Helminth protection against autoimmune diabetes in nonobese diabetic mice is independent of a type 2 immune shift and requires TGF-β

Leading hypotheses to explain helminth-mediated protection against autoimmunity postulate that type 2 or regulatory immune responses induced by helminth infections in the host limit pathogenic Th1-driven autoimmune responses. We tested these hypotheses by investigating whether infection with the filarial nematode Litomosoides sigmodontis prevents diabetes onset in IL-4-deficient NOD mice and whether depletion or absence of regulatory T cells, IL-10, or TGF-β alters helminth-mediated protection. In contrast to IL-4-competent NOD mice, IL-4-deficient NOD mice failed to develop a type 2 shift in either cytokine or Ab production during L. sigmodontis infection. Despite the absence of a type 2 immune shift, infection of IL-4-deficient NOD mice with L. sigmodontis prevented diabetes onset in all mice studied. Infections in immunocompetent and IL-4-deficient NOD mice were accompanied by increases in CD4(+)CD25(+)Foxp3(+) regulatory T cell frequencies and numbers, respectively, and helminth infection increased the proliferation of CD4(+)Foxp3(+) cells. However, depletion of CD25(+) cells in NOD mice or Foxp3(+) T cells from splenocytes transferred into NOD.scid mice did not decrease helminth-mediated protection against diabetes onset. Continuous depletion of the anti-inflammatory cytokine TGF-β, but not blockade of IL-10 signaling, prevented the beneficial effect of helminth infection on diabetes. Changes in Th17 responses did not seem to play an important role in helminth-mediated protection against autoimmunity, because helminth infection was not associated with a decreased Th17 immune response. This study demonstrates that L. sigmodontis-mediated protection against diabetes in NOD mice is not dependent on the induction of a type 2 immune shift but does require TGF-β.     

Chronic helminth infection reduces basophil responsiveness in an IL-10-dependent manner

Basophils play a key role in the development and effector phases of type 2 immune responses in both allergic diseases and helminth infections. This study shows that basophils become less responsive to IgE-mediated stimulation when mice are chronically infected with Litomosoides sigmodontis, a filarial nematode, and Schistosoma mansoni, a blood fluke. Although excretory/secretory products from microfilariae of L. sigmodontis suppressed basophils in vitro, transfer of microfilariae into mice did not result in basophil suppression. Rather, reduced basophil responsiveness, which required the presence of live helminths, was found to be dependent on host IL-10 and was accompanied by decreases in key IgE signaling molecules known to be downregulated by IL-10. Given the importance of basophils in the development of type 2 immune responses, these findings help explain the mechanism by which helminths protect against allergy and may have broad implications for understanding how helminth infections alter other disease states in people.     

Helminth infection with Litomosoides sigmodontis induces regulatory T cells and inhibits allergic sensitization, airway inflammation, and hyperreactivity in a murine asthma model

Numerous epidemiological studies have shown an inverse correlation between helminth infections and the manifestation of atopic diseases, yet the immunological mechanisms governing this phenomenon are indistinct. We therefore investigated the effects of infection with the filarial parasite Litomosoides sigmodontis on allergen-induced immune reactions and airway disease in a murine model of asthma. Infection with L. sigmodontis suppressed all aspects of the asthmatic phenotype: Ag-specific Ig production, airway reactivity to inhaled methacholine, and pulmonary eosinophilia. Similarly, Ag-specific recall proliferation and overall Th2 cytokine (IL-4, IL-5, and IL-3) production were significantly reduced after L. sigmodontis infection. Analysis of splenic mononuclear cells and mediastinal lymph nodes revealed a significant increase in the numbers of T cells with a regulatory phenotype in infected and sensitized mice compared with sensitized controls. Additionally, surface and intracellular staining for TGF-beta on splenic CD4(+) T cells as well as Ag-specific TGF-beta secretion by splenic mononuclear cells was increased in infected and sensitized animals. Administration of Abs blocking TGF-beta or depleting regulatory T cells in infected animals before allergen sensitization and challenges reversed the suppressive effect with regard to airway hyperreactivity, but did not affect airway inflammation. Despite the dissociate results of the blocking experiments, these data point toward an induction of regulatory T cells and enhanced secretion of the immunomodulatory cytokine TGF-beta as one principle mechanism. In conclusion, our data support the epidemiological evidence and enhance the immunological understanding concerning the impact of helminth infections on atopic diseases thus providing new insights for the development of future studies.     

Regulation and biological function of helminth-induced cytokine responses

The immunological hallmarks of infection with parasitic belmintbs, namely eosinophilia, mastocytosis and increased IgE synthesis, all appear to be induced by cytokines from the T_H2 subset of CD4⁺ T cells: IgE production is stimulated by interleukin 4 (IL-4), eosinophilia by IL-5 and mastocytosis by IL-3 and IL-4. Here, Fred Finkelman and colleagues argue that the functional significance of the eosinophilia-mastocytosis-IgE axis in helminth infection is unclear and suggest that in some worm infections T_H2-cell cytokines may contribute to host protection, while in others they may promote parasite survival.     

Basophils are the major producers of IL-4 during primary helminth infection

IL-4 production by leukocytes is a key regulatory event that occurs early in the type 2 immune response, which induces allergic reactions and mediates expulsion of parasites. CD4(+) T cells and basophils are thought to be the key cell types that produce IL-4 during a type 2 response. In this study, we assessed the relative contribution of both CD4(+) T cell- and basophil-IL-4 production during primary and secondary responses to Nippostrongylus brasiliensis using a murine IL-4-enhanced GFP reporter system. During infection, IL-4-producing basophils were detected systemically, and tissue recruitment occurred independent of IL-4/STAT6 signaling. We observed that basophil recruitment to a tissue environment was required for their full activation. Basophil induction in response to secondary infection exhibited accelerated kinetics in comparison with primary infection. However, total basophil numbers were not enhanced, as predicted by previous models of protective immunity. Overall, the induction and migration of IL-4-producing basophils into peripheral tissues was found to be a prominent characteristic of the primary but not memory responses to N. brasiliensis infection, in which CD4(+) T cells were identified as the major source of IL-4. Whereas basophils were the major initial producers of IL-4, we determined that normal Th2 differentiation occurs independently of basophils, and depletion of basophils led to an enhancement of inflammatory cell recruitment to the site of infection.     

Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection

Anti-helminth immunity involves CD4+ T cells, yet the precise effector mechanisms responsible for parasite killing or expulsion remain elusive. We now report an essential role for antibodies in mediating immunity against the enteric helminth Heligmosomoides polygyrus (Hp), a natural murine parasite that establishes chronic infection. Polyclonal IgG antibodies, present in naive mice and produced following Hp infection, functioned to limit egg production by adult parasites. Comparatively, affinity-matured parasite-specific IgG and IgA antibodies that developed only after multiple infections were required to prevent adult worm development. These data reveal complementary roles for polyclonal and affinity-matured parasite-specific antibodies in preventing enteric helminth infection by limiting parasite fecundity and providing immune protection against reinfection, respectively. We propose that parasite-induced polyclonal antibodies play a dual role, whereby the parasite is allowed to establish chronicity, while parasite load and spread are limited, likely reflecting the long coevolution of helminth parasites with their hosts.     

Impaired basophil induction leads to an age-dependent innate defect in type 2 immunity during helminth infection in mice

Parasitic-infection studies on rhesus macaque monkeys have shown juvenile animals to be more susceptible to infection than adults, but the immunological mechanism for this is not known. In this study, we investigated the age-dependent genesis of helminth-induced type 2 immune responses using adult (6-8-wk-old) and juvenile (21-28-d-old) mice. Following infection with the parasitic nematode Nippostrongylus brasiliensis, juvenile mice had increased susceptibility to infection relative to adult mice. Juvenile mice developed a delayed type 2 immune response with decreased Th2 cytokine production, IgE Ab responses, mouse mast cell protease 1 levels, and intestinal goblet cell induction. This innate immune defect in juvenile mice was independent of TLR signaling, dendritic cells, or CD4(+) cell function. Using IL-4-eGFP mice, it was demonstrated that the numbers of IL-4-producing basophil and eosinophils were comparable in young and adult naive mice; however, following helminth infection, the early induction of these cells was impaired in juvenile mice relative to older animals. In nonhelminth models, there was an innate in vivo defect in activation of basophils, but not eosinophils, in juvenile mice compared with adult animals. The specific role for basophils in this innate defect in helminth-induced type 2 immunity was confirmed by the capacity of adoptively transferred adult-derived basophils, but not eosinophils, to restore the ability of juvenile mice to expel N. brasiliensis. The defect in juvenile mice with regard to helminth-induced innate basophil-mediated type 2 response is relevant to allergic conditions.     

Removal of regulatory T cell activity reverses hyporesponsiveness and leads to filarial parasite clearance in vivo

Human filarial parasites cause chronic infection associated with long-term down-regulation of the host's immune response. We show here that CD4+ T cell regulation is the main determinant of parasite survival. In a laboratory model of infection, using Litomosoides sigmodontis in BALB/c mice, parasites establish for >60 days in the thoracic cavity. During infection, CD4+ T cells at this site express increasing levels of CD25, CTLA-4, and glucocorticoid-induced TNF receptor family-related gene (GITR), and by day 60, up to 70% are CTLA-4(+)GITR(high), with a lesser fraction coexpressing CD25. Upon Ag stimulation, CD4(+)CTLA-4(+)GITR(high) cells are hyporesponsive for proliferation and cytokine production. To test the hypothesis that regulatory T cell activity maintains hyporesponsiveness and prolongs infection, we treated mice with Abs to CD25 and GITR. Combined Ab treatment was able to overcome an established infection, resulting in a 73% reduction in parasite numbers (p < 0.01). Parasite killing was accompanied by increased Ag-specific immune responses and markedly reduced levels of CTLA-4 expression. The action of the CD25(+)GITR+ cells was IL-10 independent as in vivo neutralization of IL-10R did not restore the ability of the immune system to kill parasites. These data suggest that regulatory T cells act, in an IL-10-independent manner, to suppress host immunity to filariasis.     

Identification of semaphorin 4B as a negative regulator of basophil-mediated immune responses

Basophils are strong mediators of Th2 responses during helminthic infections. Recently, basophils were shown to function as APCs and promote both Th2 skewing and humoral memory responses. However, the mechanisms that regulate basophils are still unclear. In this article, we show that a class IV semaphorin, Sema4B, negatively regulates basophil functions through T cell-basophil contacts. In a screen to identify semaphorins that function in the immune system, we determined that Sema4B is expressed in T and B cells. Interestingly, Sema4B(-/-) mice had considerably increased serum IgE levels despite normal lymphocyte and dendritic cell functions. Recombinant Sema4B significantly inhibited IL-4 and IL-6 production from basophils in response to various stimuli, including IL-3, papain, and FcεRI cross-linking. In addition, T cell-derived Sema4B, which accumulated at contact sites between basophils and CD4(+) T cells, suppressed basophil-mediated Th2 skewing, suggesting that Sema4B regulates basophil responses through cognate cell-cell contacts. Furthermore, Sema4B(-/-) mice had enhanced basophil-mediated memory IgE production, which was abolished by treating with an anti-FcεRIα Ab. Collectively, these results indicate that Sema4B negatively regulates basophil-mediated Th2 and humoral memory responses.     

Parasite antigen-driven basophils are a major source of IL-4 in human filarial infections

Basophil contribution to the IL-4 pool in filarial infections was assessed using PBMC from 20 patients with active filarial infections and from 9 uninfected subjects. Patient basophils released histamine in response to Brugia malayi Ag (BmAg). They also released IL-4 within 2 h after exposure to BmAg, as assessed by intracellular cytokine flow cytometry. This IL-4 induction was Ag specific, as IL-4 was not detected in BmAg-exposed basophils obtained from uninfected subjects. Although there were, on average, 64 times more CD4(+) T cells than basophils in the peripheral circulation of filaria-infected patients, the absolute numbers of basophils and CD4(+) T cells producing IL-4 per 100000 PBMC were equivalent (geometric mean: 16 IL-4-producing basophils/100000 PBMC vs 22 IL-4-producing CD4(+) T cells/100000 PBMC). Basophils also released IL-4 in response to both low and high concentrations of BmAg, whereas CD4(+) T cells released IL-4 only after incubation with a high concentration of BmAg, raising the possibility that basophils, due to their lower threshold for activation, may actually release IL-4 more frequently than CD4(+) T cells in vivo. Furthermore, IL-4 production in vitro by Ag-stimulated purified basophils or CD4(+) T cells provided evidence that basophils release greater quantities of IL-4 per cell than CD4(+) T cells in response to BmAg. These results suggest that, when Ag-specific IgE is present in a filaria-infected individual, basophils function to amplify the ongoing Th2 response by releasing IL-4 in greater amounts and possibly more frequently than CD4(+) T cells in response to filarial Ag.     

Goblet cell-derived resistin-like molecule beta augments CD4+ T cell production of IFN-gamma and infection-induced intestinal inflammation

The secreted goblet cell-derived protein resistin-like molecule beta (RELMbeta) has been implicated in divergent functions, including a direct effector function against parasitic helminths and a pathogenic function in promoting inflammation in models of colitis and ileitis. However, whether RELMbeta influences CD4(+) T cell responses in the intestine is unknown. Using a natural model of intestinal inflammation induced by chronic infection with gastrointestinal helminth Trichuris muris, we identify dual functions for RELMbeta in augmenting CD4(+) Th1 cell responses and promoting infection-induced intestinal inflammation. Following exposure to low-dose Trichuris, wild-type C57BL/6 mice exhibit persistent infection associated with robust IFN-gamma production and intestinal inflammation. In contrast, infected RELMbeta(-/-) mice exhibited a significantly reduced expression of parasite-specific CD4(+) T cell-derived IFN-gamma and TNF-alpha and failed to develop Trichuris-induced intestinal inflammation. In in vitro T cell differentiation assays, recombinant RELMbeta activated macrophages to express MHC class II and secrete IL-12/23p40 and enhanced their ability to mediate Ag-specific IFN-gamma expression in CD4(+) T cells. Taken together, these data suggest that goblet cell-macrophage cross-talk, mediated in part by RELMbeta, can promote adaptive CD4(+) T cell responses and chronic inflammation following intestinal helminth infection.     

Intestinal helminths protect in a murine model of asthma

Underdeveloped nations are relatively protected from the worldwide asthma epidemic; the hygiene hypothesis suggests this is due to suppression of Th2-mediated inflammation by increased exposure to pathogens and their products. Although microbial exposures can promote Th2-suppressing Th1 responses, even Th2-skewing infections, such as helminths, appear to suppress atopy, suggesting an alternate explanation for these observations. To investigate whether induction of regulatory responses by helminths may counter allergic inflammation, we examined the effects of helminth infection in a murine model of atopic asthma. We chose Heligosomoides polygyrus, a gastrointestinal nematode, as the experimental helminth; this worm does not enter the lung in its life cycle. We found that H. polygyrus infection suppressed allergen-induced airway eosinophilia, bronchial hyperreactivity, and in vitro allergen-recall Th2 responses in an IL-10-dependent manner; total and OVA-specific IgE, however, were increased by worm infection. Finally, helminth-infected mice were protected against eosinophilic inflammation induced by adoptive transfer of OVA-stimulated CD4(+) cells, and transfer of cells from helminth-infected/OVA-exposed mice suppressed OVA-induced eosinophilic inflammation, suggesting a role for regulatory cells. Increased CD4(+)CD25(+)Foxp3(+) cells were found in thoracic lymph nodes of helminth-infected/OVA-exposed mice. Helminthic colonization appears to protect against asthma and atopic disorders; the regulatory cytokine, IL-10, may be a critical player.     

Cutting edge: IPSE/alpha-1, a glycoprotein from Schistosoma mansoni eggs, induces IgE-dependent, antigen-independent IL-4 production by murine basophils in vivo

During infection with the helminth parasite Schistosoma mansoni, the deposition of eggs coincides with the onset of IL-4 production and Th2 development. Although IL-4 is known as a potent inducer of Th2 differentiation, the mechanism by which schistosome eggs induce IL-4 production is not clear. In this study, we demonstrate that the S. mansoni egg Ag (SmEA) induces IgE-dependent IL-4 production by basophils derived from Heligmosomoides polygyrus-infected or OVA/alum-immunized mice in the absence of pathogen-specific IgE. The effect is mediated by the secretory glycoprotein IPSE/alpha-1, because IPSE/alpha-1-depleted SmEA no longer induces cytokine production. Conversely, recombinant IPSE/alpha-1 is sufficient to induce IL-4 production. Importantly, the injection of SmEA or recombinant IPSE/alpha-1 into H. polygyrus-infected 4get/KN2 IL-4 reporter mice rapidly induces the dose-dependent IL-4 production by basophils in the liver, a major site of egg deposition. Thus, IPSE/alpha-1 induces basophils to produce IL-4 even in the absence of Ag-specific IgE.     

Differential roles of the co-stimulatory molecules GITR and CTLA-4 in the immune response to Trichinella spiralis

We investigated the roles of the regulatory molecules glucocorticoid-induced TNF receptor family-related protein (GITR) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) in murine infection with the nematode parasite Trichinella spiralis. Expression of GITR and CTLA-4 was rapidly upregulated on cells in the mesenteric lymph nodes and spleen, with approximately 80% of CD4+ lymphocytes expressing GITR by day 7 post-infection, coinciding with release and dissemination of newborn larvae. As the infection progressed to the chronic muscle phase, expression of GITR returned to normal, whereas CTLA-4 was sustained as late as day 60. Mice treated with anti-GITR antibodies rapidly developed higher titres of parasite-specific IgG1, IgG2a, IgG2b and IgM than controls. This was accompanied by elevated background lymphocyte proliferation, but parasite establishment in the intestine or the muscle was unaffected. In contrast, treatment with anti-CTLA-4 antibody resulted in elevated serum IgE, enhanced production of interleukin-4 and interleukin-10, and lower numbers of parasites recovered from skeletal muscle. These results reveal different temporal and regulatory roles for CTLA-4 and GITR in immune responses to helminth infection.     

Anti-IL-2 treatment impairs the expansion of T(reg) cell population during acute malaria and enhances the Th1 cell response at the chronic disease

Plasmodium chabaudi infection induces a rapid and intense splenic CD4(+) T cell response that contributes to both disease pathogenesis and the control of acute parasitemia. The subsequent development of clinical immunity to disease occurs concomitantly with the persistence of low levels of chronic parasitemia. The suppressive activity of regulatory T (T(reg)) cells has been implicated in both development of clinical immunity and parasite persistence. To evaluate whether IL-2 is required to induce and to sustain the suppressive activity of T(reg) cells in malaria, we examined in detail the effects of anti-IL-2 treatment with JES6-1 monoclonal antibody (mAb) on the splenic CD4(+) T cell response during acute and chronic P. chabaudi AS infection in C57BL/6 mice. JES6-1 treatment on days 0, 2 and 4 of infection partially inhibits the expansion of the CD4(+)CD25(+)Foxp3(+) cell population during acute malaria. Despite the concomitant secretion of IL-2 and expression of high affinity IL-2 receptor by large CD4(+) T cells, JES6-1 treatment does not impair effector CD4(+) T cell activation and IFN-γ production. However, at the chronic phase of the disease, an enhancement of cellular and humoral responses occurs in JES6-1-treated mice, with increased production of TNF-α and parasite-specific IgG2a antibodies. Furthermore, JES6-1 mAb completely blocked the in vitro proliferation of CD4(+) T cells from non-treated chronic mice, while it further increased the response of CD4(+) T cells from JES6-1-treated chronic mice. We conclude that JES6-1 treatment impairs the expansion of T(reg) cell population during early P. chabaudi malaria and enhances the Th1 cell response in the late phase of the disease.     

Contrasting effects of acute and chronic gastro-intestinal helminth infections on a heterologous immune response in a transgenic adoptive transfer model

We have previously found that co-immunisation with ovalbumin (OVA) and the body fluid of the helminth Ascaris suum inhibited an OVA-specific delayed type hypersensitivity (DTH) response by reducing OVA-specific CD4+ T lymphocyte proliferation via an IL-4 independent mechanism. In the present study, we determined whether parasite infections themselves could induce similar changes to peripheral immunisation by examining the modulation of OVA-specific immune responses during acute and chronic helminth infections. Surprisingly, an acute infection with Trichinella spiralis, but not a chronic infection with Heligmosomoides polygyrus, inhibited the OVA-specific DTH reaction. Correspondingly, the T helper 1 (Th1) OVA-specific response was decreased in mice infected with T. spiralis, but not with H. polygyrus. Inhibition of the Th1 response may be a result of a shift in the Th1/Th2 balance as although both H. polygyrus and T. spiralis infected mice induced a Th2 OVA-specific response, that exhibited by T. spiralis was more potent. Furthermore, although IL-10 secretion upon OVA restimulation was similarly increased by both infections, production of this immunoregulatory cytokine may play a role in the suppression of immune responses observed with T. spiralis infection depending on the context of its release. Interestingly, analysis of the OVA-specific T lymphocyte division by carboxyfluorescein diacetate succinimidyl ester (CFSE) staining revealed that gastro-intestinal infection with the acute helminth T. spiralis, but not with chronic H. polygyrus, inhibited the systemic immune response by significantly inhibiting the antigen-specific T cell proliferation during the primary response, a mechanism similar to that observed when A. suum parasite extracts were directly mixed with the OVA during immunisation in our previous studies.     

IL-4 can correct defective IgE production in SJA/9 mice.

SJA/9 mice are unable to produce significant serum IgE responses to either primary or secondary challenge with protein Ag or helminth parasites. Previous work has shown that this defect resides the T cell rather than with the B cells. These experiments test several possible explanations for the lack of IgE production in these mice. SJA/9 mice do not appear to overproduce IFN-gamma or any other antagonist of IL-4 function. Helminth parasite infection induces normal levels of IL-5-dependent eosinophilia, suggesting that there is not a complete absence of a Th2 response in these mice. The defect, however can be explained by a substantial reduction in IL-4 production in vivo and can be corrected by infusion with IL-4. The reason for this lack of IL-4 production is not yet clear and, paradoxically, SJA/9 T cells can be stimulated in vitro to produce IL-4 levels comparable to T cells from other strains.     

IL-33, a potent inducer of adaptive immunity to intestinal nematodes

IL-33 (IL-1F11) binds ST2 (IL-1R4), both of which are associated with optimal CD4(+) Th2 polarization. Exogenous IL-33 drives induction of Th2-associated cytokines and associated pathological changes within the gut mucosa. Th2 polarization is also a prerequisite to expulsion of the intestinal-dwelling nematode Trichuris muris. In this study, we demonstrate that IL-33 mRNA is expressed early during parasite infection and susceptible mice can be induced to expel the parasite by a regime of exogenous IL-33 administration. IL-33 prevents an inappropriate parasite-specific Th1-polarized response and induces IL-4, IL-9, and IL-13. This redirection requires the presence of T cells and must occur at the initiation of the response to the pathogen. Interestingly, exogenous IL-33 also induced thymic stromal lymphopoietin mRNA within the infected caecum, an epithelial cell-restricted cytokine essential for the generation of Th2-driven parasite immunity. IL-33 also acts independently of T cells, altering intestinal pathology in chronically infected SCID mice, leading to an increased crypt length and intestinal epithelial cell proliferation, but reducing goblet cell hyperplasia. Thus, the ability of IL-33 to induce Th2 responses has functional relevance in the context of intestinal helminth infection, particularly during the initiation of the response.     

Marginal zone B cells regulate antigen-specific T cell responses during infection

Marginal zone B cells (MZB) participate in the early immune response to several pathogens. In this study, we show that in μMT mice infected with Leishmania donovani, CD8 T cells displayed a greater cytotoxic potential and generated more effector memory cells compared with infected wild type mice. The frequency of parasite-specific, IFN-γ(+) CD4 T cells was also increased in μMT mice. B cells were able to capture parasites, which was associated with upregulation of surface IgM and MyD88-dependent IL-10 production. Moreover, MZB presented parasite Ags to CD4 T cells in vitro. Depletion of MZB also enhanced T cell responses and led to a decrease in the parasite burden but did not alter the generation of effector memory T cells. Thus, MZB appear to suppress protective T cell responses during the early stages of L. donovani infection.