NADPH Oxidase Modulates Ca2+-Dependent Formation of Neutrophil Extracellular Traps

Chronic granulomatous disease (CGD) is a severe hereditary immunodeficiency associated with recurrent bacterial and fungal infections as well as aberrant inflammatory processes. The CGD phenotype depends on the deficiency of phagocytic NADPH oxidase causing the inability of phagocytes to produce reactive oxygen species (ROS). Such phagocytes have a limited ability to execute phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs) as a reaction to many receptor and pharmacological stimuli. However, neutrophil trapping in CGD patients in response to calcium ionophores has been previously described in one of the authors' studies. Some researches have shown that NADPH-oxidase-deficient neutrophils are not only incapable of generating ROS but also have major disturbances in the influx of extracellular Ca2+ due to the absence of the electrogenic function of the enzyme and the membrane depolarization during the activation and consequently multiple abnormalities in the synthesis of proinflammatory cytokines. In this study, it has been shown that the formation of NETs by neutrophils deficient in NADPH oxidase in response to calcium ionophore A23187 is accompanied by excessive accumulation of intracellular Ca2+. We propose that this violation is because of the absence of the electrogenic function in mutant NADPH oxidase that normally induces depolarization of the plasma membrane. The results have indicated the important role of phagocytic NADPH oxidase as a modulator of extracellular Ca2+ transport and that it can be used to find the cure for CGD.


INTRODUCTION
Chronic granulomatous disease (CGD) is an inherited immunodeficiency characterized by the inability of phagocytes to produce superoxide anion radicals ( ) and their derivatives [1]. The abortive generation of superoxide anion radicals is associated with the deficiency of the multicomponent enzyme complex NADPH oxidase [2], occurring due to the mutations in genes encoding its subunits (p47phox, p67phox, p40phox, gp91phox, p22phox, and Rac2 GTPase). CGD patients suffer from recurrent bacterial and fungal infections as well as aberrant inflammatory processes. Therefore, the understanding of the signaling pathways responsible for the CGD phenotype is important for finding the treatment approaches to this disease.
It has previously been shown that the stimulation of neutrophils by receptor stimuli is accompanied by an increase in the concentration of intracellular Ca 2+ participating in the activation of a number of effector functions, such as degranulation and oxidative burst [3]. The most thoroughly studied mechanism related to the increased concentration of Ca 2+ is the formation of inositol-1,4,5-triphosphate, which in turn induces the release of Ca 2+ from the intracellular storage located in the endoplasmic reticulum [4]. Depletion of Ca 2+ in cell storages results in the activation of STIM1 and STIM2 sensory proteins (stromal interaction molecule) and further stimulation of Ca 2+ -activated channels (CRAC, calcium release activated Ca 2+ channel) situated in the cytoplasmic membrane [5]. The activation of CRAC channels promotes the influx of Ca 2+ into the cell from the extracellular space. After the neutrophil activation by the chemoattractant N-formyl-methionine-leucine-phenylalanine (fMLP) and with the protein kinase C activator phorbol-12-myristate-13-acetate (PMA), the entry of extracellular Ca 2+ is suppressed. Thus, the total entry of Ca 2+ into the cell is provided by the action of two competing mechanisms: a stimulating and an inhibiting one.

RESEARCH ARTICLE
Considering that the function of NADPH oxidase in neutrophils of healthy donors consists of the transfer of a single electron from NADPH to molecular oxygen, it was suggested in 1980s that the suppression of extracellular Ca 2+ influx upon activation was associated with the electrogenic function of the enzyme provoking membrane depolarization. This theory was experimentally proven on human neutrophils showing a correlation between membrane depolarization and suppression of the entry of extracellular Ca 2+ into the cells [6]. It was demonstrated that neutrophils isolated from the blood of patients with CGD having deficient NADPH oxidase were devoid of such suppression, and the accumulation of Ca 2+ in the endoplasmic reticulum occurred much faster than in neutrophils of healthy individuals [7].
As "professional" phagocytes, neutrophils are responsible for the first line of defense against pathogens in the focus of inflammation, executing phagocytosis, degranulation, and formation of reactive oxygen species (ROS). In 2004 a new protective mechanism of neutrophils was described in the laboratory of A. Zychlinsky [8] involving the release of decondensed chromatin from cells, "decorated" with the proteins of granules, nucleus, and cytoplasm. Those structures were called neutrophil extracellular traps (NETs) because their main function was to limit the dissemination of pathogens from the primary inflammation site [8]. It should be emphasized that the formation of NETs is typically accompanied by cell death, which is why this process has been named NETosis [9].
NETosis is a multistage sequentially developing process including ROS formation mediated by NADPH oxidase and translocation of the enzymes of azurophilic granules neutrophil elastase (NE) and myeloperoxidase (MPO) into the nucleus [10]. In the nucleus-together with peptidyl arginine deaminase 4 (PAD4), citrullinating histones-NE and MPO decondense chromatin resulting in its subsequent release from the cell, i.e., NETosis [11]. It was shown that the activation of NETosis was accompanied by the increase in the concentration of intracellular Ca 2+ [12], and currently two key Ca 2+ -sensitive targets of this process are known, namely protein kinase C, responsible for the phosphorylation of NADPH oxidase, and PAD4.
It was found in some studies that, apart from the inability to generate ROS, the neutrophils of patients with CGD were unable to form NETs in response to many receptor and pharmacological stimuli (PMA) [13,14]. However, NADPH oxidase-deficient neutrophils can still form NETs under the influence of ionomycin and A23187 calcium ionophores with the help of mitochondrial ROS (mtROS) [14,15].
We hypothesized that NADPH oxidase-deficient neutrophils were not only incapable of generating oxidase-dependent ROS but also had more profound disturbances of agonist-induced signaling pathways due to the absence of membrane depolarization upon activation [7] and excessive Ca 2+ influx into the cells. According to our assumption, neutrophils isolated from the blood of healthy donors and patients suffering from CGD and stimulated to NETosis by calcium ionophore A23187 should respond differently to depletion of intracellular Ca 2+ , the verification of which was the purpose of our research. Neutrophils were isolated from the peripheral blood using centrifugation in a single-stage Ficoll-Hypaque density gradient with the density of 1.077 g/cm 3 over 25 min at 400g and room temperature as described earlier [16]. The majority of erythrocytes were removed by sedimentation in dextran. The remaining erythrocytes were lysed in hypotonic 0.2% saline during 30 s; afterwards the physiological composition was replenished by adding hypertonic 1.6% saline. Neutrophils were resuspended in complete culture medium (CCM) containing RPMI 1640 with the addition of 10 mM HEPES, 2 mM L-glutamine, and 1% inactivated fetal bovine serum (FBS). The resulting cells were represented by 98% granulocytes and their viability exceeded 99%, which was estimated by the exclusion of 0.1% trypan blue. Neutrophils were incubated for 1 h at the 4°C prior to the experiment.

MATERIALS AND METHODS
Assessment of luminol-dependent chemiluminescence (CL). Luminol-dependent CL was used to assess the concentration of total ROS, both intra-and extracellular, as previously described [17]. Freshly isolated neutrophils at a concentration of 2.5 × 10 6 cells/mL (4.5 × 10 5 cells) were incubated with BAPTA-AM at increasing concentrations for 20 min at the 37ºC and atmosphere of 5% CO 2 in the CCM (5% FBS). The CCM was then replaced by Krebs-Ringer phosphate buffer containing 120 mM NaCl, 5 mM KCl, 1.7 mM KH 2 PO 4 , 8.3 mM Na 2 HPO 4 , 10 mM glucose, 1 mM CaCl 2 , and 1.5 mM MgCl 2 at 7.3 pH. Luminol at the end concentration of 80 μM was added to 2 × 10 5 cells Vol. 75 No. 3 2020 VOROBJEVA, CHERNYAK that were then stimulated to the oxidative burst using 2.5 μM A23187, 30 nM PMA, or 800 nM fMLP. Luminol-dependent CL was analyzed immediately after the stimulation during 30 min at 37°C in a Lucy 1 microplate chemiluminometer (Anthos Labtec, Austria). The area occupied by the CL curves was calculated, and the degree of oxidative burst was calculated as a percentage of control (control: stimulated neutrophils; 100%) in the form of histograms.
Induction and fluorescent staining of NET. Fluorescence microscopy was applied to detect NET. In order to do this, freshly isolated neutrophils (2 × 10 5 cells/mL) were adhered on round coverslips in a 24-well plate in 500 μL of CCM (1% FBS) during 30 min at 37°C. Neutrophils were incubated in wells with BAPTA-AM for 20 min at 37°C and atmosphere of 5% CO 2 . NET formation was induced by 30 nM PMA or 2.5 μM A23187 for 2 h 40 min and 4 h, respectively. After the stimulation of NETosis, cells were fixated in a solution of 4% paraformaldehyde for 15 min. The samples were immersed in SYBR Green diluted in phosphate-buffer saline in a ratio of 1 : 10 000 in accordance with the manufacturer guideline and stained at room temperature for 6 min under darkroom conditions. Samples were immersed in ProLong Gold resin; cells were studied using a Leica DM LB fluorescence microscope (Leica Microsystems, Germany). Photos were taken using a Leica DC300F camera.
The total number of cells and the number of NETotic cells in each field of vision were counted followed by the assessment of the percentage of NETosis in several fields of vision. Each variant of the stimulation was provided with the minimum of three experiments and at least 500 neutrophils were analyzed for every concentration of the inhibitor.
Statistical analysis. Statistical analysis of the results was conducted using one-way ANOVA followed by a multiple comparison Bonferroni test used to evaluate the intergroup differences. The data were expressed as mean ± error of the mean. Statistically significant p-values are indicated on the graphs as * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.

RESULTS AND DISCUSSION
We previously demonstrated that neutrophils isolated from blood samples of numerous patients with CGD have an intensified spontaneous NETosis in some cases reaching 5-10%. That phenomenon indicated the preactivation of neutrophils in the organism resulting in aberrant NETosis.
If the activation of NETosis under the influence of the calcium ionophore A23187 provokes the excessive Ca 2+ influx in NADPH oxidase-deficient neutrophils similarly to the way it happens during the activation by fMLP type ligands [6], it can be expected that the incubation of such cells with the chelator of intracellu-lar Ca 2+ BAPTA-AM should not result in significant suppression of NETosis. Nevertheless, A23187induced NETosis in neutrophils of healthy individuals should be suppressed by BAPTA-AM in an effective and dose-dependent manner, as has been investigated in our study.
The CGD was diagnosed based on the absolute absence of an oxidative burst in response to zymosan in all examined patients determined by the method of registration of luminol-and lucigenin-dependent chemiluminescence at the State Research Center Institute of Immunology of Russia, as well as by genetic verification using Sanger sequencing at the Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of the Ministry of Health of Russia. All patients suffering from CGD had one of the mutations in the CYBB gene encoding the gp91phox subunit (X-linked CGD).
With the help of the registration of luminol-dependent CL, we demonstrated that the neutrophils were unable to produce ROS in response to the stimulation with PMA, fMLP, or A23187 in all patients with CGD, therefore being completely deficient in NADPH-oxidase (data are not presented). Besides, those neutrophils did not produce NETs in response to PMA (Fig. 1e) since the presence of ROS is obligatory for PMA-induced NETosis [13]. However, ionophore A23187 managed to induce NETosis after 4 h (Figs. 1c, 1e). Incubation of neutrophils of healthy donors with BAPTA-AM resulted in dose-dependent and effective suppression of NETosis induced by both A23187 and PMA (Figs. 1a, 1b), while the incubation of NADPH oxidase-deficient neutrophils with BAPTA-AM hardly led to suppression of the release of chromatin (Figs. 1c, 1e).
We presume that the absence of suppression of A23187-induced NETosis by the chelator BAPTA-AM in NADPH-oxidase-deficient neutrophils (CGD) was associated with the excessive entry of extracellular Ca 2+ into the cell. The activation of neutrophils with fMLP is associated with rapid opening of the CRAC channels and their subsequent closure due to the membrane depolarization. No depolarization of the neutrophil membrane is observed in patients with CGD and the CRAC channels remain opened providing the excessive influx of extracellular Ca 2+ [6]. Such opening of CRAC channels can be also provoked by the action of A23187 because of the release of Ca 2+ from the intracellular storages, though this effect may be weakly expressed, referring to the data of Mahomed and Anderson [18]. The transfer of Ca 2+ catalyzed by ionophores is also probably inhibited by the membrane depolarization. However, this may be also related to the fact that A23187 and ionomycin can transfer positively charged complexes of Ca 2+ across the membrane, along with the electrically neutral exchange of calcium ions to protons [19].
We were interested to find out how the depletion of intracellular Ca 2+ would react to the oxidative burst as an effector function of neutrophils. In the next series of experiments neutrophils of healthy individuals were incubated with BAPTA-AM during 20 min and later the oxidative bur was stimulated with A23187, PMA, and fMLP, a receptor activating chemoattractant. Figure 2 demonstrates that BAPTA-AM caused a dose-dependent suppression of the oxidative burst provoked by A23187 and fMLP but not PMA. Moreover, the PMA-induced oxidative burst was equally intensive in both calcium-containing and calcium-free media (data not presented). These results suggested that NADPH oxidase activation with phor-  bol ether could develop in the absence of extracellular Ca 2+ influx, which is in line with the data of other authors [20], and the phosphorylation of the enzyme complex subunits could be provided by Ca 2+ -insensitive isoforms of protein kinase C (PKCδ, PKCξ).
As for PMA-induced NETosis, Ca 2+ -sensitive enzyme PAD4 was possibly the main target of BAPTA-AM in this signaling pathway.
We have shown earlier [14] that A23187-induced NETosis involves mtROS in both neutrophils of healthy individuals and neutrophils deficient in NADPH-oxidase. At the same time, the activation of NADPH oxidase was also necessary for the induction of NETosis in normal neutrophils. Apparently, the generation of mtROS was substantially increased in the deficiency of NADPH oxidase. The results of this research contain the explanation of this phenomenon. The elevated level of intracellular Ca 2+ in NADPH oxidase-deficient neutrophils might stimulate dehydrogenases in the mitochondrial matrix and respiration, thus contributing to the increased formation of mtROS. Furthermore, we found that the A23187-dependent netotic signaling cascade is related to the activation of mitochondrial pore mPTP [14]. Increased mitochondrial level of Ca 2+ enhanced the likelihood of mPTP pore opening in NADPH oxidase-deficient neutrophils, further favoring the augmented generation of mtROS. Interestingly, the neutrophils of patients with CGD were characterized by elevated Ca 2+ -dependent production of lipid inflammatory mediator leukotriene B4 [21]. It can be proposed that the excessive level of Ca 2+ and mtROS could define both aberrant NETosis and other inflammatory processes associated with autoimmune and inflammatory diseases often affecting patients with CGD.
Thus, for the first time our experiment has shown that neutrophils of patients suffering from CGD both have a limited ability to attack a large amount of pathogens and significantly differ in calcium regulation and, therefore, disturbed NETosis as a Ca 2+ -dependent process.