NADPH oxidase priming and p47phox phosphorylation in neutrophils from synovial fluid of patients with rheumatoid arthritis and spondylarthropathy

Superoxide anion (O2°-)production by neutrophil NADPH oxidase participates in arthritic joint lesion formation. Proinflammatory cytokines such as tumor necrosis factor (TNF), interleukin 8 (IL-8) and granulocyte/macrophage-colony stimulating factor (GM-CSF) have a priming effect on neutrophil NADPH oxidase activity. NADPH oxidase activation is dependent on phosphorylation of p47phox, a cytosolic component of the enzyme. We studied O2°-production and p47phox phosphorylation in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and spondylarthropathy (SpA) according to TNF, IL-8 and GM-CSF levels. O2°-production by neutrophils isolated from SF of all the arthritis patients (RA and SpA) was higher than that of circulating resting neutrophils and when stimulated with fMLP or PMA. In addition, p47phox was partially phosphorylated in SF neutrophils compared to circulating neutrophils. High levels of TNF and IL-8 (but not GM-CSF) are detected in patient's SF (compared to circulating blood levels). TNF levels were significantly higher in RA than in SpA SF. These results suggest that increased NADPH oxidase activity could be involved in arthritic joint inflammation through increased p47phox phosphorylation. This could be the result of the presence of high levels of priming agents such as TNF and IL-8 but not GM-CSF.     

Priming of the neutrophil respiratory burst: role in host defense and inflammation

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called ‘the respiratory burst’, and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with ‘priming’ agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases.     

Genetic and molecular findings of 38 Iranian patients with chronic granulomatous disease caused by p47‐phox defect

One of the components of NADPH oxidase is p47‐phox, encoded by NCF1 gene. This study aims to find new genetic changes and clinical features in 38 Iranian patients with autosomal recessive chronic granulomatous disease (AR‐CGD) caused by NCF1 gene defect. Patients who had abnormal NBT and DHR‐1,2,3 assay with loss of p47‐phox in Western blotting were included in this study. After recording demographic and clinical data, PCR amplification was performed followed by direct sequencing for all exons and exon‐intron boundaries. The most common form of CGD in Iran was AR‐CGD due to consanguinity marriages. Among patients with AR‐CGD, NCF1 deficiency was found to be more common than other forms. Cutaneous involvements (53%), pulmonary infections (50%) and lymphadenopathy (29%) were more prevalent than other clinical manifestations of CGD. Mutation analysis of NCF1 gene identified five different mutations. Homozygous delta GT deletion (c.75_76delGT) was the most frequent mutation and was detected in more than 63% of families. Six families had a nonsense mutation in exon 7 (c.579G > A). Two novel mutations were found in exon 4 in two families, including a missense mutation (c.328C > T) and a nine‐nucleotide deletion (c.331_339delTGTCCCCAC). Genetic detection of these mutations may result in early diagnosis and prevention of possible complications of the disease. This could be useful for timely decision‐making for haematopoietic stem cell transplantation and for carrier detection as well as prenatal diagnosis of next children in the affected families. Our findings might help to predict outcomes, raise awareness and help effective treatment in these patients.     

p47phox基因第10外显子多态性与脑卒中的关联及其对血脂的影响

目的探讨p47phox基因第10外显子C923T(Ala308Val)多态性与湖南地区汉族人群脑卒中的关联及其对血脂的影响。方法采用PCR-单链构象多态技术和DNA直接测序法检测湖南地区汉族220例脑卒中患者、20个脑卒中家系成员和100名健康对照者的p47phox基因第10外显子C923T(Ala308Val)多态性;酶法测定总胆固醇、甘油三酯、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇;免疫比浊法测定apoA-Ⅰ及apoB100;ELISA法测定血清脂蛋白α水平。结果湖南省汉族人p47phox基因第10外显子C923T(Ala308Val)多态存在CC、CT、TT3种基因型,脑卒中组及其各亚型组C923T(Ala308Val)多态基因频率分布与对照组比较,差异无统计学意义(P>0.05);分析不同基因型对血脂、脂蛋白水平的影响,发现脑梗死组和对照组CT基因型血清甘油三酯水平均明显高于CC基因型(P<0.05)。结论湖南省汉族人群p47phox基因C923T(Ala308Val)多态性可能与脑卒中的易患性无关联,可能与脑梗死患者血脂代谢相关。     

Prolactin-induced activation of phagocyte NADPH oxidase in the teleost fish gilthead seabream involves the phosphorylation of p47phox by protein kinase C

The pituitary hormone prolactin (PRL) is a multifunctional polypeptide which act as a key component of the neuroendocrine-immune loop and as a local regulator of the macrophage response. The involvement of PRL in regulating monocyte/macrophage functions is suggested by the presence of PRL receptors in these cells. Recently, we reported that physiological concentrations of native PRL were able to induce the expression of the pro-inflammatory cytokines IL-1β and TNFα, and the production of reactive oxygen species (ROS) in head kidney leukocytes and macrophages from the teleost fish gilthead seabream (Sparus aurata L.). In this study, we show that the NADPH oxidase subunit p47phox becomes phosphorylated in leukocytes stimulated with PRL, an effect that is blocked when neutralizing polyclonal antibodies to PRL are added. Additionally, the pharmacological inhibition of either protein kinase C (PKC) with calphostin C or the Jak/Stat signaling pathway with AG490 impaired PKC activation, p47phox phosphorylation and ROS production in seabream leukocytes activated with PRL. Taken together, our results demonstrate for the first time the need for PKC in regulating the PRL-mediated phosphorylation of p47phox, the activation of NADPH oxidase and the production of ROS by macrophages in vertebrates.     

Expression of NOX1, a superoxide-generating NADPH oxidase, in colon cancer and inflammatory bowel disease

Reactive oxygen species (ROS) are at the centre of many physiological and pathological processes. NOX1, a ROS-producing NADPH oxidase, is highly expressed in the colon but its function in colonic physiology or pathology is still poorly understood. It has been suggested to play a role in host defence, but also in cell growth and possibly malignant transformation. In this study we characterized NOX1 expression in human colon samples derived from healthy control subjects and patients with colon cancer or inflammatory bowel disease (IBD). NOX1 mRNA expression was assessed by dot-blot hybridization, real-time PCR and in situ hybridization, using samples derived from surgical specimens from patients undergoing colon resection. In normal tissues, NOX1 expression was low in the ileum, intermediate in the right colon, and high in the left colon (p = 0.0056 right vs. left colon). NOX1 mRNA levels were not influenced by factors linked to colon tumourigenesis, such as age or sex. Moreover, there was no statistical difference in NOX1 expression between samples derived from adenomas, well differentiated or poorly differentiated colon adenocarcinomas. At a cellular level, NOX1 was highly expressed in colon epithelial cells, both within the crypts and on the luminal surface. In addition, a population of lymphocytes, particularly in the appendix, showed NOX1 expression. Lymphocytes in lesions of Crohn's disease and ulcerative colitis were also strongly positive for NOX1. In conclusion, NOX1 is an enzyme that is constitutively expressed in colon epithelium and is not associated with tumourigenesis. Its distribution in crypts and on the luminal surface, as well as its left-to-right gradient in the colon, suggests a role in host defence function. In addition to the known epithelial localization, we define lymphocytes as a novel site of NOX1 expression, where it may potentially be involved in the pathogenesis of inflammatory bowel diseases.     

Hyperinflammation in chronic granulomatous disease and anti-inflammatory role of the phagocyte NADPH oxidase

Chronic granulomatous disease (CGD) is an immunodeficiency caused by the lack of the superoxide-producing phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. However, CGD patients not only suffer from recurrent infections, but also present with inflammatory, non-infectious conditions. Among the latter, granulomas figure prominently, which gave the name to the disease, and colitis, which is frequent and leads to a substantial morbidity. In this paper, we systematically review the inflammatory lesions in different organs of CGD patients and compare them to observations in CGD mouse models. In addition to the more classical inflammatory lesions, CGD patients and their relatives have increased frequency of autoimmune diseases, and CGD mice are arthritis-prone. Possible mechanisms involved in CGD hyperinflammation include decreased degradation of phagocytosed material, redox-dependent termination of proinflammatory mediators and/or signaling, as well as redox-dependent cross-talk between phagocytes and lymphocytes (e.g. defective tryptophan catabolism). As a conclusion from this review, we propose the existence of ROS high and ROS low inflammatory responses, which are triggered as a function of the level of reactive oxygen species and have specific characteristics in terms of physiology and pathophysiology.     

Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides

Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2^−/− knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2^−/− mice died earlier than the WT strain. Immunization of the NOX2^−/− and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2^−/− mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis.     

Apocynin and enzymatically modified isoquercitrin suppress the expression of a NADPH oxidase subunit p22phox in steatosis-related preneoplastic liver foci of rats

We determined effects of the NADPH oxidase (NOX) inhibitor apocynin (APO) or the antioxidant enzymatically modified isoquercitrin (EMIQ) on an early stage of hepatocarcinogenesis in the liver with steatosis. Male rats were given a single intraperitoneal injection of N-diethylnitrosamine (DEN) and fed a high-fat diet (HFD) to subject to a two-stage hepatocarcinogenesis model. Two weeks later, rats were fed a HFD containing the lipogenic substance malachite green (MG), which were co-administered with EMIQ or APO in drinking water for 6 weeks. Three after DEN initiation, rats were subjected to a two-third partial hepatectomy to enhance cell proliferation. The HFD increased total cholesterol and alkaline phosphatase levels, which were reduced by EMIQ co-administration. APO co-administration reduced MG-increased preneoplastic liver lesions, glutathione S-transferase placental form (GST-P)-positive, adipophilin-negative liver foci, and tended to decrease MG-increased Ki-67-positive or active caspase-3-positive cells in the liver foci. EMIQ or APO co-administration reduced the expression of a NOX subunit p22phox in the liver foci, but did not alter the numbers of LC3a-positive cells, an autophagy marker. We identified no treatment-related effects on p47phox and NOX4 expression in the liver foci. The results indicated that APO or EMIQ had the potential to suppress hyperlipidaemia and steatosis-preneoplastic liver lesions, through suppression of NOX subunit expression in rats.     

Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is part of the microbicidal arsenal used by human polymorphonuclear neutrophils (PMNs) to eradicate invading pathogens. The production of a superoxide anion (O2-) into the phagolysosome is the precursor for the generation of more potent products, such as hydrogen peroxide and hypochlorite. However, this production of O2- is dependent on translocation of the oxidase subunits, including gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac2 from the cytosol or specific granules to the plasma membrane. In response to an external stimuli, PMNs change from a resting, nonadhesive state to a primed, adherent phenotype, which allows for margination from the vasculature into the tissue and chemotaxis to the site of infection upon activation. Depending on the stimuli, primed PMNs display altered structural organization of the NADPH oxidase, in that there is phosphorylation of the oxidase subunits and/or translocation from the cytosol to the plasma or granular membrane, but there is not the complete assembly required for O2- generation. Activation of PMNs is the complete assembly of the membrane-linked and cytosolic NADPH oxidase components on a PMN membrane, the plasma or granular membrane. This review will discuss the individual components associated with the NADPH oxidase complex and the function of each of these units in each physiologic stage of the PMN: rested, primed, and activated.     

Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA) activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens.     

NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL‐1β secretion but not in inflammasome activation

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin‐1 beta (IL‐1β) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL‐1β processing is still controversial and has not been previously studied in neutrophils. We report here that IL‐1β processing in human neutrophils is dependent on caspase‐1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase‐1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL‐1β; whereas ROS negatively controlled caspase‐1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL‐1β out of the cell, a role never previously described. The complex ROS‐mediated regulation of neutrophil IL‐1β secretion might constitute a physiological mechanism to control IL‐1β‐dependent inflammatory processes where neutrophils play a crucial role.     

Critical role of the NADPH oxidase subunit p47phox on vascular TLR expression and neointimal lesion formation in high-fat diet-induced obesity

Reactive oxygen species (ROS) formation is associated with inflammation and vasculature dysfunction. We investigated the potential role of the NADPH oxidase on vascular Toll-like receptor (TLR) expression and carotid neointimal formation in high-fat (HF) diet-induced obesity (DIO) model. Using mice DIO and common carotid artery flow cessation-induced lesion formation models, we examined vascular TLR2 and TLR4 expression and neointimal formation in NADPH oxidase subunit p47(phox)-deficient (p47(phox-/-)) mice. Feeding C57BL/6J mice an HF diet for 22 weeks resulted in significant increases in p47(phox), TLR2 and TLR4 expression in vascular tissues compared with mice fed a low-fat (LF) diet. Minimal changes in TLR2 and TLR4 expression was detected in p47(phox-/-) DIO mice. Furthermore, flow cessation-induced angiogenic and inflammatory response and neointimal formation were significantly attenuated in p47(phox-/-) DIO mice compared with wild-type DIO mice. In addition, exposure of endothelial cells to leptin led to ROS formation; this was accompanied by upregulation of TLR2, TLR4 expression and its downstream signaling. Leptin also increased endothelial cell migration and proliferation. Pharmacological inhibition of NADPH oxidase or genetic deletion of p47(phox) significantly diminished these alterations. Obesity increases neointimal formation via a mechanism involving p47(phox)-TLRs signaling, suggesting that the NADPH oxidase may represent a potential novel therapeutic target for the treatment of obesity-associated vascular inflammation and dysfunction.     

NADPH oxidase of human dendritic cells: role in Candida albicans killing and regulation by interferons, dectin-1 and CD206

Human monocyte-derived DC express the enzyme NADPH oxidase, responsible for ROS production. We show that Candida albicans did not activate NADPH oxidase in DC, and was poorly killed by these cells. However, Candida-killing activity increased upon DC stimulation with the NADPH oxidase activator PMA and was further enhanced by DC treatment with IFN-alpha or IFN-gamma. This fungicidal activity took place at high DC-to-Candida ratio, but decreased at low DC-to-yeast ratio, when Candida inhibited the NADPH oxidase by contrasting the assembly of the enzyme on DC plasma membrane. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated the PMA-dependent DC candidacidal capacity. Engagement of beta-glucan receptor dectin-1 induced NADPH oxidase activation in DC that was depressed by mannose-binding receptor CD206 co-stimulation. Candida was internalized by DC through mannose-binding receptors, but not through dectin-1, thus explaining why Candida did not elicit NADPH oxidase activity. Our results indicate that NADPH oxidase is involved in DC Candida-killing activity, which is increased by IFN. However, Candida escapes the oxidative damage by inhibiting NADPH oxidase and by entering DC through receptors not involved in NADPH oxidase activation.     

Macrophage‐specific NOX2 contributes to the development of lung emphysema through modulation of SIRT1/MMP‐9 pathways

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS‐producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase‐induced emphysema using NOX2‐ and NOX1‐deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2‐positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase‐induced alveolar airspace enlargement and elastin degradation were prevented in NOX2‐deficient mice, but not in NOX1‐deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase‐9 (MMP‐9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage‐restricted functional NOX2 in elastase‐induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68‐positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase‐induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage‐specific NOX2 participates in elastase‐induced emphysema through the involvement of SIRT1/MMP‐9 pathways in mice. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Hyperinflammation of chronic granulomatous disease is abolished by NOX2 reconstitution in macrophages and dendritic cells

Chronic granulomatous disease (CGD), caused by a lack of reactive oxygen species (ROS) generation by the phagocyte NADPH oxidase NOX2, leads to massively increased inflammatory responses. In order to identify the type of phagocyte which requires NOX2 activity to limit inflammation, we investigated mice with a loss of function mutation in the Ncf1 gene coding for the p subunit of NOX2 and mice with transgenic rescue of Ncf1 under control of the CD68 promoter. To induce CGD hyperinflammation, different mouse genotypes were injected intradermally with β‐glucan. Ncf1 mutant mice showed massive and prolonged hyperinflammation. Hyperinflammatory lesions were characterized by persistent neutrophilic infiltration, along with ulceration and necrosis. In contrast, in CD68 promoter rescue mice inflammation resolved within days, as seen in wild‐type animals. Measurements of ROS in rescue mice demonstrated functional NOX2 in mononuclear phagocytes (macrophages and dendritic cells) but not in neutrophils. This absence of NOX2 function was also confirmed in inflammatory tissue neutrophils. Lack of functional NOX2 in mononuclear phagocytes increased the secretion of IL‐1β at early time points and of IL‐6 and TNFα at later time points. Thus, CGD hyperinflammation is a redox dysregulation in mononuclear phagocytes, demonstrating a cell type‐specific anti‐inflammatory function of NOX2. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.