Neutrophil Reactive Oxygen Species Production Research Articles

Vimentin regulates mitochondrial ROS production and inflammatory responses of neutrophils

The intermediate filament vimentin is present in immune cells and is implicated in proinflammatory immune responses. Whether and how it supports antimicrobial activities of neutrophils are not well established. Here, we developed an immortalized neutrophil model to examine the requirement of vimentin. We demonstrate that vimentin restricts the production of proinflammatory cytokines and reactive oxygen species (ROS), but enhances phagocytosis and swarming. We observe that vimentin is dispensable for neutrophil extracellular trap (NET) formation, degranulation, and inflammasome activation. Moreover, gene expression analysis demonstrated that the presence of vimentin was associated with changes in expression of multiple genes required for mitochondrial function and ROS overproduction. Treatment of wild-type cells with rotenone, an inhibitor for complex I of the electron transport chain, increases the ROS levels. Likewise, treatment with mitoTEMPO, a SOD mimetic, rescues the ROS production in cells lacking vimentin. Together, these data show vimentin regulates neutrophil antimicrobial functions and alters ROS levels through regulation of mitochondrial activity.

BTK inhibitor-induced defects in human neutrophil effector activity against Aspergillus fumigatus are restored by TNF-α.

Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the effect of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNF-α fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNF-α did not affect ROS production in healthy neutrophils but prevented exogenous TNF-α from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNF-α was independent of transcription. Moreover, the addition of TNF-α immediately rescued ROS production in IBT-treated neutrophils, indicating that TNF-α worked through a BTK-independent signaling pathway. Finally, TNF-α restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNF-α rescued the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.

Neutrophil NADPH oxidase regulation by oxidative phosphorylation and accessory glucose metabolic pathways

Neutrophils produce reactive oxygen species (ROS) to extinguish pathogens and regulate intracellular signaling. The major producer of ROS in neutrophils is the NADPH oxidase, which is fueled by NADPH generated via the oxidative pentose phosphate pathway. It remains unclear how other accessory pathways of glucose metabolism and mitochondrial activity influence the oxidative burst in neutrophils. The respiratory burst in bone marrow-derived neutrophils was stimulated with phorbol 12-myristate 13-acetate (PMA) in the presence or absence of an inhibitor of glycogen breakdown (glycogen phosphorylase inhibitor; GPi), inhibitors of mitochondrial respiration (antimycin A and rotenone), or an inhibitor of serine biosynthesis (NCT503). Respiratory burst was also measured under conditions of limited glucose and glutamine. ROS production was measured via extracellular flux analysis or via dihydrorhodamine fluorescence. In both male and female mouse neutrophils, inhibition of glycogen breakdown by GPi delayed initiation of the oxidative burst in response to PMA (n = 6, p<0.05, Student’s T-test and Repeated Measure’s ANOVA). Inhibition of mitochondrial oxidative phosphorylation or inhibition of the serine biosynthetic pathway sustained ROS production in PMA-stimulated neutrophils (n = 6, p<0.05, Student’s T-test). Removal of glucose and glutamine showed a truncated oxidative burst (n = 6, p<0.05, Student’s T-test). Ultimately, these findings suggest that glycogen breakdown fuels the initial activation of the oxidative burst, while mitochondrial activity and the serine biosynthesis pathway control the magnitude and duration of neutrophil ROS production. Furthermore, utilization of exogenous glucose and glutamine are necessary for the entire oxidative burst. Future studies will continue to illustrate the mechanisms of neutrophil metabolic plasticity as well as connect their significance in the context of wound healing, exercise, and chronic inflammatory diseases. NIH: R01HL141191, NIH: P01 HL078825. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Vascular smooth muscle cells in response to cholesterol crystals modulates inflammatory cytokines release and promotes neutrophil extracellular trap formation

BackgroundThe formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response.MethodsHuman aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy.ResultsVSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K–AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation.ConclusionWe demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

Bidirectional regulation mechanism of TRPM2 channel: role in oxidative stress, inflammation and ischemia-reperfusion injury.

Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel that exhibits Ca2+ permeability. The TRPM2 channel is expressed in various tissues and cells and can be activated by multiple factors, including endogenous ligands, Ca2+, reactive oxygen species (ROS) and temperature. This article reviews the multiple roles of the TRPM2 channel in physiological and pathological processes, particularly on oxidative stress, inflammation and ischemia-reperfusion (I/R) injury. In oxidative stress, the excessive influx of Ca2+ caused by the activation of the TRPM2 channel may exacerbate cellular damage. However, under specific conditions, activating the TRPM2 channel can have a protective effect on cells. In inflammation, the activation of the TRPM2 channel may not only promote inflammatory response but also inhibit inflammation by regulating ROS production and bactericidal ability of macrophages and neutrophils. In I/R, the activation of the TRPM2 channel may worsen I/R injury to various organs, including the brain, heart, kidney and liver. However, activating the TRPM2 channel may protect the myocardium from I/R injury by regulating calcium influx and phosphorylating proline-rich tyrosine kinase 2 (Pyk2). A thorough investigation of the bidirectional role and regulatory mechanism of the TRPM2 channel in these physiological and pathological processes will aid in identifying new targets and strategies for treatment of related diseases.

The Effect of Voxelotor on Erythrocyte Deformability, Neutrophil Phenotype and Endothelial Activation

Introduction Sickle cell disease (SCD) is characterized by chronic intravascular hemolysis, caused by polymerization of abnormal sickle hemoglobin. The ongoing intravascular hemolysis leads to release of cell-free heme and iron, resulting in widespread oxidative damage, inflammation, neutrophil activation, endothelial activation and hypercoagulability, all contributing to vaso-occlusive events and end organ damage. Voxelotor, a small molecule reversibly binding to hemoglobin S (HbS) increases its oxygen affinity, thereby decreasing HbS polymerization and sickling, resulting in decreased hemolysis. The decrease in erythrocyte sickling might lead to improved erythrocyte deformability and decreased adhesion, while reductions in cell-free heme and labile plasma iron levels might result in decreased neutrophil activation, adhesion and aging and decreased endothelial activation. The aim of this study is to assess the effects of voxelotor on erythrocyte deformability, neutrophil activation, adhesion and aging and endothelial activation. Methods SCD patients (HbSS and HbSβ 0-thalassemia), with hemoglobin (Hb) ≤ 10.5 g/dL were treated with voxelotor 1500 mg in a single arm interventional study. Blood was collected at baseline and after 3 months of treatment. Erythrocyte deformability was assessed by ektacytometry (Lorrca, RR mechatronics), measuring elongation index (EI) and point of sickling (PoS). EI max and EI min indicate the highest and lowest erythrocyte deformability upon oxygenation and deoxygenation respectively. PoS is the oxygen tension at which 5% reduction of the EI max is observed. Antigen expression of neutrophil markers for activation, adhesion and aging were assessed with flow cytometry. Reactive oxygen species (ROS) production of neutrophils was measured by Amplex Red Assay (Molecular probes), measuring hydrogen peroxide (H 2O 2) production under unstimulated and stimulated (zymosan, streptozotocin (STZ), factor-n-formyl-MET-LEU-Phe (fMLP), Phorbol myristate acetate (PMA)) and platelet-activating factor-fMLP (PAF-fMLP)). Endothelial activation was assessed by measuring von Willebrand Factor antigen (vWF-Ag) using enzyme-linked immunosorbent assay (ELISA) (Glostrup). Prothrombin fragment 1+2 (F 1+2), thrombin-antithrombin complexes (TAT) and D-dimer were measured using ELISA. Data are presented as median with interquartile range (IQR), unless stated otherwise. A P value of <0.05 was considered significant. Results 15 SCD patients (11 HbSS, 4 HbSβ 0-thalassemia), 5 female, median age 35 years (IQR 27-46)) completed 3-month follow-up. Hb levels increased significantly after 3 months of treatment (from 7.4 (6.9 - 8.1) g/dL to 9.7 (7.9 - 11.0) g/dL, p=0.002), corresponding to significant decreases in LDH and bilirubin levels and reticulocyte counts as markers of hemolysis (p<0.05). PoS decreased significantly after 3 months (from 20.1 (17.5 - 26.1) mmHg to 16.1 (10.3 - 18.1) mmHg, p=0.006). Both EI max and EI min increased after 3 months of treatment (from 0.33 (0.24 - 0.42) to 0.38 (0.28 - 0.48), p=0.029 and from 0.13 (0.07 - 0.26) to 0.21 (0.16 - 0.41), p=0.007 respectively). Markers of neutrophil activation, adhesion and aging were generally lower after 3 months of treatment, though the differences were not statistically significant (Figure 1 A-C). Neutrophil ROS production in both unstimulated and stimulated condition remained unchanged (Figure 1D). After 3 months of treatment vWF-Ag significantly decreased (from 252 (187 - 284) % to 183 (153-205) %, p = 0.033). No changes were observed in markers of coagulation activation (F 1+2 and TAT, and D-dimer). Conclusion Voxelotor significantly improved erythrocyte deformability as suggested by decreased PoS and increased EI max and EI min, potentially leading to improved rheological properties. While markers of neutrophil activation seem to decrease, the differences are not statistically significant, possibly due to the relatively small sample size. A reduction in vWF-Ag was observed possibly reflecting diminished endothelial activation, due to a decrease in intravascular hemolysis.

Effects of intravenous administration of ascorbic acid (vitamin C) on oxidative status in healthy adult horses.

Ascorbic acid (AA) is an antioxidant that might be beneficial for adjunctive treatment of sepsis in horses. The optimal dose and effects on oxidative status are unknown. Ascorbic acid administration will increase plasma AA concentrations and decrease determinants of reactive oxygen metabolites (dROM), basal and stimulant-induced intraerythrocytic reactive oxygen species (ROS) concentrations, and stimulant-induced neutrophil ROS production, and increase plasma antioxidant capacity (PAC) in a dose-dependent manner. Eight healthy horses. Randomized placebo-controlled crossover study. Each horse received 4 single-dose IV treatments including AA at 25, 50, and 100 mg/kg and saline (placebo) with each treatment separated by ≥1 week. Blood was collected at baseline, 2 and 6 hours for assessment of plasma dROM and PAC via photometer, intraerythrocytic ROS by flow cytometry, and stimulant-induced neutrophil ROS by a fluorometric assay. Plasma AA concentrations were measured by high-performance liquid chromatography/electrochemical detection. Ascorbic acid at 100 mg/kg resulted in decreased dROM 2 hours after treatment (P = .03, 95% CI 5.51-121.2, point estimate 63.3). There was no effect of AA on basal or stimulant-induced intraerythrocytic ROS (P = .88, 95% CI -0.156 to 0.081, point estimate -0.037; P = .93, 95% CI -0.123 to 0.112, point estimate -0.006, respectively), basal or stimulant-induced neutrophil ROS (P ≥ .12, 95% CI -644.9 to 56.2, point estimate -294.4), or PAC (P ≥ .64, 95% CI -1567 to 463.4, point estimate -552.0) at any dose or timepoint. Plasma AA concentrations increased in a dose-dependent manner. High-dose administration of AA might provide antioxidant benefits in horses.

Dysregulation of neutrophil oxidant production and interleukin-1-related cytokines in granulomatosis with polyangiitis.

Neutrophils play a key role in ANCA-associated vasculitis, both as targets of autoimmunity and as facilitators of vascular damage. In granulomatosis with polyangiitis (GPA), the data regarding the production of reactive oxygen species (ROS) in neutrophils are unclear. Further, recent data suggests that ROS production could have an anti-inflammatory effect through the regulation of inflammasomes and IL-1-related cytokines. We aimed to analyse ROS production in neutrophils from patients with GPA and investigate its association with IL-1-related cytokines and the autoantigen PR3. Seventy-two GPA patients with disease flare were included in the NEUTROVASC prospective cohort study. ROS production in whole blood of patients with active GPA was evaluated and compared with that in the same patients in remission or healthy controls. Associations between ROS production, PR3 membrane expression on neutrophils, serum levels of IL-1-related cytokines as well as inflammasome-related proteins were analysed. We observed a robust defect in ROS production by neutrophils from patients with active GPA compared with healthy controls, independent of glucocorticoid treatment. Serum levels of IL-1-related cytokines were significantly increased in GPA patients, particularly in patients with kidney involvement, and levels of these cytokines returned to normal after patients achieved remission. Further, inflammasome-related proteins were significantly dysregulated in the cytosol of neutrophils as well as the serum from GPA patients. Our data suggests that ROS production and regulation of inflammasomes in neutrophils from patients with GPA are disturbed and may be a potential therapeutic target. ClinicalTrials.gov, https://www.clinicaltrials.gov, NCT01862068.

Helicobacter pylori extract induces purified neutrophils to produce reactive oxygen species only in the presence of plasma.

H. pylori is a bacterial pathogen infecting over half of the world's population and induces several gastric and extra-gastric diseases through its various virulence factors, especially cagA. These factors may be released from the bacteria during interactions with host immune cells. Neutrophils play key roles in innate immunity, and their activity is regulated by plasma factors, which can alter how these cells may interact with pathogens. The aim of the present study was to determine whether purified neutrophils could produce reactive oxygen species (ROS), one of the key functions of their anti-microbial functions, in response to extracts of cagA+ and cagA- H. pylori. Extracts from either cagA+ or cagA- H. pylori were co-cultured with human neutrophils in the presence or absence of plasma, and the neutrophil ROS production was measured. In the absence of plasma, extracts from cagA+ and cagA- H. pylori did not induce neutrophil ROS production, whereas in the presence of plasma, extracts from both cagA+ and cagA- H. pylori-induced ROS production. Furthermore, when peripheral blood mononuclear cells (PBMCs) were added to the purified neutrophils in the absence of plasma, there was no neutrophil ROS production after challenging with extracts from either cagA+ or cagA- H. pylori. Thus, it is suggested that plasma contains immunological components that change the responsiveness of neutrophils, such that when neutrophils encounter the bacterial antigens in H. pylori extracts, they become activated and produce ROS. This study also revealed a potential novel immunopathogenic pathway by which cagA activation of neutrophils contributed to inflammatory damage.

Immunosuppressive Effects of Annona muricata L. Leaf Extract on Cellular and Humoral Immune Responses in Male Wistar Rats.

Annona muricata</i> L. (Annonaceae) (AM)'s remarkable anti-inflammatory and anti-cancer activities make it a targeted plant to be explored for its immunomodulatory properties. Traditional practitioners have employed various components of AM to cure a variety of ailments, including cancer, diabetes, and inflammation. The present study evaluated the immunosuppressive effects of 80% ethanol extract of of AM leaves in male Wistar rats on different parameters of humoral and cellular immune responses. AM leaf extract (AMLE) was analyzed using UHPLC-MS/MS to profile its secondary metabolites. AMLE was rich in polyphenols which include (epi)catechin-(epi)catechin-(epi) catechin, caffeic acid, coumaroylquinic acid, hyperin, kaempferol, quinic acid and rutin. The rats were administered 100, 200 and 400 mg/kg bw of the extract daily for 14 days. The effects of AMLE on innate immune responses were determined by evaluating phagocytosis, neutrophils migration, reactive oxygen species (ROS) release, CD11b/CD18 integrin expression, and ceruloplasmin, lysozyme and myeloperoxidase (MPO) levels. The adaptive immune parameters were evaluated by immunizing the rats with sheep red blood cells (sRBC) on day 0 and administered orally with AMLE for 14 days. AMLE established significant immunosuppressive effects on the innate immune parameters by inhibiting the neutrophil migration, ROS production, phagocytic activity and expression of CD11b/CD18 integrin in a dose-dependent pattern. AMLE also suppressed ceruloplasmin, MPO and lysozyme expressions in the rat plasma dose-dependently. AMLE dose-dependently inhibited T and B lymphocytes proliferation, Th1 and Th2 cytokine production, CD4+ and CD8+ co-expression in splenocytes, immunoglobulins (IgM and IgG) expression and the sRBC-induced swelling rate of rat paw in delayed-type hypersensitivity (DTH). The strong inhibitory effects on the different parameters of humoral and cellular responses indicate that AMLE has potential to be an important source of effective immunosuppressive agents.

ROS-driven structural and functional fibrinogen modifications are reverted by interleukin-6 inhibition in Giant Cell Arteritis

BackgroundCranial and extra-cranial vascular events are among the major determinants of morbidity and mortality in Giant Cell Arteritis (GCA). Vascular events seem mostly of inflammatory nature, although the precise pathogenetic mechanisms are still unclear. We investigated the role of oxidation-induced structural and functional fibrinogen modifications in GCA. The effects of the anti-IL6R tocilizumab in counteracting these mechanisms were also assessed. Materials and methodsA cross-sectional study was conducted on 65 GCA patients and 65 matched controls. Leucocyte reactive oxygen species (ROS) production, redox state, and fibrinogen structural and functional features were compared between patients and controls. In 19 patients receiving tocilizumab, pre vs post treatment variations were assessed. ResultsGCA patients displayed enhanced blood lymphocyte, monocyte and neutrophil ROS production compared to controls, with an increased plasma lipid peroxidation and a reduced total antioxidant capacity. This oxidative impairment resulted in a sustained fibrinogen oxidation (i.e. dityrosine content 320 (204–410) vs 136 (120–176) Relative Fluorescence Units (RFU), p < 0.0001), with marked alterations in fibrinogen secondary and tertiary structure [intrinsic fluorescence: 134 (101–227) vs 400 (366–433) RFU, p < 0.001]. Structural alterations paralleled a remarkable fibrinogen functional impairment, with a reduced ability to polymerize into fibrin and a lower fibrin susceptibility to plasmin-induced lysis. In patients receiving tocilizumab, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features (p < 0.001). ConclusionsAn impaired redox status accounts for structural and functional fibrinogen modifications in GCA, suggesting a potential role of tocilizumab for cardiovascular prevention in GCA.

Escherichia coli Nissle 1917 ghosts alleviate inflammatory bowel disease in zebrafish

Escherichia coli Nissle 1917 (EcN) has become a research hotspot in inflammatory bowel disease (IBD). It has a strong targeting effect on the colon, and has some therapeutic effect on inflammatory bowel disease. EcN is prepared into EcN ghosts, which also retain EcN's biological characteristics. Consequently, EcN ghosts are used for drug delivery. This study evaluated the safety and efficacy of EcN ghosts as carriers of drugs for treating IBD in zebrafish. Caco-2 cell adhesion experiments and zebrafish intestinal adhesion experiments demonstrated that EcN ghosts was highly adherent to the intestine. Additionally, oral administration of EcN ghosts attenuated dextran sulfate sodium-induced IBD symptoms by inhibiting neutrophil chemotaxis and reactive oxygen species production in larval zebrafish. Because of the unique biological functions of EcN ghosts, it may serve as a strategy for future targeted drug delivery in IBD treatment.

Human blood neutrophils generate ROS through FcγR-signaling to mediate protection against febrile P. falciparum malaria

Blood phagocytes, such as neutrophils and monocytes, generate reactive oxygen species (ROS) as a part of host defense response against infections. We investigated the mechanism of Fcγ-Receptor (FcγR) mediated ROS production in these cells to understand how they contribute to anti-malarial immunity. Plasmodium falciparum merozoites opsonized with naturally occurring IgG triggered both intracellular and extracellular ROS generation in blood phagocytes, with neutrophils being the main contributors. Using specific inhibitors, we show that both FcγRIIIB and FcγRIIA acted synergistically to induce ROS production in neutrophils, and that NADPH oxidase 2 and the PI3K intracellular signal transduction pathway were involved in this process. High levels of neutrophil ROS were also associated with protection against febrile malaria in two geographically diverse malaria endemic regions from Ghana and India, stressing the importance of the cooperation between anti-malarial IgG and neutrophils in triggering ROS-mediated parasite killing as a mechanism for naturally acquired immunity against malaria.

New NADPH Oxidase 2 Inhibitors Display Potent Activity against Oxidative Stress by Targeting p22phox-p47phox Interactions.

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

The metabolic function of pyruvate kinase M2 regulates reactive oxygen species production and microbial killing by neutrophils

Neutrophils rely predominantly on glycolytic metabolism for their biological functions, including reactive oxygen species (ROS) production. Although pyruvate kinase M2 (PKM2) is a glycolytic enzyme known to be involved in metabolic reprogramming and gene transcription in many immune cell types, its role in neutrophils remains poorly understood. Here, we report that PKM2 regulates ROS production and microbial killing by neutrophils. Zymosan-activated neutrophils showed increased cytoplasmic expression of PKM2. Pharmacological inhibition or genetic deficiency of PKM2 in neutrophils reduced ROS production and Staphylococcus aureus killing in vitro. In addition, this also resulted in phosphoenolpyruvate (PEP) accumulation and decreased dihydroxyacetone phosphate (DHAP) production, which is required for de novo synthesis of diacylglycerol (DAG) from glycolysis. In vivo, PKM2 deficiency in myeloid cells impaired the control of infection with Staphylococcus aureus. Our results fill the gap in the current knowledge of the importance of lower glycolysis for ROS production in neutrophils, highlighting the role of PKM2 in regulating the DHAP and DAG synthesis to promote ROS production in neutrophils.

Anti-CD20 treatment and neutrophil function in central nervous system demyelinating diseases

IntroductionA contribution of neutrophil granulocytes to the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) is recognized. Anti-CD20 treatments applied in these diseases are associated with infectious complications and neutropenia. No data is available about functional characteristics of neutrophils obtained from patients with anti-CD20 treatments. MethodsIn neutrophils isolated from 13 patients with anti-CD20 treatment (9 MS, 4 NMOSD), 11 patients without anti-CD20 treatment (9 MS, 2 NMOSD) and 5 healthy controls, we analyzed chemotaxis, production of reactive oxygen species (ROS), phagocytosis, and formation of neutrophil extracellular traps (NET) in vitro. ResultsChemotaxis and ROS production were found unchanged between patients with and without anti-CD20 treatment or between patients and healthy controls. We found a higher proportion of non-phagocytosing cells in patients without anti-CD20 treatment compared to patients with anti-CD20 treatment and healthy controls. As compared to healthy controls, a higher proportion of neutrophils from patients without anti-CD20 treatments underwent NET formation, either unstimulated or stimulated with phorbol 12-myristate 3-acetate for 3 h. In about half of patients with anti-CD20 treatment (n = 7), NET formation of unstimulated neutrophils occurred already within 20 min of incubation. This was not observed in patients without anti-CD20 treatment and healthy controls. ConclusionAnti-CD20 treatment in MS and NMOSD patients does not alter chemotaxis and ROS production of neutrophils in vitro but might restore their impaired phagocytosis in these diseases. Our study reveals a predisposition to early NET formation in vitro of neutrophils obtained from patients with anti-CD20 treatment. This may contribute to associated risks of neutropenia and infections.

Targeting Myeloperoxidase Activity and Neutrophil ROS Production to Modulate Redox Process: Effect of Ellagic Acid and Analogues.

Malaria is an infectious disease caused by a Plasmodium genus parasite that remains the most widespread parasitosis. The spread of Plasmodium clones that are increasingly resistant to antimalarial molecules is a serious public health problem for underdeveloped countries. Therefore, the search for new therapeutic approaches is necessary. For example, one strategy could consist of studying the redox process involved in the development of the parasite. Regarding potential drug candidates, ellagic acid is widely studied due to its antioxidant and parasite-inhibiting properties. However, its low oral bioavailability remains a concern and has led to pharmacomodulation and the synthesis of new polyphenolic compounds to improve antimalarial activity. This work aimed at investigating the modulatory effect of ellagic acid and its analogues on the redox activity of neutrophils and myeloperoxidase involved in malaria. Overall, the compounds show an inhibitory effect on free radicals as well as on the enzyme horseradish peroxidase- and myeloperoxidase (HRP/MPO)-catalyzed oxidation of substrates (L-012 and Amplex Red). Similar results are obtained with reactive oxygen species (ROS) produced by phorbol 12-mystate acetate (PMA)-activated neutrophils. The efficiency of ellagic acid analogues will be discussed in terms of structure-activity relationships.

Sulforaphane Attenuates Neutrophil ROS Production, MPO Degranulation and Phagocytosis, but Does Not Affect NET Formation Ex Vivo and In Vitro.

Sulforaphane has several effects on the human body, including anti-inflammation, antioxidation, antimicrobial and anti-obesity effects. In this study, we examined the effect of sulforaphane on several neutrophil functions: reactive oxygen species (ROS) production, degranulation, phagocytosis, and neutrophil extracellular trap (NET) formation. We also examined the direct antioxidant effect of sulforaphane. First, we measured neutrophil ROS production induced by zymosan in whole blood in the presence of 0 to 560 µM sulforaphane. Second, we examined the direct antioxidant activity of sulforaphane using a HOCl removal test. In addition, inflammation-related proteins, including an azurophilic granule component, were measured by collecting supernatants following ROS measurements. Finally, neutrophils were isolated from blood, and phagocytosis and NET formation were measured. Sulforaphane reduced neutrophil ROS production in a concentration-dependent manner. The ability of sulforaphane to remove HOCl is stronger than that of ascorbic acid. Sulforaphane at 280 µM significantly reduced the release of myeloperoxidase from azurophilic granules, as well as that of the inflammatory cytokines TNF-α and IL-6. Sulforaphane also suppressed phagocytosis but did not affect NET formation. These results suggest that sulforaphane attenuates neutrophil ROS production, degranulation, and phagocytosis, but does not affect NET formation. Moreover, sulforaphane directly removes ROS, including HOCl.

Neisseria gonorrhoeae co-opts C4b-binding protein to enhance complement-independent survival from neutrophils.

Neisseria gonorrhoeae (Gc) is a human-specific pathogen that causes the sexually transmitted infection gonorrhea. Gc survives in neutrophil-rich gonorrheal secretions, and recovered bacteria predominantly express phase-variable, surface-expressed opacity-associated (Opa) proteins (Opa+). However, expression of Opa proteins like OpaD decreases Gc survival when exposed to human neutrophils ex vivo. Here, we made the unexpected observation that incubation with normal human serum, which is found in inflamed mucosal secretions, enhances survival of Opa+ Gc from primary human neutrophils. We directly linked this phenomenon to a novel complement-independent function for C4b-binding protein (C4BP). When bound to the bacteria, C4BP was necessary and sufficient to suppress Gc-induced neutrophil reactive oxygen species production and prevent neutrophil phagocytosis of Opa+ Gc. This research identifies for the first time a complement-independent role for C4BP in enhancing the survival of a pathogenic bacterium from phagocytes, thereby revealing how Gc exploits inflammatory conditions to persist at human mucosal surfaces.