Characterizing the Role of the E3 Ligase ITCH in Gut Mucosal Homeostasis

The mucosal barrier of the small intestine is highly dynamic, enabling the passage of nutrients that are necessary for the body’s function while simultaneously preventing a breach by harmful microorganisms that are damaging to the host. The effectiveness of the mucosal barrier is dependent on the cohesive relationship established between the luminal mucosal epithelium and the underlying immune compartment in the small intestine. The epithelium provides the first line of defense against pathogens by establishing a physical barrier separating the external environment from the body’s internal milieu, while the immune system secondarily responds to clear bacteria that have breached the epithelial barrier. The HECT E3 ubiquitin ligase ITCH is known to regulate immune responses, and loss of function of ITCH has been associated with gastrointestinal inflammatory disorders. However, the high level of ITCH expression within the intestinal epithelium suggests that it may have an important function(s) in that tissue for maintaining gut homeostasis. Indeed, we identified that global loss of ITCH (Itcha18H/a18H) in young adult animals influenced intestinal architecture characterized by increases in both crypt and villus area that were more prominent in the distal part of the small intestine. Increased crypt area was found to result from expansion of both the proliferating transit amplifying progenitor population and terminally differentiated Paneth cells. Lack of ITCH also resulted in changes in numbers of goblet cells on the villus. Epithelial cell turnover was also accelerated in Itcha18h/a18H animals, as evidenced by increases in both proliferation and apoptosis within the crypt, as well a more rapid cell migration of bromodeoxyuridine-labeled epithelial cells along the crypt-villus axis. Consistent with the observed enhancement of cellular migration, Itcha18H/a18H mice carrying the Min mutation (Itcha18H/a18H; ApcMin/+) displayed a 76% reduction in tumor burden as compared to ApcMin/+ littermates with normal levels of ITCH. To identify which aspects of these changes were cell autonomous, intestinal organoids were generated from the crypts of ITCH sufficient and ITCH deficient animals. Interestingly, epithelial cell proliferation and differentiation were not perturbed in ITCH deficient organoids, in contrast to the in vivo phenotype of the Itcha18H/a18H small intestines. However, increased cell death was observed in organoids lacking ITCH, which was also consistent with increased cleavedcaspase 3 staining in the intestines of mice lacking ITCH exclusively in the intestinal epithelium. The failure to recapitulate the Itcha18H/a18H epithelial phenotype prompted us to investigate how loss of ITCH in immune cells impacts the intestinal epithelium. Animals lacking ITCH within the myeloid cell lineage have similar defects in crypt area, as well as increases goblet and Paneth cell numbers, as compared to the Itcha18H/a18H animals, albeit delayed. These finding highlight a cell autonomous as well as non-cell autonomous function for ITCH in mediating epithelial homeostasis, and emphasize the importance of ITCH in small intestinal barrier function

Epithelial NAD+ depletion drives mitochondrial dysfunction and contributes to intestinal inflammation

IntroductionWe have previously demonstrated that a pathologic downregulation of peroxisome proliferator-activated receptor–gamma coactivator 1-alpha (PGC1α) within the intestinal epithelium contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanism underlying downregulation of PGC1α expression and activity during IBD is not yet clear.MethodsMice (male; C57Bl/6, Villincre/+;Pgc1afl/fl mice, and Pgc1afl/fl) were subjected to experimental colitis and treated with nicotinamide riboside. Western blot, high-resolution respirometry, nicotinamide adenine dinucleotide (NAD+) quantification, and immunoprecipitation were used to in this study.ResultsWe demonstrate a significant depletion in the NAD+ levels within the intestinal epithelium of mice undergoing experimental colitis, as well as humans with ulcerative colitis. While we found no decrease in the levels of NAD+-synthesizing enzymes within the intestinal epithelium of mice undergoing experimental colitis, we did find an increase in the mRNA level, as well as the enzymatic activity, of the NAD+-consuming enzyme poly(ADP-ribose) polymerase-1 (PARP1). Treatment of mice undergoing experimental colitis with an NAD+ precursor reduced the severity of colitis, restored mitochondrial function, and increased active PGC1α levels; however, NAD+ repletion did not benefit transgenic mice that lack PGC1α within the intestinal epithelium, suggesting that the therapeutic effects require an intact PGC1α axis.DiscussionOur results emphasize the importance of PGC1α expression to both mitochondrial health and homeostasis within the intestinal epithelium and suggest a novel therapeutic approach for disease management. These findings also provide a mechanistic basis for clinical trials of nicotinamide riboside in IBD patients

Intestinal epithelial CGAS dampens inflammation by upregulating autophagy

The pathogenesis of intestinal inflammation involves a complex network of cell signaling pathways. CGAS (cyclic GMP-AMP synthase) is a cytoplasmic DNA sensor that is most known for upregulating interferon-mediated inflammation. In this punctum, we discuss our novel finding that in the intestinal epithelium, CGAS binds to BECN1 (beclin 1) to subsequently induce macroautophagy/autophagy and dampen inflammation