P023 NOD2 support crypt survival and promote intestinal epithelial regeneration

NOD2 was expressed in intestinal stem cell and Paneth cells and characterised by bacterial sensor that induce an antimicrobial and inflammatory responses. Although the patients with NOD2 mutation are susceptible to Crohn’s disease (CD), NOD2 deficiency does not induce spontaneous inflammation and the most studies have reported no impact of NOD2 deficiency on intestinal inflammation. Recent research reported that NOD2 affords stem cell protection and links microbes to intestinal epithelial regeneration. We investigate whether NOD2 status is associated with crypt survival and intestinal epithelial regeneration independent upon microbiota-derived molecules. To assess crypt survival, clonogenic microcolony assay was performed. Wild-type C57BL/6J mouse and NOD2-knockout (NOD2−/−) adult mice were exposed to 9, 12, and 15 Gy of ionising irradiation and sacrificed after 72 h (n = 6). S phase cells were labelled by administering 5-ethynyl-2′-deoxyuridine (EdU) for 2 h before euthanasia. To evaluate intestinal epithelial regeneration capability, enteroid assay were underwent. Ileal crypts of wild-type C57BL/6J mouse and NOD2−/− mice were isolated and enteroid/spheroid formation was analysed. The number of survival crypts and fractional crypt survival was decreased steadily as the dosage of radiation was increased in wild-type and NOD−/− mice, although those of NOD−/− mice was more susceptible to radiation than those of wild-type mice. When the 15 Gy of radiation was exposed, the number of survival crypts (21.9 ± 6.6/5 mm of intestine vs. 9.0 ± 2.3/5 mm of intestine, p < 0.001) and fractional crypt survival (22.1% ± 3.6% vs. 3.66% ± 1.0%, p < 0.001) of the NOD−/− mice were decreased significantly. In addition, the number of EdD-positive crypts of the NOD−/− mice were decreased significantly compared with that of wild-type mice (14.7 ± 4.6/5 mm of intestine vs. 3.4 ± 2.6/5 mm of intestine, p < 0.001). Tunel-positive apoptic cells within crypts of NOD−/− mice was increased compared with those of wide-type mice. On the enteroid assay, enteroid/spheroid formation was decreased in NOD−/− mice compared with control mice, particularly in ileum. Organoid cells cultured under standard EGF, Noggin, and R-spondin-1 (ENR) conditions, the organoid formation of NOD−/− mice was decreased in number significantly (65.0 ± 46.7/100 crypts vs. 40.5 ± 21.7/100 crypts, p < 0.05) and tended to enteroid formation rather than spheroid compared with wild-type mice. Crypt survival against potentially lethal radiation damage and gut epithelial restitution is NOD2 dependent independent upon microbiota-derived molecules.