Role of IL-4Rα during acute schistosomiasis in mice.

Schistosomiasis is an important parasitic disease that causes major host morbidity and mortality in endemic areas. Research conducted in mouse models of schistosomiasis has provided great insights and understanding of how host protective immunity is orchestrated and key cellular populations involved in this process. Earlier studies using cytokine-deficient mice demonstrated the importance of IL-4 and IL-10 in mediating host survival during acute schistosomiasis. Subsequent studies employing transgenic mice carrying cell-specific deletion of IL-4Rα generated using the Cre/LoxP recombination system have been instrumental in providing more in-depth understanding of the mechanisms conferring host resistance to Schistosoma mansoni infection. In this review, we will summarize the contributions of IL-4/IL-13-responsive cellular populations in host resistance during acute schistosomiasis and their role in limiting tissue pathology.

Introduction

Schistosomiasis is a chronic parasitic disease caused by blood-dwelling trematode flatworms (flukes) of the genus Schistosoma. The disease is endemic in over 74 developing countries where it is estimated to infect approximately 200 million people 1–3. Schistosomiasis is a major cause of host morbidity and mortality in endemic areas, and 280 000 deaths per annum are attributed to the disease in sub-Saharan Africa alone; hence, the World Health Organization (WHO) has placed it amongst the top ten infectious diseases of global importance 4. The emergence of HIV/AIDS in areas where schistosomiasis is endemic has raised serious concerns about the control of schistosome infection. There is already evidence suggesting that schistosome infection affects the aetiology and transmission of HIV 5–9, tuberculosis 6,10,11 and malaria 12–14. Although schistosomiasis can effectively be treated with praziquantel, the drug does not prevent re-infection of individuals, a common occurrence in endemic areas. Thus, studying the immune biology of schistosomiasis is crucial for broadening our understanding of the disease and assisting in rational design and development of a vaccine candidate.

Schistosoma mansoni (S. mansoni) eggs lodged in the host liver and intestines provoke a dominant CD4+ T cell-dependent Th2 immune response, extensive tissue fibrosis and granulomatous inflammatory responses 15–18. Infection of gene-deficient mice with S. mansoni demonstrated an essential host protective role for Th2 cytokines such as IL-4, IL-13 and IL-10 during acute schistosomiasis 19–21. IL-4 and IL-13 signalling is mediated by heterodimeric receptor complex containing a common IL-4 receptor α (IL-4Rα) subunit 22,23. IL-4 uniquely binds and signals through the type I receptor consisting of IL-4Rα subunit and the common gamma chain (γc), while IL-13 signals through the type II receptor composed of IL-4Rα subunit and IL-13Rα1 chain 22. Furthermore, IL-13 binds to the homodimeric IL-13Rα2 receptor with high affinity 24. Initially, the IL-13Rα2 receptor was thought to act as a decoy receptor possessing no signalling abilities, but recent studies have shown that IL-13 signalling via the IL-13Rα2 induces TGF-β production and mediates fibrosis in chronic TNBS colitis 25,26. The contribution of IL-4/IL-13 signalling via IL-4Rα in mediating immune responses conferring host resistance to acute schistosomiasis has been investigated using transgenic mouse models lacking IL-4Rα expression on all haematopoietic cells.

In this review, we discuss the role played by IL-4/IL-13 signalling via the IL-4Rα in certain cellular populations during acute schistosomiasis and how it mediates host resistance or susceptibility to infection. We further discuss recent data on how cell-specific IL-4Rα expression controls granuloma formation and maintains the fine balance between a Th1/Th2 immune response, crucial for limiting the deleterious effects on the host.

Acute vs. Chronic Schistosomiasis

Schistosomiasis is characterized by two main clinical conditions – acute and chronic schistosomiasis – depending on the maturation of the parasites and their eggs. In humans, acute schistosomiasis is a debilitating febrile illness (Katayama fever) that usually occurs before the appearance of eggs in the stool and peaks 6–8 weeks after infection 27–29. Although less studied, acute illness has been associated with a predominantly T helper 1 (Th1) immune response characterized by high levels of tumour necrosis factor (TNF) in the plasma, and peripheral blood mononuclear cells (PBMCs) have been shown to produce large quantities of TNF, IL-1 and IL-6 28,30. Interestingly, the symptoms associated with acute disease seem to be uncommon in individuals living in areas where schistosomiasis is endemic compared to individuals with no previous history of exposure to infection. Chronic schistosomiasis is a more clinically relevant disease that is potentially life-threatening in individuals that develop hepatosplenic disease in response to eggs trapped in various tissues 27,28,31. This severe form of the disease is accompanied by hepatic and periportal fibrosis, portal hypertension, ascites and portosystemic shunting of venous blood 31.

The focus of this review is on murine models of schistosomiasis, which have been crucial in expanding our understanding of the host–parasite interactions and the host's immune response to S. mansoni infection. Like in humans, S. mansoni infection in mice progresses through two main stages: acute schistosomiasis and chronic schistosomiasis that are characterized by different immune response profiles. During the acute stage of infection (0–8 weeks post-infection), the immune response alters between Th1 and Th2 depending on the eliciting antigens. Immature parasite antigens elicit a moderate Th1 immune response early during infection (3–5 week post-infection), while egg antigens induce a robust Th2 immune response that peaks at 8 weeks post-infection 28. The dominant Th2 immune response and the associated pathologies are down-modulated during the chronic stages of infection (10 weeks post-infection onwards) in immunocompetent wild-type mice through a mechanism involving IL-10 32–34. It is important to mention that S. mansoni infection of mice does not evoke all aspects associated with human schistosomiasis such as portal tract fibrosis 35.

IL-4Rα-Mediated Signalling is Crucial for Host Survival During Acute Schistosomiasis

Earlier studies elucidated immunological factors involved in coordinating the mechanisms conferring host resistance or susceptibility to acute schistosomiasis using gene-deficient mice. Mice lacking IL-4 production by all haematopoietic cells (IL-4−/−) suffered from severe disease characterized by rapid cachexia coinciding with the onset of egg deposition by adult worms and eventually succumbed to S. mansoni infection by 8–10 weeks post-infection compared to wild-type control mice 19. Mortality in infected IL-4−/− mice was associated with increased production of pro-inflammatory cytokines IFN-γ and TNF-α, uncontrolled liver granuloma formation and increased intestinal inflammation that resulted in endotoxemia 19,20. In contrast, IL-13−/− mice infected with S. mansoni developed a sufficient Th2 immune response and displayed enhanced survival due to reduced hepatic fibrosis 20. Mice deficient of IL-4 and IL-13 (IL-4−/−/IL-13−/−) were found to be extremely susceptible to acute schistosomiasis, even more so than IL-4−/− mice 20. Therefore, these studies were crucial in demonstrating that IL-4 and IL-13 play distinct and contrasting pathogenic roles during S. mansoni infection in mice.

More studies were conducted using gene-deficient mice to uncover more cytokines involved in the pathogenesis of schistosomiasis. IL-4/IL-10 double-deficient mice quickly succumbed to S. mansoni infection due to increased weight loss, augmented hepatocellular damage indicated by serum aspartate transaminase (AST) levels and increased production of pro-inflammatory mediators IFN-γ, TNF-α and nitric oxide (NO), suggesting that IL-10 might be an essential immunomodulatory cytokine during acute schistosomiasis in mice 21. Unexpectedly, mice deficient in IL-10/IL-12 developed a severe wasting disease that culminated in death during the chronic stages of S. mansoni infection despite the presence of a sufficient Th2 immune response 21. This study by Hoffmann and colleagues conclusively demonstrated that excessive Th1 or Th2 cytokine responses may lead to lethal disease during S. mansoni infection in mice. Thus, regulating the polarization of the Th1 and Th2 immune response triggered by S. mansoni egg antigens is essential for host survival.

Generation of transgenic mice lacking IL-4Rα expression on all haematopoietic cells (IL-4Rα−/−) was instrumental in dissecting the role of IL-4Rα signalling in the mechanism conferring host resistance or susceptibility to acute schistosomiasis. IL-4Rα−/− mice infected with S. mansoni quickly succumbed to infection by eight weeks post-infection due to exacerbated liver hepatotoxicity indicated by increased serum AST levels, impaired egg expulsion, abrogated granuloma formation and severe gut inflammation that ultimately resulted in the leakage of lipopolysaccharides (LPS) into the bloodstream, leading to endotoxemia and septic shock 36. Treatment of IL-4Rα−/− mice with antibiotics extended survival time during S. mansoni infection, providing a mechanism responsible for mortality in these mice 36. A recent study by Herbert and colleagues showed that IL-4Rα expression by bone marrow-derived cells is required for host survival against acute schistosomiasis by limiting liver hepatotoxicity and maintaining the integrity of the gut 37. Therefore, it can be concluded that IL-4/IL-13-mediated signalling via IL-4Rα is indispensable for host survival during acute schistosomiasis.

The cellular contributions of IL-4Rα to the mechanisms providing host resistance to S. mansoni infection have been determined using novel transgenic mouse models deficient in cell-specific IL-4Rα expression generated using the Cre/loxP recombination system. Mice lacking IL-4Rα expression on macrophages and neutrophils (LysMcreIL-4Rα−/lox) were found to be highly susceptible to S. mansoni infection despite the presence of a sufficient Th2 immune response 36. Mortality in LysMcreIL-4Rα−/lox mice was caused by augmented liver damage and excessive gut inflammation which subsequently led to endotoxemia and septic shock 36. It was generally thought that the presence of classically activated macrophages that possess the ability to produce pro-inflammatory mediators is responsible for the extensive inflammation found in LysMcreIL-4Rα−/lox mice infected with S. mansoni. However, a recent study by Rani and colleagues showed that mice deficient in both classically and alternatively activated macrophages, generated by crossing MIIG transgenic mice (mice that use CD68-IVS1 promoter to drive IFN-γ-dominant negative receptor) with IL-4Rα−/− mice (MIIG × IL-4Rα−/−), quickly succumbed to S. mansoni infection due to rapid weight loss, severe liver injury and failure to upregulate the expression of indoleamine 2,3 dioxygenase (IDO), a crucial immunosuppressive enzyme 38,39. Therefore, maintaining a fine balance between IL-4Rα-responsive alternatively activated macrophages and IFN-γ-driven classically activated macrophages is crucial for prolonging host survival during acute schistosomiasis and down-modulating tissue inflammation.

Mice deficient in IL-4Rα expression specifically on CD4+ T cells (LckcreIL-4Rα−/lox) survived acute schistosomiasis by controlling egg-induced intestinal inflammation even though they developed increased granulomatous liver pathology 40. A subsequent study by Dewals and colleagues utilizing iLckcreIL-4Rα−/lox mice (pan-T cells IL-4Rα-deficient mice) showed the importance of IL-4/IL-13-responsive non-CD4+ T cells in conferring host resistance to acute schistosomiasis and limiting liver pathology 41. The specific IL-4/IL-13-responsive non-CD4+ T cell population contributing to the mechanism conferring resistance to acute schistosomiasis is yet to be determined. It has been postulated that IL-4/IL-13-responsive B cells and CD11c+ dendritic cells may be involved in mediating host resistance to S. mansoni infection.

The host protective role of IL-4Rα to S. mansoni infection is not only limited to haematopoietic cells but has recently been demonstrated in nonhaematopoietic target cells. Mice deficient in IL-4Rα expression on smooth muscle cells (SM-MHCcreIL-4Rα−/lox) were found to be highly susceptible (succumbed to infection earlier than wild-type littermate control mice) to acute schistosomiasis despite developing sufficient Th2 immune responses and the absence of intestinal inflammation and sepsis 42. Increased susceptibility of SM-MHCcreIL-4Rα−/lox mice to S. mansoni infection was associated with severe weight loss, impaired egg expulsion and decreased intestinal hypercontractility compared to wild-type littermate control mice 42. The contribution of different IL-4Rα-responsive cellular population in host survival during acute schistosomiasis is summarized in Table1.

Table 1

Schistosoma mansoni-induced pathological outcomes in cell-specific IL-4Rα-deficient mice

Mouse strain	IL-4Rα cell specificity	Mortality	AST level	Fibrosis	LPS level	Reference
IL-4Rα−/−	All cells	+	+	−	+	36
LysMcreIL-4Rα−/lox	Macrophages and neutrophils	+	+	±	+	36
CD4+IL-4Rα−/lox	CD4+ T cells	−	+	+	−	40
iLckcreIL-4Rα−/lox	Pan-T cells	+	+	±	±	41
SM-MHCcreIl-4Rα−/lox	Smooth muscle cells	+	NM	±	±	42

AST, aspartate transaminase (indicator of hepatocellular damage); LPS, lipopolysaccharides (indicator of gut destruction); +, increased compared to littermate control mice; −, decreased compared to littermate control mice; ±, equivalent to littermate control mice; NM, not measured.

IL-4/IL-13-Responsive Haematopoietic Cells Regulate Liver Granuloma Size in S. mansoni-Infected mice

Schistosoma mansoni eggs trapped in the host tissue induce a Th2-dependent granuloma formation that is characterized by the presence of T cells, eosinophils and macrophages 17,20,28,43. The absence of IL-4Rα signalling in all haematopoietic cells impaired granuloma formation during S. mansoni infection despite the presence of a sufficient Th2 response in the liver 16. In-depth analysis of cytokine production by liver granuloma-associated single cells from infected IL-4Rα−/− or STAT6−/− mice revealed that granuloma-associated Th2 cells can develop independently of IL-4Rα/Stat6 signalling in vivo and in vitro 15,18. Therefore, IL-4Rα is crucial for regulating granuloma formation in mice infected with S. mansoni.

A study by Herbert and colleagues found that mice lacking IL-4Rα expression on BM-derived cells had augmented liver granuloma size than wild-type mice, non-BM IL-4Rα−/− and IL-4Rα−/− mice 37. Specific IL-4Rα-responsive cellular populations involved in regulating liver granuloma size during S. mansoni infection in mice have been elucidated. These cellular populations are alternatively activated macrophages 36 and CD4+ T cells 40,41. Other IL-4/IL-13-responsive haematopoietic cells, such as B cells and dendritic cells, might be involved in regulating granuloma size in infected mice. However, IL-4/IL-13-responsive nonhaematopoietic cells seem to play little or no role in granuloma formation as infected SM-MHCcreIL-4Rα−/lox mice developed equivalent granulomas to littermate control mice.

In-depth analysis of liver granulomas from infected LysMcreIL-4Rα−/lox and iLcKcreIL-4Rα−/lox mice showed that alternatively activated macrophages and IL-4/IL-13-responsive T cells have distinct influences on liver granuloma microenvironment 44. Liver granulomas from LysMcreIL-4Rα−/lox mice had unaltered Th1/Th2 cytokine balance and cellular composition compared to littermate control mice, while the lack of IL-4/IL-13-responsive T cells resulted in a shift towards IFN-γ production by granuloma-associated cells and impaired cellular recruitment similarly to IL-4Rα−/− mice 44. Importantly, a small subpopulation of macrophages expressing macrophage mannose receptor (MMR) and chitinase-like molecule-1 (Ym1; MMR+Ym-1+) was found in the periphery of granulomas of infected LysMcreIL-4Rα−/lox mice, and it was absent in granulomas from iLckcreIL-4Rα−/lox mice 44. Finally, the emergence of IL-4Rα-independent MMR+Ym-1+ macrophages was shown to be induced by IL-10 signalling in macrophage-specific IL-4Rα-deficient mice 44.

Signalling via IL-4Rα is Crucial for Th2 Polarization During S. mansoni Infection

Earlier studies demonstrated the importance of IL-4 signalling through IL-4Rα in the development of Th2 cells in vitro and in vivo 45,46 and orchestrating and amplifying Th2 cytokine responses to helminth infections 19,21,47,48. Recent studies utilizing transgenic mice have elucidated IL-4Rα-responsive cellular populations that are involved in coordinating the development of Th2 immunity during acute schistosomiasis. Mice deficient of IL-4Rα signalling specifically on CD4+ T cells failed to develop a sufficient Th2 immune response indicated by reduced production of IL-4 and IL-13 by splenocytes after stimulation with schistosome egg antigen (SEA) 40. In fact, these mice developed a highly polarized Th1 immune response characterized by increased production of IFN-γ by splenocytes from infected mice 40. Moreover, impairing IL-4Rα signalling on pan-T cells abrogated Th2 cytokine production while augmenting Th1 cytokine production by total mesenteric lymph node cells and splenocytes stimulated with α-CD3 in vitro 41. Therefore, these studies demonstrated the importance of IL-4/IL-13-responsive T cells in driving optimal Th2 immunity during S. mansoni infection.

Mice deficient of alternatively activated macrophages developed a mixed Th1/Th2 cytokine responses characterized by increased Th1 cytokine production accompanied by unaltered Th2 cytokine production by splenocytes from S. mansoni-infected mice stimulated with SEA 36. Interestingly, depletion of CD11c+ dendritic cells (DCs) impaired Th2 cytokine responses in vitro and in vivo during S. mansoni infection 49. However, a subsequent study by Cook and colleagues demonstrated that DCs expressing IL-4Rα primed with SEA are not required for Th2 cytokine production in vivo, but are crucial for IFN-γ and IL-10 production 50. IL-4/IL-13-responsive smooth muscle cells do not play a role in cytokine responses during S. mansoni infection indicated by similar quantities of Th1 and Th2 cytokines as wild-type control mice 42. Therefore, IL-4/IL-13-responsive nonlymphoid cells appear not to play a role in inducing and polarizing cytokine production during S. mansoni infection in mice.

IL-4Rα Signalling Down-Modulates Liver Fibrosis During Acute Schistosomiasis

Studies have demonstrated that interfering with IL-4Rα signalling on haematopoietic cells impairs tissue fibrosis during S. mansoni infection 16,36,37,41. IL-13 was identified as a main profibrotic factor driving collagen production during S. mansoni infection 20,51. It was postulated that IL-4/IL-13-responsive alternatively activated macrophages are an important source of proline, a key ingredient in collagen formation 52. However, mice deficient of alternatively activated macrophages had similar concentrations of hydroxyproline as wild-type littermate control mice infected with S. mansoni 36. Another marker for alternatively activated macrophages is Arginase-1, a key enzyme involved in the synthesis of collagen and fibrosis 53,54. A recent study by Pesce and colleagues found that mice deficient of Arginase-1-expressing macrophages (Arg1−/flox; LysMcre) had augmented liver fibrosis during the chronic stages (12 and 22 weeks post-infection) of S. mansoni infection compared to wild-type control mice 55. This was accompanied by enlargement of the liver and increased shunting of the eggs into the lungs, the key pathological features associated with hepatosplenic form of schistosomiasis 54–56. Therefore, these studies have demonstrated that IL-4/IL-13 signalling to macrophages via IL-4Rα or macrophage-derived Arginase-1 do not mediate collagen deposition during schistosomiasis in mice.

Infection of LckcreIL-4Rα−/lox mice with S. mansoni resulted in unaltered liver hydroxyproline content compared to littermate control mice 40. Furthermore, collagen deposition was similar between pan-T cells IL-4Rα-deficient mice and littermate control mice 41. These findings were further supported by a study by Herbert and colleagues that showed that chimeric mice lacking IL-4Rα expression on bone marrow cells (BM IL-4Rα−/−) have equivalent concentration of hydroxyproline as wild-type control mice 37. In contrast, non-BM IL-4Rα−/− mice had reduced concentration of hydroxyproline compared to wild-type mice 37. Therefore, these studies demonstrated that IL-4/IL-13-responsive nonbone marrow-derived cells are a cellular source of collagen during S. mansoni infection.

Conclusion

Cell-specific IL-4Rα expression is crucial for elucidating mechanisms responsible for conferring host resistance or susceptibility to acute schistosomiasis in mice. Furthermore, cell-specific IL-4Rα expression influences the development of Th1/Th2 immune responses and regulated liver granuloma size and cellular composition. Therefore, more efforts are required to generate more cell-specific IL-4Rα-deficient transgenic mice strains to improve our insights and understanding of the immunobiology of schistosomiasis and factors contributing to host resistance.