The goal of the current R35 proposal is to understand the underlying pathogenesis of necrotizing enterocolitis (NEC), the leading cause of death from gastrointestinal disease in premature infants, and to develop novel treatments for this devastating disease. The typical patient with NEC is a premature infant who rapidly progresses from mild feeding intolerance to systemic sepsis and then death within 24 hours. NEC disproportionately affects communities of color, and half of all patients will require laparotomy, which reveals patchy intestinal inflammation and necrosis. There is no specific treatment for NEC, which increases the urgency to develop novel therapies for this devastating disease. The journey which has led me to write this proposal includes over two decades of research into NEC, including the advancement of a unifying theorem to explain NEC development, the discovery of several classes of anti-NEC agents which have been patented, and most recently, the completion as sole Editor-in- Chief of the first textbook devoted to NEC. This track record gives me a unique vantage point in which to identify the most important unanswered questions in the field, and the experience to create a plan to answer them. Prevailing thinking suggests that NEC arises from an exuberant inflammatory response to bacterial colonization in the premature intestine. In mice and human studies, I discovered that the receptor for gram negative bacterial lipopolysaccharide, namely toll-like receptor 4 (TLR4), on the intestinal epithelium, is required for NEC development. TLR4 activity is higher in the premature human and mouse intestinal lining compared to the full-term intestine, which reflects TLR4's role in governing gut development. The subsequent activation of TLR4 by colonizing bacteria leads to inflammation and NEC. These observations formed the basis for my discovery of a novel class of anti-TLR4 molecules that prevent and treat NEC in mice, piglet and human tissue ex vivo. In the current proposal, we will now address four critical knowledge gaps in the field of NEC research: 1. What causes NEC and its hallmark patchy intestinal necrosis? To answer this, we will employ ssRNA-seq in mouse and human NEC tissue and human organoids to discover and then modify TLR4- dependent genetic pathways causing NEC; 2. How can we prevent NEC? We will assess the role of diet (breast milk vs. formula) and immunometabolism on the induction of inflammation in the preterm gut and then manipulate diet-induced inflammatory pathways to reduce NEC; 3. Can we predict NEC earlier? We will assess cellular and molecular maternal factors that contribute to NEC, image blood flow in the premature gut non-invasively, and develop machine learning algorithms for prediction; 4. What causes the long term complications of NEC on the lung, brain and gut? We will perform discovery arrays in humanized organoid platforms as well as mouse and piglet NEC models to identify the link between the inflamed gu...