One of the principal features of Inflammatory Bowel Disease (IBD) is disruption of the intestinal epithelium. A variety of signaling pathways cooperate to maintain epithelial integrity and prevent cell death. A20 (TNFAIP3) and ABIN-1 (TNIP-1) are ubiquitin-interacting proteins that bind to each other and restrict TNF-induced cell death. Both genes are located at genetic risk loci for IBD. The precise mechanisms by which these 2 proteins prevent intestinal inflammation, however, are poorly understood.Methods:
To investigate the role of these proteins in intestinal epithelial cells (IECs), we crossed floxed A20 (A20FL/FL) and floxed ABIN-1 (ABIN-1FL/FL) mice with transgenic mice carrying a tamoxifen-responsive Cre-recombinase downstream of a villin promoter (villin-ER/Cre). To better understand our in vivo findings, we also generated intestinal organoids from these mice.Results:
Acute deletion of A20 or ABIN-1 alone in IECs led to no significant spontaneous phenotype. In contrast, simultaneous acute deletion of A20 and ABIN-1 in IECs led to rapid mouse lethality. This death was accompanied by significant myeloid activation, widespread epithelial denudation, and epithelial apoptosis. Interestingly, A20FL/FLABIN-1FL/+ villin-ER/Cre+ mice exhibited a less severe phenotype, suggesting a gene-dose dependent effect. Lethality was independent of MyD88 signaling on IECs, but was partially dependent on MyD88 signaling within the hematopoietic compartment. Prior studies have shown that TNF is a critical regulator of IEC death. Indeed, TNF deficiency completely rescued A20FL/FLABIN-1FL/+ villin-ER/Cre+ mice, demonstrating a central role for TNF-induced cell death in these mice. On the other hand, TNF deficiency could not rescue the lethality of the double deficient A20FL/FLABIN-1FL/FL villin-ER/Cre+ mice, indicating that TNF-independent processes also trigger IEC death in vivo. In agreement with our in vivo observations, acute simultaneous deletion of A20 and ABIN-1 in intestinal organoids sensitized the cultures to TNF-induced cell death. With regard to mechanism, IEC death after deletion of A20 and ABIN-1 could be partially rescued by inhibiting RIPK1 kinase activity. In addition, A20 and ABIN-1 deficient organoids were sensitized to both RIPK3-dependent necroptosis and caspase-dependent apoptosis.Conclusions:
These data reveal an unexpected epistatic relationship between A20 and ABIN-1, and suggest that these proteins may prevent IBD by promoting IEC survival.