First Report on Chronic Granulomatous Disease from Nepal and a Review of CYBC1 Deficiency.

Chronic granulomatous disease (CGD) primarily results from inherited defects in components of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex. These include gene defects in cytochrome B-245/558 subunit α/ß and neutrophil cytosolic factors 1, 2, and 4. Recently, homozygous loss-of-function variants in cytochrome B-245 chaperone 1 gene (CYBC1) have been discovered to cause CGD (CYBC1-CGD). Data on variant-proven CGD from low-income countries, the most underprivileged regions of the world, remain sparse due to numerous constraints. Herein, we report the first cohort of patients with CGD from Nepal, a low-income country in the Himalayas' challenging terrain. Our report includes a description of a new case of CYBC1 deficiency who was first diagnosed with CGD at our center. Only a dozen cases of CYBC1-CGD have been described in the literature thus far which have been reviewed comprehensively herein. Most of these patients have had significant infections and autoimmune/inflammatory manifestations. Pulmonary and invasive/disseminated bacterial/fungal infections were the most common followed by skin and soft-tissue infections. Inflammatory bowel disease (IBD) was the most common inflammatory manifestation (median age at diagnosis: 9 years) followed by episodes of recurrent/prolonged fever. Other autoimmune/inflammatory manifestations reported in CYBC1-CGD include acute pancreatitis, hemophagocytic lymphohistiocytosis, systemic granulomatosis, interstitial lung disease, arthritis, autoimmune hemolytic anemia, uveitis, nephritis, and eczema. Our analysis shows that patients with CYBC1-CGD are at a significantly higher risk of IBD-like illness as compared to other forms of CGD which merits further confirmatory studies in the future.

Neutrophil phagocyte oxidase activity controls invasive fungal growth and inflammation in zebrafish.

Neutrophils are primary phagocytes of the innate immune system that generate reactive oxygen species (ROS) and mediate host defense. Deficient phagocyte NADPH oxidase (PHOX) function leads to chronic granulomatous disease (CGD) that is characterized by invasive infections, including those by the generally non-pathogenic fungus Aspergillus nidulans The role of neutrophil ROS in this specific host-pathogen interaction remains unclear. Here, we exploit the optical transparency of zebrafish to image the effects of neutrophil ROS on invasive fungal growth and neutrophil behavior in response to Aspergillus nidulans In a wild-type host, A. nidulans germinates rapidly and elicits a robust inflammatory response with efficient fungal clearance. PHOX-deficient larvae have increased susceptibility to invasive A. nidulans infection despite robust neutrophil infiltration. Expression of subunit p22phox (officially known as CYBA), specifically in neutrophils, does not affect fungal germination but instead limits the area of fungal growth and excessive neutrophil inflammation and is sufficient to restore host survival in p22phox-deficient larvae. These findings suggest that neutrophil ROS limits invasive fungal growth and has immunomodulatory activities that contribute to the specific susceptibility of PHOX-deficient hosts to invasive A. nidulans infection.

Genetic Characteristics, Infectious, and Noninfectious Manifestations of 32 Patients with Chronic Granulomatous Disease.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare genetic disorder characterized by failure of phagocytic leukocytes to destroy certain microbes. We present a study on CGD patients enrolled at a single medical center concerning the infectious and noninfectious complications and genetic properties of the disease. METHODS: Icotinamide adenine dinucleotide phosphate oxidase activity and the expression of flavocytochrome b558 were measured by flow cytometry, and clinical outcomes of the patients were listed in relation to the genetic results. RESULTS: The clinical and genetic findings of 32 pediatric cases with CGD from 23 families were enrolled. Pneumonia and anemia were the most common infectious and noninfectious symptoms. Genetic analysis showed that 10 families (43.5%) carried CYBB variants and 13 families (56.5%) have autosomal recessive (AR) CGD, in which 6 families (26%) carried NCF1 variants, 4 (17.4%) carried CYBA variants, and 3 (13%) carried NCF2 variants. The median age of clinical onset was 3.3 and 48 months for patients with X-linked CGD (X-CGD) and AR-CGD, respectively. The onset of symptoms before age 1 year was 94% in X-CGD, 28.5% in AR-CGD, and 12.5% in patients with oxidase residual activity. Moreover, a de novo germline mutation at c.1415delG in CYBB (OMIM#300481) and a novel c.251_263del13bp in CYBA (OMIM#608508) were also investigated. CONCLUSIONS: Ihydrorhodamine-1,2,3 assay could not detect carrier mother in de novo case with CYBB variant. Most X-CGD patients have the onset of symptoms before age 1 year. Additionally, residual oxidase activity in AR-CGD causes a delay in onset, diagnosis, and prophylaxis. The protective role of residual activity is limited while the infection is ongoing and becoming serious.

Regulatory T cell features in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (Tregs ) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in Tregs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated Tregs from gp91phox -, p47phox - and p40phox -deficient CGD patients separately. Results show that Treg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91phox -deficiency effector Treg (eTreg ) numbers are decreased. Expression of Treg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in Treg functionality or activation state. No correlation was seen between eTreg numbers and patients' clinical phenotype. To conclude, the only difference between Tregs from CGD patients and healthy controls is a decrease in circulating eTregs in gp91phox -deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived Tregs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for Tregs in CGD-related autoinflammation/autoimmunity.

Pulmonary metastatic colonisation and granulomas in NOX2-deficient mice.

Metastasis is the leading cause of death in cancer patients, and successful colonisation of a secondary organ by circulating tumour cells (CTCs) is the rate-limiting step of this process. We used tail-vein injection of B16-F10 melanoma cells into mice to mimic the presence of CTCs and to allow for the assessment of host (microenvironmental) factors that regulate pulmonary metastatic colonisation. We found that mice deficient for the individual subunits of the NADPH oxidase of myeloid cells, NOX2 (encoded by Cyba, Cybb, Ncf1, Ncf2, and Ncf4), all showed decreased pulmonary metastatic colonisation. To understand the role of NOX2 in controlling tumour cell survival in the pulmonary microenvironment, we focused on Cyba-deficient (Cybatm1a ) mice, which showed the most significant decrease in metastatic colonisation. Interestingly, histological assessment of pulmonary metastatic colonisation was not possible in Cybatm1a mice, owing to the presence of large granulomas composed of galectin-3 (Mac-2)-positive macrophages and eosinophilic deposits; granulomas of variable penetrance and severity were also found in Cybatm1a mice that were not injected with melanoma cells, and these contributed to their decreased survival. The decreased pulmonary metastatic colonisation of Cybatm1a mice was not due to any overt defects in vascular permeability, and bone marrow chimaeras confirmed a role for the haematological system in the reduced metastatic colonisation phenotype. Examination of the lymphocyte populations, which are known key regulators of metastatic colonisation, revealed an enhanced proportion of activated T and natural killer cells in the lungs of Cybatm1a mice, relative to controls. The reduced metastatic colonisation, presence of granulomas and altered immune cell populations observed in Cybatm1a lungs were mirrored in Ncf2-deficient (Ncf2tm1a ) mice. Thus, we show that NOX2 deficiency results in both granulomas and the accumulation of antitumoural immune cells in the lungs that probably mediate the decreased pulmonary metastatic colonisation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cutting Edge: Processing of Oxidized Peptides in Macrophages Regulates T Cell Activation and Development of Autoimmune Arthritis.

APCs are known to produce NADPH oxidase (NOX) 2-derived reactive oxygen species; however, whether and how NOX2-mediated oxidation affects redox-sensitive immunogenic peptides remains elusive. In this study, we investigated a major immunogenic peptide in glucose-6-phosphate isomerase (G6PI), a potential autoantigen in rheumatoid arthritis, which can form internal disulfide bonds. Ag presentation assays showed that presentation of this G6PI peptide was more efficient in NOX2-deficient (Ncf1m1J/m1J mutant) mice, compared with wild-type controls. IFN-γ-inducible lysosomal thiol reductase (GILT), which facilitates disulfide bond-containing Ag processing, was found to be upregulated in macrophages from Ncf1 mutant mice. Ncf1 mutant mice exhibited more severe G6PI peptide-induced arthritis, which was accompanied by the increased GILT expression in macrophages and enhanced Ag-specific T cell responses. Our results show that NOX2-dependent processing of the redox-sensitive autoantigens by APCs modify T cell activity and development of autoimmune arthritis.

Depressive-Like Behaviors Are Regulated by NOX1/NADPH Oxidase by Redox Modification of NMDA Receptor 1.

Involvement of reactive oxygen species (ROS) has been suggested in the development of psychiatric disorders. NOX1 is a nonphagocytic form of NADPH oxidase whose expression in the nervous system is negligible compared with other NOX isoforms. However, NOX1-derived ROS increase inflammatory pain and tolerance to opioid analgesia. To clarify the role of NOX1 in the brain, we examined depressive-like behaviors in mice deficient in Nox1 (Nox1-/Y). Depressive-like behaviors induced by chronic social defeat stress or administration of corticosterone (CORT) were significantly ameliorated in Nox1-/Y Generation of ROS was significantly elevated in the prefrontal cortex (PFC) of mice administrated with CORT, while NOX1 mRNA was upregulated only in the ventral tegmental area (VTA) among brain areas responsible for emotional behaviors. Delivery of miRNA against NOX1 to VTA restored CORT-induced depressive-like behaviors in wild-type (WT) littermates. Administration of CORT to WT, but not to Nox1-/Y, significantly reduced transcript levels of brain-derived neurotrophic factor (bdnf), with a concomitant increase in DNA methylation of the promoter regions in bdnf Delivery of miRNA against NOX1 to VTA restored the level of BDNF mRNA in WT PFC. Redox proteome analyses demonstrated that NMDA receptor 1 (NR1) was among the molecules redox regulated by NOX1. In cultured cortical neurons, hydrogen peroxide significantly suppressed NMDA-induced upregulation of BDNF transcripts in NR1-expressing cells but not in cells harboring mutant NR1 (C744A). Together, these findings suggest a key role of NOX1 in depressive-like behaviors through NR1-mediated epigenetic modification of bdnf in the mesoprefrontal projection.SIGNIFICANCE STATEMENT NADPH oxidase is a source of reactive oxygen species (ROS) that have been implicated in the pathogenesis of various neurological disorders. We presently showed the involvement of a nonphagocytic type of NADPH oxidase, NOX1, in major depressive disorders, including behavioral, biochemical, and anatomical changes in mice. The oxidation of NR1 by NOX1-derived ROS was demonstrated in prefrontal cortex (PFC), which may be causally linked to the downregulation of BDNF, promoting depressive-like behaviors. Given that NOX1 is upregulated only in VTA but not in PFC, mesocortical projections appear to play a crucial role in NOX1-dependent depressive-like behaviors. Our study is the first to present the potential molecular mechanism underlying the development of major depression through the NOX1-induced oxidation of NR1 and epigenetic modification of bdnf.

Distinct Regulatory Effects of Myeloid Cell and Endothelial Cell NAPDH Oxidase 2 on Blood Pressure.

BACKGROUND: Hypertension caused by increased renin-angiotensin system activation is associated with elevated reactive oxygen species production. Previous studies implicate NADPH oxidase (Nox) proteins as important reactive oxygen species sources during renin-angiotensin system activation, with different Nox isoforms being potentially involved. Among these, Nox2 is expressed in multiple cell types, including endothelial cells, fibroblasts, immune cells, and microglia. Blood pressure (BP) is regulated at the central nervous system, renal, and vascular levels, but the cell-specific role of Nox2 in BP regulation is unknown. METHODS: We generated a novel mouse model with a floxed Nox2 gene and used Tie2-Cre, LysM Cre, or Cdh5-CreERT2 driver lines to develop cell-specific models of Nox2 perturbation to investigate its role in BP regulation. RESULTS: Unexpectedly, Nox2 deletion in myeloid but not endothelial cells resulted in a significant reduction in basal BP. Both Tie2-CreNox2 knockout (KO) mice (in which Nox2 was deficient in both endothelial cells and myeloid cells) and LysM CreNox2KO mice (in which Nox2 was deficient in myeloid cells) had significantly lower BP than littermate controls, whereas basal BP was unaltered in Cdh5-CreERT2 Nox2KO mice (in which Nox2 is deficient only in endothelial cells). The lower BP was attributable to an increased NO bioavailability that dynamically dilated resistance vessels in vivo under basal conditions without a change in renal function. Myeloid-specific Nox2 deletion had no effect on angiotensin II-induced hypertension, which, however, was blunted in Tie2-CreNox2KO mice, along with preservation of endothelium-dependent relaxation during angiotensin II stimulation. CONCLUSIONS: We identify a hitherto unrecognized modulation of basal BP by myeloid cell Nox2, whereas endothelial cell Nox2 regulates angiotensin II-induced hypertension. These results identify distinct cell-specific roles for Nox2 in BP regulation.

The role of NOX2-derived reactive oxygen species in collagenase-induced osteoarthritis.

OBJECTIVE: Synovitis in collagenase-induced osteoarthritis (CiOA) is driven by locally released S100A8/A9 proteins and enhances joint destruction. S100A8/A9 can induce reactive oxygen species (ROS) release by phagocytes in OA synovium via neutrophil cytosolic factor-1 (Ncf1)-regulated NOX2 activation. In the present study we investigated whether NOX2-derived ROS affect joint pathology during CiOA. METHODS: CiOA was induced in knee joints of wild type (WT) and Ncf1-deficient (Ncf1**) mice. Synovial gene expression of NOX2-subunits was measured with quantitative real-time polymerase chain reaction (qRT-PCR). Joint pathology was assessed using histology and immunohistochemistry for aggrecan neo-epitope VDIPEN. Levels of inflammatory proteins were measured with Luminex or ELISA. Phagocytes in synovium, blood, bone marrow (BM) and spleen were analyzed with flow cytometry. ROS release by phagocytes was measured with a ROS detection kit. RESULTS: CiOA induction in knee joints of WT mice caused significantly increased synovial gene expression of NOX2 subunits. On day 7 of CiOA, cartilage damage and MMP activity, as measured by VDIPEN, were comparable between WT and Ncf1** mice. Synovial thickening, synovial S100A8/A9 levels and percentages of synovial macrophages, polymorphonuclear cells (PMNs), and monocytes were not different, as were levels of inflammatory mediators in serum and phagocyte percentages in blood, BM and spleen. On day 42 of CiOA, synovitis, cartilage damage, and osteophyte formation in Ncf1** mice were unaltered when compared to WT mice. ROS detection confirmed that Ncf1** PMNs lack functional NOX2, but in vitro macrophages showed ROS production, suggesting activation of compensatory mechanisms. CONCLUSIONS: Absence of Ncf1-mediated ROS production does not alter joint pathology in CiOA.

Adipocyte-Specific Deficiency of NADPH Oxidase 4 Delays the Onset of Insulin Resistance and Attenuates Adipose Tissue Inflammation in Obesity.

OBJECTIVE: Obesity is associated with insulin resistance and adipose tissue inflammation. Reactive oxygen species (ROS) increase in adipose tissue during the development of obesity. We previously showed that in response to excess nutrients like glucose and palmitate, adipocytes generated ROS via NADPH oxidase (NOX) 4, the major adipocyte isoform, instead of using mitochondrial oxidation. However, the role of NOX4-derived ROS in the development of whole body insulin resistance, adipocyte inflammation, and recruitment of macrophages to adipose tissue during the development of obesity is unknown. APPROACH AND RESULTS: In this study, control C57BL/6 mice and mice in which NOX4 has been deleted specifically in adipocytes were fed a high-fat, high-sucrose diet. During the development of obesity in control mice, adipocyte NOX4 and pentose phosphate pathway activity were transiently increased. Primary adipocytes differentiated from mice with adipocytes deficient in NOX4 showed resistance against high glucose or palmitate-induced adipocyte inflammation. Mice with adipocytes deficient in NOX4 showed a delayed onset of insulin resistance during the development of obesity, with an initial reduction in adipose tissue inflammation that normalized with prolonged high-fat, high-sucrose feeding. CONCLUSIONS: These findings imply that NOX4-derived ROS may play a role in the onset of insulin resistance and adipose tissue inflammation. As such, therapeutics targeting NOX4-mediated ROS production could be effective in preventing obesity-associated conditions, such as insulin resistance.

Brown Adipose Tissue Regulates Small Artery Function Through NADPH Oxidase 4-Derived Hydrogen Peroxide and Redox-Sensitive Protein Kinase G-1α.

OBJECTIVE: Biomedical interest in brown adipose tissue (BAT) has increased since the discovery of functionally active BAT in adult humans. Although white adipose tissue (WAT) influences vascular function, vascular effects of BAT are elusive. Thus, we investigated the regulatory role and putative vasoprotective effects of BAT, focusing on hydrogen peroxide, nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4), and redox-sensitive signaling. APPROACH AND RESULTS: Vascular reactivity was assessed in wild-type and Nox4-knockout mice (Nox4-/-) by wire myography in the absence and presence of perivascular adipose tissue of different phenotypes from various adipose depots: (1) mixed WAT/BAT (inguinal adipose tissue) and (2) WAT (epididymal visceral fat) and BAT (intrascapular fat). In wild-type mice, epididymal visceral fat and perivascular adipose tissue increased EC50 to noradrenaline without affecting maximum contraction. BAT increased EC50 and significantly decreased maximum contraction, which were prevented by a hydrogen peroxide scavenger (polyethylene glycated catalase) and a specific cyclic GMP-dependent protein kinase G type-1α inhibitor (DT-3), but not by inhibition of endothelial nitric oxide synthase or guanylate cyclase. BAT induced dimerization of cyclic GMP-dependent protein kinase G type-1α and reduced phosphorylation of myosin light chain phosphatase subunit 1 and myosin light chain 20. BAT from Nox4-knockout mice displayed reduced hydrogen peroxide levels and no anticontractile effects. Perivascular adipose tissue from ß3 agonist-treated mice displayed browned perivascular adipose tissue and an increased anticontractile effect. CONCLUSIONS: We identify a novel vasoprotective action of BAT through an anticontractile effect that is mechanistically different to WAT. Specifically, BAT, via Nox4-derived hydrogen peroxide, induces cyclic GMP-dependent protein kinase G type-1α activation, resulting in reduced vascular contractility. BAT may constitute an interesting therapeutic target to restore vascular function and prevent vascular complications in cardiovascular diseases.

NADPH Oxidase-2 and Atherothrombosis: Insight From Chronic Granulomatous Disease.

The phagocytic cell enzyme NADPH oxidase-2 (Nox2) is critical for killing micro-organisms via production of reactive oxygen species and thus is a key element of the innate immune system. Nox2 is also detectable in endothelial cells and platelets where it has vasoconstrictive and aggregating properties, respectively. Patients with X-linked chronic granulomatous disease with hereditary Nox2 deficiency not only have impaired bacterial killing but, in association with loss of Nox2 function, also have enhanced carotid artery dilation, impaired platelet-related thrombosis, and reduced carotid atherosclerotic burden. Experimental studies corroborated these reports in chronic granulomatous disease by demonstrating (1) Nox2 is upregulated in atherosclerotic plaque, and this upregulation significantly correlates with oxidative stress and (2) pharmacological inhibition of Nox2 is associated with a delayed atherosclerotic progression in animal models. Furthermore, the role of Nox2 in platelet-associated thrombosis was substantiated by experiments showing impaired platelet activation in animals treated with a Nox2 inhibitor or impaired platelet aggregation along with reduced platelet-related thrombosis in the mouse knockout model of Nox2. Interestingly, in chronic granulomatous disease patients and in the mouse knockout model of Nox2, no defects of primary hemostasis were detected. This review analyses experimental and clinical data suggesting Nox2 is a potential target for counteracting the atherothrombotic process.

α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation.

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.

Induction of Pulmonary Granuloma Formation by Propionibacterium acnes Is Regulated by MyD88 and Nox2.

Sarcoidosis is characterized by noncaseating granulomas with an unknown cause that present primarily in the lung. Propionibacterium acnes, an immunogenic commensal skin bacterium involved in acne vulgaris, has been implicated as a possible causative agent of sarcoidosis. Here, we demonstrate that a viable strain of P. acnes isolated from a patient with sarcoidosis and instilled intratracheally into wild-type mice can generate pulmonary granulomas similar to those observed in patients with sarcoidosis. The formation of these granulomas is dependent on the administration of viable P. acnes. We also found that mice deficient in the innate immunity adapter protein MyD88 had a greater number and a larger area of granuloma lesions compared with wild-type mice administered P. acnes. Early after P. acnes administration, wild-type mice produced proinflammatory mediators and recruited neutrophils into the lung, a response that is dependent on MyD88. In addition, there was an increase in granuloma number and size after instillation with P. acnes in mice deficient in CybB, a critical component of nicotinamide adenine dinucleotide phosphate oxidase required for the production of reactive oxygen species in the phagosome. Myd88-/- or Cybb-/- mice both had increased persistence of P. acnes in the lung, together with enhanced granuloma formation. In conclusion, we have generated a mouse model of early granuloma formation induced by a clinically relevant strain of P. acnes isolated from a patient with sarcoidosis, and, using this model, we have shown that a deficiency in MyD88 or CybB is associated with impaired bacterial clearance and increased granuloma formation in the lung.

Combined NOX1/4 inhibition with GKT137831 in mice provides dose-dependent reno- and atheroprotection even in established micro- and macrovascular disease.

AIMS/HYPOTHESIS: Oxidative stress is a promising target in diabetes-associated vasculopathies, with inhibitors of NADPH oxidases (NOX), in particular isoforms 1 and 4, shown to be safe in early clinical development. We have explored a highly relevant late-stage intervention protocol using the clinically most advanced compound, the NOX1/4 inhibitor GKT137831, to determine whether end-organ damage can be reversed/attenuated when GKT137831 is administered in the setting of established diabetic complications. METHODS: GKT137831 was administered at two doses, 30 mg kg-1 day-1 and 60 mg kg-1 day-1, to ApoE -/- mice 10 weeks after diabetes induction with streptozotocin (STZ), for a period of 10 weeks. RESULTS: Consistent with Nox4 -/- mouse data, GKT137831 was protective in a model of diabetic nephropathy at both the 30 mg kg-1 day-1 and 60 mg kg-1 day-1 doses, through suppression of proinflammatory and profibrotic processes. Conversely, in diabetic atherosclerosis, where Nox1 -/y and Nox4 -/- mice have yielded qualitatively opposing results, the net effect of pharmacological NOX1/4 inhibition was protection, albeit to a lower extent and only at the lower 30 mg kg-1 day-1 dose. CONCLUSIONS/INTERPRETATION: As dose-dependent and tissue-specific effects of the dual NOX1/4 inhibitor GKT137831 were observed, it is critical to define in further studies the relative balance of inhibiting NOX4 vs NOX1 in the micro- and macrovasculature in diabetes.

NADPH Oxidase Contributes to Resistance against Aggregatibacter actinomycetemcomitans-Induced Periodontitis in Mice.

Aggregatibacter actinomycetemcomitans is a Gram-negative commensal bacterium of the oral cavity which has been associated with the pathogenesis of periodontitis with severe alveolar bone destruction. The role of host factors such as reactive oxygen and nitrogen intermediates in periodontal A. actinomycetemcomitans infection and progression to periodontitis is still ill-defined. Therefore, this study aimed to analyze the role of NADPH oxidase and inducible nitric oxide synthase (iNOS) in a murine model of A. actinomycetemcomitans-induced periodontitis. NADPH oxidase-deficient (gp91phox knockout [KO]), iNOS-deficient (iNOS KO), and C57BL/6 wild-type mice were orally infected with A. actinomycetemcomitans and analyzed for bacterial colonization at various time points. Alveolar bone mineral density and alveolar bone volume were quantified by three-dimensional micro-computed tomography, and the degree of tissue inflammation was calculated by histological analyses. At 5 weeks after infection, A. actinomycetemcomitans persisted at significantly higher levels in the murine oral cavities of infected gp91phox KO mice than in those of iNOS KO and C57BL/6 mice. Concomitantly, alveolar bone mineral density was significantly lower in all three infected groups than in uninfected controls, but with the highest loss of bone density in infected gp91phox KO mice. Only infected gp91phox KO mice revealed significant loss of alveolar bone volume and enhanced inflammatory cell infiltration, as well as an increased number of osteoclasts. Our results indicate that NADPH oxidase is important to control A. actinomycetemcomitans infection in the murine oral cavity and to prevent subsequent alveolar bone destruction and osteoclastogenesis.

Myeloid Suppressor Cells Accumulate and Regulate Blood Pressure in Hypertension.

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.

Mesenchymal Stem Cells Attenuate NADPH Oxidase-Dependent High Mobility Group Box 1 Production and Inhibit Abdominal Aortic Aneurysms.

OBJECTIVE: Abdominal aortic aneurysm (AAA) formation is characterized by inflammation, smooth muscle activation, and matrix degradation. This study tests the hypothesis that macrophage-produced high mobility group box 1 (HMGB1) production is dependent on nicotinamide adenine dinucleotide phosphate oxidase (Nox2), which leads to increase in interleukin (IL)-17 production resulting in AAA formation and that treatment with human mesenchymal stem cells (MSCs) can attenuate this process thereby inhibiting AAA formation. APPROACH AND RESULTS: Human aortic tissue demonstrated a significant increase in HMGB1 expression in AAA patients when compared with controls. An elastase-perfusion model of AAA demonstrated a significant increase in HMGB1 production in C57BL/6 (wild-type [WT]) mice, which was attenuated by MSC treatment. Furthermore, anti-HMGB1 antibody treatment of WT mice attenuated AAA formation, IL-17 production, and immune cell infiltration when compared with elastase-perfused WT mice on day 14. Elastase-perfused Nox2(-/y) mice demonstrated a significant attenuation of HMGB1 and IL-17 production, cellular infiltration, matrix metalloproteinase activity, and AAA formation when compared with WT mice on day 14. In vitro studies showed that elastase-treated macrophages from WT mice, but not from Nox2(-/y) mice, produced HMGB1, which was attenuated by MSC treatment. The production of macrophage-dependent HMGB1 involved Nox2 activation and superoxide anion production, which was mitigated by MSC treatment. CONCLUSIONS: These results demonstrate that macrophage-produced HMGB1 leads to aortic inflammation and acts as a trigger for CD4(+) T-cell-produced IL-17 during AAA formation. HMGB1 release is dependent on Nox2 activation, which can be inhibited by MSCs leading to attenuation of proinflammatory cytokines, especially IL-17, and protection against AAA formation.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis.

OBJECTIVE: Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. APPROACH AND RESULTS: NOX1(-/Y) platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2(-/-) platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1(-/Y) platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2(-/-) platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2(-/-) mice. Wild-type thrombocytopenic mice injected with NOX2(-/-) platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. CONCLUSIONS: NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

The Cytosolic NADPH Oxidase Subunit NoxO1 Promotes an Endothelial Stalk Cell Phenotype.

OBJECTIVE: Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. APPROACH AND RESULTS: A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1(-/-) when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1(-/-) lung endothelial cells (LECs) and a more tip-cell-like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1(-/-) LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1(-/-) LECs when compared with wild-type LECs. CONCLUSIONS: NoxO1 stimulates α-secretase activity probably through reactive oxygen species-mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.