Neonatal Late-Onset Sepsis (NLOS) is a clinical syndrome seen in pre-term infants characterized by non- specific sequelae from underlying bacteremia and systemic infection. While Gram-positive infections are often treatable with antibiotics, Gram-negative and fungal species still causes septic shock in 45% of patients with mortality rates reaching 20-74% and 32-50% respectively. However, the organisms cultured from blood and cerebral spinal fluid of patients with NLOS are not uniformly pathogenic. Instead, these microbes comprise normal constituents of the commensal microbiota that have gained entry to the body through either breaks in the dermis or translocation across an immature intestinal barrier. This proposal will determine how early events during postnatal colonization affect the function of Ror?t positive Innate Lymphoid Cells (ILC3s) and how this in turn shapes intestinal barrier development in newborn mammals. While much is known about how microbial colonization affects development and function of lymphocytes in adults, little is known about the process in neonates during initial colonization of the microbiota. Understanding this process in a developmentally accurate model will help uncover the mechanisms underlying susceptibility to NLOS and inform potential strategies for prevention and therapeutic intervention. This proposal employs the use of defined flora facilities and a novel murine model of NLOS in which a bioluminescent strain of Klebsiella pneumoniae is administered intra-gastrically to pups on day 5 of life. Determining alterations in the development of ILC3s from conventionally housed (CH) and minimally diverse flora (MDF) pups in early life will inform intervention strategies by which t prevent NLOS development. Our central hypothesis is that ILC3s play a critical role in early intestinal barrier development through the production of important cytokines prompted by microbial signals. Because the intestinal developmental stages of newborn mice and pre-term infants are remarkably similar, insights gleaned from these studies will have direct translational potential toward prevention strategies in the Neonatal Intensive Care Unit.