14 AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS IN THE INTESTINAL EPITHELIUM

Abstract

Previous studies revealed both genetic and environmental factors contribute to the Crohn’s disease pathogenesis. ATG16L1, an essential component of autophagy, is one of the susceptibility genes of the disease. We previously showed that mice with decreased expression of ATG16L1 and Crohn’s disease patients homozygous for the ATG16L1 risk allele develop Paneth cell abnormalities in the intestinal epithelium, and the abnormalities were dependent on murine norovirus (MNV) infection. MNV is a single stranded RNA virus that persistently infects the gastrointestinal tract. We have recently showed that MNV can provide similar benefits to the host as bacteria. However, it is still unclear how this otherwise beneficial virus induces disease pathologies in the Atg16L1 mutant setting. In this study, we investigate the role of ATG16L1 in the intestinal epithelial cell (IEC) in virally triggered inflammatory disease. To investigate the role of intestinal ATG16L1, we treated IEC specific ATG16L1-deficient mice (Atg16L1ΔIEC) with dextran sodium sulfate (DSS) in the presence or absence of MNV. We observed their survival, clinical score, and histopathology. To further examine the mechanism by which ATG16L1 functions in IEC, we derived intestinal organoids from Atg16L1ΔIEC and control mice. Finally, we investigated the effect of MNV-infection on the intestinal intraepithelial lymphocytes (IELs), T cells within the epithelial layer of intestinal mucosa with cytolytic and immunoregulatory capacities. We found that Atg16L1ΔIEC mice were more susceptible to disease following intestinal injury when they were infected with MNV (Figure 1). Instead of alterations in viral replication, this susceptibility was associated with inflammatory cytokine production and exacerbated IEC death. Intestinal organoids derived from Atg16L1ΔIEC mice displayed smaller size, fewer budding, and impaired survival, which were further exaggerated by the addition of TNFa. Among the epithelial cell types, Paneth cells were the most vulnerable to cell death in ATG16L1-deficient organoids. Mechanistic analysis revealed that TNFa induced necroptosis (programmed necrosis) in the ATG16L1-deficient IEC, which was rescued by the inhibition of RIPK1 or RIPK3-deficiency. Moreover, we found that MNV-infection altered the IELs’ subpopulations and affected their motility in vivo, and in vitro co-culture system revealed that IELs harvested from MNV-infected host killed Atg16L1ΔIEC organoids compared with those from naïve host (Figure 2). Our findings support a disease model in which intestinal ATG16L1 prevents TNFa-induced necroptosis in the presence of commensal viruses in the gut, and also suggest that necroptosis inhibition as well as TNFa blockade could be a new therapeutic option for the Crohn’s disease patients with ATG16L1 risk allele.View Large Image Figure ViewerDownload Hi-res image Download (PPT)