Necrotizing enterocolitis (NEC) is a devastating intestinal inflammatory disease that primarily affects premature infants and extremely low birth weight babies. Commonly observed risk factors for NEC are prematurity, formula feeding, intestinal dysbiosis, and infection. Previous studies strongly suggest a critical role of the inappropriate microbiome colonization and activation neonatal immune system in NEC development. However, the pathogenesis of NEC is elusive. Particularly, it remains unclear how the NEC-associated risk factors contribute to the disorder. In preliminary studies, we characterized the effect of formula-feeding on intestinal flora, gene expression, and immunobiology in neonatal mice. We also examined intestinal pathology of mouse pups which were fed with formula followed by induction of a particular antimicrobial immune response. We found that formula-feeding alone resulted in a distinct type of gut dysbiosis and pre-NEC intestinal molecular changes that predispose the neonatal gut to inappropriate microbiome colonization/infection and render intestinal mucosa to be a target of cytotoxic inflammatory cells. We further revealed that formula-fed but not dam-fed mouse pups developed NEC upon activation of a cytotoxic inflammatory cell-associated antimicrobial immune response. Thus, it appears that NEC develops following inappropriate microbiome colonization in premature infants as a consequence of “multi-hit pathophysiological events”. In this project, we will study new mechanistic insights into these events and determine how the interaction of multiple-hit events contributes to the development of NEC in two complementary aims: (1) We will first characterize the series of pathophysiological events that leads to NEC development, using a novel and pathologically relevant mouse pup model of NEC and up-to-dated in vivo experimental pathological and immunological approaches. Then, we will use RNAseq and cutting-edge bioinformatic analysis to delineate the transcriptomic response of the small intestine of mouse pups to multiple- hit challenges and to unravel the relevance of this novel mouse model of NEC for human NEC. Furthermore, we will study how major NEC risk factor-induced “multi-hit events” contribute to NEC development by focusing on inflammatory cells, mucosal inflammation-associated inflammatory mediators, and a unique signal axis that protects intestinal epithelial cells against inflammatory cell attack. We will achieve this aim using in vivo experimental approaches that draw on molecular biology and mouse genetic engineering techniques. (2) We will elucidate how formula feeding causes pre-NEC molecular changes in the small intestine of premature neonates by taking a multidisciplinary in vivo and in vitro approaches that incorporate techniques of organoid culture, molecular and cellular biology, microbiology, mouse genetic engineering and gnotobiogy. Together, our work will provide a novel mouse model relevant for human NEC, a...