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

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Abstract

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.

Introduction

Analysis of the mammalian response to infection with parasitic helminths has played a major role in unraveling immune events and effector mechanisms against extracellular parasites. Infection with parasitic helminths is a potent immune stimulus, and with a few life-cycle-specific exceptions, there is virtually universal agreement that the immune reaction mobilized against helminths is dominated by CD4+ T helper cell type-2 (TH2) cytokines (e.g. IL-4, -5 and -13) which mobilize and activate a battery of cell types (e.g. mast cells, eosinophils, goblet cells, B cells) and effector molecules (e.g. IgE, IgG1, complement) aimed at eradicating and/or destroying the parasite (Maizels and Holland, 1998, Finkelman et al., 1997, Hayes et al., 2004). However, with a few notable platyhelminth exceptions, particularly Schistosoma mansoni (Contigli et al., 1999), Taenia crassiceps (Rodriguez-Sosa et al., 2004) and Echinococcus spp. (Walker et al., 2004), elucidation of the immune response to helminths has been based on nematode-infected laboratory rodents or assessment of serum or circulating immune cells in nematode-infected humans (Artis, 2006).

In addition to immune effector cells, there are substantial data in support of a variety of immune cell phenotypes whose primary role is immunoregulatory and the prevention of disproportionate immune reactivity. Central amongst these are regulatory T cells (Tregs), classified either as natural (i.e. CD4+/CD25+/Foxp3+) or induced (TH3, Tr1) Tregs, but regulatory B cells and an alternatively activated macrophage (AAM) phenotype (arginase-1+, galactose-type C-lectin+, FIZZ1+, Ym1+) have also been implicated in homeostasis and tissue repair rather than inflammatory reactions (Allez and Mayer, 2004, Nair et al., 2005, Velazquez et al., 2005). Again, assessment of helminth-infected rodents has been important in the identification and characterization of these immuno-regulatory cells (Elliott et al., 2005). The induction of regulatory immune cells as a consequence of helminth infection is intriguing, both as a component of the hygiene hypothesis and as an explanation for the observation that helminth-infected mice or humans can be less susceptible to other auto-immune and inflammatory diseases (Yazdanbakhsh et al., 2001, Summers et al., 2005, La Flamme et al., 2003). In this context, we have shown that colitis induced by treatment with dextran sodium sulphate (DSS) or dinitrobenzene sulphonic acid (DNBS) is less severe in mice infected 8 days previously with the rat tapeworm Hymenolepis diminuta (Hunter et al., 2005, Reardon et al., 2001).

Thus, the aims of the current study were to assess the murine immune response evoked by H. diminuta infection, ascertain if mobilization of regulatory cells (i.e. Foxp+ natural Tregs and AAMs) is a component of this response, and determine if the absence of CD4+ T cells influences the protective effect of H. diminuta in the DNBS-model of chemically-induced colitis. The data show that infection with H. diminuta is accompanied by an ability to produce increased amounts of TH2 cytokines, increased expression of makers that typify natural Tregs and AAMs in the small intestine and, perhaps surprisingly, that CD4+ cells are not required for the rejection of H. diminuta from the mouse gut but are a prerequisite for the anti-colitic effect exerted by this worm in the DNBS model of colitis.

Section snippets

Helminth infection and induction of colitis

Male Balb/c, C57Bl/6 mice (6–8 weeks old; Harlan Animal Suppliers, Indianapolis, IN) and C57Bl/6 CD4 knock-out (CD4−/−) mice (6–8 weeks old; kindly provided by Dr. Jonathan Bramson, McMaster University) were housed in filter-topped cages in specific pathogen free conditions. Mice received five infective H. diminuta cysticercoids by oral gavage in 100 μl of 0.9% NaCl and were autopsied 5, 8 and 11 days p.i. (some CD4−/− mice were autopsied 21 days p.i.) (McKay et al., 1990b, McKay et al., 1991).

Results

Hymenolepis diminuta infection was confirmed by blood eosinophilia (Balb/c mice: H. diminuta infected = 5.6 ± 0.5% vs. uninfected = 1.1 ± 0.2%; C57Bl/6 mice: H. diminuta infected = 4.9 ± 0.4% vs. uninfected = 1.5 ± 0.2% (n = 4–8)). In accordance with earlier studies, all of the mice infected with H. diminuta appeared healthy and displayed normal ambulatory behavioral patterns (McKay et al., 1990a).

Discussion

Helminth parasites exert a considerable selective pressure on the immune systems of their hosts (Maizels et al., 2004). A comprehensive knowledge of this aspect of the host-parasite relationship is important from at least three perspectives: (i) the development of novel treatments for human infections or those of domesticated animals; (ii) as a means to assess effector and regulatory functions of the immune system; and (iii) in a recent development, helminth infections might serve as

Acknowledgements

This work was supported by an operating grant from the Crohn’s and Colitis Foundation of Canada (CCFC) to D.M. McKay. We thank Jun Lu for the electrophysiolgical analysis performed in this study. C. Reardon is supported by a Natural Science and Engineering Research Council (NSERC) of Canada Ph.D. studentship. D.M. McKay is recipient of a Canada Research Chair (Tier 1) in Intestinal Immunophysiology and an Alberta Heritage Foundation for Medical Research (AHFMR) Scientist.

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