STAT6 Expression and IL-13 Production in Association with Goblet Cell Hyperplasia and Worm Expulsion of Gymnophalloides seoi from C57BL/6 Mice

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.     

Stat6 signaling promotes protective immunity against Trichinella spiralis through a mast cell- and T cell-dependent mechanism

Studies in mice infected with the gastrointestinal nematode parasite Nippostrongylus brasiliensis demonstrated that IL-4/IL-13 activation of Stat6 suppresses development of intestinal mastocytosis and does not contribute to IL-4/IL-13 production, but is still essential for parasite expulsion. Because expulsion of another gastrointestinal nematode, Trichinella spiralis, unlike N. brasiliensis expulsion, is mast cell dependent, these observations suggested that T. spiralis expulsion would be Stat6 independent. Instead, we find that Stat6 activation by IL-4/IL-13 is required in T. spiralis-infected mice for the mast cell responses that induce worm expulsion and for the cytokine responses that induce intestinal mastocytosis. Furthermore, although IL-4 induces N. brasiliensis expulsion in the absence of B cells, T cells, and mast cells, mast cells and T cells are required for IL-4 induction of T. spiralis expulsion. Thus, Stat6 signaling is required for host protection against N. brasiliensis and T. spiralis but contributes to expulsion of these two worms by different mechanisms. The induction of multiple effector mechanisms by Stat6 signaling provides a way for a cytokine response induced by most gastrointestinal nematode parasites to protect against most of these parasites, even though different effector mechanisms are required for protection against different nematodes.     

Nippostrongylus brasiliensis: intestinal goblet-cell response in adoptively immunized rats.

Goblet-cell differentiation was studied in the intestinal epithelium of rats infected with the nematode Nippostrongylus brasiliensis. An increase in the proportion of goblet cells occurred at the time of worm expulsion in rats infected with 1000 or 4000 third stage larvae. Adoptive immunization of infected rats with immune-thoracic duct lymphocytes (TDL) induced extensive goblet-cell differentiation whereas the transfer of immune-TDL into normal rats had no effect. The extent of goblet-cell differentiation in adoptively immunized infected rats was proportional to the number of cells transferred. A goblet-cell response also occurred in adoptively immunized rats harboring implanted “normal” and “damaged” worms but recipients of normal worms which were not given cells were unable either to expel their worm burden or to induce a goblet-cell response. Experiments in which the parasites were expelled with an anthelmintic drug suggested that the goblet-cell increase was not simply a repair process associated with the expulsion of the parasites. In all situations where immune expulsion of the parasites occurred, there was a concomitant rise in the proportion of goblet cells. These experiments suggest that thoracic duct lymphocytes either directly or indirectly regulate the differentiation of intestinal goblet cells.     

Deficiency in Th2 Cytokine Responses Exacerbate Orthopoxvirus Infection

Ectromelia virus (ECTV) causes mousepox in mice, a disease very similar to smallpox in humans. ECTV and variola virus (VARV), the agent of smallpox, are closely related orthopoxviruses. Mousepox is an excellent small animal model to study the genetic and immunologic basis for resistance and susceptibility of humans to smallpox. Resistance to mousepox is dependent on a strong polarized type 1 immune response, associated with robust natural killer (NK) cell, cytotoxic T lymphocyte (CTL) and gamma interferon (IFN-γ) responses. In contrast, ECTV-susceptible mice generate a type 2 response, associated with weak NK cell, CTL and IFN-γ responses but robust IL-4 responses. Nonetheless, susceptible strains infected with mutant ECTV lacking virus-encoded IFN-γ binding protein (vIFN-γbp) (ECTV-IFN-γbp^Δ) control virus replication through generation of type 1 response. Since the IL-4/IL-13/STAT-6 signaling pathways polarize type 2/T helper 2 (Th2) responses with a corresponding suppression of IFN-γ production, we investigated whether the combined absence of vIFN-γbp, and one or more host genes involved in Th2 response development, influence generation of protective immunity. Most mutant mouse strains infected with wild-type (WT) virus succumbed to disease more rapidly than WT animals. Conversely, the disease outcome was significantly improved in WT mice infected with ECTV-IFN-γbp^Δ but absence of IL-4/IL-13/STAT-6 signaling pathways did not provide any added advantage. Deficiency in IL-13 or STAT-6 resulted in defective CTL responses, higher mortality rates and accelerated deaths. Deficiencies in IL-4/IL-13/STAT-6 signaling pathways significantly reduced the numbers of IFN-γ producing CD4 and CD8 T cells, indicating an absence of a switch to a Th1-like response. Factors contributing to susceptibility or resistance to mousepox are far more complex than a balance between Th1 and Th2 responses.     

Acetic acid induced ulceration in rats is not affected by infection with Hymenolepis diminuta

Analysis of rodent models of inflammatory bowel disease, airways hyper-reactivity, diabetes, and multiple sclerosis has shown that infection with helminth parasites can significantly reduce the severity of the disease. Here, we assessed whether rats infected with the tapeworm Hymenolepis diminuta were protected from gastric ulceration induced by the serosal application of acetic acid. All rats gavaged with infective cysticercoids harbored adult worms when assessed 6 wk later, and acetic acid evoked the expected gastric ulceration. However, infection with H. diminuta did not affect the degree of gastric ulceration at either 3 or 7 days post-acetic acid application, as gauged by ulcer area or histopathology. While the data do not dismiss the possibility that infection with other helminths could be anti-ulcerogenic, they illustrate that 'helminth therapy' for inflammatory disease is likely to be both disease- and helminth-specific.     

Helminth-modified pulmonary immune response protects mice from allergen-induced airway hyperresponsiveness

It has been shown that the presence of certain helminth infections in humans, including schistosomes, may reduce the propensity to develop allergies in infected populations. Using a mouse model of schistosome worm vs worm + egg infection, our objective was to dissect the mechanisms underlying the inverse relationship between helminth infections and allergies. We have demonstrated that conventional Schistosoma mansoni egg-laying male and female worm infection of mice exacerbates airway hyperresponsiveness. In contrast, mice infected with only schistosome male worms, precluding egg production, were protected from OVA-induced airway hyperresponsiveness. Worm-infected mice developed a novel modified type 2 cytokine response in the lungs, with elevated allergen-specific IL-4 and IL-13 but reduced IL-5, and increased IL-10. Although schistosome worm-only infection is a laboratory model, these data illustrate the complexity of schistosome modulation of host immunity by the worm vs egg stages of this helminth, with the potential of infections to aggravate or suppress allergic pulmonary inflammation. Thus, infection of mice with a human parasitic worm can result in reduced airway inflammation in response to a model allergen.     

Mucosal mast cell response to Hymenolepis diminuta infection in different rat strains.

Five-week-old DA male rats infected with 10 Hymenolepis diminuta cysticercoids showed significant mastocytosis 6 weeks post-infection and low persistence of worms. In F344/N rats, however, no mastocytosis and no worm loss occurred during a 6 week infection. Mucosal mast cells appear to be associated with the expulsion of H. diminuta from DA rats.     

Characterization of the immuno-regulatory response to the tapeworm Hymenolepis diminuta in the non-permissive mouse host

Hymenolepis diminuta is spontaneously expelled from mice; concomitant with worm expulsion was protection against colitis induced by dinitrobenzene sulphonic acid (DNBS). Here we examined the immune response mobilized by Balb/c and C57Bl/6 male mice in response to H. diminuta and assessed the requirement for CD4+ cells (predominantly T cells) in worm expulsion and the anti-colitic effect. Wild-type (CD4+) or CD4 knock-out (CD4-/-) mice received five H. diminuta cysticercoids and segments of jejunum and mesenteric lymph nodes (MLNs), or spleen, were excised 5, 8 and 1l days later for mRNA analysis and cytokine production, respectively. In separate experiments uninfected and infected mice received DNBS by intra-rectal infusion and indices of inflammation were assessed 3 days later (i.e. 11 days p.i.). Infection of Balb/c mice resulted in a time-dependent increase in intestinal mRNA for Foxp3, a marker of natural regulatory T cells, and markers of alternatively activated macrophages (arginase-1, FIZZ1), while concanavalin-A activation of MLN cells revealed a significant increase in T helper 2 (TH2) type cytokines: IL-4, -5, -9, -10, -13. MLN cells showed a reduced ability to induce Foxp3 expression upon stimulation. CD4-/- mice did not display this response to infection, but surprisingly did expel H. diminuta. Moreover, DNBS-induced colitis in CD4-/- mice (wasting, tissue damage, elevated myeloperoxidase) was not reduced by H. diminuta infection, whereas time-matched infected CD4+ C57Bl/6 mice had significantly less DNBS-induced inflammation. In conclusion: (i) in addition to stereotypical TH2 events, H. diminuta-infected Balb/c mice develop a local immuno-regulatory response; and (ii) CD4+ cells are not essential for H. diminuta expulsion from mice but are critical in mediating the anti-colitic effect that accompanies infection in this model.     

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.     

IL-18 regulates intestinal mastocytosis and Th2 cytokine production independently of IFN-gamma during Trichinella spiralis infection

Expulsion of the gastrointestinal nematode Trichinella spiralis is associated with pronounced mastocytosis mediated by a Th2-type response involving IL-4, IL-10, and IL-13. Here we demonstrate that IL-18 is a key negative regulator of protective immune responses against T. spiralis in vivo. IL-18 knockout mice are highly resistant to T. spiralis infection, expel the worms rapidly and subsequently develop low levels of encysted muscle larvae. The increased speed of expulsion is correlated with high numbers of mucosal mast cells and an increase in IL-13 and IL-10 secretion. When normal mice were treated with rIL-18 in vivo, worm expulsion was notably delayed, and the development of mastocytosis and Th2 cytokine production was significantly reduced. The treatment had no effect on intestinal eosinophilia or goblet cell hyperplasia but specifically inhibited the development of mastocytosis. Addition of rIL-18 to in vitro cultures of bone marrow-derived mast cells resulted in a significant reduction in cell yields as well as in the number of IL-4-secreting mast cells. In vivo treatment of T. spiralis-infected IFN-gamma knockout mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on mastocytosis and Th2 cytokine secretion is independent of IFN-gamma. Hence, IL-18 plays a significant biological role as a negative regulator of intestinal mast cell responses and may promote the survival of intestinal parasites in vivo.     

The IL-27 receptor (WSX-1) is an inhibitor of innate and adaptive elements of type 2 immunity

Although previous studies have investigated the role of IL-27/WSX-1 interactions in the regulation of Th1 responses, little is known about their role in regulating Th2-type responses. Studies presented in this work identify a direct role for IL-27/WSX-1 interactions in the negative regulation of type 2 responses independent of effects on type 1 cytokines. WSX-1-/- mice infected with the gastrointestinal helminth Trichuris muris displayed accelerated expulsion of parasites and the development of exaggerated goblet cell hyperplasia and mastocytosis in the gut due to increased production of Th2 cytokines. Enhanced mast cell activity in the absence of WSX-1 was consistent with the ability of wild-type mast cells to express this receptor. In addition, IL-27 directly suppressed CD4+ T cell proliferation and Th2 cytokine production. Together, these studies identify a novel role for IL-27/WSX-1 in limiting innate and adaptive components of type 2 immunity at mucosal sites.     

Schistosome-infected IL-4 receptor knockout (KO) mice, in contrast to IL-4 KO mice, fail to develop granulomatous pathology while maintaining the same lymphokine expression profile.

Th2 lymphocytes have been postulated to play a major role in the immunopathology induced by Schistosoma mansoni infection. Nevertheless, infected IL-4 knockout (KO) and wild-type (wt) mice develop egg granulomas comparable in size. To further investigate the function of the Th2 response in egg pathology we studied IL-4Ralpha-deficient mice, which are nonresponsive to both IL-4 and IL-13. In striking contrast to IL-4 KO animals, infected IL-4Ralpha KO mice developed only minimal hepatic granulomas and fibrosis despite the presence of CD3+ T cells in the residual egg lesions. Moreover, liver lymphokine mRNA levels in these animals and IL-4 KO mice were equivalent. In addition, infected IL-4Ralpha-deficient, IL-4-deficient, and wt animals developed similar egg Ag-specific IgG Ab titers, arguing that CD4-dependent Th activity is intact in KO mice. As expected, IFN-gamma secretion was strongly up-regulated in mesenteric lymph node cultures from both groups of deficient animals, a change reflected in increased serum IgG2a and IgG2b Ab levels. Surprisingly, Th2 cytokine production in infected IL-4Ralpha KO mice was not abolished but was only reduced and resembled that previously documented in IL-4 KO animals. This residual Th2 response is likely to explain the ability of IL-4 KO mice to generate egg granulomas, which cannot be formed in IL-4Ralpha-deficient animals because of their lack of responsiveness to the same cytokine ligands. Taken together, these findings argue that tissue pathology in schistosomiasis requires, in addition to egg-specific CD4+ lymphocytes, a previously unrecognized IL-4Ralpha+ non-T cell effector population.     

An essential role for TH2-type responses in limiting acute tissue damage during experimental helminth infection

Helminths induce potent T helper 2 (TH2)-type immune responses that can mediate worm expulsion, but the role of this response in controlling the acute tissue damage caused by migrating multicellular parasites through vital tissues remains uncertain. We used a helminth infection model in which parasitic nematode larvae migrate transiently through the lung, resulting in hemorrhage and inflammation. We found that IL-17 initially contributed to inflammation and lung damage, whereas subsequent IL-4 receptor (IL-4R) signaling reduced elevations in IL-17 mRNA levels, enhanced the expression of insulin-like growth factor 1 (IGF-1) and IL-10 and stimulated the development of M2 macrophages, all of which contributed to the rapid resolution of tissue damage. These studies indicate an essential role for TH2-type immune responses in mediating acute wound healing during helminth infection.     

Disruption of CD40-CD40 ligand pathway inhibits the development of intestinal muscle hypercontractility and protective immunity in nematode infection

In our previous studies, we demonstrated that during Trichinella spiralis infection, T helper (Th) 2 cells contribute to the development of intestinal muscle hypercontractility and worm expulsion from the gut via STAT6. In addition, we have linked the altered muscle contractility to the eviction of the parasite and thereby to the host defense. However, the initial events linking infection to the development of muscle hypercontractility are poorly understood. In this study, we examined the contribution of CD40-CD40 ligand (CD40L) interaction in the development of intestinal muscle hypercontractility, in monocyte chemoattractant protein-1 (MCP-1) production, and in the Th2 response in CD40 ligand-deficient (CD40L -/-) mice infected with T. spiralis. Expulsion of intestinal worms was substantially delayed in CD40L -/- mice compared with the wild-type mice after T. spiralis infection. Consistent with delayed worm expulsion, there was a significant attenuation of intestinal muscle contractility in CD40L -/- mice. Infected CD40L -/- mice also exhibited marked impairment in the production of MCP-1, IL-4, IL-13, IgG1, IgE, and mouse mucosal MCP 1 (MMCP-1), and in goblet cell response. These results demonstrate that CD40-CD40 ligand interaction plays an important role in MCP-1 production, Th2 response, intestinal muscle hypercontractility, and worm expulsion in nematode infection. The present data suggest that the early events leading to the generation of Th2 response include CD40-CD40 ligand interaction, which subsequently influences the production of Th2 cytokines, most likely via upregulation of MCP-1.     

Neutralizing anti-IL-10 antibody blocks the protective effect of tapeworm infection in a murine model of chemically induced colitis

There is increasing evidence that parasitic helminth infection has the ability to ameliorate other disease conditions. In this study the ability of the rat tapeworm, Hymenolepis diminuta, to modulate dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice is assessed. Mice receiving DNBS (3 mg intrarectally) developed colitis by 72 h after treatment. Mice infected 8 days before DNBS with five H. diminuta larvae were significantly protected from the colitis, as gauged by reduced clinical disease, histological damage scores, and myeloperoxidase levels. This anticolitic effect was dependent on a viable infection and helminth rejection, because no benefit was observed in mice given killed larvae or in infected STAT6 knockout mice or rats, neither of which eliminate H. diminuta. The anticolitic effect of H. diminuta was associated with increased colonic IL-10 mRNA and stimulated splenocytes from H. diminuta- plus DNBS-treated mice produced more IL-10 than splenocytes from DNBS-only treated mice. Coadministration of an anti-IL-10 Ab blocked the anticolitic effect of prophylactic H. diminuta infection. Also, mice infected 48 h after DNBS treatment showed an enhanced recovery response. Finally, using a model of OVA hypersensitivity, we found no evidence of concomitant H. diminuta infection enhancing enteric responsiveness to subsequent ex vivo OVA challenge. The data show that a viable infection of H. diminuta in a nonpermissive system exerts a profound anticolitic effect (both prophylactically and as a treatment) that is mediated at least in part via IL-10 and does not predispose to enhanced sensitivity to bystander proteins.     

Trichinella spiralis: mediation of the intestinal component of protective immunity in the rat by multiple, phase-specific, antiparasitic responses.

Rats infected orally with Trichinella spiralis developed an immunity that was induced by and expressed against separate phases of the parasite's enteral life cycle. Infectious muscle larvae generated an immune response (rapid expulsion) that was directed against the very early intestinal infection and resulted in the expulsion of worms within 24 hr. This response eliminated more than 95% of worms in an oral challenge inoculum. Developing larvae (preadults) also induced an immune response that was expressed against adult worms. The effect on adults was dependent upon continuous exposure of worms to the immune environment throughout their enteral larval development. Immunity induced by preadult T. spiralis was not expressed against adult worms transferred from nonimmune rats. While adult worms were resistant to the immunity engendered by preadults they induced an efficient immunity that was autospecific. Both “preadult” and “adult” immunities were expressed in depression of worm fecundity as well as in the expulsion of adults from the gut. However, the two reactions differed in respect to their kinetics and their efficiency against various worm burdens. Preadult immunity was directed mainly against fecundity whereas adult immunity favored worm expulsion. All responses (rapid expulsion, preadult and adult immunity, and antifecundity) acted synergistically to produce sterile immunity against challenge infections of up to 5000 muscle larvae. These findings indicate that the host protective response to T. spiralis is a complex, multifactorial process that operates sequentially and synergistically to protect the host against reinfection.     

Strongyloides ratti: Mast cell and goblet cell responses in the small intestine of infected rats

Kinetics of intestinal mast cells and goblet cells were examined in relation to worm localization at various sites in the small intestine of rats infected with 3000 filariform (stage 3) larvae of Strongyloides ratti. The most marked intestinal mastocytosis was observed on Day 20 at the anterior site of the small intestine where the majority of the worms had concentrated. The number of mast cells in the posterior small intestine increased in parallel with the posterior shift of parasites at the later stage of the infection. In contrast to the intestinal mast cell response, the number of goblet cells was not significantly affected by the infection. These results strongly suggest that intestinal mastocytosis is closely related to the presence of the worms and that mast cells may play an important role for the expulsion of S. ratti.     

Immune-mediated signaling in intestinal goblet cells via PI3-kinase- and AKT-dependent pathways

In the intestinal epithelium, activation of phosphatidylinositol 3-kinase (PI3-kinase)/AKT pathways, via growth factor-mediated signaling, has been shown to regulate cell proliferation and inhibit apoptosis. An immune-activated receptor critical for Th2 immune responses, IL-4Ralpha can also activate PI3-kinase via insulin receptor substrate (IRS)-dependent signaling. Here, using the intestinal goblet cell-specific gene RELMbeta, we investigated the effect of PI3-kinase activation via Th2 immune responses on the goblet cell phenotype. IL-13 stimulation activated PI3-kinase and AKT signal transduction in LS174T cells. Not only did pharmacological inhibition of PI3-kinase and AKT1/2 inhibit RELMbeta induction by IL-13, but AKT inhibition also significantly reduced constitutive basal expression of RELMbeta, a response reproduced by the simultaneous pharmacological inhibition of both epidermal growth factor receptor and IGF-I receptor signaling. In vivo, the disruption of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), an inhibitor of PI3-kinase activation, led to the activation of RELMbeta expression in the small intestine. Furthermore, induction of an intestinal Th2 immune response by infection with a small intestinal nematode parasite, Heligmosomoides polygyrus, led to enhanced epithelial cell proliferation, activation of AKT as demonstrated by the loss of Foxo1 nuclear localization, and robust induction of RELMbeta expression in wild-type, but not IL-4Ralpha knockout, mice. These results demonstrate that Th2 immune responses can regulate goblet cell responses by activation of PI3-kinase and AKT pathways via IL-4Ralpha.