Role of the proton channel HVCN1 in the regulation of neutrophil activation

Abstract

Excessive neutrophil activation is a feature of many inflammatory diseases. Neutrophils from affected patients are prone to release reactive oxygen species (ROS) and toxic granule proteins or to undergo NETosis, a special form of cell death characterized by the release of extracellular DNA traps (NETs). Indeed, neutrophils have been shown to play a critical role in the development of autoimmune diseases, as NETs provide autoantigens, such as double-stranded DNA in systemic lupus erythematosus (SLE) or myeloperoxidase (MPO) in ANCA-associated vasculitis. Nonetheless, the molecular switches regulating neutrophil effector functions remain to be further characterized.<br /> HVCN1 is the only voltage-gated proton channel in mammals that regulates pH and membrane potential during neutrophil activation. Its function supports the electrogenic activity of the ROS-generating enzyme NADPH oxidase. Therefore, NADPH oxidase-dependent ROS production is significantly impaired in the absence of HVCN1. However, not much is known about how HVCN1 regulates neutrophil downstream functions.<br /> Therefore, in the first part of my thesis, I further investigated the role of HVCN1 in neutrophils. I demonstrated that PMA-stimulated NETosis and MPO release were significantly increased in HVCN1^−/− neutrophils. Fittingly, HVCN1^−/− neutrophils showed atypical mobilization of calcium, increased histone citrullination, and high mitochondrial ROS caused by calcium-dependent mitochondrial depolarization. Increased NETosis, MPO release, and mitochondria ROS could be rescued by intracellular calcium chelation but not by antioxidants, suggesting that calcium is the primary determinant of increased NETosis susceptibility in HVCN1^−/− neutrophils.<br /> I further examined the consequences of HVCN1 deficiency <em>in vivo</em>. In agreement with previous reports, young HVCN1^−/− mice did not exhibit a pathogenic phenotype, however we detected a mild (auto-) inflammatory phenotype in aged, female HVCN1^−/− mice. Likewise, HVCN1^−/− mice showed more severe pathology in two models of inflammation. Correspondingly, we found HVCN1 to be downregulated in neutrophils from SLE patients in two separate datasets.<br /> This is the first study to show that HVCN1 deficiency increases the inflammatory capacity of murine neutrophils <em>in vitro</em> and <em>in vivo</em>, suggesting that downregulation of HVCN1 may have implications for human disease.