AN AUTOPHAGY PROTEIN ATG16L1 PREVENTS NECROPTOSIS BY REGULATING INTERFERON SIGNALING IN THE INTESTINAL EPITHELIUM

Abstract

Abstract Introduction ATG16L1, an essential component of autophagy, is one of the susceptibility genes of Crohn’s disease. We previously showed mice with decreased expression of Atg16L1 and Crohn’s disease patients homozygous for ATG16L1T300A risk alleles develop Paneth cell abnormalities in the intestinal epithelium. Moreover, the abnormalities were dependent on murine norovirus (MNV) infection in mice. Most recently, we reported mice with deletion of ATG16L1 in the intestinal epithelial cells (Atg16L1ΔIEC) are more susceptible to disease following intestinal injury than wild type mice when they are infected with MNV. Also, we revealed intestinal organoids derived from Atg16L1ΔIEC mouse are susceptible to TNFa-induced necroptosis. However, how ATG16L1 protects IECs against TNFa, and whether the susceptibility is preserved in human IECs harboring ATG16L1T300A risk alleles remains unclear. Aims In this study, we first examined the mechanism by which ATG16L1-deficiency renders IECs susceptible to necroptosis. Next, we investigated whether a specific ISG might be involved in the increased susceptibility. Finally, we tested whether human intestinal organoids harboring ATG16L1T300A risk alleles are susceptible to TNFa-induced necroptosis. Methods & Results We performed RNA-Seq analysis using small intestinal organoids derived from Atg16L1ΔIEC and control mouse, and found 49 genes were upregulated in naïve Atg16L1ΔIEC organoids (Figure 1). Interestingly, most of them were the genes known as interferon-stimulated genes (ISGs) representing a type I IFN (IFN-I) signature. Importantly, we found Atg16L1ΔIEC organoids pretreated with JAK-STAT inhibitor Ruxolitinib displayed better viability and decreased level of necroptosis-related proteins when they were stimulated with TNFa. Moreover, we found protein kinase R (PKR) was increased in naïve Atg16L1ΔIEC organoids, and inhibition of PKR significantly protected Atg16L1ΔIEC organoids from TNFa. Finally, we generated human intestinal organoids using biopsy specimens mostly collected from Crohn’s disease patients. We compared the viability between individuals with 2 copies vs 0 or 1 copy of ATG16L1T300A risk allele, and found organoids derived from ATG16L1T300A homozygous individuals exhibited heightened susceptibility to TNFa (Figure 2 A and B). Moreover, we tested the effect of Ruxolitinib as well as multiple necroptosis inhibitors on human organoids, and found all the inhibitors successfully protected ATG16L1T300A homozygous organoids from TNFa-induced cell death. Conclusion Our data indicate ATG16L1 prevents necroptosis by regulating interferon signaling in both mouse and human IECs, and suggest inhibition of JAK-STAT as well as necroptosis pathway could be promising therapeutic options for Crohn’s disease patients with ATG16L1T300A risk alleles.