hHv1 channels in neutrophils and the innate immune inflammatory response

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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.