Search

Strong evidence indicates critical roles of NADPH oxidase (a key superoxide-producing enzyme complex during inflammation) in activated microglia for mediating neuroinflammation and neurodegeneration. However, little is known about roles of neuronal NADPH oxidase in neurodegenerative diseases. This study aimed to investigate expression patterns, regulatory mechanisms and pathological roles of neuronal NADPH oxidase in inflammation-associated neurodegeneration. The results showed persistent upregulation of NOX2 (gp91phox; the catalytic subunit of NADPH oxidase) in both microglia and neurons in a chronic mouse model of Parkinson's disease (PD) with intraperitoneal LPS injection and LPS-treated midbrain neuron-glia cultures (a cellular model of PD). Notably, NOX2 was found for the first time to exhibit a progressive and persistent upregulation in neurons during chronic neuroinflammation. While primary neurons and N27 neuronal cells displayed basal expression of NOX1, NOX2 and NOX4, significant upregulation only occurred in NOX2 but not NOX1 or NOX4 under inflammatory conditions. Persistent NOX2 upregulation was associated with functional outcomes of oxidative stress including increased ROS production and lipid peroxidation. Neuronal NOX2 activation displayed membrane translocation of cytosolic p47phox subunit and was inhibited by apocynin and diphenyleneiodonium chloride (two widely-used NADPH oxidase inhibitors). Importantly, neuronal ROS production, mitochondrial dysfunction and degeneration induced by inflammatory mediators in microglia-derived conditional medium were blocked by pharmacological inhibition of neuronal NOX2. Furthermore, specific deletion of neuronal NOX2 prevented LPS-elicited dopaminergic neurodegeneration in neuron-microglia co-cultures separately grown in the transwell system. The attenuation of inflammation-elicited upregulation of NOX2 in neuron-enriched and neuron-glia cultures by ROS scavenger N -acetylcysteine indicated a positive feedback mechanism between excessive ROS production and NOX2 upregulation. Collectively, our findings uncovered crucial contribution of neuronal NOX2 upregulation and activation to chronic neuroinflammation and inflammation-related neurodegeneration. This study reinforced the importance of developing NADPH oxidase-targeting therapeutics for neurodegenerative diseases. [Display omitted] • Progressive, persistent neuronal NOX2 upregulation during chronic neuroinflammation. • Positive feedback mechanism between excessive ROS production and NOX2 upregulation. • Persistent neuronal NOX2 upregulation & activation sustain chronic neuroinflammation. • Persistent neuronal NOX2 upregulation and activation promote neurodegeneration. [ABSTRACT FROM AUTHOR]

Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils. X91+-CGD mutations lead to a loss of function but with a normal expression of mutated NOX2 proteins. It is therefore necessary to ensure that these mutations are indeed responsible for the loss of activity for genetic counseling. Thus to ascertain the identity of the neutrophil-like model used to study the functional impact of X91+-CGD mutations, we developed a new X-CGD cellular model using the clustered regularly interspaced short palindromic repeats associated Cas9 protein (CRISPR-Cas9) technology (all-in-one Cas9 SmartNucleaseTM Plasmid Expression System) in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. [ABSTRACT FROM AUTHOR]

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKβ1-deficient (AMPKβ1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKβ1LysM mice, whereas sodium salicylate (SS; AMPK β1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKβ1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKβ1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis. [ABSTRACT FROM AUTHOR]

Poldip2 was shown to be involved in oxidative signaling to ensure certain biological functions. It was proposed that, in VSMC, by interaction with the Nox4-associated membrane protein p22phox, Poldip2 stimulates the level of reactive oxygen species (ROS) production. In vitro, with fractionated membranes from HEK393 cells over-expressing Nox4, we confirmed the up-regulation of NADPH oxidase 4 activity by the recombinant and purified Poldip2. Besides Nox4, the Nox1, Nox2, or Nox3 isoforms are also established partners of the p22phox protein raising the question of their regulation by Poldip2 and of the effect in cells expressing simultaneously different Nox isoforms. In this study, we have addressed this issue by investigating the potential regulatory role of Poldip2 on NADPH oxidase 2, present in phagocyte cells. Unexpectedly, the effect of Poldip2 on phagocyte NADPH oxidase 2 was opposite to that observed on NADPH oxidase 4. Using membranes from circulating resting neutrophils, the ROS production rate of NADPH oxidase 2 was down-regulated by Poldip2 (2.5-fold). The down-regulation effect could not be correlated to the interaction of Poldip2 with p22phox but rather, to the interaction of Poldip2 with the p47phox protein, one of the regulatory proteins of the phagocyte NADPH oxidase. Our results show that the interaction of Poldip2 with p47phox constitutes a novel regulatory mechanism that can negatively modulate the activity of NADPH oxidase 2 by trapping the so-called "adaptor" subunit of the complex. Poldip2 could act as a tunable switch capable of specifically regulating the activities of NADPH oxidase s. This selective regulatory role of Poldip2, positive for Nox4 or negative for Nox2 could orchestrate the level and the type of ROS generated by Nox enzymes in the cells. [Display omitted] • Poldip2 down-regulates phagocyte NADPH oxidase activity. • Poldip2 has opposite effects on the activities of NADPH oxidase 2 and 4. • Poldip2 inhibition of NADPH oxidase 2 by trapping free p47phox. • The source of ROS and their relative amount in cells expressing different Nox isoforms could be regulated by Poldip2. [ABSTRACT FROM AUTHOR]

Defective superoxide production by NADPH oxidase 2 (Nox2) in phagocyte cells results in the development of chronic granulomatous disease (CGD), a hereditary disease characterized by recurrent and life‐threatening infections. The partner protein p22phox is a membrane‐spanning protein which forms a stable heterodimer with Nox2 in the endoplasmic reticulum. This interaction ensures the stability of each protein and their accurate trafficking to the cell membrane. The present paper describes the characterization of p22phox missense mutations that were identified in a patient with CGD who presented with undetectable levels of p22phox. Using a reconstitution system, it was found that p22phox expression decreased when R90Q, A117E, S118R, A124S, A124V, A125T, or E129K mutations were introduced, suggesting that these mutations destabilize the protein. In contrast, introducing an L105R mutation did not affect protein expression, but did inhibit p22phox binding to Nox2. Thus, the missense mutations discussed here contribute to the development of CGD by either disrupting protein stability or by impairing the interaction between p22phox and Nox2. [ABSTRACT FROM AUTHOR]

NADPH oxidases 2 (NOX2) is the main source of ROS in macrophages, which plays a critical role in the formation of atherosclerosis. However, effects of NOX2 inhibition on established vulnerable plaques and the potential role involved remain unclear. The purpose of this study is to investigate the latent mechanism of NOX2-triggered vulnerable plaque development. We generated a vulnerable carotid plaque model induced by carotid branch ligation and renal artery constriction, combined with a high-fat diet in ApoE−/− mice. NOX2 specific inhibitor, GSK2795039 (10 mg/kg/day by intragastric administration for 8 weeks) significantly prevented vulnerable plaque, evaluated by micro-ultrasound imaging parameters. A profile of less intraplaque hemorrhage detection, increased collagen-lipid ratio, fibrous cap thickness and less necrotic core formation were also found in GSK2795039 treated group. Mechanistically, reduced 4-HNE, in situ lesional apoptosis and enhanced efferocytosis were involved in mice treated with NOX2 inhibitor. Further analysis in mouse macrophages confirmed the role of NOX2 inhibition in enhancing macrophage efferocytosis by regulating the MertK/PI3K/AKT pathway. In summary, our data defined previously few recognized roles of NOX2 in vulnerable plaque pathogenesis and an undescribed NOX2-ROS-MerTK axis acts involved in regulating macrophage efferocytosis in the formation of rupture-prone vulnerable plaques.

Reactive oxygen species (ROS) are a family of highly reactive molecules with numerous, often pleiotropic functions within the cell and the organism. Due to their potential to destroy biological structures such as membranes, enzymes and organelles, ROS have long been recognized as harmful yet unavoidable by-products of cellular metabolism leading to ''oxidative stress'' unless counterbalanced by cellular anti-oxidative defense mechanisms. Phagocytes utilize this destructive potential of ROS released in high amounts to defend against invading pathogens. In contrast, a regulated and fine-tuned release of ''signaling ROS'' (sROS) provides essential intracellular second messengers to modulate central aspects of immunity, including antigen presentation, activation of antigen presenting cells (APC) as well as the APC:T cell interaction during T cell activation. This regulated release of sROS is foremost attributed to the specialized enzyme NADPH-oxidase (NOX) 2 expressed mainly in myeloid cells such as neutrophils, macrophages and dendritic cells (DC). NOX-2-derived sROS are primarily involved in immune regulation and mediate protection against autoimmunity as well as maintenance of self-tolerance. Consequently, deficiencies in NOX2 not only result in primary immune-deficiencies such as Chronic Granulomatous Disease (CGD) but also lead to auto-inflammatory diseases and autoimmunity. A comprehensive understanding of NOX2 activation and regulation will be key for successful pharmaceutical interventions of such ROS-related diseases in the future. In this review, we summarize recent progress regarding immune regulation by NOX2-derived ROS and the consequences of its deregulation on the development of immune disorders.

AIM: To explore whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) is expressed in fungal keratitis in mice and investigate its role in this disease. METHODS: NOX2 expression was detected in C57BL/6 mice. After testing the inhibitory effect of diphenyleneiodonium chloride (DPI) on NOX2, its impact on clinical performance, myeloperoxidase levels, the number of colonies forming units, the level of H3, the generation of reactive oxygen species (ROS) and the release of cytokines [NF-κB, interleukin-17A (IL-17A), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), Nrf2, IL-10, and TGF-β] were compared. A one-way ANOVA and an unpaired, two-tailed Student's t-test was used to determine the statistical significance. RESULTS: NOX2 expression was significantly increased after Aspergillus fumigatus injection in corneas and that this increase could be reduced by treatment with DPI. DPI treatment produced more severe inflammation and resulted in higher clinical scores, more neutrophils infiltration, a weakened ability to clear fungi, the release of fewer ROS and the formation of neutrophil extracellular traps. Treatment with DPI increased the expression of the proinflammatory cytokines NF-κB, IL-17A, IL-6, and TNF-α and decreased the expression of the anti-inflammatory cytokines Nrf2, IL-10 and TGF-β compared to their expression levels without DPI treatment. CONCLUSION: NOX2 plays an important role against Aspergillus fumigatus in the mouse cornea through killing fungi and limiting the degree of inflammation.

Cancer cells are characterised by an elevated metabolic plasticity and enhanced production of reactive oxygen species (ROS), two features acknowledged as hallmarks in cancer, with a high translational potential to the therapeutic setting. These aspects, that have been traditionally studied separately, are in fact intimately intermingled. As part of their transforming activity, some oncogenes stimulate rewiring of metabolic processes, whilst simultaneously promoting increased production of intracellular ROS. In this scenario the latest discoveries suggest the relevance of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX) to connect ROS production and metabolic control. Here we have analysed the relevance of NOX2 and NOX4 in the regulation of metabolism in chronic myeloid leukaemia (CML), a neoplasia driven by the expression of the breakpoint cluster region-Abelson fusion oncogene (BCR-ABL). Silencing of NOX2 enhances glycolysis and oxidative phosphorylation rates, together with an enhanced production of mitochondrial ROS and a decrease in mitochondrial DNA copy number, which reflects mitochondrial dysfunction. NOX4 expression was upregulated upon NOX2 silencing, and this was required to alter mitochondrial function. Our results support the relevance of NOX2 to regulate metabolism-related signalling pathways downstream of BCR-ABL. Overall we show that NOX2, through the regulation of NOX4 expression, controls metabolism and mitochondrial function in CML cells. This notion was confirmed by transcriptomic analyses, that strongly relate both NOX isoforms with metabolism regulation in CML. [Display omitted] • NOX2 silencing enhances glycolytic and OXPHOS metabolism in CML cells. • NOX2 silencing induces NOX4 upregulation, which is needed for the metabolic change. • Our results reveal a functional axis NOX2-NOX4-mitochondrial in CML cells. [ABSTRACT FROM AUTHOR]

Cardiac hypertrophy is accompanied by increased myocardial oxidative stress, and whether naringenin, a natural antioxidant, is effective in the therapy of cardiac hypertrophy remains unknown. In the present study, different dosage regimens (25, 50, and 100 mg/kg/d for three weeks) of naringenin (NAR) were orally gavaged in an isoprenaline (ISO) (7.5mg/kg)-induced cardiac hypertrophic C57BL/6J mouse model. The administration of ISO led to significant cardiac hypertrophy, which was alleviated by pretreatment with naringenin in both in vivo and in vitro experiments. Naringenin inhibited ISO-induced oxidative stress, as demonstrated by the increased SOD activity, decreased MDA level and NOX2 expression, and inhibited MAPK signaling. Meanwhile, after the pretreatment with compound C (a selective AMPK inhibitor), the anti-hypertrophic and anti-oxidative stress effects of naringenin were blocked, suggesting the protective effect of naringenin on cardiac hypertrophy. Our present study indicated that naringenin attenuated ISO-induced cardiac hypertrophy by regulating the AMPK/NOX2/MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Circulating monocytes are recruited in damaged tissues to generate macrophages that modulate disease progression. Colony-stimulating factor-1 (CSF-1) promotes the generation of monocyte-derived macrophages, which involves caspase activation. Here, we demonstrate that activated caspase-3 and caspase-7 are located to the vicinity of the mitochondria in CSF1-treated human monocytes. Active caspase-7 cleaves p47PHOX at aspartate 34, which promotes the formation of the NADPH (nicotinamide adenine dinucleotide phosphate) oxidase complex NOX2 and the production of cytosolic superoxide anions. Monocyte response to CSF-1 is altered in patients with a chronic granulomatous disease, which are constitutively defective in NOX2. Both caspase-7 down-regulation and radical oxygen species scavenging decrease the migration of CSF-1-induced macrophages. Inhibition or deletion of caspases prevents the development of lung fibrosis in mice exposed to bleomycin. Altogether, a non-conventional pathway that involves caspases and activates NOX2 is involved in CSF1-driven monocyte differentiation and could be therapeutically targeted to modulate macrophage polarization in damaged tissues. [ABSTRACT FROM AUTHOR]

Exosomes containing various biological cargoes have potential to be novel diagnostic biomarkers for metabolic diseases. In this study, retinol-binding protein 4 (RBP4) was found to be enriched in serum exosomes, and its increased levels could be considered as an independent risk factor for the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Exosomal RBP4 (exo-RBP4), primarily derived from hepatocytes, significantly enhanced the M1-like polarization of Kupffer cells (KCs) via promoting the activation of NOX2 and NF-κB and reactive oxygen species (ROS) accumulation, resulting in the over-production of inflammatory cytokines including TNF-α. Subsequently, those excess cytokines remarkably increased the levels of intracellular free fatty acid uptake and lipogenesis-related genes (FAS and SREBP-1c) but decreased fatty acid degradation-related genes (CPT-1 and PPARα) in palmitic acid-treated LO2 cells. More notably, TNF-α significantly elevated RBP4 transcription by activating STAT3 in hepatocytes, playing a positive role in NAFLD development. Intravenous injection with RBP4 (50 μg/kg) potentiated hepatic lipid accumulation, M1-type KC proportion, and serum pro-inflammatory cytokine levels in the hepatic tissues of high-fat-diet-fed mice. Collectively, these data indicated that exo-RBP4 converted KCs to M1 subtype by mediating the NOX2/ROS/NF-κB pathway, subsequently promoting de novo lipogenesis in hepatocytes by TNF-α secretion to activate the JAK2/STAT3 signaling pathway. Therefore, this study uncovered a novel intercellular communication between the inflammatory microenvironment and lipid metabolism for fostering NAFLD progression and found the potential of exo-RBP4 as a novel diagnostic biomarker and therapeutic target for NAFLD. [Display omitted] • Elevated peripheral exo-RBP4 levels were closely correlation with NAFLD progression. • Exo-RBP4 induced M1 polarization of Kupffer cells via activating NOX2/NF-κB/TNF-α pathway. • Elevated TNF-α promoted RBP4 transcription by activating STAT3 in hepatocytes. • Exo-RBP4 had potential as a new diagnostic biomarker and therapeutic target for NAFLD. • The study displayed a novel intercellular communication for fostering NAFLD progression. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a multisubunit enzyme complex that participates in the generation of superoxide or hydrogen peroxide (H2O2) and plays a key role in several biological functions. Among seven known NOX isoforms, NOX2 was the first identified in phagocytes but is also expressed in several other cell types including endothelial cells, platelets, microglia, neurons, and muscle cells. NOX2 has been assigned multiple roles in regulating many aspects of innate and adaptive immunity, and human and mouse models of NOX2 genetic deletion highlighted this key role. On the other side, NOX2 hyperactivation is involved in the pathogenesis of several diseases with different etiologies but all are characterized by an increase in oxidative stress and inflammatory process. From this point of view, the modulation of NOX2 represents an important therapeutic strategy aimed at reducing the damage associated with its hyperactivation. Although pharmacological strategies to selectively modulate NOX2 are implemented thanks to new biotechnologies, this field of research remains to be explored. Therefore, in this review, we analyzed the role of NOX2 at the crossroads between immunity and pathologies mediated by its hyperactivation. We described (1) the mechanisms of activation and regulation, (2) human, mouse, and cellular models studied to understand the role of NOX2 as an enzyme of innate immunity, (3) some of the pathologies associated with its hyperactivation, and (4) the inhibitory strategies, with reference to the most recent discoveries. [ABSTRACT FROM AUTHOR]

NADPH oxidase isoform-2 (NOX2) has been implicated in the pathophysiology of neuropathic pain (NP), mostly through the modulation of neuroinflammation. Since it is also accepted that some neuroimmune mechanisms underlying NP are sex-dependent, we aimed to evaluate the effects of early systemic treatment with the NOX2-selective inhibitor (NOX2i) GSK2795039 on behavioral responses and spinal neuroinflammation in spared nerve injury (SNI)-induced NP in male and female mice. Mechanical sensitivity was evaluated with the von Frey test, while general well-being and anxiety-like behavior were assessed with burrowing and light/dark box tests. Spinal microglial activation and cytokines IL-1β, IL-6, and IL-10, as well as macrophage colony-stimulating factor (M-CSF) were evaluated by immunofluorescence and multiplex immunoassay, respectively. NOX2i treatment reduced SNI-induced mechanical hypersensitivity and early SNI-induced microglial activation in both sexes. SNI-females, but not males, showed a transient reduction in burrowing activity. NOX2i treatment did not improve their burrowing activity, but tendentially reduced their anxiety-like behavior. NOX2i marginally decreased IL-6 in females, and increased M-CSF in males. Our findings suggest that NOX2-selective inhibition may be a potential therapeutic strategy for NP in both male and female individuals, with particular interest in females due to its apparent favorable impact in anxiety-like behavior. [ABSTRACT FROM AUTHOR]

The nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) multi-subunit complex is a highly abundant and central source of reactive oxygen species. NOX2 is a key enzyme of the innate immune system involved in antibacterial response, but excessive NOX2 activity is involved in oxidative stress and inflammation in many diseases. Inhibition of NOX2 has great potential as a therapeutic strategy. An intriguing pharmacological approach for inhibiting NOX2 is to target the p47phox subunit and thereby block the protein-protein interaction with p22phox, whereby assembling and activation of NOX2 is prevented. However, the shallow binding pocket of p47phoxmakes it difficult to develop drug-like p47phox/p22phox inhibitors. Recently, the small molecule LMH001 was reported to inhibit the p47phox/p22phox interaction, reduce endothelial NOX2 activity, and protect mice from angiotensin II-induced vascular oxidative stress. These noteworthy results could have significant impact on the field of NOX2 pharmacology, as specific and efficient inhibitors are scarce. Here, we synthesized and tested LMH001 to have it available as a positive control. We established a robust synthetic route for providing LMH001, but subsequently we experienced that LMH001 is chemically unstable in aqueous buffer. In addition, neither LMH001 nor its breakdown products were able to inhibit the p47phox/p22phox interaction in a non-cellular fluorescence polarization assay. However, LHM001 was a weak inhibitor of NOX2 in a functional cell assay, but with same low potency as one of its breakdown products. These findings question the activity and suggested mechanism of LMH001 and constitute important information for other researchers interested in chemical probes for studying NOX2 biology. [ABSTRACT FROM AUTHOR]

Proliferation, metabolism, and migration of hematopoietic stem/progenitor cells (HSPCs) are coordinated by receptors expressed on outer cell membranes that are integrated into microdomains, known as membrane lipid rafts (MLRs). These structures float freely in the cell membrane bilayer and are enriched in cholesterol and sphingolipids for their functional integrity. Receptors, if expressed in MLRs, have prolonged occupancy on the cell surface and enhanced signaling power. Based on this, we have become interested in the regulation of synthesis of MLRs components in HSPCs. To address this, we tested the effect of selected factors that promote proliferation or migration and their potential involvement in the synthesis of MLRs components in HSPCs. Based on our previous research showing that HSPCs from Nox2-KO and Nlrp3-KO mice display a profound defect in MLRs formation, we focused on the role of Nox2-ROS-Nlrp3 inflammasome in regulating lipogenesis in HSPCs. We found that while at steady state conditions, Nox2-derived ROS is required for a proper expression of enzymes regulating lipogenesis, during inflammation, this effect is augmented by Nlrp3 inflammasome. Thus, our data sheds new light on the regulation of lipogenesis in HSPCs and the involvement of the Nox2-ROS-Nlrp3 inflammasome axis that differently regulates lipogenesis at steady state conditions and in response to inflammation, modulating MLRs-mediated responsiveness of these cells to external stimuli. [ABSTRACT FROM AUTHOR]

Cardiac injury and inflammation are common findings in COVID-19 patients. Autopsy studies have revealed cardiac microvascular endothelial damage and thrombosis in COVID-19 patients, indicative of microvascular dysfunction in which reactive oxygen species (ROS) may play a role. We explored whether the ROS producing proteins NOX2, NOX4 and NOX5 are involved in COVID-19-induced cardio-microvascular endothelial dysfunction. Heart tissue were taken from the left (LV) and right (RV) ventricle of COVID-19 patients (n = 15) and the LV of controls (n = 14) at autopsy. The NOX2-, NOX4-, NOX5- and Nitrotyrosine (NT)-positive intramyocardial blood vessels fractions were quantitatively analyzed using immunohistochemistry. The LV NOX2+, NOX5+ and NT+ blood vessels fractions in COVID-19 patients were significantly higher than in controls. The fraction of NOX4+ blood vessels in COVID-19 patients was comparable with controls. In COVID-19 patients, the fractions of NOX2+, NOX5+ and NT+ vessels did not differ significantly between the LV and RV, and correlated positively between LV and RV in case of NOX5 (r = 0.710; p = 0.006). A negative correlation between NOX5 and NOX2 (r = −0.591; p = 0.029) and between NOX5 and disease time (r = −0.576; p = 0.034) was noted in the LV of COVID-19 patients. We show the induction of NOX2 and NOX5 in the cardiac microvascular endothelium in COVID-19 patients, which may contribute to the previously observed cardio-microvascular dysfunction in COVID-19 patients. The exact roles of these NOXes in pathogenesis of COVID-19 however remain to be elucidated. • The ROS producing proteins NOX2 and NOX5 are increased in COVID-19 patients. • For the first time NOX proteins were analyzed in the hearts of COVID-19 patients. • Oxidative stress plays a role in COVID-19-induced cardiac microvascular dysfunction. • These inducible NOX proteins may provide a potential target for therapy. [ABSTRACT FROM AUTHOR]

Objective To investigate the effects of liraglutide (Lir) on microglia/macrophage polarization and on the expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX2) in the ischemic brain tissue of diabetic rats with focal cerebral ischemia. Method Seventy-five rats were randomly and equally divided into 5 groups: cerebral ischemia (CI) group, diabetes+cerebral ischemia (DCI) group, insulin treatment (Ins) group, Lir treatment (Lir) group, and NOX2 agonist tetrabromocinnamic acid (TBCA) + Lir group (TBCA). On the 3rd day after cerebral ischemia, the neurological deficit scores of the rats were evaluated, and 2, 3, 5-triphenyltetrazolium chloride (TTC) staining was adopted to measure the cerebral infarct volume in each group. The co-labeling of CD16/32 and CD206 with Iba1 on microglia/macrophages were detected by immunofluorescence double-label staining, and the protein expression of NOX2, TNF-α, IL-1β, IL-10 and TGF-β were determined using Western blotting. Results As compared with the DCI group, the neurological deficit scores were decreased, and the cerebral infarction volume was reduced in the Lir group. The cell number of Iba1+/CD16/32+ microglia/macrophage (M1 type) was declined, with down-regulated expression of TNF-α, IL-1β and NOX2 in the Lir group, while the number of Iba1+/CD206+ microglia /macrophage (M2 type) was increased, with improved levels of IL-10 and TGF-β (all P < 0.05). However, the above effects were reversed by the treatment of NOX2 agonist TBCA (P < 0.05). Conclusion The neuroprotective effects of Lir on cerebral ischemic injury may be related to the inhibition of NOX2 expression and promotion of microglia/macrophages conversion to M2 type.

[Display omitted] • TNF-α and IFN-γ effectively activate NOX2. • NOX2 deficiency converts neutrophil apoptosis to GSDME-related pyroptosis. • NOX2 deficiency causes AMPK inhibition. • AMPK activators Metformin and EGCG reduce NOX2-deficient pyroptosis. Nicotinamide adenine dinucleotide phosphate oxidase complex 2 (NOX2) is an effector molecule expressed predominantly in neutrophils. Its deficiency is present in immune disorders, including systemic lupus erythematosus, chronic granulomatous disease, and rheumatoid arthritis. Recent reports indicated that NOX2 regulates autoimmunity and programmed cell death. However, the exact mechanism is unclear. In this study, we explored the effect of NOX2 on neutrophil apoptosis. We demonstrated that NOX2 deficiency caused neutrophil pyroptosis. Mechanistically, the NOX2 inhibitor GSK2795039 application or knockdown of NOX2 components resulted in increased mitochondrial ROS, inhibition of AMP-activated protein kinase (AMPK), and activation of Gasdermin E. AMPK activators, metformin and epigallocatechin gallate, inhibited deficient NOX2-induced pyroptosis. Together, these findings illustrate the involvement of NOX2 in regulating neutrophil death and emphasize its importance in autoimmunity. [ABSTRACT FROM AUTHOR]

Background/Aims Macrophages are immune cells that protect the body from external infections. Studies show that NOX2 is essential for the immune response. Macrophages can induce both inflammatory and anti-inflammatory responses: M1 macrophages induce inflammation, while M2 macrophages have anti-inflammatory effects. Macrophages can change their function based on their environment, a process known as macrophage polarization. However, the role of NOX2 in macrophage polarization is not well understood. Therefore, we investigated how NOX2 affects macrophage polarization. Methods NOX2 deficient mouse (NOX2 f/f x Lys M cre+/-) and a WT mouse (NOX2 f/f x Lys M cre-/-) were used. After isolation of BM cells, BMDM induced differentiation into M1 or M2. M1 was differentiated by treatment with LPS (10 ng/ml), and M2 was differentiated by treatment with IL-4 (10 ng/ml). Results CD38, the M1 marker, showed equivalent differentiation between WT BMDM and NOX2-deficient BMDM after 48 hours. However, within 24 hours, M1 differentiation was lower in NOX2-deficient BMDM compared to WT BMDM. In contrast, the M2 marker CD206 exhibited higher differentiation in NOX2-deficient BMDM at both 24 and 48 hours. Additionally, IL-12, an inflammatory cytokine, was expressed more highly in WT BMDM than in NOX2-deficient BMDM within 24 hours, but the levels equalized after 48 hours. Conversely, IL-6 was consistently expressed at higher levels in NOX2-deficient BMDM compared to WT BMDM. While lipid metabolism did not show significant differences, within 24 hours, LIPE (Lipase E), a lipolysis enzyme, was higher in WT BMDM compared to NOX2-deficient BMDM. Conversely, one of the FAO genes, CPT1, was significantly upregulated in NOX2-deficient M1 macrophages. Interestingly, NOX2-deficient M1 BMDM exhibited lipotoxicity in response to palmitate treatment. Conclusion This result confirmed that NOX2 of macrophage is an important factor for M1 polarization . [ABSTRACT FROM AUTHOR]

Background/Aims Nicotinamide adenine dinucleotide phosphate oxidase (NOX) family enzymes play a pivotal role in generating reactive oxygen species (ROS), which significantly contribute to the development of various cancers, including hepatocellular carcinoma (HCC). Specifically, NOX2 has been identified as being activated by palmitate in multiple cell types. However, research on NOX2 in relation to HCC has not yet been conducted. Methods The HCC cell line HepG2,Hep3B snu-449 and snu-398 was analyzed using Western blotting, PCR, and FACS. NOX2 activity was induced by palmitate. A mouse HCC model was created using transposon technology to promote HCC development. Tissues from HCC patients were analyzed by Western blotting to assess protein levels between tumor tissues (N=149) and paired normal tissues (N=149). Results Palmitate-mediated NOX2 activity induces sterol regulatory element-binding protein 2 (SREBP2) activity and generates ROS. Palmitate treatment induces cell survival, whereas NOX2 inhibition induces cell death. In HCC, inhibition of NOX2 expression led to increased fatty acid oxidation (FAO), which was particularly pronounced in HCC cell lines compared to normal hepatocyte cells. Furthermore, NOX2 activity was predominantly induced at the plasma membrane. Cell debris induces NOX2 and SREBP2 activity, indicating that lipid components are crucial in this process. This suggests that HCC acquires lipid components from dead cells, which induces NOX2 expression and activation. Overexpression of NOX2 resulted in increased migration, whereas its inhibition led to decreased migration and increased FAO. Inhibition or decreased expression of SREBP2 resulted in decreased epithelial– mesenchymal transition (EMT) signaling and migration. Additionally, depletion of NOX2 in the liver in animal models reduced both the size and number of HCCs, accompanied by decreased SREBP2 activity and EMT signaling. Moreover, an association between NOX2 or N-cadherin expression and SREBP2 activity was observed in patient samples. Conclusion In HCC, NOX2 activity induces migration through SREBP2-mediated EMT signaling, exacerbating the progression of HCC. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and its prevalence increases with age. The irregular and rapid contraction of the atria can lead to ineffective blood pumping, local blood stasis, blood clots, ischemic stroke, and heart failure. NADPH oxidases (NOX) and mitochondria are the main sources of reactive oxygen species in the heart, and dysregulated activation of NOX and mitochondrial dysfunction are associated with AF pathogenesis. NOX- and mitochondria-derived oxidative stress contribute to the onset of paroxysmal AF by inducing electrophysiological changes in atrial myocytes and structural remodeling in the atria. Because high atrial activity causes cardiac myocytes to expend extremely high energy to maintain excitation-contraction coupling during persistent AF, mitochondria, the primary energy source, undergo metabolic stress, affecting their morphology, Ca2+ handling, and ATP generation. In this review, we discuss the role of oxidative stress in activating AF-triggered activities, regulating intracellular Ca2+ handling, and functional and anatomical reentry mechanisms, all of which are associated with AF initiation, perpetuation, and progression. Changes in the extracellular matrix, inflammation, ion channel expression and function, myofibril structure, and mitochondrial function occur during the early transitional stages of AF, opening a window of opportunity to target NOX and mitochondria-derived oxidative stress using isoform-specific NOX inhibitors and mitochondrial ROS scavengers, as well as drugs that improve mitochondrial dynamics and metabolism to treat persistent AF and its transition to permanent AF.

BackgroundsCisplatin is a commonly used chemotherapeutic agent in cancer treatment. However, its high nephrotoxicity limits its therapeutic application and efficacy. Cisplatin induces nephrotoxicity mainly through oxidative stress and inflammation. Reactive oxygen species (ROS) in the kidneys mainly arise from nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 2 (NOX2), which is highly upregulated during ischemia-reperfusion injury and diabetes mellitus. However, its role in cisplatin-induced acute kidney injury (AKI) remains unknown.MethodsA 8-10-week-old NOX2 gene-knockout and wild-type mice were injected with 25 mg/kg cisplatin intraperitoneally for experiments.ResultsWe investigated the role of NOX2 in cisplatin-induced AKI and found that NOX2-mediated ROS production is a key inflammatory mediator of proximal tubular cell injury in cisplatin-induced AKI. NOX2 gene-knockout alleviated cisplatin-induced renal function decline, tubular injury score, kidney injury molecule-1(Kim-1) expression, and interleukin (IL)-6 and IL-1α levels with a reduction of ROS production. Moreover, in cisplatin-induced AKI, intercellular adhesion molecule 1 (ICAM-1) and the chemoattractant CXC ligand 1 (CXCL1) were highly expressed in association with neutrophil infiltration, which were all attenuated by deletion of NOX2.ConclusionThese data indicate that NOX2 aggravates cisplatin nephrotoxicity by promoting ROS-mediated tissue injury and neutrophil infiltration. Thus, appropriate targeting of NOX2/ROS pathway may minimize the risk of cisplatin-induced kidney injury in patients receiving cancer therapy.

IntroductionSustained neuroinflammation is a major contributor to the progression of neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s (PD) diseases. Neuroinflammation, like other cellular processes, is affected by the circadian clock. Microglia, the resident immune cells in the brain, act as major contributors to neuroinflammation and are under the influence of the circadian clock. Microglial responses such as activation, recruitment, and cytokine expression are rhythmic in their response to various stimuli. While the link between circadian rhythms and neuroinflammation is clear, significant gaps remain in our understanding of this complex relationship. To gain a greater understanding of this relationship, the interaction between the microglial circadian clock and the enzyme NADPH Oxidase Isoform 2 (NOX2) was studied; NOX2 is essential for the production of reactive oxygen species (ROS) in oxidative stress, an integral characteristic of neuroinflammation.MethodsBV2 microglia were examined over circadian time, demonstrating oscillations of the clock genes Per2 and Bmal1 and the NOX2 subunits gp91phox and p47phox.ResultsThe BV2 microglial clock exerted significant control over NOX2 expression and inhibition of NOX2 enabled the microglia to retain a functional circadian clock while reducing levels of ROS and inflammatory cytokines. These trends were mirrored in mouse bone marrow-derived primary macrophages.ConclusionsNOX2 plays a crucial role in the interaction between the circadian clock and the activation of microglia/macrophages into their pro-inflammatory state, which has important implications in the control of neuroinflammation.

Epigenetic regulation such as histone modification is implicated in the pathogenesis of myocardial ischemia/reperfusion injury (MIRI). Lysine-specific methyltransferase 2B (KMT2B) is a histone H3 lysine 4 (H3K4) methyltransferase. This study aims at exploring the role of KMT2B-mediated histone modification in MIRI. Peripheral blood samples were collected from 30 patients with acute myocardial infarction (AMI) and 30 healthy volunteers for analyses of the expression levels of KMT2B, riboflavin kinase (RFK), tumor necrosis factor (TNF)-α, and NADPH oxidase 2 (NOX2). H9C2 cardiomyocytes and Sprague-Dawley rats were utilized for developing in vitro and in vivo models. To evaluate the effects of the aforementioned molecules on cellular damage and MIRI, short hairpin RNAs or overexpression plasmids were introduced into cardiomyocytes for gene silencing or overexpression and also, they were packaged into adenovirus vectors for in vivo interventions. Immunoprecipitation assays were conducted to assess the interactions between KMT2B and RFK and among RFK, NOX2 sub-unit p22phox, and TNF receptor 1-associated death domain protein. KMT2B, RFK, TNF-α, and NOX2 were notably upregulated in AMI patients. KMT2B knockdown resulted in considerably attenuated cell apoptosis and reduced myocardial infarct area. Additionally, the release of pro-inflammatory proteins and ferroptosis were suppressed. Furthermore, KMT2B could promote RFK gene transcription by upregulating H3 methylation levels and consequently activate the TNF-α/NOX2 axis, which was the possible mechanism underlying the role of KMT2B in MIRI. KMT2B motivates MIRI-induced cellular injury and ferroptosis by inducing RFK transcription and mediating the TNF-α/NOX2 axis. [Display omitted] • KMT2B, RFK, TNF-α, and NOX2 are upregulated in peripheral blood of AMI patients. • KMT2B knockdown attenuates MIRI in rats or cardiomyocyte injury and ferroptosis. • KMT2B upregulates the H3 methylation level to promote RFK transcription. • KMT2B is involved in RFK-mediated TNF-α/NOX2 axis. • KMT2B mediates MIRI in rats or cardiomyocyte ferroptosis via TNF/RFK/NOX2 axis. [ABSTRACT FROM AUTHOR]

Background: Heart failure (HF) coincides with cardiomyocyte telomere shortening. Arterial hypertension is the most prominent risk factor for HF. Both HF and arterial hypertension are associated with dysregulation of the neurohormonal axis. How neurohormonal activation is linked to telomere shortening in the pathogenesis of HF is incompletely understood.Methods: Cardiomyocyte telomere length was assessed in a mouse model of hypertensive HF induced by excess neurohormonal activation (AngII [angiotensin II] infusion, high salt diet, and uninephrectomy), in AngII-stimulated cardiomyocytes and in endomyocardial biopsies from patients with HF. Superoxide production, expression of NOX2 (NADPH oxidase 2) and PRDX1 (peroxiredoxin 1) and HDAC6 (histone deacetylase 6) activity were assessed.Results: Telomere shortening occurred in vitro and in vivo, correlating with both left ventricular (LV) dilatation and LV systolic function impairment. Telomere shortening coincided with increased superoxide production, increased NOX2 expression, increased HDAC6 activity, loss of the telomere-specific antioxidant PRDX1, and increased oxidative DNA-damage. NOX2 knockout prevented PRDX1 depletion, DNA-damage and telomere shortening confirming this enzyme as a critical source of reactive oxygen species. Cotreatment with the NOX inhibitor apocynin ameliorated hypertensive HF and telomere shortening. Similarly, treatment with the HDAC6 inhibitor tubastatin A, which increases PRDX1 bioavailability, prevented telomere shortening in adult cardiomyocytes. To explore the clinical relevance of our findings, we examined endomyocardial biopsies from an all-comer population of patients with HF with reduced ejection fraction. Here, cardiomyocyte telomere length predicted the recovery of cardiac function.Conclusions: Cardiomyocyte telomere shortening and oxidative damage in heart failure with reduced ejection fraction induced by excess neurohormonal activation depends on NOX2-derived superoxide and may help to stratify HF therapy. [ABSTRACT FROM AUTHOR]

The aim of this research was to prospectively evaluate the impact of NOX2 gene expression profile (including NCF1, NCF2 and NCF4 genes) in peripheral blood mononuclear cells (PBMCs) on immune signatures, clinical characteristics and responsiveness to anti-TNF treatment in RA patients. Blood specimens were collected from 31 rheumatoid arthritis (RA) patients and 25 healthy controls, and 16 RA patients were followed at two timepoints during anti-TNF treatment. mRNA expression levels of selected genes and immunoregulatory cytokines concentrations were determined. We observed the significant upregulation of NCF4 and CD14 expression in RA group. The mRNA levels of NCF1 and CD14 positively correlated both in groups of RA patients and healthy controls. NOX2 gene expression profile was not associated with anti-TNF responsiveness, nor with RA clinical features. TNFα inhibition has not influenced NOX2 expression either. Notably, this study indicate the novel links between expression levels of NCF1 and monocyte differentiation antigen CD14., Competing Interests: Declaration of competing interest None declared., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The phagocyte NADPH oxidase (NOX2) is an enzyme, crucial for innate immune defense, producing reactive oxygen species necessary for pathogen destruction. Its activation requires the assembly of soluble proteins (p47 phox , p40 phox , p67 phox , and Rac) with the membrane-bound flavocytochrome b 558 (cytb 558 ). We combined circular-dichroism analyses, with decades of experimental data, to filter structural models of the NADPH oxidase complex generated by the artificial intelligence program AlphaFold2 (AF2). The predicted patterns tend to closely resemble the active states of the proteins, as shown by the compact structure of the cytb 558 , whose dehydrogenase domain is stabilized closer to the membrane. The modeling of the interaction of p47 phox with cytb 558 , which is the initial assembly and activation steps of the NADPH oxidase, enables us to describe how the C terminus of p47 phox interacts with the cytb 558 . Combining the AF2 cytb 558 -p47 phox model and its classical molecular dynamics simulations, we highlighted new hydrophobic lipid insertions of p47 phox , particularly at residues Trp80-Phe81 of its PX domain. The AF2 models also revealed the implications of intrinsically disordered regions, such as the fragment between the PX domain and the SH3 regions of p47 phox , in ensuring distant protein-protein and membrane-protein interactions. Finally, the AF2 prediction of the cytb 558 -Trimera model highlighted the importance of leaving Rac1 as a separate protein to reach an active state of the NADPH oxidase complex. Altogether, our step-by-step approach provides a structural model of the active complex showing how disordered regions and specific lipid and protein interactions can enable and stabilize the multisubunit assembly., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Hong-Miao Li,1,2 Qian Huang,1 Fen Tang,2 Tian-Ping Zhang3 1Department of Epidemiology and Biostatistics, School of Public Health, Anhui Medical University, Hefei, 230032, Anhui, People’s Republic of China; 2Anhui Chest Hospital (Anhui Provincial TB Institute), Hefei, 230022, Anhui, People’s Republic of China; 3The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, People’s Republic of ChinaCorrespondence: Tian-Ping ZhangThe First Affiliated Hospital of University of Science and Technology of China, 17 Lujiang Road, Hefei, Anhui, 230001, People’s Republic of ChinaEmail zhangtianping@ustc.edu.cnBackground: Reactive oxygen species (ROS) generated by NADPH oxidase has a pivotal role in the nonspecific innate immune response to invading microorganisms including M. tuberculosis (MTB). NCF2 and NOX2 were considered as important functional subunits of NADPH oxidase complex; hence, this study aimed to evaluate the NCF2, NOX2 mRNA expressions in PBMC of pulmonary tuberculosis (PTB) patients.Methods: A total of 79 PTB patients and 73 controls were included in our study. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to measure the NCF2, NOX2 mRNA levels, and receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic value of NCF2, NOX2 in PTB patients.Results: When compared to controls, the NCF2, NOX2 mRNA levels were significantly increased in PBMC from PTB patients (P < 0.001). However, the NCF2, NOX2 mRNA levels were not associated with major clinical and laboratory data of PTB patients. Area under curve (AUC) of ROC curve analysis for NCF2 and NOX2 were 0.686 (95% CI: 0.601, 0.770) and 0.705 (95% CI: 0.623, 0.787), respectively.Conclusion: Altered NCF2, NOX2 mRNA levels in PTB patients implied that these genes might play roles in PTB, and their expression levels might be potential biomarkers for the diagnosis of PTB.Keywords: pulmonary tuberculosis, NADPH oxidase complex, NCF2, NOX2, peripheral blood mononuclear cells

Recent work by us and others has implicated NADPH oxidase (NOX) enzymes as main producers of reactive oxygen species (ROS) following a brain insult such as status epilepticus, contributing to neuronal damage and development of epilepsy. Although several NOX isoforms have been examined in the context of epilepsy, most attention has focused on NOX2. In this present study, we demonstrate the effect of gp91ds-tat, a specific competitive inhibitor of NOX2, in in vitro epileptiform activity model as well as in temporal lobe epilepsy (TLE) model in rats. We showed that in in vitro seizure model, gp91ds-tat modulated Ca2+ oscillation, prevented epileptiform activity-induced ROS generation, mitochondrial depolarization, and neuronal death. Administration of gp91ds-tat 1 h after kainic acid-induced status epilepticus significantly decreased the expression of NOX2, as well as the overall NOX activity in the cortex and the hippocampus. Finally, we showed that upon continuous intracerebroventricular administration to epileptic rats, gp91ds-tat significantly reduced the seizure frequency and the total number of seizures post-treatment compared to the scrambled peptide-treated animals.The results of the study suggest that NOX2 may have an important effect on modulation of epileptiform activity and has a critical role in mediating seizure-induced NOX activation, ROS generation and oxidative stress in the brain, and thus significantly contributes to development of epilepsy following a brain insult.

Phagocyte oxidase plays an essential role in the first line of host defense against pathogens. It oxidizes intracellular NADPH to reduce extracellular oxygen to produce superoxide anions that participate in pathogen killing. The resting phagocyte oxidase is a heterodimeric complex formed by two transmembrane proteins NOX2 and p22. Despite the physiological importance of this complex, its structure remains elusive. Here, we reported the cryo-EM structure of the functional human NOX2-p22 complex in nanodisc in the resting state. NOX2 shows a canonical 6-TM architecture of NOX and p22 has four transmembrane helices. M3, M4, and M5 of NOX2, and M1 and M4 helices of p22 are involved in the heterodimer formation. Dehydrogenase (DH) domain of NOX2 in the resting state is not optimally docked onto the transmembrane domain, leading to inefficient electron transfer and NADPH binding. Structural analysis suggests that the cytosolic factors might activate the NOX2-p22 complex by stabilizing the DH in a productive docked conformation.

ABSTRACT Tuberculosis (TB), which is caused by the single pathogenic bacterium, Mycobacterium tuberculosis, is among the top 10 lethal diseases worldwide. This situation has been exacerbated by the increasing number of cases of multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB). Histamine is an organic nitrogenous compound that mediates a plethora of cell processes via different receptors. The expression of histamine receptor H1 (HRH1), one of the four histamine receptors identified to date was previously reported to be augmented by M. tuberculosis infection, although the underlying mechanism is unclear. In the present study, we applied confocal microscopy, flow cytometry, and Western blotting to show that HRH1 expression was enhanced in macrophages following mycobacterial infection. Furthermore, by combining techniques of gene knockdown, immunoprecipitation, intracellular bacterial burden analysis, fluorescence labeling, and imaging, we found that M. tuberculosis targeted the host HRH1 to suppress NOX2-mediated cROS production and inhibit phagosome maturation and acidification via the GRK2-p38MAPK signaling pathway. Our findings clarified the underlying mechanism of the M. tuberculosis and host HRH1 interaction and may provide useful information for the development of novel antituberculosis treatments. IMPORTANCE Once engulfed in macrophage phagosomes, M. tuberculosis adopts various strategies to take advantage of the host environment for its intracellular survival. Histamine is an organic nitrogen-containing compound that mediates a plethora of cellular processes via different receptors, but the crosstalk mechanism between M. tuberculosis and HRH1 in macrophages is not clear. Our results revealed that M. tuberculosis infection enhanced HRH1 expression, which in turn restrained macrophage bactericidal activity by modulating the GRK2-p38MAPK signaling pathway, inhibiting NOX2-mediated cROS production and phagosome maturation. Clarification of the underlying mechanism by which M. tuberculosis utilizes host HRH1 to favor its intracellular survival may provide useful information for the development of novel antituberculosis treatments.

Amebiasis is an intestinal infection caused by Entamoeba histolytica. Amebic liver abscess (ALA) is the most common extraintestinal complication of amebiasis. In animal models of ALA, neutrophils have been shown to be the first cells to come into contact with Entamoeba histolytica during the initial phase of ALA. One of the multiple mechanisms by which neutrophils exhibit amebicidal activity is through reactive oxygen species (ROS) and the enzyme NADPH oxidase (NOX2), which generates and transports electrons to subsequently reduce molecular oxygen into superoxide anion. Previous reports have shown that ROS release in the susceptible animal species (hamster) is mainly stimulated by the pathogen, in turn provoking such an exacerbated inflammatory reaction that it is unable to be controlled and results in the death of the animal model. Apocynin is a natural inhibitor of NADPH oxidase. No information is available on the role of NOX in the evolution of ALA in the hamster, a susceptible model. Our study showed that administration of a selective NADPH oxidase 2 (NOX2) enzyme inhibitor significantly decreases the percentage of ALA, the size of inflammatory foci, the number of neutrophils, and NOX activity indicated by the reduction in superoxide anion (O2−) production. Moreover, in vitro, the apocynin damages amoebae. Our results showed that apocynin administration induces a decrease in the activity of NOX that could favor a decrease in ALA progression.

Non-alcoholic fatty liver disease (NAFLD)-related liver fibrosis results in the encapsulation of injured liver parenchyma by a collagenous scar mainly imputable to hepatic stellate cells' activation. Approved pharmacological treatments against NAFLD-related fibrosis are still lacking, but natural compounds such as hydroxytyrosol (HXT) and vitamin E (VitE), are emerging as promising therapeutic opportunities. In this study, the potential anti-fibrotic effect of HXT + VitE combination therapy was investigated in vitro and in vivo. In particular, tumor growth factor (TGF)-β-activated LX-2 cells as an in vitro model, and carbon tetrachloride plus a Western diet as a mice model were employed. The effect of HXT + VitE on fibrosis was also investigated in children with biopsy-proven NAFLD. Our results demonstrated that HXT + VitE caused a reduction of proliferation, migration, contractility, and expression of pro-fibrogenic genes in TGF-β-activated LX-2 cells. HXT + VitE treatment also antagonized TGF-β-dependent upregulation of pro-oxidant NOX2 by interfering with nuclear translocation/activation of SMAD2/3 transcription factors. The mouse model of NAFLD-related fibrosis treated with HXT + VitE showed a marked reduction of fibrosis pattern by histology and gene expression. Accordingly, in children with NAFLD, HXT + VitE treatment caused a decrease of circulating levels of PIIINP and NOX2 that was supported over time. Our study suggests that HXT + VitE supplementation may improve NAFLD-related fibrosis. [ABSTRACT FROM AUTHOR]

A breach of T cell tolerance is considered as a major step in the pathogenesis of rheumatoid arthritis. In collagen-induced arthritis (CIA) model, immunization with type II collagen (COL2) leads to arthritis in mice through T cells responding to the immunodominant COL2259–273 peptide. T cells could escape from thymus negative selection because endogenous COL2259–273 peptide only weakly binds to the major histocompatibility complex class II (MHCII) molecule Aq. To investigate the regulation of T cell tolerance, we used a new mouse strain BQ.Col2266E with homozygous D266E mutations in the Col2 gene leading to a replacement of the endogenous aspartic acid (D) to glutamic acid (E) at position 266 of the COL2259–273 peptide, resulting in stronger binding to Aq. We also established BQ.Col2264R mice carrying an additional K264R mutation changed the lysine (K) at position 264 to eliminate the major TCR recognition site. The BQ.Col2266E mice were fully resistant to CIA, while the BQ.Col2264R mice developed severe arthritis. Furthermore, we studied two of the most important non-MHCII genes associated with CIA, i.e., Ncf1 and Fcgr2b. Deficiency of either gene induced arthritis in BQ.Col2266E mice, and the downstream effects differ as Ncf1 deficiency reduced Tregs and was likely to decrease expression of autoimmune regulator (AIRE) while Fcgr2b did not. In conclusion, the new human-mimicking mouse model has strong T cell tolerance to COL2, which can be broken by deficiency of Fcgr2b or Ncf1, allowing activation of autoreactive T cells and development of arthritis. [ABSTRACT FROM AUTHOR]

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases regulate production of reactive oxygen species (ROS) that cause oxidative damage to cellular components but also regulate redox signaling in many cell types with essential functions in the cardiovascular system. Research over the past couple of decades has uncovered mechanisms by which NADPH oxidase (NOX) enzymes regulate oxidative stress and compartmentalize intracellular signaling in endothelial cells, smooth muscle cells, macrophages, cardiomyocytes, fibroblasts, and other cell types. NOX2 and NOX4, for example, regulate distinct redox signaling mechanisms in cardiac myocytes pertinent to the onset and progression of cardiac hypertrophy and heart failure. Heart failure with preserved ejection fraction (HFpEF), which accounts for at least half of all heart failure cases and has few effective treatments to date, is classically associated with ventricular diastolic dysfunction, i.e., defects in ventricular relaxation and/or filling. However, HFpEF afflicts multiple organ systems and is associated with systemic pathologies including inflammation, oxidative stress, arterial stiffening, cardiac fibrosis, and renal, adipose tissue, and skeletal muscle dysfunction. Basic science studies and clinical data suggest a role for systemic and myocardial oxidative stress in HFpEF, and evidence from animal models demonstrates the critical functions of NOX enzymes in diastolic function and several HFpEF-associated comorbidities. Here, we discuss the roles of NOX enzymes in cardiovascular cells that are pertinent to the development and progression of diastolic dysfunction and HFpEF and outline potential clinical implications. [ABSTRACT FROM AUTHOR]

Current treatment of Parkinson's disease (PD) ameliorates symptoms but fails to block disease progression. This study was conducted to explore the protective effects of SVHRSP, a synthetic heat-resistant peptide derived from scorpion venom, against dopaminergic neurodegeneration in experimental models of PD. Results showed that SVHRSP dose-dependently reduced the loss of dopaminergic neuron in the nigrostriatal pathway and motor impairments in both rotenone and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid (MPTP/p)-induced mouse PD models. Microglial activation and imbalance of M1/M2 polarization were also abrogated by SVHRSP in both models. In rotenone-treated primary midbrain neuron-glial cultures, loss of dopaminergic neuron and microglial activation were mitigated by SVHRSP. Furthermore, lipopolysaccharide (LPS)-elicited microglial activation, M1 polarization and related dopaminergic neurodegeneration in primary cultures were also abrogated by SVHRSP, suggesting that inhibition of microglial activation contributed to SVHRSP-afforded neuroprotection. Mechanistic studies revealed that SVHRSP blocked both LPS- and rotenone-induced microglial NADPH oxidase (NOX2) activation by preventing membrane translocation of cytosolic subunit p47phox. NOX2 knockdown by siRNA markedly attenuated the inhibitory effects of SVHRSP against LPS- and rotenone-induced gene expressions of proinflammatory factors and related neurotoxicity. Altogether, SVHRSP protects dopaminergic neurons by blocking NOX2-mediated microglial activation in experimental PD models, providing experimental basis for the screening of clinical therapeutic drugs for PD. [Display omitted] • SVHRSP protects dopaminergic neurons in two experimental PD models. • SVHRSP blunts microglial activation and M1 polarization. • SVHRSP inhibits NOX2 activation by blocking p47phox membrane translocation. • NOX2 silence abrogates SVHRSP-afforded anti-inflammatory and neuroprotective effects. [ABSTRACT FROM AUTHOR]

Conclusions Our data demonstrate for the first time that NOX isoforms NOX1, NOX2, NOX4, and NOX5 all remained active in lung tissues of end-stage COPD patients, indicating critical roles of these enzymes in the pathogenesis of the advanced disease. Compared to the donor group, the protein expression of NOX isoforms NOX1, NOX2, NOX4, and NOX5 was significantly upregulated in lung tissue sections of patients with end-stage COPD. Chronic obstructive pulmonary disease (COPD), NOX1, NOX2, acute cigarette smoke (ACS), oxidative stress, lung inflammation, NADPH oxidase (NOX), NOX4, NOX5 Discussion In the present study, we investigated the expression profiles of NOX isoforms in end-stage COPD patients and the potential differential roles of NOX isoforms in ACS induced oxidative stress and lung inflammation in mice as a model for early COPD or early-stage COPD. Keywords: chronic obstructive pulmonary disease (COPD); NADPH oxidase (NOX); NOX1; NOX2; NOX4; NOX5; acute cigarette smoke (ACS); oxidative stress; lung inflammation EN chronic obstructive pulmonary disease (COPD) NADPH oxidase (NOX) NOX1 NOX2 NOX4 NOX5 acute cigarette smoke (ACS) oxidative stress lung inflammation N.PAG N.PAG 19 08/29/22 20220801 NES 220801 1. [Extracted from the article]

LPS was capable of enhancing the oxidative burst response in a glycolytic-dependent process in macrophages, providing evidence that a switch to glycolysis allows macrophages to have a heightened capacity to generate ROS via NOX2 oxidase. Keywords: inflammation; macrophages; NADPH oxidase; NOX2; LPS; glycolysis; pentose phosphate pathway; reactive oxygen species EN inflammation macrophages NADPH oxidase NOX2 LPS glycolysis pentose phosphate pathway reactive oxygen species N.PAG N.PAG 14 08/29/22 20220801 NES 220801 1. We concluded that glycolysis and PPP drive NOX2 oxidase ROS production in response to LPS and that this is completely independent of mitochondrial ROS and, therefore, most likely only involves TLR4-induced endosomal NOX2 oxidase ROS production. Glycolysis and the Pentose Phosphate Pathway Promote LPS-Induced NOX2 Oxidase- and IFN- -Dependent Inflammation in Macrophages. [Extracted from the article]

Glucotoxicity-induced pancreatic β-cell failure contributes to the development of type 2 diabetes mellitus (T2DM). Accumulating evidence reveals that miRNAs play a critical role in regulating pancreatic β-cell function and survival. In this study, we employed a self-assembled cell microarray (SAMcell)-based functional screening assay to identify miRNAs that are capable of regulating the dysfunction of β-cells induced by glucotoxicity. Among 62 conserved miRNAs we tested, miR-190 was identified as a candidate regulator that could effectively restore insulin expression in NIT-1 cells under high-glucose (HG) stimulation. Further analyses demonstrated that miR-190 was significantly down-regulated in HG-treated NIT-1 cells, as well as in the pancreas of diabetic mice. Mechanistic studies showed that Cybb is the direct target gene of miR-190, which encodes the gp91phox protein, a subunit of the NOX2 complex. Furthermore, both miR-190 overexpression and Cybb knockdown inhibited apoptosis and improved glucose-stimulated insulin secretion (GSIS) in HG-stimulated NIT-1 cells by attenuating the excessive production of reactive oxygen species (ROS). More importantly, a targeted delivery of mPEG-PCL-g-PDMAEMA nanoparticles/miR-190 complexes (PECgD NPs/miR-190) to the pancreas significantly ameliorated hyperglycemia, decreased fasting serum insulin levels, and improved glucose tolerance in diabetic mice. Taken together, our findings suggest that the miR-190/Cybb axis plays an important role in glucotoxicity-induced pancreatic β-cell failure. Restoring miR-190 expression levels may be a possible therapeutic strategy to protect β-cells in T2DM. [ABSTRACT FROM AUTHOR]

Simple Summary: Corilagin has anti-inflammatory and antioxidant properties. Acute lung injury is a life-threatening disease. This investigated the protective effects and mechanisms of corilagin on acute lung injury. The results show that corilagin can reduce acute lung injury via attenuation of pro-inflammatory mediators and oxidative stress. Corilagin has potential utility as a therapeutic agent for the treatment of this life-threatening respiratory disease. Acute lung injury (ALI) and acute respiratory distress syndrome are clinically life-threatening diseases. Corilagin, a major polyphenolic compound obtained from the herb Phyllanthus urinaria, has anti-inflammatory and antioxidant properties, and in this study, we sought to evaluate the protective effects and mechanisms of corilagin on lipopolysaccharide (LPS)-induced ALI in mice. ALI was induced in the mice by the intratracheal administration of LPS, and following 30 min of LPS challenge, corilagin (5 and 10 mg/kg body weight) was administered intraperitoneally. At 6 h post-LPS administration, lung tissues were collected for analysis. Corilagin treatment significantly attenuated inflammatory cell infiltration, the production of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and oxidative stress in lung tissues. In addition, corilagin inhibited the LPS-induced expression of NOX2, ERK, and NF-κB. Corilagin has anti-oxidative and anti-inflammatory effects, and can effectively reduce ALI via attenuation of the NOX2 and ERK/NF-κB signaling pathways. [ABSTRACT FROM AUTHOR]

Shengen Liao,1,&ast; Yuan Tang,1,&ast; Xin Yue,1 Rongrong Gao,1 Wenming Yao,1 Yanli Zhou,1 Haifeng Zhang2,3 1Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing, 210029, People’s Republic of China; 2Department of Cardiology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, 215002, People’s Republic of China; 3Department of Cardiology, Jiangsu Province Hospital, Nanjing, 210029, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Haifeng ZhangDepartment of Cardiology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal Hospital, Gusu School, Nanjing Medical University, Suzhou, 215002, People’s Republic of ChinaEmail haifeng_zhang@163.comBackground: This study was designed to investigate the cardioprotective role of β-hydroxybutyrate (BHB) in heart failure with preserved ejection fraction (HFpEF) and the underlying mechanism.Methods: A two-hit model with a high-fat diet (HFD) and Nω-nitrol-arginine methyl ester (L-NAME) was used as an HFpEF model. The treatment group received a weekly intraperitoneal injection of β-hydroxybutyrate (BHB). Cardiac function, inflammation, and fibrosis were evaluated. CD3+CD4+Foxp3+ positive cells within the myocardium were quantified by flow cytometry. The NADPH oxidase 2 (NOX2)/glycogen synthase kinase-3β (GSK3β) pathway was examined by immunoblot analysis.Results: BHB improved diastolic function, fibrosis and cardiac remodeling in HFpEF. Additionally, BHB inhibited cardiac inflammation and increased cardiac Treg cells, which could be due to the downregulation of the NOX2/GSK-3β pathway.Conclusion: BHB protected against the progression of HFpEF by increasing cardiac Treg cells by modulating the NOX2/GSK-3β pathway.Keywords: β-hydroxybutyrate, HFpEF, Treg cells, NOX2, GSK-3&#x03B2

Oxidative stress plays an important role in the formation of abdominal aortic aneurysm (AAA), and we have recently established a causal role of uncoupled eNOS in this severe human disease. We have also shown that activation of NADPH oxidase (NOX) lies upstream of uncoupled eNOS. Therefore, identification of the specific NOX isoforms that are required for eNOS uncoupling and AAA formation would ultimately lead to novel therapies for AAA. In the present study, we used the Ang II infused hph-1 mice to examine the roles of NOX isoforms in the development of AAA. We generated double mutants of hph-1-NOX1, hph-1-NOX2, hph-1-p47phox, and hph-1-NOX4. After two weeks of Ang II infusion, the incidence rate of AAA substantially dropped from 76.5% in Ang II infused hph-1 mice (n=34) to 11.1%, 15.0%, 9.5% and 0% in hph-1-NOX1 (n=27), hph-1-NOX2 (n=40), hph-1-p47phox (n=21), and hph-1-NOX4 (n=33) double mutant mice, respectively. The size of abdominal aortas of the four double mutant mice, determined by ultrasound analyses, was significantly smaller than the hph-1 mice. Aortic nitric oxide and H4B bioavailabilities were markedly improved in the double mutants, while superoxide production and eNOS uncoupling activity were substantially diminished. These effects seemed attributed to an endothelial specific restoration of dihydrofolate reductase expression and activity, deficiency of which has been shown to induce eNOS uncoupling and AAA formation in both Ang II-infused hph-1 and apoE null animals. In addition, over-expression of human NOX4 N129S or T555S mutant newly identified in aneurysm patients increased hydrogen peroxide production, further implicating a relationship between NOX and human aneurysm. Taken together, these data indicate that NOX isoforms 1, 2 or 4 lies upstream of dihydrofolate reductase deficiency and eNOS uncoupling to induce AAA formation. These findings may promote development of novel therapeutics for the treatment of the disease by inhibiting NOX signaling.

Islet transplantation is a promising treatment strategy for type 1 diabetes mellitus (T1DM) patients. However, oxidative stress-induced graft failure due to an insufficient revascularization is a major problem of this therapeutic approach. NADPH oxidase (NOX)2 is an important producer of reactive oxygen species (ROS) and several studies have already reported that this enzyme plays a crucial role in the endocrine function and viability of β-cells. Therefore, we hypothesized that targeting islet NOX2 improves the outcome of islet transplantation. To test this, we analyzed the cellular composition and viability of isolated wild-type (WT) and Nox2−/− islets by immunohistochemistry as well as different viability assays. Ex vivo, the effect of Nox2 deficiency on superoxide production, endocrine function and anti-oxidant protein expression was studied under hypoxic conditions. In vivo, we transplanted WT and Nox2−/− islets into mouse dorsal skinfold chambers and under the kidney capsule of diabetic mice to assess their revascularization and endocrine function, respectively. We found that the loss of NOX2 does not affect the cellular composition and viability of isolated islets. However, decreased superoxide production, higher glucose-stimulated insulin secretion as well as expression of nuclear factor erythroid 2-related factor (Nrf)2, heme oxygenase (HO)-1 and superoxide dismutase 1 (SOD1) was detected in hypoxic Nox2−/− islets when compared to WT islets. Moreover, we detected an early revascularization, a higher take rate and restoration of normoglycemia in diabetic mice transplanted with Nox2−/− islets. These findings indicate that the suppression of NOX2 activity represents a promising therapeutic strategy to improve engraftment and function of isolated islets.

Yu Tan,1,&ast; Feng Zhou,2,&ast; Dejiang Yang,1 Xiaowei Zhang,1 Meihong Zeng,1 Lei Wan1 1Department of Neurology, The Third Affiliated Hospital of Nanchang University, Nanchang City, Jiangxi Province, 330008, People’s Republic of China; 2Department of Neurology, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai City, Guangdong Province, 519000, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Yu TanDepartment of Neurology, The Third Affiliated Hospital of Nanchang University, No. 128 Xiangshan Road, Nanchang City, Jiangxi Province, 330008, People’s Republic of ChinaEmail YuTanhospital@163.comBackground: Ischemic stroke is a destructive cerebrovascular disorder related to oxidative stress; NOX2 is a major source for ROS production; and miR-126a-5p is involved in several diseases, such as abdominal aortic aneurysm. We investigated the role of miR-126a-5p in regulating NOX2 in ischemic stroke.Methods: MiR-126a-5p and NOX2 were examined in the brains of rats subjected to cerebral ischemia/reperfusion (I/R) by RT-PCR and Western blot. MiR-126a-5p agomir was delivered to examine the effects of miR-126a-5p on I/R injury. The neurological deficit, infarct volume, and brain water content were evaluated. NOX activity, ROS production, and MDA and SOD levels were detected to assess oxidative stress. H&E staining was used to examine cell state. Apoptosis was evaluated by TUNEL, caspase-3 activity, and cleaved-caspase-3 protein level. The relationship between miR-126a-5p and NOX2 was analyzed by bioinformatics and luciferase reporter assay. MiR-126a-5p mimic, miR-126a-5p inhibitor, or pcDNA-NOX2 were transfected in SH-SY5Y cells to further assess the effects of miR-126a-5p on OGD/R-induced cells injury.Results: NOX2 was upregulated and miR-126a-5p was down-regulated in the brains of I/R rats. MiR-126a-5p agomir obviously reduced the neurological deficit, infarct volume, brain water content, oxidative stress, and apoptosis in I/R rats. MiR-126a-5p targeted NOX2 directly and regulated NOX2 negatively. Moreover, miR-126a-5p mimic elevated cell viability and inhibited oxidative stress and apoptosis in OGD/R-treated SH-SY5Y cells, while miR-126a-5p inhibitor had the opposite effects. NOX2 overexpression antagonized the protective effects of miR-126a-5p mimic on OGD/R-induced cell injury.Conclusion: MiR-126a-5p is a novel potential target for ischemic stroke therapy due to its protection against cerebral I/R injury via directly targeting NOX2.Keywords: oxidative stress, NOX2, miR-126a-5p, cerebral ischemia/reperfusion

Macrophage adenosine monophosphate-activated protein kinase (AMPK) limits the development of experimental colitis. AMPK activation inhibits NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) generation, and pro-inflammatory cytokine secretion in macrophages during inflammation, while increased NOX2 expression is reported in experimental models of colitis and inflammatory bowel disease (IBD) patients. Although there are reductions in AMPK activity in IBD, it remains unclear whether targeted inhibition of NOX2 in the presence of defective AMPK can reduce the severity of colitis. Here, we investigate whether the inhibition of NOX2 ameliorates colitis in mice independent of AMPK activation. Our study identified that VAS2870 (a pan-Nox inhibitor) alleviated dextran sodium sulfate (DSS)-induced colitis in macrophage-specific AMPKβ1-deficient (AMPKβ1LysM) mice. Additionally, VAS2870 blocked LPS-induced TLR-4 and NOX2 expression, ROS production, nuclear translocation of NF-κB, and pro-inflammatory cytokine secretion in bone marrow-derived macrophages (BMDMs) from AMPKβ1LysM mice, whereas sodium salicylate (SS; AMPK β1 activator) did not. Both VAS2870 and SS inhibited LPS-induced NOX2 expression, ROS production, and pro-inflammatory cytokine secretions in bone marrow-derived macrophages (BMDMs) from wildtype (AMPKβ1fl/fl) mice but only VAS2870 inhibited these effects of LPSs in AMPKβ1LysM BMDMs. Furthermore, in macrophage cells (RAW 264.7), both SS and VAS2870 inhibited ROS production and the secretion of pro-inflammatory cytokines and reversed the impaired autophagy induced by LPSs. These data suggest that inhibiting NOX2 can reduce inflammation independent of AMPK in colitis.

Background and purpose: The NOX2 enzyme of myeloid cells generates reactive oxygen species (ROS) that have been implicated in the pathology of multiple sclerosis (MS). We aimed to determine the impact of genetic variation within CYBA, which encodes the functional CYBA/p22phox subunit of NOX2, on MS severity and progression. Methods: One hundred three MS patients with up to 49 (median = 17) years follow‐up time from first MS diagnosis were genotyped at the single nucleotide polymorphisms rs1049254 and rs4673 within CYBA. Results were matched with disease severity and time to diagnosis of secondary progressive MS (SPMS). NOX2‐mediated formation of ROS was measured by chemiluminescence in blood myeloid cells from healthy donors (n = 55) with defined genotypes at rs1049254 and rs4673. Results: The rs1049254/G and rs4673/A CYBA alleles were associated with reduced formation of ROS and were thus defined as low‐ROS alleles. Patients carrying low‐ROS alleles showed reduced multiple sclerosis severity score (p = 0.02, N = 103, linear regression) and delayed onset of SPMS (p = 0.02, hazard ratio [HR] = 0.46, n = 100, log‐rank test). In a cohort examined after 2005, patients carrying low‐ROS CYBA alleles showed >20 years longer time to secondary progression (p = 0.003, HR = 0.29, n = 59, log‐rank test). Conclusions: These results implicate NOX2 in MS, in particular for the development of secondary progressive disease, and point toward NOX2‐reductive therapy aiming to delay secondary progression. [ABSTRACT FROM AUTHOR]