue ifngamma production depends on il-12 and il-18 combined action and mediates host resistance to dengue virus infection in a nitric oxide-dependent manner a model of denv-3 infection that recapitulates severe disease and highlights the importance of ifn-gamma in host resistance to infection dengue fever activates the l-arginine-nitric oxide pathway: an explanation for reduced aggregation of human platelets the l-arginine-nitric oxide pathway: a potential therapeutic target in dengue haemorrhagic fever targeted blockade in lethal west nile virus encephalitis indicates a crucial role for very late antigen (vla)-4-dependent recruitment of nitric oxide-producing macrophages inhibition of nitric oxide synthesis increases mortality in sindbis virus encephalitis reovirus infection of the cns enhances inos expression in areas of virus-induced injury inhibition of influenza virus replication by nitric oxide inhaled nitric oxide therapy fails to improve outcome in experimental severe influenza increased inducible nitric oxide synthase expression in organs is associated with a higher severity of h5n1 influenza virus infection pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice enhanced antiviral antibody secretion and attenuated immunopathology during influenza virus infection in nitric oxide synthase-2-deficient mice inducible nitric oxide contributes to viral pathogenesis following highly pathogenic influenza virus infection in mice transactivation of inducible nitric oxide synthase gene by kruppel-like factor 6 regulates apoptosis during influenza a virus infection ccr2+ monocytederived dendritic cells and exudate macrophages produce influenzainduced pulmonary immune pathology and mortality tnf/inos-producing dendritic cells are the necessary evil of lethal influenza virus infection pioglitazone, a specific ligand of peroxisome proliferator-activated receptorgamma, accelerates gastric ulcer healing in rat evasion by stealth: inefficient immune activation underlies poor t cell response and severe disease in sars-cov-infected mice induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection suppression of herpes simplex virus type 1 (hsv-1)-induced pneumonia in mice by inhibition of inducible nitric oxide synthase (inos, nos2) early inhibition of nitric oxide production increases hsv-1 intranasal infection microglia are the major cellular source of inducible nitric oxide synthase during experimental herpes encephalitis activation of alveolar macrophages via the alternative pathway in herpesvirusinduced lung fibrosis pathological changes in the spleens of gamma interferon receptor-deficient mice infected with murine gammaherpesvirus: a role for cd8 t cells murine gammaherpesvirus-68 infection causes multi-organ fibrosis and alters leukocyte trafficking in interferon-gamma receptor knockout mice lentivirus infection causes neuroinflammation and neuronal injury in dorsal root ganglia: pathogenic effects of stat-1 and inducible nitric oxide synthase myeloid-derived suppressor cells in murine retrovirus-induced aids inhibit t-and b-cell responses in vitro that are used to define the immunodeficiency cytomegalovirus impairs antiviral cd8+ t cell immunity by recruiting inflammatory monocytes emergence of distinct multiarmed immunoregulatory antigen-presenting cells during persistent viral infection transgenic ccl2 expression in the central nervous system results in a dysregulated immune response and enhanced lethality after coronavirus infection genetic ablation of arginase 1 in macrophages and neutrophils enhances clearance of an arthritogenic alphavirus chronic but not acute virus infection induces sustained expansion of myeloid suppressor cell numbers that inhibit viral-specific t cell immunity hepatitis c virus targets over-expression of arginase i in hepatocarcinogenesis cd163/cd16 coexpression by circulating monocytes/macrophages in hiv: potential biomarkers for hiv infection and aids progression expansion of monocytic myeloid-derived suppressor cells dampens t cell function in hiv-1-seropositive individuals increased level of arginase activity correlates with disease severity in hiv-seropositive patients antiretroviral therapy abrogates association between arginase activity and hiv disease severity hiv type 1 gp120-induced expansion of myeloid derived suppressor cells is dependent on interleukin 6 and suppresses immunity arginase activity in the blood of patients with visceral leishmaniasis and hiv infection hepatitis b virus infection and immunopathogenesis in a humanized mouse model: induction of human-specific liver fibrosis and m2-like macrophages increased levels of arginase in patients with acute hepatitis b suppress antiviral t cells functional skewing of the global cd8 t cell population in chronic hepatitis b virus infection invariant nkt cells reduce the immunosuppressive activity of influenza a virus-induced myeloid-derived suppressor cells in mice and humans accumulation of cd11b(+)gr-1(+) cells in the lung, blood and bone marrow of mice infected with highly pathogenic h5n1 and h1n1 influenza viruses virus-helminth coinfection reveals a microbiota-independent mechanism of immunomodulation biochemistry of nitric oxide and its redoxactivated forms alterations of ribonucleotide reductase activity following induction of the nitrite-generating pathway in adenocarcinoma cells inhibition of tumor cell ribonucleotide reductase by macrophage-derived nitric oxide dna deaminating ability and genotoxicity of nitric oxide and its progenitors dna damage and mutation in human cells exposed to nitric oxide in vitro factor(s) present in herpes simplex virus type 1-infected cells can compensate for the loss of the large subunit of the viral ribonucleotide reductase: characterization of an icp6 deletion mutant a herpes simplex virus ribonucleotide reductase deletion mutant is defective for productive acute and reactivatable latent infections of mice and for replication in mouse cells nitric oxide donors inhibit the coxsackievirus b3 proteinases 2a and 3c in vitro, virus production in cells, and signs of myocarditis in virus-infected mice molecular basis of "suppressor" macrophages. arginine metabolism via the nitric oxide synthetase pathway selection of genetic variants of lymphocytic choriomeningitis virus in spleens of persistently infected mice massive expansion of antigen-specific cd8+ t cells during an acute virus infection viral persistence alters cd8 t-cell immunodominance and tissue distribution and results in distinct stages of functional impairment macrophage and t cell produced il-10 promotes viral chronicity innate immune cd11b+gr-1+ cells, suppressor cells, affect the immune response during theiler's virus-induced demyelinating disease clinical significance and functional studies of myeloid-derived suppressor cells in chronic hepatitis c patients myeloid suppressor cells induced by hepatitis c virus suppress t-cell responses through the production of reactive oxygen species macrophage polarization and hiv-1 infection contribution of immune activation to the pathogenesis and transmission of human immunodeficiency virus type 1 infection hepatitis b virus infection hpv16 tumor associated macrophages suppress antitumor t cell responses myeloid-derived suppressor cells: the dark knight or the joker in viral infections? murine gammaherpesvirus-induced fibrosis is associated with the development of alternatively activated macrophages work in dr. morrison's laboratory is supported by nih-niaid grants u19 ai109680 and r01 ai108725. kristina s. burrack was supported by nih-niaid training grant t32 ai052066. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-300429-b0zev8zb authors: sobocińska, justyna; roszczenko-jasińska, paula; ciesielska, anna; kwiatkowska, katarzyna title: protein palmitoylation and its role in bacterial and viral infections date: 2018-01-19 journal: front immunol doi: 10.3389/fimmu.2017.02003 sha: doc_id: 300429 cord_uid: b0zev8zb s-palmitoylation is a reversible, enzymatic posttranslational modification of proteins in which palmitoyl chain is attached to a cysteine residue via a thioester linkage. s-palmitoylation determines the functioning of proteins by affecting their association with membranes, compartmentalization in membrane domains, trafficking, and stability. in this review, we focus on s-palmitoylation of proteins, which are crucial for the interactions of pathogenic bacteria and viruses with the host. we discuss the role of palmitoylated proteins in the invasion of host cells by bacteria and viruses, and those involved in the host responses to the infection. we highlight recent data on protein s-palmitoylation in pathogens and their hosts obtained owing to the development of methods based on click chemistry and acyl-biotin exchange allowing proteomic analysis of protein lipidation. the role of the palmitoyl moiety present in bacterial lipopolysaccharide and lipoproteins, contributing to infectivity and affecting recognition of bacteria by innate immune receptors, is also discussed. geranylgeranyl cysteine thioether h-and n-ras (28) rab proteins (28) attachment of glycosylphosphatidylinositol anchor or phosphatidylethanolamine (29) to the c-terminus of proteins is also a form of lipidation but is not shown here. a n-myristoylation is in most cases co-translational, but during apoptosis caspases can cleave some proteins, such as bid, exposing their n-terminal glycine residue, which is then modified by attachment of myristate (30) . b hedgehog proteins are additionally modified by covalent attachment of cholesterol to their c-terminus (31) . c o-acylation of wnt proteins is reversed by notum of the α/β hydrolase superfamily (31) . exceeds the consumption of other saturated fatty acids and, in the usa it accounts for about 60% of the total intake of saturated fatty acids (2) . a growing body of experimental and clinical evidence points to a link between a westernized diet, including a high intake of saturated fatty acids, and chronic inflammatory diseases (3) (4) (5) . as dietary saturated and unsaturated fatty acids apparently modulate activity of immune cells, their influence on the immune responses triggered upon infection is also beginning to be investigated (6) . these facts drive the interest in palmitic acid with an aim of elucidating the molecular mechanisms of its immunomodulatory properties. in this review, we focus on s-palmitoylation of proteins crucial for the interactions of pathogenic bacteria and viruses with the host. we emphasize novel data on the role of s-palmitoylated proteins in the invasion of host cells by pathogens and those involved in the host innate immune responses to the infection, which have been obtained thanks to the application of new technical approaches. recently, substantial progress in the understanding of protein palmitoylation was made possible by the development of methods allowing high-throughput analysis of cellular/tissue palmitoyl proteomes. we begin, however, by showing how unique protein s-palmitoylation is among other protein lipidations. s-palmitoylation is a posttranslational modification of proteins consisting in a potentially reversible covalent attachment of palmitoyl chain to a cysteine residue(s) of proteins through a thioester bond ( table 1) . thus, s-palmitoylation resembles other reversible regulatory posttranslational protein modifications, including phosphorylation or acetylation, well-established factors affecting protein structure and functions. in particular, s-palmitoylation modifies cellular localization of proteins and their stability. the most dramatic changes of localization concern cytosolic proteins which upon s-palmitoylation acquire a hydrophobic anchor facilitating their docking into membranes (figure 1) . however, several integral membrane proteins also undergo s-palmitoylation. it often occurs on cysteine residue(s) located in the proximity of the junction of the transmembrane and cytoplasmic domains of the protein. s-palmitoylated transmembrane proteins occupy various cellular compartments, such as endoplasmic reticulum, golgi apparatus, and the plasma membrane. in accordance, for some proteins, such as transmembrane adaptor proteins in leukocytes, s-palmitoylation was found secondary to the length and hydrophobicity of the transmembrane domain as a determinant of plasma membrane destination (7) . s-palmitoylation also contributes to the compartmentalization of proteins to distinct domains of membranes-rafts and tetraspanin-rich microdomains. in fact, the interest in s-palmi toylation was boosted when it was found to be required for the targeting of some signaling proteins to rafts. rafts are nanodomains of the plasma membrane and some intracellular membranes, mainly of the trans-golgi apparatus, rich in sphingolipids, glycerophospholipids with saturated fatty acid chains, and cholesterol (32) . the plasma membrane nanodomains are sites of signal transduction by distinct receptors of immune cells involved in both acquired immune reactions, such as t cell receptor (tcr), fcε receptor i, fcγ receptor ii, and in innate immune responses, such as toll-like receptor 4 (tlr4) (33, 34) . rafts are also sites of virion assembly and budding, as established, e.g., for influenza a virus and human immunodeficiency virus-1 (hiv-1) (35, 36) . peripheral membrane proteins acylated with saturated fatty acids are likely to anchor preferentially between the ordered saturated lipids of rafts rather than between the disordered lipids of the surrounding membrane. it has been shown that, owing to their raft localization, s-palmitoylated kinases of the src family interact with raft-associating plasma membrane immunoreceptors and initiate signaling cascades fundamental to acquired immunity (15, 37, 38) . it is worth noting that also the acyl chains attached to proteins can affect the membrane structure. studies on model membranes have revealed that palmitic and myristic acids facilitate formation of ordered lamellar membrane regions (39, 40) . in accordance, s-palmitoylation of erythrocyte peripheral membrane protein called membrane-palmitoylated protein 1 (mpp1) was found to be required for the proper lateral organization and fluidity of erythrocyte membrane. in the absence of mpp1 s-palmitoylation, raft assembly was disturbed and erythrocyte functioning compromised leading to hemolytic anemia in patients deficient in the enzyme catalyzing this reaction (41, 42) . preferential raft association is a feature of some s-palmitoylated transmembrane proteins, e.g., adaptor proteins pag, lat, and ntal, which collaborate with the abovementioned immunoreceptors. in fact, palmitoylation is required for the raft association of most integral raft proteins (8, 43, 44) . on the other hand, s-palmitoylation does not obligatorily confer raft localization on transmembrane proteins. certain s-palmitoylated proteins, such as transferrin receptor, glycoprotein g of vesicular stomatitis virus (vsv), and anthrax toxin receptor, tumor endothelial marker 8 (tem8), are actually excluded from rafts. apparently, a combination of s-palmitoylation and the properties of the transmembrane domain of the protein contribute to its destination to the raft or non-raft environment (43, 45) . it has also been proposed that the attachment of a fatty acyl chain at the juxtamembrane cysteine(s) of a protein can induce tilting of its transmembrane fragment, determining in which part of the membrane it will accommodate to avoid a hydrophobic mismatch potentially caused by the thickness of the bilayer (46) . that not all s-palmitoylated proteins associate with rafts has been shown convincingly for macrophage-like raw264 cells, where only about half of those proteins were found in the triton x-100-resistant membrane fraction enriched in rafts (47, 48) . in accordance, proteomic data on the distribution of s-palmitoylated proteins in prostate cancer cells have revealed that several such proteins are recovered in the non-raft (triton x-100-soluble) fraction and are likely localized to microdomains enriched in scaffold proteins called tetraspanins (49) . the tetraspanins are small integral membrane proteins found in the plasma membrane and other cellular membranes, having four transmembrane helices and undergoing s-palmitoylation at several conserved cysteine residues. the tetraspanins interact with each other and with various transmembrane and cytosolic partners, often also s-palmitoylated, forming microdomains ("tetraspanin web") (50) . it has been suggested that the amino acid composition of the s-palmitoylation site in some transmembrane proteins, such as the adaptor proteins involved in acquired immune responses, determines the association of those s-palmitoylated proteins with rafts or with the tetraspanin-enriched microdomains (44) . an intriguing and still poorly addressed question concerns the relation between rafts and the tetraspanin-enriched microdomains, apparently of functional significance, e.g., during virus budding from host cells (35) . this uncertainty stems partially from the fact that s-palmitoylation of tetraspanins governs their interactions with cholesterol and gangliosides leading at certain conditions to the recovery of tetraspanins in detergent-resistant membrane fractions enriched in rafts (51, 52) . besides its involvement in targeting proteins to rafts or tetraspanin-enriched microdomain, s-palmitoylation has been found to govern accumulation of the transmembrane chaperone protein calnexin in the perinuclear domain of endoplasmic reticulum (53) . s-palmitoylation also affects protein stability through its interplay with ubiquitination or phosphorylation, as found for the anthrax toxin receptor tem8, antiviral interferon-induced transmembrane protein ifitm1, calnexin, and zdhhc6, one of palmitoyl acyltransferases described below (54) (55) (56) (57) . possibly the most intriguing is the reversible character of s-palmitoylation. enzymes catalyzing palmitoylation and depalmitoylation of proteins have been characterized (58, 59) . palmitate is transferred onto the thiol group of cysteine from cytosolic palmitoyl-coa by palmitoyl acyltransferases, enzymes containing the zinc finger dhhc domain named after the highly conserved asp-his-his-cys peptide (figure 1 ). this is a twostep reaction comprising transient autoacylation of zdhhc enzymes and transfer of the fatty acyl chain from this intermediate to a protein substrate (60) . in mammals, the zdhhc enzyme family consists of 24 proteins, and zdhhc proteins are also found in other eukaryotes but not in bacteria nor are they encoded by viral genomes. mammalian zdhhc enzymes, each having at least four transmembrane helices, are located in the plasma membrane, endoplasmic reticulum, and golgi apparatus (58) . they display some specificity toward their protein substrates and also selectivity toward fatty acyl moieties other than palmitate, which contributes to the heterogeneity of lipids attached to proteins, such as viral glycoproteins described below (61) . in the opposite process, the thioester bond is cleaved by acyl-protein thioesterases (apts) (apt1 and apt2) and palmitoyl protein thioesterases (ppts) (ppt1 and ppt2), which are localized in the cytosol and in lysosomes, respectively. apt1 and apt2 likely govern the dynamic functional changes of s-acylation of proteins (62) while ppt1 and ppt2 depalmitoylate proteins during their degradation (63, 64) . recently, serine hydrolases of the abhd17 family have also been identified as depalmitoylating enzymes, and their specific substrate proteins determined (65, 66) . of note, the zdhhcs, apt1/apt2, and abhd17 proteins are s-palmitoylated themselves, and palmitoylation of zdhhcs and depalmitoylation of apt1/2 can occur in a cascade manner (57, 62) . the dynamic cycles of palmitoylation/depalmitoylation detected for several peripheral membrane proteins are often synchronized with intracellular trafficking of those proteins. they circulate between the plasma membrane and the golgi apparatus or endosomes, as exemplified by n-and h-ras, r7-regulator of g protein and apts. in fact, it is proposed that palmitoylationdependent anchoring of apt1 in the plasma membrane allows it to depalmitoylate h-ras at this location, while subsequent autodepalmitoylation releases apt1 guiding it, alongside h-ras, for another round of palmitoylation at the golgi apparatus (62, (67) (68) (69) . cycles of palmitoylation/depalmitoylation are crucial for signaling by distinct plasma membrane receptors and for their distribution (69) (70) (71) . activation of tcr receptor or fas receptor in t cells was found to trigger quick and transient palmitoylation of lck kinase of the src family (72, 73) , but the exact meaning of the dynamic protein s-palmitoylation for processes triggered during the host-pathogen interaction awaits elucidation. it is worth mentioning that although the zddhc enzymes catalyze bulk protein palmitoylation in eukaryotic cells (74) , some proteins have a unique autopalmitoylation activity. these include bet3, a component of a multisubunit transport protein particle complex involved in vesicular trafficking, tea domain transcription factors, and also bacterial evf protein (75) (76) (77) (78) . the palmitic acid residue is attached constitutively to a specific cysteine residue of those proteins, remains buried inside a hydrophobic pocked in their core thereby affecting the tertiary structure and, thus, interactions with other proteins (75, 77) . an exhaustive discussion on the physiology of s-palmitoylated proteins in eukaryotic cells can be found in several recent reviews (46, 79, 80) . it has been established that, in addition to palmitate, various other fatty acyl moieties, such as saturated stearate (c18:0) or protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 monounsaturated palmitoleate (c16:1), and oleate (c18:1) can be attached via the thioester linkage to proteins. the early reports on the heterogeneity of the fatty acyl moieties attached to cysteines obtained by analysis of selected immunoprecipitated proteins (15, 16, (81) (82) (83) have recently been complemented by a comprehensive proteomic analysis of fatty-acylated proteins of macrophage-like raw264 cells (14) . the latter study showed that an enrichment of culture medium of cells with monounsaturated fatty acids leads to their incorporation into a similar set of proteins as those normally modified with palmitate. among them, several proteins relevant to innate immune responses were found. all these data justify the use of a broader term s-acylation rather than s-palmitoylation ( table 1 ). the physiological consequences of s-acylation of proteins with individual fatty acids are slowly being revealed. modification of fyn kinase with polyunsaturated fatty acid residue, such as arachidonate (c20:4), disturbed its raft localization and, thereby, tcr signaling (15) . a heterogeneity of s-acylation was also found in viral spike proteins, such as hemagglutinin (ha) of influenza a virus, and e1 and e2 of semliki forest virus, which are modified in host eukaryotic cells by attachment of both palmitate and stearate (9) . in ha, stearate is attached at the transmembrane cysteine while palmitate is attached to two cysteine residues in a membrane-proximal region of the protein. the stearoyl chain seems to accommodate into a groove formed by amino acids of the transmembrane helix shaping the domain in a way that facilitates its fitting into rafts (84) . s-stearoylation of human transferrin receptor 1 at the juxtamembrane cysteine residues(s) is a key factor of the signaling cascade controlling mitochondrial morphology and functioning (13) . of interest, the latter study also showed that dietary supplementation of stearic acid reversed the deleterious effects of a genetically determined mitochondria dysfunction in drosophila. taking into account that unsaturated fatty acids affect the profile of s-acylation of proteins in vitro (14, 15) , it is of outmost interest whether a similar effect of unsaturated and saturated (palmitic) fatty acids could be achieved in vivo with respect to proteins of immune cells. beside s-acylation, less frequently palmitate can also be attached to the amine group of various amino acids (glycine, cysteine, and lysine) giving n-palmitoylation or to the hydroxyl group of serine or threonine in a process called o-palmitoylation ( table 1) . as during s-palmitoylation, also other fatty acids can be utilized in these processes named then n-and o-acylation. thus, a type of protein n-acylation is n-myristoylation, a frequent modification contributing to membrane anchoring of peripheral proteins. the saturated myristate (c14:0) is transferred to the protein from myristoyl-coa by n-myristoyl transferase (two isozymes in mammals). in a vast majority of cases, myristate is attached cotranslationally to the n-terminal glycine residue (after removal of the initiator methionine) via an amide linkage ( table 1) . like most lipidations, this modification is irreversible. several viral proteins are n-myristoylated, such as gag of hiv-1 crucial for budding of newly formed virions from plasma membrane rafts of host cells, and proteins of parasitic protozoa plasmodium falciparum, trypanosoma brucei, and leishmania donovani (causing malaria, african sleeping sickness, and leishmaniosis, respectively). for this reason, n-myristoyl transferase is considered a potential drug target in the therapy of these diseases (17-19, 85, 86) . data on the n-and o-palmitoylation of proteins involved in the host-pathogen interactions are limited, but interesting conclusions can be drawn from the information concerning proteins taking part in other processes. n-palmitoylation of the n-terminal glycine of the α-subunit of a heterotrimeric g protein (gαs) has been described (20) ( table 1 ) besides the wellknown s-palmitoylation of this pivotal signaling protein. the n-palmitoylation of gαs is irreversible, and the enzyme responsible for this modification is unknown. it has been speculated that s-to n-palmitoyl migration can occur both in vivo and also in vitro during mass spectrometry analysis (20, 87) . this suggests that caution is needed in interpreting results of this methodological approach, which is used with increasing frequency to study fatty acylation of proteins in immune cells (see next sections). probably the best-characterized is the n-palmitoylation of the n-terminal cysteine residue of hedgehog proteins (sonic, indian, and desert in mammals). it determines secretion of these proteins, which regulate embryonic patterning ( table 1) . secreted wnt and ghrelin proteins are examples of o-acylation of serine residues with unusual fatty acid residues such as palmitoleate (c16:1) and octaonoate (c8:0) ( table 1 ). the fatty acylation of hedgehog, wnt, and ghrelin is catalyzed by enzymes from the multipass membrane-bound o-acyl transferases family (31) . besides these unusual fatty acid residues, attachment of palmitate to serine and threonine residues is found in secreted venom toxins of the spider plectreurys tristis, which selectively target neuronal ion channels (88) . also histone h4 is o-palmitoylated at a serine residue in the nucleus by acyl-coa:lysophosphatidylcholine acyltransferase (25) ( table 1 ). the latter is of special interest in the context of innate immune responses since histone h4 o-palmitoylation regulates transcriptional activity, which is the final outcome of the pro-inflammatory signaling pathways triggered by receptors of the innate immune system. special attention should be devoted to ε-n-acylation consisting in the attachment of a fatty acid residue to the side chain of lysine by amide linkage ( table 1) . ε-n-myristoylated are interleukin 1α (il-1α) and tumor necrosis factor α (tnfα), the pro-inflammatory cytokines crucial in combating bacterial infections (22) . the enzyme(s) catalyzing this reaction is unknown, but it has been established that sirtuins reverse this modification (89) . the ε-n-acylation affects the release of tnfα by immune cells (90, 91) . surprisingly, this rare modification is also found in toxins of so-called rtx (repeats-in-toxin) class released by some pathogenic gram-negative bacteria (23, 24) . we describe these cases in more detail in the following sections. besides s-palmitoylation and n-myristoylation, s-prenylation is another common lipidation that endows proteins with a hydrophobic moiety and contributes to their association with membranes. this modification relies on the posttranslational and irreversible attachment of either farnesyl or geranylgeranyl chains to a cysteine residue in the c-terminal caax box (alternatively also cc and cxc motifs) via a thioether linkage. the process is catalyzed by protein prenyl transferases that use polyprenylpyrophosphate as the donor of the isoprenoid group ( table 1) . in peripheral membrane proteins, the s-palmitoylation site is often located in proximity of n-myristoylation or s-prenylation sites or a polybasic motif, which all are likely to mediate initial weak binding of a protein to a membrane and thereby facilitate subsequent attachment of palmitate to the protein by the integral membrane zdhhc enzymes (31) . in contrast to s-palmitoylation, data on the role of s-prenylation of proteins key to the host-pathogen interactions are scarce (92) . however, since s-prenylation is typical for the ubiquitous small gtpases of ras superfamily, it is vital for proper functioning of b and t cells (93, 94) . a glance at table 1 indicates that palmitate can be covalently bound via oxyester, amide, and thioester linkages to respective amino acid residues creating an array of possible modifications. o-and n-palmitoylation of proteins seems to be stable, resembling in this regard the other common protein lipidations, n-myristoylation and s-prenylation. by contrast, there exist enzymes cleaving the thioester bond formed during s-palmitoylation. for a long time, our understanding of protein s-palmitoylation and its dynamics was poor in comparison with other reversible protein modifications due to technical difficulties. only recently have these difficulties been overcome with the introduction of methods allowing high-throughput identification of palmitoylated proteins, also those involved in the immune response to microbial pathogens, as discussed in the next sections. one of the basic problems hindering studies on protein s-palmitoylation lies in the fact that there is no identifiable consensus sequence for the palmitoylation site that could facilitate its prediction. from the technical point of view, the progress in a comprehensive survey of protein s-palmitoylation was also hampered by a lack of antibodies detecting this modification, with the sole exception of an antibody specific to palmitoylated psd-95 (95) . a classical method used to demonstrate protein palmitoylation is based on metabolic labeling of living cells with [ 3 h]-palmitic acid, subsequent immunoprecipitation of a selected protein and detection of the incorporated tritiated fatty acid by autoradiography (96) . a major disadvantage of this method is its low sensitivity. only a minute fraction of the radioactive palmitate is bound to proteins, the majority being incorporated into lipids, which requires lengthy film exposure (counting in days). a methodological breakthrough in the identification of palmitoylated proteins came with the development of two nonradioactive methods based on so-called click chemistry (97) (98) (99) and acyl-biotin exchange (abe) (74, 100) . these techniques have paved the way for high-throughput mass spectrometry-based proteomic analysis of protein palmitoylation in various cells and tissues and facilitated identification of new palmitoylated proteins of both pathogens and host cells involved in the innate immune responses. in the click chemistry-based method, cells are metabolically labeled with a palmitic acid analog bearing an alkyne group at the ω carbon of the fatty acyl chain, such as 17-octadecynoic acid (17odya) or alk-16 (figure 2a) , and this step resembles the classic labeling of cells with [ 3 h]-palmitic acid. however, in the click chemistry-based assay, the labeling and lysis of cells is followed by in vitro coupling of the function group of the palmitic acid analog to a reporter tag, which greatly enhances the sensitivity of detection of labeled proteins (98, 99) . thus, after cell lysis, the labeled proteins are subjected to cu (i) -catalyzed cycloaddition known as "click" reaction with an azide-bearing detection tag. in this step, a triazol is formed between the alkyne group in the palmitic acid analog and the azide of the tag (figure 2a) . the azide-bearing tags can be either fluorescent, such as tetramethylrhodamine or dyes with infrared fluorescence, or carry a biotin moiety. depending on the tag used, subsequent sds-page separation of proteins allows global visualization of palmitoylated proteins by simple in-gel fluorescence or by blotting with a streptavidin-conjugated reporter (98, 101, 102) . notably, proteins biotinylated via the click reaction can also be enriched on streptavidin-coated beads and then subjected to on-bead tryptic digestion (or in-gel digestion if eluted from the beads) followed by identification by mass spectrometry. such comprehensive click chemistry-based proteomic analysis has brought about identification of an array of palmitoylated proteins in dendritic cells (10, 103) , macrophage-like raw264 cells (14) , and t cells (99, 104, 105) . some of the s-palmitoylated proteins newly identified in those studies, such as ifitm3 and tlr2, are involved in the host-pathogen interactions regulating innate immune responses (10, 103) , while many others are known to contribute to adaptive immunity (99, 105) , as described below. recently, global profiling of toxoplasma gondii (the causative agent of toxoplasmosis) has been performed revealing that many components of the parasite's motility complex are palmitoylated (106) . similar studies on cryptococcus neoformans (the fungus causing cryptococcal meningitis) have revealed a contribution of specific zdhhc palmitoyl acyltransferase, called pfa4, to its virulence (107) . moreover, application of analogs of various saturated and unsaturated fatty acids confirmed the heterogeneous nature of the fatty acylation of proteins in raw264 cells and suggested that dietary unsaturated fatty acids, after incorporation to proteins, can change their properties and thereby affect the functioning of immune cells (14) . the major advantage of the click chemistry-based method is that it can reveal the time course of protein s-palmitoylation. by using click chemistry-based labeling in the pulse-chase mode, one can follow the dynamics of protein palmitoylation. with such an approach, it was found that the palmitate turnover on lck, an src-family tyrosine kinase, is accelerated by t cell activation (72) . additional introduction of stable isotope labeling by amino acids in cells (silac) has provided quantitative proteomic data , and after cell lysis, the click reaction is conducted with azido-tagged biotin or fluorescent probes allowing enrichment and detection of labeled proteins in various ways. biotinylated proteins can be bound on a streptavidin resin and then released using, e.g., high concentrations of urea and sds (108) . when a cleavable derivative of biotin, azido-azo-biotin, is used the labeled proteins are eluted from streptavidin beads with sodium dithionite, which cleaves the diazobenzene moiety in the linker arm of azido-azo-biotin, and analyzed by mass spectrometry or immunoblotting (109) . (b) abe method. cells or tissues are lysed, free thiol groups of proteins are blocked by alkylation, and palmitoyl moieties are released with hydroxylamine. the newly exposed protein thiol groups are subjected to labeling with biotin-hpdp allowing selective binding, elution, and analysis of the originally s-palmitoylated proteins. the proteins can also be captured without biotinylation through a direct interaction of their thiol residues with a thiol-reactive resin (acyl-rac technique). on the dynamics of protein palmitoylation in the cell (104, 110) . this approach revealed, rather unexpectedly, that in unstimulated t cell hybridoma, the palmitoylation of most protein species does not undergo turnover (104) . another advantage of the click chemistry-based assay is its high specificity, because the alkyne group introduced in the analog of palmitic acid is not normally found in cells (98, 102) . the click chemistry-based methods can also be used to follow the cellular localization of palmitoylated proteins by immunofluorescence when combined with the proximity ligation technique (111, 112) . palmitoylation of individual proteins can also be studied after their immunoprecipitation (11, 72, 73, 98 ). despite its unquestionable success, the click chemistry-based methods have limitations. they will detect only those proteins that undergo palmitoylation during the period of the metabolic labeling of cells. one should also bear in mind that the palmitic acid analog can be incorporated at s-, n-, and o-palmitoylation sites alike (111, 112) . in addition, although 17odya (alk-16) is preferentially used to mimic palmitoylation of proteins, it can also be incorporated with low efficiency at n-myristoylation sites of proteins (98, 99) . another group of proteins that will be labeled with the palmitic acid analog but are not s-palmitoylated are those bearing the glycosylphosphatidylinositol (gpi) anchor (85, 113) . most of these limitations can be overcome using various fatty acid reporters, inhibitors, and by exploiting the sensitivity of the thioester bond to hydroxylamine treatment. given the large variety of chemical reporters preferentially mimicking distinct fatty acids, recent years have witnessed a plethora of chemistry-based proteomic studies not only on palmitoylated but also myristoylated proteins and proteins bearing the gpi anchor, including those of pathogens and immune cells (10, 14, 85, 86, 114) the abe method reveals protein the abe method can be used as a complement to the click chemistry-based approach in cell studies but unlike the latter it is uniquely suitable for studying whole tissues. abe does not require metabolic labeling of proteins in living cells, thus some of the abovementioned limitations and difficulties do not apply. the abe method relies on in vitro exchange of thioester-linked palmitate to a derivative of biotin which allows subsequent affinity purification of the resulting biotin-labeled proteins on streptavidin-coated beads ( figure 2b) . the first step of the abe involves lysis of cells or tissues followed by irreversible blockage of free thiol groups in the solubilized proteins by alkylation, most often with n-ethylmaleimide. subsequently, the thioester bonds existing in s-palmitoylated proteins are broken with hydroxylamine, releasing palmitoyl moieties. the newly exposed thiol groups can now be tagged with sulfhydryl-reactive derivatives, such as biotin-hpdp, forming disulfide bonds with thiols. the biotinylated proteins are subsequently captured on streptavidin-coated beads and eluted with agents that reduce the disulfide bond between the protein and biotin-hpdp, such as β-mercapthoethanol, dtt, or tcep (49, 74, 115, 116) . as an alternative to biotinylation, in the so-called acyl-rac technique, the newly exposed protein thiol groups in hydroxylamine-treated cell lysates are captured on a resin containing sulfhydryl-reactive groups (117) . in both abe and acyl-rac, the eluted proteins can be separated by sds-page and visualized by gel staining or immunoblotting, or identified by mass spectrometry. furthermore, when the hydroxylaminereleased palmitoyl moieties are exchanged for a polyethylene glycol-maleimide derivative of a distinct molecular weight, a shift in-gel migration of tagged proteins is observed reflecting the number of fatty acyl residues originally s-bound to the protein (118, 119) . the abe method has so far been used successfully for proteomic profiling of s-acylated proteins in immune cells, such as raw264 cells (48), several types of blood cells, such as platelets, primary t cells, and immortalized b cells (120) (121) (122) , pathogenic microorganisms such as t. brucei and t. gondii (123, 124) , and tissues (125, 126) . to quantify the aberrations in protein palmitoylation in a mouse model of huntington's disease, whole animal stable isotope labeling of mammals (silam) was applied followed by tissue isolation and abe procedure (127) . in another approach, for quantitative analysis of the t-cell palmitoylome, abe was combined with labeling of proteins with various oxygen isotopes during their digestion with trypsin before mass spectrometry analysis (122) . in addition, preselection of tryptic peptides obtained by abe on streptavidin-coated or sulfhydrylreactive resins greatly facilitates the identification of s-acylation sites by mass spectrometry (49, 110, 117) . some aspects of the abe method deserve a comment. since the assay relies on the sensitivity of thioester bonds to hydroxylamine, abe detects all s-acylation without distinguishing between s-palmitoylation and the other cases. furthermore, there is a possibility of false-positive detection of proteins bearing a thioester linkage with compounds other than fatty acyl residues, such as ubiquitin in the e2 ubiquitin conjugase ubc1 (115) . another source of false-positives is proteins in which free thiol groups were not completely alkylated before biotinylation. on the other hand, insufficient deacylation of bonafide fatty-acylated proteins with hydroxylamine results in their absence in the final sample (116) . in summary, the click chemistry-based method relies on metabolic labeling of cells with a palmitic acid analog which incorporates into proteins and next tagging it with reporter molecules greatly enhancing the sensitivity of detection. it only reveals proteins undergoing s-palmitoylation during metabolic labeling of cells and allows revealing turnover of this modification. by contrast, the abe method is based on direct binding of sulfhydryl-reactive derivatives to thiol groups of cysteines unraveled by hydroxylamine treatment after lysis of cells or tissues. it allows the investigation of the whole but static palmitoylome. a comparative proteomic study of protein palmitoylation in p. falciparum found that the sets of proteins identified using these two approaches overlapped in 57.2% (113) , indicating that they provide complementary data on the cellular palmitoyl proteome. thanks to the application of the click chemistry-and abe-based methods numerous new palmitoylated proteins have been identified. in 2015, a swisspalm database was launched, (128) which provides an excellent, manually curated resource of information on palmitoylated proteins, palmitoylation sites, etc., available at http://swisspalm.epfl.ch/. all these efforts have greatly furthered our knowledge on molecular mechanisms regulating diverse aspects of cell functioning, including host-pathogen interactions and progress of infectious diseases, as highlighted below. bacteria lack protein palmitoyl acyltransferases of the zdhhc family and, therefore, are essentially devoid of s-palmitoylated proteins. yet, they have developed unique mechanisms utilizing fatty acids, such as palmitic acid, to modify their glycolipids and proteins. these modifications augment infectivity and help bacteria evade recognition by the host innate immune system. for example, the vast majority of gram-negative bacteria produce lipopolysaccharide (lps) as a part of their outer membrane. lps is composed of the variable polysaccharide o-antigen and more-conserved lipid a containing two glucosamine residues hexa-acylated with hydroxymyristic, myristic, and lauric acid. lipid a is recognized by cd14 protein and tlr4 receptor complexed with md2 protein on the plasma membrane of the host immune and some non-immune cells. activation of tlr4 triggers strong pro-inflammatory reactions aiming at eradication of the bacteria, but when exaggerated, eventually leading to sepsis (129) . incorporation of an additional palmitoyl chain into lipid a markedly diminishes its ability to activate tlr4 and to induce the host pro-inflammatory responses, which is correlated with an increased survival of bacteria forming a biofilm (130, 131) . this strategy is utilized among others by salmonella typhimurium, a causative agent of gastroenteritis, by bordetella bronchiseptica, a respiratory pathogen of human and other mammals, and by protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 yersinia pestis causing plague (132, 133) . the formation of the extra-acylated lps relies on the transfer of palmitate from phospholipids onto the hydroxymyristate chain at position 2 of glucosamine of lipid a. the reaction is catalyzed by lipid a palmitoyltransferases (pagp in salmonella and its homologs in other bacteria) localized in the outer membrane of these pathogens (134, 135) . in addition to causing steric hindrance preventing the binding to the tlr4/md2 complex, the hepta-acylation of lps also protects bacteria from the lytic activity of cationic antimicrobial peptides, most likely by reducing the fluidity of the bacterial outer membrane (136, 137) . apart from being incorporated into lps in diverse bacteria, palmitate has also been found to modify a virulence factor of gram-negative erwinia carotovora, the evf protein. the palmitoyl chain is linked via a thioester bond to the cys209 residue at the center of evf, plausibly by a self-palmitoylating activity of the protein. e. carotovora is a phytopatogen using insects such as drosophila as vectors for dissemination between plants. the palmitoylation of evf is required for infectivity of e. carotovora and its persistence in the insect gut, however, its mode of action of unknown. it has been speculated to be linked with an ability of evf to associate with lipid bilayers, but the lack of similarities between evf and any other bacterial protein of known function makes prediction on this subject difficult (79) . a number of bacterial toxins of so-called rtx class released during infection of mammals by pathogenic gram-negative bacteria undergo ε-n-acylation of the side chain of internal lysines. these toxins include adenylate cyclase of bordetella pertussis, acylated with palmitic acid, and α-hemolysin of extraintestinal (uropathogenic) escherichia coli, acylated with myristic acid and also 15-and 17-carbon fatty acids. the acylation is catalyzed by an endogenous bacterial acyltransferase which, unlike its eukaryotic counterparts, transfers the acyl chain not from acyl-coa but from acyl-carrier protein. the acylated toxins secreted by the bacteria bind to the plasma membrane of the host cells, oligomerize and form pores causing cell lysis. in the case of the toxin of b. pertussis, essential is also the delivery of the adenylate cyclase moiety to the cell interior. acylation is required for virulence possibly being involved in oligomerization of the toxins (23, 24, 138) . although lacking s-palmitoylated proteins (with the single known exception of evf), bacteria express a wide range of membrane-bound proteins modified by a complex lipidation at the n-terminus, with palmitate frequently being a component of the lipid moiety (139, 140) . the bacterial lipoproteins are synthesized in a multistep process catalyzed by a unique set of lipoprotein processing enzymes, lgt, lspa, and lnt, absent in eukaryotic cells. the formation of these lipoproteins begins with the attachment of a diacylglycerol via a thioester bond to a cysteine residue located in the so-called lipobox motif of the signal sequence of the transmembrane lipoprotein precursor. the signal sequence is then cleaved next to the lipid-modified cysteine leaving it at the n-terminus of the mature protein (141) . in gramnegative and less frequently also gram-positive bacteria, a third fatty acid residue is additionally attached via an amide linkage to the amino group of the cysteine in a reaction analogous to the n-acylation of hedgehog proteins (see table 1 ). this di-and tri-lipidation ensures membrane anchoring of the lipoproteins. all such lipoproteins of gram-positive bacteria are exposed to the milieu while in gram-negative bacteria some face the periplasm. the lipoproteins of gram-positive bacteria, e.g., streptococcus pneumoniae (causing pneumonia), mycobacterium tuberculosis (tuberculosis), and gram-negative bacteria, such as neisseria meningitidis (meningitis), y. pestis (plague), the spirochaete borrelia burgdorferi (lyme disease) and treponema pallidum (syphilis) are crucial for their virulence. they control several aspects of the host-pathogen interactions, like adhesion and entry to host cells, protection against proteolysis and oxidative stress in the host cell, and regulation of expression of genes encoding cytokines both during initiation and progress of the disease (140) (141) (142) . the surface exposure of the lipoproteins allows their involvement in the host cell invasion while on the other hand forming the so-called pattern signal recognized by the tlr2 receptor, which triggers the pro-inflammatory responses helping to combat the bacteria (143) . of interest, tlr2 is s-palmitoylated, as discussed below. the involvement of lipoproteins in pathogenesis fuels studies on their properties. one such recent work employing click chemistry to profile the lipoproteins of e. coli identified 88 lipoproteins with high/medium confidence, 70% of them predicted before by bioinformatics analysis (144) . notably, in that study a 14-carbon alkynyl fatty acid analog alk-14 rather than alk-16 was preferentially incorporated into the lipoproteins, contradicting earlier studies using gas chromatography and tlc, which found that palmitate was predominantly used for bacterial protein modification (139) . further studies are required to establish whether the fatty acid found in lipoproteins varies depending on culture conditions or is species specific. for example, 17odya labeling for click reaction confirmed incorporation of palmitate into pallilysin (tp0751), a lipoprotein of t. pallidum. pallilysin is a metalloprotease that degrades human fibrinogen and laminin. it is suggested that its exposure on the bacteria surface enables degradation of host structural proteins to facilitate rapid dissemination of this highly invasive pathogen (140) . bacteria occasionally high-jack the palmitoylation machinery of host cells to modify the environment so as to favor their internalization, survival, and replication inside the cells. bacillus anthracis (the causative agent of anthrax) is an example of such bacteria that modify s-palmitoylation of host proteins to their ends. the anthrax toxin produced by this pathogen binds to the tem8 and cmg2 (capillary morphogenesis protein-2) proteins which, under physiological conditions, are involved in cell-cell and cell-extracellular matrix interactions. they are s-palmitoylated at multiple (two to four) cysteines (54) . the s-palmitoylation of tem8 was found to inhibit its association with plasma membrane rafts preventing its ubiquitination by the raft-associated e3 ubiquitin ligase cbl. the binding of anthrax toxin drives association of the receptor-toxin complexes with rafts possibly correlated with depalmitoylation of the receptor. this allows subsequent ubiquitination of the receptor, an uptake of the receptor/toxin complexes in a clathrin-dependent manner and eventual delivery of the toxin to endosomes. these events are facilitated by s-palmitoylation of partner(s) of the receptors, most likely including kinases of the src family (54, 145, 146) . while b. anthracis utilizes palmitoylated host proteins to induce its internalization, a growing body of data suggests that protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 also bacterial proteins can undergo s-palmitoylation inside the host cells. this type of modification concerns so-called effectors, bacterial proteins that are injected into the host cell cytoplasm either across the plasma membrane or the membrane of vesicles enclosing internalized pathogens, with the help of their secretion systems. these are secretion systems type iii and type iv, homologs of which have been described for pathogens and symbionts of mammals, insects, and plants (147, 148) . the bacterial effectors can be s-palmitoylated to reach host cell membranes and thereby accumulate at a location most suitable for their activity. application of the click chemistry-based method utilizing an analog of palmitic acid (alk-16) for cell labeling has revealed s-palmitoylation of two effector proteins of salmonella enterica, such as ssph2 and ssei (149) . s. enterica invades gut endothelial cells and is a leading cause of gastroenteritis and typhoid fever. ssph2 carries an e3 ubiquitin ligase domain while ssei shows sequence homology to bacterial proteins that have a deamidase activity, and inhibits migration of salmonella-infected cells. the latter activity requires s-palmitoylation of ssei. both proteins are stably s-palmitoylated, most likely by zdhh3 and zdhh7 of the host and bind to the plasma membrane in a palmitoylationdependent manner (149) . also two effector proteins of the ipah family of shigella spp. were found to be s-palmitoylated in that study, suggesting that this modification can control the activity of effector proteins of other pathogens as well (149) . indeed, gobx and lpda, effector proteins of legionella pneumophila, the causative agent of legionnaires' disease invading macrophages and lung endothelial cells, are s-palmitoylated as was found recently using click chemistry. lpda is a phospholipase hydrolyzing various phosphatidylinositols while gobx is an e3 ubiquitin ligase. gobx is targeted in a palmitoylation-dependent manner to the golgi apparatus, and lpda to the plasma membrane and a subset of intracellular vesicles (150, 151) . thus, the diversified subcellular localization of bacterial effector proteins reflects that of eukaryotic proteins. it is worth noting that global profiling of acylated proteins with the application of click chemistry and an alkyne-functionalized analog of myristic acid, alk-14, for cell labeling was effective in reveling the mechanism of action of shigella flexneri effector protein ipaj of type iii secretion system. this is a unique protease that cleaves off the n-terminal myristoylated glycine. this proteolytic demyristoylation activity of ipaj is specific toward golgi-associated arf/arl family of gtpases regulating cargo transport through the golgi apparatus, inhibition of which is apparently pivotal for virulence of the bacteria causing diarrhea in humans (152) . in addition to the s-palmitoylation of the effectors of pathogenic bacteria of mammals mentioned earlier, double acylation, n-myristoylation and s-palmitoylation, has been reported of the so-called avirulence (avr) proteins (effectors of type iii secretion system) of pseudomonas syringae, a causative agent of diverse plant diseases. among them, avrrpm1 and arvb are n-myristoylated and s-palmitoylated by host acyltransferases at neighboring glycine and cysteine residues localized at the n-terminus of the proteins (similarly to eukaryotic kinases of the src family), while in avrpphb and two avrpphb-like effectors-orf4 and nopt, the double acylation motif is exposed after auto-cleavage of the proteins (similarly to some eukaryotic proteins cleaved by caspases). the acylation of the avr proteins ensures their anchoring in the host plasma membrane, which is required for their functioning. in disease-susceptible plants avr proteins contribute to successful infection; however, in plants expressing host resistance (r) genes they trigger plant defense signals, in both cases engaging plasma membrane-associated host proteins (153, 154) . the importance of palmitoylation of bacterial effector proteins for their infectivity is only beginning to be uncovered, in no small part owing to the development of the click chemistry-based method for detection of this protein modification. however, the strategy of high-jacking the host palmitoylation machinery to modify own proteins seems to be much more commonly employed by viruses. viruses do not encode palmitoyl acyltransferases but exploit extensively the host palmitoylation machinery to modify their proteins essential for infection of host cells and own replication. in fact, s-palmitoylation of proteins was discovered in 1979 as a modification of envelope glycoproteins of sindbis virus and vsv. in those studies [ 3 h]-palmitic acid was used for metabolic labeling of virus-infected cells and labeled proteins were identified by autoradiography (12, 155) . subsequently, a number of other viral proteins have been found to be palmitoylated using this approach. the most-studied group of viral palmitoylated proteins is those found in enveloped viruses, i.e., viruses covered by a lipid bilayer obtained during their replication from a membrane of the host cell, such as the plasma membrane or endoplasmic reticulum. influenza virus, hiv-1, hepatitis c virus (hcv), and herpes simplex virus (hsv) are the best known enveloped viruses. the envelope is rich in transmembrane, often s-palmitoylated, glycoproteins called spikes, which can bind to cognate receptors on the host cell plasma membrane triggering endocytosis of the virion, mediate subsequent fusion of the viral and cellular membranes allowing entry of the viral genome to the cytoplasm, and are also involved in the budding of newly formed virus particles from the cell. an example of such multifunctional palmitoylated transmembrane glycoproteins is ha present in the envelope of influenza virus together with another palmitoylated transmembrane protein, the matrix protein m2, which forms a proton channel earning the protein the name viroporin. as mentioned earlier, ha of influenza a virus is s-stearoylated and s-palmitoylated, respectively, at one cysteine residue located in the transmembrane domain of ha and two cysteines found in the cytoplasmic (intraviral) tail in close proximity to the membrane (156) . on the other hand, m2 is s-palmitoylated on the amphiphilic helix located in the cytoplasmic part of the protein. due to the s-palmitoylation and the presence of a cholesterol-binding motif the helix bends toward and associates with membranes (157, 158) . during infection, ha binds to sialic acid residues of glycans localized on the surface of airway and alveolar epithelial cells. the bound virions are endocytosed and next the viral and endosome membranes fuse. the membrane fusion is driven by ha, which undergoes conformational changes induced by low ph of endosomes. acidification of endosomes activates also the m2 proton channel activity, protons entering viral core facilitate dissociation of the viral genome which then moves to the nucleus where rna replication occurs. the s-palmitoylation of ha is required for the fusion of the viral and endosome membranes at least in some subtypes of the virus while the ion channel activity of m2 is not dependent on its s-palmitoylation (159) . newly synthesized viral proteins and rna are assembled into virions in the plasma membrane rafts which merge into lager platforms crucial for the virion assembly and budding off. the triple fatty acylation of ha is required for its targeting to plasma membrane rafts (160, 161) . besides s-palmitoylation, also the amino acid sequence of the transmembrane domain of ha determines its association with rafts (45) . on the other hand, among the amino acids of the cytoplasmic tail of ha no other than the two s-palmitoylated cysteines are required for viral assembly and replication, although it is still not clear whether raft targeting (in cooperation with the transmembrane fragment) is the only mechanism of their participation. it is proposed that they affect conformation of the ha tail controlling its interaction with structural matrix protein m1 lying beneath the viral envelope (162, 163) . the budding off of the virion is facilitated by m2 which localizes at the edges of rafts as a result of a combination of its s-palmitoylation, cholesterol binding, and properties of the transmembrane fragment. m2 protein can create a "wedge" altering membrane curvature thereby facilitating membrane scission and release of the virion (157, 164) . the influenza virus s-palmitoylated proteins are the archetype for many other viral proteins. thus, s-palmitoylated spike glycoproteins include s-protein of coronaviruses (e.g., severe acute respiratory syndrome virus), the fusion (f) protein of paramyxoviruses (e.g., measles virus), env of retroviruses [e.g., hiv-1, feline immunodeficiency virus (fiv)], and filoviruses (e.g., ebola). other viral proteins modified with palmitate are viroporins, such as e protein of coronaviruses, and also peripheral membrane proteins or nucleocapsid proteins absent in influenza virus. it has been found that s-palmitoylation of f13l, a peripheral protein of the envelope of vaccinia virus, controls the association of the protein with intracellular membranes, thereby the formation of the envelope (165) . the core protein of the nucleocapsid of hcv resides on the surface of lipid droplets and binds in a palmitoylation-dependent manner to membranes of the droplet-associated endoplasmic reticulum. subsequently, it recruits viral proteins and newly synthesized rna for viral particle formation (166) . besides the interest in the role of viral protein s-palmitoylation for infectivity and possible use of host zdhhc enzymes as targets of anti-influenza drugs (167) , viral proteins often serve as a model to study the consequences of fatty acylation for protein functioning and localization in distinct membrane domains (see s-palmitoylation of proteins and its influence on protein localization, trafficking, and stability of this review). readers are referred to recent exhaustive reviews that consider these topics (36, 84, 168) while we will focus here on the recent advances in the field of viral protein palmitoylation brought about mainly by proteomic studies. the click chemistry-based approach has led to the identification of s-palmitoylation in the cytoplasmic domain of the transmembrane spike protein env of fiv, considered to be the cat equivalent of hiv-1. env comprises three transmembrane gp41 glycoproteins and three associated gp120 which bind to cd4 receptor and coreceptors on the surface of t lymphocytes allowing fusion of the viral envelope and the plasma membrane and entry of viral capsid. four cysteines in fiv env are s-palmitoylated vis-a-vis two found in the env of hiv-1. the two most membrane-proximal cysteines, 804 and 811, are required for the fiv membrane-fusion activity and incorporation of env into virions (169) , in agreement with the importance of env s-palmitoylation for virion assembly of some hiv-1 strains (170) (171) (172) . the assembly of hiv-1 virions takes place in plasma membrane rafts and is driven by n-myristoylated gag protein which anchors and oligomerizes preferentially in these plasma membrane domains due to the presence of the fatty acyl chain (18) . the development of click chemistry-based methods allowed for the first time global profiling of acylated proteins in virusinfected cells. in addition to identifying acylated viral proteins this approach has also revealed how the viral infection modulates the acylation pattern of the host cell proteins. thus far, click chemistry has been used to study protein myristoylation and palmitoylation in cells infected with hiv-1 and with hsv. in the latter case, the standard metabolic labeling with alkynefunctionalized myristic and palmitic acid analogs followed by click chemistry and mass spectrometry was combined with silac to discern between the changes in the extent of protein acylation and those in their abundance following viral infection. this approach allowed an elaborate quantitative analysis of host protein acylation and has revealed an overall downregulation of the level of both host protein modifications in infected cells. while the decreased content of myristoylated proteins resulted mainly from suppression of host protein synthesis, the drop in several s-palmitoylated proteins ensued from the inhibition of their palmitoylation in infected cells. the affected proteins were localized mainly to the plasma membrane and the golgi apparatus and were involved in vesicle-mediated transport and ion transport. in addition, the study has expanded the list of hsv-encoded acylated (mostly palmitoylated) proteins that play different functions in the viral cycle, such as ge, gi, gk, us2, and us3 (110) . similar results pointing to global changes of host protein acylation were obtained upon analysis of protein myristoylation and palmitoylation in cells infected with hiv-1. in that study, the cells were labeled with analogs of palmitic or myristic acid tagged with an azide moiety for click chemistry reaction; however, the following mass spectrometry analysis did not address the relation between changes of protein acylation vs. alteration of protein level. the study identified 17 palmitoylated and 7 myristoylated proteins significantly differing in abundance between hiv-1 infected and uninfected cells. several of the proteins affected by the infection were of host origin. the abundance of myristoylated proteins was in general increased while that of the palmitoylated ones-decreased in infected cells (173) . in other words, the two studies have revealed that hsv and hiv-1 not only encode proteins that are acylated in the host cell but protein palmitoylation in host-pathogen interactions frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 2003 also alter the palmitoylation of host proteins, likely to adapt the cellular environment to favor their replication and budding. the majority of the acylated proteins affected by hiv-1 or hsv infection had not been described earlier in this context; therefore, further studies on these proteins could be crucial for better understanding of viral infection. thus, the click chemistry-based approach has been highly effective in revealing changes of the host protein palmitoylation and opening new possibilities for the identification of novel antiviral drug targets. the innate immune responses are the first line of active defense against microbial infections. the application of click chemistrybased and abe methods and their use for large-scale analysis of protein palmitoylation in murine dendritic cd2.4 cells (10, 103) , and murine macrophage-like raw264 cells (14, 48) complemented by proteomic analysis of the raft fraction of those cells (47) have contributed significantly to the understanding of the role of palmitoylation of host receptors and signaling proteins involved in innate immune responses. thus, the palmitoyl proteome analysis of murine dendritic cells unraveled s-palmitoylation of tlr2, a receptor expressed in cells of myeloidal lineage, which heterodimerizes with tlr1 or tlr6 to bind bacterial tri-or diacylated lipoproteins, respectively, and also other microbial components, such as glycolipids (e.g., lipoarabinomannan) of mycobacterium and yeast zymosan (174) . besides tlr2, two other human tlrs out of 10 ectopically expressed in hek293 cell, flagellin receptor tlr5, and tlr10, a unique tlr negatively regulating the pro-inflammatory activity of tlr2, were also found to be palmitoylated. the s-palmitoylation site of human tlr2 was mapped to cys609 adjacent to its transmembrane domain. the modification was present in unstimulated cells and was linked with up-regulation of the cell surface localization of tlr2. mutation of cys609 abolished the ability of the receptor to induce pro-inflammatory signaling in response to microbial ligands of tlr2 (10) . further studies are needed to reveal whether s-palmitoylation of tlr2 controls its association with rafts as sites of tlr2 activation (175) and/or affects endocytosis of the receptor, as found for the anthrax toxin receptor (54) . one of the most extensively studied tlrs, tlr4 activated by bacterial lps, is not palmitoylated. yet, saturated fatty acids have been indicated to trigger pro-inflammatory signaling of tlr4. thus, the tlr4/md2 receptor complex is involved in the pro-inflammatory outcome of a diet rich in palmitic acid, as was found when analyzing markers of inflammation in the heart and adipose tissue of high fat diet-fed mice (176, 177) . the molecular mechanisms underlying the pro-inflammatory properties of palmitic acid can involve its influence on the plasma membrane lipid order, hence raft organization, in a way that facilitates translocation of tlr4 (and tlr2) toward rafts (178, 179) . palmitic acid also directly binds to the tlr4-associated md2 protein (177, 180) . an influence of palmitic acid on sphingomyelin/ceramide metabolism, which enhances the lps-induced responses, has also been considered (181) . recent proteomic studies based on 17odya labeling of raw264 macrophage-like cells followed by click chemistry have revealed that stimulation of cells with lps induces profound changes of the abundance of palmitoylated proteins (182) . the data are in agreement with earlier findings showing that lps induces accumulation of s-palmitoylated lyn kinase in the raft-enriched fraction of cells, allowing it to downregulate tlr4 signaling (11) . one of the upregulated s-palmitoylated proteins was type ii phosphatidylinositol 4-kinase iiβ, which phosphorylates phosphatidylinositol to phosphatidylinositol 4-monophosphate. it was shown that palmitoylation determines the involvement of the kinase in lps-induced signaling (182) . these data suggest that s-palmitoylated proteins, including enzymes catalyzing phosphatidylinositol synthesis and turnover, are important factors affecting the pro-inflammatory responses triggered by lps. notably lps induces production of tnfα, a pro-inflammatory cytokine that is s-palmitoylated itself. tnfα is synthesized as a transmembrane 27-kda precursor (tmtnfα) transported from the endoplasmic reticulum to the plasma membrane through the golgi apparatus and recycling endosomes (183) . human tmtnfα is s-palmitoylated at cys30 located at the boundary between its transmembrane and cytosolic fragments, as was found independently by radiolabeling and by labeling with 17odya followed by click chemistry (184, 185) . poggi et al. (185) arrived at a complex model explaining how the s-palmitoylation of tnfα affects its activity ( figure 3a) . the modification was shown to favor the association of tmtnfα with rafts. upon cell activation, the extracellular domain of tmtnf is cleaved by adam17 metalloproteinase whereupon the soluble tnfα (stnfα) is released to the extracellular milieu and activates tnf receptor (tnfr) 1 and tnfr2. as adam17 localizes to both non-raft and raft regions of the plasma membrane, the s-palmitoylation of tmtnfα does not affect its cleavage and production of the soluble cytokine. however, s-palmitoylated tmtnfα interacts with tnfr1 in rafts thereby reducing the binding of stnfα and consequently reducing the sensitivity of the cell to this cytokine. in addition, the fragment of tmtnfα which remains after the release of stnfα in rafts if further processed by intramembrane sppl2a and 2b proteases giving rise to icd (intracellular domain) of an own biological activity. by contrast, the non-raft fragment of the adam17-cleaved tmtnfα is rapidly degraded (185) . the transport and maturation of tnfα are also regulated by another posttranslational acylation, ε-n-myristoylation (22) . as shown in figure 3b , myristic acid residues are attached to two lysines (lys19 and 20) of human tmtnfα. this modification is reversed by sirtuin 6 catalyzing the demyristoylation. depletion of sirtuin 6 decreases the release of stnfα since the ε-n-acylated tnfα precursor is redirected to and accumulates in lysosomes (90, 91) . it is worth noting that exogenous palmitic acid stimulates the ε-n-myristoylation of tmtnfα, thereby reducing the release of stnfα in favor of accumulation of tmtnfα in lysosomes (90, 91) . this somehow surprising anti-inflammatory effect of palmitic acid can be explained by competitive binding between long-chain fatty acids (in this case, palmitic) and myristoylated substrates of sirtuin 6 found in vitro(89) and adds a new dimension to the potential effects of palmitic acid. (a) non-palmitoylated tmtnfα is localized outside rafts while that s-palmitoylated on cys30-in rafts of the plasma membrane. tmtnfα is cleaved by adam17 protease in both these plasma membrane environments giving rise to stnfα, which subsequently activates tnf receptor (tnfr) 1 receptor leading to activation of nfκb and erk1/2. however, only the raft-residing tmtnfα is further processed by sppl2b protease to yield icd, which activates the promoter of interleukin (il)-1β and expression of il-12. on the other hand, a pool of s-palmitoylated tmtnfα interacts in rafts with tnfr1 preventing its activation by stnfα. (b) tmtnfα is transported from the endoplasmic reticulum via golgi apparatus and recycling endosomes [1, 2] to the plasma membrane [3] . in the plasma membrane, tnfα is cleaved by adam17 giving rise to stnfα [4] or is internalized [5] and either returns from the endosomes to the plasma membrane [6, 3] or is directed to lysosomes for degradation [7] . ε-n-myristoylation of tmtnfα at lys19 and lys20 facilitates its degradation [5, 7] at the expense of processing to stnfα [4] . oligomerization of tmtnfα and tnfr1 is not shown. s-palmitoylation of host proteins is also vital in antiviral defense. viral nucleic acids, which are recognized by several tlrs and also cytoplasmic pattern-recognition receptors, induce robust production of type i interferons (ifns), mainly infα and ifnβ. the ifnα and ifnβ released from cells which first encounter viruses, e.g., dendritic cells, induce an antiviral reaction in an autocrine and paracrine manner upon binding to plasma membrane ifnα/β receptor (ifnar) consisting of subunits 1 and 2. both human ifnar subunits are s-palmitoylated, as has been found by classical radiolabeling. the s-palmitoylation of ifnar1 on cys463, localized near the cytoplasmic end of the transmembrane domain, is required for downstream activation of stat1 and stat2 and the following transcription of ifnα-activated genes (186) . among the ifn-induced proteins, some have been shown to be palmitoylated, using click chemistry and abe. they include the immunity-related gtpase irgm1, bst2 also known as tetherin, and ifitm1 and 3 (10, 104) . ifitms are potent restriction factors against a wide range of enveloped viruses, e.g., influenza, west nile, dengue, and zika viruses (187, 188) . ifitms localize primarily to endolysosomal membranes where they inhibit viral replication by blocking their fusion with these membranes and also facilitate virus degradation (187) . the exact mechanism of this antiviral activity is not clear, but it seems to rely on a perturbation of the organization of endolysosomal membranes. this can be linked with the intramembrane topology of ifitms and their s-palmitoylation. ifitm1 and 3 likely possess two loops embedded in but not spanning the membrane with both the n-and c-termini facing the cytoplasm (55, 189) . s-palmitoylation of conserved cysteine residues adjacent to these loops, cys71, 72, and 105 in murine ifitm3, contributes to the membrane binding, similarly as found earlier for caveolins (119, 189) . the s-palmitoylation also facilitates clustering of ifitm3 in the membranes, which is of potential significance for its antiviral activity (103) . in support of the latter, the antiviral capacity was markedly reduced for non-palmitoylated mutant forms of ifitm3 (103, 119) . however, s-palmitoylation did not affect the endolysosomal localization or stability of ifitm3. subsequent studies have revealed that the localization and degradation of murine ifitm3, both shaping its antiviral capacity, are orchestrated by numerous posttranslational modifications comprising polyubiquitination, tyrosine phosphorylation by the src-family kinase fyn, and methylation (189, 190) . by contrast, s-palmitoylation alone of the closely related murine ifitm1 endowed it with an antiviral activity and enhanced stability by preventing proteasomal degradation (55) , which indicates diverse effects of this modification on individual ifitm isoforms. the presented data are only beginning to fill the gap which existed in our understanding of the role of protein palmitoylation in innate immune responses. for a long time, it was lagging behind that on acquired immune responses, in which a plethora of s-palmitoylated proteins have long been known to be involved. they include receptors (cd4 and cd8), tyrosine kinases of the src family, transmembrane adaptor proteins (e.g., lat, ntal, and pag/cbp), and α subunits of heterotrimeric g proteins. their s-palmitoylation in most cases targets them to rafts and is a prerequisite for their involvement in the signaling pathways triggered by immunoreceptors [tcr, b cell receptor (bcr), and fcγ and fcε receptors] crucial for the acquired immune responses. an association of some components of these signaling pathways with tetraspanin-enriched domains has also been considered. these topics are discussed in several earlier reviews (44, 79, 191, 192) . it is worth noting that large-scale proteomic analyses of fatty-acylated proteins of t cells (99, 104, 105, 122) and b cells (121) , identifying numerous new palmitoylated proteins, have been published recently. further studies will shed light on the possible engagement of those proteins in acquired immune responses and/or in the cross talk between the innate and the acquired immune system, in which phagocytic cells, such as macrophages and dendritic cells, are essential (193) . protein s-palmitoylation affects their localization, trafficking, and stability. it has long been known as an important factor controlling signal transduction by the bcr and tcr receptors involved in acquired immune responses. it is now becoming evident that palmitic acid is also a key lipid affecting the diverse processes at the host-pathogen encounter. palmitate is a component of bacterial lps and lipoproteins; s-palmitoylation of viral, some bacterial, and numerous host proteins is recognized as a crucial factor affecting both the virulence of pathogens and the innate immune reactions of the host. our understanding of the latter has benefited greatly from the development of novel methods of detection of this protein modification. their application has led to the identification of numerous proteins involved in the host-pathogen interaction. the methods have also allowed highthroughput proteomic analysis of palmitoylation of proteins in infected cells, showing widespread changes of the host cell palmitoylome. future studies will tell whether complex feedback loops comprising palmitoyl acyltransferases and acylthioesterases, similar to those of kinases and phosphatases carrying out protein phosphorylation/dephosphorylation, are involved in controlling protein s-palmitoylation in infected cells. revealing how the s-palmitoylation of particular proteins is regulated during the host-pathogen interactions should allow its modulation to favor the host defense. all authors contributed to writing and critically revised the paper. the authors thank prof. andrzej sobota from the laboratory of molecular membrane biology of the nencki institute (warsaw, poland) and dr. jan fronk from the faculty of biology, university of warsaw for helpful comments and critical discussion. the work was supported by the national science centre, poland, grant number dec-2013/08/a/nz3/00850 to kk. mechanisms of nutritional and hormonal regulation of lipogenesis atherothrombosis and coronary artery disease microbial induction of immunity, inflammation, and cancer nutritional modulation of metabolic inflammation the impact of western diet and nutrients on the microbiota and immune response at mucosal interfaces dietary lipid type, rather than total number of calories, alters outcomes of enteric infection in mice the role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins palmitoylation regulates raft affinity for the majority of integral raft proteins site-specific attachment of palmitate or stearate to cytoplasmic versus transmembrane cysteines is a common feature of viral spike proteins chemoproteomics reveals toll-like receptor fatty acylation association of lyn kinase with membrane rafts determines its negative influence on lps-induced signaling fatty acid binding to vesicular stomatitis virus glycoprotein: a new type of post-translational modification of the viral glycoprotein regulation of mitochondrial morphology and function by stearoylation of tfr1 bioorthogonal chemical reporters for monitoring unsaturated fatty-acylated proteins heterogeneous fatty acylation of src family kinases with polyunsaturated fatty acids regulates raft localization and signal transduction inhibition of protein palmitoylation, raft localization, and t cell signaling by 2-bromopalmitate and polyunsaturated fatty acids myristoylation of gag proteins of hiv-1 plays an important role in virus assembly structural basis for targeting hiv-1 gag proteins to the plasma membrane for virus assembly global profiling and inhibition of protein lipidation in vector and host stages of the sleeping sickness parasite trypanosoma brucei gα(s) is palmitoylated at the n-terminal glycine hhat is a palmitoylacyltransferase with specificity for n-palmitoylation of sonic hedgehog myristyl acylation of the tumor necrosis factor alpha precursor on specific lysine residues escherichia coli α-hemolysin (hlya) is heterogeneously acylated in vivo with 14-, 15-, and 17-carbon fatty acids internal lysine palmitoylation in adenylate cyclase toxin from bordetella pertussis acyl-coa:lysophosphatidylcholine acyltransferase i (lpcat1) catalyzes histone protein o-palmitoylation to regulate mrna synthesis ghrelin octanoylation mediated by an orphan lipid transferase monounsaturated fatty acid modification of wnt protein: its role in wnt secretion chemical-proteomic strategies to investigate cysteine posttranslational modifications phosphatidylserine in addition to phosphatidylethanolamine is an in vitro target of the mammalian atg8 modifiers, lc3, gabarap, and gate-16 posttranslational n-myristoylation of bid as a molecular switch for targeting mitochondria and apoptosis fatty acylation of proteins: the long and the short of it lipid rafts as a membrane-organizing principle membrane microdomains in immunoreceptor signaling co-operation of tlr4 and raft proteins in lps-induced pro-inflammatory signaling relationships between plasma membrane microdomains and hiv-1 assembly palmitoylation of influenza virus proteins s-acylation of lck protein tyrosine kinase is essential for its signalling function in t lymphocytes phosphorylation of fcγriia is required for the receptor-induced actin rearrangement and capping: the role of membrane rafts effects of palmitoylation on dynamics and phospholipid-bilayer-perturbing properties of the n-terminal segment of pulmonary surfactant protein sp-c as shown by 2h-nmr g proteinmembrane interactions ii: effect of g protein-linked lipids on membrane structure and g protein-membrane interactions palmitoylation of mpp1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells the role of mpp1/p55 and its palmitoylation in resting state raft organization in hel cells membrane raft association is a determinant of plasma membrane localization palmitoylated transmembrane adaptor proteins in leukocyte signaling interaction of influenza virus haemagglutinin with sphingolipid-cholesterol membrane domains via its transmembrane domain what does s-palmitoylation do to membrane proteins? quantitative proteomics analysis of macrophage rafts reveals compartmentalized activation of the proteasome and of proteasome-mediated erk activation in response to lipopolysaccharide proteomic profiling of s-acylated macrophage proteins identifies a role for palmitoylation in mitochondrial targeting of phospholipid scramblase 3 proteome scale characterization of human s-acylated proteins in lipid raft-enriched and non-raft membranes molecular interactions shaping the tetraspanin web evaluation of prototype transmembrane 4 superfamily protein complexes and their relation to lipid rafts a physical and functional link between cholesterol and tetraspanins palmitoylated calnexin is a key component of the ribosome-translocon complex receptor palmitoylation and ubiquitination regulate anthrax toxin endocytosis palmitoylation on conserved and nonconserved cysteines of murine ifitm1 regulates its stability and anti-influenza a virus activity model-driven understanding of palmitoylation dynamics: regulated acylation of the endoplasmic reticulum chaperone calnexin identification and dynamics of the human zdhhc16-zdhhc6 palmitoylation cascade intracellular localization and tissuespecific distribution of human and yeast dhhc cysteine-rich domaincontaining proteins mechanistic effects of protein palmitoylation and the cellular consequences thereof dhhc protein s-acyltransferases use similar pingpong kinetic mechanisms but display different acyl-coa specificities molecular basis of fatty acid selectivity in the zdhhc family of s-acyltransferases revealed by click chemistry dynamic palmitoylation links cytosol-membrane shuttling of acyl-protein thioesterase-1 and acyl-protein thioesterase-2 with that of proto-oncogene h-ras product and growth-associated protein-43 molecular cloning and expression of palmitoyl-protein thioesterase a cytoplasmic acyl-protein thioesterase that removes palmitate from g protein α subunits and p21 ras abhd17 proteins are novel protein depalmitoylases that regulate n-ras palmitate turnover and subcellular localization identification of psd-95 depalmitoylating enzymes an acylation cycle regulates localization and activity of palmitoylated ras isoforms differential palmitoylation regulates intracellular patterning of snap25 a mechanism regulating g protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation synaptic strength regulated by palmitate cycling on psd-95 small-molecule inhibition of apt1 affects ras localization and signaling tandem fluorescence imaging of dynamic s-acylation and protein turnover rapid and transient palmitoylation of the tyrosine kinase lck mediates fas signaling global analysis of protein palmitoylation in yeast unique self-palmitoylation activity of the transport protein particle component bet3: a mechanism required for protein stability characterization of the self-palmitoylation activity of the transport protein particle component bet3 autopalmitoylation of tead proteins regulates transcriptional output of the hippo pathway evf, a virulence factor produced by the drosophila pathogen erwinia carotovora, is an s-palmitoylated protein with a new fold that binds to lipid vesicles palmitoylation of ligands, receptors, and intracellular signaling molecules the physiology of protein s-acylation human tissue factor contains thioester-linked palmitate and stearate on the cytoplasmic half-cystine p-selectin is acylated with palmitic acid and stearic acid at cysteine 766 through a thioester linkage the n-terminal sh4 region of the src family kinase fyn is modified by methylation and heterogeneous fatty acylation: role in membrane targeting, cell adhesion, and spreading palmitoylation of virus proteins validation of n-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach global analysis of protein n-myristoylation and exploration of n-myristoyltransferase as a drug target in the neglected human pathogen leishmania donovani s-to n-palmitoyl transfer during proteomic sample preparation δ/ω-plectoxin-pt1a: an excitatory spider toxin with actions on both ca 2+ and na + channels activation of the protein deacetylase sirt6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins sirt6 regulates tnf-α secretion through hydrolysis of long-chain fatty acyl lysine lysine fatty acylation promotes lysosomal targeting of tnf-α a salmonella typhimurium effector protein sifa is modified by host cell prenylation and s-acylation machinery geranylgeranylation but not gtp-loading of rho gtpases determines t cell function inhibition of protein geranylgeranylation specifically interferes with cd40-dependent b cell activation, resulting in a reduced capacity to induce t cell immunity local palmitoylation cycles define activity-regulated postsynaptic subdomains analysis of s-acylation of proteins chemical probes for the rapid detection of fatty-acylated proteins in mammalian cells robust fluorescent detection of protein fatty-acylation with chemical reporters large-scale profiling of protein palmitoylation in mammalian cells assays of protein palmitoylation visualization and identification of fatty acylated proteins using chemical reporters nonradioactive analysis of dynamic protein palmitoylation palmitoylome profiling reveals s-palmitoylation-dependent antiviral activity of ifitm3 global profiling of dynamic protein palmitoylation proteomic analysis of fatty-acylated proteins in mammalian cells with chemical reporters reveals s-acylation of histone h3 variants global analysis of palmitoylated proteins in toxoplasma gondii a single protein s-acyl transferase acts through diverse substrates to determine cryptococcal morphology, stress tolerance, and pathogenic outcome purification of biotinylated proteins on streptavidin resin: a protocol for quantitative elution comparative analysis of cleavable azobenzene-based affinity tags for bioorthogonal chemical proteomics systems analysis of protein fatty acylation in herpes simplex virus-infected cells using chemical proteomics single-cell in situ imaging of palmitoylation in fattyacylated proteins method for cellular imaging of palmitoylated proteins with clickable probes and proximity ligation applied to hedgehog, tubulin, and ras analysis of protein palmitoylation reveals a pervasive role in plasmodium development and pathogenesis global profiling of co-and post-translationally n-myristoylated proteomes in human cells proteomic identification of palmitoylated proteins palmitoylated proteins: purification and identification site-specific analysis of protein s-acylation by resin-assisted capture proteomic analysis of fatty-acylated proteins mass-tag labeling reveals site-specific and endogenous levels of protein s-fatty acylation proteomic analysis of palmitoylated platelet proteins proteomic analysis of s-acylated proteins in human b cells reveals palmitoylation of the immune regulators cd20 and cd23 quantitative analysis of the human t cell palmitome global analysis of protein palmitoylation in african trypanosomes identification of new palmitoylated proteins in toxoplasma gondii neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation role of s-palmitoylation by zdhhc13 in mitochondrial function and metabolism in liver tracking brain palmitoylation change: predominance of glial change in a mouse model of huntington's disease protein palmitoylation database. f1000res genetic and physical mapping of the lps locus: identification of the toll-4 receptor as a candidate gene in the critical region 3-o-deacylation of lipid a by pagl, a phop/ phoq-regulated deacylase of salmonella typhimurium, modulates signaling through toll-like receptor 4 biofilms formed by gram-negative bacteria undergo increased lipid a palmitoylation, enhancing in vivo survival bordetella bronchiseptica pagp is a bvg-regulated lipid a palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract lipid a 3'-o-deacylation by salmonella outer membrane enzyme lpxr modulates the ability of lipid a to stimulate toll-like receptor 4 transfer of palmitate from phospholipids to lipid a in outer membranes of gramnegative bacteria the lipid a palmitoyltransferase pagp: molecular mechanisms and role in bacterial pathogenesis lipid a acylation and bacterial resistance against vertebrate antimicrobial peptides lps remodeling is an evolved survival strategy for bacteria activation of escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation fatty acids of treponema pallidum and borrelia burgdorferi lipoproteins bifunctional role of the treponema pallidum extracellular matrix binding adhesin tp0751 lipoproteins of bacterial pathogens high-throughput, signature-tagged mutagenic approach to identify novel virulence factors of yersinia pestis co92 in a mouse model of infection lipoproteins are critical tlr2 activating toxins in group b streptococcal sepsis rapid visualization and largescale profiling of bacterial lipoproteins with chemical reporters endocytosis of the anthrax toxin is mediated by clathrin, actin and unconventional adaptors anthrax toxin triggers the activation of src-like kinases to mediate its own uptake type iii secretion systems and disease exploitation of eukaryotic subcellular targeting mechanisms by bacterial effectors subcellular targeting of salmonella virulence proteins by host-mediated s-palmitoylation host cell-catalyzed s-palmitoylation mediates golgi targeting of the legionella ubiquitin ligase gobx legionella pneumophila effector lpda is a palmitoylated phospholipase d virulence factor myristoylome profiling reveals a concerted mechanism of arf gtpase deacylation by the bacterial protease ipaj eukaryotic fatty acylation drives plasma membrane targeting and enhances function of several type iii effector proteins from pseudomonas syringae a family of bacterial cysteine protease type iii effectors utilizes acylation-dependent and -independent strategies to localize to plasma membranes evidence for covalent attachment of fatty acids to sindbis virus glycoproteins s acylation of the hemagglutinin of influenza viruses: mass spectrometry reveals site-specific attachment of stearic acid to a transmembrane cysteine the influenza virus ion channel and maturation cofactor m2 is a cholesterol-binding protein intrinsic membrane association of the cytoplasmic tail of influenza virus m2 protein and lateral membrane sorting regulated by cholesterol binding and palmitoylation acylation-mediated membrane anchoring of avian influenza virus hemagglutinin is essential for fusion pore formation and virus infectivity role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. many raft proteins are acylated, while few are prenylated quantitative analysis of the lipidomes of the influenza virus envelope and mdck cell apical membrane influenza virus hemagglutinin (h3 subtype) requires palmitoylation of its cytoplasmic tail for assembly: m1 proteins of two subtypes differ in their ability to support assembly two cytoplasmic acylation sites and an adjacent hydrophobic residue, but no other conserved amino acids in the cytoplasmic tail of ha from influenza a virus are crucial for virus replication influenza virus m2 protein mediates escrt-independent membrane scission palmitylation of the vaccinia virus 37-kda major envelope antigen. identification of a conserved acceptor motif and biological relevance palmitoylation of hepatitis c virus core protein is important for virion production s-acylation of influenza virus proteins: are enzymes for fatty acid attachment promising drug targets? palmitoylation, pathogens and their host palmitoylation of the feline immunodeficiency virus envelope glycoprotein and its effect on fusion activity and envelope incorporation into virions palmitoylation of the hiv-1 envelope glycoprotein is critical for viral infectivity human immunodeficiency virus type 1 envelope glycoproteins that lack cytoplasmic domain cysteines: impact on association with membrane lipid rafts and incorporation onto budding virus particles the cytoplasmic domain of human immunodeficiency virus type 1 transmembrane protein gp41 harbors lipid raft association determinants bioorthogonal mimetics of palmitoyl-coa and myristoyl-coa and their subsequent isolation by click chemistry and characterization by mass spectrometry reveal novel acylated host-proteins modified by hiv-1 infection toll-like receptors and their crosstalk with other innate receptors in infection and immunity lipoteichoic acid and toll-like receptor 2 internalization and targeting to the golgi are lipid raft-dependent dietary saturated fatty acids prime the nlrp3 inflammasome via tlr4 in dendritic cells-implications for diet-induced insulin resistance saturated palmitic acid induces myocardial inflammatory injuries through direct binding to tlr4 accessory protein md2 modification of pro-inflammatory signaling by dietary components: the plasma membrane as a target mechanisms for the activation of toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid palmitic acid is a toll-like receptor 4 ligand that induces human dendritic cell secretion of il-1β acid sphingomyelinase plays a key role in palmitic acid-amplified inflammatory signaling triggered by lipopolysaccharide at low concentrations in macrophages lps upregulates palmitoylated enzymes of the phosphatidylinositol cycle. an insight from proteomic studies cytokine secretion in macrophages and other cells: pathways and mediators transmembrane tnf (pro-tnf) is palmitoylated palmitoylation of tnf alpha is involved in the regulation of tnf receptor 1 signalling palmitoylation of interferon-α (ifn-α) receptor subunit ifnar1 is required for the activation of stat1 and stat2 by ifn-α ifitm-family proteins: the cell's first line of antiviral defense the ifitms inhibit zika virus replication s-palmitoylation and ubiquitination differentially regulate interferon-induced transmembrane protein 3 (ifitm3)-mediated resistance to influenza virus phosphorylation of the antiviral protein interferon-inducible transmembrane protein 3 (ifitm3) dually regulates its endocytosis and ubiquitination greasing their way: lipid modifications determine protein association with membrane rafts protein acylation and localization in t cell signaling (review) patterns, receptors, and signals: regulation of phagosome maturation key: cord-283505-ousbar6c authors: horman, william s. j.; nguyen, thi h. o.; kedzierska, katherine; butler, jeffrey; shan, songhua; layton, rachel; bingham, john; payne, jean; bean, andrew g. d.; layton, daniel s. title: the dynamics of the ferret immune response during h7n9 influenza virus infection date: 2020-09-24 journal: front immunol doi: 10.3389/fimmu.2020.559113 sha: doc_id: 283505 cord_uid: ousbar6c as the recent outbreak of sars-cov-2 has highlighted, the threat of a pandemic event from zoonotic viruses, such as the deadly influenza a/h7n9 virus subtype, continues to be a major global health concern. h7n9 virus strains appear to exhibit greater disease severity in mammalian hosts compared to natural avian hosts, though the exact mechanisms underlying this are somewhat unclear. knowledge of the h7n9 host-pathogen interactions have mainly been constrained to natural sporadic human infections. to elucidate the cellular immune mechanisms associated with disease severity and progression, we used a ferret model to closely resemble disease outcomes in humans following influenza virus infection. intriguingly, we observed variable disease outcomes when ferrets were inoculated with the a/anhui/1/2013 (h7n9) strain. we observed relatively reduced antigen-presenting cell activation in lymphoid tissues which may be correlative with increased disease severity. additionally, depletions in cd8(+) t cells were not apparent in sick animals. this study provides further insight into the ways that lymphocytes maturate and traffic in response to h7n9 infection in the ferret model. in recent years cases of zoonotic strains of avian influenza (ai) causing severe disease in humans have caused significant global concern, with fears that these viruses may lead to devastating pandemic events in future (1, 2) . one such group of viruses are strains of h7n9 influenza virus, which have caused over 1,500 cases of infection (with a ∼40% mortality rate) in humans since their first detection in 2013 (3, 4) . of most concern with the h7n9 viruses has been the ability for this virus to present as a low pathogenicity virus in its native avian hosts and yet present with severe clinical symptoms and death in humans, without obtaining virulence factors such as multi-basic cleavage sites usually required for high pathogenicity infections. deciphering the immune cellular mechanisms associated with disease severity progression will provide a better understanding as to why h7n9 infections can produce severe disease in humans. changes in leukocyte subsets have previously been shown to correlate with more severe outcomes during ai infection, with decreases in t cell populations commonly reported in both human (h7n9 virus) and avian (h5n6 virus) hosts (5, 6) . decreases in t lymphocytes are often accompanied by upregulation of several pro-inflammatory cytokines such as interferons (ifn), most notably ifn-γ, as well as interleukins (il) such as il-6 (7). overproduction of cytokines or hypercytokinemia has also been identified as a key contributing factor in severe ai pathogenesis in both chickens and macaques, where induction of pro-inflammatory cytokines is associated with cellular apoptosis and tissue damage (8, 9) . furthermore, macrophages have been shown to be predominantly pro-inflammatory responders to h7n9 strains in a mouse model (10) , though highly pathogenic strains such as h5n1 and h7n9 display attenuated macrophage inflammation responses compared to seasonal strains such as h1n1 and h3n2 (11, 12) . while hypercytokinemia is common amongst many ai virus infections, h7n9 strains such as the human infecting a/anhui/1/2013 virus have been associated with dampened ifn responses in humans (13, 14) . furthermore, they have previously been associated with an attenuated humoral immune response in the mouse model (15) . these studies demonstrate the unpredictable nature and wide spectrum of pathogenicity of ai viruses. investigations into the pathogenesis and transmission of many human-infecting influenza viruses have been conducted in ferrets, as the clinical presentation in these animals is considered a more robust representation of human illness when compared to mice (16) . however, only a limited number of studies exist looking at influenza-specific immunity in ferrets, which have primarily focused on seasonal influenza strains (17) (18) (19) . in this study, we aimed to examine the ferret immune response to h7n9 influenza virus infection by analyzing leukocyte population variation associated with disease pathogenesis. this study found the ferret model may allow for increased knowledge of the outcomes of h7n9 infections and help in boosting our understanding of both this model and of these viruses in readiness for potential future outbreaks. all procedures described here were reviewed and approved by the commonwealth scientific and industrial research organization (csiro), australian center for disease prevention (acdp) animal ethics committee (aec#1861) and were performed in accordance with the australian code for the care and use of animals for scientific research (8th edition 2013). influenza virus a/anhui/1/2013(h7n9) used in this study was propagated by allantoic cavity inoculation of 9-11-days of embryogenesis specific-pathogen-free (spf) embryonated chicken eggs. the virus stock was titrated in chicken eggs and the 50% egg infectious dose (eid 50 ) /ml was calculated according to reed and muench (20) . all in vitro and in vivo work involving live virus was conducted within biosafety level three facilities at acdp. animal work was performed using personal protective equipment and powered air purifying respirators. fitch ferrets that were ∼6 months of age (csiro werribee animal facility) and serologically negative by hemagglutination inhibition (hi) assay for h7n9 were used in this study. prior to initiation of the study, all ferrets were free from signs of clinical disease. body temperatures were measured using an implantable subcutaneous microchip (destron fearing, delray beach, fl, usa). baseline body weights and temperatures were obtained for 3 consecutive days before challenge (i.e., day −3, −2, and −1) and on the day just prior to the challenge (day 0). six ferrets were infected intranasally with 1 × 10 6 eid 50 of virus diluted in 0.5 ml of sterile pbs, and four ferrets were mockinfected with an equivalent dilution of allantoic fluid collected from spf chicken eggs in sterile pbs as non-infected controls. following viral challenge, ferrets were monitored daily for body weight, temperature, and clinical signs of illness (including sneezing, lethargy, nasal discharge, diarrhea, and neurological dysfunction) for the duration of the study. blood samples were collected every second day. animals were anesthetized with ketamine/xylazine, and blood samples of 200-250 µl were taken from the jugular or axillary vein on days 1, 3, 5, and from the heart at the time of euthanasia for the terminal bleed. for virus titration, nasal washes were collected on days 1, 3, 5, and upon euthanasia, as described previously (6, 21) . all ferrets were euthanised on day 7 (study endpoint) or earlier due to ethical endpoints (≥10% weight loss or escalation of clinical signs). histological analysis of ferret tissues following infection was performed as previously described (22) . tissues were fixed in 10% neutral-buffered formalin for at least 24 h, processed into paraffin wax, cut and stained using haematoxylin and eosin for examination for histopathological lesions. consecutive tissue sections were stained in an immunohistochemistry (ihc) test for influenza a virus nucleoprotein (22) . lung, spleen, and mediastinal lymph nodes were harvested and processed, as previously described (17) . briefly, lung samples were manually minced using a scalpel followed by enzymatic digestion (150 u/ml collagenase i and 100 u/ml dnase i) while single-cell suspensions of spleen and lymph node samples were prepared by passing the tissue though a 70 µm strainer. peripheral blood mononuclear cells were isolated by hypotonic lysis of red blood cells using erythrocyte lysing solution (0.15 m nh 4 cl, 10 mm khco 3 , and 1mm edta ph 7.3). viral titers in tissues were measured on mdck cells by standard tcid 50 assay. relative expression of ferret immune genes was assessed using a steponeplus tm real-time pcr system and the comparative threshold cycle (ct) method according to manufacturer's instructions (applied biosystems, foster city, ca, usa). relative gene expression was calculated using mean values obtained from ct relative to the housekeeper gene (gapdh), with each ferret compared to the average of the control ferrets for each gene. primers for ferret cytokines, as well as relative gene expression calculations, were obtained from carolan et al. (23) . cells processed from ferret tissues were stained with anti-cd4 (alexafluor 488, csiro acdp sourced from wehi (24) , geelong, vic, australia), anti-cd8 (pe, clone okt8, ebioscience, ca, usa), anti-gl7 (alexafluor 647, clone gl7, bd phamingen, san diego, ca, usa), anti-mhc-ii (biotin, clone cat82a, kingfisher, saint paul, mn, usa), and anti-cd11b (alexafluor 647, clone m1/70, bd pharmingen, san diego, ca, usa). cells were stained for 1 h at 4 • c, washed in facs buffer (pbs, 4% fcs, 0.01% sodium azide) and analyzed using the lsr ii (becton-dickinson, franklin lakes, nj, usa). flow cytometry data were analyzed using flowlogic software (version 7.2.1, inivai technologies, mentone, vic, australia). to assess antibody responses, serum was collected prior to infection and at the point of euthanasia. haemagglutinin inhibition assays were performed on rde treated sera by using homologous antigen (influenza a/anhui/1/2013) as per the standard method. hi titers were expressed as the reciprocal of the highest serum dilution causing complete inhibition of hemagglutination. ferret lung and spleen cells were pelleted by centrifugation at 1,500 × g max for 5 min and washed in pbs. cells (1.25 × 10 4 /96well) were cultured with or without live h7n9 virus for 48 h at 37 • c/5% co 2 . the ferret ifn-γ elisa development kit (alp) (mabtech, stockholm, sweden) was used to determine the quantity of ifnγ secreted by cells ex vivo at endpoint and after restimulation with live h7n9 virus (moi 0.1). elisa plates were measured using a multiskan ascent plate reader with ascent software version 2.6 (thermofisher, waltham, ma, usa). ifn-γ-producing cells were detected using a ferret ifn-γ elispot assay, as per the manufacturer's instructions (mabtech, stockholm, sweden). for analyzing the infected ferrets compared to the non-infected controls, all six ferrets were grouped regardless of timepoint to give an overall view of the infection time course. for these analyses, student t-tests were conducted between the two groups. time course data was analyzed by 2-way anova. to assess the impact of h7n9 virus in ferrets, viral pathogenicity was firstly assessed based on observation of clinical signs of infected ferrets throughout the 7-day study (figure 1) . from the six h7n9-infected ferrets, three ferrets showed little to no clinical signs and survived until the study endpoint (day 7). of the remaining ferrets, two showed mild clinical signs but severe weight loss, leading to euthanasia at day 5 post-infection. one ferret, however, showed moderate to severe clinical signs consistent with those seen in highly pathogenic avian influenza infections (25) , and was euthanised at day 6 due to escalation of these clinical signs ( figure 1a ). this ferret's increase in clinical signs was identified by a play score of two, with play scores >1 signifying moderate-to-severe clinical signs ( figure 1b ). significant weight losses (>5% compared to baseline, p < 0.0001) were observed in all the infected ferrets when compared to the control ferrets from day 3 until the study endpoint ( figure 1c) . similarly, infected ferrets also showed a significant increase (p < 0.05) in body temperature at day 2 post-infection, with an average temperature of 40.1 ± 0.6 • c compared to the controls at 38.4 ± 0.4 • c ( figure 1d) . consequently, h7n9 infection in ferrets resulted in variable clinical symptoms, but overall body weight losses and heightened body temperatures at 48 h postinfection, which are within the typical timeframe for onset of influenza illness in humans. to further examine the clinical progression between the infected ferrets, viral titres from the nasal washes were determined to assess whether differences in viral replication or clearance were correlative with worsened disease progression. titres >4.0 log 10 tcid 50 /ml were obtained for all infected ferrets at day 1 postinfection and virus was still detected in all ferrets at day 3 postinfection (>3.0 log 10 tcid 50 /ml, figure 2a ). one ferret had cleared the virus by day 5 post-infection, while the other two ferrets that survived until the end of the study showed viral clearance at day 7 (figure 2a ). neither the day 5 nor the day 6 ferrets showed viral clearance by the point of euthanasia. only one ferret euthanised on day 5 showed live virus in the lung (>4.0 log 10 /ml, figure 2b ). additionally, antibody titres were measured in the ferret sera, with animals euthanized on day 7 showing the highest hi titres (all >64) and the ferret euthanized on day 6 showed a hi titer >8. the two ferrets euthanised at day 5 had no detectable hi titer ( figure 2c ). infected ferrets showed a variety of pathological outcomes following viral challenge. challenged ferrets exhibited epithelial metaplasia in the nasal turbinates (figure 3a) , with ferrets euthanised at the earlier day 5 time point exhibiting viral infection of the nasal epithelium (figure 3b) . the day 6 ferret showed the most severe lung pathology, with the lungs of this ferret showing diffuse interstitial pneumonia, with severe alveolar oedema and inflammatory cell infiltration in the alveolar spaces and around the blood vessels (figure 3c) . other infected ferrets showed broncho-interstitial pneumonia and interstitial inflammation (figure 3d ) and bronchitis, with infected epithelial cells and early stage lesions observed in one of the day 5 ferrets (figure 3e) . bronchial adenitis was also present in some of the infected ferrets, with necrosis of the bronchial glands observed along with viral antigen (figure 3f) . these changes in pathology contrast to what is seen in the healthy nasal turbinates (figure 3g) , and lung (figure 3h ) of uninfected ferrets where no lesions were present. the number of localized lesions and qualitative assessment of epithelial metaplasia was also recorded to support the histopathological findings (supplementary figure 1) . here, we observed epithelial metaplasia in the turbinates of all ferrets sampled, as well as several occurrences of bronchio-interstitial pneumonia and bronchio-adenitis. we also observed viral antigen in a number of tissues sampled (supplementary figure 1) , though interestingly, viral antigen was not observed in the diffuse interstitial pneumonia of lung lesions associated with the ferret euthanized due to clinical disease. changes in certain pro-inflammatory cytokines (including il-6, tnfα, and ifnγ) have been associated with h7n9 disease severity in humans and animal models (7). we measured levels of mrna transcripts of several pro-inflammatory cytokines commonly associated with influenza infections. in the blood there were few differences between the groups, with mcp1 the only tested cytokine showing an average fold increase >5fold compared to the controls at day 1 post-infection (data not shown). however, intriguingly the ferret euthanised on day 6 showed a large decrease in il-6 ( figure 4a , 34-fold) and ifn-γ (figure 4b , 25-fold) transcript levels on day 5. this also coincided with a lack of detectible ifn-γ in terminal serum by elisa, though this trend was consistent across all tested ferrets (data not shown). in the spleen at endpoint, there appeared to be variable outcomes for the day 7 ferrets, with somewhat increased pro-inflammatory cytokine levels such as type i ifns (ifn-α and ifn-β) in the day 5 ferrets, and decreased levels in the day 6 ferret compared to the controls (figure 4c) . in the lung, tnfα was significantly reduced (figure 4d , p < 0.05) in the infected animals, though no other consistent trends were seen. the ability to produce ifn-γ ex vivo or after restimulation with live h7n9 virus was also assessed in spleen ( figure 4e ) and lung cells (figure 4f ). there were no significant differences except for in the infected lungs, where ifn-γ-producing cells were significantly higher compared to the control ferrets ex vivo (p = 0.0389). based on these findings, our ferret infection model showed observable changes in the cytokine profiles at a key site of infection in the lung compared to non-infected controls. depletions in t cell numbers in the spleen and lungs have previously been reported for highly pathogenic avian strains h5n1 and h5n6 in both chickens and mice (6, 26) . similarly, patients that died from h7n9 infection were found to have lower t cell numbers in the blood compared to those that survived (5) . therefore, in order to assess lymphocyte involvement in disease progression we investigated the cellular responses associated with h7n9-infection both in the blood at several timepoints, and in the tissues at endpoint. here, we found that in the blood the numbers of cd4 + and cd8 + t cells in infected ferrets were generally lower than the control ferrets, particularly for the cd8 + t cell population at day 1 (p < 0.001). nevertheless, these t cell populations remained low but relatively stable over the 7 days of infection ( figure 5a) . the gl7 monoclonal antibody can be used as a marker activated t lymphocytes. however, gl7 expression was absent on the t cell subsets examined. interestingly we observed significantly higher numbers of cd4 + t cells in the spleen (p = 0.0272) and in the lungs (p = 0.0048) of infected ferrets, and a trend toward higher numbers of cd8 + t cells in the lungs (figures 5b,c) , indicating a t cell response to h7n9 infection at a key site of infection. antigen presenting cells (apcs), such as macrophages residing in the lungs, have previously been recognized as key proinflammatory responders during h5n1, h7n7, and h7n9 infections in mice (10) . cells expressing higher levels of cd11b + have been shown to directly kill virally-infected cells in humans, and act as important antigen-presenting cells by upregulating mhc class ii expression in the ferret model correlating to reduced pathology associated with influenza infection (17, 27) . in general, h7n9-infected ferrets showed higher levels of cd11b/mhc-ii dual-expressing cells (cd11b + mhc-ii + ) in the spleen, lung and lymph nodes compared to the control ferrets (figures 6a-c) . at the site of infection in the lungs, infected ferrets showed significantly higher levels of mhc-ii single-positive cells (mhc-ii + , p = 0.0286). conversely, cd11b single-positive cells (cd11b + ) from infected ferrets were found at elevated levels in the spleen and lymph nodes. individually, we did observe that the ferrets euthanised on days 5 and 6 had higher cd11b + cells in the spleen, suggesting a less activated and immature phenotype, and a trend toward lower cd11b + mhc-ii + cells in the lymph nodes when compared to the other ferrets. when analyzed by time point, there are statistically greater proportions of these cells in the ferrets that survived until trial endpoint compared to the controls (p = 0.0009) and the earlier timepoint ferrets (p = 0.0039, figure 6d ). as such, as these markers are often attributed to antigen-presenting cell maturation in the mouse model (28) , apc activation in response to h7n9 infection may be associated with disease severity seen in the ferret model. the recent sars-cov-2 outbreak has brought a sharp focus onto zoonotic viruses causing severe global pandemics. while sustained human-to-human transmission of avian-origin h7n9 viruses has yet to occur, if a zoonotic outbreak of these highconsequence viruses were to occur, the results could be a pandemic much like the sars-cov-2 outbreak but with a virus which has potential for much higher mortality rate. as such, avian influenza viruses continue to present as a major global health concern, with one of the key concerns being what makes these viruses cause such severe consequences in mammalian hosts. influenza-related symptoms of ferrets more closely resemble human clinical symptoms than that of mice (16, 29) , with outbred ferret populations giving a more accurate representation of the genetic variability seen in human populations compared to clonally inbred mouse colonies. however, husbandry requirements and a general lack of reagents for ferrets limits the scope of experimental research using these animals to date. these issues are exacerbated by biosafety level 3 (bsl3) requirements for pathogens such as h7n9, which make immunological experiments in ferrets difficult to undertake. as a result, ferret cellular immunology is still a developing field of research in the context of influenza viruses, with only a handful of studies investigating changes in leukocyte populations following influenza virus infection (17) (18) (19) . for avian-origin influenza viruses such as h7n9, little work has been done to investigate how cellular subsets are affected by viral infection, with the bulk of experiments using ai in the ferret model focusing on classifying viruses through clinical outcome-based pathogenesis and transmission studies (30) (31) (32) . thus, our study aimed to give new insight into how h7n9 affects mammalian hosts in the ferret model with a focus on cellular subsets. the ferrets in our viral challenge presented clinical outcomes with different severities. five of the six ferrets showed little to no clinical signs, nevertheless only three survived until study endpoint without complications. two ferrets reached the ethical weight loss cut-off of ≥10% by day 5 post-infection, giving us two different time points to assess the immune response. furthermore, one ferret showed a quite different disease outcome, in which the ferret presented an escalation of clinical signs and was euthanised for ethical reasons at day 6 post-infection. other studies have previously shown clinical variation between strains of h7n9 (15) , however most studies with a /anhui/1/2013 (h7n9) show ferrets presenting only mild clinical signs (1, 4, 33) . to our knowledge, this is the first reported result showing variable disease outcome in an animal study using a single low pathogenicity h7n9 strain, in which one ferret showed severe clinical outcomes. while the findings of this study are novel for this model, we were limited by the number of animals that could be used in this study and thus limited by our statistical power. furthermore, there are limitations in directly comparing ferrets euthanised on different days as, particularly for our serological data, it is difficult to discern whether the results are a function of observed severity or differences in the sampling time. as such, we believe the results from our observational study can provide a rationale for developing future ferret studies with greater numbers. it is also worth noting that ferrets used for these experiments are outbred animals, which may suggest that other combinations of host factors may be contributing to the variability seen in clinical presentation. the airway pathology in these infected ferrets was variable, with different degrees of severity observed in the six animals. these findings were generally consistent with pathology observed in other studies in which ferrets were infected with lpai h7n9 or pdm09 (h1n1) viruses (4, 31, 32, (34) (35) (36) . while the day 6 ferret which showed worsened disease progression did exhibit the most severe lung pathology, the lack of viral antigen in and around these lesions (supplementary figure 1) means they cannot conclusively be classified as being caused by the influenza infection, and thus we are hesitant to classify disease presentation in this ferret as like a high pathogenicity infection based on the histopathology findings regardless of the increase in clinical signs. hypercytokinemia has frequently been associated with worsened disease progression in cases of severe ai infections, therefore we aimed to assess the induction of pro-inflammatory cytokines in this ferret model of infection. upregulation of proinflammatory cytokines such as tnf-α and il-6 commonly observed following h7n9 infection in both cell culture (14, 37) and in severe human cases, which follow similar patterns of pathogenesis to infections with hpai h5n1 viruses (5, 38, 39) . whilst the localized pro-inflammatory cytokine response to influenza infection has been characterized for circulating influenza strains in ferrets (40) , examination into these responses for avian influenza viruses remains limited. our study found limited induction of cytokine responses at the site of infection in the lungs and in lymphoid tissues, and while attenuated ifn responses have been previously reported in human bronchial epithelial cells infected with h7n9 (14), and in ferrets severely infected with seasonal influenza strains (41) , the overall lack of cytokine induction was an unexpected finding. a limitation of this study was that these tissues could only be sampled at study endpoint, which may suggest the pro-inflammatory response had abated at the site of infection by the time of sampling given that these responses typically occur in short timeframes of 24-48 h post-infection (42) , though a lack of an evident cytokine response in the day 6 ferret still presenting clinical signs and shedding live virus was surprising. furthermore, levels of ifn-γ were below the threshold of detection by elisa in the serum of infected ferrets, and a previous study has shown ifn-γ detectable in the lungs of seasonal influenza-infected ferrets from day 5 post-infection at low levels, with greater detection observed from days 8-11 (19) . these results suggest both timing and sampling may be critical for future studies to best capture the overall ifn-γ response to h7n9, if there is such a response occurring. sampling in the blood of infected ferrets over the course of the study also found no large-scale upregulation of the cytokines tested, with only the early innate cytokine mcp1 showing an average fold increase >5fold compared to the controls at day 1 post-infection. however, in the day 6 ferret, decreases in both ifn-γ (25-fold) and il-6 (24-fold) where observed at day 5 post-infection, coinciding with an escalation in clinical signs in this animal. the decrease in il-6 in particular was unexpected, as previous studies have implicated higher levels of il-6 correlating to worsened disease progression (41) . our findings here suggest an immune dysregulation, rather than an over-activation, for worsened disease progression, and may be attributed to other factors in the cellular response. immunological work to determine the innate and adaptive responses to these viruses has mostly been conducted from infected patients or in the mouse model. human cases are often marked by leukopenia, though these measurements are routinely made via hematology rather than flow cytometric analysis (43, 44) . in other species however, we have previously described the lymphopenia for highly pathogenic strains of avian influenza, measuring splenic cd8 + t cells following infection with highly pathogenic h5n6 in chickens via facs analysis (6) . in this study, cd8 + t cells levels were relatively stable. a loss of lymphocytes detected via hematological analysis at day 1 (data not shown) was reconciled with our facs data showing a significant difference between cd8 + t cells in the blood, in which control animals had much higher levels compared to the infected animals. however, this loss was short-lived, as no further significant differences were observed across the study time points in the blood. this data is suggestive of a potential early "transient lymphopenia" of circulating leukocytes, which has previously been identified in one study during influenza infection in ferrets, and is also a phenomenon seen in humans following influenza a infection (17, 45) . our data appears to show cd8 + t cells levels in the blood of infected animals remaining relatively and consistently low across time points, whilst greater variation was observed in the uninfected control levels. furthermore, reagents for facs analysis in the ferret are still relatively novel, often not well-characterized, and heavily reliant on cross-reactivity from other species. while cd4 + and cd8 + t cells have previously been examined in influenzainfected ferrets (18) , little work has been conducted for innate cell subsets. myeloid-derived and antigen-presenting cells (apcs) have only been identified and analyzed in a handful of ferret studies, with one such study speculating that cd11b + cells are likely granulocytes such as neutrophils (17, 18) . as such, we have used these studies, together with mouse and human studies (46) (47) (48) (49) , as a basis for possible classification of apcs, with cd11b + mhc-ii + cells suggested as likely "mature" or "activated" apcs. however, further work is needed to classify these innate cell subsets in the ferret model. apcs such as alveolar macrophages have been recognized as key early responders against influenza virus infection, as they mount robust pro-inflammatory cytokine responses within 24 h of infection (27) . while our study found no significant differences in apc numbers in infected lungs compared to control numbers, there was a trend toward much greater levels of mhc-ii + apcs in the lung, especially in our cell counts. rather, it was in the peripheral tissues that we found variability in the apc subsets. cd11b + mhc-ii + "mature" cells were found in the spleens of infected ferrets at significantly higher levels (p = 0.0029), however the trend toward higher cd11b + cells in the earlier time point ferrets suggests a higher level of immature myeloid cells, or the presence of pro-inflammatory granulocytes; however, the latter is less likely due given the lack of pro-inflammatory cytokines detected in the spleen. moreover, in the draining lymph node a significant increase in cd11b + cells (p = 0.0153) was observed. interestingly, in the lymph node the 3 day seven ferrets showed noticeably higher levels of cd11b + mhc-ii + "activated" cells, with statistically greater proportions of these cells compared to the controls (p = 0.0009) and the earlier timepoint ferrets (p = 0.0039), which may suggest that these apc responses do not occur as early as day 5. this study found increased pathology in the ferret model diverges from the commonly observed pathogenesis markers such as lymphopenia and hypercytokinemia, suggesting another varied pathway that h7n9 viruses can cause severe disease in mammalian hosts. further investigation into the ferret model may allow for better characterization of these outcomes and assist in increasing our knowledge of these viruses in preparation for any potential pandemic events. all datasets generated for this study are included in the article/supplementary material. the animal study was reviewed and approved by australian animal health laboratories (aahl) animal ethics committee, csiro. characterization of h7n9 influenza a viruses isolated from humans the drivers of pathology in zoonotic avian influenza: the interplay between host and pathogen influenza at the human-animal interface: summary and assessment a highly pathogenic avian h7n9 influenza virus isolated from a human is lethal in some ferrets infected via respiratory droplets recovery from severe h7n9 disease is associated with diverse response mechanisms dominated by cd8? t cells novel reassortant h5n6 influenza a virus from the lao people's democratic republic is highly pathogenic in chickens early hypercytokinemia is associated with interferon-induced transmembrane protein-3 dysfunction and predictive of fatal h7n9 infection highly pathogenic avian influenza virus infection in chickens but not ducks is associated with elevated host immune and pro-inflammatory responses integrated clinical, pathologic, virologic, and transcriptomic analysis of h5n1 influenza virus-induced viral pneumonia in the rhesus macaque h7n9 and other pathogenic avian influenza viruses elicit a threepronged transcriptomic signature that is reminiscent of 1918 influenza virus and is associated with lethal outcome in mice highly pathogenic avian influenza viruses inhibit effective immune responses of human blood-derived macrophages infection with highly pathogenic h7 influenza viruses results in an attenuated proinflammatory cytokine and chemokine response early after infection human h7n9 virus induces a more pronounced pro-inflammatory cytokine but an attenuated interferon response in human bronchial epithelial cells when compared with an epidemiologically-linked chicken h7n9 virus suboptimal humoral immune response against influenza a(h7n9) virus is related to its internal genes the ferret as a model organism to study influenza a virus infection peripheral leukocyte migration in ferrets in response to infection with seasonal influenza virus flow cytometric and cytokine elispot approaches to characterize the cellmediated immune response in ferrets following influenza virus infection cellular immune response to human influenza viruses differs between h1n1 and h3n2 subtypes in the ferret lung a simple method of estimating fifty per cent endpoints evidence for viral interference and cross-reactive protective immunity between influenza b virus lineages the pathobiology of two indonesian h5n1 avian influenza viruses representing different clade 2.1 sublineages in chickens and ducks taqman real time rt-pcr assays for detecting ferret innate and adaptive immune responses development of an anti-ferret cd4 monoclonal antibody for the characterisation of ferret t lymphocytes predicting disease severity and viral spread of h5n1 influenza virus in ferrets in the context of natural exposure routes lymphopenia associated with highly virulent h5n1 virus infection due to plasmacytoid dendritic cell-mediated apoptosis of t cells innate immune response of human alveolar macrophages during influenza a infection differential response of respiratory dendritic cell subsets to influenza virus infection chapter 119-the ferret as an animal model of influenza virus infection pathogenesis of avian influenza a (h5n1) viruses in ferrets novel avian-origin human influenza a (h7n9) can be transmitted between ferrets via respiratory droplets infectivity, transmission, and pathology of human-isolated h7n9 influenza virus in ferrets and pigs pathogenesis and transmission of avian influenza a (h7n9) virus in ferrets and mice pathogenicity and transmissibility of north american triple reassortant swine influenza a viruses in ferrets severity of clinical disease and pathology in ferrets experimentally infected with influenza viruses is influenced by inoculum volume comparative pathology in ferrets infected with h1n1 influenza a viruses isolated from different hosts pro-inflammatory cytokine dysregulation is associated with novel avian influenza a (h7n9) virus in primary human macrophages cytokine and chemokine levels in patients infected with the novel avian influenza a (h7n9) virus in china fatal outcome of human influenza a (h5n1) is associated with high viral load and hypercytokinemia characterization of the localized immune response in the respiratory tract of ferrets following infection with influenza a and b viruses severe seasonal influenza in ferrets correlates with reduced interferon and increased il-6 induction timing and magnitude of type i interferon responses by distinct sensors impact cd8 t cell exhaustion and chronic viral infection human infections with the emerging avian influenza a h7n9 virus from wet market poultry: clinical analysis and characterisation of viral genome comparison of patients hospitalized with influenza a subtypes h7n9, h5n1, and 2009 pandemic h1n1 avian influenza virus a h7n9 infects multiple mononuclear cell types in peripheral blood and induces dysregulated cytokine responses and apoptosis in infected monocytes identification of myeloid cell subsets in murine lungs using flow cytometry distinct macrophage subpopulations characterize acute infection and chronic inflammatory lung disease all authors agreed on the final draft of the manuscript prior to submission. wh wrote the first draft and revised the manuscript. tn, kk, jbu, ss, jbi, jp, ab, and dl reviewed and edited the manuscript. wh was supported by a joint csiro/university of melbourne onehealth scholarship. this project was supported by csiro strategic funding. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.559113/full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 horman, nguyen, kedzierska, butler, shan, layton, bingham, payne, bean and layton. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-332150-j76726no authors: de stefano, ludovico; bobbio-pallavicini, francesca; manzo, antonio; montecucco, carlomaurizio; bugatti, serena title: a “window of therapeutic opportunity” for anti-cytokine therapy in patients with coronavirus disease 2019 date: 2020-10-06 journal: front immunol doi: 10.3389/fimmu.2020.572635 sha: doc_id: 332150 cord_uid: j76726no the effects of cytokine inhibition in the different phases of the severe coronavirus disease 2019 (covid-19) are currently at the center of intense debate, and preliminary results from observational studies and case reports offer conflicting results thus far. the identification of the correct timing of administration of anti-cytokine therapies and other immunosuppressants in covid-19 should take into account the intricate relationship between the viral burden, the hyperactivation of the innate immune system and the adaptive immune dysfunction. the main challenge for effective administration of anti-cytokine therapy in covid-19 will be therefore to better define a precise “window of therapeutic opportunity.” only considering a more specific set of criteria able to integrate information on direct viral damage, the cytokine burden, and the patient’s immune vulnerability, it will be possible to decide, carefully balancing both benefits and risks, the appropriateness of using immunosuppressive drugs even in patients affected primarily by an infectious disease. the coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has a wide spectrum of clinical expressiveness ranging from asymptomatic or paucisymptomatic infection to life-threating multiple organ failure. in most severe forms, sars-cov-2 infection leads to fulminant pneumonia and acute respiratory distress syndrome (ards) with a mortality rate approaching 40-50% (1) . clinical deterioration generally occurs several days after the onset of symptoms, in association with declining viral titers (2) , suggesting that part of pathophysiology may be driven by dysregulated immune responses rather than by direct viral damage. uncontrolled hyperinflammation has indeed been recognized as a pivotal pathogenetic event in , with the release of inflammatory cytokines which are injurious to host cells similarly to what happens in other hyperinflammatory syndromes characterized by cytokine storms, such as the cytokine release syndrome (crs), secondary hemophagocytic lymphohistiocytosis (shlh) and macrophage activation syndrome (mas) (4) . the known sensitivity of these syndromes to cytokine-directed therapies has fueled many expectations also for patients with covid-19. however, the precise nature and role of hyperinflammation in severe covid-19 remain poorly defined, and the risk-benefit ratio of cytokine inhibition in the different phases of the disease is largely debated (5) (6) (7) (8) . as a matter of facts, preliminary clinical results on the efficacy of therapeutic blockade of interleukin (il)-6, il-1, tumor necrosis factor (tnf), and janus kinase signaling are thus far mixed (9-23) ( table 1) , possibly also due to the different timing of drug administration in different reports. early immunosuppression during an infectious disease indeed incurs risks; on the other hand, however, suppression of pathogenic hyperinflammation may be more effective in the initial stages before clinical deterioration occurs. the identification of the correct timing of administration of immunosuppressive drugs remains therefore a research priority for the personalized management of covid-19. the health analytic platform opensafely established in the united kingdom has recently confirmed that covid-19-related deaths are strongly associated with demographic factors and comorbidities such as increasing age, male gender, obesity, cardiovascular and respiratory diseases (27) . risk prediction and tailored treatment solely based on such generic parameters is however largely inaccurate, and covid-19 outcomes likely depend on a number of other variables influencing host-pathogen interactions, including sars-cov-2 genetic variants as well as host genetic susceptibility. research in this area is only in its infancy. also building on previous experience with other coronaviridae, several studies are investigating the link between sars and genetic variants in innate immune response (28) , including mannosebinding lectin deficiency and polymorphisms, which are involved in the inactivation of a variety of respiratory pathogens through direct binding and complement activation (29) . similarly, adaptive immune dysfunction is appearing as a key, and recent studies have confirmed the role of cd3 + t cell cytopenia in determining covid-19 progression and fatality (30) . discovery of virus and host genomic factors will undoubtedly support risk stratification and targeted treatment; however, as genomic studies require long times before entering clinical practice, it is urgent to integrate easily accessible information on the dynamics and pathogenicity of the immune response during the different phases of sars-cov-2 infection. the relative contribution of viral damage and of innate and adaptive immune responses in course of covid-19 greatly varies over time (31) (32) (33) . in the early phases, common to all patients, pathogenic mechanisms are mainly driven by the cytotoxic role directly exerted by sars-cov-2, which usually affects only the upper respiratory tract. the early antiviral response mostly relying on type-i interferon (ifn) and natural killer (nk) cells, and the subsequent cd8 + t cell-mediated killing of virally infected cells as well as cd4 + t cell-dependent antibody production, allow the rapid reduction of the viral load, the paucisymptomatic character of the disease, and its recovery. however, in up to 20% of the patients, virus-induced immunosuppression occurs. this is highlighted by a marked reduction of cd4+ and cd8+ t lymphocytes in peripheral blood, defective cd8+ t cell and nk cell function, low ifn-g production, and low specific igg antibodies (34) (35) (36) (37) . the impairment of the initial antiviral defense mechanisms favors a sustained increase in the viral load capable of conditioning disease progression, especially in organs that have high angiotensin-converting enzyme 2 expression, such as the lungs but also the endothelium, the heart, the kidney, and the intestine (38, 39) . the clinical picture can evolve rapidly, with the development of interstitial pneumonia which can progress into ards (40) . in these early stages, lung damage is mainly driven by sars-cov-2 cytotoxicity, while inflammatory responses aim at eliminating the pathogen ultimately leading to tissue repair. accordingly, longitudinal immune profiling of hospitalized covid-19 cases with different outcomes has recently shown that, despite similar levels of inflammatory cytokines in the first 10 days from symptom onset, patients with less severe disease evolution also express mediators of wound healing and tissue repair (41) . in this phase, the therapeutic strategy should thus include the use of antiviral drugs and of treatments aimed at cautiously enhance immune responses. conversely, the use of drugs that compromise the efficiency of the immune system could be counterproductive, as patients with high viral loads and long virus-shedding periods are at higher risk of severe covid-19 (42) . in immunocompromised patients in whom the therapeutic strategies adopted and the presence of comorbidities do not allow adequate control of the viral load, extensive tissue damage, and subsequent uncontrolled inflammatory innate responses with exaggerated myeloid-derived cytokine production can occur (43) (44) (45) (46) . the clinical picture suddenly and unexpectedly changes with fever, respiratory failure, and ards associated with increased levels of acute phase reactants, neutrophilia, thrombocytosis, anemia, signs of coagulopathy, and cell lysis. these acute inflammatory mechanisms may precipitate tissue damage both locally (especially at the lung level) (47) and systemically, thus playing an even greater pathogenetic role than that played by direct viral damage, and significantly affecting mortality. this hyperinflammatory syndrome shares pathophysiological similarities with cytokine storms in crs, shlh and mas, conditions that sometimes complicate viral infections, systemic autoimmune and autoinflammatory diseases, hematologic diseases, and medications such as engineered t cell therapy (48) (49) (50) (51) . experience from these conditions has encouraged the use of anti-cytokine therapy also for the management of covid-19. however, the clinical picture of the cytokine storm in covid-19, especially when it is not associated with multi-organ damage secondary to de stefano et al. frontiers in immunology | www.frontiersin.org october 2020 | volume 11 | article 572635 coagulopathy, is different from that of the classic shlh/mas: it is mostly anatomically compartmentalized to the lungs, not associated with organomegaly and not accompanied by pancytopenia (5) . accordingly, serum levels of il-6 are lower in covid-19 compared to other crs (52) . furthermore, hypercytokinemia should be considered as a general marker of sars-cov-2, even in the absence of a cytokine storm, and this makes covid-19 crs not easily distinguishable from ards pathogenetically correlated only with viral cytotoxic activity, also because levels of inflammatory markers and cytokines not always correlate with outcome (53) . finally, compared to shlh/mas, the crs in covid-19 occurs in the context of immunodeficiency and immunoparalysis (5, 30, 54) , thus imposing caution on further iatrogenic immunosuppression. the identification of the correct timing of administration of anti-cytokine therapies and other immunosuppressants in covid-19 should thus take into account the intricate relationship between the viral burden, the hyperactivation of the innate immune system, and the adaptive immune dysfunction. although the therapeutic window in crs is narrow, and timely control of the cytokine storm is crucial to reduce short-term mortality, premature use of immunosuppressants could indeed further compromise viral shedding with the risk of increasing viral replication and tissue damage directly induced by the virus (43) . furthermore, iatrogenic immunosuppression could promote bacterial, fungus, or viral infectious complications, as shown with the targeting of il-6 with tocilizumab in observational studies on patients not requiring mechanical ventilation (18, 55) . also at later stages, pharmacologically induced immunosuppression in already immunocompromised patients could adversely affect the course of the disease, as suggested by the finding of significantly longer periods of hospitalization and higher rates of mortality in ventilated patients treated with tocilizumab (15, 16) or high-doses corticosteroids (56, 57) . the main challenge for effective anti-cytokine therapy in covid-19 will be therefore to better define a precise "window of therapeutic opportunity," a phase of the disease during which the benefits of cytokine inhibition are prevalent on the inevitable consequences of immunosuppression. although > 100 randomized clinical trials (rcts) on different cytokine inhibitors in covid-19 are underway, the identification of such a "window" remains elusive, and only few studies have tried to integrate further patients' characterization beyond the clinical status among the inclusion criteria ( table 2) . most of the protocols essentially require, among the inclusion criteria, the presence of pneumonia not otherwise defined if not by the extent of functional impairment, with a tendency to focus mainly on the early stages of the disease, in the absence of ards or systemic involvement. some observational studies continue to suggest that immunosuppressants such as glucocorticoids and il-6 antagonists (alone or in combination) are advantageous if administered early at hospital admission (17, 24, 58) (table 1) . however, the recovery trial has clearly shown that benefits from dexamethasone are restricted to those patients with at least 7 days of symptoms and those requiring invasive or non-invasive ventilation (59) , suggesting that, based on the clinical criterion alone, only a late phase of covid-19 is dominated by pathogenic immunity. accordingly, the use of early short-term corticosteroid therapy, even at low-doses, has been associated with worse clinical outcomes in non-severe covid-19 pneumonia in observational studies (25) ( table 1 ). in line with these findings, following preliminary results, rcts on sarilumab (another il-6 receptor antagonist) have been amended to enroll only critical patients (https://investor.regeneron.com/newsreleases/news-release-details/regeneron-and-sanofi-provideupdate-us-phase-23-adaptive), and the covacta trial on tocilizumab recently ended recruitment because neither primary nor secondary end-points were met (https://www. roche.com/media/releases/med-cor-2020-07-29.htm). better and still clinically feasible characterization of those patients with an hyperinflammatory syndrome potentially susceptible to immunosuppression could derive from the integration of markers of inflammation, tissue damage, cell lysis, and coagulopathy. this has indeed been the rationale for the development of the h-score for the diagnosis of shlh and mas, which includes, among the others, hyperferritinemia, cytopenias, and liver damage (60) . however, the many clinical and pathogenetic differences between covid-19 hyperinflammation and slhl/mas limit the generalizability of the h-score to covid-19 associated cytokine storm, and only few patients with severe covid-19 achieve diagnostic cut-offs of >169, mainly due to lower ferritin levels, absence of pancytopenia and hypofibrinogenemia (61) . similarly, the prevalence of the hyperinflammatory phenotype in ards due to covid-19 is lower compared to that observed in non-covid-19 ards despite its higher mortality (53) . however, laboratory indicators of hyperinflammation may still be of value in the identification of those covid-19 patients more at risk of escalation of respiratory support and death. using cut-off values of c-reactive protein (crp) concentrations greater than 150 mg/l or ferritin concentrations greater than 1,500 mg/l, manson jj and colleagues (62) recently described an hyperinflammatory phenotype of covid-19 characterized by poor clinical outcomes irrespective of other demographic and clinical variables. patients' stratification based on this approach could be of promise. accordingly, in an observational study on hospitalized covid-19 cases, early use of glucocorticoids was effective only in patients with high crp levels, being instead associated with increased mortality in case of low crp (26) . elevation of inflammatory markers such as crp, ferritin, and others is however non-specific for cytokine storms. cytokine assays are ready available in the clinic and testing for serum levels of cytokines known to be involved in pathogenic inflammation could help guiding the rational use of targeted immunomodulatory therapeutic strategies. accordingly, il-6 and other proinflammatory cytokines have been shown to be predictive of patient outcomes in terms of both disease severity and survival, and integrated models taking into account demographics, comorbidities, markers of inflammation and tissue damage, and cytokines show good performance with areas under the receiver operating characteristic curve approaching 0.8 (63) . several limitations however still exist even using similar approaches. apart from il-6, the trend of other cytokines whose targeting is already available in the clinic does not appear uniform in covid-19 (30, 41, 54, 63) , hampering the possibility of tailored treatments and contrasting with the apparent efficacy of some agents, such as il-1 antagonists, in preliminary observational studies (21, 22) . even in the case of il-6, no established cut-offs exist. furthermore, the release of inflammatory cytokines is also part of a well-conserved innate immune response necessary for efficient clearance of infectious agents. accordingly, levels of il-6 are similarly increased in the hyperinflammatory phenotype of non-covid-19 ards irrespective of its causative mechanism, but interventions targeting single cytokines in this setting have a long history of failure (64) . collectively, current clinical, laboratory, and even biological parameters thus do not appear optimal at distinguishing an appropriate from a dysregulated inflammatory response in the context of covid-19. ideally, in light of the very pulmonarycentric character of this condition, better knowledge could arise from the analysis of parameters deriving from bronchoalveolar lavage fluid and microscopic examination of transbronchial lung biopsies ( table 3) . these include cytological analyses with prevalence of granulocytes, macrophages, and lymphocytes; pro-inflammatory cytokine and chemokine secretion including neutrophil recruiting mediators and other attractants of monocytes and immune cells; lung infiltration by monocytes, macrophages, and neutrophils; thrombosis (65) (66) (67) (68) . the clinical, radiological, and laboratory characteristics already identified in patients with covid-19 would thus be put in the context of more specific histological, cytological, and immuneinflammatory criteria in order to identify those parameters more accurately signaling the presence of a predominantly immuno-inflammatory pathogenesis which might benefit from immunosuppression ( table 3 ). the evidence of ongoing sars-cov-2 replication late in disease would in any case support the associated use of antiviral therapy, even at a point when immunopathology is dominant (69) . in conclusion, despite the emergency situation imposes speed in the identification of the therapeutic options for patients with covid-19, better harmonization of inclusion criteria and patient stratifications for studies on immunomodulatory therapies should remain a priority. only considering a more specific set of clinical and pathological criteria, together with the extent of the viral load present in the alveolar bronchial lavage fluid and parameters useful to quantify the patient's immune vulnerability (lymphocyte count, cd4+ and cd8+ t cell levels, markers of lymphocyte exhaustion, development of specific antibodies), it will be possible to decide, carefully balancing both benefits and risks, the appropriateness of using immunosuppressive drugs even in patients affected primarily by an infectious disease. the original contributions presented in the study are included in the article/supplementary material; further inquiries can be directed to the corresponding author. all the authors contributed to the conception of the commentary. ls drafted the manuscript. all authors contributed to the article and approved the submitted version. this article was supported in part by fundings from the irccs policlinico san matteo foundation, pavia, italy. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease understanding covid-19: what does viral rna load really mean? pathological inflammation in patients with covid-19: a key role for monocytes and macrophages review: cytokine storm syndrome: looking toward the precision medicine era the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease should we stimulate or suppress immune responses in covid-19? cytokine and anticytokine interventions immunomodulatory therapy for the management of severe covid-19. beyond the anti-viral therapy: a comprehensive review cytokine storm syndrome in severe covid-19 effective treatment of severe covid-19 patients with tocilizumab pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe covid-19 tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in brescia, italy tocilizumab for treatment of severe covid-19 patients: preliminary results from smatteo covid19 registry (smacore). microorganisms efficacy and safety of tocilizumab in severe covid-19 patients: a singlecentre retrospective cohort study tocilizumab treatment for cytokine release syndrome in hospitalized covid-19 patients: survival and clinical outcomes profiling covid-19 pneumonia progressing into the cytokine storm syndrome: results from a single italian centre study on tocilizumab versus standard of care safety and efficacy of anti-il6-receptor tocilizumab use in severe and critical patients affected by coronavirus disease 2019: a comparative analysis early use of low dose tocilizumab in patients with covid-19: a retrospective cohort study with a complete follow-up tocilizumab in patients with severe covid-19: a retrospective cohort study tocilizumab among patients with covid-19 in the intensive care unit: a multicentre observational study interleukin-6 blockade with sarilumab in severe covid-19 pneumonia with systemic hyperinflammation: an open-label cohort study interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study anakinra for severe forms of covid-19: a cohort study beneficial impact of baricitinib in covid-19 moderate pneumonia; multicentre study efficacy evaluation of early, low-dose, short-term corticosteroids in adults hospitalized with non-severe covid-19 pneumonia: a retrospective cohort study effect of systemic glucocorticoids on mortality or mechanical ventilation in patients with covid-19 factors associated with covid-19-related death using opensafely the role of host genetics in the immune response to sars-cov-2 and covid-19 susceptibility and severity mannose-binding lectin in severe acute respiratory syndrome coronavirus infection viral and host factors related to the clinical outcome of covid-19 hypothesis for potential pathogenesis of sarscov-2 infection -a review of immune changes in patients with viral pneumonia covid-19: immunopathology and its implications for therapy the many faces of the anti-covid immune response clinical and immunologic features in severe and moderate forms of coronavirus disease sars-cov-2 infects t lymphocytes through its spike protein-mediated membrane fusion functional exhaustion of antiviral lymphocytes in covid-19 patients dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice tissue distribution of ace2 protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis ace2 at the centre of covid-19 from paucisymptomatic infections to severe pneumonia clinical features of patients infected with 2019 novel coronavirus in wuhan longitudinal analyses reveal immunological misfiring in severe covid-19 detectable 2019-ncov viral rna in blood is a strong indicator for the further clinical severity viral dynamics in mild and severe cases of covid-19 the role of interleukin 6 during viral infections inflammasomes and pyroptosis as therapeutic targets for covid-19 the innate immune system: fighting on the front lines or fanning the flames of covid-19? macrophage expressed ifn-b contributes to apoptotic alveolar epithelial cell injury in severe influenza virus pneumonia macrophage activation syndrome an interferon-gamma-related cytokine storm in sars patients managing cytokine release syndrome associated with novel t cell-engaging therapies current status of chimeric antigen receptor therapy for haematological malignancies prevalence of phenotypes of acute respiratory distress syndrome in critically ill patients with covid-19: a prospective observational study a dynamic covid-19 immune signature includes associations with poor prognosis tocilizumab for cytokine storm syndrome in covid-19 pneumonia: an increased risk for candidemia? frontiers in immunology | www.frontiersin.org october clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and metaanalysis early combination of tocilizumab and corticosteroids: an upgrade in anti-inflammatory therapy for severe covid dexamethasone in hospitalized patients with covid-19 -preliminary report development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome lung involvement in macrophage activation syndrome and severe covid-19: results from a cross-sectional study to assess clinical, laboratory and artificial intelligence-radiological differences covid-19-associated hyperinflammation and escalation of patient care: a retrospective longitudinal cohort study an inflammatory cytokine signature predicts covid-19 severity and survival phenotypes in acute respiratory distress syndrome: moving towards precision medicine transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in covid-19 patients heightened innate immune responses in the respiratory tract of covid-19 patients single-cell landscape of bronchoalveolar immune cells in patients with covid-19 pathological findings of covid-19 associated with acute respiratory distress syndrome histopathological findings and viral tropism in uk patients with severe fatal covid-19: a post-mortem study the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-003828-bhfghcby authors: zrzavy, tobias; kollaritsch, herwig; rommer, paulus s.; boxberger, nina; loebermann, micha; wimmer, isabella; winkelmann, alexander; zettl, uwe k. title: vaccination in multiple sclerosis: friend or foe? date: 2019-08-07 journal: front immunol doi: 10.3389/fimmu.2019.01883 sha: doc_id: 3828 cord_uid: bhfghcby multiple sclerosis (ms) is a debilitating disease of the central nervous systems (cns). disease-modifying treatments (including immunosuppressive treatments) have shown positive effects on the disease course, but are associated with systemic consequences on the immune system and may increase the risk of infections and alter vaccine efficiency. therefore, vaccination of ms patients is of major interest. over the last years, vaccine hesitancy has steadily grown especially in western countries, partly due to fear of sequelae arising from vaccination, especially neurological disorders. the interaction of vaccination and ms has been discussed for decades. in this review, we highlight the immunology of vaccination, provide a review of literature and discuss the clinical consideration of ms, vaccination and immunosuppression. in conclusion, there is consensus that ms cannot be caused by vaccines, neither by inactivated nor by live vaccines. however, particular attention should be paid to two aspects: first, in immunocompromised patients, live vaccines may lead to a stronger immune reaction with signs of the disease against which the patients have been vaccinated, albeit in weakened form. second, protection provided by vaccination should be controlled in patients who have been vaccinated while receiving immunomodulatory or immunosuppressive treatment. in conclusion, there is evidence that systemic infections can worsen ms, thus vaccination will lower the risk of relapses by reducing the risk of infections. therefore, vaccination should be in general recommended to ms patients. over the last years, especially in western countries, vaccine hesitancy has steadily grown and poses an increasing health concern (1). the recent upsurge of measles in europe is an impressive example. anti-vaccinationists argue that possible side effects weigh out the benefits (2) . especially sequelaes such as autism, multiple sclerosis (ms) and various neurological syndromes have been emphasized by the anti-vaccination lobby (3, 4) . this alarming development is even partly supported by healthcare providers including some ms neurologists, who are afraid of iatrogenic deterioration of preexisting ms. indeed, studies linking vaccination and disease onset have been published. although these studies were often underpowered and lacked an adequate design in order to provide evidence of the suspected link, they caught public awareness leading to a drop of public vaccination coverage rates (5, 6) . epidemiological studies and pharmacovigilance data have repeatedly demonstrated safety for the vast majority of vaccines. lately, a review concluded that there is no significant evidence for a causal relationship between the onset or deterioration of ms and vaccination against measles, mumps and rubella (mmr), influenza, hepatitis a, hepatitis b, human papilloma virus (hpv), diphtheria, tetanus, acellular pertussis, or meningococcal disease (7) . some studies have even indicated a decreased risk for ms and reduced disease activity in preexisting ms (8) . the aim of this review is to summarize data on vaccination and disease activity of both ms and acute disseminated encephalomyelitis (adem). moreover, vaccination-induced effects on the immune system are presented and potential interactions between ms and immunizations are discussed. vaccine-induced protection is a complex issue and depends on a cascade of mechanisms and mediators (figure 1) . eventually, protection is accomplished either by antibodies or t celldependent factors or by a combination of both including neutralizing or antitoxic antibodies, cd8 + t cells, cd4 + t cells and corresponding cytokines (e.g., interleukin (il)-2, 3, 4, 5, 9, 13, 17, 21, 22, and 26) (9) . generally, vaccines have to be capable of activating antigen-presenting cells (apcs) of the innate immune system, which subsequently present the vaccine epitope(s) to t cells-the so-called 'immunogenic potential' (10) . in this context, dendritic cells play a pivotal role due to their enhanced capability to stimulate naïve t cells (11) . the nature of vaccine-induced immunity depends on several parameters, of which the biological properties of the vaccine's epitope are of high importance (9) . live vaccines are attenuated variants of pathogens that still can activate apcs, especially immature dendritic cells, patrolling through the body. this immunogenic potential is often lost by subcellular-or subunitbased vaccines (12) , which is why these inactivated vaccine antigens are usually combined with so-called adjuvants to increase and modulate the vaccine's immunogenicity via a longer lasting and more effective activation of immune cells. one of the most widely used adjuvants are aluminum salts, which were originally thought to create a long-lasting depot of the antigen in order to provide its slow release, but have instead been shown to act on dendritic cells via prrs (pattern recognition receptors) leading to the secretion of pro-inflammatory cytokines (13) . similarly, novel adjuvants like squalens or monophosphoryl lipid a (mpla-a detoxified lipopolysaccharide) aim to enhance the innate immune response, but never reach the immunogenic potential of live attenuated vaccines (14) . adjuvants have been added to vaccines for more than 90 years and over the last decades, considerable progress has been made in understanding their mode of action and to improve safety (15) . besides the above mentioned aluminum salts, squalene and mpla, oil emulsions, saponin, toll-like receptor (tlr) agonists, enterotoxins, polysaccharides, and glycolipid adjuvants (16) are used, all of which stimulate the immune system as well. aluminum adjuvants have now been used for decades and lots of experience has been gained on its use, effectiveness, and safety and they still remain the most frequently used adjuvants. their effects on the immune system comprise stimulation of macrophages and dendritic cells via prrs, inflammasome activation, il-1β release and activation of th2 lymphocytes (15, 16) . however, besides increased immunogenicity, aluminum adjuvants also increase reactogenicity and based on data from animal models and reports on narcolepsy, silicosis, guillain-barré-syndrome (gbs) and macrophagic myofasciitis, they are also discussed to induce autoimmunity (17) . the second most commonly and long used adjuvants are oil emulsions. they have a strong reactogenic potential and can cause severe inflammatory local reactions such as ulceration and granulomas. the most well-known oil emulsion is complete freund's adjuvant. however, due to its potent reactogenicity, it is not suitable for human use. a possible association between oil emulsions and autoimmunity disorders has been hypothesized from animal models. oil emulsions are potent inducers of il-1β and il-17 (18, 19) . il-17 plays a major role in autoimmunity and ms and may trigger the migration of peripheral lymphocytes into the cns across the bbb (20, 21) . frequently, a combination of adjuvants is used to increase immunogenicity of vaccines. as03 is an adjuvant emulsion containing squalene, dl-αtocopherol, and polysorbate 80. it is e.g., used for the pandemic swine flu vaccine pandemrix r (15) or the fda-licensed h5n1 monovalent influenza vaccine. in animal studies, autoimmunity was observed in connection with as03 (22) and in humans, cases of narcolepsy have been reported (23) . oil emulsions are often combined with tlr agonists such as mpla. generally, tlr agonist adjuvants activate the inflammatory transcription factor nfκb as04 is a combination of mpla and aluminum salts and is used as adjuvant in vaccines against hepatitis b (fendrix r ) and hpv, as well as in the new recombinant vaccine against herpes zoster. most polysaccharide adjuvants activate nfκb to induce immune processes (e.g., dextran, zymosan) (24) . however, deltainulin for instance, a polysaccharide adjuvant used for advax r , acts via nfκb-independent mechanisms to enhance humoral and cellular immune responses. although the mechanisms are not yet fully understood, advax r has so far not shown inflammatory side effects and has proven safety in hepatitis b vaccination and influenza (16) . after activation of the immune cascade and stimulation of dendritic cells, the latter increase their expression of mhc molecules and chemokine receptors such as ccr7 leading to their migration toward the draining lymph nodes in order to provide co-stimulatory signals for the differentiation of naïve t cells into immune effector cells (25) . the activation of the immune cascade has various effects on t and b cells. in short, antigen-recognition by b cells leads to their activation and migration toward the t-b cell border of the lymph node, where they can subsequently receive additional stimuli by activated t helper (th) cells. these signals include cd40 interaction, secretion of cytokines by th1 or th2 cells, and finally the transformation of b cells into plasma cells predominantly secreting low affinity antibodies (26) . later, the germinal center response contributes via affinity maturation (somatic hypermutation and affinity-based selection) and isotype switch to a sustained production of high affinity antibodies by predominantly plasma cells but also memory b cells. basically, in the lymph nodes, numerous b cells with various affinity compete for the antigens presented by follicular dendritic cells. these antigens are processed and further presented via mhc ii to follicular th cells, which provide costimulatory signals (e.g., cd40, icos, and il-21) leading to survival and further proliferation of b cells with highest affinity for the antigen (27) . in conclusion, vaccination-induced immune responses, including employed cell types and mediators, vary depending on the type of vaccine administration, kind of vaccine and choice of adjuvant. while antibodies will directly prevent and reduce infections, cd4 + and cd8 + t cells rather support the organism eventually reducing, controlling and clearing the pathogens. antibodies bind to their antigen, neutralize pathogens, activate macrophages and neutrophils as well as the complement system, while cd4 + and cd8 + t cells secrete cytokines, perforins, and granzymes (9) . the choice of adjuvant seems to be critical, since some may cause problems in autoimmune diseases. thus, monitoring side effects regarding autoimmunity is essential. in the early days of vaccine development, louis pasteur used nerve tissue of infected animals to obtain a rabies virus vaccine (28) . although saving countless lives it was recognized that active sensitization with neuronal tissue could occasionally lead to neuroparalytic autoimmune complications (29) with self-limiting autoimmune encephalomyelitis that fulfilled the pathological criteria of ms (29, 30) . advances in processing techniques and increasing insights in immunology led to modern vaccines devoid of neuronal tissue. ms is a chronic disease thought to be caused by immune-mediated mechanisms. thus, immune responses caused by vaccinations will affect the immune system. however, their effects on immunology per se, but especially those in ms patients, are scarcely understood. the same means by which infections can induce autoimmunity also apply for vaccination-induced immune activation. possible structural similarities between microbial epitopes and epitopes of the cns could lead to cross-reaction of antibodies via molecular mimicry as shown for streptococcal antibodies in heart tissue (31) . additionally, epitope spreading is a mechanism leading to a broadening of the immune response from the dominant epitope to cryptic (intramolecular) or neighboring molecules (intermolecular) resulting in an increased antibody repertoire and cellular response (32) . moreover, bystander activation, a process in which activated apcs stimulate autoreactive t cells, can occur (33) . bacterial and viral infections can trigger relapses and mri activity in ms; vaccination has been proven to protect from or weaken infections, thus providing an "indirect" protection against ms disease activity (34) . several reports on neurological disorders developing after immunization have been published including several cases on encephalomyelitic disorders (impaired consciousness, ataxia and optic neuritis) as well as demyelinating lesions in a patient with transverse myelitis after active immunization against influenza (35) (36) (37) (38) . immunization against rubella was associated with diffuse myelitis and recurrent relapses with optic neuritis, paraparesis and impaired motor function (39, 40) . transverse myelitis (41) as well as optic neuritis (42, 43) were reported in patients vaccinated against measles, mumps and rubella. further cases with symptoms suggestive for disseminated encephalitis were reported after vaccination against diphtheriatetanus-poliomyelitis (dtp) (44) and after immunization against smallpox, rabies or typhus (45) . exacerbations of ms and demyelinating lesions were reported in ms patients and patients without a history of neurological conditions after immunization against hepatitis b (46) . similarly, tourbah reported on 8 patients with demyelinating lesions and clinical symptoms after vaccination against hepatitis b (47) . in contrast to these case series, a case-control study (evidence class ii) (48) including more than 440 patients with ms or optic neuritis and 950 controls without any underlying neuroimmunological disorder did not reveal an elevated risk for the development of ms or optic neuritis after immunization against hepatitis b, tetanus, influenza, measles/mumps/rubella, measles, or rubella (49) . while hernan came to same results for immunization against influenza or tetanus in a case-control study (evidence class ii), active immunization against hepatitis b was reported to pose a higher risk for ms (50) . the latter finding could, however, not be confirmed by confavreux in a large case-crossover study. additionally, no increased risk was seen for vaccination against tetanus and influenza as well (51) . similarly, other class ii case-control studies did not report on an increased risk for ms after hepatitis b vaccination (52) (53) (54) . an even decreased risk for ms was reported after tetanus immunization (8) . in a large class i study, a patient register including 789,082 females vaccinated with the quadrivalent hpv vaccine was analyzed. thereof, 4,322 patients with ms and 3,300 patients with other demyelinating disorders were studied and no increased risk for cns manifestations was seen in this large cohort (55). miller et al. performed a prospective class ii, randomized, double-blind, placebo-controlled study, which included 104 ms patients, who received either standard influenza vaccination or placebo. for a 6 months follow-up period, the occurrence of neurological symptoms or influenza was monitored and no differences were seen for relapse rates (56) . a study by langer-gould reported on an increased risk for cns demyelinating diseases within the first 30 days after vaccination. it was concluded that there is no increased risk for ms, but it seems that the transition from subclinical to overt autoimmunity in patients with existing disease is shortened (53) . two major questions arise on the topic of "ms and vaccination": (i) can vaccines cause ms and (ii) can vaccines provoke or trigger relapses in patients with ms? (i) overall, the anecdotal reports associating ms onset and vaccination had limited reliability, lacked validity and could not be replicated in larger studies. therefore, there is consensus that there is yet no evidence that ms can be caused by vaccines neither by inactivated nor by live vaccines (57). (ii) it is more difficult to assess the potency of vaccines to trigger relapses in ms patients. with respect to live vaccines it seems to be plausible that they may be able to provoke a deterioration of the disease, since they fulfill the criteria of an active infection with a replicative (although attenuated) organism. there is class iv evidence that at least the yellow fever (yf) 17d vaccine strain, which is derived from a natural occurring yf-virus and hasn't completely lost its neurotoxicity even after numerous passages, is able to provoke relapses in ms patients. however, it has to be kept in mind that the patient cohort had received immunomodulatory treatment and the sample size of this self-controlled case series study was rather small (58) . the underlying potential immunologic mechanisms, which are responsible for this elevated relapse rate, are not understood yet and larger studies are necessary to confirm this association. hypotheses may be generated based on observations after infections with helminths, mycobacteria and epstein-barr virus, or by the immunologic properties of this particular vaccine strain (59) . immunological analyzes showed that after immunization against yf, ms patients had a significantly increased mbp-and mog-specific response shown by increased numbers of cells secreting interferon, il-1α, il-1β and tumor necrosis factor compared to unvaccinated ms patients or ms patients vaccinated against influenza (58) . still, there is no evidence for other live vaccines such as mmr to deteriorate ms (57, 60) . for inactivated vaccines, there is already more evidence available that an association between ms relapses and different kinds of vaccines does not exist (7) . even for vaccines, which were publicly accused to be associated with ms disease or relapse rate, like hpv or hepatitis b vaccines, there is no evidence to support any association between vaccination and clinical course of ms, as well as for vaccines containing inactivated neurotropic viruses like tbe (53, 61) . it still remains unclear if inactivated vaccines may accelerate an upcoming relapse in patients with active ms by non-specific stimulation besides effects of vaccines on induction and the disease course of ms, potential immunological effects of adjuvants have to be considered as well. most experience on the possible induction of autoimmunity following administration of adjuvant-containing vaccines has been gained from animal models. however, results from experimental studies cannot be transferred to humans without reservation. first, the dose ratios tested in animal models are not the same as in humans and second, human immunology differs from animals. indeed, oil emulsions, aluminum salts and squalene have shown severe side effects in animal models, while they are considered to be safe in humans (17) . an analysis performed by the european medicines agency (ema) (63) investigated autoimmune disorders following vaccination against pandemic influenza a/h1n1 between october 2009 and december 2010 (64) . thirty percent of the 150 million doses of the distributed vaccines contained aluminum salts and squalene-based adjuvants. overall, the study did not suggest a significant difference in the risk for autoimmune disorders for adjuvant and non-adjuvant vaccinations. adem was reported for 10 people (adjuvant vaccines: 7, non-adjuvant vaccines: 3), ms for 21 people (adjuvant vaccines: 20, nonadjuvant vaccines: 1), ms relapses for 24 patients (adjuvant vaccines: 21, non-adjuvant vaccines: 3), and one case of relapsing remitting ms was reported for adjuvant-containing vaccination (64) . statistical analysis revealed only a nonsignificantly increased risk for gbs (15) . also, a favorable benefitrisk profile of the vaccines was demonstrated (15, 65) . in conclusion, following the reports from literature, all of the ema/fda-approved vaccines (with exception for yellow fever) and adjuvants do not show a significantly increased risk for ms and adem. constant improvement of basic immunological knowledge and technology will further improve the safety of adjuvants. table 1 gives an overview of the recommendations of standard vaccinations in the general population and in ms patients. while there is a lot of literature on vaccination and risk for ms or ms relapses available, reports on vaccination and adem are scarce. yet, adem has been discussed to be a sequelae of vaccinations (68) as well as to be preceded by infections. several cases of adem have been reported to be timely related to vaccinations against rabies (69), hpv (70, 71) , hepatitis a and b, diphtheria, tetanus and poliovirus (72) , measles, rubella and booster immunization for japanese encephalitis (73) . adem has been reported following vaccination against influenza, including eight cases after vaccination against h1n1. also, four adem cases after vaccination against yf can be found in literature (74, 75) . besides case reports, there have been some observational studies, albeit all having their limitations. in 26 out of 35 reported cases of adem, patients had infections or vaccinations prior to disease onset (76) . also, pellegrino et al. concluded a possible relation between post-vaccination adem in children and adults. four hundred four cases of adem were analyzed based on the data of the vaccine adverse event reporting system (vaers) database and the eudravigilance post-authorization module (evpm) (77) . about 60% of the cases occurred between 2 and 30 days after vaccination, most commonly against influenza and hpv. a case-control study on vaccination against hepatitis b, influenza, polio, diphtheria, pertussis, tetanus, measles, mumps, rubella, japanese encephalitis, meningitis, hepatitis a, varicella and rabies did not reveal an increased risk for the onset of adem in the time spans of 0-30 days and 61-180 days after vaccination, but between 31 and 60 days (78) . based on these reports, the risk for adem after vaccination cannot be completely ruled out. considerations on ms exacerbation and vaccination apply only for ms patients receiving no immunomodulatory/ immunosuppressive treatment. if any kind of immunosuppression is used for ms therapy, this choice of treatment will dominate the decision whether to vaccinate or not (79) . in recent years, consensus statements on vaccinations during immunosuppressive treatments were published by various national and international societies and expert panels (80) (81) (82) (83) (84) . there is consensus that inactivated vaccines will do no harm (85) even in immunosuppressed patients. however, data on the efficacy of vaccinations in combination with the various available ms medications are missing. thus, for patients either receiving more than one immunomodulatory treatment or having underlying immunomodulating condition, the outcome is difficult to predict (86). therefore, the success of vaccination should be verified by antibody testing if a valid test is available. except for a few treatments, which only lead to mild immunosuppression, live vaccines are contraindicated under immunosuppressive treatment. in some situations, risks and benefits of a live vaccine have to be weighed against each other, e.g., in varicella zoster virus (vzv)-negative ms patients under fingolimod treatment, varicella vaccination may be considered, since severe complications from natural varicella infection may outweigh the risk from this live vaccine. however, recommendations vary between different institutions even within the same country (80, 82, 83) . a recent case report on a lethal vzv infection in an immunocompromised patient after vzv live vaccination drives the discussion on this issue (87) . there is consensus about the timing of vaccination in patients, who will undergo immunosuppressive treatment: vaccinations should be given well in advance to the start of treatment (at least 2 weeks for inactivated and ≥ 4 weeks for live vaccines) and should be distinguished between primo-vaccinations and boosters. importantly, the refractory period after immunosuppression has to be considered as well, which may be up to 1 year depending on the type of medication (e.g., rituximab or alemtuzumab) (81) . vaccines will have various effects on the immune system, which greatly depend on the cell types typically engaged by the respective vaccines. the impact of immunosuppression on the various cell types (and possible mitigation of effects) should be taken into consideration. protective efficacy is mostly mediated by antibodies for the following vaccines: cholera, diphtheria toxoid, hepatitis a and b, haemophilus influenzae type b, influenza, japanese encephalitis, meningococcal ps and conjugates, papillomavirus, pneumococcal ps and conjugates, polio (sabin and salk), rabies, rotavirus, rubella, tetanus toxoid, typhoid ps, and yf. effects are solely born by t cells for tuberculosis (bcg), or by a combination of antibodies and t cells for measles and intranasal influenza vaccination. besides antibody-mediated protection, effects of t cells are discussed for pertussis (9) . for patients receiving immunosuppressive treatment, vaccination control should be performed. for diphtheria, tbe (with caution), hepatitis a, b, haemophilus influenzae type b, measles, mumps, pneumococcus, polio, rubella, tetanus, rabies and varicella, standards are available and recommended to be tested. in general, to increase the validity of vaccination control, titers should be assessed in paired samples (before and after immunization) via the same method and at highquality standards (81) . in general, patients should have received their recommended standard vaccines according to their region-specific vaccine guidelines. before certain immunosuppressive treatments are initiated, it is mandatory to exclude former infections and if necessary, vaccination should be considered according to the regulatory agencies. table 2 provides an overview on necessary vaccinations according to fda/ema guidelines (extended vaccination reflects the authors' suggestion). for many immunotherapies, a prior exclusion of an ongoing vzv infection is required and vaccination should be offered to those, who haven't gained any immunity yet. additionally, vzv-seropositive patients undergoing immunotherapy should be offered vaccination as well to prevent zoster reactivation and late effects. recently, a non-live subunit vaccine has been authorized for vzv-seropositive patients. it possesses a better risk-benefit profile compared to the live vaccine and has already been approved by many countries (88) . additionally, it should be considered to offer patients with upcoming fingolimod or alemtuzumab treatment the option of vaccination against hpv, as post-market surveillance showed increased reports of warts and cervical dysplasia due to these two ms therapies [ema; (89) ]. furthermore, pneumococcal vaccine might be considered in patients receiving b cell-depleting therapies, as severe respiratory infections during phase iii studies were seen (90, 91) . vaccine hesitancy is a major problem nowadays. the usefulness of active immunization is undisputed and has saved numerous lives. however, fear of possible, but also often unconfirmed, side effects has fostered this anti-vaccine sentiment. this has led to a recent outbreak of measles (2) and curiously some viruses and disorders, which have been assumed to be eradicated, seem to become a hot topic for western health systems again. indeed, side effects upon vaccination may occur in rare cases, however, the benefits for individual people as well as the whole population will generally outweigh adverse effects. vaccine hesitancy results in a twofold problem: (1) the missing protection for the unvaccinated people themselves but also (2) a risk for people, who are not able to get vaccinated. the missing herd immunity poses a major problem for a group of patients with fragile health. for ms patients receiving immunosuppressive treatment, an acute infection can have dangerous sequelae. thus, if possible, ms patients should be vaccinated beforehand. the possible benefits outweighdependent on the individual case-the possible risks. an additional perspective raises the possibility of vaccination against ms. indeed, early approaches exploring vaccination with synthetic peptides in experimental animal models were successful, but translation into clinical treatment was so far unsatisfying (92) (93) (94) . interestingly, it was recently shown that an anti-typhus vaccination (typhim vaccine) might have the potential to ameliorate the disease course of ms by targeting prohibitins on th17 cells. tested in an experimental ms model it led to decreased levels of il17 and increased numbers of foxp3 + regulatory t cells (95) . further investigations are needed before studies should investigate treatment options for ms patients. still, it is a good example, how immunology of vaccination might overlap with and modulate the immunology of ms. theoretically, an increased immune response against different types of vaccines, such as live attenuated viruses, inactive attenuated viruses, or portions of bacteria and viruses, could trigger increased immune response to self-antigens (45, 58, 96) , but an increased risk for ms itself or increased relapse rates after vaccination have not been show (with exception for yf) in case-control studies (7) . there is, however, evidence that infections can trigger relapses in ms (96) (97) (98) (99) (100) (101) (102) (103) (104) , which is why vaccination of ms patients should be pursued in order to reduce the risk of infections. to assure the best vaccination success, immunization and immunosuppressive treatments have to be well timed. vaccine hesitancy: definition, scope and determinants available online at a postmodern pandora's box: anti-vaccination misinformation on the internet the "urban myth" of the association between neurological disorders and vaccinations mass vaccination against hepatitis b: the french example central nervous system demyelinating diseases and recombinant hepatitis b vaccination: a critical systematic review of scientific production vaccines and multiple sclerosis: a systematic review tetanus vaccination and risk of multiple sclerosis: a systematic review vaccine immunology in: spwopokm, editor. plotkin's vaccines vaccine adjuvants: putting innate immunity to work in vivo activation of antigen-specific cd4 t cells superior immunogenicity of inactivated whole virus h5n1 influenza vaccine is primarily controlled by toll-like receptor signalling aluminum hydroxide adjuvants activate caspase-1 and induce il-1beta and il-18 release immunological mechanisms of vaccination vaccine adjuvants: from 1920 to 2015 and beyond comparative safety of vaccine adjuvants: a summary of current evidence and future needs adjuvants safety project c. safety of vaccine adjuvants: focus on autoimmunity adjuvant system as03 containing α-tocopherol modulates innate immune response and leads to improved adaptive immunity cell recruitment and cytokines in skin mice sensitized with the vaccine adjuvants: saponin, incomplete freund's adjuvant, and monophosphoryl lipid a human t h 17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation cellular mechanisms of il-17-induced blood-brain barrier disruption induction of lupus autoantibodies by adjuvants increased incidence and clinical picture of childhood narcolepsy following the 2009 h1n1 pandemic vaccination campaign in finland carbohydrate-based immune adjuvants extrafollicular antibody responses control systems and decision making for antibody production rabies vaccine prepared in human cell cultures: progress and perspectives the neuro-paralytic accidents of antirabies treatment autoimmune encephalitis in humans: how closely does it reflect multiple sclerosis? an immunological relationship between the group a streptococcus and mammalian muscle spreading of t-cell autoimmunity to cryptic determinants of an autoantigen antiviral immune responses: triggers of or triggered by autoimmunity? the risk of relapses in multiple sclerosis during systemic infections relapsing encephalomyelitis following the use of influenza vaccine ocular abnormalities after influenza immunization acute transverse myelitis after influenza vaccination: magnetic resonance imaging findings optic neuritis after influenza vaccination diffuse myelitis associated with rubella vaccination diffuse myelitis associated with rubella vaccination transverse myelitis after measles, mumps, and rubella vaccine optic neuritis complicating measles, mumps, and rubella vaccination optic neuritis following measles/rubella vaccination in two 13-year-old children relapsing acute encephalopathy: a complication of diphtheria-tetanus-poliomyelitis immunization in a young boy multiple sclerosis and vaccination central-nervous-system demyelination after immunisation with recombinant hepatitis b vaccine encephalitis after hepatitis b vaccination: recurrent disseminated encephalitis or ms? level of evidence reviews: three years of progress vaccinations and risk of central nervous system demyelinating diseases in adults recombinant hepatitis b vaccine and the risk of multiple sclerosis: a prospective study vaccines in multiple sclerosis study g. vaccinations and the risk of relapse in multiple sclerosis. vacc multiple scler study group hepatitis b vaccination and the risk of multiple sclerosis vaccines and the risk of multiple sclerosis and other central nervous system demyelinating diseases multiple sclerosis and immunological-related risk factors: results from a case-control study quadrivalent hpv vaccination and risk of multiple sclerosis and other demyelinating diseases of the central nervous system a multicenter, randomized, double-blind, placebo-controlled trial of influenza immunization in multiple sclerosis adverse effects of vaccines: evidence and causality yellow fever vaccination and increased relapse rate in travelers with multiple sclerosis serious adverse events associated with yellow fever vaccine epstein-barr virus, cytomegalovirus, and multiple sclerosis susceptibility: a multiethnic study a controlled trial of tick-borne encephalitis vaccination in patients with multiple sclerosis vaccines and multiple sclerosis summary of product characteristics autoimmune disorders after immunisation with influenza a/h1n1 vaccines with and without adjuvant: eudravigilance data and literature review available online at recommended adult immunization schedule, united states ständige impfkommission: empfehlungen der ständigen impfkommission (stiko) am robert koch-institut acute disseminated encephalomyelitis: an update biphasic demyelination of the nervous system following anti-rabies vaccination acute disseminated encephalomyelitis following vaccination against human papilloma virus two cases of acute disseminated encephalomyelitis following vaccination against human papilloma virus improvement of advanced postvaccinal demyelinating encephalitis due to plasmapheresis post-vaccination mdem associated with mog antibody in a subclinical chlamydia infected boy neurologic disease associated with 17d-204 yellow fever vaccination: a report of 15 cases longitudinal myelitis associated with yellow fever vaccination acute fulminant demyelinating disease: a descriptive study of 60 patients acute disseminated encephalomyelitis onset: evaluation based on vaccine adverse events reporting systems vaccines and the risk of acute disseminated encephalomyelitis disease-modifying therapies and infectious risks in multiple sclerosis idsa clinical practice guideline for vaccination of the immunocompromised host general best practice guidelines for immunization vaccination in immunocompromised host: recommendations of italian primary immunodeficiency network centers (ipinet) vaccination against infection in patients with multiple sclerosis tickborne encephalitis (tbe) vaccine to medically immunosuppressed patients with rheumatoid arthritis: a prospective, open-label, multi-centre study live zoster vaccination in an immunocompromised patient leading to death secondary to disseminated varicella zoster virus infection understanding the immunology of shingrix, a recombinant glycoprotein e adjuvanted herpes zoster vaccine warts and all: fingolimod and unusual hpv-associated lesions alemtuzumab versus interferon beta 1a as first-line treatment for patients with relapsing-remitting multiple sclerosis: a randomised controlled phase 3 trial ocrelizumab versus interferon beta-1a in relapsing multiple sclerosis vaccination against experimental allergic encephalomyelitis with t cell receptor peptides the ups and downs of multiple sclerosis therapeutics antigen-specific tolerization approaches in multiple sclerosis targeting prohibitins at the cell surface prevents th17-mediated autoimmunity immunization in patients with multiple sclerosis chlamydia pneumoniae infection of the central nervous system in multiple sclerosis epstein-barr virus and multiple sclerosis coronaviruses in brain tissue from patients with multiple sclerosis active human herpesvirus 6 infection in patients with multiple sclerosis cerebrospinal fluid molecular demonstration of chlamydia pneumoniae dna is associated to clinical and brain magnetic resonance imaging activity in a subset of patients with relapsing-remitting multiple sclerosis multiple sclerosis, sporadic creutzfeldt-jakob disease and bovine spongiform encephalopathy: are they autoimmune diseases evoked by acinetobacter microbes showing molecular mimicry to brain antigens? the case for rhinoviruses in the pathogenesis of multiple sclerosis epstein-barr virus infection and multiple sclerosis: a review all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2019 zrzavy, kollaritsch, rommer, boxberger, loebermann, wimmer, winkelmann and zettl. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-328011-6lf3no6u authors: zayed, hatem title: vaccine development against covid-19 prior to pandemic outbreaks, using in vitro evolution and reverse genetics date: 2020-08-14 journal: front immunol doi: 10.3389/fimmu.2020.02051 sha: doc_id: 328011 cord_uid: 6lf3no6u nan the coronavirus disease 2019 (covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which is an enveloped, non-segmented, positive-sense rna virus (1) . the complete genome of sars-cov-2 is 29.9 kb (2, 3). the virus genome contains four essential proteins that are believed to be important for the infectious ability of the virus, the glycoprotein spike (s), nucleocapsid (n), matrix (m), and small envelope (e) proteins (4) . the s glycoprotein, which mediates entry of the virus into the target cells, is the main target for host defense antibodies (5) . as of may, 2020, the covid-19 pandemic has spread to 213 countries and territories worldwide with nearly 6 million confirmed cases and ∼6% mortality (who.int). as the outbreaks spread, scientists across the globe are racing to develop vaccines against covid-19. since coronaviruses are increasing alarmingly, there is an urgent need for a safe and effective vaccine to prevent the spread of the virus during pandemic outbreaks, and stop deaths associated with the virulent covid-19. however, developing vaccines that are safe and effective requires a lot of time and testing. it is estimated that 18 months are needed to develop such a vaccine. although it is challenging to predict the severity, time, and location of future coronavirus pandemics, we can be prepared for the highly pathogenic strains that are likely to reemerge and cause future pandemics. this can be done using previous epidemiological studies on coronaviruses. for example, in 2019, chinese scientists anticipated that there would be a potential bat coronavirus that would likely emerge and infect humans, and might cause an imminent outbreak in china (6) . unfortunately, the efforts of these chinese scientists were met with no interest from the chinese government, evidenced by the lack of proper preparation for the current pandemic when it appeared in china a few months ago. we now know that sars-cov-2 shares 88% identity with two sars-like coronaviruses (bat-sl-covzxc21 and bat-sl-covzc45) that both originated in china, and use the same human angiotensin-converting enzyme 2 receptor for cell entry during the process of infection (3). if we had reacted to these predictions, then we would very likely have avoided the current crisis. in response to such forewarnings from scientists, a predictive vaccine could have been designed and developed for the potential virus pandemic. developing a vaccine during or after the pandemic outbreaks is too slow to provide timely responses against covid-19, and risks many lives. producing an efficient and safe vaccine ready for human use can take up to 18 months, according to the world health organization (who). therefore, anticipating the virus mutations responsible for the possible reemergence of highly pathogenic virulent strains may be a means by which to prepare for future, newly emerging, pandemic strains. the process of preparing a predictive vaccine can be summarized as follows: (1) the sars-cov-2 genome would be used as a template for in vitro evolution through dna shuffling techniques (7, 8) . random recombination of a viral genome in a test tube mimics the possible assortment and mutations that occur in the virus in nature, creating all possible random recombination. these changes could be incorporated into the four essential viral genes (s, n, m, and e). (2) these genes would then be subcloned individually into integrating gene delivery vehicles, such as lentiviral vectors (9) or transposons (10, 11) . (3) using reverse genetic strategies (12, 13) , the recombinant constructs would be transfected into cell lines susceptible to coronaviruses (14) , leading to secretion of viruslike particles (vlps) from the cells into the culture media. (4) the recombinant vlps could then be harvested and purified from the supernatant of the culture media. (5) vlps would then be tested for proper assembly and integrity using electron microscopy and different methods of protein quantification. (6) all possible mutant vlps would be tested using different functional assays to check for possible antigenicity. (7) the candidate vlps proven to be functional and highly immunogenic could then be used in challenge experiments using animal models and recombinant live virulent viruses believed to be highly pathogenic. (8) the vlps that are highly protective against the highly pathogenic recombinant strains would be further selected and stable cell lines made from all candidate vlp vaccines. (9) these cell lines could be expanded using bioreactors and stored for further use. lentiviral vectors could generate a stable cell line that is transgenic for the highly immunogenic antigenic determinants of covid-19 (15) , and would be able to continuously secrete vlps into the culture media (16) . thereafter, during the time of pandemic, suitable stored transgenic cell lines could be used, based on the abbreviations: covid-19, coronavirus disease 2019; sars-cov-2, severe acute respiratory syndrome coronavirus 2; vlp, virus-like particle; who, world health organization. reemergent pandemic viral mutant strain, and could be easily shipped across the globe, thawed, and manufactured on a large scale in customized large-sized bioreactors (figure 1) . vlp vaccines could be used as therapeutic vaccines and administered to infected individuals (17) , or as vaccines into healthy noninfected individuals. the immunodominant epitopes (18) of the viral mutants specific for the virus would elicit potent immune responses that could be life-saving (19) . the genomeless hollow shells would mimic the actual live virus in terms of eliciting a strong immune response; however, these shells are neither replicative nor infectious by themselves (20) . such a project should be done through international collaborations and under the supervision of the who. stocks of these vlp vaccines could be stored as vials of transgenic cell lines, able to be regularly expanded and checked for their quality and ability to generate vlp vaccines. stocks of these vials could be kept in different countries with satellite distributors managed and administered by the who. this project would require scientists with high degrees of skill that are trained in the field of vaccine design and development, and trained in several other fields such as molecular biology, virology, infectious diseases, and cell biology. the development of vlp vaccines against reemerging viral pandemics would be far affordable than the economic costs of the current covid-19 pandemic. such project requires concerted global efforts of multiple organizations, which is expected to save thousands of lives. i do believe that the time has come for all government officials and policymakers to listen very carefully to science and scientists' recommendations to ensure the health and well-being of people of our planet. hz conceptualized the study and wrote the manuscript. a novel coronavirus from patients with pneumonia in china a new coronavirus associated with human respiratory disease in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding origin and evolution of pathogenic coronaviruses structure, function, and antigenicity of the sars-cov-2 spike glycoprotein bat coronaviruses in china dna shuffling and family shuffling for in vitro gene evolution discovery of human-like lasparaginases with potential clinical use by directed evolution. sci rep membrane embedded hiv-1 envelope on the surface of a virus-like particle elicits broader immune responses than soluble envelopes a lentiviral vector expressing japanese encephalitis virus-like particles elicits broad neutralizing antibody response in pigs the sleeping beauty transposable element: evolution, regulation and genetic applications rna virus reverse genetics and vaccine design hemagglutinin amino acids related to receptor specificity could affect the protection efficacy of h5n1 and h7n9 avian influenza virus vaccines in mice sars-associated coronavirus replication in cell lines rapid generation of stable cell lines expressing high levels of erythropoietin, factor viii, and an antihuman cd20 antibody using lentiviral vectors production of virus-like particles for vaccination therapeutic vaccines against infectious diseases preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies development of rabies virus-like particles for vaccine applications: production, characterization, and protection studies lentivirus-based virus-like particles as a new protein delivery tool the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 zayed. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-319729-6lzjhn8j authors: tian, bin; zhou, ming; yang, yu; yu, lan; luo, zhaochen; tian, dayong; wang, ke; cui, min; chen, huanchun; fu, zhen f.; zhao, ling title: lab-attenuated rabies virus causes abortive infection and induces cytokine expression in astrocytes by activating mitochondrial antiviral-signaling protein signaling pathway date: 2018-01-19 journal: front immunol doi: 10.3389/fimmu.2017.02011 sha: doc_id: 319729 cord_uid: 6lzjhn8j rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acid-inducible gene i (rig-i) or melanoma differentiation-associated protein 5 (mda5). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda5, results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavsp38/nf-κb signaling pathway. these inflammatory cytokines increase the blood–brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda5 in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. rabies is an ancient disease but remains endemic in most parts of the world and causes approximately 59,000 deaths annually. the mechanism through which the causative agent, rabies virus (rabv), evades the host immune response and infects the host central nervous system (cns) has not been completely elucidated thus far. our previous studies have shown that lab-attenuated, but not wild-type (wt), rabv activates the innate immune response in the mouse and dog models. in this present study, we demonstrate that lab-attenuated rabv causes abortive infection in astrocytes, the most abundant glial cells in the cns. furthermore, we found that lab-attenuated rabv produces more double-stranded rna (dsrna) than wt rabv, which is recognized by retinoic acidinducible gene i (rig-i) or melanoma differentiation-associated protein 5 (mda5). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor molecule for rig-i and mda5, results in the production of type i interferon (ifn) and the expression of hundreds of ifn-stimulated genes, which suppress rabv replication and spread in astrocytes. notably, lab-attenuated rabv replicates in a manner identical to that of wt rabv in mavs−/− astrocytes. it was also found that lab-attenuated, but not wt, rabv induces the expression of inflammatory cytokines via the mavs-p38/nf-κb signaling pathway. these inflammatory cytokines increase the blood-brain barrier permeability and thus enable immune cells and antibodies infiltrate the cns parenchyma, resulting in rabv control and elimination. in contrast, wt rabv restricts dsrna production and thus evades innate recognition by rig-i/mda5 in astrocytes, which could be one of the mechanisms by which wt rabv evades the host immune response in resident cns cells. our findings suggest that astrocytes play a critical role in limiting the replication of lab-attenuated rabv in the cns. introduction rabies is an acute encephalomyelitis. the hallmark of rabies is that the disease is almost always fatal once clinical signs develop (1, 2) . the causative agent, rabies virus (rabv), is a negative-strand rna virus belonging to the genus lyssavirus in the family rhabdoviridae (3) . rabv enters neurons from the neuromuscular junction closest to the site of infection. after a short incubation, rabv travels to the central nervous system (cns) through sensory or motor neurons. only slight tissue damage and neuroinflammation can be observed in the brains of rabid patients (4) . in contrast, lab-attenuated rabv induces extensive inflammation and apoptosis, as well as increases in the expression levels of innate immunity-related genes in the cns of infected mice (5) (6) (7) (8) (9) (10) . these findings suggest that wt, but not lab-attenuated, rabv evades the host immune responses. the innate immune system is the first line of defense against viral invasion. viruses are usually confronted by various pattern recognition receptors (prrs), including toll-like receptors (tlrs) and retinoic acid-inducible gene i (rig-i) like helicases (rlrs) (11) . tlr family members, such as tlr3 and tlr7, are generally involved in recognizing negative-strand rna viruses (12) . tlr3 binds double-stranded rna (dsrna), whereas tlr7 recognizes single-strand rna. the main sources of dsrna in infections with single-strand rna viruses are the replicative intermediates generated by viral rna-dependent rna polymerase (13) . tlr3 and tlr7 initiate signaling though the adaptor molecules trif and myd88, respectively. both signaling cascades triggered by these proteins lead to irf3 phosphorylation in the c terminal region at serine 386, which is critical for irf3 activation by two iκb kinases (tbk-1 and ikkε) (14, 15) . activated irf3 homo-or hetero-dimerizes with irf7 and then translocates into the nucleus, to interacts with the creb binding protein cbp/p300 and stimulates the transcription of interferon (ifn)-β, as well as some ifn-stimulated genes (isgs) (15, 16) . alternatively, rna viruses can be recognized by two rlrs, rig-i, and melanoma differentiation-associated protein 5 (mda5), located in the cytoplasm (17, 18) . rig-i solely senses short and blunt dsrna of negative-strand rna viruses containing 5′ triphosphate rna in the panhandle region of their single-stranded genome (17) . unlike rig-i, mda5 preferentially binds blunt-ended dsrna, with or without 5′ triphosphate (18) . rig-i and mda5 signaling is mediated through mitochondrial antiviral-signaling protein (mavs), which is also known as ips1, visa or cardif (19) . similar to tlr signaling, rlr signaling results in irf3 activation and nuclear translocation (20) . several groups have attempted to identify the prrs that recognize rabv. prehaud et al. found that tlr3 mrna expression is upregulated following rabv infection in postmitotic human neurons (21) . furthermore, enhanced tlr3 expression has also been observed in the cerebellar cortical tissues of rabies patients (22) . the observation that tlr3 is upregulated following infection suggests that tlr3 plays a role in the innate recognition of rabv. faul et al. found that both rig-i and mda5 were responsible for inducing dendritic cell (dc) activation and type i ifn production upon rabv infection (23) . mavs, the adaptor protein for both rig-i and mda5, is essential for inducing innate immune responses in dcs. in another study, li et al. found that mice lacking tlr7 exhibited a phenotype associated with intermediate mortality rates between those of myd88−/− and control mice, indicating that tlr7 may play an important role in controlling rabv infections. however, the role of tlr7 in rabv infection is not entirely understood (24) . astrocytes are the most abundant glial cells in the cns and constitute the blood-brain barrier (bbb) along with endothelial cells and pericytes (25) . astrocytes are generally involved in regulating the cns microenvironment and also play roles in neuronal metabolic support, synaptic transmission, and neurotropism. moreover, astrocytes participate in developing and maintaining the bbb and guiding neuronal migration during development. the roles of astrocytes in innate immunity and inflammation have been reported recently (26) . astrocytes are the reservoir for many neuroinvasive viruses, such as human immunodeficiency virus, theiler's murine encephalomyelitis virus (tmev), john cunningham virus, and herpes simplex virus (hsv) (27) (28) (29) (30) . astrocytes have also been shown to play multiple roles in viral infections. specifically, they can increase bbb permeability (31) by producing cytokines and degrading tight junction proteins (32) . moreover, astrocytes have been shown to induce the innate immune response to produce ifn (33) . an early report showed that both wt and lab-attenuated rabv could successfully infect primary astrocytes in the early stages of rabv infection (34) . a recent study demonstrated that a rabv vaccine strain sad-l16 caused an abortive infection in astrocytes, but the detailed mechanism was not revealed (35) . in this study, it was found that lab-attenuated rabv produces higher level of dsrna than wt rabv, which is recognized by rig-i/mda5 and results in the activation of mavs signaling pathway. following mavs activation, the production of isg and inflammatory cytokines helps to clear the lab-attenuated rabv from the cns. in contrast, wt rabv can maintain a persistent infection in astrocytes by evading the innate recognition. mouse bend.3 cells and vero cells were obtained from the american type culture collection (atcc; manassas, va, usa) and were maintained in dulbecco's modified eagle medium (dmem) supplemented with fetal bovine serum (fbs; gibco, carlsbad, ca, usa). mouse neuroblastoma (na) cells were maintained in rpmi-1640 medium (thermo-fisher, usa) supplemented with 10% fbs. the drv-ah08 (drv) was isolated from a rabid dog in anhui province, china (36, 37) . cvs-b2c (b2c), originated from cvs-24 virus by passage in bhk-21 cells (6), has been used as a lab-attenuated rabv (38) (39) (40) . both drv and cvs-b2c were propagated in suckling icr mouse brains. all the viruses were manipulated under the standard biosecurity procedures made by the ministry of agriculture of china. a rabbit anti-ubiquitin protein c (ubc) polyconal antibody was purchased from abclonal technology (woburn, ma, usa). a rabbit anti-rig-i monoclonal antibody was purchased from enzo life technology (farmingdale, ny, usa), and rabbit polyclonal antibodies against irf7, stat1 and occludin were obtained from santa cruz biotechnology (santa cruz, ca, usa). a rabbit antiphospho-irf7 monoclonal antibody was purchased from cell signaling technology (washington, dc, usa), and rabbit anti-ifit1 and irf3 monoclonal antibodies were purchased from abcam (cambridge, ma, usa). a rabbit polyclonal anti-claudin-5 antibody, a biotinylated goat anti-rabbit or mouse 594 antibody, and an alexa fluor 488-conjugated goat antimouse or rabbit antibody were purchased from invitrogen (grand island, ny, usa). mouse monoclonal anti-zoluna occludens-1 (anti-zo-1) antibodies were obtained from sigma (st. louis, mo, usa), and mouse monoclonal anti-gapdh antibody was purchased from proteintech (wuhan, china). the mouse anti-rabv n and p monoclonal antibodies were prepared in our laboratory, and the fluorescein isothiocyanate (fitc)-conjugated anti-rabv nucleoprotein antibody used herein was obtained from female wt or mavs-knockout (mavs−/−) c57bl/6 mice (6-8 weeks old) were infected intracerebally (i.c.) with 20 µl of drv-ah08 (200 ffu), b2c (20 ffu), or mock infected with the same volume of dmem. at 7 days postinfection (d.p.i.), the mice were euthanized with co2 when moribund, and their brains were collected for immunohistochemistry analysis. blood-brain barrier permeability was determined by measuring sodium fluorescein (naf) uptake as described previously with minor modifications (41) . briefly, 100 µl of naf (100 mg/ml) was injected intraperitoneally (i.p.) into each mouse. after anesthetization, peripheral blood and brains of each mouse were collected. the fluorescence in serum and brain homogenate samples was analyzed by a spectrophotometer (biotek instruments, vt, usa) with excitation at 485 nm and emission at 530 nm. standards (125-4,000 g/ml) were prepared to calculate the naf content. naf uptake into tissue is calculated as (μg of fluorescence spinal cord/mg of tissue)/(μg of fluorescence sera/ml of blood) to normalize values for blood levels of the dye at the time of tissue collection. to detect cd45 + cell in the brain, infected mice were anesthetized with ketamine-xylazine (0.1 ml/10 g body weight), perfused with 50 ml pbs, and then the brains were transferred into 4% neutral buffered paraformaldehyde (pfa) for at least 24 h (39) . briefly, the brain sections were antigenic recovered and blocked with donkey serum, incubated with primary antibodies overnight at 4°c, and then secondary antibodies was applied. pbs was treated as a negative control by replacing primary antibodies. sections were photographed and analyzed using an olympus bx41 microscope (tokyo, japan). primary mixed glial cell cultures were established as described previously (42) . briefly, the brain cells of 1-to 3-day-old neonatal c57bl/6 mice were dissociated by repeated pipetting and then passed through a 75-nm nylon mesh (corning, ny, usa). the cells were subsequently washed once in cold pbs and cultured in dmem (with high glucose) supplemented with 10% fbs and 1% penicillin-streptomycin. the medium was changed on days 3, 5, and 7 for the astrocytes and on day 3 only for the microglia. on day 10, the flasks were shaken at 260 rpm for 2 h to remove any non-adherent cells (mainly microglia). the remaining adherent astrocytes were detached with trypsin-edta and then plated again for further experiments. the purity of the astrocyte cultures was greater than 95%. mouse neurons were obtained from embryonic mouse brains as previously described (42) and then dissociated by repeated pipetting (approximately 20 times) before being passed through a 75-nm nylon mesh. the cells were then washed once in cold pbs and cultured in dmem (with high glucose) supplemented with 5% fbs and 1% penicillin-streptomycin for 6 h, after which the medium was replaced with serum-free neural-basal medium (invitrogen, carlsbad, ca, usa) supplemented with 2% b-27 (invitrogen, carlsbad, ca, usa). viral titers were determined by direct fluorescent antibody assay (40) . na cells cultured in 96-well plates were inoculated with viruses diluted serial 10-fold and then incubated at 37°c for 48 h. then the culture supernatant was subsequently removed, and the cells were fixed and stained with fitc-conjugated anti-rabv n antibodies. the antigen-positive foci were counted under a fluorescence microscope (zeiss, germany), and the viral titers were calculated as ffu per milliliter. all titrations were conducted in quadruplicate. confocal microscopy table 1 | primers used for quantification of viral mrna, ifn-stimulated genes, chemokines, and cytokines. retinoic acid-inducible gene i (rig-i) f gcgtctcagtgcagcacatcatt qrt-pcr antibodies against dsrna, rabv n, rabv p, claudin-5, occludin, zo-1, or dapi. infected wt or mavs−/− mice were anesthetized with ketamine-xylazine (0.1 ml/10 g body weight) and then perfused with pbs followed by 10% neutral buffered formalin, as described previously (39) . three independent mouse brain samples were collected from each group and embedded in paraffin for coronal sectioning. the sections were subsequently stained with antibodies against gfap, map2, rabv p, rabv n, or dapi. after being washed, the cells or sections were incubated with an alexa fluor 488-conjugated goat antirabbit or mouse secondary antibodies or an alexa fluor 594-conjugated goat antirabbit or mouse secondary antibodies for 1 h at room temperature. staining was visualized with a nikon a1 confocal laser microscope system equipped with nis-elements imaging software (nikon, melville, ny, usa) and was quantified using fiji, an imagej distribution package manufactured by nih (http://imagej.net/introduction). mean fluorescence intensity (mfi) was quantified using the region of interest, which encompassed the entire cell to include the membrane, and background staining was quantified using three negatively stained regions per cell. these regions were subtracted from the total mfi. for the protein synthesis inhibition tests, primary astrocytes were pretreated with cycloheximide (chx) (invivogen, san diego, ca, usa) for 1 h at a dose of 50 µg/ml, infected with drv or b2c at an moi of 0.1 and then continued to be incubated with chx for another 24 h. for the tbk1 activation blockage assays, bx795 (invivogen, san diego, ca, usa) at a dose of 1 µm was used to treat primary astrocytes. for the inflammatory pathway blockage assays, the primary astrocytes were pretreated with a p38 inhibitor (skepinone-l; sellek, houston, tx, usa), a jnk inhibitor (jnk inhibitor ix; sellek, houston, tx, usa), an nf-κb inhibitor (sc75741; sellek, houston, tx, usa), or dmso as control at a dose of 5 µg/ml for 1 h. then the cells were infected with drv or b2c at moi 1 and incubated with the above inhibitors for 48 h. the concentrations of tnf-α and il-6 in the supernatant of astrocytes were measured by the commercial elisa kits according to the manufacturer's instruction (abclonal technology, woburn, ma, usa). rna was isolated with trizol ® reagent (invitrogen), according to the manufacturer's instructions, and qrt-pcr was performed as described previously (39) . briefly, 800 ng of total rna (from either cells or tissue) was transcribed into cdna in a reaction mixture with a total of volume of 20 µl using a superscript iii reverse transcription kit (toyobo). the reaction mixture comprised 1 μl of cdna combined with 5 µl of iq5 sybr green mix (biorad, hercules, ca, usa), 3 µl of diethyl pyrocarbonatetreated water, and 0.5 µl of primer mix (the concentration of each primer was 10 mm). the cdna was amplified using an iq5 icycler (bio-rad), and the cycle threshold (ct) values were recorded. the ct value was inversely correlated with the mrna concentration, and each ct unit represented a twofold change in the mrna concentration. basal mrna expression levels were expressed as δ ct values and were normalized to β-actin mrna expression levels [ δ ct ct (isg)/ δ ct (β-actin)]. induced mrna expression levels were expressed as fold changes relative to mock-infection levels using the 2 δδct method. all the primer sequences are listed in (table 1) . to quantify cellular rabv n rna levels, we transcribed the total rna using avian myeloblastosis virus reverse transcriptase xl (takara, kusatsu, japan) and a primer specific for the rabv n genomic sequence. a standard curve was generated from serially diluted plasmids carrying a rabv n gene and the copy numbers of n mrna were normalized to 1 mg of total rna. the cells were lysed with ripa buffer containing protease inhibitors (roche), and the protein concentrations were measured using a dc protein assay kit (bio-rad). equal quantities of protein were resolved by 12 or 15% sds-page and then transferred to polyvinylidene difluoride membranes (bio-rad), which were blocked with 5% nonfat milk before being incubated with primary antibodies against rig-i, phosphorylated irf7 (p-irf7), stat1, ifit1, rabv n, claudin-5, occludin, zo-1, or gapdh and then probed with the appropriate secondary antibodies. the blots were then visualized using ecl reagent (ge, pittsburgh, pa, usa) and detected under an intelligent dark box ii (ge, pittsburgh, pa, usa). the 590-gain pmt scan generated the optimal standard curve, and the results of this scan were analyzed using q-analyzer software for qam-cyt-1 (raybiotech). transendothelial permeability assay was performed according to the methods described previously (43), with some modifications. b.end3 cells were grown on 3-μm-pore transwell filter inserts until they reached 100% confluency. the medium was then treated with uv-inactivated cell culture supernatants collected from rabv-infected astrocytes. after the cells had incubated for 48 h, they were treated apically with dextran-fitc at a dose of 0.1 µg/ml for 30 min. the samples were then removed from the lower chamber and subjected to fluorescence measurement, which were performed using a fluorimeter (biotek, winooski, vt, usa; the excitation wavelength was 492 nm, and the emission wavelength was 520 nm). the fluorescence values for the experimental cells were subsequently compared to corresponding values for a control cell monolayer. data are expressed as the mean and standard error of the mean (sem), and the significance of the differences between groups was evaluated by student's t test or one-way analysis of variance followed by tukey's post hoc test. the survival ratios were analyzed by log-rank (mantel-cox) test. the asterisks indicated statistical significance (*, p < 0.05; **, p < 0.01; ***, p < 0.001). graphs were plotted and analyzed using graphpad prism software, version 6.0 (graphpad software, la jolla, ca, usa). results comparison of the pathogenicity of drv-ah08 and cvs-b2c in mice first, the pathogenicity of the wt rabv strain drv-ah08 (drv) and lab-attenuated strain cvs-b2c (b2c) used in this study were compared in a mouse model. c57/bl6 mice were i.c. inoculated with 20 ffu b2c or drv and the development of rabies was observed. as expected, drv-infected mice displayed development of the diseases at 5 d.p.i. and all moribund at 9 d.p.i., earlier than b2c-infected mice which all succumb to rabies at 10 d.p.i. (figure 1a) . we found that b2c replicated faster than drv at the early stage of the infection in the cns. to ensure that the viral load in the brains is similar, c57/bl6 mice were i.c. inoculated with 20 ffu b2c or 200 ffu drv. then the viral load in the mice brain were determined at 3, 5, 6, 7, 8, and 9 d.p.i. the results showed that the genomic rna of drv was lower than that of b2c at 3 and 5 d.p.i., but reached the same level as that of b2c at 6 and 7 d.p.i. (figure 1b) . previous studies have shown that the lab-attenuated rabv infection enhances the bbb permeability and induces inflammation in the brain (8, 38) . thus, the na-fluorescence (na-f) uptake of both strains at 7 d.p.i. was measured, and the data showed that na-f uptake in b2c-infected brain was significantly higher than that of drv or mock-infected brains ( figure 1c) . consistently, there were more cd45 + lymphocytes in b2c-infected brains than drv-infected mouse brains (figures 1d,e) . all these results are consistent with the previous findings related to the cns inflammation and bbb permeability change during comparison of the pathogenicity between wt and lab-attenuated rabv (6, 8, 39) . astrocytes play an important role in the induction of innate immunity in the cns. to investigate the role of astrocytes in the pathogenesis of rabv, we isolated primary astrocytes and neurons from suckling mice and infected them with drv or b2c. the growth kinetics of both viruses in astrocytes was assessed by virus titration and immunofluorescence assay (ifa). the viral loads in the cell culture supernatants infected with b2c quickly reached peak at 1 d.p.i., and then gradually decreased until 15 d.p.i. in contrast, the virus titer of drv was initially relatively low but subsequently increased steadily until the endpoint (figure 2a) . to be noted, the viral titer of drv in the cell supernatant maintained at a relatively low level than that of b2c in astrocyte at 3 and 5 d.p.i., but n mrna transcription levels of drv was significantly higher than that of b2c at 3 and 5 d.p.i. (figure 2c) . the ifa results showed that drv persistently replicated in astrocytes and large immunofluorescence foci could be observed at 7 d.p.i., while no obvious immunofluorescence foci could be found in b2c-infected cells ( figure 2d) . as a control, virus replication kinetics in primary neurons was also compared. it was found that viral titers of b2c were always higher than those of wt rabv at the indicated time points (figure 2b) . the ifa results also showed that both drv and b2c could efficiently replicate in neuron ( figure 2d) . to confirm these observations in vivo, c57bl/6 mice were i.c. infected with 20 ffu b2c or 200 ffu drv. infected mice were euthanized when moribund and the brains were harvested for fluorescence ihc analysis. gfap was a well-known surface marker for astrocytes (44) , and the gfap staining demonstrated drv could effectively infect astrocytes in the brains ( figure 2e) . however, few infected astrocytes could be observed in b2cinfected mouse brains (figure 2e ). in contrast, neuron was intensively infected by both drv and b2c ( figure 2f) . together, these results show that the b2c causes abortive infection in astrocytes both in vitro and vivo. previous studies have demonstrated that rabv can be recognized by rig-i and mda5, which share a common adaptor mavs in dcs (23) . to assess innate immune responses in astrocytes, cells were infected with drv or b2c at an moi of 0.1 and the expression of several proteins involved in the mavs signaling pathway, namely, rig-i, p-irf7, stat1 and ifit1 (isg56), was measured by western blot. the ubiquitin ligase trim25 mediates lysine 63-linked ubiquitination of rig-i's n-terminal card domains is indispensable to induce type i ifn production and antiviral immunity (45) . thus, the immunoprecipitation of rig-i was carried out and then resolved by western blot by using an anti-ubiquitin antibody. as expected, rig-i was much more robustly ubiquitinated in astrocytes infected with b2c than that in astrocytes infected with drv. consistently, the expression levels of rig-i, p-irf7, stat1, and ifit1 in b2c-infected astrocytes were higher than those in drv-infected cells ( figure 3a) . next, we attempted to determine which viral product (rna or protein) activates mavs signaling pathway in astrocytes. primary astrocytes were treated with chx, a protein synthesis inhibitor, to inhibit viral protein synthesis. it was found that b2c and drv n transcription levels were similar between chxtreated and mock-treated astrocytes at 24 h p.i. (figure 3b) . however, the expression levels of the genes involved in the mavs signaling pathway, namely, rig-i, mda5, mavs, irf7, ifn-β, stat1, and ifit1, were significantly upregulated by b2c compared with drv in both chx-treated and mock-treated astrocytes, indicating that viral rna rather than proteins activates ifn pathway depending on mavs (figures 3c-i) . taken together, these data demonstrate that the lab-attenuated rabv, but not wt rabv, activates the mavs signaling pathway by viral rna, resulting in the production of ifn as well as isgs in astrocytes. retinoic acid-inducible gene i and mda5 activation is induced mostly by dsrna, which is produced during viral replication (13) . to compare the amount of dsrna produced by wt and labattenuated rabv, primary astrocytes were infected with drv or b2c at an moi of 0.1. at 1 and 3 d.p.i., dsrna was stained with specific antibody and observed by a confocal fluorescence microscope. the results demonstrate that more dsrna is synthesized in b2c-infected astrocytes than those in drv-infected cells ( figure 4a) . the dsrna intensity per cell in b2c-infected cell was significantly higher than that in drv-infected cells (figure 4b) , considering the similar rabv intensity per cell (figure 4c) . the ratio of dsrna intensity to rabv intensity in b2c-infected astrocyte was significantly higher than that in drv-infected cells ( figure 4d) . these results suggest that lab-attenuated rabv produces more viral dsrna than wt rabv during viral replication, (20 ffu) . (e) at 7 d.p.i., mice were euthanized, perfused with pbs and then fixed with 4% pfa. the brains were subsequently coated with paraffin, and the brain sections were stained with antibodies against gfap (red), rabv p protein (green), or dapi (blue). the white arrows indicate rabv-infected astrocytes. the scale bars represent 50 µm. (f) the same brain sections as (e) were stained with antibodies against map2 (green), rabv p protein (red), or dapi (blue), then visualized under a confocal microscope. the scale bars represent 50 µm. notably, b2c titers were significantly increased in mavs−/− astrocytes compared with wt astrocytes (figures 5a,b) . moreover, the cell numbers of immunofluorescence plaques in mavs−/− astrocytes caused by b2c infection were significantly more than those in wt astrocytes (figures 5e,f) . in contrast, mavs deficiency had no significant influence on drv replication and spread in astrocytes (figures 5a,e) . tbk1 is an iκb kinase downstream of the mavs signaling pathway and is critical for irf3 phosphorylation. treatment with the tbk1 specific inhibitor bx795 significantly increased b2c titers in astrocytes (figures 5c,d) . to verify these observations in vivo, mavs−/− mice were i.c. infected with drv or b2c, and virus infection of astrocytes was determined by ifa. we found that both drv and b2c could efficiently infected mavs−/− astrocytes (figures 5g,h) . taken together, these findings suggest that mavs signaling significantly restricts the replication and spread of lab-attenuated but not wt rabv in astrocytes. recent studies have shown that tlr7 may be another innate immune molecule that recognizes rabv (24) . thus, the role of tlr7 in rabv replication was investigated in astrocytes. astrocytes from tlr7−/− and control mice were isolated and then infected with drv or b2c at an moi of 0.01. at different time points p.i., viral titers (figures 5a,b ) and the formation of viral immunofluorescence foci was analyzed (figures 5e,f) . the results demonstrated that tlr7 deficiency did not significantly affect the replication and spread of either drv or b2c in astrocytes, suggesting that tlr7 does not play a role in containing rabv replication and spread in astrocytes. astrocytes are one of the major sources of inflammatory cytokines in the cns post viral infection, and these cytokines play an important role in regulating bbb permeability. to investigate rabv-induced cytokine production, primary astrocytes from wt and mavs−/− mice were prepared and infected with drv or b2c at an moi of 0.01. at indicated time points, the cell culture supernatants were harvested and analyzed with a cytokine array kit. the results showed that b2c induced significantly higher levels of cytokine expression, namely, tnf-α, il-6, il-1β, ifn-γ, il-17, and vegf expression, than drv in wt and mavs−/− astrocytes. the levels of cytokine expression in mavs−/− astrocytes were significantly lower than those in wt astrocytes, indicating that rabv-induced inflammatory cytokine production in astrocytes is dependent on the mavs signaling pathway (figures 6a-f) . the specific pathway through which rabv induces cytokine production was subsequently identified in astrocytes. a previous study demonstrated that rabv induces cytokine production in macrophages mainly through p38, jnk, and nf-κb pathways (46, 47) . thus, primary astrocytes were treated with p38, jnk, and nf-κb pathway inhibitors skepinone-l, jnk ix, and sc75741, respectively, and the concentrations of tnf-α and il-6 in the cell supernatant were measured by elisa. the results showed that skepinone-l and sc75741 caused greater reductions in tnf-α ( figure 6g ) and il-6 ( figure 6h ) protein levels than jnk ix in b2c-infected astrocytes. none of these inhibitors significantly altered the expression levels of tnf-α and il-6 in drv-infected astrocytes. these findings suggest that lab-attenuated rabv induces cytokine expression in astrocytes mainly through the p38 and nf-κb pathways and that wt rabv suppresses cytokine production in astrocytes. our previous studies demonstrated that the chemokines/ cytokines induced by rabv infection are responsible for reducing tj protein expression and enhancing bbb permeability (39) . to investigate the effect of these cytokines on bbb permeability, a mouse brain microvascular endothelial cell line b.end3, was cocultured with uv-inactivated supernatants infected with drv or b2c and collected them at different time points after infection. treatment with the supernatants from b2c-infected astrocytes for 48 h (figure 7a ) induced significant increase in dextran-fitc infiltration from 48 to 96 h p.i. notably, treatment with the supernatants from b2c-infected mavs−/− astrocytes elicited significant increases in dextran-fitc permeability at 48 h p.i. (figure 7b) . no significant increases in dextran-fitc permeability were observed after treatment with the supernatants from wt or mavs−/− astrocytes infected with drv. to gain an insight into the mechanisms by which labattenuated rabv enhances bbb permeability, the expression levels of the indicated tj proteins (claudin-5, occludin, and zo-1) were assessed in astrocytes by western blot. it was found that the expression levels of claudin-5 and occludin were unchanged, while zo-1 was significantly reduced after the cells were treated with the supernatants from b2c-infected wt astrocyte ( figure 7c) . however, this reduction was attenuated in cells treated with the supernatants from mavs−/− astrocytes infected with b2c. no significant changes in zo-1 expression were observed in b.end3 cells treated with supernatants from wt or mavs−/− astrocyte infected with drv ( figure 7d) . similarly, ifa results showed that zo-1 expression was significantly decreased in b.end3 cells cocultured with the supernatants from b2c-infected astrocytes collected at 48 h p.i. (figure 7e) . zo-1 fluorescence intensity was only slightly decreased in b.end3 cells cocultured with the supernatants from b2c-infected mavs−/− astrocytes collected at 48 h p.i., indicating that cell-to-cell contact was not significantly decreased in these cells. moreover, no zo-1 degradation was observed in b.end3 cells cocultured with the supernatants from wt or mavs−/− astrocytes infected with drv. to be noted, no significant changes in claudin-5 and occludin expression were detected in either wt or mavs−/− astrocytes upon rabv infection (figures 7c,e) . together, these results illustrate that lab-attenuated, but not wt, rabv upregulates the production of inflammatory cytokines in astrocytes, resulting in zo-1 degradation in bmecs and subsequent increases in bbb permeability. furthermore, these results indicate that cytokine production is dependent on mavs signaling pathway. astrocytes play critical roles in host defense during viral infections of the cns (7) . prr activation in astrocytes results in the expression of many immune mediators, including type i ifns and inflammatory cytokines (6, 7) . during infections caused by pathogens for which glia are not susceptible targets, activation of the innate immune system caused by pathogen recognition in astrocytes may promote antiviral immune responses in susceptible neurons, as well as cns leukocyte trafficking (3, 5, 7) . in an early report, primary murine, feline, and human astrocytes were infected with wt (srv) and lab-attenuated rabv (era) (34), after which viral loads and replication were assessed by infectivity assay and immunofluorescence. the results showed that astrocytes can be infected by rabv, suggesting that astrocytes may play a role in viral spread and persistence and/or neuronal dysfunction (34) . however, in that study, viral loads and replication were evaluated only at the early time point after infection (3 d.p.i.) . in this present study, the growth of drv with that of b2c, which were used as a pair of wt and lab-attenuated rabv in previous studies (38) (39) (40) , was compared in a long-term experiment. surprisingly, we found that lab-attenuated rabv, but not wt rabv, caused abortive replication in astrocytes, a feature that may be associated with the ability of the virus to evade the innate immune response. productive infection of the astrocyte is critical for neurotropic pathogens to induce encephalitis. astrocytes sensed viral entry into the cns and mounted a type i ifn response, which rapidly restricts the virus after neuronal transport into the cns. previous studies demonstrated that tlr3−/− astrocytes were more permissive to hsv infection and caused severe symptom of encephalitis and tissue damage, which was due to impaired type i ifn production in the absence of tlr3 (30) . a recently work found that abortively infected astrocytes are the major producers of ifn-β after infection of the brain with diverse neurotropic viruses, including tmev, rabv, and vesicular stomatitis virus (vsv) (35) . consistent with these studies, we also found that the abortive infection of lab-attenuated rabv in astrocytes was related to its ability to activate ifn signaling pathway. basal isg expression levels are an important determinant of susceptibility to viral infection. we have found that astrocytes have higher basal expression levels of the mrnas encoding isg proteins, such mda5 and stat1, and other molecules crucial for recognizing viral invasion and creating an more antiviral environment than neurons (9) , which may explain why lab-attenuated rabv activates the innate immune response to a degree sufficient to restrict viral replication in astrocytes but not in neurons. double-stranded rna is a viral product that plays an essential role in inducing innate immunity, which leads to the production of type i ifns and the activation of hundreds of isgs. early biochemical studies of viral replication suggested that most viruses produce dsrnas (13) . however, in 2006, weber et al. reported that dsrna could be detected by ifa in cells infected with positive-stranded rna viruses, but not with negative-stranded rna viruses (48) . this notion was challenged by two other studies on dsrna production in cells infected with negative-stranded rna viruses (13, 48) . moreover, a recent study demonstrated the dsrna formation in cells infected with several negative-stranded rna viruses, such as vsv, measles virus (mev), and influenza a virus, although the intensity of the staining of dsrna tended to be weaker in cells infected with negative-stranded rna viruses when compared with those infected with positive-stranded rna viruses (13) . consistent with this finding, the production of dsrna was detected in both lab-attenuated and wt rabv-infected astrocytes, although lab-attenuated rabv produced significantly more dsrna in the cytoplasm than wt rabv. we attempted to investigate why lab-attenuated rabv produces more dsrna than wt rabv. pfaller et al. found that dsrna expression was much lower in cells infected with wt mev than in cells infected with a mutant mev whose c protein was knocked out which is known to control genome replication and transcription (49) . similarly, takeuchi et al. observed dsrna formation in cells infected with sendai virus (50) with c protein knocked out but not in cells infected with wt sev, indicating that the c protein limits or masks dsrna production (51) . both mev and sev are within the family paramyxoviridae, thus it is possible that their c protein possess the similar function to subvert the production of dsrna. ebola virus protein vp35 adopts a unique strategy to mask key cellular recognition sites on dsrna (52) . a recently study showed that the coronavirus endonuclease (endou) activity is the key to prevent early induction of dsrna. replication of endou-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of virus infection (53) . in this present study, by exchanging viral genes between wt and lab-attenuated rabv, we found that single n, p, and g of wt rabv could not suppress dsrna formation (data not shown). however, multiple viral proteins of rabv may work together to limit the production of dsrna. further studies are needed to elucidate the mechanism through which wt rabv restricts dsrna formation and thus evades recognition by the innate immune system in infected cells. the bbb, which is composed of specialized bmecs joined by tjs and ensheathed by astrocytes and pericytes, plays an important role in protecting the cns. our previous studies have shown that rabv does not infect bmecs, nor does it modulate tj protein expression in bmecs (39) . however, brain extracts prepared from mice infected with lab-attenuated rabv but not wt rabv reduced tj protein expression in bmecs, indicating that the above enhancements of bbb permeability and reductions in tj protein expression are not caused by rabv infection. rather, they are caused by virus-induced inflammatory chemokines/ cytokines. the innate immune mechanisms that regulate bbb function in the setting of infectious diseases have been appreciated only recently. multiple inflammatory cytokines, including tnf-α, il-6, il-1β, ifn-γ, il-17, and vegf, disrupt bbb and tj integrity in bmecs (46, 50, (54) (55) (56) , and inflammatory cytokine signaling at the bbb during infection facilitates leukocyte trafficking into the cns, which is essential for the clearance of many pathogens (39, 41) . our present study demonstrates that lab-attenuated rabv induces production of several inflammatory cytokines in astrocytes, especially tnf-α, il-6, il-1β, ifn-γ, il-17, and vegf, which cause disruption of the bbb and tj integrity. our findings suggest that astrocytes play a critical role in regulating bbb permeability as a major source of cytokines during viral infection. furthermore, we found that the production of inflammatory cytokines in astrocytes by lab-attenuated rabv was dependent on mavs signaling pathway, underscoring the critical role of mavs signaling in defensing against rabv infection in cns. 8 | the proposed model for the mechanism through which wild-type (wt) and lab-attenuated rabies virus (rabv) infect astrocytes. during infection, lab-attenuated rabv produces double-stranded rna (dsrna), which is recognized by retinoic acid-inducible gene i (rig-i)/melanoma differentiation-associated protein 5 (mda5). activation of mitochondrial antiviral-signaling protein (mavs), the common adaptor protein for rig-i and mda5, leads to enhanced production of interferon, as well as some ifn-stimulated genes, which limit rabv replication and spread. mavs activation stimulates the p38 and nf-κb signaling pathways and induces cytokine production in astrocytes. inflammatory cytokines promote blood-brain barrier (bbb) permeability, enabling peripheral inflammatory cells and antibodies to infiltrate into the central nervous system, thereby facilitating rabv clearance. in contrast, wt rabv prevents activation of the mavs signaling pathway by restricting dsrna production. conclusion based on the results, we propose the following model: labattenuated rabv produces dsrna recognized by rig-i, mda5, or both, resulting in the activation of the mavs signaling pathway in astrocytes. ifn expression induces the transcription of hundreds of isgs to inhibit rabv replication in astrocytes and causes the abortive infection by lab-attenuated rabv. the inflammatory cytokines induced by lab-attenuated rabv enhance bbb permeability, enabling immune cells and antibodies to infiltrate the cns and facilitate rabv clearance. conversely, wt rabv restricts dsrna production and then evades recognition by the innate immune system, resulting in persistent viral replication in astrocytes (figure 8 ). current status of rabies and prospects for elimination the cell biology of rabies virus: using stealth to reach the brain rhabdoviruses: rabies virus role of apoptosis in rabies viral encephalitis: a comparative study in mice, canine, and human brain with a review of literature silver-haired bat rabies virus variant does not induce apoptosis in the brain of experimentally infected mice attenuated rabies virus activates, while pathogenic rabies virus evades, the host innate immune responses in the central nervous system rabies virus-induced apoptosis involves caspase-dependent and caspase-independent pathways failure to open the bloodbrain barrier and deliver immune effectors to central nervous system tissues leads to the lethal outcome of silver-haired bat rabies virus infection the roles of chemokines in rabies virus infection: overexpression may not always be beneficial neuronal apoptosis does not play an important role in human rabies encephalitis toll-like receptors in innate immunity toll-like receptor function and signaling double-strand rna is detected by immunofluorescence analysis in rna and dna virus infections including those by negative-strand rna viruses myd88 functions as a negative regulator of tlr3/trif-induced corneal inflammation by inhibiting activation of c-jun n-terminal kinase regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 involvement of the ubiquitin-like domain of tbk1/ikk-i kinases in regulation of ifn-inducible genes recognition of 5' triphosphate by rig-i helicase requires short blunt double-stranded rna as contained in panhandle of negative-strand virus structural basis of double-stranded rna recognition by the rig-i like receptor mda5 mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response induction of irf-3 and irf-7 phosphorylation following activation of the rig-i pathway virus infection switches tlr-3-positive human neurons to become strong producers of beta interferon toll-like receptor 3 (tlr3) plays a major role in the formation of rabies virus negri bodies rabies virus infection induces type i interferon production in an ips-1 dependent manner while dendritic cell activation relies on ifnar signaling the role of toll-like receptors in the induction of immune responses during rabies virus infection blood-brain barrier biology and methodology immune players in the cns: the astrocyte susceptibility to theiler's virus-induced demyelinating disease correlates with astrocyte class ii induction and antigen presentation longlasting astrocyte reaction to persistent junin virus infection of rat cortical neurons astrocyte infection by hiv-1: mechanisms of restricted virus replication, and role in the pathogenesis of hiv-1-associated dementia tlr3 deficiency renders astrocytes permissive to herpes simplex virus infection and facilitates establishment of cns infection in mice potential mechanisms for astrocyte-timp-1 downregulation in chronic inflammatory diseases viral disruption of the blood-brain barrier astrocyte response to junin virus infection rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes abortively infected astrocytes appear to represent the main source of interferon beta in the virus-infected brain complete genome sequence of a street rabies virus isolated from a rabid dog in china rabies viruses leader rna interacts with host hsc70 and inhibits virus replication role of chemokines in the enhancement of bbb permeability and inflammatory infiltration after rabies virus infection enhancement of blood-brain barrier permeability and reduction of tight junction protein expression are modulated by chemokines/cytokines induced by rabies virus infection the inability of wild-type rabies virus to activate dendritic cells is dependent on the glycoprotein and correlates with its low level of the de novo-synthesized leader rna expression of neuronal cxcl10 induced by rabies virus infection initiates infiltration of inflammatory cells, production of chemokines and cytokines, and enhancement of blood-brain barrier permeability cell-type-specific type i interferon antagonism influences organ tropism of murine coronavirus infection of pericytes in vitro by japanese encephalitis virus disrupts the integrity of the endothelial barrier regional astrocyte ifn signaling restricts pathogenesis during neurotropic viral infection influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i overexpression of tumor necrosis factor alpha by a recombinant rabies virus attenuates replication in neurons and prevents lethal infection in mice rabies virus-induced activation of mitogen-activated protein kinase and nf-kappab signaling pathways regulates expression of cxc and cc chemokine ligands in microglia doublestranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses measles virus c protein impairs production of defective copyback double-stranded viral rna and activation of protein kinase r production of il-8, il-17, ifn-gamma and ip-10 in human astrocytes correlates with alphavirus attenuation sendai virus c protein plays a role in restricting pkr activation by limiting the generation of intracellular double-stranded rna ebolavirus vp35 coats the backbone of double-stranded rna for interferon antagonism early endonuclease-mediated evasion of rna sensing ensures efficient coronavirus replication astrocytes produce and release interleukin-1, interleukin-6, tumor necrosis factor alpha and interferon-gamma following traumatic and metabolic injury cellular mechanisms of il-17-induced blood-brain barrier disruption expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type i interferon the authors wish to thank the staff members of the animal facility at huazhong agricultural university for caring for the mice. this work was partially supported by the national natural science foundation of china (31720103917 and 31330078 to zf and 31372419 and 31522057 to lz); the national program on key research project of china (2016yfd0500400 to lz). key: cord-312955-gs65c3fy authors: schreiber, gideon title: the role of type i interferons in the pathogenesis and treatment of covid-19 date: 2020-09-30 journal: front immunol doi: 10.3389/fimmu.2020.595739 sha: doc_id: 312955 cord_uid: gs65c3fy type i interferons (ifn-i) were first discovered over 60 years ago in a classical experiment by isaacs and lindenman, who showed that ifn-is possess antiviral activity. later, it became one of the first approved protein drugs using heterologous protein expression systems, which allowed its large-scale production. it has been approved, and widely used in a pleiotropy of diseases, including multiple-sclerosis, hepatitis b and c, and some forms of cancer. preliminary clinical data has supported its effectiveness against potential pandemic pathogens such as ebola and sars. still, more efficient and specific drugs have taken its place in treating such diseases. the covid-19 global pandemic has again lifted the status of ifn-is to become one of the more promising drug candidates, with initial clinical trials showing promising results in reducing the severity and duration of the disease. although sars-cov-2 inhibits the production of ifnβ and thus obstructs the innate immune response to this virus, it is sensitive to the antiviral activity of externally administrated ifn-is. in this review i discuss the diverse modes of biological actions of ifn-is and how these are related to biophysical parameters of ifn-i–receptor interaction and cell-type specificity in light of the large variety of binding affinities of the different ifn-i subtypes towards the common interferon receptor. furthermore, i discuss how these may guide the optimized use ifn-is in combatting covid-19. type i interferons (ifn-i) are a family of cytokines that bind the type i interferon receptor, constituted of two transmembrane subunits, ifnar1 and ifnar2 ( figure 1 ). the two receptors are constituted of an extracellular domain, which binds ifn-i, a transmembrane helix and an unstructured intracellular domain (icd) that binds jaks and stats (1, 2) . jak1 is associated with ifnar2 and tyk2 with ifnar1. stat1 and stat2 (and maybe also other stats) were found to be constitutively bound to the icd of ifnar2 (3) (4) (5) . binding results in close proximity of the intracellularly associated jaks, jak1 and tyk2, resulting in their activation through cross phosphorylation ( figure 1 ) (6, 7) . this also results in receptor phosphorylation, which role is still under debate (3, (8) (9) (10) . the phosphorylated stats dissociate from the receptor and form homo and hetero dimers, which are transported to the nucleus, where they serve as transcription factors for a large number of genes. the most prominent effects are associated with stat1/stat2 heterodimerization, which together with irf9 form the interferon-stimulated gene factor 3 (isgf3), which bind a distinct group of target genes harboring the interferonstimulated response elements (isre). in addition to this, ifn-i drives stat1/stat1 and stat3/stat3 homodimerization, the formation of a stat2/irf9 binary complex and more (6, (10) (11) (12) (figure 2 ). this leads to the transcription activation or suppression of over 1,000 genes, which drive a wide range of innate and adaptive immune functions. these, in turn respond against various pathogens, act as important regulators in tumor immunity and have a role in pathophysiology and autoimmune diseases (10, (13) (14) (15) (16) (17) (18) . stat2 knockout cells still activate a stat1/stat1 response mediated by irf1, while stat1 knockout cells activate a stat2/irf9-induced response (10) . surprisingly, no change in the gene induction relative to wildtype cells was observed in stat3 knockout hela cells, despite the strong ifn-i-induced phosphorylation of stat3. however, as ifn-i responses are cell-type specific, a stat3/stat3induced response may still be found in other cells than hela. due to this wide range of physiological responses, ifn-i has provided therapeutic benefits for multiple diseases, including multiple sclerosis, some cancers and viral diseases (hepatitis b and c) (19) (20) (21) . due to the efficient activation of antiviral activities by ifn-is, most viruses have contemplated mechanisms to avoid its actions (22) (23) (24) . for example, the ebola virus, which outbreak in central africa killed tens of thousands of people (25, 26) , avoids ifn-i activity by producing the vp24 protein that binds the karyopherin alpha nuclear transporter. thereby, it inhibits the nuclear transport of phosphorylated stat1, rendering cells refractory to ifn-is. another example of viral mechanisms that evolved to eliminate ifn-i functions in inducing innate immunity is given by the sars corona virus, where both the production of ifnb and the ifn-i induced signaling are attenuated. recently, a more infective version of sars has emerged, sars-cov-2 (which causes the covid-19 disease). covid-19 cases have been first reported by the end of 2019 in china, and rapidly became a world-wide epidemic with unprecedented consequences (27, 28) . sars-cov-2 seems to have originated from horseshoe bats. similar virus strains that circulate in bats in hubei province in china may in the future cause further new zoonotic outbreaks (29) . sars-cov-2 has 83% homology to the sars-cov virus that also spread from china in 2002 (30) . sars-cov-2 proved to be much more infectious compared to the original sars virus, resulting in a global epidemic. as ifn-i drives strong antiviral activities, the mechanisms sars-cov and sars-cov-2 combat ifn-i activities has been a matter of intense research, with at least 6 proteins being identified to counteract ifn-i functions in the sars-cov virus (31) . in addition, ifn-is were implicated in contributing to the severity of the cytokine storm, which is a major complication of sars-cov and sars-cov-2 and can lead to respiratory distress syndrome (ards) and death (31, 32) . in this review i will describe our current knowledge on the involvement of ifn-is in the development of the covid-19 disease, and how this relates to the different activities associated with type i interferons. type i interferon receptors are found on all cell types, and are a major component of the innate immune system. human type i interferons include 13 similar ifnas with 80% homology between them and single ifnw, k, ϵ and b, with lower homology (30-50%). all of them bind the receptor complex, composed of ifnar1 and ifnar2 at the same proximal location (1, 2, 33) . despite structural similarities among the ternary ifn-i-ifnar1-ifnar2 complexes, ifn-is drive a range of different activities, dependent on the cell type and the interferon subtype (34) . this apparent paradox has major implications for understanding the role of ifn-i in health and disease and its varied applications as a drug against a pleiotropy of diseases. ifn-i signaling is initiated by binding of ifn-i to its receptor. it has been suggested that cytokine receptors are pre-associated, with ligand binding activating signaling through the induction of conformational changes (35) . however, more recent singlemolecule receptor tracking on life cells has clearly shown that for many of the cytokines, its role is to bring the receptors into close proximity, which drives signaling (36) . this seems to be the case also for ifn-i induction, as shown both using single receptor tracking and mutational analysis ( figure 1 ) (37, 38) . while structurally, the ternary ligand-receptor complex seems to be the same for all ifn-is, the binding affinity differs by many orders of magnitude. the tightest binding ifn-i is ifnb, which binds ifnar1 with 100 nm affinity and ifnar2 with subnanomolar affinity. the different ifna subtypes bind ifnar1 with 0.5 to 5 µm affinity and ifnar2 with 1 to 100 nm affinity, with ifna1 being the weakest binding ifna (39, 40) . even weaker binding was measured for ifnϵ, with~100-fold reduced affinity relative to ifna proteins (15) . interestingly, ifnϵ is constitutively expressed by the reproductive tract epithelium and is regulated by hormones during the estrus cycle, reproduction, menopause and by exogenous hormones. thus, its mode of action is different from other ifn-is (41) . these large differences in binding affinity between ifn-i subtypes were suggested to result in major differences in biological activity. to obtain a better insight into the molecular mechanisms of their actions, ifna2 was engineered to cover the whole range of binding affinities of natural ifn-is to both the high affinity (ifnar2) and low affinity (ifnar1) receptor chains (1) . these studies have shown that indeed, the binding affinity to both receptors is a major determinant of ifn-i activity (42) . using both natural and engineered ifn-is has shown that even weak binding ifn-is activate the cellular antiviral program at very low (pm) concentrations (39) . moreover, the antiviral program was activated in all cell-lines tested. despite the 50-fold higher affinity of ifnb over ifna2 towards binding ifnar receptors, its potency to elicit an antiviral response is similar. for example, in wish cells (originally thought to be of amniotic origin, but later found to be a hela (cervix cancer) contaminant) the ec 50 for antiviral activity of ifna2 is 0.3 pm, while the ec 50 for ifnb is 0.15 pm (43) . wish cells have been extensively used to characterize ifn-i activity, including for definition of ifn-i unit activity. an upper limit for antiviral potency was further verified by engineering an ifna2 variant, yns-a8-tail, with 50fold tighter binding to ifnar1 and 15-fold tighter binding to ifnar2 in comparison to ifna2 (thereby surpassing the receptor binding affinity of natural ifnb). still, the ec 50 for antiviral activity is only 3-fold lower in comparison to ifna2 (44, 45) . conversely to antiviral activity, ifnb is much more potent in activating the antiproliferative program relative to ifna2, a result that was also verified using the ifna2 variant, yns-a8tail (45) . the ec 50 for antiproliferative activity on wish cells is 2 nm for ifna2, 50 pm for ifnb and 20 pm for yns-a8-tail. a similar increase in antiproliferative potency was observed also for ovcar3 and hela cells. interestingly, while antiviral activity was observed in all cell lines tested, some cell lines were not susceptible to ifn-i induced antiproliferative activity (for example t47d and k562), independent on the concentration and subtype of ifn-i (45) . to better understand the molecular basis for this finding, ifn-i induced gene expression was monitored using various ifn-i subtypes or engineered mutants on the background of different cell-lines. these experiments showed that low concentrations of weaker binding interferons activate the expression of mostly antiviral genes. higher concentrations of interferons activate also other genes, many of them related to immune-modulation (45) . examples for such genes are chemokines such as cxcl10 and 11, which are involved in chemotaxis of t cells and natural killer cells, induction of apoptosis, regulation of cell growth and more. we gave the term of "robust" for the common ifn-i induced program (including its antiviral activity) and "tunable" for the other programs induced by ifn-is, which include between others antiproliferative and immunomodulatory activities (34) . further investigations into these two programs has shown that cells with low receptor numbers activate only the robust program, and that not all cell types execute the tunable program, conversely to the robust program that is common to all cells (46) . tighter binding ifn-is at higher concentrations are essential for the activation of the tunable program. genes upregulated by the robust program are mostly classical antiviral genes, such as mx1 and mx2, oas1 and 2, pkr, ifit1, 2 and 3, isg15, and many more. figure 3a according to string and go analysis, the commonly upregulated genes have a strong antiviral signature. the top go terms (fdr <10 −25 ) are response to type i interferon, innate immune response, response to virus, defense response and immune system process. it is interesting to note that antiviral genes constitute most of the upregulated genes common to all 4 cell lines. antiviral genes are also the majority of upregulated genes in k562 and t47d cells. conversely, ovcar3 and hela cells have many unique upregulated genes, many of them related to immunomodulatory functions, cell cycle, apoptosys and more. ifn-i. the diagram shows that 53 genes are commonly upregulated by all 4 cell-lines. figure 3b shows string protein interaction analysis of these common genes. clearly, these form a tightly interacting mesh of gene products. gene ontology analysis shows these genes to have an extremely high signature for antiviral activity and ifn-i activation. promoter analysis of common isgs has shown them to be driven by the classical isre promoter sequence (45) . conversely, for tunable genes no clear promoter sequence was identified. the exact mechanism of how tunable genes are upregulated by ifn-i is thus not yet fully understood. from an immunological point of view, ifn-is have three major functions: 1. to activate an antiviral state in infected and neighboring cells that limits spread of infection. 2. modulate innate immune responses, including antigen presentation and natural killer cell functions while restraining pro-inflammatory pathways. 3. activating the adaptive immune system for the development of high-affinity antigen-specific t and b cell responses (47) . as ifn-is are highly active molecules, their expression and signaling potency is highly regulated. opposing augmenting and suppressive signals are induced by host factors. suppressive pathways include ifn-i activation of usp18, an isg that suppresses signal transduction by reducing the ability of ifn-is to form an active receptor complex (38, 48) . a second inhibitory mechanism is the induction of socs1 and socs3, which kir domain block the substrate binding groove on jak, thereby inhibiting stat phosphorylation (49) . a third mechanism is by rapid endocytosis and subsequent lysosomal degradation of activated ifnar complexes (50, 51) resulting in reduced receptor numbers ( figure 1 ). it has been demonstrated that a mutant in ifnar1 (s535a and s526a in human and mouse respectively), which fails in ifnar1 endocytosis through blocking its ubiquitination result in high incidence of inflammation (51, 52) . at the transcriptional level, ifn-i response can also be regulated by mir-155, which is highly induced by pattern recognition receptors and inflammatory signaling, and suppresses the expression of over 100 genes. between them genes related to the interferon pathway. it was shown that mir-155-deficient cd8(+) t cells had enhanced type i interferon signaling and were more susceptible to interferon's antiproliferative effect (53) . high basal ifn-i levels are implicated in various immunological diseases, such as systemic lupus erythematosus and more (18, 54, 55) . however, ifn-i has also anti-inflammatory effects, as best demonstrated by their ability to suppress multiple-sclerosis (56) . it is important to note that beneficial results in treating multiplesclerosis were observed only for ifnb but not for ifna treatment (56) . to see whether this relates to the higher receptor binding affinity of ifnb, we established a transgenic mouse harboring the human interferon-receptors extracellular domains fussed to the mouse intracellular domains and compared the severity of eae in a mice model upon treatment with ifna2, ifnb and the high-affinity engineered ifn-yns-a8-tail. we found that the ifn-yns-a8-tail had the strongest suppressive effect on the development of eae (57) . the effect was further enhanced by pasylation of ifn-yns-a8-tail, which extends it plasma half-life by 10-fold. interestingly, we found a tight relation between the increased levels of expression of pd-l1 in mice and the severity of the disease. these data show that tight binding ifn-is induce preferential anti-inflammatory responses, at least in this ms mouse model. another example for the immunosuppressive activity of ifn-i was shown for lcmv infection, which induces consistent ifn-i production including the immunosuppressive factors il-10 and pd-l1 (58) . in addition to the above, interferons contribute to inflammasome activation through several different mechanisms, including caspase-11 expression and the ifn-i inducible gbp protein expression, which was reported to have an important role in caspase-11 activation and pyroptotic cell death (59) . ifn-is have important roles in protecting the lung from spread of respiratory viruses. in addition to their direct role, ifn-is have also been found to be critical in initiating lung inflammatory responses, by inducing recruitment and activation of immune responses, which have to be kept under control. ifn-is have been shown to result in the production of chemokines such as ccl2 and cxcl10, which play important roles in the recruitment of monocytes/macrophages, t cells, nk cells, and dcs, therefore directly influencing inflammation in the lung (60) . this varied effect of type i ifns on t cells is partly dependent on the different stats induced by type i ifns. in the absence of ifn-is, the detection of accumulating viral rna and downstream processing of the signal is compromised, leading to viral spread and also to reduced inflammation in the lung. interestingly, there is an age-related reduction of ifn-i production and isg induction after viral infection, which may be related to the higher susceptibility of elderly population to lung infections (61). viruses have developed many strategies to interfere with the synthesis of ifn-is or the ifn-i induced responses. one of them, is the stimulation of turnover of the interferon receptors. among other viruses implicated in accelerating the turnover of ifnar1 are ebv, herpes simplex virus, hepatitis c and b viruses, vesicular stomatitis virus and the sars coronavirus (62, 63) . sars-cov has been shown to suppress ifn-i responses in the host through multiple mechanisms. a subdued ifn-i response diminishes antigen presentation and reduces the antiviral adaptive th-1 immune response. ifn-is communicate between cells against pathogens and have a critical role in the immune system, such as activating natural killer (nk) cells and macrophages. in addition, ifn-is cause flu-like symptoms, which are observed in various diseases. these symptoms may have a role in alerting a person of his/her sickness, in order to limit disease-spread to other individuals. in sars-cov and mers-cov, the induction of ifnb is suppressed altogether. this dampening approach is highly associated with the disease severity and increased mortality (64) . in the lethal cases of sars-cov or mers-cov infections, the increased influx of inflammatory cells is always observed. in a mouse model of sars-cov infection, imbalance in ifn-i and inflammatory cells were shown as the main cause of fatal pneumonia (65) . in addition to these, sars-cov implements strategies to evade the immune response by antagonizing ifn-i induced signaling pathways. the orf6 protein blocks the expression of stat1activated genes (66) . sars-cov and mers-cov encode papainlike protease (plp) that is able to impede the immune response function (67) . in addition, sars-cov interacts with isg15 and antagonizes the ifn-i-mediated antiviral response (68) . the mers-cov orf4b antagonizes the antiviral ifnb production by inhibiting irf3 and irf7 (69) . also sars-cov inhibits activation of irf3/7, slowing ifnb production upon infection (70) . while irf3 is expressed in many different cell types, plasmacytoid dendritic cells are the only cells constitutively expressing irf7 (47) . ifn-i treatment has been studied against mers-cov and sars-cov in numerous experiments, both in vitro and in vivo, and in combination or not with lopinavir/ritonavir, ribavirin, remdesivir, corticosteroids, or ifng. while ifna and b were efficient in vitro and in certain animal models, their success in humans was less convincing [for review see, (71, 72) ]. it should be noted that reduction in ards mortality (not related to sars) was also found to be at best marginal upon treatment with ifn-i (73) . still, one has to consider that mice studies have shown the timing of ifn-i administration to be critical, with positive effects being observed if ifn-i was administered shortly after infection. conversely, ifn-i failed to inhibit viral replication and resulted in unwanted side-effects when administered later in the disease circle (74, 75) . these include elevated lung cytokine/chemokine levels, vascular leakage, and impaired virus-specific t cell responses. it is interesting to note that a knockout of the ifn-i receptor in mice resulted in its protection from lethal sars-cov infection. these findings have major implications on how to treat humans against sars and mers, and could have affected the outcome of the clinical studies. the covid-19 pandemic started in december 2019 in wuhan, china. by the summer of 2020, thirty million cases were reported worldwide, with over 900,000 fatalities. as covid-19 is closely related to the sars-cov virus, the interest in the effect of interferons on its disease progression, and its potential as a drug was immediate. disease progression of covid-19 goes through a number of stages. the initial stage, which last from 2 to 14 days (usually 5-6 days) from infection is asymptomatic. a certain proportion of patients never produce any symptoms (the percentage of those is under debate, but a range of 30-50% is most likely). of those who develop symptoms, they are mostly mild (80% of those who develop symptoms). from the remaining 20%, about half will develop severe symptoms, which require hospitalization in intensive care units. the mortality rate, from those developing symptoms is 2% to 5%. the numbers given above are average, and change dramatically with age. at young age most of the infected people will be asymptomatic, while over the age of 70 about 80% will have symptoms. moreover, as the age progresses, symptom severity increases (76) . the major complication of severe infection is pneumonia, which can develop into acute respiratory distress syndrome (ards). in addition, covid-19 has been linked to cardiovascular sequelae, such as myocardial injury, arrhythmias, cardiomyopathy and heart failure, acute kidney injury, neurological complications, and acute ischemic stroke (28) . developing severe symptoms and death is strongly related to background conditions. the strongest relation is to age, with the risk to people under 50 being very small, while the risk peaks for people over the age of 75. in addition, chronic kidney disease, chronic obstructive pulmonary disease, immunocompromised state, obesity, heart conditions and type 2 diabetes are linked to higher incidents of sever disease (76) . cov-2 is presumed to infect people mostly though inhalation of viral particles, which can be airborne, in droplets or otherwise through infection through touching infected surfaces. the spike protein on the cov-2 surface binds to the human ace2 protein, which serves as its receptor ( figure 4) . the homotrimeric spike glycoprotein is made from s1 and s2 subunits. its binding and subsequent cleavage by the host protease tmprss2 results in the fusion between cell and viral membranes and cell entry (77) . blocking the ace2 receptors by specific antibodies voids viral entry (77) (78) (79) . interestingly, cov-2 receptor-binding domain (rbd) exhibited significantly higher binding affinity to ace2 than the sars-cov rbd, which was speculated to relate to the higher infectivity of covid-19 in relation to sars. after membrane fusion, the virus enters through the endosomal pathway and the viral rna is released into the host cell. the viral rna is then translated into viral polyproteins, which are cleaved into small products by viral proteases (papain-like protease [plpro] and the main protease [mpro]). viral proteins and genome rna are subsequently assembled into virions in the er and golgi and then transported and released out of the cell. the exact mechanism of viral self-assembly is still under intense investigation (80, 81) . investigating ace2 and the viral entry-associated protease tmprss2 expression levels in lung tissue and trachea has shown that tmprss2 is expressed in both tissues, while ace2 is predominantly expressed in a transient secretory cell type (82) . in addition, ace2 and tmprss2 co-expressing cells were found within lung type ii alveolar cells (which also release pulmonary surfactant), enterocytes, and nasal goblet secretory cells (83) . using single-cell rna-sequencing, ace2 and tmprss2 were found to be highly expressed also in the nasal goblet and ciliated cells (84) . the inhaled virus likely binds to epithelial cells in the nasal cavity and starts replicating. the virus propagates and migrates down the respiratory tract along the conducting airways, and a more robust innate immune response is triggered. for about 80% of the infected patients, the disease will be mild and mostly restricted to the upper and conducting airways. unfortunately, about 20% of the infected patients will progress to more severe disease and will develop pulmonary infiltrates and some of them will develop ards (85). like many other viruses, also sars-cov and sars-cov-2 have evolved mechanisms to reduce their exposure to ifn-i. in both viruses, mechanisms to block the production of ifnb were identified. while the antiviral potency of ifn-is on sars-cov is moderate, sars-cov-2 seems to be highly sensitive to ifn-i. this is evident by the significant reduction in viral replication observed following ifn-i treatment at both 24 and 48 h postinfection (86) . in sars-cov-2-infected cells, ifn-i results in elevated stat1 levels and isg production (in contrast to sars-cov infected cells). this raises the question of why the innate immune system fails to combat sars-cov-2? the apparent answer to this is in the inhibition of ifnb production by proteins of the sars-cov-2 virus. within cells, rna viruses are sensed by the innate immune system through three major classes of pattern recognition receptors (prrs): toll-like receptors (i.e. tlr-3, -7, -8), rig-i-like receptors (rlrs), and nod-like receptors (nlrs) (87) . to identify the molecular mechanisms that block ifnb production through activation of irf3/7, several research groups transfected cells individually with all the cov-2 viral genes and with either rig i, mda5, or mavs (88, 89) . among the 27 cov-2 proteins transfected to cells, they identified nsp14 and orf6 as competent suppressors of ifnb. yuen et al. also identified nsp13 and 15, while lei et al. identified nsp1, nsp12 and the m protein as potent inhibitors of the mavs pathway, leading to inhibition of ifnb production ( figure 4) . orf6 was between the strongest suppressors of ifnb production in both studies. orf6 was also the only sars-cov-2 gene suppressing the activity of an interferon-stimulated response element (isre) promoter in both studies. lei et al. also identified nsp1 and nsp14 as potent inhibitors of the induction of an isre promotor. in another study, li et al. showed that the viral orf6, orf8, and nucleocapsid proteins were strong inhibitors of ifnb production, and through this of the ifn-i innate immune response (90) . in this study, orf6 and orf8 also inhibited induction of transcription an isre promotor driving a luciferase as reporter, following ifnb treatment. in addition to the above-mentioned sars-cov-2 genes, orf3b was implicated by konno et al. as being a potent antagonist towards ifn-i production (91) . an interesting civet in this study is the finding that a natural variant, with a longer orf3b reading frame increased disease severity in two patients. in light of the much higher than expected coding capacity of the sars-cov-2 genome, where many more proteins than genes were identified (92), we may find even more proteins and peptides being involved in eliminating the innate immune response, including through inhibition of ifn-i activities. another mechanism by which sars-cov-2 inhibit antiviral functions of the cell is thought the activity of the papain-like protease (plpro), which is essential for viral polyprotein processing. this gene was found to preferentially cleave the ubiquitin-like modifier interferon-stimulated gene 15 (isg15), figure 4 | sars-cov-2 has multiple effects on the immune system, including inhibition of ifnb production, which results in isgs not to be produced, cd4+ and cd8+ exhaustion and increased levels of pro-inflammatory proteins (tnfa, il6, nf-kb). currently, the most promising drugs against covid-19 include ifn-is, antiinflammatory and antiviral drugs, protease inhibitors, antibodies, sars-cov2 -ace2 (receptor) binding inhibitors and more. which is an ifn-i induced gene with strong antiviral activity (93) . this represents another layer of attenuation of ifn-i responses by sars-cov-2 and is similar to the mechanism previously identified for sars-cov (68) . inhibition of ifnb production by cov-2 got further confirmation from measuring the levels of different cytokines in sars-cov-2-infected patients. an integrated immune analysis, including immune cell analysis, whole-blood transcriptomics and cytokine quantification on covid-19 patients at 8 to 12 days after disease onset has shown an impaired ifn-i response that is a result of low ifn-i levels (94) . this, in turn results in the low production of interferon-stimulated genes. conversely, high levels of il6 and tnfa were measured ( figure 4) (95, 96) . this is in contrast to what is seen in patients infected with highly pathogenic influenza viruses. the high production of pro-inflammatory cytokines and low production of ifn-is during sars-cov-2 infection suggests effective activation of nf-kb but not irf3 and irf7 (95) . impaired ifn-i production during severe covid-19 may also lead to an imbalance in the pro-inflammatory versus pro-repair functions of airway macrophages. this was indeed seen in severely ill patients with covid-19. other innate immune cells such as natural killer (nk) cells are also regulated by ifn-is during coronavirus infection. severe covid-19 is associated with exhaustion of cd4+ and cd8+ t cells (97) , which may be a result of deficient ifn-i production, as ifn-is promote survival of t cells. an important issue to consider is that early production of ifn-is promote efficient t cell responses, while a delayed response may inhibit t cell proliferation or their exit from lymphoid organs and thus cause their functional exhaustion. indeed, t reg cell counts in covid-19 patients inversely correlate with disease severity (98, 99) . interestingly, transcriptomic analysis of blood, lung, and airways of cov-2-infected patients showed that while ifnb was indeed not highly expressed in either, a number of ifnas were highly upregulated in the lung and airways but not in blood (100) . moreover, a clear ifn-i-induced gene expression profile was also detected for lung and airways, but not for blood (pbmcs). a similar finding of elevated ifna but not ifnb, during covid-19 infection was also found by wei et al. (101) . in this study, the elevated ifn-i response was restricted to the stage in the disease were patients were in intensive care. in another study of 26 patients, of whom 5 did not produce ifn-i, those patients had higher viral load, required more aggressive medical intervention and their time of stay in the intensive care unit was longer that ifn-i producing patients (102) . pdcs are the most rapid and abundant ifn-i producers. pdcs express tlr7 and tlr9 which are important in sensing viruses. the response of pdcs to viruses, particularly ifn-i production, is significantly impaired with ageing while secretion of all other pro-inflammatory cytokines was comparable to that of younger individuals (103) . this may relate to the master regulator for ifn-i production, irf7, which expression, phosphorylation and nuclear translocation decreases with age. in addition, local neutrophil-mediated inflammation is increased with age, while cytotoxicity of nk cells induced by type i ifn-is decreases in aged mice (104) . in addition to age, other factors were also associated with reduced interferon responses. one of them is obesity, which is related to impaired ifna and ifnb responses, which may relate to inadequate response of obese people against viral infections (105) . clinical trials of using ifn-i for treating corona viruses has a long history. already in 1983, intranasal human ifna2 was given both before and after corona virus challenge, a strain that is causing common cold. the incidence of colds, the severity of symptoms and signs, and virus replication were all reduced in subjects receiving interferon as compared with those given placebo (106) . for sars-cov, no randomized placebo-controlled trials have been performed to test the efficacy of ifn-is, however, comparing the clinical outcome of patients treated with ifn-a (infacon-1) with patients at different locations (not a control group) that were not treated, has suggested clinical benefits (107) . these studies have raised the hope that ifn-i may be a potent drug also against covid-19. this hope was further exuberated by the observation that externally administrated ifn-i induced a strong antiviral response, much more than that observed for sars-cov (86) . while some of the sars-cov-2 proteins may affect isg production (most notably, orf6 and 8, see above), the main defense of sars-cov-2 against ifn-i innate immunity seems to be the prevention of ifnb production, which can be substituted by external administration. a major problem in assessing the efficiency of ifn-i against covid-19 is the lack of a good small animal model. while such models are now under development, they are still not perfect. in a recent study, mice were infected with a replication-deficient adenovirus containing human ace2, and then infected with sars-cov-2. these mice developed pneumonia, severe pulmonary pathology, and high-titer virus replication in lungs. to test the role of ifn-i in disease development, ifnar1 ko mice were infected with sars-cov-2, showing higher viral titer over time. next, the mice were treated prior to infection with poly i:c, a strong inducer of ifn-i. this resulted in significantly diminished clinical disease and induced more rapid virus clearance (108) . these results suggest that at least in a mice model, ifn-i may benefit disease recovery. due to the lack of a good animal model, and the availability of clinically approved ifn-i therapies, multiple clinical studies have been conducted administrating different subtypes of ifn-is using different routes of administration (for summary see table 1 ). in a preventive study, nasal drops of ifna1 were given to 2,944 healthy medical staff in shiyan city hospital, hubei province for 28 days to prevent sars-cov-2 infections. none of them developed serious side effects or was infected with cov-2. while the study lacked a control group from the same city, overall in hubei province 3,387 medical staff were diagnosed with covid-19 (109) . the study thus gives an indication that ifn-i may help in preventing infection for high risk medical personal. to test the benefit of subcutaneous injection of ifnb on early stage patients, an open clinical trial was conducted with 127 patients, 86 were assigned to the combination of lopinavir, ritonavir, ribavirin, and three doses of 8 million international units of ifnb, while the control group of 41 patients were given all the above except ifnb. the median number of days from symptom onset to start of study treatment was 5 days. patients given also ifnb had a significantly shorter median time from the start of treatment to negative nasopharyngeal swab (5-11 days) in comparison to the control group (8-15 days) . moreover, ifnb reduced viral load and number of significantly ill patients relative to the control group, this without significant side-effects (110) . in a medical study on the effects of treatment with ifna2b in a cohort of confirmed covid-19 patients, some of the 77 participants were given nebulized ifna2b with or without arbidol while others were given only arbidol. treatment with ifna2b with or without arbidol reduced the duration of detectable virus in the upper respiratory tract and reduced duration of elevated blood levels of il6 and c-reactive protein, which are inflammatory markers (111) . while the study did not include a standard care group, and all patients recovered, it still provides an indication of ifn-i efficiency. the efficiency of ifnb1a subcutaneously injected three times weekly for 2 weeks for treatment of severe covid-19 was tested in a randomized clinical trial. all the patients (including the control group) received standard of care, including a range of other medicines (hydroxychloroquine, antibiotics, antiviral medicine and more). while the clinical response was not significantly different between the ifnb1 and the control groups, the 28-day overall mortality was significantly lower (19% vs. 44%) in the ifnb1 treated group (112) . in a retrospective study of patients receiving ifna2 through inhalation, alone or in combination with other drugs at a relative early versus late stage of the infection, it was found that those receiving ifna2 at an early stage had a significantly lower rate of mortality. in contrast, late interferon therapy increased mortality and delayed recovery (113) . the study suggests a relation between the time of ifn-i treatment and its efficiency. synairgen, a uk-based company, performed a controlled clinical trial of inhaled ifnb on 221 patients and reported that compared with placebo the odds of developing severe disease during the treatment period decreased by 79% for hospitalized patients receiving sng001, and that patients who received sng001 were more than twice as likely to recover from the virus during the treatment period versus those randomized to placebo. these are between the best results achieved so far in curing covid-19. more clinical trials are now under way to evaluate ifn-i efficiency, but clearly the initial trials have been encouraging. moreover, due to the many years of experience in treating patients with ifn-is, the availability of the drug and its relatively modest cost make it an excellent candidate for mass treatment, once approved. however, critical questions remain concerning the use of ifn-is for covid-19 and other diseases ( figure 4) . these questions relate to the optimal ifn-i subtype, drug-concentration, duration of treatment, mode of treatment and at which frequency should it be given. ample experience exists with subcutaneously administration, which is almost the only route ifn-is were used in the clinic. here, non-modified ifn-is are usually administrated two to three times weekly, while pegylated ifn-is are administrated once per week or less. injection of ifn-is will result in a systemic response, where ifn-is were shown to have antiviral functions as well as pro and anti-inflammatory functions. contrary, if given by inhalation, it will directly target the epithelial, and thus replace the ifnb, which production is inhibited by the virus. administration as nasal drops of ifna may be an excellent prophylactic method for people at high risk. ideally, these questions could be answered using animal models. the problem is that the disease in those is not equivalent to that observed in humans. due to the severity of the disease and the high proven safety of ifn-is, more clinical trials on humans, testing the many open questions related to its best mode of administration may be the fastest way forwards. the subtype to use is another important question. for multiple-sclerosis, ifnb has been used for many years (114) , as it seems to provide a better anti-inflammatory response than ifnas. this may relate to its higher binding affinity to the interferon receptors, as has been demonstrated using a tight binding ifna mutant (yns-a8 tail), which binding affinity even surpasses that of ifnb [see above (57) ]. for combating viral disease, most notable hepatitis c, ifna2 has been most commonly used (115) , which was later replaced by pegylated (long plasma half-life) ifna2 (116) . also, for cancers ifnas were mostly used (117) . a good clinical explanation of why specific ifn-i subtypes were used is often missing, and decisions of which interferon to use may often relate to availability rather than to efficacy. moreover, due to the specie specificity of ifn-is, one cannot deduce from mouse experiments, which ifn-i to use in humans, as the data are not transferable (57, 118) . the main difference between ifnas and ifnb is that the later has a stronger potency to induce antiproliferative and immunomodulatory responses (tunable), while ifna will provide a cleaner antiviral response (robust) without the additional responses associated with ifnb. the open question is which is desired for covid-19 treatment, where complications arise from the exuberated immune response. another, important parameter is the time of intervention by ifn-i, in early or late-stage covid-19 disease. in a recent study in mice it has been shown that prolonged ifn-i and iii signaling interferes with lung repair during influenza recovery, probably through p53 induction, which reduces epithelial proliferation and healing, while early treatment protects mice (119) . in sars-cov-2 this is further complicated by the "cytokine-storm" symptoms of severe covid-19, as indicated by elevated il6 and tnf-alpha levels. whether ifn-administration, particularly ifnb suppresses or exacerbate the sars-cov-2 cytokine storm needs to be urgently determined, as to provide a guide for future application of ifn-i therapy in sars-cov-2 treatment. the author confirms being the sole contributor of this work and has approved it for publication. structural linkage between ligand discrimination and receptor activation by type i interferons structural and dynamic determinants of type i interferon receptor assembly and their functional interpretation affinity of stat2 for the subunits of the interferon alpha receptor stat3 activation by type i interferons is dependent on specific tyrosines located in the cytoplasmic domain of interferon receptor chain 2c. activation of multiple stats proceeds through the redundant usage of two tyrosine residues stat2 is an essential adaptor in usp18-mediated suppression of type i interferon signaling the unique role of stat2 in constitutive and ifn-induced transcription and antiviral responses the jak-stat pathway at twenty phosphorylated interferon-alpha receptor 1 subunit (ifnar1) acts as a docking site for the latent form of the 113 kda stat2 protein functional subdomains of stat2 required for preassociation with the alpha interferon receptor and for signaling crispr/cas9-based knockout strategy elucidates components essential for type 1 interferon signaling in human hela cells stat2/irf9 directs a prolonged isgf3-like transcriptional response and antiviral activity in the absence of stat1 statphosphorylation-independent induction of interferon regulatory factor-9 by interferon-beta the yin and yang of type i interferon activity in bacterial infection interferome: the database of interferon regulated genes defining the distinct, intrinsic properties of the novel type i interferon, ifnϵ interferon b-1a for the treatment of ebola virus disease: a historically controlled, single-arm proof-of-concept trial interferon: current status and future prospects in cancer therapy regulation of type i interferon signaling in immunity and inflammation: a comprehensive review interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures redefining the role of interferon in the treatment of malignant diseases interferon-beta-1b-induced short-and long-term signatures of treatment activity in multiple sclerosis inverse interference: how viruses fight the interferon system type-i interferon responses: from friend to foe in the battle against chronic viral infection mechanisms underlying the inhibition of interferon signaling by viruses ebola virus vp24 targets a unique nls binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated stat1 ebolaviruses: new roles for old proteins covid-19 infection: origin, transmission, and characteristics of human coronaviruses covid-19 outbreak: an overview evolutionary origins of the sars-cov-2 sarbecovirus lineage responsible for the covid-19 pandemic the proteins of severe acute respiratory syndrome coronavirus-2 (sars cov-2 or n-cov19), the cause of covid-19 type i and type iii interferons -induction, signaling, evasion, and application to combat covid-19 covid-19: pathogenesis, cytokine storm and therapeutic potential of interferons structural basis of a unique interferon-beta signaling axis mediated via the receptor ifnar1 the molecular basis for differential type i interferon signaling understanding cytokine and growth factor receptor activation mechanisms mechanism of homodimeric cytokine receptor activation and dysregulation by oncogenic mutations type i interferon signaling is decoupled from specific receptor orientation through lenient requirements of the transmembrane domain receptor dimerization dynamics as a regulatory valve for plasticity of type i interferon signaling binding and activity of all human alpha interferon subtypes inquiring into the differential action of interferons (ifns): an ifn-alpha2 mutant with enhanced affinity to ifnar1 is functionally similar to ifn-beta properties and functions of the novel type i interferon epsilon the stability of the ternary interferon-receptor complex rather than the affinity to the individual subunits dictates differential biological activities new structural and functional aspects of the type i interferon-receptor interaction revealed by comprehensive mutational analysis of the binding interface an interferon {alpha}2 mutant optimized by phage display for ifnar1 binding confers specifically enhanced antitumor activities multifaceted activities of type i interferon are revealed by a receptor antagonist stochastic receptor expression determines cell fate upon interferon treatment regulation of type i interferon responses usp18 establishes the transcriptional and anti-proliferative interferon alpha/ beta differential kinase inhibition, competitive binding and proteasomal degradation: resolving the molecular function of the suppressor of cytokine signaling (socs) proteins scf (hos) ubiquitin ligase mediates the ligand-induced down-regulation of the interferon-alpha receptor triggering ubiquitination of ifnar1 protects tissues from inflammatory injury therapeutic elimination of the type 1 interferon receptor for treating psoriatic skin inflammation the microrna mir-155 controls cd8(+) t cell responses by regulating interferon signaling interferons in autoimmune and inflammatory diseases: regulation and roles type i interferons in host defence and inflammatory diseases how type i interferons work in multiple sclerosis and other diseases: some unexpected mechanisms enhanced in vivo efficacy of a type i interferon superagonist with extended plasma half-life in a mouse model of multiple sclerosis persistent lcmv infection is controlled by blockade of type i interferon signaling the role of interferons in inflammation and inflammasome activation alpha/beta interferon receptor signaling amplifies early proinflammatory cytokine production in the lung during respiratory syncytial virus infection type i interferons as regulators of lung inflammation ubiquitination-mediated regulation of interferon responses viral dedication to vigorous destruction of interferon receptors immunopathogenesis of coronavirus infections: implications for sars pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane coronavirus papain-like proteases negatively regulate antiviral innate immune response through disruption of sting-mediated signaling structural insights into the interaction of coronavirus papain-like proteases and interferon-stimulated gene product 15 from different species middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets dynamic innate immune responses of human bronchial epithelial cells to severe acute respiratory syndrome-associated coronavirus infection a review of treatment modalities for middle east respiratory syndrome sars: systematic review of treatment effects effect of intravenous interferon b-1a on death and days free from mechanical ventilation among patients with moderate to severe acute respiratory distress syndrome: a randomized clinical trial dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice ifn-i response timing relative to virus replication determines mers coronavirus infection outcomes age-dependent effects in the transmission and control of covid-19 epidemics sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor cryo-em structure of the 2019-ncov spike in the prefusion conformation characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine specific viral rna drives the sars cov-2 nucleocapsid to phase separate the sars-cov-2 nucleocapsid protein is dynamic, disordered, and phase separates with rna sars-cov-2 receptor ace2 and tmprss2 are primarily expressed in bronchial transient secretory cells sars-cov-2 receptor ace2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes pathogenesis of covid-19 from a cell biology perspective sars-cov-2 is sensitive to type i interferon pretreatment sensing of rna viruses: a review of innate immune receptors involved in recognizing rna virus invasion sars-cov-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists activation and evasion of type i interferon responses by sars-cov-2 the orf6, orf8 and nucleocapsid proteins of sars-cov-2 inhibit type i interferon signaling pathway sars-cov-2 orf3b is a potent interferon antagonist whose activity is further increased by a naturally occurring elongation variant the coding capacity of sars-cov-2 papain-like protease regulates sars-cov-2 viral spread and innate immunity impaired type i interferon activity and inflammatory responses in severe covid-19 patients imbalanced host response to sars-cov-2 drives development of covid-19 dysregulation of type i interferon responses in covid-19 reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) dysregulation of the immune response affects the outcome of critical covid-19 patients dysregulation of immune response in patients with covid-19 in wuhan, china comprehensive transcriptomic analysis of covid-19 blood viral invasion and type i interferon response characterize the immunophenotypes during covid-19 infection type i ifn immunoprofiling in covid-19 patients mechanisms and implications of age-associated impaired innate interferon secretion by dendritic cells: a mini-review age-dependent dysregulation of innate immunity decreased interferon-a and interferon-b production in obesity and expression of suppressor of cytokine signaling intranasal interferon as protection against experimental respiratory coronavirus infection in volunteers interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study generation of a broadly useful model for covid-19 pathogenesis, vaccination, and treatment an experimental trial of recombinant human interferon alpha nasal drops to prevent coronavirus disease 2019 in medical staff in an epidemic area triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial interferon-a2b treatment for covid-19 a randomized clinical trial of the efficacy and safety of interferon oe≤-1a in treatment of severe covid-19 retrospective multicenter cohort study shows early interferon therapy is associated with favorable clinical responses in covid-19 patients the mechanism of action of interferon-b in relapsing multiple sclerosis pharmacokinetics of alphaifn-2b in chronic hepatitis c virus patients undergoing chronic hemodialysis or with normal renal function: clinical implications use of peginterferon alfa-2a (40 kd) (pegasys ® ) for the treatment of hepatitis c biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons bridging the species divide: transgenic mice humanized for type-i interferon response type i and iii interferons disrupt lung epithelial repair during recovery from viral infection the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 schreiber. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-304619-tpv76833 authors: chatterjea, devavani title: teaching immunology as a liberal art date: 2020-07-14 journal: front immunol doi: 10.3389/fimmu.2020.01462 sha: doc_id: 304619 cord_uid: tpv76833 a complex, rapidly evolving biomedical field that is of critical relevance to human health and well-being, immunology provides important and substantive opportunities to practice and teach the central tenets of a liberal arts curriculum. it's one of those "end of semester" days in december-i am looking forward to wrapping up the term, the familiar mix of exhaustion and anticipation in my bones. the junior and senior biology majors in my immunology survey course at an undergraduate liberal arts college in the midwest are setting up their immunology-themed art presentations. a pile of "plushies"-giant stuffed fabric white blood cells decorated with their known surface markers invites tactile exploration, and an impromptu game of toss. an immune cell synapse wired with leds lights up in series as "activation" switches are flicked on. students flock to the edible displays. a towering croquembouche "lymph node" of choux pastries invites them to pull out individual ones to taste-flavored with different fillings, the pastries represent the different cells in a lymph node. as the puffs get eaten, the spun sugar matrix of the tower loses shape, much as a lymph node matrix would without resident cells. the hematopoiesis cookie table is a hit. the student who set it up explains how a basic set of ingredients is flexibly transformed into different kinds of cookies-at which points commitments to certain final products occur and when and how steps become irreversible; class-mates sample some of the finished products and take turns building cookies of different "lineages" with nuts, fruit, chocolate chips, bits of candy sparking a spontaneous discussion about food allergies, routes of exposure and safe handling practices. a student clears their throat and the hum of chatter subsides. a self-described "non-artist, " they have chosen instead to deliver a "testimony to congress" to advocate for robust funding for immunological research inspired by the advocacy of members of the american association of immunologists (1). as standin lawmakers, we listen attentively to the evidence-based arguments for the importance of basic immunology research for a healthy society. there are tough but respectful questions on animal research ethics, a plausible timeline for a universal flu vaccine and the structural inequities of access to cutting edge cancer therapies such as car-t cells. after the q&a, students read each other's artist's statements, take turns trying to sit on the fold-out monocyte chair without falling, and play with the stick and string co-stimulation maze which can only be solved with 3 manipulations in the correct sequence! over 20 years ago, i was an undergraduate in an immunology class, irresistibly drawn to the discipline despite the confounding maze of nomenclatures, the alphabet soup of transgenic tcr names and the flood of cell types and molecules that went over my head. through graduate and post-doctoral work, my love for the field endured and i realized that i wanted an undergraduate liberal arts curriculum to be the canvas for my immunology teaching and research. i don't think that, at the time, i could have foreseen a class period quite like the one i just described: students making the material their own in inventive and surprising ways, going confidently into the heart of foundational and cutting-edge concepts and using their intellectual and practical engagement with the material to connect their study of immunology with their lives. teaching and learning immunology as a liberal art together with my students has been transformative for all of us. macalester college is an urban small undergraduate liberal arts college with ∼2,000 students−30% students of color, 14% international, and 16% first-generation. biology is one of the top 5 majors. i teach an immunology survey course with laboratory for undergraduates who have taken cell biology and genetics. though the human immune system is the primary focus of the course, we study amoeba, social insects, bacteria, plants, and jawless fish to better understand the evolution of protective responses. students write multipledraft review papers with graphical abstracts, volunteer with the immune deficiency foundation, present art/performance works and write weekly reflective essays connecting their immunology learning to other parts of their academic or personal lives. my immunological methods course is embedded in my research program investigating the connections between environmental toxins, allergic responses, and chronic pain; students participate in scientific conversations and critique scaffolded by preparatory writing assignments, map metaarguments from sub-fields of published literature, cooperatively design, and execute experiments, and write a collaborative scientific paper. i use my upper-level seminar courses-neuroimmunology and cancer immunology to teach more advanced students about public communication of science. in our college's first year course program i offer semester-long immunology-themed courses: aids/influenza/malaria -ancient pathogens in a brave new world explores the persistence and reemergence of infections and inflammatory diseases in vulnerable populations around the world and bodies on fire centers on the global pandemic of inflammatory diseases. these courses do not have pre-requisites and are structured around connecting patient/physician memoirs, popular science books, and science journalism with the scientists and scientific discoveries they describe and typically ask students to explore these connections through writing, movement, and art. historian william cronon describes the essence of liberal education as "gaining the power and the wisdom, the generosity, and the freedom to connect"-through the acts of listening, reading, writing, talking, solving puzzles, seeking complex truths, seeing other perspectives, working in a community and being willing to both lead and follow in honest and imaginative ways (2) . structurally, a liberal arts education connects the natural and physical sciences, humanities, social sciences, quantitative thinking, and artistic inquiry. even as they engage deeply with methods and analyses in particular areas of study, students learn to appreciate different ways of making meaning of our world with tools from different disciplines. they learn to recognize and interrogate the societal structures and deep assumptions that drive the ways in which such bodies of knowledge are constructed within and across academic disciplines. immunology is a perfect fit for a liberal arts education. while traditional practices such as variolation and uses of immunomodulatory foods and botanical medicines have existed for thousands of years in societies around the globe, the constructs of cellular and circulating immune mechanisms have been articulated in the context of academic biomedicine only as recently as the late 1800s. and within these 200 years, paradigms have been swiftly proposed, critiqued, modified and transformed into an ever more complex and nuanced understanding of immunity (3). concepts of preservation of self over "non-self " have morphed into understandings of danger, disruption, repair, and memory embedded deep within cell lineages, epigenetic imprints and tissue architectures. mechanisms once described more bluntly as "killing pathogens" are now understood as highly regulated, selective, tunable responses to commensal and non-commensal microbes that constitute the multi-species ecosystems of multicellular hosts. while the immune system gives us critical protection for survival, virtually every global health concern from emerging infections, allergies and asthma, autoimmunity, chronic pain, and other psychiatric, cardiovascular, and metabolic imbalances are all fueled by these same mechanisms of inflammation, shifted by context to become harmful and pathological. author chimamanda ngozi adichie, in her ted talk "the danger of a single story, " warns that assuming a single story about a people leads to dangerous misconceptions, and learning to listen for the many different stories is essential for cross-cultural understanding (4) . immune responses, with their double-edged nature, provide a natural set of case studies in the importance of "many stories." immune responses demand careful contextual analyses, and to study them closely is to learn to grapple with complexity and uncertainty-an essential skill in today's rapidly changing, connected yet divergent world. another advantage of studying immunology is its immediate personal and social relevance. students only have to look at their own bodies, experiences of well-being and illnesses, and their environments for applications of what they learn. for many students, one immunology-related class might be their only sustained experience with the discipline, but the lessons they draw from it have the potential to remain relevant and useful in their lives. as a powerful example of this, i have observed my neuroimmunology students particularly resonate with learning about the role inflammation plays in mental health. students on college campuses are experiencing anxiety and depression at unprecedented levels, and managing neurological diagnoses while removed from their families and support systems (5) . understanding the roles of pathological inflammation intertwined with these mental health conditions, exploring the connections of stress, diet, and rest to these neuro-inflammatory pathways are empowering for students; appreciating the "bodily" basis of psychological challenges appears to make them seem more tractable. while these lessons do not take the place of the counseling and/or psychiatric support they or their peers need and receive, i have observed that students do find this scientific parsing of the mind-body connection to be of practical use. many immunology students are drawn to careers in biomedical research and its applications in the practices of medicine and/or public health. immunological researchdiscovery, translational, academic, clinical, industrial-and its applications in drug development, medical technologies, and public health interventions are at once scientific and social endeavors. countering anti-vaccine movements, crafting community, and government public health responses to disease outbreaks, regulating environmental toxicants in food, water, and air all contain important immunological arguments at their core. being able to understand and speak the language of immunology and tell its stories to specialist as well as general audiences so they can be truly heard is an important skill for students to practice. iteratively learning to read the often dense and technical immunological literature and synthesizing and communicating these findings in their own written and spoken words is both preparation for future work in biomedical fields and a core tenet of a liberal arts education-the importance of listening, reading, speaking, arguing, and writing. these skills are not unique to the study of immunology, but immunology offers undergraduates and their professors in a liberal arts context a rich and pragmatic field within the biomedical sciences in which to practice them. students in my courses and research laboratory write literature reviews, give talks and present posters on their research at conferences, and collaborate with me on writing papers and grant proposals for scientific audiences. however, they also write white papers and reflective essays connecting their learning in immunology to other disciplines, prepare educational materials for community organizations, teach secondary school students and mentor younger peers and, in doing so, practice translating the technical jargon of scientific communication into information that their audiences need and can use. a spacious liberal arts education makes room for multidisciplinary training, provides opportunities for immersive learning and community engagement and asks students to connect their learning to the world in different ways, giving them opportunities to make this complicated and compelling field their own. the perceived "difficulty" of immunology can be deconstructed in this more permissive, integrative environment to allow creative strategies for making meaning and finding purposeful engagement with the subject. immune systems are synergistic wholes of interconnected parts continuously stirred up by new discoveries that complicate existing knowledge and demand new ideas and interpretations; this has been so since paul ehrlich sketched his intricate visions of cells shedding neutralizing anti-toxins and butted heads with ilya metchnikoff 's cheeky but utterly prescient observation that immunity might just look like hungry amoeba out to forage (6). in the last two decades, our view of the immune system has been transformed by newly discovered innate cell subsets, the regulation of immunity by microbial and viral symbionts, the control of immune responses by metabolic switches, and the realization that all cells, not just the ones that we recognize as immune cells, participate in and regulate immune responses of multicellular organisms. this framework of synergistic interactions and multi-factorial outcomes can provide our students with maps and metaphors useful beyond immunology, for broader understandings of complex social and planetary processes. the precarious balance of protective vs. harmful immune responses is a mirror of the collateral costs of inequities, statesanctioned violence, and xenophobia in our societies. chronic inflammation and accompanying adverse health outcomes are materially correlated with lower socio-economic status, lack of access to nutritious foods, stressful living conditions and unstable access to healthcare (7) . that any immune response takes a toll on the living tissue it is trying to protect from real or imagined threats parallels the effect that xenophobic, reactive intolerance, and unregulated violence can have on a community or society at large. just as our own cells and those of our commensal symbionts maintain a collaborative understanding that we disrupt at our peril, our local and global communities are sensitive to the behavior of individuals and cooperation between the diverse populations who live in them. tolerance, balance, homeostasis, repair are technical terms with specific immunological meanings that are just as relevant to our social fabric as they are to our understanding of healthy and disease states of our bodies. and likewise, jingoistic militarized language about the immune system vanquishing pathogens can echo intolerant social rhetoric. the nuance and care required to understand and modulate immune responses and their outcomes serve as object lessons in how we speak and act as individual and collectives in social and political arenas. an immunological framework can also be applied to the relationship of humans with our planet as a whole. humaninduced climate change has driven our planet and its inhabitants to a perilous state of imbalance, with rapid rise in temperature and sea levels, catastrophic weather events, heat stress, food shortages, displacement of peoples, biodiversity loss, emerging pathogens (such as sars-cov2), and exacerbation of poverty and conflict, all of which create negative health outcomes for those who are most vulnerable and have the least access to resources. the united nations intergovernmental panel on climate change (8) advocates for immediate, massive, and collective action to mitigate this crisis if we are to survive. our students are joining their climate activist peers-greta thunberg, isra hirsi, xiye bastida, and others in climate strikes and actions to emphasize the urgency of the situation. the literal health effects of climate change are, and will be marked by inflammatory processes in our individual bodies, and sharp increases in global disease burdens; it is as if the entire planet is in a state of chronic inflammatory distress. everything is connected and what we do individually, and collectively, to our bodies and to our world comes back to us. teaching about our immune systems in integrative, socially relevant ways can help our students make meaningful connections between the content of their learning and the larger global context in which they live. in her book teaching to transgress (9), feminist author and educator bell hooks says: to educate as the practice of freedom is a way of teaching that anyone can learn. that learning process comes easiest to those of us who teach who also believe that there is an aspect of our vocation . . . is not merely to share information but to share in the intellectual and spiritual growth of our students. in information-dense, rapidly evolving fields of study, it is natural to feel overwhelmed by the responsibility to share information as accurately and comprehensively as possible before our limited time with any one group of students comes to a close. i am grateful that immunology-the beautiful, maddening, messy field that it is-keeps me humble and honest about the work i really want to do with my students and the way in which i want to do it. it resolutely refuses to be told as a "single story" and any arcane details memorized for exams are known to have modest shelf lives in any case. so with each passing year, i am challenged to re-imagine how i can best help my students be as prepared as possible to hear and understand all of the immunological stories that have not been written yet-to be able to know the workings of their future bodies and minds a little better, to understand and appreciate why a pandemic coronavirus can ravage one body it infects and leave another unscathed, to be able to use these stories to build healthy lives and communities, and make new discoveries. in her more recent book, teaching community: a pedagogy of hope (10), bell hooks says: "it is imperative that we (teachers) maintain hope even when the harshness of reality suggests otherwise." i take these words to heart. much of western biomedical science has been built around concepts of illness rather than wellness and i wonder whether it is simply too overwhelming to keep coming back to narratives and mechanisms of morbidity, dysfunction, and imbalance. here again, the spaciousness of a liberal arts framework allows both instructors and students to be more open to leavening the difficult topics with moments of beauty and fun. psychologist alison gopnik has demonstrated that children who "pretend play, " in elaborate, unreal scenarios with the aid of language, props and gestures, are able to respond correctly to counterfactual questions about a novel real-world causal relationship (11) . while the evolutionary imperative for play may well be to develop robust cognitive functions, children play because it is a lot of fun. the paradox of play is that in order to be able to reach a variety of practical benefits in the long run, one must be somewhat less concerned with immediate accomplishments of goals in the short run. eating a cardamom and orange cream-filled choux bun pulled out of a patisserie "lymph node" might not immediately seem central to learning about immune systems but it is delicious and it distills the joy of learning and sharing in a way that sticks in our brains and hearts-both my students' and mine. a liberal arts education with its emphasis on connective and integrative inquiry aims to be transformative, to crack the world open a little bit wider for every student with every course of study, every class, every discipline. but it is not only the student who is transformed, it is also the teacher. teaching immunology as a liberal art has made me a more curious, capable and happier immunologist than i had known i could be. the original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author. the goals of a liberal education immunology's coming of age the danger of a single story who world mental health surveys international college student project: prevalence and distribution of mental disorders microbe hunters reissued socioeconomic status and inflammation: a meta-analysis the intergovernmental panel on climate change. global warming of 1.5 0 c teaching to transgress: education as the practice of freedom teaching community: a pedagogy of hope the power of possibility: causal learning, counterfactual reasoning and pretend play the author confirms being the sole contributor of this work and has approved it for publication. i thank my students at macalester college for being my collaborators on this journey since 2006, and my fellow immunologists at liberal arts institutions for their inspiring conversations and support. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 chatterjea. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-319774-mkz7z38o authors: hou, dongni; chen, cuicui; seely, eric john; chen, shujing; song, yuanlin title: high-throughput sequencing-based immune repertoire study during infectious disease date: 2016-08-31 journal: front immunol doi: 10.3389/fimmu.2016.00336 sha: doc_id: 319774 cord_uid: mkz7z38o the selectivity of the adaptive immune response is based on the enormous diversity of t and b cell antigen-specific receptors. the immune repertoire, the collection of t and b cells with functional diversity in the circulatory system at any given time, is dynamic and reflects the essence of immune selectivity. in this article, we review the recent advances in immune repertoire study of infectious diseases, which were achieved by traditional techniques and high-throughput sequencing (hts) techniques. hts techniques enable the determination of complementary regions of lymphocyte receptors with unprecedented efficiency and scale. this progress in methodology enhances the understanding of immunologic changes during pathogen challenge and also provides a basis for further development of novel diagnostic markers, immunotherapies, and vaccines. introduction the adaptive immune system is composed of b and t cells that form a highly selective guard against evolving pathogens. the foundation of the adaptive immune response is based on the enormous diversity of t and b cell antigen receptors that can recognize epitopes from a near infinite number of different internal and external antigens. this profound diversity of t (tcrs) and b cell receptors (bcrs) is generated by v-d-j gene recombination of the tcr/bcr locus and subsequent somatic hypermutation and class-switching recombination of b cells after antigen stimulation. thus, study of the immune repertoire, portrayed as the antigen-specific information within lymphocytes, has been a key to understanding the response of adaptive immunity during infection. despite extensive efforts using traditional techniques, analysis of the immune repertoire with high resolution has remained difficult. several sequencing strategies, for example, sanger sequencing, have been implemented to determine cdna segments encoding variable regions of immunoglobulin (or tcrs) (1, 2) . however, these low-throughput techniques lack the power to provide a broad picture of the full immune repertoire. during the past two decades, however, technical advances in high-throughput sequencing (hts), also known as next-generation sequencing (ngs), along with evolving bioinformatic and statistical tools, have provided a new approach capable of analyzing the immune repertoire at the single sequence level. these methods create an unprecedentedly high-resolution picture of the immune repertoire and also provide massive data that cover each lymphocyte from the sample, in theory, dispensing with limitation of sequencing quantity (3) . considering the extremely important role of the adaptive immune system in defending against infectious agents, hts has great potential to aid in the discovery novel infectious agents and also offers new approaches for antibody or vaccine development. in this review, we introduce the implementation of hts to the study of the immune repertoire and review the associated bioinformatic tools required for data processing and analysis. we then focus on the success of this technology in facilitating the exploration of infection-related immune repertoires for clinical diagnosis, treatment, and prevention. amazing diversity makes the immune system the most effective system to fight against a broad scope of disease causing pathogens. this repertoire is generated by a complex series of genetic events (4) . for t cells, the variable region of each tcr chain consists of three complementary determining regions (cdrs) and four frame regions (frs) . cdrs are the variable portion of the receptor and determine the antigen specificity. while cdr1 and cdr2 are formed by variable (v) gene, cdr3 is generated by random selection and recombination of variable (v), diversity (d), and joining (j) gene segments in the heavy chain (v and j region gene segments in light chain) (5, 6) (figure 1) . thus, cdr3 is the most diverse component of a receptor, which binds mhc molecules and (or) antigens. construction of the tcr with an alpha chain and a beta chain is also a process that contributes to receptor diversity. the formation and revision of the t and b cell lymphocyte receptor repertoire is a highly dynamic process. the number of each lymphocyte clone changes dramatically and depends on cell specificity and the history of antigen exposure. when encountering exogenous antigens, t cells that express receptors capable of binding to a specifically compatible peptide-mhc (pmhc) complex will expand, resulting in a massive population of antigen-specific t cells that initiate the adaptive immune response (7) (8) (9) (10) (11) . this antigen-driven proliferation process of t cells is distinct between cd4+ and cd8+ t cells after initial antigenic stimulus. although these two types of t cells show comparable protein expression, proliferation rate, and transcriptome features after 2 days of non-infective stimulation, subsequent division of t cells differently depends on continuous existence of self-pmhc complexes. cd4+ t cells proliferate in a limited pattern, and its subsequent response requires persistent stimulation from antigen-presenting cells. cd8+ t cell is "programed" to extensive expansion after short stimulation even when transferred into antigen-free hosts (12) . the post-antigen stimulation response of b cells is more complicated because it is accompanied by somatic hypermutation and class-switch recombination that offer additional diversification of the b cell repertoire (6) . somatic hypermutation is the process of introducing point mutations at cdr1, cdr2, cdr3, and fr3 to produce b cells of higher affinity to target antigens. these higher affinity clones are then selected and expanded, which is called affinity maturation. additionally, in class-switch recombination, the gene loci encoding the c region of bcrs are excised and replaced by a series of new constant gene segments, resulting in functional differences of igg, ige, or iga that participate in different immune mechanisms during pathogen elimination. traditional strategies for studying the immune repertoire prior to hts, many strategies were developed to explore postinfection immune repertoires (13). immunoscope spectratyping has been used to investigate tcr/bcr repertoires since the 1990s (5, 14) . in this technique, using one (for b cell) or more (for t cell) v or j gene specific primer pairs, the length of cdr3 can be determined (15) . cdr3 length in healthy population shows a bellshaped pattern, indicating a polyclonal repertoire. however, the unusual peaks in infected patients imply a perturbed oligoclonal repertoire with clonal expansion. as such, cdr3 spectratyping provides robust information on the complexity and stability of circulating t/b cell repertoires and insights into the immune repertoire after infection (16) (17) (18) (19) (20) . even if it is relatively easy and cheap, the nucleotide sequences of cdr3 remain obscure, and the extent of heterogeneity within a particular cdr3 length cannot be assessed. detailed nucleotide sequences of gene segments encoding the variable region can be determined by traditional dna sequencing techniques such as sanger sequencing (21, 22) . flow cytometry and cdr3 spectratyping help to isolate t/b cells of interest, which complements weaknesses of sanger sequencing in quantity limitation. single-cell sequencing is able to identify sequences of several b cells that produce monoclonal antibodies specific to certain virus, which contributes greatly to analyzing genetic features of the antibodies in the process of antibody discovery (2, (22) (23) (24) . after collecting peripheral blood samples, the b cells or memory b cells are isolated and immortalized to produce antibodies. according to the hai titers and neutralizing titers determined by elisa, the virus-specific b cells can be identified, which helps to narrow b cell candidates for sequencing. these functional test-based antibody discovery strategies are successful but laborious. despite this, these strategies are well designed for targeted antibody searching; however, it is insufficient for creating a high-resolution picture of the human immune repertoire. high-throughput sequencing has recently become a novel and powerful tool to investigate the immune repertoire. the depth and comprehensiveness of high-throughput immune repertoire sequencing are greater than ever, and the enormous sequencing data of disease-specific tcr/bcr clones provide great potential for the revealing dynamic changes in clonality during infectious states. establishing a lymphocyte repertoire database starts from sample collection from carefully selected populations and isolation of interested t cell or b cell subgroups. due to the well-acknowledged heterogeneity of tcrs and bcrs between individuals, longitudinal studies tracking dynamic alterations in certain population help to reduce difficulties in data interpretation at unraveling the infection course. classification of subgroups of t cell and b cells, e.g., naive and memory t/b cells, cd4+, and cd8+ t cells, is necessary if distinct behavior of these subgroups is considered in detail. the methodology of library preparation and amplification need careful design since it affects accuracy of the ultimate sequencing data. due to the difference of v and j gene segments, a common primer does not apply to sequencing of cdr3. multiplex pcr is capable to amplify multiple loci simultaneously and, however, is likely to introduce bias. it is because of non-specific amplification, primer-dimer formation, and uneven reaction conditions. more precise and quantitative multiplex pcr may be achieved through primer concentration adjustment and bias filtering using amplification bias among the templates as controls (23) . another alternative pcr method is 5′race pcr, which provides a less biased pcr library using primers that bind downstream of the variable domain (24) . sequencing techniques are evolving continuously to be deeper and more precise, and there are three prevalent hts platforms available today. the comparison of mechanisms, sequencing depth, and other critical features of each platform is shown in table 1 . the illumina and roche 454 platforms have been most commonly used during immune repertoire analysis. the outputs of each platform must be analyzed with caution because of notable platform-specific sequencing error (25) (26) (27) (28) . insertions and deletions of nucleotides, resulting from imperfect interpretation of homopolymeric stretches, are considerable for the roche 454 platform (29) , while substitution errors are predominant in illumina platform (30, 31) . the overall error rate of illumina platform is lowest while that of ion torrent is highest among the three (32) . in an attempt to correct sequencing errors, three algorithms are most commonly used including k-mer spectrum, multiple sequence alignment, and suffix tree (26) . based on (27) . pcr and sequencing errors inevitably result in overestimate of repertoire diversity. the common statistical strategy for both pcr and sequencing error removal is eliminating low abundance and low-quality sequences (with low phred score), but it leads to a great loss of sequencing information. to rescue these sequences, low-quality cdr3 sequences with no more than three low-quality nucleotides can be mapped to "core clonotypes" derived from high-quality sequences with allowed mismatches at low-quality position. then, the low-abundance core clonotypes are merged with the high-abundance core clonotypes with less than three allowed mismatches at v (≤2 mismatches), d (≤1 mismatches), and j (≤2 mismatches) gene segments to correct pcr errors. this integrated algorithm based on sequence quality abundance could efficiently correct artificial errors and avoid information loss, thus providing more reliable estimation of repertoire diversity (28) . unique molecular identifiers that label each starting molecule help to reduce both pcr and sequencing errors (24, 41) . combined with this experimental strategy, molecular identifier groups-based error correction (migec) corrects pcr and sequencing errors more efficiently than other quality-and frequency-based strategies (42) . determination of the v-d-j gene segments from which the cdr3s are rearranged, as well as identification of point mutations, is often achieved using the immunogenetics database (http:// www.imgt.org) (43) , despite the controversies about its validity (44) (45) (46) . new v-d-j gene annotation tools based on various algorithms are reported, such as igblast (47), ihmmune-align (48) , and decombinator (49, 50) ( table 2 ). in addition, many integrated bioinformatic tools (mitcr, lymanalyzer, change-o, etc.) for data processing are developed recently (51-62) ( table 2) , which provides various statistical approaches for diversity estimation, repertoire comparison, clustering analysis, and somatic hypermutation analysis ( table 3) . despite these tools, standardized bioinformatic analysis and visualization strategy is lacking, which remains the main obstacle for comparison of researches from different investigators. high-throughput sequencing techniques have revolutionized the study of the immune repertoire. utilizing hts, many important insights into mechanisms of immune response have been gained. it is also the cornerstone for potential clinical applications of repertoire analysis, including identification of diagnostic biomarkers, design of therapeutic antibodies, and development of new vaccines. estimating the diversity of a tcr/bcr repertoire is necessary for estimating the theoretical size of the repertoire and for tracking changes in clonal populations during the clinical course of infection. several different methods may be used to describe the diversity of lymphocyte repertoires at different levels -vdj recombination diversity (90), simpson diversity index (91, 92) , and some non-parametric methods. decrease in the overall diversity of the immune repertoire have been observed after various antigen exposures, including hiv, influenza, and human herpes virus, which implies expansion of particular t/b cell clones (67, 88, 92, 93) . our group compared changes in the diversity of the tcr beta chain and bcr heavy chain after h7n9 virus infection. interestingly, these results show that the diversity of the bcr heavy chain starts to increase 2 weeks after h7n9 infection, while the tcr beta chain repertoire continues to contract. in addition, a more diverse bcr repertoire and a less diverse tcr beta chain repertoire in convalescent phase correlate with improved prognoses, implying differences in the response process of humoral and cellular immunity. the immune response to vaccination has been used as an ideal model for antibody repertoire research due to the convenience drawing blood samples at well-defined time points (94) . studies using vaccines, such as influenza and tt, have revealed dynamic changes in the size and diversity of antibody repertoires before and after antigen stimulation (41, 90, 95, 96) . comparison of post-vaccination responses suggests divergent repertoire properties among individuals, different age groups, and successive immunization of the same individual with different influenza vaccines (tiv and laiv) (66, 90, 95) . the maximum clonal response has been found to occur 7 days after vaccination, but the magnitude of response varies between individuals despite an identical immune challenge, which may be influenced by previous exposure, age, and other concurrent immune responses. in addition, study of b cell memory can be achieved by repeated sequencing of samples taken from the same individual in separate immune responses to an antigen. vollmers and colleagues identified a group of b-cell clones as a recall response to two substantially different vaccine compositions, implying the possibility of identifying cross-specific antibody using repertoire analysis (41) . immune responses recorded by sequencing data have also been useful in testing the role of adjuvants in eliciting broad-spectrum antibodies. wiley and co-workers tested the immune response of mice immunized with malaria vaccine by analyzing igg repertoires. they found that tlr agonist used as adjuvant increases the diversity of igg variable region, which is related to improved ability of the antibodies to recognize a broad spectrum of epitopes (97) . these studies exemplify a new level of details assessing vaccine response and pioneered hts implications in vaccine design. in infected patients, antigen-specific t/b cell repertoires form in response to antigen exposure in both circulation and peripheral tissues. immune repertoire sequencing provides broad information including crucial antigen-specific clones, which have the potential to halt the spread of pathogens (98) . diagnostic marker discovery using sequencing data relies on these antigen-specific clones with stereotyped features in the post-infection repertoires. these features are assessed at different levels such as gene rearrangement, identical or similar cdr3 sequence overlap, and certain cdr3s length. after influenza h1n1 vaccination, the dominant clonotype of ig heavy chains has the same v-j gene rearrangement, cdr3 length, and somatic mutation position in cdr1 and cdr3 with previously reported influenza antibodies (66) . however, in this study, the convergent dominant sequence is only found in one individual. further researches in a broader population including non-dominant sequences are needed. a more successful example is reported in ig repertoires related to dengue virus infection. using cross validation and other approaches, stereotyped cdr3 sequences or cdr3 lengths that have high prevalence in the acute dengue samples are found to be specific to acute dengue infection, which are absent or of low prevalence in healthy and post-convalescent population (88) . identification of pathogen-specific sequences also helps in differential diagnosis between infectious and non-infectious diseases. according to dziubianau et al., comparing pbmcs-derived t cell clonotypes specific to a given virus with t cells from different origins (allograft-derived and urine-derived lymphocytes) provides a new methodology for differential diagnosis of two post-transplant complications -bkv-associated nephropathy and acute cellular rejection, which shows a glimpse of applications of t cell sequencing in diagnosis (99) . in addition, a recent study searched sequencing data for cdr3 amino acid motifs that have been reported to be specific for a particular pathogen and succeeded in identifying cdr3 sequences identical or similar to these motifs in post-vaccination volunteers (100) . according to these results, interestingly, it is low frequency sequences that possess the probability of becoming promising biomarkers instead of the dominant ones. however, immune responses show dramatic differences in cdr3 sequences responding to same pathogens across individuals and age groups. this intrinsic divergence between individuals is the major obstacle in finding "public" sequences as optimal biomarkers. nevertheless, we hold a promise for the application of hts data in differential diagnosis because it provides a large number of candidate sequences for biomarker investigation. instead of using single biomarkers such as psa or afp in diagnosis, a combination panel of selected sequences may establish a pathogen-specific sequence library for diagnosis, which holds the potential of unprecedented sensitivity and specificity. recombinant monoclonal antibodies have great potential in the treatment of specific infections. in recent years, several strategies, including phage display libraries, single b cell expression, and b cell immortalization, have been used to discover antibodies against specific antigens (101) . hts of the antibody repertoire, combined with subsequent bioinformatic tools or traditional screening tools, has facilitated the identification of antigenspecific sequences (102) (103) (104) (105) . the methodology predicting antigen specificity completely from analysis of bcr sequences has not been possible yet, albeit it has considerable potential in immune repertoire studies. nevertheless, there have been many efforts made in mining the hts data for functional antibodies (106) . a relatively direct method for identifying such sequences is based on the similarity of amino acid sequence to previously reported antibodies. researchers have successfully found sequences of high identity with the broadly neutralizing antibodies and strain-specific antibodies from established antibody repertoires of patients with influenza infection or vaccination (66) . some of these sequences have proven to have neutralizing activity, validating the potential of deep sequencing-based antibody identification. success of this work also suggests the possibility of monoclonal antibody synthesis without cell cloning for treatment (66) . furthermore, another method using the frequency rank of heavy chain and light chain sequences to predict the function of antibody sequences has been reported successful in mouse models (107) . exciting work by kwong and coworkers demonstrates the feasibility of identifying neutralizing active clones through bioinformatic analysis from hiv patients (108, 109) . they established several steps for interrogating variants of known neutralizing antibody classes from hiv-infected patients, with or without previous knowledge if the patient had antibodies belonging to this family. first, the heavy or light chain sequences, derived from the germline ighv or iglv gene same as template antibody, are isolated from a new donor. then, these sequences were compared with the germline gene for "divergence" and the template antibody sequence for "identity, " generating a contour plot called "divergence/identity plot. " the sequences segregate into clusters in the plot, from which the high divergence and high identity sequences were selected as candidates for neutralizing antibodies. this process is called "grid-based strategy. " then, the germline sequence, candidate sequences, and template antibody sequences of the same class are merged to build a phylogenetic tree rooted by the template sequence, which is called "cross-donor analysis. " the heavy chain sequences from new donor clustered in the subtree of the template sequences are then expressed with template light chain to generate antibodies. most of these sequences have neutralizing activity to hiv-1. the outstanding efficiency of this method was also demonstrated when compared to sequence-based strategy and prevalence-based strategy (108, 109) . lu et al. also validated this phylogenetic method in identifying functional anti-staphylococcus aureus antibodies (110) . these strategies start from previously reported antibody sequences. however, such antibody sequences are not always available, especially during poorly characterized viral infections such as h7n9. pairing the heavy and light chains as an integrated antibody has been another challenge for hts-based immune repertoire analysis. in most cases, researchers only focus on the heavy chain, which causes a critical loss of antibody integrity and leads to problems in following synthesis of artificial monoclonal antibody. two strategies have been reported to correctly pair the heavy chain and light chain sequences based on the frequency or evolution models. reddy and colleagues (107) have pioneered pairing based on the frequency ranks, using plasma cells isolated from bone marrow of immunized mice and matching the two chains of similar rank order. monoclonal antibodies expressed in this way did show antigen specificity. due to the linkage of heavy chain and light chain as an integrated protein, their evolution undergoes the same enzymatic mutation process, and they evolve together to bind the same antigen with high affinity. based on this theoretical foundation, phylogenic analysis has been used as another method to compare the evolutionary topography of the heavy chain and light chain after bioinformatic identification of transcripts related to a known hiv neutralizing antibody (109, 111) . reconstituted novel antibodies consist of phylogenetically matched chains showing similar neutralizing function but less auto-reactivity compared to the mismatched ones. several groups have recently achieved advances in the technology of paired sequencing of antibodies. single-cell pcr has been utilized to create a two-dimensional bar-coded primer matrix to link two chains of the bcr (112) . using this technique, busse and coworkers analyzed paired sequences of over 46,000 b cells in one experiment and accomplished subsequent antibody gene cloning and expression. at the same time, turchaninova and coworkers performed pioneering research in emulsion-based technology for sequencing antibody repertoires of paired chains (113) . they used water-in-oil emulsions for cell-based overlap expansion rt-pcr, although its yield was relatively low yield. another high-throughput paired sequencing method by dekosky et al. used micro well plates to isolate b cells and magnetic beads to capture mrnas (114) . very recently, dekosky's group combined and improved these previous techniques, and developed a costeffective and efficient methodology to establish a more precisely paired repertoire (115) . predicting t cell specificity based on tcr heterodimer sequence is more difficult than antibodies because of the highly variable nature of each of the components of the tcr-peptide-mhc complex (116) . due to the challenges posed by the highly variable cdr3 loop of the tcr and the complexity of predicting protein-protein interactions (117, 118) , experimental functional tests for mining antigen-specific t cells might be a more fruitful approach (119). recent advances in hts-based antibody sequencing may provide the biggest benefit for the field of vaccine development. over the years, efforts to elicit protective immune responses to hiv by immunization have not been successful. during acute viral infections, high-affinity neutralizing antibodies develop in just weeks. however, generating effective broadly neutralizing antibodies during chronic infections, such as hiv, takes significantly longer time. furthermore, the neutralizing power of these antibodies is often variable due to impairment of the host immune function, unusual features of env, and co-evolution of the virus in response to the host antibody response (120, 121) . deep sequencing analysis has identified rare variants of known hiv-neutralizing antibodies and has elucidated the ontogeny of these neutralizing antibodies (108, 109, 111, 122) . these findings have cast a light on antibody guided vaccine development. in following studies, the hts-based phylogenetic strategy greatly facilitated study in co-evolution of neutralizing antibodies and virus mutants (123) . combined with long-term follow-up studies, these results illustrate how mutations in some sites allow the virus to escape some neutralizing antibodies, and how the virus, with the help of secondary neutralizing antibodies, becomes sensitive to the neutralizing antibody (98, 124, 125) . these studies suggest a promising pathway to elicit broadly neutralizing antibodies by sequential immunization with selected immunogens (123, 126) . furthermore, structure studies of the neutralizing antibody family provide candidates for future vaccine designs (127) . high-throughput sequencing has been a breakthrough technology for the study of the immune repertoire and has already had a profound effect on our knowledge of the immune systems physiology during health and disease. in particular, hts has transformed our understanding of immune repertoire formation during infection, malignancy, and autoimmunity. advances in this filed of research will rely on progress of similar laboratory techniques. collection of more precise sequencing data can be anticipated with consistent improvement of sequencing techniques and error correction strategies. an increasing number of researchers have shown interest in this area in recent years, and this has provided vast quantities of data that could provide answers to important existing questions. these data repositories should be effectively utilized. establishing a public database and collecting deep sequencing data for collective collaboration will facilitate information exchange and the investigation of the varieties of repertoires across gender, age, race, and healthy state. in addition, development of standardized bioinformatic tools will be indispensable for harnessing hts output. although the expected potential of immune repertoire studies in clinical use is enormous, more work remains to be done to incorporate the observed dynamic changes and sequence signatures with clinical features and outcomes. questions remain about how the severity of certain infections is related to alterations of the immune repertoire response and various manifestations of cdr3 sequences, and how to predict abundance of protective immunoglobulins or t cell from a given sequence library. in terms of therapeutic discoveries, identification and production of functional antibodies and t cells will promote the development of passive immune therapies and vaccines. traditional and recently reported large-scale screening strategies may contribute greatly to this process. advances in hts of the immune repertoire during health and disease will provide comprehensive views of the adaptive immune response in very near future and will open the door to more rationale immunotherapy for infection. dh was involved in study design, wrote the first draft of the manuscript, conducted the literature search, reviewed the abstracts, performed the analysis, and contributed to the final draft; cc and sc were involved in study design and reviewed the abstracts. es revised the manuscript. ys designed and supervised the study, revised the final draft, and contributed to the analysis. all authors have read and approved the final manuscript. analysis of the b-cell progenitor compartment at the level of single cells tools to therapeutically harness the human antibody response sequence analysis of t-cell repertoires in health and disease immunology: the roots of antibody diversity the sizes of the cdr3 hypervariable regions of the murine t-cell receptor beta chains vary as a function of the recombined germ-line segments somatic generation of antibody diversity dynamic programming of cd8+ t lymphocyte responses a cellular calculus for signal integration by t cells selective expansion of high-or low-avidity cytotoxic t lymphocytes and efficacy for adoptive immunotherapy t cell affinity maturation by selective expansion during infection cutting edge: cd4 and cd8 t cells are intrinsically different in their proliferative responses differential kinetics of antigen dependency of cd4+ and cd8+ t cells the past, present, and future of immune repertoire biology -the rise of next-generation repertoire analysis circulating t cell repertoire complexity in normal individuals and bone marrow recipients analyzed by cdr3 size spectratyping. correlation with immune status t-cell repertoire diversity and clonal expansions in normal and clinical samples rapid t cell receptor delineation reveals clonal expansion limitation of the magnitude of the hiv-1-specific cd8+ t cell response t lymphocyte repertoire in theiler's virus encephalomyelitis: the nonspecific infiltration of the central nervous system of infected sjl/j mice is associated with a selective local t cell expansion conserved t cell receptor repertoire in primary and memory cd8 t cell responses to an acute viral infection vaccination-induced changes in human b-cell repertoire and pneumococcal igm and iga antibody at different ages a profound alteration of blood tcrb repertoire allows prediction of cerebral malaria human immunoglobulin (ig)m+igd+ peripheral blood b cells expressing the cd27 cell surface antigen carry somatically mutated variable region genes: cd27 as a general marker for somatically mutated (memory) b cells tracing b cell development in human germinal centres by molecular analysis of single cells picked from histological sections using synthetic templates to design an unbiased multiplex pcr assay toward a more accurate view of human b-cell repertoire by next-generation sequencing, unbiased repertoire capture and single-molecule barcoding identification of errors introduced during high throughput sequencing of the t cell receptor repertoire a survey of error-correction methods for next-generation sequencing denoising dna deep sequencing data-high-throughput sequencing errors and their correction next generation sequencing for tcr repertoire profiling: platform-specific features and correction algorithms accuracy and quality assessment of 454 gs-flx titanium pyrosequencing a tale of three next generation sequencing platforms: comparison of ion torrent, pacific biosciences and illumina miseq sequencers evaluation of genomic high-throughput sequencing data generated on illumina hiseq and genome analyzer systems characterizing and measuring bias in sequence data bfc: correcting illumina sequencing errors hitec: accurate error correction in high-throughput sequencing data lighter: fast and memory-efficient sequencing error correction without counting reptile: representative tiling for short read error correction echo: a reference-free short-read error correction algorithm accurate determination of microbial diversity from 454 pyrosequencing data rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions hector: a parallel multistage homopolymer spectrum based error corrector for 454 sequencing data genetic measurement of memory b-cell recall using antibody repertoire sequencing towards error-free profiling of immune repertoires imgt, the international immunogenetics database(r) reconsidering the human immunoglobulin heavy-chain locus: 1. an evaluation of the expressed human ighd gene repertoire many human immunoglobulin heavy-chain ighv gene polymorphisms have been reported in error the reported germline repertoire of human immunoglobulin kappa chain genes is relatively complete and accurate igblast: an immunoglobulin variable domain sequence analysis tool ihmmune-align: hidden markov model-based alignment and identification of germline genes in rearranged immunoglobulin gene sequences decombinator: a tool for fast, efficient gene assignment in t-cell receptor sequences using a finite state machine soda2: a hidden markov model approach for identification of immunoglobulin rearrangements model for comparative analysis of antigen receptor repertoires personalized, sequencing-based immune profiling spurs startups preparing unbiased t-cell receptor and antibody cdna libraries for the deep next generation sequencing profiling change-o: a toolkit for analyzing large-scale b cell immunoglobulin repertoire sequencing data immunexplorer (imex): a software framework for diversity and clonality analyses of immunoglobulins and t cell receptors on the basis of imgt/highv-quest preprocessed ngs data lymanalyzer: a tool for comprehensive analysis of next generation sequencing data of t cell receptors and immunoglobulins tcr: an r package for t cell receptor repertoire advanced data analysis mitcr: software for t-cell receptor sequencing data analysis imonitor: a robust pipeline for tcr and bcr repertoire analysis scireptor: analysis of single-cell level immunoglobulin repertoires vdjtools: unifying post-analysis of t cell receptor repertoires characterizing immune repertoires by high throughput sequencing: strategies and applications junctional and allele-specific residues are critical for mers-cov neutralization by an exceptionally potent germline-like antibody longitudinal analysis of the peripheral b cell repertoire reveals unique effects of immunization with a new influenza virus strain imgt/v-quest, an integrated software program for immunoglobulin and t cell receptor v-j and v-d-j rearrangement analysis human responses to influenza vaccination show seroconversion signatures and convergent antibody rearrangements effects of aging, cytomegalovirus infection, and ebv infection on human b cell repertoires antibody light-chain-restricted recognition of the site of immune pressure in the rv144 hiv-1 vaccine trial is phylogenetically conserved lower igg somatic hypermutation rates during acute dengue virus infection is compatible with a germinal center-independent b cell response diverse antibody genetic and recognition properties revealed following hiv-1 envelope glycoprotein immunization tracking global changes induced in the cd4 t-cell receptor repertoire by immunization with a complex antigen using short stretches of cdr3 protein sequence dynamic perturbations of the t-cell receptor repertoire in chronic hiv infection and following antiretroviral therapy neutralizing antibodies against west nile virus identified directly from human b cells by single-cell analysis and next generation sequencing high-resolution analysis of the human t-cell receptor repertoire automated analysis of high-throughput b-cell sequencing data reveals a high frequency of novel immunoglobulin v gene segment alleles maximum entropy models for antibody diversity highthroughput sequencing of the zebrafish antibody repertoire precise determination of the diversity of a combinatorial antibody library gives insight into the human immunoglobulin repertoire in-depth assessment of within-individual and inter-individual variation in the b cell receptor repertoire partitioning diversity into independent alpha and beta components estimating the richness of a population when the maximum number of classes is fixed: a nonparametric solution to an archaeological problem origin and t cell receptor diversity of foxp3+cd4+cd25+ t cells estimating the number of species in a stochastic abundance model diversity and clonal selection in the human t-cell repertoire bayesian multivariate poisson abundance models for t-cell receptor data estimation of t-cell repertoire diversity and clonal size distribution by poisson abundance models method for assessing the similarity between subsets of the t cell receptor repertoire convergent antibody signatures in human dengue comparing a few snp calling algorithms using low-coverage sequencing data high-resolution antibody dynamics of vaccine-induced immune responses antiretroviral therapy reduces the magnitude and t cell receptor repertoire diversity of hiv-specific t cell responses without changing t cell clonotype dominance complex t-cell receptor repertoire dynamics underlie the cd8+ t-cell response to hiv-1 immune surveillance by cd8alphaalpha+ skin-resident t cells in human herpes virus infection studying the antibody repertoire after vaccination: practical applications lineage structure of the human antibody repertoire in response to influenza vaccination identification and characterization of the constituent human serum antibodies elicited by vaccination targeting tlrs expands the antibody repertoire in response to a malaria vaccine sequencing the functional antibody repertoire -diagnostic and therapeutic discovery tcr repertoire analysis by next generation sequencing allows complex differential diagnosis of t cell-related pathology identification of antigen-specific b cell receptor sequences using public repertoire analysis selecting and screening recombinant antibody libraries synthetic antibodies designed on natural sequence landscapes construction of a rationally designed antibody platform for sequencing-assisted selection shaping of human germline igh repertoires revealed by deep sequencing deep sequencing of phage display libraries to support antibody discovery the application of next generation sequencing to the understanding of antibody repertoires monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells de novo identification of vrc01 class hiv-1-neutralizing antibodies by next-generation sequencing of b-cell transcripts mining the antibodyome for hiv-1-neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains identifying functional anti-staphylococcus aureus antibodies by sequencing antibody repertoires of patient plasmablasts somatic populations of pgt135-137 hiv-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics single-cell based high-throughput sequencing of full-length immunoglobulin heavy and light chain genes pairing of t-cell receptor chains via emulsion pcr high-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire in-depth determination and analysis of the human paired heavy-and light-chain antibody repertoire beyond model antigens: high-dimensional methods for the analysis of antigen-specific t cells cdr3 loop flexibility contributes to the degeneracy of tcr recognition deconstructing the peptide-mhc specificity of t cell recognition combinatorial hla-peptide bead libraries for high throughput identification of cd8(+) t cell specificity broadly neutralizing antibodies and the search for an hiv-1 vaccine: the end of the beginning human antibodies that neutralize hiv-1: identification, structures, and b cell ontogenies focused evolution of hiv-1 neutralizing antibodies revealed by structures and deep sequencing co-evolution of a broadly neutralizing hiv-1 antibody and founder virus cooperation of b cell lineages in induction of hiv-1-broadly neutralizing antibodies maturation and diversity of the vrc01-antibody lineage over 15 years of chronic hiv-1 infection b-cell-lineage immunogen design in vaccine development with hiv-1 as a case study structural repertoire of hiv-1-neutralizing antibodies targeting the cd4 supersite in 14 donors key: cord-266085-914y3je0 authors: correa, isabel; ilieva, kristina m.; crescioli, silvia; lombardi, sara; figini, mariangela; cheung, anthony; spicer, james f.; tutt, andrew n. j.; nestle, frank o.; karagiannis, panagiotis; lacy, katie e.; karagiannis, sophia n. title: evaluation of antigen-conjugated fluorescent beads to identify antigen-specific b cells date: 2018-03-23 journal: front immunol doi: 10.3389/fimmu.2018.00493 sha: doc_id: 266085 cord_uid: 914y3je0 selection of single antigen-specific b cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. to establish this, we selected folate receptor alpha (frα) as a model antigen and a mouse b cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (mov18 igg1) specific for frα, as test antibody-expressing cells. beads were conjugated to frα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-frα. bead-bound cells were single cell-sorted and processed for single cell rna retrotranscription and pcr to isolate antibody heavy and light chain variable regions. variable regions were then cloned and expressed as human igg1/k antibodies. like the original clone, engineered antibodies from single cells recognized native frα. to evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (pbmcs) were spiked with test antibody-expressing cells. antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. in pbmc pools, beads conjugated to recombinant antigens frα and her2 bound antigen-specific anti-frα mov18 and anti-her2 trastuzumab antibody-expressing cells, respectively. from melanoma patient-derived b cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. this approach may be further developed to facilitate analysis of b cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. the selection and study of single antigen-specific b cells and the identification of their expressed antibodies can help shed light on the nature and functions of the human humoral immune response. in our study, we present an antigen-conjugated fluorescent bead methodology designed to identify and isolate single antibody-expressing cells and to derive and clone matched heavy and light chain antibody variable regions into full length antibodies. we believe that our single cell-to-functional antibody strategy falls within the scope of this research topic, as it opens the way for opportunities to analyze b cells and their antibody profiles at the single cell level and may be potentially applied to help unravel diverse humoral immune repertoires in different health and disease states. the study of humoral immunity may be key to understand human health, aging, and disease, as well as to predict or monitor host immune responses to pathogen challenge and disease progression (1) . moreover, dissecting b cell responses could help inform medical interventions such as preventative vaccines or therapeutic treatments (2, 3) . currently, therapeutic monoclonal antibodies are part of the standard care of treatment for various conditions including diabetes, autoimmune and allergic diseases, and various types of cancer. a big proportion of these antibodies are fully mouse or mouse-human chimeras (4) . therefore, identification of fully human, heavy (h), and light (l) variable regionmatched antibodies may be of interest for clinical applications, as administration of fully human antibodies is considered less likely to induce antidrug antibody responses compared with chimeric or humanized antibodies (5) . immune monitoring of human b cells recognizing specific antigens, alongside identification, cloning, and production of their cognate monoclonal antibodies, may, therefore, be of substantial value to help evaluate reactivity and immunological response to these antigens. analyses of the antibody repertoires of b cells have been reported in the human setting and in animal models, with the most common applications in the study of autoimmune diseases (6) , viral infections, and b cell malignancies (7) (8) (9) . some studies have employed high-throughput sequencing of heavy chain variable regions or of paired h and l variable region sequences (10, 11) . ultra-high-throughput sequencing techniques have also been developed, capable of analyzing repertoires of thousands of b cells from small human blood samples, with some allowing the mapping of paired h and l variable regions, in a few hours (12) (13) (14) . b cells may also be a source of antigen-specific antibodies for clinical diagnostics or for therapeutic applications. existing methods to identify antigen-specific b cells and generate monoclonal antibodies include ex vivo b cell culture approaches, which could promote the survival and expansion of certain b cell subsets, screening of the culture supernatants to identify b cell reactivity and fluorescent-activated cell sorting (15) (16) (17) (18) (19) (20) . an essential element in the process of selecting antigen-specific b cells is detection of antibodies with a certain degree of specificity. this could be achieved by screening cell culture supernatants through elispot assays or elisa-based methods using immobilized recombinant antigens or cells (16, 20) . screening cell culture supernatants by elisa, although highly sensitive, represents only a surrogate parameter and antigen reactivity should ultimately be confirmed only after sequencing and expression of the selected clone. for all these applications, the gold standard of identifying antigen-specific antibodies remains the expression of the recombinant antibody and further evaluation of its antigen recognition properties. workflows to facilitate selection of single human b cells without ex vivo growth, stimulation, and clone expansion, and which do not require sampling of cell culture supernatants could offer additional tools for the study of human b cell immunity. novel approaches to address these requirements involve the use of modified fluorescent tetramers for direct b cell screening by fluorescent-activated cell sorting (21, 22) . in this study, we describe the design of a bead-based methodology to identify single antibody-expressing b cells, and to clone and produce antigen-specific antibodies. the workflow features bead-based identification and isolation of specific b cells using direct fluorescent-activated cell sorting, sequencing, and cloning of matched heavy and light chain variable regions in a single full sequence antibody expression vector system, and expression and testing the antigenic reactivity of the antibody clone. the workflow is designed to avoid ex vivo b cell expansion and secondary clone selection and to facilitate antibody generation and downstream evaluation. human immune cells were isolated from venous blood of healthy volunteers and patients with malignant melanoma. specimens were collected with informed written consent in accordance with the declaration of helsinki. the study was conducted at king's college london, king's college london, guy's and st thomas' nhs foundation trust (08/h0804/139 approved by london bridge nres committee; 16/lo/0366 approved by london-central nres committee). human peripheral blood mononuclear cells (pbmc) were isolated from 40 ml blood using ficoll ® paque plus density centrifugation (ge healthcare). cell culture was performed using aseptic technique at 37°c in a humidified atmosphere in 5% co2, unless otherwise specified. the human ovarian carcinoma cell line igrov1 naturally over-expressing folate receptor alpha (frα) was grown in rpmi 1640 glutamax™ medium (thermo scientific) supplemented with 10% fetal calf serum (fcs). the human breast cancer cell line mda-mb-231 was grown in dmem glutamax™ medium (thermo scientific) supplemented with 10% fcs. the permanently transfected murine myeloma cell lines sp2/0-mov18 specific for frα and sp2/0-sf25, recognizing a colon carcinoma antigen (23) , were cultured in dulbecco's modified eagle's medium plus 10% fcs as previously described (24) . the human embryonic kidney cell lines, expi293f cells, were cultured in serum-free expi293 expression medium (thermo scientific) on a stuart orbital shaker at 125 rpm at 8% co2. expi293f cells were transfected with pvitro1-hygro-mcs antibody constructs using the expifectamine293 transfection kit (thermo scientific) as per manufacturer's instructions. the anti-human epidermal growth factor receptor 2 (her2) and the melanomaassociated antigen-specific chondroitin sulfate proteoglycan (cspg4) antibody constructs were previously described (25, 26) . different avidin-or streptavidin-coated fluorescent beads of different sizes were used (table s1 in to biotinylate proteins of melanoma cell lines, confluent cell monolayers were washed with pbs and borate buffer ph 9. then, sulfo-nhs-lc-biotin at 1 mg/ml in pbs was added and incubated for 20 min on ice. the reaction was quenched with 200 mm tris, 120 mm nacl ph 7.4, and the cells were subsequently extensively washed with pbs. cell lysis buffer (cell signaling technology) containing 0.1% triton and supplemented with 2 mm pmsf was added to the monolayer and the cells were harvested by scrapping. lysates were briefly sonicated and incubated on an orbital rocker for 30 min at 4 c. the lysates were cleared by centrifugation at 900 × g for 15 min, followed by a second centrifugation at 12,000 × g for 30 min. supernatants were either used immediately or stored at −80°c. recombinant folate receptor α (frα) (r&d systems, cat no 5646-fr) and human epidermal growth factor receptor 2 (erbb2/her2) fc chimera (r&d systems, cat no 1129-er) were reconstituted in pbs at 0.1 mg/ml as recommended by the manufacturer. biotins with three different arms were used: (i) sulfo-nhs-lc-biotin with a spacer arm of 22.4 å; (ii) sulfo-nhs-lc-lc-biotin with a spacer arm of 30.5 å, and (iii) nhs-peg12-biotin with a spacer arm of 56 å (all from pierce-thermo fisher scientific). the three forms of biotin were reconstituted following the manufacturer's instructions and mixed with the recombinant proteins in pbs at a 20:1 (biotin:recombinant protein) molar ratio and incubated for 90 min at room temperature (rt). free biotin was removed by dialysis in pbs. for binding of biotin-conjugated frα to lumavidin 5.6 µm microspheres, 100 µl of microsphere solution was washed with 1 ml of 1% bsa-pbs-0.05% sodium azide, spun at 8,000 × g for 2 min, and pellets were resuspended in 100 µl of 1% bsa-pbs-0.05% sodium azide. then, 37.5 µg of biotin-conjugated recombinant protein (in 300 µl of pbs-1% bsa) was added to the beads and mixed, by rotation, for up to 16 h at rt. beads were then washed three times with 1 ml of 1% bsa-pbs-0.05% sodium azide and resuspended in 100 µl of 1% bsa-pbs-0.05% sodium azide. for binding of biotin-conjugated frα to all sphero fluorescent particles, 100 µl of beads were washed with 1 ml of 1% bsa-pbs-0.05% sodium azide, spun at 13,000 × g for 20 min at rt, and the pellet was resuspended in the original volume of beads with 1% bsa-pbs-0.05% sodium azide. then, 25 µg of biotin-conjugated recombinant protein was added and mixed, by rotation, for 2-16 h at rt. after binding, beads were washed three times with 1 ml 1% bsa-pbs-0.05% sodium azide and resuspended in 100 µl of 1% bsa-pbs-0.05% sodium azide. for binding of biotin-conjugated cell extracts to lumavidin 5.6 µm microspheres, 200 µl of microsphere solution were washed with 1 ml of 1% bsa-pbs-0.05% sodium azide, spun at 8,000 × g for 2 min, and pellets were resuspended in 200 µl of 1% bsa-pbs-0.05% sodium azide. then, 300 µl of biotinconjugated cell extracts were added to the beads and mixed, by rotation, for up to 16 h at rt. after binding, beads were washed three times with 1 ml of 1% bsa-pbs-0.05% sodium azide and resuspended in 200 µl of 1% bsa-pbs-0.05% sodium azide. for each test, 15 µl beads were used. to check that frα/her2 proteins were attached on the bead surface, conjugated beads were stained with specific antibodies for frα/her2. conjugated beads (10 µl) were incubated in 200 µl of facs buffer (5% fcs-pbs) for 20 min at rt, then, 1 µg of the monoclonal antibody mov18 (anti-frα), anti-her2 [previously described in ref. (27) ] or isotype control antibodies were added. in all described experiments, the hapten-specific in-house produced monoclonal antibody anti-nip igg was used as an isotype control. the beads were incubated for 30 min at 4°c, washed with 2 ml of facs buffer, and incubated with anti-human immunoglobulin (ig) antibody conjugated to fitc (vector laboratories) for another 30 min at 4°c. after a final wash with facs buffer, stained beads were analyzed on a facscanto™ flow cytometer (bd biosciences). beads coated with melanoma cell line extracts were tested by staining with a monoclonal igg antibody recognizing the melanoma-associated antigen cspg4. sp2/0 cells expressing mov18 igg or the control antibody sf25 were prestained with anti-mouse mhc class i-pe antibody to be used with streptavidin sa-blue 1.1 µm or lumavidin 5.6 µm beads (table s1 in ig variable region cloning pcr tubes containing sorted cells in lysis buffer were thawed and reverse transcription was performed in a thermal cycler using the superscript vilo cdna synthesis kit (thermo fis her scientific) following the manufacturer's recommendations. variable regions of ig heavy and light chains were amplified by two rounds of pcr amplification in 20 µl mix, using 10 µl of phusion flash polymerase mix per reaction (finnzymes), each primer at 0.5 µm, 2 µl of the reverse transcriptase reaction as template for the first table 1) , and the nested pcr protocol were previously described (28) . pcr products were separated on agarose gels, purified using the qiaquick gel extraction kit (qiagen), and cloned using the zeroblunt pcr cloning kit (life technologies). six colonies were grown for each pcr band, plasmid dna purified using qiaprep spin miniprep kit (qiagen) and sequenced by source bioscience using the m13 forward primer. sequences were analyzed using imgt/v-quest (29). heavy and light chain variable regions were cloned in pvitrohygro-1 (invivogen) using polymerase incomplete primer extension (pipe) cloning as previously described (25) . in brief, pvitro-hygro-1, containing pre-cloned human antibody heavy and light constant region cassettes (gamma 1/kappa) was used as a template in two separate pipe pcr reactions to amplify two linear plasmid fragments with partially single-stranded 5″ ends. similarly, the ig heavy and light chain variable regions were pipe pcr-amplified, using the pcr-blunt constructs, generated previously, as pcr templates. next, the pcr products were diluted four times with ddh2o and mixed in a ratio of 1 we designed a process to allow the identification of antigenspecific antibody-expressing b cells, which can be performed without prior ex vivo growth or secondary screening of b cells. to establish this system, we selected folate receptor alpha (frα) as a model antigen. as the test antibody-expressing cells, we selected a b cell line expressing both the soluble and the membranebound [b cell receptor (bcr)] form of a human/mouse chimeric antibody (mov18 igg1 clone) specific for frα. the workflow (figure 1 ) entails coupling of fluorescent polystyrene beads with the nominal antigen, folate receptor alpha (frα), followed by binding of frα-coated beads to anti-frα antibody-expressing b cells. single bead-conjugated cells were identified and isolated by facs sorting directly onto microplates containing lysis buffer. rna released from single cells was converted to cdna using reverse transcription followed by a semi-nested rt-pcr with ig specific primers. matched variable h and l chains were sequenced and cloned into a single expression vector containing the h and l constant igg1 region sequences as previously reported (25, 26) . the full antibody was then expressed in a human expression host. the antibody was purified and antibody specificity for the antigen was tested on target antigen-expressing cells. we first established the workflow using apc fluorochrome lumavidin microspheres of 5.6 µm diameter (lumavidin 5.6 µm; table s1 in supplementary material). prior to interrogating the ability of these beads to bind antibody-expressing cells, we used flow cytometric evaluations to confirm that fluorescent beads could be successfully coupled to antigens. we tested the antigens frα and her2 conjugated to fluorescent beads. the anti-frα antibody mov18 igg bound to frα-conjugated beads, and the her2-specific antibody trastuzumab bound to her-2conjugated beads. on the other hand, the hapten-specific monoclonal antibody nip igg showed only background binding to frα-conjugated or to her-2-conjugated beads, and no binding was detected on beads incubated with secondary antibody alone (figure 2a) . we also interrogated different coupling agents with varying biotin lengths (lc-biotin, 22.4 å; lc-lc-biotin, 30.5 å; peg12-biotin, 56 å) to assess whether the distance between bead and antigen from the bead surface could affect antigen recognition by anti-frα-expressing b cells. we found no significant differences in mov18 igg antibody binding to bead-conjugated frα with respect to any of these coupling agents ( figure 2b) . we then established a model system using microspherecoupled frα and anti-frα antibody-expressing b cells. we confirmed that a small proportion of frα+ lumavidin 5.6 µm microspheres recognized single anti-frα antibody-expressing b cells (sp2/0-mov18) ( figure 3a) . when single bead-bound cells were isolated into lysis buffer-containing microplate wells by flow cytometric sorting, single cell pcr yielded matched h and l chain variable region dna products were detected to be of the expected size ( figure 3b) . antibody variable region sequences were cloned into a pvitro1 vector containing human igg1/k constant region cassettes and the antibody was expressed in expi293f™ cells. subsequently, the cloned igg1 antibody was used to assess binding to igrov1 cells, which express native cell surface human frα. flow cytometric evaluations confirmed that the anti-frα antibody cloned from a sorted single antibody-expressing b cell was able to specifically recognize antigen-expressing igrov1 cells. the cloned anti-frα antibody did not recognize mda-mb-231 breast carcinoma cells that do not express frα (figure 3c) . these findings confirmed that it is possible to extract matched variable region sequences from single antibody-expressing cells selected by recognition of specific antigen-coated microspheres. selection of single b cells allowed the identification of the expected matched variable regions and permitted the cloning and production of the corresponding monoclonal antibody from a single cell. this antibody was able to recognize natively expressed cell surface frα. influence of microsphere diameter and biotin length on specific recognition of antigen-expressing b cells since we observed that frα+ microspheres were able to bind only a small proportion of single anti-frα antibody-expressing b cells figure 1 | workflow for identification of antigen-specific monoclonal antibodies derived from single cell cloning. biotinylated antigen (folate receptor alpha, frα) was conjugated to fluorescent beads conjugated to streptavidin or avidin. antigen-coated beads could bind cells expressing an antigen-specific immunoglobulin (ig) and bead-bound cells were purified using cell sorting. rna from sorted cells was reverse transcribed and ig variable regions were amplified by nested pcr, sequenced, and cloned using the dual expression plasmid pvitro1 containing human heavy and light chain constant region cassettes (gamma1/kappa). antibodies were expressed in a human cell expression system, purified, and tested for antigen binding. (sp2/0-mov18), we investigated whether microsphere diameter or the frα-biotin on the beads could influence specific recognition of antibody-expressing cells. we confirmed that sp2/0-mov18 igg cells expressing mov18 igg on the cell surface could be recognized by human recom binant frα ( figure 4a ). as observed with recombinant antibody binding to frα-coated microspheres (figure 2) , the distance between bead and antigen from the bead surface did not affect antigen recognition by anti-frα antibody-expressing sp2/0-mov18 cells ( figure 4b) . however, the proportion of the sp2/0-mov18 cells recognized by fluorescent beads coated with frα differed with bead size or type: microspheres of smaller bead diameters (sa-red 0.5 µm and sa-blue 1.1 µm) appeared to bind a higher proportion of b cells (70.3 and 54.9%, respectively) compared to the lumavidin 5.6 µm beads (1.4%) (figures 4c,d) . the length of the biotin arm had minor effects on the binding of the smaller sized (a-red 0.8 µm and sa-blue 1.1 µm) microspheres to sp2/0-mov18 cells. the peg12-biotin arm showed a slightly higher proportion of microsphere-attached sp2/0-mov18 cells (69-71% of cells) compared with beads attached to frα via lc-lc-biotin (59-61%) or lc-biotin (54-64%) arms ( figure 4e) . binding of frα-coated fluorescent beads appeared to be specific for antibody-expressing b cells, since frα-coated beads bound 54-71% of sp2/0-mov18 igg cells, while frα-coated beads bound 6-9% of sp2/0 non-specific antibody-expressing cells used as controls ( figure 4e) . these findings suggest that bead size or type could influence the proportion of possible antibody-expressing b cells recognized by antigen-conjugated fluorescent beads. since alongside specific binding of beads to antibody-expressing b cells, we also detected non-specific binding of frα-coated beads to control sp2/0 cells (figure 4e) , we further investigated the background binding of fluorescent beads to freshly isolated human pbmcs. a proportion (~1%) of human pbmcs (top panel) and also of human b cells (cd19+ cells, lower panel) could bind to frα-coated beads in most likely a non-specific manner ( figure 5a) . different blocking agents did not appear to reduce the levels of background binding (table s2 in supplementary material). furthermore, a-red 0.8 µm, sa-blue 1.1 µm, and lumavidin 5.6 µm frα-coupled (peg12-biotin) beads were incubated with pbmcs and binding was directly compared to recognition of sp2/0-mov18 igg cells (used as positive controls). frα-coupled a-red 0.8 µm microspheres showed 54.3% specific and 0.56% non-specific b cell recognition, while sa-blue 1.1 µm microspheres showed 53.4% specific and 1.36% non-specific recognition of b cells. on the other hand, the larger sized lumavidin 5.6 µm microspheres showed 8.7% specific and 0.07% non-specific binding ( figure 5b) . this suggested that the smaller diameter beads were more likely to select antibody-expressing cells. in order to analyze whether, and at what frequency, anti-frα antibody-expressing b cells in pbmc samples may be detected by frα-coupled beads, we spiked human pbmc with sp2/0-mov18 cells at different frequencies and we detected double-positive (frα+/sp2/0+) events by flow cytometry (figure 6a) . frα-coupled a-red 0.8 µm fluorescent beads were incubated with pbmc (1 µl beads/10 6 pbmc, table s1 in supplementary material) in the presence of serially diluted (1:50, 1:500, 1:5,000) sp2/0-mov18 cells pre-labeled with an anti-cd45 antibody. the actual dilution was calculated post acquisition ( figure 6b) . actual positive events were defined as anti-frα antibody-expressing b cells among all bead-bound cells and used to evaluate potential specific selection of real antigen-specific cells. the actual true events were defined as antigen-specific b cells bound to beads among all possible antibody-specific sp2/0+ events and used to estimate the selection of antigen-binding cells compared to all specific cells in a sample. we observed that frα+ beads were able to identify 49% actual positive events (b cells) when the specific b cell frequency was ~1:50. the proportion of actual positive events (anti-frα antibody-expressing b cells) was lower when the specific b cell frequencies in the pbmc pool were reduced (figures 6b,c) . the actual true events (b cells bound to beads among all possible antibody-expressing sp2/0+ events) ranged between 9.4 and 18% (figures 6b,c) . beads of different types and diameter sizes varied in ability to detect actual positive events, and the proportion of actual positive events (among all bead-bound cells) decreased when the frequencies of frα+/sp2/0+ b cells were lower among pbmcs. between 5 and 75% of antibodyexpressing b cells recognized by beads were antigen-specific when b cells were found in high frequencies (≥1:100) in human blood. sa-blue 1.1 µm lc-lc biotin beads detected the highest proportion (75%) when b cells were found at a frequency of 1:43 ( figure 6d ; table s3 in supplementary material). to confirm selection of antibody-expressing cells by specific antibody, pbmc spiking showed that frα-coated beads were more likely than her2-coated beads to be recognized by frα-specific sp2/0-mov18 cells (figure 7a ; figure s1 in supplementary material). anti-sf25 antibody-expressing sp2/0 cells were also less likely to be detected by frα-coated beads in pbmc samples (figure 7a) . similarly, her2-coated beads were more likely to bind anti-her2 (trastuzumab)expressing expi293f cells compared with anti-cspg4 control antibody-expressing expi293f cells. her2-coated beads were also more likely to bind anti-her2 (trastuzumab)-expressing expi293f cells compared with frα-coated beads, which showed low binding to anti-her2 (trastuzumab)-expressing expi293f cells (figure 7b) . human ig expression on the surface of expi293f cells is shown in figure s1 in supplementary material. together, these data suggest that antibody-expressing cells in pbmc preparations are able to recognize beads conjugated to their specific antigens. to interrogate this approach in the human setting, we evaluated whether fluorescent beads conjugated to melanoma cell surface antigens were able to identify antigen-reactive b cells. we prepared fluorescent beads coated with antigens extracted from human melanoma sk-mel-28 cells, which natively express the tumor-associated antigen chondroitin sulfate proteoglycan 4 (cspg4). melanoma antigen-coated beads were recognized by an anti-cspg4 antibody but not by mov18 antibody specific for the antigen frα not expressed by sk-mel-28 cells ( figure s2 in supplementary material), suggesting that antigen-reactive antibodies can specifically recognize antigen bound on these beads. we then screened for antigen-reactive antibodies from human b cells. pbmcs from patients with melanoma incubated with melanoma antigen-coated beads were screened to identify mature cd19/cd22+ b cells recognizing antigen-coated beads. single bead-bound b cells were isolated by single cell sorting ig heavy and light chain sequences were amplified, sequenced, and antibodies were cloned (figures 8a,b ). an antibody derived from a single bead-bound b cell could recognize melanoma antigen-coated fluorescent beads compared with secondary only or a non-specific antibody control. the b cell-derived clone showed binding comparable to that of a monoclonal antibody specific for the melanoma-associated antigen cspg4 (figure 8c) . taken together, these findings suggest that antigen-reactive b cells in human blood could be recognized by antigen-conjugated fluorescent beads and could be sorted by flow cytometry for the expression of monoclonal antibodies. in this report, we present the development of a fluorescent bead method for the selection of single antigen-reactive b cell clones and the production of the cloned antigen-specific antibodies for downstream testing. the workflow comprises: (a) conjugation of fluorescent microspheres with a recombinant antigen of interest; (b) identification of fluorescently labeled single antibodyexpressing b cells by flow cytometry using antigen-conjugated beads; (c) flow cytometric sorting of bead-bound single cells directly into lysis buffer; (d) single cell retrotranscription and sequencing of matched heavy and light chain antibody variable regions; (e) cloning and expression using a vector containing human constant region cassettes; (f) confirmation of antigen reactivity of the produced full-length antibody. we designed this process to allow the identification of antigen-specific antibodyexpressing b cells without the requirement of prior ex vivo growth or secondary screening of b cells, and we aimed to conduct the workflow from clone identification to antibody production and characterization in a timeframe of approximately 23 days. to design this protocol, we employed a model system featuring human recombinant folate receptor alpha (frα) as the target antigen, a b cell line expressing both the soluble and the membranebound forms of mov18 igg1, a human/mouse chimeric antibody specific for frα. we showed that fluorescent microspheres can be coupled with the recombinant antigen via biotin-streptavidin/ avidin bridging, and that immobilized antigens could be readily detected by antigen-specific monoclonal antibodies. recognition of the epitope on frα by the test antibody mov18 is thought to be dependent on the native folding of the target antigen. here, we demonstrated that it is possible to use antigen-coupled fluorescent beads of different sizes to identify and isolate single b cells expressing cell surface-expressed antibodies that recognize this conformational epitope. antigen-antibody recognition could be improved by evaluating beads with different characteristics and diameters, while varying the lengths of the avidin coupling agents did not significantly influence antibody-expressing cell recognition by the beads. the latter observation also suggested that engagement of the antigen to the bead surface via different biotins did not mask epitope recognition by specific antibody either in solution or when the antibody was expressed on the surface of a b cell. a key feature of this streptavidin-biotin bead-based approach is that, in principle, it may enable the coupling of virtually any known native or recombinant antigen to fluorescent beads and facilitate the detection of b cells reactive to this antigen. our strategy may offer different features compared with those of other detection methods. for instance, antibodies used for detection of bcr on b cells may also interact not only with bcrs but also with fc receptors on human b cells (30) . tetramer technologies may be applicable to the selection of antigen-specific b cells, but fluorophores have been known to be released from antigen complexes and to bind non-specifically to immune cells, yielding false positive events (31) . our protocol is also specifically designed to avoid the requirement for b cell culture and ex vivo expansion or for antibody selection from culture supernatants prior to sub-cloning, limiting dilution and isolation of antigen-specific b cell clones. as well as being laborious and lengthy, these steps may also be limited by specific expansion of distinct b cell populations not always representative of the original b cell repertoire, and which could suffer from potential loss of the antigen-specific clone during the cell ex vivo culture processes (17) . following flow sorting of single cells directly into lysis buffer, we confirmed that it is possible to extract matched h and l chain variable region sequences from single antibody-expressing cells selected by specific antigen-coated beads. employing the frα-mov18 sp2/0 b cell model, we confirmed that b cell selection by frα+ fluorescent beads allowed the identification of the matched variable regions and permitted the cloning and production of the corresponding monoclonal antibody from a single cell. this was greatly expedited with the use of a single vector expi293f™ cell line-based expression system, which permitted antibody production within a few days (25, 26) . while the vector used in this study contained the constant regions of human igg1, in principle, this platform could be used for engineering of b cell-derived antibodies with constant regions of any isotype or species desired, potentially facilitating a wide range of downstream applications for this technology. importantly, we showed that cloned and expressed full-length antibody with variable regions extracted from a single b cell could recognize native cell surface-expressed human frα. the specificity of an identified antibody could ultimately be confirmed only by the expression of the antibody and subsequent testing against the natively expressed antigen. previous published studies report the number of detected b cells and the antigenic reactivity of antibodies secreted in supernatants of ex vivo b cell cultures without cloning, production, or testing of the derived antibody clones (16, 32) . a similar b cell identification process to the one reported in our study described the frequency of antigen-specific b cells detected using a modified bead-based method (31) . with this tool, it was possible to monitor the frequencies of anti-hla, anti-tetanus toxin-, and anti-ebna1-committed b cells in different individuals. however, antibodies from selected b cells are often not cloned and expressed subsequently in order to analyze their ability to recognize natively expressed cognate antigens. our data confirming antibody sequencing, cloning, expression, and antigen recognition provide an early proof of principle that functional ig sequences could be recovered with this methodology. we ascertained that binding of frα-coated fluorescent beads can specifically single out antibody-expressing b cells. we found that frα-coated beads bound up to 71% of possible sp2/0-mov18 igg cells. however, alongside enhanced recognition of possible antigen-reactive b cells, frα-coated beads also bound to 6-9% of non-specific sp2/0 b cells. furthermore, a proportion (~1%) of human circulating b cells could bind to frα-coated beads in most likely a non-specific manner. together, these suggest that background non-specific binding of beads to cells may be a limitation of this methodology. we employed two approaches to evaluate whether antigen-coupled beads could detect antigen-reactive antibody-expressing cells in pbmc samples. our first approach was to "spike" human pbmc with sp2/0-mov18 b cells at different frequencies. we demonstrated that frα + fluorescent beads could identify antibody-expressing cells among pbmc populations when these antigen-reactive cells were present at higher frequencies in human blood. we also found that different bead types had different specific recognition and background recognition profiles. the proportion of actual positive b cells among all bead-bound cells decreased with lower frequencies of frα+/sp2/0+ b cells among pbmcs. when b cells were found in high frequencies (≥1:100) in human blood, the proportion of actual positive events, the specific b cells recognized by the beads, ranged from 5 to 75% of the total bound cells, with the sa-blue 1.1 µm lc-lc biotin beads able to detect the highest proportion (75%). although fluorescent activated cell sorting could enable separating a single cell from a heterogeneous population, the detection of cell populations with a low frequency remains challenging and presents a technical limitation with different protocols (33, 34) . consistent with previous studies, here, we observed great variability in the acquired numbers of cells when analyzing samples with very low antigen-specific b cell frequencies. this implies that only active and high frequency humoral responses may be readily studied in the present context, while the detection of low frequency b cells represents an inherent limitation of this methodology and requires further optimization. as a next step, we asked whether using this protocol, fluorescent beads coated with melanoma cell line antigens may be able to identify antigen-reactive b cells in individuals suffering of malignant melanoma. using melanoma cell line surface antigen-coated fluorescent beads, we expressed a monoclonal antibody that bound to melanoma cell line protein-coated beads. this suggested that antigen-reactive b cells in human blood may be singled out using fluorescent beads. the selection of single antigen-specific b cells and the identification of their expressed antibodies is critical to gaining a deeper understanding of the nature and functions of active human humoral immune responses. identification of b cells, as well as cloning and production of their heavy and light chain matched antigen-specific monoclonal antibodies thus remain highly desirable in immunology research and antibody discovery. consequently, discovery of antigen-specific b cell clones forms the focus of numerous high-and low-throughput approaches (12) (13) (14) . our bead-based protocol may provide an alternative, readily applicable means for exploring human b cells by facilitating the study of single b cell-derived antibodies and their functional profiles. since the frequency of antigen-specific b cells in the human circulation remains ≤1% of the total b cell populations (35) , increasing the probability for more specific selection of such low-frequency b cells remains a major challenge with this and many other available technologies. future efforts may help improve specific selection by incorporating additional selection markers such as beads of multiple fluorophores (31), or b cell activation markers to single out bcr-activated cells more likely to be matured antibody-expressing clones (36, 37 ). an alternative strategy may entail an additional imaging tool to verify selection of single antigen-reactive b cells subsequent to cell sorting (38) . furthermore, adjusting the cloning process to identify antibodies of different subclasses or specificities or from specific b cell subsets could improve the chance of detecting clones and clonal families in certain diseases, in which immunological conditions may promote specific antibody profiles (39) (40) (41) (42) . individually or combined, such approaches may help increase the chances of clonal selection. in summary, we describe the establishment of a methodology to identify single antibody-expressing cells and to produce and test their sequenced recombinant antibodies in a workflow that may be readily applicable in any basic and translational immunology laboratory setting. this single cell-to-functional antibody strategy may open the way for new opportunities to analyze b cells and their antibody profiles at the single cell level and may be potentially applied to help unravel diverse humoral immune repertoires in blood and tissues and in different health and disease conditions. human immune cells were isolated from the venous blood of human volunteers. specimens were collected with informed written consent in accordance with the declaration of helsinki. author contributions sk, pk, kl, and fn conceived the study, and ic, ki, sc, pk, kl, fn, at, and sk designed the methodology. ic, ki, pk, sc, sl, mf, and ac acquired data, generated materials, or helped with the data analysis and interpretation. sk, pk, ki, ic, and sc wrote the manuscript. ic, ki, sc, sl, mf, ac, js, at, fn, pk, kl, and sk discussed and interpreted the data and edited the manuscript. sk supervised the study, led and coordinated the project. we thank all volunteers who participated in this study. we acknowledge the biomedical research centre immune monitoring core facility team at guy's and st. thomas' nhs foundation trust and the nikon imaging centre at kings college london for assistance. assessment of b cell repertoire in humans age-related changes in human peripheral blood igh repertoire following vaccination vaccination-induced changes in human b-cell repertoire and pneumococcal igm and iga antibody at different ages monoclonal antibodies: a review monoclonal antibody therapeutics: history and future detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct rt-pcr method neutralizing antibodies against west nile virus identified directly from human b cells by single-cell analysis and next generation sequencing immunoglobulin kappa variable region gene selection during early human b cell development in health and systemic lupus erythematosus influence of seasonal exposure to grass pollen on local and peripheral blood ige repertoires in patients with allergic rhinitis igg subclass switching and clonal expansion in cutaneous melanoma and normal skin novel method for high-throughput full-length ighv-d-j sequencing of the immune repertoire from bulk b-cells with single-cell resolution ultrahigh-throughput sequencing of the immune receptor repertoire from millions of lymphocytes high-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire high-throughput single-cell analysis of b cell receptor usage among autoantigen-specific plasma cells in celiac disease profiling human antibody responses by integrated single-cell analysis monitoring the systemic human memory b cell compartment of melanoma patients for anti-tumor igg antibodies an efficient method to make human monoclonal antibodies from memory b cells: potent neutralization of sars coronavirus a method for the isolation and characterization of functional murine monoclonal antibodies by single b cell cloning cloning and expression of murine ig genes from single b cells rapid cloning of high-affinity human monoclonal antibodies against influenza virus antigen-specific single b cell sorting and expression-cloning from immunoglobulin humanized rats: a rapid and versatile method for the generation of high affinity and discriminative human monoclonal antibodies ex vivo characterization and isolation of rare memory b cells with antigen tetramers chimeric murine-human antibodies directed against folate binding receptor are efficient mediators of ovarian carcinoma cell killing comparison of ige and igg antibody-dependent cytotoxicity in vitro and in a scid mouse xenograft model of ovarian carcinoma a tool kit for rapid cloning and expression of recombinant antibodies functionally active fc mutant antibodies recognizing cancer antigens generated rapidly at high yields characterisation of an engineered trastuzumab ige antibody and effector cell mechanisms targeting her2/neu-positive tumour cells allergen specificity of igg(4)-expressing b cells in patients with grass pollen allergy undergoing immunotherapy immunoglobulin and t cell receptor genes: imgt((r)) and the birth and rise of immunoinformatics isolation of antigenspecific b cells characterization of antigen-specific b cells using nominal antigen-coated flow-beads ex-vivo clonally expanded b lymphocytes infiltrating colorectal carcinoma are of mature immunophenotype and produce functional igg functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (np)-specific, igg1+ b cells from antibody-secreting and memory b cell pathways in the c57bl/6 immune response to np antigenspecific b cell memory: expression and replenishment of a novel b220(-) memory b cell compartment isolation and characterization of human antigen-specific b lymphocytes role of syk in b-cell development and antigen-receptor signaling kinetics of b cell receptor signaling in human b cell subsets mapped by phosphospecific flow cytometry assurance of monoclonality in one round of cloning through cell sorting for single cell deposition coupled with high resolution cell imaging igg4 subclass antibodies impair antitumor immunity in melanoma elevated igg4 in patient circulation is associated with the risk of disease progression in melanoma a therapeutic antibody for cancer, derived from single human b cells human regulatory b cells in health and disease: therapeutic potential the authors are solely responsible for study design, data collection, analysis, decision to publish, and preparation of the manuscript. key: cord-280941-ds6x0yym authors: kim, young-seok; son, ahyun; kim, jihoon; kwon, soon bin; kim, myung hee; kim, paul; kim, jieun; byun, young ho; sung, jemin; lee, jinhee; yu, ji eun; park, chan; kim, yeon-sook; cho, nam-hyuk; chang, jun; seong, baik l. title: chaperna-mediated assembly of ferritin-based middle east respiratory syndrome-coronavirus nanoparticles date: 2018-05-17 journal: front immunol doi: 10.3389/fimmu.2018.01093 sha: doc_id: 280941 cord_uid: ds6x0yym the folding of monomeric antigens and their subsequent assembly into higher ordered structures are crucial for robust and effective production of nanoparticle (np) vaccines in a timely and reproducible manner. despite significant advances in in silico design and structure-based assembly, most engineered nps are refractory to soluble expression and fail to assemble as designed, presenting major challenges in the manufacturing process. the failure is due to a lack of understanding of the kinetic pathways and enabling technical platforms to ensure successful folding of the monomer antigens into regular assemblages. capitalizing on a novel function of rna as a molecular chaperone (chaperna: chaperone + rna), we provide a robust protein-folding vehicle that may be implemented to np assembly in bacterial hosts. the receptor-binding domain (rbd) of middle east respiratory syndrome-coronavirus (mers-cov) was fused with the rna-interaction domain (rid) and bacterioferritin, and expressed in escherichia coli in a soluble form. site-specific proteolytic removal of the rid prompted the assemblage of monomers into nps, which was confirmed by electron microscopy and dynamic light scattering. the mutations that affected the rna binding to rbd significantly increased the soluble aggregation into amorphous structures, reducing the overall yield of nps of a defined size. this underscored the rna-antigen interactions during np assembly. the sera after mouse immunization effectively interfered with the binding of mers-cov rbd to the cellular receptor hdpp4. the results suggest that rna-binding controls the overall kinetic network of the antigen folding pathway in favor of enhanced assemblage of nps into highly regular and immunologically relevant conformations. the concentration of the ion fe(2+), salt, and fusion linker also contributed to the assembly in vitro, and the stability of the nps. the kinetic “pace-keeping” role of chaperna in the super molecular assembly of antigen monomers holds promise for the development and delivery of nps and virus-like particles as recombinant vaccines and for serological detection of viral infections. introduction various types of viral vaccines have been developed over the last century with a wide spectrum of efficacy and safety (1, 2) . the manufacturing of most conventional vaccines-live attenuated, inactivated, or subunit vaccines-invariably require the culturing of infectious viruses in cell substrates (3) . despite dedicated efforts, conventional cell culture often fails to produce sufficient amounts of virus for evaluating the immunogenicity, protective efficacy, and safety of viral vaccines. moreover, some emerging viruses cause high-mortality rates, without options for treatment or prophylaxis, necessitating their manipulation, and manufacture under stringent bio-safety environment (4) . not surprisingly, alternative technologies that circumvent these limitations are a high priority in the areas of vaccine development and production. nanoparticles (nps), virus-like particles (vlps), and assembly of multimeric peptides each provide attractive platforms for vaccine design (5) . virus-like particles and nps structurally resemble infectious virions, but are non-infectious due to the lack of viral genomes. recombinant surface antigens from natural virions are assembled into highly ordered conformations as empty particles devoid of genetic material. antigenic epitopes are presented on the multivalent and highly repetitive outer structure of the nps, which leads to the crosslinking of b-cell receptors and the induction of long-lasting immune responses (6) (7) (8) . by mimicking the morphology of the natural infectious virions, the regularly assembled particles are highly immunogenic, and are amenable to diagnostic and prophylactic exploitation. among the simplest targets are the vlps of non-enveloped viruses, such as hepatitis e virus or human papilloma virus, and are composed purely of viral capsid proteins (9) (10) (11) . in contrast to non-enveloped viruses, where virion assembly is exclusive to capsid proteins, enveloped viruses (e.g., coronavirus or flavivirus), require an additional membrane component for assembly into mature virions. consequently, in enveloped vlps, the assembly of matrix proteins provides a molecular scaffold, and viral antigens are embedded into lipid membranes. different types of glycoproteins may be embedded in the lipid membrane as target antigens for generating immunological responses (12) . however, this process requires multiple proteins (surface antigens and matrix proteins), and the enveloped vlps are not structurally uniform and are difficult to characterize. a promising alternative is to present the target antigens on the surfaces of self-assembled nps, which, in lieu of lipid membranes, serve as the macromolecular scaffold for the presentation of the antigens of interest. in developing np vaccines, consideration should be given regarding the selection of a robust and faithful system for np assembly that enables the cost-effective development and delivery of vaccines in a timely manner. structure-based approaches in silico and their underlying principles are relatively advanced for np assembly (13) (14) (15) . most of the approaches consider the thermodynamic stability of the final assembled nps, without due recognition for the kinetic complexities controlling regular assemblage over random interactions that lead to misfolded aggregations. therefore, it is not surprising that most engineered nps are refractory to soluble expression, which presents practical challenges in production, both at a laboratory-scale and in commercial manufacturing processes. this problem becomes augmented when expressed in bacterial hosts because of a lack of folding assistance in the bacterial cytoplasm for viral antigens. therefore, due to advantages in assisted folding, posttranslational modifications, and the possibility of generating multiple-component nps and vlps, eukaryotic hosts such as yeast, insects, and mammalian cells have been favored over bacterial hosts (16) (17) (18) . however, these systems are significantly more expensive than bacterial systems, are more time-consuming, and the down-stream processes are usually more complex. moreover, the purification of vlps from insect cell systems poses a challenge due to similar physicochemical properties between the vlps and the baculoviruses (1, 16) . bacterial systems, if available, would provide a cost-effective means to develop and deliver vaccines, as well as sero-diagnostic antigen kits used to diagnose-specific infection diseases. middle east respiratory syndrome (mers) was first reported in saudi arabia in 2012 and has caused multiple cases of infection with high mortality in europe and asia (19, 20) . mers is caused by mers-coronavirus (mers-cov), which can be transmitted from camels to humans, and from humans to other humans (21, 22) . worldwide transmission is increasing in direct household and community-wide transmission, as well as in nosocomial settings, as exemplified in a 2015 outbreak in korea (23, 24) . neither effective vaccines nor therapeutic interventions are currently available. because of this, assembly of mers-cov antigens into immunologically relevant conformation as nps would be of interest and may be helpful in developing vaccines, sero-diagnostic tools, and therapeutic monoclonal antibodies. in the current study, we present a novel bacterial np of mers-cov antigen using ferritin as a molecular scaffold for self-assembly. ferritin, which is present in most living organisms, has 24 identical subunits that spontaneously self-assemble and form np complexes with internal and external diameters of 8 and 12 nm, respectively (25, 26) . previous studies show that ferritins of helicobacter pylori from a human isolate can be used as scaffold for hiv and influenza np vaccines, using eukaryotic host cells such as human embryonic kidney cells (hek293f or hek293s) (27, 28) . likewise, bacterioferritin (fr), which self-assembles into nanocages with octahedral symmetry, has also been evaluated as a potential drug delivery system (29) . however, viral antigens of human pathogens are prone to misfolding into aggregates, which necessitates chemical refolding of the insoluble aggregates in order to regain solubility and to allow regular assembly of the antigen (30, 31) . in addition, displaying antigens on the surface of multi-molecularly assembled scaffolds in bacterial hosts remains a daunting challenge. we hypothesized that nps displaying the receptor-binding domain (rbd) of the spike protein from mers-cov could be produced in a bacterial system by harnessing the function of a molecular chaperone. conventionally, protein folding and the prevention of non-functional aggregation have been ascribed to molecular chaperones (32) (33) (34) . recently, it has been shown that rna molecules are able to provide novel functions as molecular chaperones (35) (36) (37) . based on novel findings, the concept of chaperna (chaperone + rna) function was established (38) . in this report, chaperna function was harnessed for the folding and assembly of hybrid ferritin monomers into nps using a bacterial expression system. we also demonstrated that the biophysical properties, including solubility, yield, and stability of mers-cov nps, could be improved by properly controlling the rna-binding affinity, and the concentrations of fe 2+ and salts. the chapernabased np assembly may prove to be a versatile tool for developing and delivering recombinant vaccines and for serological detection of emerging/re-emerging viruses. the expression vector pge-hrid(3) was constructed from the parental vector pge-lysrs (3) (39) . the pge-lysrs(3) vector was enzymatically cut with ndei and kpni. the pcr product of hrid, which carries the tev protease cleavage site and a 6-histidine tag at the c-terminus, was cut using the same restriction enzymes and the digested fragment inserted into the vector to generate pge-hrid(3). fr (genebank accession no. nc_000913.3) dna was synthesized by, and purchased from, cosmo genetech (korea). the dna was cleaved with sali and hindiii, and inserted into pge-hrid(3) to generate hrid(3)-fr. the receptor binding domain (rbd), n-terminal residues 367-606, of the mers-cov s protein (genbank accession no. afs88936.1), was generated by gene synthesis, cut with kpni and sali, and inserted into hrid-fr to generate pge-hrid(3)-rbd-fr. linker ssg or asg was inserted into the c-terminus of the rbd using overlapping pcr, cleaved with kpni and sai, and ligated into hrid-fr, generating pge-hrid(3)-rbd-[ssg]-fr or pge-hrid(3)-rbd-[asg]-fr, respectively. the schematic diagrams of each expression vector are illustrated in figure 1b . the genes of mutant hrid(2 m) (k19a and k23a) and hrid(9 m) (k19a, k23a, r24a, k27a, k30a, k31a, k35a, k38a, and k40a) were generated by gene synthesis, cleaved with ndei and kpni, and inserted into pge-hrid(3)-rbd-fr, generating pge-hrid(2 m)-rbd-fr and pge-hrid(9 m)-rbd-fr, respectively. the mutation sites and amino acid sequences of the mutants are shown in table s1 in supplementary material. the resulting expression vectors were transformed into the escherichia coli strain shuffle ® t7. the cells were grown in 50 ml of lb medium with ampicillin (50 µg/ml) at 30°c overnight. each type of transformant was inoculated into 500 ml of lb medium with ampicillin, grown at 30°c until an optical density (od600) of 0.6-0.8 was reached. protein expression was induced with 1 mm iptg for 12 h. each sample was harvested by centrifugation, lysed by sonication in lysis buffer (50 mm tris-hcl, ph 7.5; 10% glycerol; 2 mm 2-mercaptoethanol; and 0.1% tween-20). the soluble fraction of each lysate was purified on a ni-affinity histrap™ hp column by atka prime (ge healthcare) and concentrated with centriprep™ (merck millipore ltd.). the purified proteins were treated with tev protease to remove the fusion partner hrid. the assembled nps were purified by gel filtration on 10/300 superose™ 6 increase columns (ge healthcare). to examine the size and structure of the purified nps, microscopic evaluations using tem and cryo-em were performed. for tem analysis, a drop of the nps was placed onto a formvar/carboncoated tem grid (spl). the grid was negatively stained with 2% uranyl acetate, dried, and examined using a jem-1011 electron microscope (jeol) at an accelerating voltage of 80 kv. the particle sizes were calculated using camera-megaview iii (soft imaging system-germany) for measuring the nps in random image fields. for cryo-em, the nps were placed onto plasma-treated formvar/ carbon 200 copper grid (ems) and negatively stained with 2% uranyl acetate. the grid was accelerated at 200 kv with an fei cryotecnai f20 cryo-em microscope made available through the korean institute of science and technology. the nps were examined and photographed in high resolution. nanoparticle samples (3 ml) were placed into a dispo-h cell, and analyzed using a zeta-potential & particle size analyzer (els(37, 30, and 18°c ) and the cell lysates were separated into total (t), soluble (s), and insoluble (p) fractions by centrifugation (left panel). the solubility of each protein expressed at 18°c was measured by a gel densitometer and the data were summarized and shown in the right panel (n = 3). statistical significance (**p < 0.01, ***p < 0.001) was indicated for the samples compared with the control using a two-tailed student's t-test. (d) illustration of mers-cov rbd-fr nps using the chaperna-based hrid fusion partner. the hrid facilitated folding of the aggregation-prone rbd-fr through interaction with rna. the monomer of rbd-fr formed a properly folded trimeric structure by cleaving hrid with tev protease. eight trimers assembled and formed into mers-cov-like nps. red triangles indicate the rbd trimer on the fr nps. of the nps was measured twice at 25°c in water as a solvent with the sample accumulation time at 200 s. effect of salt and fe 2+ concentrations on np assembly and stability cultured cells (3 ml) were lysed with lysis buffer in the presence of various concentrations of nacl (0, 50, 100, 150, 200, 225, 250, 275 , and 300 mm) to evaluate the intracellular proteins. all samples were performed in triplicate. the cell lysates were separated into soluble and insoluble fractions by centrifugation, and the protein stabilities analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page). thus, the proteins from cell lysates (500 ml culture) were purified using hispur™ ni-nta resin (thermo fisher scientific) in buffer a depending on nacl concentration (0-300 mm). to evaluate the effects on fe 2+ on np formation, cells were cultured in lb media with various concentrations of fe 2+ (0, 200, 500, and 1,000 µm). np formation was examined by size exclusion chromatography (sec), sds-page, tem, and dls at the various concentrations of nacl or fe 2+ . the cells were harvested, sonicated with lysis buffer, and separated into soluble and pellet fractions by centrifugation. target proteins in the soluble fraction were purified using hispur™ ni-nta resin (thermo fisher scientific), following the manufacturer's instruction. t (total lysate), s (soluble fraction), p (pellet fraction), w (wash fraction), and e (the elution fraction were analyzed by sds-page. co-purification of the nucleic acids and proteins in the wash and elute were analyzed on a native agarose gel. the nucleic acids were visualized with ethidium bromide (etbr), and the proteins with coomassie staining. cultured cells (10 ml) were harvested using the same method described above. the cells were lysed with 500 µl of protein extraction reagent b-per™ ii (thermo scientific) and separated into soluble and pellet fractions by centrifuged 12,000 rpm for 10 m. a 200 µl aliquot of each soluble fraction was further treated with 250 µg/ml of rnase a (intron biotechnology) and incubated at 37°c for 15 min. the nuclease treated samples were clarified by centrifugation at 12,000 rpm for 15 min and the soluble supernatants and the pelleted precipitates were analyzed on an sds-page gel followed by western blot analysis. to confirm the proper folding of rbd-fr and its variant (rbd-[ssg]-fr), the binding of the purified proteins with the mers-cov receptor hddp4 was performed by elisa. fr only and phosphate-buffered saline (pbs) were used as negative controls. nunc 96-well microtiter immunoplates (thermo fisher scientific) were coated with 100 ng/well of hdpp4 proteins (abcam) and incubated at 4°c overnight. the plates were washed and blocked with 150 µl/well of blocking buffer (1% bsa) for 1 h at room temperature. rbd (ssg linker, wt, 2 m, or 9 m)-fr (100 ng/ well) were added for 2 h at 37°c. an anti-penta his antibody (100 µl/well; qiagen) was serially diluted (1/100 to 1/12,800) in tbst [50 mm tris-cl (ph 7.4), 0.05% tween-20], added to the wells, and incubated for 1 h at 37°c. a secondary goat anti-mouse igg antibody conjugated with hrp in a 100-µl volume (1:5,000, sigma-aldrich) was added and incubated for 1 h at 37°c. the plates were washed three times with tbst at the end of each step. after washing, 100 µl/well of substrate tmb solution (bd biosciences) were added to the well and the plates were incubated at 37°c for 30 min in the dark. 50 µl of stop solution (2 n h2so4) was added to the well to stop the colorimetric reaction, and the absorbance at 450 nm was measured using an elisa reader, fluostar optima (bmg labtech). . the coating antigens were removed, and the wells were blocked with pbst (5% skim milk in pbs and tween-20) for 1 h at 37°c. after 2 h, the blocking solution was removed. twofold serially diluted sera from four patients (cnnh-0709, 0809, 1009, and 1309) were added to each well and incubated at 37°c for 2 h. the antigencoated wells were incubated with peroxidase-conjugated goat anti-human igg antibody (kpl, seracare life sciences, milford, ma, usa) at 37°c for 1 h. the primary antibody was removed and 3,3′,5,5′-tetramethylbenzidine (tmb; sigma-aldrich) was added to each well as colorimetric substrate. immediately after treatment of the reactions with stopping solution (sigma-aldrich), the od was read at 450 nm. six-week-old female balb/c mice were immunized with 20 µg/ mouse of the rbd-fr, rbd-[ssg]-fr, or rbd protein generated as described above, or with commercially available mers-cov rbd protein (mers-rbd-005p; eenzyme) as antigen in bsl-2 facility in ylarc. antigens were diluted in pbs. for the first group, equal volume of mf59 adjuvant (addavax, cat. no vacadx-10) (43) was mixed by pipetting. for the other group, equal volume of antigens and alum adjuvant (thermo fisher scientific) were mixed by pipetting following the manufacturers' protocol. pbs plus adjuvant and fr were used as negative controls. the immunized mice were boosted twice with intramuscular injections on days 14 and 28. mice were anesthetized on days 27 and 41 for ocular bleeding from the orbital sinus ( figure s10 in supplementary material). immune sera were processed by centrifugation of the collected blood at 12,000 × g for 30 min. the spleen and the balf (bronchoalveolar lavage) were obtained at 7 days after the last immunization from sacrificed mice. balf was taken by washing the airways with 1 ml of pbs. t-cell population from immunized mice were analyzed by flow cytometric analysis (43, 44) . the spleens were taken at 7 days after the last immunization from the sacrificed mice. to obtain single-cell suspensions, the tissues were homogenized and passed through 70 µm cell strainers (spl). after centrifugation, erythrocytes were removed by red blood cell lysing buffer (sigma). the cells were washed and resuspended in iscove's modified dulbecco's media containing 10% fbs. for intracellular cytokine staining, the splenocytes were stimulated with 10 µg/ml rbd protein or phorbol myristate acetate/ionomycin in the presence 10 ng/ml recombinant human il-2 (biolegend) and brefeldin a (1:1,000; ebioscience) at 37°c for 5 h. after stimulation, the cells were blocked with rat anti-mouse cd16/cd32 (bd biosciences) and surface stained with anti-cd8 (fitc, clone 53-6.7; biolegend) and anti-cd4 (pe/cy7, clone gk1.5; biolegend) at 4°c for 30 min. the stained cells were fixed in facs lysing solution (bd biosciences) at room temperature for 20 min, and permeabilized with facs buffer (0.5% fbs, 0.1% nan3 in pbs) containing 0.5% saponin (sigma) at room temperature for 15 min. then, the cells were stained with anti-ifn-γ (pe, clone xmg1.2; biolegend) and anti-tnf-α (apc, clone mp6-xt22; biolegend) at room temperature for 40 min. all data were collected by bd lsr fortessa (bd biosciences) and analyzed with flowjo software (tree star inc., ashland, or, usa). competition elisa was performed to determine whether mers-cov antigen [rbd-[ssg]-fr, rbd-fr, rbd, and fr (negative control)]-immunized mouse serum inhibited binding of rbd protein to hdpp4 receptor (45, 46) . 500 ng/well hdpp4 protein (abcam) was coated on nunc 96-well microtiter immunoplates (thermo fisher scientific) and incubated overnight at 4°c. plates were washed and blocked with 150 µl/well of blocking buffer [5% skim milk in pbs and tween-20 (pbst)] for 1 h at 37°c. at the same time, mouse sera immunized with rbd, rbd-[ssg]-fr, rbd-fr, and fr were serially diluted (1/10 to 1/160) with 500 ng/well rbd protein (mers-rbd-005p; eenzyme) in tbst [50 mm tris-cl (ph 7.4), 0.05% tween-20], added to new wells, and incubated for 1 h at 37°c. 100 µl solution was added to each well at 37°c and incubated for 2 h. after that, 100 µl of anti-6xhis tag antibody conjugated with horseradish peroxidase (1:1,000, thermo fisher scientific) was added to each well and incubated for 1 h at 37°c. plates were washed three times with tbst, and 100 µl/well of substrate tmb solution (bd biosciences) was incubated at 37°c for 30 min in the dark. 50 µl of stop solution (2 n h2so4) was added to the well to stop the color reaction and measure the absorbance at 450 nm using an elisa reader fluostar optima (bmg labtech). the hrid facilitated the solubility of mers-cov rbd-fr the spike glycoprotein (s) of mers-cov was used for the generation of mers-cov-like nps. s protein forms trimers, resulting in large spikes on the virus envelope (47) . it is challenging to express the full-sized s protein (~200 kda) in e. coli. thus, the s1 domain of s protein (~80 kda), which includes the receptor-binding ability, was used. our initial attempt to express the s1 domain, either as s1 or as an s1-fr fusion protein, failed; the expression level and solubility of the protein was below the lower limit of detection by sds-page and western blotting ( figure s1 in supplementary material). we therefore used the rbd (367-606 a.a.) of the s1 protein, which has a pivotal function as illustrated in figure 1b (48, 49) . when expressed alone in e. coli, the rbd is not able to form the trimeric assembly (unpublished observation), due to the lack of the hr2 domain within the s2 domain (50) . to overcome this problem, fr was used as scaffold for the assembly. fr is a spherical np whose subunits form trimers that subsequently result in octahedral structures composed of 24 identical subunits (51) . we therefore performed computational modeling to evaluate the potential of fr as scaffold for trimer formation of the rbd. possible trimer formation was analyzed by computational modeling using modeler (13, 52) and cluspro (41, 42) . various linkers, including ssg, asg, and d6, were introduced between the rbd and fr with a goal to minimize steric hindrance between the two domains so as to enhance trimer and np formation. in silico analysis showed energy-stable trimeric models of rbd-fr, rbd-[ssg]-fr, and rbd-[asg]-fr, whereas rbd-d6-fr failed to form a trimeric structure ( figure 1a) . the rbd-[ssg]-fr was predicted to be the most stable and well-structured compared with rbd-fr and rbd-[asg]-fr. initial testing of the rbd-fr constructs without hrid fusion showed that none of the constructs were solubly expressed, even under low-temperature culture conditions ( figure 1c ) ( figure 1b) . we previously confirmed that by using chaperna, the globular domain of influenza hemagglutinin (ha) is efficiently assembled into a trimeric complex with an immunologically relevant conformation (yang et al., in press) . as shown in figure 1c , the hrid fusion significantly increased the solubility of both rbd-fr (59.1%) and rbd-[ssg]-fr (62.83%), indicating that the chaperna platform effectively increased both the solubility and the folding of its fused target proteins. because of the poor expression level and low solubility of the rbd-[asg]-fr construct (figure 1c) , further experiments were performed using only the rbd-fr and rbd-[ssg]-fr constructs. after purification of the soluble proteins ( figure s2 in supplementary material), we determined the potential effects of using hrid as a fusion partner for the self-assembly of the nps. as shown in figure s3 in supplementary material, hrid-rbd-fr failed to form nps. because of this, we performed tev protease cleavage of the hrid. removal of the hrid domain facilitated the self-assembly of the rbd-fr monomers, and also eliminated the immune response against the non-self hrid domain in balb/c mice ( figure s4 in supplementary material). after hrid cleavage, rbd-fr and rbd-[ssg]-fr were purified using sec (figure 2a) . as expected, rbd-[ssg]-fr assembled into properly formed nps (1,080 kda) more efficiently than did rbd-fr nps, which were mainly detected in the void-volume fractions, suggesting they were irregularly assembled soluble aggregates. the size of the rbd-[ssg]-fr nps was further confirmed by tem. tem images of the rbd-[ssg]-fr np structures showed hollow, spherical particles that were more compact than the rbd-fr nps. the average diameter of the rbd-[ssg]-fr nps was 28-30 nm (figure 2b) . in contrast, dls analysis of the rbd-fr np structure without the ssg linker appeared to be smaller with an average intensity diameter of 26.3 nm, and this compared with rbd-[ssg]-fr that had an average intensity distribution diameter of 30.5 nm (figure 2c) . consistent with the sec analysis, rbd-fr without a fusion partner was mostly produced in a soluble aggregated form. therefore, we identified that the protein folding did not occur properly without hrid, and the formation of nps was confirmed by both sec and sds-page analyses. as shown in figure s5 in supplementary material, the purified nps retained their stability over an extended period of time at various temperatures (25, 4 , and −20°c). thus, these results indicate that the ssg linker allowed the rbd-fr to generate properly assembled nps. it should also be noted that the efficiency of protein folding and nps formation may be further enhance through appropriate linker selection. it has been reported that ionic strength plays an important role in the stability and self-assembly of ferritins (53, 54) . we examined the effect of salt concentration on the formation and stability of the rbd-[ssg]-fr nps at various concentrations (0-300 mm). consistent with the previous studies, the stability of the protein was highly affected by the concentration of nacl in the lysis buffer by sds-page ( figure 3a ) (n = 3). the solubility of the protein significantly decreased as the concentration of nacl increased from 0 to 100 mm, with the solubility being about 8.79-fold lower at 100 mm compared with 0 mm. unlike previous studies, the solubility of the protein was gradually recovered at higher nacl concentrations (>100 mm); the solubility at 300 mm was 1.45-fold higher than at 0 mm. furthermore, the yield of soluble of protein per liter of culture increased in a salt concentrationdependent manner (figure 3b) . to further investigate the effect of salt concentration, the physicochemical and morphological properties of the rbd-[ssg]-fr protein were examined by sec, tem, and dls. in 50 mm nacl, most of the protein was aggregated during the purification process, and the purified protein failed to form spherical structures, but instead, existed predominantly as 45 kda monomers (figures 3c,d; figure s6 in supplementary material). in contrast, the protein that was lysed in 0 mm nacl and purified in 200 mm nacl, developed well-structured nps according to tem and dls analyses (figures 3c,d; figure s6 in supplementary material). however, based on sec analysis, at high-salt concentrations (>250 mm), the protein failed to form stable structures with the proteins being eluted predominantly in the void volume, suggesting they were soluble aggregates under the high-salt concentrations ( figure 3c) . transmission electron microscopy images under the various salt concentrations clearly supported the conclusion, showing that the tendency for aggregation was dependent on the salt concentration ( figure 3d) . taken together, the results underscored the importance of salt concentration on the solubility of monomers and the quality of multimeric assembly of hybrid nps. ferritin has an intrinsic ability to interact with fe 2+ to form ferritin-iron cores (55) . thus, it was worth investigating the effect of fe 2+ on the assembly and stability of rbd-[ssg]-fr nps. cells were grown in lb medium with various concentrations of fe 2+ . as shown in figure 4a , the yield of purified protein was significantly increased from cultures with 500 µm fe 2+ , reflecting a 2.7-fold increase compared with similar cultures 0 µm fe 2+ . the cell growth and purification yield at 1,000 µm fe 2+ were slightly decreased, presumably due to the toxicity of ferric acid. np formation under the various concentrations of fe 2+ was analyzed by sec ( figure 4b) . consistent with the previous results, the proteins were eluted mainly in the fractions expected for the size of assembled nps (1,080 kda). of note, the ratio between nps and soluble aggregates in the sec analysis showed that np formation was facilitated at high concentrations of fe 2+ (figure 4b) . the formation of rbd-[ssg]-fr nps at an fe 2+ concentration of 1,000 µm was confirmed by tem ( figure 4c ) and dls ( figure 4d) . the tem analysis clearly showed that the morphology of the proteins was more compact, and probably highly stable, when assembled at high fe 2+ concentrations (500 µm) than at lower concentrations (0 µm) ( figure 4c ). as shown in figure 4d , the average diameter of nps examined by dls was 25.1 nm at high fe 2+ concentration (500-1,000 µm) and 27.7-32.2 nm at lower concentration (0-200 µm). these results suggest that both fe 2+ and salts concentrations influenced the efficiency and quality of the regular assembly of hybrid ferritin monomers into nps. our previous studies show that an rna-protein interaction is crucial for transducing the chaperone function of rna into the folding of client proteins (38) . consistent with that, our present study showed that rna facilitated the folding of its interacting proteins. the solubility of hrid(wt)-rbd-fr was 5.69-fold higher than rbd-fr without hrid fusion (figure 1) , strongly supporting the previous studies. in addition, the solubility of rbd alone was completely insoluble (figure 5b ; figure s7 in supplementary material). it has been shown that the positively charged residues of lysine moieties in hrid contribute to trna binding (56) . in the current study, the trna binding induced the intrinsically disordered protein (idp) status of hrid to form alpha-helical structures ( figure 5a) . thus, two rna-binding (table s1 in supplementary material). the total e. coli lysate (t) was fractionated into the soluble fraction (s) and the pellet fraction (p) by centrifugation. as expected, both rbd and rbd-fr without fusion to hrid domain, were refractory to being produced as soluble proteins ( figure 5b) . interestingly, the solubility of the rna-binding mutants did not decrease, but actually increased to 75.3% for the 2 m mutant and 93.4% for the 9 m mutant compared with wild-type protein at 60.1% (figure 5b) . considering that hrid is relatively unstructured in the absence of trna binding, the results are consistent with previous reports that the fusion with idps promotes the solubility of target proteins (57) (58) (59) . following purification of wild-type hrid-rbd-fr (hrid(wt)-rbd-fr), electrophoretic mobility shift assays showed that greater amounts of nucleic acids were co-purified with hrid(wt)-rbd-fr protein than with the mutant hrid-rbd-frs (2 and 9 m) under non-denaturing conditions ( figure 5c ). the relative ratio of nucleic acid based on etbr staining and proteins based on coomassie staining in the eluted fraction confirmed the reduced affinity of mutants to nucleic acids. to test if rna had a role in maintaining the stability of the target proteins, the lysates were treated with rnase a to eliminate rna, and the solubility of each protein was analyzed by sds-page and western blotting. the soluble fractions of the lysates (s) were incubated at 37°c in the presence and absence of rnase a and the samples were further separated into soluble fraction (ss′) and insoluble fraction (sp′) by centrifugation. as shown in the left panel of figure 6 , rnase a treatment completely abolished the effect of rna on protein solubility as compared with the control (rnase a−) or with samples prior to rnase treatment. parallel experiments with the 2 and 9 m mutants showed much less rna co-purified with the proteins, confirming the reduced affinity to nucleic acids and the complete depletion of rna by rnase a treatment (figure 6, left panel) . remarkably, the solubility of hrid(wt)-rbd-fr was greatly reduced by depletion of rna as reflected in the ratio of [ss′]/ [sp′] [0.1 and 0.4 for rnase (+) and rnase (−), respectively] by both coomassie staining and western blot analyses (figure 6 , right panel). however, the solubility of the mutants (2 and 9 m), was not significantly affected by rnase a treatment, probably due to their lower affinity to rna (figure 6, right panel) . taken together, the results demonstrate that hrid(wt)-rbd-fr maintained a strong affinity for rna, and that affinity was pivotal for maintaining the solubility of the protein. to further define the rna dependence of solubility of the ferritin hybrids (figure 6) , we investigated if the rna binding had a role in the formation of nps. rbd-fr and the various hrid-rbd-fr (wt, 2, and 9 m) proteins were purified by nickel-affinity chromatography ( figure s2 in supplementary material) and their physicochemical properties analyzed by sec (figure 7a) , tem (figure 7b) , and dls ( figure 7c) . the soluble yields of rbd-fr (hrid fusion) was approximately 1.6 mg/l of culture, representing greater than 1,000-fold higher levels than its hrid (−) counterpart (~15 μg/l culture), again confirming the role of hrid as a robust enhancer for solubility and assembly. it was striking to note that the two mutant proteins, despite high solubility (figure 5b) , were detected at disproportionately higher amounts in the void fractions of sec, indicating that they failed to form nps of a defined size, and existed predominantly as soluble aggregates ( figure 7a) . however, hrid(wt)-rbd-fr predominantly formed nps of a defined size (~1,080 kda). it is also interesting to note that there was a slight shift of the rna-binding mutants (2 and 9 m) in the elution pattern, suggesting a larger size of nps compared with wild-type nps. overall, the ratio between soluble aggregates in the void volume and the nps of defined size clearly showed that rna binding was crucial for assembly of the monomers into nps. as a control, rbd-fr (without hrid fusion) existed predominantly as soluble aggregates ( figure s8 in supplementary material). consistent with these results, em analysis confirmed well-structured nps by hrid(wt)-rbd-fr, compared to largely aggregated structures by the mutant proteins ( figure 7b) . even if multi-molecular structure was formed, the structure becomes unstable, mostly as soluble aggregates. consistently, the intensity distribution diameter of the wild-type protein, as estimated by dls analysis, was 25 nm compared with larger sizes of hrid(2 m) at 34.2, 519.2 nm and hrid(9 m) at 52, 717.7 nm (figure 7c ; figure s9 in supplementary material). it is conceivable that soluble aggregates may shield the exposed 6-histidine tag, resulting in a decreased binding affinity to nickel resins and elution in earlier fractions compared with wt protein ( figure s2 in supplementary material). taken together, the data demonstrate that rna binding prevented aggregation into irregular conformations and guided the self-assembly of the hybrid ferritin monomers into nps of a stable structure. the immunological properties of ferritin nps were analyzed by elisa. the hddp4 (human dpp4) receptor has been previously identified as the receptor for mers-cov human infection (46) . therefore, using hdpp4 as a coating antigen, elisa-binding assays between rbd nps and the receptor were performed (figure 8) . fr without rbd fusion failed to bind, and was similar to the pbs negative control. strikingly, the binding ability increased in the same order as the rna-binding ability (hrid(wt) > 2 m > 9 m), with highest absorbance observed in the wt with the ssg linker (hrid(wt)-rbd-[ssg]-fr). the results show that the conformation of rbd in the wt nps better resembled the protective antigen of mers-cov rbd from 293 cells, compared with the rna-binding mutants 2 and 9 m. again, judicious choice of linker between the ferritin carrier and the antigen was important for receptor binding and was reflected in its importance for np assembly into a stable conformation (figure 2) . finally, the elisa results for np against human patients was investigated using the sera from four mers-cov-infected patients (figure 9) . six different proteins, including five recombinant nps (hrid(wt)-rbd-fr, hrid(2 m)-rbd-fr, hrid(9 m)-rbd-fr, hrid(wt)-rbd-[ssg]-fr, and fr), and mers-cov rbd protein were compared by elisa using them as capture antigens. strong elisa signals were detected for the four recombinant nps and mers-cov rbd from 293 cells (positive control). the wt form consistently showed a higher response than the rnabinding mutants (hrid(wt) > 2 m > 9 m), with hrid(wt)-rbd-[ssg]-fr being the best binder among constructs tested. these results address to the utility of the e. coli assembled mers-cov rbd-fr nps as useful tools for sero-diagnosis of mers-cov infection. taken together, the results confirmed the immunologically relevant conformation of the mers-cov rbd displayed on the hybrid ferritin particles, and the crucial role of rna in controlling the kinetic pathway for the assembly of viral antigen monomers into stable nps. to evaluate the immunogenicity of ferritin-based nps, balb/c mice (n = 5) were immunized with rbd, rbd-fr, and rbd-[ssg]-fr nps antigens. the trnas were found to be removed from the hrid protein during the purification process. before immunization, potential rna contamination in the purified proteins was determined by gel electrophoresis. as shown in figure s11 in supplementary material, rna was below detection level, if any, after several purification steps, compared with the proteins purified in the first step. previously, mf59-adjuvated and alum-adjuvated mers-cov antigen have been reported to increase the antibody and t-cell responses in mice (44, 60) . thus, the first group and second group were immunized twice with 20.0 µg of antigen containing the equal volume of alum figure s2 in supplementary material and size exclusion chromatography was used to explore hdpp4 receptor-binding affinity to the protein. all data are shown as mean ± sd from triplicate samples. fr alone and phosphate-buffered saline were used as negative controls. figure s2 in supplementary material were used as coating antigens. fr alone and infected cell lysates were used as negative and positive controls, respectively. virus-infected sera from four patients were serially diluted from 1:100 (twofold dilution). all data are presented as mean ± sd of duplicate samples. higher than rbd, respectively. the antibody responses by rbd-fr and rbd-[ssg]-fr nps were much stronger than the rbd in all antibody subtypes tested (igg1, igg2a, and igg2b) (figures 10b-d) . as a test of mucosal immune responses, the rbd-specific iga antibody levels from balf were also analyzed by elisa (figure 10e) . mf59 adjuvanted rbd-[ssg]-fr nps presented significantly higher od values than rbd and fr (negative control). these results suggested that rbd-[ssg]-fr nps induces local mucosal immune response stronger than rbd. in addition, it was confirmed that antibody responses of igg, igg1 (th1), igg2a, and igg2b (th2) against mf59-adjuvated antigens were higher than those from alum-adjuvated antigens. in contrast, pbs and fr control groups failed to, or only weakly induce an antibody response against rbd protein. these results suggest that fr-based nps significantly enhance various antibody responses than monomeric antigens. the cellular immune responses were investigated in mice immunized with protein (rbd, rbd-fr, rbd-[ssg]-fr) and fr (negative control). splenocytes of mice (n = 4) were harvested 1 week after the last immunization, stimulated with | nanoparticles-immunized mouse serum inhibited interaction between middle east respiratory syndrome-coronavirus receptor-binding domain (rbd) and hdpp4 receptor. competition enzyme-linked immunosorbent assay showed that anti-rbd mouse sera (1:10, from mice immunized with rbd-[ssg]-fr, rbd-fr, and rbd) blocked binding between rbd (5 µg/ml) and hdpp4 receptor (5 µg/ml). fr-immunized mouse serum (1:10) was used as a negative control. all sera were serially diluted from 1:10 (twofold dilution). all data are presented as mean ± sd (n = 5) and p-values were obtained using student's two-tailed tests (***p < 0.001). figure 10 | immune responses in receptor-binding domain (rbd) nanoparticles (nps) immunized mice (n = 5). endpoint titer of igg (a), igg1 (b), igg2a (c), and igg2b (d) antibody binding to middle east respiratory syndrome-coronavirus rbd were detected using mice serum after two immunizations. rbd-specific antibodies were detected after immunizations of rbd nps, rbd, fr with adjuvant (alum and mf59) using enzyme-linked immunosorbent assay. (e). rbd-specific iga antibodies were detected using balf (diluted 1:8) after immunization of protein with mf59. od, optical density. each endpoint titer was shown by individual. all error bars were shown as mean ± sd (n = 5) and all p-values were obtained using student's two-tailed tests (**p < 0.01, ***p < 0.001). rbd protein, and analyzed for cytokines by flow cytometry. in the rbd-immunized group, ifn-γ and tnf-α-producing cd4 + t-cell responses were detected at low levels. however, ifn-γ and tnf-α-producing cd4 + t cells were significantly increased in rbd-fr and rbd-[ssg]-fr-immunized groups compared with rbd and fr-immunized group ( figure s12 in supplementary material) . these results demonstrated that the rbd nps vaccination induced antigen-specific cd4 + t cells that produced ifn-γ and tnf-α upon antigen stimulation. anti-nps serum effectively blocked rbd protein binding to the hdpp4 receptor middle east respiratory syndrome-coronavirus infection is mediated by the interaction of rbd and the host receptor hdpp4 (45, 46) . as a correlate of protection, a competition elisa was performed to investigate whether antibodies generated from nps immunization were able to interfere with the binding to hdpp4. thus, after incubation of rbd protein with mouse serum (1:10), the binding of serum-mixed samples to hdpp4 protein was measured. as shown in figure 11 , rbd-[ssg]-fr, rbd-fr, and rbd-immunized sera strongly abolished the binding of rbd to hdpp4 receptor (93.3, 82.2, and 75.67%, respectively). interestingly, the relative efficiency of interference correlates with that of np assemblage (figure 11 ). in contrast, the fr-immunized mouse serum (negative control) failed to inhibit the interaction. taking together, these results demonstrate that immunization of nps greatly stimulates mers-covspecific antibody response that effectively interferes with the cellular receptor binding, suggesting its possibility as a vaccine. however, protection efficacy should ultimately be tested in a live virus challenge model. having key immunologic features, like a highly repetitive nanostructure, provides a designing principle for nps in inducing potent and long-lasting antibody responses. for vlps of non-enveloped viruses, assembly is made purely by capsid proteins. for enveloped viruses, however, additional membrane components and matrix proteins are required to display the target antigens on the surface of assembled vlps. a promising alternative is to present target antigen on the surfaces of selfassembled nps, which, in lieu of lipid membranes and matrix proteins, serve as a macromolecular scaffold for the presentation of antigens of interest (61) . ferritins, as a substitute for matrix proteins and membranes, have been used as scaffold for the regular assembly of target antigens. however, ferritin-based nps have been produced only in host cells of mammalian or insect origin (28, 62) . previously, we showed that influenza ha could be assembled in a soluble, trimeric, and immunologically relevant conformation by exploiting chaperna activity (63) . the present study is the first report of using rnas as molecular chaperone for supra-molecular structures. here, we present a novel bacterial system for np assembly of hybrid ferritin displayed surface antigens from mers-cov. the nps reacted strongly with sera derived from mers-cov-infected patients (figure 9 ) confirming their utility in sero-diagnosis of infection. moreover, the antisera, generated from immunization of mice, were able to interfere with the binding to the cellular receptor hdpp4 (figure 8 ), in part of essential protective immune responses. the efficiency of receptor-binding inhibition (figure 11) , as well as the ability for inducing the mucosal responses (figure 10e) , correlated with the regular assembly of nps as examined by dls or em (figure 2) , confirming that presentation of antigenic epitopes on a multivalent and highly repetitive structure is indeed important for the quality of immune responses. overall, the quality of nps and consequent immune responses were governed by the rna-mediated assembly of antigens. we hypothesized that chaperna function could be harnessed for presenting target antigens as highly repetitive nanostructures ( figure 1d) . the hrid is the n-terminal domain of hlysrs and was previously identified as a nucleic acid-binding domain ( figure 5 ) (63) . in this report, the hrid was exploited as a transducer for chaperna function (tcf) by serving as a docking-tag for cellular rna for the folding/assembly of the hybrid fr containing client antigen proteins [rbd of mers-cov (figure 1d) ]. the advantage of using hrid as a tcf could be many fold. first, hrid is small (8.3 kda), monomeric, and was flexible enough to allow the access of site-specific protease for the removal of hrid ( figure s3 in supplementary material). of note, hrid belongs to idps, which switches into stabile α-helixes upon binding with trnas. second, the bound rna, due to its highly negative charge, may resist uncontrolled intermolecular interactions among monomers into amorphous aggregation. finally, even the naked hrid (in the absence of rna binding), due to its intrinsically flexible nature, may not pose physical hindrance to multiple interactions among monomers, enabling assembly into stable super-structures, upon removal of the hrid. thus, the potential "pace-making" function harnessed with the rna molecule, allows a regular assembly of monomers as highly repetitive nanostructures. consequently, in the current study, hybrid fr was produced in soluble forms, could be purified by one-step affinity chromatography, and most remarkably, assembled into nps of defined sizes upon removal of the hrid ( figure s2 in supplementary material). consistent with the principles of design, the loss of rna binding by hrid significantly hampered the regular assembly of the ferritin monomers and increased the amount of non-functional misfolded proteins as soluble aggregates (figure 7) . thus, the overall yield, as well as the quality of nps, were dependent on the chaperna function transduced by the hrid, which in turn was mediated by interaction with cellular rnas (likely to be trnas). the driving and controlling factors for de novo assembly of biomolecules are poorly understood. historically, host factors like groel/s were initially discovered as molecular chaperones for supporting viral growth in e. coli and supporting the assembly of viral capsid proteins (64, 65) . moreover, groel/s also cooperates with rbcx in plant cells for the assembly of multicomponent rubisco, which is the most abundant protein in the biosphere responsible for photosynthesis (66) . therefore, it is intriguing that rna could provide such a robust folding/ assembly of a supra-molecular structure. we recently confirmed that the present strategy could be successfully applied to the assembly of bacterially synthesized monomers of norovirus into vlps composed of 180 monomers (unpublished observation, seong, b.l.). whether rna can substitute for, or collaborate with pre-existing protein-based molecular chaperones remains an exciting avenue for future investigations. it should be noted that the defined versatile functions are being expanded for rna molecules. as an engineered system for harnessing chaperna function, the present report may prove to be the tip of an iceberg for pivotal function of rna molecules as chaperones for the folding and supra-molecular assembly of proteins in living organisms (36, 38) . various factors were identified as important for efficient assembly of mers-cov nps. as an extrinsic factor, the binding affinity of hrid to cellular rnas was crucial for the assembly and the quality of the assembled nps (figure 7) . as intrinsic factors, the concentration of salts and fe 2+ also influenced the assembly and stability of nps (figures 3 and 4) . the ionic strength played an important role in the stability and self-assembly of ferritins, and aggregation increased with increasing concentrations of nacl (54) . the assembly of the hybrid mers-cov nps revealed an interesting change in salt dependence, with 200-225 mm nacl buffer as optimal condition as confirmed by em and dls analyses (figure 3) . the change in salt dependence was probably due to the presence of electrostatic interactions among rbd domains (54, 67) . the dependence on fe 2+ was not surprising considering that ferritin has an intrinsic ability to interact with fe 2+ to form ferritin-iron cores (55) . based on our experience, to enhance the quality of nps, it is advisable to control fe 2+ concentrations, both during the culturing of the bacterial cells and during the purification of the soluble monomer proteins (figure 4) . first, the yield of the purified protein was increased in the presence of 500 µm fe 2+ (figure 4b) , up to 2.7-fold greater compared with the control conditions lacking fe 2+ . second, the ratio between nps and soluble aggregates in sec showed that nps formation was facilitated at high concentrations of fe 2+ , and resulted in a more compact morphology under em (figures 4b,c) . thus, both the overall yield and the quality of nps were governed by their intrinsic ability to interact with fe 2+ . finally, our data show that the presence and the nature of the linker between the ferritin and the rbd antigen was also important to the assembly of nps. it is possible that a linker with flexibility and sufficient length would accommodate the steric requirements for assembly of multimeric nps. however, it is difficult to precisely predict the effect of the linker, and therefore it is advisable to screen multiple constructs during the early stages of testing the assembly of nps displaying antigens of interest. in conclusion, the chaperna-based antigen assembly platform holds promise for the development and delivery of np-based vaccines to enhance rbd-specific antibody responses, and the serological detection of emerging viruses. various types of designing principles have advanced the structure-based approaches to np assembly (61, 68) . however, most of the in silico methods consider the thermodynamic stability of the final assembled nps, but not necessarily the kinetic pathways leading to their successful folding into regular assemblages. consequently, most nps are refractory to soluble expression and fail to assemble as designed, resulting in significant, and practical challenges in the manufacturing process. the chaperna-mediated folding and the "pace-keeping" assembly of monomers into higher ordered structures will enable faithful production of np and vlp-based vaccines against emerging and re-emerging viral infections. this study was carried out in accordance with the recommenda figure 7 | elucidation of rna-mediated nanoparticle (np) formation of receptor-binding domain (rbd)-fr. (a) size exclusion chromatography analysis of rbd-fr nps purified from the tev protease-cleaved hrid(wt, 2, or 9 m)-rbd-fr. the fractions (11-12 ml) estimated as nps were further analyzed by transmission electron microscopy (b) and dynamic light scattering (c) exploiting virus-like particles as innovative vaccines against emerging viral infections traditional and new influenza vaccines vaccine manufacturing: challenges and solutions management of accidental exposure to ebola virus in the biosafety level 4 laboratory vaccine delivery using nanoparticles vaccine delivery: a matter of size, geometry, kinetics and molecular patterns virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development the influence of antigen organization on b cell responsiveness structure of the hepatitis e virus-like particle suggests mechanisms for virus assembly and receptor binding self-assembly of human papillomavirus type 1 capsids by expression of the l1 protein alone or by coexpression of the l1 and l2 capsid proteins how will hpv vaccines affect cervical cancer? virus-like particles as universal influenza vaccines comparative protein structure modeling using modeller structure-based design of peptides that self-assemble into regular polyhedral nanoparticles comparative protein structure modeling of genes and genomes construction and characterization of virus-like particles: a review self-assembling protein nanoparticles in the design of vaccines differences in the post-translational modifications of human papillomavirus type 6b major capsid protein expressed from a baculovirus system compared with a vaccinia virus system isolation of a novel coronavirus from a man with pneumonia in saudi arabia middle east respiratory syndrome coronavirus (mers-cov) serology in major livestock species in an affected region in jordan human infection with mers coronavirus after exposure to infected camels, saudi arabia interhuman transmissibility of middle east respiratory syndrome coronavirus: estimation of pandemic risk an outbreak of middle east respiratory syndrome coronavirus infection in south korea mers outbreak in korea: hospital-to-hospital transmission the composition and the structure of bacterioferritin of escherichia coli self-assembly in the ferritin nano-cage protein superfamily presenting native-like trimeric hiv-1 antigens with self-assembling nanoparticles hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection ferritin nanocages: great potential as clinically translatable drug delivery vehicles? refolding of recombinant proteins advances in refolding of proteins produced in e. coli the role of molecular chaperones in protein folding molecular chaperones in the cytosol: from nascent chain to folded protein the hsp70 and hsp60 chaperone machines rna-mediated chaperone type for de novo protein folding m1 rna is important for the in-cell solubility of its cognate c5 protein: implications for rna-mediated protein folding rnas as chaperones the folding competence of hiv-1 tat mediated by interaction with tar rna protein solubility and folding enhancement by interaction with rna modeling of loops in protein structures cluspro: a fully automated algorithm for protein-protein docking the cluspro web server for protein-protein docking universal vaccine against respiratory syncytial virus a and b subtypes optimization of antigen dose for a receptor-binding domain-based subunit vaccine against mers coronavirus a truncated receptor-binding domain of mers-cov spike protein potently inhibits mers-cov infection and induces strong neutralizing antibody responses: implication for developing therapeutics and vaccines dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-emc middle east respiratory syndrome coronavirus (mers-cov) entry inhibitors targeting spike protein molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 structure of mers-cov spike receptor-binding domain complexed with human receptor dpp4 structure-based discovery of middle east respiratory syndrome coronavirus fusion inhibitor stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of rhodobacter capsulatus bacterioferritin and the native protein evaluation of comparative protein structure modeling by modeller-3 salt-dependent aggregation and assembly of e coli-expressed ferritin ferritin protein nanocages use ion channels, catalytic sites, and nucleation channels to manage iron/oxygen chemistry a peptide from the extension of lys-trna synthetase binds to transfer rna and dna sweeping away protein aggregation with entropic bristles: intrinsically disordered protein fusions enhance soluble expression intrinsically disordered proteins and intrinsically disordered protein regions intrinsically unstructured proteins and their functions identification of an ideal adjuvant for receptor-binding domain-based subunit vaccines against middle east respiratory syndrome coronavirus protein/peptide-templated biomimetic synthesis of inorganic nanoparticles for biomedical applications self-assembling influenza nanoparticle vaccines elicit broadly neutralizing h1n1 antibodies harnessing an rna-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation host participation in bacteriophage lambda head assembly properties of a mutant of escherichia coli defective in bacteriophage lambda head formation (groe). i. initial characterization coupled chaperone action in folding and assembly of hexadecameric rubisco modeling the influence of salt on the hydrophobic effect and protein fold stability rational design of an epstein-barr virus vaccine targeting the receptor-binding site key: cord-296585-yfh5d4io authors: su, yu-ching; jalalvand, farshid; thegerström, john; riesbeck, kristian title: the interplay between immune response and bacterial infection in copd: focus upon non-typeable haemophilus influenzae date: 2018-11-05 journal: front immunol doi: 10.3389/fimmu.2018.02530 sha: doc_id: 296585 cord_uid: yfh5d4io chronic obstructive pulmonary disease (copd) is a debilitating respiratory disease and one of the leading causes of morbidity and mortality worldwide. it is characterized by persistent respiratory symptoms and airflow limitation due to abnormalities in the lower airway following consistent exposure to noxious particles or gases. acute exacerbations of copd (aecopd) are characterized by increased cough, purulent sputum production, and dyspnea. the aecopd is mostly associated with infection caused by common cold viruses or bacteria, or co-infections. chronic and persistent infection by non-typeable haemophilus influenzae (nthi), a gram-negative coccobacillus, contributes to almost half of the infective exacerbations caused by bacteria. this is supported by reports that nthi is commonly isolated in the sputum from copd patients during exacerbations. persistent colonization of nthi in the lower airway requires a plethora of phenotypic adaptation and virulent mechanisms that are developed over time to cope with changing environmental pressures in the airway such as host immuno-inflammatory response. chronic inhalation of noxious irritants in copd causes a changed balance in the lung microbiome, abnormal inflammatory response, and an impaired airway immune system. these conditions significantly provide an opportunistic platform for nthi colonization and infection resulting in a “vicious circle.” episodes of large inflammation as the consequences of multiple interactions between airway immune cells and nthi, accumulatively contribute to copd exacerbations and may result in worsening of the clinical status. in this review, we discuss in detail the interplay and crosstalk between airway immune residents and nthi, and their effect in aecopd for better understanding of nthi pathogenesis in copd patients. the lungs are vital organs involved in gas exchange between the vascular system and the external environment, thus they are greatly exposed to the environment-derived microorganisms, including fungi, viruses, and bacteria. the bronchial tree and parenchymal tissues of the lungs, that until recently were considered as sterile, are colonized by phylogenetically-diverse microbes. the genera of firmicutes, bacteroidetes, and proteobacteria are the most common phyla identified and represent 60% of the total bacterial microbiome in the healthy airway (1, 2) . the majority of the lung microbiota belongs to the normal flora that play an important role in the pulmonary epithelial integrity, colonization resistance, and homeostasis of the immune system in the respiratory tract (3) . a small fraction of them are, however, potentially pathogenic microorganisms that are involved in a variety of lung diseases, as exemplified by the genus haemophilus. non-typeable haemophilus influenzae (nthi) is a gram-negative coccobacillus that are commonly residing in the human airways. uniquely and yet unexplained, nthi is a commensal when colonizing the nasopharynx or throat, but pathogenic in the lower airways triggering a robust inflammatory response [for reviews see (4, 5) ]. nthi is considered a potential opportunistic pathogen as it frequently infects the lower respiratory tract of lungs with structural damage as a consequence of non-infectious lung diseases or mechanical injuries. moreover, nthi occasionally causes bronchitis and pneumonia (6) . in addition, lower airway colonization by nthi has been associated with disease progression of several more or less non-infectious lung diseases such as bronchiectasis (7), cystic fibrosis (8) , interstitial lung diseases (9, 10) , but mostly in chronic obstructive pulmonary disease (copd) (11, 12) . copd is a severe inflammatory lung disease characterized by airflow limitation with a range of pathological changes. both genetics and environmental factors trigger the onset of copd, however, microbes including nthi play an important role in the acute exacerbations. this review describes the disease progression of copd in the context of host immuneinteractions linked to nthi, and the overall impact in disease exacerbation. copd is the third leading cause of morbidity and mortality worldwide expected to affect more than 210 million people by 2030 (13, 14) . according to the global initiative for chronic obstructive lung disease (gold), copd is a pulmonary disease that is manageable, but significant exacerbations and co-morbidities may, however, contribute to the overall severity in individual patients (15) . copd is characterized by chronic airflow limitation of the peripheral airways with a range of pathological changes in the lung that are not fully reversible, and usually become progressively worse over time. the progression of copd is associated with an abnormal inflammatory response of the lung to noxious particles or gases. from a pathological point of view, copd comprises a group of pulmonary abnormalities related to the inflammatory reaction of the airways, alveoli, and pulmonary vessels (16) (17) (18) (19) . these include (i) pulmonary emphysema, (ii) chronic bronchitis, and (iii) disease in the small airways. the pulmonary abnormalities progressively affect all parts of the lung, resulting in increased resistance of the conducting airways and thus chronic airflow obstruction that eventually will lead to a declined lung function. emphysema is a permanent loss of elastic lung recoil caused by elastolytic destruction and enlargement of the alveolar wall distal to the terminal bronchioles. this consequently results in the loss of alveolar attachments to the small airways and thus limitation of airflow and gaseous exchanges. chronic bronchitis is characterized by consecutive and chronic cough with expectorations that last for more than 3 months within 2 years. it is associated with inflammation of the bronchial walls with increased inflammatory infiltrates, hyperplasia of goblet cells, hypertrophy of tracheobronchial submucosa, increased mucous secretion and, finally, dilatation of the airway ducts (airways of about 2-4 mm in internal diameter). the majority of the ciliated epithelium lining the airways are also either compromised or dysfunctionnal, and may be replaced by nonciliated squamous epithelial cells. small airway diseases, on the other hand, involve hyperplasia and metaplasia of mucosal glands and goblet cells, hypersecretion of intraluminal mucus, macrophage bronchiolitis, and accumulation of lymphocytes in the small bronchioles (airways of ∼2 mm or less in diameter and terminal bronchioles). in addition, distortion, fibrosis, stenosis, tortuosities, hyperplasia, and hypertrophia of the small airway smooth muscles also contribute to the loss of elasticity in the lung parenchyma. although copd mainly affects the lungs, it also produces significant extrapulmonary consequences as a results of an escalated inflammatory response orchestrated by airway cells and immune mediators (20, 21) . the comorbidities are commonly seen in copd patients despite the actual mechanism responsible for the systemic inflammation remains to be elucidated. the development of copd is multifactorial, with cigarette or tobacco smoking being the primary cause of copd (22, 23) . other risk factors that may promote the onset and progression of copd includes prolonged occupational exposure to particles/gases in mining and textile industries, air pollution resulting from biomass combustion, and bronchial hyperresponsiveness (16, 18, 24) . the variability of copd incidences among smokers is also explained by a genetic predisposition, such as α1-antitrypsin deficiency and cutis laxa [mutation of the elastin gene (eln)] (25, 26) . the α1-antitrypsin deficiency is caused by deleterious homozygous mutations in serpina1 which contributes to 1-2% of copd cases. the deficiency results in increased neutrophil elastase activity that ultimately leads to the degradation and collapse of the alveoli. importantly, meta-analyses of genome-wide association studies (gwas) and other genotyping studies have revealed that multiple single nucleotide polymorphism (snps) in at least 34 genes from different pulmonary genomic loci are associated with copd susceptibility (27) (28) (29) (30) . airway epithelium exposed to cigarette or tobacco smoke has compromised tight junctions and delayed epithelial wound repair (31) (32) (33) (34) . moreover, cigarette smoke alters basal cell differentiation and subepithelial extracellular matrix (ecm) composition, and thus causes airway remodeling (i.e., goblet cell hyperplasia and small airway squamous metaplasia) (35) (36) (37) . this results in mucus hypersecretion, impaired mucocilliary clearance, and airway obstruction. tobacco or cigarette smoke also enhances proliferation and ecm deposition by activating the extracellular signal related kinase (erk) and the p38 signaling pathway (38). the alteration of major ecm components are widespread in all lung compartments in copd patients with a total increase of type i and iii collagens, fibronectin, and laminin in parallel with reduced concentrations of proteoglycans, perlecan decorin, versican, biglycan, tenascin and elastin (39, 40). cigarette induced-overexpression of matrix metalloproteases (mmps1, 2, 7, 9, 12, and 28) and elastase has also been reported, and may contribute to the airway tissue destruction and fibrosis (41-43). in addition, harmful volatile chemicals derived from cigarette smoke (i.e., acetaldehyde, acrolein, and crotonaldehyde) are prone to form carcinogen adducts with dna and various proteins (i.e., apoliprotein e and surfactant protein a). they also dysregulate airway epithelial ion transport, disrupt the phagocytic activity of airway phagocytes, and diminish the airway surface liquid volume (44-46). numerous proteomics and transcriptomic analyses have unveiled the crucial impact of cigarette or tobacco smoke and copd disease progression on airway gene expression (47, 48). the differential gene expression studies were done using copd experimental models or clinical samples [i.e., bronchial epithelial cells, sputum, plasma, blood, and bronchoalveolar lavage (bal) fluid]. collectively, most of the altered genes are involved in oxidative stress, xenobiotic metabolism, antioxidant responses, dna repair, ecm remodeling, inflammatory responses, and immune defenses, which the latter two are our major interest of discussion in this review. the omics data aid in the increased knowledge of molecular mechanisms in copd. they may reflect the dynamic response and attempts by the airway epithelial cells to repair the cytotoxic injury primarily triggered by inhaled irritants. deleterious and irreversible alterations occurring and interfering with the airway epithelial homeostasis and immune defense may promote copd development and progression. notably, gene alterations in phagosomal-and leukocyte transendothelial migration pathways (ltm) are significantly correlated with the level of t cells and airway obstruction in smokers (49). the ltm, however, were found to be further dysregulated in copd patients. hence, in addition to clinical/physiology variables, a number of gene products with significant differential gene expression may be targeted as specific proteomic signatures or biomarkers for early copd detection, patient monitoring, disease subgrouping, and finally treatment selection (50, 51). tobacco or cigarette smoke regulates airway gene expression via two main mechanisms, by altering the status of (i) chromatin remodeling, and (ii) dna methylation of the target genes (figure 1 ) (52-54). chromatin remodeling is a result of a disrupted balance in histone acetylation/deacetylation (55). excessive activation of more than 20 transcription factors including nf-κb, and lipoprotein peroxidation products (peroxinitrite, acrolein, and 4hne from tobacco smoking) contributes to such anomaly. nf-κb is a key inflammatory and redox-sensitive transcription factor that plays a direct role in cigarette smoke-induced airway inflammation. nf-κb has been described as a "smokesensor" due to its sensitive activation by tobacco residues (56). stimulation of multiple signaling cascades [p38 mitogenactivated protein (mapk) kinases, mitogen and stress-activated kinase 1 (msk1), protein kinase c zeta (pkcζ), and iκb kinase (ikk) complex (ikkα, ikkβ, and nemo)] by tobacco residues promotes the activation and nuclear translocation of transcription factor nf-κb rela/p65 (54, 57-64). this is followed by a complex formation of nf-κb/cbp-p300 [coactivator, crebbinding protein (cbp) or cbp/p300] at target dna sequences. it should be noted that cbp/p300 also has intrinsic histone acetyltransferase (hat) activity. subsequent acetylation and phosphorylation of the subunit p65 in the nf-κb/cbp-p300 complex by the activated msk1/pkcζ-signaling pathways (and other 11 different kinases), and cbp/p300, respectively, are required for the full activation of nf-κb (57, 60, 63). this enhances the dna binding affinity of the complex. histones h3 and h4 in the chromatin complex of target sequences are then being acetylated (histone h3 at lys9; h4 at lys8 and lys12) and phosphorylated (histone h3 at ser10) by the subunit cbp of the nf-κb/cbp-p300 complex, and the activated msk1 and pkcζ, respectively. the hyperacetylated core histones, however, fail to be neutralized or deacetylated by a dysfunctional histone deacetylase (hdac2). peroxinitrite nitrates the tyrosine residues of the hdac2 and causes inhibition of activation and reduced expression of the protein. of note, peroxinitrite is a by-product generated from the immune cell-derived nitrite oxide (no) and reactive oxygen species (ros) of cigarette smoke (65, 66). cigarette or tobacco smoke disturbs the dna methylation status of target genes through several mechanisms. firstly, dna damage caused by cigarette smoke stimulates the dna methyltransferase 1 (dnmt) to actively induce cpgs methylation at the damaged site (67). the hypermethylation is prone to introduce error of methylation in some target genes, resulting in reduced gene expression. secondly, activation of nicotine signaling pathway by tobacco smoke causes camkii/iv and erk/mapk pathway activation that subsequently induces the activity of cbp to suppress the expression of dnmt1. this may result in reduced dna methylation and thus altered level of gene repression by dnmt (68-70). finally, enhanced activities of transcription factors such as hypoxia inducible factor 1 due to the high level of carbon monoxide and hypoxia have also been reported to influence airway gene expression (71) . consequently, the combinatorial effect from both aberrant acetylation of histone and dna methylation promotes the transformation of chromatin from a condensed structure to an activated open conformation. this facilitates irregular accessibility of dna for transcription machineries, hence figure 1 | cigarette and tobacco smoke has several effects on gene regulation. nicotine and other compounds in the smoke alter gene expression by two pathways, firstly, chromatin remodeling (left) and secondly, dna methylation (right). chromatin remodeling involves activation of kinases signaling pathways, activation and nuclear translocation of transcription factor nf-κb (rela/p65), and complex formation with cbp/p300 on specific dna sites. cbp/p300 is intrinsically a histone acetyltransferase (hat). subunit p65 is further phosphorylated at ser276 and ser311, respectively, by msk1 and pkcζ, whereas cbp acetylates p65 at lys310. the phosphorylation and acetylation enhance the interaction within the nf-κb/cbp/p300 complex while stabilizing the dna binding of nf-κb. the complex of nf-κb/cbp/p300 then modifies the histones through cbp-mediated acetylations of histone h3 (at lys9) and h4 at lys 8 and lys12, and phosphorylation of h3 at ser10 by msk1 and pkcζ. this results in the structure change of chromatin, from a condensed structure (repressed) to an activated open conformation. the transcription of target genes is therefore increased. in the second mechanism, several side effects resulting from cigarette smoking such as dna damage and nicotine signaling could trigger the hypermethylation or decreased methylation of target dna. this may lead to dna methylation anomalies and thus altered dna expression. resulting hypoxia due to high concentrations of carbon monoxide also contributes to altered gene expression. the aberrant gene expression by cigarette smoke mostly occurs in pro-inflammatory genes with resulting increased production of inflammatory mediators, and amplified inflammation in the copd lung upon exposure. irregular gene expression by various cell types in the airway. the mechanisms reported are responsible for increased expression of nf-κb-dependent proinflammatory gene products [i.e., il-1β, il-6, il-8, ccl-5 cyclooxygenase (cox)-2, and mip-2/cxcl2] in both pulmonary structural cells (bronchial, small airway, and alveolar epithelial cells) and immune cells (alveolar macrophages), increased vegf and inos in nasal fibroblasts and lymphocytes (jurkat t cells), respectively, and decreased activity of antioxidant transcription factor nrf2 and α1-antitrypsin in bronchial epithelial cells (54, 56, 57, 59, 62-64, [72] [73] [74] [75] [76] [77] [78] [79] . these may contribute to the anatomical anomalies in the airway and excessive inflammatory responses among smokers during the course of copd. copd is associated with chronic inflammation in the peripheral airways orchestrated by both innate and adaptive immune responses that are interconnected via dendritic cells (80) . increasing numbers of inflammatory cells (neutrophils, macrophages, t and b lymphocytes, mast cell, eosinophils, and dendritic cells) and inflammatory mediators are accumulated in the airway lumen/wall in the lung parenchyma (19, 81) . these immune cells and inflammatory mediators can hence be detected in the sputum and bal fluid of copd patients. the level of accumulation is positively correlated with disease severity. an increasing number of studies using animal models and clinical tissues have reported the nature of excessive airway inflammatory responses in copd. despite this, the heterogeneity in symptoms progression among copd patients remain unexplained. the overall mechanism of copd inflammatory immune response is depicted in figure 2 . the first line of defense in the lung-the innate immunity and inflammasome lung structural cells (epithelial and endothelial cells, fibroblasts, and airway smooth muscle cells) are activated by inhaled irritants through the stimulation of several pattern recognitions receptors (prrs), with toll-like receptor (tlr)-4 being reported as the key player in most of the inflammatory responses (82) (83) (84) (85) . this causes an increased expression and release of an array of pro-inflammatory mediators and chemokines through the oxidative pathway by the activated bronchial epithelial cells and immune cells (alveolar macrophages). the inflammatory mediators [(interleukin (il)-1β, il-6, il-8, il-33, c-x-c motif chemokine ligand (cxcl) 10, granulocyte-macrophage colonystimulating factor (gm-csf), granulocyte-colony stimulating factor (g-csf), tumor necrosis factor (tnf)-α, fibroblast growth factor 1 and 2 (fgf1/2), transforming growth factor (tgf)-β1, c-c motif chemokine ligand (ccl) 2, ccl20, and thymic stromal lymphopoietin (tslp)] act on recruited immune cells and resident cells to initiate a series of innate immune responses (23, (86) (87) (88) (89) (90) . meanwhile, activated alveolar macrophages, which are usually patrolling the lung parenchyma, further release more pro-inflammatory mediators and chemokines [il-1β, il-6, il-8, il-23, tnf-α, ccl1, cxcl1, cxcl5 (ena-78), cxcl9, cxcl10, cxcl11, ccl2, leukotriene b4 (ltb 4 )], ros, elastolytic enzyme [matrix metalloprotease protein (mmp)-2,−9, and−12; and cathepsin-k,-l, and -s], gm-csf, and g-csf (23, 91, 92) . the enhanced levels of ccl2 and cxcl1 result in recruitement of blood monocytes expressing ccr2 and cxcr2 (receptors for ccl2 and cxcl1, respectively), to the lung and differentiate locally into macrophages. interestingly, there are higher expression levels of the ccr2 and cxcr2 found on blood monocytes in copd subjects (93) . this may explain the rapid recruitment and excessive accumulation of monocyte-derived interstitial macrophages in the lung tissue of copd patients (94, 95) . upregulation of neutrophil chemoattractors (ltb 4 , cxcl1, cxcl5, il-8, and tnf-α) induces a massive migration of circulating neutrophils into the lung parenchyma (96) . the transmigration of blood neutrophils occurs through adherence of the granulocytes to e-selectin of endothelial cells that is found to be upregulated in copd (97) . this results in airway neutrophilia in several copd patients (96, 98, 99) . the recruited neutrophils (to the lung) are then activated to secrete granule proteins [myeloperoxidase (mpo) and neutrophil lipocalin] while releasing its own il-8 for further neutrophilic recruitment and amplification of the inflammation (100) . in addition to the macrophage-derived proteases, neutrophils also secrete serine proteases [neutrophil elastase (ne), cathepsin g, proteinase-3, mmp-8, and mmp-9] that are associated with serious alveolar destruction in emphysema (101) . the protease activity may be further enhanced in conditions with genetic deficiencies or suppressed expression of α1-antitrypsin by tobacco smoke. in addition, ne, cathepsin g, and proteinase-3 are involved in the stimulation of mucus secretion from submucosal glands and goblet cells, resulting in airway mucus hypersecretion and airway obstruction in copd (101) . the nlrp3 (nlrp3: nucleotide-binding domain, leucinerich-containing family, pyrin domain-containing-3 or nod-like receptor protein 3) inflammasome is a cytosolic multi-protein complex (consisting of the inflammation sensor protein nlrp3, adapter protein asc, and the effector protein caspase-1) (102). the nlrp3 inflammasomes are involved in the copd airway inflammation by regulating the production of pro-inflammatory cytokines il-1α, il-1β, and il-18. these cytokines are important for neutrophil survival and activation of t helper (th) 17 cells (103) . interestingly, local airway nlrp3 inflammasome activation is positively correlated with acute exacerbations and lower airway microbial colonization in copd patients (103, 104) . moreover, in an elastase-induced emphysema model, the nlrp3 inflammasome is activated in addition to hyperproduction of mucin muc5ac by diesel extract particles, extracellular atp, and inflammatory protein s100 (105, 106) . the adaptive immunity is initiated at a later stage, and is recognized by the increased number of t and b lymphocytes and pulmonary dendritic cells. dendritic cells are the major antigen-presenting cells (apc) in the airways, and link the innate and adaptive immunity. circulating dendritic cells (expressing receptors ccr2 and ccr6) are recruited to the airway via dendritic chemoattractants ccl2 and ccl20 released by activated airway epithelial cells in response to cigarette smoke (107, 108) . dendritic cells act by endocytosis of inhaled irritants that subsequently are processed into antigen peptides during maturation and further migration to lymph nodes. uncommitted t lymphocytes are thereafter primed by the presented antigen. these important cells are activated by il-12 released from dendritic cells for subsequent commitment into antigen-specific t cell lineages, i.e., t helper 1 (th1; cd3 + cd4 + ) cells, whereas immature dendritic cells in the airway promote th2 differentiation (23, 109) . interestingly, in copd patients, pulmonary th and cytotoxic t cells (tc; cd3 + cd8 + ) express more cxcr3 receptors compared to healthy individuals (110, 111) . this enhances their migration toward chemoattractants cxcl9, cxcl10, and cxcl11 that are actively released by alveolar macrophages in copd subjects. activated cd8 + t cell subset type 1 (tc1) releases perforins, granzyme b, and tnf-α to induce alveolar cells apoptosis, contributing to the emphysema (112) . in parallel, pulmonary th17 t cells are activated by alveolar macrophage-derived il-6 and il-23 to secrete il-17a and il-22 causing neutrophilic inflammation (113, 114) . inflammatory cytokines are also released by type 3 figure 2 | non-typeable h. influenzae-dependent immune responses in the lower airway of copd patients result in inflammation. airway epithelium exposed to cigarette or tobacco smoke display an increased permeability with compromised tight junctions, and airway remodeling (goblet cell hyperplasia and small airway squamous metaplasia). cigarette smoke causes the activation of airway epithelium and alveolar macrophages. the activated airway structural and resident immune cells release an array of chemotactic factors responsible for recruitment of inflammatory and immune cells to the lung. activated epithelium produces tgf-β and fgf that triggers the production of ecm molecules by fibroblasts. increased deposition of ecm causes progression of fibrosis and air flow limitation. the chemokines cxcl1 and il-8, and ltb 4 attract the circulating neutrophils through the receptors cxcr2 and blt 1 , respectively. meanwhile, cxcl1 and ccl2 targeting the receptors cxcr2 and ccr2 on blood monocytes are also released. recruited blood monocytes differentiate into macrophages in the airway tissue. activated alveolar macrophage and epithelium cell also release inflammasome (1l-1β and il-18) for neutrophils survival and activation of helper t cells th17. the chemokine il-23 are released by macrophages to attract t helper cell subset th17, and ilc3. both th17 and ilc3 will release il-17 and il-22 that will act on the alveolar epithelium to release cxcl1 and il-8 for enhanced recruitment of neutrophils, resulting in neutrophilic inflammation. activated neutrophils are thereafter degranulated and release myeloperoxidase (mpo), lipocalin, neutrophil elastase (ne), cathepsin-g (cg), proteinase-3 (prot-3), and matrix metalloprotease (mmp) 8 and 9. the granulated products are proteolytic and elastilolytic to aveolar, causing alveolar destruction and emphysema. in addition, ne, cg, and prot-3 are also targeting goblet cells and submucosal glands to induce hypersecretion of mucus. dendritic cells carrying the receptors ccr2 and ccr6 are recruited to airway tissue via chemottractants ccl2 and ccl20. the dendritic cells uptake the antigen (smoke residues), and present the antigens to the naïve t cells at lymph nodes. uncommitted t lymphocytes are thereafter primed to the presented antigen and activated by il-12 derived from dendritic cells (professional antigen presenting cells; apc). mature/activated t cells expressing receptor cxcr3 are chemotactic toward cxcl9, cxcl10, and cxcl11 and are recruited to the lung tissue. cytotoxic cd8+ t cell subtype tc1 releases perforin and granzyme b resulting in epithelial apoptosis contributing to emphysema progression. for the humoral immune response, b cells are activated by th2, enter the circulation via high-endothelial venule (hev)-like vessel and transported to lung tissue, and organized into lymphoid follicles at peripheral airway. b cell-derived plasma cells from lymphoid follicles release iga, and secreted into airway lumen as secretory iga (siga) via the polymeric immunoglobulin receptor. mucosal antibodies play an important role to eradicate pathogens and noxious antigens via immune exclusion. however, the airway defense by siga is diminished by nthi iga protease that degrade the antibodies. tlr2 and tlr4 of the airway phagocytes and epithelium following exposure to cigarette smoke are not responding to p6 and los of nthi. this results in defective phagocytosis and delayed bacterial clearance from the airway. the suppressed tlr4 induction in t cells has also lead to th2 predominant immune response, with low production of ifn-γ and reduced t cell-mediated immune killing of nthi. moreover, nthi downregulates foxp3 of tregs and thus impairs the anti-inflammatory/pro-inflammatory balance of tregs. the extensive immunosuppressive activity by tregs diminishes the response of effector t to (continued) figure 2 | nthi stimulation. lastly, plasma cells from copd patients fail to produce nthi-specific antibodies and compromised immunoglobulin class switching. the impairment of the host immune response in copd toward nthi infection are labeled in blue. in total, nthi infection in copd lung adversely reduces the production of il-1β, il-6, il-8, cxcl-10, il-22, tnf-α, antimicrobial peptide (amp), and ifn-γ. this may explain the inefficient eradication of airway pathogens in copd patients whereby persistent nthi infection concomitantly escalates the inflammation and thus exacerbation in copd. innate lymphoid cells (ilc3) (115) . the ilcs are involved in the homeostasis of lung immunity and are regulated by epithelially produced il-33 and tslp (116, 117) , and are further stimulated in response to cell damage. the accumulation of b lymphocytes in the peripheral airway and within lymphoid follicles is associated with airway autoimmunity in the progression of copd (118). airway tissue damage in conjunction with impaired t-regulatory cells (tregs), both related by cigarette smoke, contributes to the formation of autoantibodies against airway components. autoantibodies against elastin, epithelial, endothelial, carbonylated, and citrullinated proteins are found in the circulation of copd patients (119) (120) (121) (122) (123) (124) . the generation of autoantibodies might activate plasma exudate-derived complement components resulting in a chronic inflammation, and consequently damage of the airways with emphysema progression (124) (125) (126) (127) . from a physiological point of view, a modulated inflammatory process is important for a protective and optimal immune response. however, the prolonged airway inflammation in copd as a results of impaired homeostasis leads to serious side effects since it amplifies the tissue damage and impairs the local immune defenses. the abrogated local immune system may make the airways of copd patients susceptible for opportunistic or recurrent infections by viruses and bacteria that in turn might exacerbate the disease. acute exacerbations of copd (aecopd) are episodes of acute symptom worsening that usually are associated with both respiratory (increased airway inflammation) and non-respiratory (system inflammation/co-morbidities) effects (128) (129) (130) . the typical symptoms of an aecopd include increased production of purulent sputum, dyspnea, cough, wheezing, and symptoms of a cold that may last from 7 days up to 12 weeks (15, 130, 131) . it commonly occurs in patients with advanced copd and results in additional therapy based on the level of exacerbations. exacerbations are classified in three levels according to gold. there is the mild disease that can be treated with short acting bronchodilators (sab); moderate disease with sab combined with antibiotics and/ or oral corticosteroids; and finally severe exacerbations with acute respiratory failure which requires emergency room visit and eventually hospitalization (15, 130) . aecopd is a complex yet multifactorial consequence of copd. most of the exacerbations could be triggered by infectious (up to 80%) or non-infectious agents (∼10%) (aecopd with known etiology), whereas up to 30% of cases are of unknown etiology (132, 133) . respiratory tract infections are the major causes for aecopd with known etiology and are mainly attributed to infections by viruses, bacteria, and atypical bacteria (not detected with conventional gram-staining) (11, 134, 135) . non-infectious causes of aecopd include air pollution, environmental factors, meteorological effects, and comorbidities of the patients, all of which are partially contributing to copd exacerbations (133, 135, 136) . respiratory viral infections are often the primary cause in the infection-dependent aecopd, and virus was identified as single or multiple infecting strains from up to 64% of copd patients with exacerbations recorded between years 2001-2017 (137) (138) (139) (140) (141) (142) (143) (144) (145) . the most common infecting viruses are, by far, human rhinovirus, influenza virus a, and respiratory syncytial virus, whereas parainfluenza virus, coronavirus, echovirus, human metapneumovirus, and adenovirus are considerably rare. bacterial infections contribute to an average of 50% of infective acute exacerbations with a prevalence being reported ranging from 26 to 81% (132, 135, (146) (147) (148) . the most commonly pathogenic bacterial species isolated from the lower airway of copd patients during aecopd are nthi, moraxella catarrhalis, streptococcus pneumoniae, staphylococcus aureus, pseudomonas aeruginosa, and klebsiella pneumoniae (11, 129, 133, 136, (149) (150) (151) (152) . it has been suggested that infection with new strains of the infecting species, rather than a new species, is highly associated with an increased risk of exacerbation (11, 153, 154) . atypical bacteria that cause exacerbations are chlamydia spp., legionella pneumophilia, and mycoplasma spp. in contrast to viral infections that are diagnosed in 5-45% of copd patients with stable disease and increase to 39.3-64% during copd exacerbations, bacterial colonization in the airways are more common with the same species during both stable disease (25-86%) and exacerbations (58.8-81%) (11, 132, 136, 137, 142, (155) (156) (157) (158) . hence the precise or direct role of bacterial infection as the primary cause in triggering aecopd remains controversial although a significantly increased bacterial load is observed during exacerbation in several patients. this further suggests that bacteria might be more involved as secondary invaders after an initial viral infection. viral infections have been reported to cause several physiological changes in the lung that in turn facilitates secondary bacterial invasion. the mechanism of bacterial superinfection has been described for h. influenzae, s. pneumoniae, s. aureus, and many other airway pathogens (159) (160) (161) . firstly, viral infections destroy the tight junctions of the airway epithelial barrier while inducing epithelium apoptosis. this results in the onset of airway epithelium lining repair whereby the sloughed off dead cells would become a rich nutrient source for growth of infecting bacteria. the damaged epithelium lining also enables bacterial adherence to the exposed basement membrane and ecm. secondly, the demolished ciliated clearance as a result of the virus-damaged airway epithelium lining further promotes bacterial colonization and subsequent epithelial transmigration into deeper tissues (162) (163) (164) . lastly, viral infections are also detrimental to the airway immune defense by causing degradation of antimicrobial peptides (amp), and by triggering ifn-γ secretion by immune cells. this results in suppressed macrophage and neutrophil responses to infecting bacteria, and thus enables bacterial evasion of the airway immune defense (165) (166) (167) (168) . nevertheless, viral and bacterial coinfection have greater impact in the aecopd airway inflammatory responses than bacteria or virus infection alone (168, 169) . this is in parallel with the co-isolation of both respiratory viruses and bacteria from 6 to 30% of aecopd patients (129, 133, 136, (170) (171) (172) (173) (174) . infective aecopd is also attributed to impaired functions of amp, macrophages, and neutrophils triggered by inhaled irritants such as tobacco smoke. expression of microbialinduced amp (human β-defensin 2) is suppressed in airway epithelial cells when exposed to cigarette smoke (175, 176) . both the alveolar and monocyte-derived macrophages in patients with copd are defective in phagocytosis of bacteria such as h. influenzae and s. pneumoniae (177, 178) , and in efferocytosis of apoptotic neutrophils and epithelial cells. in addition, neutrophils from copd patients are aberrant in chemotactic response with defective accuracy (179) . all these factors contribute to the failure to resolve inflammation in copd leading to facilitated chronic microbial colonization, also during exacerbations. the low number of cultivable bacteria found in healthy individuals previously led to the conclusion that healthy and normal lungs are virtually sterile. this hypothesis is currently being revised, since the introduction of 16s rdna based molecular diagnostics has shown that even healthy lungs have a distinct microbial community, different from that seen in the upper respiratory tract (180, 181) . this has led to the concept of a core human lung microbiome which can be altered in copd stable disease and during exacerbations (182) . the role of the lung microbiome in the pathogenesis of copd by influencing host immune response has also been suggested (151, (183) (184) (185) (186) (187) (188) . the stability of the lung microbiome has profound impact on maintaining local immune homeostasis (189) . according to the "vicious circle" hypothesis, airway inflammation and impaired immune defenses caused by either viral infections or irritant inhalation have ecological influence on the airway microenvironment and growth conditions that would eventually lead to dysbiosis of the lung microbiota (182, 190) . the changed lung microbiome would then cause a maladaptive immunological response resulting in further inflammation and damage of the lung immune defenses, and additional alteration of the lung microbiome. the chain of events thus generates a vicious circle that contributes to copd progression and exacerbation. several studies have documented that copd progression from stable state to an exacerbation could induce microbiota shift in the lower airway (bronchioles), sputum, and throat (151, (190) (191) (192) (193) (194) (195) (196) . alteration in the microbiome complexity or richness is associated with the inflammatory process and changes in ecm protein expression in the lung, as observed in copd (185, 197) . declined diversity in the lung microbiome has been reported to be related to disease severity, inflammation and decreased lung functions in copd. this includes the increased emphysematous destruction, bronchial tissue remodeling, lymphoid follicle formation, elevated autoantibodies, and il-17a production, and finally increased neutrophil extracellular traps (net) formation in the airway of animal models or aecopd patients (198) (199) (200) (201) . it has recently been reported that lung microbiome diversity is also associated with genetic factors. mannose-binding lectin (mbl) deficiency has also been associated with disease severity and exacerbations in patients with cystic fibrosis and bronchiectasis (202) . however, copd patients with a genetic deficiency in mbl are less susceptible to haemophilus spp. colonization, lowering the risk of exacerbations while their lung microbiota is more diverse than normal copd patients (203) . in this review we will focus on nthi, one of the dominant genera that is relatively abundant in the total copd-dependent lung microbiome, due to its role of infection in copd immunological responses (136, 149, 192-195, 198, 204-206) . the microbiology of h. influenzae has recently been reviewed in detail by our group and others (4, 5, 207) . it is a gramnegative coccobacillus that commonly colonizes the human nasopharynx, and is typed as capsulated (type a-f) or nonencapsulated strains (nthi). h. influenzae may cause both invasive and mucosal disease (208) . since the introduction of capsule polysaccharide conjugate vaccines against type b (hib), nthi dominate, followed by capsule type f (hif) (209, 210) . mucosal infections, including acute otitis media, sinusitis, and exacerbations in copd, are nowadays mainly associated with nthi. there has also been a significant shift in the epidemiology of severe invasive disease, from hib infections in small children to nthi in adults (210, 211) . the most common principal infection focus by h. influenzae is now community acquired pneumonia (cap), whereas the incidence of historically common diagnoses such as meningitis and epiglottitis have significantly decreased (210, 211) . patients with underlying conditions, notably copd, seem to be at higher risk for invasive infections (209) . there is consensus that h. influenzae is one of the key bacterial pathogens involved in pathogenesis of both stable copd disease and acute exacerbations (207) . however, the relative abundance and significance of nthi in copd varies between different studies. several factors, such as sampling methodology, choice of microbiological analysis and, if the patient has a stable disease or an exacerbation, or has been subject to previous antibiotic therapies, tend to affect the outcome of the studies (212) . common sampling methods from the lower respiratory tract include both bronchoscopy techniques such as protected specimen brush (psb) and collection of bal fluid as well as non-invasive methods like sputum sampling (213) . all of these methods, particularly sputum, are to some extent subject to the risk of contamination from the normal microbial flora of the oro-and nasopharynx, which might reduce their specificity (213) . however, several studies still show a distinct association between lower respiratory tract samples and clinical parameters in copd patients, making the information valuable (214) . cultivable bacteria are seldom found in the lower airways of healthy individuals (215) , whereas copd patients show bacterial growth in 30-50% of cases even during stable disease ( table 1) . on top of that, several studies have shown a significant increase in the proteobacteria phylum, which includes haemophilus spp., in individuals with both stable disease and aecopd ( table 2) . nthi is consistently one of the predominating bacterial species isolated in those cultures; other important pathogens include s. pneumoniae, m. catarrhalis, and p. aeruginosa (219) . during aecopd, the bacterial load is increased even further, and nthi continues to be the predominating species (208) . furthermore, acquisition of a new nthi strain has, in one study, been linked to the onset of aecopd (153) . moreover, the growth and dominance of h. influenzae following rhinovirus infection was observed in the sputum microbiome of patients with copd (190) . the chronic inflammation that characterizes copd pathogenesis causes significant changes to the pulmonary tissue. the lower respiratory tract of patients suffering from this disease is marked by epithelial denuding, hypersecretion of mucus, disproportionate phagocyte presence and imbalances in antioxidant/oxidants (220) . this altered milieu selects for specific bacterial species that are genetically equipped to competently address these environmental stressors (151, 195, 221) . nthi is the most common pathogen isolated from the sputum of copd patients, and the primary cause of exacerbations (212) , indicating a unique ability to colonize and persist in the chronically inflamed lower respiratory tract. in recent years, great efforts have been made in understanding how nthi colonizes the pulmonary tissue. in addition to the regular arsenal of virulence factors associated with nthi (5), the bacterial pathogen undergoes specific adaptations to increase its fitness in the copd setting. specific genetic islands that include ureabcefgh, lic2b, hgba, iga, hmw1, and hmw2 have been reported to be enriched in nthi strains isolated from copd patients compared to commensal nthi (222) . these genes are involved in raising the ph of the environment, lipooligosaccharide (los) synthesis, iron uptake, immune evasion, and attachment to host tissue. the validity of these findings is strengthened by previous work identifying upregulation of many of the same bacterial gene products during growth in copd sputum (223) . moreover, peroxiredoxinthioredoxin, an antioxidant enzyme, was found to be one of the most enriched proteins in nthi during growth in copd sputum, suggesting that the bacteria upregulate oxidative stress-countermeasures when facing oxidative imbalances in the diseased lung (223) . oxidative stress resistance has previously been shown to be vital for nthi survival in infection models (224) . in a seminal investigation by pettigrew et al., wholegenome sequencing (wgs) was conducted to follow the in vivo adaptation of nthi to the copd environment over time (225) . several interesting findings were reported in this work. firstly, the median duration of persistence by the pathogen was found to be 161 days, but it could persist in patients for up to as many as 1,422 days. secondly, slipped-strand mispairing-mediated phase variation was identified as the primary genetic adaptation to the niche. poignantly, the genes affected by the regulation mechanism encoded for (among others) the hmw adhesins, los biosynthesis, and iron uptake, that is, the same processes identified in the previous studies as important for copd adaptation (222, 223) . thirdly, and somewhat surprising, it was observed that a very limited number of genes were gained/lost during persistent colonization, meaning that selection for strains that thrive in the inflamed lower airways occurs at the very onset of colonization. finally, the authors reported that genetic changes occurred in 8 of the 12 investigated vaccine antigens during persistent infections, a fact that might be taken into consideration for potential vaccine development against nthi. another virulence factor that has been reported by murphy and co-workers to play a pivotal role for nthi survival in copd settings is iga-protease, a hydrolytic enzyme that cleaves secretory iga (siga) antibodies in the mucosal epithelium (226) (227) (228) . four genes encode for the same number of different variants of the endopeptidase with various cleavage site specificities: two igaa (igaa1 and igaa2) and two igab (igab1 and igab2). the igaa is present in all nthi whereas igab is present in ∼40% of the strains (226) . the igab1 gene has been reported to be more prevalent in copd exacerbation-causing strains, although the in vivo expression levels did not differ from asymptomatic colonization strains that also carried the gene (226) . however, iga-protease b1 and b2 have been found to promote the intracellular survival of nthi in human epithelial cells, providing a secondary function (in addition to hydrolysis of iga antibodies) that could facilitate nthi growth in inflamed environments (227) . while a majority of the persistent nthi strains that dwell in copd patients continuously express one or more variants of the enzyme, it has recently been found that a phase variation to an off-state can occur via slipped-strand mispairing over time (228) . this suggests that during certain conditions, there is a fitness benefit in not expressing iga in the airways of copd patients, albeit the specifics of this process are currently unknown. another interesting aspect of nthi colonization of copd patients is with regard to biofilm formation (229) . nthi strains that colonize the eustachian tube causing otitis media are known to build up biofilms in situ (230) . however, strains isolated from copd patients tend to have significantly diminished ability to form biofilm compared to invasive strains or those isolated from otitis media patients (229) , suggesting that this mechanism is not important for survival in the copd niche. as biofilms tend to protect the bacterial community from external assaults, these findings could indicate that the hypermucoid milieu in the copd airways is severely impaired in its ability to deliver an apt immune response for optimal clearance of residing microorganisms. in light of this impairment, biofilm formation might not be necessary for nthi to persist in this particular environment. infections with nthi have also been shown to reduce cellular levels of e-cadherin, a protein required for tight junction formation and epithelial cell integrity in human cells (231) . considering that perturbations in the epithelial cell barrier caused by the loss of e-cadherin is a common symptom of copd, nthi-mediated exacerbations likely contribute to this step of copd pathogenesis. the subsequent denuding of the epithelium could facilitate microbial colonization of the basal lamina, a well-established virulence mechanism employed by nthi and other pathogens (232) . it is currently unknown which bacterial virulence factor(s) that induce the reduction of e-cadherin levels in the host. in summary, investigations from recent years show that the environment of the lower respiratory tract of copd patients selects for nthi strains that can upregulate adhesins, modify los biosynthesis pathways, increase antioxidant stress responses and cellular invasion strategies, and, finally, trigger tolerance against acidic ph. these important colonization mechanisms thus provide researchers with viable targets for developing novel therapies. nthi is a commensal in the nasopharyngeal site but is often associated with strong inflammatory responses in the lower respiratory airways, especially in patients with copd, bronchiectasis, cystic fibrosis, pneumonia, or idiopathic pulmonary fibrosis (11, 233) . colonization and subsequent infection of nthi in the lower airways of copd patients elicits episodes of immune responses orchestrated by both the innate and adaptive immunity. nthi infection is thus commonly associated with inflammation that is mainly mediated by transcription factor nf-κb-dependent production of proinflammatory mediators. the activation of nf-κb requires induction of cross-signaling networks and cascades via activation of prrs (pattern recognition receptors) of host innate immune cells (234) . unresolved or prolonged (chronic) inflammation or failure to restore the homeostatic inflammatory status potentially contributes to exacerbations. this is clearly shown in murine copd simulation models with nthi-triggered inflammation (235-237). mice exposed to nthi lysates display inflamed airways loaded with increased levels of inflammatory mediators and phagocyte infiltrates. moreover, multiple exposures to bacterial lysates which may represent a chronic nthi infection caused extremely high infiltration of phagocytes and lymphocytes in the airways of this particular mouse model. in addition, the airway walls of the infected animals were also thickened due to increased collagen deposition (fibrosis) that reflects the typical copd features. the host immune response and specific interactions during nthi infection in copd is summarized in figure 2 . the epithelium and alveolar macrophages are predominant cell types in the airway compartment. they comprise the first line of defense in the cellular immune response against potential inhaled pathogens and antigens. the sensing of bacteria, and particularly nthi in the lower airways is initiated via prrs expressed on innate immune cells and endothelium in addition to epithelial cells (238) (239) (240) . tlrs are prrs that sense stimulation by nthiderived pathogen-associated molecular patterns (pamps), and play a primary role in initiating effector cellular responses and intracellular signaling for nf-κb activation (238) . among the different tlrs, most of the studies on nthi infection have by far been focused on tlr2 and 4. lipoproteins including nthi p6, and los are potent immunomodulators for activation of tlr2 and tlr4, respectively, and has been described in several studies on airway epithelial cells and alveolar macrophages (241) (242) (243) (244) . interaction of nthi lipoprotein p6 with tlr2 on human epithelial cells [type ii alveolar a549 and human middle ear epithelial cells (hmee)] causes nf-κb-dependent activation via two distinct tlr-signaling pathways, that is, the nf-κb translocation-dependent, and -independent pathways (242) . the nf-κb nuclear translocation-dependent pathway requires activation of nf-κb-inducing kinase ikk complex. in the second pathway, the mkk3/6-p38 mapk signaling cascade is recruited for direct nuclear phosphorylation, and thus activation of nf-κb. the branching of both pathways may occur at the tgf-β activated kinase 1 (tak1) signaling junction. nthi stimulation via tlr2 and downstream activation of p38 mapk/nf-κb-dependent pathways result in expression of cox-2 and prostaglandin (e2) (pge2) that promote inflammatory responses (245) . tlr4 stimulation by nthi los also contributes to the activation of nf-κb via two signaling pathways, the primary activating pathway of myd88 cascade and the alternative pathway of toll/il-1r domain-containing adapter-inducing interferon-β (trif). both pathways activate nf-κb through phosphorylation and degradation of inhibitor iκbα (243, 246) . nthi-tlr4 signaling mediates an effective innate immune response that leads to upregulation of tnf-α, il-1β, il-6, macrophage-inflammatory protein (mip)-1α, mip-2, and neutrophil infiltration in the airways of mice. the tlr4 response promotes efficient pulmonary clearance of bacteria in tlr4expressing animals compared to cd14/tlr4 knockout mice (243, 244) . a recent study by jungnickel et al. revealed that, in parallel with the infection-induced pulmonary neutrophilic inflammation, nthi-dependent stimulation of both tlr2 and tlr4 in a transgenic mouse [(kras la1 ) with oncogenic kras allele in the lung epithelium] additionally promotes the proliferation of kras-induced early adenomatous lesion in the lung in an tlr-dependent manner (247) . the association or role of nthi-induced airway inflammation in lung cancer progression, however, is not supported by another recent cohort study showing the lack of differences in nthi specific-antibodies between cancer-and non-cancer copd patients (12) . lastly, dectin-1 and the epidermal growth factor receptor (efgr) pathway also have proinflammtory effects upon interaction with nthi (248, 249) . activation of the dectindependent proinflammatory response requires nthi-induced phosphorylation of the dectin-1 hem-immunoreceptor tyrosinebased activation motif (hemitam) (248) . direct activation of efgr in alveolar cells and hmee by nthi-derived egflike factor has been shown to contribute to nf-κb activation. the efgr-dependent nf-κb activation is mediated via an nf-κb nuclear translocation-independent pathway, which involves both mkk3/6-p38 and pi3k/akt signaling pathways (249) . surprisingly, the interaction of efgr and nthi also results in negative regulation and suppression of the induction of tlr2 via the src-mkk3/6-p38 α/β map kinase-dependent signaling cascade, and this in turn may facilitate nthi infection (250) . the actual components of nthi that exhibit the egf-like factor activity have, however, yet to be defined. the efgrdependent negative regulation of tlr2 may thus suggest a novel mechanism targeted by nthi for immune evasion by attenuating the responses of host prr, despite the contradicted role of efgr in proinflammatory and innate immune responses of the airway epithelium (251) . nthi infection also upregulates the nrlp3-inflammasome during nthi-induced inflammation in the airway epithelium and alveolar macrophages, leading to increased secretion of il-1β and il-8, and thus neutrophilic influx to the lung (252) . some of the endogenous inflammatory mediators that are produced in response to nthi infection, including tnf-α, il-1α, and tgf-β1, may act synergetically with nthi on the airway epithelial and immune cells. the synergetic interaction drives a positive feedback loop to amplify the nf-κb transcriptional activity on proinflammatory genes and further augments airway inflammation. the synergetic activation of nf-κb by nthi and tnf-α in hmee and normal human bronchial epithelial (nhbe) cells occurs via nf-κb nuclear translocation-dependent and independent pathways. the latter pathway involves mapk/extracellular signal regulated kinase kinase kinase 1 (mekk1)-dependent activation of mapk kinase 3/6-p38 mapk pathway (253) . however, the synergetic action of nthi with tgf-β1 is mediated by another mechanism which involves smad3/4-protein kinase a (pka)-p300-dependent signaling cascade. the pathway components, pka and p300, phosphorylates residue ser276 and acetylates lys221 of the nf-κb subunit p65, respectively. this results in enhanced dna-binding activity of nf-κb (254) . the synergetic action of nthi with both tnf-α and tgf-β1 enhances the production of tnf-α, il-1β, and il-8 from airway epithelial cells and interstitial polymorphonuclear infiltrates. recently, it has been reported that co-infection of human rhinovirus and nthi on the airway epithelial cells (nhbe cells and the beas-2b cell line) also results in synergetic induction of ccl20 and il-8, albeit the exact mechanism remains to be elucidated (255) . of note, activated macrophages also release increased concentrations of tnf-α and il-1α (256) , further enhancing the inflammatory synergetic effect of surrounding immune cells. finally, il-1α acts synergetically with nthi to upregulate the expression of amp β-defensin 2 (defb-4) via the p38/mapk pathway (257) . of note, il-1α could also act individually to upregulate the expression of defb-4 via the src-dependent mek1/2-erk1/2 signaling pathway (258) . taken together, the synergetic action may aid in the expansion of the inflammatory response and in some cases worsen the clinical outcome. alveolar macrophages located in the air-parenchyma interface are the primary professional phagocytes in the lung (259, 260) . these cells are responsible for infection eradication through its phagolysosomal machinery while releasing a plethora of inflammatory cytokines and chemokines for promoting a local inflammatory response and recruitment of neutrophils. neutrophils are the first responder cells recruited from circulation to the airway for efficient killing of pathogens through an array of microbicidal strategies (261, 262) . during nthi lung infection, both alveolar macrophages and neutrophils are the main innate immune cells involved in the pulmonary bacterial clearance through phagocytosis. they are also an important source of cytokine secretion required for induction of other immune cells and enhanced bacterial killing. eradication of nthi by alveolar macrophages involves adhesion or contact, phagocytosis and phagolysosomal processing of bacteria, in addition to secretion of tnf-α. phagocytic clearance of nthi by alveolar macrophages is orchestrated through actin polymerization, plasma membrane lipid rafts, and phosphatidylinositol 3-kinase (pi3k) signaling cascade upon induction of macrophage prrs by nthi (256) . interestingly, in response to nthi infection, human alveolar macrophages, and blood neutrophils produce extensive amount of intracellular and extracellular ros as a component of the antimicrobial defense. this leads to the formation of macrophage and neutrophil extracellular traps (mets and nets, respectively), with co-expression of mmp-12 for enhanced bacterial killing (263, 264) . nevertheless, the overexpression of mmps may adversely result in a protease imbalance and contribute to alveolar emphysematous destruction and bronchiectasis in copd (265) . moreover, excessive endogenous ros production could also introduce airway oxidative stress that is detrimental by causing chronic inflammation and tissue damage in the lung, and thus contributing to the copd exacerbation (266, 267) . the net formation is elicited mainly by nthi los in addition to other haemophilus pamps (264) . several studies by king et al. have revealed that t cellmediated adaptive immune responses against nthi airway infection in patients with idiopathic bronchiectasis and copd has been predominated by a th2/tc2 response (268) (269) (270) . the activated t cells produce reduced level of the cd40 ligand and ifn-γ, and increased levels of tnf-α, il-13, and il-17, as well as altered igg subclass production by plasma cells. it is to be noted that the th2/tc2-mediated immune response is less effective in suppressing nthi infection. redirecting the th2/tc2-mediated immune response to th1/tc1 dominant (which is more protective) by adding the th1/tc1 mediators (cd40 ligand and ifn-γ) has helped to restore the t cellmediated immune killing of nthi (269) . however, a separate study in a copd mouse model by lu et al. reported that nthi infection causes increased production of airway type 1 interferon (1-ifn) (271) . it was further reported that dna of nthi acts as a pamp in stimulating the sting/tbk1/irf3 pathway, and thus the production of 1-ifn. the impact of the bacterial dnainduced 1-ifn in host immune/inflammatory response, which may potentially induce a th1/tc1 response requires further investigations. copd patients also have abnormally higher number of treg cells, myeloid-derived suppressor cells (mdsc), and exhausted effector t cells (pd-1 + ) than healthy individuals (272, 273) . cigarette smoke-induced anti-inflammatory activity of tregs in a copd model is further suppressed by nthi infection. the pathogen causes downregulation of foxp3 (biomarker of tregs), and thus impairs the anti-inflammatory/pro-inflammatory balance of tregs (273, 274) . this may lead to the extensive immunosuppressive activity by tregs on the proliferation of nthi p6-specific effector t cells, causing a diminished response of effector t cells to sputum il-6 and il-8 induction, and increased levels of il-10 and tgf-β1 (272, 275) . recently, it has been reported that mucosal-associated invariant t cells (mait) from copd patients are more effective in response to nthi stimulation and thus produce increased levels of ifn-γ, 3-, to 10-fold more than the copd th (cd4 + ) and tc (cd8 + ) cells (276) . however, the pulmonary mait cell immune responses are compromised in the presence of corticosteroids that are commonly used for the treatment of copd. this may potentially prone the t cell-mediated immunity to a th2/tc2 response in copd patients treated with corticosteroids (277) . interestingly, antigen-specific th17 cells from nthi-immunized non-copd mice model recognize both homologous and heterologous strains of nthi, and are able to confer protection upon adoptive transfer (278) . however, it is unclear whether the th17 cell which is prone to the inflammatory response could be "trained" to counteract the nthi infection in copd patients, particularly during exacerbations. during the systemic humoral immune response in nthiinfected copd patients, greater concentrations of nthi-specific igg, iga, igm, and ige serum antibodies are produced compared to non-infected controls (12, (279) (280) (281) . some of the nthispecific serum immunoglobulins are specific to p2, p5, and p6 (12, 282, 283) . however, decreased mucosal antibodies associated with siga deficiency, or decreased total igg in the small airways have been reported in copd patients, and might be associated with disease severity (283, 284) . importantly, nthi-specific mucosal siga has been found to be lower in the airways of nthi-infected copd patients than the non-colonized patients (285, 286) . the epithelial polymeric immunoglobulin receptor (pigr) is essential for the generation of mucosal siga. it is, however, downregulated in copd patients with a positive correlation to disease severity and increased level of tgf-β (287) . the combinatorial effects of downregulated plgr and elevated tgf-β1 contribute to an impaired mucosal iga immunity in copd patients. a mouse model lacking the pigr ( −/− ) is therefore devoid of siga and are susceptible to airway stimulation by an nthi lysate resulting in increased inflammation and airway neutrophilia. interestingly, introduction of exogenously added siga mitigated the airway inflammation (288) . nthiinfected copd patients with greater airway inflammation have also decreased nthi-specific mucosal igg1 in the bal fluid compared to the non-colonized patients (283) . interestingly, the phenomenon with decreased nthi-specific antibodies seems to be restricted to the airways, since the specific serum antibodies are not affected. therefore, the reduced mucosal igg is unlikely to be associated with hypogammaglobulinemia (igg deficiency), despite the latter was reported as a contributing factor in nthi infection (289) . decreased airway iga might be attributed to the expression of iga proteases by nthi. the bacterial iga protease degrades the local airway iga during airway colonization to avoid immune exclusion by siga (226, 228) . reduced mucosal antibodies might promote host immune evasion and resistance to complement-mediated killing of nthi, thus enable persistent colonization of nthi in the airways of copd patients, in addition to a plethora of various other virulence mechanisms (4, 5, 207) . in a cohort study of stable copd patients, augmented airway inflammation and plasma fibrinogen, but not systemic inflammation, were found to be constantly correlated with the increased bacterial load (233) . higher numbers of nthi has a greater impact than s. pneumoniae and m. catarrhalis in triggering inflammatory responses as measured by the augmented levels of inflammatory cytokines in sputum including il-8, mpo, and 1l-1β. the increased inflammatory response in affected patients is potentially attributed to the persistent colonization of nthi in the lower airway (207, 233) . the compromised innate immune response in copd, particularly the decreased microbicidal activity, has been regarded as one of the culprits for persistent airway colonization by nthi, and is highly associated with copd exacerbations (figure 2 ). whilst the role of macrophage extracellular traps (met) for killing of nthi remains unknown, it has been reported that blood neutrophils and net from copd patients are defective in the killing of planktonic or biofilm/net-entrapped nthi, respectively (263, 264, 290) . a series of studies by berenson et al. revealed that alveolar macrophages derived from copd patients are basically dysfunctional in eradication of nthi (177, (291) (292) (293) . intriguingly, tlr2 and tlr4 expressed on alveolar macrophages from copd patients are intrinsically unresponsive to the potent immunomodulatory lipoprotein p6 and los, respectively. this causes decreased los/p6-induced expression of tlrs, reduced nf-κb nuclear activation and consequently diminished il-8, tnf-α, and il-1β responses by alveolar macrophages from copd patients. the compromised tlr expression and signaling potentially contribute to the defective complement-dependent and independent phagocytosis of nthi. the defective phagocytosis is greater for nthi than for m. catarrhalis, and correlates with disease severity. interestingly, the phagocytosis disability was not detected in monocyte-derived macrophages in copd. in contrast, however, taylor et al. reported that monocyte-derived macrophages from copd patients are also defective in phagocytosis of nthi and s. pneumoniae. the author also suggested that the defective monocytederived macrophages are not attributed to the alteration in cell surface tlr2 or tlr4 expression, macrophage receptor with collagenous structure (marco), cd163, cd36 or the mannose receptor (178) . the unresponsive tlr2 and tlr4 in copd alveolar macrophages to nthi lipoprotein and los might be explained by the recently reported phenomenon of tlr tolerance (294) . repetitive stimulation of copd alveolar macrophages with the same tlr ligands, pam3csk4 and lps desensitizes the tlr2 and tlr4, respectively, and generates tlr tolerance. moreover, the repetitive tlr stimulation further reduced the production of tnf-α, ccl5, and il-10 without affecting the constantly augmented level of il-6 and il-8 in alveolar macrophages. this may provide alternative explanations for diminished immune responses against the recurrent/repetitive infection by nthi. the intrinsically reduced expression of tlrs in copd patients may also contribute to the impaired pulmonary immune response thus facilitating nthi persistent colonization. expression of tlr2 or tlr4 are found to be lower on sputum neutrophils, alveolar macrophages, nasal epithelium, and t cells in copd patients despite high concentrations of il-8 and mmp-9 (295) (296) (297) (298) . the lack of the more protective th1/tc1 immune response in copd patients against nthi infection might be attributed to upregulated antagonists (a20, irak-m, and myd88s) of the myd88/irak/mapk signaling pathway in copd t cells (295) . it should be noted that the myd88/irak/mapk pathway is required for expression of tlr4 in th1, whereas production of ifn-γ in th1/tc1 is tlr4-dependent via the tlr4/trif/ikke/tbk1 signaling pathway. the antagonists prevent the nthi los-induced tlr4 expression in th1 and tc1 and thus a reduced secretion of ifn-γ. in addition, unusual high numbers of tregs in copd patients have also contributed to effector t cell dysfunction or a th2/tc2 predominant immune response (272) . however, freeman et al. reported that tc (cd8 + ) cells from copd patients have increased expression of tlr1, tlr2, tlr4, tlr6, and tlr2/1 as well as tc1 cytokines (ifnγ and tnf-α) compared to healthy individuals that may imply the auto-aggressive response of lung tc cells in copd lung inflammation (299) . however, the copd tc cells can only be stimulated by ligands for tlr2/1 (pam3csk4) yet tolerant to other agonists, indicating the dysfunctional tlrs or tlr tolerance on t cells despite their high level of receptor expression. inversely, peripheral blood neutrophils isolated from copd patients have increased expression of tlr2, tlr4, and nlrp3 (298, 300) . nevertheless, the increased tlrs expression might not improve the microbicidal ability of copd peripheral neutrophils probably due to the inaccurate responses to cytokines (179) . in addition, certain types of snps (snps) in tlr2 and tlr4 have also been associated with decreased lung function, enhanced inflammatory responses and increased immune cell infiltration in copd (301) . interestingly, the diminished il-8 responsiveness of copd alveolar macrophage to nthi infection has a strong association with the carriage of tlr9 (t1237c) polymorphism instead of tlr2 (arg753gln), tlr4 (thr399ile; asp299gly), and tlr9 (t1486c) (302) . the carriage of tlr9 (t1237c) is also positively correlated with diminished lung function. of note, the activation of tlr9-signaling cascade in pro-inflammatory cytokine response requires stimulation from microbial dna (303) . the microbicidal malfunction in both innate and adaptive immune cells is also potentially linked to the deleterious effect of tobacco smoke, the major risk factor for copd. it has been reported that, exposure of tobacco or cigarette smoke can impair phagocytosis/engulfment of nthi by alveolar macrophages isolated from copd patients (256, 304) . moreover, the chemical exposure also suppressed the tlr-induced tnfα, il-6, and il-10 production in copd alveolar macrophages that have been pre-stimulated with tlr2, 4, or 5 ligands (pam3csk4, lps, or phase i flagellin, respectively), or whole nthi bacteria (305) . this may potentially delay the macrophagedependent bacterial clearance. the suppressive effect of cigarette smoke in macrophage-dependent phagocytosis is due to the suppression of the pi3k signaling cascade which is required for optimal phagocytic activity and movement (256) . meanwhile, the cigarette smoke also inhibits the activation of the p38-erk signaling pathway and p65/nf-κb, thus dampens the nthi losinduced cytokine production of copd alveolar macrophages (305) . the diminished alveolar macrophage responsiveness could also be related to anticholinergic agents used by copd patients that results in lower concentrations of nthi-induced tnfα (306). nevertheless, the impaired phagocytosis of nthi by copd alveolar macrophages could be improved in the presence of nuclear erythroid related factor 2 and microrna mir-328 (307, 308) . interestingly, in addition to the constant exacerbated inflammatory effect observed in different murine model studies, gaschler et al. observed a rapid pulmonary clearance of nthi in mice upon exposure to cigarette smoke, and this was positively correlated with an increased neutrophilia in the animal bal fluid (236, (309) (310) (311) . however, in other copd animal studies, cigarette smoke also impaired the il-22 production that has a potential anti-bacterial activity while delaying the airway clearance of nthi (311) (312) (313) . interestingly, il-22 might play a protective role in copd exacerbation as supplementation of il-22 manages to restore the homeostasis of airway immune response and improve nthi clearance (313) . the increased airway neutrophilia might be due to the enhanced production of pulmonary il-17 triggered by cigarette smoke (152, 236, 311, 314) . this may imply the important microbicidal role of neutrophils (neutrophilia) in compensating the copd-or cigarette smoke-associated dysfunctional alveolar macrophages (96, 315) . however, such compensation may not be adequate to provide optimal immune defense to eradicate persistent nthi lower airway colonization, since the cigarette smoke also has profound suppressive effect on the host adaptive immunity, thus constantly risking the copd patients to episodes of exacerbation and relapsed infection. in adaptive immunity, cigarette smoke impairs the antigen-specific b and t cells responses to nthi infection. it suppresses the secretion of ifnγ and il-4 by nthi-specific t cells. antibody production by b cells has also been attenuated, with lower levels of specific anti-p6 antibodies and compromised igg1, igg2a, and iga class switching (311, 312) . a recent and some previous cohort studies revealed that the level of airway antimicrobial cathelicidin (hcap18/ll-37) in copd patients increase gradually from the stable disease to exacerbation states (176, 316) . moreover, higher levels of cathelicidin are positively associated with nthi airway colonization, sputum neutrophilia, and higher concentrations of il-8, particularly in the nthi-infected copd patients. of note, cathelicidin and other amps play important roles in the innate immune defense against different pathogens and persist immunomodulatory properties (317) (318) (319) . ironically, it is plausible that the increased level of cathelicidin could diminish or alter the balance in lung microbiota, and the immune/inflammatory response. this might contribute to the "vicious circle, " thus considerably increasing the risk for nthi infection during copd exacerbations (214, 320) . moreover, the microbicidal property of cathelicidin could be compromised by the inflammatory conditions in the airway, such as low ph, or the effect of cigarettes that causes peptide citrulination and modification (321, 322) . finally, expression of amps (human beta defensin 2 and s100a7) by copd airway epithelium in response to nthi infection, is also disturbed by cigarette smoke. the insulted airway cells have also a reduced expression of tlr4 and il-8, and impaired nthi-induced nf-κb activation (175, 296) . thus, a large body of evidence exists on the deleterious effects of tobacco smoke. antibiotic treatment of aecopd has been shown to significantly reduce the risk of treatment failure, especially for in-patients with severe exacerbations and patients requiring intensive care (323) . the efficacity of antibiotic treatment for out-patients with exacerbations is less clear (323, 324) . recommendations on which empirical treatment to use for aecopd varies between different countries, but common antimicrobial agents that are frequently used as definitive therapy against nthi include aminopenicillins (with or without a beta-lactamase inhibitor), tetracyclines, trimethoprimsulfamethoxazole, and fluoroquinolones. in addition, the clinical and laboratory standards institute (clsi) has developed clinical breakpoints for the macrolides azithromycin and clarithromycin (325) , whereas the european committee on antimicrobial susceptibility testing (eucast) have not set any clinical breakpoints against this class of antibiotics due to lack of clinical data (326) . one study shows that nthi frequently develops resistance to macrolides during prolonged treatment and that treatment failure may occur, making fluoroquinolones more reliable for eradication in copd-patients (327) . as for aminopenicillins, resistance is also common, with up to 10-20% of nthi isolates expressing beta-lactamases and an additional 10-20% of the isolates having amino acid substitutions in penicillin-binding protein 3 (pbp3), which reduces their susceptibility to these agents (328, 329) . the fraction of isolates expressing beta-lactamases has been stable during the last years, whereas an increase has been seen in isolates displaying altered pbp3 (330, 331) . this is worrisome, since some of these amino acid substitutions also confer resistance to third generation cephalosporins (332) . moreover, there seems to be a correlation between isolates expressing altered pbp3 and increased invasiveness. studies have shown that strains that express a mutated pbp3 with certain key amino acid substitution have a significantly higher rate of invasion of bronchial epithelial cells compared to strains with a wild type pbp3 (333) . however, when such mutated pbp3 was cloned into a susceptible wild type strain, invasion efficacy did not increase, suggesting that pbp3 is only indirectly linked to invasion (334) . besides using antibiotics for acute management of copd exacerbations, some studies have considered the use of continuous prophylactic antibiotics in the management of patients with copd (212) . there is some evidence that continuous administration of macrolide antibiotics would prevent future exacerbations in a selected population of the most severely ill patients, but a cochrane review revealed no support for a reduced all-cause mortality or less hospital readmissions (335) . however, more recent studies have shown a significant decrease in both the frequency of exacerbations and hospitalizations when long-term azithromycin treatment was chosen (336) . the fact that macrolide antibiotics display not only antimicrobial effects, but also have anti-inflammatory and immunomodulatory properties, has made them interesting to use as prophylactic therapy (212) . it has been shown that azithromycin inhibits mucus hypersecretion in the respiratory tract by significantly inhibiting tnf-α induction of the muc5ac mucin secretion from human nasal epithelial cells (337) . more specifically, it has been shown that azithromycin can reduce the nthi-dependent induction of muc5ac expression by suppressing the transcription factor activator protein-1 (338) . apart from affecting mucus secretion, it also seems that low-dose azithromycin has the ability to improve phagocytosis of bacteria by airway macrophages (339) . one study showed that azithromycin concentrations that were unable to kill nthi still increased the uptake rate of the bacteria into alveolar macrophages by enhancing their phagocytic function (340) . however, the risk of development of antimicrobial resistance limits the use of low-dose azithromycin solely for its immunological properties. this has triggered an interest in finding new macrolide substances that lack antibiotic effect and solely interact with the airway immune system (341) . the considerable clinical problems caused by nthi with regard to copd exacerbations and otitis media has prompted the scientific community to investigate whether a vaccine can be developed against the pathogen (5, 58, 342) . the search has been intensified due to a steady increase in antibiotic resistance and a trend of more invasive infections caused by nthi over the last decade (5) . whereas, a highly efficient glycoconjugate vaccine has previously been developed against hib, an identical strategy cannot be employed against nthi due to the lack of a polysaccharide capsule. vaccine developments efforts have thus been concentrated on identifying nthi surface structures that are immunogenic, have low antigenic variability, and are conserved across this genetically highly heterogeneous species. several promising vaccine candidates have been identified in the last 25 years, as excellently reviewed elsewhere (58, 342). two of these antigens, fused into one protein, protein e-pila, are together with protein d currently being tested by glaxosmithkline in a phase iib proof-of-concept clinical trial (randomized, observer-blind, placebo-controlled, and multicentric) for infection prophylaxis in copd patients (50-70 years old) (5) . notably, the m. catarrhalis ubiquitous surface protein a2 (uspa2) is also included in the vaccine so that an immune response against both exacerbation-causing pathogens could be elicited by the same preparation. this clinical study (nct03281876) is the only one currently being conducted on nthi (and m. catarrhalis) according to clinicaltrials.gov, and as the investigations are on-going, the results are currently unknown. due to an increase in the difficulty to treat nthi infections, an efficient and protective nthi vaccine likely considerably raises the quality of life of copd patients. since nthi-mediated exacerbations contribute to the progression of the disease and a steady deterioration of the pulmonary capacity of those patients, prevention against nthi infections potentially slows down the debilitating effect of the disease. it is therefore critical to continue this line of research until such a vaccine has been obtained. it could also be worth targeting non-conventional structures with a vaccine, such as the secreted enzymes urease and iga1protease that have proven important for nthi infections in copd patients in several studies (222) . copd is a multifaceted airway disease. several factors influence the clinical outcome of copd. importantly, the crosstalk between intrinsic factors (the stability and integrity of the airway immune response and structure in addition to hereditary factors), and the extrinsic factors (lung microbiome, viral and bacterial infections, meteorological factors, and noxious inhalation) determines the fate of lower airway opportunistic infection by h. influenzae. intriguingly, nthi has been one of the most isolated pathogens at both stable and exacerbation states of copd. such persistent airway colonization of nthi costs virulence fitness to counteract with the bactericidal effect of the host immune response. adversely, the impaired defense mechanisms in copd are not only unable to protect the lung structure from inhaled physical assaults, but they also fail to suppress nthi infection. the disoriented immune response in copd instead allows the pathogen to cause more harm and inflammation in the airways. the currently used bronchodilator and inhaled corticosteroid therapies have limited efficacy in preventing disease progression in copd. moreover, the inhaled corticosteroid therapies might have side effects that may weaken the immune response. hence, more investigations are needed to garner a more adequate knowledge regarding the variabilities in immune networking of copd. this knowledge will be an important platform for a more efficient drug design. in addition, a vaccine targeting nthi is another important approach in controlling the infective exacerbations in copd as the antibiotic treatment is also getting dampened by the emergence of nthi antibiotic resistance. y-cs coordinated and drafted the major part of the manuscript, and prepared the figures; fj participated in the literature study of virulence and vaccine research of nthi; jt prepared the review section for nthi epidemiology in copd and antibiotic studies; y-cs and kr edited and critically revised the manuscript. all authors read and approved the final manuscript. topographical continuity of bacterial populations in the healthy human respiratory tract bacterial topography of the healthy human lower respiratory tract the microbiota of the respiratory tract: gatekeeper to respiratory health host-pathogen interactions of nontypeable haemophilus influenzae: from commensal to pathogen update on non-typeable haemophilus influenzaemediated disease and vaccine development the evidence for non-typeable haemophilus influenzae as a causative agent of childhood pneumonia bronchiectasis in children: current concepts in immunology and microbiology. front pediatr haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation the role of infection in interstitial lung diseases: a review microbial communities in the respiratory tract of patients with interstitial lung disease infection in the pathogenesis and course of chronic obstructive pulmonary disease nontypeable haemophilus influenzae detection in the lower airways of patients with lung cancer and chronic obstructive pulmonary disease the global burden of respiratory disease epidemiology and prevalence of chronic obstructive pulmonary disease multifaceted mechanisms in copd: inflammation, immunity, and tissue repair and destruction inflammation in copd: pathogenesis, local and systemic effects chronic obstructive pulmonary disease chronic obstructive pulmonary disease complex chronic comorbidities of copd copd: a multifactorial systemic disease global burden of copd: risk factors, prevalence, and future trends tobacco smoke induces and alters immune responses in the lung triggering inflammation, allergy, asthma and other lung diseases: a mechanistic review bronchial asthma: progress in diagnosis and treatments. topics: ii. pathogenesis and pathophysiology; 1. the dutch hypothesis and british hypothesis alpha-1 antitrypsin deficiency a novel elastin gene mutation resulting in an autosomal dominant form of cutis laxa risk loci for chronic obstructive pulmonary disease: a genome-wide association study and meta-analysis association between the length of the muc8-minisatellite 5 region and susceptibility to chronic obstructive pulmonary disease (copd) human airway branch variation and chronic obstructive pulmonary disease exomewide analysis of rare coding variation identifies novel associations with copd and airflow limitation in mocs3, ifit3 and serpina12 exposure to cigarette smoke extract and lipopolysaccharide modifies cytoskeleton organization in bronchial epithelial cells airway epithelial barrier dysfunction in chronic obstructive pulmonary disease: role decarboxylase 67 promoter methylation in gabaergic interneurons nicotinic acetylcholine receptor-mediated calcium signaling in the nervous system nicotine addiction cigarette smoke reversibly activates hypoxia-inducible factor 1 in a reactive oxygen species-dependent manner decreased histone deacetylase 2 impairs nrf2 activation by oxidative stress cigarette smoke extract increases vascular endothelial growth factor production via tlr4/ros/mapks/nf-kappab pathway in nasal fibroblast receptor for advanced glycation end-products signals through ras during tobacco smoke-induced pulmonary inflammation oxidation of z alpha1-antitrypsin by cigarette smoke induces polymerization: a novel mechanism of early-onset emphysema role of proteases in chronic obstructive pulmonary disease cigarette smoke induces proinflammatory cytokine release by activation of nf-kappab and posttranslational modifications of histone deacetylase in macrophages cigarette smoke and its component acrolein augment il /cxcl8 mrna stability via p38 mapk/mk2 signaling in human pulmonary cells mechanisms underlying cigarette smoke-induced nf-kappab activation in human lymphocytes: the role of reactive nitrogen species dendritic cells in pathogenesis of copd immunology of asthma and chronic obstructive pulmonary disease new insights into the immunology of chronic obstructive pulmonary disease immune dysfunction in patients with chronic obstructive pulmonary disease murine tlr4 is implicated in cigarette smoke-induced pulmonary inflammation tlr2 and tlr4 mediated host immune responses in major infectious diseases: a review barrier epithelial cells and the control of type 2 immunity cigarette smoking and inflammation: cellular and molecular mechanisms cigarette smoke induces the release of cxcl-8 from human bronchial epithelial cells via tlrs and induction of the inflammasome long-term il-33-producing epithelial progenitor cells in chronic obstructive lung disease mediators of chronic obstructive pulmonary disease alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases, cysteine, and serine proteases impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients with chronic obstructive pulmonary disease increased levels of the chemokines groalpha and mcp-1 in sputum samples from patients with copd the nature of small-airway obstruction in chronic obstructive pulmonary disease severity of airflow limitation is associated with severity of airway inflammation in smokers advanced role of neutrophils in common respiratory diseases upregulation of adhesion molecules in the bronchial mucosa of subjects with chronic obstructive bronchitis the neutrophil in chronic obstructive pulmonary disease increased metabolic activity of neutrophils in patients with chronic obstructive pulmonary disease granulocyte activation markers in induced sputum: comparison between chronic obstructive pulmonary disease, asthma, and normal subjects airway mucus function and dysfunction molecular mechanisms regulating nlrp3 inflammasome activation role of the inflammasome in chronic obstructive pulmonary disease (copd) nlrp3 inflammasome involves in the acute exacerbation of patients with chronic obstructive pulmonary disease inflammatory mediators atp and s100a12 activate the nlrp3 inflammasome to induce muc5ac production in airway epithelial cells the activation of nlrp3-inflammsome by stimulation of diesel exhaust particles in lung tissues from emphysema model and raw 264.7 cell line accumulation of dendritic cells and increased ccl20 levels in the airways of patients with chronic obstructive pulmonary disease cigarette smoke-induced pulmonary emphysema in scid-mice. is the acquired immune system required? dendritic cells in human lung disease: recent advances increased expression of the chemokine receptor cxcr3 and its ligand cxcl10 in peripheral airways of smokers with chronic obstructive pulmonary disease cxcr3 and ccr5 chemokines in induced sputum from patients with copd lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema t helper type 17-related cytokine expression is increased in the bronchial mucosa of stable chronic obstructive pulmonary disease patients regulation of il-17 in chronic inflammation in the human lung characterization and quantification of innate lymphoid cell subsets in human lung increased il-33 expression in chronic obstructive pulmonary disease expression and cellular provenance of thymic stromal lymphopoietin and chemokines in patients with severe asthma and chronic obstructive pulmonary disease antielastin autoimmunity in tobacco smoking-induced emphysema antiendothelial cell antibodies in patients with copd autoantibodies in patients with chronic obstructive pulmonary disease neutrophil priming by cigarette smoke condensate and a tobacco anti-idiotypic antibody identification of cytokeratin 18 as a bronchial epithelial autoantigen associated with nonallergic asthma autoimmunity, t-cells and stat-4 in the pathogenesis of chronic obstructive pulmonary disease hypothesis: does copd have an autoimmune component? oxidative stress-induced antibodies to carbonyl-modified protein correlate with severity of chronic obstructive pulmonary disease what is (and what is not) a copd exacerbation: thoughts from the new gold guidelines copd exacerbations: defining their cause and prevention exacerbations of copd time course and recovery of exacerbations in patients with chronic obstructive pulmonary disease acute exacerbation of copd role of nontypeable haemophilus influenzae in exacerbations and progression of chronic obstructive pulmonary disease exacerbations of chronic obstructive pulmonary disease a prospective, observational cohort study of the seasonal dynamics of airway pathogens in the aetiology of exacerbations in copd respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease virus-induced exacerbations in asthma and copd prevalence of viral infection detected by pcr and rt-pcr in patients with acute exacerbation of copd: a systematic review viruses are frequently present as the infecting agent in acute exacerbations of chronic obstructive pulmonary disease in patients presenting to hospital the role of viral infections in exacerbations of chronic obstructive pulmonary disease and asthma respiratory viruses in acute exacerbations of chronic obstructive pulmonary disease worldwide prevalence of viral infection in aecopd patients: a meta-analysis. microb pathog respiratory viral infections in chronic lung diseases analysis of viral infection and biomarkers in patients with acute exacerbation of chronic obstructive pulmonary disease predicting early mortality in acute exacerbation of chronic obstructive pulmonary disease using the curb65 score vulnerability, beliefs, treatments and economic burden of chronic obstructive pulmonary disease in rural areas in china: a cross-sectional study microbiologic determinants of exacerbation in chronic obstructive pulmonary disease lung microbiome dynamics in copd exacerbations pneumonic and non-pneumonic exacerbations in bronchiectasis: clinical and microbiological differences longitudinal profiling of the lung microbiome in the aeris study demonstrates repeatability of bacterial and eosinophilic copd exacerbations copd is characterized by increased detection of haemophilus influenzae, streptococcus pneumoniae and a deficiency of bacillus species new strains of bacteria and exacerbations of chronic obstructive pulmonary disease role of infections acute exacerbations of chronic obstructive pulmonary disease: identification of biologic clusters and their biomarkers human rhinovirus infection during naturally occurring copd exacerbations respiratory viral detection and small airway inflammation in lung tissue of patients with stable, mild copd bacterial-viral load and the immune response in stable and exacerbated copd: significance and therapeutic prospects rhinovirus attenuates non-typeable haemophilus influenzae-stimulated il-8 responses via tlr2-dependent degradation of irak-1 haemophilus influenzae increases the susceptibility and inflammatory response of airway epithelial cells to viral infections exposure to common respiratory bacteria alters the airway epithelial response to subsequent viral infection rhinovirus disrupts the barrier function of polarized airway epithelial cells rhinovirus enhances various bacterial adhesions to nasal epithelial cells simultaneously mechanisms of severe mortality-associated bacterial co-infections following influenza virus infection. front cell infect microbiol rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages human β-defensin-2 production upon viral and bacterial co-infection is attenuated in copd the co-pathogenesis of influenza viruses with bacteria in the lung viral-bacterial interactions in the respiratory tract viral, bacterial or both? regardless, we need to treat infection in copd viral and bacterial infection in acute asthma and chronic obstructive pulmonary disease increases the risk of readmission infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations effect of interactions between lower airway bacterial and rhinoviral infection in exacerbations of copd detection of multiple viral and bacterial infections in acute exacerbation of chronic obstructive pulmonary disease: a pilot prospective study serum inflammatory biomarkers and clinical outcomes of copd exacerbation caused by different pathogens antibacterial defense of human airway epithelial cells from chronic obstructive pulmonary disease patients induced by acute exposure to nontypeable haemophilus influenzae: modulation by cigarette smoke effects of bacterial infection on airway antimicrobial peptides and proteins in copd phagocytic dysfunction of human alveolar macrophages and severity of chronic obstructive pulmonary disease defective macrophage phagocytosis of bacteria in copd behavioral and structural differences in migrating peripheral neutrophils from patients with chronic obstructive pulmonary disease bacterial microbiome of lungs in copd new opportunities for managing acute and chronic lung infections role of the lung microbiome in the pathogenesis of chronic obstructive pulmonary disease airway microbial metagenomics. microbes infect the consequence of matrix dysfunction on lung immunity and the microbiome in copd the lung microbiome, immunity, and the pathogenesis of chronic lung disease microbiome in chronic obstructive pulmonary disease biological exacerbation clusters demonstrate asthma and chronic obstructive pulmonary disease overlap with distinct mediator and microbiome profiles the transient but not resident (tbnr) microbiome: a yin yang model for lung immune system outgrowth of the bacterial airway microbiome after rhinovirus exacerbation of chronic obstructive pulmonary disease influence of lung ct changes in chronic obstructive pulmonary disease (copd) on the human lung microbiome comparing microbiota profiles in induced and spontaneous sputum samples in copd patients characterization of throat microbial flora in smokers with or without copd severity-related changes of bronchial microbiome in chronic obstructive pulmonary disease airway microbiome dynamics in exacerbations of chronic obstructive pulmonary disease exacerbation induces a microbiota shift in sputa of copd patients microbiome effects on immunity, health and disease in the lung neutrophil extracellular traps are associated with disease severity and microbiota diversity in patients with chronic obstructive pulmonary disease microbiota promotes chronic pulmonary inflammation by enhancing il-17a and autoantibodies the lung tissue microbiome in chronic obstructive pulmonary disease host response to the lung microbiome in chronic obstructive pulmonary disease mannose-binding lectin deficiency and disease severity in noncystic fibrosis bronchiectasis: a prospective study genetic mannose binding lectin deficiency is associated with airway microbiota diversity and reduced exacerbation frequency in copd jtd special edition 'hot topics in copd'-the microbiome in copd analysis of the lung microbiome in the "healthy" smoker and in copd disordered microbial communities in asthmatic airways insights on persistent airway infection by non-typeable haemophilus influenzae in chronic obstructive pulmonary disease non-typeable haemophilus influenzae, an under-recognised pathogen clinical and molecular epidemiology of haemophilus influenzae causing invasive disease in adult patients invasive disease caused by haemophilus influenzae in sweden 1997-2009; evidence of increasing incidence and clinical burden of non-type b strains invasive disease caused by nontypeable haemophilus influenzae nontypeable haemophilus influenzae and chronic obstructive pulmonary disease: a review for clinicians optimal sampling sites and methods for detection of pathogens possibly causing community-acquired lower respiratory tract infections the role of the microbiome in exacerbations of chronic lung diseases lung microbiology and exacerbations in copd airway inflammation and bronchial bacterial colonization in chronic obstructive pulmonary disease nontypeable haemophilus influenzae in the lower respiratory tract of patients with chronic bronchitis bronchial microbiome of severe copd patients colonised by pseudomonas aeruginosa lower airway colonization and inflammatory response in copd: a focus on haemophilus influenzae differential adaptation of microbial pathogens to airways of patients with cystic fibrosis and chronic obstructive pulmonary disease bacterial adaptation during chronic respiratory infections nontypeable haemophilus influenzae genetic islands associated with chronic pulmonary infection proteomic expression profiling of haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses genome-wide fitness profiling reveals adaptations required by haemophilus in coinfection with influenza a virus in the murine lung haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease expression of iga proteases by haemophilus influenzae in the respiratory tract of adults with chronic obstructive pulmonary disease immunoglobulin a protease variants facilitate intracellular survival in epithelial cells by nontypeable haemophilus influenzae that persist in the human respiratory tract in chronic obstructive pulmonary disease changes in iga protease expression are conferred by changes in genomes during persistent infection by nontypeable haemophilus influenzae in chronic obstructive pulmonary disease increased biofilm formation by nontypeable haemophilus influenzae isolates from patients with invasive disease or otitis media versus strains recovered from cases of respiratory infections nontypeable haemophilus influenzae biofilms: role in chronic airway infections nontypeable haemophilus influenzae (nthi) directly interfere with the regulation of e-cadherin in lung epithelial cells. microbes infect human pathogens utilize host extracellular matrix proteins laminin and collagen for adhesion and invasion of the host inflammatory thresholds and the species-specific effects of colonising bacteria in stable chronic obstructive pulmonary disease nf-κb signaling in inflammation haemophilus influenzae lysate induces aspects of the chronic obstructive pulmonary disease phenotype combined exposure to cigarette smoke and nontypeable haemophilus influenzae drives development of a copd phenotype in mice cigarette smokeinduced lung disease predisposes to more severe infection with nontypeable haemophilus influenzae: protective effects of andrographolide the role of tlr2 in infection and immunity toll-like receptor signaling pathways a comparative review of toll-like receptor 4 expression and functionality in different animal species outer membrane protein p6 of nontypeable haemophilus influenzae is a potent and selective inducer of human macrophage proinflammatory cytokines activation of nf-kappa b by nontypeable hemophilus influenzae is mediated by toll-like receptor 2-tak1-dependent nik-ikk alpha /beta-i kappa b alpha and mkk3/6-p38 map kinase signaling pathways in epithelial cells toll-like receptor 4 mediates innate immune responses to haemophilus influenzae infection in mouse lung the myd88-dependent, but not the myd88-independent, pathway of tlr4 signaling is important in clearing nontypeable haemophilus influenzae from the mouse lung nontypeable haemophilus influenzae induces cox-2 and pge2 expression in lung epithelial cells via activation of p38 mapk and nf-kappa b beyond myd88 and trif pathways in toll-like receptor signaling nontypeable haemophilus influenzae-promoted proliferation of kras-induced early adenomatous lesions is completely dependent on toll-like receptor signaling dectin-1 is expressed in human lung and mediates the proinflammatory immune response to nontypeable haemophilus influenza activation of epidermal growth factor receptor is required for nthiinduced nf-kappab-dependent inflammation epidermal growth factor receptor acts as a negative regulator for bacterium nontypeable haemophilus influenzae-induced toll-like receptor 2 expression via an src-dependent p38 mitogen-activated protein kinase signaling pathway multiple tlrs activate egfr via a signaling cascade to produce innate immune responses in airway epithelium nontypeable haemophilus influenzae infection upregulates the nlrp3 inflammasome and leads to caspase-1-dependent secretion of interleukin-1β -a possible pathway of exacerbations in copd synergistic activation of nf-kappab by nontypeable haemophilus influenzae and tumor necrosis factor alpha tgf-beta induces p65 acetylation to enhance bacteria-induced nf-kappab activation rhinovirus-bacteria coexposure synergistically induces ccl20 production from human bronchial epithelial cells nontypeable haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke synergistic effect of interleukin 1 alpha on nontypeable haemophilus influenzae-induced upregulation of human beta-defensin 2 in middle ear epithelial cells activation of a src-dependent raf-mek1/2-erk signaling pathway is required for il-1alpha-induced upregulation of beta-defensin 2 in human middle ear epithelial cells macrophages and polymorphonuclear neutrophils in lung defense and injury lung macrophage phenotypes and functional responses: role in the pathogenesis of copd neutrophils: between host defence, immune modulation, and tissue injury how neutrophils kill microbes nontypeable haemophilus influenzae induces sustained lung oxidative stress and protease expression nontypeable haemophilus influenzae initiates formation of neutrophil extracellular traps matrix metalloproteinases in destructive lung disease oxidative stress in copd: sources, markers, and potential mechanisms oxidative stress in the lung -the essential paradox adaptive immunity to nontypeable haemophilus influenzae effect of interferon gamma and cd40 ligation on intracellular monocyte survival of nontypeable haemophilus influenzae lung t-cell responses to nontypeable haemophilus influenzae in patients with chronic obstructive pulmonary disease nontypeable haemophilus influenzae dna stimulates type i interferon expression via sting signaling pathway t-regulatory cells and programmed death 1+ t cells contribute to effector t-cell dysfunction in patients with chronic obstructive pulmonary disease the role of regulatory t cell in nontypeable haemophilus influenzae-induced acute exacerbation of chronic obstructive pulmonary disease earlylife intranasal colonization with nontypeable haemophilus influenzae exacerbates juvenile airway disease in mice lymphocyte proliferative response to p6 of haemophilus influenzae is associated with relative protection from exacerbations of chronic obstructive pulmonary disease steroid-induced deficiency of mucosal-associated invariant t cells in the chronic obstructive pulmonary disease lung. implications for nontypeable haemophilus influenzae infection inhaled corticosteroids in chronic obstructive pulmonary disease. a two-edged sword recognition of conserved antigens by th17 cells provides broad protection against pulmonary haemophilus influenzae infection immunoglobulin concentrations in serum and nasal secretions in chronic obstructive pulmonary disease. a matched-pair study haemophilus influenzae infections in patients with chronic obstructive pulmonary disease despite specific antibodies in serum and sputum mucosal immunization: a realistic alternative mapping of a strain-specific bactericidal epitope to the surface-exposed loop 5 on the p2 porin protein of non-typeable haemophilus influenzae relationships between mucosal antibodies, non-typeable haemophilus influenzae (nthi) infection and airway inflammation in copd bronchial secretory immunoglobulin a deficiency correlates with airway inflammation and progression of chronic obstructive pulmonary disease specific iga and metalloproteinase activity in bronchial secretions from stable chronic obstructive pulmonary disease patients colonized by haemophilus influenzae emphysematous lung destruction by cigarette smoke. the effects of latent adenoviral infection on the lung inflammatory response polymeric immunoglobulin receptor down-regulation in chronic obstructive pulmonary disease. persistence in the cultured epithelium and role of transforming growth factor-β airway bacteria drive a progressive copd-like phenotype in mice with polymeric immunoglobulin receptor deficiency hypogammaglobulinaemia: cumulative experience in 49 patients in a tertiary care institution evasion of neutrophil extracellular traps by respiratory pathogens impaired phagocytosis of nontypeable haemophilus influenzae by human alveolar macrophages in chronic obstructive pulmonary disease impaired innate immune alveolar macrophage response and the predilection for copd exacerbations impaired alveolar macrophage response to haemophilus antigens in chronic obstructive lung disease the effects of repeated toll-like receptors 2 and 4 stimulation in copd alveolar macrophages the t-helper cell type 1 immune response to gram-negative bacterial infections is impaired in copd epithelial expression of tlr4 is modulated in copd and by steroids, salmeterol and cigarette smoke toll-like receptor expression in smokers with and without copd expression of toll-like receptor 2 and 4 in peripheral blood neutrophil cells from patients with chronic obstructive pulmonary disease lung cd8+ t cells in copd have increased expression of bacterial tlrs increased neutrophil expression of pattern recognition receptors during copd exacerbations toll-like receptor (tlr2 and tlr4) polymorphisms and chronic obstructive pulmonary disease impaired innate copd alveolar macrophage responses and tolllike receptor-9 polymorphisms tlr9 as a key receptor for the recognition of dna impact of cigarette smoke on clearance and inflammation after pseudomonas aeruginosa infection effects of cigarette smoke on toll-like receptor (tlr) activation of chronic obstructive pulmonary disease (copd) macrophages the impact of exogenous factors on respiratory pathogen-induced innate alveolar macrophage responses in copd targeting nrf2 signaling improves bacterial clearance by alveolar macrophages in patients with copd and in a mouse model antagonism of mir-328 increases the antimicrobial function of macrophages and neutrophils and rapid clearance of non-typeable haemophilus influenzae (nthi) from infected lung bacteria challenge in smoke-exposed mice exacerbates inflammation and skews the inflammatory profile mechanisms of clearance of nontypeable haemophilus influenzae from cigarette smoke-exposed mouse lungs cigarette smoke exposure exacerbates lung inflammation and compromises immunity to bacterial infection secondhand smoke induces inflammation and impairs immunity to respiratory infections interleukin-22 protects against non-typeable haemophilus influenzae infection: alteration during chronic obstructive pulmonary disease il-17a and the promotion of neutrophilia in acute exacerbation of chronic obstructive pulmonary disease chronic obstructive pulmonary disease, neutrophils and bacterial infection: a complex web involving il-17 and il-22 unravels antimicrobial peptide levels are linked to airway inflammation, bacterial colonisation and exacerbations in chronic obstructive pulmonary disease antimicrobial peptides and innate lung defenses: role in infectious and noninfectious lung diseases and therapeutic applications secretory leukocyte protease inhibitor and elafin/trappin-2: versatile mucosal antimicrobials and regulators of immunity regulation and function of antimicrobial peptides in immunity and diseases of the lung host-microorganism interactions in lung diseases ph modulates the activity and synergism of the airway surface liquid antimicrobials beta-defensin-3 and ll-37 peptidylarginine deiminases present in the airways during tobacco smoking and inflammation can citrullinate the host defense peptide ll-37, resulting in altered activities antibiotics for exacerbations of chronic obstructive pulmonary disease doxycycline for outpatient-treated acute exacerbations of copd: a randomised double-blind placebo-controlled trial performance standards for antimicrobial susceptibility testing breakpoint tables for interpretation of mics and zone diameters. version effect of fluoroquinolones and macrolides on eradication and resistance of haemophilus influenzae in chronic obstructive pulmonary disease multilocus sequence typing and ftsl sequencing: a powerful tool for surveillance of penicillin-binding protein 3-mediated beta-lactam resistance in nontypeable haemophilus influenzae antimicrobial resistance in haemophilus influenzae increase of β-lactam-resistant invasive haemophilus influenzae in emergence of clonally related multidrug resistant haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins ampicillin-resistant non-beta-lactamaseproducing haemophilus influenzae in spain: recent emergence of clonal isolates with increased resistance to cefotaxime and cefixime an amino acid substitution in pbp-3 in haemophilus influenzae associate with the invasion to bronchial epithelial cells genotypically defined beta-lactamase-negative ampicillin-resistant isolates of non-typable haemophilus influenzae are associated with increased invasion of bronchial epithelial cells in vitro prophylactic antibiotic therapy for chronic obstructive pulmonary disease (copd) clinical and safety outcomes of long-term azithromycin therapy in severe copd beyond the first year of treatment azithromycin inhibits mucus hypersecretion from airway epithelial cells azithromycin inhibits nontypeable haemophilus influenzae-induced muc5ac expression and secretion via inhibition of activator protein-1 in human airway epithelial cells low-dose azithromycin improves phagocytosis of bacteria by both alveolar and monocyte-derived macrophages in chronic obstructive pulmonary disease subjects relationship between azithromycin susceptibility and administration efficacy for nontypeable haemophilus influenzae respiratory infection nonantibiotic macrolides restore airway macrophage phagocytic function with potential anti-inflammatory effects in chronic lung diseases vaccines for nontypeable haemophilus influenzae: the future is now we thank the following funding agencies for their financial support during the preparation of the manuscript. they are the alfred österlund, the anna and edwin berger, the swedish medical research council (grant number k2015-57x-03163-43-4, www.vr.se), the physiographical society (forssman's foundation and, endowments for the natural sciences, medicine and technology), skåne county council's research and development foundation, and heart lung foundation (kr: grant number 20150697, www.hjart-lungfonden.se). the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2018 su, jalalvand, thegerström and riesbeck. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-302295-nblmshni authors: savva, athina; roger, thierry title: targeting toll-like receptors: promising therapeutic strategies for the management of sepsis-associated pathology and infectious diseases date: 2013-11-18 journal: front immunol doi: 10.3389/fimmu.2013.00387 sha: doc_id: 302295 cord_uid: nblmshni toll-like receptors (tlrs) are pattern recognition receptors playing a fundamental role in sensing microbial invasion and initiating innate and adaptive immune responses. tlrs are also triggered by danger signals released by injured or stressed cells during sepsis. here we focus on studies developing tlr agonists and antagonists for the treatment of infectious diseases and sepsis. positioned at the cell surface, tlr4 is essential for sensing lipopolysaccharide of gram-negative bacteria, tlr2 is involved in the recognition of a large panel of microbial ligands, while tlr5 recognizes flagellin. endosomal tlr3, tlr7, tlr8, tlr9 are specialized in the sensing of nucleic acids produced notably during viral infections. tlr4 and tlr2 are favorite targets for developing anti-sepsis drugs, and antagonistic compounds have shown efficient protection from septic shock in pre-clinical models. results from clinical trials evaluating anti-tlr4 and anti-tlr2 approaches are presented, discussing the challenges of study design in sepsis and future exploitation of these agents in infectious diseases. we also report results from studies suggesting that the tlr5 agonist flagellin may protect from infections of the gastrointestinal tract and that agonists of endosomal tlrs are very promising for treating chronic viral infections. altogether, tlr-targeted therapies have a strong potential for prevention and intervention in infectious diseases, notably sepsis. sepsis is one of the leading causes of death worldwide. incidence of severe sepsis is increasing and mortality rates remain significantly high despite early care management (1) . moreover, more than 30% of survivors develop long-term functional disabilities and cognitive impairments (2) . the surviving sepsis campaign is a global initiative incepted in early 2000s with the aim to improve sepsis diagnosis and treatment in order to enhance the awareness of sequelae and to decrease high mortality rates associated with sepsis 1 . in collaboration with many countries in europe and the united states, the surviving sepsis campaign suggests evidencebased guidelines and bundles. the most recent guidelines recommend acute resuscitation of septic patients, administration of antibiotics and support of organ failure. yet, no treatment targeting the underlying mechanism of sepsis is actually available (3) . recombinant human activated protein c (rhapc, xigris®, eli lilly), the only drug specifically registered for sepsis, has recently been withdrawn from the market following the negative results from the prowess-shock study that did not show reduction in mortality at 28 or 90 days in patients with septic shock (4) . it is generally admitted that sepsis results from a dysregulated host response to an initial insult, characterized by inflammation mediating tissue damage and organ failure and an immune suppression state responsible for the development of secondary infections (5) (6) (7) . the immune response to an infection is initiated by the sensing of microbial structures through families of receptors collectively called pattern recognition receptors (prrs). the most well-described families comprise toll-like receptors (tlrs), nucleotide binding oligomerization domains (nods)-like receptors (nlrs), c-type lectin receptors (clrs, such as dectin-1, dectin-2, dc-sign), rig-i-like receptors (rlrs, rig-i, and mda5), and intra-cytosolic dna sensors. prrs are expressed by innate immune cells like dendritic cells and macrophages. the binding of microbial ligands to prrs promotes the release of mediators, among which cytokines, that initiate and regulate the inflammatory response necessary to eliminate invasive pathogens and coordinate the development of the adaptive immune response (8, 9) . innate immune cells are also triggered by damage (or danger)associated molecular patterns (damps), known as alarmins. damps are endogenous components commonly released by injured or stressed cells, such as nucleic acids, histones, uric acid crystals, atp, cytochrome c, s100 molecules, and hmgb1. damps are primarily sensed through the nlrp3 inflammasome, which controls the secretion of il-1β and il-18 (10) . the concept of prrs sensing microbial-associated molecular patterns (mamps) and discriminating self from non-self molecular structures was proposed by janeway (11) . two major cornerstone discoveries largely confirmed janeway's concept. the first one was the demonstration of the essential antifungal role of www.frontiersin.org the toll protein in drosophila (12) . the second one arose from the positional cloning linking lps (commonly called endotoxin) unresponsive phenotype of c3h/hej and c57bl/10sccr strains of mice to missense and null mutations of the toll-like receptor 4 (tlr4) gene (13) . the importance of these discoveries and of the role of dendritic cells as central regulators of innate and adaptive immunity has been acknowledged by the 2011 nobel prize in physiology or medicine attributed to bruce a. beutler and jules a. hoffmann "for their discoveries concerning the activation of innate immunity" and to ralph m. steinman "for his discovery of the dendritic cell and its role in adaptive immunity" (14) . toll-like receptors belong to the most studied family of prrs, due to their central role in host defenses and involvement in a number of pathological processes that include sepsis. tlrs are type i trans-membrane proteins composed of an extracellular leucine-rich repeat (lrr) domain involved in ligand recognition, a trans-membrane domain, and a toll-interleukin 1 receptor (tir) domain involved in signaling (9, 15) . about 10 functional human tlrs (tlr1-10) and 12 functional mouse tlrs (tlr1-9, tlr11-13) have been described, each one being involved in the sensing of distinct microbial products ( table 1) . expressed at the cell surface, tlr4 detects lps from gram-negative bacteria. tlr4 shuttles to late endosome to induce alternative signaling following lps sensing. tlr2 as heterodimers in association with either tlr1 or tlr6 (and possibly tlr10) senses a variety of microbial products, such as lipopeptides, lipoproteins, peptidoglycan, porins, β-glucan, glycosylphosphatidylinositol (gpi) anchors, and glycoproteins from gram-positive bacteria, gram-negative bacteria, mycoplasma, mycobacteria, fungi, parasites, and viruses. tlr5 senses flagellin of bacterial flagella. tlr3, tlr7, tlr8, and tlr9 are strategically expressed in endosomal compartments to recognize microbial nucleic acids: double-stranded rna (dsrna) by tlr3, single-stranded rna (ssrna) by tlr7 and tlr8, and unmethylated cpg motif containing dna by tlr9 ( table 1) . it is therefore not surprising that endosomal tlrs have been primarily involved in host defenses against viruses, whereas tlr1, tlr2, and tlr4-6 have been mainly involved in host response to bacterial and fungal infection. tlrs cooperate with other molecules to recognize microbial ligands. for example, tlr2 requires cd14, cd36, and dectin-1 for the recognition of peptidoglycan, lipopeptides, and β-glucan, respectively. of note, tlrs are also triggered by damps released by injured or stressed cells during infection (16) ( table 1) . tlr activation enables pathogen elimination by promoting bactericidal activity of leukocytes, and maturation and function of antigen presenting cells, thus orchestrating the development of adaptive immune responses (17) . the signaling pathways resulting from tlr triggering engage adaptors that are recruited by tir/tir domain interactions ( table 1) : myeloid differentiation primary response gene (18) (myd88), tir domain-containing adaptor protein (tirap, also known as mal), tir domain-containing adaptor inducing interferon (ifn)β (trif), and trif related adaptor molecule (tram) (19) . myd88 is essential for signaling through all tlrs except tlr3 and is involved in early nuclear factor-κb (nf-κb) and mitogen-activated protein kinases (mapks) activation and pro-inflammatory gene expression. tirap serves as a bridge to recruit myd88 to tlr2 and tlr4. trif initiates myd88independent ifn regulatory factor 3 (irf3) and late nf-κb activation involved in the production of type i ifns and ifn-inducible genes (19, 20) . trif is recruited to the cytoplasmic domain of tlr3 and, in late endosome, through tram that bridges trif to tlr4. a fifth tir domain-containing adaptor, sterile α-, and armadillo-motif containing protein (sarm) acts as a negative regulator of tlr3 and tlr4 signaling. sarm interacts with trif and inhibits the induction trif-dependent genes. the signaling pathways activated downstream tlrs have some redundancy. yet, the engagement of multiple tlrs, especially myd88 and trif-dependent tlrs, have synergistic effects on host responses (21, 22) . intracellular cross talk between signaling pathways may also occur when different families of prrs are involved. for example, dectin-1 synergizes with tlr2 and tlr4 and increases cytokine production through canonical and noncanonical nf-κb pathways (23, 24) . moreover, a single mamp can be detected by different prrs. this differential sensing is primarily depending on the localization of the mamp. indeed, flagellin is sensed by tlr5 expressed at the cell surface and by the naip5/nlrc4 (also known as ipaf) inflammasome when localized in the cytoplasm (25) (26) (27) . similarly, peptidoglycans stimulate membrane tlr2 and intra-cytosolic nod1/nod2dependent cell activation (28) . interestingly, some cpg and non-cpg oligodeoxynucleotides directly stimulate and polarize t-cells through tlr9 and myd88-independent mechanisms (29), possibly through intracellular dna sensors. recently, hagan et al. and kayagaki et al. demonstrated non-canonical tlr4-independent recognition of intracellular lps through an uncharacterized receptor (30, 31) . this unconventional mode of lps sensing activates caspase-11-dependent il-1β secretion and sensitizes mice to endotoxic shock. all these observations indicate that the host has evolved different strategies to sense invading microorganisms. ideally, all possible interactions should be characterized and/or anticipated, so that the effect of treatment application can be predicted and/or translated. this mandates carefully planned experiments that represent real-life conditions and a detailed knowledge of compound's mode of action. obviously, the redundancy of microbial sensing pathways should be taken into consideration when developing or applying targeted-treatment strategies to a single prr. experimental animal models and human clinical studies support a crucial role for tlrs in infectious diseases. the first evidence came from the observation that tlr4 defective c3h/hej and c57bl/10sccr mice are hyporesponsive to lps and susceptible to otherwise non-lethal infection with escherichia coli and salmonella typhimurium. subsequent studies with mice knockout in tlrs or tlr adaptor molecules have demonstrated the importance of the tlr pathway in host defenses. for example, tlr2 knockout mice are highly susceptible to infections by staphylococcus aureus and streptococcus pneumoniae (32) . more recently, human association studies have linked polymorphisms affecting tlr expression or tlr structure with an augmented propensity to develop infections (32) (33) (34) (35) . the discovery of tlrs and their involvement in innate immune responses has attracted much interest into the development of drugs for controlling infections and improving sepsis management. this field of research has been very dynamic, and (36) . these two particular aspects of the tlr-targeting field will not be addressed in this review. herein, we will review the most popular agonist (tlr3, tlr5, tlr7, tlr8, tlr9) and antagonist (tlr2, tlr3, tlr4, tlr9) agents used in pre-clinical and clinical models of acute and chronic infections, including sepsis. relevant registered clinical trials 2 are listed in table 2 . frontiers in immunology | microbial immunology lps is the main pro-inflammatory molecule anchored in the outermembrane of gram-negative bacteria (51) . neutralization of bacterial lps, inhibition of its recognition by host cells or inhibition of signaling downstream lps binding to its receptor has long been considered a promising approach for the treatment of severe sepsis and septic shock. interestingly, endotoxemia is prevalent in septic patients, not only in those with gram-negative infection. indeed, translocation of viable bacteria and lps from the gastrointestinal tract has been proposed to participate in the pathophysiology of sepsis. tlr4 was identified 15 years ago as the signal-transducing molecule of the lps receptor complex (13) , which also comprises md-2 and cd14. thus, tlr4 is regarded as a primary target for treating sepsis (52) . tlr4 expression is increased in human monocytes of healthy volunteers challenged with lps (53), as well as in patients with sepsis (54) . moreover, polymorphisms in the tlr4 gene have been associated with gramnegative sepsis (33, 35) . in the following sections, we present the most advanced tlr4 antagonists developed for the treatment of sepsis. strategies to inhibit lps-mediated toxic effects have been initiated years before the discovery of tlr4 (13) and the unraveling of the crystal structure of the tlr4-md-2-lps complex (55) . lipid a, the toxic moiety of lps, is highly conserved among endotoxins and constitutes an ideal therapeutic target (56) . e5531, developed by eisai research institute of boston (andover, ma, usa), was the first-generation lipid a antagonist derived from rhodobacter capsulatus endotoxin. e5531 conferred protection in experimental models of endotoxemia and lethal infection with e. coli (57) . the protective effect likely occurred through the binding of e5531 to the tlr4-md-2 complex and the inhibition of the interaction between lps and tlr4-md-2 (58) . e5531 also blocked endotoxin response in human healthy volunteers challenged intravenously with lps (59). e5531 development went through phase 2 clinical trial, but was stopped due to issues of bioavailability. a second-generation lps antagonist drug candidate developed by eisai is eritoran tetrasodium (known as eritoran or e5564), a synthetic lipid a analog of rhodobacter sphaeroides (60) . eritoran blocked lps-induced cytokines in vitro and in experimental animal models (61) (62) (63) . in a phase 1 clinical trial enrolling healthy volunteers challenged with lps, eritoran inhibited pro-inflammatory cytokine production and diminished clinical symptoms of sepsis, including fever, chills, tachycardia, and headache. additionally, creactive protein levels and white blood counts were significantly decreased (64) (65) (66) (67) . the only adverse event observed was a dosedependent phlebitis, due to the fact that high doses of eritoran were used to achieve stable activity of the drug over time. a phase 2 randomized control trial recruiting critically ill septic patients as assessed by the acute physiology and chronic health evaluation ii (apache ii) score disclosed a trend toward decreased mortality in the eritoran treated group (37) . phase 3 access (a controlled comparison of eritoran and placebo in patients with severe sepsis) clinical trial for severe sepsis started in 2006, and results were published in 2013. about 1304 patients were treated with eritoran and 657 patients with placebo within 12 h after the onset of the first organ dysfunction. unfortunately, analyses did not reveal reduced all-cause mortality in primary and secondary end-points (i.e., 28 days and 1 year mortality) (38) . eisai (tokyo, japan) waived to submit eritoran to marketing authorization for the treatment of severe sepsis in january 2011, based on preliminary results of the access trial. several reasons may account for the lack of efficacy of eritoran (68) (69) (70) . for instance, patients were not enrolled or monitored based on the circulating levels of lps, questioning about the appropriateness of inclusion criteria. it is also possible that eritoran would be more efficient if administrated rapidly, before septic shock is underway, pointing the early and aggressive sepsis management as a possible interfering factor. other factors to take into account include the heterogeneity of patients for genetic background, underlying diseases, inflammatory and immune status, sepsis severity, infectious agent, and site of infection. as mentioned earlier, intracellular lps sensed in a tlr4-independent manner sensitizes mice to endotoxic shock (30, 31) . this non-canonical lps detection may have limited the efficacy of the anti-tlr4 strategy. moreover, upon infection, innate immune cells will likely sense several mamps via several tlrs and non-tlr prrs. for example, gram-negative bacteria express mamps that may trigger redundant inflammatory pathways through tlr2 (lipopeptides), tlr4 (lps), tlr5 (flagellin), tlr7 (ssrna), and tlr9 (bacterial dna). all these observations suggest that blocking one single pathway may be insufficient to interfere with the deleterious cascade of events observed in sepsis. the positive side of the access trial failure was a rethink of the design of sepsis clinical trials (68) (69) (70) . clearly, a drug like eritoran should be tested in selected patients and treatment efficacy examined and adjusted according to predefined appropriate biomarkers (such as lps blood levels and genetic polymorphisms affecting the tlr4 pathway). a rigorous approach combining the power of "omics" technologies would allow the selection of homogeneous cohorts and the follow-up of the response to treatment, both of which are mandatory for the successful development of anti-sepsis drugs. another anti-sepsis agent that exhibited promising therapeutic properties is tak-242 [ethyl-(6r)-[n -(2-chloro-4-fluorophenyl) sulfamoyl] or resatorvid] from takeda pharmaceutical company (osaka, japan). tak-242 was originally characterized as a suppressor of nitric oxide (no) and cytokine production by lpsstimulated macrophages and during endotoxic shock in mice (71) . tak-242 binds to cysteine 747 in the intracellular domain of tlr4, thereby inhibiting both myd88-dependent and myd88independent pathways activated by lps (72) . when administered in conscious guinea pigs following lps challenge, tak-242 significantly improved septic shock symptoms, decreasing hmgb1 systemic levels, and increasing survival in a dose-dependent manner (73) . tak-242 also increased survival rates from 17 to 50% and improved organ dysfunction when co-administered with antibiotics in a mouse model of cecal ligation and puncture (clp). no effect on circulating bacterial counts was observed (74) . a double-blind, randomized, placebo-controlled trial was initiated with tak-242 (39) . inclusion criteria comprised symptoms of severe sepsis accompanied with either shock and/or respiratory frontiers in immunology | microbial immunology failure. the study was stopped prematurely due to failure to achieve significant decrease of systemic cytokine levels at stage 1 of the analysis (39) . a phase 3 clinical study was designed but never launched based on business decision and not due to safety or efficacy concerns. antibodies directed against tlr4 or the tlr4-md-2 complex have been generated and showed promising results in several pre-clinical studies. we engineered a soluble chimeric protein composed of the n-terminal and central domains of mouse tlr4 (amino acid 1-334) fused to the fc domain of human igg1 (75) . the chimeric molecule was used to generate high titer anti-mouse tlr4 rabbit polyclonal antibodies. the anti-tlr4 antibodies powerfully inhibited nf-κb and mapk activation and cytokine production by lps-stimulated cells in vitro. the antibodies also hampered cytokine production and protected mice from lethal endotoxemia when administered both prophylactically and therapeutically 4 h after lps. prophylactic administration of anti-tlr4 antibodies blunted tnf production and strikingly increased survival in e. coli sepsis, from 0% in the control antibody group to 80% in the anti-tlr4 group. even more impressive, anti-tlr4 therapy initiated as much as 13 h after the onset of infection in a model of e. coli peritoneal infection improved survival from 30 to 75% (75) . our studies demonstrate that anti-tlr4 antibodies are efficient as adjunctive therapy for e. coli sepsis, with a window of clinical application comprising prophylactic and therapeutic intervention opportunities. several anti-tlr4 monoclonal antibodies have been produced. the group of miyake (university of tokyo, japan) reported in 2000 the generation of mts510, the first rat monoclonal antibody specific of the mouse tlr4-md-2 complex (76) . mts510 was shown to inhibit lps-induced nf-κb activation and tnf production by macrophages. 5e3 is a rat monoclonal antibody produced by novimmune sa (geneva, switzerland) that reacts with the tlr4-md-2 complex (77). 5e3 inhibited lps-induced cell activation, and protected mice from lethal endotoxemia when injected up to 7 h after lps challenge. moreover, administration of 5e3 at the time of surgery improved the outcome of mice with colon ascendens stent peritonitis, a model of polymicrobial abdominal sepsis. finally, the rat monoclonal antibody 1a6, that recognizes both mouse and human tlr4-md-2 complexes, conferred protection in a model of e. coli sepsis, but not salmonella enterica, sepsis (78) . although some tlr4 inhibitors have entered clinical and pre-clinical trials, others remain in the developmental stage. lps-trap-fc antibodies (comprising the extracellular domain of mouse tlr4 fused with md-2 and linked to human igg fc) dose-dependently decreased il-6 release by macrophages, opsonized gram-negative bacteria, and enhanced phagocytosis and complement-mediated bacterial killing (79) . cell-penetrating peptides comprising the translocating segment of drosophila antennapedia homeodomain fused with bb loop sequences of tlr4 (i.e., tlr4-bb peptides) inhibited lps-induced nf-κb and mapk activation and cytokine production (80, 81) . further studies will be required before advancing these products toward the clinical level. altogether, the experimental data reported above provided strong support for the concept of tlr4-targeted therapy for gram-negative sepsis. in the gloomy context following the withdrawn of rhapc and eritoran from the sepsis field, it is hopeful that ni-0101 has entered clinical development. ni-0101 is an anti-tlr4 monoclonal antibody produced by novimmune able to block tlr4 dimerization and tlr4-mediated signaling triggered by lps and endogenous and chemical ligands of tlr4. data from pre-clinical studies in models of arthritis, respiratory inflammation, and organ injury have highlighted the potential favorable action of this agent 3 . a phase 1 clinical study is currently recruiting participants to evaluate drug safety and tolerance in healthy volunteers before and after ex vivo and in vivo lps challenge. pharmacokinetics and pharmacodynamics will also be assessed. results from these studies are eagerly awaited. albeit less well characterized, tlr4 has been implicated in the sensing of non-bacterial microorganisms such as viruses and fungi. tlr4 recognizes o-linked mannan from candida albicans, and human studies have linked asp229gly tlr4 polymorphism with susceptibility to bloodstream candidiasis and pulmonary aspergillosis (82) (83) (84) . in a model of disseminated infection with c. albicans, c3h/hej tlr4-deficient mice exhibited a 10-fold increased fungal load in the kidneys, which was associated with reduced production of the chemokines kc and mip-2 and an impaired recruitment of neutrophils (85) . treatment with hta 125, an anti-human tlr4 mouse monoclonal antibody (86), interfered with neutrophil-mediated protection against c. albicans invasion and cell injury in an in vitro epithelial model of oral candidiasis (87) and inhibited tnf production by human pbmcs stimulated with aspergillus hyphae (88) . it is still unclear whether targeting tlr4 may be beneficial in the context of fungal infections. a more clear yet unexpected picture has arisen from viral infection studies. reactive oxygen species (ros) produced by the nadph oxidase generates oxidized host phospholipids that stimulate tlr4 and the production of cytokines involved in acute lung injury (89) . using a mouse model of lethal infection with influenza, the group of stephanie vogel (university of maryland, baltimore, ma, usa) reported that eritoran significantly increases survival in a dose-dependent manner even when administered 6 days after viral challenge. lung pathology and clinical symptoms were improved while viral titers and influenza-induced cytokine gene expression in lung homogenates were decreased compared to the placebo-treated group. these data suggest that the therapeutic effect of eritoran in a more practical timing of severe sepsis treatment remains substantial (90) . they also suggest that, despite the failure of eritoran in the access trial, new therapeutic potentials might still emerge for this agent. toll-like receptor 2 has been implicated in the recognition of an amazingly broad spectrum of microbial ligands originating from bacteria, fungi, viruses, and parasites (91) . this property is at least partly due to the fact that tlr2 forms heterodimers with tlr1, tlr6, and possibly tlr10 (9, 15, 92) . the biological relevance of tlr2 homodimers is controversial. indeed, some ligands have been reported to trigger cells through tlr2 independently of tlr1 and tlr6. yet, only tlr2/tlr1 and tlr2/tlr6 heterodimers have been successfully crystallized (93, 94) . tlr2 represents an interesting target for numerous conditions, but clinical development of tlr2-targeting drugs has been less extensive than that of tlr4. t2.5 is a tlr2 neutralizing mouse monoclonal antibody. t2.5 blocked pam 3 csk 4 lipopeptide (a tlr1/tlr2-ligand)stimulated nf-κb nuclear translocation and mapk phosphorylation in vitro. in models of pam 3 csk 4 -induced toxic shock and microbial challenge with a high inoculum of heat-inactivated bacillus subtilis, t2.5 prevented lethal shock-like syndrome and increased survival when administered 1 h before or up to 3 h after infection (95) . furthermore, t2.5 used in combination with the 1a6 anti-tlr4/md-2 antibody and antimicrobial therapy protected mice from sepsis caused by s. enterica and e. coli (78) . intracellular antibodies, i.e., intrabodies, have been designed to block the intracellular translocation of tlrs from the endoplasmatic reticulum to the cell surface. αt2ib is a functional anti-tlr2 scfv intrabody comprising the variable domains of the heavy and light chains of t2.5 linked together by a synthetic (gly 4 ser) 3 amino acid sequence. αt2ib bound intracellularly to tlr2 and led to retention and accumulation of tlr2 inside the endoplasmatic reticulum. adenovirus-mediated expression of αt2ib in raw 264.7 macrophages and mouse bone marrow derived macrophages inhibited tlr2 surface expression and tlr2-ligand-driven tnf production (96) . these data suggest for a therapeutic potential of t2.5 or αt2ib in microbial infections. many studies have attempted to elucidate the pathogenesis of acute kidney injury associated with sepsis, which involves mechanisms similar to those occurring during ischemia/reperfusion (97) . damps released during infection are detected through tlr2 by immune cells recruited to the ischemic tissue and/or by cells of the ischemic tissue itself, amplifying the inflammatory response and inducing injury upon reperfusion (98, 99) . blocking tlr2 under these conditions may be cytoprotective (18) . opn-305 is a humanized anti-tlr2 igg4 monoclonal antibody [derived from opn-301 (98) developed by opsona therapeutics (dublin, ireland)]. opn-305 reduced tlr2-driven pro-inflammatory cytokine production through blocking of tlr2/1 and tlr2/6 mediated signaling. in a porcine model of myocardial ischemia/reperfusion injury, pretreatment with opn-305 or administration of opn-305 1 h after ischemia was associated with a 50% decrease in infarct size (100) . results from a first in human phase 1 trial evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics of ascending doses of opn-305 given intravenously in healthy adult subjects have just been released (101) . tlr2 occupancy and inhibition of il-6 secretion induced by heat-killed listeria monocytogenes were assessed in whole blood collected up to 90 days after treatment with either the antibody or placebo. opn-305 was well tolerated, with no significant toxicity even at the highest dose tested. impressively, opn-305 at doses of 0.5 and 10 mg/kg occupied 100% of tlr2 molecules expressed on monocytes collected 14 and 90 days after challenge, respectively. il-6 release was inhibited in a parallel manner. these results suggest that treatment with opn-305 could provide short-term protection against ischemia/reperfusion and be adjusted to confer long-lasting blockage in the case of tlr2-mediated chronic diseases (101) . a phase 2 trial assessing safety, tolerability, and efficacy of opn-305 in kidney transplant patients has been initiated (nct01794663). new techniques are continuously implemented to facilitate the identification of therapeutic targets for adjunctive treatment in sepsis. immunoprecipitation with systematic evolution of ligands by exponential enrichment (selex) was developed to screen and identify high-affinity dna and rna molecules that bind to tlr2 and could be used to detect other molecules influencing tlrdriven activity. a most promising candidate, ap-177, was shown to interact with tlr2, thereby obstructing ligand binding to the receptor and inhibiting tlr2-ligand-induced nf-κb activity and il-6 and il-8 production in thp-1 and hek 293 cells (102) . cellpenetrating tlr2-bb peptides have been generated and shown to interfere with tlr2-ligand-induced activation of nf-κb and mapk and cytokine production (80) . whether these compounds will undergo clinical evaluation is unknown. toll-like receptor 3 is an endosomal prr that senses dsrna typically produced during viral infection (103) . experimental models comparing tlr3 wild-type and tlr3 knockout mice revealed either a protective role (west nile virus, encephalomyocarditis virus, poliovirus, coxsackievirus, murine cytomegalovirus, herpes simplex virus), a deleterious role (west nile virus, influenza a virus, phlebovirus), or no influence (lymphocytic choriomeningitis virus, vesicular stomatitis virus, murine cytomegalovirus, reovirus) of tlr3 on anti-viral responses (104) . therefore, tlr3 agonists and antagonists might be efficient adjunctive therapies for viral infections depending on the context. in the following sections, we describe the development of synthetic dsrna tlr3 agonists (see tlr3 agonists) and of synthetic ssdna tlr3 antagonists and anti-tlr3 neutralizing antibodies (see tlr3 antagonists). the dsrna synthetic analog polyinosinic:polycytidylic acid [poly(i:c)] is a potent immunostimulant. for clinical development, poly(i:c) was stabilized with polylysine and carboxymethylcellulose (poly-iclc) (hiltonol, oncovir, washington, dc, usa) and used to generate poly(i:c 12 u) (rintatolimod, tradename ampligen, hemispherx biopharma, philadelphia, pa, usa) by substituting an uridylic acid at a molar ratio of 12:1 in the synthesis of the polycytidylic acid strand (105) . poly(i:c) and its derivatives have been tested in several clinical trials as adjuvants for vaccines (for both infectious diseases and cancer) and complement to haart (highly active anti-retroviral therapy) in human immunodeficiency virus (hiv) infected patients, topics that we do not discuss here. poly(i:c 12 u) is highly specific for tlr3 and, unlike its parental molecule poly(i:c), does not require melanoma differentiationassociated protein 5 (mda5, a cytosolic prr for viruses), for the induction of the signaling cascade leading to type i ifn production (106) . poly(i:c 12 u) has some anti-viral activity against hiv, hepatitis b virus (hbv), coxsackie b3 virus, and several flaviviruses. results from animal models of lethal respiratory viral infection by severe acute respiratory syndrome coronavirus (sars-cov) and punta toro virus highlighted the favorable impact of intranasal treatment with poly(i:c) or poly(i:c 12 u) on survival and viral loads in infected mice (107, 108) . the mode of action of poly(i:c) in the respiratory tract was linked to the induction of caspase-mediated apoptosis and ros, which are involved in the cleavage and shedding of soluble tnf receptor blocking tnf bioactivity (109) . interestingly, poly(i:c) protected against bacterial infections in the respiratory tract as well as in the central nervous system (cns). in a mouse model of pseudomonas aeruginosa pneumonia secondary to clp, intranasal administration of poly(i:c) improved immune activation and lowered bacterial load in the lungs compared to the untreated animals (110) . corroborating results showing enhanced phagocytosis and killing of e. coli by microglial cells suggest that tlr3 activation is crucial for the immune response of cns against invading pathogens (111, 112) . these data support the development of tlr3 agonists as adjuvant therapies to prevent or reduce the severity of respiratory tract infections caused by viruses and possibly bacteria. in connection with that particular field, a phase 1 safety, tolerability, and pharmacokinetic trial of nasally applied poly-iclc in human volunteers is ongoing and will explore immune activation markers. a phase 1/2 clinical trial is assessing the immunogenicity and safety of flumist®(live attenuated influenza vaccine, med-immune, gaithersburg, md, usa) intranasal influenza vaccine administered with and without a poly(i:c 12 u). more recently, tlr3 antagonists have been developed taking into consideration that tlr3 over-activation by viral dsrna may have detrimental consequences in some situations. indeed, it has been reported that administration of poly(i:c) in mice prior to intratracheal challenge with s. pneumoniae impaired bacterial clearance and increased mortality. excessive production of type i ifn was involved in this phenomenon (113) . single-stranded dna oligonucleotides (ssdna odns) efficiently competed with dsrna for binding to tlr3, thus inhibiting cytokine production and costimulatory molecule expression by epithelial cells, pbmcs, and dendritic cells (114, 115) . the efficacy of ssdna odns was demonstrated in cynomolgus macaques, where intranasal injection of ssdnas odns inhibited poly(i:c)-induced cytokine production in nasal secretions (115) . in addition to microbial ligands, tlr3 senses damps released from injured tissue during inflammation, for example rna from necrotic cells, promoting an excessive inflammatory response. interestingly, administration of a tlr3 neutralizing antibody to mice reduced cecal damage induced by gut ischemia and improved survival of animals with polymicrobial sepsis when the antibody was given 6 and 24 h after clp surgery (116) . collectively, these data demonstrate that tlr3 works as an endogenous sensor of necrosis and a regulator of the immune response, pointing to receptor modulation as a possible adjuvant therapy for sepsis. work remains to be done to clearly delineate the precise role of tlr3 in viral and bacterial infections and to appraise the benefit afforded by tlr3 agonistic or antagonistic strategies for infectious diseases, especially septic shock. a single tlr5 agonist has had clinical development, namely cblb502. flagellin is the only ligand of tlr5 described to date. detailed structural basis of flagellin recognition by tlr5 has been obtained through crystallographic analyses, unraveling a unique mode of interaction between the two molecules as depicted from stoichiometry, ligand arrangement, and binding interfaces (117) . the role of structural constraints for induction of the nf-κb signaling cascade downstream tlr5 was supported by structure-guided mutagenesis and deletion analyses on cblb502 (entolimob), a therapeutic agent derivative of s. enterica flagellin implemented by cleveland biolabs (buffalo, ny, usa). cblb502 is currently tested in a phase 1 trial in late stage cancer patients (nct01527136). several clinical trials have investigated the safety and adjuvant efficacy of recombinant flagellin in a number of vaccine settings (against influenza virus, helicobacter pylori, campylobacter, yersinia pestis, west nile virus, etc) (118) . cblb502 plays a protective role against radiation-induced tissue injury, probably by suppressing apoptosis, attenuating ros generation, and promoting tissue regeneration (119) . these properties could explain the beneficial effect of this agonist in a murine model of acute ischemic renal failure when administered 30 min after reperfusion (120) . highlighting the favorable role of flagellin against tissue damage, results from two mouse studies suggested that protection and repair of the intestinal mucosa that serves as a first line defense barrier is the key mode of action of flagellin. in the first study, flagellin was shown to induce the expression of regiiiγ, a c-type lectin with bactericidal activity, and to restrict small intestine colonization with vancomycin-resistant enterococcus (vre) in animals inoculated with vre via oral gavage (121) . in the second study, treatment with flagellin reduced intestinal epithelium destruction induced by dextran sodium sulfate (a chemical used to induce severe acute colitis) and increased survival of mice inoculated with s. typhimurium by oral gavage (122) . these data suggest that flagellin or tlr5 agonists may represent attractive tools for treating pathologies that injure the intestinal tract, including severe sepsis. no tlr5 antagonists have been reported. toll-like receptor 9 agonists tested in clinical trials are synthetic cpg oligodeoxynucleotides (cpg odns) among which cpg10101, imo-2125, sd-101, and cpg 7909 that mimic unmethylated cpg dinucleotide-rich sequences enriched in microbial dna. cpg odns are powerful immunostimulants, exploited for their adjuvant properties in vaccines against infectious diseases (flu, malaria, hiv infection, pneumococcal and meningococcal diseases) and cancer (melanoma, leukemia, glioblastoma, and colorectal, prostate and breast cancer) (123) . the adjuvant properties of cpg odns have been used to enhance www.frontiersin.org the phagocytosis and the killing of bacteria (s. typhimurium and s. pneumoniae) by phagocytic cells (124, 125) . interestingly, a recent study showed that cpg odn given 1 h prior to clp surgery prevented clp-induced cardiac dysfunction in mice. the authors proposed that targeting tlr9 could be a useful approach for the management of cardiovascular dysfunction in severe sepsis patients (126) . therapeutic strategies focusing on tlr9-mediated immunomodulation are currently being implemented for chronic viral infections, such as chronic hepatitis c (hcv). plasmacytoid dendritic cells are the main cells producing type i ifn and are therefore considered to play an important role in viral infections. tlr9 agonists stimulate plasmacytoid dendritic cells to produce large amounts of type i ifn, especially ifnα, which is the backbone of therapy for hcv. indeed, ifnα powerfully inhibits viral replication and promotes innate and adaptive host immune responses. moreover, ifn production appears to be impaired in plasmacytoid dendritic cells of hcv patients (127, 128) . cpg10101 was originally developed under the trade name actilon by coley pharmaceuticals (wellesley, ma, usa), a company recently incorporated by pfizer (new york city, ny, usa). cpg10101 has undergone two phase 1 studies with promising results. a phase 1a study for drug safety and pharmacokinetics conducted in 48 healthy volunteers revealed well tolerated immunostimulatory effects without serious adverse events even when using high doses of cpg10101 (129) . cpg10101 was also tested in 60 hcv patients, 50 infected with genotype 1 hcv. cpg10101 was administered subcutaneously to four randomized groups at different doses twice per week for 4 weeks alone or in combination with pegylated ifnα and ribavirin. the tlr9 agonistic effect of cpg10101 was associated with the induction of ifnγ and ifnα and the decrease of viral loads. the only serious adverse events were urticaria and pruritis, without manifestation of respiratory complications (130) . a phase 2 study enrolling 113 non-responders genotype 1 hcv patients has been completed, but results have not been released yet. imo-2125, manufactured by idera pharmaceuticals (cambridge, ma, usa), has undergone two phase 1 trials: one for dose estimation and one for safety, pharmacokinetics, and pharmacodynamics, enrolling 60 and 40 treatment-naïve genotype 1 hcv patients, respectively. imo-2125 administration dosedependently decreased viral loads, increased the production of anti-viral cytokines and chemokines especially ifnα, and activated nk and t-cell responses (41, 42) . a phase 2 trial was planned, but in april 2011 the company postponed its initiation. the decision was made based on histological data from a 26-week non-clinical toxicology study of imo-2125 in rodents and non-human primates 4 . preliminary analyses suggested evidence of atypical lymphocytic proliferation, although no adverse events were reported in humans. thorough analysis results are pending. a phase 1b study sponsored by dynavax (berkeley, ca, usa) investigated the safety and efficacy of sd-101 in chronic hcv. sd-101 was administered as monotherapy or in combination with ribavirin to 34 chronically infected, treatment-naïve, genotype 1 hcv patients. results released in 2010 indicated that sd-101 was well tolerated and safe without any serious adverse events. the drug had significant anti-viral activity based on dose-dependent anti-viral response, with 100% of patients at the highest dose showing more than one log reduction in viral load, and increased expression of type i ifn-dependent anti-viral genes (ip-10, mcp-1 mx-b, isg-54k). these data comfort results from in vitro studies showing that sd-101 stimulated human pbmcs to produce 20-fold higher levels of both ifnα and ifnλ in comparison with first-generation tlr9 agonists (40) . bacterial dna released during infection is a mamp, and exuberant activation of tlr9 may participate to the sepsis pathophysiology. hence, drug inhibitors of tlr9 may have therapeutic potential in human sepsis. as a proof of concept, administration up to 12 h after surgery of a single dose of an inhibitory cpg odn blocking tlr9 signaling protected mice from polymicrobial sepsis following clp (131) . hmgb proteins are essential for triggering nucleic acid receptor-mediated innate immune responses. hmgb-binding non-immunogenic-odns have been designed to inhibit hmgb-mediated pathologies. a non-immunogenic odn termed ism odn was tested in a mouse model of endotoxemia. impressively, 70% of mice treated with ism odn survived up to 72 h after lps challenge, while all mice from the control group died within 48 h (132). these data argue for a possible use of non-immunogenic-odns in therapeutic interventions. antimalarial drugs such as chloroquine are well known for their anti-inflammatory properties in autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (133) . chloroquine blunted cytokine production and protected mice from toxic shock induced by cpg odn and lps. chloroquine down-regulated the expression of both tlr9 and tlr4, suggesting that it acts at multiple levels to inhibit inflammation (134, 135) . additionally, when administered 6 h after clp in elderly mice treated with fluids and antimicrobials, chloroquine significantly improved survival, strengthened renal function and protected from multiple organ dysfunction (136) . these results support clinical evaluation of chloroquine in patients with severe sepsis, especially those presenting with acute renal failure. based on its anti-inflammatory activity and because it inhibits endosomal acidification which are important for cell infection by viruses, chloroquine is also tested in multiple trials for prevention and/or treatment of viral infections (hiv, influenza, dengue, chikungunya). yet, the mode of action of chloroquine is multi-factorial and not only through tlr9. toll-like receptor 7 and 8 are closely related tlrs well known for their capacity to recognize ssrna from viruses such as hcv, hbv, hiv, influenza virus, herpes simplex virus, epstein-barr virus, vesicular stomatitis virus, papilloma virus, respiratory syncytial virus, and sendai virus. in agreement, tlr7 is primarily expressed in plasmacytoid dendritic cells. more recently, tlr7 and tlr8 were shown to sense bacterial rna released within phagosomal vacuoles (137) . tlr7 and tlr8 triggering induces potent antiviral immune responses characterized by the production of type frontiers in immunology | microbial immunology i ifns and nf-κb-dependent cytokines. tlr7/8 agonists are primarily developed for treating viral diseases, but also as adjuvants for cancer and infectious disease vaccines. imiquimod (aldara, originally developed by 3m pharmaceuticals, maplewood, mn, usa) is the only tlr7 agonist marketed for anti-viral treatment, i.e., external ano-genital warts caused by human papilloma virus. numerous tlr7/8 agonists are in clinical development, like cl097, isatoribine, ana975, ana773, pf-04878691 (852a), r-848 (resiquimod), and gs-9620. although therapeutic strategies for hcv have evolved in the recent years, quest of new immunomodulatory targets remains mandatory. treatment with new agents such as protease inhibitors appears to be efficient but presents with issues of resistance in the long run (138) . moreover, current protocol therapy with ifnα provides replenishment with a specific subtype of this cytokine. however, use of tlr7 agonists is able to induce a variety of ifn subtypes, possibly providing a more radical and integrated anti-viral activity (139) . finally, administration of tlr7/8 agonists may overcome the adverse events caused by ifnα, like the suppression of granulocyte colony stimulating factor (g-csf) leading to neutropenia. indeed, cl097 reversed ifnα-mediated inhibition of g-csf production by pbmcs obtained from hcv patients and healthy volunteers (140) . cl097 also restored defective cytokine production by myeloid dendritic cells from hiv patients (141) . isatoribine (anadys pharmaceuticals, san diego, ca, usa) was one of the first guanosin analog selective tlr7 agonists to be implemented and tested on humans (142) . intravenous administration of isatoribine to 12 hcv patients during a 7day treatment plan resulted in reduced plasma concentration of hcv rna, regardless virus genotype. adverse events comprised dose-dependent joint pain, decreased white blood cells, and platelets counts, insomnia, and headache (143) . the development of an oral prodrug that could lack the detrimental effects of isatoribine, especially in the gastrointestinal tract, pointed to a new candidate, ana975. preliminary results from a study conducted with ana975 were promising. oral administration of ana975 presented with elevated plasma levels of isatoribine at a concentration able to reduce hcv rna in the plasma of infected patients (144) . unfortunately, anadys pharmaceuticals and novartis (basel, switzerland) announced in 2007 discontinuation of drug development due to unacceptable toxicity in pre-clinical animal studies (145) . subsequent elaboration of an oral prodrug of isatoribine by anadys pharmaceuticals led to the generation of ana773. ana773 exhibits efficient induction of endogenous type i ifns. a double-blind, placebo-controlled study was conducted in 34 patients with chronic hcv, either treatment-naïve or relapsed from ifn-based therapy. interestingly, ana773 was safe and well tolerated and presented only with grade 1 and 2 adverse events. moreover, ana773 dose-dependently decreased hcv rna levels (48) . in another study, repeated treatment with ana773 was associated with transient decrease of myeloid and plasmacytoid dendritic cells and increased levels of ifnα and ip-10 in the blood of patients achieving a reduction in the viral load, suggesting an impairment in ifnα production in the case of non-responders (49) . pf-04878691, formerly known as 852a, is a tlr7 agonist generated by pfizer and implemented so that repeated low doses of the drug would be accompanied with the benefits of the agonistic activity without adverse events. a proof of concept study was conducted to evaluate safety and tolerability of the drug and to determine pharmacokinetics and pharmacodynamics. twentyfour healthy volunteers received orally increasing doses of pf-04878691. pf-04878691 induced immune response in a dosedependent and frequency-related manner. however, two of the subjects exhibited severe lymphopenia along with flu-like symptoms and hypotension (50) . in an attempt to decide on the future perspectives of this compound, a model was used to predict the safety and efficacy of pf-04878691 in hcv patients. this model exploited clinical results from the former study in healthy volunteers along with those reported from the use of cpg10101. further optimization will be required before entering the drug in a phase 2 study (146) . other tlr7/8 agonists have reached phase 3 trials, but demonstrated lack of efficacy and serious adverse effects. when monocytes from hiv patients were stimulated with resiquimod, il-12 secretion was augmented while tnf production was decreased compared to the control group. additionally, hiv replication in cultured monocytes was inhibited (147) . these promising in vitro results were reproduced in a phase 2 trial that enrolled patients with herpes simplex virus type 2. topical application of resiquimod protected from viral lesion spreading (148) . however, a phase 3 trial disclosed lack of efficacy of the drug and, although a phase 2 study for the treatment of hcv demonstrated decreased viral loads, adverse side effects similar to those resulting from ifnα treatment were of serious concern (149). two phase 1 clinical trials for the safety and pharmacokinetics of a novel compound, gs-9620 (gilead sciences, foster city, ca, usa), are currently enrolling treatment-naïve and viral suppressed hbv patients respectively, while another one enrolling hcv patients has been recently approved. gs-9620 is a tlr7 agonist tested originally in hbv infected chimpanzees. drug administration was associated with reduced viral loads both in plasma and liver, probably through enhanced apoptosis of hepatocytes (150) . testing of ascending dosages of the drug in healthy volunteers presented with flu-like adverse events at a dose of 8-12 mg, but cytokine induction was achieved even at administration of 2 mg pointing to a promising adjunctive treatment for chronic viral infections (151). all the above data illustrate the efforts devoted to the development of tlr7/8 agonists for treating viral pathologies. taking into account that bacterial rna triggers tlr7 and tlr8 pathways, one may speculate that tlr7/8 agonists would impact on bacterial sepsis. unfortunately, there are almost no data available on that subject. in one report, intravenous injection of r-848 prior to sepsis induction using the colon ascendens stent peritonitis model increased cytokine release and bacterial clearance, but the effect on survival was not reported (152) . finally, considering that excessive tlr activation induced by viruses or bacteria may have www.frontiersin.org detrimental effects for the host, it might be of interest to generate tlr7/8 antagonists to counteract overwhelming immune activation in conditions of severe infections. targeting tlrs is a promising field for sepsis management and infection control. tlr agonists are widely used to optimize vaccine efficacy, taking advantage of their powerful adjuvancity. whether tlr agonists and antagonists will have such success for treatment therapies, especially for sepsis, has to be established. agonists of tlr3 and tlr7-9 yielded very promising results for treating viral infections. only few studies tested antagonistic drugs. mainly for historical reasons and because of their ligand specificity, tlr4 and to a lesser extent tlr2 are the favorite targets for developing antisepsis drugs. this domain of research has monopolized important resources, but unfortunately many drugs tested in animal models have not entered human studies. those that proceeded, like eritoran and tak-242, did not achieve primary endpoint goal and/or were accompanied with manifestation of adverse events. animal models provide invaluable information about the role of tlrs and the mechanism underlying infection pathogenesis, but lack complexity in terms of comorbidities and host response compared to human disease (153) . so, how to explore more efficiently new treatment strategies? improving the design of clinical studies is mandatory. we should discriminate those patients that could benefit from therapy based on their genotype (for example affecting tlr pathways) and the expression of the targeted molecule, and select homogeneous, well-described population bearing the same underlying conditions and disease severity (68) (69) (70) 154) . ideally, sepsis studies should use specific biomarkers to help patient enrollment and weigh treatment efficacy in realistic conditions (155) . the failure of the recent trials in patients with severe sepsis has taught us valuable lessons regarding patient selection, time of intervention, and follow-up (68) (69) (70) . blocking one single mediator may also not be sufficient to interfere with sepsis. developing sepsis-specific tlr-targeted therapies for patients is a path strewn with obstacles, but an exciting and promising area of research. these studies offer new possibilities for prevention and intervention in infectious diseases, but also for other conditions, such as cancer, allergy, asthma and autoimmune diseases either directly or through improvement of vaccines. benchmarking the incidence and mortality of severe sepsis in the united states long-term cognitive impairment and functional disability among survivors of severe sepsis surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock drotrecogin alfa (activated) in adults with septic shock epigenetics in sepsis: targeting histone deacetylases sepsis: something old, something new, and a systems view harmful molecular mechanisms in sepsis regulation of adaptive immunity by the innate immune system toll-like receptors and their crosstalk with other innate receptors in infection and immunity inflammasomes and their roles in health and disease approaching the asymptote? evolution and revolution in immunology the dorsoventral regulatory gene cassette spatzle/toll/cactus controls the potent antifungal response in drosophila adults defective lps signaling in c3h/hej and c57bl/10sccr mice: mutations in tlr4 gene bridging innate and adaptive immunity newly described pattern recognition receptors team up against intracellular pathogens dampening inflammation by modulating tlr signalling toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system renal-associated tlr2 mediates ischemia/reperfusion injury in the kidney the family of five: tir-domain-containing adaptors in toll-like receptor signalling pattern recognition receptors and inflammation synergy and cross-tolerance between toll-like receptor (tlr)2-and tlr4-mediated signaling pathways selected toll-like receptor agonist combinations synergistically trigger a t helper type 1-polarizing program in dendritic cells syk kinase is required for collaborative cytokine production induced through dectin-1 and toll-like receptors dectin-1 directs t helper cell differentiation by controlling noncanonical nf-kappab activation through raf-1 and syk cytosolic flagellin requires ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages innate immune recognition of bacterial ligands by naips determines inflammasome specificity the nlrc4 inflammasome receptors for bacterial flagellin and type iii secretion apparatus signalling pathways and molecular interactions of nod1 and nod2 cpg and non-cpg oligodeoxynucleotides directly costimulate mouse and human cd4+ t cells through a tlr9-and myd88-independent mechanism cytoplasmic lps activates caspase-11: implications in tlr4-independent endotoxic shock noncanonical inflammasome activation by intracellular lps independent of tlr4 how important are toll-like receptors for antimicrobial responses? innate immunogenetics: a tool for exploring new frontiers of host defence human genetic susceptibility to infectious disease genetic variation in toll-like receptors and disease susceptibility targeting toll-like receptors: emerging therapeutics? phase 2 trial of eritoran tetrasodium (e5564), a toll-like receptor 4 antagonist, in patients with severe sepsis effect of eritoran, an antagonist of md2-tlr4, on mortality in patients with severe sepsis: the access randomized trial a randomized, double-blind, placebo-controlled trial of tak-242 for the treatment of severe sepsis phase 1b dose-secalation study of sd-101, a novel therapeutic tlr-9 agonist, in treatment-naive chronic hepatitis c patients a phase 1, multi-center, randomized, placebo-controlled, dose-escalation study of imo-2125, a tlr9 agonist, in hepatitis c-nonresponders imo-2125 plus ribavirin gives substantial first-dose viral load reductions, cumulative anti-viral effect, is well tolerated in naive genotype 1 hcv patients: a phase 1 trial chloroquine for influenza prevention: a randomised, double-blind, placebo controlled trial chloroquine use improves dengue-related symptoms imiquimod 3.75% cream applied daily to treat anogenital warts: combined results from women in two randomized, placebo-controlled studies imiquimod 5% cream for external genital or perianal warts in human immunodeficiency virus-positive patients treated with highly active antiretroviral therapy: an open-label, noncomparative study first-line therapy for human cutaneous leishmaniasis in peru using the tlr7 agonist imiquimod in combination with pentavalent antimony randomised clinical trial: anti-viral activity of ana773, an oral inducer of endogenous interferons acting via tlr7, in chronic hcv potent immune activation in chronic hepatitis c patients upon administration of an oral inducer of endogenous interferons that acts via toll-like receptor 7 the innate immune response, clinical outcomes, and ex vivo hcv antiviral efficacy of a tlr7 agonist (pf-4878691) innate immune sensing and its roots: the story of endotoxin toll-like receptor 4 modulation as a strategy to treat sepsis expression of tumour necrosis factor receptor and toll-like receptor 2 and 4 on peripheral blood leucocytes of human volunteers after endotoxin challenge: a comparison of flow cytometric light scatter and immunofluorescence gating increased expression of tolllike receptor-2 and -4 on leukocytes from patients with sepsis the structural basis of lipopolysaccharide recognition by the tlr4-md-2 complex bacterial endotoxin: molecular relationships of structure to activity and function lipopolysaccharide interaction with cell surface toll-like receptor 4-md-2: higher affinity than that with md-2 or cd14 a lipid a analog, e5531, blocks the endotoxin response in human volunteers with experimental endotoxemia eritoran tetrasodium (e5564) treatment for sepsis: review of preclinical and clinical studies inhibition of endotoxin response by e5564, a novel toll-like receptor 4-directed endotoxin antagonist toll-like receptor 4 antagonist (e5564) prevents the chronic airway response to inhaled lipopolysaccharide physiologic, biochemical, and imaging characterization of acute lung injury in mice pharmacokinetics of e5564, a lipopolysaccharide antagonist, in patients with impaired hepatic function extended in vivo pharmacodynamic activity of e5564 in normal volunteers with experimental endotoxemia safety, pharmacokinetics, pharmacodynamics, and plasma lipoprotein distribution of eritoran (e5564) during continuous intravenous infusion into healthy volunteers continuous pharmacodynamic activity of eritoran tetrasodium, a tlr4 antagonist, during intermittent intravenous infusion into normal volunteers sepsis studies need new direction recombinant human activated protein c as a therapy for severe sepsis: lessons learned? trial watch: sepsis study failure highlights need for trial design rethink discovery of novel and potent small-molecule inhibitors of no and cytokine production as antisepsis agents: synthesis and biological activity of alkyl 6-(n-substituted sulfamoyl)cyclohex-1-ene-1-carboxylate analysis of binding site for the novel small-molecule tlr4 signal transduction inhibitor tak-242 and its therapeutic effect on mouse sepsis model the novel selective toll-like receptor 4 signal transduction inhibitor tak-242 prevents endotoxaemia in conscious guinea-pigs combination of imipenem and tak-242, a tolllike receptor 4 signal transduction inhibitor, improves survival in a murine model of polymicrobial sepsis protection from lethal gram-negative bacterial sepsis by targeting toll-like receptor 4 cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-md-2 complex on mouse peritoneal macrophages tlr4/md-2 monoclonal antibody therapy affords protection in experimental models of septic shock tlr4-induced ifn-gamma production increases tlr2 sensitivity and drives gramnegative sepsis in mice lipopolysaccharide-trap-fc, a multifunctional agent to battle gram-negative bacteria cutting edge: differential inhibition of tlr signaling pathways by cell-permeable peptides representing bb loops of tlrs targeting tlr4 signaling by tlr4 toll/il-1 receptor domain-derived decoy peptides: identification of the tlr4 toll/il-1 receptor domain dimerization interface immunity to fungal infections interplay between candida albicans and the mammalian innate host defense genetic susceptibility to candida infections the role of toll-like receptor (tlr) 2 and tlr4 in the host defense against disseminated candidiasis md-2, a molecule that confers lipopolysaccharide responsiveness on toll-like receptor human epithelial cells establish direct antifungal defense through tlr4-mediated signaling involvement of cd14 and toll-like receptors in activation of human monocytes by aspergillus fumigatus hyphae identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury the tlr4 antagonist eritoran protects mice from lethal influenza infection the role of tlr2 in infection and immunity human tlr10 is a functional receptor, expressed by b cells and plasmacytoid dendritic cells, which activates gene transcription through myd88 crystal structure of the tlr1-tlr2 heterodimer induced by binding of a tri-acylated lipopeptide recognition of lipopeptide patterns by toll-like receptor 2-toll-like receptor 6 heterodimer antagonistic antibody prevents toll-like receptor 2-driven lethal shock-like syndromes generation of anti-tlr2 intrabody mediating inhibition of macrophage surface tlr2 expression and tlr2-driven cell activation searching for mechanisms that matter in early septic acute kidney injury: an experimental study myocardial ischemia/reperfusion injury is mediated by leukocytic tolllike receptor-2 and reduced by systemic administration of a novel anti-toll-like receptor-2 antibody ischaemia-induced up-regulation of toll-like receptor 2 in circulating monocytes in cardiogenic shock treatment with opn-305, a humanized anti-toll-like receptor-2 antibody, reduces myocardial ischemia/reperfusion injury in pigs randomized, double-blind, placebo-controlled, dose-escalating phase i, healthy subjects study of intravenous opn-305, a humanized anti-tlr-2 antibody identification and characterization of oligonucleotides that inhibit toll-like receptor 2-associated immune responses toll-like receptor, rig-i-like receptors and the nlrp3 inflammasome: key modulators of innate immune responses to double-stranded rna viruses antiviral responses induced by the tlr3 pathway structural requirements of the ri n-rc n complex for induction of human interferon essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus tlr3 is essential for the induction of protective immunity against punta toro virus infection by the double-stranded rna (dsrna), poly(i:c12u), but not poly(i:c): differential recognition of synthetic dsrna molecules intranasal treatment with poly(i*c) protects aged mice from lethal respiratory virus infections double-stranded rna induces shedding of the 34-kda soluble tnfr1 from human airway epithelial cells via tlr3-trif-rip1-dependent signaling: roles for dual oxidase 2-and caspasedependent pathways tlr3 agonist improves survival to secondary pneumonia in a double injury model the viral tlr3 agonist poly(i:c) stimulates phagocytosis and intracellular killing of escherichia coli by microglial cells microglia recognize doublestranded rna via tlr3 poly i:c enhances susceptibility to secondary pulmonary infections by gram-positive bacteria single-stranded oligonucleotides can inhibit cytokine production induced by human toll-like receptor 3 singlestranded dna oligonucleotides inhibit tlr3-mediated responses in human monocyte-derived dendritic cells and in vivo in cynomolgus macaques tlr3 is an endogenous sensor of tissue necrosis during acute inflammatory events structural basis of tlr5-flagellin recognition and signaling flagellin as an adjuvant: cellular mechanisms and potential an agonist of toll-like receptor 5 has radioprotective activity in mouse and primate models tlr5 agonist inhibits acute renal ischemic failure bacterial flagellin stimulates toll-like receptor 5-dependent defense against vancomycin-resistant enterococcus infection flagellin treatment protects against chemicals, bacteria, viruses, and radiation cpg still rocks! update on an accidental drug tlr 9 activation in dendritic cells enhances salmonella killing and antigen presentation via involvement of the reactive oxygen species toll-like receptor stimulation enhances phagocytosis and intracellular killing of nonencapsulated and encapsulated streptococcus pneumoniae by murine microglia the toll-like receptor 9 ligand, cpg oligodeoxynucleotide, attenuates cardiac dysfunction in polymicrobial sepsis, involving activation of both phosphoinositide 3 kinase/akt and extracellular-signal-related kinase signaling rational design of new cpg oligonucleotides that combine b cell activation with high ifn-alpha induction in plasmacytoid dendritic cells a class c cpg toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis c safety, pharmacokinetics and immune effects in normal volunteers of cpg 10101 (actilon), an investigational synthetic toll-like receptor 9 agonist phase 1b, randomized, double-blind, dose-escalation trial of cpg 10101 in patients with chronic hepatitis c virus toll-like receptor 9 inhibition reduces mortality in polymicrobial sepsis suppression of immune responses by nonimmunogenic oligodeoxynucleotides with high affinity for high-mobility group box proteins (hmgbs) mechanism of endosomal tlr inhibition by antimalarial drugs and imidazoquinolines chloroquine inhibits proinflammatory cytokine release into human whole blood chloroquine protects mice from challenge with cpg odn and lps by decreasing proinflammatory cytokine release chloroquine and inhibition of toll-like receptor 9 protect from sepsisinduced acute kidney injury detection of prokaryotic mrna signifies microbial viability and promotes immunity telaprevir and pegylated interferon-alpha-2a inhibit wild-type and resistant genotype 1 hepatitis c virus replication in patients stimulation of interferon and cytokine gene expression by imiquimod and stimulation by sendai virus utilize similar signal transduction pathways interferon-alpha suppressed granulocyte colony stimulating factor production is reversed by cl097, a tlr7/8 agonist tlr7/tlr8 activation restores defective cytokine secretion by myeloid dendritic cells but not by plasmacytoid dendritic cells in hiv-infected pregnant women and newborns molecular basis for the immunostimulatory activity of guanine nucleoside analogs: activation of toll-like receptor 7 isatoribine, an agonist of tlr7, reduces plasma virus concentration in chronic hepatitis c infection discovery of ana975: an oral prodrug of the tlr-7 agonist isatoribine masked oral prodrugs of toll-like receptor 7 agonists: a new approach for the treatment of infectious disease use of modelling and simulation techniques to support decision making on the progression of pf-04878691, a tlr7 agonist being developed for hepatitis c r-848 triggers the expression of tlr7/8 and suppresses hiv replication in monocytes topical resiquimod 0.01% gel decreases herpes simplex virus type 2 genital shedding: a randomized, controlled trial oral resiquimod in chronic hcv infection: safety and efficacy in 2 placebocontrolled, double-blind phase iia studies gs-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis b virus in chronically infected chimpanzees safety, pharmacokinetics and pharmacodynamics of gs-9620, an oral toll-like receptor 7 agonist stimulation of tlr7 prior to polymicrobial sepsis improves the immune control of the inflammatory response in adult mice the search for effective therapy for sepsis: back to the drawing board? sepsis: time to reconsider the concept biomarkers in sepsis we would like to thank all our collaborators that participated in our studies. thierry roger is supported through grants from the swiss national science foundation (310000_114073, 310030_132744, 310030_138488, and 310030_146838), an interdisciplinary grant from the faculty of biology and medicine of the university of lausanne (switzerland) and the european com the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. frontiers in immunology | microbial immunology key: cord-328549-r56lih8j authors: okamoto, masaaki; kouwaki, takahisa; fukushima, yoshimi; oshiumi, hiroyuki title: regulation of rig-i activation by k63-linked polyubiquitination date: 2018-01-05 journal: front immunol doi: 10.3389/fimmu.2017.01942 sha: doc_id: 328549 cord_uid: r56lih8j rig-i is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded rna (dsrna). influenza a virus, hepatitis c virus, and several other pathogenic viruses are mainly recognized by rig-i, resulting in the activation of the innate immune responses. the protein comprises n-terminal two caspase activation and recruitment domains (2cards), an rna helicase domain, and the c-terminal domain (ctd). the ctd recognizes 5′-triphosphate viral dsrna. after recognition of viral dsrna, the protein harbors k63-linked polyubiquitination essential for rig-i activation. first, it was reported that trim25 ubiquitin ligase delivered k63-linked polyubiquitin moiety to the 2cards. the polyubiquitin chain stabilizes a structure called the 2card tetramer, in which four 2cards assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (mavs) protein on mitochondria. mavs aggregation then triggers the signal to induce the innate immune responses. however, subsequent studies have reported that riplet, mex3c, and trim4 ubiquitin ligases are also involved in k63-linked polyubiquitination and the activation of rig-i. mex3c and trim4 mediate polyubiquitination of the 2cards. by contrast, riplet ubiquitinates the ctd. the physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. in this review, we summarize recent findings related to k63-linked polyubiquitination and propose a model that could reconcile current contradictory theories. we also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection. rig-i is a pattern recognition receptor and recognizes cytoplasmic viral double-stranded rna (dsrna). influenza a virus, hepatitis c virus, and several other pathogenic viruses are mainly recognized by rig-i, resulting in the activation of the innate immune responses. the protein comprises n-terminal two caspase activation and recruitment domains (2cards), an rna helicase domain, and the c-terminal domain (ctd). the ctd recognizes 5′-triphosphate viral dsrna. after recognition of viral dsrna, the protein harbors k63-linked polyubiquitination essential for rig-i activation. first, it was reported that trim25 ubiquitin ligase delivered k63-linked polyubiquitin moiety to the 2cards. the polyubiquitin chain stabilizes a structure called the 2card tetramer, in which four 2cards assemble and make a core that promotes the aggregation of the mitochondrial antiviral-signaling (mavs) protein on mitochondria. mavs aggregation then triggers the signal to induce the innate immune responses. however, subsequent studies have reported that riplet, mex3c, and trim4 ubiquitin ligases are also involved in k63-linked polyubiquitination and the activation of rig-i. mex3c and trim4 mediate polyubiquitination of the 2cards. by contrast, riplet ubiquitinates the ctd. the physiological significance of each ubiquitin ligases has been shown by knockout and knockdown studies, but there appears to be contradictory to evidence reported in the literature. in this review, we summarize recent findings related to k63-linked polyubiquitination and propose a model that could reconcile current contradictory theories. we also discuss the physiological significance of the ubiquitin ligases in the immune system against viral infection. keywords: rig-i, ubiquitin, innate immunity, virus, signaling pathway introduction pattern recognition receptors (prrs) recognize viral nucleic acids and trigger a signal to induce the innate immune responses during viral infection (1, 2) . rig-i is a cytoplasmic rna helicase and a prr that recognizes cytoplasmic 5′ tri-or diphosphate double-stranded rna (dsrna) (3) (4) (5) . rig-i binds relatively short dsrna (<1 kbp) and is involved in the recognition of various viral infections, such as influenza a and b viruses, japanese encephalitis virus, hepatitis c virus (hcv), dengue virus, and west nile virus (6) (7) (8) . after recognition of viral rna, rig-i associates with an adaptor protein, mitochondrial antiviral-signaling (mavs) protein, also called ips-1, cardif, and visa (9) (10) (11) (12) , resulting in the aggregation of mavs on the outer membrane of mitochondria. this ubiquitination of rig-i frontiers in immunology | www.frontiersin.org january 2018 | volume 8 | article 1942 aggregation triggers a signal to induce the expression of type i interferon (ifn) and other inflammatory cytokines (13) . the rig-i protein comprises two caspase-activation and recruitment domains (2cards) at the n-terminal region, an rna helicase domain, and a c-terminal domain (ctd) (14) (15) (16) . viral dsrna binds to the rna helicase domain and the ctd, and 5′ tri-and diphosphate are recognized by the ctd (16, 17) . the n-terminal 2cards are responsible for the association with mavs and, therefore, are required for triggering downstream signaling (5) . in resting cells, the c-terminal region, which includes the ctd and the linker region between the ctd and the helicase domain, suppresses the n-terminal 2cards (14, 18) . binding of the ctd to dsrna induces the conformational change of the rig-i protein, resulting in the release of the 2cards (14) . subsequently, the proteins assemble along viral dsrna and form a nucleoprotein filament (19) . the released 2cards also assemble and form a 2card tetramer structure (20) . the structure functions as a core for mavs aggregation on mitochondria (21) . the rig-i protein harbors lys 63-linked (k63-linked) polyubiquitination required for its activation (22) . trim25 is a ubiquitin ligase and delivers k63-linked polyubiquitin moiety to the rig-i 2cards (22, 23) . the polyubiquitin chains stabilize the 2card tetramer structure (21) . the physiological significance of trim25 in rig-i activation has been shown by several studies (22) (23) (24) (25) (26) (27) . however, recent studies have reported three other ubiquitin ligases, ring finger protein leading to rig-i activation (riplet), mex-3 rna-binding family member c (mex3c), and trim4, which are required for the polyubiquitination and activation of rig-i (28-30). ubiquitin ligases add a ubiquitin chain at k but not r residues of the target protein. there are 18 k residues in the rig-i 2cards, and mass spectrometry analysis revealed that the 2cards fragment carried k63-linked polyubiquitin chains at k99, k169, k172, k181, k190, and k193 (22) . knockdown of trim25 abrogated polyubiquitination of the 2cards fragment, suggesting that trim25 mediates k63-linked polyubiquitination at the 6 k residues of the 2cards (22) . the 2cards fragment has an ability to bind to mavs, and overexpression of the 2cards fragment leads to auto-activation of signaling (5, (9) (10) (11) (12) . an amino acid substitution assay revealed that the substitution of k172, but not of other k residues, with r abrogated the signaling induced by the 2cards fragment (22) . knockout of trim25 severely reduced rig-i-mediated type i ifn production during viral infection. these observations indicate the importance of trim25-mediated k172 ubiquitination (22) . evidence also suggests that trim25 produces unanchored k63-linked polyubiquitin chains in response to viral infection and delivered them to rig-i (23) . the same study also showed that the k172 residue of rig-i was important for non-covalent binding of rig-i with unanchored polyubiquitin chains (23) . considering that mass spectrometry analysis revealed the covalent binding of rig-i with k63-linked polyubiquitin chains, these observations indicate that either covalent or non-covalent binding with polyubiquitin chains is sufficient for rig-i 2cards activation (23, 26) . a structural study of rig-i 2cards tetramer provided evidence that both covalent and non-covalent binding of polyubiquitin chains promotes the formation of the 2card tetramer structure (21, 26) . the trim25 activity itself is regulated by the physical interaction between the trim25 spry domain and rig-i 2cards (31) . the cooperative assembly of trim25 and rig-i facilitates the dimerization of the trim25 ring domain, which is required for trim25 to make polyubiquitin chain (31) . an accumulating body of evidence has shown that trim25 delivers k63-linked polyubiquitin moiety to rig-i 2cards for rig-i activation and that the k172 residue is important for the binding of rig-i to polyubiquitin chains (26, 27, 32) . however, subsequent studies revealed that not only k172 but also other k residues are also important for the binding of rig-i to k63linked polyubiquitin chains. first, shigemoto et al. reported that the expression of the rig-i k172r full-length protein could compensate for a defect in rig-i knockout mouse embryonic fibroblasts (mefs) after sendai virus infection (33) . second, two other ubiquitin ligases, mex3c and trim4, were reported to mediate polyubiquitination of the rig-i 2cards at other k residues (29, 30) . kuniyoshi (30) . mass spectrometry analysis has also revealed the ubiquitination at k48, k96, k170, as well as k172 and k190 of the 2cards (34) . they reported that simultaneous amino acids substitutions at k48, k96, and k172 substantially reduced the polyubiquitination of rig-i (34). these observations suggest that k63-linked polyubiquitination at these k residues can compensate for the loss of k172 binding to the ubiquitin chain under certain conditions (figure 1 ). riplet, another ubiquitin ligase, is also involved in k63-linked polyubiquitination and activation of rig-i. riplet was first isolated by our yeast two-hybrid screening using the rig-i ctd fragment as a bait (28) . an immunoprecipitation assay then confirmed that the protein bound to the ctd fragment, and our studies also indicated that riplet mediates k63-linked polyubiquitination of the rig-i ctd (28) . our mutation analysis indicated that k849 and k851 of the ctd were important for riplet-mediated polyubiquitination, and riplet also targeted other k residues, including k888, k907, and k909 of the ctd and k788 in the linker region between the ctd and the helicase domain (24, 28) (figure 1) . to assess the physiological significance of the protein, we generated riplet knockout mice. knockout of riplet severely impaired the type i ifn and il-6 production in mef, macrophages, and conventional dendritic cells following influenza a virus and an rig-i c-terminal fragment (735-925 aa region), which includes the linker region and the ctd, suppresses the 2card activation (14) , and kageyama et al. reported that the linker region (746-801 aa) was responsible for the auto-suppression (18) . riplet targets the k788 in the linker region. therefore, it is possible that the ubiquitination of the linker region disrupts the auto-suppression. the structure analysis revealed that k849, k851, k858, and k888 of the ctd bind to 5′ triphosphate of dsrna ends (36) . the k849, k851, and k888 of the ctd are targeted by riplet. binding of the ctd to 5′ triphosphate of dsrna was reported to induce conformational change of the rig-i protein (16) . although several rig-i molecules assemble along dsrna, only one rig-i molecule binds the 5′ triphosphate at the dsrna end (19) (figure 2a) . therefore, riplet could access the k849, k851, and k888 of the ctds of the rig-i molecules associating with dsrna (but not the end of dsrna) to induce conformational change of rig-i. these k residues are located at the edge of the ctd basic cleft, which is an rna-binding site (16) . further studies are required to reveal underlying mechanism of riplet-mediated rig-i activation. despite the identification of four ubiquitin ligases, we found that knockout of riplet alone could abolish the polyubiquitination of the endogenous rig-i protein (24, 35) . recently, shi et al. also reported that knockout of riplet is sufficient to abolish the polyubiquitination of rig-i and, therefore, claimed that riplet is a primary ubiquitin ligase and mediates k63-linked polyubiquitination of the 2cards (34) . however, their model appears to be contradict to previous papers showing that trim25 plays a crucial role in rig-i activation. previously, we have postulated a sequential ubiquitination model that riplet-mediated polyubiquitination of rig-i c-terminal region is a prerequisite for the polyubiquitination of the 2cards (figure 2) (24) . this model could explain the apparent discrepancy in the literature, because due to the initial failure to polyubiquitinate the c-terminal region, this would obstruct the subsequent polyubiquitination of the 2cards by other ubiquitin ligases. this indicates that knockout of riplet alone is sufficient to abolish the polyubiquitination of the ctd, the linker region, and the 2cards. in a previous study, we have shown that riplet promotes the binding of trim25 to rig-i (24). this observation supports the sequential model. it is expected that riplet-mediated polyubiquitination leads to the release of auto-suppression and/or conformational change of rig-i, which would allow the access of trim25 to the 2cards and/or promote rig-i assembly along dsrna (figure 2b) . considering that higher-order oligomerization of trim25 with the 2cards is required to induce trim25-mediated polyubiquitination (31), it is not surprising that riplet-mediated c-terminal ubiquitination is a prerequisite for the second ubiquitination by trim25 (figure 2b) . mex3c or trim4 might compensate for the defect of trim25 under certain experimental conditions, because these two ubiquitin ligases target the 2cards in a similar way (figure 1) . although we failed to detect an interaction between riplet and the 2cards fragment, other groups have reported that riplet bound to the 2cards fragment and was involved in the k63-linked polyubiquitination of the 2cards (34, 37) . these observations do not conflict with the sequential ubiquitination model because several ubiquitin ligases can compensate for the loss of trim25 in some conditions. we do not exclude the possibility that riplet is not only involved in the primary ubiquitination of the ctd and the linker region but also the secondary ubiquitination of the 2cards (figure 2 ). type i ifn exhibits a strong antiviral effect, and hence several viruses have evolved to suppress the type i ifn production. hcv is a major cause of hepatocellular carcinoma and persistently infects hepatocytes over several decades without exclusion by the host immune system. a viral ns3-4a protease is required to cleave viral polypeptides and produce mature viral proteins; however, it is also important to suppress the host innate immune response. ns3-4a of hcv cleaves mavs, which results in the release of mavs from mitochondria (12) . several reports have shown that released mavs protein fails to trigger signaling to induce type i ifn production (9) . accordingly, ns3-4a-mediated cleavage of mavs abrogates rig-i-mediated type i ifn production. ns3-4a protein also targets the riplet protein. the ring finger domain is a catalytic domain of the riplet protein, and viral ns3-4a protease cleaves the domain and destabilizes the protein (24) . ns1 protein of influenza a virus also has the ability to suppress type i ifn production (38) . although several mechanisms have been postulated, gack et al. reported that viral ns1 bound to trim25 and inhibited trim25-mediated polyubiquitination of rig-i (39) . in further study, they reported that ns1 protein also targeted the riplet protein and inhibited rig-i polyubiquitination (40) . severe acute respiratory syndrome coronavirus (sars-cov) also interferes trim25 function (41) . the nucleocapsid protein of sars-cov physically interacted with trim25 and inhibited the binding of trim25 to rig-i, resulting in the attenuation of rig-i-signaling (41) . these data imply that viruses obtained the ability to suppress the ubiquitin ligases to escape innate immune responses. conversely, these data indicate the importance of the two ubiquitin ligases, trim25 and riplet, for the antiviral innate immune response. trim25 is also called efp, and it has been shown that trim25/ efp mediates the polyubiquitination of 14-3-3σ and promotes its proteolysis to suppress the growth of breast tumor cells (42) . although other targets of riplet have not been reported, it was shown that mutations on human riplet genes (also called rnf135) are linked to learning disabilities and several neuropsychiatric disorders (43, 44) . thus, it is expected that riplet targets the proteins involved in these conditions. there are several reports that riplet and trim25 are involved in tumorigenesis (42, 45, 46) . as several viruses have the ability to abrogate the described ubiquitin ligases, it is expected that viral protein-mediated inhibition of the ubiquitin ligases affects both innate immunity and other phenomena, such as virus-induced tumorigenesis and neuropsychiatric disorders. there are two protein families related to rig-i called rig-ilike receptors (rlrs). lgp2 is an rlr, and the ctd structure of the protein is similar to that of rig-i (14) . initial studies reported that lgp2 is a negative regulator for rig-i signaling (15, 47) . however, knockout and biochemical studies have revealed that lgp2 functions as a positive regulator of the rig-i pathway (48, 49) . lgp2 is also expressed in cd8 + t cells and is required for cd8 + t cell proliferation (50) . it remains unclear whether lgp2 carries a ubiquitin chain. considering the conservation of the ctd between rig-i and lgp2, it is possible that riplet also targets the ctd of lgp2 and affects lgp2-mediated rig-i activation and cd8 + t cell proliferation. further studies are required to fully elucidate the role of the ubiquitin ligases in the antiviral immune response. ho and mo wrote the manuscript. tk and yf helped the discussion. immune signaling by rig-i-like receptors toll-like receptors and their crosstalk with other innate receptors in infection and immunity antiviral immunity via rig-i-mediated recognition of rna bearing 5'-diphosphates 5'-triphosphate rna is the ligand for rig-i the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene distinct rig-i and mda5 signaling by rna viruses in innate immunity differential roles of mda5 and rig-i helicases in the recognition of rna viruses identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction visa is an adapter protein required for virus-triggered ifn-beta signaling cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 shared and unique functions of the dexd/h-box helicases rig-i mda5, and lgp2 in antiviral innate immunity nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the c-terminal regulatory domain is the rna 5'-triphosphate sensor of rig-i 55 amino acid linker between helicase and carboxyl terminal domains of rig-i functions as a critical repression domain and determines inter-domain conformation rig-i forms signaling-competent filaments in an atp-dependent, ubiquitin-independent manner structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 structural basis for ubiquitin-mediated antiviral signal activation by rig-i trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity a distinct role of ripletmediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses hepatitis c virus reveals a novel early control in acute immune response post-translational control of intracellular pathogen sensing pathways viral evasion of intracellular dna and rna sensing riplet/rnf135, a ring finger protein, ubiquitinates rig-i to promote interferon-beta induction during the early phase of viral infection pivotal role of rna-binding e3 ubiquitin ligase mex3c in rig-i-mediated antiviral innate immunity trim4 modulates type i interferon induction and cellular antiviral response by targeting rig-i for k63-linked ubiquitination mechanism of trim25 catalytic activation in the antiviral rig-i pathway ubiquitin-mediated modulation of the cytoplasmic viral rna sensor rig-i identification of loss of function mutations in human genes encoding rig-i and mda5: implications for resistance to type i diabetes ube2d3 and ube2n are essential for rig-i-mediated mavs aggregation in antiviral innate immunity the ubiquitin ligase riplet is essential for rig-i-dependent innate immune responses to rna virus infection visualizing the determinants of viral rna recognition by innate immune sensor rig-i reul is a novel e3 ubiquitin ligase and stimulator of retinoic-acid-inducible gene-i viral infection switches non-plasmacytoid dendritic cells into high interferon producers influenza a virus ns1 targets the ubiquitin ligase trim25 to evade recognition by the host viral rna sensor rig-i species-specific inhibition of rig-i ubiquitination and ifn induction by the influenza a virus ns1 protein the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth mutations in rnf135, a gene within the nf1 microdeletion region, cause phenotypic abnormalities including overgrowth mutation screening of the ubiquitin ligase gene rnf135 in french patients with autism ring finger protein, promotes the proliferation of human glioblastoma cells in vivo and in vitro via the erk pathway the e3 ubiquitin ligase rnf135 regulates the tumorigenesis activity of tongue cancer scc25 cells loss of dexd/h box rna helicase lgp2 manifests disparate antiviral responses lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses the innate immune sensor lgp2 activates antiviral signaling by regulating mda5-rna interaction and filament assembly the rig-i-like receptor lgp2 controls cd8(+) t cell survival and fitness the authors thank dr. t. seya and dr. m. matsumoto for their helpful discussion. this work was supported in part by grantsin-aid from ministry of education, science, and culture, and presto jst. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-321568-okvt1fg3 authors: alberca, ricardo wesley; teixeira, franciane mouradian emidio; beserra, danielle rosa; de oliveira, emily araujo; andrade, milena mary de souza; pietrobon, anna julia; sato, maria notomi title: perspective: the potential effects of naringenin in covid-19 date: 2020-09-25 journal: front immunol doi: 10.3389/fimmu.2020.570919 sha: doc_id: 321568 cord_uid: okvt1fg3 coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2), was declared a pandemic by the world health organization in march 2020. severe covid-19 cases develop severe acute respiratory syndrome, which can result in multiple organ failure, sepsis, and death. the higher risk group includes the elderly and subjects with pre-existing chronic illnesses such as obesity, hypertension, and diabetes. to date, no specific treatment or vaccine is available for covid-19. among many compounds, naringenin (nar) a flavonoid present in citrus fruits has been investigated for antiviral and anti-inflammatory properties like reducing viral replication and cytokine production. in this perspective, we summarize nar potential anti-inflammatory role in covid-19 associated risk factors and sars-cov-2 infection. the respiratory diseases named coronavirus disease 2019 (covid-19) is generated by a respiratory infection with severe acute respiratory syndrome coronavirus-2 (sars-cov-2) (1, 2) . due to the rapidl viral transmission, the disease was declared a pandemic by the world health organization a few months after the first diagnosed case (3, 4) . besides the similar clinical manifestations to previous severe acute respiratory syndrome coronavirus-1 (sars-cov-1), sars-cov-2 infection presents a much lower death rate (5). approximately 5% of patients progress to a severe covid-19, developing mainly severe acute respiratory syndrome, with 3% needing assisted respiratory mechanic ventilation. coronavirus disease 2019 can progress to septic shock and multiple organ failure (6) and exhibits a death rate of approximately 2% (7). the sars-cov-2 can infect human cells by entry via the angiotensin-converting enzyme 2 (ace2) receptor and transmembrane serine protease 2 (tmprss2) (8). although this process is wildly accepted, other possible infective routes are being explored such as antibody-dependent enhancement (ade) (7) and via cd147 (9). angiotensin-converting enzyme 2 expression is one of the main explanations for the higher airway infection, as it is highly expressed in the respiratory tract such as epithelial cells of the alveoli, trachea, and bronchi, some bronchial glands and alveolar macrophages (10). however, ace2 is also expressed in the ileo, kidney, adipose tissue, heart, brain, blood vessels, stomach, liver, and oral and nasal mucosas (11), which could corroborate the systemic inflammatory profile in covid-19. upon viral entry, the virus induces the host to increase the production and release of inflammatory cytokines, which can lead to greater immune activation and tissue damage (12). hypothetically, the reduction of inflammation could aid covid-19 patients (13). several compounds have been associated with antiviral and anti-inflammatory properties and could impact covid-19 development such as vitamin d (14), vitamin e (15), vitamin b12 (16), omega-3 (17), and flavonoids (18). naringenin (nar) is an important natural flavonoid present in citrus fruits, like grapefruit (43.5 mg/100 ml) and oranges (2.13 mg/100 ml) (19), with a high analgesic, anti-oxidant, anti-inflammatory, anti-tumoral, and anti-viral effect (20-23) (figure 1) . the consumption of 8 ml/kg of orange juice increases nar plasma levels from 0 to 300 µg/l 4 h after ingestion (24). the antiviral effect of nar has been studied in several viruses, such as dengue (25, 26), hepatitis c (27), zika (28), chikungunya (29), semliki forest (30), herpes simplex 1 and 2 (31), yellow fever (32), and human immunodeficiency virus (33). several in vitro studies have highlighted nar's antiviral effect in pre-infection and post-infection (28). similar to other natural compounds, nar has extensively been investigated in in vitro, but has very limited results in in vivo models of viral infection (34, 35) ( figure 1b) . nevertheless, the in vitro and in vivo anti-inflammatory potential of nar has been highlighted in several animal models, including respiratory syndromes (35, 36) . in this perspective, we highlight the mechanism in which nar may present an important anti-inflammatory role in covid-19. inflammation can be characterized by the regulation of proand anti-inflammatory mediators in resident cells and leukocytes recruited from the blood (37). there are strong pieces of evidence of the role of nar under inflammatory conditions due to a wide range of mechanisms. the immunomodulatory properties of nar are associated with the regulation of key signaling pathways, like nuclear factor kappa-light-chainenhancer of activated b cells (nf-κb) (38), pi3k/akt (23), and mitogen-activated protein kinases (mapk) (39) in different cell types ( figure 1a) . macrophages are an important cell in the covid-19 pathology, being able to sense and respond to pathogens and produce inflammatory cytokines and chemokines (40). in murine macrophages, nar can reduce inflammatory mediators production induced by lps, and in a murine endotoxemia model reduces the mortality rates from 60 to 0% (41) . murine macrophages infected with a gram-negative bacteria (chlamydia trachomatis) nar reduced the production of il-1β, il1α, il-6, tnf, il-12p70, and il-10 in a dose-dependent manner (42) . moreover, nar's anti-inflammatory effects have been demonstrated in vivo (41) , in macrophage and ex vivo human whole-blood models, reducing il-1β, il-6, il-8, and tnf upon lps stimulus to close to non-stimulated levels (43) (figure 2) . barnes et al. described that the cytokine storm developed by severe covid-19 patients is related to an exacerbation of neutrophil activation (44) . it is clear the central role of neutrophils in covid-19, as neutrophilia and neutrophil-tolymphocyte ratio in covid-19 patients is associated with disease severity (45, 46) . lung biopsies have also identified an infiltration of neutrophils (47) and the formation of neutrophil extracellular traps in covid-19 patients (48) (figure 1a) . although some animals like cats, ferrets, mice, hamsters, and macaques can be infected by sars-cov-2, the usage of animal models in covid-19 is currently limited (49) . in an animal model of acute respiratory distress syndrome (ards), a syndrome with an increase in il-6, tnf, and neutrophils in the lungs, nar supplementation can reduce neutrophils infiltration and oxidative stress, greatly reducing airway inflammation and lung injury (50) . naringenin reduction of oxidative stress is partially mediated by a curb in the anion superoxide production (51, 52) (figure 2) . naringenin can suppress inflammatory molecules production through both transcriptional and post-transcriptional mechanisms (18). in a lps-induced model of inflammation in a mouse model, nar suppressed tnf and il-6 production by macrophages and t lymphocytes without interfering in the toll-like receptor (tlr) cascade but by increasing intracellular cytokine degradation through lysosome-dependent mechanisms (23). these data indicate a potential role in the control of inflammation and oxidative stress-related to airway inflammatory insults (figure 2a) . these anti-inflammatory and anti-oxidant effects are also described in chronic comorbidities like in diabetes mellitus (53, 54) , dyslipidemia, hyperinsulinemia, and being overweight (55) , which are all risk factors associated with severe covid-19 (4, 56, 57) ( figure 1c) . in animal experimental models, nar was able to modulate different inflammation syndromes and at different sites, such as colitis (58) , hepatitis (59), obesity (60), cancer (61) , and acute respiratory syndrome (36). this is particularly important in covid-19, because sars-cov-2 infection induces a systemic inflammation and can infect many different organs including lungs, heart, liver, brain, kidneys, and the intestines (62) . in addition, nar can promote lysosome-dependent cytokine protein degradation, which may be important in covid-19 (63, 64) , considering the systemic and cytokine storm during severe covid-19 (65) . in fact, nar-induced immunomodulation has been demonstrated in airway inflammatory disorders. in a murine asthma model, treatment with nar reduced airway hyperactivity and airway inflammation, with a reduction in the levels of il-4 and il-13 in bronchoalveolar lavage and serum ige levels as well improvement in lung function assay (66) (67) (68) . overall, the treatment with nar reduced lung eosinophilia to similar levels to non-asthmatic group (66) (67) (68) . in lung fibrosis induced by infection with mycoplasma pneumoniae, nar reduced autophagy-mediated airway inflammation and lung fibrosis (69, 70) , and, in a chronic obstructive pulmonary disease (copd) model, nar was able to mitigate lung inflammation, reduce the expression of tgf-β, and increase glucocorticoid receptor expression (gcr) nar reduces viral entry, assembly, and replication via modulation of surface molecules, production of antiviral components, inflammatory molecules and/or direct interaction with viral components. (c) nar can influence the development and severity of many different diseases, in different organs, such as cancer, hepatitis, colitis, and severe acute respiratory syndrome. (71) . naringenin anti-inflammatory effect was also verified in radiation-induced lung injury, reducing lung inflammation and il-1β levels (72) . the nar anti-inflammatory effect is thus not directly mediated to a type 2 or type 1/17 immune response but a regulation of the immune response. studies have highlighted the increase in t regulatory cells and transforming growth factor-β after nar consumption via aryl hydrocarbon receptor-mediated pathway (73) . nevertheless, the excessive regulation of the inflammatory response could impair anti-viral immune response, that has not been previously observed with nar supplementation. naringenin can also activate the interferon-stimulated response element and enhance ifn-i production via an increase in the expression of irf7 (74) and increase nk cell activity via enhanced nkg2d ligand expression (75) . considering the crucial role of nk cells and ifn-i in the anti-viral immune response, nar may also contribute to the viral load control. overall, these previous studies demonstrated, in vivo and in vitro, that nar is a strong candidate as an adjuvant in reducing airway and systemic inflammation. two coronaviruses have been responsible for recent epidemics. in 2002, the sars-cov-1 epidemic caused 8,098 cases, with 774 deaths in 11 countries (76) (77) (78) . in 2012, in the middle east, another coronavirus also caused severe acute respiratory syndrome, being named mers-cov (79) . until 2020, mers-cov had caused 2,494 cases, with 858 associated deaths (77) . clinical manifestation of sars-cov-1 and mers-cov is similar. patients report clinical symptoms such as fever, cough, body pain, headache, and less commonly, diarrhea and nausea (80) . however, the need for intensive care and mechanical ventilation is greater in mers-cov than in sars-cov-1 (81, 82) . similarly to mers-cov and sars-cov-1, sars-cov-2 infection is mainly transmitted by respiratory droplets expelled from an infected person during sneezes or coughs (83, 84) . severe acute respiratory syndrome coronavirus-2 surface glycoprotein spike (s protein) binds to ace2 on the surface of the host's cell surface. this invading process is the same used by sars-cov-1 (85) . in comparison, mers-cov uses dipeptidyl peptidase 4 (dpp4), a multifunction surface protein to entry into cells (85) . dipeptidyl peptidase 4 is mainly expressed on the kidney, intestine, liver, prostate, and activated leukocytes. dipeptidyl peptidase 4 is expressed on the lower respiratory tract, glands located in submucosa of the upper respiratory tract, lung macrophages, and alveolar epithelial cells (86) . after these coronaviruses (mers-cov, sars-cov-1, and sars-cov-2) invade the host's cell, polypeptides are released from the polyproteins by proteolytic processing. the proteolytic process is mediated by papain-like protease (pl pro ) and 3chymotrypsin-like protease (3cl pro ). the 3cl pro cleaves the polyprotein to generate various non-structural proteins, crucial for viral replication (87, 88) . due to the main role of 3cl in coronaviruses viral cycle, inhibitors of 3cl could potentially be used in covid-19. flavonoid inhibition of the 3cl protease has been described in mers-cov (89) and sars (90), but nar was not among the flavonoids investigated. nevertheless, in silico analysis demonstrated that nar has the potential to inhibit sars-cov-2 3cl pro (91) . a recent study verified that sars-cov-1 and sars-cov-2 share 99.02% of genetic similarity of 3cl, with only 12 punctual mutations (88), leading to the possible inhibition of 3cl by nar and other flavonoids. another possible mechanism is the inhibition of the twopore ionic channel (tpc1 and tpc2) (92) . inhibition of tpc1 and tpc2 reduces mers-cov infectivity, intracellular traffic (93) , and viral replication (93, 94) . due to sars-cov-2 viral genome sequencing similarities with mers-cov and sars-cov-1 (95), it is possible that similar mechanism of inhibition of tpc1 and tpc2 channel be effective in covid-19, aiding in the reduction of viral replication (96) .interestingly, nar can inhibit the activity of tpc1 and tpc2 both in humans and plants (97) . nar is a hydrophilic substance with a higher affinity for the cytoplasmic membrane generating intracellular accumulation of nar (98) . therefore, this affinity probably enhances intracellular signaling and the modulation of and tpc1 and tpc2 (27). therefore, the tpc1 and tpc2's modulation by nar should be further investigated as a possible anti-coronavirus intervention. several reports of natural compounds with anti-sars-cov-2 potential are currently being investigated. substances that may compete with the ace2 receptor or reduces the ace2 expression may present an alternative or adjuvant therapy in covid-19 (99) . in fact, nar consumption has been associated with a reduction in ace2 expression in the kidneys of rats (100) and could bind directly to the ace2 receptor (101) . however, nutritional interventions aiming to regulate sars-cov-2 entry receptor ace2 need to be carefully evaluated, as downregulating of ace2 could also lead to greater inflammation and lung damage (102, 103) . previous reports demonstrated that the oral consumption of nar can reduce acute lung injury in a mouse model (50) and reduce the production of proinflammatory cytokines (18). this is extremely relevant, as a part of covid-19 lung injury can be classified as ards (104) . coronavirus disease 2019 can also lead to cytokine storm, progress to septic shock, and cause death (105, 106) . modulating the cytokine storm is thus a vital process for treating covid-19. naringenin has been used in experimental models to regulate the production of il-6 and tnf (23), cytokines that are increased in covid-19 and further increased in severe cases (107, 108) . also in an animal model of septic shock, the consumption of nar has been demonstrated to reduce kidney damage via an increase in antioxidant enzymes (109) . studies verified a direct role of nar in abrogating viral replication in human cells, before (21) and after infection (30). in sars-cov2, in silico analysis demonstrated that nar has the potential to inhibit sars-cov-2 3cl pro and consequently inhibit viral replication (91) , which still needs to be further verified experimentally. the consumption of nar via citrus fruits (110) or supplementation (111) can rapidly increase circulating levels of nar and increase intracellular levels of nar (98, 111) . an increase in the concentration of nar in plasma samples can be observed 20 min oral consumption and peaking around 4 h post-consumption (112) . in addition, in vitro models have also demonstrated a long-term anti-viral benefit, even after discontinuation of supplementation with nar (21), although there is little evidence of in vivo antiviral activity (35). previous clinical trials with the consumption of 500 ml/day for 8 weeks of orange juice, rich in nar, has demonstrated an adjuvant effect in antiviral therapy (34). the consumption of 340 ml of grapefruit juice per day (containing approximately 210 mg of nar) also improved cardiac-related measurements in post-menopause women (113) . although nar is one of the most important naturaly occurring flavonoids, there is a lack of clinical trials and data on pharmacokinetic aspects, metabolic fate, and chemical stability that may limit the usage of this bioactive compound in humans (35). a caveat of nar is the oral consumption. although widely accepted by patients, it could be a barrier in severe covid-19 patients. therefore, nar may be better applied as a prophylactic intervention or on the onset of sars-cov-2 infection. the possible effect of nar on the ace2 receptor also needs to investigated, as ace2 reduction could lead to greater inflammation (102, 103) . naringenin is mostly absorbed in the small intestine (114) , and differences in microbiota may thus also present an important inter-individual variable (24, 112) . another caveat is the nar poor aqueous solubility and bioavailability; currently, the usage of liposomes, nanoparticles, and other formulations may present itself as a solution (115) (116) (117) (118) . furthermore, nar interactions with the cytochrome p450 (cyp) system need to be evaluated, as nar can affect drugmetabolizing enzymes and pharmacokinetic of important drugs that may be of regular use or specific in covid-19 patients (119) (120) (121) . in conclusion, nar potential as an anti-inflammatory nutritional intervention has been demonstrated in many different diseases, such as sars-cov-1 and mers-cov. further investigations and clinical trials are needed to help understand the role of nar consumption in humans during a viral infection, especially in sars-cov-2 infection and covid-19. all datasets generated in this study are included in the article/supplementary material. ra: conception, writing, and review. ft: writing, drawing, and review. db, eo, ma, ap, and ms: writing and review. all authors contributed to the article and approved the submitted version. what is covid-19? front young minds covid-19: immunopathology and its implications for therapy covid-19, ace2, and the cardiovascular consequences the citrus flavonoid naringenin confers protection in a murine endotoxaemia model through ampk-atf3-dependent negative regulation of the tlr4 signalling pathway flavonoid naringenin: a potential immunomodulator for chlamydia trachomatis inflammation naringenin has anti-inflammatory properties in macrophage and ex vivo human whole-blood models targeting potential drivers of covid-19: neutrophil extracellular traps clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan neutrophil-to-lymphocyte ratio predicts severe illness patients with 2019 novel coronavirus in the early stage. medrxiv a pathological report of three covid-19 cases by minimally invasive autopsies pulmonary and cardiac pathology in covid-19: the first autopsy series from new orleans. medrxiv susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2 naringenin ameliorates lpsinduced acute lung injury through its anti-oxidative and anti-inflammatory activity and by inhibition of the pi3k/akt pathway naringenin inhibits superoxide anioninduced inflammatory pain: role of oxidative stress, cytokines, nrf-2 and the no-cgmp-pkg-katp channel signaling pathway naringenin inhibits uvb irradiation-induced inflammation and oxidative stress in the skin of hairless mice anti-diabetic, anti-inflammatory, and anti-oxidant effects of naringenin in an in vitro human model and an in vivo murine model of gestational diabetes mellitus naringenin neutralises oxidative stress and nerve growth factor discrepancy in experimental diabetic neuropathy naringenin prevents dyslipidemia, apolipoprotein b overproduction, and hyperinsulinemia in ldl receptor-null mice with diet-induced insulin resistance risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan, china obesity as a risk factor for covid-19: an overview protective effect of naringenin against experimental colitis via suppression of toll-like receptor 4/nf-κb signalling therapeutic efficacy of silymarin and naringenin in reducing arsenic-induced hepatic damage in young rats naringenin prevents obesity, hepatic steatosis, and glucose intolerance in male mice independent of fibroblast growth factor 21 naringenin reduces lung metastasis in a breast cancer resection model new understanding of the damage of sars-cov-2 infection outside the respiratory system disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in crohn's disease naringenin as a potential immunomodulator in therapeutics covid-19 cytokine storm: the interplay between inflammation and coagulation naringenin chalcone suppresses allergic asthma by inhibiting the type-2 function of cd4 t cells naringin protects ovalbumininduced airway inflammation in a mouse model of asthma naringenin inhibits allergeninduced airway inflammation and airway responsiveness and inhibits nf-κb activity in a murine model of asthma naringenin: a potential immunomodulator for inhibiting lung fibrosis and metastasis the protective effect of naringenin on airway remodeling after mycoplasma pneumoniae infection by inhibiting autophagymediated lung inflammation and fibrosis naringenin attenuates inflammation in chronic obstructive pulmonary disease in cigarette smoke induced mouse model and involves suppression of nf-κb naringenin ameliorates radiation-induced lung injury by lowering il-1b levels dietary flavonoid naringenin induces regulatory t cells via an aryl hydrocarbon receptor mediated pathway flavanones common to citrus fruits activate the interferon-stimulated response element by stimulating expression of irf7 naringenin enhances nk cell lysis activity by increasing the expression of nkg2d ligands on burkitt's lymphoma cells a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury coronavirus covid-19 has killed more people than sars and mers combined, despite lower case fatality rate coronavirus pathogenesis middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease sars and mers: recent insights into emerging coronaviruses clinical aspects and outcomes of 70 patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study sars-cov-2, sars-cov, and mers-cov: a comparative overview comparative pathogenesis of covid-19, mers, and sars in a nonhuman primate model genome composition and divergence of the novel coronavirus (2019-ncov) originating in china differential expression of the middle east respiratory syndrome coronavirus receptor in the upper respiratory tracts of humans and dromedary camels coronavirus main proteinase (3clpro) structure: basis for design of anti-sars drugs structural basis of sars-cov-2 3clpro and anti-covid-19 drug discovery from medicinal plants characteristics of flavonoids as potent mers-cov 3c-like protease inhibitors inhibition of sars-cov 3cl protease by flavonoids potential inhibitor of covid-19 main protease (m pro) from several medicinal plant compounds by molecular docking study pairing phosphoinositides with calcium ions in endolysosomal dynamics: phosphoinositides control the direction and specificity of membrane trafficking by regulating the activity of calcium channels in the endolysosomes naadp-dependent ca2+ signaling regulates middle east respiratory syndrome-coronavirus pseudovirus translocation through the endolysosomal system coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner could an endo-lysosomal ion channel be the achilles heel of sars-cov2? cell calcium the trinity of covid-19: immunity, inflammation and intervention a perspective on the modulation of plant and animal two pore channels (tpcs) by the flavonoid naringenin accumulation of a nod gene inducer, the flavonoid naringenin, in the cytoplasmic membrane of rhizobium leguminosarum biovar viciae is caused by the ph-dependent hydrophobicity of naringenin potential natural compounds for preventing sars-cov-2 (2019-ncov) infection. preprints naringenin ameliorates renovascular hypertensive renal damage by normalizing the balance of reninangiotensin system components in rats citrus fruits are rich in flavonoids for immunoregulation and potential targeting ace2 angiotensin-converting enzyme 2 protects from severe acute lung failure hypothesis: angiotensin-converting enzyme inhibitors and angiotensin receptor blockers may increase the risk of severe covid-19 covid-19 does not lead to a "typical" acute respiratory distress syndrome cytokine storm and sepsis disease pathogenesis cytokine storm in covid-19 and treatment clinical characteristics of 140 patients infected with sars-cov-2 in wuhan, china clinical and immunologic features in severe and moderate forms of coronavirus disease 2019. medrxiv protective effects of naringenin in a rat model of sepsis-triggered acute kidney injury via activation of antioxidant enzymes and reduction in urinary angiotensinogen does consumption of red grapefruit juice alter naringenin concentrations in milk produced by breastfeeding mothers? plos one the citrus-derived flavonoid naringenin exerts uterotrophic effects in female mice at human relevant doses pharmacokinetics of the citrus flavanone aglycones hesperetin and naringenin after single oral administration in human subjects flavanones protect from arterial stiffness in postmenopausal women consuming grapefruit juice for 6 mo: a randomized, controlled, crossover trial bioavailability is improved by enzymatic modification frontiers in immunology | www.frontiersin.org of the citrus flavonoid hesperidin in humans: a randomized, doubleblind, crossover trial enhanced solubility and bioavailability of naringenin via liposomal nanoformulation: preparation and in vitro and in vivo evaluations pvp-coated naringenin nanoparticles for biomedical applications -in vivo toxicological evaluations self-nanoemulsifying drug delivery system (snedds) of the poorly water-soluble grapefruit flavonoid naringenin: design, characterization, in vitro and in vivo evaluation formulation and evaluation of naringenin nanosuspensions for bioavailability enhancement enantiomers of naringenin as pleiotropic, stereoselective inhibitors of cytochrome p450 isoforms naringenin and interindividual variability in interaction of coumarin with grapefruit juice the fate of naringin in humans: a key to grapefruit juice-drug interactions? the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 alberca, teixeira, beserra, de oliveira, andrade, pietrobon and sato. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-328003-yovp8squ authors: duan, liangwei; zheng, qianqian; zhang, hongxia; niu, yuna; lou, yunwei; wang, hui title: the sars-cov-2 spike glycoprotein biosynthesis, structure, function, and antigenicity: implications for the design of spike-based vaccine immunogens date: 2020-10-07 journal: front immunol doi: 10.3389/fimmu.2020.576622 sha: doc_id: 328003 cord_uid: yovp8squ the ongoing pandemic of coronavirus disease 2019 (covid-19), caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), poses a grave threat to global public health and imposes a severe burden on the entire human society. like other coronaviruses, the sars-cov-2 genome encodes spike (s) glycoproteins, which protrude from the surface of mature virions. the s glycoprotein plays essential roles in virus attachment, fusion and entry into the host cell. surface location of the s glycoprotein renders it a direct target for host immune responses, making it the main target of neutralizing antibodies. in the light of its crucial roles in viral infection and adaptive immunity, the s protein is the focus of most vaccine strategies as well as therapeutic interventions. in this review, we highlight and describe the recent progress that has been made in the biosynthesis, structure, function, and antigenicity of the sars-cov-2 s glycoprotein, aiming to provide valuable insights into the design and development of the s protein-based vaccines as well as therapeutics. the coronavirus disease 2019 (covid-19) global pandemic represents an unprecedented public health, social and economic challenge (1, 2) . the etiological agent of covid-19 is a new member of the coronaviridae family that is closely related to severe acute respiratory syndrome coronavirus (sars-cov) and was recently referred to as sars-cov-2 by the coronavirus study group of the international committee on taxonomy of viruses (3) . the virus has spread rapidly and sustainably around the global resulting in over twenty-one million cases and more than 750,000 deaths as of august 15, 2020 (4) . coronaviruses (covs) are enveloped positive-sense rna viruses (5) . enveloped covs entering host cells and initiating infection is achieved through the fusion of viral and cellular membranes (6, 7) . membrane fusion is mediated by the large type i transmembrane s glycoprotein on the viral envelope and the cognate receptor on the surface of host cells (8) (9) (10) . the surfaceexposed location of the s glycoprotein not only allows it to carry out membrane fusion but also renders it a direct target for host immune responses, making it the major target of neutralizing antibodies (11) . because of its central roles in viral infection and eliciting protective humoral and cell-mediated immune responses in hosts during infection (10) , the s protein is the primary target for vaccine design as well as antiviral therapeutics (12) . here, we provide a comprehensive overview of the wealth of research related to the sars-cov-2 s glycoprotein biosynthesis, structure, function, and antigenicity, aiming to provide useful insights into the design and development of the s protein-based vaccines as well as therapeutics to prevent or treat the ongoing global spread of sars-cov-2/covid-19. the sars-cov-2 s glycoprotein is synthesized as a 1273-amino acid polyprotein precursor on the rough endoplasmic reticulum (rer) (figure 1 ) (13) . the unprocessed precursor harbors an endoplasmic reticulum (er) signal sequence located at the n terminus, which targets the s glycoprotein to the rer membrane and is removed by cellular signal peptidases in the lumen of the er (14, 15) . a single stop-transfer, membrane-spanning sequence located at the c terminus of the s protein prevents it from being fully released into the lumen of the er and subsequent secretion from the infected cell (16, 17) . co-translationally, n-linked, highmannose oligosaccharide side chains are added during synthesis (18, 19) . shortly after synthesis, the s glycoprotein monomers trimerize, which might be thought to facilitate the transport from the er to the golgi complex. once in the golgi complex, most of the high-mannose oligosaccharide side chains are modified to more complex forms (20, 21) , and o-linked oligosaccharide side chains are also added (22, 23) . in the trans-golgi network, the sars-cov-2 s glycoprotein is proteolytically cleaved by cellular furin or furin-like proteases at the s1/s2 cleavage site, comprising multiple arginine residues that are not found in the closely related sars-cov (24, 25) . cleavage at the s1/s2 site yields a surface subunit s1, which attaches the virus to the host cell surface receptor, and a transmembrane subunit s2, which mediates the fusion of viral and host cell membranes (10) . the s1 and s2 subunits remain associated through noncovalent interactions in a metastable prefusion state (11) . furin-like cleavage is essential for the sprotein mediated cell-cell fusion and viral infectivity, and is required for efficient sars-cov-2 infection of human lung cells (24) and airway epithelial cells (26) . following cleavage, an er retrieval signal (errs) consisting of a conserved kxhxx motif (27) located at the extreme c terminus ensures that the mature sars-cov-2 s protein accumulates near the er-golgi intermediate compartment (ergic) (27, 28) , where driven by interactions with another structural protein, the membrane (m) protein, the s protein participates in virus particle assembly and is incorporated into virus envelope ( figure 1 ) (29, 30) . besides, a fraction of mature sars-cov-2 s proteins travel through the secretory pathway to the plasma membrane, where they can mediate fusion of infected with uninfected cells to form multinucleated giant cells (syncytia) (24, 31) . this may allow direct spreading of the virus between cells and potentially alter the virulence of sars-cov-2 (24) . notably, a deletion of~20 amino acid containing the errs from the cytoplasmic tail of the sars-cov-2 s protein has been shown to increase the infectivity of single-cycle vesicular stomatitis virus (vsv)-s pseudotypes (9) and replicationcompetent recombinant vsvs bearing the s glycoprotein (32, 33) , which likely could be translated to single-cycle human immunodeficiency virus (hiv)-s or other retrovirus-s pseudotypes straightforward (33) . presumably, this deletion may enhance the cell surface expression of the sars-cov-2 s glycoprotein (32) , thereby facilitating the s protein incorporation into pseudovirions and replication-competent virions. as mentioned above, the sars-cov-2 s glycoprotein plays pivotal roles in viral infection and pathogenesis. mature s glycoprotein on the viral surface is a heavily glycosylated trimer, each protomer of which is composed of 1260 amino acids (residues 14-1273) ( figure 2a) . the surface subunit s1 is composed of 672 amino acids (residues 14-685) and organized into four domains: an n-terminal domain (ntd), a c-terminal domain (ctd, also known as the receptor-binding domain, rbd), and two subdomains (sd1 and sd2) ( figure 2a ) (34) . the transmembrane s2 subunit is composed of 588 amino acids (residues 686-1273) and contains an n-terminal hydrophobic fusion peptide (fp), two heptad repeats (hr1 and hr2), a transmembrane domain (tm), and a cytoplasmic tail (ct), arranged as fp-hr1-hr2-tm-ct ( figure 2a ) (34) . as a typical class i viral fusion protein (35) , the sars-cov-2 s glycoprotein shares common structural, topological and mechanistic features with other class i fusion proteins, including hiv envelope (env) glycoprotein and influenza virus haemagglutinin (ha) (36) (37) (38) . like other class i viral fusion proteins, the sars-cov-2 s glycoprotein is also a conformational machine that mediates viral entry by rearranging from a metastable unliganded state, through a prehairpin intermediate state, to a stable postfusion state (38, 39) . since the first genome sequence of sars-cov-2 became publicly available (40) , a number of structures have been determined for the sars-cov-2 s glycoprotein trimer fragments in both the prefusion and postfusion states ( figures 2b-d) (11, 34, 41) . the overall architecture of the prefusion sars-cov-2 s ectodomain stabilized by two consecutive proline mutations in two conformations determined by single particle cryo-electron microscopy (cryo-em) is a~160 å long trimer with a triangular cross-section, with the s1 subunit adopting a "v" shape contributing to the overall triangular appearance and the s2 subunit forming the stalk ( figures 2b, c) (11, 34) . the structural difference between these two conformations only lies in the position of one of the three s1 rbds ( figures 2b, c) (11) . when all three rbds are in the "down" position, the resulting s ectodomain trimer assumes a closed conformation, in which the receptor-binding surface of the s1 rbd is buried at the interface between protomers and cannot be accessible by its receptor ( figure 2b ) (11) . the s ectodomain trimer with one single rbd in the "up" position assumes a partially open conformation and represents the functional state, as the receptorbinding surface of the "up" rbd can be fully exposed ( figure 2c ) (11, 34) . the structural information provides a blueprint for structure-based design of vaccine immunogens and entry inhibitors of sars-cov-2. in the closed sars-cov-2 s ectodomain trimer, interprotomer interactions occur through the s1 ctd packed against the other two s1 ctds and one ntd from an adjacent protomer because of domain swapping and through s2, primarily between helical interactions formed by the upstream and central helices from each subunit around the trimer axis ( figure 2b ) (11) . the s1 subunits rest above the s2 trimer, the life cycle of sars-cov-2 begins with membrane fusion occurring at the plasma membrane or within acidified endosomes after endocytosis, which is mediated by conformational changes in the s glycoprotein triggered by angiotensin-converting enzyme 2 (ace2) binding. following viral entry, sars-cov-2 releases its genomic rna into the host cell cytoplasm. genome rna is first translated into viral replicase polyproteins (pp1a and 1ab), which are further cleaved by viral proteases into a total of 16 nonstructural proteins. a replication-transcription complex (rtc) is formed based on many of these nonstructural proteins. in the process of genome replication and transcription mediated by rtc, the negative-sense (− sense) genomic rna is synthesized and used as a template to produce positive-sense (+ sense) genomic rna and subgenomic rnas. the nucleocapsid (n) structural protein and viral rna are replicated, transcribed, and synthesized in the cytoplasm, whereas other viral structural proteins, including the s protein, membrane (m) protein and envelope (e) protein, are transcribed and then translated in the rough endoplasmic reticulum (rer) and transported to the golgi complex. in the rer and golgi complex, the sars-cov-2 glycoprotein is subjected to co-translational and post-translational processing, including signal peptide removal, trimerization, extensive glycosylation and subunit cleavage. the n protein is subsequently associated with the positive sense genomic rna to become a nucleoprotein complex (nucleocapsid), which together with s, m, and e proteins as well as other viral proteins, is further assembled and followed by budding into the lumen of the er-golgi intermediate compartment (ergic) to form mature virions. finally, the mature virions are released from the host cell, waiting for a new life cycle to start. this figure is adapted from the template in biorender (https://biorender.com/). stabilizing the later in the prefusion conformation ( figure 2b ) (11) . when the s ectodomain trimer adopts a partially open conformation, the rbd in the "up" position will abolish the contacts with the s2 subunit of an adjacent protomer, destabilizing the partially open conformation ( figure 2c ) (11, 34) . this will be beneficial to the dissociation of the s1 subunit and facilitate conformational rearrangements that the s2 trimer undergoes to mediate viral entry. prefusion structures of human coronavirus hku1 (hcov-hku1) and mouse hepatitis virus s protein ectodomains without two consecutive proline mutations reveal only fully closed conformation (37, 42) , similar to that observed for a full-length, wild-type prefusion form of the sars-cov-2 s glycoprotein (41) . notably, it is well established that trimeric prefusion hiv-1 env primarily resides in a closed configuration that is conformationally masked to evade antibody-mediated neutralization (43, 44) and can spontaneously sample a transient, functional configuration (45) . it can thus be speculated that native cov s glycoproteins on mature and infectious virions share a similar conformational masking feature (46) , concealing the receptor-binding surface (for those utilizing ctds as rbds) ( figure 2c ), which is further discussed below. several lines of research have established that angiotensinconverting enzyme 2 (ace2) is an entry receptor for sars-cov-2 (47) (48) (49) . detailed interactions between the sars-cov-2 rbd and its receptor ace have been revealed by several structures of ace2 in complex with rbd (50) (51) (52) (53) . structurally, rbd consists of two subdomains: a core and an external subdomain (51, 52) . an extended loop (residues 438-506), which lies on one edge of the core subdomain, presents a gently concave surface to cradle the n-terminal helix (a1) of ace2. analysis of the interface between the sars-cov-2 rbd and ace2 reveals that a total of 17 residues in rbd are in contact with 20 amino acids in ace2, forming a network of hydrophilic interactions that are suggested to predominate the virus-receptor engagement (51) . outside this extended loop, residue lys417 located in helix a3 of the core subdomain, was shown to form ionic interactions with asp30 of ace2. as the extended loop contains almost all the amino acids of the sars-cov-2 rbd that contact ace2, it is referred to as the receptor-binding motif (rbm) (51) . it has been proposed that inhibiting the interaction between rbd and ace2 might be useful in treating sars-cov-2 infection. recombinant soluble ace2 (54) and ace2-fc (55, 56) have been shown to have potential applications in the prevention and treatment of sars-cov-2 infection in vitro. as the interaction between the rbd and ace2 is extensive, small molecules probably cannot be used as entry inhibitors to effectively block the virus entry by targeting the interaction interface. however, peptides would be able to engage most of the residues belonging to rbm (57) . a pioneering study demonstrated that a 23-amino acid peptide (residues 21-43), derived from the n-terminal helix (a1) of ace2, specifically associates with the sars-cov-2 rbd with low nanomolar affinity and disables receptor interactions (57), representing a promising strategy for preventing the virus from invading human cells. in another study, a 65-amino acid peptide (residues 19-83), derived from the n-terminal back-to-back helices (a1 and 2) and composed of most of the residues of ace2 that mediate interactions with the s protein, shows a similar but probably more potent inhibitory effect (58) . the formation of a trimer-of-hairpins structure (also known as six-helix bundle) comprising hr1 and hr2 in the postfusion conformation is a unifying feature of class i viral fusion proteins (37) . the crystal structure of a protein construct in which sars-cov-2 hr1 and hr2 were connected by a six-residue hydrophilic flexible linker was determined to be a canonical six-helix bundle structure with a rod-like shape ∼115 å in length and ∼25 å in diameter (59) . three hr1 helices form a parallel central coiled-coil with three hr2 helices packing in an oblique, antiparallel manner against deep hydrophobic grooves on the surface of the central coiled-coil (59) . notably, when a full-length s protein construct bearing the native furin-like cleavage site was transiently expressed by expi293f cells, the purified s proteins contained the dissociated s2 trimer in the postfusion conformation (41) . the cryo-em structure of this trimeric postfusion s2 shows that the central helix (ch) extended regular helices from the central coiled-coil, oriented toward target cells ( figure 2d ) (41) , which forms the longest central triple helical coiled-coil (~180å) among all known class i transmembrane subunit structures. the sars-cov-2 s trimer in the pre-hairpin intermediate state is very unstable and is just transiently present in vivo after triggering by ace2 engagement, stymieing structural characterization of the s protein in this state (60) . however, although this fusion-intermediate phase is very short, it is enough for inhibitory peptides to associate with the prehairpin intermediate and block the six-helix bundle formation (39) . furthermore, it has already been shown that the hr1 regions in various human covs are highly conserved (61) , and therefore could serve as an attractive target for the design and development of potent and broad-spectrum inhibitors of pan-covs, including sars-cov-2. a highly potent pan-coronavirus fusion inhibitor, ek1c4, has been reported to have good prophylactic and therapeutic potential against sars-cov-2 infection (59). as mentioned earlier, the sars-cov-2 s proteins are heavily decorated by heterogeneous n-linked glycans projecting from the s trimer surface. the sars-cov-2 s sequence encodes up to 22 n-linked glycan sequons per protomer, which likely plays an important role in protein folding (19) and host immune evasion as a glycan shield (62) . of the 22 potential n-linked glycosylation sites on the s protein, 14 were identified to be predominantly occupied by processed, complex-type glycans (63) . the remaining eight sites were found to be dominated by oligomannose-type glycans, which are divergent from those founded on host glycoproteins (63) . although glycosylation sites (n165, n234, n343) proximal to the receptor-binding sites on the sars-cov-2 s protein can be observed, ace2 bound to the glycosylated and deglycosylated s ectodomains with nearly identical affinity (1.7 nm vs 1.5 nm) determined by a biolayer interferometry binding assay (64) . this observation suggests that the high binding affinity between the sars-cov-2 s protein and ace2 does not depend on the s protein glycosylation. when the site-specific n-linked glycans are mapped onto the prefusion structure of the sars-cov-2 s ectodomain (63), the resulting model exhibited substantially higher levels of glycanfree surface than that revealed by structures of fully glycosylated, trimeric hiv-1 env ectodomains (65, 66) . this suggests that the sars-cov-2 s protein is covered by a less dense and less effective glycan shield compared to viral glycoproteins from hiv-1 (36, 66) and lassa virus (67) , which may be beneficial for the induction of humoral immunity and could be good news for a sars-cov-2 vaccine (68) . notably, it has been shown that multiple major viral surface antigens have neutralizing epitopes that are partly or even exclusively composed of carbohydrate moieties (69, 70) , exemplified by the hiv-1 env spike, which could be recognized by a large number of carbohydrate-binding antibodies, including 2g12, pg9, pg16, ch04, pgt121, pgt128, pgt135, and pgt145 (70, 71) . in the case of sars-cov-2, more recently a potent neutralizing antibody against both sars-cov and sars-cov-2, s309, has been shown to recognize a highly conserved glycan-containing rbd epitope (72) . these observations suggest that carbohydrate moieties could be immunogenic and highlight the need for immunogens to display the glycans important for the recognition of neutralizing antibodies (73) ; in support of this, specific n-linked glycans on hemagglutinin has been shown to be essential for the elicitation of broadly neutralizing antibodies against influenza (74) . accordingly, there has been mounting interest in exploring the potential of immunogenic glycan moieties as vaccine candidates against multiple viruses, including sars-cov-2 (75, 76) . membrane fusion and viral entry of sars-cov-2 is initiated by binding of rbd in the viral s glycoprotein transiently sampling the functional conformation to ace2 on the surface of target cells (figure 1 ) (10). after receptor engagement at the plasma membrane or ensuing virus endocytosis by the host cell (8), a second cleavage (s2′ cleavage site) is generated, which is mediated by a cellular serine protease tmprss2 (48) or endosomal cysteine proteases cathepsins b and l (10) (figure 1) . protease cleavage at s2′ site frees the fusion peptide from the new s2 n-terminal region, further destabilizes the sars-cov-2 s glycoprotein and may initiate s2-mediated membrane fusion cascade. following the second cleavage, the fusion peptide at the n terminus of the s2 trimer is inserted into the host membrane (8) , forming the pre-hairpin intermediate state (39) . since the pre-hairpin intermediate state is extremely unstable, the s2 fusion protein is refolded quickly and irreversibly into the stable postfusion state (39, 77) . these large conformational rearrangements pull the viral and host cell membrane into close proximity, leading ultimately to the membrane fusion (8, 39) . since sars-cov-2 was identified as the causative agent of covid-19, and its first genome sequence was released immediately and freely by a chinese research group (40), sars-cov-2 vaccine candidates based on various vaccine platforms, such as inactivated or live attenuated vaccines, dna and mrna vaccines, viral vector-based vaccines, and recombinant protein-based vaccines, have been developed (12, 78) . most of these vaccine strategies are based on the full-length s glycoprotein, the major viral surface antigen (12) . when a vaccine strategy requires that the sars-cov-2 s protein be recombinantly expressed in the human body, the errs should be omitted to enhance the cell surface expression level of the resulting protein. theoretically, the native hiv-1 env trimer present on the surface of intact virions is thought to be a most ideal immunogen (60) , as most of the neutralizing antibodies thus far described could recognize and bind to the prefusion form of trimeric hiv-1 env, although it is with great difficulty that such neutralizing antibodies against this glycan-covered, sequence-variable native form are induced (36) . for sars-cov-2, different lines of research have shown that convalescent sera from sars-cov and sars-cov-2 patients showed no or limited crossneutralization activity against these two viruses by pseudotyped and authentic viral infection assays, despite significant crossreactivity in binding to the s glycoproteins of both viruses (9, (79) (80) (81) . similar results were also observed in infected or immunized animals (48, 79, 81) . together with the finding that although the sars-cov-2 s protein shares a high degree of amino acid sequence identity with that of sars-cov (~76% overall), the rbm is less conserved (~47% identity) than any other functional region or domain (82) , it can thus been surmised that the rbm has the most immunodominant neutralizing epitope(s) of the whole s protein, capable of readily eliciting strong neutralizing antibody responses. however, the native trimeric sars-cov-2 s protein could conceal each of its immunodominant rbms by adopting the closed conformation (41, 83) . therefore, sars-cov-2 evades immune surveillance also through conformational masking, which is well-documented for hiv-1 (43, 44) ; while at the same time, the s protein could transiently sample the functional state to engage ace2, consistent with the notion that the fusion glycoprotein of highly pathogenic viruses have evolved to perform its functions while evading host neutralizing antibody responses. another concern for vaccine candidates based on the fulllength s glycoprotein of sars-cov-2 is raised by the observation that the s1 subunit could spontaneously dissociate from the s glycoprotein probably as a trimer that still assumes the rbd closed conformation, leaving only the postfusion s2 trimer (41) . the resulting s1 and s2 subunits might expose immunodominant, nonneutralizing epitopes that are utilized by sars-cov-2 to serve as decoys to distract the host immune system, inducing a large proportion of ineffective antibody responses, as documented for hiv-1 (60) and respiratory syncytial virus (rsv) (84) . it should be noted that although vaccine candidates based on the full-length s protein of the closely related sars-cov could elicit neutralizing antibody responses against infection of sars-cov, they may also induce harmful immune responses, including liver damage of the vaccinated animals, infection of human immune cells by sars-cov, and antibody-dependent enhancement of sars-cov infection (85) (86) (87) (88) (89) . therefore, although the s proteins of both sars-cov and sars-cov-2 are thought to be promising vaccine immunogens for generating protective immunity, optimizing antigen design is critical to ensure an optimal immune response through exposing more neutralizing epitopes and displaying fewer potentially weakly or non-neutralizing epitopes (90) . vaccines containing or expressing the full-length s protein or its soluble ectodomain form should thus be engineered to sample a rbd(s) "up" conformation while the rest is still kept in the prefusion state (91, 92) . apart from recombinant, soluble, stabilized ectodomains that are engineered to expose the immunodominant rbd by adapting the rbd(s) "up" conformation, rbd proteins of sars-cov and sars-cov-2 have also been widely used as recombinant protein-based vaccines (85, (93) (94) (95) . the rbd of sars-cov is highly immunogenic (96, 97) and is targeted by most of the neutralizing monoclonal antibodies that have been characterized (98) . based on the observation that a 193-amino acid fragment (residues 318-510) was previously identified to be the minimal rbd region of sars-cov (99), a corresponding 194-amino acid fragment (residues 331-524) can be readily selected as the minimal rbd region of sars-cov-2 and has already been characterized (100) . this minimal form of rbds of both viruses could serve as a vaccine candidate (100) . however, a conserved cysteine residue is located immediately upstream of the minimal rbd fragments of both viruses and always forms a disulfide bond in nearly all published structures containing this residue (101, 102) ; this is also the case for middle east respiratory syndrome coronavirus (mers-cov) (103, 104) and hcov-hku1 (37), consistent with the observation that all rbds of these viruses share a conserved structural core. the disulfide bond contributes to stabilization of the rbd structure and likely modulates the protein immunogenicity. this notion is consistent with the observation that mice immunized with a longer form of the sars-cov rbd (residues 318-536) produced a higher titer of neutralizing antibodies compared with mice immunized with the minimal rbd region (residues 318-510) (105) . therefore, when each of the minimal rbd fragments of sars-cov and sars-cov-2 is used as vaccine candidates, the critical cysteine residue should not be ignored and thus should be included (106) . besides the rbd, which has been shown to a major target for human neutralizing antibody responses (107), the ntd was recently identified to be a new vulnerable site of the sars-cov-2 s protein for antibody neutralizing and therefore could also serve as a recombinant protein-based vaccine (108) (109) (110) . as expected, ntd-specific neutralizing antibodies could target the s protein in both closed and open conformations (108) . in addition, the apparent accessibility of the fusion peptide and hr1 region in published structures of the sars-cov-2 s ectodomain trimer as well as their high sequence conservation among covs suggests that they would be good immunogen candidates for epitope-focused vaccine design aimed at raising broadly cov neutralizing antibodies (46) . the epitope-focused vaccine design has proven to be successful in generating neutralizing antibodies against rsv fusion glycoprotein (111). however, neutralizing antibodies targeted against these two regions still need to be isolated in infected individuals to support this notion. unlike wild-type full-length s protein of sars-cov-2, the above monomeric fragments do not induce any infection-enhancing antibodies or harmful immune or inflammatory responses (106, 112) , all of which could be potentially avoided through structure-based immunogen design to improve immunogenicity (113, 114) . however, wide-type full-length or soluble ectodomain form of the sars-cov-2 s protein could trigger stronger cellular immune responses (115) , which have been demonstrated to play an important role in controlling diseases caused by covs (116, 117) , including sars-cov-2 (118) , and are probably also an important determinant of effective vaccines against sars-cov-2 (115, 119) . additionally, when more than one rbd of the s protein trimer is engineered to be locked in the "up" conformation (120, 121) , the antigenicity and immunogenicity of the resulting rbds would be significantly enhanced compared to monomeric rbd form (97, 122) . moreover, improved protection is likely to be achieved when vaccinated with full-length or soluble ectodomain form of the sars-cov-2 s protein in that both forms can elicit neutralizing antibodies directed against non-rbd sites, as observed for mers-cov (123) . genetic variation has been used by many viruses that have rna genomes (124) , including hiv and influenza, as a mechanism to avoid antibody-mediated immunity, and is partially responsible for the great difficulty in developing effective and durable vaccines against these viruses (36) . as an rna virus, however, sars-cov-2 has a very low mutation rate overall (125) likely because covs have a genetic proofreading mechanism (126) . all reported variations occurred in the sars-cov-2 s glycoprotein have a prevalence of no more than 1% (127) , with an exception of d614g, which has become the most prevalent genotype in the global covid-19 pandemic (127) . fortunately, although the d614g mutation of the sars-cov-2 s protein has been shown to enhance viral infectivity (128) (129) (130) , until now there is no evidence that infection with sars-cov-2 carrying the g614 mutant will be associated with disease severity (127, 131) . furthermore, assays using both monoclonal and polyclonal antibodies generated from individuals naturally infected with d614-or g614-carrying viruses demonstrated that the d614g mutation retains or even increases viral susceptibility to neutralization (127, 130, 132, 133) . this suggests that the d614g mutant maintains or favors an open, functional conformational state (134) . although at an extremely low frequency, natural variations, including l452r a475v, v483a, and f490l that render the s glycoprotein resistant to certain neutralizing antibodies targeting the rbd, emerged under no selection pressure exerted by approved vaccines or neutralizing antibodies or entry inhibitors (127, 132) . however, it has been shown that sars-cov-2 escape mutants could be easily selected and quickly amplified under the selection pressure of single antibody treatment (135) . these observations suggest that a combination of at least two neutralizing antibodies that recognize and bind to distinct and non-overlapping epitopes on the sars-cov-2 s glycoprotein (e.g., rbd and ntd, as well as hr and glycan) is required to restrict the possible occurrence of viral escape mutants and potential subsequent loss of single antibody-mediated neutralization (135) (136) (137) (138) . when these observations are taken into consideration for vaccine design and development, an ideal sars-cov-2 immunogen should contain as many exposed neutralizing epitopes as possible, although the rbd also possesses extra epitope(s) besides the epitope in the rbm region (72, (139) (140) (141) . sars-cov-2 is a highly contagious pathogen that continues to spread quickly around the globe, causing covid-19 to be one of the worst pandemics in recorded history. a safe and efficacious vaccine represents one of the best ways to reduce or eliminate the covid-19 pandemic (142) . unfortunately, no vaccines for any of the known human covs have been licensed (143, 144) , although several potential sars-cov and mers-cov vaccines have advanced into human clinical trials for years (117, 145) , suggesting the development of effective vaccines against human covs has always been challenging. however, it has been shown that both sars-cov and sars-cov-2 could readily induce neutralizing antibodies following natural infection or immunization (146) (147) (148) (149) . moreover, a growing number of neutralizing monoclonal antibodies targeting the sars-cov-2 s glycoprotein with high potency have been isolated from plenty of convalescent donors (33) as well as humanized mice (136, 141) , some of which have been shown to afford protection against sars-cov-2 challenge in animal models. it thus seems that vaccine candidates designed to elicit such neutralizing antibodies are feasible. it is widely accepted that the s protein of sars-cov-2 is a most promising immunogen for producing protective immunity (150) . however, it is likely that the s protein has evolved to perform its functions while evading host neutralizing antibody responses and thus should be engineered to ensure an optimal immune response (151, 152) . the immunogen design strategies described in this review based on the wealth of the sars-cov-2 s glycoprotein research related to its biosynthesis, structure, function, antigenicity as well as immunogenicity will likely contribute to the ultimate success of safe and efficacious vaccines against sars-cov-2/covid-19. covid-19: emergence, spread, possible treatments, and global burden highlight of immune pathogenic response and hematopathologic effect in sars-cov, mers-cov, and sars-cov-2 infection coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying 2019-ncov and naming it sars-cov-2 an interactive web-based dashboard to track covid-19 in real time origin and evolution of pathogenic coronaviruses viral membrane fusion cell entry mechanisms of sars-cov-2 coronavirus membrane fusion mechanism offers a potential target for antiviral development characterization of spike glycoprotein of sars-cov-2 on virus entry and its immune cross-reactivity with sars-cov mechanisms of coronavirus cell entry mediated by the viral spike protein structure, function, and antigenicity of the sars-cov-2 spike glycoprotein sars-cov-2 vaccines: status report composition and divergence of coronavirus spike proteins and host ace2 receptors predict potential intermediate hosts of sars-cov-2 n-linked protein glycosylation in the endoplasmic reticulum protein folding in the endoplasmic reticulum important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry snapshot: n-glycosylation processing pathways across kingdoms n-linked protein glycosylation in the er intracellular functions of n-linked glycans mechanisms and principles of n-linked protein glycosylation glycosylation quality control by the golgi structure the proximal origin of sars-cov-2 snapshot: o-glycosylation pathways across kingdoms a multibasic cleavage site in the spike protein of sars-cov-2 is essential for infection of human lung cells the spike glycoprotein of the new coronavirus 2019-ncov contains a furin-like cleavage site absent in cov of the same clade tmprss2 and furin are both essential for proteolytic activation and spread of sars-cov-2 in human airway epithelial cells and provide promising drug targets intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site the intracellular sites of early replication and budding of sarscoronavirus the cytoplasmic tail of the severe acute respiratory syndrome coronavirus spike protein contains a novel endoplasmic reticulum retrieval signal that binds copi and promotes interaction with membrane protein the contribution of the cytoplasmic retrieval signal of severe acute respiratory syndrome coronavirus to intracellular accumulation of s proteins and incorporation of s protein into viruslike particles properties of coronavirus and sars-cov-2 a replication-competent vesicular stomatitis virus for studies of sars-cov-2 spike-mediated cell entry and its inhibition measuring sars-cov-2 neutralizing antibody activity using pseudotyped and chimeric viruses cryo-em structure of the 2019-ncov spike in the prefusion conformation the coronavirus spike protein is a class i virus fusion protein: structural and functional characterization of the fusion core complex structure and immune recognition of trimeric pre-fusion hiv-1 env pre-fusion structure of a human coronavirus spike protein common features of enveloped viruses and implications for immunogen design for next-generation vaccines viral membrane fusion a new coronavirus associated with human respiratory disease in china distinct conformational states of sars-cov-2 spike protein cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer hiv-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites conformational masking and receptor-dependent unmasking of highly conserved env epitopes recognized by non-neutralizing antibodies that mediate potent adcc against hiv-1 conformational dynamics of single hiv-1 envelope trimers on the surface of native virions immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen a pneumonia outbreak associated with a new coronavirus of probable bat origin sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structural basis of receptor recognition by sars-cov-2 structure of the sars-cov-2 spike receptor-binding domain bound to the ace2 receptor structural and functional basis of sars-cov-2 entry by using human ace2 structural basis for the recognition of sars-cov-2 by full-length human ace2 inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 neutralization of sars-cov-2 spike pseudotyped virus by recombinant ace2-ig novel ace2-igg1 fusions with increased activity against sars-cov-2. biorxiv the first-in-class peptide binder to the sars-cov-2 spike protein ace2 fragment as a decoy for novel sars-cov-2 virus inhibition of sars-cov-2 (previously 2019-ncov) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion insights into vaccine development for acquired immune deficiency syndrome from crystal structures of human immunodeficiency virus-1 gp41 and equine infectious anemia virus gp45 insights into the design of sars-cov-2 spike-based immunogens a pancoronavirus fusion inhibitor targeting the hr1 domain of human coronavirus spike glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy site-specific glycan analysis of the sars-cov-2 spike mass spectrometry analysis of newly emerging coronavirus hcov-19 spike s protein and human ace2 reveals camouflaging glycans and unique post-translational modifications vulnerabilities in coronavirus glycan shields despite extensive glycosylation trimeric hiv-1-env structures define glycan shields from clades a, b, and g structure of the lassa virus glycan shield provides a model for immunological resistance site-specific nglycosylation characterization of recombinant sars-cov-2 spike proteins using high-resolution mass spectrometry recent strategies targeting hiv glycans in vaccine design protein and glycan mimicry in hiv vaccine design structure and immune recognition of the hiv glycan shield cross-neutralization of sars-cov-2 by a human monoclonal sars-cov antibody vaccination with glycan-modified hiv nfl envelope trimer-liposomes elicits broadly neutralizing antibodies to multiple sites of vulnerability n-linked glycans and k147 residue on hemagglutinin synergize to elicit broadly reactive h1n1 influenza virus antibodies targeting host-derived glycans on enveloped viruses for antibody-based vaccine design coronaviruses' sugar shields as vaccine candidates cryo-em structures of mers-cov and sars-cov spike glycoproteins reveal the dynamic receptor binding domains the covid-19 vaccine development landscape lack of antibodymediated cross-protection between sars-cov-2 and sars-cov infections lack of cross-neutralization by sars patient sera towards sars-cov-2 cross-reactive antibody response between sars-cov-2 and sars-cov infections phylogenetic analysis and structural modeling of sars-cov-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop in situ structural analysis of sars-cov-2 spike reveals flexibility mediated by three hinges structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus the spike protein of sars-cov-a target for vaccine and therapeutic development sars vaccine development from sars-cov to sars-cov-2: safety and broad-spectrum are important for coronavirus vaccine development role of antibodydependent enhancement (ade) in the virulence of sars-cov-2 and its mitigation strategies for the development of vaccines and immunotherapies to counter covid-19 a perspective on potential antibody-dependent enhancement of sars-cov-2 developing covid-19 vaccines at pandemic speed sars-cov-2 protein subunit vaccination elicits potent neutralizing antibody responses sars-cov-2 mrna vaccine design enabled by prototype pathogen preparedness the sars-cov-2 vaccine pipeline: an overview roadmap to developing a recombinant coronavirus s protein receptor-binding domain vaccine for severe acute respiratory syndrome subunit vaccines against emerging pathogenic human coronaviruses the receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in sars-cov-2 patients a universal design of betacoronavirus vaccines against covid-19, mers, and sars neutralizing antibodies against sars-cov-2 and other human coronaviruses a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody structural insights into coronavirus entry molecular basis of binding between novel human coronavirus mers-cov and its receptor cd26 unexpected receptor functional mimicry elucidates activation of coronavirus fusion yeast-expressed sars-cov recombinant receptor-binding domain (rbd219-n1) formulated with alum induces protective immunity and reduces immune enhancement a vaccine targeting the rbd of the s protein of sars-cov-2 induces protective immunity convergent antibody responses to sars-cov-2 in convalescent individuals a potent neutralizing human antibody reveals the n-terminal domain of the spike protein of sars-cov-2 as a site of vulnerability potent neutralizing antibodies from covid-19 patients define multiple targets of vulnerability potent neutralizing antibodies directed to multiple epitopes on sars-cov-2 spike proof of principle for epitope-focused vaccine design the sars-cov-2 receptor-binding domain elicits a potent neutralizing response without antibody-dependent enhancement what are the most powerful immunogen design vaccine strategies? a structural biologist's perspective structural vaccinology for viral vaccine design targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals t-cell immunity of sars-cov: implications for vaccine development against mers-cov recent advances in the vaccine development against middle east respiratory syndrome-coronavirus divergent sars-cov-2-specific t and b cell responses in severe but not mild covid-19 preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies controlling the sars-cov-2 spike glycoprotein conformation structure-based design of prefusion-stabilized sars-cov-2 spikes high epitope density in a single protein molecule significantly enhances antigenicity as well as immunogenicity: a novel strategy for modern vaccine development and a preliminary investigation about b cell discrimination of monomeric proteins evaluation of candidate vaccine approaches for mers-cov quasispecies theory and the behavior of rna viruses coast-to-coast spread of sars-cov-2 during the early epidemic in the united states insights into rna synthesis, capping, and proofreading mechanisms of sars-coronavirus tracking changes in sars-cov-2 spike: evidence that d614g increases infectivity of the covid-19 virus the d614g mutation in the sars-cov-2 spike protein reduces s1 shedding and increases infectivity the d614g mutation of sars-cov-2 spike protein enhances viral infectivity sars-cov-2 spike protein variant d614g increases infectivity and retains sensitivity to antibodies that target the receptor binding domain making sense of mutation: what d614g means for the covid-19 pandemic remains unclear the impact of mutations in sars-cov-2 spike on viral infectivity and antigenicity d614g spike mutation increases sars cov-2 susceptibility to neutralization. medrxiv the sars-cov-2 spike variant d614g favors an open conformational state antibody cocktail to sars-cov-2 spike protein prevents rapid mutational escape seen with individual antibodies studies in humanized mice and convalescent humans yield a sars-cov-2 antibody cocktail neutralizing antibodies against sars-cov-2 and other human coronaviruses perspectives on the development of neutralizing antibodies against sars-cov-2 a highly conserved cryptic epitope in the receptor binding domains of sars-cov-2 and sars-cov structural basis for neutralization of sars-cov-2 and sars-cov by a potent therapeutic antibody a human monoclonal antibody blocking sars-cov-2 infection updated approaches against sars-cov-2 vaccines for covid-19: perspectives, prospects, and challenges based on candidate sars, mers, and animal coronavirus vaccines antibodies and vaccines against middle east respiratory syndrome coronavirus vaccines against coronaviruses: the state of the art. vaccines (basel) (2020) 8:309 immunogenic profile of sars-cov-2 spike in individuals recovered from covid-19. medrxiv immunogenicity of a dna vaccine candidate for covid-19 dna vaccine protection against sars-cov-2 in rhesus macaques antibody signature induced by sars-cov-2 spike protein immunogens in rabbits progress and prospects on vaccine development against sars-cov-2. vaccines (basel) (2020) 8:153 what are the most powerful immunogen design vaccine strategies? reverse vaccinology 2.0 shows great promise structure-based vaccine antigen design all authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication. we would like to thank prof. xinqi liu for critical reading of the manuscript; and drs. yanbin feng, mengyuan xu, jing ma and jianrong feng for helpful comments and discussions on the manuscript. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 duan, zheng, zhang, niu, lou and wang. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-308433-vrkdtrfz authors: roberts, ceri a.; durham, lucy e.; fleskens, veerle; evans, hayley g.; taams, leonie s. title: tnf blockade maintains an il-10(+) phenotype in human effector cd4(+) and cd8(+) t cells date: 2017-02-15 journal: front immunol doi: 10.3389/fimmu.2017.00157 sha: doc_id: 308433 cord_uid: vrkdtrfz cd4(+) and cd8(+) effector t cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine il-10. however, the underlying cellular mechanisms that regulate expression of il-10 in different t cell subpopulations are not yet fully elucidated. we recently showed that tnf inhibitors (tnfi) promote il-10 expression in human cd4(+) t cells, including il-17(+) cd4(+) t cells. here, we further characterized the regulation of il-10 expression via blockade of tnf signaling or other cytokine/co-stimulatory pathways, in human t cell subpopulations. addition of the tnfi drug adalimumab to anti-cd3-stimulated human cd4(+) t cell/monocyte cocultures led to increased percentages of il-10(+) cells in pro-inflammatory il-17(+), ifnγ(+), tnfα(+), gm-csf(+), and il-4(+) cd4(+) t cell subpopulations. conversely, exogenous tnfα strongly decreased il-10(+) cell frequencies. tnf blockade also regulated il-10 expression in cd4(+) t cells upon antigenic stimulation. using time course experiments in whole peripheral blood mononuclear cell (pbmc) cultures, we show that tnf blockade maintained, rather than increased, il-10(+) cell frequencies in both cd4(+) and cd8(+) t cells following in vitro stimulation in a doseand time-dependent manner. blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated co-stimulation did not significantly regulate il-10 expression within cd4(+) or cd8(+) t cell subpopulations. we show that tnf blockade acts directly on effector cd4(+) t cells, in the absence of monocytes or cd4(+) cd25(high)cd127(low) regulatory t cells and independently of il-27, resulting in higher il-10(+) frequencies after 3 days in culture. il-10/il-10r blockade reduced the frequency of il-10-expressing cells both in the presence and absence of tnf blockade. addition of recombinant il-10 alone was insufficient to drive an increase in il-10(+) cd4(+) t cell frequencies in 3-day cd4(+) t cell/monocyte cocultures, but resulted in increased il-10 expression at later time points in whole pbmc cultures. together, these data provide additional insights into the regulation of il-10 expression in human t cells by tnf blockade. the maintenance of an il-10(+) phenotype across a broad range of effector t cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy. tnf blockade maintains an il-10 + phenotype in human effector cd4 + and cd8 + t cells ceri a. roberts cd4 + and cd8 + effector t cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine il-10. however, the underlying cellular mechanisms that regulate expression of il-10 in different t cell subpopulations are not yet fully elucidated. we recently showed that tnf inhibitors (tnfi) promote il-10 expression in human cd4 + t cells, including il-17 + cd4 + t cells. here, we further characterized the regulation of il-10 expression via blockade of tnf signaling or other cytokine/co-stimulatory pathways, in human t cell subpopulations. addition of the tnfi drug adalimumab to anti-cd3-stimulated human cd4 + t cell/monocyte cocultures led to increased percentages of il-10 + cells in pro-inflammatory il-17 + , ifnγ + , tnfα + , gm-csf + , and il-4 + cd4 + t cell subpopulations. conversely, exogenous tnfα strongly decreased il-10 + cell frequencies. tnf blockade also regulated il-10 expression in cd4 + t cells upon antigenic stimulation. using time course experiments in whole peripheral blood mononuclear cell (pbmc) cultures, we show that tnf blockade maintained, rather than increased, il-10 + cell frequencies in both cd4 + and cd8 + t cells following in vitro stimulation in a dose-and time-dependent manner. blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated co-stimulation did not significantly regulate il-10 expression within cd4 + or cd8 + t cell subpopulations. we show that tnf blockade acts directly on effector cd4 + t cells, in the absence of monocytes or cd4 + cd25 high cd127 low regulatory t cells and independently of il-27, resulting in higher il-10 + frequencies after 3 days in culture. il-10/il-10r blockade reduced the frequency of il-10-expressing cells both in the presence and absence of tnf blockade. addition of recombinant il-10 alone was insufficient to drive an increase in il-10 + cd4 + t cell frequencies in 3-day cd4 + t cell/monocyte cocultures, but resulted in increased il-10 expression at later time points in whole pbmc cultures. together, these data provide additional insights into the regulation of il-10 expression in human t cells by tnf blockade. the maintenance of an il-10 + phenotype across a broad range of effector t cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy. keywords: tumor necrosis factor, anti-tnf, tnf inhibitors, adalimumab, interleukin-10, cd4 + t cell polarization, cd8 + t cell polarization, il-10 regulation introduction the treatment of immune-mediated inflammatory diseases has improved considerably over the last 20 years with the advent of biological therapeutics. tnfα was the first cytokine to be fully validated as a therapeutic target in ra (1) . tnfα inhibitors (tnfi) have revolutionized treatment of ra and have been used in over a million patients worldwide (2) . despite the good clinical response observed in many patients, tnfα blockade does not offer a curative treatment; approximately one-third of patients do not respond and loss of efficacy is frequently observed (3) . importantly, it is currently not possible to predict which patients will respond to tnfi therapy. in addition, in some inflammatory diseases such as sjögren's syndrome (4, 5) and multiple sclerosis (6), tnfi have not shown clinical efficacy. furthermore, and paradoxically, some patients treated with tnfi develop de novo autoimmune diseases (7) . these observations indicate that the underlying mechanisms relating to tnf blockade in humans are incompletely understood and require further exploration. the effects of tnfi are more wide-ranging than simply neutralizing the biological activity of soluble and membrane-bound tnfα (mtnfα). for example, by binding mtnfα, anti-tnf mabs can mediate cell death by complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (8) (9) (10) (11) . tnfα inhibitors have also been shown to affect downstream cytokine pathways (il-1, il-6, and il-8) (2), modulate apc function (12) , and promote regulatory t cell (treg) expansion (13) (14) (15) although opposite findings regarding the latter have been reported (16) (17) (18) (19) . recent data from our laboratory demonstrated that tnf blockade promotes il-10 expression in human cd4 + t cells (20) . it was shown both cross-sectionally and longitudinally that inflammatory arthritis patients on tnfi therapy have an increased frequency of peripheral blood (pb) il-10 + cd4 + t cells. these in vivo findings were reproduced in vitro by coculturing cd4 + t cells from healthy donors with autologous cd14 + monocytes and anti-cd3 mab, in the presence of different tnfi drugs (adalimumab, infliximab, etanercept, or certolizumab) (20) . furthermore, we showed an increase in the percentage of il-10 co-expressing il-17 + cd4 + t cells, suggesting that otherwise pro-inflammatory cells displayed anti-inflammatory potential. indeed, re-sorted tnfi-exposed il-17 + cd4 + t cells secreted increased levels of il-10, which was biologically active and could modulate markers of monocyte activation (20) . although il-17 + cd4 + t cells are recognized as an important cell population in inflammatory disease, other cd4 + t cell subsets also contribute to inflammation (21) (22) (23) (24) , as well as cd8 + t cells which can also be potent producers of proinflammatory cytokines (25) (26) (27) (28) (29) . in this study, we therefore investigated in vitro whether tnf blockade regulates il-10 expression in other pro-inflammatory cytokine-producing t cell subsets, whether blockade of other cytokines or t cell activation pathways also drives il-10 expression, and how tnf blockade may manifest its il-10-regulating effect on t cells. peripheral blood samples were obtained from healthy adult volunteers. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphoprep™ (axis-shield, oslo, norway). cd14 + monocytes and cd4 + t cells were isolated by magnetic-activated cell sorting (macs) according to the manufacturer's instructions (miltenyi biotec, bergisch-gladbach, germany), and purity was confirmed by flow cytometry. monocytes (average purity 98%) were isolated by positive selection using anti-cd14 microbeads. cd4 + t cells were isolated via negative depletion (average purity 95%), and in some experiments, cd45ro + cd4 + t cells were subsequently enriched by positive selection using cd45ro microbeads (average purity 87%). in some experiments, cd4 + t cells were sorted to very high purity (> 99%) and part of the cells depleted of cd4 + cd25 high cd127 low tregs by facs-sorting after labeling cells with cd4 percp cy5.5 (sk3), cd25 pe (m-a251), cd127 alexa fluor 488 (a019d5) mabs (all from biolegend, cambridge, uk). the study was approved by the bromley research ethics committee (06/q0705/20), and written informed consent was obtained from all participants. cells were cultured at 37°c with 5% co2 in culture medium [rpmi 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (lot# 07f7435k, south american origin)] and 1% penicillin, streptomycin, and l-glutamine (all life technologies, carlsbad, ca, usa). freshly isolated bulk cd4 + or memory (cd45ro + )-enriched t cells (0.5 × 10 6 ) and cd14 + monocytes (0.5 × 10 6 unless otherwise indicated) were cocultured with 100 ng/ml anti-cd3 mab (clone okt3, janssen-cilag ltd., high wycombe, uk). cocultures were incubated at 37°c with 5% co2 for 3 days. in some experiments, macs-isolated cd4 + t cells were cultured alone with anti-cd3/cd28 stimulation. anti-cd3 (okt3) was coated onto culture plates at 1.25 µg/ml in pbs for a minimum of 2 h at 37°c. wells were then washed three times with pbs before adding cd4 + t cells in culture medium. anti-cd28 (clone cd8.2; bd biosciences) was added to the well at a final concentration of 1 µg/ml. in some experiments, whole pbmcs were cultured with 100 ng/ml anti-cd3 mab for up to 5 days. where indicated, the following recombinant cytokines, neutralizing antibodies, or other reagents (from r&d systems unless otherwise indicated) were added at the start of the culture period: recombinant human (rh) tnfα (10 ng/ml; biosource, camarillo, ca, usa), rhil-27 (10-100 ng/ml), rhil-10 (10 ng/ml; rhil-10 used to generate data in figure s5 in supplementary material from peprotech, rocky hill, nj, usa), neutralizing/blocking antibodies to ifnγ (clone 25718, 10 µg/ml), il-17 (clone 41809, 10 µg/ml), il-1r1 (polyclonal, 2 µg/ml), il-10 (clone 23738, 10 µg/ml), il-10r (clone 8516, 10 µg/ml), and il-27 (polyclonal, 5 µg/ml). the following isotype control antibodies were used at an assay-appropriate concentration: human igg1 (abcam, cambridge, uk), mouse igg1, mouse igg2a, mouse igg2b, goat igg (all r&d systems). clinical grade biologics were purchased anti-tnf maintains il-10 + cd4 + /cd8 + t cells . antibodies to detect cd4 and/ or cd3 were added intracellularly, to allow for staining of these markers following our t cell culture conditions. stained cells were acquired using a facscantoii or lsrfortessa (bd biosciences); in most experiments, 100,000 t cell events were recorded. the gating strategies used to analyze intracellular cytokine expression in cd4 + t cells are described in figure s1 in supplementary material. all flow cytometry data were analyzed using flowjo software (version 7.6.1 or 10, tree star, inc., ashland, or, usa). il-10 was measured in cell culture supernatants using the il-10 elisa max kit (biolegend), according to manufacturer's instructions. il-27 was measured in cell culture supernatants using the duoset human il-27 elisa (ebioscience), according to manufacturer's instructions. microwell absorbance was read at 450 nm using a wallac 1420 microplate reader (perkin elmer, waltham, ma, usa). concentrations of the analytes were determined based on the standard curve included on each plate. statistical testing was performed with graphpad prism 5.0 or 6.0 (graphpad, san diego, ca, usa). data sets were tested for normality using the d' agostino and pearson omnibus normality test, followed by statistical significance testing using the appropriate tests as indicated in figure legends. data sets with n values <8 were tested non-parametrically. p values < 0.05 were considered statistically significant. tnf blockade regulates il-10 expression in human cd4 + and cd8 + t cells to investigate whether tnf blockade regulates il-10 expression in different pro-inflammatory cytokine-producing cd4 + t cells, we isolated cd4 + t cells from pb of healthy donors and cocultured the cells with cd14 + monocytes and anti-cd3 mab (100 ng/ml) in the absence or presence of the anti-tnf mab adalimumab (1 µg/ml), as previously described (20) . after 3 days, cells were restimulated with pma and ionomycin in the presence of golgistop for 3 h and stained for cytokine expression (gating strategy shown in figure s1 in supplementary material). in agreement with our previous data (20) , tnf blockade led to a significant increase in il-10 + cells within total cd4 + t cells as well as in the il-17 + cd4 + t cell subset. in addition, we found a strong increase in il-10-expressing cells within ifnγ + , tnfα + , gm-csf + , and il-4 + cd4 + t cell populations (figures 1a,b) . tnf blockade did not alter the frequencies of ifnγ + , tnfα + , or gm-csf + cd4 + t cell populations but did induce a modest increase in il-17 + cd4 + t cells, as previously shown (20) , and in most donors in il-4 + frequencies, although this did not reach statistical significance ( figures s2a, b in supplementary material). addition of an isotype control human igg1 mab did not promote il-10 expression in cd4 + t cells ( figure s3 in supplementary material). contrary to the effects of tnf blockade, addition of rhtnfα to cocultures of cd4 + cd45ro + t cells, monocytes and anti-cd3 mab led to a striking decrease in the percentage of il-10 + cells within total cd4 + t cells and within il-17 + , ifnγ + , and tnfα + cd4 + t cell subsets (gm-csf + and il-4 + cd4 + t cells were not tested) ( figure 1c ). tnfα addition did not significantly alter the frequencies of il-17 + , ifnγ + , or tnfα + cd4 + t cell subsets ( figure s2c in supplementary material). since pro-inflammatory cytokine-expressing cd8 + t cells also contribute to immune-mediated inflammatory diseases (27) (28) (29) , we investigated whether tnf blockade can regulate il-10 expression in cd8 + t cells. we adapted the culture system to stimulate whole pbmc with anti-cd3 mab (100 ng/ml) in the absence or presence of a dose range of adalimumab (0.01-10 µg/ml). after 3 days, the cultures were restimulated with pma/ionomycin in the presence of golgistop and cytokine expression was assessed within either the cd4 + or the cd8 + t cell populations. significant increases in the percentages of il-10 + cells within both cd4 + and cd8 + t cell populations were observed, including in the il-17 + and ifnγ + subpopulations (figure 2) . the regulation of il-10 expression by tnf blockade was dose responsive in both cd4 + and cd8 + t cell subsets, with significantly increased il-10 + frequencies following culture with either 1 or 5 µg/ml adalimumab ( figure 2c ). these doses reflect figure 1 | tnf blockade promotes, while tnfα impairs il-10 expression in pro-inflammatory cd4 + t cell subsets. cd4 + t cells (open symbols) or cd4 + cd45ro + t cells (filled symbols) were cocultured with autologous cd14 + monocytes and anti-cd3 mab (100 ng/ml) in the absence or presence of the anti-tnf mab adalimumab (1 µg/ml) (a,b) or rhtnfα (10 ng/ml) (c). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. cd4 + t cells were gated as described in figure s1 in supplementary material. (a,b) representative dot plots (a) and cumulative data (b) showing the percentages of il-10 + cells within total cd4 + t cells (n = 35), or within il-17 + cd4 + (n = 35), ifnγ + cd4 + (n = 24), tnfα + cd4 + (n = 27), gm-csf + cd4 + (n = 12), or il-4 + cd4 + (n = 8) cells after culture in the absence or presence of anti-tnf. data were analyzed using wilcoxon matched-pairs signed rank test. (c) cumulative data showing the percentages of il-10 + cells within total cd4 + cd45ro + t cells or within il-17 + (n = 8), ifnγ + (n = 8), or tnfα + (n = 8) cd4 + cd45ro + t cells after culture in the absence or presence of rhtnfα. data were analyzed using paired t test. each connecting line represents an individual donor (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). figure 2 | tnf blockade regulates il-10 expression in cd4 + and cd8 + t cells in a dose-dependent manner. (a,b) freshly isolated peripheral blood mononuclear cells were cultured with anti-cd3 mab (100 ng/ml) in the absence (control) or presence (anti-tnf) of adalimumab (1 µg/ml). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. cells were gated on live single cd3 + cells and il-10 expression was assessed within cd4 + (a) or cd8 + (b) t cell subsets. representative dot plots show the percentages of il-10 + cells within total cd4 + or cd8 + t cells, or within il-17 + or ifnγ + subsets after culture in the absence or presence of anti-tnf. (c) cells were cultured as described above in the presence of increasing doses of adalimumab (0-10 μg/ml). box-whisker plots represent data from n = 6 individual donors; whiskers show minimum to maximum values. data were analyzed by friedman test with comparison to control by dunn's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). the clinically effective trough levels for adalimumab in human serum following treatment (30, 31) . in agreement with data from the cd4 + t cell/monocyte cocultures, addition of rhtnfα (100 ng/ml) to anti-cd3-stimulated pbmc led to a reduction in il-10 + cell frequencies, in both cd4 + and cd8 + t cells (n = 3, data not shown). since these in vitro studies of t cell responses to tnf blockade all relied on monoclonal anti-cd3 stimulation, we tested whether tnf blockade could still elicit increased il-10 + t cell responses in the context of a more physiological antigenic stimulation. pediacel is a pentavalent vaccine consisting of purified diphtheria toxoid, purified tetanus toxoid, acellular pertussis vaccine, inactivated poliovirus, and haemophilus influenzae type b polysaccharide. revaxis is a booster vaccine containing purified diphtheria and tetanus toxoids and inactivated poliovirus. each vaccine was added to cd4 + t cell/monocyte cocultures (1:1,000 dilution) in the presence or absence of tnf blockade. after 6 days, antigen-stimulated cd4 + t cells cultured in the presence of adalimumab demonstrated elevated il-10 + frequencies as compared to cells that were not exposed to tnf blockade ( figure s4 in supplementary material). together these data demonstrate that in vitro tnf blockade provided at a physiological concentration and in a physiological setup can promote il-10 expression in cd4 + and cd8 + t cells, including in subsets expressing pro-inflammatory cytokines. tnf blockade maintains il-10 expression in cd4 + and cd8 + t cells thus far, assessment of cytokine expression following in vitro tnf blockade was carried out after 3 days of culture. time course experiments were performed to assess the kinetics of il-10 expression in cd4 + and cd8 + t cells. pbmc were cultured with anti-cd3 mab in the absence or presence of adalimumab, and the frequencies of il-10 expressing cells were analyzed 4-120 h after stimulation. the data show that early after tcr stimulation (18-24 h) the frequencies of il-10 expressing cells within cd4 + and cd8 + t cells increased rapidly irrespective of the presence of tnf blockade. however, at later time points (from 42 to 120 h), higher frequencies of il-10 + cells were sustained within cd4 + and cd8 + t cells in the presence of tnf blockade (figure 3) . these experiments suggest that tnf blockade maintains, rather than directly induces, an il-10 + phenotype in cd4 + and cd8 + t cells following tcr stimulation. blockade of tnfα, but not il-17, ifnγ, il-6r, or cd80/cd86-mediated costimulation, regulates il-10 in human cd4 + t cells our previous work demonstrated that in addition to adalimumab, other tnfα inhibitors (etanercept, infliximab, or certolizumab) as well as tnfr1/2 blocking mabs were capable of increasing frequencies of il-10-expressing il-17 + cd4 + t cells (20) . we investigated whether blockade of additional pro-inflammatory pathways could promote il-10 expression in cd4 + t cells. blockade of il-17a did not enhance the frequencies of il-10 + cells in any of the cd4 + t cell populations tested (figure 4a) . blockade of ifnγ did not affect the percentage of il-10 + cells within total cd4 + t cells, or within ifnγ or tnfα + subpopulations, but led to modestly increased frequencies of il-10 + expressing cells within the il-17 + population, although this effect was much weaker than the effect of tnf blockade in parallel cultures ( figure 4a) . addition of tocilizumab (il-6r blockade) or abatacept (ctla4-ig, which blocks cd80/cd86-mediated co-stimulation), both of which are biologic drugs routinely used in the clinic to treat rheumatoid arthritis, did not increase il-10 + frequencies in cd4 + t cells, il-17 + , ifnγ + , or tnfα + cd4 + t cell subpopulations (figure 4b) . to determine whether blockade of these pathways might regulate il-10 expression with different kinetics to tnf blockade, il-10 + frequencies were analyzed within both cd4 + and cd8 + t cells at different time points in anti-cd3-stimulated pbmc cultures exposed to these antibodies or drugs. il-10 + cd4 + and il-10 + cd8 + t cell frequencies were not regulated at any time point by blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated co-stimulation ( figure s5 in supplementary material). blockade of il-1r1 in cd4 + t cell/monocyte cocultures resulted in a significantly increased proportion of il-10 + cells within total cd4 + t cells and within il-17 + , ifnγ + , or tnfα + subpopulations ( figure 4b) . however, this effect was not replicated in either cd4 + or cd8 + t cells in whole pbmc cultures ( figure s5 in supplementary material), indicating that the capacity of il-1 blockade to regulate il-10 expression may be dependent on the in vitro culture conditions. together these data indicate that il-10 expression in cd4 + t cells and cd8 + t cells can be regulated by blocking tnfα signaling, but not by blocking ifnγ, il-17, il-6r, or cd80/cd86-mediated co-stimulation, at least in vitro. since cd14 + monocytes are major producers of tnfα, we explored whether the presence of monocytes was required for the effects of tnf blockade on regulating il-10. cd4 + t cells were sorted to a very high purity (>99%) and stimulated with platebound anti-cd3 and soluble anti-cd28 mab in the absence of monocytes or placed in coculture with monocytes and anti-cd3 mab, with or without addition of anti-tnf mab. in the cd4 + t cell only cultures, tnf blockade still brought about increased il-10 + cell frequencies within the total cd4 + population and also within il-17 + , ifnγ + , or tnfα + subpopulations, similar to the increased il-10 + frequencies observed in cd4 t cell/monocyte cocultures (figures 5a,b) . analysis of supernatants from anti-cd3/cd28-stimulated cd4 + t cells by elisa confirmed that levels of secreted il-10 were increased during culture in the presence of tnf blockade (figure 5c) . one potential interpretation of the observed increased frequencies of il-10 + cells following tnf blockade could be that there is an expansion of a pre-existing population of tregs. our previous work indicated that in three donors, macs-depletion of cd4 + cd25 + cells did not impair the anti-tnf-mediated increase in il-10 + frequencies within il-17 + cd4 + t cells and that no increase in foxp3 + cell frequencies was apparent upon tnf blockade (20) . to investigate this further, macs-isolated cd4 + t cells were facs sorted to very high purity (>99%) to yield either total cd4 + t cells or effector cd4 + cd25-cd127 + t cells (teff; depleted of cd25 high cd127 low treg). these cells were then cultured with anti-cd3/cd28 mabs in the absence of monocytes, or placed in coculture with monocytes and anti-cd3 mab, in the absence or presence of adalimumab. our data show that tnf blockade still resulted in increased frequencies of il-10-expressing cells in effector t cells in the absence of cd25 high cd127 low tregs (figure 6 ). together these data indicate that tnf blockade regulates il-10 expression directly in cd4 + effector t cells and does not require the presence of cd14 + monocytes or cd4 + cd25 high cd127 low tregs. (a) cd4 + t cells (n = 6) or cd45ro + enriched cd4 + t cells (n = 2) were cocultured 1:1 with autologous monocytes in the presence of anti-cd3 (100 ng/ml), without (control) or with anti-tnf mab (adalimumab, 1 μg/ml) or neutralizing antibodies to ifnγ or il-17 (10 μg/ml) or the appropriate isotype control antibodies (migg2a and migg2b, respectively). (b) cd45ro + enriched cd4 + t cells (n = 9) were cocultured 1:1 with autologous monocytes in the presence of anti-cd3 (100 ng/ml), without (control) or with anti-tnf mab (adalimumab, 1 μg/ml), tocilizumab (50 µg/ml), abatacept (5 µg/ml) or anti-il-1r1-blocking ab (2 μg/ml). (a,b) after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. data show frequencies of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + , or tnfα + cd4 + t cell subpopulations. data were analyzed by wilcoxon matched-pairs signed rank test with comparison to the appropriate control condition (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). in support of our findings that tnf blockade can exert its effects on cd4 + t cells in the absence of monocytes, we did not find evidence for a role of the monocyte-derived anti-inflammatory mediator il-27 (32) in mediating il-10 expression in the context of tnf blockade. this was demonstrated by the findings that tnf blockade did not induce il-27 secretion, rhil-27 enhanced il-10 secretion but did not result in increased percentages of il-10 + cd4 + t cells, and il-27 blockade did not impair the increased il-10 + cd4 + t cell frequencies brought about by tnf blockade ( figure s6 in supplementary material). il-10 partly contributes to tnf blockade-mediated il-10 regulation finally, we explored whether modulation of il-10 expression in cd4 + t cells by tnf blockade was dependent on il-10 signaling itself. neutralizing antibodies against il-10 and il-10r were added to cd4 + t cell/monocyte cocultures stimulated with anti-cd3 mab, in the presence or absence of tnf blockade, and intracellular cytokine expression was assessed after 3 days. blockade of il-10 signaling in the absence of adalimumab led to significantly reduced il-10 + frequencies within total cd4 + t cells and within il-17 + , ifnγ, or tnfα + subpopulations ( figure 7a) . when adalimumab and il-10/il-10r blocking mabs were added in combination to the cocultures, a decrease in il-10 + cd4 + t cell frequencies was observed as compared to the condition treated with adalimumab alone; however, an increase was still observed as compared to il-10/il-10r blockade alone. these data suggest that pathways additional to il-10 signaling could play a role in the regulation of il-10 expression by tnf blockade. indeed, when we tested whether addition of 6) showing frequencies of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + or tnfα + cd4 + t cell subpopulations. (c) after 3 days in culture but prior to re-stimulation, supernatants were harvested from cd4 + t cell only cultures (n = 15) and analyzed by enzyme-linked immunosorbent assay for il-10 secretion. data were analyzed by wilcoxon matched-pairs signed rank test (*p < 0.05, ****p < 0.0001). roberts et al. anti-tnf maintains il-10 + cd4 + /cd8 + t cells frontiers in immunology | www.frontiersin.org february 2017 | volume 8 | article 157 exogenous il-10 (10 ng/ml) could drive il-10 expression in cd4 + t cell/monocyte cocultures, we found that this by itself was not sufficient to increase il-10 + cell frequencies in total cd4 + t cells, or within il-17 + , ifnγ + , or tnfα + subpopulations. addition of rhil-10 even decreased the percentage of il-10 + cells in total cd4 + t cells and tnfα + cd4 + t cells (figure 7b) . the biological activity of the recombinant il-10 was validated by culturing freshly isolated monocytes with 0, 10, or 100 ng/ml rhil-10 for 20 h and confirming that rhil-10 addition resulted in increased cd14 and cd163 and reduced hla-dr expression by flow cytometry, in accordance with previous studies (33, 34) (n = 2, data not shown). we next investigated the regulation of il-10 in response to rhil-10 in both cd4 + and cd8 + t cells using whole pbmc cultures assessed for il-10 expression over a 5-day time course (figure s5 in supplementary material). we found that at earlier time points (18-24 h) , rhil-10 addition reduced il-10 + cd4 + t cell frequencies (n = 6, p = 0.03, wilcoxon matched-pairs signed rank test), while at later time points (66-72 and 114-120 h), rhil-10 slightly increased il-10 + cd4 + t cell frequencies (n = 6, p = 0.03, wilcoxon matched-pairs signed rank test). il-10 + cd8 + t cell frequencies remained very low throughout, regardless of rhil-10 addition. together, these data indicate that il-10 signaling contributes in part to tnf blockade-mediated regulation of il-10 expression in cd4 + t cells, but that other factors also play a role. the effects of exogenous il-10 on regulating il-10 expression in cd4 + t cells appear to be dependent on both time and in vitro culture conditions. here, we show that t cell stimulation in the presence of tnf blockade maintains the proportion of cells expressing the antiinflammatory cytokine il-10. this phenomenon is observed in total cd4 + and cd8 + t cells as well as within a variety of pro-inflammatory cytokine-expressing (il-17 + , ifnγ + , tnfα + , gm-csf + or il-4 + ) subpopulations. tnf blockade regulates il-10 expression whether cd4 + t cells are stimulated in the presence or absence of monocytes, and when an antigenic stimulus is used in place of monoclonal anti-cd3 stimulation. we found that blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated costimulation did not regulate il-10 expression in cd4 + or cd8 + t cell subpopulations. blocking antibodies to il-1r1 resulted in increased il-10 + cd4 + t cell frequencies when added to cd4 + t cell/monocyte cocultures, but this effect was not replicated in whole pbmc cultures. one explanation could be that additional cell types are present in pbmc cultures that can produce or respond to il-1. neither cd4 + cd25 high cd127 low tregs nor il-27 was required for tnf blockade to exert its il-10-promoting effects. our data confirm and extend our previous work showing that tnf blockade promotes il-10 expression in cd4 + t cells (20) and support other findings in the literature. a small study of ra patients found that frequencies of il-10 + pbmc were increased following treatment with infliximab (14) . however, in the three patients studied, it was not confirmed which cell subset(s) expressed il-10. ebert later showed that supernatants from cultures of either monocytes (adherent pbmc) or t cells (non-adherent, cd14 − cd20 − hla-dr − pbmc), contained increased il-10 following exposure to infliximab in vitro (35) . antiga et al. demonstrated increased percentages of il-10 + cd4 + t cells in t cell blasts isolated from skin lesions of psoriasis patients, following treatment with etanercept (36), and pb il-10 + cd4 + t cells were also increased in posterior uveitis patients following treatment with a p55 tnfα receptor fusion protein (37) . earlier studies assessing mouse transgenic t cells (38) or human cd4 + t cell clones (39) also found that tnf blockade led to increased il-10 production in cell culture supernatants. in further support of our data, boks et al. showed that blockade of tnfα during in vitro priming of naïve cd4 + figure 7 | il-10 expression in cd4 + t cells is inhibited by il-10 blockade, in both the absence and presence of tnf blockade. (a) cd45ro + enriched cd4 + t cells were cocultured 1:1 with autologous monocytes in the presence of anti-cd3 (okt3,100 ng/ml), with or without anti-tnf mab (adalimumab, 1 μg/ml), with or without neutralizing antibodies to il-10 and il-10r (both 10 μg/ml). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. box-whisker plots show frequency of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + or tnfα + subpopulations; whiskers show minimum to maximum values. data were analyzed by repeated measures anova, with comparison between selected conditions by sidak's multiple comparison test (n = 9). (b) cd45ro + enriched (filled symbols, n = 5) or bulk (open symbols, n = 1) cd4 + t cells were cocultured 1:1 with autologous monocytes and stimulated with anti-cd3 mab (100 ng/ml), with or without recombinant human il-10 (rhil-10; 10 ng/ml). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. data show frequencies of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + , or tnfα + subpopulations and were assessed by wilcoxon matched-pairs signed rank test (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). t cells with tolerogenic dcs favored the development of il-10 + cd4 + t cells (40) . thus, tnfi-mediated regulation of il-10 in cd4 + t cells has been indicated by a growing number of in vitro and ex vivo studies. the presence of il-10-expressing cd8 + t cells has been reported in several mouse models of infection, including acute influenza infection (41) , chronic mycobacterium tuberculosis infection (42) , and coronavirus-induced encephalitis (43) . furthermore, il-10 + cd8 + t cells have been found in patients with hiv (44) and chronic hepatitis c infection (45) . il-10 expression in cd8 + t cells can be induced by il-4, either alone (46) or in combination with il-12 (47) , or dexamethasone (48) . however, to the best of our knowledge, this is the first report showing that tnf blockade can regulate il-10 expression in cd8 + t cells. il-27 can stimulate il-10 production by various effector cd4 + t cell subsets (32, 49, 50) . our data suggest however that the capacity of tnf blockade to affect il-10 expression in cd4 + t cells is not dependent on il-27. in agreement with other work on human t cells (51, 52) , we found that addition of recombinant il-27 to anti-cd3/cd28-stimulated cd4 + t cells resulted in increased il-10 secretion, although this was not strictly dosedependent. however, in contrast to these other studies, which used naïve cd4 + t cells, our analysis of intracellular cytokine expression did not demonstrate increased il-10 + cd4 + t cell frequencies after 3 days in culture with il-27. this is possibly anti-tnf maintains il-10 + cd4 + /cd8 + t cells frontiers in immunology | www.frontiersin.org february 2017 | volume 8 | article 157 due to different kinetics of il-10 expression in a bulk cd4 + t cell population. it is of note that although in cd4 + t cell/monocyte cocultures il-10 blockade led to reduced il-10 + frequencies in both the presence and absence of tnf blockade, il-10 addition by itself was not sufficient to drive il-10 expression in cd4 + t cells. these data are in line with previous work showing that il-10 is necessary but not sufficient to enhance development of il-10-producing tr1 cells following in vitro differentiation in the presence of vitamin d3 plus dexamethasone (53) . however, when added to anti-cd3-stimulated whole pbmc cultures, rhil-10 did slightly increase il-10 + cd4 + t cell frequencies after 3 and 5 days in culture, indicating that the regulation of il-10 expression in cd4 + t cells is dependent on in vitro culture conditions. in inflammatory diseases, such as ra, the normal balance of immune regulation is disturbed and t cell-derived proinflammatory mediators can contribute to disease pathogenesis in an uncontrolled manner. this study indicates that a potentially immunoregulatory il-10 + phenotype is broadly maintained in effector t cells following exposure to tnf blockade, which may represent an underappreciated mechanism of action for this widely used therapeutic strategy. we previously provided evidence that the il-10 secreted by il-17 + cd4 + t cells following tnf blockade is biologically active (20) ; however, further work is required to investigate whether the increase in il-10 + frequencies across different t cell subpopulations is functionally relevant in the context of inflammatory disease. additional insights into the underlying molecular mechanisms via which tnf blockade maintains il-10 expression could identify potential targets for novel therapeutic strategies. author contributions cr, ld, and vf designed and performed experiments and analyzed and interpreted data. lt and he conceived the study, contributed to experimental design, and interpreted data. cr and lt wrote and revised the manuscript. all authors edited the manuscript. development of anti-tnf therapy for rheumatoid arthritis anti-tnf biologic agents: still the therapy of choice for rheumatoid arthritis direct comparison of treatment responses, remission rates, and drug adherence in patients with rheumatoid arthritis treated with adalimumab, etanercept, or infliximab: results from eight years of surveillance of clinical practice in the nationwide danish danbio registry inefficacy of infliximab in primary sjögren's syndrome: results of the randomized, controlled trial of remicade in primary sjögren's syndrome (tripss) lack of efficacy of etanercept in sjögren syndrome correlates with failed suppression of tumour necrosis factor α and systemic immune activation the university of british columbia ms/mri analysis group. tnf neutralization in ms: results of a randomized, placebo-controlled multicenter study biologics-induced autoimmune diseases differences in binding and effector functions between classes of tnf antagonists comparisons of affinities, avidities, and complement activation of adalimumab, infliximab, and etanercept in binding to soluble and membrane tumor necrosis factor mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor α-expressing cells: comparison among infliximab, etanercept, and adalimumab mechanism of action of certolizumab pegol (cdp870): in vitro comparison with other anti-tumor necrosis factor alpha agents tumour necrosis factor alpha blockade impairs dendritic cell survival and function in rheumatoid arthritis anti-tnf-alpha therapy induces a distinct regulatory t cell population in patients with rheumatoid arthritis via tgf-beta compromised function of regulatory t cells in rheumatoid arthritis and reversal by anti-tnfα therapy anti-tnf drives regulatory t cell expansion by paradoxically promoting membrane tnf-tnf-rii binding in rheumatoid arthritis number and phenotype of rheumatoid arthritis patients' cd4+cd25hi frontiers in immunology | www regulatory t cells are not affected by adalimumab or etanercept influence of anti-tumor necrosis factor therapy (adalimumab) on regulatory t cells and dendritic cells in rheumatoid arthritis critical role for tnf in the induction of human antigen-specific regulatory t cells by tolerogenic dendritic cells pathogenic t cells have a paradoxical protective effect in murine autoimmune diabetes by boosting tregs tnf-α blockade induces il-10 expression in human cd4+ t cells the interplay between monocytes/ macrophages and cd4+ t cell subsets in rheumatoid arthritis th17 and regulatory t cell balance in autoimmune and inflammatory diseases proinflammatory cytokines in the pathogenesis of inflammatory bowel diseases t cells in multiple sclerosis and experimental autoimmune encephalomyelitis il-17+ cd8+ t cells: differentiation, phenotype and role in inflammatory disease cd8(+) t cells in human autoimmune arthritis: the unusual suspects il-17+cd8+ t-cells are enriched in the joints of patients with psoriatic arthritis and correlate with disease activity and joint damage progression cd8+ t cells in the lesional skin of atopic dermatitis and psoriasis patients are an important source of ifn-γ, il-13, il-17, and il-22 overrepresentation of il-17a and il-22 producing cd8 t cells in lesional skin suggests their involvement in the pathogenesis of psoriasis tumor necrosis factor antagonist mechanisms of action: a comprehensive review significant associations of antidrug antibody levels with serum drug trough levels and therapeutic response of adalimumab and etanercept treatment in rheumatoid arthritis interleukins 27 and 6 induce stat3-mediated t cell production of interleukin 10 il-10) and viral il-10 strongly reduce antigen-specific human t cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression human monocytes express cd163, which is upregulated by il-10 and identical to p155 infliximab and the tnf-α system etanercept downregulates the th17 pathway and decreases the il-17+/il-10+ cell ratio in patients with psoriasis vulgaris anti-tnfα therapy modulates the phenotype of peripheral blood cd4+ t cells in patients with posterior segment intraocular inflammation the role of tnf alpha and related cytokines in the development and function of the autoreactive t-cell repertoire chronic exposure to tumor necrosis factor (tnf) in vitro impairs the activation of t cells through the t cell receptor/cd3 complex; reversal in vivo by anti-tnf antibodies in patients with rheumatoid arthritis inhibition of tnf receptor signaling by anti-tnfα biologicals primes naïve cd4+ t cells towards il-10+ t cells with a regulatory phenotype and function effector t cells control lung inflammation during acute influenza virus infection by producing il-10 clonal expansions of cd8+ t cells with il-10 secreting capacity occur during chronic mycobacterium tuberculosis infection highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirus-induced encephalitis hiv-specific il-10-positive cd8+ t cells are increased in advanced disease and are associated with decreased hiv-specific cytolysis intrahepatic virus-specific il-10-producing cd8 t cells prevent liver damage during chronic hepatitis c virus infection il-4 induces a suppressive il-10-producing cd8+ t cell population via a cdkn2a-dependent mechanism cytokine-induced il-10-secreting cd8 t cells represent a phenotypically distinct suppressor t-cell lineage glucocorticoids drive human cd8(+) t cell differentiation towards a phenotype with high il-10 and reduced il-4, il-5 and il-13 production suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated t cells a dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory t cells il-27 is a key regulator of il-10 and il-17 production by human cd4+ t cells il-27 induces the differentiation of tr1-like cells from human naive cd4+ t cells via the phosphorylation of stat1 and stat3 vitro generation of interleukin 10-producing regulatory cd4+ t cells is key: cord-297960-4x1j0iqg authors: bösl, korbinian; ianevski, aleksandr; than, thoa t.; andersen, petter i.; kuivanen, suvi; teppor, mona; zusinaite, eva; dumpis, uga; vitkauskiene, astra; cox, rebecca j.; kallio-kokko, hannimari; bergqvist, anders; tenson, tanel; merits, andres; oksenych, valentyn; bjørås, magnar; anthonsen, marit w.; shum, david; kaarbø, mari; vapalahti, olli; windisch, marc p.; superti-furga, giulio; snijder, berend; kainov, denis; kandasamy, richard k. title: common nodes of virus–host interaction revealed through an integrated network analysis date: 2019-10-04 journal: front immunol doi: 10.3389/fimmu.2019.02186 sha: doc_id: 297960 cord_uid: 4x1j0iqg viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses are one of the major causes of acute and chronic infectious diseases and thus a major contributor to the global burden of disease. several studies have shown how viruses have evolved to hijack basic cellular pathways and evade innate immune response by modulating key host factors and signaling pathways. a collective view of these multiple studies could advance our understanding of virus-host interactions and provide new therapeutic perspectives for the treatment of viral diseases. here, we performed an integrative meta-analysis to elucidate the 17 different host-virus interactomes. network and bioinformatics analyses showed how viruses with small genomes efficiently achieve the maximal effect by targeting multifunctional and highly connected host proteins with a high occurrence of disordered regions. we also identified the core cellular process subnetworks that are targeted by all the viruses. integration with functional rna interference (rnai) datasets showed that a large proportion of the targets are required for viral replication. furthermore, we performed an interactome-informed drug re-purposing screen and identified novel activities for broad-spectrum antiviral agents against hepatitis c virus and human metapneumovirus. altogether, these orthogonal datasets could serve as a platform for hypothesis generation and follow-up studies to broaden our understanding of the viral evasion landscape. viruses continue to be a major contributor to the global burden of disease through acute and chronic infections that cause substantial economic impact in addition to increased mortality and morbidity (1) . despite the tremendous improvement in the understanding of the antiviral immune response and the availability of therapeutics, existing and emerging viral diseases are an ever-growing problem, particularly in developing countries. development of antiviral resistance of hepatitis c virus (hcv), influenza a virus (iav), herpes simplex virus (hsv), human cytomegalovirus (hcmv), human immunodeficiency virus (hiv), and other viruses is a major concern (2-4). one of the main reasons for increasing resistances is the accumulation of mutations in the viral genome caused by multiple factors including the polymerase infidelity (5, 6) . therefore, the world health organization (who) and the united nations have urged for better control of viral diseases. this has led to turning the focus on the host for therapeutic intervention. targeting the host factors has been proven to be useful for restricting viral infections (7, 8) . the small molecule ccr5 inhibitor maraviroc and the anti-cd4 monoclonal antibody ibalizumab are examples of successful use of host-directed therapies for combating hiv in clinic (9) (10) (11) . viruses have evolved to evade the host antiviral response at various stages starting from viral sensing to antiviral proinflammatory responses (12) (13) (14) . multiple studies attempted to understand global principles of the viral evasion employed by various viruses, including dengue virus (denv), ebola virus (ebov), iav, and hiv (15) (16) (17) (18) (19) (20) . global systems-level approaches including functional rnai screens, interactome mapping technologies such as affinity-purification mass spectrometry (ap-ms), quantitative proteomics, and crispr/cas9-based screens have provided unparalleled details and insights into the dynamics of host proteome in immune cells (21) (22) (23) (24) , host-virus interactome (15-17, 25, 26) , and also identified important host dependency factors of various viruses (25, 27, 28) . meta-analyses of such high-dimensional datasets have been crucial for identifying novel host factors as drug targets such as ubr4 in iav infection (29) . moreover, some of these factors represent drug targets for multiple viruses (30) . we hypothesized that combining a meta-analysis of host-virus protein-protein interactions of multiple viruses and functional rnai screens would provide novel insights for developing broadspectrum antiviral strategies. for this, we assembled a host-virus protein-protein interactome of 5,781 host-virus interactions (hereafter referred to as "hvppi") covering 183 viral proteins from 17 different viruses and 2,381 host proteins. we performed extensive bioinformatics and network analysis and integrated this dataset with genome-wide or druggable-genome rnai screen data from published studies. this resulted in the assembly of critical nodes of viral evasion and identification of core cellular processes and druggable nodes that were verified by a drug re-purposing screen using broad-spectrum antivirals. host-virus protein-protein interactions were downloaded from published studies (15-17, 25, 31-34) we performed gene-set enrichment analysis using david bioinformatics resources [version 6.8, (38) ]. for all enrichment analysis, a p-value cutoff of ≤0.01 was used as significant. protein disorder analysis was performed using iupred2a software. we used the offline version with protein sequences downloaded from uniprot. statistical analysis of disordered region distribution was performed by kolmogorov-smirnov test in r statistical environment. annotation of human proteins was mapped from uniprot id to ensembl using ensdb.hsapiens.v86. the index of subcellular localization of interaction partners of single viral proteins was calculated for all viral proteins with ≥ five host targets. localization of host targets was mapped using compartments (39) , filtered for a minimum evidence score of 3 in the knowledge channel, excluding non-experimental based localization predictions. evidence for all protein was subsequently divided by the absolute number of host-targets per viral protein. multiple sequence alignment was performed using clustal x [version 2.0, (40)]. genome-wide rnai screen data for hcv (41) and hpv18 (42) , through genomernai database [(43)-gr00197], as well as druggable rnai screen data for hpv16 (44) , vacv (45) , and sv40 (46) were integrated in the existing network as z-scores. drug-gene interaction data was downloaded from dgidb and drugbank. the identifiers were mapped to uniprot ids and then compared with hvppi. for the hmpv nl/1/00-gfp screen, approximately 4×10 4 human retinal pigment epithelial (rpe) cells were seeded per well in 96-well plates. human non-malignant rpe cell line represents excellent model system for studying replication of many viruses including respiratory (30, 47, 48) . the cells were grown for 24 h in dmem-f12 medium supplemented with 10% fbs, 0.35% nahco 3 , and 100 µg/ml streptomicine and 100 iu/ml penicillin. the medium was replaced with virus growth medium (vgm) containing 0.2% bovine serum albumin (bsa), 2 mm l-glutamine, 0.35% nahco 3 , and 1 µg/ml l-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin in dmem-f12. hcv screen-associated cell culture conditions are described in kim et al. (49) . the compounds were added to the cells in 3fold dilutions at seven different concentrations starting from 50 µm. no compounds were added to the control wells. the cells were mock-or virus-infected at a multiplicity of infection (moi) of one. after 48 h of infection, the medium was removed from the cells. to monitor cell viability, celltiter-glo reagent was added (30 µl per well). this assay quantifies atp, an indicator of metabolically active living cells. the luminescence was measured with a plate reader. to determine compound efficacy, hmpv nl/1/00-mediated gfp expression was measured. the half-maximal cytotoxic concentration (cc 50 ) and the half-maximal effective concentration (ec 50 ) for each compound were calculated after non-linear regression analysis with a variable slope using graphpad prism software version 7.0a. the relative effectiveness of the drug was quantified as the selectivity index (si = cc 50 ec 50 ). cytotoxicity and antiviral activity of the compounds against gfp-expressing hcv in huh-7.5 cells was determined as previously described (49). to provide new and critical insights into viral evasion mechanisms we performed a comprehensive meta-analysis of the host-virus interaction landscape. we assembled the hostvirus protein-protein interaction data ("hvppi") from published studies ( figure 1a ) (15-17, 25, 31-34) . this dataset comprised of protein-protein interactions from two different types of experimental methods-affinity purification mass spectrometry (ap-ms) and yeast two-hybrid screens (y2h). altogether, this combined dataset includes 183 viral proteins, 2,381 host proteins, and 5,781 protein-protein interactions ( figure 1b and figure s1 ). many interactome networks including yeast and human are scale-free networks, where a large portion of the nodes (e.g., a protein in the network) have few interactions and only a few nodes have large number of interactions. the latter are often referred to as "hubs" which are crucial in keeping the network intact (34) . we performed network topology analysis to infer the properties of the host proteins targeted by the viral proteins in the context of the human protein interactome. we considered two important parameters-relative betweenness centrality (which reflects the amount of information that passes through this protein in the human interactome) and degree (number of binding partners in the human interactome) of the host proteins targeted by each virus. the targets of all the viruses showed higher betweenness centrality and degree as compared to an average protein in the human interactome (figures 1c,d) . this shows that viruses, by targeting "hubs" and proteins that serves as key communication nodes, have evolved the best way to disrupt the scale-free human interactome. this topological property thereby shows how viruses having small genomes achieve the maximal effect in rewiring the human interactome to benefit viral survival and replication. our analysis is in agreement with several previous studies, which have highlighted this property (15, 16, 31, 50, 51) . we propose that this could be a general principle for all viruses. proteins typically fold into stable three-dimensional structures that mediate specific functions. in addition, there are substructures in proteins termed "intrinsically disordered regions (idrs)" which lack stable structures under normal physiological conditions. idrs are required for multiple cellular functions even though they lack these defined structures (52) . many studies have highlighted the presence of such idrs in viral proteins (53) (54) (55) , such as e6 from human papilloma virus, that are crucial for hijacking the cellular machinery. we analyzed the host proteins from the hvppi for presence of idrs using the prediction software iupred (56) . it is estimated that the human proteome more than 100,000 short linear binding motifs in idrs (57, 58) . proteins with idrs are often signaling hubs and might form dynamic complexes with other proteins through specific motif based interactions (58) . we found a statistically significant enrichment (p-value < 6.246 × 10 −06 ) of idrs in the host proteins targeted by viruses (figure 2a and figure s2 ). we then identified the subnetwork in the hvppi which contained the top host targets with high disorderness score ( figure 2b) . the top five proteins with large idrs include cd44 antigen (cd44), serine/arginine repetitive matrix protein 2 (srrm2), myristoylated alaninerich c-kinase substrate (marcks), bag family molecular chaperone regulator 3 (bag3), and mitochondrial antiviralsignaling protein (mavs; figure 2c ). cd44 is a marker of exhausted cd8+ t cells (59) and replication of hcv in t cells was shown to decrease cell proliferation by inhibiting cd44 expression and signaling (60) . srrm2 is a serine/arginine-rich protein involved in rna splicing (61) . srrm2 is differentially phosphorylated in hiv-1 infected cells and absence of srrm2 lead to increased hiv-1 gene expression, since it regulates the splicing of hiv-1 (62) . in the hvppi, srrm2 is targeted by multiple viral proteins including the tat protein from hiv-1. tat protein has an important role in the stimulation of the transcription of the long terminal repeat (ltr) (63) . in addition, ns1 protein from influenza b virus has also been reported to interact with srrm2 (64) . proteins of the marcks family are involved in a range of cellular processes including cell adhesion and migration (65) . marcks is a negative regulator of lipopolysaccharide (lps)-induced toll-like receptor 4 (tlr4) signaling in mouse macrophages (66) . mavs is an adaptor protein in the rig-i signaling pathway involved in the sensing of rna. ablasser et al. (67) reported that double-stranded dna serves as a template for rna polymerase ii and is transcribed into a 5 ′ triphosphate containing double-stranded rna, which activates the rig-i signaling pathway. in the hvppi, mavs is targeted by several proteins from dsdna viruses such as ebv and hpv. altogether, our analysis shows that the idr-high part of the human proteome is an essential part of the viral evasion strategy and some of the selected targets highlighted here could show novel insights into the viral evasion mechanisms. however, the very flexible protein structure of disordered proteins also makes them also difficult to target with drugs. to assess the signaling pathways and cellular processes within the hvppi, we identified highly connected subnetworks within hvppi network. we constructed a host-host interaction network based on the host targets in the hvppi and identified a number of highly connected subnetworks/clusters (figure 3) . we then performed a gene-set enrichment analysis of significantly enriched biological processes. we found one or more enriched processes for each of this subnetwork including core cellular processes such as proteasome, spliceosome, protein translation, protein/rna transport, and cell cycle. next we listed the viruses that target one or more of these processes, and found that almost all the core pathways and processes are targeted by all the 17 viruses that are part of the hvppi (figure s3 ). this analysis highlights the core components of the cellular process subnetworks which are targeted as part of the viral evasion strategies and thus could be broad-spectrum antiviral hot-spots from a therapeutic point of view. given that the viral proteins were interacting with a large number of host proteins, we analyzed the sub-cellular location of the host proteins. we performed gene-set enrichment analysis of sub-cellular localization information provided by uniprot database. we binned the localization into 11 compartments and estimated the percent of host proteins in a given compartment as compared to the total number of host proteins targeted by a given figure 3 | clusters of hvppi involved in core cellular processes. network view of the "clusters" or highly-connected sub-networks and their associated cellular processes. each cluster is marked in a unique color. virus. we found that the viral targets were distributed across multiple subcellular compartments with cytoplasm being the most common ( figure s4a ). the hvppi includes two different strains of iav-pr8 (h1n1) and udorn (h3n2). the subcellular localization analysis showed that both strains were enriched for nuclear proteins. non-structural protein 1 (ns1) from both the strains had the highest number of nuclear targets but their targets were very different ( figure 4a) . ns1 of udorn was enriched for a large number of histones as compared to ns1 of pr8 that had large number of heterogeneous nuclear ribonucleoproteins (hnrnps), such as hnrnpu−a known restriction factor for many viruses. this corroborates with the observation that ns1 protein has short linear histone mimicry motifs that can suppress the host antiviral response (68) . in our analysis, we found that it is ns1 of udorn that has a histone mimicry motif "arsk" (figure s4b) . similarly, hpv11 and hpv18 e5 proteins interact more often with host proteins located in the endoplasmic reticulum (er). we found both common and specific subsets of er proteins targeted by the e5 protein ( figure 4b ). hpv18 e5 protein er targets were enriched for phospholipid biosynthesis as well as gpi anchor related proteins, such as phosphatidylinositol glycan anchor biosynthesis class s/t/u (pigs, pigt, and pigu), glycosylphosphatidylinositol anchor attachment 1 (gpaa1) and phosphatidylserine synthase 2 (ptdss2). hpv11 e5 protein er targets were enriched for er-associated ubiquitindependent protein catabolism involving host proteins such as er degradation enhancing alpha-mannosidase-like protein 3 (edem3) and er lipid raft associated 1 (erlin1). er targets common to hpv18 and hpv11 e5 protein were enriched for unfolded protein response, n-linked glycosylation and protein folding involving host proteins such as srp receptor alpha/beta subunit (srpra/srprb) and catalytic subunits of the oligosaccharyltransferase complex (stt3a and stt3b). two independent crispr/cas9 screening studies identified multiple er associated components including stt3a and stt3b as host factors for denv, zika virus (zikv) and japanese encephalitis virus (jev) (27, 28) . the non-canonical function of stt3a and stt3b is required for denv replication and that ns1 protein of denv interacts with these proteins (28) . our orthogonal approach can lead to the identification of critical host factors, and similar functions of er components, such as stt3a and stt3b, are used by hpv11 and hpv18 as well. thus, targeting the non-canonical function of stt3a and stt3b could be a broad antiviral strategy. overall, the enrichment analysis clearly shows that there is commonality and specificity in the subcellular targets of the viral proteins and that detailed interrogation of these targets can give vital clues into the viral evasion mechanisms. rnai screens have been a powerful high-throughput method to identify various cellular functions, including for identification of host restriction factors of viruses (69) . in order to explore the functional relevance of the host targets in the hvppi, we integrated it with five published rnai screens that performed genome-wide or druggable-genome-wide rnai screens for identifying host factors of hcv (41), hpv18 (43), hpv16 (44), sv40 (46) , and vacv (45) . we found that host targets from the hvppi were spread across the spectrum of genes with proviral as well as antiviral phenotype (figure s5) , thus showing that targeting of the host protein by the virus could lead into any direction that favors the virus. we then investigated the top 50 proviral genes that are also targeted by the viral proteins as seen in the hvppi. we identified 42 host proteins ( figure 5a ) that were significantly enriched for coatomer protein complex 1 (copi), protein translation/transport and proteasome ( figure 5b) . this further substantiates the findings from the earlier section on the core cellular processes targeted by the viruses. network analysis of these top hits showed high level on connectivity and crosstalk-for example between the translation and proteasome machinery (figure 5c ). vesicle carriers are involved in the transport of membranes and proteins. copi system is one of the three vesicular carrier systems that is involved in the early secretory pathway (70) (71) (72) . moreover, it has been pointed out that there is a strong similarity between vesicular transport and viral transport [viral entry to budding process, (73) ]; thus making copi system important for the viral life cycle. in addition to the findings of the present study, sirna-based silencing of copi lead to a decrease in entry and subsequent gene expression of iav, vsv, lcmv, and hpiv3 and disruption of the copi complex inhibited the production of infectious progeny virus (74, 75) . copi coatomer inactivation results in a direct decrease of vsv attachment and uptake, but not for membrane fusion or rnp release; however the direct mechanism remains unclear (76) . altogether, these top hits including the copi system could serve as targets for developing therapeutic antiviral intervention strategies for a broad group of viruses. our analyses pointed out that viral evasion mechanism observed in one virus could also be relevant for other viruses. to test this, we obtained known drug-gene interactions from dgidb (77). we selected 28 investigational/experimental/approved antivirals compounds (30) which had dgidb annotated targets that are part of the hvppi. we added 12 agents as controls (table s1 and figure s6) . we tested 40 broad-spectrumantivirals against gfp-expressing human metapneumovirus (hmpv) nl/1/00 strain (78) . seven different concentrations of the compounds were added to hmpv or mock-infected cells. hmpv-induced gfp expression and cell viability was measured and after 48 h to determine compound efficiency and toxicity. after the initial screening, we identified five compounds, which lowered gfp-expression without detectable cytotoxicity (with si > 3). we repeated the experiment with these compounds. the experiments revealed novel activity of azacytidine, lopinavir, nitazoxanide, itraconazole, and oritavancin against hmpv ( figure s7a and table 1, table s2 ). similarly, we examined toxicity and antiviral activity of broad-spectrum-antivirals against gfp-expressing hcv in huh-7.5 cells using previously described procedures (49) . our test identified azithromycin, cidofovir, oritavancin, dibucaine, gefitinib, minocycline, and pirlindole mesylate as novel anti-hcv agents with si > 3 ( figure s7b and table 1, table s2 ). in summary, our metaanalysis approach of the hvppi could provide novel and faster approaches for the re-purposing of existing drugs as antiviral agents. using integrative analysis of orthogonal datasets our study provides a comprehensive view of viral evasion mechanisms. in particular, our analysis of the hvppi network revealed that all the viruses have evolved to target proteins that are central and have strong control over the human interactome. host proteins targeted by viruses contain a high proportion of intrinsically disordered regions. we identified the core cellular processes and associated proteins that are targeted by all viruses. detailed comparative analysis of the subcellular localization of the host proteins showed commonality and specificity both between viral proteins from different strains of the same virus; and between viruses. integrating hvppi with functional rnai screens showed that 28% of the hvppi are host factors of one or more virus. hvppi data-based drug re-purposing screen identified novel activities for various broad-spectrum antivirals against hmpv and hcv. this unique dataset can be used for further detailed interrogation of the mechanisms behind viral evasion. this could serve as a starting point for identifying novel host targets and generating hypothesis in the context of viral evasion and development of pan-viral therapeutic intervention strategies. the methods described here also provide unique ways of dissecting the orthogonal datasets. various analyses from this study have highlighted the existence on pan-viral evasion points that could be utilized for the development of host-directed antiviral therapies. it is also intriguing to see that there is commonality and specificity at the level of sub cellular localization of the viral targets. our analyses have underlined some salient features in the context of iav, hpv, denv, and hcv. further detailed analysis in this context along with protein sequence features, such as short linear motifs [slims; (79) ] would provide novel insights as well as deeper understanding of how small sequence features are involved in the hijacking of the host machinery. integration of such data with known drug-gene interactions provides a clear estimate of the druggable proportion in the hvppi. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. our meta-analysis approach of the hvppi could provide novel avenues of re-purposing existing drugs for antiviral targeting strategies. to prove the concept, we tested 40 bsas against hmpv, hcv, sindbis virus (sinv), cytomegalovirus (cmv), and hepatitis b virus (hbv). importantly, 28 bsas have dgidb annotated targets that are part of the hvppi, whereas 12 were used as controls. these safein-man drugs have already been used as investigational agents or experimental drugs in different virus infections (table s2) . we demonstrated novel antiviral effects of azacytidine, itraconazole, lopinavir, nitazoxanide, and oritavancin against hmpv, as well as cidofovir, dibucaine, azithromycin, gefitinib, minocycline, oritavancin, and pirlindole against hcv. azithromycin, is an fda-approved antibiotic of the macrolide family. it is also an investigational agent against rsv and experimental agent against ebov, hrv-a, zikv, and rsv. cidofovir is an fda-approved anti-cmv drug. it is also investigational agent against adv, bkv, hpv, hsv-1, hsv-2, and experimental drug against b19v. dibucaine is an fdaapproved amide local anesthetic. in addition, it is experimental anti-hev-a, hev-b, hev-d, and ebov agent. gefitinib is an fda approved anticancer drug. it has also antiviral activity against bkv, cmv, and vacv. minocycline is a broad-spectrum antibiotic and experimental anti-denv, hiv-1, and wnv agent. oritavancin is a semisynthetic glycopeptide antibiotic used for the treatment of gram-positive bacterial skin infections. it also inhibits ebov, mers-cov, and sars-cov infections. pirlindole is an antidepressant, which is also experimental anti-hev-a, hev-b, and hev-d agent. azacitidine is a chemical analog of cytidine, which is used in the treatment of myelodysplastic syndrome. it is also an experimental anti-adv, the measurements were repeated three times (p < 0.05). fluav, rvfv, hiv-1, and hiv-2 agent. itraconazole is an antifungal medication. it is also used as experimental anti-hev-b, hrv-b, hrv-a, par-a3, and safv agent. nitazoxanide is a broad-spectrum antiparasitic drug, which is also investigational agent against fluav and hcv and experimental anti-chikv, rsv, hbv, hiv-1, vacv, rv, jev, mers-cov, nov, ruv, and zikv agent. lopinavir is an fda-approved antiretroviral of the protease inhibitor class. it is also investigational anti-mers-cov and experimental anti-zikv agent (table s2 ). in addition to inhibition of viral proteases (table s2) , lopinavir was reported to induce host rnasel production in infected and non-infected cells (80) . rnasel is endoribonuclease that is a part of interferon (ifn) antiviral response, which is the most critical node of virus-host interactions. although, the antiviral mechanisms of action of other compounds are still unknown, these agents could inhibit steps of viral infections, which precede reporter protein expression from viral rna. in summary, our results indicate that existing bsas could be re-purposed to other viral infections. to further expand a spectrum of their activities, these bsas could be tested against other viruses. re-purposing these and other safe-inman antiviral therapeutics could save resources and time needed for development of novel drugs to quickly address unmet medical needs, because safety profiles of these agents in humans are available. effective treatment with broad-spectrum-antivirals may shortly become available, pending the results of further pre-clinical studies and clinical trials. this, in turn, means that some broad-spectrum-antivirals could be used for rapid management of new or emerging drug-resistant strains, as well as for first-line treatment or for prophylaxis of acute virus infections or for viral co-infections. the most effective and tolerable compounds could expand the available therapeutics for the treatment of viral diseases, improving preparedness and the protection of the general population from viral epidemics and pandemics. all datasets used for this study are accessible as stated in the materials and methods section 2.1. we thank christian sinzger and group for the egfp-expressing tb40e cmv strain. we thank vironovative and erasmus mc for the gfp-expressing hmpv nl/1/00 strain. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02186/full#supplementary-material redefining chronic viral infection antiviral drug resistance as an adaptive process emerging virus diseases: can we ever expect the unexpected? emerg microbes infect herpes simplex virus resistance to acyclovir and penciclovir after two decades of antiviral therapy mechanisms of viral mutation complexities of viral mutation rates host-directed therapies for infectious diseases: current status, recent progress, and future prospects host-directed therapies for bacterial and viral infections maraviroc: a ccr5-receptor antagonist for the treatment of hiv-1 infection determination of hiv tropism and its use in the clinical practice ibalizumab: an anti-cd4 monoclonal antibody for the treatment of hiv-1 infection viral evasion and subversion of patternrecognition receptor signalling when the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases ten strategies of interferon evasion by viruses interpreting cancer genomes using systematic host network perturbations by tumour virus proteins global landscape of hiv-human protein complexes comparative flavivirus-host protein interaction mapping reveals mechanisms of dengue and zika virus pathogenesis protein interaction mapping identifies rbbp6 as a negative regulator of ebola virus replication multi-omics studies towards novel modulators of influenza a virus-host interaction quantitative temporal viromics: an approach to investigate host-pathogen interaction high-definition analysis of host protein stability during human cytomegalovirus infection reveals antiviral factors and viral evasion mechanisms a time-resolved molecular map of the macrophage response to vsv infection a temporal proteomic map of epstein-barr virus lytic replication in b cells a physical and regulatory map of host-influenza interactions reveals pathways in h1n1 infection human host factors required for influenza virus replication a crispr screen defines a signal peptide processing pathway required by flaviviruses genetic dissection of flaviviridae host factors through genome-scale crispr screens meta-and orthogonal integration of influenza "omics" data defines a role for ubr4 in virus budding novel activities of safe-in-human broad-spectrum antiviral agents hepatitis c virus infection protein network a physical interaction network of dengue virus and human proteins analysis of vaccinia virus-host protein-protein interactions: validations of yeast two-hybrid screenings interactome networks and human disease biogrid: a general repository for interaction datasets cytoscape: a software environment for integrated models of biomolecular interaction networks an automated method for finding molecular complexes in large protein interaction networks systematic and integrative analysis of large gene lists using david bioinformatics resources compartments: unification and visualization of protein subcellular localization evidence clustal w and clustal x version 2.0 a functional genomic screen identifies cellular cofactors of hepatitis c virus replication genome-wide sirna screen identifies smcx, ep400, and brd4 as e2-dependent regulators of human papillomavirus oncogene expression genomernai: a database for cell-based and in vivo rnai phenotypes, 2013 update large scale rnai reveals the requirement of nuclear envelope breakdown for nuclear import of human papillomaviruses rnai screening reveals proteasome-and cullin3-dependent stages in vaccinia virus infection singlecell analysis of population context advances rnai screening at multiple levels obatoclax, saliphenylhalamide, and gemcitabine inhibit influenza a virus infection saliphenylhalamide and gemcitabine inhibit zika virus infection in vitro and differentially affect cellular signaling, transcription and metabolism benzothiazepinecarboxamides: novel hepatitis c virus inhibitors that interfere with viral entry and the generation of infectious virions a review on computational systems biology of pathogen-host interactions structural principles within the human-virus proteinprotein interaction network intrinsically disordered proteins and intrinsically disordered protein regions structural disorder in viral proteins understanding the role of intrinsic disorder of viral proteins in the oncogenicity of different types of hpv how do intrinsically disordered viral proteins hijack the cell? iupred2a: context-dependent prediction of protein disorder as a function of redox state and protein binding a million peptide motifs for the molecular biologist intrinsically disordered proteins in cellular signaling and regulation molecular and cellular insights into t cell exhaustion hepatitis c virus infection of t cells inhibits proliferation and enhances fas-mediated apoptosis by down-regulating the expression of cd44 splicing variant 6 the srm160/300 splicing coactivator subunits quantitative phosphoproteomics reveals extensive cellular reprogramming during hiv-1 entry the hiv-1 tat protein has a versatile role in activating viral transcription human interactome of the influenza b virus ns1 protein cross-talk unfolded: marcks proteins marcks as a negative regulator of lipopolysaccharide signaling rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate suppression of the antiviral response by an influenza histone mimic the use of rnai-based screens to identify host proteins involved in viral replication the copi system: molecular mechanisms and function bi-directional protein transport between the er and golgi copi-mediated transport viruses as vesicular carriers of the viral genome: a functional module perspective dissecting the role of copi complexes in influenza virus infection rnai screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand rna viruses genetic inactivation of copi coatomer separately inhibits vesicular stomatitis virus entry and gene expression dgidb 3.0: a redesign and expansion of the drug-gene interaction database an improved plaque reduction virus neutralization assay for human metapneumovirus how viruses hijack cell regulation lopinavir up-regulates expression of the antiviral protein ribonuclease l in human papillomavirus-positive cervical carcinoma cells key: cord-254809-o454k6ae authors: he, bing; wang, jun; wang, yudie; zhao, juan; huang, juan; tian, yu; yang, cheng; zhang, heng; zhang, mingxia; gu, lixing; zhou, xiaocui; zhou, jingjiao title: the metabolic changes and immune profiles in patients with covid-19 date: 2020-08-28 journal: front immunol doi: 10.3389/fimmu.2020.02075 sha: doc_id: 254809 cord_uid: o454k6ae to explore the metabolic changes and immune profiles in patients with covid-19, we analyzed the data of patients with mild and severe covid-19 as well as young children with covid-19. of the leukocytes, 47% (iqr, 33–59) were lymphocytes [2.5 × 10(9)/l (iqr, 2.2–3.3)], and monocytes were 0.51 × 10(9)/l (iqr, 0.45–0.57) in young children with covid-19. in 32 mild covid-19 patients, circulating monocytes were 0.45 × 10(9)/l (iqr, 0.36–0.64). twenty-one severe patients had low po(2) [57 mmhg (iqr, 50–73)] and so(2) [90% (iqr, 86–93)] and high lactate dehydrogenase [580 u/l (iqr, 447–696)], cardiac troponin i [0.07 ng/ml (iqr, 0.02–0.30)], and pro-bnp [498 pg/ml (iqr, 241–1,726)]. serum d-dimer and fdp were 9.89 mg/l (iqr, 3.62–22.85) and 32.7 mg/l (iqr, 12.8–81.9), and a large number of rbc (46/μl (iqr, 4–242) was presented in urine, a cue of disseminated intravascular coagulation (dic) in severe patients. three patients had comorbidity with diabetes, and 18 patients without diabetes also presented high blood glucose [7.4 mmol/l (iqr, 5.9–10.1)]. fifteen of 21 (71%) severe cases had urine glucose +, and nine of 21 (43%) had urine ketone body +. the increased glucose was partially caused by reduced glucose consumption of cells. severe cases had extraordinarily low serum uric acid [176 μmol/l (iqr, 131–256)]. in the late stage of covid-19, severe cases had extremely low cd4(+) t cells and cd8(+) t cells, but unusually high neutrophils [6.5 × 10(9)/l (iqr, 4.8–9.6)], procalcitonin [0.27 ng/ml (iqr, 0.14–1.94)], c-reactive protein [66 mg/l (iqr, 25–114)] and an extremely high level of interleukin-6. four of 21 (19%) severe cases had co-infection with fungi, and two of 21 (9%) severe cases had bacterial infection. our findings suggest that, severe cases had acute respiratory distress syndrome (ards) i–iii, and metabolic disorders of glucose, lipid, uric acid, etc., even multiple organ dysfunction (mods) and dic. increased neutrophils and severe inflammatory responses were involved in ards, mods, and dic. with the dramatical decrease of t-lymphocytes, severe cases were susceptible to co-infect with bacteria and fungi in the late stage of covid-19. in young children, extremely high lymphocytes and monocytes might be associated with the low morbidity of covid-19. the significantly increased monocytes might play an important role in the recovery of patients with mild covid-19. in december 2019, an unknown viral pneumonia emerged in wuhan, china, and it then escalated into an unprecedented outbreak (1) . chinese authorities have identified a new type of coronavirus named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (2). on february 11, 2020 , the infectious disease caused by this viral strain was officially named covid-19 (coronavirus disease 2019) by the world health organization (who) (3) . by march 12, covid-19 had swept into at least 117 countries and killed more than 4,000 people, and who officially announced a pandemic of covid-19 viral disease (4) . as of june 21, 2020, covid-19 cases have been confirmed in 214 countries and territories, and the total was up to 8,708,008, including 461,715 deaths (5) . so far, according to reported patients' data, some remarkable phenomena have been observed. first, only 1% of patients with covid-19 were infants and young children, and very few young patients have developed severe covid-19 (6) . leukocytes are the main immune cells to fight against pathogens, and the total leukocyte count is higher in young children than in adults (7) . moreover, the thymus gland of an infant is large and continues to grow throughout childhood. thus, the thymus produces more than enough matures tlymphocytes throughout the child's life (8) . we explored whether the count and differential of leukocytes in infants and young children are associated with very low morbidity rates of covid19. second, from the epidemiology and clinical characteristics of covid-19, 81% of patients were diagnosed as mild cases, and most mild cases can recover from covid-19 infection (9) . so, it could be that specific leukocytes contributed to the recovery of patients with mild covid-19. monocytes are important immune sentinel cells critical in the defense against viral infection in the blood. they achieve this via diverse mechanisms that include the detection of viruses, migration into infected tissues, differentiation into macrophages and dendritic cells, and pathogen clearance by phagocytosis and intracellular killing (10, 11) . besides monocytes, the effect of lymphocytes on mild covid-19 cases is still unclear. in this study, 32 mild patients have been examined to explore the potential roles of monocytes and lymphocytes in the recovery of patients with mild covid-19. third, according to an analysis of nearly 45,000 confirmed cases, 19% of patients with covid-19 have been identified as severe cases and critically ill cases, involving severe pneumonia and metabolic disorders, developing into acute respiratory distress syndrome (ards), multiple organ dysfunctions (mods), and even septic shock and death (9, 12) . some studies suggested that the immunopathogenesis after viral infection has been linked to the development of the disease into severe cases (13, 14) . to explore the potential roles of immunopathogenesis in the progress of covid-19 infection, 21 severe covid-19 patients have been investigated to explore how the immunopathogenesis was involved in ards and metabolic disorders, even mods, disseminated intravascular coagulation (dic), and death. in this study, we investigated mild cases and severe cases infected with sars-cov-2, as well as healthy young children and adults. our multiple comparative analysis showed that not only is leukocyte composition different in healthy groups, these differences can also be found during various stages of sars-cov-2 infection. our study suggests that monocytes, neutrophils, and t-lymphocytes are associated with the onset and progress of covid-19 infection, and immunopathogenesis was involved in ards, metabolic disorders, and mods in severe cases. this study increases our understanding of the immune responses during covid-19 infection and provides support to develop novel, feasible, and effective treatments for covid-19 infection. research sources: covid-19 patients and healthy individuals covid-19 infection was rapidly endemic in wuhan, china, in january, 2020. renmin hospital of wuhan university is at the very forefront of the fight against covid-19. we collected the data of patients with covid-19, including the clinical records, laboratory results and chest computed tomography (ct) scan images of mild and severe cases in the hospital. for comparison with covid-19 cases, the data of 35 healthy adults and 31 young children have been collected from the physical examination center of the hospitals. these healthy individuals have no significant medical condition and were in stable physical condition at that time. the data of patients with covid-19 and healthy persons have been all reviewed by a group of professional doctors from the hospitals, including basic features, nucleic acid tests, clinical data, laboratory results, co-infection with other pathogens, ct images, and other primary data. the study design has been approved by the ethics committee of the hospital. nasopharyngeal swab samples were collected from patients, and tested as soon as possible to increase the detection rate of sars-cov-2. reverse transcription polymerase chain reaction (rt-pcr) kit (daan gene, shenzhen, china) was used to detect the conserved genes of sars-cov-2, such as orf1ab gene, n gene, and e gene with lightcycler 480 system (roche, switzerland). if two or more of these three targeted genes has been detected as positive or one gene has been detected positive in two different samples from the same patient, the result is considered as positive for sars-cov-2. meanwhile, the results can also be analyzed in conjunction with the patient's chest ct images. blood samples were collected from patients for laboratory tests. serum biochemical tests, including aspartate aminotransferase (ast), alanine aminotransferase (alt), creatine kinase (ck) and lactate dehydrogenase (ldh) were determined with cobas c501 testing system (roche, germany). procalcitonin (pct) and cardiac troponin i (ctn i) were analyzed by cl-2000i chemiluminescence immunoassay system (mindray, shenzhen, china). coagulation indicators were detecting with acl top 700 hemostasis testing systems (werfen, usa). all the blood samples from healthy persons were used for comparison. to study the count and differential of lymphocytes, the blood samples from covid-19 patients were stained with cd3, cd4, cd8, cd19, cd16, and cd56 antibodies (bd multi-test imk kit, usa) and were analyzed by bd facscanto ii flow cytometer (bd, usa). th1/th2 kit (bd, usa) was used to quantitatively measure il-2, il-4, il-10, tnf, and ifn-γ protein levels. to examine the effect of sars-cov-2 on the patients' humoral immune function, immunoglobulins (igm, igg, iga, and ige), complement 3 (c3) and complement 4 (c4) were tested (siemens healthineers, usa). c-reactive protein (crp) and interleukin (il-6) were measured for covid-19 patients (mindray, shenzhen, china). serum samples of patients were collected and tested for the igm of respiratory tract pathogens with pneumoslide igm kit (vircell, spain), including human respiratory syncytial virus, influenza a virus (subtypes h1n1 and h3n2), influenza b virus, parainfluenza virus 1/2/3, metapneumovirus, common coronavirus, epstein-barr virus, cytomegalovirus, rhinovirus, adenovirus, and bocavirus, as well as legionella pneumophila serum type i, mycoplasma pneumonia, and chlamydia pneumoniae. nasopharyngeal secretions were tested for nucleic acids of 13 respiratory pathogens (health gene technologies, ningbo, china). sputum culture was performed to identify bacterial and fungal co-infection. the fungal examination was performed with fungus (1-3)-β-d-glucan kit (dynamiker biotechnology, tianjin, china) and platelia aspergillus ag kit (bio-rad, usa). continuous measurements have been presented as median and interquartile range (iqr) and categorical variables as percentages. for assessing laboratory results, we also assessed whether the measurements were outside the normal range. unpaired t-test with welch's correction was used for comparison, and p < 0.05 and <0.01 were considered statistically significant and highly statistically significant, respectively. graphpad prism 8.0.2 (san diego, ca, usa) and spss25.0 (ibm, armonk, ny, usa) were used for all analyses. patients with fever and/or cough were admitted to hospital after february 1, 2020. chest ct images indicated multiple patchy, ground-glass opacity in the lungs ( figure 1a) . thirty-two patients were further diagnosed as infected with sars-cov-2 by real-time rt-pcr. there were 17 men and 15 women, and the median age of these mild cases was 42. the clinical characteristics of mild patients were presented in supplementary table s1 . compared with healthy adults, the count of leukocytes and neutrophils in mild covid-19 patients did not increase, but the median percentage and count of lymphocytes were 26% (iqr, 19-34) and 1.2 × 10 9 /l (iqr, 1.1-1.6), respectively, which were significantly less than those of healthy adults, 34% (iqr, 29-39) and 2.0 × 10 9 /l (iqr, 1.8-2.5), respectively (p < 0.001). interestingly, the median percentage and count of monocytes were 8.2% (iqr, 7.1-9.2) and 0.45 × 10 9 /l (iqr, 0.36-0.64), which were significantly higher than those of healthy adults 6.3% (iqr, 5.5-7.1) and 0.39 × 10 9 /l (iqr, 0.35-0.42) (p ≤ 0.001) ( table 1 ). the significantly increased number of monocytes could play an important role in the recovery of patients with mild covid-19. to investigate why infants and young children have low morbidity of covid-19, we analyzed the clinical characteristics of young children with covid-19, and collected the data of circulating leukocytes of young children with/without covid-19. comparative analyses showed that young children have much higher leukocyte counts [6.9 × 10 9 /l (iqr, 6.1-8.1)] than adults. of note, 51% (iqr, 42-58) of leukocytes are lymphocytes [3.4 × 10 9 /l, (iqr, 2.5-4.6)] in young children. the median count of monocytes in young children is 0.46 × 10 9 /l (iqr, 0.41-0.67), which is much higher than that of adults [0.39 × 10 9 /l (iqr, 0.35-0.42)] (p = 0.001). lymphocytes of young children with covid-19 was a little lower than those of healthy children, but remained at a high level [2.5 × 10 9 /l (iqr, 2.2-3.3)]. young children with covid-19 had a high level of monocytes [0.51 × 10 9 /l (iqr, 0.45-0.57)] as well ( table 2) . such a high number of lymphocytes and monocytes has benefit to fight against sars-cov-2, which might be associated with the low morbidity of covid-19 in young children. we collected and compared the data of 21 severe cases and 32 mild cases. chest ct images of severe cases indicated that there was critically diffuse ground-glass opacity in both lungs. the critically ill patient's bedside chest x-ray showed the lung texture enhanced and the translucency decreased, and multiple patchy shadows in both lungs. (d) serum il-6 concentration between mild patients (n = 32) and severe patients (n = 21). the normal range of il-6 is ≤10 pg/ml. **p < 0.01. (e) the analysis of th1/th2 cytokine panel between mild patients (n = 32) and severe patients (n = 21). the normal range of il-2, ifn-γ, il-4, and il-10 are ≤11.4 pg/ml, 18 pg/ml, 12.9 pg/ml, and 5.9 pg/ml, respectively. *p < 0.05. a representative ct image is presented in figure 1b . in bedside chest x-ray results of the critically ill patients, the translucency of both lungs was diffusely decreased, and a large area of patchy shadow appeared with uneven density. tracheal intubation can be observed in the trachea and the heart shadow outline ( figure 1c) . the clinical characteristics of severe patients were presented in supplementary table s1 . these ct and x-ray images showed that the primary and most significant changes were in the lower respiratory tract of patients with severe covid-19. among the respiratory indicators we measured, severe cases had lower partial pressure of oxygen (po 2 ) and oxygen saturation (so 2 ), 57 mmhg (iqr, 50-73) and 90% (iqr, 86-93), respectively, and suffered from different degrees of ards, i to iii ( table 3) . table 4) . increased glucose and low uric acid in blood should be noted here. the level of blood glucose was 5. table 3) . the total of leukocytes was 7.6 × 10 9 /l (iqr, 5.5-11.3) in the peripheral blood of severe cases, which were much more than those in mild cases. compared with mild cases, severe cases had low levels of monocytes [0.37 × 10 9 /l (iqr, 0.21-0.51)]. however, the percentage and count of lymphocytes in severe cases were only 7% (iqr, 4-10) and 0.5 × 10 9 /l (iqr, 0.3-0.8) respectively, which were significantly lower than those in mild cases ( table 1) . the subsets of lymphocytes were examined by flow cytometry, including natural killer (nk) cells (cd16 + cd56 + ), b cells (cd19 + ), and t cells (cd3 + ). the results showed that severe cases had nk cells [63/µl (iqr, 26-109)] and b cells [91/µl (iqr, 54-181)], which was not a significant difference from the mild cases (p > 0.05). in addition, the functions of b cells and complements were tested, including igm, igg, iga, ige, c3, and c4, for both mild and severe covid-19 cases. for severe cases, the values of igm, c3, and c4 were slightly lower than those in mild cases, but these values were still within the normal range. however, compared with mild cases, severe cases had much lower levels of cd4 + t cells and cd8 + t cells, 146/µl [iqr, 107-277] and 59/µl (iqr, 33-109), respectively. the decrease of cd8 + t cells was much more than that of cd4 + t cells, and the ratio of cd4 + t cells/cd8 + t cells increased by 2.38 (iqr, 1.62-4.63) ( table 4 ). further examination of th1/th2 cytokines also indicated that severe patients had normal levels of il-2, and ifnγ, as well as il-4 in peripheral blood, but the level of il-10 in severe patients was 4 times higher than normal (figure 1e ). in this study, the clinical course of severe cases was over 3 weeks, and severe cases had a potentially high risk of co-infection with other pathogens due to critical exhaustion of cd4 + and cd8 + t cells. the respiratory tract pathogens were tested in severe cases, including 10 viruses, legionella pneumophila, mycoplasma pneumoniae, and chlamydia pneumoniae, which were all negative. the fungal examinations, g assay and gm assay, were also performed in severe cases. the results of bacterial and fungal examinations indicated that four of 21 (19%) severe cases had co-infection with fungi, and two of 21 (9%) severe cases had co-infection with bacteria. a high number of white blood cells (wbc) [18/µl (iqr, ] was found in the urine of severe cases ( table 4 ). further examinations showed that the median pct was 0.27 ng/ml (iqr, 0.14-1.94) in severe cases, a cue of potential sepsis/septic shock. among the inflammatory factors tested in severe cases, the median of crp was 66 mg/l (iqr, 25-114), which was much higher than those in mild cases ( table 4) . il-6 slightly increased in mild cases, but exceptionally high level of il-6 presented in severe cases, even 40 times higher than normal in some critically ill cases (figure 1d) . the release of the inflammatory factors triggered by sars-cov-2 replication and/or co-infection with bacteria and fungi, played important roles in the progress of covid-19 infection. in the late stage of the disease in severe covid-19 cases, 88% (iqr, 84-92) of leukocytes were neutrophils [6.5 × 10 9 /l (iqr, 4.8-9.6)] ( table 1) . previous studies showed that largely number of neutrophils triggered inflammatory responses and caused excessive organ injury in acute inflammatory disease, such as sepsis (15, 16) . exceptionally high neutrophil numbers might be involved in severe inflammatory responses and might be associated with ards, mods, and even sepsis/septic shock, dic, and death during the late stage of severe covid-19 infection. in this study, we first analyzed the clinical features and leukocyte differential of mild covid-19 patients admitted to the hospital after february 1, 2020. thirty-two mild cases, with a median age of 42 years, had recovered from covid-19 infection. our data showed that compared with healthy adults, patients with mild covid-19 had lower lymphocytes in the acute stage, which was consistent with previous studies (12) . however, mild covid-19 cases had high counts of circulating monocytes [0.45 × 10 9 /l (iqr, 0.36-0.64)]. in addition, mild patients had normal level of il-4 and il-10 in peripheral blood, but they had a 1-2-fold increase of il-6. monocytes/macrophages play very important roles in fighting against invading foreign viruses. literature from the past 30 years has emphasized links among il-6 and innate immune response, such as mononuclear phagocytes (10, 11, 17) . for patients with mild covid-19, a high monocyte count and slight increase of il-6 might be helpful for eradicating the sars-cov-2 infection and were associated with recovery from covid-19. based on the epidemiology and clinical characteristics of covid-19, young children under six have the lowest morbidity rate, and very few young children with covid-19 develop severe cases (6, 18) . according to our comparative analysis, young children under six have highly circulating monocytes, and 51% (iqr, 42-58) of leukocytes are lymphocytes had a high level of monocytes [0.51 × 10 9 /l (0.45-0.57)] as well. the intricate process of t-lymphocyte development in the thymus is essential in the formation and maintenance of the peripheral t-lymphocytes. the thymus of a young child is big, and has the function of maintaining the large amounts of t-lymphocytes in the peripheral blood (19, 20) . extremely high levels of circulating lymphocytes and monocytes would benefit to fight against sars-cov-2 infection, which might be associated with the low morbidity of covid-19 in young children. to explore the metabolic changes and immune responses in the progress of covid-19 cases, we investigated 21 patients with severe covid-19 infection. the median age of these patients was 57, and the clinical course was more than 3 weeks. ct scan images showed multiple patchy ground-glass shadows in the left and right lungs. bedside chest radiography of critically ill patients indicated that the brightness of both lungs was decreased and multiple patchy shadows were observed. these clinical characteristics of severe cases are very similar to those reported in previous studies (21, 22) . the 21 severe covid-19 cases had ards i to iii, and had extremely high levels of ctni, ldh, and pro-bnp, a marker of severe cardiac dysfunction and even heart failure. besides that, an extraordinarily low level of serum uric acid [176 µmol/l (131-256)] was found in severe cases. uric acid is synthesized mainly in the liver and other tissues, which usually dissolves in the blood, and is removed from the body through urine. the extraordinarily low level of serum uric acid might indicate that potential liver and/or rental metabolism dysregulated in severe patients. among 21 severe cases, three patients had the comorbidity of diabetes, and other patients also had very high blood glucose we further investigated immune responses in patients with severe covid-19. first, different subpopulations of lymphocytes were investigated. the percentage and count of b cells and nk cells did not obviously change, which is consistent with the results from a previous report (23) . the results of igm/igg/iga/ige, c3 and c4 also indicated that b cells and complements held normal functions. however, compared with mild cases, severe covid-19 cases had lower levels of cd4 + t cells [146/µl (iqr, 107-277)] and an even more significant reduction in cd8 + t cells [only 59/µl (iqr, 33-109)], which has a sharper drop than cd4 + t cell. we further analyzed th1/th2 panel, in severe patients, th1 cytokines (il-2 and ifn-γ) were in the normal range, but il-10, one of th2 cytokines, was about four times higher than normal. previous studies presented that in severe patients, cd4 + t cells and cd8 + t cells highly expressed the exhaustion markers, including nkg2a, pd-1, and tim-3 (24, 25) . the dramatical decrease and functional exhaustion of cd4 + t cells and cd8 + t cells represents an important immunological characteristic of severe covid-19 infection. following the exhaustion of t cells, severe cases had high potential for co-infection with other pathogens. in this study, 4 of 21 (19%) severe patients had coinfection with fungi, and two of 21 (9%) severe patients had bacterial co-infection. twenty-one severe cases had a high level of pct and crp, 0.27 ng/ml (iqr, 0.14-1.94) and 66 mg/l (iqr, 25-114), respectively. il-6 was much higher than normal in severe cases, even 40 times higher than normal in some critically ill cases. with sars-cov-2 replication and/or coinfection with bacteria and fungi, severe inflammatory responses played important roles in the progress of severe covid-19 infection. in the late stage of severe covid-19, 88% (iqr, 84-92) of leukocytes were neutrophils [6.5 × 10 9 /l (iqr, 4.8-9.6). a high number of wbc [18/µl (iqr, ] was presented in urine of severe patients. previous studies suggest that, in sepsis, a large number of neutrophil and the formation of neutrophil extracellular traps (net) triggered severe inflammatory responses and excessive tissue damage (15, 16, 26) . the significant increase in neutrophils might be involved in severe inflammatory responses and mods, even dic and death in severe covid-19 patients. additionally, uric acid is the predominant anti-oxidant molecule in the plasma and respiratory tract, and is necessary for induction of type 2 immune responses. uric acid plays a pivotal role in protecting against pathogen infections and autoimmune diseases (27, 28) . whether the decrease of serum uric acid is associated with the inflammatory responses in severe covid-19 cases need to be explored. there are several limitations to this study. first, we investigated 16 young children with covid-19 and 53 adult cases, including 32 mild cases and 21 severe cases. more cases will need to be collected for comparative analysis of the difference between severe and critically ill patients. second, more inflammatory cytokines and chemokines will be analyzed for severe and critically ill patients and will be further evaluated for inflammatory storm mediated ards, dic, mods, and coagulation disorders. third, the mechanisms by which sars-cov-2 infection causes the reduction and functional exhaustion of cd4 + t cells and cd8 + t cells are still unclear. in-vitro and in-vivo experiments need to be performed to explore the mechanisms of t cell exhaustion. in summary, our findings suggest that extremely high level of lymphocytes and monocytes could help hamper sars-cov-2 replication, which might be associated with the low morbidity of covid-19 in infants and young children. a high number of monocytes would be helpful for removing sars-cov-2 and play an important role in the recovery of patients with mild covid-19. in the late stage of the disease, severe cases suffered from ards, metabolic disorders, mods and coagulation disorders. with dramatical decrease of cd4 + t cells and cd8 + t cells, extraordinarily increased neutrophils and severe inflammatory responses are involved in ards, mods, and coagulation disorders and can even lead to dic and death in severe cases. whether the decrease of serum uric acid is associated with the inflammatory responses in severe covid-19 cases needs to be further explored. these findings can not only greatly improve our understanding of metabolic and immunological characteristics, but also provide a mechanistic basis for the prevention and treatment of covid-19 infection. all datasets generated for this study are included in the article/supplementary material. the studies involving human participants were reviewed and approved by renmin hospital of wuhan university. written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china novel coronavirus (2019-ncov) situation report-22 world health organization. coronavirus disease 2019 (covid-19) situation report-52 world health organization characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention leukocyte counts in 7,739 healthy black persons: effects of age and sex changes in thymic regulatory t-cell maturation from birth to puberty: differences in atopic children world health organization. coronavirus disease 2019 (covid-19) situation report-41 the phenotype and function of preterm infant monocytes: implications for susceptibility to infection monocytes, viruses and metaphors: hanging the trojan horse epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study pathological findings of covid-19 associated with acute respiratory distress syndrome clinical features of patients infected with 2019 novel coronavirus in wuhan the role of neutrophils in immune dysfunction during severe inflammation circulating mitochondrial damps cause inflammatory responses to injury il-6 switches the differentiation of monocytes from dendritic cells to macrophages early epidemiological analysis of the coronavirus disease 2019 outbreak based on crowdsourced data: a population-level observational study a cell atlas of human thymic development defines t cell repertoire formation combined tcrg and tcra trec analysis reveals increased peripheral t-lymphocyte but constant intra-thymic proliferative history upon ageing clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients. medrxiv functional exhaustion of antiviral lymphocytes in covid-19 patients reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) neutrophil extracellular traps in immunity and disease physiological functions and pathogenic potential of uric acid: a review barrier epithelial cells and the control of type 2 immunity the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2020.02075/full#supplementary-material the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 he, wang, wang, zhao, huang, tian, yang, zhang, zhang, gu, zhou and zhou. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-353217-gmc3qrci authors: de miranda santos, isabel kinney ferreira; costa, carlos henrique nery title: impact of hydroxychloroquine on antibody responses to the sars-cov-2 coronavirus date: 2020-08-04 journal: front immunol doi: 10.3389/fimmu.2020.01739 sha: doc_id: 353217 cord_uid: gmc3qrci nan recent large observational studies indicate that hydroxychloroquine (hy) does not affect outcomes of patients hospitalized with covid-19 (1, 2) and may even be harmful (3) . results of double-blind, randomized studies to assess efficacy of hy more rigorously are still not available. in spite of these facts, officials are currently advocating use of hydroxychloroquine (hy) for treatment and even prevention of covid-19. in view of this situation and of the importance of correct interpretation of antibody profiles for planning preventive measures for covid-19, we would like to bring the attention of readers to studies that raise concerns about the possible impact of hy upon antibody responses to sars-cov-2. hy and chloroquine are lysosomotropic drugs that increase the ph of the lysosome, thus affecting functions of proteins involved in antigen presenting pathways and in b-cell activation (7) . to the best of our knowledge, there are no new facts in the scientific and medical literature that indicate that the same mechanism could not operate in hy-treated patients suffering from covid-19 and negatively impact their sars-cov-2-specific antibody responses. indeed, recent findings indicate that some individuals, including hospitalized patients, who have recovered from covid-19 have not made vigorous igg antibody responses. however, the most comprehensive publications addressing antibody responses, wherein study subjects presented viability in levels of igg antibody responses, have not detailed the treatment regimens delivered to the subjects (8) (9) (10) (11) . plans for employing immunity profiles against sars-cov-2 to relax social distancing and other epidemic mitigation measures and to create "immunity passports" to control spread of covid-19 have recently been questioned by the world health organization because of uncertainty regarding antibody responses (12) . as more needs to be learned about the role of antibodies in recovery from and protection against infection with sars-cov-2, the impact of hy and other treatment regimens on antibody responses requires systematic evaluation. observational study of hydroxychloroquine in hospitalized patients with covid-19 association of treatment with hydroxychloroquine or azithromycin with in-hospital mortality in patients with covid-19 in new york state hydroxychloroquine or chloroquine with or without a macrolide for treatment of covid-19: a multinational registry analysis antibody response to preexposure human diploid-cell rabies vaccine given concurrently with chloroquine humoral immune response to tetanus-diphtheria vaccine given during extended use of chloroquine or primaquine malaria chemoprophylaxis effect of antimalarial drugs on the immune response to intramuscular rabies vaccination using a postexposure prophylaxis regimen mechanisms of action of hydroxychloroquine and chloroquine: implications for rheumatology temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infections by sars-cov-2: an observational cohort study detection of sars-cov-2-specific humoral and cellular immunity in covid-19 convalescent individuals evaluation of the euroimmun anti-sars-cov-2 elisa assay for detection of iga and igg antibodies a systematic review of antibody mediated immunity to coronaviruses: antibody kinetics, correlates of protection, and association of antibody responses with severity of disease available online at: www.who.int/newsroom/commentaries/detail/immunity-passports-in-the-context-of-covid-19 all authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. key: cord-336924-7xcbtn3q authors: borghi, maria orietta; beltagy, asmaa; garrafa, emirena; curreli, daniele; cecchini, germana; bodio, caterina; grossi, claudia; blengino, simonetta; tincani, angela; franceschini, franco; andreoli, laura; lazzaroni, maria grazia; piantoni, silvia; masneri, stefania; crisafulli, francesca; brugnoni, duilio; muiesan, maria lorenza; salvetti, massimo; parati, gianfranco; torresani, erminio; mahler, michael; heilbron, francesca; pregnolato, francesca; pengo, martino; tedesco, francesco; pozzi, nicola; meroni, pier luigi title: anti-phospholipid antibodies in covid-19 are different from those detectable in the anti-phospholipid syndrome date: 2020-10-15 journal: front immunol doi: 10.3389/fimmu.2020.584241 sha: doc_id: 336924 cord_uid: 7xcbtn3q background: critically ill patients with coronavirus disease 2019 (covid-19) have a profound hypercoagulable state and often develop coagulopathy which leads to organ failure and death. because of a prolonged activated partial-thromboplastin time (aptt), a relationship with anti-phospholipid antibodies (apls) has been proposed, but results are controversial. functional assays for apl (i.e., lupus anticoagulant) can be influenced by concomitant anticoagulation and/or high levels of c reactive protein. the presence of anti-cardiolipin (acl), anti-beta2-glycoprotein i (anti-β(2)gpi), and anti-phosphatidylserine/prothrombin (aps/pt) antibodies was not investigated systematically. epitope specificity of anti-β(2)gpi antibodies was not reported. objective: to evaluate the prevalence and the clinical association of apl in a large cohort of covid-19 patients, and to characterize the epitope specificity of anti-β(2)gpi antibodies. methods: elisa and chemiluminescence assays were used to test 122 sera of patients suffering from severe covid-19. of them, 16 displayed major thrombotic events. results: anti-β(2)gpi igg/iga/igm was the most frequent in 15.6/6.6/9.0% of patients, while acl igg/igm was detected in 5.7/6.6% by elisa. comparable values were found by chemiluminescence. aps/pt igg/igm were detectable in 2.5 and 9.8% by elisa. no association between thrombosis and apl was found. reactivity against domain 1 and 4-5 of β(2)gpi was limited to 3/58 (5.2%) tested sera for each domain and did not correlate with acl/anti-β(2)gpi nor with thrombosis. conclusions: apl show a low prevalence in covid-19 patients and are not associated with major thrombotic events. apl in covid-19 patients are mainly directed against β(2)gpi but display an epitope specificity different from antibodies in antiphospholipid syndrome. critically ill patients with coronavirus disease 2019 (covid19) have a profound hypercoagulable state and often develop thrombosis in veins, arteries and in the microcirculation (1, 2) . a recent analysis showed several coagulation abnormalities in these patients, including prominent elevation of fibrin/fibrinogen degradation products (i.e., d-dimer) and a prolonged activated partial-thromboplastin time (aptt). while high levels of d-dimer are consistent with sustained activation of the clotting and fibrinolytic cascades, the combination of prolonged aptt and both arterial and venous thrombosis was, however, surprising, and it is reminiscent of a clinical scenario known as antiphospholipid syndrome (aps) (3) . looking at the causes of aptt prolongation, recent studies have shown that lupus anticoagulant (la) can be detected in a significant percentage of covid-19 samples (4) (5) (6) . since la is often caused by anti-phospholipid antibody (apl), these findings support the idea that apl may play a role in covid-19 (7) . however, it is important to point out that la is a very sensitive assay and its outcome can be influenced by several factors, most notably heparin administration (8) and a profound inflammatory state characterized by high levels of c reactive protein (crp) (9, 10) . both of them are present in covid-19 patients (11) . another method to detect apl that is in principle insensitive to anticoagulation and other confounding agents relies on the detection and quantification of autoantibodies using solid-phase assays (3) . using this method, the presence of apl was recently reported in a handful of case reports and small cohorts of patients (4, 6, 7, 12, 13) . while encouraging, this data is limited and its interpretation remains controversial, with some investigators proposing an important role of apl in covid-19 patients (7) while others suggesting a very poor correlation between apl and thrombotic events (14) . there is no information on the antigen specificity of covid-19 apl in comparison with aps antibodies. such information and a larger study, possibly multicenter, may be instrumental to clarify the real clinical value of these autoantibodies. a total of 122 patients were enrolled from two covid-19 referral centers in lombardia. all patients tested positive to sars-cov-2, and classified as severe or critical covid-19 (11) . the mean age was 68.5 (± sd 16.4) years; 77 were men and 45 women. no diagnosis of previous autoimmune diseases was made; six patients had a thrombotic event (three arterial and three venous) in the past clinical history. the presence of antinuclear antibodies (anas) was investigated in 58 patients at istituto auxologico italiano by hep2-iif and solid phase ctd screening following the guidelines described in agmon-levin et al. (15) . of the 58 samples, none was positive for ana. eighty-seven patients suffering from aps were also tested for anti-cardiolipin (acl) and anti-b 2 gpi igg/igm (16) .the study was approved by the ethics committees (istituto auxologico italiano 3-04-2020 -milan and asst spedali civili np4187 -brescia). acl and anti-b 2 gpi igg/iga/igm were detected by chemiluminescence immunoassay (cia; quanta flash, inova, san diego, ca, us) and a home-made elisa as described (16, 17) . anti-b 2 gpi domain 1 igg (anti-d1) were detected by cia (16, 17) , igg anti-d4-5 by a home-made elisa, as described (16, 17) . detailed methods are reported in the supplementary material. anti-phosphatidylserine/prothrombin (aps/pt) igg/igm were detected by a commercial elisa as reported (18) . blood samples were collected in the first week after hospital admission. data were analyzed using r v3.4.0. descriptive statistics was used to summarize data. associations and differences between categorical or continuous variables were tested by fisher's exact test and non-parametric mann-whitney test, respectively. a p-value < 0.05 was considered statistically significant. table 1 reports the median with minimum and maximum values for different coagulation and inflammation parameters in 122 severe or critical covid-19 patients. in particular, prolonged aptt (>30 s) was found in 57.6% while pt inr values were above the cut-off in 24.8% of the cases. most of the patients (120/ 122) were on anticoagulation with low molecular weight heparin (70% on therapeutic and the remaining on prophylactic dosage). despite anticoagulation, we observed sixteen thrombotic events (13.1%, 8 in veins and 8 in arteries). these statistics are in agreement with previous reports (2, (19) (20) (21) (22) (23) (24) and document a systemic inflammation and a coagulopathy in our patients. in the aps field, testing for la is not recommended when patients are on heparin, since the presence of heparin, even if neutralized, may lead to false-positive results (8) . likewise, high levels crp, such as those found in our cohort of patients, have been shown to prolong aptt independently from the presence of apl (9, 10). on these bases, the presence of apl was researched using solid-phase assays, and not la. first, we investigated the presence of acl and anti-b 2 gpi, two aps classification criteria (3) . testing was independently performed in milan and brescia, using harmonized methodologies (25) . the prevalence of covid-19 patients positive for acl and anti-b 2 gpi igg/iga/ igm detected by elisa and cia is summarized in table 2 . the elisa raw data are shown in figure 1 . we found igg/igm acl in 5.7/6.6% of patients, whereas anti-b 2 gpi igg/iga/igm were found in 15.6/6.6/9.0% of patients. similar values were obtained for acl antibodies using cia ( table 2) , whereas a slightly lower sensitivity was obtained for anti-b 2 gpi antibodies (26) . the positivity for acl and anti-b 2 gpi antibodies was at medium/ low titer in contrast with the medium/high titers found in the control group of primary aps ( figure 1 ). there is no association between apl positivity and thrombotic events. fifty-eight sera were also tested with d1 and d4-5-coated plates in order to characterize their epitope specificity. figure 2b shows that three out of 58 samples reacted with d1, while in figure 2c , three samples tested positive for d4-5. none of the sera was positive for both domains and all displayed a weak reactivity with no association with thrombosis. prolonged aptt (>30 s) was found in 57.6% of the patients. although aps/pt are not included in the aps classification laboratory tests, they can be associated with a prolonged aptt and with the presence of la (18) . consequently, we looked at the presence of aps/pt antibodies in our cohort and we found fifteen out of 122 sera positive for aps/pt (12.3%), mostly of the igm isotype (12 out 15) and at a low titer (figure 3 ). there was no association between prolonged aptt and the presence of aps/pt antibodies nor with thrombotic events in our covid-19 cohort. taken together, our data show a low prevalence of classification criteria apl in covid-19 patients. in this regard, our study confirms recent studies obtained with smaller cohorts of patients (4, 14, 24) . importantly, our data also shows that apl are slightly more reactive towards b 2 gpi-coated plates as compared to clcoated ones and that, regardless of the nature of apl, there is no association between apl positivity and thrombotic events (p = 1). a striking difference between the autoantibody profile in covid-19 patients as compared to the one in aps concerned the titers of apl. medium/low apl titers were consistently found in patients with covid-19. by contrast, medium/high titers are usually found in aps patients (figure 1) . this difference suggests that apl found in covid-19 may be different from apl found in aps and led us to further investigate the epitope specificity of antib 2 gpi antibodies. we focused on autoantibodies directed against the n-terminal domain 1 (anti-d1) or the c-terminal domains 4-5 (anti-d4-5) of the molecule (17) (figure 2a) . this is because anti-d1 antibodies are associated with an increased risk of thrombosis and pregnancy complications in aps (16, 17, 28) . by contrast, anti d4-5 antibodies are associated neither with vascular nor obstetric aps manifestations (16, 29) . furthermore, anti d4-5 antibodies are also reported at high levels in the so called asymptomatic apl carriers and are frequently found in non-aps (e.g., patients with leprosy, atopic dermatitis, atherosclerosis, and in children born to mothers with systemic autoimmune diseases) (29) . we found that three out of 58 samples reacted with d1, and three samples tested positive for d4-5. none of the sera was positive for both domains and all displayed a weak reactivity. although the number of the investigated sera is relatively small, this finding is quite different from the results found in aps in which almost all the sera positive for the whole b 2 gpi molecule also reacted with domain d1 at high titer (16, 28) . furthermore, at variance with aps patients, none of the anti-d1 positive patients displayed thrombotic events (28) . approximately, 57% of covid-19 patients have prolonged aptt. yet, only a small proportion of covid-19 patients carry acl and anti-b 2 gpi antibodies. this suggests that other factors must be responsible for the prolonged aptt phenomenon and likely for the la activity. la may be affected by the concomitant heparin treatment and the high crp levels. although more sensitive and specific diagnostic algorithms have been suggested (30), we followed the isth guidelines available at the beginning of the study (31) . since aps/pt can be associated with a prolonged aptt and with the presence of la (18), we tested our cohort for aps/pt antibodies. we found a small percentage (12.3%) of positive sera, mostly of the igm isotype (12 out 15) and at a low titer. again, there was no association between prolonged aptt and the presence of aps/pt antibodies nor with thrombotic events in our covid-19 cohort. this indicates that aps/pt are not responsible for the prolongation of aptt nor are predictors of adverse clinical outcomes. furthermore, in contrast to what we would have expected in aps (32), we found no associations between the presence of aps/ pt, acl, and anti-b 2 gpi antibodies. this data is in line with the unusual epitope specificity of anti-b 2 gpi antibodies documented in figure 2 , supporting the hypothesis that apl found in covid-19 patients are different from apl found in aps patients. whether covid-19 apl are similar to the ones found in other infectious diseases such as hcv, hbv and hiv (33) remains to be determined. despite heparin treatment, 13.1% of our patients displayed thrombotic events. although we cannot exclude that treatment could be protective, the prevalence of vascular events was in line with that reported by other studies as recently reviewed (34) . in conclusion, while the medium/high apl titers with d1 specificity are associated with vascular events in aps, low antibody titers with reactivity against b 2 gpi epitope(s) different from d1 or d4,5 can be found in covid-19. this may explain the lack of association with thrombotic events in covid-19. in addition, our data do not support the hypothesis that apl can be the main cause of prolonged aptt in these patients. although low titer apl are not predictive of vascular events in the aps, it is important to keep in mind that covid-19 patients suffer from an acute form of systemic inflammation with complement activation (35) , which may be responsible for endothelial perturbation. in this context, since b 2 gpi can accumulate on the activated endothelium at high density, even low titers of apl may become pathogenic thus potentiating or even triggering thrombus formation, especially when anticoagulation is suspended. a comparable condition in which low titers of apl can cause substantial damage is seen in obstetric aps, where high levels of b 2 gpi can be found in the placenta (36) . hence, while transitory apl are likely to be clinically irrelevant in covid-19 patients as in other infections (33) , detection of apl may be useful for identifying patients potentially at risk of thrombosis after the hospital discharge. accordingly, anticoagulant prophylaxis or therapies affecting cell signaling involved in inflammatory and coagulation responses could be justified before a confirmatory assay (3, 37) . the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the studies involving human participants were reviewed and approved by istituto auxologico italiano 3-04-2020 -milan and asst spedali civili np4187 -brescia. the ethics committee waived the requirement of written informed consent for participation. this article has been released as a pre-print at [medrxiv pulmonary post-mortem findings in a series of covid-19 cases from northern italy: a two-centre descriptive study abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia diagnosis and management of the antiphospholipid syndrome lupus anticoagulant is frequent in patients with covid-19 lupus anticoagulant and abnormal coagulation tests in patients with covid-19 high frequency of antiphospholipid antibodies in critically-ill covid-19 patients: a link with hypercoagulability? coagulopathy and antiphospholipid antibodies in patients with covid-19 frequent false-positive results of lupus anticoagulant tests in plasmas of patients receiving the new oral anticoagulants and enoxaparin lupus anticoagulant (lac) testing in patients with inflammatory status: does c-reactive protein interfere with lac test results? when to screen for lupus anticoagulant? influence of testing during acute phase and consequences for clinical practise clinical and immunological features of severe and moderate coronavirus disease 2019 prothrombotic antiphospholipid antibodies in covid-19. medrxiv (2020) brief report: antiphospholipid antibodies in critically ill patients with coronavirus disease 2019 (covid-19) antiphospholipid antibodies are not elevated in patients with severe covid-19 pneumonia and venous thromboembolism international recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies beyond thrombosis: anti-b2gpi domain 1 antibodies identify late pregnancy morbidity in anti-phospholipid syndrome new insight into antiphospholipid syndrome: antibodies to b2glycoprotein i-domain 5 fail to induce thrombi in rats antiprothrombin antibodies: a comparative analysis of homemade and commercial methods. a collaborative study by the forum interdisciplinare per la ricerca nelle malattie autoimmuni (firma) venous thrombosis among critically ill patients with coronavirus disease 2019 (covid-19) deep vein thrombosis in hospitalized patients with coronavirus disease 2019 (covid-19) in wuhan, china: prevalence, risk factors, and outcome incidence of thrombotic complications in critically ill icu patients with covid-19 venous and arterial thromboembolic complications in covid-19 patients admitted to an academic hospital in incidence of venous thromboembolism in hospitalized patients with covid-19 characteristics of ischaemic stroke associated with covid-19 are the current attempts at standardization of antiphospholipid antibodies still useful? emerging technologies signal a shift in direction a clinical approach for defining the threshold between low and medium anti-cardiolipin antibody levels for quanta flash assays the jelongated conformation of beta2-glycoprotein i predominates in solution: implications for our understanding of antiphospholipid syndrome prevalence and thrombotic risk assessment of anti-b2 glycoprotein i domain i antibodies: a systematic review clinical characterization of antiphospholipid syndrome by detection of igg antibodies against b2 -glycoprotein i domain 1 and domain 4/5: ratio of antidomain 1 to anti-domain 4/5 as a useful new biomarker for antiphospholipid syndrome nine-test panel has superior sensitivity to detect antiphospholipid antibody syndrome in patients with or without sle subcommittee on lupus anticoagulant/antiphospholipid antibodies. laboratory criteria for antiphospholipid syndrome: communication from the ssc of the isth tetra positive thrombotic antiphospholipid syndrome: major contribution of antiphosphatidyl-serine/prothrombin antibodies to lupus anticoagulant activity infections and the antiphospholipid syndrome coagulation abnormalities and thrombosis in patients infected with sars-cov-2 and other pandemic viruses complement activation in patients with covid-19: a novel therapeutic target eureka algorithm predicts obstetric risk and response to treatment in women with different subsets of anti-phospholipid antibodies metabolic pathways mediate pathogenesis and offer targets for treatment in rheumatic diseases antiphospholipid antibodies in covid-19 are different from those detectable in the anti-phospholipid syndrome. version 2 medrxiv the authors would like to thank n. carabellese and g. martini (department of laboratory diagnostics; asst spedali civili, brescia, italy) for their valuable collaboration; all the physicians of the covid-19 units of the irccs istituto auxologico the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2020. 584241/full#supplementary-material conflict of interest: mm was employed by inova diagnostics, inc.the remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 borghi, beltagy, garrafa, curreli, cecchini, bodio, grossi, blengino, tincani, franceschini, andreoli, lazzaroni, piantoni, masneri, crisafulli, brugnoni, muiesan, salvetti, parati, torresani, mahler, heilbron, pregnolato, pengo, tedesco, pozzi and meroni. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-338261-tyimwctm authors: farr, laura; ghosh, swagata; moonah, shannon title: role of mif cytokine/cd74 receptor pathway in protecting against injury and promoting repair date: 2020-06-23 journal: front immunol doi: 10.3389/fimmu.2020.01273 sha: doc_id: 338261 cord_uid: tyimwctm wound healing after an injury is essential for life. an in-depth understanding of the healing process is necessary to ultimately improve the currently limited treatment options for patients suffering as a result of damage to various organs and tissues. injuries, even the most minor, trigger an inflammatory response that protects the host and activates repair pathways. in recent years, substantial progress has been made in delineating the mechanisms by which inflammatory cytokines and their receptors facilitate tissue repair and regeneration. this mini review focuses on emerging literature on the role of the cytokine macrophage migration inhibitory factor (mif) and its cell membrane receptor cd74, in protecting against injury and promoting healing in different parts of the body. whenever an injury occurs, the body needs to repair it efficiently in order to protect from further damage and restore function. from minor scratches to myocardial infarction, we continually experience traumatic events throughout life. therefore, the healing process is essential for survival. further understanding of the mechanisms that promote healing could lead to new therapeutic opportunities to improve the lives of individuals with illnesses that resulted from organ and tissue injury (1, 2) . in addition to protecting against invading pathogens, an appropriate inflammatory response activates repair pathways that are essential for healing, without causing unwanted damage to the host tissue. cytokines play a crucial role in inflammation-driven repair. cytokines act by binding to specific receptors on certain cell types triggering downstream signaling events that ultimately promote the healing process (3, 4) . this review focuses on the recent advances that have greatly contributed to our current understanding of the link between the signaling pathways activated upon binding of macrophage migration inhibitory factor cytokine to its membrane receptor cd74 and wound healing in different body parts (figure 1 ). macrophage migration inhibitory factor (mif) is one of the first described cytokines, identified as a soluble immune cell-derived factor over 50 years ago in 1966. similar to cytokines such as tumor necrosis factor (tnf), mif's range of functions has exceeded what is implied by the historical name (5, 6) . the mif gene was cloned in 1989, and subsequent studies have demonstrated a wide range of roles for mif. mif is a truly pleiotropic inflammatory cytokine that is expressed by a variety of cells, and is a critical upstream mediator of innate immunity. given its important role in immunity, it is not surprising that excess mif expression has been linked to exaggerated inflammation and immunopathology. in addition, mif demonstrates well-documented proliferative properties. mif is secreted by many different types of cells and interacts with several receptors, which helps to explain the variety of biological functions. receptors that interact that bind mif include cd74, and chemokine receptors cxcr2 and cxcr4 (7) (8) (9) (10) (11) (12) (13) (14) (15) . cd74 is a type ii transmembrane protein consisting of an nterminal cytosolic tail, a short transmembrane region, and a long c-terminus luminal region. human cd74 is encoded on chromosome 5 and consists of four isoforms. isoforms p33 and p41 are generated by alternative splicing, that is, the p33 isoform is created by excluding exon 6b from p41 cd74 transcript. isoforms p35 and p43 originate from an alternative start site (16) (17) (18) (19) (20) (21) . while cd74 was first discovered in 1979 through coimmunoprecipitation of the major histocompatibility class ii antigen (mhcii), it wasn't until 1989 the antigen presentation function of cd74 was recognized. cd74 is expressed on classical antigen presenting cells (apcs), such as dendritic cells and macrophages, acts as a chaperone that binds mhcii, and is commonly referred to as the class ii invariant chain (ii) (16, 18, 22, 23) . subsequently, a growing body of evidence supported the concept that cd74 could have additional functions as a receptor. surface expression of cd74 occurred independently of concomitant mhcii expression. additionally, cd74 expression was found on the surface of non-apcs such as endothelial cells, and epithelial cells in the kidney, lung, gut, and skin (24, 25) . the receptor that mediated mif activity remained elusive until a study in 2003, which utilized a cdna library and fluorescently conjugated mif to screen for a receptor and identified cd74 as the mif receptor. the authors described that mif bound to the extracellular domain of cd74, resulting in extracellular signal-regulated kinase (erk) pathway activation (25) . mif-induced erk activation through cd74 appears to depend on cd74 forming a complex with co-receptor cd44 (cd74/cd44) (26, 27) . in addition to erk, stimulation of cd74 has been shown to trigger activation of the pi3k-akt signal transduction cascade, nf-κb, and the amp-activated protein kinase (ampk) pathways. these pathways play important roles in cell proliferation and survival (28) . d-dopachrome tautomerase (d-dt, mif-2) was recently described as a member of the mif protein superfamily, demonstrating overlapping inflammatory and proliferative properties with mif. d-dt and mif genes are located in close proximity on chromosome 22, ∼80 kb apart. the amino acid sequence of human mif and d-dt shows 34% identity, however, the structure of the two proteins is highly conserved. d-dt binds cd74 and initiates similar signaling pathways (29, 30) . mif homologs are also expressed by parasites. these mif homologs are structurally and functionally similar to human mif and interact with cd74. while it may seem counter-intuitive for protozoans to secrete mif, parasite mif appears to contribute to immune evasion and invasion (31) (32) (33) . regulation of mif-cd74 interactions occurs at several levels. mif is constitutively expressed with increased mif secretion occurring early in the inflammatory response. triggers of increased mif release include lipopolysaccharide (lps) and cell injury. secreted mif then interacts with cd74 to carry out some if its functions (10, 14) . cd74 activity is regulated by changes in expression, proteolytic processing, and mifinteracting proteins that prevent binding to cd74. similar to mif, cd74 is expressed on multiple cells: immune cells (e.g., b lymphocytes, macrophages, dendritic cells) and non-immune cells including epithelial cells. information on the regulation of cd74 expression in these different cells remains limited. increased cd74 expression is observed in injury, inflammation, and cancer. ifn-γ, a cytokine crucial to both innate and adaptive immunity, increases cd74 expression in a variety of cells (34) (35) (36) . intracellular binding partners released in the extracellular space can regulate cytokine activity. both ribosomal protein s19 (rps19) and c-jun activation domain binding protein 1 (jab1) were shown to have regulatory effects by binding to mif, inhibiting its interaction with cd74 (37, 38) . cd74 also exists in a soluble cd74 ectodomain form which results from proteolytic shedding of the ectodomain region. however, the molecular mechanism including the protease responsible for releasing cd74 ectodomain remains poorly understood. ectodomain shedding decreases the amount of cd74 surface receptors available to interact with mif. also, cd74 ectodomain regulates mif activity by acting as a decoy receptor, sequestering free mif to negatively regulate mif signaling (39) (40) (41) . another proteolytic step involves signal peptide peptidase-like 2a (sppl2a), which is an aspartic intramembrane protease. sppl2a has shown to play an important role in cd74 proteolysis (42, 43) . yet, the exact role of sppl2a-mediated cd74 proteolysis in mif signaling and whether modulating sppl2a enzyme activity affects mif proinflammatory and proliferative functions remain to be fully investigated (16) . in the following sections, we summarize the recent data supporting the reparative role of mif-cd74 signaling in different organs and tissues during injury. the role of cd74 in other disease processes, antigen presentation, and cancer has been well-reviewed elsewhere (16, 16-18, 28, 44-48) . inflammatory bowel disease (ibd), exemplified by crohn's disease (cd) and ulcerative colitis (uc), is a growing public health challenge and socio-economic problem that affect millions with rapidly increasing incidence worldwide (49, 50) . mucosal healing has been established as an important treatment predictor of sustained clinical remission and resection-free survival in ibd (51) . unfortunately, a significant number of ibd patients do not respond to current treatment (including corticosteroids or biologics), and as many as 70% of cd and 25% of uc patients require surgical resection of affected regions of their intestine (52) . current therapeutic strategies focus on limiting inflammation, thus, there is an urgent need to develop new approaches that also facilitate tissue repair and mucosal healing. our understanding of the genetic contributions to ibd has seen significant advances over the past few decades. genome-wide association studies (gwas) have identified new single nucleotide polymorphisms (snps) associated with ibd predisposition and treatment failure (53, 54) . a recent study aimed at determining genetic factors associated with poor response to anti-tnf therapy, found that a strong association between a cd74 polymorphism and anti-tnf failure in patients with ulcerative colitis. the rs7709772 snp is located in the cd74 promoter region. the odds ratio for non-response to anti-tnf therapy with this snp was relatively high at 22 (55) . cd74 gene expression is increased in patients with ibd (56, 57) , which occurs in the inflamed areas compared with noninflamed and healthy intestine (figure 2 ). cd74 overexpression was most noticeable in proliferating crypt epithelial cells of patients with ibd and amebic colitis, a condition often misdiagnosed as ibd (58, 59) . cd74 is almost undetectable in the epithelium of non-inflamed human and mice intestine when analyzed by immunohistochemistry (58, 60, 61) . therefore, it was not too surprising to find that cd74 deficient mice had normal colon, histology, and barrier integrity, and lacked spontaneous colitis in the absence of pathologic insults (58) . on the other hand, mif is expressed by epithelial cells that line the intestine and mif-deficient mice have impaired intestinal barrier integrity (62) . using a combination of genetic knock-out, bone marrow chimera mice, chemical, non-chemically-induced, acute, and chronic mouse models of colitis, cd74 was found to be essential for mucosal healing in colitis-associated injury. at the cellular level, mif stimulation of cd74 on intestinal epithelial cells increased cell proliferation and wound closure, an effect that was lost in cd74-deficient cells. mechanistically, mif, which also is increased in colitis, stimulated the cd74 receptor, activating proproliferative akt and erk pathways (58) . so while dispensable in steady state conditions, cd74 appears to be necessary for reparative inflammation. based on these findings, enhancing the cd74 pathway might represent a unique treatment approach for promoting healing in ibd. though, finding the right ligand to stimulate cd74 may present a challenge. that is, stimulation of cd74 with exogenous mif might lead to an excessive inflammatory state, as mif is capable of stimulating cxcr2 and cxcr4 receptors in addition to cd74. cxcr2 and cxcr4 receptors when activated promote influx of neutrophils and lymphocytes, respectively (63, 64) . lung injury arises from a wide variety of insults, which include pulmonary infections, such as bacterial and viral pneumonia caused by influenza and coronavirus, vaping-associated pulmonary illness (vapi), ischemia-reperfusion-induced lung injury, and ventilator-induced lung injury (65) (66) (67) . in the 2018-2019 season, influenza caused around 500,000 hospitalizations and 34,000 deaths (68) . the emerging covid-19 has an increasing impact through infections and deaths as well as the economic impacts of quarantines and event cancellations to reduce infection spread (69, 70) . lung injury causes damage to the epithelium. the alveolar epithelial barrier consists of two main cell types: alveolar epithelial type i and type ii cells. type i cells are flat cells through which gas exchange takes place and occupies most of the alveolar surface area. type ii cells serve as progenitor cells for the alveolar epithelium. type i cells are more sensitive to injury and are predominantly destroyed during lung damage. type ii cells proliferate and differentiate into type i cells, thus actively reforming the alveolar epithelium after damage and promoting alveolar repair (71) . type ii cells express cd74 on their surface. during acute injury such as viral infection, type i cells release mif. extracellular mif binds to cd74 on adjacent type ii epithelial cells, activating akt and erk pathways, resulting in cell proliferation and differentiation to restore the alveolar barrier (72) . lung endothelial cells display almost undetectable amounts of cd74 at baseline. a recent study found that chronic hyperoxia led to cd74 upregulation in endothelial cells (73) . hyperoxia is common in patients with adult respiratory distress syndrome (ards), which is due to the requirement for high levels of supplemental oxygen. endothelial injury is a key feature of hyperoxic acute lung injury (74) . mif-cd74 activation was found to protect from oxidative stress in an animal model. mif and cd74 genetic knock-outs, and pharmacological inhibition of cd74 resulted in loss of the protective effects of cd74. this led to increases in inflammatory cytokines, apoptosis, and mortality. at the molecular level, cd74 activation during hyperoxia induced proliferative and pro-survival effects through erk and akt activation (73) . neutrophils appear to play a significant role in tissue damage and the development of acute lung injury (75) . it is important to mention that excess mif was shown to correlate with neutrophil accumulation into the lung (76) . however, it remains unclear how much mif-cxcr2 interaction is contributing to leukocyte recruitment. acute kidney injury (aki) remains a significant medical problem and is associated with increased hospital mortality, length of stay, and costs. individuals who survive an aki hospitalization are likely to fail renal function recovery and go on to develop chronic kidney disease and hypertension (77) . most cases of aki are due to ischemia, but our kidneys are also vulnerable to damage by toxins, infection, and immune-mediated insults. ischemic aki, for example, results in significant renal tubular cell damage. free radicals formed during ischemia and reperfusion (i/r) also contribute to renal damage. surviving cells undergo epithelium regeneration to restore healthy renal function (78, 79) . a better understanding of the repair processes underlying kidney repair will facilitate therapies that will prevent injury, promote recovery, and minimize the progression to chronic kidney disease. cd74 is expressed on the surface of renal tubular epithelial cells. also, these cells express low levels of mif which is increased following aki to ensure adequate supplies at the site of damage (80, 81) . a spontaneous pathological renal phenotype is absent mif knock-out mice, suggesting little to no effect on healthy organs (82) . however, high mif levels can be found in the serum of patients following cardiac surgery and correlates with protection from aki (81) . in a murine model of experimental ischemia-reperfusion injury, mif, mif-2, and cd74 knock-out mice had worse tubular injury compared to wild type control mice. mif-2 improved the recovery of injured epithelial cells by enhancing cell regeneration through secretory leukocyte proteinase inhibitor (slpi) and activating transcription factor (atf) 4-dependent mechanisms (83) . slpi has proliferative, antioxidant and cytoprotective properties, and is being evaluated as a biomarker for aki after surgery (84) (85) (86) . while mif/mif-2 are likely protective in ir, this might not be the case for all renal diseases depending on the underlying pathology. for example, mif has been linked to injury and inflammation in models of glomerular diseases (87) (88) (89) . therefore, additional studies are required to determine which patient conditions would benefit from blockade vs. stimulation strategies. cardiovascular disease is the leading cause of death in the united states. risk factors for cardiovascular disease include smoking, obesity, and hypertension. myocardial infarction, or heart attack, occurs in one american every 40 s (90) . treatment for mi is composed of anti-coagulant medication, thrombolytics, and surgical intervention to restore normal blood flow. however, damage to cardiomyocytes caused by ischemia is not addressed in the standard treatment regimen and can lead to heart failure. targeting repair of heart tissue during mi may improve patient outcomes and prevent chronic disease. cd74 signaling was shown to have protective effects in cardiomyocytes in cardiac i/r injury animal model. mif is secreted from the cardiomyocytes during i/r and acts in an autocrine-paracrine manner, stimulating cell surface cd74 receptor. activation of cd74 with exogenous mif-2 improved cell survival and infarct size both in wild-type control and conditional mif-2 knockout mice, while cd74 deletion led to worse injury. mechanistically, mif-2 binding to cd74 quickly activates the amp-activated protein kinase (ampk) cascade via a calcium dependent kinase, cammk2 (91) . activation of the ampk pathway in cardiomyocytes decreases apoptosis, necrosis, and contractile dysfunction following ischemia (91, 92) . mif-2 in contrast to mif appears to lack the necessary cxcr-interacting motifs necessary for activation, and it is believed to exert a more selective action in activating the tissue-protective cd74 signaling pathway. that said, mif triggers the cd74/cd44/ampk receptor signaling pathway, which promotes glucose uptake in cardiomyocytes and protects the heart during ischemia-reperfusion injury (93, 94) . further studies are required to determine the potential of mif/mif-2 as a treatment strategy to protect the heart against ischemic injury. impaired wound healing in the setting of non-healing surgical or traumatic wounds, pressure ulcers, diabetic foot ulcers, venous, and ischemic ulcers, presents a substantial healthcare burden. chronic non-healing wounds contribute to significant healthcare costs, poor quality of life, and serious outcomes such as amputations (95, 96) . following injury, several cytokines play important roles during tissue repair and promote cutaneous wound healing by the classic stages of wound repair: inflammation, new tissue formation, and remodeling (97, 98) . therefore, cytokine pathways have been targeted when designing regenerative strategies to promote chronic wound repair (99) . gene expression studies have been valuable for identifying cytokines expressed during the inflammatory process in a wound setting (100) . a study analyzing gene expression profiles in patients with punch biopsies found mif gene expression increased during cutaneous wound healing (101) . the role of mif in promoting wound healing was investigated using an animal model of skin injury. mif levels were elevated early after injury and facilitated proliferation and migration of keratinocytes from the edge of the wound (102) . these results support a reparative response of mif to cutaneous injury. in addition, transcriptomic analysis revealed cd74 upregulated in pressure ulcers in a neuropathic ulcer mouse model (103) . it is plausible that the mif-cd74 pathway promotes cutaneous wound repair, however, further studies will be required to characterize the role of cd74 signaling in cutaneous wound healing. cd74 signaling has also been found to play a potential role in healing in other tissues such as the nervous system and liver. sciatica is a chronically painful disease caused by injury to the sciatic nerve. schwann cells express cd74, and mif is upregulated following sciatic nerve injury. mif-stimulated cd74 activation of the erk pathway led to schwann cell proliferation and subsequent nerve regeneration. also, in vitro studies show that mif facilitates schwann cell migration. both schwann cell proliferation and migration promote nerve regeneration (104) . a separate in vitro study demonstrated that cd74 activation by mif promoted cell survival and proliferation of neural progenitor cells (105) . further studies will be required to determine if mif-induced proliferation of neural progenitor cells can be a therapeutic option in brain disorders. in the liver, cd74-mif signaling plays a protective role in nonalcoholic fatty liver disease (nafld) by enhancing ampk (106) . while this review focuses on the protective role of mif-cd74 signaling, it should be noted that this is not the case for all diseases (18, 44, 107) . the complex pathological processes that result in disease combined with cd74's expression on a variety of cell types, and its multiple co-receptors with diverse downstream signaling pathways contribute to these varied outcomes. for example, lupus nephritis is inflammation of the kidney that is caused by the autoimmune disease systemic lupus erythematosus (sle) (108) . b cells participate in sle immunopathogenesis (109) . b lymphocytes express elevated levels of cd74 in mouse models of sle and lupus-prone mouse strains have elevated mif. both mif and cd74 elevated expression positively correlated with worsening inflammation. mif inhibition and cd74 deficiency protected against glomerulonephritis in lupusprone mice (110, 111) . despite these results that suggest mif-cd74 pathway plays a role in lupus pathology, a phase 1 clinical trial of an anti-mif monoclonal antibody in lupus nephritis was terminated early for unclear reasons (112) . these findings suggest that mif-cd74 functions with differential outcomes occur in a context-and cell type-dependent manner. given this complexity, additional research is needed to determine when and how to inhibit or stimulate the mif-cd74 pathway to achieve benefit. also, whether disease associations are a result of different coreceptor involvement on different cell types should be a focus of future research. mif's proinflammatory effects involve enhancing the expression of various cytokines such as tnf-α, il-6, il-8 (14) . cytokines like il-6 are now recognized for their roles triggering tissue repair and regeneration (4, 113) . while these downstream proinflammatory mif effects have been linked to immune disorders, it remains possible that they play a role in the healing effects of mif-cd74 signaling. this would be an interesting area for future investigation as balancing the positive and negative effects of mif appears to be key. discussed above is the recurrent observations of the protective effects of mif-cd74 signaling in wound-healing. recent studies have furthered our understanding of the mechanisms by which cd74 stimulation leads to tissue repair in multiple parts of the body involving some of the most important diseases. despite these advances, key questions remain unanswered. for example, although there is mechanistic overlap, the downstream pathways that are important for cd74-mediated repair appear to vary with the tissue or cell type. in epithelial cells, such as those that line the gut and alveoli of the lungs, mif-cd74 interaction triggers the activation of pro-survival and proliferative akt and erk pathways. in contrast, activation of the pro-survival kinase ampk seems to play a more significant role in cardiomyocytes and hepatocytes. the molecular reason for the different downstream signaling pathways beyond differences in cell types is not fully understood and present worthy unknowns to be solved by future studies. furthermore, a selective agonist that will stimulate cd74-mediated repair with little or no unwanted side effects remains poorly defined. the answers to such questions may allow us to translate these recent scientific discoveries into clinical interventions, and ultimately benefit those suffering as a result of injury to various organs and tissues. lf, sg, and sm wrote, edited and reviewed the manuscript. all authors contributed to the article and approved the submitted version. this work was supported by national institutes of health (nih) r01ai026649-s1, k08ai119181, uva seed grant, and the robert wood johnson foundation-harold amos medical faculty development program award. tissue repair: the hidden drama wound repair and regeneration inflammation and metabolism in tissue repair and regeneration reparative inflammation takes charge of tissue regeneration mechanism of a reaction in vitro associated with delayed-type hypersensitivity delayed hypersensitivity in vitro: its mediation by cell-free substances formed by lymphoid cell-antigen interaction mif is a pituitary-derived cytokine that potentiates lethal endotoxaemia molecular cloning of a cdna encoding a human macrophage migration inhibitory factor a proinflammatory cytokine inhibits p53 tumor suppressor activity macrophage migration inhibitory factor: a regulator of innate immunity cytokine patterns in patients with cancer: a systematic review macrophage migration inhibitory factor: a probable link between inflammation and cancer. immunity il-8 secreted in a macrophage migration-inhibitory factor-and cd74-dependent manner regulates b cell chronic lymphocytic leukemia survival rediscovering mif: new tricks for an old cytokine enhanced expression of macrophage migration inhibitory factor in prostatic adenocarcinoma metastases the multifaceted roles of the invariant chain cd74-more than just a chaperone the integral role of cd74 in antigen presentation, mif signal transduction, and b cell survival and homeostasis mif-and cd74-dependent, mechanisms alternative splicing and alternative initiation of translation explain the four forms of the ia antigen-associated invariant chain one gene encodes two distinct ia-associated invariant chains an alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin detection of a common polypeptide chain in ia and ie sub-region immunoprecipitates a role of laassociated invariant chains in antigen processing and pressentation surface expression of the invariant chain (cd74) is independent of concomitant expression of major histocompatibility complex class ii antigens mif signal transduction initiated by binding to cd74 cd44 is the signaling component of the macrophage migration inhibitory factor-cd74 receptor complex the chondroitin sulfate form of invariant chain can enhance stimulation of t cell responses through interaction with cd44 the biological function and significance of cd74 in immune diseases the d-dopachrome tautomerase (ddt) gene product is a cytokine and functional homolog of macrophage migration inhibitory factor (mif) d-dopachrome tautomerase (d-dt or mif-2): doubling the mif cytokine family parasite-produced mif cytokine: role in immune evasion, invasion, and pathogenesis the non-mammalian mif superfamily targeting parasite-produced macrophage migration inhibitory factor as an antivirulence strategy with antibiotic-antibody combination to reduce tissue damage cell surface cd74-mif interactions drive melanoma survival in response to interferon-γ the untold story of ifn-γ in cancer biology pleiotropic role of macrophage migration inhibitory factor in cancer ribosomal protein s19 interacts with macrophage migration inhibitory factor and attenuates its pro-inflammatory function interaction between parasite-encoded jab1/csn5 and macrophage migration inhibitory factor proteins attenuates its proinflammatory function cd74 is a member of the regulated intramembrane proteolysisprocessed protein family interaction of mif family proteins in myocardial ischemia/reperfusion damage and their influence on clinical outcome of cardiac surgery patients the role of macrophage migration inhibitory factor in autoimmune liver disease the intramembrane protease sppl2a promotes b cell development and controls endosomal traffic by cleavage of the invariant chain substrate determinants of signal peptide peptidase-like 2a (sppl2a)-mediated intramembrane proteolysis of the invariant chain cd74 cd74: an emerging opportunity as a therapeutic target in cancer and autoimmune disease cd74 in antigen presentation, inflammation, and cancers of the gastrointestinal tract cd74-nrg1 fusions in lung adenocarcinoma cd74: a new candidate target for the immunotherapy of b-cell neoplasms worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population-based studies the global, regional, and national burden of inflammatory bowel disease in 195 countries and territories, 1990-2017: a systematic analysis for the global burden of disease study current and emerging therapeutic targets for ibd update on the surgical treatment of inflammatory bowel disease inflammatory bowel disease: genetics, epigenetics, and pathogenesis genetics of inflammatory bowel diseases colonic phenotypes are associated with poorer response to anti-tnf therapies in patients with ibd ulcerative colitis and crohn's disease: distinctive gene expression profiles and novel susceptibility candidate genes colonic epithelial cell diversity in health and inflammatory bowel disease cd74 signaling links inflammation to intestinal epithelial cell regeneration and promotes mucosal healing fulminant amebic colitis after corticosteroid therapy: a systematic review invariant chain expression in colon neoplasms influence of major histocompatibility complex class i and ii antigens on survival in colorectal carcinoma the role of macrophage migration inhibitory factor in the function of intestinal barrier mif is a noncognate ligand of cxc chemokine receptors in inflammatory and atherogenic cell recruitment structural determinants of mif functions in cxcr2-mediated inflammatory and atherogenic leukocyte recruitment pathology of vaping-associated lung injury ischemia-reperfusioninduced lung injury ventilator-induced lung injury annual estimates of the burden of seasonal influenza in the united states: a tool for strengthening influenza surveillance and preparedness early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia first case of 2019 novel coronavirus in the united states biology of alveolar type ii cells surface expression of cd74 by type ii alveolar epithelial cells: a potential mechanism for macrophage migration inhibitory factor-induced epithelial repair endothelial cd74 mediates macrophage migration inhibitory factor protection in hyperoxic lung injury hyperoxic acute lung injury neutrophil recruitment and function in health and inflammation macrophage cd74 contributes to mif-induced pulmonary inflammation trends in hospitalizations for acute kidney injury -united states cellular pathophysiology of ischemic acute kidney injury update on mechanisms of schemic acute kidney injury an essential mif-cd74 signaling axis in kidney tubular regeneration, with prospects for precision medicine and pharmacological augmentation the protective role of macrophage migration inhibitory factor in acute kidney injury after cardiac surgery macrophage migration inhibitory factor limits renal inflammation and fibrosis by counteracting tubular cell cycle arrest mif-2/d-dt enhances proximal tubular cell regeneration through slpi-and atf4-dependent mechanisms slpi-a biomarker of acute kidney injury after open and endovascular thoracoabdominal aortic aneurysm (taaa) repair secretory leukocyte protease inhibitor (slpi)-a novel predictive biomarker of acute kidney injury after cardiac surgery: a prospective observational study secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes a smallmolecule macrophage migration inhibitory factor antagonist protects against glomerulonephritis in lupus-prone nzb/nzw f1 and mrl/lpr mice macrophage migration inhibitory factor deficiency attenuates macrophage recruitment, glomerulonephritis, and lethality in mrl/lpr mice antimacrophage migration inhibitory factor reduces transforming growth factor-β1 expression in experimental iga nephropathy heart disease and stroke statistics-2019 update: a report from the american heart association the vestigial enzyme ddopachrome tautomerase protects the heart against ischemic injury ampk: energy sensor and survival mechanism in the ischemic heart macrophage migration inhibitory factor stimulates amp-activated protein kinase in the ischaemic heart exosomes from mesenchymal stem cells overexpressing mif enhance myocardial repair human skin wounds: a major and snowballing threat to public health and the economy the humanistic and economic burden of chronic wounds: a protocol for a systematic review regulation of wound healing by growth factors and cytokines exploring the role of stem cells in cutaneous wound healing immune regulation of skin wound healing: mechanisms and novel therapeutic targets wound repair at a glance gene expression profiling of cutaneous wound healing enhancement of macrophage migration inhibitory factor (mif) expression in injured epidermis and cultured fibroblasts transcriptomic analysis of cutaneous inflammatory biomarkers in a mouse model of small fiber neuropathy mif/cd74 axis participates in inflammatory activation of schwann cells following sciatic nerve injury macrophage migration inhibitory factor (mif) promotes cell survival and proliferation of neural stem/progenitor cells protective role of macrophage migration inhibitory factor in nonalcoholic steatohepatitis contribution of the macrophage migration inhibitory factor superfamily of cytokines in the pathogenesis of preclinical and human multiple sclerosis: in silico and in vivo evidences update on lupus nephritis mechanisms of b cell autoimmunity in sle cd74 deficiency mitigates systemic lupus erythematosus-like autoimmunity and pathological findings in mice a role for the b-cell cd74/macrophage migration inhibitory factor pathway in the immunomodulation of systemic lupus erythematosus by a therapeutic tolerogenic peptide anti-macrophage migration inhibitory factor (anti-mif) antibody in lupus nephritis a gp130-src-yap module links inflammation to epithelial regeneration we thank richard bucala md, ph.d. (yale university) for the support. we apologize to those colleagues whose work could not be included due to space limitations. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 farr, ghosh and moonah. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-312075-asbt0mcj authors: schulz, katharina s.; mossman, karen l. title: viral evasion strategies in type i ifn signaling – a summary of recent developments date: 2016-11-11 journal: front immunol doi: 10.3389/fimmu.2016.00498 sha: doc_id: 312075 cord_uid: asbt0mcj the immune system protects the organism against infections and the damage associated with them. the first line of defense against pathogens is the innate immune response. in the case of a viral infection, it induces the interferon (ifn) signaling cascade and eventually the expression of type i ifn, which then causes an antiviral state in the cells. however, many viruses have developed strategies to counteract this mechanism and prevent the production of ifn. in order to modulate or inhibit the ifn signaling cascade in their favor, viruses have found ways to interfere at every single step of the cascade, for example, by inducing protein degradation or cleavage, or by mediate protein polyubiquitination. in this article, we will review examples of viruses that modulate the ifn response and describe the mechanisms they use. the mammalian immune system evolved to detect and fight viral infections effectively. the induction of type i interferon (ifn), predominantly ifn-α and ifn-β, forms the first line of defense. the type i ifn response consists of two parts. first, the cell produces type i ifn, when triggered by a viral stimulus. the ifn is then secreted and, in the second part of the response, it is sensed by the producing, as well as neighboring cells, resulting in the production of ifn-stimulated genes (isgs) [reviewed in ref. (1) ]. viruses, which have coevolved with their host, develop strategies to counteract the signaling cascades of the innate immune system and ensure their replication. recently, several reviews were published, describing the innate immune evasion strategies of individual viruses or virus families, such as influenza virus (2, 3) , phleboviruses (4), herpes viruses (5-7), coronaviruses severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) (8) , human immunodeficiency virus (hiv) (9, 10) , as well as multiple rna viruses (11, 12) . moreover, there are recent articles that review how viruses prevent detection by pathogen recognition receptors (prrs) (13, 14) and how viruses modulate innate immune signaling by use of viral deubiquitinases (15) . in this review, we will compare the different strategies viruses have developed to suppress innate immune signaling of individual components of the innate immune signaling cascade. due to the tremendous amount of data in this field, we will focus on recent discoveries. older studies were summarized in ref. (16, 17) . (14) ]. the most important viral markers for the innate immune system are viral nucleic acids. the detection of viral dna through the cgas-sting pathway and the counter measurements taken by viruses have been reviewed recently (18) and are not part of this review. viral rnas, which are mostly double-stranded (ds-)rna, are recognized by three prrs: the endosomal toll-like receptor 3 (tlr3), the cytoplasmic retinoic acid-inducible gene i (rig-i)like receptors (rlrs), and the nucleotide-oligomerization domain (nod)-like receptors (nlrs) (19) . tlr3 and the rlrs are important for inducing the type i ifn response, whereas nlrs have been shown to regulate interleukin-1β (il-1β) maturation through activation of caspase-1 (20) . the group of rlrs consists of rig-i, melanoma differentiation-associated gene 5 (mda5), and laboratory of genetics and physiology 2 (lgp2). the three receptors have a similar structure, all containing a caboxyterminal domain, which functions as a repressor domain (rd) in rig-i and lgp2 (21) and a central helicase domain, but lgp2 lacks the caspase activation and recruitment domains (cards) that function in signaling [reviewed in ref. (19, 22) ]. both the helicase and the carboxy-terminal domain are required for rna binding. rig-1 and mda-5 detect specific viral rna pamps, while lgp2 negatively regulates rig-i signaling and promotes rna binding to mda5 [reviewed in detail in ref. (14) ]. in unstimulated cells, rig-i and mda-5 are kept in a repressed state due to phosphorylations on serine and threonine residues in the cards and carboxy-terminal domains (23, 24) . upon binding of rna, both rig-i and mda-5 undergo conformational changes, resulting in release of their cards (25, 26) . recruited phosphatases remove the phosphate residues, and e3 ubiquitin ligases attach lys63-linked ubiquitin polymers onto the cards and c-terminal domain of rig-i, which are important for rig-i tetramerization (27) (28) (29) (30) (31) . rna-bound rig-1 then interacts with 14-3-3ε, a mitochondrial trafficking protein, and the trim25 ubiquitin ligase, which together transport rig-i to the mitochondria (32) . there the cards of rig-i or mda-5 interact with the card of the mitochondrial activator of virus signaling (mavs, also known as ips-1, visa, and cardif), which is an essential signaling adaptor protein. the activation of mavs has recently been reviewed in detail in ref. (33) . tlr3 interacts with trif, which serves as a molecular platform and forms physical interactions with several adaptor molecules (34) . by interacting with upstream adaptors, trif undergoes conformational changes and recruits the downstream tnf receptor-associated factor (traf)3 and traf6 [reviewed in ref. (35) ]. the kinase receptor-interacting protein-1 (rip-1) is part of both the signaling pathways downstream of tlr3 and rig-i. it can interact with trif to induce nfκb activation (36) . moreover, the dsrna-activated tlr3 can recruit trif, rip-1, and caspase-8 and induce apoptosis (37) . also, rip-1 and its adaptor protein fas-associated protein with death domain (fadd) are part of the signaling cascade downstream of rig-i and mda-5 and involved in the activation of the transcription factors interferon regulatory factor (irf)3 and irf7 (38) . traf3 serves as a linker between the upstream adaptor proteins (trif or myd88 for tlrs and mavs for rlrs) and the downstream signaling kinases tbk1/ikkε or irak1/ikkα. the recruitment of traf3 to the tlr or rlr signaling complexes activates the e3 ligase activity of traf3, which then catalyzes its own k63-linked ubiquitinylation. subsequent traf3 activates tbk1/ikkε or irak/ikkα [reviewed in ref. (39) ] (figure 1) . viruses target rig-i directly or indirectly to block the type i ifn response. the phlebovirus toscana virus expresses a non-structural protein, which directly interacts with rig-i and induces its proteasomal degradation (40, 41) . foot-and-mouth disease virus (fmdv) proteins l pro , 3c pro , and 2b increase the rig-i mrna expression but decrease the protein expression of rig-i. l pro and 3c pro both induce rig-i degradation, whereas the mechanism of how 2b reduces rig-i protein levels has not been solved yet (42) . other viruses target rig-i indirectly. hepatitis b virus (hbv) induces mir146a, which then posttranscriptionally inhibits the expression of rig-i and suppresses the production of type i ifn (43) . the dengue virus ns3 protein binds to 14-3-3ε and prevents the translocation of rig-i to mavs. the binding site on ns3 is a highly conserved phosphomimetic motif, which was verified by generation of a virus containing a mutation in this motif (44) . it has been proposed that in certain cell types rig-i requires sentinels, such as the protein ddx60, which associates with rig-i and promotes the rig-i rna-binding activity (45, 46) . other studies question ddx60 acting as a broadly active enhancer of antiviral responses (47, 48) and instead suggest that ddx60 only functions in the antiviral response to specific viruses, such as hepatitis c virus (47) . however, there are data indicating that influenza a virus and hepatitis c virus attenuate ifnβ-promoter activation by targeting the sentinel ddx60. both viruses activate the epidermal growth factor (egf) receptor, which in turn phosphorylates ddx60 on tyr-793 and tyr-796. this results in the attenuation of ddx60-dependent rig-i activation. in addition, independent of its role as sentinel for rig-i viral rna recognition, ddx60 plays a role in viral rna degradation (46) (figure 1 ). mitochondrial activator of virus signaling is blocked by different viruses in various ways. the dengue virus protein ns4a targets mavs, and the interaction prevents the binding of mavs to rig-i (49) . the porcine reproductive and respiratory syndrome virus (prrsv) 3c-like protease (3clsp), by contrast, cleaves mavs in a proteasome-and caspase-independent manner at glu268 (e268/g269). both cleavage products fail to activate the type i ifn response (50) . likewise, the hepatitis c virus protein ns3-4a (51, 52) , as well as the highly pathogenic porcine reproductive and respiratory syndrome virus (hp-prrsv) protein nsp4 (53) have been shown to cleave mavs and block rlr signaling. the porcine epidemic diarrhea virus (pedv) also targets mavs in small intestinal epithelial cells (iecs). however, the exact mechanism has not been solved yet (54) (figure 1) . the sars coronavirus protein orf9b not only influences antiviral signaling but also alters host cell mitochondria morphology by inducing degradation of the dynamin-like protein (drp1). mavs becomes concentrated into small puncta in the presence (56) . in addition to mavs, also the levels of traf3 and traf6 are reduced by orf9b. however, it is unlikely that traf3 and traf6 are targeted directly. more likely, they are degraded due to their interaction with mavs (55) (figure 1) . human t-cell lymphotropic virus type i (htlv-1) protein tax disrupts innate immune signaling in multiple ways: it binds to the rip homotypic interaction motif (rhim) domains of rip-1 and disrupts the interaction between rip-1 and rig-i or mda-5 and the activation of the type i ifn promoter. tax also binds to trif and thereby interrupts the tlr3 signaling cascade. finally, tax blocks the association between rip-1 and irf7, which resulted in repression of the irf7 activity (57) (figure 1) . middle east respiratory syndrome coronavirus m protein interacts with traf3 and disrupts the interaction between traf3 and tbk1, which ultimately leads to a reduced irf3 activation. for the interaction with traf3, the n-terminal transmembrane domain of the mers-cov m protein is sufficient (58) , similar to what has been shown for sars-cov before (59) (figure 1 ). triggering of the tlr3-and rlr-signaling cascade results in the activation of the transcription factors nfκb and irf3/irf7. in its inactive state, the transcription factor nfκb is complexed with its inhibitor iκb (60) . upon stimulation, iκb is phosphorylated by the iκb kinase (ikk) complex, which is composed of two catalytic subunits, such as ikkα and ikkβ, and a regulatory subunit, such as nfκb essential modulator (nemo) (61) . the phosphorylation of iκbα induces its polyubiquitination through the e3 ubiquitin ligase β-transducin repeat-containing protein (β-trcp) and subsequent proteasomal degradation (62) , allowing nfκb to translocate into the nucleus and induce the expression of target genes (63) (figure 2) . encephalomyocarditis virus (emcv) protein 3c cleaves traf family member-associated nfκb activator (tank), which inhibits traf6-mediated nfκb activation, on gln291. as a result, nfκb is activated and the unstable c-terminal fragment of tank is subjected to proteasomal degradation (64) . also, other viruses express proteases that cleave tank, although on other residues, such as porcine reproductive and respiratory syndrome virus (prrsv) (tank is cleaved by nsp4), fmdv (protease 3c cleaves tank), and equine arteritis virus (eav) (tank is cleaved by nsp4). thus, tank seems to be a common target of several positive rna viral proteases (64) (figure 2) . several viruses have been shown to disrupt ifn signaling by cleaving nemo. pedv 3c-like protease, nsp5, cleaves nemo at gln231 (65), whereas the hepatitis a virus 3c protease (3c pro ) cleaves nemo at gln304 (66) and the picornavirus fmdv protease 3c pro at gln383, removing the c-terminal zinc finger domain from the protein (67) . the human rotavirus has developed another way. its non-structural protein 1 (nsp1) has been shown to inhibit the nfκb pathway by degrading β-trcp and consequently stabilizing iκb (68) (figure 2) . tank-binding kinase 1 (tbk1) and inhibitor of κb kinase ε (ikkε) are classified as non-canonical serine/threonine kinases and are both able to induce irf3 and irf7 phosphorylation and subsequent dimerization (69) (70) (71) (72) . however, while tbk1 is constitutively expressed in most cell types, the expression of ikkε is more restricted (73) . upon stimulation, tbk1 and ikkε are recruited by adaptor proteins to signaling complexes to be activated by phosphorylation on ser172 and both have been shown to be subjected to k63-linked polyubiquitination [reviewed in ref. (73, 74) ]. for tbk1, k63-linked polyubiquitination seems to be important for tlr-and rlr-induced ifn production, as ubiquitin chains might serve as a platform for the assembly of tbk1 signaling complexes. moreover, deubiquitinases are able to terminate the tbk1-mediated pathway by cleaving the k63linked ubiquitin chains [reviewed in ref. (74, 75) ]. activated tbk1/ikkε phosphorylates irf3 and/or irf7 in the cytosol at specific serine residues. this phosphorylation results in homo-or heterodimerization of irf3 and irf7 and nuclear translocation (76, 77) . interestingly, while irf3 is constitutively expressed, irf7 is expressed at low levels in most cell types and expression is induced upon ifn signaling. therefore, in most cells, irf7 strongly enhances the production of ifn [reviewed in ref. (78) ]. once phosphorylated irf3 and/or irf7 dimers have translocated into the nucleus, they bind to the transcription coactivator creb-binding protein (cpb)/p300 (79, 80) . together with other factors, such as nfκb, they form the enhanceosome on the ifnβ promoter and induce the expression of type i ifn [reviewed in ref. (76) ]. the viral proteins that target tbk1 act by either blocking activation of tbk1 by mavs or by inhibiting activation of irf3 by tbk1. the mers-cov protein orf4b blocks ifnβ production by binding to tbk1 and ikkε and suppressing the formation of a mavs/ikkε complex (81) . in addition to inhibiting tbk1/ikkε activation, orf4b can also inhibit the production of ifnβ in the nucleus; however, the mechanism has not been solved yet (81) . recently, two herpes simplex virus proteins have been shown to target tbk1/ikkε and inhibit the phosphorylation of irf3: icp27 (82) and vp24 (83) . also, dengue virus serotype 4 non-structural proteins ns2a and ns4b, as well as the ns2a and ns4b proteins of other dengue viruses, inhibit the phosphorylation of tbk1 (84) and pedv n protein has been shown to interact with tbk1, hampering the association of tbk1 with irf3 and preventing the activation of irf3 activation (85) . the human t-cell leukemia virus type 1 oncoprotein tax has been shown to also interact with tbk1. however, studies came to contradicting results on how that influences the production of ifnβ. while one group showed that tax activates tbk1 and the production of ifnβ (86), another group showed that tax suppresses the ifn production by interaction with tbk1 (87) . interestingly, when a recent study tested how the rabies virus p protein of street strains behaves compared to laboratory-adapted strains with regard to the induction of type i ifn, they found that both street strains and laboratory strains inhibit tbk1-mediated signaling, but only the p protein of street strains also interacts with and inhibits ikkε-inducible irf3dependent ifnβ expression (88) (figure 1) . interferon regulatory factor 3 is targeted by many viruses to impair innate immune signaling. most viruses inhibit the phosphorylation and thereby also the dimerization and translocation of irf3, such as the porcine deltacoronavirus (89) or poliovirus (90) . hepatitis e virus protein orf3 also suppresses irf3 phosphorylation, but in an indirect way. it activates the signal regulator protein α (sirp-α), which negatively regulates type i ifn induction (91) . in contrast, porcine bocavirus (pbov) np1 protein does not affect irf3 expression, phosphorylation, or nuclear translocation. instead, it interacts with the dna-binding domain of irf3 and inhibits the dna-binding activity (92) . a very interesting way of how to circumvent the host innate immune response was found when studying gammaherpesviruses kaposi's sarcoma-associated herpesvirus (kshv) and rhesus macaque rhadinovirus (rrv). they express several viral homologs to the irfs, called viral irfs (virfs). these virfs have found multiple ways to suppress type i ifn production. for kshv, different strategies have been reviewed in ref. (6) . recently, the rrv virf r6 has been shown to interact with the transcriptional coactivator cbp in the nucleus, similar to the kshv virf1. as a result, cbp cannot form a complex with the phosphorylated irf3, and the ifn expression is not induced (93) (94) (95) . interestingly, rrv r6 is the first virf for which an association with the viron could be shown. therefore, virf v6 can shut down the type i ifn response shortly after the cell was infected, rendering the cell more susceptible to infection (95) . the pedv protein nsp1 also targets cbp. nsp1 induces cbp degradation in a proteasome-dependent manner and thereby interrupts enhanceosome assembly and the production of type i ifn (96) (figure 1) . for most of these interactions, the molecular mechanisms have not been unraveled yet. a protein that has been shown to interact with and induce proteasomal degradation of irf3 some time ago is classical swine fever virus (csfv) npro (97, 98) . recently, the molecular mechanism has been published. irf3 and npro interact direct and form a soluble 1:1 complex. moreover, it was shown that npro interacts with the full-length irf3, not with individual domains, and that npro binds the constitutively active form of irf3 in the presence of cpb. thus, npro interacts with both the monomer and the active irf3 dimer and likely targets both species for ubiquitinylation and proteasomal degradation (99) . interferon regulatory factor 7 is targeted by two human enteroviruses, such as enterovirus 71 and enterovirus 68. they downregulate irf7 by cleaving it with their protease 3c, leaving the cleavage products unable to induce ifn expression. while enterovirus 71 cleaves irf7 once at gln189-ser190 (100), enterovirus 68 cleaves it twice, the cleavage sites being gln167 and gln189 (101) . moreover, megalocytivirus, a dna virus that infects marine and freshwater fish, induces the expression of the host microrna pol-mir-731, which then specifically suppresses the expression of irf7 (102) (figure 1) . the type i ifns act in an autocrine, paracrine, or systemic manner to stimulate antiviral responses. they are recognized by the ifnα/β receptor (ifnar), which consists of the subunits ifnar1 and ifnar2 expressed on virtually all cell types (103) . the interaction of type i ifn with the receptor results in the phosphorylation and activation of the ifnar1-and ifnar2-associated tyrosine kinases tyrosine kinase 2 (tyk2) and janus kinase 1 (jak1), which then phosphorylate ifnar tyrosine residues, resulting in the recruitment and activation of signaling molecules, such as the signal transducer and activator of transcription (stat) family of transcription factors (104, 105) . upon activation, stat1 and stat2, together with irf9, form the ifn-stimulated gene factor 3 (isgf3), which then translocates into the nucleus to induce transcription of isgs [reviewed in detail in ref. (106) (107) (108) ]. several viruses target ifnar to prohibit ifn binding and signaling. influenza virus induces the degradation of ifnar1. hemagglutinin (ha) triggers the phosphorylation and ubiquitinylation of ifnar1, thus promoting protein degradation (109) . encephalitic flaviviruses, such as tick-borne encephalitis virus or west nile virus, inhibit ifnar1 surface expression. their protein ns5 binds the cellular dipeptidase prolidase (pepd), which is involved in ifnar1 maturation and accumulation, activation of ifnβ-stimulated gene induction, and ifn-dependent viral control. this interaction inhibits ifnar1 intracellular trafficking and glycosylation but does not promote ifnar1 degradation (110) (figure 3) . both stat1 and stat2 are targeted by many viruses to suppress isg induction. pedv induces stat1 ubiquitinylation and targets it for degradation in the proteasomes (111) . (112) . similarly, human metapneumovirus (hmpv) protein sh impairs stat1 expression, phosphorylation, and activation (113) . simian varicella virus not only inhibits stat2 phosphorylation but also promotes degradation of irf9 in a proteasome-dependent manner through its protein orf63 (114) . also, infectious bronchitis virus (ibv) inhibits phosphorylation and nuclear translocation of stat1. however, despite detailed analyses, it is unclear which viral protein is responsible. it was, however, shown that the accessory protein 3a contributes to ibv resistance to type i ifn, although the target is unknown as well (115) . in case of the human parvovirus b19, it becomes evidently clear that both the virus and the immune system constantly evolve to prevail. while its protein ns1 suppresses stat phosphorylation, the immune system senses the protein and triggers the production of type i ifn (116) . sftsv, an emerging tick-borne pathogen, developed multiple ways to prevent isg induction. the viral non-structural protein ns impairs stat1 expression, phosphorylation, and activation (117) and interacts with stat2 and sequesters stat1 and stat2 into viral inclusion bodies, where they are trapped (118) (figure 3) . the jak-stat signal transduction pathway is negatively regulated by the suppressor of cytokine signaling (socs) family of proteins in form of a classical feedback loop (119, 120) . some viruses induce the expression of socs to take advantage of this mechanism to minimize the induction of isgs. japanese encephalitic virus (jev) downregulates the expression of micro-rna mir-432, which then results in upregulated socs5 levels (121) . varicella-zoster virus (vzv) infection induces the expression of socs3 (122) and respiratory syncytial virus (rsv) nonstructural proteins ns1 and ns2 induce upregulation of socs1 and socs3, which also inhibited the induction of chemokines (123) (figure 3 ). viruses fully depend on the translation machinery of the host cell for replication. accordingly, they have evolved multiple ways to hamper host protein synthesis [reviewed in ref. (124) ]. one way is to shut off host protein synthesis. for some time, it was thought that gamma-and deltacoronaviruses do not induce host shutoff, such as alpha-and betacoronaviruses do. however, a recent study showed that the infectious bronchitis gammacoronavirus induces host shutoff using its protein 5b. it seems like 5b is a functional equivalent of nsp1, the host shutoff protein of alpha-and betacoronaviruses (125) . viruses evolved to have various strategies to circumvent the innate immune response by blocking the production of type i ifn or the expression of isgs. while these diverse strategies may appear contradictory between viruses, several factors require consideration. for example, the use of clinical isolates versus viral evasion of interferon pathways frontiers in immunology | www.frontiersin.org november 2016 | volume 7 | article 498 laboratory-passaged strains could yield different results, particularly with rna viruses that rapidly accumulate mutations due to error-prone rna-dependent rna polymerases. moreover, the choice of cell line can greatly influence experimental outcomes, as many immortalized or transformed continual cell lines harbor mutations in critical innate immune signaling (126) . likewise, the use of genetic knockout versus knockdown cell lines or organisms can influence experimental outcomes, as can the experimental procedures themselves, particularly when endogenous interactions are disrupted with the use of overexpression approaches. studying the mechanisms used by viruses to prevent an immune response is of great importance for the development of new strategies to limit the sequelae of viral infections. identification of key immune evasion proteins allows development of antivirals to target these proteins. alternatively, identification of key cellular antiviral pathways allows development of strategies to enhance these pathways to overwhelm incoming viruses. information on key immune evasion factors further facilitates the engineering of safe and effective vaccine strains and designing strategies to target new emerging viruses from the same or closely related family. ks and km conceptualized the scope of the review article. ks wrote the review with input from km. this work was supported by a postdoctoral fellowship from the deutsche forschungsgemeinschaft (schu3011/1-1). work in the mossman laboratory on innate antiviral signaling is supported by the canadian institutes for health research. induction and function of ifnbeta during viral and bacterial infection functions of the influenza a virus ns1 protein in antiviral defense to conquer the host, influenza virus is packing it in: interferon-antagonistic strategies beyond ns1 phleboviruses and the type i interferon response the tiers and dimensions of evasion of the type i interferon response by human cytomegalovirus herpesviruses: interfering innate immunity by targeting viral sensing and interferon pathways evasion of host antiviral innate immunity by hsv-1, an update middle east respiratory syndrome and severe acute respiratory syndrome innate antiviral immune signaling, viral evasion and modulation by hiv-1 hiv replication: a game of hide and sense strategies of highly pathogenic rna viruses to block dsrna detection by rig-i-like receptors: hide, mask, hit molecular mechanisms of innate immune inhibition by non-segmented negative-sense rna viruses cytosolic sensing of viruses viral evasion of intracellular dna and rna sensing regulation of cellular innate antiviral signaling by ubiquitin modification recent advances in understanding viral evasion of type i interferon devasthanam as. mechanisms underlying the inhibition of interferon signaling by viruses the cgas-sting defense pathway and its counteraction by viruses rig-i in rna virus recognition intracellular nod-like receptors in host defense and disease regulation of innate antiviral defenses through a shared repressor domain in rig-i and lgp2 an evolving arsenal: viral rna detection by rig-i-like receptors phosphorylationmediated negative regulation of rig-i antiviral activity phosphorylation of rig-i by casein kinase ii inhibits its antiviral response structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna a structure-based model of rig-i activation trim25 ringfinger e3 ubiquitin ligase is essential for rig-i-mediated antiviral activity reconstitution of the rig-i pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity conventional protein kinase c-alpha (pkc-alpha) and pkc-beta negatively regulate rig-i antiviral signal transduction a distinct role of riplet-mediated k63-linked polyubiquitination of the rig-i repressor domain in human antiviral innate immune responses dephosphorylation of the rna sensors rig-i and mda5 by the phosphatase pp1 is essential for innate immune signaling the mitochondrial targeting chaperone 14-3-3epsilon regulates a rig-i translocon that mediates membrane association and innate antiviral immunity how rig-i like receptors activate mavs antiviral responses induced by the tlr3 pathway a unique host defense pathway: trif mediates both antiviral and antibacterial immune responses rip1 is an essential mediator of toll-like receptor 3-induced nf-kappa b activation dsrna induces apoptosis through an atypical death complex associating tlr3 to caspase-8 a fadd-dependent innate immune mechanism in mammalian cells traf3: a novel tumor suppressor gene in macrophages. macrophage (houst) (2015) 2:e1009 toscana virus nss protein inhibits the induction of type i interferon by interacting with rig-i truncation of the c-terminal region of toscana virus nss protein is critical for interferon-beta antagonism and protein stability foot-and-mouth disease virus viroporin 2b antagonizes rig-i mediated antiviral effects by inhibition of its protein expression hepatitis b virus inhibits intrinsic rig-i and rig-g immune signaling via inducing mir146a a phosphomimetic-based mechanism of dengue virus to antagonize innate immunity ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling ddx60 is involved in rig-i-dependent and independent antiviral responses, and its function is attenuated by virus-induced egfr activation a diverse range of gene products are effectors of the type i interferon antiviral response mouse superkiller-2-like helicase ddx60 is dispensable for type i ifn induction and immunity to multiple viruses dengue virus subverts host innate immunity by targeting adaptor protein mavs porcine reproductive and respiratory syndrome virus 3c protease cleaves the mitochondrial antiviral signalling complex to antagonize ifn-beta expression hepatitis c virus protease ns3/4a cleaves mitochondrial antiviral signaling protein off the mitochondria to evade innate immunity hepatitis c virus ns3-4a inhibits the peroxisomal mavs-dependent antiviral signalling response highly pathogenic porcine reproductive and respiratory syndrome virus nsp4 cleaves visa to impair antiviral responses mediated by rig-i-like receptors porcine epidemic diarrhea virus inhibits dsrna-induced interferon-beta production in porcine intestinal epithelial cells by blockade of the rig-i-mediated pathway sars-coronavirus open reading frame-9b suppresses innate immunity by targeting mitochondria and the mavs/traf3/traf6 signalosome pcbp2 mediates degradation of the adaptor mavs via the hect ubiquitin ligase aip4 oncogenic human t-cell lymphotropic virus type 1 tax suppression of primary innate immune signaling pathways middle east respiratory syndrome coronavirus m protein suppresses type i interferon expression through the inhibition of tbk1-dependent phosphorylation of irf3 suppression of innate antiviral response by severe acute respiratory syndrome coronavirus m protein is mediated through the first transmembrane domain a firm hand on nfkappab: structures of the ikappabalpha-nfkappab complex ikappab kinases: key regulators of the nf-kappab pathway inducible degradation of ikappabalpha by the proteasome requires interaction with the f-box protein h-betatrcp shaping the nuclear action of nf-kappab encephalomyocarditis virus 3c protease relieves traf family member-associated nf-kappab activator (tank) inhibitory effect on traf6-mediated nf-kappab signaling through cleavage of tank porcine epidemic diarrhea virus 3c-like protease regulates its interferon antagonism by cleaving nemo hepatitis a virus 3c protease cleaves nemo to impair induction of beta interferon foot-and-mouth disease virus 3c protease cleaves nemo to impair innate immune signaling nsp1 of human rotaviruses commonly inhibits nf-kappab signalling by inducing beta-trcp degradation ikkepsilon and tbk1 are essential components of the irf3 signaling pathway triggering the interferon antiviral response through an ikk-related pathway regulation and function of ikk and ikk-related kinases involvement of the ubiquitin-like domain of tbk1/ikk-i kinases in regulation of ifn-inducible genes ikappab kinase epsilon (ikkepsilon): a therapeutic target in inflammation and cancer regulation of tbk1 activity by optineurin contributes to cell cycle-dependent expression of the interferon pathway negative regulation of tbk1-mediated antiviral immunity irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors the irf family transcription factors in immunity and oncogenesis the irf family transcription factors at the interface of innate and adaptive immune responses direct triggering of the type i interferon system by virus infection: activation of a transcription factor complex containing irf-3 and cbp/p300 mechanism for transcriptional synergy between interferon regulatory factor (irf)-3 and irf-7 in activation of the interferon-β gene promoter middle east respiratory syndrome coronavirus orf4b protein inhibits type i interferon production through both cytoplasmic and nuclear targets hsv-1 icp27 targets the tbk1-activated sting signalsome to inhibit virus-induced type i ifn expression herpes simplex virus 1 serine protease vp24 blocks the dna-sensing signal pathway by abrogating activation of interferon regulatory factor 3 dengue virus ns proteins inhibit rig-i/mavs signaling by blocking tbk1/irf3 phosphorylation: dengue virus serotype 1 ns4a is a unique interferon-regulating virulence determinant porcine epidemic diarrhea virus nucleocapsid protein antagonizes beta interferon production by sequestering the interaction between irf3 and tbk1 htlv-1 tax protein recruitment into ikkepsilon and tbk1 kinase complexes enhances ifn-i expression suppression of type i interferon production by human t-cell leukemia virus type 1 oncoprotein tax through inhibition of irf3 phosphorylation contribution of the interaction between the rabies virus p protein and i-kappa b kinase to the inhibition of type i ifn induction signalling porcine deltacoronavirus (pdcov) infection suppresses rig-i-mediated interferon-beta production proteolysis of mda5 and ips-1 is not required for inhibition of the type i ifn response by poliovirus hepatitis e virus infection activates signal regulator protein alpha to down-regulate type i interferon porcine bocavirus np1 protein suppresses type i ifn production by interfering with irf3 dnabinding activity functional analysis of human herpesvirus 8-encoded viral interferon regulatory factor 1 and its association with cellular interferon regulatory factors and p300 viral interferon regulatory factor 1 of kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) binds to, and inhibits transactivation of, creb-binding protein a rhesus rhadinovirus viral interferon (ifn) regulatory factor is virion associated and inhibits the early ifn antiviral response suppression of type i interferon production by porcine epidemic diarrhea virus and degradation of creb-binding protein by nsp1 classical swine fever virus npro interacts with interferon regulatory factor 3 and induces its proteasomal degradation the npro product of classical swine fever virus and bovine viral diarrhea virus uses a conserved mechanism to target interferon regulatory factor-3 pestivirus npro directly interacts with interferon regulatory factor 3 (irf3) monomer and dimer cleavage of interferon regulatory factor 7 by enterovirus 71 3c suppresses cellular responses 3c protease of enterovirus d68 inhibits cellular defense mediated by interferon regulatory factor 7 pol-mir-731, a teleost mirna upregulated by megalocytivirus, negatively regulates virus-induced type i interferon response, apoptosis, and cell cycle arrest the interferons and their receptors -distribution and regulation jak-stat signaling: from interferons to cytokines stats get their move on transcriptional regulation by stat1 and stat2 in the interferon jak-stat pathway stat2 and irf9: beyond isgf3. jakstat (2013) dynamic control of type i ifn signalling by an integrated network of negative regulators hemagglutinin of influenza a virus antagonizes type i interferon (ifn) responses by inducing degradation of type i ifn receptor 1 flavivirus antagonism of type i interferon signaling reveals prolidase as a regulator of ifnar1 surface expression porcine epidemic diarrhea virus infection inhibits interferon signaling by targeted degradation of stat1 la piedad michoacan mexico virus v protein antagonizes type i interferon response by binding stat2 protein and preventing stats nuclear translocation human metapneumovirus small hydrophobic (sh) protein downregulates type i ifn pathway signaling by affecting stat1 expression and phosphorylation varicella viruses inhibit interferon-stimulated jak-stat signaling through multiple mechanisms infectious bronchitis coronavirus inhibits stat1 signaling and requires accessory proteins for resistance to type i interferon activity nonstructural protein (ns1) of human parvovirus b19 stimulates host innate immunity and blunts the exogenous type i interferon signaling in vitro suppression of type i and type iii ifn signalling by nss protein of severe fever with thrombocytopenia syndrome virus through inhibition of stat1 phosphorylation and activation disruption of type i interferon signaling by the nonstructural protein of severe fever with thrombocytopenia syndrome virus via the hijacking of stat2 and stat1 into inclusion bodies the role of suppressors of cytokine signaling (socs) proteins in regulation of the immune response regulation of the immune system by socs family adaptor proteins japanese encephalitis virus exploits the microrna-432 to regulate the expression of suppressor of cytokine signaling (socs) 5 suppressor of cytokine signaling 3 expression induced by varicella-zoster virus infection results in the modulation of virus replication respiratory syncytial virus nonstructural proteins upregulate socs1 and socs3 in the different manner from endogenous ifn signaling viral subversion of the host protein synthesis machinery infectious bronchitis coronavirus limits interferon production by inducing a host shutoff that requires accessory protein 5b deregulation of interferon signaling in malignant cells the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-312438-zr9zx7pv authors: hoo, regina; nakimuli, annettee; vento-tormo, roser title: innate immune mechanisms to protect against infection at the human decidual-placental interface date: 2020-09-10 journal: front immunol doi: 10.3389/fimmu.2020.02070 sha: doc_id: 312438 cord_uid: zr9zx7pv during pregnancy, the placenta forms the anatomical barrier between the mother and developing fetus. infectious agents can potentially breach the placental barrier resulting in pathogenic transmission from mother to fetus. innate immune responses, orchestrated by maternal and fetal cells at the decidual-placental interface, are the first line of defense to avoid vertical transmission. here, we outline the anatomy of the human placenta and uterine lining, the decidua, and discuss the potential capacity of pathogen pattern recognition and other host defense strategies present in the innate immune cells at the placental-decidual interface. we consider major congenital infections that access the placenta from hematogenous or decidual route. finally, we highlight the challenges in studying human placental responses to pathogens and vertical transmission using current experimental models and identify gaps in knowledge that need to be addressed. we further propose novel experimental strategies to address such limitations. the human placenta is the temporary extra-embryonic organ that is present only during pregnancy and is the anatomical boundary between the mother and fetus. it has a range of functions including transport of nutrients and gases, and hormonal production (1) . the placenta forms a physical, selective barrier between the maternal and fetal circulations, preventing transfer of pathogens. the uterine mucosal lining, the endometrium, is transformed into the decidua during early pregnancy (2) . a range of innate immune mechanisms can respond to pathogens in both the decidua and the placenta (3, 4) . the maternal-fetal interface is a protective barrier against pathogens, but some pathogens can transfer from the mother to fetus by different routes and cause fetal infection (3, 4) . vertical transmission during pregnancy can occur on distinct boundaries between the mother and the fetus: (i) the intervillous space (ivs), where placental villi is in direct contact with the maternal blood, (ii) the implantation site or decidua basalis, where maternal cells are in direct contact with the invading fetal trophoblast, and (iii) the fetal membranes, which are in direct contact with the uterine cavity (5) . defense mechanisms in the cervix, such as the production of mucus and antimicrobial peptides (amp), limit ascending infection from pathogens present in the lower genital tract, that otherwise may access the uterine cavity (6) . however, some pathogens can escape antimicrobial strategies at the cervix and ascend to the uterus, where they can bypass the fetal membranes and lead to the inflammation of the membranes-also known as chorioamnionitis-and infection of the amniotic fluid (7, 8) . pathologic and immune features of chorioamnionitis and intra-amniotic infection are generally associated with bacterial invasion and inflammation [refer to (8, 9) for a comprehensive review on these mechanisms]. here, we focus on infections and innate immune mechanisms at the uterine-placental interfacecases (i) and (ii) (figure 1) . infections at the uterine-placental interface are commonly associated with viruses, parasites and few bacteria ( table 1) . viral pathogens such as human cytomegalovirus (hcmv), zika (zikv), and rubella virus are the most common vertically transmitted pathogens through the decidual-placental interface ( table 1 ) (26, 27) . non-viral pathogens, such as toxoplasma gondii and listeria monocytogenes, can cross the placental barrier via cell-to-cell transmission ( table 1 ) (28, 29) . fetal infection can result in various forms of congenital anomalies in humans ( table 1) . understanding the pathogenic mechanisms used by infectious agents is central to preventing vertical transmission and controlling infection during pregnancy. how the innate immune cells and mechanisms in the placenta and the uterus recognize and respond to protect both the fetus and mother remains controversial due to technical and ethical constraints. however, there are several different models currently used to interrogate the uterine-placental interface in pregnancy. firstly, mice are frequently used as a pregnancy model for infection. although the murine models have provided important insights into the pathogenesis of various infection agents in the context of pregnancy, there are still limitations with this approach. the anatomy of placentation, length of gestation, and use of inbred strains, make extrapolation to humans problematic (30, 31) . secondly, a range of human trophoblast and choriocarcinoma cell lines are used as in vitro models for infection with pathogens. in contrast to the first trimester trophoblast in vivo, these cell lines do not recapitulate normal human trophoblast characteristics such as expression of the human leukocyte antigen (hla) class i and methylation of elf5 (32, 33) . thirdly, human primary placental explants are frequently used. the syncytium dies rapidly in these cultures and it is virtually impossible to standardize the types of villi sampled (30) . therefore, these in vitro experimental factors should be taken into careful consideration when interpreting studies of infection of trophoblast. in this review, we cover the innate immune features of the decidual-placental interface throughout gestation. we identify the gaps in knowledge and highlight the limitations of current studies and experimental models. finally, we discuss novel experimental strategies for understanding how infection affects pregnancy in humans. the trophoblasts of the placenta are the barrier between fetal and maternal tissues. they are derived from the trophectoderm, the outer layer of the blastocyst that forms an inner mononuclear layer with an outer primary syncytium following implantation (34) . the trophoblast in contact with the maternal cells can be: (i) syncytiotrophoblast (sct), a single layer multinucleated, syncytial layer formed by fusion of the underlying villous cytotrophoblast (vct), and (ii) extravillous trophoblast (evt), that invade from the cytotrophoblast shell and anchoring villi into the transformed maternal endometrium, the decidua (2) . the function of evt is to transform the uterine spiral arteries so that maternal blood is delivered to the intervillous space at low pressure. the arteries are surrounded by interstitial evt that destroys the smooth muscle cells of the arterial media, known as "fibrinoid" change (35, 36) . subsequently, endovascular evt (eevt) moves down the spiral arteries from the placentadecidua boundary (35) . these eevt form a plug of cells, limiting surges of arterial blood from damaging the delicate villi. evt invasion transforms the arteries to support optimal regulation of blood flow into the placenta during fetal development (36) . the plugs dissipate between 8 and 10 weeks of gestation when the full hemochorial circulation is established (37) . maternal blood then flows into the ivs, and establishes direct contact with the sct allowing for proper nutrient and gas exchange between the mother and the fetus. hofbauer (hb) cells are fetal macrophages of the human placenta (38) . hb cells can be detected in the placental villous stroma as early as 3 weeks post-conception and are present throughout pregnancy (1, 39) . they are likely to have a variety of functions including control of villous remodeling and differentiation, hormonal secretion, and trophoblast turnover (1, 40) . several lines of evidence have led to the postulation that hb cells may have a role in infection during pregnancy. hb cells with zikv viral particles detected intracellularly have been shown (41, 42). human immunodeficiency virus 1 (hiv-1) has also been detected in hb cells from first trimester infected placenta (43) . whether the hb cells can serve as a reservoir or limit virus replication is still unknown. isolated hb cells from healthy term placenta show elevation of pro-inflammatory cytokines such as il-6, mcp-1, ip-10, and ifn-α upon in vitro infection with zikv (44) . hb cells from the first trimester placenta are also permissive for zikv infection and replication (23) . however, this must be interpreted with caution because in vitro culture of hb cells do not entirely recapitulate the complexity of villous stromal microenvironment, such as presence of hormone and growth factors, all of which will influence the function and activity of hb cells (45) . the sct is the barrier between maternal blood and the placental core as it separates the ivs from the underlying fetal villous stroma. blood-borne pathogens such as viruses and parasites can potentially be transmitted through the sct barrier (figure 1) . how can pathogens cross the sct barrier and the vct to infect the villous stroma? although the sct is an efficient barrier due to its stiff, highly dense actin cytoskeleton network and continuous membrane (46), the syncytium undergoes continuous breaks or gaps and dynamic repair processes (47) . breaks in the syncytium could potentially lead to transmission of pathogens into the underlying vct. our recent work showed that a novel population of maternal macrophages (m3) is associated with the sct in early pregnancy and might be involved in repairing the breaks in the syncytium (10). it is intriguing that m3 macrophages infected with intracellular pathogens could possibly gain access to the underlying vct via the syncytial breaks (figure 2) . only a few viral entry receptors on the sct are described. notably, the sct lacks expression of zikv entry receptors, axl, and tyro3 (48) and the hcmv entry co-receptor integrin α/β (49) . this is further supported by the transcriptomic expression of viral receptors in placental cells (10, 50, 51). expression of surface receptors commonly used by zikv such as axl and hcmv such as nrp2 and pdgfra are lowly expressed by the sct (50) . in addition, there is minimal co-expression of ace2, the receptor gene for human severe acute respiratory syndrome coronavirus 2 (sars-cov-2), and tmprss2, the viral spike protein serine protease gene (50, 52) . in line with this, there is no conclusive and direct evidence of vertical transmission of sars-cov-2 in a placenta from a healthy individual. there are some reports showing sars-cov-2 is predominantly localized at the sct of the second trimester placenta (53, 54) and can lead to severe inflammatory infiltrate in the ivs (55) . however, these findings are presented in a very small number of patients with severe disease or pre-existing pregnancy complications (54, 55) . alternative transplacental mechanisms have been postulated at the syncytial barrier. neonatal fc receptor (fcrn) is expressed on the apical surface of the sct and functions to selectively figure 2 | toll-like receptors and potential inflammatory response at the sct-blood interface. predominant tlrs found in the human placenta from early and term pregnancies. tlr2 and tlr4 are expressed in human placenta sct, vct, and in hb cells. infiltration of infected maternal blood, infected immune cells, or release of pathogenic determinant such lipopolysaccharide (lps), peptidoglycan, or parasite materials such as hemozoin or gpi (glycosylphosphatidylinositol) into the ivs will activate tlr-mediated signaling, leading to the production of a wide range of cytokines and chemokines. severe infection is characterized by massive immune cell infiltration including monocytes and neutrophils from systemic circulation and overproduction of inflammatory cytokines upon tlr activation. this may lead to sct inflammation and damage. sct also secretes antimicrobial peptides as innate immune mechanisms. figure is created by biorender.com. frontiers in immunology | www.frontiersin.org transport maternal igg (56) . fcrn could be exploited by certain viruses to enter the placenta including zikv, hiv-1, and hcmv (19, 57, 58) . transferrin receptor 1 (tfr1) is expressed on the apical end of the sct, and functions as the primary iron transporter into the basal side of the sct to provide sufficient iron stores into fetal circulation (59) . tfr1 has been associated with viral entry into a broad host cell range, including hepatitis c virus (60, 61) suggesting a possible mechanism of viral transport across the sct barrier. some pathogens, although unable to cross the sct barrier, can still adhere to the syncytium and cause further pathology. for instance, plasmodium falciparum infected red blood cells can bind with high affinity to chondroitin sulfate a expressed on the sct, resulting in local inflammation, syncytial breaks, and damage (62) (63) (64) . although the sct is an effective barrier to most pathogens, local inflammation, tissue damage, and fcrn or tfr1-mediated viral entry at the sct can potentially allow pathogen to breach the syncytial barrier, giving opportunity for transmission from maternal blood into placental villi (figure 2 ). during the first trimester of pregnancy, fetal evt invades deeply into the uterus. the decidua basalis, the region located at the implantation site, is populated at this time by a distinctive subset of innate lymphocytes, decidual natural killer cells (dnk), which constitute up to 70% of leukocytes. we have identified three major populations of dnk by single-cell rna-sequencing with unique phenotypes and functions in early pregnancy (10). in addition, there are populations of decidual macrophages (dms) (∼20%), conventional dendritic cells (dcs) and small proportions of t cells (∼10-15%), whereas b cells, plasma cells, mast cells, and granulocytes are virtually absent (10) (figure 1) . the proportion of immune cells will vary throughout pregnancy, with an increase in the proportion of t cells at term (51) . systemic infections will reach all organs including the decidua. whether pathogens can also access the decidua via the cervix is still unclear. chlamydia trachomatis, a common sexuallytransmitted intracellular bacteria, was detected in glandular epithelial cells and unidentified decidual cells in decidual biopsies (11) . this suggests the possibility of infections ascending and spreading from cell-to-cell from the lower genital tract into endometrial glands and vascular endothelium. the decidua basalis is in close contact with fetal cells and the maternal vasculature (figure 1) . first trimester dms and decidual stromal cells are susceptible to zikv infection and replication ex vivo (23) . hence, infection could possibly spread from infected maternal immune and non-immune cells at the decidua, into uninfected vct in the columns of the anchoring villi, and finally into the fetal compartment. however, this is likely to be limited to certain microorganisms which are capable of cell-to-cell spread, have an intracellular host niche, and are able to escape host innate defense mechanisms (table 1) . hcmv, the most common cause of congenital infection, is mostly reported to infect from the decidua (11, 65) . women with primary hcmv infection and first pregnancy are more likely to transmit the virus to their fetus, compared to multiparous women with previous infection and demonstrable antibodies (66) (67) (68) . low affinity maternal antibodies against hcmv correlate with higher viral loads detected in the decidua, whereas patients with intermediate to high neutralizing antibodies have minimal viral replication (65) , suggesting that maternal immunity against hcmv reduces risk of vertical transmission. hcmv protein was also detected in a range of cells within the decidua including endothelial, decidual stromal cells, dcs and macrophages (11, 65) , suggesting that that infected maternal leukocytes could initiate transmission through contact and infection of endothelial cells that line decidual blood vessels. despite the evidence of decidual infection, the mechanism of vertical transmission for hcmv is still in debate. dnks have been proposed to play a protective role against hcmv infection through several mechanisms including modulation of their cytotoxic effector function (69) and the interactions between the killer-cell immunoglobulin receptors (kirs) expressed by dnk and hla molecules expressed in the infected cells (70, 71) . activating kir2ds1 by dnks has been demonstrated to be more cytolytic against hla-c2 hcmv-infected maternal decidual stromal cells (70) . similar cytotoxic response was also observed when peripheral blood nk cells expressing kir2ds1 were exposed to hcmv-infected fibroblasts (71) . hence, this implies that in the decidua, dnks are capable of eliminating harmful infection depending on the combination of kir/hla interactions between dnk and infected cells. dnks are also able to control hiv-1 infection in vitro through production of ifn-γ (72) . the role of dnk in controlling viral infection may protect against potential risk of vertical transmission from the decidua. pattern recognition receptors (prr) are encoded in the germ-line and recognize specific, conserved pathogen-associated molecular patterns (pamps). these include gram-negative bacteria lipopolysaccharide (lps), gram-positive bacteria lipoteichoic acids, lipoprotein, dna, rna, glucans, and peptidoglycans (73, 74). pathogen recognition is not only an essential component of the innate immune response against infection, but also plays an important role in bridging the innate and adaptive systems by toll-like receptors (tlr) activation of antigen presenting cells by up-regulation of major histocompatibility complex (mhc) and co-stimulatory molecules (75) . tlrs, the most studied family of prr, are type i transmembrane proteins with large extracellular domains containing leucine-rich repeats that are expressed at the cell surface or intracellularly (76) . each tlr recognizes distinct pamps, leading to the activation of the transcription factor nf-κb and/or the interferon-regulatory factor (irf) family, and the production of a wide range of cytokines and chemokines, including type i ifns (76, 77) expression of tlrs is dynamic and changes in response to different pathogens and cytokines (74) . tlr2 (which recognizes bacterial proteoglycan) and tlr4 (which recognizes bacterial lps) are the most well-studied, with immunohistochemical evidence of expression in healthy primary sct at term (78) (79) (80) . in contrast, in the first trimester, tlr2 and tlr4 proteins are expressed in vct and evt, but minimally in sct (81, 82) (figure 3) . there is therefore variation in tlr2 and tlr4 expression in the different trophoblast lineages across pregnancy. why and how such dynamic regulation of tlr expression occurs during gestation requires further investigation in a broader range of human placental samples (different donors, gestation stages, genetic background, sampling regions). it is likely that alteration in cytokines profiles in the microenvironment as pregnancy progresses (83) may result in the variation in the expression of tlrs in the placenta. current evidence is only limited to in vitro tlr2/4 stimulation studies using placental explants and primary first trimester trophoblast cells, which drives the expression of figure 3 | toll-like receptors and potential inflammatory response at the decidua. predominant tlrs found in the human placenta from early and term pregnancies. tlr2 and tlr4 are expressed in evt. dm and dnk also express a wide range of tlr families, where stimulation of tlr agonists lead to the production of a variety of cytokines and chemokines. infiltration of infected cells and release of pamps in the decidua, which will activate tlr-mediated signaling. overproduction of inflammatory cytokines at the decidua may lead to local inflammation. figure is created by biorender.com. pro-inflammatory cytokines il-6, il-8, tnf-α, and ifn-γ (78, 80, 81) . tlr2 and tlr4 proteins are expressed in hb cells, confirmed by co-expression of cd68 in healthy term placentas (78) . in early pregnancy, our findings indicate that only tlr4 but not tlr2 transcripts are expressed in steady-state hb cells (10) (figure 4) . enhancement of il-6 and il-8 secretion upon stimulation of isolated first trimester hb cells with tlr4 agonist, lps (84), does suggest a role for tlrs on hb cells in bacterial recognition and placental inflammation during early pregnancy. hb cells are postulated to have a role in viral replication (41, 42), however evidence on the expression and function of viral nucleic acid sensing receptors tlr3, tlr7, tlr8, and tlr9 in hb cells is lacking. our findings show that tlr7, which recognizes viral single-strand rna (ssrna) (85) is expressed in steady-state hb cells (figure 4) (10) . other tlrs have also been shown to be expressed in decidua cells. dms and dnks isolated from first trimester pregnancies show steady state level expression of tlr1-9 transcripts and respond to a broad range of pamps, including heat-killed bacteria, microbial membranes, and nucleic acids (86) . stimulating primary dms with these pamps produces high levels of tnf-α, il-1β, il-6, il-8, il-12, il-10, and il-1ra, whereas dnks secrete il-6, il-8, and ifn-γ (86) . this study suggests that, in addition to the physiological roles of dms and dnks in accommodating the uterus for placentation, dms and dnks may play a role in pathogen recognition and antimicrobial response via activation of tlr signaling (figure 3) . the extent to which subsets of dms or dnks population (10) are critical for tlr-mediated response at the decidua is currently unknown. in malaria endemic populations, single nucleotide polymorphisms (snps) within the tlr4 coding and tlr9 promoter regions are associated with variation in disease severity and parasitemia control (87, 88) . in the case of pregnancy malaria, primiparous infected mothers with common tlr4 and tlr9 polymorphic variants are correlated with severe complications such as low birth weight and maternal anemia (89) . this highlights the importance of studies involving large cohorts of individuals which include genotyping from pregnant mothers living in malaria endemic regions (see section on "challenges and future perspective"). animal models have also been used to study the functional role of tlr signaling, particularly for pathogens that are intracellular at some stage of their life cycle ( table 1) . tlr4 and tlr9 are strongly activated by malaria parasite pamps such as glycosylphosphatidylinositol (gpi), dna, and hemozoin (90, 91) (figure 2) . in a mouse model of placental malaria, tlr4, and myd88 signaling activation resulted in placental expression of pro-inflammatory markers, such as il-6 and tnf-α (92, 93) . these studies also demonstrated that malaria parasite infection and inflammation in the mouse placenta lead to reduced fetus growth rate and disorganization of the vascular space in the placenta (92, 93) . however, tlr-mediated inflammation and pathology in the human placenta upon malaria infection is unknown and remains to be further investigated. studies of congenital toxoplasmosis are also currently limited to animal models. tlr2 and tlr4 are associated with recognition of t. gondii's infection in mice (94) . engagement of the t. gondii ligand by tlr2 and tlr4 at the sct-blood or in the evt-decidua compartments is plausible, although there is still no direct evidence for such host-parasite interaction in humans. tlr11 has a role in controlling t. gondii infection in mice (95, 96) , however in humans tlr11 is a pseudogene and is not expressed (97) . cytosolic prrs play an important role in fighting against viral infection by eliciting host type i interferons (ifn) antiviral response through recognition of single and double stranded rna (ssrna and dsrna) (98, 99) . examples of prrs are the cytosolic retinoic acid-inducible gene-i-like (rig-i) and the melanoma differentiation-associated protein 5 (mda5) receptors, both expressed in the sct and vct of term placenta (100) . in the human placenta, there is limited information on the function of rig-1 and mda5, but they may play a crucial role in recognizing a variety of rna viruses, including zikv and dengue virus (101) . the nucleotide binding oligomerization domain-like receptors (nod-like receptors; nlr) recognizes intracellular pathogen products which have entered into the host cytoplasmic compartment (74) . both nod-1 and nod-2 receptors, which are known to detect intracellular bacterial peptidoglycans (102) , are expressed in the sct in the first trimester and term placentas (103, 104) . the nlr pyrin-containing 1 and 3 proteins (nlrp1 and nlrp3) form the major inflammasome complexes, which contribute to activation of inflammatory caspases and pathogen clearance (105, 106) . activation of nlrp3 and aim2 inflammasomes, together with high expression of il-1r, il-1β, and caspase-1 was recently shown in the placental tissue of mothers infected with p. falciparum with significant pathology (107) . in a murine model of intra-amniotic inflammation induced by bacterial lps, tissue sections from the decidua basalis region expressed high levels of nlrp3, but negligible caspase-1 activation suggesting a possible non-canonical activation of the nlrp3 inflammasome (108) . our analysis shows that decidual dm1 expresses high levels of nlrp3 transcript at steady state compared to other cell types (10) (figure 4) , thus dm1 may play a role in nlrp3-mediated pathogen recognition during early pregnancy. amp secreted by epithelial and immune cells are small peptides that bind and destroy most groups of pathogens-bacteria, yeasts, fungi, and viruses (109) . in addition to direct killing of pathogens, amps can rapidly modulate innate host immune responses by recruiting myeloid cells and lymphocytes to the site of infection and mediating activation of tlr (110, 111) . the human placenta expresses high levels of β-defensins, a family of broad spectrum antimicrobial peptides which participate in direct bactericidal and anti-viral activity (112) . specific subtypes of β-defensins (hbd-1, 2, and 3) are expressed in scts (112) , suggesting these amps can target potentially bacterial or viral infection from the maternal blood. recognition of pamps by prrs during infection leads to production of pro-inflammatory cytokines that can aid in clearing the pathogen (74) . studies on the direct role of proinflammatory cytokines on the placenta in the case of infection is limited. inflammatory mediators can directly influence infection outcome and fetal development, but they can also cause damage to the placenta if produced in excess (113). amongst the proinflammatory cytokines associated with uterine-placental infection during pregnancy, the antiviral ifn are the most well-characterized. ifns are secreted by a variety of cell types as the first line of defense against viral infection (114) . type i ifns, including ifn-α and ifn-β, are potent antiviral cytokines. ifn-α and ifn-β bind to the ifnar1/2 receptor and lead to expression of ifn stimulated genes (isgs), which control virus infection through a variety of mechanisms (114) . loss of ifnar in the placenta leads to vertical transmission and fetal mortality in murine herpesvirus-68 (mhv68) infected mice (115) . in the mouse model of zikv infection, type i ifn-mediated signaling is essential for the control of viral replication in the placenta, but can also lead to significant placental pathology and fetal mortality (116, 117) . the mechanism of type i ifn-mediated placental pathology has been recently elucidated. ifn-induced transmembrane (ifitm) protein, which normally blocks viral entry into host cells, impairs syncytin-mediated fusion of vct to form sct, leading to aberrant placental development (118) . type ii ifn, ifnγ, predominantly produced by nk and cd4+ t cells is crucial in controlling parasitic infection, such as t. gondii in mice (94, 119) . however, elevated levels of ifnγ in response to t. gondii infection can lead to pathological effects during pregnancy including fetal demise (119, 120) . severe placental pathology and fetal death have also been associated with elevation of ifnγ during pregnancy in a murine model of malaria (121). hence, proper regulation of type i and ii ifn-mediated signaling at the uterine-placental interface is crucial in limiting pathogen replication, whilst preserving a balanced environment for normal placental development (122) . type iii ifn, ifnλ, are constitutively secreted by the human sct, which presumably confers antiviral effects against zikv infection (123) (124) (125) . tryptophan metabolism by ido indoleamine 2,3-dioxygenase (ido) is a host intracellular enzyme which metabolizes the amino acid tryptophan (126) . ido has been associated with maternal immunoregulation during pregnancy (127) . it also plays a key role in the control of bacterial and viral replication, through limiting the bioavailability of tryptophan (128) . ido also inhibits the replication of several parasitic pathogens including t. gondii in human fibroblasts (129) and leishmania spp in human macrophages (130) . mouse infection with l. monocytogenes showed that ido is elevated in an ifn-γ-dependent manner in stromal cells of the metrial gland and decidua basalis; a crucial process to resolve bacterial infection in the mouse placenta (131) . our findings also show ido1 expression is enriched in epithelial glandular and dc1 cell type in the first trimester decidua (10) (figure 4) . the presence of ido in decidua suggests that the enzyme might have a central role in limiting parasitic, viral, and bacterial replication, thus preventing their spread to the fetus. research on how the human placenta safeguards itself against infections is challenging due to obvious logistical and ethical issues in obtaining tissue from early in gestation (box 1). although animal experimental models have provided important insights relating to the immune responses to pathogenic infection, major differences between human and animal placentas must be considered (30, 31) . likewise, differences between strains of pathogens adapted for mice compared with human clinical isolates should be taken into account as this may lead to variation in pathogenesis and cellular response. one such example is the use of mouse cmv, which is unable to cross the mouse placental barrier, unlike the hmvc counterpart which can be transmitted transplacentally in humans (132) . therefore, all data obtained from studies of infection in pregnant animals needs careful interpretation and consideration prior to translation to clinical infection in humans. inherent properties of trophoblast cell lines, primary cultures or explants vary between donors, and are likely to be confounded by the area of the placenta that is sampled and as well as stage of gestation (133) . for instance, villous placental explants will vary depending on the types of villi sampled and the presence of box 1 | perspective of vertical transmission and innate immune function during pregnancy and infection. a variety of maternal infections can lead to vertical transmission ( table 1) . the exact mechanisms these pathogens use to escape host defense and cross the placental barrier into the fetal compartment are not entirely known. experimental models that recapitulate infection of the human placenta and thus vertical transmission are challenging to set up. more data and representative experimental models are needed to answer these questions: (i) how do different pathogens escape or modulate the maternal-fetal host innate immune barrier (ii) why do some pathogens lead to congenital infection but not others? studying infected human placentas will be essential in understanding this but access to these samples is difficult especially in low and middle-income countries (lmic) where maternal infection is particularly prevalent (who, maternal mortality index 2019). despite evidence of expression in primary placental tissue, functional studies on important innate immune features such as tlrs, amps, rig-i, mda5, nlrs, and ido during infection and pregnancy are lacking. understanding how different cell types at the uterine-placental interface (hb cells, dnks, and dms) respond to pathogen challenge is essential, but remains under-researched. a critical obstacle is to also extrapolate the protective and pathological mechanisms of cytokines from mouse to human infection. therefore, systematic comparison of the innate immune effector mechanisms across gestation, in the placenta and decidua from natural human infection vs. healthy pregnancy, will provide a more accurate representation in clinical settings. attached decidual tissue (133) . caution is therefore needed when interpreting data using these experimental models. to overcome such limitations, population-based cohort studies of women with infection during pregnancy with extensive tissue sampling should be performed. these need to include and focus on lmic where infection is still a major cause of maternal and fetal mortality and morbidity. cohort studies and epidemiological surveillance on maternal infections can offer significant insights into disease pathogenesis and accelerate clinical interventions (134) . collaborations between clinicians and researchers for population-based cohort collection and sample processing will be instrumental to achieving this goal. biological samples such as blood or placenta collected from controls and infected pregnant individuals could be stored and cryopreserved retrospectively. to capture the overall heterogeneity of infected and non-infected placenta samples, sampling, and biobanking criteria of different regions of placenta should be considered (135) . protocols are now available to use frozen tissue processed for single-cell/nuclei and spatial genomics (136, 137) . hence, application of single-cell "omics" on infected vs. healthy human placental and decidual samples will enable us to evaluate cellular heterogeneity in response to infection. the capacity to detect transcripts specific to host or pathogen mrna from the same tissue using in situ nucleic acid hybridization methods will provide direct quantification of infection burden and identification of potential target host cells within the same tissue (138) . recent advances in spatial transcriptomics methods have also allowed gene expression signatures to be quantified and resolved from individual tissue sections (139) . combination of these emerging technologies with new methods to integrate single-cell and spatial data computationally (140) will provide an unbiased approach to characterize and profile the transcriptome of individual cells in situ from the placenta and decidua in response to infections. we anticipate that high-throughput datasets generated from cohort sampling studies will unravel novel cell states and tissue spatial localization associated with placental infections and inflammation. this will also allow us to better characterize not only the innate immune response or makers of infection, but also other adaptive immune states in the human placenta (box 1). the use of in vitro models will also further define host responses to infection. the recent generation of human trophoblast stem cells (htscs) (141) and three-dimensional (3d) trophoblast organoids (142, 143) offer a great opportunity to study infections in early pregnancy where the access to first trimester placental samples is a concern. more importantly, the htscs and trophoblast organoids fulfill the criteria characteristic of human first trimester trophoblast in vivo (32) . both htscs and trophoblast organoids can differentiate in vitro into sct and evt with appropriate media (142, 143) allowing infection experiments on both the major trophoblast subpopulations present at the two major sites of contact between maternal and fetal cells. sequencing of both host and pathogen transcriptomes from infected trophoblast at single-cell resolution will also advance our understanding on host-pathogen interactions in placentas (144, 145) . further refinement of the trophoblast organoid and htscs culture system is needed to address key biological questions unanswered by current models. these include studying the effect of infection on cellular crosstalk between trophoblast and other primary placental cells such as hb cells, or decidual cells in culture, such as dnk or decidual stromal cells. adaptation of crispr/cas9 genome editing technology for the trophoblast organoids or htscs will offer novel insights into essential host genes required for vertical transmission and placental defense mechanisms in humans. major maternal and fetal complications as a result of infection are still a concern, especially in lmic with highest prevalence reported in countries of sub-saharan africa (who, maternal mortality index 2019). profound limitations on current study models and ethical regulations on studying human placenta have significantly delayed the development of therapies and vaccines for maternal-fetal infection. how vertical transmission occurs and how the uterine-placental innate immune system reacts to infection remain as major unresolved questions. revolutionary advances in single-cell genomics, imaging, computational, and stem cell biology methods are currently underway to study the molecular and cellular mechanisms of human diseases. therefore, it is now an exciting time to apply these transformative technologies to comprehensively address fundamental questions on host-pathogen interaction at the human uterine-placental interface. rh, an, and rv-t wrote and edited the manuscript. all authors contributed to the article and approved the submitted version. rh and rv-t were supported by wellcome sanger core funding (wt206194). an was supported by a sanger international fellowship, a nurture fellowship (d43tw010132) and a group leader award supported through the deltas africa initiative (107743/z/15/z). the deltas africa initiative is an independent funding scheme of the african academy of science (aas), alliance for accelerating excellence in science in africa (aesa), supported by the new partnership for africa's development planning and coordinating agency (nepad agency) with funding from the wellcome trust (grant no. 107743), and the uk government. cellular basis of interaction between trophoblast and uterus at implantation immune mechanisms at the maternal-fetal interface: perspectives and challenges immune responses at the maternal-fetal interface zika virus -reigniting the torch antimicrobial peptides in the female reproductive tract: a critical component of the mucosal immune barrier with physiological and clinical implications evidence that intra-amniotic infections are often the result of an ascending invasion -a molecular microbiological study acute chorioamnionitis and funisitis: definition, pathologic features, and clinical significance single-cell reconstruction of the early maternal-fetal interface in humans viral and bacterial pathogens at the maternal-fetal interface perinatal group b streptococcal infections: virulence factors, immunity, and prevention strategies listeria monocytogenes: a multifaceted model q fever during pregnancy: a public health problem in southern france treponema pallidum invades intercellular junctions of endothelial cell monolayers congenital transmission of trypanosoma cruzi: a review about the interactions between the parasite, the placenta, the maternal and the fetal/neonatal immune responses maternal antibodies enhance or prevent cytomegalovirus infection in the placenta by neonatal fc receptor-mediated transcytosis immunolocalization and distribution of rubella antigen in fatal congenital rubella syndrome recent advances in replication and infection of human parvovirus b19 consequences of varicella and herpes zoster in pregnancy: prospective study of 1739 cases zika virus reveals broad tissue and cell tropism during the first trimester of pregnancy asian zika virus strains target cd14+ blood monocytes and induce m2-skewed immunosuppression during pregnancy cd14+cd16+ monocytes are the main target of zika virus infection in peripheral blood mononuclear cells in a paediatric study in nicaragua pereira l. congenital viral infection: traversing the uterine-placental interface understanding how listeria monocytogenes targets and crosses host barriers transmission of toxoplasma gondii from infected dendritic cells to natural killer cells development of the human placenta animal models of human placentation -a review what is trophoblast? a combination of criteria define human first-trimester trophoblast elf5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta the human placenta uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy trophoblast invasion maternal arterial connections to the placental intervillous space during the first trimester of human pregnancy: the boyd collection revisited the cytology of hofbauer cells a three-dimensional study of the normal human placental villous core zika virus infection at different pregnancy stages: anatomopathological findings, target cells and viral persistence in placental tissues zika virus rna replication and persistence in brain and placental tissue hiv-1 in trophoblastic and villous hofbauer cells, and haematological precursors in eight-week fetuses zika virus infects human placental macrophages placental syncytium forms a biophysical barrier against pathogen invasion apoptotic changes occur in syncytiotrophoblast of human placental villi where fibrin type fibrinoid is deposited at discontinuities in the villous trophoblast zika virus targets different primary human placental cells, suggesting two routes for vertical transmission developmental regulation of human cytomegalovirus receptors in cytotrophoblasts correlates with distinct replication sites in the placenta does the human placenta express the canonical cell entry mediators for sars-cov-2? elife single cell transcriptional signatures of the human placenta in term and preterm parturition sars-cov-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes visualization of sars-cov-2 virus invading the human placenta using electron microscopy sars-cov-2 infection of the placenta probable congenital sars-cov-2 infection in a neonate born to a woman with active sars-cov-2 infection an igg-transporting fc receptor expressed in the syncytiotrophoblast of human placenta maternal immunity and antibodies to dengue virus promote infection and zika virus-induced microcephaly in fetuses the neonatal fc receptor (fcrn) enhances human immunodeficiency virus type 1 (hiv-1) transcytosis across epithelial cells identification and localization of divalent metal transporter-1 (dmt-1) in term human placenta identification of transferrin receptor 1 as a hepatitis c virus entry factor viral infection and iron metabolism malaria in pregnancy: pathogenesis and immunity adherence of plasmodium falciparum to chondroitin sulfate a in the human placenta syncytiotrophoblast degradation and the pathophysiology of the malaria-infected placenta human cytomegalovirus transmission from the uterus to the placenta correlates with the presence of pathogenic bacteria and maternal immunity interval between births and risk of congenital cytomegalovirus infection maternal immunity and prevention of congenital cytomegalovirus infection properties and mechanisms of immunoglobulins for congenital cytomegalovirus disease human cytomegalovirus infection elicits new decidual natural killer cell effector functions expression of kir2ds1 by decidual natural killer cells increases their ability to control placental hcmv infection modulation of human leukocyte antigen-c by human cytomegalovirus stimulates kir2ds1 recognition by natural killer cells nk cells control hiv-1 infection of macrophages through soluble factors and cellular contacts in the human decidua pathogen recognition and innate immunity dendritic cells and the control of immunity toll-like receptor signaling pathways irfs: master regulators of signalling by toll-like receptors and cytosolic pattern-recognition receptors cell type-specific expression and function of toll-like receptors 2 and 4 in human placenta: implications in fetal infection expression and subcellular localization of tlr-4 in term and first trimester human placenta expression and regulation of the pattern recognition receptors toll-like receptor-2 and toll-like receptor-4 in the human placenta differential expression of toll-like receptors in the human placenta across early gestation toll-like receptors and pregnancy: trophoblast as modulators of the immune response toll-like receptor-mediated responses by placental hofbauer cells (hbcs): a potential pro-inflammatory role for fetal m2 macrophages tlr7 is involved in sequence-specific sensing of singlestranded rnas in human macrophages human decidual macrophages and nk cells differentially express toll-like receptors and display distinct cytokine profiles upon tlr stimulation variants in the toll-like receptor signaling pathway and clinical outcomes of malaria toll-like receptor (tlr) polymorphisms in african children: common tlr-4 variants predispose to severe malaria common polymorphisms of toll-like receptors 4 and 9 are associated with the clinical manifestation of malaria during pregnancy tolllike receptor 9 mediates innate immune activation by the malaria pigment hemozoin induction of proinflammatory responses in macrophages by the glycosylphosphatidylinositols ofplasmodium falciparum myd88 signaling is directly involved in the development of murine placental malaria tlr4-mediated placental pathology and pregnancy outcome in experimental malaria innate immunity to toxoplasma gondii infection tlr11 activation of dendritic cells by a protozoan profilin-like protein toxoplasma profilin is essential for host cell invasion and tlr11-dependent induction of an interleukin-12 response a toll-like receptor that prevents infection by uropathogenic bacteria viral rna detection by rig-i-like receptors length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene 5 human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors rig-i recognizes the 5 ′ region of dengue and zika virus genomes nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan expression and function of nod-like receptors by human term gestation-associated tissues nod protein expression and function in first trimester trophoblast cells the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-beta sensing and reacting to microbes through the inflammasomes inflammasome activation and il-1 signaling during placental malaria induce poor pregnancy outcomes intra-amniotic inflammation induces preterm birth by activating the nlrp3 inflammasome † the role of cationic antimicrobial peptides in innate host defences betadefensins: linking innate and adaptive immunity through dendritic and t cell ccr6 plasmacytoid dendritic cells sense self-dna coupled with antimicrobial peptide yockey lj, iwasaki a. interferons and proinflammatory cytokines in pregnancy and fetal development interferon-inducible antiviral effectors cutting edge: fetal/placental type i ifn can affect maternal survival and fetal viral load during viral infection type i interferons instigate fetal demise after zika virus infection zika virus infection during pregnancy in mice causes placental damage and fetal demise ifitm proteins inhibit placental syncytiotrophoblast formation and promote fetal demise interferon-gamma: the major mediator of resistance against toxoplasma gondii toxoplasma gondii-induced foetal resorption in mice involves interferongamma-induced apoptosis and spiral artery dilation at the maternofoetal interface contributions of maternal and fetal antiviral immunity in congenital disease organotypic models of type iii interferon-mediated protection from zika virus infections at the maternal-fetal interface type iii interferons produced by human placental trophoblasts confer protection against zika virus infection gestational stage and ifn-λ signaling regulate zikv infection in utero indoleamine 2,3 dioxygenase and metabolic control of immune responses prevention of allogeneic fetal rejection by tryptophan catabolism new insights into ido biology in bacterial and viral infections interferon gamma blocks the growth of toxoplasma gondii in human fibroblasts by inducing the host cells to degrade tryptophan role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages indoleamine 2,3-dioxygenase is regulated by ifn-gamma in the mouse placenta during listeria monocytogenes infection immunobiology of congenital cytomegalovirus infection of the central nervous system-the murine cytomegalovirus model high incidence of contaminating maternal cell overgrowth in human placental mesenchymal stem/stromal cell cultures: a systematic review the burden of vaccine-preventable diseases in pregnancy in low-resource settings optimising sample collection for placental research a single-cell and single-nucleus rna-seq toolbox for fresh and frozen human tumors fin-seq: transcriptional profiling of specific cell types from frozen archived tissue of the human central nervous system rnascope: a novel in situ rna analysis platform for formalin-fixed, paraffin-embedded tissues visualization and analysis of gene expression in tissue sections by spatial transcriptomics spatial mapping of cell types by integration of transcriptomics data derivation of human trophoblast stem cells trophoblast organoids as a model for maternalfetal interactions during human placentation self-renewing trophoblast organoids recapitulate the developmental program of the early human placenta scdual-seq: mapping the gene regulatory program of salmonella infection by host and pathogen single-cell rna-sequencing we would like to thank ashely moffett for the useful discussions and critical review of the manuscript. we are also very grateful to sarah aldridge, loren gibson, damiana alvarez, carlos talavera-lopez, and anna arutyunyan for their insightful comments and corrections. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 hoo, nakimuli and vento-tormo. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-293736-nyvwv31m authors: méry, geoffroy; epaulard, olivier; borel, anne-laure; toussaint, bertrand; le gouellec, audrey title: covid-19: underlying adipokine storm and angiotensin 1-7 umbrella date: 2020-07-21 journal: front immunol doi: 10.3389/fimmu.2020.01714 sha: doc_id: 293736 cord_uid: nyvwv31m severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the third coronavirus leading to a global health outbreak. despite the high mortality rates from sars-cov-1 and middle-east respiratory syndrome (mers)-cov infections, which both sparked the interest of the scientific community, the underlying physiopathology of the sars-cov-2 infection, remains partially unclear. sars-cov-2 shares similar features with sars-cov-1, notably the use of the angiotensin conversion enzyme 2 (ace2) as a receptor to enter the host cells. however, some features of the sars-cov-2 pandemic are unique. in this work, we focus on the association between obesity, metabolic syndrome, and type 2 diabetes on the one hand, and the severity of covid-19 infection on the other, as it seems greater in these patients. we discuss how adipocyte dysfunction leads to a specific immune environment that predisposes obese patients to respiratory failure during covid-19. we also hypothesize that an ace2-cleaved protein, angiotensin 1-7, has a beneficial action on immune deregulation and that its low expression during the sars-cov-2 infection could explain the severity of infection. this introduces angiotensin 1-7 as a potential candidate of interest in therapeutic research on cov infections. coronavirus (cov) is a single-stranded rna virus involved in human and animal diseases. the rare event of its transmission from avian and mammalian reservoirs (mostly bats) to the human host has led to widespread epidemics in recent years (1) . indeed, over the last two decades, three cov outbreaks have forced human populations to change their perspectives regarding the control of emerging diseases and the importance of public health in general. the first outbreak caused by severe acute respiratory syndrome coronavirus 1 (sars-cov-1) occurred between november 2002 and july 2003, originating from china and then spreading worldwide (2) . although the symptoms of sars-cov-1 infection were in most cases non-specific, including lethargy, myalgia, and headache, the high mortality rate of 10% in case series was mostly related to respiratory failure due to acute respiratory distress syndrome (ards) (3, 4) . the physiopathology underlying the severity of sars-cov-1 infection remained unclear after the epidemic due to insufficient sampling. a second cov epidemic occurred in 2012 with middle east respiratory syndrome (mers)-cov, which has mostly led to small-size outbreaks in the years ever since (5) . although it did not reach a pandemic status, mers-cov continues to infect humans, and the world health organization identified more than 850 patients who have died of related complications since its discovery (6) . indeed, mers-cov has a higher mortality rate in case series (case fatality rate of ∼30%), mostly from respiratory failure, which has led to the identification of unique strategies of cov infections to escape the immune response. due to the ending of the sars-cov-1 epidemic and the somewhat limited number of cases of mers-cov in the recent years, understanding the mechanisms of cov infections in humans has proven to be complex, and the conclusions drawn from in vitro experiments and animal models remain difficult to extrapolate. in november 2019, cases of a pneumonia with atypical features were reported in wuhan, china; in january 2020, sars-cov-2 was identified as the cause of this new cov-induced disease , which became a worldwide pandemic in the following months (7) . although the mortality rates of this new covid-19 are still being debated, ranging between 0.3 and 1.5%, it is still lower than those associated with sars-cov-1 and mers-cov infections. patients suffering from severe sars-cov-2 infection could be healthy or only have mild comorbidities such as hypertension or diabetes (8) . most of all, severe cases due to respiratory failure occur 7-12 days after the first symptoms (9) . studies on covid-19 have progressively stressed its similarities with previous cov infections, mostly sars-cov-1, with the same unanswered questions regarding its physiopathology. one notable feature of this disease, already observed in previous cov infections, is the high prevalence of obese patients among the most severe cases. here we seek to explore what underlies the link between immune response and respiratory failure in cov infections on the one hand, and the current observation of obesity as a risk factor for severe outcome in covid-19 on the other. most of the time, the need for intensive care during covid-19 is secondary to the onset of ards (9), as defined by the berlin criteria (bilateral shadowing on lung radiology, rapid deterioration of symptoms, and objective hypoxemia on blood samples). in the first published series, 30% of these ards cases were accompanied by septic shock or other organ dysfunction (8, 10) . the nature of covid-19-induced ards is still under discussion. interleukin (il) dosages are usually very high, and hypoxemia is severe in covid-19-induced ards, which matches the hyperinflammatory profile described by calfee et al. (11) . sars-cov-1-induced ards was associated with vascular leakage and neutrophilic alveolitis (12) , both of which are compatible with a hyper-inflammatory profile. in covid-19, some experts observed ventilatory abnormalities suggestive of microcirculatory involvement such as hypoxic pulmonary vasoconstriction or distal thrombosis (13, 14) . this points to the contribution of several factors in respiratory failure, with experts also citing the possible involvement of genuine viral pneumonia as well as capillary thrombosis by neutrophil extracellular traps (nets) (15) . the reason for this respiratory outcome is most likely a complex interplay of multiple factors, which derive directly from cov virulence. the membrane protein angiotensin-converting enzyme 2 (ace2) is used as an entry receptor by sars-cov-1 and sars-cov-2 (16, 17) . it has been reported that sars-cov-2 has a greater affinity for ace2 than sars-cov-1 due to the specific amino-acid composition in the receptor-binding domain of the spike protein (18) . ace2 is expressed at varying levels by most cells in the body but primarily in the small intestine, testis, kidney, heart, thyroid, and adipose tissue cells (19) . the expression of ace2 in adipocytes seems to be promoted by high fat diets (20) . in the lungs, it is expressed by 2% of epithelial cells, increasing with cell differentiation, and it is mainly located on the apical (or luminal) pole, serving as an accessible anchor point to airborne contaminants (19) . ace2 is a key enzyme of the renin-angiotensin system, converting angiotensin 2 (ang2) into ang1-7. ang2 binds to a receptor, the angiotensin type 1 receptor (at1r), a transmembrane g protein-coupled receptor, which is found in a large variety of cells, ranging from smooth muscle cells, endometrium, and myocardium to blood cells, renal interstitial, and glomeruli. the activation of at1r has several effects: for example, vasoconstriction, vascular permeability, macrophage maturation, and pro-inflammatory cytokine release. during the resolution phase of the inflammation, ang2 promotes tumor growth factor beta production and fibroblast proliferation, leading to fibrosis and inadequate healing of the wounded tissue (21) . an antagonistic pathway of the ang2-derived effects results from the binding of ang1-7 to the mitochondrial assembly (mas) receptor. mas receptor is a ubiquitous g-protein-coupled receptor, implicated, among others, in retina development (22) , muscle wasting (23) , and benign prostate hyperplasia (24) . activation of the mas receptor by ang1-7 induces vasodilatation by a nitric-oxide-dependent mechanism (25, 26) and reduces oxidative stress induced by ang2 in vascular injuries (27) . in macrophages, it promotes an anti-inflammatory profile (28), for example, by lowering pro-inflammatory cytokine production, notably il-6 and tumor necrosis factor alpha (tnfα). ang1-7 has also shown beneficial effects in inflammation resolution and fibrosis, notably in kidney and myocardial disease (21, 29) . the binding of ace2 by sars-cov-2 prevents it from exerting its enzymatic activities, resulting in decreased anti-inflammatory ang1-7 production and the accumulation of pro-inflammatory ang2 (16, 17) . this results in high cytokine titers, neutrophil infiltration, and endothelial dysfunction in the lungs, potentially predisposing for ards. as early as 2004, ace2 tampering was suggested to be an important mechanism in sars-cov-1 infection (30, 31) . it was only later discovered that cov possesses very specific mechanisms to escape the host's immunity (32) . these mechanisms, in addition to the pro-inflammatory response secondary to ace2 binding, might act as a trigger for a sustained and uncontrolled inflammatory response, leading to ards. in general, an efficient antiviral response is driven by t-helper lymphocytes (lth) with a specific polarization such as lth1 and lth2. lth1 refers to a polarization in which lth primarily promotes cytotoxic lymphocytes (ctl) and natural killers (nk) for the control and destruction of infected cells as well as the release of specific cytokines, such as type 1 interferon (inf-1) by innate immune cells. inf-1 is produced by infected cells and innate immune cells after recognizing the viral pathogen-associated molecular patterns (pamps), such as single-strained or uncapped rna, using cytoplasmic pattern-recognition receptors (prr). in particular, toll-like receptor 3 (tlr3) induces toll/interleukin-1 receptor domain-containing adapter-inducing interferon-β (trif). hosts deficient in either tlr3 or trif are more susceptible to viral injuries and thus more at risk of developing ards during cov infections (33) . inf-1 activates the janus kinase-signal transducers and activators of transcription (jak-stat) pathway, resulting in the modulation of hundreds of interferon-sensitive genes and notably in the synthesis of specific cytokines, preferably oriented toward viral control and clearance (34) . most of these steps, involved in inf-1 signaling, are blocked by cov infections. this evolution trait is probably due to the presence of a constitutive inf-1 production in bats (principal reservoir of cov). cov infections are expert evaders of this antiviral response (35) . their escape plan revolves around three main mechanisms: -first, hiding viral rna from cytoplasmic prr. after entering the cell, sars-cov-1 shields its rna by forming, inside the host's endoplasmic reticulum, a large network of doublemembrane vesicles isolated from the cytosol (36, 37) . the modified capping of the viral rna 2 ′ -o-methylation also prevents the binding to an important cytosolic prr (38) . -next, direct tampering of the prr-related enzymes. for example, the papain-like protein in cov can modify the ubiquitinylation profile of tlr7 (39) or other antiviralrelated prr (40) . moreover, s protein triggers il-1rassociated kinase and peroxisome proliferator-associated receptor gamma, subsequently downregulating interferon regulatory factor 7 activity (41) . in addition, the jamming of tlr3 phosphorylation reduces the prr activity, while blocking most of the inf-1 production pathways. -lastly, the non-structural protein 1 in both mers and sars-cov-1 can selectively degrade host rna via endonucleolytic activity against which the viral rna is protected (42, 43) . the many mechanisms used by cov probably leave the infected cells in a defensive cul-de-sac where they are incapable of developing an efficient antiviral response. on the one hand, viral pamps do not result in inf-1 production. on the other hand, non-viral pamps such as debris from cell lysis still stimulate the immune response. this could lead to inappropriate cytokine environments that lack inf-1 and are thus less effective against viruses, as seen in covid-19 (44) . indeed, during covid-19 infection, most patients exhibit a specific cytokine profile, associating innate immunity chemokines (such as monocyte chemoattractant protein 3 and interferon gamma-induced protein 10 (ip-10), which are suggestive of macrophage activation and epithelial suffering), and pro-inflammatory macrophage-produced cytokines such as il-6 (45). moreover, cov infections can directly induce the activation of nuclear factor kappa b (nfkb), notably by tampering with the tnf receptor-associated factor 3 pathway (traf3) via its open reading frame 3a. activation by ubiquitination of traf3 also promotes the de novo development of the nod-like receptor pyrin domain containing protein 3 (nlrp3) inflammasome and the production of il-1β and il-18 (46) . this cytokine production promotes macrophage activation and inf-3, although it does not salvage a deficient polarization of the adaptive immunity toward lth1 and its subsequent efficient antiviral response. high plasma levels of il-6 and the absence of inf-1 have been noted in severe patients (47) , illustrating a sustained innate response that fails to achieve viral clearance and triggers ards. however, this sustained inflammation without lth polarization might not be the only profile to bypass the antiviral cul-de-sac. some patients infected by mers-cov demonstrated a polarization of the immune profile toward a lth17-mediated response. faure et al. compared two cases of mers with different outcomes (48) ; the patient with a fatal outcome had an early increase in il-17 and il-23 titers (hallmarks of lth17 polarization), whereas the surviving patient had a spike in inf-1 but no indication of lth17 polarization. lth17 are effective actors in the clearance of extracellular microorganisms such as fungi and bacteria, but poorly effective against viral pathogens (49) . in general, viral pamps do not usually polarize the immune response to lth17. the association of severe outcome and inappropriate cytokine environment in cov infection suggests a link with immune polarization, as a result of the "cul-de-sac" of antiviral response induced by the cov escaping strategies. the resulting inefficient immune profile leads to a sustained viral exposure and persistent inflammatory state. in addition to the pro-inflammatory signals mediated by ace2 inhibition, this sustained and inappropriate immune activation might be strongly involved in the development of ards. obesity is a common condition, affecting up to 30% of adults in western countries. it is defined by a body mass index (bmi) >30 kg/m 2 , irrespective of the location of the adipose tissue. however, all profiles of obesity are not equivalent in terms of their consequences. indeed, abdominal (or visceral) obesity (estimated by the waist circumference or waist-to-hip ratio), in which visceral fat predominates, is more associated with metabolic disorders such as type 2 diabetes or hypertension, compared to "metabolically healthy" obesity, in which subcutaneous fat predominates. early observations in the sars-cov-2 epidemics suggested obesity to be a risk factor to covd-19, or at least to severe forms of the disease (50) . in our retrospective cohort, we observed more than 60% of patients with overweight or obesity (n = 155) (figure 1) . in a retrospective cohort, simonnet et al. showed an increasing risk of intensive care unit (icu) admission in covid-19 patients as bmi increased, independently of other metabolic disorders (51) , which was subsequently confirmed by other teams (52, 53) . thus, obesity appears to be a risk factor for presenting a severe form of covid-19. it should be mentioned that once in icu, obesity is known to confer a survival advantage, termed the "obesity paradox" (54) . patients with a bmi > 25 kg/m 2 seem to survive mechanical ventilation and severe septic states significantly better than patients with a normal or low bmi (55, 56) , presumably due to their elevated muscle mass, which represents a metabolic reserve in the hypermetabolic state of icu patients (54, 57) . it is not yet known whether once admitted to icu, obese covid-19 patients also present a better prognosis than patients of normal body weight. the scientific observations of the last two decades have placed obesity in a complex pathological framework centered around the deregulation of adipocyte, which is far from the naive idea of a simple diet-induced condition (58). white adipose tissue (wat) is now recognized as an independent endocrine organ, whose main role is to regulate and store the energy provided by food. however, the hormones released by wat, specific to the adipocyte and known as adipokines, reach a large variety of organs and modulate an extensive range of functions, from appetite control to inflammatory response (29) . leptin is the leading adipokine, whose anorexigen properties regulate satiety and food intake. leptin levels in blood are proportionate to the amount of wat and increase with bmi. interestingly, the leptin receptor (lepr) on immune cells mostly activates jak-stat and nfkb dependent pathways, except in neutrophils, macrophages, and antigen presenting cells, which all express a particular form of lepr. leptin promotes migration in the wat of resident macrophages and induces their polarization toward a pro-inflammatory profile or a classical activated macrophage (m1) profile, and unbalances the lth profiles, by reducing regulatory t-cells and promoting lth17 polarization (59) . adiponectin is another adipokine, whose levels increase in proportion to subcutaneous fat but decrease with visceral fat accumulation. it favors whole-body insulin sensitivity, fatty-acid oxidation and diminishes the hepatic neo-glucogenesis pathways (60) . adiponectin promotes primarily lth1 polarization, hence antiviral inflammation. other adipokines, such as lipocalin-2, down-regulate inflammatory lth altogether by promoting regulatory lymphocytes. adipokines form a large family regularly counting new members over the last few years, all of which reveal complex and multiple implications in the regulation of energy storage and release, adipose tissue regulation and rather ubiquitous cellular metabolism (61) . unlike subcutaneous fat, visceral fat accumulation, also described as "abdominal obesity, " is characterized by a dysfunctional profile of adipokines associated with a rise in pro-inflammatory signals. the triggers of this dysfunction is believed to be a metabolic stress in the presence of nutrient excess and a hypoxic stress caused by hypertrophic visceral adipocytes, due to an increase in cells' size and low neovascularization, via a mobilization of hypoxia inducible factor 1 (62) . unlike visceral fat, subcutaneous fat expansion is hyperplasic and is not correlated with low-grade inflammation (63) . in severe abdominal obesity, the adipokine profile is unbalanced in favor of leptin production and low-grade inflammation at the expense of adiponectin, or lipocalin-2. this deregulation of the adipokine profile links various disorders associated with metabolic diseases, such as insulin-resistance, to inflammatory manifestations, as described in rheumatoid arthritis (64) . ang1-7 takes an active role in regulating the effects of adipokines. its involvement was reviewed by lelis et al., with an exhaustive approach and emphasis on other adipokines that will not be described here, such as sirtuin and resistin (29) . a strong interest in ang1-7 has already arisen from these observations, particularly in the field of atherosclerosis and non-alcoholic fatty liver disease, in which ang1-7 seems beneficial. in a concise article, mori et al. hypothesized that the disruption of the reninangiontensin system by the virus could impair the energetic functions of these pathways during sars-cov infections (65) . we suggest that the tampering with such pathways could also lead to abnormalities in the inflammatory response observed in severe cov infections through their influence on immune regulation and cytokine production. adipocyte dysfunction in visceral fat is correlated to lowgrade persistent inflammation, known as meta-inflammation, which is suspected to be the starting point or an early factor in metabolic disorders associated with severe obesity (63) . this meta-inflammation is mostly driven by the leptinactivated m1 macrophages in wat. wat-resident macrophages exhibit pro-inflammatory behavior, producing il-1β, il-6, and tnfα. the precursor of il-1β is cleaved into bioactive il-1β by the nlrp3 inflammasome, as a result of the nfkb pathway activation, which is induced by both pro-inflammatory and hypoxic signals originating from the adipocytes (58). adiponectin can inhibit nfkb activation, but as mentioned above, depending on the obesity severity and profile, the effects of adiponectin can easily be overwhelmed by those of leptin (66) . leptin also polarizes hematopoiesis directly in the bone marrow, promoting granulocyte, and erythroblast lines (the latter probably acts as a protective mechanism against hypoxia) at the expense of lymphocytes (67) . when neutrophils are mature and circulating, leptin also promotes their survival on a dosedependent scale (68) . higher levels of neutrophils have thus been observed in obese patients, possibly making the neutrophil recruitment during an inflammatory process more potent than in patients with a normal bmi (69) . besides suffering from a pro-inflammatory environment, which favors macrophage activation and neutrophil production, obese patients exhibit abnormal responses to viral infection. as summarized by honce et al., during influenza infections, obese patients tend to have greater neutrophil activation and net development, contributing to capillary damage and thrombosis. such phenomena have been extensively found in covid-19 patients (70) . their inflammatory response is also characterized by a lack of inf-1 production as well as a strong cytokine production, notably il-6, ip-10, and type 3 inf, which are elevated in severe covid-19. interestingly, patients with visceral fat accumulation also tend to have a lower tlr3 expression in adipocytes, muscle cells, and adipose tissue-resident macrophages, as well as a concomitant lower production of cytokines following exposure to viral pamps (71) (72) (73) . this suggests that their baseline profile resembles that found in severe cov infections, in which the antiviral response is less efficient, but the overall inflammation is higher than in other viral infections. finally, both obesity and metabolic disorders are associated with vascular dysfunction. at the acute phase of lung infection, this could result in microcirculatory abnormalities, as suggested by intensive care physicians, and increased lung edema. patients with visceral fat accumulation, type 2 diabetes (74), and hypertension are not the only subjects at a higher risk of severe sars-cov-2 infection. when considering metabolic disorders separately, diabetes, non-alcoholic liver disease, and obstructive sleep disorders have been recently reported as risk factors for a severe outcome (74) (75) (76) . this suggests that the metabolic dysfunction associated with these disorders more than obesity alone might be involved in the severity of the disease in these patients. when comparing the effects of ang1-7 and the inflammatory environment of patients with adipocyte dysregulation and metabolic disorders, an interesting pattern emerges. all the immunological features arising from the adipocyte dysfunction-(i.e., m1 macrophage polarization with il-6 and tnfα production), and neutrophil promotion-may contribute to the development of ards and thus be countered by the activation of the ang1-7/mas receptor axis. ang1-7 also favors figure 2 | impact of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) on pathways promoting acute respiratory distress syndrome (ards). by inactivating the angiotensin conversion enzyme 2 (ace2), sars-cov-2 leads to an accumulation of angiotensin 2 and a lower dosage of angiotensin 1-7, respectively resulting in the higher promotion and lower inhibition of pro-inflammatory signals. a strong capillary barrier and a beneficial oxidative profile, which are altered in patients with visceral fat activation and could help to prevent ards. this leads us to two hypotheses: either patients with metabolic disorders, primarily visceral fat accumulation, have a constitutional lower titer of ang1-7, as suggested by some observations (77) , and a resulting higher inflammation; or the ang1-7 levels in these patients are preserved and restrain the baseline inflammation. in the first case, the inappropriate inflammatory response, added to the diminished activation of tlr3 in obese patients, leads to unrestrained inflammation. however, if ang1-7 is present in these patients and limits the meta-inflammation, acting as a guardrail, the antagonization of ace2 by sars-cov-1 and 2 in addition to the lack of de novo ang1-7 production could exacerbate the meta-inflammation and contribute to the severe septic states of obese patients with covid-19, as illustrated in figure 2 . in both cases, the supplementation of ang1-7 in these patients might improve fitness upon sars-cov infection. ace2 deficiency has already been explored by some research teams to better understand the potential metabolic benefits of conversion enzyme inhibitors used in hypertension, among others. their studies highlighted the association between ace2 deficiency and higher titers of pro-inflammatory cytokines in obese mice, as well as in mice with glucose intolerance (78) , which is closely correlated with metainflammation (79) . other studies correlate ace2 deficiency with epicardial inflammation (80) . this suggests that the ang1-7/mas axis allows a better control of inflammation in obese patients. tlr4 is a receptor to lps and leads to nfkb activation and (among others) hepatic inflammation. when administered orally to rats fed with a high-fat diet, ang1-7 lowered hepatic inflammation, notably through a modulation of a metabolic pathway involving tlr4 (81) . moreover, promoting the effects of the ang1-7/mas receptor axis using medication also improves the aforementioned cytokines and oxidative stress in obese mice, with a protective effect against diabetic cardiomyopathy (82) . ang1-7 is already in the spotlight of scientific research given its beneficial effects in preventing the development of metabolic disorders and obesity (83) . we believe that our literature review highlights the beneficial effects of ang1-7 on metainflammation in preexisting obesity and its potential involvement in inflammatory response and viral clearance, notably against sars-cov-2. modulation of the renin-angiotensin system has been mentioned by others to explain the severity of covid-19. a recent study found a lower mortality and intubation risk during covid-19 among elderly patients treated with nifedipine or amlodipine (84) , although the study sample was small and most of the accessible data do not suggest a strong connection (85, 86) . however, these drugs interfere with at1r and not with the genuine production of ang1-7. in obese patients with covid-19, this hypothesis should be considered. oral or parenteral ang1-7 supplementation could be a therapeutic option to diminish the low-grade systemic inflammation due to adipocyte dysfunction and attenuate the severity of ace2-mediated injuries consecutive to sars infection. parenteral ang1-7 has already been used in human research on account of its property to enhance acetylcholine-mediated vasodilatation in endothelia, with safe outcomes (87) . covid-19 is a viral disease with remarkable characteristics given its high severity in obese patients and its ability to tamper ace2 metabolism. we believe that more than being just an incentive to accelerate research on viral infection, covid-19 also presents an opportunity to respond to questions that were previously considered to be too intricate or complex, such as non-septic inflammation or the immune system communication underlying metabolic disorders. understanding the multiple and interrelated factors linking sars-cov-2 infection, angiotensin metabolism, global inflammation, and metabolic disorders such as type 2 diabetes and obesity should provide us with a better insight into the way in which these conditions and physiological states interact outside of an acute aggression. the raw data supporting the conclusions of this article will be made available by the authors within respect of general data protection regulation, without undue reservation. the studies involving human participants were reviewed and approved by comité ethique du centre investigation clinique (cecic). subjects were all informed and did not oppose, written consent for participation was not required for this study in accordance with the national legislation and the institutional requirements. gm and al conceptualized the idea, provided discussion, feedback, and organize the plan of the article. gm, a-lb, oe, and al wrote the paper. gm, a-lb, oe, bt, and al reviewed the different version of paper. all authors contributed to the article and approved the submitted version. this work was supported by the foundation of grenoble alpes university, grenoble, france. severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection severe acute respiratory syndrome) coronavirus as a possible cause of severe acute respiratory syndrome sars: clinical features and diagnosis mers coronavirus: diagnostics, epidemiology and transmission available online at a novel coronavirus from patients with pneumonia in china clinical features of patients infected with 2019 novel coronavirus in wuhan intensive care management of coronavirus disease 2019 (covid-19): challenges and recommendations epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials dysregulated type i interferon and inflammatory monocyte-macrophage responses cause lethal pneumonia in sars-cov-infected mice covid-19 pneumonia: ards or not? clinical phenotypes of sars-cov-2: implications for clinicians and researchers targeting potential drivers of covid-19: neutrophil extracellular traps a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor characterization of the receptor-binding domain (rbd) of 2019 novel coronavirus: implication for development of rbd protein as a viral attachment inhibitor and vaccine expression of the sars-cov-2 cell receptor gene ace2 in a wide variety of human tissues ace2 is expressed in mouse adipocytes and regulated by a high-fat diet ace2, angiotensin-(1-7) and mas receptor axis in inflammation and fibrosis the expression of masreceptor of the renin-angiotensin system in the human eye expression of the mas receptor is upregulated in skeletal muscle wasting embarking effect of ace2-angiotensin 1-7/mas receptor axis in benign prostate hyperplasia clinical characteristics of allergic bronchopulmonary aspergillosis mas receptor is involved in the estrogen-receptor induced nitric oxide-dependent vasorelaxation mas receptor mediates cardioprotection of angiotensin-(1-7) against angiotensin ii-induced cardiomyocyte autophagy and cardiac remodelling through inhibition of oxidative stress role of the receptor mas in macrophage-mediated inflammation in vivo angiotensin-(1-7), adipokines and inflammation sars: future research and vaccine pathogenesis of severe acute respiratory syndrome chemokine up-regulation in sars-coronavirus-infected, monocyte-derived human dendritic cells toll-like receptor 3 signaling via trif contributes to a protective innate immune response to severe acute respiratory syndrome coronavirus infection regulation of type i interferon responses chapter seven -interaction of sars and mers coronaviruses with the antiviral interferon response sars-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro sars-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum attenuation and restoration of severe acute respiratory syndrome coronavirus mutant lacking 2 ′ -o-methyltransferase activity sars coronavirus papain-like protease inhibits the tlr7 signaling pathway through removing lys63-linked polyubiquitination of traf3 and traf6 the severe acute respiratory syndrome coronavirus nucleocapsid inhibits type i interferon production by interfering with trim25-mediated rig-i ubiquitination middle east respiratory syndrome corona virus spike glycoprotein suppresses macrophage responses via dpp4-mediated induction of irak-m and pparγ sars coronavirus nsp1 protein induces template-dependent endonucleolytic cleavage of mrnas: viral mrnas are resistant to nsp1-induced rna cleavage middle east respiratory syndrome coronavirus nsp1 inhibits host gene expression by selectively targeting mrnas transcribed in the nucleus while sparing mrnas of cytoplasmic origin severe acute respiratory syndrome coronavirus orf6 antagonizes stat1 function by sequestering nuclear import factors on the rough endoplasmic reticulum/golgi membrane plasma ip-10 and mcp-3 levels are highly associated with disease severity and predict the progression of covid-19 a tug-of-war between severe acute respiratory syndrome coronavirus 2 and host antiviral defence: lessons from other pathogenic viruses the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? interleukin-17a (il-17a), a key molecule of innate and adaptive immunity, and its potential involvement in covid-19-related thrombotic and vascular mechanisms early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia high prevalence of obesity in severe acute respiratory syndrome coronavirus-2 (sars-cov-2) requiring invasive mechanical ventilation prevalence of obesity among adult inpatients with covid-19 in france obesity and its implications for covid-19 mortality initiation of nutritional support is delayed in critically ill obese patients: a multicenter cohort study obesity survival paradox in pneumonia: a meta-analysis obesity paradox" in acute respiratory distress syndrome: asystematic review and meta-analysis high-fat feeding protects mice from ventilator-induced lung injury, via neutrophil-independent mechanisms leptin in the interplay of inflammation, metabolism and immune system disorders adiponectin regulation and function adipokines: linking metabolic syndrome, the immune system, and arthritic diseases oxygen deprivation and the cellular response to hypoxia in adipocytes-perspectives on white and brown adipose tissues in obesity an update on immune dysregulation in obesity-related insulin resistance adipokines and inflammation: is it a question of weight? sars-cov-2 perturbs the renin-angiotensin system and energy metabolism recombinant adiponectin inhibits inflammation processes via nf-kb pathway in acute pancreatitis a role for leptin in sustaining lymphopoiesis and myelopoiesis apoptotic pathways are inhibited by leptin receptor activation in neutrophils neutrophil infiltration and systemic vascular inflammation in obese women impact of obesity on influenza a virus pathogenesis, immune response, and evolution defects in tlr3 expression and rnase l activation lead to decreased mnsod expression and insulin resistance in muscle cells of obese people decreased tlr3 in hyperplastic adipose tissue, blood and inflamed adipocytes is related to metabolic inflammation decreased interferon-α and interferon-β production in obesity and expression of suppressor of cytokine signaling diabetes is a risk factor for the progression and prognosis of covid-19 metabolic associated fatty liver disease increases covid-19 disease severity in non-diabetic patients coronavirus disease 2019 and obstructive sleep apnea syndrome cross talk between angiotensin-(1-7)/mas axis and sirtuins in adipose tissue and metabolism of high-fat feed mice deficiency of ace2 in bone-marrow-derived cells increases expression of tnf-α in adipose stromal cells and augments glucose intolerance in obese c57bl/6 mice tlr4 links innate immunity and fatty acid-induced insulin resistance ace2 deficiency worsens epicardial adipose tissue inflammation and cardiac dysfunction in response to diet-induced obesity oral angiotensin-(1-7) prevented obesity and hepatic inflammation by inhibition of resistin/tlr4/mapk/nf-κb in rats fed with high-fat diet azilsartan ameliorates diabetic cardiomyopathy in young db/db mice through the modulation of ace-2/ang 1-7/mas receptor cascade angiotensin 1-7: a peptide for preventing and treating metabolic syndrome nifedipine and amlodipine are associated with improved mortality and decreased risk for intubation and mechanical ventilation in elderly patients hospitalized for covid-19 interactions of coronaviruses with ace2, angiotensin ii, and ras inhibitors-lessons from available evidence and insights into covid-19 renin-angiotensin-aldosterone system inhibitors in patients with covid-19 favorable vascular actions of angiotensin-(1-7) in human obesity acknowledgments authors thank mrs. victoria grace, from english publications, for the careful editing of the publication. we kindly thank p. audoin, c. clape, a. metz, and i el amrani for their support regarding the reglementary procedures. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 méry, epaulard, borel, toussaint and le gouellec. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-283382-t890r7xp authors: coughlan, lynda title: factors which contribute to the immunogenicity of non-replicating adenoviral vectored vaccines date: 2020-05-19 journal: front immunol doi: 10.3389/fimmu.2020.00909 sha: doc_id: 283382 cord_uid: t890r7xp adenoviral vectors are a safe and potently immunogenic vaccine delivery platform. non-replicating ad vectors possess several attributes which make them attractive vaccines for infectious disease, including their capacity for high titer growth, ease of manipulation, safety, and immunogenicity in clinical studies, as well as their compatibility with clinical manufacturing and thermo-stabilization procedures. in general, ad vectors are immunogenic vaccines, which elicit robust transgene antigen-specific cellular (namely cd8(+) t cells) and/or humoral immune responses. a large number of adenoviruses isolated from humans and non-human primates, which have low seroprevalence in humans, have been vectorized and tested as vaccines in animal models and humans. however, a distinct hierarchy of immunological potency has been identified between diverse ad vectors, which unfortunately limits the potential use of many vectors which have otherwise desirable manufacturing characteristics. the precise mechanistic factors which underlie the profound disparities in immunogenicity are not clearly defined and are the subject of ongoing, detailed investigation. it has been suggested that a combination of factors contribute to the potent immunogenicity of particular ad vectors, including the magnitude and duration of vaccine antigen expression following immunization. furthermore, the excessive induction of type i interferons by some ad vectors has been suggested to impair transgene expression levels, dampening subsequent immune responses. therefore, the induction of balanced, but not excessive stimulation of innate signaling is optimal. entry factor binding or receptor usage of distinct ad vectors can also affect their in vivo tropism following administration by different routes. the abundance and accessibility of innate immune cells and/or antigen-presenting cells at the site of injection contributes to early innate immune responses to ad vaccination, affecting the outcome of the adaptive immune response. although a significant amount of information exists regarding the tropism determinants of the common human adenovirus type-5 vector, very little is known about the receptor usage and tropism of rare species or non-human ad vectors. increased understanding of how different facets of the host response to ad vectors contribute to their immunological potency will be essential for the development of optimized and customized ad vaccine platforms for specific diseases. adenoviruses (ad) represent a promising vector platform for the development of vaccines for infectious disease, largely due to their safety and ability to stimulate robust cellular and/or humoral immune responses in multiple species (1) (2) (3) (4) (5) (6) (7) (8) , as compared with other genetic vaccine platforms (5, (9) (10) (11) (12) . adenoviruses derived from humans and non-human primates (nhp) belong to the family adenoviridae and the genus mastadenoviridae, and are further subdivided into species a-g (i.e., for species a viruses, these are denoted hadv-a followed by the type number). accounting for the inclusion of many ad recombinants (13, 14) , ∼103 human ads (http://hadvwg.gmu. edu/) and >200 non-human ad serotypes have been identified to date. adenoviruses are non-enveloped viruses which contain a double-stranded dna genome. the virion exterior is composed of three major structural proteins, the fiber, the penton base and the hexon [ (15) (16) (17) (18) ; figure 1 ]. recombinant ad (rad) vectors can easily be rendered replication-incompetent (non-replicating) through deletion of the essential viral gene e1 from their genome and can be vectorized for easy manipulation (16, (19) (20) (21) . further improvements to these first-generation ad vectors have been developed in which the e3 region is also deleted, to accommodate a larger heterologous transgene capacity of ∼7.5 kbp. ad vectors display a number of desirable characteristics which make them particularly well-suited to prophylactic vaccine applications. their genome is stable and easy to manipulate, they can be amplified and produced to high titers using various complementing cell lines which adhere with good clinical practice (gcp) procedures (22) , and they have an outstanding track-record as safe and immunogenic vaccines in numerous human clinical trials (1) (2) (3) (4) (5) (6) (7) (23) (24) (25) . historically, the most commonly used rad has been human mastadenovirus c, human adenovirus type-5 (hadv-c5, referred to ad5 throughout this manuscript). however, despite its well-characterized biology and robust immunogenicity, high seroprevalence has limited its widespread use in humans and has prompted the development and investigation of novel ad species, either rare species human ads (6, 20, 26) or those derived from nhps (5, 8, 27) , many of which have very low seroprevalence (27) (28) (29) (30) (31) . for the purpose of this review, the nomenclature of ad types will use reference to the vertebrate species from which the vector was derived (i.e., h for human or ch for chimpanzee) followed by the virus type number, as outlined in table 1 . human ad vectors will include their assigned adenovirus species group (i.e., a-g). this nomenclature has been proposed to ictv by dr. don seto, george mason university and dr. james chodosh, harvard university (personal communication). the use of non-replicating viral vectors as a vaccine platform has several advantages over other vaccine formulations (i.e., recombinant protein, inactivated particles). viral vectored vaccines retain some characteristics of a live attenuated vaccine in terms of their ability to enter target cells, engage intracellular trafficking pathways to deliver their genome and facilitate antigen (ag) expression and subsequent ag-presentation in vivo, but (17) and was created with ©biorender -biorender.com. proteins are not to scale. possess additional safety features. furthermore, in order to drive the expression of substantial quantities of transcripts which correspond to the encoded vaccine ag, non-replicating ad vectors make use of powerful exogenous promoters, such as the cytomegalovirus (cmv) promoter (32). unlike recombinant protein or inactivated vaccines in which antigen quantity is limited to the input vaccine dose, the use of exogenous promoters facilitates more sustained transgene antigen expression in vivo. in general, ad vectors are well-established to stimulate cd8 + t cell responses directed toward transgene ag, with selected ad types confirmed to elicit robust cellular immunity in both animal models (8, (32) (33) (34) (35) (36) and humans [(1, 3, 6, 23, 33, 37) ; see table 2 ]. memory cd8 + t cell responses elicited following vaccination with ad vectors exhibit an extended contraction phase (38) . importantly, the persistent ag expression following immunization with ad vaccines enables the induction of sustained immune responses (5, 36, (39) (40) (41) , making them very attractive vaccine vectors for conferring long-lasting immunity. it is believed that the prolonged expression of vaccine ag facilitates the maintenance of effector cd8 + t cells while simultaneously permitting their differentiation into central memory populations (36) . improved understanding into how ad vectors prime and maintain such long-lived responses will be crucial not only in designing improved ad vaccines, but also other vaccine adenovirus classification for human and non-human ad vectors referred to in this review. the nomenclature of ad vectors derived from non-human primates, including chimpanzees, is not standardized, resulting in the confusing use of multiple names assigned by individuals who vectorized these constructs. in this review text, we propose to follow current standards for human ad vectors, such as hadv-c5, as outlined by ictv, for descriptions of ad vectors derived from chimpanzees or non-human primates. abbreviated "alternative" names are used in table 2 platforms which are optimized for diverse disease targets. however, the precise factors which contribute to the robust immunogenicity associated with particular ad-vectored vaccines are currently unclear. it is widely appreciated in the ad vaccine field that ad vectors can act as a "self-adjuvant, " allowing the stimulation of multiple innate immune signaling pathways upon viral entry, which can augment the immunogenicity of the encoded ag (although conversely, stimulation of certain signaling pathways can also be detrimental to their immunogenicity, as discussed below). although we have some understanding of how individual pathways work in vitro in defined cell types (i.e., dendritic cells, macrophages), understanding how these pathways intersect, or cooperate in the development of protective immunity in vivo, is complex and our understanding is incomplete. additionally, it is apparent that there is a clear hierarchy of immunological potency when evaluating distinct ad species and serotypes as vaccine vectors in animal models. although a few selected vectors display robust immunogenicity in vivo which is comparable to that of ad5, most are less immunogenic (5, 8, 15, 29, 31) , and there are considerable differences in the phenotype and functionality of immune response elicited (8, 42, 43) . in recent years, investigators have begun to identify several crucial factors which could contribute to these profound disparities in immunological potency. it is now believed that differences in (i) cellular receptor and/or co-receptor usage, viral entry, trafficking, endosomal escape, and in vivo tropism can contribute to the (ii) differential activation of innate immune signaling which influences subsequent immune responses (44) . in addition, it is apparent that the (iii) magnitude and persistence of transgene expression can also shape the ensuing immune response (33) and all of these factors are in turn affected by the (iv) vaccine dose (8) and route of administration (45) . increased understanding of, and implementation of efforts to overcome these striking differences in immunological potency or quality, will absolutely be required for the development of optimal ad vectors for clinical use. as a result of extensive study over the past few decades, we have a clear understanding of the in vitro tropism determinants of vectors derived from species c hadvs (i.e., hadv-c5/ad5) (18) . the classical entry pathway of rad5-based vectors in non-immune cells is mediated by binding of the fiber knob domain (figure 2 ) to the coxsackie and adenovirus receptor (car). following this "docking" interaction, viral internalization is facilitated through interactions between the arginine-glycineaspartate (rgd) motif within the viral penton base and cellular integrins (namely αvβ3 and αvβ5) on the surface of cells (46, 47) . adenovirus capsid disassembly then proceeds in a systematic and stepwise process. initial binding to car is a motile interaction, whereas the subsequent interaction with immobile αv integrins results in the ripping or shedding of fibers from the virion, initiating partial disassembly of the virion at the plasma membrane (48, 49) . it was previously believed that endosomal escape was ph-dependent (50) . however, it has subsequently been demonstrated that exposure of protein vi from the capsid interior (51) at the cell surface, as a result of mechanical strain induced by the antagonistic car:integrin interaction (48) , facilitates access to the cytoplasm through the action of its ph-independent membrane lytic activity (52) (53) (54) . following endosomal escape, the virion is transported to the nuclear pore complex via the microtubule network (50, 52) . once the virion has docked at the nuclear pore complex, interactions with cellular proteins trigger further capsid disassembly and allow the viral dna to extrude into the nucleus for subsequent gene expression (18) . however, unlike ad5, adenoviral types derived from species b or d viruses such as hadv-b35, hadv-b3, hadv-d11, or hadv-d26 can use alternative binding/entry factors or receptors to car, such as cd46 (55-57), desmoglein-2 (dsg-2) (58), or sialic acid (59) . the post-entry steps of these rare species ad viruses in diverse cell types are not as well-characterized as the car-mediated entry of ad5. however, it is considered that the use of alternative entry pathways or different receptors can not only result in differences in endosomal escape, trafficking to the nucleus and subsequent transgene expression (60) , but can also impact on the in vivo tropism of the vector following different routes of vaccine administration (i.e., intramuscular vs. intranasal). as a result, triggering of innate immune signaling pathways may also differ at each step of the entry process (33, 61) . less efficient trafficking pathways could result in weak or limited induction of cytokines/chemokines (62), or increased uptake in cell types which result in vector degradation with minimal transgene expression (63) (64) (65) (66) . consequently, such differences between diverse ad vectors can impact on the magnitude and phenotype of the ensuing adaptive immune response when they are used as a vaccine platform (8, 33, 44) . macrophages. in addition to the classical in vitro entry pathways described above, ad vectors can infect mononuclear phagocytes efficiently both in vitro and in vivo, independently of their described surface receptors (i.e., car, dsg-2) (67), which are absent on murine macrophages. in vivo interactions with tissue resident macrophages, such as kupffer cells in the liver or alveolar macrophages in the lung, can result in scavenging and degradation of significant amounts of input ad vector (64) (65) (66) . not only can these interactions result in limited transgene expression which could affect the therapeutic efficacy, but the phagocytosis of ad particles can trigger inflammatory responses (68, 69) , leading to undesirable off-target toxicity (70) . this is a particularly important consideration for therapeutic applications which require systemic administration or are designed for use in immunocompromised individuals (i.e., oncolytic viral therapy for disseminated metastases) (18) . as a result, efforts have been made to characterize the mechanisms of ad viral entry in macrophages and to better understand how these interactions contribute to the induction of inflammatory responses within defined anatomical compartments following different routes of administration (i.e., intramuscular, intranasal vs. intravenous). opsonization of ad viral particles by complement or antibodies (natural or anti-viral) can bridge entry into macrophages by engaging fc-receptors (fcrs) or complement receptors (71) (72) (73) . additionally, a role for scavenging receptors in facilitating viral entry into murine macrophages, both in vitro and in vivo, has been outlined (62, 67, 74) . scavenging receptors are a heterogenous and structurally diverse family of receptors capable of interacting with endogenous proteins and lipids, microbial ligands, and non-opsonized particles, including viruses (75) . in addition to contributing to the clearance of particulate ag, scavenging receptors have been implicated in innate immune sensing, due to their ability to recognize pathogen-associated molecular patterns (pamps). murine sr-a1 (74), sr-aii (76) , and marco (sr-a6) (67) have been described as receptors for rad vectors, and marco + marginal zone macrophages in the spleen have been shown to accumulate ad5-based vectors following intravenous (i.v.) delivery in mice (28, 77) . the fiber knob (i.e., sr-a1), or hexon protein has been implicated in mediating these interactions (i.e., sr-aii and sr-a6). interestingly, sr-a6 (marco) was shown to not only facilitate entry and efficient gene transduction with ad5, but also with hadv-c2, hadv-b35, and hadv-d26 (67). the mechanism of interaction between ad and sr-aii/sr-a6 was proposed to involve the negative charge conferred by specific hypervariable regions (hvrs) of the viral hexon, namely hvr1. in support of this, preferential scavenging of negatively charged particles has previously been shown to contribute to the differential recognition of ad vectors by macrophages in vivo (76, 78, 79) . dendritic cells. dendritic cells (dcs) are a specialized subset of professional ag presenting cells which are central to the development of protective immunity. dcs can process ag using several methods; (i) direct presentation, in which an infected dc presents peptide:major histocompatibility complex class (mhc) complexes directly to t cells, (ii) cross-presentation in which ag derived from other infected cells is phagocytosed by dcs, processed and then presented to t cells, and (iii) cross-dressing (80) , in which peptide:mhc complexes are acquired from other professional or non-professional antigen presenting cells (apcs) and are transferred to the dc through a process of trogocytosis, in which fragments of the plasma membrane containing the mhc complex merges with the recipient cell. cross-dressing is also hypothesized to occur through the intercellular transfer of pre-formed peptide:mhc complexes by extracellular vesicles, such as exosomes [(81-84); figure 3 ]. a critical role for dcs in the robust induction of cd8 + t cell responses following immunization with rad vectors has been demonstrated (86, 87) . it has been shown that cd8 + , but not cd4 + t cell responses elicited by ad vaccines, are dependent on cross-presentation by specific sub-populations of dcs, including cd8α + dcs (33) . in support of this, ag-specific cd8 + t cell responses in batf3-deficient mice (batf3 −/− ), which preclude the development of the latter dc population (cd8α + dcs), were shown to be ∼80% lower following immunization with several ad vectors (33) . dcs infected with ads upregulate mhc, as well as co-stimulatory molecules including cd40, cd80, or cd86, leading to the activation or maturation of dcs (88), in a manner which is believed to be dependent on nuclear factor kappa-lightchain-enhancer of activated b cells (nf-κb) signaling (89) . the maturation of murine dcs has been proposed to be mediated by the fiber knob domain of ad5 (90) . in vivo experiments in mice have shown that uptake of rad5-based vaccines in draining lymph nodes (dlns) following intramuscular (i.m.) or subcutaneous (s.c.) vaccination is highest in cd11c + cd8 − b220 − dcs, although cd11c + cd8 + b220 − dcs were the most potent for eliciting naïve ag-specific t cell proliferation (86) . as previous studies have shown that targeting vaccine ag to defined populations of dcs can improve immunity and vaccine efficacy (91, 92) , efforts are ongoing to better understand the precise interactions between diverse ad types and dc subpopulations in vivo, as well as how we can engineer ad vectors which are targeted to specific receptors on the surface of dcs (93, 94) . receptors on the surface of dendritic cells (dcs) can permit entry of ads independently of the classical car receptor, which is largely absent on dcs (95) . receptors proposed to be involved in ad viral entry into human or murine dcs include dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (dc-sign) (95), cd46 (61, 96, 97) , or cd80/cd86 (98) . a vector based on chimpanzee ad vector 1, chadv-1, was shown to efficiently transduce cd46-expressing murine dcs in vitro (97) . recombinant ad5 vectors pseudotyped with the fiber knob from ad3 (99) or porcine adenovirus type 4 (100), can increase entry into human dcs, via cd80/cd86 and surface glycans, respectively. similarly, pseudotyping rad5 with the fibers from species b, hadv-b16 or species d, hadv-d37 displayed increased entry into murine dcs compared with unmodified rad5 (101) . interestingly, in the latter study, increased entry into dcs was not associated with improved cellular immunity following subcutaneous immunization. in support of this, species b ad vectors, including ad35, were long considered to hold great potential as potently immunogenic vaccine vectors due to their increased ability to target both myeloid and plasmacytoid human dcs via cd46. however, these vectors were subsequently shown to be some of the least potent ad vaccines in vivo (5, 8) . these observations and findings are important in highlighting that multiple parameters, such as post-entry intracellular trafficking kinetics or differential activation of innate immune signaling pathways, not just viral tropism, likely play key roles in the induction of robust immunogenicity. the ability of diverse rad vaccines to elicit robust cellular immune responses and confer protective immunity in animal models makes them an attractive vector platform for vaccine development ( table 2 ). in addition to this, their safety in clinical applications, compatibility with clinical-grade manufacturing and scale-up (102, 103) and their suitability for long-term storage (104) , or thermo-stabilization (105) (106) (107) and stockpiling for cold-chain free storage, has solidified their appeal as vaccines for major infectious diseases (108) . it is this clear translational potential which has emphasized the importance of improving our understanding of the mechanisms which underlie differences in the immunological potency of diverse ad types. ad vectors are capable of triggering multiple innate immune sensors at several steps in the viral entry pathway (109, 110) , in a process which does not require viral replication or gene expression (111) . viral penton rgd:cellular integrin-mediated internalization and subsequent escape from the endosome is considered to be a crucial step in activating many innate immune responses to ad vaccines (112) . it is considered that preferential stimulation (or avoidance) of defined innate immune signaling pathways could impact on the downstream immunogenicity of distinct ad vectors. with regard to assessing the differential stimulation of innate immune signaling pathways, this is complicated by the fact that many vectors have not been compared side-byside, and published data proposing roles for these pathways in the immunological potency of different ad vectors are often contradictory. however, a number of pathways which have been implicated in innate immune sensing of ad vectors and the caveats associated, are described below. recombinant ad vectors contain pamps which can be sensed by cell-surface or endosomal pattern recognition receptors (prrs) such as the toll-like receptors (tlrs). tlrs implicated in the sensing of ad vectors include tlr2 (113), tlr4 (114) , and endosomally located tlr9 (61, 113) , which can trigger the down-stream activation and transcription of anti-viral genes including nf-κb, mitogen-activated protein kinases (mapk) and interferon-regulatory factors (irfs). the intracellular adaptor protein myd88 has been reported to play a major role in the induction of ag-specific cellular immune responses following tlr-mediated sensing of ad vaccines (109, 113) . importantly, the ability to engage multiple myd88-dependent signaling pathways simultaneously, is believed to contribute to the robust immunogenicity associated with ad vaccines. however, their immunological potency is also attributed to the fact that innate immune activation by recombinant ad vaccines can occur not only via tlr-dependent mechanisms, but also through numerous tlr-independent pathways (86, 109, 110, 113, 114) . the viral dna itself can play a crucial role in triggering innate immune responses. in recent years it has been demonstrated that following rupture of the endosomal membrane, ad viral dna can also be sensed by the cytosolic dna sensor cgas (115, 116) . the engagement of cgas triggers a signaling cascade involving the adaptor sting (117) and activation of the kinase tbk1, which initiate the induction of irf3-responsive genes (115) , such as type i interferons (ifns). it has been shown that the absence of cgas or sting results in reduced activation of early innate immunity (i.e., ifn-β, cytokines, chemokines) but does not impact adaptive anti-vector immune responses in mice. however, the latter studies were performed in the context of i.v. delivery and anti-vector, not transgene-specific immunity (116) , and as such, the relative importance of dna sensor pathways in the immunogenicity of ads as vaccine vectors is less clear. it has recently has been suggested that ag expression is a more crucial predictor of ag-specific memory t cells (33) , as abrogation of sting and type i ifn responses during ad vaccination in mice merely altered the early kinetics of cd8 + t cells, but did not impair the magnitude of t cell memory responses (33) . in the latter study, it was shown that sting could act as a dominant innate prr sensor for many ad vectors. interestingly, abrogation of sting accelerated the kinetics of ag-specific t cell responses following vaccination with chadv-63, a chimpanzee ad vector, but was dispensable for the early induction of cd8 + t cell responses for ad5 and rare species hadv-d28based vaccines (33) . this supports the idea that a complex interplay between multiple prr-mediated signaling pathways exists, and that different ad vectors are differentially impacted by these pathways. our understanding of this is confounded by differences in receptor usage, in vivo tropism, engagement of prrs in diverse hematopoietic and non-hematopoietic cells, and by differences in putative pamps on diverse ad particles. in addition to inducing the expression of anti-viral genes, infection with ad vectors also triggers pro-inflammatory responses through cytosolic dna-sensing mechanisms which are independent of tlr9 and irfs (111) . in macrophages, recognition of ad viral dna has been shown to be mediated by the innate cytosolic molecular complex, or inflammasome, in a process involving nlrp3 and (asc), which is independent of viral gene expression or replication (117) . the multi-protein inflammasome complex mediates caspase-1 activity, resulting in the processing of pro-interleukin-1β into its active and secreted form. il-1β subsequently induces signaling cascades of proinflammatory cytokines and chemokines through the il-1ri both in vitro and in vivo in response to ad infection. however, alternative and contradictory mechanisms of immune activation in macrophages by ads have also been identified which are independent of the nlrp3 inflammasome and its components. di paolo et al. showed that direct interactions between the rgd motif within the penton base of the ad virion and the β3 subunit of integrins on the surface of macrophages were responsible for activating il-1α. the authors also proposed that il-1α, not il-1β, was the predominant activator of innate immune responses to ad5 in vivo (77) . one very important caveat which complicates our ability to systematically investigate how innate immune responses contribute to downstream adaptive immunity to ad vaccines, is that many studies are performed in vitro, using defined non-immune epithelial/endothelial cells or cultured immune cells including macrophages or dendritic cells (110, 115, 118) . these cell type-specific findings often contradict subsequent in vivo studies using transgenic mice in which these "critical" mediators of innate immunity are knocked-out (77, 116) . for example, despite numerous reports describing an important role for tlr9 in vitro, comparisons of wildtype and tlr9 −/− mice have demonstrated that the impact of tlr9 in innate immune sensing of ad particles in vivo is minimal (77), at least for i.v. administration of ad5. similarly, although cgas or sting were shown to be pivotal in early immune sensing of ad in vivo, studies using cgas −/− or sting −/− mice showed that these molecular effectors have little impact on subsequent adaptive immunity and antibody production (116) . these discrepancies are obviously further complicated by the known capacity of ad vectors to engage multiple innate signaling pathways simultaneously, rendering individual pathways at least partially redundant in vivo (110) . in addition to this, differences in the route of ad vector delivery (i.e., i.m, i.v., or intranasal) and access to different cell types, the multiplicity of infection (moi) or injected dose, the timing or method of analysis reported in published work and the use of non-human or rare species ad vectors with differential receptor usage, also limits our ability to fully dissect out the key contributing pathways (118, 119) . collectively, these factors highlight the many challenges facing the field and explain why we currently lack consensus on precisely which innate signaling pathways could contribute to protective immunity following vaccination with diverse ad vectors. it has been proposed that minimal induction of type i ifns (44, 102, 120) , in conjunction with sustained transgene expression (33) , are hallmarks of potently immunogenic ad vaccine vectors in vivo. excessive stimulation of type i ifn pathways at early time-points following immunization has been shown to lead to decreased transgene expression and subsequently reduced ag-specific antibody (ab) responses, following immunization with a chimpanzee ad vector, chadv-68 (120) . the authors demonstrated that these effects could be reversed by immunizing mice which have a defective type i ifn receptor ifnar −/− , resulting in an increased ab response, thereby confirming that type i ifn stimulation can have a detrimental impact on humoral immunity directed toward the ad-encoded transgene ag (120) . in support of these findings, quinn et al. also showed that abrogation of type i ifn and sting could increase transgene expression from rad vaccines, and that the development of protective cellular immunity correlated with this increased transgene expression (33) . following immunization with rare species and non-human ad vectors, the authors used a systems biology-based, gene expression analysis approach at several time-points to confirm the differential modulation of ifn responsive genes. they determined that type i and type ii ifns were upregulated at 8 h post-immunization, which was followed by the induction of isgs by 24 h. interestingly, the most protective rads identified in their study (i.e., ad5 and chimpanzee ad, chadv-3) exhibited the weakest transcriptional activation of these pathways. however, only the impact of innate gene activation on cd8 + t cell responses, but not transgene-specific abs, was investigated (33) . nonetheless, collectively, these studies support the concept of robust, persistent ag expression combined with low innate gene stimulation in contributing to the potency of rad vaccines (33, 102) . this is also supported by the knowledge that relative to rare species human and non-human ad vectors (33, 120) , immunization with the potently immunogenic ad5 vector is well-established to result in robust, persistent ag expression (33, 36, 41, 121, 122) , while triggering minimal type i ifn responses in vivo. future studies which aim to comprehensively characterize the contribution of early innate immune activation and correlate this with the downstream immunological potency and efficacy of lead ad vaccine platforms will be required. non-replicating ad5-based vectors are well-established for their ability to confer robust transgene expression following immunization (36) . furthermore, low level transgene expression can persist long-term (41, 122) , with transcriptionally active ad vector genomes being maintained in muscle at the injection site, or within draining lymph nodes (36, 122) , depending on the route of administration. as previously outlined, it has been proposed that it is this magnitude and persistence of transgene ag expression which is crucial for the induction of robust and protective t cell responses following ad vaccination (33). as described above, quinn et al. demonstrated that strong activation of innate immune gene expression profiles in the draining lymph nodes (dlns) correlated with limiting ad-mediated transgene expression for many rare or non-human ad types (33) . the ad vectors with the highest levels of transgene ad expression in dlns, ad5, and chimpanzee ad vector chadv-3, also had the most attenuated ifn induction. the latter vectors were both potently immunogenic following immunization in mice. in agreement with this, similar comparative immunogenicity studies in mice have shown that transgene expression levels within muscle and dlns are lower following immunization with a chimpanzee ad vector, chadox1, when compared with ad5, and that this translates to superior immunogenicity observed with ad5 (123) . to directly address the question of how persistence of ag contributes to the induction of the robust immunogenicity of ad vaccines, finn et al. constructed an ad vaccine with a doxycycline-regulated transgene expression cassette (121) . by switching off transgene expression at early vs. late time-points post-immunization, the authors confirmed the importance of presence of ag in expanding and maintaining memory t cell responses up to d30, but showed that the maintenance of memory responses at later time-points (d60) is independent of transgene expression. as discussed above, it is apparent that a combination of multiple parameters influences the extent of transgene expression. these factors include the receptor usage and cellular tropism of each ad vector, the presence and accessibility of specific cell types at the site of injection, in addition to differences in the induction of early innate immunity by diverse ad vectors. collectively, these parameters shape subsequent adaptive immune responses. the successful transduction of cells at the site of vaccine administration, and subsequent engagement of defined and desirable prrs which result in robust transgene expression, depend on the cell type and the specific ad vector being studied. with this in mind, much of the evidence to date has focused on characterizing the induction of innate immune responses in vitro in apcs such as dcs or macrophages, which play important roles in initiating anti-viral immune responses. it is considered that inflammation induced at the injection site, can lead to an influx of apcs (monocytes or dcs) which carry ag to the dlns (124) . immature dcs residing at the site of vaccination respond to innate immune signals (i.e., stimulation of tlr pathways) by undergoing maturation, upregulating co-stimulatory molecules and migrating to dlns where they present ag to naïve t cells (124) . however, non-professional apcs expressing mhc i (125, 126) , such as tissue-specific epithelial or endothelial cells, could also contribute to the immune sensing of ad vectors at the site of injection (39) . therefore, it is clear that the route of vaccine administration (45, 127, 128) , the abundance and accessibility of cell types at those locations, as well as the surface expression of suitable entry receptors, will profoundly affect the immunological potency and protective efficacy of a chosen ad vector. as a result of the long history of experimental use of ad vectors as oncolytic agents aimed at treating disseminated metastases, a significant amount of information exists in the literature regarding interactions between ad vectors, immune cells (28, 77, 129) , parenchymal cells (18, 63, 113, (130) (131) (132) (133) (134) , and stromal cells (130) following i.v. delivery of ad vectors (18, 112, 135) . however, i.v. vaccination would be impractical for widespread population use and so immunization by i.m. or i.n. administration is preferable, particularly for vaccination against respiratory pathogens. unfortunately, less is known about the precise interactions which occur at the site of injection or within dlns in vivo following i.m. or i.n. immunization with ads in animal models, and these questions are even more difficult to address in humans, without the use of invasive procedures (such as fine needle aspirates of lymph nodes) (136) . the cell types present when vaccine is administered i.m. include myocytes, skeletal muscle cells, fibroblasts and endothelial cells, with apcs such as dcs or macrophages representing a minority when compared with the abundance of murine skeletal muscle cells (87) . early studies by mercier et al. demonstrated that transduction of different cell types can modulate the outcome and phenotype of the humoral immune response following ad vaccination. the authors transduced dcs (professional apcs), myoblasts (progenitor cells which give rise to muscle cells) and endothelial cells ex vivo with ad5 expressing a model antigen β-galactosidase (β-gal) (87) , and vaccinated mice i.m. with the ad-transduced cells. the authors found that all transduced cell types elicited humoral immune responses to the β-gal transgene to a similar extent (albeit with differences in their temporal kinetics), but that the igg isotype subclass profile differed. injection of transduced dcs or endothelial cells resulted in production of ag-specific abs which were exclusively igg 2a , whereas vaccination with ad-transduced myoblasts elicited a more balanced ab response with equivalent igg 1 :igg 2a . interestingly, only mice immunized with ad-transduced dcs elicited robust cd8 + t cell responses, as did vaccination with control virus ad-β-gal, suggesting that ad interactions with different cell types in vivo could influence divergent arms of the adaptive immune response. bassett et al. also demonstrated that ag presentation by non-lymphoid cells, in cooperation with hematopoietic apcs, contributes to the kinetics of primary cd8 + t cell expansion, the maintenance of memory responses and to the functional phenotype of the cellular immune response following ad vaccination (39) . through a series of investigations, the authors showed that although dlns act as the site of immunological priming in response to ad vaccination, primary expansion of the ag-specific cd8 + t cell response requires a source of sustained ag expression outside of dlns (39) . they hypothesized that this cell type was of non-hematopoietic origin, due to their prior findings that a radioresistant population of cells was capable of priming cd8 + t cell responses in mice leukopenic mice (137) . therefore, it is feasible that several modes of ag presentation take place following i.m. immunization with ad vectors, all of which contribute to different facets of the ensuing immune response. these interactions are summarized in figure 4 , showing that ag presentation could take place not only within dlns, but also at the site of injection, with or without the involvement of professional apcs. it is important to note that the majority of detailed mechanistic studies into the immunogenicity of ad vectors have been performed using ad5-based vaccines and as such, similar information regarding the in vivo cellular tropism of rare species or non-human ad vectors, is much more limited (123) . it will therefore be crucial to outline the precise factors which confer a hierarchy of potency between ad vectors in the future, as many ad vectors are already under clinical investigation, or are advancing rapidly into clinical trials. this improved knowledge would allow us to engineer optimal platform vectors which combine multiple attributes associated with potent immunogenicity and long-lived protective efficacy. mucosal i.n. or aerosolized (a.e.) delivery of ad vectors is particularly attractive for the development of vaccines against respiratory pathogens (108, 139, 140) . ad vectors are capable of eliciting both humoral and cellular immune responses following i.n. (4, (140) (141) (142) and a.e. (143) vaccination in animal models, both in the bronchoalveolar lavage (bal) and within the lung interstitium (15, 144) . many wildtype adenoviruses are common respiratory pathogens (i.e., hadv-c5, hadv-e4), highlighting their potential suitability for targeting vaccine ag to mucosal surfaces within the respiratory tract. however, several chimpanzee ad vectors, which are not associated with respiratory infections, have displayed superior immunogenicity to ad5 when non-professional apcs such as parenchymal cells at the site of injection (muscle cells shown as an example) can present antigenic peptide on mhc i to infiltrating cd8 + t lymphocytes, outside of secondary lymphoid organs. figure is updated from coughlan et al. (108) and holst and thomsen (138) and was created with ©biorender -biorender.com. administered by the i.n. route in mice, including a chadv-68based vaccine against pulmonary tuberculosis (tb) (144) and a chadv-7 vaccine against pseudomonas aeruginosa (145) . a major limitation in the application of rad vaccines to mucosal respiratory surfaces may be the rapid scavenging and degradation of ad vaccine vector particles by tissue resident alveolar macrophages in the lung, as it has previously been estimated that ∼70% of ad vector genomes are lost within the first 24h following lung delivery in mice (intratracheal) (66) . furthermore, alveolar macrophages were found to be the predominant cell type responsible for initial inflammatory cytokine induction (68) . however, depending on the balance of innate immune stimulation and the retention of a certain level of transgene expression within the respiratory tract, these interactions could be beneficial for the induction of subsequent adaptive immune responses. alveolar macrophages may play a role in trafficking to dlns to facilitate ag priming, or the inflammatory cytokines they release could signal the recruitment of lymphoid cells which could further potentiate immune responses to ad-encoded transgene ag. the many studies which have confirmed the induction of protective immunity following i.n. vaccination with ad vectors support this possibility, but the precise mechanistic factors which underlie these effects and the specific cell types which contribute to vaccine efficacy are not extensively described. as differences in the phenotype and trafficking potential of cd8 + t cells has been observed when comparing i.m. vs. i.n. vaccination of mice with ad vectors, it will be important to identify the optimal ad vaccine platform which elicits the correct correlates of protection for a specific disease target, before deciding on the ideal route of vaccine administration (128) . the ability to engage and activate multiple, diverse innate immune signaling pathways simultaneously (excluding type i ifn) can allow rad vectors to act as an effective selfadjuvant, relative to other vaccination platforms (109, 110, 146) . these attributes suggest that ad virions themselves possess several pamps. in fact, it is considered that the native receptor determinants or entry factors of a particular ad vector may, at least in part, regulate innate immune activation. in support of this, binding of the fiber knob domain to car is sufficient to activate p38mapk, p44/42mapk (erk1/2), and nf-κb pathways (147) , resulting in the transcription of proinflammatory cytokines both in vitro and in vivo (148) . similarly, ad vectors which are capable of using cd46 as a receptor have been shown to display preferential activation of tlr9 to carbinding ad vectors in vitro (61) . however, cd46 distribution is limited to the expression in the testes in mice, rats and guinea pigs, in contrast to its widespread expression in humans (149) . therefore, this likely has little contribution to the immunological potency of cd46-using ad vectors in murine models. the fiber knob domain of ad5-based vectors has been shown to contribute to the maturation of murine dcs, as recombinant knob protein, full-length fiber protein and penton capsomers (penton plus fiber), but not hexon or penton alone, were capable of exerting this effect (90) . in support of this, teigler et al. previously suggested that fiber-receptor interactions were important for triggering innate immune responses to rare species ad vectors (26) . furthermore, studies using hadv-b35 vectors pseudotyped with the fiber knob from ad5 were shown to be more immunogenic in mice and nhp, suggesting that the fiber knob domain could contribute substantially to immunological potency (150) . however, similar studies in which the entire fiber and penton rgd motif from rad5 were swapped into chimpanzee ad vector chadox1, failed to increase the immunogenicity of chadox1 relative to ad5, suggesting that factors beyond fiber/penton capsomer interactions confer immune responses to chadox1 (123) . more recently it has been shown that a member of the vitamin-k dependent protein family, growth arrest specific protein (gas6), can bind differentially to the fiber of rad vectors (151), interacting with a putative epitope which is outside of the fiber knob domain, such as the fiber shaft. interestingly, gas6 bound efficiently to the fiber of car-binding ad5, without affecting its ability to enter cells, but significantly reduced the induction of type i ifns, resulting in prolonged transgene expression. conversely, gas6 failed to bind to the fibers of non-ad5 and non-car binding viruses, such as a species d ad vectors hadv-d28, which has been shown to display reduced immunogenicity in mice when compared with rad5 (152) . in support of this, hadv-d28 has been shown to elicit robust ifn-α and had increased stimulation of ifn-responsive genes when compared with ad5 (44) . importantly, immunization of ifnr1 −/− mice with hadv-d28 resulted in robust cellular immune responses, comparable to ad5 (44) . these data support the hypothesis by several other groups that ad vectors which trigger robust type i ifn responses exhibit reduced transgene expression and impaired vaccine immunogenicity (33, 120) . therefore, one strategy to overcome stimulation of ifn-α could be to identify the precise amino acid binding epitopes for gas6 and genetically engineer this region into the fiber of non-gas6 binding ad vectors in an effort to increase their immunogenicity (151) . in addition to interactions with car, interactions between the rgd motif within the penton base and cellular integrins can contribute to the induction of innate immune signaling pathways both in vitro (153) and in vivo (77) . following i.v. administration of ad5, interactions between the β3 subunit of cellular integrins and the rgd motif on marco + marginal zone macrophages in the spleen were required for triggering il-1α-dependent innate immune signaling in mice (77) . however, it is unclear how these interactions affect immune responses following immunization by other routes of administration. finally, the ad virion itself and its major capsid protein, the hexon, has been described as a potent adjuvant, capable of activating cd4 + and cd8 + cellular immune responses to a model immunogen mixture in mice (146) . although the precise mechanism underlying this effect has not been identified, subsequent studies have suggested that the hvr regions, particularly hvr1, are involved in enhancing interactions with scavenging macrophages (76, 78) . one approach to identifying lead vaccine candidates is to perform head-to-head comparisons of immunogenicity in animal models to identify the most immunogenic vectors, followed by detailed and systematic experimental studies to try to identify the underlying mechanisms which contribute to immunological potency (i.e., transgene expression magnitude and duration, innate immune stimulation). however, in the interim, efforts are ongoing to try to maximize immune recognition of the ad encoded ag, in an effort to compensate for the reduced immunogenicity associated with certain ad vectors which are otherwise an ideal platform (i.e., low seroprevalence, high titer growth, stability). furthermore, this type of modification could permit dose-sparing, which would have cost saving effects, as well as minimizing any vector-induced reactogenicity, without compromising on immunogenicity. such approaches include the use of molecular or genetic adjuvants, namely in the form of fusion proteins with the vaccine antigen of interest, which help to potentiate immunogenicity by enhancing ag presentation or dissemination [reviewed in detail by neukirch et al. (154) ]. one such approach which has previously been described includes the generation of fusion-ag cassettes which enhance mhc presentation. this can be achieved by fusing vaccine ag to β-microglobulin for enhanced mhc i presentation (155) , or to the invariant chain of mhc ii (156) (157) (158) (159) . these approaches were capable of enhancing antigen-specific cd8 + t cells responses in mice following immunization with an ad vector encoding the ii or β2-microglobulin fusion antigen. in a separate approach, we recently have shown that encoding vaccine ag cassettes in which a model antigen, enhanced green fluorescent protein (egfp), is fused to a protein domain known to tether proteins to the surface of extracellular vesicles (evs), can dramatically improve the humoral immunogenicity of both ad5 and chimpanzee ad vector chadox1 in mice following i.m. and i.n vaccination. evs play important roles in regulating immune responses and are conveyors of cellular communication signals (15) . as evs are frequently implicated in host-pathogen interactions and have been shown to transfer antigenic material to apcs (81, 82) , we reasoned that directed targeting of a vaccine ag to their surface could potentiate immune responses. although cellular immunity was largely unaffected, ag-specific humoral immune responses were increased up to 400-fold when compared with unmodified egfp (15) . the choice of molecular or genetic adjuvant will depend on the predicted correlates of protection for a specific disease target: in certain cases, a robust humoral immune response will be more important than a cellular immune response. further to this, some adjuvanting technologies will work for one ag but not for another, and structural constraints may limit the ability to modify complex, multimeric ags. this will require the optimization and selection of different components to be combined and assembled into customized ad vectors which are tailored to specific pathogens. challenges a number of promising animal studies solidified the appeal of ad5 as a platform vaccine vector for infectious diseases. in particular, a study by sullivan et al. demonstrated that a singleshot, low dose (1 × 10 10 vp) immunization with ad5 expressing ebola virus glycoprotein (gp) could provide 100% protection from challenge in nhps (160) . similar studies using ad5 based vectors expressing siv gag demonstrated its superiority to plasmid dna or modified vaccinia ankara (mva) in attenuating viremia following virus challenge (12) . on the basis of these promising early results, a multicenter phase ii clinical trial called the merck step study was initiated, in which a vaccine composed of a mixture of ad5 vectors expressing hiv-1 gag, pol, and nef genes was administered to participants at high risk for hiv-1 acquisition (161, 162) . phase i safety and immunogenicity studies in healthy, hiv-seronegative adults showed that this vaccine could elicit antigen-specific ifn-γ elispot responses in both ad5 seronegative and seropositive individuals (163) . however, the phase ii study was terminated prematurely due to futility and failure to meet pre-defined endpoints: an interim analysis determined that the vaccine would not be efficacious in preventing hiv-1 infection, or in reducing viral-load setpoint in seroconverters, despite eliciting t cell responses in most participants (162, 163) . subsequent to this, a post-hoc analysis of the study suggested an association between vaccination with the ad5 vaccine and increased acquisition of hiv-1 (161). on multivariate analyses, this increase was largely restricted to a defined sub-set of participants: uncircumcised men with high baseline antibody titers against ad5. several hypotheses were proposed to explain the increase in hiv-1 acquisition, including the formation of immune complexes (ic) between anti-ad5 antibodies and dcs, which were shown to enhance hiv-1infection of t cells in dc-t cell co-cultures in vitro (164) . an alternative hypothesis suggested that ad5 immunization induced the expansion of ad-specific memory cd4 + t cells which upregulate ccr5 expression and/or homing markers for mucosal sites, thereby increasing the pool of hiv-1 susceptible cells at the site of infection (165) . although the latter hypothesis has been challenged (166) , the precise mechanisms underlying the increased acquisition of hiv-1 in the merck step trial remain inconclusive (166) . however, it is important to note that the effects were shown to wane over time (167) . nonetheless, this outcome led to a dampening in enthusiasm for the broad application of ad5-based vaccines for major infectious diseases, prompting the investigation of novel rare species human or non-human ad viruses as alternative vaccine platforms. several rare species or non-human ad vaccines are now leading the way in human clinical trials, namely species c vector hadv-c6 and species d vector hadv-26, as well as chimpanzee ad vectors chadv-3, panadv-3, chadv-63, and chadox1, which cluster phylogenetically with species c or e human ads (see table 1 ). many of these vector candidates had previously been identified in animal studies as being potently immunogenic, and in some cases were comparable to the potency of ad5 [(5, 168); table 2 ]. in particular, hadv-c6 and chadv-3 appear to possess attributes which make them an attractive platform vector (22%, 12% seroprevalence, respectively) (2, 5), and as a result, have been developed for clinical testing as vaccines against major global pathogens, hepatitis c virus (hcv) (2) and hiv-1 (169) . both hadv-c6 and chadv-3 were shown to be safe and immunogenic in humans when used as a vaccine to elicit immunity against hcv, although hadv-c6 appeared to be superior in its ability to cellular immune responses with increased magnitude and breadth at lower doses (i.e., 5 × 10 8 vp) (2) . with regard to chadv-3, part of its appeal includes its ability to elicit long-lived cellular and humoral immune responses directed toward the encoded vaccine antigen. immunization of nhps with chadv-3 expressing hiv gag resulted in cellular immune responses which persisted for more than 5 years (5). a heterologous boost with panadv-3 at week 299 facilitated an expansion of gag-specific ifn-γ secreting t cells, in addition to boosting antibodies to hiv-1 gag. as such, there is broad interest in using these rare vectors in heterologous prime:boost vaccination regimens in an effort to confer long-lived protective immunity against challenging pathogens. in support of this, a phase i clinical trial to test the hadv-c6 and chadv-3 vectors expressing hcv antigens demonstrated that heterologous boost immunizations resulted in long-lived, polyfunctional effector, and central memory t cell responses which were sustained for up to 1 year in humans (2). panadv-3 expressing respiratory syncytial virus (rsv) fusion (f), nucleocapsid (n), and matrix (m2-1) antigens has also been tested in humans following i.n. and i.m. administration (37) . neutralizing antibodies to rsv f were increased following i.m., but not i.n. prime immunization. similar trends were observed for antigen-specific t cell responses to the vaccine inserts, although increases were minimal following panadv-3 i.m. prime only. this vaccine was also evaluated in older adults (60-75 years) who are at increased risk of severe rsv disease, with similar results (170) . immune responses elicited by panadv-3 were improved upon boosting with an mva vector which also encoded the rsv transgene antigen. chadv-63, also identified as a clinically viable ad vector with low seroprevalence which displayed protective efficacy in animal studies ( table 2) , has been shown to be safe and immunogenic in children and adults (171) (172) (173) (174) . when chadv-63 has been used in a prime:boost vaccination regimen with mva expressing malaria antigens, promising efficacy was observed in uk and kenyan adults (175, 176) , but has recently been associated with disappointing efficacy in field trials in young children in burkina faso, a highly endemic malaria transmission region (177) . chadox-1 is a species e chimpanzee ad vector developed by the jenner institute at university of oxford which has been tested clinically as a vaccine for influenza virus as a standalone vector or for use in prime:boost with mva (1, 3) , and for several other infectious disease targets such as chikungunya fever (nct03590392), malaria (nct03203421), and tuberculosis (nct01829490). numerous additional trials are currently ongoing or actively recruiting participants, including a recently initiated study to test a novel vaccine for covid-19 (nct04324606). in addition to vectors derived from nhps, promising advances have been the development of hadv-d26 vectors. with a number of clinical trials registered on clinicaltrials.gov, this platform has advanced into clinical studies as a vaccine against ebola virus (178) (179) (180) , rsv (181), hiv-1 (6, 182, 183) , and has also very recently been announced as a candidate vectored vaccine against covid-19. the first-in-human testing of hadv-d26 expressing hiv-1 env demonstrated that the vaccine was safe and well-tolerated and elicited env-specific antibodies and antigen-specific elispot responses in all participants (182) . although hiv-1 specific neutralizing antibodies were not detected, the study reported multi-functional readouts for the non-neutralizing antibodies elicited, including effector functions such as antibody-dependent cell-mediated phagocytosis (adcp), antibody-dependent cell-mediated virus inhibition (adcvi) (183) . this vaccine platform was subsequently improved upon by encoding polyvalent "mosaic" hiv-1 antigens, env, gag and pol, representing computationally optimized sequences aimed at maximizing recognition of t cell epitopes (184) . evaluation of the hadv-d26 platform in various prime:boost regimens is ongoing and preliminary data suggest that it is immunogenic, capable of eliciting env-specific antibodies which exhibit adcp and cellular immune responses out to week 50. importantly, these assays were found to be correlates of protection in a parallel shiv challenge model in rhesus monkeys (macaca mulatta) (6). the hadv-d26 mosaic hiv-1 vaccine is currently in phase iib efficacy studies in sub-saharan africa (nct03060629). based on the literature, it appears that ad vectors derived from species c, d, or e are most likely to be immunogenic vectors. requirements for selecting specific vectors will vary depending on whether the required application is as a stand-alone vaccine or as part of a prime:boost regimen. for standalone vaccination regimens aimed at eliciting a rapid, protective response during an emerging pandemic, the magnitude of response to an identified correlate of protection following a single shot is crucial. secondary considerations for an evolving pandemic scenario would be rapid immunogenicity at a low dose, and the capacity for lyophilization or stabilization, to facilitate dose-sparing, vaccine cost-effectiveness and pandemic preparedness. in this regard, of the ad vectors evaluated to date, chadv-3 and hadv-c6 appear promising. for protection against more complex pathogens which require long-lived polyfunctional responses, or sustained humoral immunity with extensive breadth (i.e., universal influenza vaccine, hiv-1 or hcv), heterologous prime:boost vaccination regimens should be evaluated using diverse ad vectors, or ads in combination with mva or protein based immunogens at different intervals. it is difficult to predict which ad vectors should take precedence as the encoded antigen will need to be tailored to elicit the correct phenotype of immunity against a defined correlate of protection for each specific disease target. however, underpinning the evaluation of all ad-based vaccines in pre-clinical animal studies, should be the inclusion of species c ad5 vector controls to represent a benchmark of immunological potency. in addition, ad vaccine candidates should be compared at several doses to evaluate the maintenance of vaccine potency upon dose de-escalation. the field should also make efforts to improve our understanding of the basic biology of many of these novel ad vectors, as insights into the receptor usage, interactions of ad vectors with different cell types following immunization and subsequent stimulation of differential innate signaling pathways will all impact on their downstream immunogenicity and ability to confer protective efficacy. unfortunately, one major challenge in performing these types of head-to-head comparisons is the lack of widespread availability of many of these rare species or non-human ad vectors to academic investigators, as many of these are being developed by large pharmaceutical companies. it is clear that a hierarchy exists in the immunological potency observed between rare species human and non-human ad vectors in various animal species. as outlined above, ad vector immunogenicity is most likely dependent on a complex combination of factors, rather than any particular factor in isolation. an ideal ad vaccine platform will combine the following attributes; (i) low seroprevalence in humans, (ii) robust immunogenicity with potential for dose-sparing, and (iii) suitable manufacturing characteristics (i.e., growth to high titers, genome stability). better understanding of the mechanisms which define effective vaccines, will enable us to design and customize improvements to existing ad vaccine vectors, enhancing their potential for future clinical use. lc: writing and editing-original and final draft. clinical assessment of a novel recombinant simian adenovirus chadox1 as a vectored vaccine expressing conserved influenza a antigens novel adenovirus-based vaccines induce broad and sustained t cell responses to hcv in man heterologous two-dose vaccination with simian adenovirus and poxvirus vectors elicits long-lasting cellular immunity to influenza virus a in healthy adults safety and immunogenicity of novel respiratory syncytial virus (rsv) vaccines based on the rsv viral proteins f, n and m2-1 encoded by simian adenovirus (panad3-rsv) and mva (mva-rsv); protocol for an open-label, doseescalation, single-centre, phase. 1 clinical trial in healthy adults vaccine vectors derived from a large collection of simian adenoviruses induce potent cellular immunity across multiple species evaluation of a mosaic hiv-1 vaccine in a multicentre, randomised, double-blind, placebo-controlled, phase 1/2a clinical trial (approach) and in rhesus monkeys (nhp 13-19) a monovalent chimpanzee adenovirus ebola vaccine boosted with mva comparative analysis of the magnitude, quality, phenotype, and protective capacity of simian immunodeficiency virus gag-specific cd8+ t cells following human-, simian-, and chimpanzee-derived recombinant adenoviral vector immunization comparative immunogenicity in rhesus monkeys of dna plasmid, recombinant vaccinia virus, and replication-defective adenovirus vectors expressing a human immunodeficiency virus type 1 gag gene adenoviruses as vaccine vectors recombinant adenovirus serotype 26 (ad26) and ad35 vaccine vectors bypass immunity to ad5 and protect nonhuman primates against ebolavirus challenge replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency-virus immunity molecular evolution of human adenoviruses overreliance on the hexon gene, leading to misclassification of human adenoviruses targeting antigen to the surface of evs improves the in vivo immunogenicity of human and non-human adenoviral vaccines in mice an engineered virus library as a resource for the spectrum-wide exploration of virus and vector diversity adenoviruses: update on structure and function tropism-modification strategies for targeted gene delivery using adenoviral vectors novel vector construction based on alternative adenovirus types via homologous recombination generation and characterization of a novel candidate gene therapy and vaccination vector based on human species d adenovirus type 56 characterization of a family of chimpanzee adenoviruses and development of molecular clones for gene transfer vectors adenoviral producer cells safety and immunogenicity of an oral, replicating adenovirus serotype 4 vector vaccine for h5n1 influenza: a randomised, double-blind, placebo-controlled, phase 1 study efficacy, immunogenicity, and safety of an oral influenza vaccine: a placebocontrolled and active-controlled phase 2 human challenge study clinical advances in viral-vectored influenza vaccines vaccination with adenovirus serotypes 35, 26, and 48 elicits higher levels of innate cytokine responses than adenovirus serotype 5 in rhesus monkeys a novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity ad48 hexon hypervariable region substitutions lead to toxicity and increased inflammatory responses following intravenous delivery comparative seroprevalence immunogenicity of six rare serotype recombinant adenovirus vaccine vectors from subgroups b and d a review of 65 years of human adenovirus seroprevalence a bovine adenoviral vector-based h5n1 influenza -vaccine provides enhanced immunogenicity and protection at a significantly low dose antigen expression determines adenoviral vaccine potency independent of ifn and sting signaling detailed analysis of the cd8+ tcell response following adenovirus vaccination cd8+ cellular immunity mediates rad5 vaccine protection against ebola virus infection of nonhuman primates adenoviral vectors persist in vivo and maintain activated cd8+ t cells: implications for their use as vaccines chimpanzee adenovirus-and mva-vectored respiratory syncytial virus vaccine is safe and immunogenic in adults adenovirus-triggered innate signalling pathways cd8+ t-cell expansion and maintenance after recombinant adenovirus immunization rely upon cooperation between hematopoietic and nonhematopoietic antigen-presenting cells increased glucose disposal induced by adenovirus-mediated transfer of glucokinase to skeletal muscle in vivo longterm humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector comparative analysis of simian immunodeficiency virus gag-specific effector and memory cd8+ t cells induced by different adenovirus vectors alternative serotype adenovirus vaccine vectors elicit memory t cells with enhanced anamnestic capacity compared to ad5 vectors type i ifn induced by adenovirus serotypes 28 and 35 has multiple effects on t cell immunogenicity quality of the transgenespecific cd8+ t cell response induced by adenoviral vector immunization is critically influenced by virus dose and route of vaccination analysis of the interaction between rgd-expressing adenovirus type 5 fiber knob domains and alphavbeta3 integrin reveals distinct binding profiles and intracellular trafficking integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internalization but not virus attachment a direct and versatile assay measuring membrane penetration of adenovirus in single cells drifting motions of the adenovirus receptor car and immobile integrins initiate virus uncoating and membrane lytic protein exposure stepwise dismantling of adenovirus 2 during entry into cells difference imaging of adenovirus: bridging the resolution gap between x-ray crystallography and electron microscopy a capsid-encoded ppxy-motif facilitates adenovirus entry spatiotemporal dynamics of adenovirus membrane rupture and endosomal escape adenovirus protein vi mediates membrane disruption following capsid disassembly localization of regions in cd46 that interact with adenovirus the human membrane cofactor cd46 is a receptor for species b adenovirus serotype 3 cd46 is a cellular receptor for group b adenoviruses desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14 human adenovirus type 26 uses sialic acid-bearing glycans as a primary cell entry receptor the interaction between the fiber knob domain and the cellular attachment receptor determines the intracellular trafficking route of adenoviruses preferential activation of toll-like receptor nine by cd46-utilizing adenoviruses key role of the scavenger receptor marco in mediating adenovirus infection and subsequent innate responses of macrophages. mbio analysis of adenovirus sequestration in the liver, transduction of hepatic cells, and innate toxicity after injection of fiber-modified vectors sequestration of adenoviral vector by kupffer cells leads to a nonlinear dose response of transduction in liver pu.1 redirects adenovirus to lysosomes in alveolar macrophages, uncoupling internalization from infection role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithelial surface of the respiratory tract lung macrophage scavenger receptor sr-a6 (marco) is an adenovirus type-specific virus entry receptor internalization of adenovirus by alveolar macrophages initiates early proinflammatory signaling during acute respiratory tract infection rapid kupffer cell death after intravenous injection of adenovirus vectors a hemodynamic response to intravenous adenovirus vector particles is caused by systemic kupffer cell-mediated activation of endothelial cells clearance of adenovirus by kupffer cells is mediated by scavenger receptors, natural antibodies, and complement neutrophils interact with adenovirus vectors via fc receptors and complement receptor 1 antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response scavenger receptor a: a new route for adenovirus 5 scavenger receptors in homeostasis and immunity identification of adenovirus serotype 5 hexon regions that interact with scavenger receptors virus binding to a plasma membrane receptor triggers interleukin-1 alpha-mediated proinflammatory macrophage response in vivo blood clearance rates of adenovirus type 5 in mice hepatic uptake of negatively charged particles in rats: possible involvement of serum proteins in recognition by scavenger receptor dendritic cells cross-dressed with peptide mhc class i complexes prime cd8+ t cells indirect activation of naive cd4+ t cells by dendritic cell-derived exosomes membrane vesicles as conveyors of immune responses follicular dendritic cells carry mhc class ii-expressing microvesicles at their surface cd8+ dendritic cells use lfa-1 to capture mhc-peptide complexes from exosomes in vivo immunology: cross-dressers turn on t cells cd8+ t cell responses following replication-defective adenovirus serotype 5 immunization are dependent on cd11c+ dendritic cells but show redundancy in their requirement of tlr and nucleotidebinding oligomerization domain-like receptor signaling distinct roles of adenovirus vector-transduced dendritic cells, myoblasts, and endothelial cells in mediating an immune response against a transgene product effect of adenovirus gene transfer vectors on the immunologic functions of mouse dendritic cells recombinant adenovirus induces maturation of dendritic cells via an nf-kappab-dependent pathway the maturation of murine dendritic cells induced by human adenovirus is mediated by the fiber knob domain efficient targeting of protein antigen to the dendritic cell receptor dec-205 in the steady state leads to antigen presentation on major histocompatibility complex class i products and peripheral cd8+ t cell tolerance inhibition of melanoma growth by targeting of antigen to dendritic cells via an anti-dec-205 single-chain fragment variable molecule enhanced gene transfer to mouse dendritic cells using adenoviral vectors coated with a novel adapter molecule high efficiency transduction of dendritic cells by adenoviral vectors targeted to dc-sign adenovirus serotype 5 infects human dendritic cells via a coxsackievirus-adenovirus receptor-independent receptor pathway mediated by lactoferrin dc-sign myeloid and plasmacytoid dendritic cells are susceptible to recombinant adenovirus vectors and stimulate polyfunctional memory t cell responses a cd46-binding chimpanzee adenovirus vector as a vaccine carrier adenovirus serotype 3 utilizes cd80 (b7.1) and cd86 (b7.2) as cellular attachment receptors improved induction of anti-melanoma t cells by adenovirus-5/3 fiber modification to target human dcs incorporation of porcine adenovirus 4 fiber protein enhances infectivity of adenovirus vector on dendritic cells: implications for immune-mediated cancer therapy in vitro dendritic cell infection by pseudotyped adenoviral vectors does not correlate with their in vivo immunogenicity chimpanzee adenoviral vectors as vaccines -challenges to move the technology into the fast lane production of adenovirus vectors and their use as a delivery system for influenza vaccines stability and suitability for storage and distribution of ad26.zebov/mva-bn(r)-filo heterologous prime-boost ebola vaccine spray dried human and chimpanzee adenoviral-vectored vaccines are thermally stable and immunogenic in vivo long-term thermostabilization of live poxviral and adenoviral vaccine vectors at supraphysiological temperatures in carbohydrate glass a simian-adenovirus-vectored rabies vaccine suitable for thermostabilisation and clinical development for low-cost single-dose pre-exposure prophylaxis adenoviral vectors as novel vaccines for influenza multiple innate immune pathways contribute to the immunogenicity of recombinant adenovirus vaccine vectors innate immune response to adenoviral vectors is mediated by both toll-like receptor-dependent and -independent pathways the inflammasome recognizes cytosolic microbial and host dna and triggers an innate immune response adenovirus sensing by the immune system adenovirus vector-induced innate inflammatory mediators, mapk signaling, as well as adaptive immune responses are dependent upon both tlr2 and tlr9 in vivo tlrs differentially modulate several adenovirus vector-induced immune responses adenovirus detection by the cgas/sting/tbk1 dna sensing cascade diminished innate antiviral response to adenovirus vectors in cgas/sting-deficient mice minimally impacts adaptive immunity sting is a direct innate immune sensor of cyclic di-gmp adenovirus vector induced innate immune responses: impact upon efficacy and toxicity in gene therapy and vaccine applications innate immunity to adenovirus type i interferon inhibits antibody responses induced by a chimpanzee adenovirus vector persistence of transgene expression influences cd8+ t-cell expansion and maintenance following immunization with recombinant adenovirus the cd8+ t cell population elicited by recombinant adenovirus displays a novel partially exhausted phenotype associated with prolonged antigen presentation that nonetheless provides long-term immunity differential immunogenicity between hadv-5 and chimpanzee adenovirus vector chadox1 is independent of fiber and penton rgd loop sequences in mice translating innate immunity into immunological memory: implications for vaccine development antigen presentation by nonhemopoietic cells amplifies clonal expansion of effector cd8 t cells in a pathogen-specific manner presentation of toxoplasma gondii antigens via the endogenous major histocompatibility complex class i pathway in nonprofessional and professional antigen-presenting cells immunity against heterosubtypic influenza virus induced by adenovirus and mva expressing nucleoprotein and matrix protein-1 route of adenovirus-based hiv-1 vaccine delivery impacts the phenotype and trafficking of vaccine-elicited cd8+ t lymphocytes combined fiber modifications both to target alpha(v)beta(6) and detarget the coxsackievirus-adenovirus receptor improve virus toxicity profiles in vivo but fail to improve antitumoral efficacy relative to adenovirus serotype 5 biodistribution and retargeting of fx-binding ablated adenovirus serotype 5 adenovirus binding to blood factors results in liver cell infection and hepatotoxicity adenovirus serotype 5 hexon mediates liver gene transfer biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor x innate immunity to adenovirus: lessons from mice normal human lymph node t follicular helper cells and germinal center b cells accessed via fine needle aspirations recombinant adenovirus vaccines can successfully elicit cd8+ t cell immunity under conditions of extreme leukopenia harnessing the potential of adenovirus vectored vaccines overcoming barriers in the path to a universal influenza virus vaccine superior immune responses induced by intranasal immunization with recombinant adenovirus-based vaccine expressing full-length spike protein of middle east respiratory syndrome coronavirus single intranasal immunization with chimpanzee adenovirusbased vaccine induces sustained and protective immunity against mers-cov infection comparison of systemic and mucosal immunization with replicating single cycle adenoviruses unique cellular and humoral immunogenicity profiles generated by aerosol, intranasal, or parenteral vaccination in rhesus macaques novel chimpanzee adenovirus-vectored respiratory mucosal tuberculosis vaccine: overcoming local anti-human adenovirus immunity for potent tb protection protective anti-pseudomonas aeruginosa humoral and cellular mucosal immunity by adc7-mediated expression of the p. aeruginosa protein oprf adenovirus hexon protein is a potent adjuvant for activation of a cellular immune response interaction of adenovirus type 5 fiber with the coxsackievirus and adenovirus receptor activates inflammatory response in human respiratory cells influence of fiber detargeting on adenovirus-mediated innate and adaptive immune activation molecular cloning of rat and mouse membrane cofactor protein (mcp, cd46): preferential expression in testis and close linkage between the mouse mcp and cr2 genes on distal chromosome 1 immunogenicity of recombinant fiber-chimeric adenovirus serotype 35 vector-based vaccines in mice and rhesus monkeys inhibition of type i interferon responses by adenovirus serotype-dependent gas6 binding potent immune responses and in vitro pro-inflammatory cytokine suppression by a novel adenovirus vaccine vector based on rare human serotype 28 activation of p38 and erk signaling during adenovirus vector cell entry lead to expression of the c-x-c chemokine ip-10 the potential of adenoviral vaccine vectors with altered antigen presentation capabilities rapid and sustained cd4(+) t-cell-independent immunity from adenovirus-encoded vaccine antigens enhanced and sustained cd8+ t cell responses with an adenoviral vector-based hepatitis c virus vaccine encoding ns3 linked to the mhc class ii chaperone protein invariant chain enhanced vaccine-induced cd8+ t cell responses to malaria antigen me-trap by fusion to mhc class ii invariant chain adenovirus-based vaccine against listeria monocytogenes: extending the concept of invariant chain linkage development of a molecular adjuvant to enhance antigen-specific cd8(+) t cell responses immune protection of nonhuman primates against ebola virus with single low-dose adenovirus vectors encoding modified gps efficacy assessment of a cell-mediated immunity hiv-1 vaccine (the step study): a double-blind, randomised, placebo-controlled, test-of-concept trial hiv-1 vaccine-induced immunity in the test-of-concept step study: a case-cohort analysis safety and immunogenicity of a replication hiv-1 clade b gag/pol/nef vaccine in healthy adults activation of a dendritic cell-t cell axis by ad5 immune complexes creates an improved environment for replication of hiv in t cells adenovirus vector vaccination induces expansion of memory cd4 t cells with a mucosal homing phenotype that are readily susceptible to hiv-1 adenovirus-specific immunity after immunization with an ad5 hiv-1 vaccine candidate in humans extended follow-up confirms early vaccine-enhanced risk of hiv acquisition and demonstrates waning effect over time among participants in a randomized trial of recombinant adenovirus hiv vaccine (step study) a novel adenovirus type 6 (ad6)-based hepatitis c virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques safety and immunogenicity of the merck adenovirus serotype 5 (mrkad5) and mrkad6 human immunodeficiency virus type 1 trigene vaccines alone and in combination in healthy adults novel genetically-modified chimpanzee adenovirus and mvavectored respiratory syncytial virus vaccine safely boosts humoral and cellular immunity in healthy older adults safety and immunogenicity of malaria vectored vaccines given with routine expanded program on immunization vaccines in gambian infants and neonates: a randomized controlled trial. front immunol viral vector malaria vaccines induce high-level t cell and antibody responses in west african children and infants assessment of novel vaccination regimens using viral vectored liver stage malaria vaccines encoding me-trap safety and immunogenicity of chad63 and mva me-trap in west african children and infants evaluation of the efficacy of chad63-mva vectored vaccines expressing circumsporozoite protein and me-trap against controlled human malaria infection in malaria-naive individuals prime-boost vaccination with chimpanzee adenovirus and modified vaccinia ankara encoding trap provides partial protection against plasmodium falciparum infection in kenyan adults first field efficacy trial of the chad63 mva me-trap vectored malaria vaccine candidate in 5-17 months old infants and children a prophylactic multivalent vaccine against different filovirus species is immunogenic and provides protection from lethal infections with ebolavirus and marburgvirus species in non-human primates safety and immunogenicity of a 2-dose heterologous vaccination regimen with ad26.zebov and mva-bn-filo ebola vaccines: 12-month data from a phase 1 randomized clinical trial in uganda and tanzania safety and immunogenicity of novel adenovirus type 26-and modified vaccinia ankara-vectored ebola vaccines: a randomized clinical trial adenovectors encoding rsv-f protein induce durable and mucosal immunity in macaques after two intramuscular administrations firstin-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 hiv-1 env vaccine (ipcavd 001) characterization of humoral and cellular immune responses elicited by a recombinant adenovirus serotype 26 hiv-1 env vaccine in healthy adults (ipcavd 001) mosaic hiv-1 vaccines expand the breadth and depth of cellular immune responses in rhesus monkeys thanks to prof. peter palese, ismms, for helpful and constructive discussion of this review. figures were created with ©biorender -biorender.com. the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 coughlan. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-323756-atnrw9ew authors: vabret, nicolas; blander, j. magarian title: sensing microbial rna in the cytosol date: 2013-12-25 journal: front immunol doi: 10.3389/fimmu.2013.00468 sha: doc_id: 323756 cord_uid: atnrw9ew the innate immune system faces the difficult task of keeping a fine balance between sensitive detection of microbial presence and avoidance of autoimmunity. to this aim, key mechanisms of innate responses rely on isolation of pathogens in specialized subcellular compartments, or sensing of specific microbial patterns absent from the host. efficient detection of foreign rna in the cytosol requires an additional layer of complexity from the immune system. in this particular case, innate sensors should be able to distinguish self and non-self molecules that share several similar properties. in this review, we discuss this interplay between cytosolic pattern recognition receptors and the microbial rna they detect. we describe how microbial rnas gain access to the cytosol, which receptors they activate and counter-strategies developed by microorganisms to avoid this response. when janeway formulated the theory of pattern recognition in 1989, he proposed that host cells could sense microbial infection owing to receptors able to recognize invariant molecular structures defined as pathogen-associated molecular patterns (pamps). these patterns would be present in groups of pathogens, but absent in the host (1) . years later, janeway and medzhitov described the activity of the first mammalian member of the toll-like receptor (tlr) family, toll-like receptor 4 (2) . tlrs comprise a family of transmembrane proteins able to recognize conserved microbial features and activate the immune response (3) . once activated, tlrs and others pattern recognition receptors (prrs) initiate several intracellular pathways, including those mediated by nuclear factor-κb (nf-κb), mitogen-activated protein kinases (mapks), and interferon regulatory factors (irfs). another outcome of activation of distinct members of cytosolic prrs is their oligomerization into multimeric cytosolic structures called inflammasomes, which activate the cysteine protease caspase-1, subsequently leading to the production of biologically active forms of pro-inflammatory cytokines (4) . initially thought to detect exclusively microbial derived ligands, prrs were later shown to recognize host derived danger signals, which are released in response to stress conditions such as cellular damage or tissue injury (3) . under normal physiological conditions, these ligands are not accessible to their respective prrs and do not activate the immune system. conversely, it was first suggested that self-dna artificially introduced into the cytoplasm by transfection could activate nf-κb and the mapk pathway (5) . evidence that any dna, regardless of its origin, can engage innate immune receptors when localized outside of the nucleus was further confirmed by the identification of several endosomal and cytosolic dna sensors [reviewed in ref. (6) ]. in contrast to cytosolic dna, rna sensing in the cytoplasm raises many questions on the mechanisms used by the innate system to specifically distinguish non-self-rna from self-rna. during infection, microbial rnas share the cytosolic cellular compartment with several host rna species, including messenger rna (mrna), transfer rna (trna), ribosomal rna (rrna), microrna, and other small regulatory rnas. as a consequence, cytosolic sensors must display a high affinity for specific microbial features to avoid activation by host molecules that would otherwise elicit autoimmune responses. despite this apparent challenge, efficient detection of foreign rna in the cytosol is essential for innate immunity. during certain viral infections, rna may be the only microbial pamp produced throughout most of the replication cycle. additionally, our laboratory previously showed that recognition of bacterial mrna in the cytosol was critical to elicit a robust innate response against bacterial infection (7) . finally, cytosolic sensing of pathogen invasion by non-immune infected cells provides the very first steps of innate response against infection, before phagocytosis-competent immune cells are recruited to the site of infection. in this review, we summarize the current understanding of cytosolic rna sensing. we describe instances in which microbial rnas gain access to the cytosol, the prrs they activate, their corresponding ligands and strategies developed by microorganisms to conceal their rnas. rna entry into host cells generally takes place during the first steps of a microbial infection. we distinguish four processes leading to the presence of microbial rna in the cytosol of eukaryotic cells, where it can engage host prrs (figure 1 ). figure 1 | cytosolic recognition of microbial rna. genomic rna from rna viruses access the cytosol immediately after the cell entry step of the replication cycle, where it may be amplified by viral rna-dependent rna polymerase (rdrp). genomic dna from dna viruses is transcribed by viral or cellular rna polymerase, including the cytosolic rna polymerase iii. bacterial rna can access the cytosol through the activity of auxiliary secretion systems or during passive leakage of phagosomal products. once in the cytosol, microbial rna binds different families of prrs classified as rlrs, non-rlr helicases, and other receptors. downstream signaling pathways include activation of mavs, trif, and the nlrp3 inflammasome. black arrows, rna entry; red arrows, signaling pathways. step of the replication cycle. viruses can directly release their genome at the plasma membrane after binding to a receptor. alternatively, they can be first internalized through endocytosis or macropinocytosis. endocytosed virus particles will typically traffic through endosomal vesicles by actin-dependent and/or microtubule-dependent transport (8) . specific environmental triggers like endosomal ph acidification induce either fusion of enveloped virus with the endosome, or membrane penetration of viral proteins, allowing viral genetic material to be released into the cytoplasm (8) . alternatively, viruses can spread by direct cell-cell contact (9) . cell-to-cell transmission of viral material can activate cytoplasmic innate pathways, as exemplified with hepatitis c virus (10), lymphocytic choriomeningitis virus (11) , or human immunodeficiency virus transmission (12) . other viral rna pamps can be produced during viral replication. david baltimore has defined a classification of viruses based on the mechanism of mrna production (13) . viruses are clustered in seven groups depending on their genomes (dna, rna), strandedness (single or double), sense or antisense, and method of replication. the type of rna ligands produced during viral replication will depend on the type of viral genome and the strategy used to generate mrna. rna ligands can be generated by dna viruses and retroviruses via genome transcription, or by synthesis of mrna and replication intermediates by rna-dependent rna polymerases (rdrps) of rna viruses (8) . it has been shown that ligands generated in phagolysosomes after phagocytosis of bacteria by immune cells can engage cytosolic innate immune receptors (14) . similarly, we showed that rna from escherichia coli could activate receptors in the cytosol after phagocytosis by macrophages (7) . we demonstrated that phagosomes carrying e. coli exhibit intrinsic leakiness, suggesting a mechanism by which bacterial rna, irrespective of the activity of virulence factors, can gain access to the cytoplasm (7) . alternatively, bacteria express secretion systems to translocate products outside of the bacterial cell wall. in the case of intracellular bacteria, auxiliary secretion systems like seca2 in listeria monocytogenes have been shown to actively translocate listeria rna into the cytoplasm, resulting in activation of cytosolic sensors (15, 16) . similarly, another study proved that cytosolic rna sensors participate in the type 1 interferon (ifn-i) response to legionella pneumophila. although the authors did not demonstrate the translocation of legionella rna into the cytosol of infected cells, they discuss their data through a model where it would be the case (17) . future studies looking for additional secreted rna will likely provide additional insights on their interaction with the innate immune system. two independent groups have demonstrated that cytoplasmic dsdna triggers ifn-i production via rna polymerase iii, which transcribes dna into 5'-triphosphate (5 -ppp) rna, subsequently recognized by cytosolic rna receptors (18, 19) . this pathway has been involved in the sensing of dna viruses, like epstein-barr virus, or intracellular bacteria, like l. pneumophila (18, 19) . although the rna intermediate produced is not sensu stricto microbial, its generation is due to the activity of a microbial invader. the best-studied cytosolic rna sensors are the three members of rig-i-like receptors (rlrs), a subfamily of the dexd/hbox family of helicases. they consist of retinoic acid-inducible gene i (rig-i), melanoma differentiation factor 5 (mda5), and laboratory of genetics and physiology 2 (lgp2). they share a similar organization with three distinct domains: (i) a c-terminal repressor domain (rd) embedded within the c-terminal domain (ctd); (ii) a central atpase containing dexd/h-box helicase domain able to bind rna; and (iii) a n-terminal tandem card domain that mediates downstream signaling, and which is present in rig-i and mda5 but absent in lgp2. upon activation by rna ligands, rig-i and mda5 are subsequently recruited to the adaptor protein mitochondrial antiviral signaling (mavs) via a card-card interaction and lead to activation of nf-κb and irfs (20) (21) (22) (23) . in contrast to tlr expression that is predominantly expressed in specialized immune cells such as macrophages and dendritic cells (dcs), rlrs are found in the cytosol of most cell types and are strongly induced in response to ifn-i (24, 25) . the rig-i ligand has been characterized as an rna molecule with two distinct features: (i) a 5 -ppp moiety (26, 27) and (ii) bluntend base pair at the 5'-end (28, 29) . blunt-end base pairs can be found in double-stranded rna (dsrna) and secondary rna structures such as hairpin or panhandle conformations (28, 29) . recent structural studies have contributed toward a better understanding of ligand binding and activation of rig-i. specificity of 5 -ppp binding is conferred by the ctd, and the helicase domain binds the double-stranded part of the rna. rig-i is normally held in an auto-repressed conformation, and ligand binding results in a conformational change, releasing the card domain which can subsequently initiate signaling by association with mavs (30) (31) (32) . despite the increasing amount of high-resolution crystal data, the consensus definition of rig-i ligand remains controversial. other rig-i ligands have been indeed described in the literature including long (33) or short dsrna (34) (35) (36) lacking the 5 -ppp. however, thermodynamic analysis have shown that the full-length human rig-i protein binds 5'-ppp dsrna with 126-fold higher affinity than 5'-oh dsrna, and dsrna with a 361-fold higher affinity than short single stranded rna (ssrna) lacking secondary structure (37) . many viral families display blunt-end base-paired rna with a 5 -ppp, directly in their genomic rna or in replication intermediates. consistent with this notion, rig-i has been shown to be involved in the recognition of many viruses, either antisense (−)ssrna viruses (38, 39) or sense (+)ssrna/dsrna viruses (40, 41) . notably rig-i can detect panhandle structures found in lacrosse viral particles (39) or in influenza genomic rna (28, 38) . sendai virus (sev) and other mononegavirales produce defective interfering (di) viral genomes containing panhandle structures that activate rig-i in infected cells (42) . retinoic acid-inducible gene i recognition has not been limited to rna virus since rig-i is involved in recognition of dna viruses, such as epstein-barr virus or adenovirus through the rna polymerase iii pathway (18, 43, 44) . moreover, rig-i is also able to detect bacterial infections. bacterial mrna are not capped and it has been estimated that approximately 40% of rna oligonucleotides in e. coli have a 5 -ppp (45) . reports in the literature describe sensing of l. monocytogenes secreted rna (15, 16) or purified legionella (17) and helicobacter pylori rna (46) by rig-i. finally, rig-i can also sense shigella flexneri infection in macrophages through the rna polymerase iii pathway (47) . melanoma differentiation factor 5 ligand is less characterized than rig-i. using poly(i:c) as a synthetic dsrna mimic, studies have shown that mda5 binds long, but not short dsrna (35, 40, 48) . structural analyses have demonstrated that mda5 specifically recognizes the internal duplex structure of dsrna and uses it as a platform to stack along dsrna in a head-to-tail arrangement. this mechanism promotes stochastic assembly of the tandem card oligomers that activates the signaling adaptor mavs (49) . melanoma differentiation factor 5 detects infection by viral families known to produce long dsrna structures during their replication cycle, including (+)ssrna viruses like picornaviruses, dsrna viruses like reoviruses, or dna viruses like poxviruses (35, (50) (51) (52) (53) . in the case of (+)ssrna virus infection, fluorescent imaging studies have confirmed that mda5 recognizes preferentially the dsrna generated during the replication of these viruses, but not the genomic ssrna (54) . prior to the structural study mentioned above, multiple observations raised the possibility that there may exist additional mda5 ligands, different from the consensus long dsrna. thus, a study has shown that mda5 cooperates with the ribonuclease rnase l to induce ifn-i in response to a viral mrna from parainfluenza 5 virus (55) . interestingly, rnase l converts rna into small rna products, with shorter length than the current mda5 www.frontiersin.org ligand definition (56) . another work published the same year has shown that mrna lacking 2'-o-methylation at their 5' cap structure induces production of ifn-i through mda5 activation (57) . however, the data published, which focus on coronavirus infection, did not elucidate whether the absence of methylation was directly recognized by mda5 or via another intermediate (57) . after binding to their specific ligands, both rig-i and mda5 activate mavs to trigger a common signaling pathway. the majority of mavs is localized on the mitochondrial membrane and its engagement by rlrs causes a conformational change that propagates to adjacent un-activated mavs in a prion-like behavior (58) . the formation of these very large mavs aggregates results in a largescale amplification of the signaling cascade. this cascade involves the recruitment of cytosolic adaptor molecules, followed by the activation of the canonical ikks, ikk-α, ikk-β, and ikk-γ, the mapk and the non-canonical ikk-related kinase, tbk1 and ikki/ε. ultimately, specific transcription factors, such as irf3, nf-κb, and depending on the cell type irf5 and irf7, are translocated to the nucleus where they promote the expression of ifn-i genes and pro-inflammatory cytokines [reviewed in ref. (59) ]. finally, mavs has been recently shown to interact with nodlike receptor family, pyrin domain containing 3 (nlrp3) and promote its recruitment to the mitochondria. the authors emphasize the central role of mavs in innate immune signaling events by showing its importance in the functioning of nlrp3 inflammasome and the production of il-1β (60) . of note, mavs independent activation of the nlrp3 inflammasome by rig-i has also been reported (61, 62) . the third member of rlrs, lgp2, is able to bind dsrna (63, 64) , however, its role in immune activation is poorly understood. lgp2 was proposed to be a modulator of rlr signaling. studies showed that lgp2 was required for rig-i and mda5 activity, in particular during picornaviral infection (65) (66) (67) . another work proposed that lgp2 would inhibit rig-i through competition with its ligand (64) . it is however unclear whether lgp2 binds microbial rna in an infectious context, and what specific features of the rna it would recognize. further studies will be required to clarify the precise role of lgp2. apart from rlr, several recent studies have highlighted the importance of other dexd/h-box helicases in microbial rna sensing. rna helicases of the dead box family are involved in various different steps of rna metabolism [reviewed in ref. (68) ]. they share eight conserved motifs that are involved in atp binding, atp hydrolysis, nucleic acid binding, and rna unwinding activity. additionally, most dexd/h-box helicases contain auxiliary nand c-terminal domains that confer on them functional specificities, such as an ability to induce downstream signaling or to bind specific rna targets (69) . ddx3 (ddx3x) can bind poly(i:c) or vesicular stomatitis virus (vsv) rna and was shown to enhance the ifn-i response to vsv infection by interaction with the rlr-mavs complex. overexpression assays suggest that ddx3 precipitates with rig-i and mda5 (70) . since ddx3 is easily detected in resting cells, the authors propose a sentinel role for this helicase, the activity of which would be required during the initial steps of viral infection. another study showed that upon sev infection, ddx3 interacts with ikkε, an essential component of the irf3 signaling pathway, increasing the induction of the ifn-β promoter (71) . moreover, ddx3 is targeted by vaccinia virus protein k7 (71) , an inhibitor of ifn-β production, and by hcv core protein, which can disrupt its interaction with mavs (72) . these observations highlight the importance of ddx3 in efficient viral sensing. using overexpression and knock-down experiments, dhx9 was shown to be required for the production of ifn-i and proinflammatory cytokines in response to poly(i:c), influenza virus, and reovirus by a murine splenic dc line and bone-marrow derived dcs. dhx9 can bind dsrna via its dsrna-binding motif and interact with mavs through both its helicase c-terminal domain and ha2-duf (73). myeloid dcs have also been shown to express a complex composed of ddx1, ddx21, and dhx36 that triggers an antiviral program in response to poly(i:c), in a pathway dependent of the adapter molecule tir-domain containing adapter-inducing interferon-β (trif). ddx1 binds to poly(i:c) via its helicase a domain, while dhx36 and ddx21 bind the tir domain of trif via their ha2-duf and prk domains, respectively. this complex seems to be required for the innate response against influenza or reovirus infection (74) . notably, a separate study also characterized dhx36 and dhx9 as a sensor for the dsdna species cpg-a and -b, respectively. in this case, both dhx36 and dhx9 activate the cytosolic adapter protein myeloid differentiation primary response gene 88 (myd88) by binding to its tir domain (75) . another recent study by yong-jun liu's group identified another helicase, dhx33, as a cytosolic rna receptor able to activate the nlrp3 inflammasome (76) . dhx33 is involved in inflammasome activation after sensing cytosolic rna such as poly(i:c) or reoviral rna when directly delivered by lipofection to the cytoplasm of a macrophage cell line or human monocyte-derived macrophages. additional experiments suggested that dhx33 could also possibly be involved in detection of cytosolic bacterial rna. the authors showed that dhx33 can bind to dsrna through its helicase c domain and to nlpr3 through its dead domain (76) . a few months later, another study performed on myeloid dcs confirmed the role of dhx33 in the sensing of cytosolic poly(i:c) and reoviral rna. surprisingly, in this case, poly(i:c)-induced activation of mapk, nf-κb, and irf3 was mediated by mavs, which binds the helicase c domain of dhx33 (77) . ddx60 has also been shown to enhance the ifn-i response to rna and dna stimulation through formation of complexes with frontiers in immunology | molecular innate immunity rig-i, mda5, and lgp2 but not with mavs. this complex formation has been deciphered with overexpression assays in the case of mda5 and lgp2, and with endogenous rig-i during vsv infection. ddx60 expression is induced by viral infection and its helicase domain can bind ds-or ss-vsv rna generated in vitro, independently of the 5 -ppp (78) . interestingly, ddx60 can also bind dsdna, and was shown to play role in ifn-i expression after infection with herpes simplex virus-1, a dna virus. this ability to bind both dsrna and dna raises the question of the feature ddx60 recognizes. it should be finally noted that the role of ddx60 in the ifn-i pathway has been questioned (79) . several other cytoplasmic receptors have been shown to play a role in microbial rna recognition. this is the case for the cytoplasmic protein kinase r (pkr), which is important for antiviral activity. pkr is activated by dsrna from viruses and is a component of mapk and nf-κb signaling pathways [reviewed in ref. (80) ]. activation of pkr can also be mediated by short 5 -ppp rnas containing limited secondary structures (81) . proteins from the interferon-induced protein with tetratricopeptide repeats (ifits) family, such as ifit1 and 5, bind 5 -ppp of viral rna (82) . using short in vitro transcribed oligonucleotides, crystal structure studies have demonstrated that ifit proteins contain a positively charged cavity designed to engage, without any particular sequence specificity, ssrna with a 5 -ppp end. contrary to rig-i, ifit proteins cannot bind blunt-ended 5 -ppp dsrna, and owing to the limitations imposed by their rna-binding pockets, ifit1 and ifit5 require 5'-overhangs of at least 5 or 3 nt, respectively (83) . using a 2 -o-methyltransferase mutant of japanese encephalitis virus, another study showed that ifit1 preferentially binds to 5 capped 2 -o-unmethylated mrna (84) , confirming previous findings showing that 2 -o-methylation of viral mrna caps promotes ifit1 evasion (85, 86) . the mechanism of ifit1 antiviral action is not completely understood, and it has been proposed that ifit might sequester viral rnas (82) or inhibit viral mrna translation (84) . the crystal structure of ifit2 (known as isg54) was also described. ifit2 specifically binds adenylate uridylate (au)rich rnas in vitro, independently of the presence of a 5 -ppp (87) . the authors showed that rna-binding capacity of this protein mediates its antiviral properties, using a model of hek293t cells infected by newcastle disease virus or sev (87) . nucleotide-binding oligomerization domain containing protein 2 (nod2) is a member of the nod1/apaf-1 family and encodes a protein with two card domains and six leucinerich repeats (lrrs). nod2 is primarily known for its ability to recognize bacterial peptidoglycan, but it also plays a role in the antiviral response. nod2 has been shown to activate mavs after stimulation with viral ssrna or human respiratory syncytial virus infection (88) . nlrp3 is involved in cytosolic rna sensing. caspase-1 cleavage triggered by influenza virus, sev, or bacterial mrna is dependent on nlrp3 inflammasome activation (7, 89, 90) . however, direct binding of nod2 or nlrp3 to microbial rna has not been established. leucine-rich repeat flightless-interacting protein 1 (lrrfip1) contributes to the production of ifn-β induced by vsv and l. monocytogenes in macrophages (91) . mostly located in the cytosol, lrrfip1 can also be found in rna-containing lysosomes (92) . lrrfip1 can bind both dsrna and dsdna and subsequently induce ifn-i expression through β-catenin phosphorylation. activated β-catenin is translocated to the nucleus and increases ifn-β expression by binding to the c-terminal domain of the transcription factor irf3 and promoting the recruitment of the acetyltransferase p300 to the ifnb1 promoter. several other microbial rna features have been suspected or proposed to act as potential signals for cytosolic sensing, suggesting the existence of receptors detecting these characteristics. a computational analysis identified cpg motifs in an au-rich rna as an immunostimulatory feature. this sequence motif is underrepresented in both ssrna viruses and host innate immune gene mrna, and its frequency in influenza virus genomes has decreased throughout evolution (93) . since this evolutionary pressure seems to also be applied on host mrna, the implication of a cytosolic receptor is possible, although experimental studies identified endosomal tlr7 as a potential prr (94) . another study identified the nucleotide bias of a-rich hiv-1 genome as a strong inducer of ifn-i and potent mediator of lentiviral pathogenicity. the authors showed that the ability of rna sequences derived from the hiv-1 genome to induce an interferon response correlated with their nucleotide bias and that codon-optimized sequences lost their stimulatory activity (95) . the experimental procedure used in this study consisted of direct delivery via lipofection of in vitro transcribed rna sequences into the cytosol of a reporter cell line, suggesting a potential role for a cytoplasmic rna sensor (95) . recently, our group identified bacterial mrnas as an activator of the nlrp3 inflammasome. polyadenylation of these rnas abrogated their immunostimulatory activities, suggesting that features at the 3 end of mrna, rather than the 5 end, could engage cytoplasmic cellular sensors (7) . philip bevilacqua's group has shown that different nucleoside modifications on rna, such as base or sugar internal modifications, suppress their intrinsic ability to activate immune sensors, notably pkr. the authors propose that self-rna editing could be a mechanism used by the innate immune system to discriminate self-transcripts from "unmodified" microbial rnas (96, 97) . conversely, microbial rna editing by cellular deaminase enzymes such as dsrna-specific adenosine deaminase (adar) have been shown to enhance its recognition by cytosolic sensors (98) . other host transcript specificities, like association to cellular components that prevent prr binding, or specific tertiary structure such as the eukaryotic mrna closed loop conformation (99) , could be determinants for the differentiation of host mrnas from microbial rnas. identification of receptors able to recognize such features are lacking so far. infectious microorganisms have developed several strategies to evade cytosolic sensing. one of these strategies, which we only mention briefly here, is the direct targeting by microbial proteins www.frontiersin.org some (−)ssrna viruses edit the 5 -ppp moieties in their genomes as well as replication intermediates into 5 mono-phosphates to avoid recognition by rlrs (101) . arenaviruses produce rna panhandle structures with a 5 -ppp containing a gtp overhanging nucleotide. this viral structure is suggested to act as a rig-i ligand decoy, by trapping rig-i but not activating it (102) . we are beginning to understand how eukaryotic cells use nucleoside modifications in order to protect self-rnas from innate sensing. for example, higher eukaryotes have acquired the ability to 2 -omethylate their mrnas, allowing cellular receptors to distinguish self from unmethylated non-self mrna through specific types of antiviral sensors such as mda5 and ifits (57, 85) . consistent with the red queen hypothesis (103) , which postulates that parasites have to constantly evolve in order to adapt to their host species, the same immune escape strategy has been mimicked by several pathogens, like flaviviruses (84, 86) . similarly, 2 -o-methylation of g18 (gm18) on bacterial trna suppresses activation of the immune response in plasmacytoid dcs (104, 105) . flaviviruses and other viruses are also known to induce cellular membrane reorganization that allows them to replicate in subcellular compartments, creating new replication-dependent organelles (106) . thus, tick-borne encephalitis virus or japanese encephalitis virus have been shown to rearrange endoplasmic reticulum membranes to provide a compartment where viral dsrna is concealed from prr recognition. this hijacking of internal cell membrane induces a delayed cytosolic exposure of viral rna to innate receptors and accordingly, ifn-i responses are only measured late in the replication cycle (107) (108) (109) . the ns1 protein from influenza virus can prevent rna sensing through the formation of a chain of ns1 molecules along the influenza dsrna backbone (110) . picornaviruses mask their 5ppp with a viral encoded protein, vpg, which functions as a 5 cap and as a primer during rna synthesis. interestingly, studies have shown that vpg could be used to evade rig-i recognition (111) . similarly, ebola virus vp35 assembles into dimmers to cap the ends of viral dsrna and hide the specific rig-i recognition site (112) . while one vp35 monomer binds the terminus and backbone of dsrna, the other vp35 monomer binds only the phosphate backbone of the dsrna, displaying a unique mode of dsrna concealing from prr (112) . another hemorrhagic fever virus, lassa fever virus, uses the 3'-5' exonuclease activity of its nucleoprotein (np) to degrade stimulatory dsrna (113) . this activity seems to be shared by other arenaviruses (114) . finally, the protein c from human parainfluenza virus type 1 (hpiv1), a paramyxoviridae, has been shown to limit the accumulation of dsrna. cell infection by a virus mutant defective for the c protein displays higher accumulation of several viral rnas, including viral genome, antigenome, and mrna, eventually leading to the accumulation of dsrna. thus, by limiting intracytosolic quantities of viral dsrna, the c protein of hpiv1 avoids dsrna triggering of mda5 and pkr in infected cells (115) . the multiplicity of prr pathways is an essential determinant of the immune system's ability to sense with precision the level of frontiers in immunology | molecular innate immunity microbial threat and to respond accordingly (4) . however, as far as cytosolic rna sensors are concerned, it is striking to observe the contrast between the high number of prrs that have been isolated and the similarities of the pamps they recognize ( table 1) . while 5 -ppp and dsrna are undoubtedly powerful triggers of the innate immunity, they cannot account for the diversity of responses that the organism is able to elicit against a wide range of pathogens. our understanding of how the immune system distinguishes between foreign and self-nucleic acids will continue to improve over time. this will help us better define the precise role played by cytosolic rna sensors in the global immune response against pathogens. approaching the asymptote? evolution and revolution in immunology a human homologue of the drosophila toll protein signals activation of adaptive immunity approaching the asymptote: 20 years later beyond pattern recognition: five immune checkpoints for scaling the microbial threat activation of target-tissue immune-recognition molecules by double-stranded polynucleotides immune sensing of dna detection of prokaryotic mrna signifies microbial viability and promotes immunity principles of virology cell-to-cell transmission of viruses plasmacytoid dendritic cells sense hepatitis c virus-infected cells, produce interferon, and inhibit infection human pdcs sense lcmv infected cells in vitro innate sensing of hiv-infected cells expression of animal virus genomes bacterial ligands generated in a phagosome are targets of the cytosolic innate immune system rig-i detects infection with live listeria by sensing secreted bacterial nucleic acids rig-i detects triphosphorylated rna of listeria monocytogenes during infection in non-immune cells identification of host cytosolic sensors and bacterial factors regulating the type i interferon response to legionella pneumophila rig-i-dependent sensing of poly(da:dt) through the induction of an rna polymerase iii-transcribed rna intermediate rna polymerase iii detects cytosolic dna and induces type i interferons through the rig-i pathway ips-1, an adaptor triggering rig-i-and mda5-mediated type i interferon induction cardif is an adaptor protein in the rig-i antiviral pathway and is targeted by hepatitis c virus identification and characterization of mavs, a mitochondrial antiviral signaling protein that activates nf-kappab and irf 3 visa is an adapter protein required for virus-triggered ifn-beta signaling expression analysis and genomic characterization of human melanoma differentiation associated gene-5, mda-5: a novel type i interferon-responsive apoptosis-inducing gene the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses 5'-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing 5'-phosphates recognition of 5' triphosphate by rig-i helicase requires short blunt doublestranded rna as contained in panhandle of negative-strand virus 5'-triphosphate rna requires base-paired structures to activate antiviral signaling via rig-i structural basis of rna recognition and activation by innate immune receptor rig-i structural basis for the activation of innate immune pattern-recognition receptor rig-i by viral rna structural insights into rna recognition by rig-i molecular mechanism of signal perception and integration by the innate immune sensor retinoic acid-inducible gene-i (rig-i) a structural basis for discriminating between self and nonself double-stranded rnas in mammalian cells length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-i and melanoma differentiation-associated gene nonself rna-sensing mechanism of rig-i helicase and activation of antiviral immune responses the thermodynamic basis for viral rna detection by the rig-i innate immune sensor rig-i detects viral genomic rna during negative-strand rna virus infection incoming rna virus nucleocapsids containing a 5'-triphosphorylated genome activate rig-i and antiviral signaling differential roles of mda5 and rig-i helicases in the recognition of rna viruses innate immunity induced by composition-dependent rig-i recognition of hepatitis c virus rna preference of rig-i for short viral rna molecules in infected cells revealed by next-generation sequencing epstein-barr virus-encoded small rna induces il-10 through rig-i-mediated irf-3 signaling adenovirus virus-associated rnas induce type i interferon expression through a rig-i-mediated pathway the 5' ends of rna oligonucleotides in escherichia coli and mrna degradation extracellular and intracellular pattern recognition receptors cooperate in the recognition of helicobacter pylori ifngamma inhibits the cytosolic replication of shigella flexneri via the cytoplasmic rna sensor rig-i essential role of mda-5 in type i ifn responses to polyriboinosinic:polyribocytidylic acid and encephalomyocarditis picornavirus structural basis for dsrna recognition, filament formation, and antiviral signal activation by mda5 double-stranded rna is produced by positive-strand rna viruses and dna viruses but not in detectable amounts by negative-strand rna viruses activation of mda5 requires higher-order rna structures generated during virus infection mda5 detects the double-stranded rna replicative form in picornavirus-infected cells innate immune response after adenoviral gene delivery into skin is mediated by aim2, nalp3, dai and mda5 visualisation of direct interaction of mda5 and the dsrna replicative intermediate form of positive strand rna viruses activation of ifn-&#946; expression by a viral mrna through rnase l and mda5 new insights into the role of rnase l in innate immunity ribose 2'-o-methylation provides a molecular signature for the distinction of self and non-self mrna dependent on the rna sensor mda5 mavs forms functional prion-like aggregates to activate and propagate antiviral innate immune response intracellular pathogen detection by rig-i-like receptors the adaptor mavs promotes nlrp3 mitochondrial localization and inflammasome activation recognition of rna virus by rig-i results in activation of card9 and inflammasome signaling for interleukin 1 beta production type i ifn triggers rig-i/tlr3/nlrp3-dependent inflammasome activation in influenza a virus infected cells the rig-i-like receptor lgp2 recognizes the termini of double-stranded rna the regulatory domain of the rig-i family atpase lgp2 senses doublestranded rna lgp2 is a positive regulator of rig-i-and mda5-mediated antiviral responses atp hydrolysis enhances rna recognition and antiviral signal transduction by the innate immune sensor, laboratory of genetics and physiology 2 (lgp2) lgp2 plays a critical role in sensitizing mda-5 to activation by double-stranded rna from unwinding to clamping -the dead box rna helicase family dexd/h-box rna helicases as mediators of anti-viral innate immunity and essential host factors for viral replication dead/h box 3 (ddx3) helicase binds the rig-i adaptor ips-1 to up-regulate ifn-beta-inducing potential viral targeting of dead box protein 3 reveals its role in tbk1/ikkepsilon-mediated irf activation hepatitis c virus core protein abrogates the ddx3 function that enhances ips-1-mediated ifn-beta induction dhx9 pairs with ips-1 to sense double-stranded rna in myeloid dendritic cells ddx1, ddx21, and dhx36 helicases form a complex with the adaptor molecule trif to sense dsrna in dendritic cells aspartateglutamate-alanine-histidine box motif (deah)/rna helicase a helicases sense microbial dna in human plasmacytoid dendritic cells the dhx33 rna helicase senses cytosolic rna and activates the nlrp3 inflammasome the interaction between the helicase dhx33 and ips-1 as a novel pathway to sense double-stranded rna and rna viruses in myeloid dendritic cells ddx60, a dexd/h box helicase, is a novel antiviral factor promoting rig-i-like receptor-mediated signaling cytosolic sensing of viruses interferon-inducible antiviral effectors 5'-triphosphate-dependent activation of pkr by rnas with short stem-loops ifit1 is an antiviral protein that recognizes 5'-triphosphate rna structural basis for viral 5'-ppp-rna recognition by human ifit proteins ifit1 inhibits japanese encephalitis virus replication through binding to 5' capped 2'-o unmethylated rna 2'-o methylation of the viral mrna cap evades host restriction by ifit family members 2'-o methylation of the viral mrna cap by west nile virus evades ifit1-dependent and -independent mechanisms of host restriction in vivo crystal structure of isg54 reveals a novel rna binding structure and potential functional mechanisms activation of innate immune antiviral responses by nod2 critical role for cryopyrin/nalp3 in activation of caspase-1 in response to viral infection and double-stranded rna the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna the cytosolic nucleic acid sensor lrrfip1 mediates the production of type i interferon via a betacatenin-dependent pathway characterization of lrrfip1 patterns of oligonucleotide sequences in viral and host cell rna identify mediators of the host innate immune system oligonucleotide motifs that disappear during the evolution of influenza virus in humans increase alpha interferon secretion by plasmacytoid dendritic cells the biased nucleotide composition of hiv-1 triggers type i interferon response and correlates with subtype d increased pathogenicity nucleoside modifications modulate activation of the protein kinase pkr in an rna structure-specific manner native tertiary structure and nucleoside modifications suppress trna's intrinsic ability to activate the innate immune sensor pkr inosine-containing rna is a novel innate immune recognition element and reduces rsv infection the mechanism of eukaryotic translation initiation and principles of its regulation targeting of immune signalling networks by bacterial pathogens processing of genome 5' termini as a strategy of negative-strand rna viruses to avoid rig-i-dependent interferon induction short doublestranded rnas with an overhanging 5' ppp-nucleotide, as found in arenavirus genomes, act as rig-i decoys a new evolutionary law identification of modifications in microbial, native trna that suppress immunostimulatory activity the 2'-o-methylation status of a single guanosine controls transfer rna-mediated toll-like receptor 7 activation or inhibition viral reorganization of the secretory pathway generates distinct organelles for rna replication tick-borne encephalitis virus delays interferon induction and hides its double-stranded rna in intracellular membrane vesicles delayed cytosolic exposure of japanese encephalitis virus double-stranded rna impedes interferon activation and enhances viral dissemination in porcine cells formation of membrane-defined compartments by tick-borne encephalitis virus contributes to the early delay in interferon signaling x-ray structure of ns1 from a highly pathogenic h5n1 influenza virus the genome-linked protein vpg of vertebrate viruses -a multifaceted protein ebolavirus vp35 uses a bimodal strategy to bind dsrna for innate immune suppression structure of the lassa virus nucleoprotein reveals a dsrna-specific 3' to 5' exonuclease activity essential for immune suppression structures of arenaviral nucleoproteins with triphosphate dsrna reveal a unique mechanism of immune suppression the c proteins of human parainfluenza virus type 1 limit double-stranded rna accumulation that would otherwise trigger activation of mda5 and protein kinase r the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. key: cord-342776-hkjhqgie authors: jewett, anahid title: the potential effect of novel coronavirus sars-cov-2 on nk cells; a perspective on potential therapeutic interventions date: 2020-07-10 journal: front immunol doi: 10.3389/fimmu.2020.01692 sha: doc_id: 342776 cord_uid: hkjhqgie coronavirus-induced disease-2019 (covid-19) continues to cause significant morbidity and mortality worldwide. while studies on sars-cov-2 effects on immune cell function continue to progress, we know very little about the significance of depletion of key immune effectors by the virus in the mortality and morbidity of the disease. this commentary outlines what is the reported literature thus far on the effect of virus on nk cells known to kill virally infected cells. it also underscores the necessity for the future comprehensive studies of nk cells in sars-cov-2 infected individuals and animal models to better understand the role and significance of reported nk cell depletion and functional inactivation in disease morbidity and mortality, in hope to design effective therapeutic interventions for the disease. coronavirus-induced disease-2019 (covid-19) poses a great public health threat, and presents a complex challenge for epidemiologists and public health professionals around the planet, as the disease has shifted from a regional epidemic to a worldwide pandemic in a short period of time. the toll that the disease has had on the global level continues to increase as the virus reaches all continents, except antarctica, afflicting more than 180 countries. initial reports of covid-19 disease came from wuhan, china in late december 2019, as patients began complaining about unexplained respiratory infections, which later was coined as "pneumonia of unknown etiology" (1) . shortly after surfacing of the virus several independent laboratories identified the causative agent of covid-19 disease, ultimately naming it as severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (2, 3) . while the search is continuing to uncover the infectious path of sars-cov-2, several key findings have led the infectious disease experts to partly uncover the mechanisms of the original spread to humans. by phylogenetically comparing sars-cov-2 to other coronaviruses, it was noted that the new virus was highly identical to other coronaviruses that had originated from bats (3, 4) . however, to date the complete transmission route remains elusive. despite the novelty of this particular strain of coronavirus, the sars-cov-2 is not without precedent. outbreaks in the past decades, such as severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), identified viruses that fall into the same category of coronaviruses, which are single-stranded rna viruses (+ssrna) that morphologically have been determined to express crown-like spikes on their surfaces. however, the difference seen between prior species of coronaviruses and sars-cov-2 partly lies in their respective symptom presentations in patients. compared to sars and mers, the symptoms of covid-19 disease are not presented earlier in the infectious cycle, which may be a reason for the greater ability of viral transmission in patients (4) . the incubation period of the sars-cov-2 is relatively longer than those of sars and mers (7-14 days vs. 5.0-6.9 and 4.4-6.9, respectively) (4). in addition to its longer incubation period, the mean reproductive number (r 0 ) of sars-cov-2 has also been estimated to range from 2.20 to 3.58, indicating that each infected patient can on average transmit the disease to two to three other individuals (5, 6) . according to the available covid-19 clinical data, most patients fall into the range of 30-79 years of age, although several cases have been identified in younger individuals and in children recently (7) . for infected patients, severity of symptoms has been classified as mild, severe, and critical. this spectrum of disease widely varies, as clinical presentation in infected individuals have ranged from asymptomatic infection to severe respiratory failure (2) . asymptomatic transmission of sars-cov-2 poses a great public health challenge in containment efforts, as previous reports have noted as much as 12.6% of case reports to be presymptomatic transmission (8) . however, the main characteristic symptoms of covid-19 disease have included fevers, fatigue, dry cough and respiratory distress. the number of sars-cov-2 infected cases will certainly continue to rise worldwide especially now that many countries have chosen to relax the rules of social distancing and isolation due to the reopening of the economy and the work force. one of the most troubling factors about this disease is the lack of adequate understanding of the virus and the mechanisms by which it mediates the underlying pathology in humans. the problem has been compounded by the limited ability of the research laboratories to conduct studies due to the implementation of social distancing since many academic university laboratories have either been shut down or been operating at a minimum capacity. although the existing novel therapeutic strategies and research on potential vaccines are important directions, they will not be sufficient to provide adequate progress to fully understand the potential of the virus to infect individuals and the underlying mechanisms by which the virus causes pathology. containment efforts, through quarantines and social distancing, hand washing and wearing mask are important directions to mitigate the spread of sars-cov-2 infections. however, at the moment, we do not have the capability of large scale testing which would be necessary for the identification and isolation of asymptomatic and symptomatic patients to halt the chain of viral transmission. therefore, until the existing public health measures are able to curtail the transmission and bring the disease somewhat under control, the research laboratories will not be able to fully engage in the studies of covid-19 disease worldwide, thereby slowing the discoveries of more effective treatments and vaccines. progression models of covid-19 disease paint a dismal forecast for the duration of the outbreak, and therefore, warrant the discovery of novel treatments to alleviate disease symptoms that will supplement containment efforts. researchers continue to search for viable treatments to alleviate symptoms of the disease and to eradicate the spread of the disease. the search for effective treatments for covid-19 disease has featured studies advocating for the potential use of the anti-malarial drugs, hydroxychloroquine. the placebo controlled studies of hydroxychloroquine have not shown any efficacy, and indeed, in certain cases they have exhibited dangerous side effects in patients. others have advocated the immunologic path in support of monoclonal antibody therapy (9, 10) . on the other hand remdesivir, an antiviral drug, was shown to be superior to placebo in shortening the time to recovery in adults hospitalized with covid-19 disease with the evidence of lower respiratory tract infection (11) . the development of a sars-cov-2 vaccine would utilize the adaptive immune system to combat symptoms in patients, along with providing a preventative measure for healthy individuals. however, current timelines estimate that vaccine development could take up to anywhere from 12 to 18 months. while studies on sars-cov-2's effects on immune functions continue to progress, published studies concerning other coronaviruses may shed some light on how the immune system may be employed to mitigate covid-19 disease. based on experiences with coronaviruses in sars and mers, it has been suggested that the sars-cov-2 infection may also trigger major immunological changes, such as delayed or suppressed type 1 ifn response and the influx of activated neutrophils and inflammatory monocytes/macrophages (12) . in addition, the effect of virus on host following infection has demonstrated severe changes in the proportion of different immune effectors (13) . in particular, in the peripheral blood of patients that were infected with sars, it was noted that there were significantly lower numbers of natural killer (nk) cells compared to healthy subjects (14) . such a profile has also been extended to the immune responses of covid-19 individuals. a study of 452 covid-19 individuals demonstrated that the numbers of nk, b and t cells were significantly decreased, with more severe cases being associated with greater decreases in the numbers (15) , please see the references (16) (17) (18) (19) for reviews on the potential mechanisms of inactivation and loss of nk cells. upon admission, the neutrophil counts were remarkably higher in patients with severe covid-19 disease than in the mild cases, whereas the total lymphocyte counts were significantly lower in severe cases when compared to the mild cases (15) . the numbers of nk cells and total t cells as well as cd8+ t cells were significantly lower in patients exhibiting severe symptoms when compared to those with the mild symptoms and healthy controls (20) . thus, a correlation could be seen with severe decrease in nk and t cell numbers and the extent of severity of the disease. furthermore, the function of nk and cd8+ t cells was found to be suppressed along with the increased expression of nkg2a in covid-19 individuals (20) . more importantly, in patients convalescing after therapy, the numbers of nk and cd8+ t cells were restored with reduced expression of nkg2a. in addition, these results suggested that the functional exhaustion of cytotoxic lymphocytes was directly associated with severity of sars-cov-2 infection. hence, sars-cov-2 infection is likely to paralyze the antiviral immunity at an early stage and contribute to progression and severity of the disease (20) (figure 1) . in patients infected with sars-cov-2, nkg2a expression was increased significantly on nk and cd8+ t cells compared to those in healthy controls (20) . lower percentages of cd107a+ nk, ifn-γ+ nk, il-2+ nk, and tnf-α+ nk cells and decreased mean fluorescence intensities (mfi) of granzyme b+ nk cells were also reported in covid-19 individuals when compared to healthy controls (20) . although these preliminary results are of significance, they still would need to be reproduced by other laboratories, however, they clearly point to the potential contribution of functional exhaustion of cytotoxic lymphocytes in covid-19 disease (20) . thus, future studies should be focused to fully understand the contribution of dysfunctional nk cells in disease pathogenesis. in addition, the underlying mechanisms of depletion of nk cells such as viral infection of nk cells, mobilization and homing of nk cells to the tissues from the peripheral blood, and activation induced cell death would need to be investigated (figure 1) . neutrophils are the most abundant white blood cells in the lung, and they are critical effectors against infections, in particular against bacterial infections, however they are also capable of inducing life-threatening morbidities. moreover, natural killer cells are the most abundant lymphocytes in the lung, and therefore, play an important role not only in curtailment of infection but also in exertion of significant regulatory effect (21) . however, little is known regarding the specific mechanisms by which nk cells maintain local homeostasis. by using lung-intravital microscopy to directly visualize and quantify neutrophil and natural killer cell interaction within the lung of live mice, the authors reported in a preliminary study that nk cells were greatly responsible for the slower pace of scanning of endothelium by neutrophils over a large area, and they were able to reduce the number of neutrophils that accumulated in an lps-triggered inflammatory challenge (21) . indeed, depletion of nk cells in mice exhibited severe respiratory distress associated with protein-rich, high-permeability alveolar edema accompanied by neutrophil infiltration in myocardial infarction model (22) . thus, nk cells are important effectors in not only combating the infection directly but also indirectly by activating the local inflammatory processes to curtail infection. in addition, they are also able to limit local immune activation in the lung to a manageable level without causing or allowing significant pathologies to be induced by other immune effectors (figure 1) . thus, nk cells are important in keeping the balance of immune activation in such a way that sufficient levels of activation will ensue to remove the infection in the presence of finely tuned inflammatory processes to avoid local tissue damage. as mentioned above the infectious agent of covid-19 disease depletes nk cells in the peripheral blood, and potentially even in the lung tissues of patients, thereby, disabling and depleting the core immune effectors necessary to remove the virus and regulate uncontrolled immune activation. indeed, nk cells are the army generals of the immune effectors which bring order and discipline to the infected tissue microenvironment. without the nk cells it is likely that immune anarchy may ensue and result in the uncontrolled expansion and activation of other immune effectors (16) (17) (18) (19) . we have previously shown that nk cells also curtail the numbers of cd4+ t cells and expand cd8+ t cells (23, 24) and (manuscript submitted). therefore, lack of nk cells may also result in the decrease expansion of cd8+ t cells as seen in covid-19 individuals (16) (17) (18) (19) (figure 1) . thus, it is no surprise that patients suffering from covid-19 disease have greater cd4/cd8 ratios as reported previously. the covid-19 individuals suffer from increased viral replication as well as uncontrolled inflammation resulting in cytokine storm and widespread tissue and organ damage (25) (26) (27) . nk cells are known to mediate cytotoxicity, and regulate both the innate and adaptive immune functions through the release of many pro-and anti-inflammatory growth factors, cytokines and chemokines (16-19, 28, 29) . they constitute 5-15% of the peripheral blood mononuclear cells (pbmcs), and are cytotoxic effectors in the blood of healthy individuals with the ability to recognize and lyse virally infected cells, including sars-cov-2 infected cells and a number of different cancer stem cells (cscs) and undifferentiated or poorly differentiated tumors which constitute the most aggressive subpopulations of the tumors. morphologically, nk cells are large granular lymphocytes that develop in the bone marrow. after development, the majority of nk cells are found in peripheral blood as the third largest lymphocyte population, next to b and t cells (30) . moreover, nk cells are also found in the tissues such as in healthy skin, gut, lung, liver, lymphoid organs and uterus during pregnancy (31) . nk cells have two different effector functions: cytotoxicity and cytokine release. in contrast to cd8+ cytotoxic t lymphocytes, nk cells do not need priming with antigen in order to kill their target cells. their function is regulated by the sum of interactions between activating and inhibitory receptors on their surface and the ligands on the target cells (32) . ligands for nk activating receptors are expressed on fast proliferating cells that are virally infected or malignantly transformed. cytotoxic activity of nk cells is executed by two distinct mechanisms. one is regulated by the cytotoxic granules containing perforin and granzymes. after the formation of the immune synapse between nk and target cells, cytotoxic granules are released. perforin alters the permeability of the target cell membrane, allowing the entry of granzymes. the second pathway is interaction of ligands on nk cells with their respective cell death receptors on target cells. target cell death can also be induced through antibody dependent cellular cytotoxicity (adcc) (16) (17) (18) (19) . indeed, nk cell mediated adcc is likely one of the key mechanisms by which antibodies induced by the virus in recovered patients known as convalescent plasma or serum are able to alleviate the symptoms and improve the disease outcomes in infected and recovering patients (33) (figure 1) . the second key effector function of nk cells is the release of cytokines and chemokines. two major cytokines released by nk cells are ifn-γ and tnf-α (16) (17) (18) (19) 34) . released cytokines not only affect the function of innate and adaptive immune cells, but it can also impact the differentiation of both healthy and cancer cells (figure 1) . similar to sars-cov-2 infection in patients, decreased nk cell function in the tumor microenvironment, and peripheral blood of cancer patients as well as down-modulation of cd16 receptors on the surface of nk cells have been reported previously (23, (35) (36) (37) (38) (39) (40) . decreased function of nk cells is associated with increased viral infection and cancer risk, whereas higher function was correlated with prevention of establishment and progression of infection and cancer (16) (17) (18) (19) . indeed, older patients and those with immunosuppression are more susceptible to severe form of sars-cov-2 infection, and are likely to die from it. thus, it is no surprise that the same subsets of population of individuals are found to have lower expansion and functions of nk cells as reported previously (41, 42) . decreased nk and t cell numbers and function can be due to the activation induced cell death and/or direct infection of the immune cells by the virus (figure 1) . indeed, recent studies indicated that similar to mers-cov infection, sars-cov-2 also infects t cells through receptor-dependent, s-protein mediated membrane fusion and that the infection can be inhibited by ek1 peptide (43) . furthermore, the infection is abortive since sars-cov-2 does not have the capability to replicate in the t cells (43) . with promising studies demonstrating significant decreases and functional deficiencies in immune cell populations in particular the nk cells in covid-19 patients, immunotherapybased treatments using nk cells and cd8+ t cells and/or enhancement of the proliferation and function of nk cells in patients may present a significant and viable avenue toward mitigating disease establishment and progression. the original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. aj prepared the manuscript. evaluation and treatment coronavirus (covid-19) coronavirus disease 2019: what we know genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding covid-19: what has been learned and to be learned about the novel coronavirus disease preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak preliminary estimation of the novel coronavirus disease (covid-19) cases in iran: a modelling analysis based on overseas cases and air travel data characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention serial interval of covid-19 among publicly reported confirmed cases hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial perspectives on monoclonal antibody therapy as potential therapeutic intervention for coronavirus disease-19 (covid-19) remdesivir for the treatment of covid-19 -preliminary report immune responses in covid-19 and potential vaccines: lessons learned from sars and mers epidemic characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia the involvement of natural killer cells in the pathogenesis of severe acute respiratory syndrome dysregulation of immune response in patients with covid-19 in wuhan, china editorial overview: tumour immunology: are we on the path to win the battle against cancer through immunotherapy? nk cells shape pancreatic and oral tumor microenvironments; role in inhibition of tumor growth and metastasis natural killer cells target and differentiate cancer stem-like cells/undifferentiated tumors: strategies to optimize their growth and expansion for effective cancer immunotherapy natural killer cells: diverse functions in tumor immunity and defects in pre-neoplastic and neoplastic stages of tumorigenesis functional exhaustion of antiviral lymphocytes in covid-19 patients pulmonary natural killer cells control neutrophil intravascular motility and response to acute inflammation lung natural killer cells play a major counter-regulatory role in pulmonary vascular hyperpermeability after myocardial infarction super-charged nk cells inhibit growth and progression of stem-like/poorly differentiated oral tumors in vivo in humanized blt mice; effect on tumor differentiation and response to chemotherapeutic drugs probiotic-treated super-charged nk cells efficiently clear poorly differentiated pancreatic tumors in hu-blt mice association of coronavirus disease 2019 (covid-19) with myocardial injury and mortality the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease cytokine release syndrome in severe covid-19 nk cell-based immunotherapy for cancer the broad spectrum of human natural killer cell diversity the biology of human natural killer-cell subsets natural killer cell distribution and trafficking in human tissues activating and inhibitory receptors of natural killer cells treatment of 5 critically ill patients with covid-19 with convalescent plasma natural killer cells: development, maturation, and clinical utilization alterations in expression and function of signal-transducing proteins in tumor-associated t and natural killer cells in patients with ovarian carcinoma clinical significance of decreased zeta chain expression in peripheral blood lymphocytes of patients with head and neck cancer natural cytotoxic activity of peripheral-blood lymphocytes and cancer incidence: an 11-year follow-up study of a general population progressive natural killer cell dysfunction associated with alterations in subset proportions and receptor expression in soft-tissue sarcoma patients novel strategy to expand super-charged nk cells with significant potential to lyse and differentiate cancer stem cells: differences in nk expansion and function between healthy and cancer patients deficiencies in natural killer cell numbers, expansion, and function at the preneoplastic stage of pancreatic cancer by kras mutation in the pancreas of obese mice the impact of ageing on natural killer cell function and potential consequences for health in older adults effect of aging on nk cell population and their proliferation at ex vivo culture condition retracted article: sars-cov-2 infects t lymphocytes through its spike protein-mediated membrane fusion the author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 jewett. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-253862-jl1zhg13 authors: khalaf, khalil; papp, natalia; chou, jadzia tin-tsen; hana, doris; mackiewicz, andrzej; kaczmarek, mariusz title: sars-cov-2: pathogenesis, and advancements in diagnostics and treatment date: 2020-10-06 journal: front immunol doi: 10.3389/fimmu.2020.570927 sha: doc_id: 253862 cord_uid: jl1zhg13 the emergence and rapid spread of sars-cov-2 in december 2019 has brought the world to a standstill. while less pathogenic than the 2002–2003 sars-cov, this novel betacoronavirus presents a global threat due to its high transmission rate, ability to invade multiple tissues, and ability to trigger immunological hyperactivation. the identification of the animal reservoir and intermediate host were important steps toward slowing the spread of disease, and its genetic similarity to sars-cov has helped to determine pathogenesis and direct treatment strategies. the exponential increase in cases has necessitated fast and reliable testing procedures. although rt-pcr remains the gold standard, it is a time-consuming procedure, paving the way for newer techniques such as serologic tests and enzyme immunoassays. various clinical trials using broad antiviral agents in addition to novel medications have produced controversial results; however, the advancement of immunotherapy, particularly monoclonal antibodies and immune modulators is showing great promise in clinical trials. non-orthodox medications such as anti-malarials have been tested in multiple institutions but definitive conclusions are yet to be made. adjuvant therapies have also proven to be effective in decreasing mortality in the disease course. while no formal guidelines have been established, the multitude of ongoing clinical trials as a result of unprecedented access to research data brings us closer to halting the sars-cov-2 pandemic. coronaviruses are widely known virulent pathogens affecting mammalian and avian species. previously, six globally distributed species of the virus have been identified to cause illness in humans. they are: human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), severe acute respiratory syndrome coronavirus (sars-cov), and middle east respiratory syndrome coronavirus (mers-cov) ( table 1) . sars-cov first emerged in [2002] [2003] in china, presenting as an atypical pneumonia with febrile state, headaches, and a marked cough that may rapidly deteriorate into respiratory failure. the sars-cov outbreak was the first to be declared a pandemic, and ultimately infected 8,000 persons in 29 countries, with a mortality rate of 10% (5). epidemiological analysis posited an infectious zoonotic agent, and further serological studies identified the intermediate host to be the palm civet (paguma larvata). spillover from animals to humans was hypothesized to be via the horseshoe bat (genus rhinolophus). an adaptation of the sars-cov virus was later proven to be due to interspecies dwelling (6) . a combination of ribavirin and pulse prednisolone showed positive results early in the outbreak and was adopted as the standard protocol. eventually, ribavirin was demonstrated to have increased cytotoxicity and lacked efficient antiviral action in vitro, and while pulse prednisolone showed efficacy in managing critically ill patients, its high dose administration was associated with disseminated fungal infections (7) . later, the combination of interferon-alpha1 (inf-α1) and corticosteroids was found to yield a better prognosis, but did not prove beneficial for patients in the late stage of the disease (8) . protease inhibitors produced an outcome similar to that of inf-α1 (9) . a novel coronavirus later named mers-cov emerged in saudi arabia in 2012. similar to sars, infected patients presented with a variety of clinical courses, from mild upper respiratory symptoms to fulminant pneumonia and multi-organ system failure. phylogenic and sequencing studies have proposed a bat origin, and surface protein modification was found to be derived from the intermediate hosts, dromedary camels. from 2012 to 2015, more than 2,000 confirmed cases were reported, mainly in middle eastern countries, with a mortality rate of 35% (10) . as with sars-cov, no definitive protocol was implemented, and most patients were treated with supportive therapy to preserve organ integrity (11) . in a cohort study conducted by omrani et al., a treatment protocol involving ifn-α2 and ribavirin was initiated, and while survival improved significantly at 14 days after treatment, this was not seen at day 28, necessitating further treatment options (12) . sporadic outbreaks occur to this day, with patients undergoing largely supportive therapy. the sars-cov-2 outbreak began in december 2019 in wuhan, china where clusters of atypical pneumonia yielded evidence of a novel strain of coronavirus. as of august 24, 2020, 16,036,308 cases had been reported worldwide, with a fatality rate of 5%. although this novel virus is less severe than the first sars-cov outbreak, human-to-human transmission remains very high and the number of cases continues to rise exponentially in major urban areas, highlighting the urgent need to develop new containment, diagnostic, and treatment protocols. as time is of the essence, the pathogenic mechanism cannot be studied with the rigor and comprehension otherwise expected, and the opportunities to compare therapeutic protocols are few. origin of the virus during the initial outbreak, liu et al. obtained samples of blood, bronchoalveolar lavage fluid, and anal and oral swabs from patients in wuhan, china suffering from a pneumonialike illness resembling the 2002-2003 sars-cov. a pan-cov pcr was performed on samples from five patients, and were positive for a pathogen from the coronaviridae family. this was compared with a full-length sequence of viral rna from a bat coronavirus (bat-covratg13), and demonstrated 96.2% similarity. thus, it is probable that the bat is the main reservoir of the novel coronavirus. identification of the intermediate host is an essential step in controlling the spread of disease, and became a priority for research teams. unfortunately, this was complicated by the many species of wild animals sold at the huanan seafood market, where the first cases were reported to have had contact. in 2019, a sars-cov-like pathogen known to be widely distributed in the malayan pangolin samples was discovered. the receptorbinding domain (rbd) present on the spike protein (s) is a crucial determinant in host range, as its interaction with the host receptor is responsible for the infection. rbd sequences from bat-covratg13, pangolin-sars-like cov and the novel sarslike pathogen were aligned. ninety three percentage similarity was demonstrated between the novel sars-like pathogen and the pangolin sars-like cov, and 89% similarity was demonstrated between the novel sars-like pathogen and the bat-covratg13. thus, on the basis of the rbd, the pangolin-sars-like cov is determined to be more likely than the bat-covratg13 to infect humans, making this the possible intermediate host (13) . xiao et al. conducted another study in which the pangolin-sars-like cov was isolated and amino acid sequence was compared to sars-cov-2. this yielded 100, 98.6, 97.8, and 90 .7% similarity with the s, m, e, and n proteins, respectively, of the novel sars-cov, strengthening the previous assumption that the pangolin was the intermediate host (14) . pathogenic classification is used to determine whether the pathogen is new or recurring in order to best implement safety and treatment protocols. while serological reactivity to viral proteins had been the mainstay of viral classification in the past, the process today now depends on replicated protein sequences. the international committee on taxonomy of viruses (ictv) maintains a study group for each viral family (15) . after analysis, the novel virus was assigned to the order nidovirales on the basis of the following domains: polyprotein protease (3clpro), catalytic domain of rna polymerase (rdrp), nidovirus-associated rdrp (niran), zinc binding domain (zbd), and helicase (hel1) (16) . subsequent next generation sequencing and phylogenic analysis placed the novel pathogen within the subgenus sarbecovirus of the genus betacoronavirus (17) ( table 2) . like other coronaviruses, the viral envelope is composed of a lipid bilayer derived from host cellular material, and the structural proteins are spike proteins composed of a trimeric glycoprotein projecting from the envelope like a crown. cryo-electron microscopy was used to compare the structural differences between the spike (s) proteins of sars-cov and sars-cov-2. unlike the previous sars-cov which possessed a single arginine targeted by a trypsin protease, sars-cov-2 possesses an s1/s2 protease cleavage site which is recognized by furin proteases. neuman et al. demonstrated the high level of protein organization and interaction within the envelope: the s protein was shown to be aligned with the npc, a crucial component of viral organization in protein-protein interactions that ensures high specificity and effectiveness for host invasion (19) . sars-cov-2 is an enveloped virus with a 29,904 bp positivesense non-segmented rna genome. the non-structural proteins (nsps) are described in table 3 . the remainder of the genome is responsible for accessory and structural proteins such as s, m, e and n. host cell recognition is the first and most essential step in viral pathogenesis. studies on the 2002-2003 sars-cov outbreak uncovered key interactions between the spike (s) protein, the rbd and angiotensin-converting enzyme 2 (ace-2). due to the previously mentioned similarities between sars-cov-2 and its predecessor, a viral infectivity study was performed using hela cells with and without the ace-2 receptor, which showed that only cells possessing the ace-2 receptor were infected (21) . the spike trimer is a class i fusion protein; upon infection the spike is cleaved by host proteases at the s1/s2 site for division of the two domains. the s1 domain possesses the rbd which recognizes and binds the ace-2 receptor in a prefusion state. structural rearrangement of the s protein subsequently exposes a furin cleavage site on the s2 domain which enables viral entry by means of fusion after the s1 domain is shed (22) . viral replication was hypothesized to occur via a process called autophagy: an evolutionary cellular process in which cytoplasmic proteins create isolation membranes surrounding materials destined for degradation (23) . evidence of coronavirus autophagy was first demonstrated by prentice et al., who showed that in coronavirus mouse hepatitis viruses, atg5-induced autophagy was required for the formation of double membrane vesicles (dmv) and for replication (23) . another study confirmed the use of atg5dependent autophagy of betacoronaviruses through nsp 6 (24). chen et al. previously showed that both sars-cov and mers-cov employ the use of papain-like proteases (plpro) to induce the formation of autophagosomes, but their fusion to lysosomes is inhibited which promotes the replication process (25) . however, other studies have demonstrated that key autophagy proteins atg5 or atg7 were not required for coronavirus mouse hepatitis or sars-cov viral replication (26) . reggiori et al. also yielded comparable results showing that the replication and release processes are not dependent on autophagy, but that the dmv's were coated with (lc3)-i (non-lipidated microtubule associated protein i light chain 3), thus showing the importance of this protein for viral replication (27) . a recent study by benveunto et