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Aims: Increased oxidative stress and vascular inflammation are implicated in increased cardiovascular disease (CVD) incidence with age. We and others demonstrated that NOX1/2 NADPH oxidase inhibition, by genetic deletion of p47phox, in Apoe(-/-) mice decreases vascular reactive oxygen species (ROS) generation and atherosclerosis in young age. The present study examined whether NOX1/2 NADPH oxidases are also pivotal to aging-associated CVD., Results: Both aged (16 months) Apoe(-/-) and Apoe(-/-)/p47phox(-/-) mice had increased atherosclerotic lesion area, aortic stiffness, and systolic dysfunction compared with young (4 months) cohorts. Cellular and mitochondrial ROS (mtROS) levels were significantly higher in aortic wall and vascular smooth muscle cells (VSMCs) from aged wild-type and p47phox(-/-) mice. VSMCs from aged mice had increased mitochondrial protein oxidation and dysfunction and increased vascular cell adhesion molecule 1 expression, which was abrogated with (2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) treatment. NOX4 expression was increased in the vasculature and mitochondria of aged mice and its suppression with shRNA in VSMCs from aged mice decreased mtROS levels and improved function. Increased mtROS levels were associated with enhanced mitochondrial NOX4 expression in aortic VSMCs from aged subjects, and NOX4 expression levels in arterial wall correlated with age and atherosclerotic severity. Aged Apoe(-/-) mice treated with MitoTEMPO and 2-(2-chlorophenyl)-4-methyl-5-(pyridin-2-ylmethyl)-1H-pyrazolo[4,3-c]pyridine-3,6(2H,5H)-dione had decreased vascular ROS levels and atherosclerosis and preserved vascular and cardiac function., Innovation and Conclusion: These data suggest that NOX4, but not NOX1/2, and mitochondrial oxidative stress are mediators of CVD in aging under hyperlipidemic conditions. Regulating NOX4 activity/expression and using mitochondrial antioxidants are potential approaches to reducing aging-associated CVD.

Background & Aims: Dual oxidase 2 (DUOX2), a hydrogen-peroxide generator at the apical membrane of gastrointestinal epithelia, is up-regulated in patients with inflammatory bowel disease (IBD) before the onset of inflammation, but little is known about its effects. We investigated the role of DUOX2 in maintaining mucosal immune homeostasis in mice., Methods: We analyzed the regulation of DUOX2 in intestinal tissues of germ-free vs conventional mice, mice given antibiotics or colonized with only segmented filamentous bacteria, mice associated with human microbiota, and mice with deficiencies in interleukin (IL) 23 and IL22 signaling. We performed 16S ribosomal RNA gene quantitative polymerase chain reaction of intestinal mucosa and mesenteric lymph nodes of Duoxa(-/-) mice that lack functional DUOX enzymes. Genes differentially expressed in Duoxa(-/-) mice compared with co-housed wild-type littermates were correlated with gene expression changes in early-stage IBD using gene set enrichment analysis., Results: Colonization of mice with segmented filamentous bacteria up-regulated intestinal expression of DUOX2. DUOX2 regulated redox signaling within mucosa-associated microbes and restricted bacterial access to lymphatic tissues of the mice, thereby reducing microbiota-induced immune responses. Induction of Duox2 transcription by microbial colonization did not require the mucosal cytokines IL17 or IL22, although IL22 increased expression of Duox2. Dysbiotic, but not healthy human microbiota, activated a DUOX2 response in recipient germ-free mice that corresponded to abnormal colonization of the mucosa with distinct populations of microbes. In Duoxa(-/-) mice, abnormalities in ileal mucosal gene expression at homeostasis recapitulated those in patients with mucosal dysbiosis., Conclusions: DUOX2 regulates interactions between the intestinal microbiota and the mucosa to maintain immune homeostasis in mice. Mucosal dysbiosis leads to increased expression of DUOX2, which might be a marker of perturbed mucosal homeostasis in patients with early-stage IBD., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Maintaining the redox balance between generation and elimination of reactive oxygen species (ROS) is critical for health. Disturbances such as continuously elevated ROS levels will result in oxidative stress and development of disease, but likewise, insufficient ROS production will be detrimental to health. Reduced or even complete loss of ROS generation originates mainly from inactivating variants in genes encoding for NADPH oxidase complexes. In particular, deficiency in phagocyte Nox2 oxidase function due to genetic variants (CYBB, CYBA, NCF1, NCF2, NCF4) has been recognized as a direct cause of chronic granulomatous disease (CGD), an inherited immune disorder. More recently, additional diseases have been linked to functionally altered variants in genes encoding for other NADPH oxidases, such as for DUOX2/DUOXA2 in congenital hypothyroidism, or for the Nox2 complex, NOX1 and DUOX2 as risk factors for inflammatory bowel disease. A comprehensive overview of novel developments in terms of Nox/Duox-deficiency disorders is presented, combined with insights gained from structure-function studies that will aid in predicting functional defects of clinical variants., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Chronic granulomatous disease (CGD) is an inherited immunodeficiency linked with mutations in the multi-subunit leucocyte NADPH oxidase. Myeloid-derived phagocytic cells deficient in NADPH oxidase fail to produce sufficient levels of reactive oxygen species to clear engulfed pathogens. In this study we show that oxidase also influences B-cell functions, including responses to single-stranded RNA or unmethylated DNA by endosomal Toll-like receptors (TLRs) 7 and 9. In response to TLR7/9 ligands, B-cell lines derived from patients with CGD with mutations in either the NADPH oxidase p40(phox) or p47(phox) subunits produced only low levels of reactive oxygen species. Remarkably, cytokine secretion and p38 mitogen-activated protein kinase activation by these oxidase-deficient B cells was significantly increased upon TLR7/9 activation when compared with oxidase-sufficient B cells. Increased TLR responsiveness was also detected in B cells from oxidase-deficient mice. NADPH oxidase-deficient patient-derived B cells also expressed enhanced levels of TLR7 and TLR9 mRNA and protein compared with the same cells reconstituted to restore oxidase activity. These data demonstrate that the loss of oxidase function associated with CGD can significantly impact B-cell TLR signalling in response to nucleic acids with potential repercussions for auto-reactivity in patients., (© 2015 John Wiley & Sons Ltd.)

Rationale: Chronic inflammation is a major contributor to the progressive pathology of hypertension, and T-cell activation is required for the genesis of hypertension. However, the precise role of myeloid cells in this process is unclear., Objective: To characterize and understand the role of peripheral myeloid cells in the development of hypertension., Methods and Results: We examined myeloid cells in the periphery of hypertensive mice and found that increased numbers of CD11b(+)Gr1(+) myeloid cells in blood and the spleen are a characteristic of 3 murine models of experimental hypertension (angiotensin II, L-NG-nitroarginine methyl ester, and high salt). These cells express surface markers and transcription factors associated with immaturity and immunosuppression. Also, they produce hydrogen peroxide to suppress T-cell activation. These are characteristics of myeloid-derived suppressor cells (MDSCs). Depletion of hypertensive MDSCs increased blood pressure and renal inflammation. In contrast, adoptive transfer of wild-type MDSCs to hypertensive mice reduced blood pressure, whereas the transfer of nicotinamide adenine dinucleotide phosphate oxidase 2-deficient MDSCs did not., Conclusion: The accumulation of MDSCs is a characteristic of experimental models of hypertension. MDSCs limit inflammation and the increase of blood pressure through the production of hydrogen peroxide., (© 2015 American Heart Association, Inc.)

This investigation explored the mechanism for inhibition of β2 integrin adhesion molecules when neutrophils are exposed to nitric oxide ((•)NO). Roles for specific proteins were elucidated using chemical inhibitors, depletion with small inhibitory RNA, and cells from knock-out mice. Optimal inhibition occurs with exposures to a (•)NO flux of ∼ 28 nmol/min for 2 min or more, which sets up an autocatalytic cascade triggered by activating type 2 nitric-oxide synthase (NOS-2) and NADPH oxidase (NOX). Integrin inhibition does not occur with neutrophils exposed to a NOX inhibitor (Nox2ds), a NOS-2 inhibitor (1400 W), or with cells from mice lacking NOS-2 or the gp91(phox) component of NOX. Reactive species cause S-nitrosylation of cytosolic actin that enhances actin polymerization. Protein cross-linking and actin filament formation assays indicate that increased polymerization occurs because of associations involving vasodilator-stimulated phosphoprotein, focal adhesion kinase, and protein-disulfide isomerase in proximity to actin filaments. These effects were inhibited in cells exposed to ultraviolet light which photo-reverses S-nitrosylated cysteine residues and by co-incubations with cytochalasin D. The autocatalytic cycle can be arrested by protein kinase G activated with 8-bromo-cyclic GMP and by a high (•)NO flux (∼ 112 nmol/min) that inactivates NOX., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The phagocyte NADPH oxidase NOX2 produces reactive oxygen species (ROS) and is a well-known player in host defence. However, there is also increasing evidence for a regulatory role of NOX2 in adaptive immunity. Deficiency in phagocyte NADPH oxidase causes chronic granulomatous disease (CGD) in humans, a condition that can also be studied in CGD mice. Clinical observations in CGD patients suggest a higher susceptibility to autoimmune diseases, in particular lupus, idiopathic thrombocytopenic purpura and rheumatoid arthritis. In mice, a strong correlation exists between a polymorphism in a NOX2 subunit and the development of autoimmune arthritis. NOX2 deficiency in mice also favours lupus development. Both CGD patients and CGD mice exhibit increased levels of immunoglobulins, including autoantibodies. Despite these phenotypes suggesting a role for NOX2 in specific immunity, mechanistic explanations for the typical increase of CGD in autoimmune disease and antibody levels are still preliminary. NOX2-dependent ROS generation is well documented for dendritic cells and B-lymphocytes. It is unclear whether T-lymphocytes produce ROS themselves or whether they are exposed to ROS derived from dendritic cells during the process of antigen presentation. ROS are signalling molecules in virtually any cell type, including T- and B-lymphocytes. However, knowledge about the impact of ROS-dependent signalling on T- and B-lymphocyte phenotype and response is still limited. ROS might contribute to Th1/Th2/Th17 cell fate decisions during T-lymphocyte activation and might enhance immunoglobulin production by B-lymphocytes. In dendritic cells, NOX2-derived ROS might be important for antigen processing and cell activation.

The NADPH oxidase 2 (NOX2) complex is responsible for the production of ROS in phagocytic cells. Genetic defects in NOX2 lead to opportunistic infections and inflammatory manifestations such as granulomas in humans, also known as chronic granulomatous disease (CGD). This condition is mirrored in mice with defective ROS production and interestingly both species are predisposed to autoimmune diseases. An unresolved question is whether the hyper-inflammation and tendency to develop autoimmunity are secondary to the increased infections, or whether these are parallel phenomena. We generated germ-free ROS deficient Ncf1 mutant mice that when reared in specific pathogen-free condition, are highly susceptible to collagen-induced arthritis compared with wild-type mice. Strikingly, arthritis incidence and severity was almost identical in germ-free and specific pathogen-free ROS-deficient mice. In addition, partial reduction of the microbial flora by antibiotics treatment did not alter the disease course. Taken together, this shows that ROS has a clear immune regulatory function that is decoupled from its function in host defence., (© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Malformed α-Synuclein (α-syn) aggregates in neurons are released into the extracellular space, activating microglia to induce chronic neuroinflammation that further enhances neuronal damage in α-synucleinopathies, such as Parkinson's disease. The mechanisms by which α-syn aggregates activate and recruit microglia remain unclear, however. Here we show that α-syn aggregates act as chemoattractants to direct microglia toward damaged neurons. In addition, we describe a mechanism underlying this directional migration of microglia. Specifically, chemotaxis occurs when α-syn binds to integrin CD11b, leading to H2O2 production by NADPH oxidase. H2O2 directly attracts microglia via a process in which extracellularly generated H2O2 diffuses into the cytoplasm and tyrosine protein kinase Lyn, phosphorylates the F-actin-associated protein cortactin after sensing changes in the microglial intracellular concentration of H2O2. Finally, phosphorylated cortactin mediates actin cytoskeleton rearrangement and facilitates directional cell migration. These findings have significant implications, given that α-syn-mediated microglial migration reaches beyond Parkinson's disease.

Aims: Sphingosylphosphorylcholine (SPC) elicits vasoconstriction at micromolar concentrations. At lower concentrations (≤1 µmol/L), however, it does not constrict intrapulmonary arteries (IPAs), but strongly potentiates vasoreactivity. Our aim was to determine whether this also occurs in a systemic artery and to delineate the signalling pathway., Methods and Results: Rat mesenteric arteries and IPAs mounted on a myograph were challenged with ∼25 mmol/L [K+] to induce a small vasoconstriction. SPC (1 µmol/L) dramatically potentiated this constriction in all arteries by ∼400%. The potentiation was greatly suppressed or abolished by inhibition of phospholipase C (PLC; U73122), PKCε (inhibitory peptide), Src (PP2), and NADPH oxidase (VAS2870), and also by Tempol (superoxide scavenger), but not by inhibition of Rho kinase (Y27632). Potentiation was lost in mesenteric arteries from p47(phox-/-), but not NOX2(-/-), mice. The intracellular superoxide generator LY83583 mimicked the effect of SPC. SPC elevated reactive oxygen species (ROS) in vascular smooth muscle cells, and this was blocked by PP2, VAS2870, and siRNA knockdown of PKCε. SPC (1 µmol/L) significantly reduced the EC50 for U46619-induced vasoconstriction, an action ablated by Tempol. In patch-clamped mesenteric artery cells, SPC (200 nmol/L) enhanced Ba2+ current through L-type Ca2+ channels, an action abolished by Tempol but mimicked by LY83583., Conclusion: Our results suggest that low concentrations of SPC activate a PLC-coupled and NOX1-mediated increase in ROS, with consequent enhancement of voltage-gated Ca2+ entry and thus vasoreactivity. We speculate that this pathway is not specific for SPC, but may also contribute to vasoconstriction elicited by other G-protein coupled receptor and PLC-coupled agonists., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Chronic granulomatous disease (CGD) is an autosomal recessive or X-linked disorder caused by NADPH oxidase deficiency leading to an impaired ability of reactive superoxide anion and metabolite formation and recurring severe bacterial and fungal infections, with a high mortality rate. Diarrhea, colitis, ileus, perirectal abscess formation and anal fissures are reported gastrointestinal findings in these patients. We report a case of intractable colitis associated with CGD in a young girl.

Hepatic fibrosis in nonalcoholic steatohepatitis (NASH) is the common pathophysiological process resulting from chronic liver inflammation and oxidative stress. Although significant research has been carried out on the role of leptin-induced NADPH oxidase in fibrogenesis, the molecular mechanisms that connect the leptin-NADPH oxidase axis in upregulation of transforming growth factor (TGF)-β signaling have been unclear. We aimed to investigate the role of leptin-mediated upregulation of NADPH oxidase and its subsequent induction of micro-RNA 21 (miR21) in fibrogenesis. Human NASH livers and a high-fat (60% kcal) diet-fed chronic mouse model, where hepatotoxin bromodichloromethane was used to induce NASH, were used for this study. To prove the role of the leptin-NADPH oxidase-miR21 axis, mice deficient in genes for leptin, p47phox, and miR21 were used. Results showed that wild-type mice and human livers with NASH had increased oxidative stress, increased p47phox expression, augmented NF-κB activation, and increased miR21 levels. These mice and human livers showed increased TGF-β, SMAD2/3-SMAD4 colocalizations in the nucleus, increased immunoreactivity against Col1α, and α-SMA with a concomitant decrease in protein levels of SMAD7. Mice that were deficient in leptin or p47phox had decreased activated NF-κB and miR21 levels, suggesting the role of leptin and NADPH oxidase in inducing NF-κB-mediated miR21 expression. Further miR21 knockout mice had decreased colocalization events of SMAD2/3-SMAD4 in the nucleus, increased SMAD7 levels, and decreased fibrogenesis. Taken together, the studies show the novel role of leptin-NADPH oxidase induction of miR21 as a key regulator of TGF-β signaling and fibrogenesis in experimental and human NASH., (Copyright © 2015 the American Physiological Society.)

Kawasaki disease (KD) is an acute self-limiting form of vasculitis that afflicts infants and children and manifests as fever and signs of mucocutaneous inflammation. Children with KD show various laboratory inflammatory abnormalities, such as elevations in their white blood cell (WBC) count, C-reactive protein (CRP) level, and erythrocyte sedimentation rate (ESR). We here performed a genome-wide association study (GWAS) of 178 KD patients to identify the genetic loci that influence 10 important KD laboratory markers: WBC count, neutrophil count, platelet count, CRP, ESR, hemoglobin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and total protein. A total of 165 loci passed our arbitrary stage 1 threshold for replication (p < 1 × 10(-5)). Of these, only 2 SNPs (rs12068753 and rs4786091) demonstrated a significant association with the CRP level in replication study of 473 KD patients (p < 0.05). The SNP located at the CRP locus (rs12068753) demonstrated the most significant association with CRP in KD patients (beta = 4.73 and p = 1.20 × 10(-6) according to the stage 1 GWAS; beta = 3.65 and p = 1.35 × 10(-8) according to the replication study; beta = 3.97 and p = 1.11 × 10(-13) according to combined analysis) and explained 8.1% of the phenotypic variation observed. However, this SNP did not demonstrate any significant association with CRP in the general population (beta = 0.37 and p = 0.1732) and only explained 0.1% of the phenotypic variation in this instance. Furthermore, rs12068753 did not affect the development of coronary artery lesions or intravenous immunoglobulin resistance in KD patients. These results indicate that common variants in the CRP promoter can play an important role in the CRP levels in KD.

ANCA-activated phagocytes cause vasculitis and necrotizing crescentic GN (NCGN). ANCA-induced phagocyte NADPH oxidase (Phox) may contribute by generating tissue-damaging reactive oxygen species. We tested an alternative hypothesis, in which Phox restrains inflammation by downregulating caspase-1, thereby reducing IL-1β generation and limiting NCGN. In an antimyeloperoxidase (anti-MPO) antibody-mediated disease model, mice transplanted with either gp91(phox)-deficient or p47(phox)-deficient bone marrow showed accelerated disease with increased crescents, necrosis, glomerular monocytes, and renal IL-1β levels compared with mice transplanted with wild-type bone marrow. IL-1β receptor blockade abrogated aggravated NCGN in gp91(phox)-deficient mice. In vitro, challenge with anti-MPO antibody strongly enhanced caspase-1 activity and IL-1β generation in gp91(phox)-deficient and p47(phox)-deficient monocytes compared with wild-type monocytes. This enhanced IL-1β generation was abrogated when caspase-1 was blocked. ANCA-induced superoxide and IL-1β generation were inversely related in human monocytes. Furthermore, transplantation of gp91(phox)/caspase-1 double-deficient bone marrow rescued the accelerated NCGN phenotype in gp91(phox) bone marrow-deficient mice. These results suggest that Phox-generated reactive oxygen species downregulate caspase-1, thereby keeping the inflammasome in check and limiting ANCA-induced inflammation. IL-1 receptor blockade may provide a promising strategy in NCGN, whereas our data question the benefit of antioxidants., (Copyright © 2015 by the American Society of Nephrology.)

Background: Deficient production of reactive oxygen species (ROS) by the phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase in patients with chronic granulomatous disease (CGD) results in susceptibility to certain pathogens secondary to impaired oxidative killing and mobilization of other phagocyte defenses. Peroxisome proliferator-activated receptor (PPAR) γ agonists, including pioglitazone, approved for type 2 diabetes therapy alter cellular metabolism and can heighten ROS production. It was hypothesized that pioglitazone treatment of gp91(phox-/-) mice, a murine model of human CGD, would enhance phagocyte oxidant production and killing of Staphylococcus aureus, a significant pathogen in patients with this disorder., Objectives: We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense., Methods: Wild-type and gp91(phox-/-) mice were treated with the PPARγ agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated., Results: As demonstrated by 3 different ROS-sensing probes, short-term treatment of gp91(phox-/-) mice with pioglitazone enhanced stimulated ROS production in neutrophils and monocytes from blood and neutrophils and inflammatory macrophages recruited to tissues. Mitochondria were identified as the source of ROS. Findings were replicated in human monocytes from patients with CGD after ex vivo pioglitazone treatment. Importantly, although mitochondrial (mt)ROS were deficient in gp91(phox-/-) phagocytes, their restoration with treatment significantly enabled killing of S aureus both ex vivo and in vivo., Conclusions: Together, the data support the hypothesis that signaling from the NADPH oxidase under normal circumstances governs phagocyte mtROS production and that such signaling is lacking in the absence of a functioning phagocyte oxidase. PPARγ agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD., (Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The production of microRNAs (miRNA) is influenced by various stimuli, including environmental stresses. We hypothesized that reactive oxygen species (ROS)-associated stress could regulate macrophage miRNA synthesis. miRNAs undergo unique steps of maturation processing through either one of two pathways of cytoplasmic processing. Unlike the canonical pathway, the regulation of alternative cytoplasmic processing of miRNA has not been fully elucidated yet. We cultured bone marrow derived macrophages (BMDM) from wild type (WT) and p47(phox-/-) mice and profiled miRNA expression using microarrays. We analyzed 375 miRNAs including four endogenous controls to normalize the data. At resting state, p47(phox-/-) BMDM has the markedly reduced expression of miR-451 compared to WT BMDM, without other significant differences. Unlike majority of miRNAs, miR-451 goes through the unique alternative processing pathway, in which Ago2 plays a key role. In spite of significant reduction of mature miR-451, however, its precursor form, pre-mir-451, was similar in both BMDMs, suggesting that the processing of pre-mir-451 is impaired in p47(phox-/-) BMDM. Moreover, p47(phox-/-) BMDM expressed significantly reduced level of Ago2. In contrast, Ago2 mRNA levels were similar in WT and p47(phox-/-) BMDM, suggesting a post-transcriptional defect of Ago2 production in p47(phox-/-) macrophages, which resulted in impaired processing of pre-miR-451. In order to examine the functional significance of miR-451 in macrophages, we cultured BMDMs from miR-451 knock-out mice. Of interest, miR-451-deficient BMDM exhibited reduced ROS generation upon zymosan stimulation, compared to WT BMDM. Our studies suggest functional crosstalk between ROS and miR-451 in the regulation of macrophage oxidant stress.

Prion infections cause neurodegeneration, which often goes along with oxidative stress. However, the cellular source of reactive oxygen species (ROS) and their pathogenetic significance are unclear. Here we analyzed the contribution of NOX2, a prominent NADPH oxidase, to prion diseases. We found that NOX2 is markedly upregulated in microglia within affected brain regions of patients with Creutzfeldt-Jakob disease (CJD). Similarly, NOX2 expression was upregulated in prion-inoculated mouse brains and in murine cerebellar organotypic cultured slices (COCS). We then removed microglia from COCS using a ganciclovir-dependent lineage ablation strategy. NOX2 became undetectable in ganciclovir-treated COCS, confirming its microglial origin. Upon challenge with prions, NOX2-deficient mice showed delayed onset of motor deficits and a modest, but significant prolongation of survival. Dihydroethidium assays demonstrated a conspicuous ROS burst at the terminal stage of disease in wild-type mice, but not in NOX2-ablated mice. Interestingly, the improved motor performance in NOX2 deficient mice was already measurable at earlier stages of the disease, between 13 and 16 weeks post-inoculation. We conclude that NOX2 is a major source of ROS in prion diseases and can affect prion pathogenesis.

Background: Patients with chronic granulomatous disease (CGD) experience immunodeficiency because of defects in the phagocyte NADPH oxidase and the concomitant reduction in reactive oxygen intermediates. This may result in a reduction in atherosclerotic injury., Methods and Results: We prospectively assessed the prevalence of cardiovascular risk factors, biomarkers of inflammation and neutrophil activation, and the presence of magnetic resonance imaging and computed tomography quantified subclinical atherosclerosis in the carotid and coronary arteries of 41 patients with CGD and 25 healthy controls in the same age range. Univariable and multivariable associations among risk factors, inflammatory markers, and atherosclerosis burden were assessed. Patients with CGD had significant elevations in traditional risk factors and inflammatory markers compared with control subjects, including hypertension, high-sensitivity C-reactive protein, oxidized low-density lipoprotein, and low high-density lipoprotein. Despite this, patients with CGD had a 22% lower internal carotid artery wall volume compared with control subjects (361.3±76.4 mm(3) versus 463.5±104.7 mm(3); P<0.001). This difference was comparable in p47(phox)- and gp91(phox)-deficient subtypes of CGD and independent of risk factors in multivariate regression analysis. In contrast, the prevalence of coronary arterial calcification was similar between patients with CGD and control subjects (14.6%, CGD; 6.3%, controls; P=0.39)., Conclusions: The observation by magnetic resonance imaging and computerized tomography of reduced carotid but not coronary artery atherosclerosis in patients with CGD despite the high prevalence of traditional risk factors raises questions about the role of NADPH oxidase in the pathogenesis of clinically significant atherosclerosis. Additional high-resolution studies in multiple vascular beds are required to address the therapeutic potential of NADPH oxidase inhibition in cardiovascular diseases., Clinical Trial Registration Url: http://www.clinicaltrials.gov. Unique identifier: NCT01063309., (© 2014 American Heart Association, Inc.)

Objective: Although nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) is reportedly essential for phagocyte host defenses, it has been found to aggravate atherosclerosis in apolipoprotein E (Apoe)-null mice through excess production of superoxide. We therefore assessed the role of NOX2 in an experimental model of abdominal aortic aneurysm (AAA) and assessed the mechanism of NOX2 action in AAA., Approach and Results: AAA was induced in low-density lipoprotein receptor-null (Ldlr(-/-)) mice by infusing angiotensin II. Nox2 expression was elevated in the abdominal aortae of these mice during infusion of angiotensin II, with enhanced Nox2 expression mainly because of the recruitment of NOX2-enriched macrophages into AAA lesions. Unexpectedly, systemic Nox2 deficiency promoted AAA development but reduced the level of reactive oxygen species in AAA lesions. Nox2 deficiency stimulated macrophage conversion toward the M1 subset, enhancing expression of interleukin (IL)-1β and matrix metalloproteinase-9/12 mRNA. Administration of neutralizing antibody against IL-1β abolished AAA development in Nox2-deficient mice. Bone marrow transplantation experiments revealed that AAA aggravation by Nox2 deficiency is because of bone marrow-derived cells. Isolated bone marrow-derived macrophages from Nox2-null mice could not generate reactive oxygen species. In contrast, IL-1β expression in peritoneal and bone marrow-derived macrophages, but not in peritoneal neutrophils, was substantially enhanced by Nox2 deficiency. Pharmacological inhibition of Janus kinase/signal transducers and activators of transcription signaling inhibited excess IL-1β expression in Nox2-deficient macrophages, whereas matrix metalloproteinase-9 secretion was constitutively stimulated via nuclear factor-κB signals., Conclusions: Nox2 deficiency enhances macrophage secretion of IL-1β and matrix metalloproteinase-9, disrupting tissue-remodeling functions in AAA lesions. These actions are unfavorable if NOX2 is to serve as a molecular target for AAA., (© 2014 American Heart Association, Inc.)

Objectives: Platelets generate oxidized LDL (ox-LDL) via NOX2-derived oxidative stress. We investigated if once generated by activated platelets ox-LDL can propagate platelet activation., Methods: Experiments were performed in platelets from healthy subjects (HS), hyper-cholesterolemic patients and patients with NOX2 hereditary deficiency., Results: Agonist-stimulated platelets from HS added with LDL were associated with a dose-dependent increase of reactive oxidant species and ox-LDL. Agonist-stimulated platelets from HS added with a fixed dose of LDL (57.14 μmol/L) or added with homogenized human atherosclerotic plaque showed enhanced ox-LDL formation (approximately +50% and +30% respectively), which was lowered by a NOX2 inhibitor (approximately -35% and -25% respectively). Compared to HS, ox-LDL production was more pronounced in agonist-stimulated platelet rich plasma (PRP) from hyper-cholesterolemic patients but was almost absent in PRP from NOX2-deficient patients. Platelet aggregation and 8-iso-PGF2α-ΙΙΙ formation increased in LDL-treated washed platelets (+42% and +53% respectively) and PRP (+31% and +53% respectively). Also, LDL enhanced platelet-dependent thrombosis at arterial shear rate (+33%) but did not affect platelet activation in NOX2-deficient patients. Platelet activation by LDL was significantly inhibited by CD36 or LOX1 blocking peptides, two ox-LDL receptor antagonists, or by a NOX2 inhibitor. LDL-added platelets showed increased p38MAPK (+59%) and PKC (+51%) phosphorylation, p47(phox) translocation to platelet membrane (+34%) and NOX2 activation (+30%), which were inhibited by ox-LDL receptor antagonists., Conclusion: Platelets oxidize LDL, which in turn amplify platelet activation via specific ox-LDL receptors; both effects are mediated by NOX2 activation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the genes encoding subunits of the phagocytic NADPH oxidase system. Patients can present with severe, recurrent infections and noninfectious conditions. Among the latter, inflammatory manifestations are predominant, especially granulomas and colitis. In this article, we systematically review the possible mechanisms of hyperinflammation in this rare primary immunodeficiency condition and their correlations with clinical aspects.

Objective and Design: We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1β release to better understand inflammasome signaling in macrophages., Material or Subjects: Human volunteer blood donors were recruited to isolate monocytes to differentiate them into macrophages. Wild-type or DUOX1-deficient C57/B6 mice were used to prepare bone marrow-derived macrophages., Treatment: Murine or human macrophages were treated in vitro with NLRP3 inflammasome agonists (ATP, silica crystals) or calcium agonists (thapsigargin, ionomycin) in calcium-containing or calcium-free medium., Methods: Intracellular calcium changes were followed by measuring FURA2-based fluorescence. Gene expression changes were measured by quantitative real-time PCR. Protein expression was assessed by western blotting. Enzymatic activity was measured by fluorescence caspase-1 activity assay. IL-1β release was determined by ELISA. ELISA data were analyzed by ANOVA and Tukey's post hoc test., Results: Our data show that calcium is essential for IL-1β release in human macrophages. Increases in cytosolic calcium alone lead to IL-1β secretion. Calcium removal blocks caspase-1 activation. Human macrophages express Duox1, a calcium-regulated NADPH oxidase that produces reactive oxygen species. However, Duox1-deficient murine macrophages show normal IL-1β release., Conclusions: Human macrophage inflammasome activation and IL-1β secretion requires calcium but does not involve NADPH oxidases.

Objectives: An elevated plasma aldosterone level is an independent cardiovascular risk factor. Although excess aldosterone promotes cardiovascular disease, no studies have examined the effect of increased plasma aldosterone on the cerebral circulation. A major source of vascular reactive oxygen species (ROS) during cardiovascular disease is the NADPH oxidases. Because Nox2-containing NADPH oxidase (Nox2 oxidase) is highly expressed in the cerebral endothelium, we postulated that it might contribute to ROS generation and vascular dysfunction in response to aldosterone. Here, we examined the effect of aldosterone and Nox2 oxidase on ROS production and endothelial dysfunction in the cerebral circulation, and whether the effects of aldosterone are exacerbated in aged mice., Methods and Results: In adult (average age ∼24-25 weeks) wild-type and Nox2-deficient (Nox2(/y)) mice, neither vehicle nor aldosterone (0.28  mg/kg per day for 14 days) affected blood pressure (measured using tail-cuff). By contrast, aldosterone treatment reduced dilation of the basilar artery (measured using myography) to the endothelium-dependent agonist acetylcholine in wild-type mice (P < 0.05), but had no such effect in Nox2(/y) mice (P > 0.05). Aldosterone increased basal and phorbol dibutyrate-stimulated superoxide production (measured using L-012-enhanced chemiluminesence) in cerebral arteries from wild-type but not from Nox2(/y) mice. In aged wild-type mice (average age ∼70 weeks), aldosterone treatment increased blood pressure, but had a similar effect on cerebral artery superoxide levels as in adult wild-type mice., Conclusion: These data indicate that Nox2 oxidase mediates aldosterone-induced increases in ROS production and endothelial dysfunction in cerebral arteries from adult mice independently of blood pressure changes. Aldosterone-induced hypertension is augmented during aging.

Oxidative stress plays a key pathogenic role in experimental and human heart failure. However, the source of ROS (reactive oxygen species) is a key determinant of the cardiac adaptation to pathological stressors. In the present study, we have shown that human dilated cardiomyopathy is associated with increased NOX2 (NADPH oxidase 2) levels, increased oxidative stress with adverse myocardial remodelling and activation of MAPKs (mitogen-activated protein kinases). Advanced heart failure in mice was also associated with increased NOX2 levels. Furthermore, we have utilized the pressure-overload model to examine the role of NOX2 in advanced heart failure. Increased cardiomyocyte hypertrophy and myocardial fibrosis in response to pressure overload correlated with increased oxidative stress, and loss of NOX2 prevented the increase in oxidative stress, development of cardiomyocyte hypertrophy, myocardial fibrosis and increased myocardial MMP (matrix metalloproteinase) activity in response to pressure overload. Consistent with these findings, expression of disease markers revealed a marked suppression of atrial natriuretic factor, β-myosin heavy chain, B-type natriuretic peptide and α-skeletal actin expression in pressure-overloaded hearts from NOX2-deficient mice. Activation of MAPK signalling, a well-known mediator of pathological remodelling, was lowered in hearts from NOX2-deficient mice in response to pressure overload. Functional assessment using transthoracic echocardiography and invasive pressure-volume loop analysis showed a marked protection in diastolic and systolic dysfunction in pressure-overloaded hearts from NOX2-deficient mice. Loss of NOX2 prevented oxidative stress in heart disease and resulted in sustained protection from the progression to advanced heart failure. Our results support a key pathogenic role of NOX2 in murine and human heart failure, and specific therapy antagonizing NOX2 activity may have therapeutic effects in advanced heart failure.

The role of NADPH oxidase (Nox) in both the promotion and impairment of compensatory collateral growth remains controversial because the specific Nox and reactive oxygen species involved are unclear. The aim of this study was to identify the primary Nox and reactive oxygen species associated with early stage compensatory collateral growth in young, healthy animals. Ligation of the feed arteries that form primary collateral pathways in rat mesentery and mouse hindlimb was used to assess the role of Nox during collateral growth. Changes in mesenteric collateral artery Nox mRNA expression determined by real-time PCR at 1, 3, and 7 days relative to same-animal control arteries suggested a role for Nox subunits Nox2 and p47(phox). Administration of apocynin or Nox2ds-tat suppressed collateral growth in both rat and mouse models, suggesting the Nox2/p47(phox) interaction was involved. Functional significance of p47(phox) expression was assessed by evaluation of collateral growth in rats administered p47(phox) small interfering RNA and in p47(phox-/-) mice. Diameter measurements of collateral mesenteric and gracilis arteries at 7 and 14 days, respectively, indicated no significant collateral growth compared with control rats or C57BL/6 mice. Chronic polyethylene glycol-conjugated catalase administration significantly suppressed collateral development in rats and mice, implying a requirement for H2O2. Taken together, these results suggest that Nox2, modulated at least in part by p47(phox), mediates early stage compensatory collateral development via a process dependent upon peroxide generation. These results have important implications for the use of antioxidants and the development of therapies for peripheral arterial disease., (Copyright © 2014 the American Physiological Society.)

Background: Chronic sleep fragmentation (SF) without sleep curtailment induces increased adiposity. However, it remains unclear whether mobilization, proliferation, and differentiation of adipocyte progenitors (APs) occurs in visceral white adipose tissue (VWAT), and whether nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (Nox2) activity plays a role., Methods: Changes in VWAT depot cell size and AP proliferation were assessed in wild-type and Nox2 null male mice exposed to SF and control sleep (SC). To assess mobilization, proliferation, and differentiation of bone marrow mesenchymal stem cells (BM-MSC), Sca-1+ bone marrow progenitors were isolated from GFP+ or RFP+ mice, and injected intravenously to adult male mice (C57BL/6) previously exposed to SF or SC., Results: In comparison with SC, SF was associated with increased weight accrual at 3 w and thereafter, larger subcutaneous and visceral fat depots, and overall adipocyte size at 8 w. Increased global AP numbers in VWAT along with enhanced AP BrDU labeling in vitro and in vivo emerged in SF. Systemic injections of GFP+ BM-MSC resulted in increased AP in VWAT, as well as in enhanced differentiation into adipocytes in SF-exposed mice. No differences occurred between SF and SC in Nox2 null mice for any of these measurements., Conclusions: Chronic sleep fragmentation (SF) induces obesity in mice and increased proliferation and differentiation of adipocyte progenitors (AP) in visceral white adipose tissue (VWAT) that are mediated by increased Nox2 activity. In addition, enhanced migration of bone marrow mesenchymal stem cells from the systemic circulation into VWAT, along with AP differentiation, proliferation, and adipocyte formation occur in SF-exposed wild-type but not in oxidase 2 (Nox2) null mice. Thus, Nox2 may provide a therapeutic target to prevent obesity in the context of sleep disorders.

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a leading contributor to mortality during recent influenza pandemics. The mechanism for this influenza-induced susceptibility to secondary S. aureus infection is poorly understood. In this study, we show that innate antibacterial immunity was significantly suppressed during the recovery stage of influenza infection, even though MRSA superinfection had no significant effect on viral burdens. Compared with mice infected with bacteria alone, postinfluenza MRSA-infected mice exhibited impaired bacterial clearance, which was not due to defective phagocyte recruitment, but rather coincided with reduced intracellular reactive oxygen species levels in alveolar macrophages and neutrophils. NADPH oxidase is responsible for reactive oxygen species production during phagocytic bacterial killing, a process also known as oxidative burst. We found that gp91(phox)-containing NADPH oxidase activity in macrophages and neutrophils was essential for optimal bacterial clearance during respiratory MRSA infections. In contrast to wild-type animals, gp91(phox-/-) mice exhibited similar defects in MRSA clearance before and after influenza infection. Using gp91(phox+/-) mosaic mice, we further demonstrate that influenza infection inhibits a cell-intrinsic contribution of NADPH oxidase to phagocyte bactericidal activity. Taken together, our results establish that influenza infection suppresses NADPH oxidase-dependent bacterial clearance and leads to susceptibility to secondary MRSA infection.

Psoriasis is a chronic inflammatory skin disease resulting from immune dysregulation. Regulatory T cells (Tregs) are important in the prevention of psoriasis. Traditionally, reactive oxygen species (ROS) are known to be implicated in the progression of inflammatory diseases, including psoriasis, but many recent studies suggested the protective role of ROS in immune-mediated diseases. In particular, severe cases of psoriasis vulgaris have been reported to be successfully treated by hyperbaric oxygen therapy (HBOT), which raises tissue level of ROS. Also it was reported that Treg function was closely associated with ROS level. However, it has been only investigated in lowered levels of ROS so far. Thus, in this study, to clarify the relationship between ROS level and Treg function, as well as their role in the pathogenesis of psoriasis, we investigated imiquimod-induced psoriatic dermatitis (PD) in association with Treg function both in elevated and lowered levels of ROS by using knockout mice, such as glutathione peroxidase-1(-/-) and neutrophil cytosolic factor-1(-/-) mice, as well as by using HBOT or chemicals, such as 2,3-dimethoxy-1,4-naphthoquinone and N-acetylcysteine. The results consistently showed Tregs were hyperfunctional in elevated levels of ROS, whereas hypofunctional in lowered levels of ROS. In addition, imiquimod-induced PD was attenuated in elevated levels of ROS, whereas aggravated in lowered levels of ROS. For the molecular mechanism that may link ROS level and Treg function, we investigated the expression of an immunoregulatory enzyme, indoleamine 2,3-dioxygenase (IDO) which is induced by ROS, in PD lesions. Taken together, it was implied that appropriately elevated levels of ROS might prevent psoriasis through enhancing IDO expression and Treg function.

Patients with chronic granulomatous disease (CGD) have a mutated NADPH complex resulting in defective production of reactive oxygen species; these patients can develop severe colitis and are highly susceptible to invasive fungal infection. In NADPH oxidase-deficient mice, autophagy is defective but inflammasome activation is present despite lack of reactive oxygen species production. However, whether these processes are mutually regulated in CGD and whether defective autophagy is clinically relevant in patients with CGD is unknown. Here, we demonstrate that macrophages from CGD mice and blood monocytes from CGD patients display minimal recruitment of microtubule-associated protein 1 light chain 3 (LC3) to phagosomes. This defect in autophagy results in increased IL-1β release. Blocking IL-1 with the receptor antagonist (anakinra) decreases neutrophil recruitment and T helper 17 responses and protects CGD mice from colitis and also from invasive aspergillosis. In addition to decreased inflammasome activation, anakinra restored autophagy in CGD mice in vivo, with increased Aspergillus-induced LC3 recruitment and increased expression of autophagy genes. Anakinra also increased Aspergillus-induced LC3 recruitment from 23% to 51% (P < 0.01) in vitro in monocytes from CGD patients. The clinical relevance of these findings was assessed by treating CGD patients who had severe colitis with IL-1 receptor blockade using anakinra. Anakinra treatment resulted in a rapid and sustained improvement in colitis. Thus, inflammation in CGD is due to IL-1-dependent mechanisms, such as decreased autophagy and increased inflammasome activation, which are linked pathological conditions in CGD that can be restored by IL-1 receptor blockade.

Francisella tularensis is a category A biodefence agent that causes a fatal human disease known as tularaemia. The pathogenicity of F. tularensis depends on its ability to persist inside host immune cells primarily by resisting an attack from host-generated reactive oxygen and nitrogen species (ROS/RNS). Based on the ability of F. tularensis to resist high ROS/RNS levels, we have hypothesized that additional unknown factors act in conjunction with known antioxidant defences to render ROS resistance. By screening a transposon insertion library of F. tularensis LVS in the presence of hydrogen peroxide, we have identified an oxidant-sensitive mutant in putative EmrA1 (FTL&#95;0687) secretion protein. The results demonstrate that the emrA1 mutant is highly sensitive to oxidants and several antimicrobial agents, and exhibits diminished intramacrophage growth that can be restored to wild-type F. tularensis LVS levels by either transcomplementation, inhibition of ROS generation or infection in NADPH oxidase deficient (gp91Phox(-/-)) macrophages. The emrA1 mutant is attenuated for virulence, which is restored by infection in gp91Phox(-/-) mice. Further, EmrA1 contributes to oxidative stress resistance by affecting secretion of Francisella antioxidant enzymes SodB and KatG. This study exposes unique links between transporter activity and the antioxidant defence mechanisms of F. tularensis., (© 2014 John Wiley & Sons Ltd.)

Background: The NADPH oxidase family (Nox) produces reactive oxygen species by adding the electron donated by NADPH to oxygen. Excessive reactive oxygen species production under a variety of pathological conditions has been attributed to increased Nox activity. Here, we aimed at investigating the role of Nox in cardiac ischemic injury through gain- and loss-of-function approaches., Methods and Results: We modulated Nox activity in the heart by cardiac-specific expression of Nox4 and dominant negative Nox4. Modulation of Nox activity drastically changes the cellular redox status. Increasing Nox activity by cardiac-specific overexpression of Nox4 imposed oxidative stress on the myocardium [increased NAD(P)(+)/NAD(P)H and decreased glutathione/glutathione disulfide ratio] and worsened cardiac energetics and contractile function after ischemia-reperfusion. Overexpression of the dominant negative Nox4 (DN), which abolished the Nox function, led to a markedly reduced state [decreased NAD(P)(+)/NAD(P)H and increased glutathione/glutathione disulfide ratio] at baseline and paradoxically promoted mitochondrial reactive oxygen species production during ischemia resulting in no recovery of heart function after reperfusion. Limiting the generation of reducing equivalent through modulating carbon substrates availability partially restored the NAD(+)/NADH ratio and protected dominant negative Nox4 hearts from ischemic injury., Conclusions: This study reveals an important role of Nox in cardiac redox regulation and highlights the complexity of developing therapies that affect the intricately connected redox states.

The final control of renal water reabsorption occurs in the collecting duct (CD) and relies on regulated expression of aquaporin-2 (AQP2) in principal CD cells. AQP2 transcription is primarily induced by type 2 vasopressin receptor (V2R)-cAMP-protein kinase A (PKA) signaling but also by other factors, including TonEBP and NF-κB. NAPDH oxidase 4 (NOX4) represents a major source of reactive oxygen species (ROS) in the kidney. Because NOX-derived ROS may alter PKA, TonEBP and NF-κB activity, we examined the effects of NOX4 depletion on AQP2 expression. Depleted NOX4 expression by siRNA (siNOX4) in mpkCCDcl4 cells attenuated increased AQP2 mRNA expression by arginine vasopressin (AVP) but not by hypertonicity, which induces both TonEBP and NF-κB activity. AVP-induced AQP2 expression was similarly decreased by the flavoprotein inhibitor diphenyleneiodonium. siNOX4 altered neither TonEBP nor NF-κB activity but attenuated AVP-inducible cellular cAMP concentration, PKA activity and CREB phosphorylation as well as AQP2 mRNA expression induced by forskolin, a potent activator of adenylate cyclase. The repressive effect of siNOX4 on AVP-induced AQP2 mRNA expression was abolished by the non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and was significantly decreased by selective PDE antagonists cilostamide and rolipram, but not vinpocetine, which respectively target PDE3, PDE4 and PDE1. Thus, by inhibiting PDE3 and PDE4 activity NOX4-derived ROS may contribute to V2R-cAMP-PKA signaling and enhance AQP2 transcription.

Uncontrolled fibrosis in organs like heart, kidney, liver and lung is detrimental and may lead to end-stage organ failure. Currently there is no effective treatment for fibrotic disorders. Transforming growth factor (TGF)-β has a fundamental role in orchestrating the process of fibrogenesis; however, interventions directly targeting TGF-β would have undesired systemic side effects due to the multiple physiological functions of TGF-β. Further characterization of the downstream signaling pathway(s) involved in TGF-β-mediated fibrosis may lead to discovery of novel treatment strategies for fibrotic disorders. Accumulating evidence suggests that Nox4 NADPH oxidase may be an important downstream effector in mediating TGF-β-induced fibrosis, while NADPH oxidase-dependent redox signaling may in turn regulate TGF-β/Smad signaling in a feed-forward manner. It is proposed that pharmacological inhibition of the Nox4 function may represent a novel approach in treatment of fibrotic disorders.

Cell death implies morphological changes that may contribute to the progression of this process. In astrocytes, the mechanisms involving the cytoskeletal changes during cell death are not well explored. Although NADPH oxidase (NOX) has been described as being a critical factor in the production of ROS, not much information is available about the participation of NOX-derived ROS in the cell death of astrocytes and their role in the alterations of the cytoskeleton during the death of astrocytes. In this study, we have evaluated the participation of ROS in the death of cultured cerebellar astrocytes using staurosporine (St) as death inductor. We found that astrocytes express NOX1, NOX2, and NOX4. Also, St induced an early ROS production and NOX activation that participate in the death of astrocytes. These findings suggest that ROS produced by St is generated through NOX1 and NOX4. Finally, we showed that the reorganization of tubulin and actin induced by St is ROS independent and that St did not change the level of expression of these cytoskeletal proteins. We conclude that ROS produced by a NOX is required for cell death in astrocytes, but not for the morphological alterations induced by St.

Significance: Reactive oxygen species (ROS) are signaling molecules that are important in physiological processes, including host defense, aging, and cellular homeostasis. Increased ROS bioavailability and altered redox signaling (oxidative stress) have been implicated in the onset and/or progression of chronic diseases, including hypertension., Recent Advances: Although oxidative stress may not be the only cause of hypertension, it amplifies blood pressure elevation in the presence of other pro-hypertensive factors, such as salt loading, activation of the renin-angiotensin-aldosterone system, and sympathetic hyperactivity, at least in experimental models. A major source for ROS in the cardiovascular-renal system is a family of nicotinamide adenine dinucleotide phosphate oxidases (Noxs), including the prototypic Nox2-based Nox, and Nox family members: Nox1, Nox4, and Nox5., Critical Issues: Although extensive experimental data support a role for increased ROS levels and altered redox signaling in the pathogenesis of hypertension, the role in clinical hypertension is unclear, as a direct causative role of ROS in blood pressure elevation has yet to be demonstrated in humans. Nevertheless, what is becoming increasingly evident is that abnormal ROS regulation and aberrant signaling through redox-sensitive pathways are important in the pathophysiological processes which is associated with vascular injury and target-organ damage in hypertension., Future Directions: There is a paucity of clinical information related to the mechanisms of oxidative stress and blood pressure elevation, and a few assays accurately measure ROS directly in patients. Such further ROS research is needed in humans and in the development of adequately validated analytical methods to accurately assess oxidative stress in the clinic.

Chronic granulomatous disease (CGD) is a primary immunodeficiency (PID) characterized by the inability of phagocytes to produce reactive oxygen intermediates (ROIs) due to a defect in the NADPH oxidase complex. Recent studies have revealed that ROIs are involved in inflammatory signaling in phagocytes, illuminating the underlying mechanisms of hyper-inflammation in CGD. CGD patients frequently suffer from CGD-associated bowel inflammation, granuloma, and life-threatening infections. Based on the discovery of the regulatory function of ROIs in the immune response, therapeutic methods for excessive inflammation focusing on inflammatory cytokines are being developed for CGD. Although hematopoietic stem cell (HSC) transplantation (HSCT) is a curative therapy for CGD, successful transplants greatly depend on HSC source selection and the degree of matching of potential donors. Gene therapy trials for PID have been performed on over 120 patients with no HLA identical donor for HSCT, and have demonstrated clinical benefits. Genotoxicity in HSC gene therapy trials has expanded our knowledge on the mechanisms of vector-associated clonal expansion of gene-modified cells, which will advance gene therapy development using self-inactivating retrovirus and lentivirus vectors. We will discuss the complications of HSCT for CGD. We will then outline the status of gene therapy approaches in the treatment of CGD.

Background: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play important roles in vascular activation. The p22(phox) subunit is necessary for the activity of NADPH oxidase complexes utilizing Nox1, Nox2, Nox3, and Nox4 catalytic subunits., Methods: We assessed p22(phox)-deficient mice and human tissue for altered vascular activation., Results: Mice deficient in p22(phox) were smaller than their wild-type littermates but showed no alteration in basal blood pressure. The wild-type littermates were relatively resistant to forming intimal hyperplasia following carotid ligation, and the intimal hyperplasia that developed was not altered by p22(phox) deficiency. However, at the site of carotid artery ligation, the p22(phox)-deficient mice showed significantly less vascular elastic fiber loss compared with their wild-type littermates. This preservation of elastic fibers was associated with a reduced matrix metallopeptidase (MMP) 12/tissue inhibitor of metalloproteinase (TIMP) 1 expression ratio. A similar decrease in the relative MMP12/TIMP1 expression ratio occurred in human coronary artery smooth muscle cells upon knockdown of the hydrogen peroxide responsive kinase CK1αLS. In the ligated carotid arteries, the p22(phox)-deficient mice showed reduced expression of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), suggesting reduced activity of CK1αLS. In a lung biopsy from a human patient with p22(phox) deficiency, there was also reduced vascular hnRNP-C expression., Conclusions: These findings indicate that NADPH oxidase complexes modulate aspects of vascular activation including vascular elastic fiber loss, the MMP12/TIMP1 expression ratio, and the expression of hnRNP-C. Furthermore, these findings suggest that the effects of NADPH oxidase on vascular activation are mediated in part by protein kinase CK1αLS., (© 2013.)

The NADPH oxidase 2 (NOX2) complex is a professional producer of reactive oxygen species (ROS) and is mainly expressed in phagocytes. While the activity of the NOX2 complex is essential for immunity against pathogens and protection against autoimmunity, its role in the development of malignant tumors remains unclear. We compared wild type and Ncf1 (m1J) mutated mice, which lack functional NOX2 complex, in four different tumor models. Ncf1 (m1J) mutated mice developed significantly smaller tumors in two melanoma models in which B16 melanoma cells expressing a hematopoietic growth factor FLT3L or luciferase reporter were used. Ncf1 (m1J) mutated mice developed significantly fewer Lewis Lung Carcinoma (LLC) tumors, but the tumors that did develop, grew at a pace that was similar to the wild type mice. In the spontaneously arising prostate carcinoma model (TRAMP), tumor growth was not affected. The lack of ROS-mediated protection against tumor growth was associated with increased production of immunity-associated cytokines. A significant increase in Th2 associated cytokines was observed in the LLC model. Our present data show that ROS regulate rejection of the antigenic B16-luc and LLC tumors, whereas the data do not support a role for ROS in growth of intrinsically generated tumors.

Rationale: Autophagy is an essential survival mechanism during energy stress in the heart. Oxidative stress is activated by energy stress, but its role in mediating autophagy is poorly understood. NADPH oxidase (Nox) 4 is an enzyme that generates reactive oxygen species (ROS) at intracellular membranes. Whether Nox4 acts as a sensor of energy stress to mediate activation of autophagy is unknown., Objective: We investigated whether Nox4 is involved in the regulation of autophagy and cell survival during energy stress in cardiomyocytes., Methods and Results: Production of ROS in cardiomyocytes was increased during glucose deprivation (GD) in a Nox4-dependent manner. Protein levels and the ROS-producing activity of Nox4 were increased in the endoplasmic reticulum (ER), but not in mitochondria, in response to GD. Selective knockdown of Nox4, but not Nox2, or selective reduction of ROS in the ER with ER-targeted catalase, but not mitochondria-targeted perioxiredoxin 3, abrogated GD-induced autophagy. Nox4 promoted autophagy during GD through activation of the protein kinase RNA-activated-like ER kinase pathway by suppression of prolyl hydroxylase 4. The decrease in cell survival during GD in the presence of Nox4 knockdown was rescued by reactivation of autophagy by Atg7 overexpression, indicating that the effect of Nox4 on cell survival is critically mediated through regulation of autophagy. Nox4 was activated during fasting and prolonged ischemia in the mouse heart, where Nox4 is also required for autophagy activation and cardioprotection., Conclusions: Nox4 critically mediates autophagy in response to energy stress in cardiomyocytes by eliciting ROS in the ER and stimulating the protein kinase RNA-activated-like ER kinase signaling pathway.

ROS are implicated in bone diseases. NADPH oxidase 4 (NOX4), a constitutively active enzymatic source of ROS, may contribute to the development of such disorders. Therefore, we studied the role of NOX4 in bone homeostasis. Nox4(-/-) mice displayed higher bone density and reduced numbers and markers of osteoclasts. Ex vivo, differentiation of monocytes into osteoclasts with RANKL and M-CSF induced Nox4 expression. Loss of NOX4 activity attenuated osteoclastogenesis, which was accompanied by impaired activation of RANKL-induced NFATc1 and c-JUN. In an in vivo model of murine ovariectomy–induced osteoporosis, pharmacological inhibition or acute genetic knockdown of Nox4 mitigated loss of trabecular bone. Human bone obtained from patients with increased osteoclast activity exhibited increased NOX4 expression. Moreover, a SNP of NOX4 was associated with elevated circulating markers of bone turnover and reduced bone density in women. Thus, NOX4 is involved in bone loss and represents a potential therapeutic target for the treatment of osteoporosis.

Chronic Granulomatous Disease (CGD) is a rare primary immunodeficiency due to a defect in one of the NADPH oxidase complex subunits; 70 % of cases are X-linked, due to a CYBB mutation, resulting in defective production of gp91PHOX. Female carriers of X-linked CGD have previously been considered to be unaffected. It is increasingly recognized that they may suffer from similar problems to CGD patients. This review will examine the literature about clinical manifestations of disease in X-linked carriers of CGD.

Background and Purpose: Systemic oxidative stress associated with dietary calorie overload plays an important role in the deterioration of vascular function in middle-aged patients suffering from obesity and insulin resistance. However, effective therapy is still lacking., Experimental Approach: In this study, we used a mouse model of middle-aged obesity to investigate the therapeutic potential of pharmaceutical inhibition (apocynin, 5 mM supplied in the drinking water) or knockout of Nox2, an enzyme generating reactive oxygen species (ROS), in high-fat diet (HFD)-induced obesity, oxidative stress, insulin resistance and endothelial dysfunction. Littermates of C57BL/6J wild-type (WT) and Nox2 knockout (KO) mice (7 months old) were fed with a HFD (45% kcal fat) or normal chow diet (NCD, 12% kcal fat) for 16 weeks and used at 11 months of age., Key Results: Compared to NCD WT mice, HFD WT mice developed obesity, insulin resistance, dyslipidaemia and hypertension. Aortic vessels from these mice showed significantly increased Nox2 expression and ROS production, accompanied by significantly increased ERK1/2 activation, reduced insulin receptor expression, decreased Akt and eNOS phosphorylation and impaired endothelium-dependent vessel relaxation to acetylcholine. All these HFD-induced abnormalities (except the hyperinsulinaemia) were absent in apocynin-treated WT or Nox2 KO mice given the same HFD., Conclusions and Implications: In conclusion, Nox2-derived ROS played a key role in damaging insulin receptor and endothelial function in dietary obesity after middle-age. Targeting Nox2 could represent a valuable therapeutic strategy in the metabolic syndrome., (© 2013 The British Pharmacological Society.)

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.

Unlabelled: Advanced glycation endproducts (AGEs) accumulate in patients with diabetes, yet the link between AGEs and inflammatory and fibrogenic activity in nonalcoholic steatohepatitis (NASH) has not been explored. Tumor necrosis factor alpha (TNF-α)-converting enzyme (TACE) is at the center of inflammatory processes. Because the main natural regulator of TACE activity is the tissue inhibitor of metalloproteinase 3 (Timp3), we hypothesized that AGEs induce TACE through nicotinamide adenine dinucleotide phosphate reduced oxidase 2 (NOX2); and the down-regulation of Sirtuin 1 (Sirt1)/Timp3 pathways mediate fibrogenic activity in NASH. The role of NOX2, Sirt1, Timp3, and TACE was evaluated in choline-deficient L-amino acid defined (CDAA) or Western diet (WD)-fed wild-type (WT) and NOX2(-/-) mice. To restore Timp3, mice were injected with adenovirus (Ad)-Timp3. Sirt1 and Timp3 expressions were studied in livers from NASH patients, and we found that their levels were significantly lower than in healthy controls. In WT mice on the CDAA or WD, Sirt1 and Timp3 expressions were lower, whereas production of reactive oxidative species and TACE activity significantly increased with an increase in active TNF-α production as well as induction of fibrogenic transcripts. Ad-Timp3 injection resulted in a significant decline in TACE activity, procollagen α1 (I), alpha smooth muscle actin (α-SMA) and transforming growth factor beta (TGF-β) expression. NOX2(-/-) mice on the CDAA or WD had no significant change in Sirt1, Timp3, and TACE activity or the fibrosis markers assessed. In vitro, AGE exposure decreased Sirt1 and Timp3 in hepatic stellate cells by a NOX2-dependent pathway, and TACE was induced after exposure to AGEs., Conclusion: TACE activation is central to the pathogenesis of NASH and is mediated by AGEs through NOX2 induction and down-regulation of Sirt1/Timp3 pathways., (Copyright © 2013 by the American Association for the Study of Liver Diseases.)

Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder with multi-systemic manifestations, caused by a heterozygous segmental deletion of 1.55-1.83 Mb at chromosomal band 7q11.23. The deletion can include the NCF1 gene that encodes the p47(phox) protein, a component of the leukocyte NADPH oxidase enzyme, which is essential for the defense against microbial pathogens. It has been postulated that WBS patients with two functional NCF1 genes are more susceptible to occurrence of hypertension than WBS patients with only one functional NCF1 gene. We now describe two extremely rare WBS patients without any functional NCF1 gene, because of a mutation in NCF1 on the allele not carrying the NCF1-removing WBS deletion. These two patients suffer from chronic granulomatous disease with increased microbial infections in addition to WBS. Interestingly, one of these patients did suffer from hypertension, indicating that other factors than NADPH oxidase in vascular tissue may be involved in causing hypertension.

Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4(+) T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P < 0·01) and humans (P = 0·02), compared to their healthy counterparts. CD4-depleted murine models suggested that the role of T cells might be redundant because resistance to aspergillus infection was conserved in CD4(+) T cell-depleted mice, similar to wild-type animals. In contrast, mice depleted of neutrophils alone or neutrophils and CD4(+) T cells developed clinical and pathological evidence of pulmonary aspergillosis and increased mortality (P < 0·05 compared to non-depleted animals). Our findings that T cells in CGD have a robust aspergillus CD4(+) T cell response suggest that CD4(+) T cell-based immunotherapy for this disease is unlikely to be beneficial., (© 2013 British Society for Immunology.)

Granulibacter bethesdensis is a Gram-negative pathogen in patients with chronic granulomatous disease (CGD), a deficiency in the phagocyte NADPH oxidase. Repeated isolation of genetically identical strains from the same patient over years, and prolonged waxing and waning seropositivity in some subjects, raises the possibility of long-term persistence. G. bethesdensis resists killing by serum, CGD polymorphonuclear leukocytes (PMN), and antimicrobial peptides, indicating resistance to nonoxidative killing mechanisms. Although G. bethesdensis extends the survival of PMN, persistent intracellular bacterial survival might rely on longer-lived macrophages and their precursor monocytes. Therefore, we examined phagocytic killing by primary human monocytes and monocyte-derived macrophages (MDM). Cells from both normal and CGD subjects internalized G. bethesdensis similarly. G. bethesdensis stimulated superoxide production in normal monocytes, but to a lesser degree than in normal PMN. Normal but not CGD monocytes and MDM killed G. bethesdensis and required in vitro treatment with IFN-γ to maintain this killing effect. Although in vitro IFN-γ did not enhance G. bethesdensis killing in CGD monocytes, it restricted growth in proportion to CGD PMN residual superoxide production, providing a potential method to identify patients responsive to IFN-γ therapy. In IFN-γ-treated CGD MDM, G. bethesdensis persisted for the duration of the study (7 d) without decreasing viability of the host cells. These results indicate that G. bethesdensis is highly resistant to oxygen-independent microbicides of myeloid cells, requires an intact NADPH oxidase for clearance, and can persist long-term in CGD mononuclear phagocytes, most likely relating to the persistence of this microorganism in infected CGD patients.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, a multienzyme complex, is the major source for production of reactive oxygen species (ROS). ROS are increased in allergic diseases, such as asthma, but the role of ROS in disease pathogenesis remains uncertain. We hypothesized that mice unable to generate ROS via the NADPH oxidase pathway would have decreased allergic airway inflammation. To test this hypothesis, we studied gp91phox(-/-) mice in a model of allergic airway inflammation after sensitization and challenge with ovalbumin. Serum, bronchoalveolar lavage fluid, and lungs were then examined for evidence of allergic inflammation. We found that mice lacking a functional NADPH oxidase complex had significantly decreased ROS production and allergic airway inflammation, compared with wild-type (WT) control animals. To determine the mechanism by which allergic inflammation was inhibited by gp91phox deficiency, we cultured bone marrow-derived dendritic cells from WT and gp91phox(-/-) mice and activated them with LPS. IL-12 expression was significantly increased in the gp91phox(-/-) bone marrow-derived dendritic cells, suggesting that the cytokine profile produced in the absence of gp91phox enhanced the conditions leading to T helper (Th) type 1 differentiation, while inhibiting Th2 polarization. Splenocytes from sensitized gp91phox(-/-) animals produced significantly less IL-13 in response to ovalbumin challenge in vitro compared with splenocytes from sensitized WT mice, suggesting that NADPH oxidase promotes allergic sensitization. In contrast, inflammatory cytokines produced by T cells cultured from WT and gp91phox(-/-) mice under Th0, Th1, Th2, and Th17 conditions were not significantly different. This study demonstrates the importance of NADPH oxidase activity and ROS production in a murine model of asthma.