T-bet controls intestinal mucosa immune responses via repression of type 2 innate lymphoid cell function.

Innate lymphoid cells (ILCs) play an important role in regulating immune responses at mucosal surfaces. The transcription factor T-bet is crucial for the function of ILC1s and NCR+ ILC3s and constitutive deletion of T-bet prevents the development of these subsets. Lack of T-bet in the absence of an adaptive immune system causes microbiota-dependent colitis to occur due to aberrant ILC3 responses. Thus, T-bet expression in the innate immune system has been considered to dampen pathogenic immune responses. Here, we show that T-bet plays an unexpected role in negatively regulating innate type 2 responses, in the context of an otherwise intact immune system. Selective loss of T-bet in ILCs leads to the expansion and increased activity of ILC2s, which has a functionally important impact on mucosal immunity, including enhanced protection from Trichinella spiralis infection and inflammatory colitis. Mechanistically, we show that T-bet controls the intestinal ILC pool through regulation of IL-7 receptor signalling. These data demonstrate that T-bet expression in ILCs acts as the key transcriptional checkpoint in regulating pathogenic vs. protective mucosal immune responses, which has significant implications for the understanding of the pathogenesis of inflammatory bowel diseases and intestinal infections.

Extracellular vesicles induce protective immunity against Trichuris muris.

Gastrointestinal nematodes, such as Trichuris trichiura (human whipworm), are a major source of morbidity in humans and their livestock. There is a paucity of commercially available vaccines against these parasites, and vaccine development for T. trichiura has been impeded by a lack of known host protective antigens. Experimental vaccinations with T. muris (murine whipworm) soluble Excretory/Secretory (ES) material have demonstrated that it is possible to induce protective immunity in mice; however, the potential for extracellular vesicles (EVs) as a source of antigenic material has remained relatively unexplored. Here, we demonstrate that EVs isolated from T. muris ES can induce protective immunity in mice when administered as a vaccine without adjuvant and show that the protective properties of these EVs are dependent on intact vesicles. We also identified several proteins within EV preparations that are targeted by the host antibodies following vaccination and subsequent infection with T. muris. Many of these proteins, including VWD and vitellogenin N and DUF1943-domain-containing protein, vacuolar protein sorting-associated protein 52 and TSP-1 domain-containing protein, were detected in both soluble ES and EV samples and have homologues in other parasites of medical and veterinary importance, and as such are possible protective antigens.

A sticky end for gastrointestinal helminths; the role of the mucus barrier.

Gastrointestinal (GI) nematodes are a group of successful multicellular parasites that have evolved to coexist within the intestinal niche of multiple species. It is estimated that over 10% of the world's population are chronically infected by GI nematodes, making this group of parasitic nematodes a major burden to global health. Despite the large number of affected individuals, there are few effective treatments to eradicate these infections. Research into GI nematode infections has primarily focused on defining the immunological and pathological consequences on host protection. One important but neglected aspect of host protection is mucus, and the concept that mucus is just a simple barrier is no longer tenable. In fact, mucus is a highly regulated and dynamic-secreted matrix, underpinned by a physical hydrated network of highly glycosylated mucins, which is increasingly recognized to have a key protective role against GI nematode infections. Unravelling the complex interplay between mucins, the underlying epithelium and immune cells during infection are a major challenge and are required to fully define the protective role of the mucus barrier. This review summarizes the current state of knowledge on mucins and the mucus barrier during GI nematode infections, with particular focus on murine models of infection.

Trichinella spiralis antigens prime mixed Th1/Th2 response but do not induce de novo generation of Foxp3+ T cells in vitro.

Many parasitic helminth infections induce Th2-type immune responses and engage the regulatory network. In this study, we specifically investigated the influence of antigens derived from different life stages of the helminth Trichinella spiralis on the polarization of naive CD4(+) T cells by dendritic cells. Results obtained from C57BL/6 mice showed that T. spiralis derived antigens have the capacity to induce bone marrow-derived dendritic cells to acquire an incompletely mature phenotype that promotes a significant proliferation of naive CD4(+) T cells and a mixed Th1/Th2 cytokine profile with the predominance of Th2 cytokines. Increased production of IL-4, IL-9, IL-10 and IL-13 accompanied increased IFN-γ. Furthermore, dendritic cells pulsed with T. spiralis antigens did not induce an increase in the population of Foxp3(+) T regulatory cells. Although other helminth antigens have demonstrated the capacity to induce de novo generation of Foxp3(+) T regulatory cells, here our in vitro studies provide no evidence that T. spiralis antigens have this capacity.

Changes in the mucosal barrier during acute and chronic Trichuris muris infection.

The intestinal mucosal barrier, part of the innate immune defence, is responsive to the external environment and changes in response to infection. There is disparate evidence for the epithelial and goblet cell products within the intrinsic barrier being part of a response to resolve infection. We comprehensively analysed the changes of mucosal glycoconjugates during acute and chronic infection by utilising the Trichuris muris (T. muris) model. Transcription factors, atonal homolog 1 (Math-1) and SAM pointed domain containing ETS transcription factor (Spdef) were activated during acute infection, which promoted stem cell fate towards a secretory cell phenotype. The thickness of the intermediate barrier, the carbohydrate-rich glycocalyx, composed of cell surface mucins increased with exposure to T. muris, with an increase in Muc4, Muc13 and Muc17. Overall, hypersecretion of glycoproteins into the extrinsic barrier (mediated by IL-13) via the gamma amino-butyric acid-α3 receptor (GABA-α3), was observed during acute infection. Furthermore, altered glycosylation was observed during acute and chronic infection; mucins were more highly charged during acute infection than during chronic infection. This study readdresses the changes within the mucosal barrier, in particular in the cell surface and secreted mucins during acute and chronic nematode infection.

Colonic transcriptional profiling in resistance and susceptibility to trichuriasis: phenotyping a chronic colitis and lessons for iatrogenic helminthosis.

BACKGROUND: Helminth therapy is advocated to restore and maintain control of inflammatory responses, particularly chronic colitis. However, helminths can induce chronic colitis in susceptible individuals. Susceptibility has an immunogenetic basis: defining this is essential if nematode therapy is to be successfully and safely targeted in inflammatory bowel disease (IBD). To validate a preclinical mouse model we phenotyped the response to Trichuris muris in mice. We determined colonic transcriptional activity in naïve and infected mice and linked differential gene expression to mechanistic pathways. METHODS: T. muris-infected resistant (BALB/c) and susceptible (AKR) mice were studied to a chronic colitic timepoint (day 35). Colonic genome-wide expression was performed by microarray. Significant transcriptional changes were analyzed by cluster and gene ontology filtering and KEGG pathway mapping. RESULTS: Day 35 infected AKR displayed chronic diarrhea, weight loss, and transmural colonic inflammation; BALB/c remained asymptomatic, cleared the infection, and demonstrated normal histology. Compared to BALB/c mice, infected AKR upregulated gene expression clusters were overrepresented by immune response, chemotaxis, and apoptosis pathways. Cellular/tissue homeostasis and tight junction pathways dominated downregulated AKR expression clusters. Infected AKR T-helper cell development/polarization markers demonstrated predominant T(H) 1/T(H) 17 transcriptional activity. Colitic AKR data mirrored established murine models and human colitis. CONCLUSIONS: T. muris infection in the mouse shows striking phenotypic and transcriptional similarities to widely used models of IBD and human IBD. This preclinical mouse model presents a platform to examine biological commonalities among chronic colitides. However, these data urge caution in untargeted therapeutic helminth use until risk/benefit in susceptible individuals is more fully understood.

Exploitation of the intestinal microflora by the parasitic nematode Trichuris muris.

The inhabitants of the mammalian gut are not always relatively benign commensal bacteria but may also include larger and more parasitic organisms, such as worms and protozoa. At some level, all these organisms are capable of interacting with each other. We found that successful establishment of the chronically infecting parasitic nematode Trichuris muris in the large intestine of mice is dependent on microflora and coincident with modulation of the host immune response. By reducing the number of bacteria in the host animal, we significantly reduced the number of hatched T. muris eggs. Critical interactions between bacteria (microflora) and parasites (macrofauna) introduced a new dynamic to the intestinal niche, which has fundamental implications for our current concepts of intestinal homeostasis and regulation of immunity.

The intestinal epithelium: sensors to effectors in nematode infection.

The role of the intestinal epithelium as part of the physical barrier to infection is well established alongside its central roles in food absorption, sensing nutrients, and water balance. Nematodes are one of the most common types of pathogen to dwell in the intestine. This article reviews recent data that have identified crucial roles for intestinal epithelial cells in sensing these kinds of pathogens and initiating innate responses, which qualitatively influence adaptive immune responses against them. Moreover, it is now clear that the epithelium itself--in addition to the cells that lie within it--are key to many of the protective mechanisms that result in expulsion of these large multicellular parasites from the intestine. An understanding of the IEC and intraepithelial leukocyte response is crucial to both development of mucosal vaccines, and the mechanisms that underlie the emerging use of intestinal dwelling helminths for therapeutic treatments of inflammatory and autoimmune disease.

The role of TNF-alpha in Trichuris muris infection I: influence of TNF-alpha receptor usage, gender and IL-13.

Th1 and Th2 responses to the gut-dwelling nematode Trichuris muris have been well established in mouse models of infection, with Th2 responses clearly playing an important role in resistance. TNF-alpha has previously been shown to play an undefined role in resistance, although it is not a typical Th2 cytokine. However, the relative importance of the two TNF-alpha receptors, p55 and p75, has not previously been investigated. We demonstrate that p55 is the dominant TNF-alpha receptor during T. muris infection as p55-/- mice are more susceptible to infection than p75-/- mice. Moreover, p75 clearly plays a role in negatively regulating TNF-alpha. We also demonstrate that a gender difference influences the immune response of p55-/- and p75-/- mice in response to T. muris infection, with female mice fully expelling by day 35 post-infection (p.i.) and male mice harbouring chronic infections. Further, this gender difference can be reversed with recombinant IL-13 (rIL-13) in male gene-deficient mice or IL-13R2.Fc treatment in female gene-deficient mice.

The role of TNF-alpha in Trichuris muris infection II: global enhancement of ongoing Th1 or Th2 responses.

Host resistance to Trichuris muris is driven by Th2 responses. However, TNF-alpha has also been shown to play a role in protection. As TNF-alpha has a variety of actions, the exact role of TNF-alpha in immunity to T. muris is yet to be established. Here we demonstrate that although blocking TNF-alpha has been shown to abrogate resistance, rTNF-alpha treatment does not promote resistance. Further, we show that TNF-alpha functions to enhance the ongoing immune response. AKR animals that typically respond to infection with a polarized Th1 response produce greater levels of Th1 cytokines when treated with TNF-alpha and BALB/c animals that normally respond with a polarized Th2 response produce higher levels of Th2 cytokines. Crucially, blocking TNF-alpha in the strong Th2 responder strain BALB/c does not prevent expulsion of T. muris, thus supporting its role as a biological enhancer. TNF-alpha does increase transcription of both IFN-gamma and IL-13 in vitro but can also act synergistically with IL-13 in vitro to promote production of RELMbeta, which has also been shown to play a role in resistance to T. muris. Thus, this data demonstrates that TNF-alpha acts to enhance an ongoing immune response but is not necessary for a strong protective Th2 response.

Immune control of food intake: enteroendocrine cells are regulated by CD4+ T lymphocytes during small intestinal inflammation.

BACKGROUND AND AIMS: Gastrointestinal inflammation reduces food intake but the biological mechanisms explaining suppressed feeding during inflammation are unknown. We have used a model of upper gut infection (Trichinella spiralis in the mouse) to study the effect of inflammation on food intake, and explored the role of a key enteroendocrine cell (EEC) in the regulation of feeding by the immune response. METHODS: Food intake of NIH mice infected with the intestinal nematode Trichinella spiralis was measured. Duodenal cholecystokinin (CCK) cells were counted. Plasma CCK was measured. Infected mice were treated with a specific CCK1 receptor antagonist, and food intake reassessed. The influence of the immune response on food intake and CCK was mechanistically examined by treating mice with CD4 or mast cell neutralising antibodies. The role of the T helper 2 response was further explored in mice genetically deficient for interleukin (IL)-4, IL-13, or IL-4Ralpha (receptor alpha subunit). RESULTS: Food intake of infected mice was significantly reduced at the temporal peak of intestinal inflammation. CCK expressing EEC were upregulated in infected mice, and plasma CCK levels were increased. A CCK1 receptor antagonist restored the food intake of infected mice to a significant degree. Furthermore, suppression of food intake was completely abolished in the absence of CD4+ T lymphocytes or IL-4Ralpha. CONCLUSIONS: The data show for the first time that intestinal inflammation results in reduced food intake due to upregulation of CCK. Moreover, following infection, food intake and CCK expressing cells are under the specific control of CD4+ T-cells, via release of IL-4 and IL-13.

Cytokine response profiles predict species-specific infection patterns in human GI nematodes.

This study investigated associations between pre-treatment cytokine expression and infection patterns, before and after de-worming, in humans exposed to two gastrointestinal nematode species. Quantitative measures of Ascaris lumbricoides and Trichuris trichiura infection (based on faecal egg counts) were estimated immediately before and 8-9 months after treatment in a Cameroonian population. Whole blood cytokine responses to parasite-derived antigens were assayed immediately pre-treatment. An overall measure of the tendency towards species-specific infection (increasing with A. lumbricoides faecal egg counts and decreasing with T. trichiura faecal egg counts) was significantly positively related to IL-10 levels in older (14-57 year) hosts. There was a significant negative influence of IL-5 on reinfection probability in T. trichiura but not A. lumbricoides. This effect coincided with reduced reinfection success in T. trichiura compared to A. lumbricoides. T(H)2 cytokine expression by younger hosts (4-13 year) was negatively associated with contemporary A. lumbricoides faecal egg counts before treatment. Following treatment, the pre-treatment T(H)2 cytokine expression data for younger hosts (now reflecting responsiveness 8-9 months in the past) were negatively associated with T. trichiura faecal egg counts. Taken together, these observations suggest a successional interaction between T(H)2-driven immune responses and species infection over time. However, any differential effects of the measured immune responses on species-specific recruitment, maturation and mortality were superimposed upon (and outweighed by) the effects of other factors favouring coinfection.

The role of CD8+ cells in the establishment and maintenance of a Trichuris muris infection.

Chronic infection by the caecal-dwelling intestinal murine nematode Trichuris muris occurs if given as a high-dose infection to 'susceptible' AKR mice or as a low-dose infection to the normally 'resistant' C57BL/6 mouse strain. Both regimes result in a type 1 cytokine response, i.e. high levels of IFN-gamma and IL-12. Here we show this susceptible response is associated with a large population of CD8(+) IFN-gamma(+) cells within the mesenteric lymph nodes and numerous CD8(+) cells infiltrating the caecal mucosa. Despite this, the in vivo abolition of CD8(+) cells within AKR and C57BL/6 mice, either prior to infection or once infection has become established, failed to affect chronicity, implying that CD8(+) T cells are not essential for the initiation or maintenance of the susceptible response to T. muris. Interestingly, the percentage of IFN-gamma(+) CD4(+) cells increased in treated groups, perhaps in a compensatory role. The majority of antigen-specific cytokine responses were comparable in both treated and control groups, although IL-5 was fivefold higher in animals receiving anti-CD8 mAbs and IFN-gamma was also raised in treated mice. Mastocytosis was unaltered by CD8 depletion, however, paradoxically, eosinophilia within the caecum was reduced in treated mice. Together these data clearly demonstrate that CD8(+) T cells are associated with chronic T. muris infection; however, these cells are dispensable for both the early and late phases of this response, but do appear to play a role in the regulation of certain cytokines and caecal eosinophilia.

Modulation of intestinal muscle contraction by interleukin-9 (IL-9) or IL-9 neutralization: correlation with worm expulsion in murine nematode infections.

Immune responses associated with intestinal nematode infections are characterized by the activation of T-helper 2 (Th2) cells. Previous studies demonstrated that during Trichinella spiralis infection, Th2 cells contribute to the development of intestinal muscle hypercontractility and to worm eviction from the gut, in part through signal transducer and activator of transcription factor 6 (Stat6). Interleukin-9 (IL-9), a Th2-cell-derived cytokine, has pleiotropic activities on various cells that are not mediated through Stat6. In this study, we investigated the role of IL-9 in the generation of enteric muscle hypercontractility in mice infected with the intestinal parasite T. spiralis and the cecal parasite Trichuris muris. Treatment of mice with IL-9 enhanced infection-induced jejunal muscle hypercontractility and accelerated worm expulsion in T. spiralis infection. These effects were associated with an up-regulation of IL-4 and IL-13 production from in vitro-stimulated spleen cells. In addition, increases in the level of intestinal goblet cells and in the level of mouse mucosal mast cell protease 1 (MMCP-1) in serum were observed in infected mice following IL-9 administration. However, the neutralization of IL-9 by anti-IL-9 vaccination or by anti-IL-9 antibody had no significant effect on worm expulsion or muscle contraction in T. spiralis-infected mice. In contrast, the neutralization of IL-9 significantly attenuated T. muris infection-induced colonic muscle hypercontractility and inhibited worm expulsion. The attenuated expulsion of the parasite by IL-9 neutralization was not accompanied by changes in goblet cell hyperplasia or the MMCP-1 level. These findings suggest that IL-9 contributes to intestinal muscle function and to host protective immunity and that its importance and contribution may differ depending on the type of nematode infection.

The effect of challenge and trickle Trichuris muris infections on the polarisation of the immune response.

In the field, determination of mechanisms of immunity to geohelminths are problematic due to the variation in infection exposure, host genetics, nutrition and co-infection. This study uses a well defined laboratory model, Trichuris muris in the mouse to study immune responses to challenge and trickle infections. The rationale is thus to study parasite acquisition under more natural antigen dose exposure. Antigen dose has previously been shown in this system to affect the outcome of infection with low antigen doses favouring type 1 responses (and susceptibility) and high antigen doses favouring type 2 responses (and resistance). A high level challenge infection could be established in a normally resistant host but only following priming of the immune response by a low level infection. Once type 2 responses were initiated it was impossible to switch an ongoing type 2 response even using IL-12 which is a potent stimulus of type 1 responses. Trickle infections resulted in no clear polarisation of the immune response. It was possible to build up the level of infection to a threshold level beyond which type 2 responses and expulsion were initiated. This threshold level was dependent upon host genetic background. Our results reveal a complex spectrum of responses and demonstrate that resistance and type 2 responses can be built up with increasing parasite exposure. The data provide compelling evidence to support a role for acquisition of acquired immunity to gastro-intestinal nematodes under complex infection patterns such as those found in the field.

Leucocyte recruitment during enteric nematode infection.

Resolution of infection with the intestinal nematode Trichinella spiralis depends on the host mounting a T helper 2 (Th2) response. It is known that both mast cells and T cells play a crucial role. We have previously shown that efficient migration of mast cells to the gut during infection depends on their expression of the integrin beta 7. beta 7 forms a heterodimer complex with either alpha E or alpha 4 integrin chains, alpha E beta 7 binding to E-cadherin expressed by epithelial cells and alpha 4 beta 7 binding to mucosal addressin cell adhesion molecule (MAdCAM-1) on the endothelium. We were interested to know whether dysfunctional mast cell localization to the gut in the absence of beta 7 was due to the failure of alpha 4 beta 7 to bind to MAdCAM-1 or the failure of alpha E beta 7 to bind to E-cadherin. We used blocking monoclonal antibodies against alpha E (M290) or alpha 4 (PS2) or beta 7 (HB293) during T. spiralis infection of C57BL/6 mice and found that all antibody treatments reduced mastocytosis. In contrast, none of the antibody treatments prevented the migration of CD3(+) T cells into the intestine. These results indicate that during inflammation (a) there is integrin redundancy for lymphocytes but not for mast cells and (b) both alpha E beta 7 and alpha 4 beta 7 are crucial either for the entry of mast cells into the gut or for their maturation once they have entered.

Interleukin (IL)-18 promotes the development of chronic gastrointestinal helminth infection by downregulating IL-13.

Expulsion of the gastrointestinal nematode Trichuris muris is mediated by a T helper (Th) 2 type response involving interleukin (IL)-4 and IL-13. Here we show that Th1 response-associated susceptibility involves prior activation of IL-18 and caspase-1 followed by IL-12 and interferon (IFN)-gamma in the intestine. IL-18-deficient mice are highly resistant to chronic T. muris infection and in vivo treatment of normal mice with recombinant (r)IL-18 suppresses IL-13 and IL-4 secretion but does not affect IFN-gamma. In vivo treatment of T. muris-infected IFN-gamma-deficient mice with rIL-18 demonstrated that the inhibitory effect of IL-18 on IL-13 secretion is independent of IFN-gamma. Hence, IL-18 does not function as an IFN-gamma-inducing cytokine during chronic T. muris infection but rather as a direct regulator of Th2 cytokines. These results provide the first demonstration of the critical role of IL-18 in regulating Th cell responses during gastrointestinal nematode infection.

Cytokine regulation of resistance and susceptibility to intestinal nematode infection - from host to parasite.

Intestinal nematode infection is one of the most common forms of parasite infection worldwide. Both man and domestic stock suffer considerable morbidity from these infectious agents. The majority of our current understanding of the host parasite relationship to gut dwelling nematodes comes from well-defined laboratory models. One of the most informative over recent years has been study of whipworm infection in the mouse, Trichuris muris (T. muris). Infection in inbred strains of mouse shows a spectrum of response phenotypes reflecting the variation observed under natural conditions in the wild. Resistance and worm expulsion is mediated by CD4+ T helper two cells with a dominant role for interleukin (IL)-13. The effector mechanisms responsible for worm expulsion remain undefined but new evidence suggests a role for tumour necrosis factor alpha (TNF-alpha). Susceptibility to chronic infection is mediated through a T helper 1 (Th1) response characterised through the secretion of interferon gamma (IFN-gamma). A major new role for IL-18 has been defined in induction of a Th1 response through a novel down-regulation of IL-13. Moreover, progression to chronic infection may involve the parasite itself. T. muris secretes a protein that shares epitopes with host IFN-gamma, which may interfere with host protective cytokine, mediated protection and thus, promotes its own survival.

Expression of interleukin-9 leads to Th2 cytokine-dominated responses and fatal enteropathy in mice with chronic Schistosoma mansoni infections.

Mice infected with Schistosoma mansoni develop Th2 cytokine-mediated granulomatous pathology that is focused on the liver and intestines. In this study, transgenic mice constitutively expressing IL-9 were infected with S. mansoni and the outcome of infection was determined. Eight weeks after infection, transgenic mice with acute infections had a moderate increase in Th2 cytokine production but were overtly normal with respect to parasite infection and pathological responses. Transgenic mice with chronic infections died 10 weeks after infection, with 86% of transgenic mice dead by week 12 of infection, compared to 7% mortality in infected wild-type mice. Stimulation of mesenteric lymph node cells from infected transgenic mice with parasite antigen elicited elevated interleukin-4 (IL-4) and IL-5 production and reduced gamma interferon and tumor necrosis factor alpha production compared to the responses in wild-type mice. Morbid transgenic mice had substantial enlargement of the ileum, which was associated with muscular hypertrophy, mastocytosis, eosinophilia, goblet cell hyperplasia, and increased mucin expression. We also observed that uninfected transgenic mice exhibited alterations in their intestines. Although there was hepatic mastocytosis and eosinophilia in infected transgenic mice, there was no hepatocyte damage. Death of transgenic mice expressing IL-9 during schistosome infection was primarily associated with enteropathy. This study highlights the pleiotropic in vivo activity of IL-9 and demonstrates that an elevated Th2 cytokine phenotype leads to death during murine schistosome infection.

Gastrointestinal nematode expulsion in IL-4 knockout mice is IL-13 dependent.

Female IL-4 knockout (KO) mice on a C57BL/6 background (F4KOC57) are susceptible to infection with the cecal-dwelling nematode Trichuris muris whereas wild-type C57BL/6 mice are resistant and expel the parasite. In this study we show that in sharp contrast, female IL-4 KO mice on a BALB/c background (F4KOB/c) are resistant to infection as are wild-type BALB/c mice. Although susceptible F4KOC57 make negligible levels of all type 2 cytokines, resistant F4KOB/c were capable of producing significant levels of antigen-specific IL-13 (a cytokine shown to be critical in resistance to T. muris). To examine if the IL-13 in F4KOB/c mice was of functional importance, it was neutralized in vivo using a fusion protein, A25 (sIL-13 R.Fc). The results presented here clearly demonstrate that neutralization of IL-13 in vivo did indeed prevent T. muris expulsion in normally resistant F4KOB/c mice. In addition, administration of recombinant mouse IL-13 to normally susceptible male IL-4KO BALB/c mice (M4KOB/c) caused an 87.85 % reduction in worm burden. Collectively, these data show that IL-13 is important in the poorly understood effector mechanisms resulting in the expulsion of T. muris from the gut. Moreover, the present data highlight the functional importance of gender and background strain in interpretation of studies using gene-targeted animals.