The Effect of Irgm 1 on Intestinal Stem Cell Function

Crohn’s Disease (CD), an inflammatory bowel disease characterized by areas of transmural inflammation anywhere in the GI tract (from mouth to anus) affects over 700,000 patients in the US alone, many of whom will require lifelong immunosuppression and/or surgical resection to maintain remission of their disease (1). CD pathogenesis is rooted in genetic susceptibility but requires an environmental trigger before establishing its pervasive, chronic GI inflammation. Genome-wide association studies have identified numerous risk CD risk alleles, many of which are related to the cellular recycling process known as autophagy, which is particularly important in stressful or nutrient-deficient settings (2,3). Among these genes is immunity-related GTPase M (IRGM); knocking out its murine homolog (Irgm1) generates increased susceptibility to ileocolitis (4). Moreover, in the intestinal epithelium, the Paneth cells of Irgm1 KO mice have been shown to exhibit decreased functional innate immunerelated defenses. Previous work suggests that dysfunctional Paneth cells may predispose intestinal dysbiosis (5). In addition to regulating the intestinal microbiota, Paneth cells also play a role as niche cells for intestinal stem cells (ISCs). Given the improved outcomes among patients who are able to achieve epithelial healing as a clinical endpoint (6), Dr. Gulati’s lab is investigating whether ISC biology can be manipulated to optimize epithelial proliferation and, thereby, mucosal healing. Using mouse models lacking particular autophagy-related and CD susceptibility genes, such as Irgm1, the role of autophagy in the ISC milieu can be investigated. The intestine possesses two putative stem cell populations: 1) actively cycling Crypt Basement Columnar cells (CBCs) expressing such markers as Lgr5, Olfm4, and Ascl2; and 2) quiescent, “backup” stem cells at the “+4” position up from the base of the crypt, expressing such markers as mTERT, HopX, and Bmi1. In situations of injury or active stem cell depletion, “+4” cells are thought to be recruited to action in order to maintain the integrity of the intestinal epithelium (7). Actively cycling stem cells bear the experimental boon of exquisite radiation sensitivity. Potten et al. (8) characterized these distinct populations and developed the “Microcolony Assay,” which utilizes total body irradiation (TBI) as the modality for investigating changes in the number of surviving crypts. While radiation enteritis is not intended as a faithful model of CD, the irradiation of Irgm1 KO vs. WT mice should provide real insight into the role Irgm1 plays in ISC biology. Further, preliminary work by the Gulati lab has demonstrated that ex vivo enteroid cultures from Irgm1 KO mice show decreased proliferation and survival in culture, compared to WT enteroids. Enteroid systems are grown from actual intestinal crypts harvested from our mice of interest; when supplemented with a pro-proliferative medium, they reproducibly bud into enterospheres on day 1 and into functionally manipulable “mini-gut” systems by day 7 (9). Even though enteroids only comprise intestinal epithelial cells, these data suggest that the proposed in vivo model merits further exploration.