Time profiling molecular toxicity AgNP ISJ 10: 69-76, 2013 ISSN 1824-307X REVIEW Earthworm’s immunity in the nanomaterial world: new room, future challenges Y Hayashia,b ,P Engelmannc a Department of Bioscience - Terrestrial Ecology, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark b iNANO Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark c Department of Immunology and Biotechnology, Clinical Center, University of Pécs, Szigeti u. 12, Pécs H-7643, Hungary Accepted August 22, 2013 Abstract Since the advent of the nanotechnology era, the environmental sink has been continuously receiving engineered nanomaterials as well as their derivatives. Our current understanding of the potential impact of nanomaterials on invertebrate immunity is limited to only a handful of initial studies including those on earthworms. Recently, we reported selective accumulation of silver nanoparticles in the amoebocyte population of Eisenia fetida coelomocytes in vitro. In this review, we give an overview of available literature on the life-history impacts on earthworms, and what we have learnt of the immune responses to nanoparticles with references to other invertebrate species and vertebrate counterparts. We discuss the significant contribution of amoebocytes as nanoparticle scavengers and suggest a possibility of studying inter-cellular communications in coelomocytes. Implications from the leading researches in vertebrate models tell us that study of the nanoparticle recognition involved in cellular uptake as well as sub- and inter-cellular events may uncover further intriguing insights into earthworm’s immunity in the nanomaterial world. Key Words: nanomaterials, silver nanoparticles, immunogenicity, earthworms, coelomocytes, uptake   Introduction It was not until the advent of the nanotechnology era that earthworms were on the verge of encountering the “unknown” delivered by the modern human society. The current definition of a nanomaterial adopted by European Commission states “A natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or as an agglomerate and where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm - 100 nm”. Naturally occurring ultrafine particles of the same size range have existed long before the emergence of nanotechnology. Yet, already in 2005 the extensive review by Oberdörster and colleagues (2005) pointed out possible scenarios of engineered nanomaterials posing threats to ecosystem health. The major concern stemming from rapid development and commercialisation of nanotechnology products is uncertainty of such novel formulations in the modes of action and routes of exposure to living organisms. ___________________________________________________________________________ Corresponding author: Yuya Hayashi Aarhus University, the iNANO house Gustav Wieds Vej 14, 8000 Aarhus C, Denmark. E-mail: yuya@inano.au.dk The environmental sink has received, and is expected to continue receiving, commercially produced nanomaterials as well as their derivatives, environmental transformations and the fate of which have yet to be elucidated in particular in the soil milieus (reviewed in Tourinho et al., 2012). Immunity is a vital function to maintain organism’s well-being, and represents a sensitive physiological indicator that may be affected even at low concentrations of nanomaterials exposure. Only a handful of studies exist so far to aid the current understanding of immune responses to nanomaterials in invertebrates, particularly earthworms. This includes our recent in vitro study on Eisenia fetida exposed to silver nanoparticles (AgNPs) (Hayashi et al., 2012) supporting molecular responses observed in vivo (Hayashi et al., in press). Studies on other earthworm species have been reported by van der Ploeg and co-workers, where Lumbricus rubellus was exposed to the carbon-based nanoparticle C60 fullerene in vivo (2013) and in vitro (2012) and likewise exposed to AgNPs (unpublished), as well as partially by the work of Hooper et al. (2011) with E. veneta exposed to zinc oxide NPs. In this review we seek to recapitulate what has been learnt from the initial studies on the effects of 69   mailto:yuya@inano.au.dk nanoparticles (NPs) on earthworm immunity, with references to other invertebrate species and vertebrate counterparts. The upshot is that different types of immunocytes may respond to NPs in a distinct manner intimately linked to the cell’s ability, and that previously uncharacterised aspects of earthworm immunity are emerging in the light of the nanotechnology era. Sub-lethal impacts of nanomaterials on earthworms Limited numbers of studies are currently available in the literature on the impact of nanomaterials on earthworms. Carbon-based nanomaterials can affect the life-history traits of E. veneta (Scott-Fordsmand et al., 2008), E. fetida (Li and Alvarez, 2011) and L. rubellus (van der Ploeg et al., 2011). Common to all was reduced reproduction, which could result in a significant decrease in the population growth rate (van der Ploeg et al., 2011). C60 fullerenes are also suggested to bioaccumulate in earthworms (E. fetida) following soil exposure (Li et al., 2010), whereas carbon nanotubes did not accumulate in E. fetida (Petersen et al., 2008a) or L. variegatus (Petersen et al., 2008b) when depuration of the gut content was allowed. Ecotoxicological screening of metal-based nanomaterials indicated significant reproductive failure in E. fetida exposed to silver or copper NPs (Heckmann et al., 2011). Different types (size and coatings) of AgNPs disturb earthworm’s reproductive capacity at 500-1000 mg/kg soil in E. fetida (Heckmann et al., 2011; Shoults-Wilson et al., 2011c; 2011b), and in L. rubellus significant reproductive toxicity was observed at concentrations as low as 154 mg/kg soil when the AgNPs were well dispersed in soil (van der Ploeg et al., unpublished). Bioaccumulation of AgNPs in earthworms seems relatively low (Coutris et al., 2012; Shoults-Wilson et al., 2011c; 2011b; van der Ploeg et al., unpublished), however, more sensitive endpoints indicate that earthworm’s physiological traits are affected already at sub-100 mg/kg soil, for example, avoidance (Shoults-Wilson et al., 2011a), enzyme activities (Hu et al., 2012), oxidative stress responses (Tsyusko et al., 2012) and tissue apoptosis (Lapied et al., 2010). Other types of NPs (e.g. alumina, titania and zinc oxide) have also been tested on earthworms and reviewed elsewhere (Tourinho et al., 2012), and two types of NPs (copper and gold) were observed to biodistribute across the tissues, partly if not all, persisting their nanoparticulate forms (Unrine et al., 2010a; 2010b). Scarcely studied, however, is the potential immunological effect of nanomaterials; which leads us to limit our focus of the review on carbon-based NPs and AgNPs, where insightful observations were revisited in this context. Immunogenicity of nanomaterials Recognition, uptake and inflammation: cutting-edge studies in vertebrates Before remarking rather limited knowledge of the effects of nanomaterials on earthworm’s immunity, we begin by capturing potentially relevant prospects that arise from studies of mammalian cells. In general to all eukaryotic cellular machineries size/shape and surface chemistry of nanomaterials are the central parameters in the interaction with immune systems, for example, via biological ligand-receptor signalling (reviewed in Dobrovolskaia and McNeil, 2007). Within the past few years the concepts of dynamic assembly of NPs and biomolecules were established (Shemetov et al., 2012) and this has shed light on how the cells “see” and interact with NPs in the context of receptor-mediated responses (reviewed in Monopoli et al., 2012). Supporting this notion, evidence of NP uptake via conventional endocytic (e.g. Lesniak et al., 2013; Wang et al., in press) and phagocytic (e.g. Lunov et al., 2011) pathways is emerging. This has a direct implication to innate cellular immunity, which relies mainly on non-self pattern recognition and macromolecule-marking (opsonisation) of particles for phagocytic clearance. On the contrary, relatively less explored is the inflammatory potential of NPs as a result of direct receptor activation (e.g. Bastús et al., 2009). This is beautifully vindicated by the activation of integrin receptor signalling in THP-1 cells (a human acute monocytic leukemia cell line) through binding of fibrinogen unfolded upon interactions with NPs (Deng et al., 2011). Inflammatory responses can also be induced indirectly resulting from oxidative stress, a frequently described mode of action of NPs (e.g. Hayashi et al., 2012). AgNPs are a good example of nanomaterials that involve oxidative stress (Foldbjerg et al., 2012; Hayashi et al., 2012; Jiang et al., 2013) and modulate pro-inflammatory cytokines including a catalogue of interleukins and TNF-α (reviewed in Klippstein et al., 2010). Earthworm’s immune responses to nanomaterials The relative simplicity of invertebrate immunity offers a potentially sensitive and accessible means of disentangling the complex interactions of NPs and immune cells. To address this challenge, we have developed an in vitro model of E. fetida coelomocytes. In our recent report, we observed at the molecular level a cascade of stress responses initiating from oxidative stress genes to immune genes downstream following short-term exposure to AgNPs in coelomocytes and in THP-1 cells (Hayashi et al., 2012). A similar set of genes was affected with a temporal shift when the worms were exposed in vivo to the AgNPs in a soil matrix (Hayashi et al., in press). From evolutionary perspectives, a similarity between E. fetida coelomocytes and THP- 1 cells in the expression patterns of catalase (repressed over time), a well-known oxidative stress response gene, and myeloid differentiation factor 88 (MyD88, induced over time), which encodes a central adaptor protein of Toll-like receptors, was an intriguing observation that may comprise a part of immune responses to AgNPs in earthworms but also across the animal kingdom (Hayashi et al., 2012). Signal transduction, primarily through mitogen activated protein kinase (MAPK) pathways, appears to coordinate the cross-talk between oxidative stress and immune responses to AgNPs, as implicated in the expression profiles of MEK kinase 1 (MEKK1) gene both in coelomocytes and THP-1 cells (Hayashi et al., 2012), as well as in vivo 70   Fig. 1 Light (A) and scanning electron (B, C and D) micrographs of Eisenia fetida coelomocytes. The light micrograph was imaged at x200 magnification with a phase-contrast microscopy. The scanning electron micrographs were imaged on the coelomocytes after paraformaldehyde fixation and gold-sputtering (nominal 30 nm in thickness). From morphological observations, panels B and C appear to be amoebocyte populations while panel D is most likely a chloragocyte (characterised by the granule-rich feature of chloragosomes). in earthworms (Hayashi et al., in press). Phosphorylation states of MAPK families in earthworm coelomocytes were not examined, whereas in vitro studies of NPs using haemocytes from Mytilus species suggest rapid activation of MAPK cascade members, namely p38 and JNKs, with subsequent nitric oxide production (Canesi et al., 2008; 2010a). The authors further reported effects on other related traits, such as oxidative burst and lysozyme activity. Interestingly, these observations were made with the NP concentrations at which lysosomal membrane stability, a sensitive indicator of cell viability, was not affected. When the molluscs were exposed in vivo to NPs in an aquatic system, the digestive gland appeared as the likely target of NPs with signatures of oxidative stress and haemocyte damages (Canesi et al., 2010b). Similarly, haemocytes from freshwater mussels (Dreissena sp.) exposed in vivo to titania NPs accumulated the NPs intracellularly and showed reduced phagocytic activity as well as activation of ERK1/2 and p38 MAPK families (Couleau et al., 2012). van der Ploeg and colleagues (2013) exposed L. rubellus earthworms in vivo to C60 fullerenes for two different exposure durations (4-weeks and lifelong), and in both cases observed suppression of heat shock protein 70 (HSP70) gene while enzymes involved in antioxidant mechanisms were unaffected. Of particular interest is significant suppression of coelomic cytolytic factor 1 (CCF1) gene in the lifelong experiment (van der Ploeg et al., 2013); CCF1 is a known pattern recognition receptor in earthworm’s immunity (for review see Bilej et al., 2010). Tissue injuries without histological signatures of inflammation were observed both in 4 71   weeks- and, albeit less severely, lifelong-exposed worms in parallel with decreased CCF1 expression (van der Ploeg et al., 2013). On this basis, the authors suggested immunosuppressive effects of C60, rather than a result of coelomocyte mortality. This was supported by their in vitro work, in which coelomocytes from L. rubellus were viable at a wide range of C60 concentrations while CCF1 was down- regulated in concurrence with reduced phagocytic activity (van der Ploeg et al., 2012). Although these initial studies are only indicative of the extent to which the nanomaterials may interfere with the function of earthworm’s immune systems, early warnings are already given; that nanomaterials are a new class of environmental contaminants posing potential threats to earthworms. Dissecting earthworm’s innate immunity Earthworm immune system consists of cellular (coelomocytes) and humoral components (antimicrobial, cytolytic and pattern recognition molecules) directed towards non-self materials in a natural non-specific manner (reviewed in Cooper et al., 2002). Cytochemical, immunological and functional approaches characterised three major subpopulations (morphological observations by light and electron micrographs are shown in Fig. 1), among which hyaline and granular amoebocytes participate in the cellular effector mechanisms (e.g. phagocytosis and encapsulation) while chloragocytes (eleocytes) contribute more to homeostasis and humoral immunity (Engelmann et al., 2004; 2005; Opper et al., 2010). Fig. 2 illustrates the potential room for future challenges in facing the nanotechnology era, supported with evidence in earthworms or other multicellular organisms. Amoebocytes as the scavenger of nanomaterials Coelomocytes of L. rubellus, largely lacking free-circulating chloragocytes (Cholewa et al., 2006), proved capable of internalising polymeric NPs (hydrodynamic diameter of 45 ± 5 nm) apparently involving energy-dependent transport mechanisms (clathrin- and caveolin-mediated endocytosis pathways) (van der Ploeg et al., 2012). Our recent study suggests that the hyaline sub- group of amoebocytes and PMA-differentiated macrophage-like THP-1 cells, but not the monocytic phenotype of THP-1 cells, can accumulate AgNPs of the primary particle sizes around 83 ± 22 nm (Hayashi et al., 2012). Amongst the coelomocyte populations, the hyaline amoebocytes are known to adhere and engulf bacteria (Engelmann et al., 2005), and may thus be considered as the invertebrate counterpart of macrophages. Although NP uptake mechanisms are largely unknown in coelomocytes, the macrophage-like THP-1 cells appear to effect macropinocytosis for the uptake of negatively-charged NPs (Lunov et al., 2011). In mammals, macropinocytosis initiates with cell membrane ruffling via actin rearrangement, suggesting an intriguing possibility of passive uptake of NPs that are membrane-adhered (Fig. 2). Amongst invertebrates, ascidian haemocytes are able to engulf particles via a RGD motif-dependent macropinocytosis (Ballarin and Burighel, 2006), however, such mechanisms are not yet known in earthworms. Another potential phagocytic pathway is via scavenger receptor class A that is expressed by both human macrophages and macrophage-like THP-1 cells, but not by monocytic THP-1 cells (Lunov et al., 2011). Scavenger receptors are conserved pattern recognition receptors known to bind lipids (lipopolysaccharides and modified low- density lipoproteins) and polyanions for phagocytosis. In particular, a macrophage receptor with collagenous structure (MARCO) is known to recognise and associate with NPs for phagocytic clearance in mammalian cells (Kanno et al., 2007). In invertebrates, haemocytes from insects (Franc et al., 1996) and molluscs (Liu et al., 2011) are known to effect scavenger receptor-mediated uptake of pathogens and apoptotic cells. To date, scavenger receptors are yet to be identified in earthworms; however, their ubiquitous presence suggests an unequivocally conserved role in innate immune recognition that may be involved in NP uptake as in vertebrate counterparts (Fig. 2). Toxicological implications arising from selective cellular uptake of nanomaterials are profound. Of metal-based nanomaterials that readily dissolve and liberate bioactive metal ions, AgNPs represent the most well-studied type of NPs (e.g. Liu et al., 2010). Free Ag+ ions, the product of oxidative dissolution, itself is highly biologically active and reacts with biomolecules (e.g. proteins and DNA) of the cellular components in a similar manner as reactive oxygen species (ROS). AgNPs and Ag+ ions co-exist extracellularly and/or intracellularly, indicating a multitude of stress pathways not limited to those for the nanoparticulate form but including contribution of liquid-phase silver (e.g. Beer et al., 2012; Yang et al., 2011). Intracellular uptake of AgNPs is likely to involve subsequent fusion with lysosomes that may accelerate oxidative dissolution of AgNPs under the acidic milieu (Jiang et al., 2013). This implies that AgNPs may have a targeted impact on amoebocytes as a result of preferential accumulation and subsequent in situ molecular damages by liberated Ag+ ions (Hayashi et al., 2012; see Fig. 2). Time-course profiling of representative gene expressions, in parallel with flow-cytometric analysis of the intracellular ROS level, favour the view that the amoebocyte populations are under oxidative stress that can signal-transduce to immune cascades downstream (Hayashi et al., 2012; and Fig. 2). Recently, the amoebocyte populations, but not chloragocytes, were found to recruit calcium for activation (e.g. Homa et al., 2013) and that they may possess a similar biochemistry of calcium signalling as in higher organisms linking stress responses to activation of immune systems (Opper et al., 2010). Studies on how AgNPs affect calcium signalling in amoebocytes may further illuminate the cross-talk between stress and immune responses as known for another highly-conserved signal transduction cascade, MAPK pathways (Fig. 2). 72   Fig. 2. Schematic illustrating the room and future challenges to progress further in our understanding of the effects of nanomaterials on earthworm’s immunity. See the main text for references to each heading. Headings with a question mark show subjects that are relatively less understood in coelomocytes and in vertebrates. Drawings are not to scale. Me+; metal ions, ROS; Reactive oxygen species. Inter-cellular communications: an indirect effect? Phagocytes secrete cytokines as a biological means of cellular communication to initiate e.g. inflammation but also other immunological functions, such as acute phase responses by liver cells. As described earlier in this review, cytokine secretion/modulation is a documented effect of NPs while much less is known for secondary impacts related to altered cytokine profiles (Fig. 2). Direct evidence, however, has not been discovered for conserved and novel cytokines in earthworms. In contrast, other invertebrate organisms (especially insects) have already provided sufficient data for cytokine-mediated immune functions (e.g. haematopoiesis) (Malagoli, 2010; Malagoli et al., 2012; Söderhäll et al., 2005). The conservation and existence of proinflammatory cytokines in earthworms are not a fairy-tale; earlier we observed positive reactions of coelomocytes to monoclonal antibodies targeted for mammalian TNF-α (Engelmann et al., 2002), and recently the work of Fuller-Espie and colleagues (2008) supports enhanced phagocytic activity of hyaline amoebocytes treated with mammalian cytokines (notably IL-1β, IL-2 and TNF-α). The coelomic fluid of earthworms is sometimes assumed in the immunological context equivalent to blood plasma in mammals, both representing a protein-rich immune-competent circulatory system. Distinct from the mammalian counterpart is the existence of (migratory and sessile) chloragocytes involved in the regulation of essential minerals, haemoglobins and metallothioneins in response to natural stressors (Molnár et al., 2012). This is probably by functional analogy with the hepatic/renal systems of vertebrates, and chloragocytes may contribute to regulation of the total protein balance in the coelomic fluid. For example in echinoderm, immunostimulants and invertebrate cytokines were able to regulate secretion of acute phase proteins from coelomocytes (Beck et al., 2002). Its relation to immunostimulation remains unclear, but in Eisenia earthworms the expression and secretion of the cytolytic/antimicrobial molecule lysenin was increased in migratory chloragocytes upon Gram- 73   positive bacterial challenge of total coelomocytes (Opper et al., 2013). Given that these characteristics partly resemble those of vertebrate hepatic cells, acute phase response in coelomocytes may await further investigations using nanomaterials as a tool to selectively target amoebocytes and modulate cytokine profiles. This also suggests an interesting feature of using coelomocytes as a mixed- population model integrating the cell-to-cell signalling events (especially of immune effector cells to hepatic-like cells) that is otherwise difficult to study in vertebrate in vitro models of monocultured cell lines. We also note that secretion of humoral factors and deposition of chloragosomes (specialised lysosome-like structures of chloragocytes) may lead to dynamic interactions of NPs and these biomolecules (Fig. 2). Understanding immune responses to nanomaterials: the challenges In the light of our current understanding in nanomaterials and their immunogenicity, we have in this review given an account for a summary of available literature on the life-history impacts of nanomaterials on earthworms, and what we have learnt of the immune responses to nanomaterials. The phagocytic population of the coelomocytes, namely hyaline amoebocytes, seems a susceptible target of nanomaterials, as a result of which indirect responses by chloragocytes are conceivable. Environmental toxicants (such as pesticides and heavy metals) compromise the host’s immune system. These pollutants inhibit the cellular immune functions (e.g. phagocytosis) while humoral components (e.g. lysozyme production) are elevated. At the dawn of knowledge about the impact of nanomaterials on invertebrate immunity, it is still obscured whether they influence cellular and/or humoral arms of immunity in a fashion that has not been previously documented. We have discussed a significant contribution of amoebocytes as NP scavengers and proposed a possibility of studying inter-cellular communications in coelomocytes. Implications from the leading researches in vertebrate models tell us that study of the NP recognition involved in cellular uptake as well as sub- and inter-cellular events may uncover further intriguing insights into earthworm’s immunity in the nanomaterial world. Acknowledgements We are grateful to three anonymous reviewers for their valuable comments on the manuscript. We acknowledge the financial support of the Danish Research Council (FUU 1-5971-10000377) to YH, and of the Medical Faculty Research Foundation, University of Pécs (PTE ÁOK-KA 2013/09) as well as Berde Botond visiting fellowship to PE. 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