hHv1 channels in neutrophils and the innate immune inflammatory response

概要/摘要（30行） 与公共卫生的相关性。多形性白细胞（PMN，中性粒细胞）释放反应性 氧自由基（ROS）对抗感染，但这种炎症反应也可以启动和促进， 门肺损伤急性肺损伤（ALI）及其严重形式，急性呼吸窘迫综合征 在40%的患者中是致命的急性呼吸窘迫综合征（ARDS）的特征是在肺组织中积聚富含白蛋白的液体。 肺气隙专注于下游细胞因子作用的药物治疗未能改善 发病率或死亡率;我们假设，并提供证据，针对人类电压门控蛋白， 吨通道（hHv 1）在早期步骤可以更有效。我们建议以hHv 1为目标，因为（i）改变- 中性粒细胞中的nel启动并维持炎症反应，（ii）C6，一种独特的hHv 1抑制剂， 抑制人PMN ROS产生，和（iii）C6抑制ALI小鼠模型中的肺损害。 简要背景。这个应用程序建立在我们创建第一个 hHv 1（C6肽）高亲和力和特异性直接阻断剂，并使用它来显示，首先，人 精子需要hHv 1介导的H+流出来启动获能，允许顶体反应， 卵母细胞受精，第二，人PMN中的hHv 1是产生和维持 在先天性免疫炎症反应期间，炎症因子，包括ROS和蛋白酶。 独特的功能和创新。我们的试验数据揭示了途径中的第二个靶点：白蛋白 (Alb)是激活人PMN中的hHv 1所必需的，我们描述了一种肽（L*），其阻断ALB-激活- 和ROS的产生。支持我们的驱动假说，我们在这里表明，C6和L* 抑制， 人PMN中的hHv 1，减少ROS的产生，C6在ALI小鼠模型中保护， 肺顺应性，并减少ROS，促炎细胞因子，蛋白质，和中性粒细胞在肺， 肺泡灌洗液我们采用我们的新的膜栓系（T肽）的方法，以加快结构， 功能研究和肽设计显示，二价C6（C62）完全抑制开放的hHv 1变异， nels，受益于先进的生物物理和体内方法，以及两位专家合作者。 三个具体目标。(1)Alb对hHv 1的激活作用是为了寻找这种作用的结构和机制基础。 Alb和更有效的天然代谢物的作用。(2)白蛋白对中性粒细胞炎症反应的调节 sponse试图用C6、C62和L* 以及肽作用的基础来描述hHv 1在PMN中的作用。 (3)用Hv 1抑制剂抑制急性肺损伤研究WT和Hv 1 KO小鼠的ALI模型。 意义这项工作解决了一个未满足的医疗需求，最近更明显的广告- COVID-19相关的ALI/ARDS的排放，并具有更广泛的影响，因为PMN和其他噬菌体中的Hv 1， 细胞参与其它急性和慢性炎性疾病。我们建议采用独特的 hHv 1抑制剂和创新的方法来理解和抑制这种病理生理学。

SUMMARY/ABSTRACT (30 lines) Relevance to public health. Polymorphonuclear leukocytes (PMN, neutrophils) release reactive oxygen species (ROS) to combat infection, but this inflammatory response can also initiate and propa- gate lung damage. Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS) that is fatal in 40% of patients, are characterized by accumulation of albumin-rich fluid in the pulmonary air spaces. Drug therapies focused on downstream cytokine actions have failed to improve morbidity or mortality; we hypothesize, and offer evidence, that targeting the human voltage-gated pro- ton channel (hHv1) at early steps can be more effective. We propose to target hHv1 because (i) the chan- nel in PMN initiates and sustains the inflammatory response, (ii) C6, a unique blocker of hHv1 sup- presses human PMN ROS production, and (iii) C6 suppresses lung compromise in an ALI mouse model. Brief background. This application builds on advances in the last period when we created the first high-affinity and specific direct blocker of hHv1 (C6 peptide) and used it to show, first, that human sperm require hHv1-mediated H+ efflux to initiate capacitation, allowing the acrosomal reaction, and oocyte fertilization and, second, that hHv1 in human PMN is required to produce and sustain release of inflammatory agents, including ROS and proteases, during the innate immune inflammatory response. Unique features and innovation. Our pilot data reveal a second target in the pathway: albumin (Alb) is required to activate hHv1 in human PMN and we describe a peptide (L*) that blocks Alb-activa- tion and ROS production. Supporting our driving hypothesis, we show here that both C6 and L* inhibit hHv1 in human PMN, decreasing ROS production, and that C6 protects in an ALI mouse model, restor- ing lung compliance, and decreasing ROS, proinflammatory cytokines, protein, and PMN in bron- choalveolar lavage fluid. We employ our novel membrane tethered (T-peptide) method to speed struc- ture-function studies and peptide design, show a bivalent C6 (C62) that fully inhibits open hHv1 chan- nels, benefit from advanced biophysical and in vivo methods, and two expert collaborators. Three specific aims. (1) Alb activation of hHv1 seeks the structural and mechanistic basis for the action of Alb and a more potent natural metabolite. (2) Alb regulation of the PMN inflammatory re- sponse seeks to delineate the role of hHv1 in PMN using C6, C62 and L* and the basis for peptide action. (3) Inhibiting acute lung injury with Hv1 inhibitors studies an ALI model in WT and Hv1 KO mice. Significance. This work addresses an unmet medical need, recently made more apparent by the ad- vent of COVID-19-related ALI/ARDS and has broader influence because Hv1 in PMN and other phago- cytes is complicit in additional acute and chronic inflammatory disorders. We propose to apply unique hHv1 inhibitors and innovative methods to understand and suppress this pathophysiology.