Correspondence: Kathryn J. Else, Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK (e-mail: email@example.com).
Gastrointestinal (GI) nematodes are incredibly successful parasites. Choosing to live in an exposed extracellular niche, in confrontation with a potentially hostile environment, their persistent, chronic lifestyle is persuasive evidence in itself for their profound ability to modulate their hosts’ immune response. Modulation is essential to avoid their own destruction but also subtly balanced to avoid compromising host survival. This review describes the early circumstantial evidence that gave clues to the immunomodulatory capabilities of the GI nematodes, the roles that T regulatory cells and alternatively activated macrophages play in this immunomodulation and provides examples of the types of specific parasite-derived factors that are known to modulate host immunity, potentiating parasite survival.
Successful parasitism requires the parasite to survive within its host long enough to reproduce. Having co-evolved with the host's immune system, it is perhaps not surprising that parasites have developed rather unique relationships with their hosts in order to support their sophisticated parasitic lifestyle. Gastrointestinal (GI) nematode parasites have exploited the intestinal niche and chosen the gut as a habitat regardless of its potentially powerful immune system. Despite the hostility of this environment, these worms survive and indeed thrive, living in equilibrium with host immunity.
Epidemiological studies reveal clearly that GI nematode infections are chronic and associated with Th2-related responses such as elevated levels of IgE (1,2). It is also clear that helminth infections exert non-specific immunomodulator effects on the immune system (3,4), with the most prominent and provocative recent data in this area relating to the treatment of inflammatory bowel disease using intestinal nematodes (5–7).
LABORATORY MODELS OF GI NEMATODE INFECTION
There are four main models of GI nematode infection in the mouse: Trichuris muris, Trichinella spiralis and the hookworms Heligmosomoides polygyrus and Nippostrongylus brasiliensis. Data from these four models reveal these helminths to be potent Th2 inducers (8,9). These Th2 responses are clearly protective allowing the host to efficiently expel the parasite. In most cases, however, the types of effector mechanisms orchestrated by these Th2 cells are still not known. Induction of potentially hostile Th2 responses appears a precarious strategy for GI nematodes to adopt, and it is only really with the emergence of the concept of the ‘modified Th2 phenotype’ (10,11) that the Th2-inducing power of these parasites coupled with the chronic nature of these infections is explained. Thus, some of these parasites may be able to constrain or ‘modify’ the antiparasitic Th2 response via induction of, for instance, alternatively activated macrophages (AAMφ) and T regulatory cells (see below) in order to survive. Of the four GI nematodes only T. muris (in certain mouse strains) and H. polygyrus are truly chronic at high dose infections (12,13). Infections with N. brasiliensis only develop to chronicity if low-dose infections are given (14). T. spiralis does not survive for long in any mouse strain and appears to have adopted an extraordinary life-cycle strategy quite distinct to those of the other three parasites, rapidly developing to maturity and reproducing prior to Th2-mediated expulsion. Thus, it is perhaps within the GI nematode species, which are of a more chronic nature (T. muris and H. polygyrus) that one might expect to uncover the clearest evidence of successful immunomodulatory strategies. Interestingly, these two parasites have adopted quite different approaches to surviving within their hosts –H. polygyrus dysregulates the Th2 response so that it is not effective, down-regulating specific cytokines to depress the mucosal mast cell response (15). Further, recent data also suggest that this helminth can induce the development of T regulatory cells (16). In contrast, T. muris quickly down-regulates an early Th2 response in favour of a strong Th1 response, which is unable to orchestrate the effector responses within the host that would normally culminate in worm expulsion (17). Perhaps this is the ultimate parasite survival strategy: rather than modify a potentially protective Th2 response actually promote the ‘wrong’ type of response with respects to worm elimination. Although Th1-mediated inflammation is itself potentially damaging, perhaps the Th1 response that accompanies chronic T. muris infection is itself ‘modified’ and/or regulated. Certainly chronic T. muris infection is accompanied by increased levels of the regulatory cytokine IL-10, and heavily infected susceptible mouse strains show no overt signs of severe immunopathology. In addition, IL-10-deficient mice are susceptible to infection and do suffer severe pathology (18). Thus, IL-10 is important in both resistance to infection and in the regulation of Th1-mediated pathology. Furthermore, we have shown that the gut tissue of susceptible mice is dominated by the expression of genes involved in tryptophan metabolism (indoleamine 3,3 dioxygenase, tryptophanyl tRNA synthetase), which is associated with the induction of T-cell anergy in vitro and in vivo (19). Thus, the local gut environment of T. muris-infected mice may well be regulated, generating mellow conditions conducive to parasite survival and avoiding host-damaging pathology. Why the strategy of diverting the host immune response away from the protective Th2 type towards a non-protective Th1 type has not been more widely adopted is unclear. Promoting Th1 responses may ultimately be safer, at least in terms of avoiding worm expulsion, but perhaps modifying an ongoing and potentially protective Th2 response is a less risky strategy, not requiring the precision in timing required to redirect an early Th2 towards a Th1 before the antiparasitic Th2 response gains dominance.
IMMUNOMODULATION BY GI NEMATODES
Studies in the 1970s and 1980s are testimony to the profound effects parasites and/or their extracts can have on host immunity. For instance, N. brasiliensis infection is prolonged in the presence of H. polygyrus (20). Heligmosomoides polygyrus delays the expulsion of an otherwise acute T. spiralis infection (21) and suppresses the immune responses to a variety of heterologous antigens (22). MLNC from N. brasiliensis-infected mice are unable to respond to TNP-BGG (23). Nematode infections are able to prolong rat kidney allograft survival (24). The later larval stage of T. muris appears to be extremely immunomodulatory with curtailment of a primary infection prior to the third larval (L3) stage priming for resistance to challenge but primary exposure of the host to stages beyond the L3 prime for susceptibility to any subsequent challenge (25). In the context of T. muris infection, three isolates of this species exist; the J, S and E isolates, with the S isolate being profoundly immunomodulatory, surviving in mouse strains that expel the other two isolates (26). The successful survival strategy of the S isolate, like the E isolate, involves the development of a Th1 response in the absence of a Th2 response (27). However, unlike infections with the E isolate, which even when they survive to chronicity trigger local cellular responses in the gut such as mastocytosis, there is a dearth of, for instance, mast cells in the mucosal tissue of S-infected mice (28). As with the E isolate, survival in the host depends on intracellular signalling through MyD88 (C. E. Johnston and K. J. Else, unpublished), and for the E isolate, at least survival requires the presence of toll receptor (TLR) 4 (29). This implies at the mechanistic level that the survival strategy of T. muris includes interactions between parasite-derived products and toll receptor bearing cells such as dendritic cells or intestinal epithelial cells. These interactions may then shape the quality of the adaptive immune response via, for instance, influencing the local cytokine environment. Although as yet no differences between the isolates in the ability of their excretory secretory products to stimulate dendritic cells have been identified (C. E. Johnston, J. E. Bradley, J. M. Behnke and K. J. Else, unpublished), these isolates represent powerful tools in the elucidation of the mechanisms underlying parasite immunomodulation. Intriguingly, intestinal epithelial cells do respond innately to E isolate antigen by releasing a range of cytokines (M. L. deSchoolmeester, H. Manku and K. J. Else, submitted); therefore, it is possible that the invading larvae start to subtly subvert the host immune response within hours after infection to generate local conditions that favour survival.
Over the last few years there has been real progress in understanding the mechanisms that underlie the numerous examples of immune modulation by GI nematodes. These mechanisms can be broadly divided into two overlapping subgroups: the induction of immunomodulatory cell types and the production of parasite-derived immunomodulatory molecules, which target key parts of the host immune system.
INDUCTION OF IMMUNOMODULATORY CELLS TYPES
The alternatively activated macrophage (AAMØ)
Clues to the mechanisms underlying parasite-mediated immunomodulation came from studies on antigen-presenting cells. T- and B-cell activation is reliant on costimulatory signals sourced from the antigen-presenting cell and so these cells represent attractive targets for modulation by the parasites. The concept of the ‘nematode-induced’ alternatively activated macrophage (AAMØ) as a suppressor APC in the context of Th2-dominated infections has been best characterized in the context of the tissue-dwelling filarial parasites (30,31), where these AAMØ induced by nematode infection were shown to suppress the ability of lymphocytes to proliferate by a cell contact-dependent mechanism. Their presence in other parasitic infections (including Taenia crassiceps and trypanosomes) and how they influence disease outcome has been reviewed recently (32,33). Classical activation of macrophages by IFN-gamma leads to their development into a highly microbicidal state, with the up-regulation of the enzyme inducible nitric oxide synthase leading to nitric oxide production. In Th2 settings the AAMØ develops which up-regulate the enzyme arginase and the products of the genes Ym1 and Fizz1 (32,33). In the context of GI nematodes Ym1, Fizz1 and two other chitinase and Fizz family members (ChaFFs), acidic mammalian chitinase (AMCase) and Fizz2 have been shown to be induced at the site of infection with N. brasiliensis (34). The functional importance of the AAMØin vivo is beginning to emerge with three roles proposed: immunoregulatory, thus, holding in check the inflammatory response to the parasite, tissue repair and effector cells, actually releasing molecules that damage the parasite (11). Although it is unclear as yet as to whether the induction of AAMØ by GI nematodes benefits the host, the parasite, both, or indeed neither, the immunoregulatory role is intriguing. Thus, in the N. brasiliensis study, both Ym1 and Fizz1 were expressed in the draining lymph nodes of infected mice, as well as at the site of infection, implying the potential to immunoregulate. Indeed, it has previously been shown that a soluble N. brasiliensis extract modulates B-cell proliferation via affecting macrophage function (35). In addition, using a T-cell transgenic adoptive transfer system Boittelle et al. (36) showed that Ascaris suum body fluid (ABF) reduces the proliferative capacity of OVA specific cells during a primary immune response in vivo, and hypothesized that ABF may be inducing a suppressor APC in draining lymph nodes.
Th2-promoting dendritic cells
Effects of GI nematode parasite products on dendritic cells are also beginning to emerge. Thus, the maturation of dendritic cells with excretory secretory antigens from N. brasiliensis promotes the development of a Th2-driving dendritic cell population (37). This is consistent with both the potent Th2 responses seen after infection with GI helminths and the evolution by the host of mechanisms to ‘recognize’ the type of infecting pathogen subsequent to the generation of an ‘appropriate’ Th2-based protective immune response. The pressures on N. brasiliensis to evolve antigens that promote the development of Th2-inducing dendritic cells are unclear as this will favour the generation of antiparasitic Th2 responses. However, if the ensuing Th2 responses are themselves locally regulated in the gut environment, perhaps by the AAMØ, the rationale might seem clearer.
T regulatory cells
Epidemiological studies in man suggest that helminth infections provide protection from both Th2-driven allergies and Th1-mediated autoimmune disease. For instance, populations with heavy helminth infections seem to have reduced skin reactivity to common allergens. This has been shown for schistosome infections (38) and GI nematodes (39). In addition, recently T. suis was proposed as an effective treatment for inflammatory bowel disease (5–7). The mechanism underlying these observations is still debated with two competing but not necessarily mutually exclusive hypotheses presented: immune regulation and immune deviation (40). Immune regulation involves the induction by helminths of T regulatory cells, which dampen down Th1- or Th2-mediated disease via, for instance, the anti-inflammatory cytokines IL-10 or TGF-beta. Immune-deviation proposes diminished Th1 responses due to the induction of a Th2 response by helminths. The epidemiological observations are supported by studies in laboratory models, which show that nematode parasites suppress experimentally induced asthma and colitis (41–43). The concept that helminth infections not only trigger modified Th2 responses but also promote the production of T regulatory cells would allow for prolonged parasite survival and protect the host from potentially fatal immunopathology; thus, it is consistent with parasite survival strategies. The direct existence of T regulatory cells in helminth infections has perhaps best been shown for schistosomes (44) where IL-10 production by CD25+ was shown to protect mice from immunopathology triggered by immune response to the egg. In addition, T regulatory cells have been cloned from people infected with the filarial nematode onchocerciasis (45). In the context of GI nematodes, however, the evidence is less clear, although Elliott et al. (16) demonstrated that H. polygyrus infection inhibited colitis in IL-10 knockout mice. This inhibition was associated with an increase in the expression of Foxp3 in draining lymph nodes, with Foxp3 being a transcription factor that drives T-regulatory-cell development. Other evidence for a role for T regulatory cells in GI nematode infection is beginning to emerge. For instance, modulation of immune responses to ovalbumin, mediated by Ascaris suis body fluid may be mediated via the immunoregulatory cytokine IL-10, perhaps suggesting a role for the T-regulatory-cell network (46). Further, unpublished work, reported in Maizels et al. (11), describes experiments using H. polygyrus to depress airway allergy followed by the removal of this protective effect by depleting CD25+ cells, thus, implicating the T regulatory cell in the protective mechanism. Over the coming years, the T-regulatory-cell story will no doubt reveal more secrets, particularly in the context of the GI nematodes where the story is by no means complete. It is likely that both regulation and the Th1/Th2-cell subset balance play roles in the remarkable and exploitable ability of helminths to protect their hosts against allergies and autoimmune disease.
PARASITE-DERIVED IMMUNOMODULATORY MOLECULES UNDERLYING GI NEMATODE LONGEVITY
Nematode secretions include a multitude of products with the potential to modulate host immunity. These products interfere with the immune system at all levels, from disrupting antigen presentation, through driving polar T-cell responses to disarming the final effectors of antiparasite immunity. Some of these products are described below and further reflect the subtle and complex interactions, which have evolved between parasites and their hosts.
The evolution of host-like molecules to modulate immunity
It has long been established that susceptibility to T. muris involves both host genetics and parasite-induced modulation. The immunomodulatory effects of the later larval stages and adult stages of the parasite mean that host strains that are genetically slow in mounting a protective immune response are exposed to increasing levels of immunomodulation (25). This is exemplified by some inbred mouse strains, e.g. B10D2/n, where the time of expulsion coincides with the moult to the third larval stage and thus genetically identical individuals that are kinetically slightly slower in developing a protective Th2 response, in relation to the parasite growth rate, become susceptible to infection. Their littermates, in contrast, are able to expel the parasite and mount a protective immune response (47). The mechanisms behind this immune modulation have not been fully characterized; however, parasite molecules have been shown to share epitopes with host IFN-gamma and bind to the IFN-gamma receptor (48). The production of an IFN-gamma homologue by T. muris would promote the development of a non-protective Th1 response and so favour parasite survival. Equally T. muris extracts contain a macrophage-migration-inhibitory factor (MIF) orthologue (49) involved in macrophage activation and which may play a role in the development of the alternatively activated phenotype exhibited by macrophages during nematode infections (50). The evolution of molecules similar or identical to those of the host in order to modulate immunity is also evident in the survival strategy of A. suum, which possesses a chemokine-like neutrophil chemoattractant (51) and extraordinarily appears to produce morphine to enhance its survival within its host (52).
Human hookworms have been particularly well studied in the context of the ability of their secretions to modify immune responses. In contrast to the IFN-gamma-like homologue of T. muris, Necator americanus secretes a protein that binds to natural killer cells inducing the production of IFN-gamma (53). Thus, these two GI nematodes use slightly different strategies to reach the same end point of redirecting potentially host-protective Th2 responses.
Impairment of effector cell recruitment and shielding from effector molecules
Necator americanus also produces factors that impair the recruitment of potentially antiparasitic eosinophils to the site of infection. These factors have been characterized as metalloproteases, which specifically cleave the eosinophil chemoattractant CCL3 (eotaxin) (54). Adopting a similar strategy, N. brasiliensis secretes a platelet-activating factor (PAF) acetylhydrolase, which inactivates PAF thus down-regulating inflammation in the gut (55). The production of oxygen radicals is thought to play an important role in the host effector response against parasites. Necator possesses a superoxide dismutase (56) and a glutathione S-transferase (57), as defence against products of the respiratory burst such as the reactive oxygen species and products of lipid peroxidation. Indeed the possession of primary defence enzymes such as superoxide dismutase and glutathione S-transferase is common amongst the GI nematodes (58–60). This reflects the importance of detoxifying reactive oxygen radicals as part of the defence strategy of GI nematodes, the tactic here being to disarm the final immune effector response should the initial subversion of the Th2 response fail. Also, armed with a calreticulin capable of interfering with the complement cascade (61) and the ability to induce T-cell apoptosis (62), the evasion strategies employed by Necator to support its chronic lifestyle are clearly subtle and sophisticated.
Also common amongst the GI nematodes is the possession of protease inhibitors, and these too may modulate the end stages of the host's immunological effector response to promote parasite survival. Thus, most GI nematodes infections are characterized by an inflammatory cell infiltrate in the gut with local increases in, for instance, mast cells. Mast cells release a variety of serine proteases (chymases, tryptases) as do neutrophils (cathepsin G, elastase) and macrophages (cathepsin G). Hence the ability to disarm these potent chemical mediators is valuable and indeed protease inhibitors have been characterized in, for instance, T. suis (63), Ascaris (64,65) and Ancylostoma ceylanicum (66) and immunomodulatory roles proposed.
Modulation of antigen presentation
The generation of an antigen-specific adaptive immune response to GI nematodes requires parasite antigens to be processed by antigen-presenting cells and presented to T cells in the context of MHC class II. Cysteine proteases are involved in several steps of this process, for instance, they are important in the initial degradation of proteins in the endosomal-lysosomal compartment of the APC. It is becoming clear that nematodes are able to interfere with this pathway by producing cysteine protease inhibitors or cystatins (67). In terms of GI nematodes, a particularly good example of this is the cystatin of N. brasiliensis, which prevents the processing of ovalbumin by lysosomal cysteine proteases (68). Similarly, a cystatin has been identified in Haemonchus contortus (69).
Subversion of intracellular signalling pathways?
The evolutionary pressures exerted on GI nematodes to develop survival strategies may be different according to the niche of the gut within which they live. Most GI nematodes are luminal dwelling. However T. spiralis and T. muris have adopted a more intimate association with their hosts, living intracellularly, within intestinal epithelial cells. Have they developed novel modulatory mechanisms more akin to those employed by intracellular protozoan parasites in order to ensure survival in this unusual habitat? Certainly it is known that T. spiralis secretes serine/threonine protein kinases (70) and a nucleotide diphosphate kinase (71), an enzyme important in maintaining intracellular nucleotide pools. Thus, T. spiralis has the potential to modulate host cell function and, although these sorts of enzymes may not be unique to intracellular GI nematodes (72), we have recently shown that secretions from T. muris also have protein kinase activity, fitting well with survival within an intracellular habitat (K. Gounaris, C. E. Johnston, J. E. Bradley, J. M. Behnke, K. J. Else, unpublished).
GI nematodes possess broad ranges of immunoregulatory mechanisms ranging from modifying the phenotype of the host immune response to one that is in their favour, through to preventing immune attack by incapacitating the effector response. Some of these strategies are represented in Figure 1 and summarized in Table 1. Importantly, although many of these parasite-dependent modifications of the host immune response have the potential to favour parasite survival, there are few instances where the benefit to the parasite has been directly proven. Even in host parasite relationships where a modified host immune response has been shown to underlie worm persistence (the Th1 response of mice infected with T. muris (25–28,47,73)), the parasite-derived molecules, which promote the modification, remain speculative (48). Indeed to critically address this would require the treatment of the host with antibodies to deplete modified cell populations or to block the immunomodulatory factors, or the development of transgenic parasites lacking putative parasite survival molecules.
Table 1. Summary of the types of strategies employed by GI nematode parasites to modify the immune system of their hosts. In most cases the benefit of the modified immune response to the parasite is implied by the study rather than proven
The continued building of our understanding of the immunomodulatory strategies employed by GI nematode parasites will bring two broad benefits. Thus, we may be able to generate vaccines against the immunomodulatory molecules to allow the antiparasitic effector response to proceed without provoking pathology. In addition, we may be able to use the immunomodulatory factors, evolved as weapons against us, to develop novel strategies to control allergy and autoimmune disease. So have GI nematodes outwitted the immune system? Rather than outwitting the immune system these parasites have co-evolved with the immune system and live in harmony with it (5), through the production of a range of immunomodulatory agents that support a successful host–parasite relationship.
I would like to thank my colleagues who have allowed me to describe their unpublished data, Dr Matthew deSchoolmeester for his comments on the manuscript and the Wellcome Trust (grant number 044494) for supporting my research.