Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis

Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H ions reducing the intracellular pH (pH ). Nevertheless, how extracellular pH regu...

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Published in:	Frontiers in medicine Vol. 5; p. 19
Main Authors:	Khan, Meraj A, Philip, Lijy M, Cheung, Guillaume, Vadakepeedika, Shawn, Grasemann, Hartmut, Sweezey, Neil, Palaniyar, Nades
Format:	Journal Article
Language:	English
Published:	Switzerland Frontiers Media S.A 13-02-2018
Subjects:	Gram-negative bacteria-induced NETosis lipopolysaccharide-induced NETosis Medicine NADPH-dependent NETosis neutrophil extracellular trap formation spontaneous NETosis NADPH-dependent NETosis Gram-negative bacteria-induced NETosis lipopolysaccharide-induced NETosis neutrophil extracellular trap formation Gram-positive bacteria-induced NETosis pH spontaneous NETosis correcting pH
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Summary:	Neutrophils migrating from the blood (pH 7.35-7.45) into the surrounding tissues encounter changes in extracellular pH (pH ) conditions. Upon activation of NADPH oxidase 2 (Nox), neutrophils generate large amounts of H ions reducing the intracellular pH (pH ). 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 pH (ranging from 6.6 to 7.8; every 0.2 units) increased pH of both activated and resting neutrophils within 10-20 min (Seminaphtharhodafluor dual fluorescence measurements). Since Nox activity generates H ions, pH 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 lipopolysaccharide-, (Gram-negative)-, and (Gram-positive)-induced NETosis. Thus, higher pH promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pH -mediated regulation of Nox-dependent NETosis. Raising pH either by sodium bicarbonate or Tris base (clinically known as Tris hydroxymethyl aminomethane, tromethamine, or THAM) increases NETosis. Each Tris molecule can bind 3H ions, whereas each bicarbonate HCO3 ion binds 1H ion. Therefore, the amount of Tris solution required to cause the same increase in pH level is less than that of equimolar bicarbonate solution. For that reason, regulating NETosis by pH with specific buffers such as THAM could be more effective than bicarbonate in managing NET-related diseases.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Edited by: Evi X. Stavrou, Case Western Reserve University, United States Specialty section: This article was submitted to Hematology, a section of the journal Frontiers in Medicine Reviewed by: Kimberly Martinod, KU Leuven Kulak, Belgium; Jaehyung Cho, University of Illinois at Chicago, United States
ISSN:	2296-858X 2296-858X
DOI:	10.3389/fmed.2018.00019