Alkaline pH Promotes NADPH Oxidase-Independent Neutrophil Extracellular Trap Formation: A Matter of Mitochondrial Reactive Oxygen Species Generation and Citrullination and Cleavage of Histone

Abstract

pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10–20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics.

Frontline Science: Aggregated neutrophil extracellular traps prevent inflammation on the neutrophil-rich ocular surface

“…This was further confirmed by the anion channel inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid disodium salt hydrate inhibiting the bicarbonate‐induced NET release in a time‐ and dose‐dependent manner . The effect of pH on capacity of neutrophils to form NETs was later confirmed by other researchers . Furthermore, e.c.…”

“…7 The effect of pH on capacity of neutrophils to form NETs was later confirmed by other researchers. [53][54][55] Furthermore, e.c. acidification prolongs the neutrophil survival, ROS production is inhibited, and phagocytosis is increased, but phagolysosomal killing is decreased.…”

To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps

“…Also, no real consensus yet exists about which medium to use for the storage of neutrophils prior to or during assessing NET formation in vitro, although the composition of the culture medium strongly influences the propensity to form NETs [153,154] (st. 80). Also, NET assays must be performed under controlled CO 2 /HCO 3 -/pH balance [155][156][157] (st. 81). For the future, the introduction of standardized buffers to assess NET formation is desirable (st. 82).…”

NETosis: Molecular Mechanisms, Role in Physiology and Pathology

“…Under moderately alkaline conditions and at reduced CO 2 /HCO 3 – ratio, the level of NETosis caused by PMA, Ca 2+ ionophore, uric acid microcrystals, or LPS was increased [ 54 ]. An increase of pH in neutrophil cytoplasm caused a raise in the concentration of Ca 2+ and enhanced production of ROS by both NADPH oxidase and mitochondria [ 55 ]. A decrease of pH in the medium causes inhibition of NETosis, possibly due to inhibition of glycolysis [ 56 ].…”