IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine

Chronic mucosal inflammation and tissue damage predisposes patients to the development of colorectal cancer (CRC)1. This association could be explained by the hypothesis that the same factors and pathways important for wound healing also promote tumorigenesis. A sensor of tissue damage should induce these factors to promote tissue repair and regulate their action to prevent development of cancer. IL-22, a cytokine of the IL-10 superfamily, plays an important role for colonic epithelial cell repair, and is increased in the blood and intestine of IBD patients2, 3. This cytokine can be neutralized by the soluble IL-22 receptor, known as the IL-22 binding protein (IL-22BP, IL-22RA2), however the significance of endogenous IL-22BP in vivo and the pathways that regulate this receptor are unknown4, 5. We describe herein that IL-22BP plays a crucial role in controlling tumorigenesis and epithelial cell proliferation in the colon. IL-22BP is highly expressed by dendritic cells (DC) in the colon in steady state conditions. Sensing of intestinal tissue damage via the NLRP3 or NLRP6 inflammasomes led to an IL-18-dependent down regulation of IL-22BP, thereby increasing the ratio of IL-22/IL-22BP. IL-22, which is induced during intestinal tissue damage, exerted protective properties during the peak of damage, but promoted tumor development if uncontrolled during the recovery phase. Thus the IL-22-IL-22BP axis critically regulates intestinal tissue repair and tumorigenesis in the colon.

Mice were cared for in accordance with institutional animal care and use committeeapproved protocols at the Yale University School of Medicine animal facility.

Tumor induction
Mice were injected intraperitoneally with AOM (Sigma) at a dose of 7.5mg/kg body weight. After 5 days, mice were fed 2.5% DSS (MP biomedicals, M.W. =36,000-50,000 Da) in the drinking water for 5 days, followed by 16 days of regular water. This cycle was repeated twice times 33 . Mice were sacrificed on day 80 of the experiment.

DSS colitis
For acute DSS colitis induction mice were administered 2.5% DSS in the drinking water for 7 days, followed by 3 days of regular water. For chronic DSS colitis induction mice were fed 2.5% DSS for 5 days followed by 16 days of regular water. This cycle was repeated three times. According to the animal protocol mice were sacrificed, if they lost more then 20% of their initial body mass.

Endoscopic Procedures
-20 -Colonoscopy was performed in a blinded fashion for colitis and tumor monitoring using the Coloview system (Karl Storz, Germany) as previously described 6 . Briefly: Colitis scoring was based on granularity of mucosal surface, stool consistence, vascular pattern, translucency of the colon and fibrin visible (0-3 points for each). Tumor sizes were graded from 1 to 5. Tumors observed during endoscopy were counted to obtain the total number of tumors per animal. The total tumor score per mouse was calculated as sum of all tumor sizes. The colon was wounded using the endoscopy and a biopsy forceps as described previously 34,8 . A rebiopsy was taken two days after the wounding either close to the initial biopsy or in a distance of about 0.5 cm.

Histopathology procedures
Colons were evaluated and were assigned scores by investigators blinded to experimental manipulation. Each section was evaluated by a semi quantitative criterionbased method (score 0-5) essentially as described before 35 . For immunohistochemistry Paraffin-embedded sections were stained with anti-BrdU (Sigma) or anti-Ki67 (Thermo (Lab Vision)). DAKO EnVision™ System was used for detection. All sections were counterstained with hematoxylin. TUNEL staining was performed using ApoAlert® DNA Fragmentation Assay Kit according to the manufacturer's instruction. BrdU was injected four hours before the animals were sacrificed. were purified using MACS. In a second step CD45 positive IEL and LPL cells were further purified using a MoFlo. The purity of CD45 positive cells was > 95%, EC were >98% CD45 negative.

Colon explant culture
One centimeter of the distal colon was removed, washed with PBS, scaled, and cultured for three days in X-Vivo medium containing Penicillin, Streptomycin and Tetracycline. IL-6 was measured using CBA.

RNA Analysis
Total RNA was extracted from colon tissue, tumors, colon biopsies or cells using

Human subjects
Informed consent was obtained from healthy participants per protocol approved by the institutional review board at Yale University.