P-315 The Necroptosis Adaptor RIPK3 Induces Tissue Repair in Response to Intestinal Injury

Background: The serine/threonine kinase receptor interacting protein kinase 3 (RIPK3) is an essential signal adaptor for necroptosis, a recently identified form of regulated necrosis that promotes inflammation. The initiator caspase, caspase 8, cleaves and inactivates RIPK3 and its upstream activator RIPK1. Hence, necroptosis is optimally induced when caspase 8 is inhibited. Importantly, caspase 8 deficiency in intestinal epithelial cells (IECs) led to aggressive RIPK3-dependent necroptosis, inflammation and mortality in mice. This result suggests that RIPK3-mediated necroptosis may be a driver of detrimental inflammation in inflammatory bowel disease. However, caspase 8 deficiency is a rare event. Therefore, we asked whether RIPK3 similarly drives intestinal inflammation in mice with intact caspase 8. Methods: We generated a RIPK3-GFP reporter mouse model and RIPK3 tissue-specific deletion model. We challenged these mice with dextran sodium sulfate (DSS) to induce injury and inflammation in the intestine. We measure RIPK3 expression, severity of colitis and cytokine expression in these mice. Results: Contrary to the prevailing model and the result in caspase 8-deficient mice, we found that RIPK3-deficient mice were hypersensitive to chemical (dextran sodium sulfate [DSS])-induced colitis. Surprisingly, this hypersensitivity was due to failure to properly repair the damaged IECs, but not due to changes in cell death. To further determine the underlying mechanism, we generated a RIPK3-GFP reporter mouse model in which RIPK3 expression can be tracked by green fluorescent protein (GFP) fluorescence. We found that distinct populations of mononuclear phagocytes (MNPs) in the intestinal lamina propria express high levels of RIPK3. Moreover, tissue specific deletion of RIPK3 in these MNPs fully recapitulated the DSS hypersensitivity of germline RIPK3-deficient mice, indicating that these MNPs signal through RIPK3 to mediate intestinal tissue repair. In the absence of RIPK3, expression of repair-associated cytokines including IL-23, IL-1b and IL-22 was severely compromised. Conclusions: These results indicate that the cell death inducer RIPK3 has an unexpected function in tissue repair and homeostasis, and that it can promote detrimental as well as beneficial inflammation through distinct cell types and mechanisms.