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 (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 10–20 min (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 Escherichia coli lipopolysaccharide-, Pseudomonas aeruginosa (Gram-negative)-, and Staphylococcus aureus (Gram-positive)-induced NETosis. Thus, higher pHe promoted Nox-dependent ROS production, protease activity, and NETosis; lower pH has the opposite effect. These studies provided mechanistic steps of pHe-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.

inTrODUcTiOn Neutrophils infiltrating tissues during an inflammatory response encounter a range of levels of pH (1)(2)(3). Nevertheless, how extracellular pH (pHe) regulates NETosis is not clearly understood. In response to different agonists, neutrophils undergo two major types of NETosis (4)(5)(6). It has been well-established that bacterial endotoxin, Gram-negative bacteria (e.g., Pseudomonas aeruginosa), and Gram-positive bacteria (e.g., Staphylococcus aureus) induce NADPH oxidase 2 (Nox) and subsequent ROS production (7)(8)(9). We have recently shown that neutrophils undergo increased Nox-dependent NETosis in response to higher doses of lipopolysaccharide (LPS) and increasing microbial load (7). By contrast, increasing intracellular calcium concentration induces citrullination of histones and thereby facilitates Nox-independent NETosis (5,9). In this study, we address the mechanism by which pH regulates Nox-dependent NETosis. Paradoxically, neutrophils carry two sets of NETosis-related enzymes, with either acidic or basic pH optima. The pH optima for neutrophil proteases (e.g., elastase, proteinase 3, and cathepsins) and myeloperoxidase (MPO) are basic (pH 7.5-8.5) and acidic (pH 4.7-6.0), respectively (10)(11)(12). A recent study showed that acidic pH lowers neutrophil extracellular trap (NET) formation induced by phorbol myristate acetate (PMA) and immune complexes (13). However, the effect of pH on LPS, Gram-positive, and Gram-negative bacteria are unknown. Therefore, we here studied the effect of pH on Nox-dependent NETosis induced by agonists such as PMA, LPS, P. aeruginosa, and S. aureus. We show that altered pHe rapidly affects neutrophilic intracellular pH (pHi), Nox-mediated reactive oxygen species (ROS) production, protease-mediated histone cleavage, and subsequent NETosis. Raising pH through a range from 6.6 to 7.8 increases the level of NETosis. These findings help explain how pH regulates NETosis and demonstrate that pH adjustment could potentially be used to regulate NETosis of migrating neutrophils. Tris is a more effective pH regulator than bicarbonate, therefore, Tris-based buffers could be better for correcting pH and regulating NET-related diseases.

MaTerials anD MeThODs research ethics Board approval
This study protocol for using human blood samples was approved by the ethics committee of The Hospital for Sick Children, Toronto. All the procedures including healthy human volunteer recruitment for blood donation were performed in accordance with the ethics committee guidelines. All the volunteers participating in this study gave their signed consent prior to blood draw.

Buffer and reagent Preparation
SYTOX ® Green Nucleic Acid Stain dye, DHR123, and pHi indicator Seminaphtharhodafluor (SNARF) were obtained from Molecular Probes (Thermo Fisher Scientific, Waltham, MA, USA). All buffers, agonists, inhibitors, and other reagents were purchased from Sigma-Aldrich unless otherwise stated. The standard medium of these experiments was RPMI 1640 medium (Invitrogen) supplemented with 10 mM HEPES buffer.
The amount of HCl and NaOH was predetermined to change the pH of RPMI media to 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, or 7.8, and these isotonic RPMI media with different pHs were added to the neutrophil suspension for further experiments.

Primary human neutrophils isolation
The primary human neutrophils were isolated by using a slight modification of the PolymorphPrep (Axis-Shield) protocol as previously reported (4)(5)(6)(7). Briefly, the peripheral blood from the healthy male donors was collected in K2 EDTA blood collection tubes (Becton, Dickinson and Co.). Equal volumes of blood were layered over the PolymorphPrep (Axis-Shield) and spun for 35 min at 600 × g, 25°C to separate the neutrophil band. After washing the neutrophil band, red blood cells were lysed with a 0.2% (w/v) NaCl hypotonic solution for 30 s followed by an addition of an equal volume of 1.6% (w/v) NaCl solution with 20 mM HEPES buffer to provide the isotonic condition. Isolated neutrophils were resuspended in RPMI medium (Invitrogen) containing 10 mM HEPES (pH 7.2). Neutrophil counting and viability check were done by trypan blue and using a hemocytometer. Furthermore, the purity of neutrophils was determined by Cytospin preparation and imaging. Only neutrophil preparations with >95-98% live and pure were used in the experiments. For each experiment, multiple donors were used to get enough replicates; see details in specific figure legends.

snarF Preparation ph i Detection
Isolated neutrophils were treated with 10 µM SNARF ® -4F 5-(and-6)-Carboxylic Acid (Thermo Fisher Scientific) pH indicator dye and incubated at 37°C for 15 min. After incubation, the cells were washed and suspended in fresh RPMI medium at 1 × 10 6 /mL cells. A volume of 50 µL, containing 50,000 neutrophils was seeded into a 96-well clear bottom black plate (BD Biosciences). Equal volumes (50 µL) of the isotonic RPMI with predetermined pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8) were added into the wells to adjust the pH of the corresponding wells. Neutrophils buffered with different pHs were stimulated either with only media (−ve control) or PMA. The dual emission spectra of SNARF were measured after adding media (−ve control) and PMA and considered as the zero time point reading. Further pH changes were recorded for every 10 min up to 60 min with an Omega fluorescence microplate reader. The emission spectrum of SNARF undergoes a pH-dependent wavelength shift, and therefore, the ratios (580/640 nm) of the fluorescence intensities from the dye at two emission wavelengths were used for pHi determinations. Carboxy SNARF-4F is typically used by exciting the dye at one wavelength (between 488 and 530 nm) while monitoring the fluorescence emission at two wavelengths, typically at 580 and 640 nm. The fluorescence response of the SNARF indicators has been calibrated with pH-controlled RPMI (6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8)  with DHR123 (20 µM) for 10 min as per manufacturer's instructions with brief modification as we reported earlier (5,7,14). After washing the extracellular DHR123 dye, cells were resuspended in fresh RPMI (10 mM HEPES) media and 50 µL of 50,000 cells, seeded into 96-well plates. Furthermore, DHR123preloaded neutrophils media were adjusted for pHs (6.6, 6.8, 7.0, 7.2, 7.4, 7.6, and 7.8) by adding the equal volume (50 µL) of isotonic RPMI calibrated for respective pHs. These cells were activated with either only media (negative control), PMA, or LPS for another 30 min. The fluorescence was measured every 10 min by an Omega fluorescence microplate reader (900 data points per well) to assess the kinetics of the ROS generation in different conditions. Each condition was tested with a technical duplicate. Biological replicates (n-values; donors) of independent experiments were reported in figure legends. Confocal images were acquired as described earlier by counter staining of the nuclei with DAPI in different pH conditions.

Flow cytometry
Flow cytometry was used for validating the ROS production in neutrophils at different pHe conditions. Cells were treated with 20 µM DHR123 for 10 min at 37°C. These preloaded cells were washed and resuspended in fresh media, and their pHs were adjusted to 6.6, 7.4, and 7.8. These cells with adjusted pHes were activated with either media only (−ve control) or PMA for 30 min, and the ROS production was analyzed by counting 10

immunoblot analysis
For the immunoblot analysis, the tubes containing 1 × 10 6 cells with adjusted pHs (6.6, 7.0, 7.4, and 7.8) in each experimental condition were activated either by negative control (only media) or by PMA for 120 min. After incubation, the tubes were placed on ice for 10 min and then centrifuged at 20,000 rcf at 4°C for 10 min. The supernatant was then discarded, and the cell pellets were lysed using the lysis buffer containing 1% (w/v) Triton X-100, 25 mM NaF, 50 mM Tris, 10 mM KCl, 10 µg/mL aprotinin, 2 mM PMSF, 1 mM levamisole, 1 mM NaVO3, 0.5 µM EDTA, 25 µM leupeptin, 25 µM pepstatin, one protease inhibitor cocktail tablets per 5 mL (Roche), and one phosphatase inhibitor cocktail tablet per 10 mL (Roche). The samples were then vortexed for 10 s followed by three times of sonication using an aquasonic sonicator (VWR, model 50D at the highest power setting), at 8-10°C, 3 min each. A quarter volume of 5 × loading dye [125 mM Tris HCl at pH 6.8, 6% (w/v) SDS, 8% (v/v) β-mercaptoethanol, 18% (v/v) glycerol, 5 mM EDTA, 5 mM EGTA, 10 µg/mL leupeptin, 10 µg/mL pepstatin, 10 µg/mL aprotinin, 10 mM NaF, 5 mM NaVO3, and 1 mM levamisole] was added followed by 10 min of heating at 95°C with 350 rcm shaking. The samples were separated in a 5% (w/v) stacking and 10% (w/v) resolving gel at 100 V and transferred on a nitrocellulose membrane for 90 min at 400 mA. After transfer, the membranes were blocked with 5% (w/v) milk or BSA in 0.05% phosphate-buffered saline (PBS) with 0.1% Tween (PBST) for 1 h at room temperature. The membranes were incubated with the primary antibody at 4°C overnight followed by three washes with PBST for 30 min. The antibodies used were as follows: anti-Histone H4 (ab16483; Abcam) rabbit pAb at 1:1,000 and anti-GAPDH (FL-335; Santa Cruz) rabbit pAb at 1:2,500. The membranes were then incubated in the secondary antibody solution for 1 h and then washed three times with 0.1% PBST for 30 min. The secondary antibodies used were as follows: donkey anti-rabbit IgG-HRP (31458; Thermo Fisher) at 1:7,500. The densitometry analysis of the blots was done using the Image Studio software (LI-COR Biotechnology) and normalized to the GAPDH.

Bacterial culture
Pseudomonas aeruginosa and S. aureus were selected from single colonies in LB-agar plates and grown overnight in sterile LB broth. Overnight bacterial growth culture was diluted by a factor of 10 and subcultured for 3 h to eliminate the dead colonies or bacteria. Bacterial culture was harvested and washed three times in 5 mL of PBS (pH 7.4) by centrifugation at 5,000 × g for 5 min at 4°C. The bacterial concentration was determined by taking optical density (OD) at 600 nm. Furthermore, the multiplicity of infection was calculated by using the colony-forming unit (CFU) formula established in the laboratory by empirical methods:

statistical analysis
Statistical analysis was performed using GraphPad Prism statistical analysis software (Version 5.0a). Student's t-test was used for comparing two groups, and for more than two groups, ANOVA with Bonferroni's posttest or Dunnett's test was used where appropriate. The technical repeats and applied statistics are mentioned in each Figure legends. A p-value of ≤0.05 was considered to be statistically significant. All data are presented as mean ± SEM.

resUlTs high ph Promotes spontaneous and PMa-induced neTosis
We first determined the effect of pH on spontaneous and Noxdependent NETosis. We incubated purified peripheral blood neutrophils (PMNs) at pH above or below physiological blood pH (6.6-7.8; in 0.2 pH increments) with or without the prototypic agonist PMA to induce Nox-dependent NETosis. Monitoring Sytox green fluorescence (a proxy for extracellular NET DNA) every 30 min for 4 h showed that raising pH increased the rate and amount of NETosis (Figures 1A,B). To confirm true NETosis, we conducted confocal immunofluorescence microscopy. These images showed colocalization of MPO and NET DNA and confirmed that resting and PMA-stimulated neutrophils formed greater amounts of NETs with increasing pH (Figures 1C,D; Figure S1 in Supplementary Material). A regression line at the final time point showed a clear increase in Sytox Green readings with increasing pH (Figures 1A,B, inset). The slope of spontaneous NETosis was less steep than that of PMA-mediated NETosis, indicating that the rate of spontaneous NETosis was lower than that of PMA-mediated NETosis. Overall, pH affected both spontaneous and PMA-mediated NETosis; pH above the normal blood pH of ~7.4 promoted NETosis, whereas a more acidic pH suppressed NETosis.
changes in ph e rapidly altered ph i To determine the mechanism that governs pH-mediated NETosis, we next tested how pHe changes affect pHi. We used a pH-sensitive dual-wavelength SNARF dye for this purpose. SNARF-loaded neutrophils were suspended in media with six different levels of pHe ranging from 6.6 to 7.8, in the presence or absence of PMA. Changes in pHi were monitored by SNARF wavelength ratios every 10 min up to 1 h. We limited our analysis to 1 h because neutrophils in both control and PMA conditions are viable up to this time point (Figure 1; Sytox assays also indicate cell permeability), avoiding the extracellular buffers entering any dead cells. Within 10-20 min, the changes in pHi of the neutrophils reflected the changes in pHe in both control and PMA conditions (Figures 2A,B). The pHi values of PMA-treated neutrophils were lower than in control neutrophils (the SNARF ratio difference was 0.62; p = 0.001 and p = 0.002) over the entire pHe range studied; however, the pHi did not change substantially after 10 min (slope of the graphs at 10 and 60 for controls: 0.124 vs. 0.126; for PMA: 0.093 vs. 0.095) (Figures 2C,D). Activated Nox catalyzes the oxidation of NADPH and generates NADP + , electron (e − ), and H + , concomitantly transports e − out of the cytoplasm but leaves the NADP + and H + in the cytoplasm; hence, pHi should be lower in PMA-activated neutrophils than control neutrophils. The data obtained in this set of experiments indicate that activated neutrophils had an acidic pHi, which was further reduced by low pHe.
higher ph stimulated rOs Production in both resting and PMa-stimulated neutrophils The e − generated by Nox reacts with molecular O2 to generate superoxide among other ROS, which are important for NETosis (15,16). Therefore, we measured intracellular ROS by DHR123 as previously described (5,7,14). Neutrophils were preloaded with DHR123 and activated with or without PMA at six different pH levels. Generation of oxidized green florescence R123 in these neutrophils was monitored every 10 min for 30 min. Raising pH stimulated ROS production in both control and PMA-treated neutrophils (Figures 3A,B). The magnitude and rates of ROS production were higher for PMA-treated than control neutrophils (see insets). To confirm increased ROS production, we imaged these cells as well as performed flow cytometry analyses at 30 min where ROS production was high in PMA-treated cells. These data sets show that increasing pH increased ROS production. Elevating pH also increased production of ROS in control neutrophils, albeit to a lesser degree (Figures 3C,D). Therefore, raising pH increased ROS production in neutrophils, and the pH effect was enhanced in the presence of the activators of neutrophils.    (17,18). To determine whether pH-dependent NETosis is attributable to Nox-mediated ROS production, we repeated the NETosis assays, in the presence or absence of the Nox inhibitor diphenyleneiodonium (DPI). Sytox Green plate reader assays (Figures 4A,B; Figure S2 in Supplementary Material) and confocal microscopy (Figures 4C,D) indicated that DPI suppresses both spontaneous and PMA-mediated NETosis. Therefore, like PMA-mediated NETosis, spontaneous NETosis was also dependent on Nox activity. Taken together, these results (Figures 1-4) showed that alterations in pHe led to pHi adjustments within 10-20 min; elevating pH stimulated Nox activity and subsequent ROS production and thereby enhancing spontaneous and PMA-mediated NETosis (PMA > > no PMA). Higher pH facilitated these steps, whereas low pH exerted the opposite effect, suppressing both spontaneous and PMA-mediated NETosis.

higher ph Promoted histone cleavage and Modification during neTosis
In addition to ROS generation, cleavage of histones by granular proteases is a key step in Nox-dependent NETosis. Therefore, we determined H4 cleavage by Western blot analysis during spontaneous and PMA-mediated NETosis across a range of pH conditions. No substantial differences were detected during spontaneous NETosis. By contrast, a clear pH-dependent increase in H4 cleavage was detected during PMA-mediated NETosis. Standardizing H4 cleavage with GAPDH confirmed the pH-dependent increase in histone cleavage (Figures 5A,B). The intensity of all protein bands decreased with increasing pH, suggesting that neutrophil proteases were more active at higher pH. Therefore, neutrophil proteases that are more active at higher pH promote histone cleavage in PMAmediated NETosis under more alkaline conditions.  . (a,B) The R123-based ROS generation kinetics showed that elevating pH increased ROS production in both control and PMA-treated neutrophils. As shown in the inset regression plot, the magnitude and the rate of ROS production were higher for PMA-treated neutrophils than control neutrophils (n = 3-4; *p < 0.05, between pH 6.6 and 7.8 conditions at respective time points; two-way ANOVA with Bonferroni's posttest conducted at each time point; best fit non-linear polynomial second-order regression analysis; p-value in each inset graph shows whether the slope is different than 0; error bars represent SEM). (c) The ROS generation in neutrophils activated with either control or PMA at pH 6.6, 7.4, or 7.8 was imaged by confocal microscopy. The R123 (green) and DNA (blue) fluorescence staining at 30 min showed more ROS at higher pH in both control and PMA-activated neutrophils, although the amount was greater in PMA-treated cells (n = 3; scale bar 20 µm). (D) Flow cytometry analyses were performed to detect the ROS production in each cell. DHR123-preloaded neutrophils were activated either by media (−ve control) or PMA for 30 min at different pH conditions (pH 6.6, 7.4, and pH 7.8). Mean fluorescence intensities (percentage of maximum) showed higher ROS production at higher pH (n = 3; *p < 0.05, between pH 6.6 and 7.8 conditions; one-way ANOVA with Dunnett's posttest). We also examined the degree of CitH3 formation by immunocytochemistry to examine the relevance of PAD4 in the pHdependent increase in Nox-dependent NETosis. Some degree of CitH3 formation (based on the qualitative examination of CitH3 immunostaining) was detected at higher pH conditions. The few NETotic neutrophils present at higher pH showed CitH3, whereas the intact neutrophils did not ( Figure S3 in Supplementary Material). Nevertheless, CitH3 is hardly detectable in PMA-mediated NETosis  compared to the positive controls (neutro phils activated with calcium ionophore A23187). As expected, the degree of CitH3 formation was much less in PMA-mediated Nox-dependent NETosis than calcium ionophore-mediated NETosis (4,5). Therefore, raising pH induced a modest degree of CitH3 formation, but the contribution of CitH3 to Nox-dependent NETosis is low. The densitometry data of each H4 band were normalized to the total intensities of both bands. The densitometry data showed the increased pH gradient promotes H4 cleavage in PMA-mediated NETosis (n = 3; *p < 0.05, comparing between bands I and II at their respective pH conditions; one-sample t-test compared to each band). See Figure S4 in Supplementary Material, for the full Western blot images. ph changes had similar effects on lPs-and Bacteria-induced neTosis Gram-negative bacteria, their cell wall component LPS, and Gram-positive bacteria induce Nox-dependent NETosis (5,7,19,20). Therefore, to determine the effect of pH on biologically relevant NETosis-inducing agonists, we first tested the effect of pH on LPS-mediated NETosis. Sytox Green plate reader assays and imaging showed that raising pH stimulated LPSmediated NETosis (Figure 6A). Regression analyses conducted at the last time point showed a clear pH-dependent effect of NETosis (inset). Immunofluorescence confocal microscopy and quantifying neutrophil with different nuclear morphology and extracellular DNA confirmed the Sytox Green plate reader assay findings (Figures 6B,C). Therefore, as with PMA-mediated Nox-dependent NETosis, elevating pH stimulated LPS-mediated NETosis.
We also tested the effect of pH on NETosis induced by pathogens such as the Gram-negative bacterium P. aeruginosa and Gram-positive bacterium S. aureus. Like PMA and LPS conditions, more NETosis occurred in response to bacteria under more alkaline conditions (Figure 7). Collectively, these data (Figures 1-7) showed that raising pH increased ROS production, protease activity, and NETosis induced under baseline conditions, as well as by PMA, LPS, Gram-negative, and Gram-positive bacteria. The pH effect was much higher when the NETosis was induced by an agonist.

correcting low ph by sodium Bicarbonate and Tris Base corrects neTosis
Different compounds can be used for adjusting pH. Bicarbonate and Tris are two compounds that can be used to increase pH in humans. Theoretically, each Tris molecule can bind to three H + ions, whereas each bicarbonate ion can neutralize one H + ion. Hence, we tested the efficiency of these compounds in modulating NETosis. To increase the pH of the media from 6.6 to various pHs, Tris solutions required smaller volumes than the equimolar bicarbonate solutions (Figure 8A). Neutrophils in low pH (6.6) media were activated by negative control (medium), PMA, or LPS to induce NETosis. After 30 min, the pH was adjusted by adding precalculated amounts of sodium bicarbonate or Tris to 7.4, and NETosis kinetics were recorded. The percentage of DNA release graphs show that the pH elevation from 6.6 to 7.4 enabled these neutrophils to undergo effective NETosis (Figures 8B-D). Therefore, both bicarbonate and Tris base (or THAM) could be used for increasing the pH and subsequently promoting NETosis. Tris requires much smaller volumes than bicarbonate solutions to adjust the pH, hence, this trivalent molecule may be a better treatment option than bicarbonate.

DiscUssiOn
Infection and inflammation can alter the pH of affected tissues. Open wounds, tumors, ducts from several glands, and airway secretions show variable baseline pH, which change under disease conditions (21)(22)(23)(24). Neutrophils that extravasate into infected and/or inflamed tissues or body fluids will be exposed to various pH conditions [e.g., pH is high in pancreatitis; low in cystic fibrosis (CF) airways; and moderate in arthritis joints] (25)(26)(27)(28)(29). How the pHe regulates Nox-dependent NETosis was not clearly established. In this study, we showed that modulation of pHe led to pHi adjustments and that elevating pH promoted spontaneous, as well as PMA-, LPS-and bacteria-induced, Noxdependent NETosis. Our data provide key mechanistic details and show that high pH increased ROS production and histone cleavage to promote NETosis (Figure 9). These findings clarify the regulation of NETosis in various organs during infectious and inflammatory conditions and indicate that pH modification may alter neutrophil function in such conditions. We and others have reported varying levels of background NETosis (4)(5)(6)(7)30). However, the factors and mechanisms that regulate spontaneous NETosis are not well understood. Our present study showed that pH is a key regulator of spontaneous NETosis (Figure 1). It is interesting that modulation of pHe rapidly altered the pHi of resting neutrophils (Figure 2). pHe changed the pHi at a rate of 0.05 pH unit/min, under different buffer conditions (31)(32)(33)(34). At this rate, pHe from 7.34 to 6.6 and 7.8 would equilibrate with pHi within 14.8 and 9.2 min, respectively. Therefore, the values obtained in our experiments match with the rate of ~0.05 pH unit/min. Accumulation of the product of the NADPH oxidase reaction (H + ions or acidic pH) inhibits the activity of the enzyme (35,36), and the pH optimum for Nox activity is slightly basic (pH ~7.5) (35)(36)(37). Although the amount of ROS generated at baseline is low, raising pH increased ROS in unstimulated neutrophils, albeit in smaller quantities (Figure 3). DPI is an inhibitor of Nox activity (5, 7) and suppressed spontaneous NETosis (Figure 4; Figure S2 in Supplementary Material); hence, Nox regulated the baseline NETosis. Therefore, increased baseline NETosis due to an elevation of pH was attributable to increased Nox activity and subsequent ROS production. Citrullination of H3 can facilitate the chromatin decondensation necessary for NETosis when intracellular calcium concentrations are high (4,5,38). In resting neutrophils, CitH3 formation occurred only in the subpopulation of cells undergoing NETosis. This was apparent at higher pH ( Figure  S3 in Supplementary Material), indicating that PAD4 activity increased with increasing pH (39,40); however, the levels of CitH3 formation was much lower in these cells compared to the conditions that increase intracellular calcium (4,5,7). While our manuscript is in preparation, studies showed that CitH3 increases with elevating pH in pancreas via increased PAD4 activity (27) and with increased bicarbonate concentrations (34). Our recent studies further showed that in response to calcium ionophores, the levels of intracellular calcium, mitochondrial ROS, CitH3 formation, and subsequent Nox-independent NETosis drastically increase with increasing pH (41). Therefore, raising pH stimulated spontaneous NETosis via Nox-dependent ROS production and, to a limited extent, due to the activity of citrullination of histones. The percentage of DNA release kinetics show that pH correction either by sodium bicarbonate or Tris base, clinically known as THAM (from 6.6 to 7.4), increases NETosis compared to low pH (6.6) condition. Collectively, the data show that the pH correction (from 6.6 to 7.4) helps neutrophils to undergo NETosis (n = 3; *p < 0.05, comparing between pH 6.6 and Tris base (6.6-7.8) conditions; two-way ANOVA with Bonferroni's posttest conducted at each time point; one-sample t-test). See Figure S5  The magnitudes of the effects of pH on activated neutrophils were substantially different from resting neutrophils (Figure 1). PMA-activated neutrophils produced much higher NETosis compared to resting cells, and the effect of raised pH on NETosis was also greater. Based on morphology, MPO immunostaining and quantifying NETosis using images clearly showed typical NETosis (4,5,7). Therefore, pH appeared to exert different effects on PMA-mediated NETosis compared to spontaneous NETosis. SNARF fluorescence analyses showed that pHe changed pHi within 10 min, just as in resting neutrophils (Figure 2). However, the slope of the PMA regression line was lower than for resting neutrophils (0.093 vs. 0.124 SNARF ratio increase/pH unit). PMA-treated neutrophils are known to activate Nox, which generates H + ions and reduces pH in the cytoplasm during ROS production (42,43). Lower pH in PMA-activated compared to resting neutrophils reflected activation of Nox. Nevertheless, pHe changes pHi regardless of the activation status of the cells.
Reactive oxygen species levels in PMA-activated neutrophils are several orders of magnitude higher than in resting neutrophils (~3-5-fold). The pH-mediated change in ROS production was also much higher in PMA-stimulated cells than in resting neutrophils (non-linear slope with the positive factors of 268x + 1.7x 3 for resting control vs. 616x + 3.9x 3 for PMA stimulation; see the full equation on inset of Figure 3). These equations show that the effect of pH on ROS is higher at a high pH. DPI, a known inhibitor of Nox (5, 7), suppressed ROS production over the entire pH range tested. Therefore, Nox was the major contributor of ROS in these neutrophils (Figure 4). The effect of pH on Nox activity and ROS production reported here was consistent with previous studies on the effect of pH on ROS production (44)(45)(46) and recent reports on NETosis (13,34), except an early study that reported the opposite effect of pH on NETosis (47). Therefore, pH was a key regulator of ROS that controls Nox-dependent NETosis.
Histone cleavage and transcription facilitate chromatin decondensation (4,19,48,49). Although resting neutrophils did not show changes in histone cleavage with increasing pH, PMAmediated NETosis showed a clear increase in histone cleavage at higher pH ( Figure 5). The pH optimum of neutrophil proteases is alkaline (50,51), and granular proteins enter the nuclei during NETosis (42). Histone H4 cleavage was reported during NETosis and is considered to be an important step involved in chromatin FigUre 9 | Elevated pHi stimulates reactive oxygen species (ROS) production and histone H4 cleavage to regulate spontaneous and Nox-dependent NETosis. As the NADPH oxidase (Nox-2) activity triggered, electrons from cytoplasmic NADPH are translocated inside the phagosome or extracellularly, to form superoxide anions O 2 −⋅ from O2 molecules. For each electron shifted into the phagosome, one proton is left in the cytoplasm, decreasing intracellular pH (pHi) and increasing phagosome pH. This helps in myeloperoxidase (MPO) activity to produce HOCl. Here, the increased extracellular pH alters the pHi within ~10 min. The increase in pHi increases ROS production and promotes histone H4 cleavage. Collectively, the increase in ROS and H4 cleavage regulate the pH-dependent spontaneous and phorbol myristate acetate (PMA)-mediated Nox-dependent NETosis. decondensation (19,52). CitH3 formation is not a major event during Nox-dependent NETosis (4,5), and immunostaining showed only a slight increase in CitH3 formation at higher pH ( Figure S3 in Supplementary Material). Therefore, neutrophil proteases could effectively cleave histones at increasing pH to facilitate chromatin decondensation during agonist-induced NETosis. This is a key finding that may explain the relevance of higher pH for increasing NETosis. Although acidic pH suppresses NETosis, either sodium bicarbonate or Tris base, Tris hydroxymethyl aminomethane, is clinically referred to as THAM, effectively corrects the pH and increase NETosis (Figure 8). Correcting pH could be a promising approach for correcting the low pH-mediated suppression of NETosis. Several studies including our own showed that LPS and bacteria can induce NETosis (4,7,8,30,53). We have recently shown that increasing LPS concentrations and bacterial load promote Nox-dependent NETosis (7). The effect of higher pH increasing NETosis is not restricted to the prototypic Nox-dependent NETosis-inducing agonist PMA. The same pH effect was seen for biologically relevant ligands such as LPS and bacteria (Figures 7  and 9). Therefore, pH was an important factor affecting NET induction by both Gram-negative (Escherichia coli LPS and P. aeruginosa) and Gram-positive (S. aureus) bacteria.
The findings reported in this study may suggest that regulating pH could be a therapeutic option for treating inflammatory conditions and NET-related diseases. For example, nasal and airway surface liquid in patients with CF is acidic (pH of ~5.2-7.1) compared to healthy people (pH of ~7.1-7.9) (44,54,55), and CF airways are often chronically infected with P. aeruginosa or S. aureus (1,44,56). Large numbers of neutrophils infiltrate the CF lung and accumulate in the lumen of the airways (1,37). Although CF neutrophils undergo NETosis ex vivo, it remains unclear whether NETosis is compromised in CF in vivo. Elevating pH in CF airways could be a potential intervention aiming to improve NETosis and neutrophil homeostasis.
In summary, our present study showed that raising pH in neutrophils stimulated Nox activity and ROS production essential for NETosis, particularly during agonist-induced Nox-dependent NETosis. Neutrophil proteases have been shown to be important for NETosis (57,58). At higher pH, proteases that entered NETotic nuclei could cleave histones more effectively due to their high pH optima. Therefore, high pH facilitates NETosis whereas low pH suppresses NETosis (Figure 9). Clinically used compounds such as sodium bicarbonate and THAM effectively raise pH and promote NETosis, suggesting the possibility of these compounds correcting defective NETosis in vivo.

eThics sTaTeMenT
This study protocol for using human blood samples was approved by the ethics committee of The Hospital for Sick Children, Toronto. All the procedures including healthy human volunteer recruitment for blood donation were performed in accordance with the ethics committee guidelines. All the volunteers participating in this study gave their signed consent prior to the blood draw. aUThOr cOnTriBUTiOns MK, LP, GC, and SV conducted experiments; MK and LP interpreted the data, prepared figures, and the manuscript; NS and HG interpreted the data and edited the manuscript. NP is the principal investigator, conceived the idea, planned experiments, supervised the study, interpreted the data, and prepared and edited the manuscript.