PROJECT SUMMARY Early life adversity (ELA) induces long-lasting chronic low-grade inflammation and increases the risk for chronic inflammatory diseases (CIDs) like obesity, diabetes, and atherosclerosis later in life. Current literature on the adult immune phenotype following ELA has shown hyperinflammatory leukocyte signaling as a driver of chronic inflammation. However, the intracellular mechanisms through which ELA alters definitive hematopoiesis and developmentally programs leukocyte inflammatory signaling remain unclear. Mast cells (MCs) are well-established orchestrators of immune responses and leukocyte recruiters, and their heightened activity is unsurprisingly characteristic of ELA associated diseases. Using murine and porcine models of ELA, we have previously demonstrated the MC as an effector cell of the inflammatory and behavioral responses to acute and chronic stress. We used the murine ELA model of neonatal maternal separation with early weaning (NMS) to perform a preliminary characterization of the ELA-associated, MC inflammatory phenotype. Bone marrow resident hematopoietic stem cells (HSCs) and progenitors from adult NMS and normally handled (NH) control mice of both sexes were differentiated in culture into primary bone marrow derived MCs (BMMCs). In comparison to BMMCs from NH mice of the same sex, BMMCs from NMS mice exhibited greater IgE-mediated release of dense core secretory granule (DCSG) proinflammatory mediators and LPS- and IL33-induced release of de novo synthesized cytokines. Our preliminary data revealed that BMMCs from NMS mice exhibited upregulation of cholesterol-related genes, with greater pathway upregulation in NMS females. Further, inhibition of cholesterol biosynthesis coincided with decreased DCSG mediators. The primary objective of this proposal is to define the mechanisms through which ELA alters MC programming, specifically focusing on cholesterol metabolism, to developmentally heighten the MC inflammatory phenotype and enhance host susceptibility to disease. Our primary hypothesis is that ELA modulates the long-term MC inflammatory phenotype and MC programming through the upregulation of intracellular cholesterol metabolism, which is critical for increased granulogenesis and MC proinflammatory activity. To test this hypothesis, we will utilize primary BMMC cultures and tissue MCs from NMS and NH mice of both sexes as in vitro and in vivo models, respectively, of the functional and developmental effects of ELA on immunity. Aim 1 will investigate the impact of ELA on MCp and MC cholesterol metabolism and their link to granulogenesis, while Aim 2 will examine the role of cholesterol metabolism and granulogenesis in the ELA-heightened MC inflammatory phenotype. These experiments will define intracellular cholesterol metabolism as a target mechanism driving the long-term effects of ELA on MC programming and MC inflammatory phenotype. This proposed research has far-reaching implications for understanding how ...