OX40 interactions in gastrointestinal nematode infection

Abstract

The immune expulsion of gastrointestinal nematode parasites is usually associated with T helper type 2 (Th2) responses, but the effector mechanisms directly responsible for parasite loss have not been elucidated. The intestinal inflammatory response accompanying infection with gastrointestinal helminths is thought to be a contributory factor leading to the expulsion of the parasite. However, we have shown that the intestinal inflammation, which is controlled by interleukin (IL)-4, is not required for parasite expulsion. OX40-OX40 ligand (L) signals have been shown to be important for the development of Th2 immune responses but are also involved in a number of inflammatory diseases including those of the intestine. Here, we have investigated the effect of OX40 and OX40L fusion protein treatment on the induction of protective Th2 responses and enteropathy following infection with the gastrointestinal nematode Trichinella spiralis. Treatment with an OX40-immunoglobulin (Ig) blocking fusion protein resulted in enhanced expulsion of the parasite and an increase in the accompanying mastocytosis, despite unaltered levels of Th2 cytokines. Furthermore, there was a delay in the villus atrophy and crypt hyperplasia usually associated with this infection. In contrast, levels of Th2 cytokines were greatly up-regulated in mice treated with an OX40L-Ig activating fusion protein, yet the expulsion of the parasite and the enteropathy were unaffected. Therefore, OX40 ligation potentiates the Th2 response without enhancing host protective immune responses, whereas blocking the OX40-OX40L interaction enhances host protection without promoting Th2 cytokine responses during Trichinella spiralis infection.

Figure 1

In vivo treatment with OX40–immunoglobulin (Ig) significantly enhanced worm expulsion from the gut. Trichinella spiralis-infected mice were treated with either OX40 or OX40 ligand (OX40L)–Ig fusion protein. Mice were killed at 7 and 14 days post infection and adult worms within the small intestine were counted. *, Significantly different from uninfected mice; †, significantly different from infected untreated mice (P < 0·05). Data are presented as the mean gut burden + the standard error of the mean for five mice per group. Results shown are representative of two independent experiments.

Figure 2

Up-regulation of T helper type 2 (Th2)-associated cytokines in the mesenteric lymph node (MLN) during Trichinella spiralis infection following in vivo treatment with OX40 ligand (OX40L)–immunoglobulin (Ig) fusion protein. MLNs were removed, single cell suspensions prepared and MLN cells stimulated with 50 µg/ml T. spiralis antigen. Supernatants were analysed by sandwich enzyme-linked immunosorbent assay (ELISA) for the presence of interleukin (IL)-4, (b) IL-13, (c) interferon (IFN)-γ and (d) IL-18. *, Significantly different from uninfected mice; †, significantly different from infected untreated mice (P < 0·05). Data are presented as the mean cytokine concentration (pg/ml) + the standard error of the mean for five mice per group. Results shown are representative of two independent experiments.

Figure 3

In vivo treatment with OX40 ligand (OX40L)–immunoglobulin (Ig) fusion protein enhanced IgE synthesis in T. spiralis-infected mice but did not affect IgG1 or IgG2a production. Total serum IgE (a) and T. spiralis antigen-specific IgG1 (b) and IgG2a (c) concentrations were determined by enzyme-linked immunosorbent assay (ELISA). IgE concentrations are expressed as the mean + the standard error of the mean for five mice per group. T. spiralis antigen-specific IgG1/IgG2a levels are expressed as mean optical density (OD) + the standard error of the mean for five mice per group. *, Significantly different from uninfected mice; †, significantly different from infected untreated mice (P < 0·001). Results shown are representative of two independent experiments.

Figure 4

In vivo treatment with OX40–immunoglobulin (Ig) significantly delayed intestinal pathology during early infection with T. spiralis. The lengths of villi and crypts (a) were measured and the mean number of mitotic figures per crypt (b) determined at 7 and 14 days post infection (d.p.i.). The entire small intestine from infected and uninfected mice was weighed at 7 and 14 d.p.i. (c). *, significantly different from uninfected mice; †, significantly different from infected untreated mice (P < 0·05). Data are presented as mean villus/crypt length + standard error of the mean (SEM), mean number of mitotic figures + SEM and mean wet weight (g) + SEM, respectively. Five mice were used per group and the results shown are representative of two independent experiments.

Figure 5

In vivo treatment with OX40–immunoglobulin (Ig) fusion protein, but not OX40 ligand (OX40L)–Ig, enhanced mucosal mastocytosis during T. spiralis infection. Carnoy's fixed gut tissue from uninfected and infected mice at 14 days post infection was stained with 0·5% toluidine blue to reveal mucosal mast cells (MMCs). MMCs were counted in at least 10 randomly selected villus crypt units (VCU). Data are expressed as mean number of MMCs per VCU + standard error of the mean for five mice per group. *, Significantly different from uninfected mice; †, significantly different from infected untreated mice (P < 0·05). Results shown are representative of two independent experiments.