Colitis and intestinal inflammation in IL10-/- mice results from IL-13Rα2-mediated attenuation of IL-13 activity

Abstract

Background & aims: The cytokine interleukin (IL)-10 is required to maintain immune homeostasis in the gastrointestinal tract. IL-10 null mice spontaneously develop colitis or are more susceptible to induction of colitis by infections, drugs, and autoimmune reactions. IL-13 regulates inflammatory conditions; its activity might be compromised by the IL-13 decoy receptor (IL-13Rα2).

Methods: We examined the roles of IL-13 and IL-13Rα2 in intestinal inflammation in mice. To study the function of IL-13Rα2, il10(-/-) mice were crossed with il13rα2(-/-) to generate il10(-/-)il13rα2(-/-) double knockout (dKO) mice. Colitis was induced with the gastrointestinal toxin piroxicam or Trichuris muris infection.

Results: Induction of colitis by interferon (IFN)-γ or IL-17 in IL-10 null mice requires IL-13Rα2. Following exposure of il10(-/-) mice to piroxicam or infection with T muris, production of IL-13Rα2 increased, resulting in decreased IL-13 bioactivity and increased inflammation in response to IFN-γ or IL-17A. In contrast to il10(-/-) mice, dKO mice were resistant to piroxicam-induced colitis; they also developed less severe colitis during chronic infection with T muris infection. In both models, resistance to IFN-γ and IL-17-mediated intestinal inflammation was associated with increased IL-13 activity. Susceptibility to colitis was restored when the dKO mice were injected with monoclonal antibodies against IL-13, confirming its protective role.

Conclusions: Colitis and intestinal inflammation in IL10(-/-) mice results from IL-13Rα2-mediated attenuation of IL-13 activity. In the absence of IL-13Rα2, IL-13 suppresses proinflammatory Th1 and Th17 responses. Reagents that block the IL-13 decoy receptor IL-13Rα2 might be developed for inflammatory bowel disease associated with increased levels of IFN-γ and IL-17.

Figure 1. Deletion of IL-13Rα2 suppresses IL-17A and IFNγ production and protects il10^−/− mice from piroxicam-induced colitis

Mice were fed piroxicam infused food for 14 days, with colitis assessed on day 14. Five animals per group were used with 1 of 2 experiments shown. Mean±SEM are shown with * p<0.05 considered statistically significant. A- 5μm sections of paraffin-embedded colon were stained with H&E and assessed for pathological abnormalities. B- Animal weights and circulating PMN’s measured in whole blood after 14 days of piroxicam-infused food. C- Mesenteric lymph node (MLN) cells stimulated with PMA and ionomycin in the presence of BFA and stained with anti-mouse CD4, CD25, IL-17A and IFNγ and Foxp3. D- Lamina propria lymphocytes isolated and quantified at day 14 were stimulated with PMA and ionomycin in the presence of BFA and stained with anti-mouse CD4, IL-17A and IFNγ. E- RNA was extracted from the colon of mice, 14 days post piroxicam exposure with ym-1, arg1 and il22 gene transcripts quantified and expressed relative to HPRT.

Figure 2. Lethal intestinal helminth infection in il10^−/− mice is reversed by deleting IL-13Rα2

Mice were infected with 200 T. muris eggs and monitored for survival or euthanized and assessed on day-15. Five animals per group were used with 1 of 3 experiments shown. Mean±SEM are shown with p<0.05 considered statistically significant. A- Survival of mice with evidence of infection at day 103. B- 5μm sections of paraffin-embedded caecum were stained with AB-PAS and assessed for goblet cell frequency and gob5 gene expression. C- Inflammation, Eosinophilia and mucus-producing cells was scored from 5μm sections of paraffin-embedded caecum. D- Mesenteric lymph node cells re-stimulated with 10μg of T. muris antigen were cultured for 4 days. IL-4, IL-10, IL-17A and IFNγ production were measured by ELISA.

Figure 3. In vitro Th17 cells possess a functional IL-13 receptor that regulates IL-17A production

FACS-purified naïve CD4⁺CD62L^hiCD44^lo T cells were stimulated under Th1, Th2 or Th17 conditions. One of 4 experiments is shown. Mean±SEM are shown with p<0.05 considered statistically significant. A- Canonical Th1 (IFNγ), Th2 (IL-4) and Th17 (IL-17A) cytokines were measured in culture supernatants. mRNA transcripts for il13Rα1, il4rα and γC were measured from polarized cells. B- Th17-polarized cells cultured with rIL-13. Th17 frequency and IL-17A secretion were measured by flow cytometry and ELISA, respectively.

Figure 4. IL-13 regulates Th17 and Th1 responses and protects mice from a lethal infection

Mice were infected with 200 T. muris eggs and monitored for weight loss and survival or euthanized and assessed at day-21. Animals were treated with 500μg of anti-IL-13 mAb per week starting on day 1. Five animals per group were used with 1 of 2 experiments shown. Mean±SEM are shown with p<0.05 considered statistically significant. A- Weight monitored daily. B- Survival of mice. C- Mesenteric lymph node cells re-stimulated with 10μg of T. muris antigen were cultured for 4 days. IL-13, IFNγ and IL-17A production was measured by ELISA. D- RNA isolated from the caecum was reverse transcribed and assessed for gob5, ifnγ, il13rα2 and il17a transcripts. E- 5μm sections of paraffin-embedded caecum were stained with Giemsa and AB-PAS (shown) and assessed for submucosal inflammation and mucus-producing cell frequency.