Neutrophils are crucial immune cells with key functions including phagocytosis, granule release, and NETosis (NET formation). NETs are extracellular webs of DNA, histones, and antimicrobial proteins that trap and kill pathogens. Two main NETosis pathways exist: NOX-dependent, requiring reactive oxygen species (ROS) production by NADPH oxidase; and NOX-independent, utilizing mitochondrial reactive oxygen species (mROS). NOX-independent NETosis, induced by calcium ionophores like A23187 and ionomycin, is rapid. Peptidylarginine deiminase 4 (PAD4), which citrullinates histones, is crucial for NOX-independent NETosis, leading to chromatin decondensation. pH significantly impacts enzymatic reactions in cells, including neutrophils. PAD4 and neutrophil elastase (NE), both involved in NET formation, have alkaline pH optima. While the influence of intracellular pH on neutrophil function is known, its effects on NET formation's various steps remain unclear. This study aimed to elucidate the mechanism by which alkalinization regulates NOX-independent NETosis, focusing on calcium influx, mROS production, and histone modifications.

Previous research established the existence of NOX-dependent and NOX-independent pathways for NET formation. NOX-independent NETosis, induced by calcium ionophores, is a rapid process. The role of PAD4 in citrullination of histones, leading to chromatin decondensation and NET formation, has been well-documented. Studies have also highlighted the impact of pH on various neutrophil functions, but a comprehensive understanding of pH's role in regulating NETosis, particularly the NOX-independent pathway, was lacking. Existing literature indicated that lower intracellular pH impaired neutrophil function, while the effects of pH on NET formation were only beginning to be explored, with some studies suggesting that alkaline pH could enhance NET formation.

The study used human neutrophils isolated from peripheral blood. Extracellular pH was manipulated using HCl and NaOH to create media with pH values ranging from 6.6 to 7.8. NET formation was assessed using a Sytox Green assay, measuring DNA release. Intracellular pH was measured using the SNARF-4F dye. Intracellular calcium levels were determined using Fluo-4-AM. mROS production was measured with MitoSOX Red. Immunofluorescence confocal microscopy was employed to visualize PAD4, citrullinated histone 3 (CitH3), and myeloperoxidase (MPO). Western blotting was used to analyze histone 4 cleavage. Statistical analysis involved one-way ANOVA, two-way ANOVA, and t-tests, with p<0.05 considered significant. The study included technical and biological replicates using neutrophils from multiple donors.

Alkaline pH significantly enhanced both spontaneous and calcium ionophore-induced NET formation. Calcium ionophores caused a dramatic increase in intracellular pH. Higher pH increased intracellular calcium levels in both resting and stimulated neutrophils. Alkaline pH promoted mROS production, and the mROS scavenger MitoTempo significantly suppressed NET formation, particularly at higher pH. Alkaline pH increased PAD4 activity (indicated by increased CitH3), and confocal microscopy showed co-localization of PAD4 and CitH3 with DNA in NETs. Finally, increased pH promoted histone 4 cleavage, particularly during ionomycin-induced NETosis.

The study's findings demonstrate a clear link between alkaline pH and enhanced NOX-independent NET formation. The observed increase in intracellular calcium, mROS production, PAD4 activity, and histone cleavage at higher pH values directly contributes to the increased NET formation. The crucial role of mROS in this process is supported by the significant reduction in NET formation observed with the mROS scavenger MitoTempo. The results are consistent with the optimal alkaline pH for PAD4 and NE activity. The differences observed between A23187 and ionomycin-induced NETosis, particularly regarding histone cleavage, suggest nuanced mechanisms of action for these calcium ionophores. This research offers mechanistic insights into how pH influences NOX-independent NET formation, particularly relevant in inflammatory microenvironments often characterized by pH shifts.

This study provides novel mechanistic insights into the regulation of NOX-independent NET formation by pH. Alkaline pH promotes NET formation via increased calcium influx, mROS generation, PAD4 activation (resulting in increased CitH3), and histone 4 cleavage. These findings have implications for understanding the role of NETs in sterile inflammation and other inflammatory conditions characterized by pH changes. Future research could explore the therapeutic potential of pH modulation in managing NET-related diseases. Further investigation is needed to fully elucidate the interplay between different proteases and the specific roles of different histone modifications in pH-dependent NETosis.

The study focused on in vitro experiments using isolated human neutrophils. The findings may not directly translate to the complex in vivo environment where other factors influence NET formation. The study primarily used two calcium ionophores and additional studies are needed to confirm that this pathway is representative of all stimuli leading to NOX-independent NETosis. Further research is required to investigate the precise impact of pH on the various NET formation-related enzymes and their interactions.