Summary: The main objective of this proposal is to understand how neutrophils migrate across the intestinal epithelia, toward the eventual goal of manipulating this process in pathologic conditions where it can become excessive as in the context of idiopathic inflammatory intestinal disease as well as enteric bacterial infection. In particular, occurrence and severity of colitis appears to be correlated with the extent of neutrophil transmigration across the colonic epithelium and recent studies have shown that neutrophil migration can incite the migration of other cells that mediate inflammation. Thus, neutrophil transepithelial migration and accumulation at mucosal surfaces is a hallmark of many inflammatory conditions, and this process correlates directly with clinical disease activity and epithelial injury. Currently, the mechanisms that define neutrophil-epithelial interactions during an inflammatory response are not completely understood. To fill this gap in knowledge, we have uniquely shown that secretion of the eicosanoid hepoxilin A3 (HxA3) through the apically expressed efflux pump known as MRP-2 establishes the chemotactic gradient across the intestinal epithelium that is required for neutrophils from the submucosal space to move into the colonic lumen at times of inflammation. Additionally, we identified the N-acyl ethanolamine (NAE) class of eCBs to function in suppressing neutrophil transepithelial migration, and that these molecules are secreted through the apical efflux pump known as P-glycoprotein (P-gp). We hypothesize that these pro- and anti-inflammatory pathways communicate to provide a responsive, integrated mechanism to control inflammation status and that these are dysregulated in disease settings. To test this central hypothesis, we aim to identify the HxA3 receptor(s) on the cell surface of neutrophils (Aim 1), determine the nature of NAE-type ECBs/P-gp and HxA3/MRP2 signaling (Aim 2), and to explore crosstalk points between these two pathways that drive the inflammatory function of neutrophils (Aim 3). Successful completion of these Aims will provide a consolidated picture of mechanisms controlling neutrophil transmigration across the intestinal epithelium that will lead to both novel biological principles and therapeutic intervention strategies.