O-012 IL-36 Signaling Controls the Induced Regulatory T Cell—TH9 Cell Balance and Gut Inflammation

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Abstract

Background:

In the United States over 1.4 million people suffer from inflammatory bowel disease (IBD). Therefore, there is a pressing need for the development of novel molecular therapeutics targeting pro-inflammatory mediators. Recently a novel interleukin-1 family member, IL-36γ was shown to be up-regulated in patients with inflammatory bowel disease (IBD) and to play an important role in healing of acute intestinal damage in mice, however, the function of IL-36γ with regards to T helper (TH) cell differentiation during gut inflammation remains undefined. Given that regulatory and effector TH cells are abundant in the intestine, where they control the critical balance between tolerance and intestinal inflammation, we investigated the role of IL-36γ/IL-36 receptor (IL-36R) signaling in TH cell-mediated immunity and gut inflammation.

Methods:

TH cells and dendritic cells were sorted from mice deficient in IL-36R, MyD88, NFκBp50, IL-4 and signal transducer and activator of transcription (STAT)-6 and the role of IL-36γ in modulating induced regulatory T cell (iTreg) cell and effector T cell (Teff) cell differentiation was analyzed in vitro by using flow cytometry, enzyme linked immunosorbent assay (ELISA) and PCR array analysis. The oxazolone model of TH2/9 colitis was induced in mice deficient in IL-36γ and its receptor. CD45RBhi model of TH1 colitis was induced by transferring CD45RBhi naive T cells from WT mice or IL-36R−/− mice into Rag1−/− recipients. Mice were followed for clinical indicators of colitis, including weight loss and loose stool, and assessed for histological signs of inflammation by H&E staining of colonic tissue. Colonic lamina propria cells were isolated to assess CD4+ T cell differentiation and cytokine production by using flow cytometry and ELISA.

Results:

Both IL-36γ- and IL-36R-deficient mice were protected from oxazolone-induced colitis and exhibited increased colonic iTreg and diminished IL-9 producing (TH9) cells. Similarly, Rag1−/− mice transferred with IL-36R-deficient CD45RBhi cells exhibited significantly reduced weight loss and colonic inflammation when compared to Rag1−/− mice transferred with IL-36R-sufficient CD45RBhi cells. Mechanistically, in vitro T/DC co-cultures revealed that IL-36γ signaling via IL-36R, MyD88, and NFκBp50 directly in CD4+ T cells potently inhibited Foxp3-expressing iTreg cell differentiation, while concomitantly promoting the development of TH9 cells through the involvement of STAT transcription factors.

Conclusions:

Our findings reveal a fundamental contribution for the IL-36γ/IL-36R axis in promoting TH2/9-dependent colonic inflammation. Further insight into the functions of specific IL-36 ligands and IL-36R during intestinal inflammation may contribute to the development of novel therapeutic strategies manipulating this cytokine axis during distinct phases of human IBD.

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