E. MULTILOCULARIS METACESTODE METABOLITES

The E. multilocularis metacestode actively secretes or expresses molecules that putatively have potent effects on the immune system of the murine host. The production of these molecules and their chemical compositions might depend on the stage of the parasite (oncosphere, early vesicle or fully mature metacestode). However, little is known about biological effector molecules arising metabolically or somatically from the intrahepatic stages of the metacestode, although various E. multilocularis antigens, their epitopes and respective genes have been characterized. Among the main antigens described, a major carbohydrate named Em2 (G11) localizes on the surface of the laminated layer of the metacestode (Gottstein et al. Reference Gottstein, Dai, Walker, Stettler, Muller and Hemphill1992). Another polysaccharide-containing antigen C has been isolated and characterized from crude metacestode extract (Sato and Furuya, Reference Sato and Furuya1994). Similar investigations have yielded the finding of EmP2 (Ingold et al. Reference Ingold, Gottstein and Hemphill1998), a high molecular mass glycan as a part of the major structural elements of the laminated layer (Ingold et al. Reference Ingold, Gottstein and Hemphill2000). Biological activity of a high molecular mass carbohydrate molecule, called Em492, was demonstrated by Walker et al. (Reference Walker, Baz, Dematteis, Stettler, Gottstein, Schaller and Hemphill2004), from which they concluded that the Em492-antigen could be one of the factors contributing to immunosuppressive events that occur at the host-parasite interface. Another neutral glycosphingolipid has been identified as suppressor of human PBMCs proliferation following stimulation by phytohemagglutinin (Persat et al. Reference Persat, Bouhours, Mojon and Petavy1992, Reference Persat, Vincent, Schmitt and Mojon1996). Structure determination of this glycosphingolipid fraction had revealed that it belongs to the neogala series (Galβ1→6Gal). Huelsmeier and co-workers (Reference Huelsmeier, Gehrig, Geyer, Sack, Gottstein, Deplazes and Kohler2002) had then isolated novel mucin-type glycoforms from the metacestode of E. multilocularis, and these glycoforms contained mucin-type core-I type and core-II type structures that were further diversified by addition of GlcNAc or Gal residues. Recently, Koizumi et al. (Reference Koizumi, Hada, Kaburaki, Yamano, Schweizer and Takeda2009) reported on the synthesis of the glycan portions of a glycoprotein antigen of E. multilocularis in order to elucidate the interactions between oligosaccharides and sera of AE by enzyme-linked immunosorbent assay (ELISA). Stereocontrolled synthesis of branched tri-, tetra-, and pentasaccharides displaying a Galβ1→3GalNAc core in the glycan portion of the glycoprotein antigen was achieved, which may become an interesting tool for further studies on their putative biological function.

With regard to metabolized proteins, an E. multilocularis protoscolex-associated antigen of 62 kDa (Auer et al. Reference Auer, Hermentin and Aspöck1988), two 70 and 90 kDa proteins (Korkmaz et al. Reference Korkmaz, Inceboz, Celebi, Babaoglu and Uner2004), and several recombinant E. multilocularis-proteins (such as antigen II/3 (Vogel et al. Reference Vogel, Gottstein, Müller and Seebeck1988) and its subfragments II/3-10 (Müller et al. Reference Müller, Gottstein, Vogel, Flury and Seebeck1989) and Em18 (Ito et al. Reference Ito, Schantz and Wilson1995), EM10 (Frosch et al. Reference Frosch, Frosch, Pfister, Schaad and Bitter-Suermann1991)), have all been published and discussed in view of a potential biological role. However, these antigens were mainly used to investigate respective immune responses with emphasis on immunodiagnosis of AE, and their biological functions have not been appropriately studied. Siles and coworkers (Reference Siles Lucas, Felleisen, Hemphill, Wilson and Gottstein1998) identified and cloned a 14-3-3-gene of E. multilocularis, which appeared to play a key role in basic cellular events related to cellular proliferation, including signal transduction, cell-cycle control, cell differentiation and cell survival (Siles et al. Reference Siles Lucas, Felleisen, Hemphill, Wilson and Gottstein1998; Siles and Gottstein, Reference Sher, Pearce and Kaye2003). E. multilocularis rec14-3-3 protein, used as a vaccine, was highly protective (97%) against primary challenge infection with E. multilocularis eggs (Siles et al. Reference Siles-Lucas and Gottstein2003). Gauci et al. (Reference Gauci, Merli, Muller, Chow, Yagi, Mackenstedt and Lightowlers2002) identified an E. multilocularis cDNA encoding an antigen (designated EM95), which demonstrated that a respective EM95 recombinant protein could be used to induce significant levels of protection against challenge infection with E. multilocularis eggs in mice. In a similar context, Kouguchi et al. (Reference Kouguchi, Matsumoto, Katoh, Oku, Suzuki and Yagi2007) identified a cDNA clone, designated as EMY162 that encoded a putatively secreted protein. EMY162 shared structural features with the EM95 antigen, e.g. 31% amino acid sequence identity to EM95. RT-PCR analysis revealed that EMY162 gene expression was significantly higher than EM95 at each life cycle stage. Recombinant EMY162 antigen induced a significant level of host protection (74·3%) upon experimental challenge infection with E. multilocularis eggs in mice.

HOST RESPONSE TO METABOLITES AND SOMATIC PARASITE MOLECULES OF E. MULTILOCULARIS

In murine infections with E. multilocularis, the involvement of cellular immunity in controlling the infection is strongly suggested by the intense granulomatous infiltration observed in the periparasitic area of lesions (Bresson-Hadni et al. Reference Bresson-Hadni, Liance, Meyer, Houin, Bresson and Vuitton1990; Emery et al. Reference Emery, Liance, Deriaud, Vuitton, Houin and Leclerc1996). E. multilocularis appears to induce skewed Th2-responses (Emery et al. Reference Emery, Liance, Deriaud, Vuitton, Houin and Leclerc1996). Based on in vitro and in vivo studies, Th2 dominated immunity was associated with increased susceptibility to disease, while Th1 cell activation through IL-12 (Emery et al. Reference Emery, Liance, Deriaud, Vuitton, Houin and Leclerc1996), IFNγ (Jenne et al. Reference Jenne, Kilwinski, Radloff, Flick and Kern1998; Liance et al. Reference Liance, Aicard-Blum, Emery, Houin and Vuitton1998), TNFα (Amiot et al. Reference Amiot, Vuong, Defontaines, Pater, Dautry and Uance1999) and IFNα (Godot et al. Reference Godot, Harraga, Podoprigora, Liance, Bardonnet and Vuitton2003) was suggested to induce protective immunity in AE (Emery et al. Reference Emery, Leclerc, Sengphommachanh, Vuitton and Liance1998; Vuitton, Reference Vuitton2003). The intense periparasitic granulomatous infiltration indicates an intense host-parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell-deficient mouse strains (Dai et al. Reference Dai, Waldvogel, Siles-Lucas and Gottstein2004). At the time of initial encounter with its murine host, the metacestode might modulate the immune response; the changes that it induces are dynamic and depend on the stage of development, e.g. ranging from oncosphere, to early stage vesicles up to a fully mature and fertile metacestode. Dendritic cells (DCs) and macrophages (MØs) are among the first cells encountered by the parasite, which, by secreting and expressing certain molecules, has evolved mechanisms to suppress the major inflammatory and thus immunopathological pathway. Interaction of parasite metabolites with Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) that are expressed largely, but not exclusively, on DCs and MØs is assumed to result in phenotypic changes and modification of the cytokine profiles produced by these cell types, but this has not yet been shown experimentally at the early post-oncospheral stage of infection of murine AE.

DENDRITIC CELLS AND MACROPHAGES (MØ)

DCs, the most important antigen-presenting cells (APCs) in the initiation of a type 1 or type 2 immune response, in dependence of the nature of the antigen(s) (Foti et al. Reference Foti, Granucci, Pelizzola, Beretta and Ricciardi-Castagnoli2006), range among the first players in the elaboration of a specific immune response. In the frame of a Th1 immune orientation, it is largely accepted that DCs are activated mostly by bacterial or viral pathogens via Toll-like receptor (TLR) ligation to produce IL-12 and TNF-α, both pro-inflammatory cytokines inducing a Th1 oriented response (Boonstra et al. Reference Boonstra, Asselin-Paturel, Gilliet, Crain, Trinchieri, Liu and O'Garra2003; Takeda et al. Reference Takeda, Kaisho and Akira2003). Th1-associated DC activation by microbial products evokes rapid phenotypic changes, including up-regulation of MHC class II, CD80, CD86 and CD40 (Reis e Sousa et al. Reference Reis e Sousa, Sher and Kaye1999; Romagnoli et al. Reference Romagnoli, Nisini, Chiani, Mariotti, Teloni, Cassone and Torosantucci2004). Thereafter, DCs have the ability to fully activate effector T cells. There is no mirror-image signature of cytokine and surface ligands that DCs express to stimulate Th2 differentiation. However, exposure of DCs to some helminthic antigens, including the products of filarial Acanthocheilonema viteae (ES-62), Schistosoma mansoni soluble egg antigen (SEA), and the schistosome-associated glycan lacto-N-ficopentaose III (LNFPIII), was found to pulse DCs to prime CD4+ T cells into Th2 type cells, and this occurred in the absence of increased MHC class II expression and co-stimulation molecule up-regulation (Whelan et al. Reference Whelan, Harnett, Houston, Patel, Harnett and Rigley2000; MacDonald et al. Reference MacDonald, Straw, Bauman and Pearce2001; Thomas et al. Reference Thomas, Carter, Atochina, Da'Dara, Piskorska, McGuire and Harn2003). Ingold et al. (Reference Ingold, Gottstein and Hemphill2000) had revealed the presence of high molecular mass glycans that form the major structural elements on the laminated layer of the metacestode of E. multilocularis. Whether exposure of DCs to these AE-glycans would pulse them to prime naïve CD4+ T into Th2 differentiated cells needs to be addressed.

Those helminth antigens mentioned above (ES-62, SEA, glycan LNFPIII) do not appear to induce DCs to produce IL-12 (MacDonald et al. Reference MacDonald, Straw, Bauman and Pearce2001). Thus, the Th2 response could result as a default-pathway that occurs in the absence of IL-12 production (Sher et al. Reference Sher, Pearce and Kaye2003). DCs did not display any new phenotype following stimulation with the respective parasite antigens. Thus DC-dependent Th2 immunity appeared to result from antigen presentation in the absence of DC activation and/or maturation (Maizels et al. Reference Maizels, Balic, Gomez-Escobar, Nair, Taylor and Allen2004). Indeed, it has been previously shown that immature DCs did not mature upon exposure to unfractionated crude metacestode antigen of E. multilocularis (Maizels et al. Reference Maizels, Balic, Gomez-Escobar, Nair, Taylor and Allen2004). DCs that can induce tolerance may need to be resistant to maturation-inducing factors (Morelli and Thomson, Reference Morelli and Thomson2007). A primary early source of IL-4 is needed to drive the priming of naïve CD4+ T cells into differentiated Th2 type cells (Abbas et al. Reference Abbas, Murphy and Sher1996; O'Garra, Reference O'Garra1998). It has been shown earlier in several models of ecto- or endo-parasitic infections that IL-4 might be produced early by different cell types, including DCs and other cells such as keratinocytes, Tγδ, mast cells and basophils (Mbow et al. Reference Mbow, Christe, Rutti and Brossard1994; Falcone et al. Reference Falcone, Dahinden, Gibbs, Noll, Amon, Hebestreit, Abrahamsen, Klaucke, Schlaak and Haas1996). Aumüller et al. (Reference Aumüller, Schramm, Gronow, Brehm, Gibbs, Doenhoff and Haas2004) used extracts from metacestodes of E. multilocularis to induce basophil degranulation, as well as the secretion of histamine, IL-4 and IL-13, in a dose-dependent manner. They concluded that E. multilocularis induces a Th2 response upon IL-4 release from basophils.

Macrophages from AE-infected mice (AE-MØ) as APCs exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ from non-infected mice (Mejri and Gottstein, Reference Mejri and Gottstein2006). This obstructed activity in antigen presentation of AE-MØ appeared to trigger an unresponsiveness of T cells, which in turn led to the suppression of their clonal expansion during the chronic phase of AE infection. In a similar context it was shown that high periparasitic NO production by peritoneal exudate cells, mainly AE-MØs, also contributed to periparasitic immunosuppression (Dai and Gottstein, Reference Dai and Gottstein1999; Andrade et al. Reference Andrade, Siles-Lucas, Espinoza, Pérez Arellano, Gottstein and Muro2004). Parasite-derived molecules also interfered with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. This correlated with the marked depression of the cell mediated immune response that had been observed in chronic AE (Devouge and Ali-Khan, Reference Devouge and Ali-Khan1983; Kizaki et al. Reference Kizaki, Kobayashi, Ogasawara, Day, Good and Onoé1991, Reference Kizaki, Ishige, Bingyan, Day, Good and Onoe1993).

T AND OTHER CELLS

Cells of the innate immune system are not the only targets of these immunomodulatory parasite-derived molecules. Endothelial cells (in the skin, lungs, intestine and liver) can also be induced to express and secrete anti-inflammatory mediators, such as IL-10 and prostaglandins (Zaccone et al. Reference Zaccone, Burton and Cooke2008). In this way, the parasite not only reduces its likelihood of elimination but can also minimize local host-tissue damage, with coincidental and paradoxical benefits for the host. By inducing functional changes in DCs and MØs, the metacestode can achieve important shifts in T cell subsets. An initial acute inflammatory Th1 response is subverted gradually to a Th2 response during the chronic phase of AE. Cytokines, such as IL-4, IL-5, IL-9 and IL-13, secreted largely by immune cell types in response to parasite antigens, not only down-modulate the Th1 response but can also promote parasite expulsion and tissue renewal and repair (Pennock and Grencis, Reference Pennock and Grencis2006). The metacestode most likely achieves the Th2 expansion through the induction of regulatory cytokines, such as IL-10 and TGF-β (Zhang et al. Reference Zhang, Hüe, Sène, Penfornis, Bresson-Hadni, Kantelip, Caillat-Zucman and Vuitton2008). As mentioned above, in murine AE, the host cell mediated immune response plays an important role in controlling the metacestode proliferation.

In the past decade, the Th1–Th2 paradigm has been revisited continually and alternative T cell lineages have been proposed. In particular, the recent discovery of the IL-17 cytokine family has added a new dimension to the balance of inflammation and tolerance during parasite infections. The presence of IL-17-secreting CD4+ T (Th17) lymphocytes correlates with high hepatic pathology in murine schistosomiasis (Rutitzky et al. Reference Rutitzky, Hernandez, Yim, Ricklan, Finger, Mohan, Peter, Wakeland and Stadecker2005), which prompts a more detailed similar investigation in murine AE. Another member of the IL-17 cytokine family, IL-25 (or IL-17E), also has an important role in parasitic infections. Fallon et al. (Reference Fallon, Ballantyne, Mangan, Barlow, Dasvarma, Hewett, McIlgorm, Jolin and McKenzie2006) demonstrated that IL-25 was important for mounting an appropriate Th2 response to Nippostrongylus brasilensis, and also for efficient parasite clearance. The sources of IL-25 include activated Th2 cells, mast cells and a non-B non-T cells (Fort et al. Reference Fort, Cheung, Yen, Li, Zurawski, Lo, Menon, Clifford, Hunte, Lesley, Muchamuel, Hurst, Zurawski, Leach, Gorman and Rennick2001). IL-25 as a new player will have to be the focus of respective investigations in murine AE, too.

EOSINOPHILS

One of the striking features observed in experimental murine AE (and also in naturally acquired AE of humans) is the absence of any eosinophilia. The mobilization of eosinophils is known to be a crucial immunological event that plays an important role in the host defence against helminths. Eotaxin, a CC-proinflammatory chemokine, is one of several described chemo-attractants for eosinophils. In addition, also IL-5 may mobilize these cells (Yamaguchi et al. Reference Yamaguchi, Hayashi, Sugama, Miura, Kasahara, Kitamura, Torisu, Mita, Tominaga and Takatsu1988) but its role remains controversial. In many examples of nematode infections, eosinophilia is a marked characteristic, and eosinophils directly cause profound damage to the worm tegument, such as in Strongyloides ratti and in T. spiralis, in which a marked reduction of fertility and longevity was observed (Machado et al. Reference Machado, Ueta, Lourenço, Anibal, Sorgi, Soares, Roque-Barreira, Medeiros and Faccioli2005). On the other hand, IL-5 and eosinophils had no detectable effects on the infection with Mesocestoides corti, Hymenolepis diminuta and Fasciola hepatica (Ovington and Behm, Reference Ovington and Behm1997). An extravasation of eosinophils causing eosinophilia in the peritoneal cavity has been demonstrated to be beneficial for the host by causing damage to the immigrant immature Fasciola hepatica, resulting in the erosion of the tegumental syncytium (Burden et al. Reference Burden, Bland, Hammet and Hughes1983). Eosinophils possess granules containing a variety of toxic molecules (major basic protein (MBP), peroxidase, neurotoxin, histaminase and others) which are active against many multicellular parasites, in particular helminths (Saraswathi et al. Reference Saraswathi, Nalinkumar, Ranganathan, Umadevi and Elizabeth2003). To elucidate the situation in experimental murine AE, we demonstrated that metacestode antigens (VF and E/S) exhibit proteolytic activity on eotaxin in vitro (Mejri and Gottstein, Reference Mejri and Gottstein2009). Inhibition of eotaxin activity may suppress the mobilization of eosinophils into the peritoneal cavity of intraperitoneally AE-infected mice. Eotaxin is considered as one of the main activator and chemo-attractant of resident eosinophils secreted principally by epithelial cells of the intestine (Mishra et al. Reference Mishra, Hogan, Lee, Foster and Rothenberg1999). A putative inhibition of granulocytic eosinophil infiltration may be relevant for parasite survival, as this type of cells is particularly important in the defence against helminths (Ovington and Behm, Reference Ovington and Behm1997). In experimental murine AE, the detected eotaxin inactivation by VF and E/S products may contribute to explain the absence of eosinophils within the peritoneal cavity of AE-secondary infected mice. Absent eosinophils thus may be a part of a series of events that maintain a low level of inflammation displayed within the peritoneal cavity of experimentally infected mice.

FROM MURINE TO HUMAN AE

The conventional course of AE as a disease in humans resembles strongly that of the naturally infected mouse, in that untreated AE will, in many but not all cases, finally lead to fatality. The first detection of naturally ‘aborted’ calcified liver lesions in E. multilocularis-infected persons (Godot et al. Reference Godot, Harraga, Beurton, Deschaseaux, Sarciron, Gottstein and Vuitton2000; Gottstein et al. Reference Gottstein, Saucy, Deplazes, Reichen, Demierre, Zürcher, Busato and Pugin2001) have indicated that not all infected individuals permit E. multilocularis metacestode development (Vuitton, Reference Vuitton2003). That cell mediated immunological parameters play a crucial role also in human AE became clinically obvious when immunosuppressive status such as after liver transplantation (Bresson-Hadni et al. Reference Bresson-Hadni, Koch, Miguet, Gillet, Mantion, Heyd and Vuitton2003) or during AIDS (Sailer et al. Reference Sailer, Soelder, Allerberger, Zaknun, Feichtinger and Gottstein1997; Zingg et al. Reference Zingg, Renner-Schneiter, Pauli-Magnus, Renner, van Overbeck, Schläpfer, Weber, Weber, Opravil, Gottstein and Speck2004), resulted in a dramatically increased disease severity. Most studies so far have stressed a role for CD8+ T cells and for Interleukin-10 in the development disease susceptibility (Vuitton et al. Reference Vuitton2003). A spontaneous secretion of IL-10 by PBMCs seemed to be the immunological hallmark of patients with progressive forms of AE. IL-10-induced inhibition of effector macrophages but also of antigen-presenting dendritic cells may be operating to protect the parasitic growth and survival (Vuitton et al. Reference Vuitton2003). Susceptibility to infection in humans associates with predominantly TH2-related immunity (Wellinghausen et al. Reference Wellinghausen, Gebert and Kern1999), including IL-10 (Godot et al. Reference Godot, Harraga, Deschaseaux, Bresson-Hadni, Gottstein, Emilie and Vuitton1997; Reference Godot, Harraga, Beurton, Deschaseaux, Sarciron, Gottstein and Vuitton2000), IL-4 (Kilwinski et al. Reference Kilwinski, Jenne, Jellen-Ritter, Radloff, Flick and Kern1999), IL-5 (Sturm et al. Reference Sturm, Menzel, Gottstein and Kern1995) production, especially during chronic stage of infection. Thus, in terms of Th polarization and associated cytokine expression, man and mouse appear to respond to infection quite similarly. Kocherscheidt et al. (Reference Kocherscheidt, Flakowski, Grüner, Hamm, Dietz, Kern and Soboslay2008) studied chemokine responses in AE patients at different states of infection (progressive, stable and cured AE). The production of CC and CXC chemokines which are associated with inflammation (MIP-1 alpha/CCL3, MIP-1 beta/CCL4, RANTES/CCL5 and GRO-alpha/CXCL1) was constitutively larger in all groups of AE patients than in controls (Kocherscheidt et al. Reference Kocherscheidt, Flakowski, Grüner, Hamm, Dietz, Kern and Soboslay2008). A disparate cellular responsiveness was observed in all groups of AE patients to viable E. multilocularis vesicles; cluster 1 (GRO-alpha/CXCL1, MCP-3/CCL7, MCP-4/CCL13, TARC/CCL17, LARC/CCL20) and cluster 2 chemokines (PARC/CCL18, MDC/CCL22, MIG/CXCL9) were down-regulated, while cluster 3 chemokines (MIP-1 alpha/CCL3, MIP-1 beta/CCL4, RANTES/CCL5) appeared up-regulated (Kocherscheidt et al. Reference Kocherscheidt, Flakowski, Grüner, Hamm, Dietz, Kern and Soboslay2008). The fact that E. multilocularis metacestodes selectively suppressed cellular chemokine production in AE patients may constitute an immune escape mechanism which reduces inflammatory host responses, prevents tissue destruction and organ damage, but may also facilitate parasite persistence.

FROM AE TO CE

In tandem with AE, the host response to infection with E. granulosus (cystic echinococcosis, CE) exhibits some similarities, but also striking dissimilarities. E. granulosus evokes an immune response, which is involved in the formation of a host-derived adventitious capsule. This often calcifies uniquely in the periphery of the cyst, one of the typical features found in imaging procedures, and a marked difference to AE. On differentiation into the hydatid cyst, mechanisms inhibiting complement activation on the cyst wall have been elucidated, contributing to the understanding of how the inflammatory response is controlled during CE. Similarly to AE, immunoregulatory events have been linked to the generation of T suppressor populations and to impairing the accessory action of macrophages in lymphoproliferative responses (Riley and Dixon, Reference Riley and Dixon1987). E. granulosus was also shown to be a polyclonal activator of B cells inducing both transformation and differentiation, and the effect was thymus independent (Cox et al. Reference Cox, Marshall-Clarke and Dixon1989). The mechanism by which the hydatid cyst regulates potentially larvicidal effector mechanisms appeared to be based on the production of lymphokines suppressive for metacestode killing (Jenkins et al. Reference Jenkins, Dixon, Rakha and Carter1990). Data obtained from experimental infections of E. granulosus supported the hypothesis that early IL-10, secreted by B cells in response to non-protein antigens, may favour parasite-survival and the establishment of a polarized type-2 cytokine response (Baz et al. Reference Baz, Ettlin and Dematteis2006). The coexistence of elevated quantities of interferon gamma (IFN-g), interleukin (IL)-4, IL-5, IL-6 and IL-10 observed in most of CE patients supports Th1, Th17 and Th2 cell activation in CE. In particular, Th1 cell activation seemed to be more related to protective immunity, whereas patients with active and transitional cyst presented a rather mixed Th1/ Th2 and Th0 orientation (Rigano et al. Reference Rigano, Buttari, De Falco, Profumo, Ortona, Margutti, Scottà, Teggi and Siracusano2004). The latter may be actively triggered by the parasite itself, as demonstrated by the fact that the hydatid cyst secretes and exposes numerous immunomodulatory molecules to the host's immune system, similar to AE (Siracusano et al. Reference Siracusano, Rigano, Ortona, Profumo, Margutti, Buttari, Delunardo and Teggi2008).

MODULATION OF HOST RESPONSE BY E. MULTILOCULARIS METABOLITES

The larval infection with E. multilocularis begins with the intrahepatic post-oncospheral development of a metacestode. In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the metacestode laminated layer (Gottstein et al. Reference Gottstein, Dai, Walker, Stettler, Muller and Hemphill2002). For instance, the laminated layer has been proposed to protect the germinal layer from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exudate cells contributes to periparasitic immunosuppression (Dai and Gottstein, Reference Dai and Gottstein1999; Andrade et al. Reference Andrade, Siles-Lucas, Espinoza, Pérez Arellano, Gottstein and Muro2004), explaining why iNOS-deficient mice exhibit a significantly lower susceptibility towards experimental infection (Dai et al. Reference Dai, Waldvogel, Jungi, Stettler and Gottstein2003).

Carbohydrate components of the laminated layer, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host, i.e. the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta+CD4+ T cells, and in the absence of interactions between CD40 and CD40 ligand (Dai et al. Reference Dai, Hemphill, Waldvogel, Ingold, Deplazes, Mossmann and Gottstein2001). Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 (Walker et al. Reference Walker, Baz, Dematteis, Stettler, Gottstein, Schaller and Hemphill2004) as a purified parasite metabolite suppresses ConA and antigen-stimulated splenocyte proliferation.

Interesting insights into immunomodulation by the parasite were obtained with regard to human AE. Hübner et al. (Reference Hübner, Manfras, Margos, Eiffler, Hoffmann, Schulz-Key, Kern and Soboslay2006) examined the production of cytokines, chemokines and the expression of CD molecules on peripheral blood mononuclear cells (PBMC) from AE patients and healthy controls in response to E. multilocularis metacestode culture supernatant, viable metacestode vesicles and vesicle fluid antigen in vitro. After 48 h of co-culture, the antigens depressed the release of the proinflammatory cytokine interleukin (IL)-12 by PBMC. This effect was dose-dependent and a suppression of tumour necrosis factor (TNF)-α and IL-12 was observed even when PBMC were activated with lipopolysaccharide (LPS). Comparing proinflammatory cytokine release by AE patients and controls showed that the release of IL-12 and TNF-α was reduced in AE patients, which was accompanied by an increased number of CD4+CD25+ cells and a reduced release of the Th2 type chemokine CCL17 (thymus and activation regulated chemokine, TARC), suggesting an anti-inflammatory response to the metacestode in human AE patients (Hübner et al. Reference Hübner, Manfras, Margos, Eiffler, Hoffmann, Schulz-Key, Kern and Soboslay2006). Instead, the production of IFN-γ and the expression of CD28 on CD4+ T cells were increased in PBMC from AE patients when compared to controls. This was accompanied by a higher release of the Th2-type chemokine CCL22 (macrophage derived chemokine, MDC) supporting that E. multilocularis also generates proinflammatory immune responses. These results indicate that E. multilocularis antigens modulated both, regulatory and inflammatory, Th1 and Th2 cytokines and chemokines.

Sako et al. (Reference Sako, Yamasaki, Nakaya, Nakao and Ito2007) isolated two cDNA clones from E. multilocularis metacestodes encoding cysteine peptidases (EmCLP1 and EmCLP2). The authors showed that EmCLP1 and EmCLP2 are capable of degrading a variety of proteins, including components of the extracellular matrix, albumin and also IgG. The degradation of IgG appeared to interfere with the cytotoxic activity of infiltrating neutrophils and macrophages. These cells were attracted by IL-8 and MCP-1, respectively, following activation in vitro of PMN and PBMC from AE patients by parasite vesicles. Such production of chemokines in the presence of specific antibodies may not enhance cellular attacks on the parasite, but stimulate further emigration of effector cells into the inflammatory lesions surrounding proliferating E. multilocularis metacestodes (Dreweck et al. Reference Dreweck, Soboslay, Schulz-Key, Gottstein and Kern1999). The degradation of humoral molecules might be closely related to the pathogenesis of intrahepatic AE. It has also been shown that the metacestode development in the murine liver is triggered by cell signaling originating from the intermediate host (Brehm et al. Reference Brehm, Spiliotis, Zavala-Góngora, Konrad and Frosch2006). The phosphorylation of EmMPK1, a parasitic orthologue of the extracellular signal-regulated kinase (ERK) MAPK, is specifically induced in in vitro cultured E. multilocularis metacestode vesicles, in response to exogenous host serum, hepatic cells and/or human epidermal growth factor (EGF). The E. multilocularis metacestode is thus able to ‘sense’ host factors which results in an activation of the parasite MAPK cascade (Spiliotis et al. Reference Spiliotis, Konrad, Gelmedin, Tappe, Brückner, Mösch and Brehm2006). The fact the intrahepatic metacestode expresses signaling systems with significant homologies to those of the host raises the interesting question whether cross-communication between cytokines and corresponding receptors of host and parasite can occur during an infection, i.e. whether the parasite may also influence signaling mechanisms of host cells through the secretion of various molecules that might bind to host cell surface receptors. Such interactions could contribute to immunomodulatory activities of E. multilocularis or be involved in mechanisms of organotropism and/or in host tissue destruction or regeneration during parasitic development. Lin et al. (Reference Lin, Wang, Lu, Zhou, Mantion, Wen, Vuitton and Richert2009) have recently described three mitogen-activated protein kinases (MAPKs), namely p38, JNK and ERK1/2, that become activated in primary cultures of rat hepatocytes upon exposure to metacestode vesicle fluid. JNK activation by host-free supernatant of E. multilocularis cultures suggested that liver cell signaling pathways are actually activated by parasitic components. Hepatic proliferation in AE could thus be induced through a direct influence of the parasite and not only linked to the usual reaction of hepatic cells to the occupying process that takes place in the liver (Lin et al. Reference Lin, Wang, Lu, Zhou, Mantion, Wen, Vuitton and Richert2009).

One prominent pathway in the cross-talk between E. multilocularis metacestodes in infected tissues and the immune effector cells involves the activating killer cell lectin-like receptor (NKG2D) and its ligands (major histocompatability complex class I chain–related molecules A and B [MICA/B] and UL16-binding proteins) (Bahram, Reference Bahram2000; Groh et al. Reference Groh, Rhinehart, Randolph-Habecker, Topp, Riddell and Spies2001). The germinal layer of the parasite and especially its inner germinal layer, demonstrated strong staining with the anti-MICA/B Mab, implying its presence within these compartments. Hepatic cells of the liver parenchyma surrounding the metacestode expressed MICA/B, even in areas distant from the parasitic vesicles. Because parasite and host cells expressed high amounts of MICA/B proteins, they represent putative targets of liver-infiltrating CD8+ T cells and/or NK cells that constitutively express NKG2D on their surface. The strong expression of MICA/B in the liver of AE patients contrasted with the low number of NK cells and the lack of NKG2D expression on the numerous CD8+ T lymphocytes of the periparasitic infiltrate (Zhang et al. Reference Zhang, Hüe, Sène, Penfornis, Bresson-Hadni, Kantelip, Caillat-Zucman and Vuitton2008). Some conventional cancer patients presented elevated levels of tumour-derived soluble MICA in their serum (Jinushi et al. Reference Jinushi, Takehara, Tatsumi, Hiramatsu, Sakamori, Yamaguchi and Hayashi2005). This soluble MICA reduced surface expression of NKG2D and impaired NK and CD8+ T cell functions (Holdenrieder et al. Reference Holdenrieder, Stieber, Peterfi, Nagel, Steinle and Salih2006). Sustained expression of NKG2D ligands on tissue targets could also down-regulate surface expression of NKG2D and reduce general cytotoxicity (Doubrovina et al. Reference Doubrovina, Doubrovin, Vider, Sisson, O'Reilly, Dupont and Vyas2003; Wu et al. Reference Wu, Wei, Zhang, Zhang and Tian2005). Therefore, although soluble MICA was absent in the serum of AE patients, the strong sustained expression of MICA/B molecules by E. multilocularis metacestodes and host cells might lead to down-regulation of NKG2D, with subsequent inhibition of NKG2D-dependent CD8+ T cell–mediated cytotoxicity. This could contribute to the sustained growth of the parasite. Additionally, TGF-β, present in most T cells of the granuloma, down-regulate surface expression of NKG2D and contribute to the reduction of general cytotoxicity. In previous studies, TGF-β has been shown to down-regulate expression of NKG2D in cancer patients, thereby impairing NKG2D-mediated immune surveillance and mediating immune escape of tumours (Castriconi et al. Reference Castriconi, Cantoni, Della Chiesa, Vitale, Marcenaro, Conte, Biassoni, Bottino, Moretta and Moretta2003). It would not be surprising if similar events occur in AE.

PROSPECTIVE STUDIES AND APPLICATION

The elucidation of the peritoneal cytokine profile characterizing the chronic infection mode of murine AE is crucial to explain some mechanisms behind the impairment of the immune response against the infective larval stage of E. multilocularis. The role of DCs, known to orchestrate the immune response, needs to be understood in more detail. Therefore, one needs to address the question whether peritoneal DCs are activated by monitoring their abundance and gene expression levels of cytokines such as TGF-β, IL-10 and IL-12 following AE-infection. It has been previously shown that immature DCs did not mature upon exposure to unfractionated crude metacestode antigen of E. multilocularis (Jenne et al. Reference Jenne, Arrighi, Sauter and Kern2001), and lymphocytes in periparasitic infiltrates of AE-infected liver strongly expressed TGF-β, a pleitropic immunosuppressive cytokine (Zhang et al. Reference Zhang, Hüe, Sène, Penfornis, Bresson-Hadni, Kantelip, Caillat-Zucman and Vuitton2008). TGF-β is able to attenuate CIITA gene expression and consequently inhibits HLA-DRA expression. Moreover, it inhibits the expression of co-stimulatory molecules CD80, CD86 and CD40 on APCs (Rojas et al. Reference Rojas, Balaji, Subramanian and Boom1999). Therefore, one needs to study the integrity of the pathway used by MHC class II molecules within the AE-DCs and the formation of MHC class II-peptide complexes on the surface of AE-DCs, providing the first signal following recognition by CD4+ T helper cells (Germain, Reference Germain1994). The gene expression levels of the different molecules implicated in the formation of MHC class II-peptides complexes, including CIITA, I-aβ chain, invariant chain (li), non classical class II molecule (H-2M) and cathepsin S enzyme (Cat-S) (Weenink et al. Reference Weenink, Averdunk, Boston, Boswarva, Guery, Adorini, Mellins, McCluskey and Gautam1997) have to be assessed as well. Studies on the phenotype, in particular the surface expression of co-stimulatory molecules such as B7-1, B7-2 and CD40, will show whether AE-DCs provide the secondary signal required for T cell activation.

Several subpopulations of regulatory T cells have been described in other infection models, including suppressive CD8+ T cells (Honey, Reference Honey2005), two types of regulatory T cells (Tr1) and T helper 3 (Th3) that might be induced at the mucosal surface to maintain tolerance. Both of them mediated regulation by secretion of soluble factors IL-10 (Tr1) and/or TGF-β (Th3). These regulatory cells were often found within the intestinal mucosa (Groux et al. Reference Groux, O'Garra, Bigler, Rouleau, Antonenko, de Vries and Roncarolo1997; Faria and Weiner, Reference Faria and Weiner2005). Another key regulatory T cell is the natural CD4+CD25+ T cell. These cells express Foxp3, a transcription factor protein that inhibits IL-2 production (Hori et al. Reference Hori, Nomura and Sakaguchi2003; Ghiringhelli et al. Reference Ghiringhelli, Puig, Roux, Parcellier, Schmitt, Solary, Kroemer, Martin, Chauffert and Zitvogel2005). Herein it will interesting to investigate the generation and implication of regulatory CD4+ and CD8+ T cells in the suppression of the immune response of AE-infected mice. As it had already been reported that naïve CD4+CD25+ T cells do not proliferate unless supplemented with IL-2 or IL-4, the Th2 immune response, characterized by high expression levels of IL-4, provides, in the presence of TGF-β, favourable conditions to promote the propagation of regulatory AE-CD4+CD25+ pT cells. To clarify this issue, it will be interesting to determine the proportion of CD4+CD25+ reg T cells in the peritoneal cavity of AE-infected mice, and to elucidate whether these cells produce TGF-β.

We can by no means provide a complete picture of the range of molecules that may be produced by the E. multilocularis metacestode with the potential to modulate immune responses. It is assumed commonly that the production of such molecules serves to facilitate the growth and survival of the parasite. For example, the induction of TGF-β may serve two functions. Internal maturation of the metacestode tissue might depend on signaling through a TGF-β receptor, although this cytokine can also modulate the host immune response.

In terms of application of these findings to human AE, increasing our knowledge on how the parasite modulates the host immune response is crucially linked to the identification and mechanistic understanding of those parasite and host factors that interact with each other, serve in functions such as host-parasite communication, immunological cross-talk, and metacestode growth and proliferation. These are the metabolites that determine whether an infection leads to survival or death, and they represent a crucial prerequisite for the development of novel immunotherapeutic tools. Such tools could be applied for either prevention of infection and/or disease at an early stage of infection, or for the elimination of established infections, possibly in combination with appropriate chemotherapeutical measures.