Programming of PMN host-defense function during transendothelial migration

项目摘要/摘要 多形核中性粒细胞(PMN或中性粒细胞)通过快速清除入侵的 病原体。这些循环细胞通过内皮细胞黏附连接(AJ)进入 被感染的组织和清除病原体。对PMN所受机械力的作用知之甚少 跨内皮细胞转归的体验。我们的支持数据描述了潜在的 黏附连接在激活PMN宿主防御功能中的重要作用。我们观察到激活了 PMN在细胞旁变性诱导钙内流时表达Piezo1，而钙内流又迅速 稳定HIF1α和上调中性粒细胞中NADP4的表达以编程这些细胞 高效的病原体“杀手”。这些发现首次将PMN表达的Piezo1与Piezo1联系起来 宿主防御功能，引出了一个根本的问题：钙是如何通过Piezo1程序进入PMN的 PMN的宿主防御功能？在目标1中，我们将确定PMN表达的Piezo1信号的作用 激活移行中性粒细胞宿主防御功能的途径。在这里，我们将描述信号 Piezo1激活下游的通路，迅速稳定中性粒细胞中的HIF1α，并编程使其成为 更有效的细菌“杀手”。这些研究将涉及Piezo1-HIF1α信号的遗传分析 移行中性粒细胞，如PMN特异性Piezo1和Hif1α基因敲除小鼠。我们还将确定是否 Piezo1的药理激活或功能获得突变体在PMN中的表达足以 激活相关铜绿假单胞菌肺炎模型中的PNM防御系统。在目标2中，我们将 确定PMN表达的NOX4在调节PMN的氧化和裂解特性中的作用 有效清除肺部病原体。这些研究将探讨NOX4在PMN中的作用。 先天免疫防御程序的机制。利用体外和体内实验，我们将确定如何 缺氧诱导因子1α诱导NOX4基因激活及NOX4对吞噬酶体氧化和裂解特性的调节 和高效杀灭病原体。拟议的研究对于理解PMN的监管将是必不可少的 宿主防御功能，目的是确定Piezo1激活剂的治疗潜力。

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.