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

The study investigates how extracellular pH modulates NOX-independent neutrophil extracellular trap (NET) formation induced by calcium ionophores (A23187 and ionomycin). Prior work established two NETosis pathways: NOX-dependent and NOX-independent, with the latter relying on mitochondrial ROS (mROS) and rapid kinetics. PAD4-driven histone citrullination is crucial for NOX-independent NETosis, and key enzymes such as PAD4 and neutrophil elastase (NE) have alkaline pH optima. While pH is known to influence neutrophil functions, its impact on specific steps of NOX-independent NETosis—calcium influx, mROS generation, histone citrullination, and histone cleavage—remained unclear. The authors hypothesized that alkalinization enhances calcium influx, increases mROS, boosts PAD4 activity (CitH3), and augments histone cleavage, thereby promoting NOX-independent NET formation.

Background highlights include: (1) NETs identified as antimicrobial structures, with two established NETosis pathways (NOX-dependent vs. NOX-independent). (2) NOX-independent NETosis is rapid and uses mROS, not NADPH oxidase-derived ROS. (3) PAD4-mediated histone citrullination is pivotal for chromatin decondensation and NET formation; PAD4 translocates to the nucleus upon calcium binding. (4) Enzyme pH optima: PAD4 (~7.6–8.0) and NE (~8.0–8.5) are alkaline, suggesting higher activity in alkaline environments. (5) Acidic pH impairs multiple neutrophil functions; limited and emerging studies linked pH to NET formation, including bicarbonate-dependent effects on NET capacity and extracellular acidification inhibiting ROS-dependent NETs. The literature underscores a mechanistic gap regarding how pH regulates sequential steps of NOX-independent NETosis.

Human peripheral blood neutrophils were isolated from healthy male donors using PolymorphPrep gradient separation and RBC lysis, achieving >95–98% viability and purity. Cells were maintained in RPMI with 10 mM HEPES. Media pH was adjusted (6.6–7.8) using 5 M HCl or 5 M NaOH. NET formation was quantified by SYTOX Green fluorescence over 4 h, normalizing to 0.5% Triton X-100 lysis (100% DNA release); controls confirmed pH did not affect dye fluorescence. Intracellular pH was measured using SNARF-4F (excitation 488–530 nm; emission 580/640 nm), recording ratios (640/580) at 0, 10, and 20 min after exposure to different pH media with or without ionophores. Intracellular Ca2+ was assessed with Fluo-4 AM in cells preloaded in Ca2+-free HBSS, then exposed to RPMI at pH 6.6, 7.2, or 7.8 and stimulated with A23187 or ionomycin; fluorescence was recorded every 30 s up to 20 min; controls verified pH did not alter Fluo-4 fluorescence. mROS was measured with MitoSOX Red (4 µM) in cells at pH 6.6, 7.2, or 7.8 stimulated with ionophores; readings every 4 min up to 120 min; dye-only controls assessed pH effects. The mROS scavenger MitoTEMPO (200 µM, 15 min preincubation) was used to test dependence of mROS and NET formation on pH and ionophore stimulation. Immunofluorescence confocal microscopy assessed PAD4 and CitH3 at 30 min, and MPO, CitH3, and DNA co-localization at 120 min across pH 6.6, 7.2, and 7.8. Western blotting probed histone H4 cleavage after 60 min stimulation at pH 6.6, 7.2, or 7.8, normalizing to GAPDH. Statistical analyses included one-way or two-way ANOVA with appropriate post-tests and t-tests; significance at p<0.05.

- NET formation vs. pH: Alkaline pH increased, and acidic pH decreased, NETosis. SYTOX Green kinetics and confocal imaging confirmed higher NETs at pH 7.8 vs. 6.6 in both spontaneous and ionophore-stimulated conditions. Regression slopes at 120 min highlighted pH sensitivity: spontaneous ≈3.754 vs. A23187 ≈35.92 and ionomycin ≈32.92, indicating a much stronger pH effect under ionophore stimulation. - Intracellular pH: SNARF ratios (640/580) increased with extracellular pH and rose markedly upon A23187 or ionomycin stimulation within 20 min. Polynomial fits at 20 min demonstrated a steep increase, particularly beyond pH 7.6, for ionophore-stimulated cells compared to controls. - Calcium influx: Higher pH (7.8 vs. 6.6) elevated cytosolic Ca2+ in resting and stimulated neutrophils; the stepwise increase between pHs was ∼0.2-fold, with absolute Ca2+ levels substantially higher under ionophore stimulation. Imaging corroborated plate-reader findings. - mROS: Alkaline pH enhanced mROS production in resting and especially in A23187- or ionomycin-stimulated neutrophils within 30 min. MitoTEMPO significantly reduced mROS and concomitantly suppressed NOX-independent NET formation. mROS was not a major contributor to PMA-induced (NOX-dependent) NETosis. - PAD4 activity and CitH3: Immunofluorescence showed increased PAD4 staining and histone H3 citrullination at higher pH in ionophore-stimulated cells. MPO, DNA, and CitH3 co-localized more prominently at alkaline pH, consistent with enhanced NET formation. - Histone cleavage: Histone H4 cleavage was minimal in unstimulated cells across pHs but increased with pH under A23187 or ionomycin stimulation, with a stronger and more reproducible effect for ionomycin. Densitometry normalized to GAPDH showed significant pH-dependent increases (two-way ANOVA; p<0.05 to p<0.001 depending on condition). - Controls: pH did not directly affect fluorescence of SYTOX Green, Fluo-4 AM, or MitoSOX dyes. Overall, alkalinization promotes a cascade—higher intracellular pH, enhanced Ca2+ influx, increased mROS, elevated PAD4-mediated histone citrullination, and greater histone H4 cleavage—culminating in increased NOX-independent NETosis.

The findings resolve key mechanistic questions on how pH regulates NOX-independent, calcium ionophore-induced NETosis. Elevated extracellular pH rapidly raises intracellular pH, augmenting Ca2+ influx. Ionophore Ca2+ binding efficacy increases with pH, particularly for ionomycin, amplifying intracellular calcium signals. This promotes mROS production, a critical mediator of NOX-independent NETosis, as evidenced by MitoTEMPO’s suppression of both mROS and NET formation. In parallel, PAD4’s alkaline optimal activity supports increased histone H3 citrullination, facilitating chromatin decondensation. NE-driven histone H4 cleavage, which also has an alkaline pH optimum, is enhanced, especially under ionomycin, further aiding chromatin relaxation. Conversely, MPO’s acidic pH optimum suggests it is less central to NOX-independent NETosis in alkaline conditions. Collectively, these pH-sensitive steps explain the robust enhancement of NET formation at alkaline pH. Pathobiologically, local pH shifts in tissues—acidic in inflamed cores or alkaline in contexts like pancreatic ducts—could regulate NET formation spatially. Modulating pH might thus represent a strategy to control NET-mediated tissue damage in sterile inflammation or diseases such as pancreatitis and cystic fibrosis.

Alkaline extracellular pH enhances NOX-independent NET formation by driving a coordinated increase in intracellular pH, calcium influx, mitochondrial ROS generation, PAD4-mediated histone H3 citrullination, and histone H4 cleavage. These mechanistic insights clarify how pH regulates calcium ionophore-induced NETosis and suggest that therapeutic manipulation of local pH could modulate NET formation in sterile inflammatory conditions and mitigate damage from agents like ionomycin. Future work could assess in vivo relevance across disease models and evaluate targeted pH modulation or combined interventions (e.g., mROS scavenging, PAD4 or NE inhibition) to regulate pathological NETosis.

The authors note methodological constraints affecting interpretation: (1) Intracellular pH and mROS measurements emphasized early time points (e.g., 20–30 min) to ensure high cell viability and avoid confounding from dye interactions with intracellular components in dying cells; MitoSOX signals at later times can be confounded. (2) Controls verified that pH did not directly alter the fluorescence properties of SYTOX Green, Fluo-4 AM, or MitoSOX, but measurements remain dependent on dye-based proxies. No explicit additional limitations were stated.