338 ISJ 15: 338-345, 2018 ISSN 1824-307X SHORT COMMUNICATION Distinct immune- and defense-related molecular fingerprints in sepatated coelomocyte subsets of Eisenia andrei earthworms K Bodó1, D Ernszt2, P Németh1, P Engelmann1,* 1Department of Immunology and Biotechnology, Clinical Center, Medical School, University of Pécs 2Department of Physiology, Medical School, University of Pécs Accepted September 4, 2018 Abstract During phylogenesis different types of immunocytes such as amoebocytes and eleocytes have developed in earthworms to defend the host against microbial pathogens. Previously we applied a cell sorting-based approach to untangle the morphological and functional properties of these aforementioned coelomocyte subsets. In order to compare their constitutive gene expression patterns, cell-sorting was performed and followed by semiquantitative RT-PCR in the distinct, separated coelomocyte subpopulations of unmanipulated Eisenia andrei earthworms. We targeted a variety of genes with diverse functions ranging from pattern recognition through intracellular signaling to oxidative stress. Several immune-related genes (CCF, TLR, lumbricin, LuRP, MyD88) were only manifested in the amoebocytes. In contrast, other immune response genes (lysozyme, lysenin), lysosomal hydrolases (cathepsin L and cathepsin C) and cystatin B were expressed in both subpopulations. In addition, cell signaling molecules (MyD88, PKC1) and oxidative stress-related genes (Cu/ZnSOD, MnSOD) were mainly observed in amoebocytes, while other stress-related genes (Cd-metallothionein, catalase) were apparent in both subsets. We conclude that these characteristic differences of the molecular signatures manifest in the functional heterogeneity of distinct coelomocyte subtypes. Key Words: Eisenia andrei, coelomocytes, cell sorting, gene expression, immune response, oxidative stress Introduction Evolutionary conserved immune mechanisms are reported from diverse invertebrate organisms (Loker et al., 2004). A surprising complexity and close cooperation between cellular and humoral immune components can be observed in several invertebrate models including earthworms (Cooper et al., 2002; Cooper and Roch 2003; Bilej et al., 2010; Engelmann et al., 2016b). Earthworm coelomocytes are divided into amoebocyte and eleocyte subpopulations. Similarly to other invertebrate immunocytes, these cells are derived from the mesoderm (eleocytes are considered to be originated from the gut surface- located, liver equivalent chloragogenous tissue), and possess various functions during the immune response (Engelmann et al., 2005). In this regard, ___________________________________________________________________________ Corresponding author: Péter Engelmann Department of Immunology and Biotechnology Clinical Center, Medical School, University of Pécs Pécs, H-7643, Szigeti u. 12, Hungary E-mail: engelmann.peter@pte.hu amoebocytes are mainly involved in the phagocytosis and encapsulation (Fuller-Espie, 2010), while eleocytes have no phagocytic properties, but they produce a handful of bioactive molecules (Stein et al., 1977; Valembois et al., 1985). Recently we applied a cell-sorting-based approach to separate these distinct coelomocyte subsets upon their light-scatter properties (from the perspective of physical parameters; size and granularity). After separation we characterized their differences in morphological, cytochemical, functional and lectin-binding properties (Engelmann et al., 2016a). In the last two decades several immune proteins have been identified in earthworms; however, little is known about their differential gene expression in the coelomocyte subgroups (Bilej et al., 2010; Engelmann et al., 2016b). Aware of the phenotypic and functional differences in the coelomocyte subsets, we aimed to analyze the distinct expression patterns of several immune and stress-related target genes in the separated amoebocytes and eleocytes. mailto:engelmann.peter@pte.hu 339 Table 1 List of primers were applied for semi-quantitative RT-PCR experiments Target Gene Gene Bank accession # Sequence (5'-3')a Amplicon size (bp) TLR JX898685 ATT GTG TCA AAC GCC TTC GC 123 GTC GGC GAT CTC TTC CAA CA CCF AF030028 CAT TAA GCC GAC GTT GCT GG 145 CGT CCT GTA GCA TCC GTT GT LBP/BPI JQ407018 GGT TCG ACC TCC GAC GAT AC 107 GGT CAA CAG GGC GTC CAT TA Lysozyme DQ339138 GTC GCA TGG ATG TCG GAT CT 120 GCG AGC AGT CCA TCT GAG TT Lumbricin KX816866 ACT CGG AAC GCA AGA ACC AA 139 GGT TCT GCG TGA CCT CCT TC LuRP KX816867 GGT CGA GAG AAT CAA CCC AAC TA 133 CTT GTG AGC GAT GTC GGC TA Lysenin D85846 TGA TCC ACA CTG GTG CTT CC 117 CAG GTG CCA AGG AGA AGA AG MyD88 EH670202 TGC GAG TAC AGG CTC GTT AAC 100 CGT GCA GAT GTG GTT TAG GA MEKK 1 EH672240 CAA GGA ACG ATC CCA TTC AT 147 GTA TCA TGG TGC AAC CAA CG PKC1 DQ286716 TTT TAT GCG GCC GAA GTC A 120 GTC GGC GAT TTT GCA GTG A Mt AJ236886 CTT GTT GCT GCA CAA ACT GC 129 TTT CCA CAT TTG CCC TTC TC Catalase DQ286713 TAC AAA CTG GTG AAC GCC GA 139 AAA GGT CAC GGG TCG CAT AG Cu/ZnSOD KR106132 TGC CAA GTT TGA AGT GAC GG 103 TTT GCC AAG ATC GTC CAC CA MnSOD KU057379 CCG AAG AAA AGC TGG CTG AA 91 TGT CCT CCG CCG TTG AAT Cystatin B BP524680 TGG AGG GGA TGC TTT GCA TT 123 ACG CAG ACA AGG TAC GAA GA Cathepsin B HO001247 TCC TGC CTT TCC AGA TTC ATT T 90 GAA CCA CAG GAG CCC TGA TC Cathepsin C GR228740 CGG CTA CTT CCG CAT CGT T 120 AGC GCC TGC TCA GAA GGA Cathepsin L EY892565 CAA CGG CTG TTT CCT ATC CAA 110 GAA AAC ACA CGA TGC AAT GCA COI HQ534065 GGA TTT GGA AAC TGA CTT C 312 TCG TTC TAG TCG AAG CCC AC 18S AY365460 ATT AAG CCA TGC ATG TCT AAG CAC 135 CTT TGT GGC ATG TAT TAG CTC CAG RPL17 BB998250 GCA GAA TTC AAG GGA CTG GA 159 CTC CTT CTC GGA CAG GAT GA β-actin JQ038870 ATG TGG ATC AGC AAG CAG GAG TA 90 ATC GCC GAG ATC GGA ATC TT aUpper and lower sequences represent forward and reverse primers Materials and methods Earthworm husbandry Adult Eisenia andrei earthworms were maintained in breeding stocks at standard conditions (Molnár et al., 2012). Prior to coelomocyte harvesting, earthworms were placed onto moist tissue paper allowing defecation to avoid contamination during coelomocyte collections. Coelomocyte harvesting Coelomocytes were isolated as we described earlier (Engelmann et al., 2004) and enumerated by 0.14% trypan-blue dye-based exclusion. Cell sorting and flow cytometry Collected coelomocytes were resuspended in Lumbricus balanced salt solution (LBSS) (Engelmann et al., 2005) supplemented with 1% fetal bovine serum (FBS, Biowest, Nuaillé, France) and 5 mM EDTA (Sigma-Aldrich, Hungary) to prevent cell aggregation. Coelomocytes were sorted according to their basic forward and side scatter (FSC/SSC) characteristics reflecting their cell size and granularity, respectively. Sorting procedure was performed by a FACSAria III (BD Biosciences) cell sorter as we described earlier (Engelmann et al., 2016a). The efficacy of sorting and the coelomocyte viability was controlled by 7-amino-actinomycin D (7-AAD) using a FACSCalibur flow cytometer. 340 Hematoxylin-eosin staining Sorted coelomocyte subsets (80 µl of 5 x 105/ml) were spread onto glass sides using Cytospin 3 (SHANDON, ThermoScientific, Waltham, MA, USA) apparatus. Hematoxylin-eosin staining was employed following standard protocols. RNA isolation, cDNA synthesis and semiquantitative RT-PCR Total RNA was isolated from unseparated coelomocyte, sorted amoebocyte and eleocyte samples using NucleoSpin® RNA isolation kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s protocol. The quality and quantity of RNA samples were measured by NanoDrop 1000 spectrophotometer (ThermoScientific). Following DNAse I digestion (Sigma-Aldrich) the reverse transcription reaction was performed by Hi-Capacity Reverse Transcription Kit applying random hexamers (ThermoScientific). DNAse I-treated total RNA was reverse transcribed and subsequently used in the PCR reactions. Gene specific primers were designed based on the available sequences from NCBI GenBank Database and their major characteristics are detailed in Table 1. The following PCR conditions were applied: an initial denaturation step at 95 oC for 10 min, followed by 35 cycles of denaturation at 95 oC for 30 s, annealing at 54 oC for 30 s, and elongation at 72 oC for 30 s. Amplification cycles were terminated by a final extension at 72 oC at 10 min. Finally, PCR mixtures were analyzed on 1% (w/v) agarose gel, and PCR products were visualized by GelRed (Biotium, Inc., Fremont, CA, USA) dye. Gel pictures were photographed by GelDoc XR system (BioRad, Hercules, CA, USA). Results and Discussion Pattern recognition receptors (PRRs) are attributed to separated amoebocytes To analyze the distinct gene expression patterns we separated the coelomocyte subpopulations based on their physical manifestation (size and granularity) (Fig. 1a). Post- sort cell viability measurements (7-AAD staining) indicated a high survival rate (82-85%) for sorted amoebocytes, while we were not able to evaluate the ratio of alive/dead eleocyte due to their high autofluorescence. Hematoxylin and eosin staining was performed to check the efficacy of the sorting process. Majority of the sorted population was composed by hyaline amoebocytes (Fig. 1b), whereas a small percentage of granular amoebocytes appeared as well. Separated eleocytes (Fig. 1c) were easily perceptible by their small nucleus and the cytoplasm filled with chloragosomes. Fig. 1 Pre- and post-sorting analyses of coelomocyte subsets and cytochemical properties of sorted populations. Total coelomocyte population (a) was separated to amoebocyte (b) and eleocyte (c) subpopulations upon their physical manifestations. Post-sort analyses demonstrated that amoebocytes were mainly undamaged; however, eleocytes showed a certain fragility evidenced by the increased amount of debris. Hematoxilin-eosin staining revealed mixed total coelomocytes prior to sorting while homogenous amoebocytes (b) and eleocyte subpopulations (c) can be observed following the separation. Scale bars: 100 µm. Representative dot-plots are presented from three independent experiments. 341 Innate immunity operates with a panel of PRRs to discriminate between non-self and self structures. Recently, some unique and evolutionary conserved PRR molecules have been identified in E. andrei earthworms (Bilej et al., 2010; Engelmann et al., 2016b). First, we investigated the expression of pattern recognition receptor (PRR) genes including coelomic cytolytic factor (CCF), toll-like receptor (TLR), and LPS-binding protein/bacterial permeability-increasing protein (LBP/BPI). CCF is an unique LPS, peptidoglycan and β-1,3- glucan/N,N’-diacetylchitobiose-binding protein that is expressed at higher level in the chloragogenous tissue and lower level in large coelomocytes (Beschin et al., 1998). In the case of coelomocyte subsets, we found that CCF was only expressed in separated amoebocyte subpopulation, but it was not present in sorted eleocytes (Fig. 2a). First evidence of annelid TLRs was emerged from the analyses of polychaete and hirudean species (Davidson et al., 2008; Cuviller-Hot et al., 2011). Shortly, the coding sequence of TLR was identified in E. andrei (EaTLR). EaTLR expression level was relatively low in coelomocytes (Škanta et al., 2013) and -according to our observation- only occurred in amoebocytes (Fig. 2a), however its presence is not restricted exclusively to immunocompetent tissues (Škanta et al., 2013; Engelmann et al., 2016b). Further genomic investigations have shown the high diversity of TLRs in annelid earthworms (Fjøsne et al., 2015). Recently, a homologue of evolutionarily conserved LBP/BPI molecule was isolated from E. andrei. The highest expression level of EaLBP/BPI was observed in coelomocytes, seminal vesicles, while the lowest level appeared in the intestine (Škanta et al., 2016). We observed that LBP/BPI expression only occurred in amoebocytes, but not in eleocytes (Fig. 2a). These findings confirmed the notion that molecular recognition of pathogens is dedicated mainly to the amoebocyte subpopulation. In comparison to the earlier functional studies (Valembois et al., 1985; Valembois and Lasségues, 1995), our results support the concept that amoebocyte subpopulation is priorly involved in the pathogen-triggered phagocytic response. Distinct antimicrobial molecular fingerprints of coelomocyte subsets Nowadays, antimicrobial proteins (AMPs) are recognized as the first line of defense against microbial pathogens (Boman, 1991; Zasloff, 2002). Lysozyme is a highly conserved AMP present in many different organisms ranging from plants to human. Previous investigations have revealed that E. andrei lysozyme represents strong sequence similarity with other invertebrate lysozymes (Josková et al., 2009). Furthermore its expression is increased in coelomocytes upon Gram-positive and Gram-negative bacteria exposure. We found that both separated coelomocyte subpopulation expressed this AMP, however we observed a more augmented lysozyme expression in separated amoebocytes compared to eleocytes (Fig. 2b). Lumbricin is a distinctive earthworm AMP that was initially isolated from Lumbricus rubellus. So far several additional lumbricin homologues have been described from other annelid species (Cho et al., 1998; Wang et al., 2003; Schikorski et al., 2008; Li et al., 2011). Its expression was observed in several tissues, but not in coelomocytes (Li et al., 2011). Recently, we have identified the coding sequence of lumbricin and its novel-related peptide (LuRP) in E. andrei. Lumbricin and LuRP show close relationship with other lumbricin homologues (Bodó et al., 2019). Additionally, we observed that lumbricin and LuRP exert ubiquitous expression in several earthworm tissues (including coelomocytes), but their highest expression was evidenced in the foregut. Among other tissues Lumbricin and LuRP expression was the lowest in coelomocytes. As for their distribution in the subpopulations we observed that only amoebocyte subpopulation turned out to be weakly positive, but eleocytes were negative for these genes (Fig. 2b). A species-specific bioactive molecule lysenin has been described from Eisenia earthworms. Lysenin protein family consists of sphingomyelin- (and phosphocholine)-binding molecules, and possess diverse biological activities (cytotoxicity, antimicrobial activity, and opsonization) (Engelmann et al., 2016a). Our immunohistochemical analysis showed that mostly eleocytes were the lysenin expressing cells (Opper et al., 2013), in contrast to previous observations where chloragocytes were suggested as the major players in lysenin production (Ohta et al., 2000). Lysenin expression was manifested in both subpopulations (Fig. 2b) that is concordant with our previous flow cytometry- based observations (Opper et al., 2013). Conserved signaling molecules in coelomocyte subsets Our knowledge is very limited concerning on the intracellular signaling in earthworm immunity (Engelmann et al., 2011). We gained recent knowledge of different signaling pathways and we tested their expression pattern in the separated coelomocyte subsets. Certain signaling pathways such as MAPK cascade are fundamental and evolutionarily conserved; since several publications are available from various organisms (Sakaguchi et al., 2004; Ragab et al., 2011). Hayashi et al., 2012 observed down-regulation of MEKK1 level in silver nanoparticle (AgNP)-exposed Eisenia coelomocytes. We measured that MEKK1 expression is present in the isolated amoebocytes, besides we found a very weak signal in the eleocyte population (Fig. 2c). Innate immunity is largely dependent on the engagement of TLRs. Following PAMP recognition intracellular molecular events are initiated by the cytosolic components of TLR signaling pathway, one such is the MyD88. Hayashi et al., 2012 have observed a delayed induction of MyD88 gene in coelomocytes upon AgNP exposure. MyD88 was only expressed in the amoebocyte subpopulation that is relevant with the amoebocyte-restricted TLR expression (Fig. 2c). Protein kinase C (PKC) has fundamental functions in cellular homeostasis, and its role is well implicated in the immune response (Larsen et al., 2002). Earthworm PKC1 and PKC2 were recently 342 Fig. 2 Expression patterns of (a) pattern recognition receptors; PRRs, (b) antimicrobial peptides AMP, (c) signaling pathway genes, (d) metal-, oxidative stress-induced molecules, (e) hydrolytic proteases, and (f) housekeeping genes in coelomocytes of Eisenia andrei. (a) TLR, CCF, and LBP/BPI; (b) Lysozyme, Lumbricin, LuRP and Lysenin; (c) MEKK 1, MyD88, and PKC1; (d) Mt, Cat, Cu/ZnSOD and MnSOD; (e) Cyst B, Cath B, Cath C, and Cath L; (f) COI, 18S, 7 RPL17 and β-actin. Total coelomocytes (T), and separated eleocytes (E) and amoebocytes (A), with H2O as PCR negative control. Representative images are presented from three independent experiments. partially cloned (Brulle et al., 2006). Homa et al., (2013) found that a phorbol ester (PMA), a potent activator of PKC caused the proliferation of earthworm coelomocytes. Interestingly, in the course of in vitro PMA administration we observed that PKC is not involved in the Ca2+-dependent activation of coelomocytes (Opper et al., 2010; Engelmann et al., 2011). Among coelomocyte subgroups only the separated amoebocytes evidenced the expression of PKC (Fig. 2c). In fact, the amoebocyte subpopulation has a crucial role in the pathogen recognition, and in the downstream inflammatory response evidenced by the selective expression of signal transduction molecules and antimicrobial factors. Expression of oxidative, metal stress genes in separated coelomocyte subsets In addition to immune response-related genes, we examined the expression patterns of other defense-related genes involved in metal- and oxidative stress. Metallothioneins (Mt) are intensively studied metal-sequestrating proteins and ubiquitously expressed in a wide variety of organisms including earthworms (Calisi et al., 2014; Kowald et al., 2016). In recent years, earthworms are frequently applied as a sentinel organism to evaluate metal contaminations in soil (Calisi et al., 2014). Homa et al., (2005) previously described that E. fetida coelomocytes are able to accumulate various metal ions. Earlier results demonstrated that 343 Mt expression is mostly attributed to chloragocytes (Morgan et al., 2004); however we found that Mt manifestation occurred in both separated amoebocyte and eleocyte (free-floating chloragocyte) subpopulations. In contrast to the previous data we observed weak signal in eleocytes, while amoebocytes had a stronger Mt expression (Fig. 2d). All living organisms possess a diversity of antioxidant defense mechanisms (Wang et al., 2015). Catalase (Cat) is one conserved key enzyme of oxidative stress, and it exists in many different cell types including earthworm coelomocytes (Brulle et al., 2006). Cat expression was observed in both sorted coelomocyte subpopulations (Fig. 2d). Amoebocytes are involved in the early immune response against pathogens, but probably both subpopulations participate in maintaining the normal cellular homeostasis. Another oxidative stress-related enzyme, Cu/Zn-SOD has been recently cloned and characterized in E. fetida (Xiong et al., 2012). The predicted amino acid sequence was excavated that genetic distance of Cu/Zn-SOD in E. fetida was far from other invertebrate SOD molecules. Indeed, it showed strong sequence similarity with homologue sequences from Tubifex tubifex and L. rubellus (Xiong et al., 2012). In addition, the sequence of MnSOD has been recently assessed in E. andrei (Roubalová et al., 2018). Interestingly, its role in innate immune responses now has been elucidated (Wang et al., 2015). In contrast to Mt and Cat expression Cu/Zn-SOD and MnSOD were only manifested in separated amoebocyte subpopulation (Fig. 2d). Indeed, following the phagocytosis the intracellular “killing” in amoebocytes is mediated by the free reactive oxygen (and nitrogen) species that needs to be terminated by certain antioxidative enzymes (e.g. Cat, SOD). Hydrolytic endopeptidases and their inhibitor are present in coelomocyte subsets Cathepsins (Cath) are ubiquitous lysosomal proteases involved in many aspects of the cell life cycle. Cath B, Cath L and Cath C have been identified in several invertebrate organisms. Undoubtedly, Cath B is one of the most typical member of this molecular family that takes essential part in the immune response against bacterial infections (Balaji et al., 2002). Cath B is involved in the regulation of apoptosis, and this lysosomal protease is implicated to be involved in the immune mechanisms of the echinoderm, Apostichopus japonicus (Chen et al., 2016). Cath L is also identified in several invertebrates including the leech Theromyzon tessulatum. Cath L was involved in the phagocytic responses of leeches (Lefebvre et al., 2008). Cystatin B (Cyst B) is an endogenous cathepsin inhibitor, which localization was observed in the cytosol, mitochondria and nucleus (Kopitar- Jerala, 2015). Previously, cystatin B gene was described in the leech T. tessulatum, and upregulated after bacterial challenge (Lefebvre et al., 2004). Interestingly, leech coelomocytes possess a differential expression of cathepsin L (in chloragocytes and amoebocytes) and cystatin B (only in chloragocytes) (Lefebvre et al., 2008). According to our results all of the observed cathepsins (Cath B, C and L) were present in the sorted amoebocyte subpopulation, while only Cath C is absent from the sorted eleocytes (Fig. 2e). Interestingly, Cath C expression evidenced a low expression in total coelomocytes, while it appeared relatively higher in isolated amoebocytes. The inhibitor of these lysosomal proteases, Cyst B was expressed in both coelomocyte subpopulations (Fig. 2e). Their immune function in E. andrei is still unknown; however we cannot rule out the option that these proteases might be the possible regulators of lysenin-mediated cell lysis and also involved in the phagocytic machinery (Engelmann et al., 2016a). We have chosen four „housekeeping” genes to prove the intact RNA quality of the sorted coelomocyte subsets. All of tested genes including COI, 18S, RPL17 and β-actin expressed in both populations, however RPL17 and β-actin genes showed a lower level in eleocytes (Fig. 2f). Conclusions Taken together, hereby we report initially the differential expression patterns of immune and defense-related genes in sorted coelomocyte subsets of E. andrei. Our results verify the previously observed cytochemical, immunological and functional differences of coelomocyte subsets at the molecular level. Accordingly, amoebocytes are the main effector cells participating in pathogen recognition, and elimination. Eleocyte subpopulation is mainly involved in the stress response and production of bioactive molecules. These results provide fine details about the substantial molecular functions of separated coelomocyte subsets. Acknowledgement We acknowledge the financial support of Medical School Research Foundation (PTE-ÁOK- KA 2017/04), University of Pécs, the GINOP-232- 15-2016-00050 and EFOP-361-16-2016-00004 grants. P.E. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. The present scientific contribution is dedicated to the 650th anniversary of the foundation of the University of Pécs, Hungary. References Balaji KN, Schaschke N, Machleidt W, Catalfamo M, Henkart PA. Surface cathepsin B protects cytotoxic lymphocytes from self-destruction after degranulation. J. Exp. Med. 196: 493-503, 2002. Beschin A, Bilej M, Hanssens F, Raymakers J, Van Dyck E, Revets H, et al. Identification and cloning of a glucan-and lipopolysaccharide- binding protein from Eisenia foetida earthworm involved in the activation of prophenoloxidase cascade. J. Biol. Chem. 273: 24948-24954, 1998. Bilej M, Procházková P, Silerová M, Josková R. Earthworm immunity. Adv. Exp. Med. 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