AUTHOR=Khan Meraj A. , Philip Lijy M. , Cheung Guillaume , Vadakepeedika Shawn , Grasemann Hartmut , Sweezey Neil , Palaniyar Nades TITLE=Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis JOURNAL=Frontiers in Medicine VOLUME=5 YEAR=2018 URL=https://www.frontiersin.org/journals/medicine/articles/10.3389/fmed.2018.00019 DOI=10.3389/fmed.2018.00019 ISSN=2296-858X ABSTRACT=
Neutrophils migrating from the blood (pH 7.35β7.45) into the surrounding tissues encounter changes in extracellular pH (pHe) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H+ ions reducing the intracellular pH (pHi). Nevertheless, how extracellular pH regulates neutrophil extracellular trap (NET) formation (NETosis) is not clearly established. We hypothesized that increasing pH increases Nox-mediated production of reactive oxygen species (ROS) and neutrophil protease activity, stimulating NETosis. Here, we found that raising pHe (ranging from 6.6 to 7.8; every 0.2 units) increased pHi of both activated and resting neutrophils within 10β20βmin (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H+ ions, pHi is lower in neutrophils that are activated compared to resting. We also found that higher pH stimulated Nox-dependent ROS production (R123 generation; flow cytometry, plate reader assay, and imaging) during spontaneous and phorbol myristate acetate-induced NETosis (Sytox Green assays, immunoconfocal microscopy, and quantifying NETs). In neutrophils that are activated and not resting, higher pH stimulated histone H4 cleavage (Western blots) and NETosis. Raising pH increased