Project Summary/Abstract Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in premature infants and is characterized by an uncontrolled inflammatory response. The lack of understanding of the mechanisms that regulate the vicious inflammatory cascade, the inability to determine which infants are susceptible to NEC and a lack of therapeutic targets all contribute to the persistently high mortality rate. The goals of the proposed research are to determine the mechanisms that regulate the pathologic immune response during NEC and use this knowledge to design novel epithelial-or immune-specific strategies for this devastating disease. Our laboratory has recently discovered that the interleukin-22 (IL-22) signaling pathway plays a critical role in attenuating the inflammatory response during NEC, as mice lacking the receptor for IL-22 (IL-22Ra1) on the intestinal epithelium demonstrate accelerated mortality in a neonatal mouse model of NEC- like intestinal injury. We demonstrate that mice subjected to experimental NEC develop gross evidence of small intestinal ischemia and necrosis, that can be completely rescued in mice with intact IL-22 signaling by administering recombinant IL-22. In seeking to determine the mechanisms mediating this protection, we have demonstrated that treatment with recombinant IL-22 decreases the pro-inflammatory Th17 lymphocytic infiltrate, which we have shown contributes to NEC pathogenesis. Furthermore, we also determined that IL-22 signaling is important in the regulation of intestinal stem cell differentiation, as mice deficient in IL-22Ra1 in the intestine demonstrate an abnormally profound phenotype characterized by decreased numbers of secretory cells in the intestine as well as decreased expression of critical genes involved in intestinal stem cell development and host defense. Based on these findings, we hypothesize that IL-22 signaling through the receptor IL-22Ra1 attenuates NEC by 1) enhancing intestinal stem cell function, 2) increasing goblet cell differentiation and 3) shifting the immune cell repertoire towards an anti-inflammatory phenotype. We will complete our aims of this project by bringing together a multi-disciplinary team with expertise in epithelial biology, mucosal immunology, mass cytometry, next-generation sequencing, high-resolution confocal and intravital microscopy as well as microfluidics and engineering for the derivation of in vitro models of human intestinal function using gut-on-a-chip devices. These studies will make a significant conceptual advance in understanding the signaling pathways involved in attenuating NEC, explaining the unique susceptibility of the premature infant to NEC based on a defect in IL-22 signaling, and we will evaluate a novel therapeutic strategy for NEC by introducing the anti-inflammatory cytokine IL-22 in the intestinal milieu.