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.

Methods for the identification of chemical respiratory allergens in rodents: comparisons of cytokine profiling with induced changes in serum IgE.

No validated or widely recognized test methods are currently available for the prospective identification of chemicals with the potential to cause respiratory allergy. The cellular and molecular mechanisms that result in the induction of chemical sensitization of the respiratory tract are unclear, although there is evidence for the selective development of T helper 2 (Th2)-type responses and, in some cases, the production of IgE antibody. We have therefore examined the utility of cytokine profiling using BALB/c mice, together with the measurement of induced increases in the total serum concentration of IgE in the Brown Norway (BN) rat, as markers for the prospective identification of chemical respiratory allergens. Responses provoked by the reference respiratory allergen trimellitic anhydride (TMA) have been compared with those stimulated by the respiratory sensitizing diisocyanates toluene diisocyanate (TDI) and hexamethylene diisocyanate (HDI) and by the acid anhydride hexahydrophthalic anhydride (HHPA). Topical exposure of BN rats to TMA, TDI and HHPA each provoked marked immune activation (increases in lymph node cellularity and proliferation). However, only treatment with TMA stimulated vigorous increases in the total serum concentration of IgE. In contrast, exposure to HHPA, TDI or HDI failed to provoke significant changes in serum IgE concentration or induced only transient and relatively weak increases in serum IgE levels. In parallel experiments using BALB/c strain mice, however, topical application of all four chemical respiratory allergens provoked a marked Th2-type cytokine secretion profile in draining lymph node cells. These data suggest that the measurement of induced changes in serum IgE is not sufficiently sensitive for the robust identification of chemical respiratory allergens. Furthermore, irrespective of the reasons for variations in TMA-induced IgE production among BN rats, doubts remain regarding the utility of these animals for the characterization of immune responses to chemical allergens. Cytokine profiling using the BALB/c strain mouse apparently provides a more robust method for the hazard assessment of chemical respiratory allergens.

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.

Beta7 integrin-deficient mice: delayed leukocyte recruitment and attenuated protective immunity in the small intestine during enteric helminth infection.

The ontogeny and function of gut-associated-lymphoid tissue is known to be critically dependent on the beta7 integrin subfamily. We have investigated the development of intestinal inflammation and pathogen-specific protective immunity to enteric helminth infection in beta7 integrin knockout (KO) mice. During Trichinella spiralis infection of the small intestine there was a significant delay and reduction in the magnitude of intestinal eosinophilia and mastocytosis in the absence of P7 integrin, resulting in impaired host protection. Aberrant distribution of mast cells was also observed in the small intestine of infected KO mice. Adoptive transfer of primed wild-type mesenteric lymph node cells into T. spiralis-infected beta7 KO mice did not restore the intestinal mast cell response, suggesting that the defect in intestinal mastocytosis is due to the absence of beta7 expression on this population rather than an indirect consequence of reduced T cell numbers. In contrast, no impairment in leukocyte recruitment or protection against Trichuris muris infection of the large intestine was observed in KO mice. Taken together the data provide the first description of reduced leukocyte homing and attenuated protective immunity against helminth infection in beta7 KO mice. Furthermore, these results suggest that beta7 integrin-independent adhesion molecule interactions are deployed in the large but not small intestine during intestinal inflammation.

Anti-IL-9 vaccination prevents worm expulsion and blood eosinophilia in Trichuris muris-infected mice.

Production of neutralizing anti-IL-9 antibodies was induced in mice by immunization with mouse IL-9 coupled to ovalbumin. In the six mouse strains tested, a strong and long-lasting anti-IL-9 response developed with seric inhibitory titers of 10(-3) to 10(-5), as measured in an in vitro IL-9-dependent cell proliferation assay. In vivo, this immunization completely abrogated the increase in mast-cell protease-1 levels as well as the eosinophilia observed in mice after implantation of an IL-9-secreting tumor. We took advantage of this method to assess the role of IL-9 in infections with nematode Trichuris muris, where IL-9 production correlates with the resistant phenotype. C57BL/6 mice, which normally expel the parasite, became susceptible after anti-IL-9 immunization, demonstrating that IL-9 plays a critical role in this model. In addition, neutralization of IL-9 also inhibited parasite-induced blood eosinophilia. Taken together, the present data demonstrate the potency of our strategy to antagonize IL-9 in vivo and shows that this cytokine plays a major role in resistance against T. muris infection.

Tumor necrosis factor alpha is a critical component of interleukin 13-mediated protective T helper cell type 2 responses during helminth infection.

In vivo manipulation of cytokine and/or cytokine receptor expression has previously shown that resistance to infection with the caecum-dwelling helminth Trichuris muris is dependent on interleukin (IL)-4 and IL-13 while susceptibility is associated with a T helper cell type 1 (Th1) cytokine response. Using gene-targeted mice deficient in tumor necrosis factor (TNF) receptor signaling and anti-TNF-alpha monoclonal antibody treatment, we have extended these studies to reveal a critical role for TNF-alpha in regulation of Th2 cytokine-mediated host protection. In vivo blockade of TNF-alpha in normally resistant mice, although not altering IL-4, IL-5, or IL-13 production in the draining lymph node, significantly delayed worm expulsion for the duration of treatment. IL-13-mediated worm expulsion in IL-4 knockout (KO) mice was also shown to be TNF-alpha dependent, and could be enhanced by administration of recombinant TNF-alpha. Furthermore, TNF receptor KO mice failed to expel T. muris, producing high levels of parasite-specific immunoglobulin G2a and the generation of a predominantly Th1 response, suggesting that the absence of TNF function from the onset of infection dramatically alters the phenotype of the response. These results provide the first demonstration of the role of TNF-alpha in regulating Th2 cytokine-mediated responses at mucosal sites, and have implications for the design of rational therapies against helminth infection and allergy.