PROJECT SUMMARY / ABSTRACT Polymorphonuclear neutrophil (PMN or neutrophil) maintain human health by rapidly eliminating the invading pathogens. These circulating cells transmigrate across the endothelial adherens junctions (AJs) to enter into the infected tissue and to clear the pathogens. Little is known about the role of mechanical forces which PMN experience during transmigration across the endothelium. Our Supporting Data describe the potentially important role of adherens junctions in activating PMN’s host defense function. We observed that activation of PMN-expressed Piezo1 during paracellular transmutation induced calcium influx, which in turn, promptly stabilized Hif1α and upregulated expression of NADPH oxidase 4 (Nox4) in PMN to program these cells to efficient “killers” of the pathogens. These findings have for the first time linked PMN-expressed Piezo1 to the host-defense function, leading to the fundamental question “how Ca2+ influx in PMN via Piezo1 program the host-defense function of PMN?” In Aim 1, we will determine the role of PMN-expressed Piezo1 signaling pathway in activating the host-defense function of transmigrating PMN. Here we will delineate the signaling pathways downstream of Piezo1 activation that promptly stabilizes Hif1α in PMN and programs PMN to become more efficient bacterial “killers”. The studies will involve genetic analysis of Piezo1- Hif1α signaling of the transmigrating PMN such as PMN-specific Piezo1 and Hif1α knockout mice. We will also determine whether pharmacological activation of Piezo1 or expression of gain-of-function Piezo1 mutant in PMN is sufficient to activate the PNM defense system in the relevant P. aeruginosa-induced pneumonia model. In Aim 2, we will determine the role of PMN-expressed Nox4 in regulating oxidative and lytic properties of PMN and in the efficient elimination of pathogens in lung. These studies will address the function of Nox4 in PMN in the mechanism of innate immune defense program. Using in vitro and in vivo experiments, we will determine how Hif1α induces activation of Nox4 gene and how Nox4 regulates oxidative and lytic properties of phagolysosome and efficient pathogen killing. The proposed studies will be essential for understanding the regulation of PMN host-defense function with the goal of identifying therapeutic potential of Piezo1 activators.