Heligmosomoides polygyrus promotes regulatory T-cell cytokine production in the murine normal distal intestine

Abstract

Helminths down-regulate inflammation and may prevent development of several autoimmune illnesses, such as inflammatory bowel disease. We determined if exposure to the duodenal helminth Heligmosomoides polygyrus establishes cytokine pathways in the distal intestine that may protect from intestinal inflammation. Mice received 200 H. polygyrus larvae and were studied 2 weeks later. Lamina propria mononuclear cells (LPMC) were isolated from the terminal ileum for analysis and in vitro experiments. Mice with H. polygyrus were resistant to trinitrobenzenesulfonic acid (TNBS)-induced colitis, a Th1 cytokine-dependent inflammation. Heligmosomoides polygyrus did not change the normal microscopic appearance of the terminal ileum and colon and minimally affected LPMC composition. However, colonization altered LPMC cytokine profiles, blocking gamma interferon (IFN-gamma) and interleukin 12 (IL-12) p40 release but promoting IL-4, IL-5, IL-13, and IL-10 secretion. IL-10 blockade in vitro with anti-IL-10 receptor (IL-10R) monoclonal antibody restored LPMC IFN-gamma and IL-12 p40 secretion. IL-10 blockade in vivo worsened TNBS colitis in H. polygyrus-colonized mice. Lamina propria CD4(+) T cells isolated from colonized mice inhibited IFN-gamma production by splenic T cells from worm-free mice. This inhibition did not require cell contact and was dependent on IL-10. Heligmosomoides polygyrus colonization inhibits Th1 and promotes Th2 and regulatory cytokine production in distant intestinal regions without changing histology or LPMC composition. IL-10 is particularly important for limiting the Th1 response. The T-cell origin of these cytokines demonstrates mucosal regulatory T-cell induction.

FIG. 1.

Mice colonized with H. polygyrus are protected from TNBS colitis. Mice were inoculated with H. polygyrus, while control mice received only sham inoculation. Two weeks later, they received TNBS in 50% ethanol given rectally to induce colitis. Colitis severity was assessed 4 days later. The inflammation was scored on a 0-to-4 scale. Previous experiments determined that the height of the inflammation occurred at day 4. Data are means ± SE from 10 animals studied in 2 separate experiments. Control versus H. polygyrus, P < 0.01.

FIG. 2.

H. polygyrus inhibits mucosal IFN-γ and IL-12 (p40) responsiveness. Healthy C57BL/6 mice were inoculated with 200 H. polygyrus larvae by oral gavage, while control mice received only sham inoculation. Two weeks later, LPMC from the terminal ileum of either H. polygyrus-colonized or control animals (H. poly versus Control) were cultured (0.5 × 10⁶ cells/well) for 48 h with or without anti-CD3/CD28 (αCD3) to stimulate IFN-γ production (A) or with CpG oligonucleotide (0.6 μg/ml) to stimulate IL-12 (p40) secretion (B). Supernatants were assayed for IFN-γ and IL-12 (p40) content using ELISAs. Data are means ± SE of nine determinations from three independent experiments. Control versus H. polygyrus: anti-CD3-stimulated IFN-γ, P < 0.01; CpG-stimulated IL-12 p40, P < 0.01.

FIG. 3.

T cells are the source of anti-CD3/CD28-stimulated IFN-γ in LPMC from uninfected mice. (A) LP T cells (T cells) or LPMC depleted of T cells (Non T) derived from uninfected WT mice were cultured (5 × 10⁵ cells/well) for 48 h with or without adherent anti-CD3/CD28 MAbs (αCD3). Culture supernatants were assayed for IFN-γ after the incubation. Data are means ± SE of nine determinations from three independent experiments. (B) Unfractionated LPMC from uninfected WT mice were cultured as described in the legend for Fig. 2 and then assayed for expression of IFN-γ by intracellular flow cytometry using a lymphoid gate. Results are representative of two independent experiments.

FIG. 4.

Heligmosomoides polygyrus promotes mucosal IL-4, IL-5, and IL-13 production. C57BL/6 mice were inoculated with 200 H. polygyrus larvae (H. poly) and LPMC cultured as described in the legend for Fig. 2. Control mice did not receive H. polygyrus. Next, supernatants were assayed for IL-4, IL-5, and IL-13 content using ELISAs. Data are means ± SE of nine determinations from three independent experiments. Control versus H. polygyrus: IL-4, P < 0.05; IL-5, P < 0.01; IL-13, P < 0.01.

FIG. 5.

LP T cells are the source of anti-CD3/CD28 (αCD3)-stimulated IL-4 in colonized mice. C57BL/6 mice were inoculated with 200 H. polygyrus larvae by oral gavage. (A) Two weeks later, LP T cells (T Cells) or LPMC depleted of T cells (Non-T) (5 × 10⁵ cells/well) were cultured as described in the legend for Fig. 2. Data are means ± SE of nine determinations from three independent experiments. (B) Unfractionated LPMC from H. polygyrus-colonized WT mice were cultured as described in the legend for Fig. 3 and then assayed for expression of IL-4 by intracellular flow cytometry using a lymphoid gate as described in the legend for Fig. 3. Results are representative of two independent experiments.

FIG. 6.

Heligmosomoides polygyrus induces mucosal IL-10 production. Mice were inoculated with H. polygyrus, while control mice received only sham inoculation. Two weeks later, unfractionated LPMC (Cells) from either H. polygyrus-colonized or control animals (H. poly versus Control) were cultured as described in the legend for Fig. 2 with or without anti-CD3/CD28 MAbs (αCD3). Supernatants then were assayed for IL-10 by ELISA. In other experiments, LP T cells or LPMC depleted of T cells from mice bearing H. polygyrus were cultured as described in the legend for Fig. 2 to study IL-10 secretion (B). Data are means ± SE for six determinations from two independent experiments. Control versus H. polygyrus anti-CD3-stimulated IL-10, P < 0.01 for either unfractionated or T-cell groups.

FIG. 7.

Anti-IL-10R MAb (αIL10R) reverses both IFN-γ and IL-12 blockade. Mice were inoculated with H. polygyrus. Two weeks later, unfractionated LPMC from either control mice (Control) or H. polygyrus-colonized animals (H. poly) were cultured (0.5 × 10⁶ cells/well) for 48 h with anti-CD3/CD28 MAbs to stimulate IFN-γ production (A) or CpG oligonucleotides (0.6 μg/ml) to promote IL-12 p40 secretion (B). Some wells also contained blocking anti-IL-10R MAb. After the incubation, supernatants were assayed for IFN-γ and IL-12 p40. Data are means ± SE of eight determinations from four independent experiments. Control IFN-γ, P < 0.05; H. polygyrus IFN-γ, P < 0.01; control IL-12, P < 0.05; H. polygyrus IL-12, P < 0.01.

FIG. 8.

Treatment with monoclonal anti-IL-10R antibody worsens TNBS colitis in H. polygyrus-colonized mice. Mice were inoculated with H. polygyrus, while control mice received only sham inoculation. Two weeks later, they received TNBS as described in the legend for Fig. 1 and were given either rat IgG (0.5 mg) (IgG Rx) or monoclonal rat anti-mouse IL-10R blocking antibody (0.5 mg) (αIL10R) by intraperitoneal injection. Colitis severity was assessed 4 days later as described in the legend for Fig. 1. Shown are representative photomicrographs from helminth-naive mice treated with isotype control antibody (A), helminth-naive mice treated with monoclonal anti-IL-10R antibody (B), helminth-colonized mice treated with isotype control antibody (C), or helminth-colonized mice treated with monoclonal anti-IL-10R antibody (D). Data are means ± SE from three separate experiments, each with four to five mice per group.

FIG. 9.

LP T cells from colonized mice inhibit splenic T-cell IFN-γ production. Transwell experiments were performed with 96-well Transwell plates. Splenic T cells (Spl T) (3 × 10⁴ cells/well) were mixed with equal numbers of irradiated APC and placed in the outer chamber. LP T cells (6 × 10⁴ cells/well) from H. polygyrus-infected mice were either added directly to the outer chamber or placed in the inner chamber of the Transwell system. Some chambers also received blocking anti-IL-10R (αIL10R) MAb (1 mg/ml). The ratio of splenic T cells to the LP T cells was 1:2. Cells were cultured for 48 h. IFN-γ was measured in culture supernatants by ELISA. Data are means ± SE of eight determinations from two independent experiments. *, P < 0.05.

FIG. 10.

LP CD4⁺ but not CD8⁺ T cells inhibit splenic T-cell IFN-γ production. LP T cells from H. polygyrus-infected mice were enriched for the CD8⁺ or CD4⁺ subset as described in Materials and Methods. These cells were mixed with the splenic responder T cells (Spl T) at a 2:1 ratio. Cells were cultured for 48 h, and then IFN-γ was measured in culture supernatants by ELISA. Data are means ± SE of 12 determinations from 3 independent experiments. Spl T versus Spl T + CD4⁺ LP, P < 0.01; Spl T versus Spl T + CD8⁺ LP, P > 0.05.