Colonization with Heligmosomoides polygyrus suppresses mucosal IL-17 production

Abstract

Helminth exposure appears to protect hosts from inappropriate inflammatory responses, such as those causing inflammatory bowel disease. A recently identified, strongly proinflammatory limb of the immune response is characterized by T cell IL-17 production. Many autoimmune type inflammatory diseases are associated with IL-17 release. Because helminths protect from these diseases, we examined IL-17 production in helminth-colonized mice. We colonized mice with Heligmosomoides polygyrus, an intestinal helminth, and analyzed IL-17 production by lamina propria mononuclear cells (LPMC) and mesenteric lymph node (MLN) cells. Colonization with H. polygyrus reduces IL-17A mRNA by MLN cells and inhibits IL-17 production by cultured LPMC and MLN cells. Helminth exposure augments IL-4 and IL-10 production. Blocking both IL-4 and IL-10, but not IL-10 alone, restores IL-17 production in vitro. Colonization of colitic IL-10-deficient mice with H. polygyrus suppresses LPMC IL-17 production and improves colitis. Ab-mediated blockade of IL-17 improves colitis in IL-10-deficient mice. Thus, helminth-associated inhibition of IL-17 production is most likely an important mechanism mediating protection from inappropriate intestinal inflammation.

Figure 1

IL17 mRNA expression, cytokine production, and Th17 frequency is reduced in H. polygyrus-colonized mice. A) Total RNA was isolated from freshly isolated unstimulated MLN cells of C57BL/6 WT naïve (control) or 2 week helminth-colonized mice. IL17A mRNA content was determined by real-time PCR using HPRT mRNA as reference. Data are from 3 separate experiments. B) MLN cells were isolated from B6WT mice colonized for 2 weeks with H. polygyrus. Control MLN cells were from helminth-naïve B6WT mice. MLN cells were cultured for 48hr at 5x10⁵ cells/well with or without anti-CD3 stimulation. Culture supernatant IL17A content was measured by ELISA. Data are means ± SE from 3 separate experiments. C) MLN cells from helminth-naïve and 2 week colonized B6WT mice were isolated and cultured with PMA/ionomycin then Golgi-plug. Then cells were fixed, labeled and prepared for intra-cytoplasmic flow cytometry. Cells were stained for IL17 and CD4 expression. Results presented are representative of 3 experiments. D) LPMC were isolated from B6WT mice colonized for 2 weeks with H. polygyrus or helminth-naïve B6WT mice and cultured for 48 hr at 5x10⁵ cells/well in the absence or presence of anti-CD3/anti-CD28 stimulation. Culture supernatant IL17 content was measured by ELISA. Data are means ± SE from 2 separate experiments

Figure 2

Simultaneous blockade of IL10 and IL4 signaling restores in vitro MLN cell IL17 production. B6WT MLN cells were isolated and cultured as in Figure 1B with anti-CD3 stimulation. Some cultures included blocking anti-IL10R (1B1.3, 2.5μg/ml), anti-IL4 (11B11, 2.5μg/ml) or isotype control (RIg) antibody. IL17A production was determined by ELISA. Data mean ± SE from 2 (A,B) or 3 (C) separate experiments.

Figure 3

Heligmosomoides polygyrus colonization inhibits IL17 in IL10^−/− mice. and IL17 blockade improves established IL10^−/− colitis. A) MLN cells were isolated from helminth-naïve or H. polygyrus-colonized IL10^−/− mice and cultured as in Figure 1B with anti-CD3 stimulation. Some cultures included blocking anti-IL4 (11B11, 2.5μg/ml) or isotype control (RIg) antibody. IL17 production was determined by ELISA. Data mean ± SE from 2 separate experiments. B) MLN cells were isolated from helminth-naïve IL10^−/− mice and cultured as in Fig 1B with anti-CD3 stimulation in the absence and presence of recombinant IL10 and/or IL4. IL17A production was determined by ELISA. Data mean ± SE from 3 separate experiments.

Figure 4

Heligmosomoides polygyrus colonization inhibits IL17 in colitic mice and IL17 blockade improves established IL10^−/− colitis. A) Some IL10^−/− mice were rendered colitic with piroxicam then colonized with H. polygyrus. Controls were given sham gavage. Other IL10^−/− mice did not receive piroxicam. After 2 weeks, LPMC were isolated and cultured for 48hr at 5x10³ cells/well. IL17A was measured by ELISA. Data are mean ± SE of 2 experiments. For the piroxicam untreated groups, Control vs. H.poly (*) p<0.05. B) IL10^−/− mice were rendered colitic with piroxicam then treated with two injections of blocking anti-IL17 mAb (TC11 18H10, BD Pharmingen) given one week apart at 0.5mg/mouse or with isotype control antibody. One week after the last injection colons were removed for histology. Colitis was scored using a 0–4 point scale by readers blinded to the experimental group. Data are mean ± SE from 2 experiments.