Immune responses to Schistosoma haematobium ...

Parasite Immunology, 2014, 36, 428–438

DOI: 10.1111/pim.12084

Review Article

Immune responses to Schistosoma haematobium infection J. I. ODEGAARD1 & M. H. HSIEH2 1 Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA, 2Department of Urology, Stanford University School of Medicine, Stanford, CA, USA

SUMMARY Urogenital schistosomiasis is one of the greatest single infectious sources of human morbidity and mortality known. Through a complex cycle of infection, migration and eventual maturation and mating, S. haematobium (the aetiological agent of urogenital schistosomiasis) deposits highly immunogenic eggs within the bladder and other pelvic organs, activating a wide range of immune programs that determine both infection outcome as well as downstream immunopathology. In this review, we discuss the experimental and observational bases for our current understanding of these immune programs, focusing specifically on how the balance of type 1 and type 2 responses governs subsequent immunopathology and clinical outcome. Keywords immune modulation, immunoepidemiology, Schistosoma haematobium, Schistosoma spp, schistosomiasis, urogenital

INTRODUCTION With well over one-half billion people affected worldwide, schistosomiasis is one of the most wide-reaching clinically significant infections on the planet (1,2). Moreover, Schistosoma haematobium, as the most prevalent member of the genus, is one of the greatest single sources of human morbidity and mortality known with an African disease burden that equals and may possibly exceed even that of malaria (3). Despite such impressive infamy, much of S. haematobium’s biology and pathology remains surprisingly enigmatic, due in perhaps equal parts to silent infections, a decades-long clinical course, historically inatCorrespondence: Michael H. Hsieh, Department of Urology, Stanford University School of Medicine, Stanford, CA 94305, USA (e-mail: [email protected]). Disclosures: None. Received: 24 May 2013 Accepted for publication: 20 October 2013

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tentive funding agencies and the difficulties inherent in studying fastidious organisms with complicated lifecycles. Nevertheless, recently refocused philanthropic and governmental funding has enabled several critical advancements in our ability to experimentally manipulate S. haematobium, opening the species-specific biology of this important organism to systematic study for the first time. In this review, we discuss the recent evolution of S. haematobium-specific reagents and techniques and how these complement traditional clinical studies to describe the unique pathology of S. haematobium infection. More specifically, we will focus on the complex immune response to urogenital egg deposition and discuss how the parasitehost interaction evolves to either parasite elimination or progressive clinical disease.

SCHISTOSOMA HAEMATOBIUM BIOLOGY Like other schistosomes, S. haematobium occupies a complex and specific biological niche (reviewed in (4)). The lifecycle begins in fresh water aquatic environments, where eggs hatch into miracidia and infest various species of freshwater snail (Bulinus), which act as the intermediate host. Upon maturation, S. haematobium emerges as freeswimming cercariae that traverse the water column until they come into contact with human skin, into which they burrow. With access to the dermal vasculature, the cercariae mature to schistosomulae, migrate through the host circulation, mature and eventually lodge in specific venous plexuses within the body as adult worm mating pairs and begin to lay eggs. Importantly, this migration is not at all random and instead demonstrates remarkable tissue specificity; S. haematobium, for example, migrates to venous plexuses in the pelvis (primarily those draining the bladder and female genital tract), whereas S. mansoni and S. japonicum lodge in the portal circulation. This anatomical particularity is crucial to understanding the varied clinical manifestations associated with schistosome infection as it tethers subsequent pathology to specific tissues, © 2013 John Wiley & Sons Ltd

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setting the stage for hepatosplenic (as in S. mansoni and S. japonicum infection) or urogenital disease (as in S. haematobium infection). Once in place, adult worm pairs may persist for many fecund years and produce thousands of eggs. Though many eggs eventually pass into the intestinal or bladder lumens and are excreted, many become trapped within the host tissue, evoking a potent immune response that, over months and years, results in dramatic tissue remodelling and long-term pathological consequences. Much of our knowledge of the pathophysiology of schistosome infections is derived from two basic approaches: clinical observation and animal models. Clinical observation has formed the basis for urogenital schistosomiasis research since its inception, generally in the form of serological/urinary analyses (antibody responses or hematuria, for example), ultrasound examination, or in monitoring end pathology (such as renal failure or urothelial carcinoma), all of which lend valuable insights into the natural history of chronic infection including the various possible immunopathological outcomes. Moreover, studies involving urine and serum analysis provide a serial view of infection evolution over time. Neither of these approaches, however, is able to provide the standardization or experimental manipulability necessary for rigorous definition or mechanistic investigation. Moreover, the absence of acute clinical stigmata of infection largely precludes identification of recently infected individuals and, thus, study of the nascent phases of infection including those early determinants of resistance to chronic infection. Where heterogeneous patient backgrounds and environments, unknown infection duration and ethical restrictions on intervention complicate human studies, animal models are relatively free of these shortcomings as their genetic backgrounds, environmental exposures and infection timing may be rigorously controlled and manipulated. Because of this tractability, animal models are indispensable in infectious agent research; however, care must be taken to ensure faithful recapitulation of human disease. Importantly, animal model fidelity has proven to be particularly problematic in the study of urogenital schistosomiasis. For most disease research, mice are the favoured model organism—their genetics are both defined and manipulable, they breed and subsist successfully in constrained lab environments, and a bevy of species-specific tools and experience is available for them. In an unfortunate contrast to human disease, however, S. haematobium worm pairs exhibit a tropism for the portal circulation in the mouse and other commonly employed rodents, eliciting pathology quite distinct from urogenital disease. Indeed, researchers have had to resort to costly and ethically fraught primate models to approximate human urogenital pathology, none of which have proven tenable for © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 428–438

Immune responses to S. haematobium infection

routine use. Due to this experimental intransigence, much of schistosome-directed research has focused instead on rodent models of S. mansoni and, to a lesser extent, S. japonicum, both of which approximate human hepatosplenic disease in the mouse. As these models have established important paradigms that apply to S. haematobium as well, mounting evidence suggests that species- and tissuespecific factors make such approaches poor surrogates in the study of urogenital schistosomiasis. For example, the effects of egg expulsion are very different in the bacteriarich environment of the gut lumen when compared with the sterile bladder, just as the biochemical functionality of the liver is affected differently than the biomechanical function of the bladder. Indeed, even within the same pathological context of hepatosplenic disease, S. haematobium, S. mansoni and S. japonicum infections all result in different histological appearances, pathological sequelae and disease kinetics. As S. haematobium fails to establish urogenital disease in all but a small fraction of infected mice (5,6), studies of mouse hepatic schistosomiasis demonstrate that the basic host immune response is intact and similar to that of human hepatosplenic disease, strongly supporting the general applicability of this model of infection. To circumvent S. haematobium’s lack of pelvic organ involvement in the mouse, our laboratory developed a novel model of urogenital schistosomiasis in which S. haematobium eggs—the primary determinants of the host immune responses—are surgically implanted into the mouse bladder lamina propria (the layer of loose connective tissue between the urothelium and the smooth muscle in which eggs lodge in human infection), effectively simulating bladder oviposition in urogenital disease (7). Importantly, this model faithfully recapitulates the salient architectural, pathological and immunological sequelae of human disease, thus offering the first experimentally tractable model of urogenital schistosomiasis. This model in combination with recent in vitro technological advances (such as S. haematobium-directed short interfering RNA technology (8)) and bioinformatic resources (such as the publication of the S. haematobium genome (9)) has, for the first time, placed many of the fundamental questions in schistosomal urogenital immunity and pathology within reach.

INNATE IMMUNE RESPONSES TO SCHISTOSOME INFECTION As discussed above, schistosome infections begin with cercarial penetration of the skin, migration into the dermal vasculature and eventual deposition in a terminal venous plexus, the location of which determines the specifics of the eventual clinical disease. During this weeks-long

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migration, cercariae mature into adult worms and form male-female pairs, offering the host immune system a potentially broad array of antigens. Despite this antigenic diversity, however, the host immune response to cercarial and adult worm antigens is, by comparison to later eggtriggered disease, muted (10–13). Indeed, aside from rare cases of cercarial dermatitis (swimmer’s itch) and Katayama syndrome (hypersensitivity responses to acute and mid-stage infection, respectively, both of which are especially rare in S. haematobium infection relative to other schistosome species), S. haematobium infection is rarely clinically apparent until egg deposition occurs (as highlighted in (4)). Importantly, the immune response to adult worms, while less intense than that to tissue-embedded eggs, is the primary determinant of infection outcome, with those individuals who mount robust antibody (particularly IgE) responses to adult worm antigens more likely to resist reinfection than others with more tepid responses (14). In contrast to the relatively ineffectual response to viable cercariae and adult worms, the eggs produced by adult S. haematobium evoke a robust immune response. Unfortunately, early immune responses to egg deposition are largely undefined in the human due to our inability to identify and study newly infected individuals. The advent of the mouse model of direct oviposition (7), however, opens this response accessible to study. In this context, S. haematobium eggs elicit an immediate (within 24 h) and brisk host response from local and recruited innate immune cells. Interestingly, the character of the initial response is mixed, with induction of inflammatory mediators (such as TNF-a) as well as cytokines associated with type 2 responses (such as CCL11). As with many other infectious stimuli, such immediate responses have two salient effects—additional innate immune cells are recruited to maintain and amplify the initial response, whereas the adaptive immune system is recruited and activated. Innate immune cell recruitment is, of course, a hallmark of many immune responses; however, schistosome egg-elicited responses are distinguished by their degree and persistence—indeed, the recruitment of innate immune cells to sites of schistosome egg deposition results in the development of a highly specialized secondary immune tissue known as a granuloma (Figure 1), an especially impressive feat in the bladder given the relative paucity of leucocytes in uninfected tissue. At time points as early as 4 days after oviposition in the mouse model (7), S. haematobium eggs are segregated from surrounding tissue by a dense but loosely cohesive aggregate of newly recruited macrophages and eosinophils. Over the subsequent 7 days, this highly cellular innate immune admixture organizes into a cohesive granuloma centred on the inciting schistosome egg(s).

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(a)

(b)

Figure 1 Schistosoma haematobium egg granuloma structure. (a) Within 4 days, the immune response to surgically implanted S. haematobium eggs (arrows) forms a loosely organized mass lesion (dashed line) within the bladder lamina propria dominated by dyshesive macrophages admixed with eosinophils, lymphocytes and neutrophils. (b) Over time, this response evolves into a highly organized granuloma (dashed line), which comprises a core of multinucleated syncytial macrophages (giant cells) and tightly cohesive single macrophages surrounding the now-calcified eggs (arrows) with admixed eosinophils and peripheral lymphoid aggregates (asterisks). These features are histologically identical to those observed in human infection.

Characteristically, eggs are engulfed by multinucleated syncytial macrophages (giant cells), which are in turn surrounded by an organized and cohesive cuff of epithelioid macrophages with admixed eosinophils and, to varying degrees, neutrophils concentrated around the granuloma periphery. Within the mouse urogenital model, this architecture persists for more than 100 days after oviposition with minimal but progressive loss of cellularity similar to what is observed in mouse models of hepatosplenic disease. Most importantly, this response is histologically identical to the egg-centred granulomata observed in human © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 428–438



disease (15–19), although the polypoid lesions occasionally seen in human disease (20) are not apparent.

Macrophages As the primary structural constituent of the granuloma, macrophages unsurprisingly play a central role initiating and guiding the overall innate immune response. In vitro and in vivo experiments have long ago established the macrophage’s ability to mount robust type 2 responses to schistosome eggs including expression of high levels of ARG1, MRC and CHI3L3 (reviewed in (21)). Indeed, S. mansoni infection of mice deficient in type 2 macrophage responses (such as mice lacking IL-4Ra in macrophages) results in fatal inflammatory responses due to their inability to form proper granulomata to segregate eggs from surrounding tissue (22). Moreover, numerous observations of hepatic disease models have implicated type 2 macrophages in the initial fibrotic stabilization of the granuloma and subsequent associated tissue fibrosis (23–25). Ex vivo interrogation of granuloma-derived leucocytes in the mouse model of urogenital disease, however, unexpectedly revealed elevated expression of type 1 macrophage activation markers (such as NOS2 and CD40) concurrent with expression of type 2-associated genes (such as ARG1, MRC and CHI3L3) (26), suggesting that egg deposition in this context involves either a mixed phenotype or a mixed population of type 1 and type 2 macrophages. Moreover, expression analyses of developing granulomata have described extensive expression of both type 1- and type 2-associated cytokine and chemokine signalling pathways (26), further suggesting that urogenital oviposition triggers both macrophage activation programs.

Eosinophils Eosinophil infiltration and eosinophiluria are cardinal histological and clinical signs of parasitic infection (27,28); however, much of this cell’s functional significance remains elusive. In the pre-egg stages of the S. haematobium lifecycle, eosinophils are important effector cells capable of killing cercariae and adult worms in a degranulation- and IgE-dependent manner (29). In fact, the ability of rats to clear schistosome infections without developing chronic disease may be attributable, in part, to the ease with which rat eosinophils degranulate in response to IgE-decorated worms relative to eosinophils from mice, an animal susceptible to chronic infection (30). (This particular observation may also be due, in part, to the absence of high-affinity IgE receptor I—a surface receptor on human eosinophils involved in IgE-mediated anti-schistosome activity in vitro (31)—on mouse © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 428–438

eosinophils; however, the overall significance of the rat infection phenotype is unclear and may, in fact, be an idiosyncrasy of the model itself.) Upon urogenital oviposition, however, eosinophils rapidly migrate to the bladder, presumably in response to CCL5/RANTES), CCL11/ eotaxin-1 and CCL24/eotaxin-2 (although CCL11 is the only one of these chemokines known to be highly expressed in the immediate response), where they accumulate as the most numerous cell type within the granuloma (although due to their larger size, macrophages occupy far more volume) (7). Indeed, in schistosome-infected humans, eosinophils are even detectable (albeit at much reduced numbers) near old, long-calcified egg remnants in longstanding lesions where much of the granulomatous response has long since disappeared (18,19) (Odegaard, unpublished observations). Though their functional contribution in urogenital schistosomiasis has not yet been described, eosinophils serve as an important source of IL4 prior to the emergence of the Th2 response in many other immunological contexts (such as in adipose tissue and skeletal muscle) (32,33) and remain one of the primary sources of IL-13 throughout the evolution of hepatic granulomatous responses (34). Indeed, eosinophil-derived IL-13 has been suggested to be an important driver of fibrosing responses in hepatosplenic schistosomiasis both directly through activation of hepatic stellate cells and indirectly through the promotion of type 2 macrophage activation (34). Congruent with this hypothesis, mice deficient in IL-5 demonstrate smaller S. mansoni granulomata and significantly reduced liver fibrosis (34). More recently, however, the role of the eosinophil has been called into question by the absence of any substantial difference between wild type and DdblGATA or TgPHIL mice (both of which entirely lack eosinophils but demonstrate preserved IL-5 production) during infection with S. mansoni (35). Though the absence of a phenotype is striking, these studies examined hepatic egg-induced pathology only, which does not exclude a role in the elimination of adult worms and cercariae.

Dendritic cells As the principal antigen-presenting cell, dendritic cells constitute a critical bridge between early innate and later adaptive immune responses to S. haematobium infection; however, the specifics of this role have not yet been described. Indeed, even in well-studied models of S. mansoni infection, dendritic cell activation remains somewhat enigmatic. Dendritic cells (broadly—if inaccurately— defined in many studies by CD11c expression) are necessary for proper Th2 responses to schistosome egg deposition (36); however, numerous experiments have

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demonstrated that even highly immunoreactive schistosome antigen preparations (soluble egg antigen, for example) fail to elicit canonical maturation of dendritic cells or expression of activating cytokines or costimulatory molecules in vitro (37–39). Despite this apparent apathy, however, these cells demonstrate a surprisingly potent capacity to induce an activated Th2 phenotype in na€ıve T cells in a CD40-, OX40L- and NFjB activation-dependent manner (39–42). These apparently contradictory findings have generated multiple proposed novel maturation pathways; however, in general, little evidence exists to support their in vivo relevance. One such recently proposed mechanism involves omega-1, a T2 RNase abundant in SEA and secreted by viable S. mansoni eggs (43). When internalized by immature dendritic cells, this particular molecule drives Th2 polarizing capacity through an ill-defined mechanism involving RNase-dependent nonspecific degradation of host mRNA and rRNA pools (43). How nonspecific RNA degradation is linked to T cell stimulation and Th2 polarization capacity is unclear; however, this finding suggests a novel mechanistic link between the observed in vitro and in vivo effects of schistosome antigens. Whether such mechanisms govern dendritic cell participation in urogenital disease is entirely unclear (43), as initial studies failed to detect omega-1 in S. haematobium infection (44).

Natural killer cells As the primary and secondary outcomes of schistosome infections correlate strongly with the relative balance between type 1 and type 2 immune responses, there is considerable interest in how these activation programs are initiated, how their relative contributions are regulated and in what ways might they be manipulated for therapeutic effect. As a major source of IFN-c and other type 1-biasing cytokines, natural killer (NK) cells are of natural interest in this context; however, the data supporting their role in schistosome infection is limited to three direct observations. First, NK cells are recruited to schistosome eggassociated hepatic granulomata (45), where their presence corresponds to locally increased levels of IL-12 (although they do not appear to contribute significantly to IFN-c levels within the granuloma or serum) (46). Second, depletion of NK cells results in increased schistosome egg-associated granuloma volume and fibrosis, whereas their activation (along with many that of several other lineages) by TLR3 ligands has the opposite effect (46–48). Finally, activated NK cells are capable of killing activated hepatic stellate cells—a key fibrogenic cell in hepatic fibrosis—in vitro and in vivo in the context of toxin-induced fibrosis (49). Though these observations are compelling and suggest a role in limiting the granulomatous response, the

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true contribution of NK cells even in relatively well-studied hepatic schistosomiasis is unclear, and their relevance to urogenital disease is entirely unknown.

Basophils Despite the past use of basophil degranulation as a diagnostic assay for urogenital schistosomiasis (50), the contribution of basophils to human urogenital schistosomiasis is entirely unknown (as is true in much of basophil biology). The ability of basophils to produce substantial amounts of IL-4 in response to S. mansoni infection in mice long supported the belief that these cells innately produce IL-4 in response to schistosome exposure and initiate the observed type 2 bias in the schistosome-associated immune response. The description of IL-4 inducing principle from S. mansoni eggs (IPSE)/alpha-1—an aptly-named protein secreted from viable schistosome eggs that triggers IL-4 release from basophils—seemed to support this hypothesis (51); however, careful dissection of this putative pathway in mice using multiple helminth species including S. mansoni recently debunked this theory through three major observations. First, basophil deletion yielded no defect in Th2 responses or any other demonstrable infection-related phenotype (51). Second, basophils were incapable of IL-4 production in the absence of T cells (52). Lastly, interactions between basophils and T cells were shown to be kinetically incompatible with antigen presentation and were demonstrated instead to represent a necessary licensing process only after which basophils were capable of IL-4 production (52). However, basophils were able to rescue the phenotype of mice lacking IL-4 and IL-13 in their T cells, suggesting that though it is not required in wildtype mice, basophil-derived IL-4 can functionally replace IL-4 production by Th2 cells during S. mansoni infection (52). As such, the basophil lineage remains a potential contributor to schistosome-associated immune responses and immunopathology; however, the nonredundant functions of this putative role remain unclear.

Mast cells Due to the strong correlation between serum IgE levels and resistance to S. haematobium, investigators have long proposed mast cells (one of the primary effector cell types for IgE-dependent parasite killing) as potential mediators of IgE’s protective effects. Observational studies in individuals vocationally exposed to S. mansoni, however, demonstrated that circulating mast cell precursor numbers were, in fact, negatively correlated with resistance (53), although verification of this effect and mechanistic explanation are absent. Interestingly, mast cell-derived IL-10 © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 428–438


was recently implicated in chronic bacterial urinary tract infection (54), suggesting a possible role for this lineage in the regulation of chronic inflammatory responses in the genitourinary tract and, perhaps, in the eventual induction of regulatory T cell responses. Unsurprisingly, however, little direct data are available regarding the potential role of mast cells in urogenital schistosomiasis.

ADAPTIVE IMMUNE RESPONSES TO SCHISTOSOME INFECTION T cells Though macrophages form the structural bulk of the granuloma, multiple lines of evidence suggest that T lymphocytes play a critical role in both the local and the systemic responses to infection. First, T cells rapidly infiltrate eggexposed tissues and are already present in large numbers in the nascent granuloma at day 4 in the mouse model of bladder oviposition (7). In later time points and in human samples, these cells are found scattered throughout the granuloma as well as concentrated in lymphoid follicles in the granuloma’s periphery. Second, T cell-deficient mouse models of S. japonicum and S. mansoni hepatic disease demonstrate egg-centred zones of hepatocyte necrosis accompanied by an exaggerated neutrophil response (55,56) suggesting that T cells are required to restrain the inflammatory response. Similarly, mouse models of S. mansoni infection require an effective Th2 response to sequester highly phlogistic eggs from surrounding tissue and prevent lethal inflammation (22). Third, both T cells present locally and in the draining lymph node robustly express Th2-associated cytokines (e.g. IL-4 and IL-13) well in advance of systemic Th2 responses (7), supporting the hypothesis that this activation bias is established by early T cell responses in the granuloma. Lastly, interference with key Th2-restricted factors (such as IL-4) by either genetic deletion in mice or polymorphisms in human populations results in severe restriction of granuloma-forming responses and exacerbated disease (22,57–59). These data in aggregate support a model in which the initial innate immune response generates Th2 cells, which then support granuloma formation through IL-4 production, recruit and maintain eosinophils via IL-5, and promote fibrosing responses via IL-13. In contrast, Th1 and Th17 inflammatory responses are largely suppressed after an initial spike in the mouse urogenital oviposition model (7). Indeed, S. mansoni infection in mice deficient in IFN-c, IL-12, or IL-17 is little different than in wild-type controls (60–62). Congruently, systemic levels of type 2 cytokines increase markedly by day 7 post-oviposition and remain elevated for at least © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 428–438


28 days, whereas Th1- and Th17-associated mediators remain equivalent to controls (7). This pattern further holds in mouse models of hepatic disease where the Th1 response acts to limit granuloma size and fibrotic stabilization through both direct and indirect effects (22). Importantly, serological studies in humans consistently report similar elevations in Th2 cytokines with suppression of their Th1/Th17 counterparts (63). One important distinction between the human data and the mouse model of oviposition, however, resides in the regulatory T cell (Treg) response. In the mouse model, markers of Treg cells and responses are suppressed (7), as human reports describe increased levels of IL-10 and Treg responses, although at levels much lower than those of Th2 mediators (63). This discrepancy is likely to be due to idiosyncratic rather than species-specific effects as mouse models of S. mansoni infection recapitulate Treg induction, which are required here for controlling inflammation throughout the process of egg expulsion into the gut lumen (21). Regardless, there is ample evidence in both mice and humans that schistosome soluble egg antigens induce IL-10, which in turn suppresses T cell responses (64–69). These immunomodulatory pathways may ultimately serve to reduce the host pathophysiology associated with schistosomiasis. Congruent with observations from mouse models, the human data strongly suggest that induction of Th1/Th17 mediators, as it is not uniformly correlated with infection, is strongly associated with increased urogenital morbidity and end-stage pathology, whereas Th2 responses are associated with decreased disease sequelae (63). Congruently, polymorphisms that either impair type 2 (e.g. hypomorphic variants in IL13 and STAT6) or enhance type 1 responses (e.g. hypomorphic variants in CTLA4) are both associated with increased S. haematobium infection intensity in endemic populations (70,71). Taken in aggregate, these data suggest that the balance of type 1 responses relative to type 2 is an important determinant of the local control of urogenital disease and contributes to infection resistance.

Natural killer T cells Natural killer T (NKT) cells have not been well studied in the context of schistosome infection; however, studies utilizing CD1d!/! mice, which lack all NKT cells, suggest that these cells play a role in type 2/regulatory responses to S. mansoni infections, although direct measures of infection (such as worm and egg counts and hepatic pathology) are unaffected (72). Interestingly, Ja18!/! mice, which specifically lack only type I NKT cells, demonstrate impaired type 1 responses whereas type 2/regulatory responses remain intact (72). Moreover, these mice

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Schistosoma haematobium infection evokes a complex array of immune responses targeting either the egg or adult worm/cercarial stages. As responses to the latter appear to

govern resistance to infection, parasite clearance and susceptibility to re-infection, the proximal determinant of S. haematobium-related morbidity and mortality is the eggdirected immune response. In productive (from S. haematobium’s point of view) oviposition, the cascade of immune responses detailed above facilitate the segregation of immunogenic eggs from the host and shepherd them to the organ lumen, where they may be expelled from the body and perpetuate the helminth’s life cycle. For unclear reasons, however, a subset of eggs fails to be expelled and instead persists within the tissue, chronically activating the host immune system. This activation comprises a remarkable diversity of immune pathways; however, these may be broadly classified as type 1, type 2, or regulatory responses and are typified by their associated T lymphocyte phenotypes with Th1, Th2 and Treg activation. Though the human data are far from clear and are confounded by cohort effects and other biases, the eventual outcome of S. haematobium infection is thought to be directly related to the balance between these various immune programs and the tissue responses they direct—Th1 responses are associated with tissue damage and heightened infection intensity in many human studies, whereas Th2 bias is correlated with decreased egg burden and infection resistance (56). It should be noted, however, that these are general trends and studies exist in each category suggesting contrasting interpretations. The generalized consequences of regulatory responses are similarly unclear (although control of type 1 responses has been proposed). Moreover, it remains unclear whether these responses direct the disease course or are merely markers thereof. What is clear, however, is that as some egg-directed immune responses are necessary to protect the host from deleterious inflammation and tissue damage during egg transit and expulsion, chronic activation can have marked pathological consequences. One of the primary sources of morbidity and mortality in S. haematobium-infected individuals is egg-induced urinary tract fibrosis. Here, chronic activation of egg-directed immune responses leads to recruitment and activation of myofibroblast-like cells and the progressive accumulation of collagen, which culminates in tissue fibrosis (80). Unlike schistosome-associated hepatic fibrosis, however, where fibrosis has been linked to the hepatic stellate cell (30,44), the origin of myofibroblast-like cells in urogenital disease is unclear. Regardless, the direct biomechanical consequences of fibrosis include stricture, obstruction and general urinary dysfunction, which can progress to obstructive renal pathology, renal failure and death (81,82). Secondary consequences of chronic infection also include hematuria, increased propensity to renal and urinary tract coinfections and the progressive accumulation of urothelial damage/atypia that culminates in squamous

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demonstrate larger granulomata, enhanced IgE responses and slightly lower egg counts (72), suggesting that NKT cell promotion of type 2 responses may be specifically due to type 2 NKT cells and that type 1 cells may favour type 1 responses. This is in contrast to other immunological contexts (such as in adipose tissue and liver metabolism) and other studies of schistosome infection (73) in which type I NKT cells have been reported to favour type 2/regulatory responses. As such, the actual contribution of this lineage to S. mansoni infection is unclear, whereas their role in S. haematobium infection remains entirely undescribed.

B cells Though T lymphocytes play a critical role in managing schistosome egg-associated pathology, B lymphocytes are more directly involved in infection clearance and resistance. As in T cells, high levels of S. haematobium-specific type 2 B cell effector molecules (primarily IgE) have been associated with reduced susceptibility to reinfection (14); however, B cell-deficient animals are still able to sequester deposited S. japonicum eggs within granulomata, although with somewhat delayed kinetics (74). In human populations where S. haematobium is endemic, however, B cell responses are critical to the development of parasite resistance (14). In these populations, exposure is nearly universal as defined by the ubiquity of S. haematobium-specific IgM (75); however, chronic disease and end-stage pathology is restricted to a minority of individuals with a substantial subgroup evincing no demonstrable evidence of disease. This population is distinguished by several B cell-related criteria including high titres of S. haematobium-specific IgE, anti-AWA (adult worm antigen) IgG1 and anti-Sh13 IgG3 as well as high IgE:IgG4 (63,76–78). Importantly, these resistance-associated immunoglobulin responses are directed not against immunopathogenic egg antigens but against the adult worm. It is also important to note, however, that these responses generally tend to increase with age and that these specific antigens are unlikely to represent the only immunologically productive targets, as anti-S. haematobium antibody repertoire diversity beyond these specific targets remains a powerful predictor of resistance (79). This observation has important implications for the nascent efforts to develop effective schistosome-specific vaccines.

IMMUNOPATHOLOGY OF UROGENITAL SCHISTOSOMIASIS



cell carcinoma, an especially aggressive type of bladder cancer, as well as urothelial carcinoma (83,84). In addition to the bladder pathology, 30% to 75% (depending on the population studied and examination techniques) of S. haematobium-infected females develop genital pathology detectable on examination as sandy patches (physical stigmata of tissue-entrapped eggs) on the cervical mucosa with the vagina, upper tract and external genitalia less commonly affected (85–90). Importantly, as sandy patches of the cervix are pathognomonic for FGS in women, the early appearance and subsequent evolution of cervical lesions in girls with FGS remains unknown. In addition, as the basic pathophysiology here is much the same as in the bladder, the tissue-specific consequences include increased susceptibility to sexually transmitted infections (especially HIV), dyspareunia and infertility (85–90). Importantly, infection and genital disease is often established during childhood, long before the onset of sexual maturity, leading to lifelong pathological consequences (91). Less commonly, S. haematobium can involve the male genital organs, where it generally manifests as hematospermia and possibly infertility (92). Though S. haematobium infection physically localizes to the urogenital tract, its infectious sequelae may systemically manifest. Indeed, a major source of S. haematobium’s disease burden manifests as developmental delay of chronically infected children both as a consequence of nutritional deficiencies (most commonly due to iron deficiency associated with chronic inflammation and hematuria) and repeated urinary tract coinfections (93–98). Moreover, S. haematobium-infected individuals demonstrate increased susceptibility to Plasmodium species due, in large part, to IL-10’s influence on cytokine and antibody responses (99,100). These same alterations in immune timbre, however, provide one of the few potentially positive effects of infection—infected individuals demonstrate substantially reduced off-target immunoreactivity such as occurs in atopic and autoimmune conditions. Given the potent type 2 immune deviation associated with schistosomal infection, it is unsurprising that disease severity in such type 1-driven immune pathologies as Crohn’s disease, type 1 diabetes and multiple sclerosis would be significantly reduced (reviewed in (101)); however, similarly beneficial effects are observed in type 2-driven pathologies such as asthma and atopic disease (102,103), suggesting that disease amelioration might be due to regulatory influences rather than type 1-type 2 antagonism. Indeed, schistosome-infected individuals demonstrate enhanced basal and stimulated IL-10 production capable of effectively inhibiting both autoimmune and atopic pathologies (104). Congruently, studies of asthma in S. mansoni-endemic populations demonstrate that infected asthmatics not only © 2013 John Wiley & Sons Ltd, Parasite Immunology, 36, 428–438

demonstrated lower disease severity than uninfected controls but that this difference vanished on treatment with the schistosomicide praziquantel (105). Interestingly, functionally significant IL10 promoter polymorphisms correlate with atopic disease and total IgE levels but not with schistosome infection susceptibility or outcome (106), suggesting that IL-10 exercises a more potent influence over atopic immunopathology than schistosome-associated disease. The mechanisms underlying this observation are unclear; however, a leading hypothesis involves schistosome-specific IgE out-competing allergen-specific IgE for mast cell surface receptor occupancy. It is perhaps more likely, however, that schistosome-elicited IL-10 directly inhibits the pro-atopic activity of target lineages, as has been demonstrated in basophils (107). As in other aspects of human schistosome studies, the significance of these effects is often unclear as effect magnitude and even direction varies between reports.

CONCLUSION Urogenital schistosomiasis comprises a significant and under-appreciated source of human morbidity and mortality; however, very little is known of the host or pathogen determinants of disease. This lack of clarity is, in part, due to the relatively enigmatic nature of the aetiological agent, S. haematobium, and the historical lack of experimentally tractable disease models. With the recent advent of S. haematobium-specific tools and a mouse model of urogenital infection, however, the field is poised to dissect urogenital disease away from its better-studied hepatosplenic cousin and define the unique immunopathology that governs our interaction with this deadly schistosome. To facilitate the study of S. haematobium infection, we present below a summary of areas of particular interest to the field. Gleaning insights in these areas will further our understanding of fibrosing disease (both urogenital and otherwise), cancer and immunopathology in general and, hopefully, further arm us to intervene therapeutically therein.

OPPORTUNITIES FOR SCHISTOSOMA HAEMATOBIUM INFECTION RESEARCH

• • • •

Development of animal models of S. haematobium infection conducive to the study of the role of IgE. Identification of major secreted products of S. haematobium eggs and comparisons of their structure and function to homologs in other schistosome species. Determination of how and by what mechanisms S. haematobium adult worms track to the pelvic circulation. Elucidation of how inflammatory cervicovaginal lesions evolve during female genital schistosomiasis.

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