Inhibition of gp91(phox) contributes towards normobaric hyperoxia afforded neuroprotection in focal cerebral ischemia.
ABSTRACT: Oxygen therapy is a promising treatment strategy for ischemic stroke. One potential safety concern with oxygen therapy, however, is the possibility of increased generation of reactive oxygen species (ROS), which could exacerbate ischemic brain injury. Our previous study indicated that normobaric hyperoxia (NBO, 95% O(2) with 5% CO(2)) treatment during ischemia salvaged ischemic brain tissue and significantly reduced ROS generation in transient experimental stroke. In this follow-up study, we tested the hypothesis that suppression of NADPH oxidase is an important mechanism for NBO-induced reduction of ROS generation in focal cerebral ischemia. Male Sprague-Dawley rats were given NBO (95% O(2)) or normoxia (21% O(2)) during 90-min filament occlusion of the middle cerebral artery, followed by 22.5-hour reperfusion. NBO treatment increased the tissue oxygen partial pressure (pO(2)) level in the ischemic penumbra close to the pre-ischemic value, as measured by electronic paramagnetic resonance (EPR), and led to a 30.2% reduction in magnetic resonance imaging (MRI) apparent diffusion coefficients (ADC) lesion volume. Real time PCR and western blot analyses showed that the mRNA and protein expression of NADPH oxidase catalytic subunit gp91(phox) were upregulated in the ischemic brain, which was significantly inhibited by NBO. As a consequence of gp91(phox) inhibition, NBO treatment reduced NADPH oxidase activity in the ischemic brain. Our results suggest that NBO treatment given during ischemia reduces ROS generation via inhibiting NADPH oxidase, which may serve as an important mechanism underlying NBO's neuroprotection in acute ischemic stroke.
Project description:Sepsis syndrome is characterized by a dysregulated inflammatory response to infection. NADPH oxidase-dependent reactive oxygen species (ROS) play significant roles in the pathophysiology of sepsis. We previously showed that disruption of Nrf2, a master regulator of antioxidant defenses, caused a dysregulation of innate immune response that resulted in greater mortality in a polymicrobial sepsis and LPS shock model; however, the underlying mechanisms are unclear. In the current study, compared with wild-type (Nrf2(+/+)) macrophages, we observed greater protein kinase C-induced NADPH oxidase-dependent ROS generation in Nrf2-disrupted (Nrf2(-/-)) macrophages that was modulated by glutathione levels. To address the NADPH oxidase-mediated hyperinflammatory response and sepsis-induced lung injury and mortality in Nrf2(-/-) mice, we used double knockout mice lacking Nrf2 and NADPH oxidase subunit, gp91(phox) (Nrf2(-/-)//gp91(phox-/-)). Compared with Nrf2(+/+) macrophages, LPS induced greater activation of TLR4 as evident by TLR4 surface trafficking and downstream recruitment of MyD88 and Toll/IL-1R domain-containing adaptor in Nrf2(-/-) macrophages that was diminished by ablation of gp91(phox). Similarly, phosphorylation of IkappaB and IFN regulatory factor 3 as well as cytokine expression was markedly higher in Nrf2(-/-) macrophages; whereas, it was similar in Nrf2(+/+) and Nrf2(-/-)//gp91(phox-/-). In vivo studies showed greater LPS-induced pulmonary inflammation in Nrf2(-/-) mice that was significantly reduced by ablation of gp91(phox). Furthermore, LPS shock and polymicrobial sepsis induced early and greater mortality in Nrf2(-/-) mice; whereas, Nrf2(-/-)//gp91(phox-/-) showed prolonged survival. Together, these results demonstrate that Nrf2 is essential for the regulation of NADPH oxidase-dependent ROS-mediated TLR4 activation and lethal innate immune response in sepsis.
Project description:Carbon monoxide (CO), a byproduct of heme catabolism by heme oxygenase (HO), confers potent antiinflammatory effects. Here we demonstrate that CO derived from HO-1 inhibited Toll-like receptor (TLR) 2, 4, 5, and 9 signaling, but not TLR3-dependent signaling, in macrophages. Ligand-mediated receptor trafficking to lipid rafts represents an early event in signal initiation of immune cells. Trafficking of TLR4 to lipid rafts in response to LPS was reactive oxygen species (ROS) dependent because it was inhibited by diphenylene iodonium, an inhibitor of NADPH oxidase, and in gp91(phox)-deficient macrophages. CO selectively inhibited ligand-induced recruitment of TLR4 to lipid rafts, which was also associated with the inhibition of ligand-induced ROS production in macrophages. TLR3 did not translocate to lipid rafts by polyinosine-polycytidylic acid (poly(I:C)). CO had no effect on poly(I:C)-induced ROS production and TLR3 signaling. The inhibitory effect of CO on TLR-induced cytokine production was abolished in gp91(phox)-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91(phox). Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase-dependent ROS generation.
Project description:Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91(phox)/p22(phox)) and the cytoplasm (p47(phox), p67(phox), and p40(phox)). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa-) Gc does not stimulate neutrophil ROS production. Using constitutively Opa- and OpaD-positive (OpaD+) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa- Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD+ Gc. gp91(phox) and p22(phox) were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa- Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47(phox) "organizing" subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.
Project description:Myeloperoxidase (MPO) is an important enzyme involved in the genesis and development of atherosclerosis. Vascular peroxidase 1 (VPO1) is a newly discovered member of the peroxidase family that is mainly expressed in vascular endothelial cells and smooth muscle cells and has structural characteristics and biological activity similar to those of MPO. Our specific aims were to explore the effects of VPO1 on endothelial cell apoptosis induced by oxidized low-density lipoprotein (ox-LDL) and the underlying mechanisms. The results showed that ox-LDL induced endothelial cell apoptosis and the expression of VPO1 in endothelial cells in a concentration- and time-dependent manner concomitant with increased intracellular reactive oxygen species (ROS) and hypochlorous acid (HOCl) generation, and up-regulated protein expression of the NADPH oxidase gp91(phox) subunit and phosphorylation of p38 MAPK. All these effects of ox-LDL were inhibited by VPO1 gene silencing and NADPH oxidase gp91(phox) subunit gene silencing or by pretreatment with the NADPH oxidase inhibitor apocynin or diphenyliodonium. The p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor DEVD-CHO significantly inhibited ox-LDL-induced endothelial cell apoptosis, but had no effect on intracellular ROS and HOCl generation or the expression of NADPH oxidase gp91(phox) subunit or VPO1. Collectively, these findings suggest for the first time that VPO1 plays a critical role in ox-LDL-induced endothelial cell apoptosis and that there is a positive feedback loop between VPO1/HOCl and the now-accepted dogma that the NADPH oxidase/ROS/p38 MAPK/caspase-3 pathway is involved in ox-LDL-induced endothelial cell apoptosis.
Project description: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) ? 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.We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense.Wild-type and gp91(phox-/-) mice were treated with the PPAR? agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated.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.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. PPAR? agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD.
Project description:Flavocytochrome b(558), the catalytic core of the phagocyte NADPH oxidase (NOX2), mediates electron transfer from NADPH to molecular oxygen to generate superoxide, the precursor of highly ROS for host defense. Flavocytochrome b(558) is an integral membrane heterodimer consisting of a large glycosylated subunit, gp91(phox), and a smaller subunit, p22(phox). We recently showed in murine macrophages that flavocytochrome b(558) localizes to the PM and Rab11-positive recycling endosomes, whereas in primary hMDMs, gp91(phox) and p22(phox) reside in the PM and the ER. The antimicrobial activity of macrophages, including ROS production, is greatly enhanced by IFN-?, but how this is achieved is incompletely understood. To further define the mechanisms by which IFN-? enhances macrophage NADPH oxidase activity, we evaluated changes in flavocytochrome b(558) expression and localization, along with NADPH oxidase activity, in IFN-? stimulated RAW 264.7 cells and primary murine BMDMs and hMDMs. We found that enhanced capacity for ROS production is, in part, a result of increased protein expression of gp91(phox) and p22(phox) but also demonstrate that IFN-? induced a shift in the predominant localization of gp91(phox) and p22(phox) from intracellular membrane compartments to the PM. Our results are the first to show that a cytokine can change the distribution of macrophage flavocytochrome b(558) and provide a potential, new mechanism by which IFN-? modulates macrophage antimicrobial activity. Altogether, our data suggest that the mechanisms by which IFN-? regulates antimicrobial activity of macrophages are more complex than previously appreciated.
Project description:The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91(phox), p22(phox), p47(phox), p67(phox), p40(phox), and rac-2. Mutations in each of the phox components, except p40(phox), have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40(phox) by investigating the NADPH oxidase responses of neutrophils isolated from p40(phox-/-) mice. In the absence of p40(phox), the expression of p67(phox) is reduced by approximately 55% and oxidase responses to tumor necrosis factor alpha/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by approximately 97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40(phox-/-) neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40(phox) as an essential component in bacterial killing.
Project description:NADPH oxidases are important for neuronal function but detailed subcellular localization studies have not been performed. Here, we provide the first evidence for the presence of functional NADPH oxidase 2 (NOX2)-type complex in neuronal growth cones and its bidirectional relationship with the actin cytoskeleton. NADPH oxidase inhibition resulted in reduced F-actin content, retrograde F-actin flow, and neurite outgrowth. Stimulation of NADPH oxidase via protein kinase C activation increased levels of hydrogen peroxide in the growth cone periphery. The main enzymatic NADPH oxidase subunit NOX2/gp91(phox) localized to the growth cone plasma membrane and showed little overlap with the regulatory subunit p40(phox) . p40(phox) itself exhibited colocalization with filopodial actin bundles. Differential subcellular fractionation revealed preferential association of NOX2/gp91(phox) and p40(phox) with the membrane and the cytoskeletal fraction, respectively. When neurite growth was evoked with beads coated with the cell adhesion molecule apCAM, we observed a significant increase in colocalization of p40(phox) with NOX2/gp91(phox) at apCAM adhesion sites. Together, these findings suggest a bidirectional functional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones, which contributes to the control of neurite outgrowth. We have previously shown that reactive oxygen species (ROS) are critical for actin organization and dynamics in neuronal growth cones as well as neurite outgrowth. Here, we report that the cytosolic subunit p40(phox) of the NOX2-type NADPH oxidase complex is partially associated with F-actin in neuronal growth cones, while ROS produced by this complex regulates F-actin dynamics and neurite growth. These findings provide evidence for a bidirectional relationship between NADPH oxidase activity and the actin cytoskeleton in neuronal growth cones.
Project description:BACKGROUND/AIMS:Activated stellate cells are considered the principal mediators of chronic alcoholic pancreatitis/fibrosis. However the mechanisms of alcohol action on pancreatic stellate cells (PaSCs) are poorly understood. The aims of this study were to determine the presence and role of the NADPH oxidase system in mediating alcohol effects on PaSCs with specific emphasis on proliferation. METHODS:PaSC NADPH oxidase components mRNA and protein were determined by RT-PCR and Western blot. The NADPH oxidase activity was measured by detecting the production of reactive oxygen species using lucigenin-derived chemiluminescence assay. PaSC DNA synthesis, a measure of proliferation, was performed by determining the [3H] thymidine incorporation into DNA. RESULTS:mRNA for NADPH oxidase components Nox1, gp91(phox), Nox4, p22(phox), p47(phox) and p67(phox) and protein for NADPH oxidase subunits gp91(phox), p22(phox), p47(phox) and p67(phox) are present in PaSCs. Treatment with platelet-derived growth factor (PDGF) significantly increased the NADPH oxidase activity and DNA synthesis in cultured PaSCs. Alcohol treatment markedly augmented both the NADPH oxidase activity and the DNA synthesis caused by PDGF, which was prevented by antioxidant N-acetyl-L-cysteine, ROS scavenger tiron, and the NADPH oxidase inhibitor diphenylene iodium. The effects of PDGF on NADPH oxidase activity and DNA synthesis were prevented in PaSCs isolated from the pancreas of mice with a genetic deficiency of p47(phox). CONCLUSIONS:Ethanol causes proliferation of stellate cells by augmenting the activation of the cell's NADPH oxidase system stimulated by PDGF. These results provide new insights into the mechanisms of alcohol-induced fibrosing disorders.
Project description:<h4>Rationale</h4>The role of NADPH oxidase activation in pneumonia is complex because reactive oxygen species contribute to both microbial killing and regulation of the acute pulmonary infiltrate. The relative importance of each role remains poorly defined in community-acquired pneumonia.<h4>Objectives</h4>We evaluated the contribution of NADPH oxidase-derived reactive oxygen species to the pathogenesis of pneumococcal pneumonia, addressing both the contribution to microbial killing and regulation of the inflammatory response.<h4>Methods</h4>Mice deficient in the gp91(phox) component of the phagocyte NADPH oxidase were studied after pneumococcal challenge.<h4>Measurements and main results</h4>gp91(phox)(-/-) mice demonstrated no defect in microbial clearance as compared with wild-type C57BL/6 mice. A significant increase in bacterial clearance from the lungs of gp91(phox)(-/-) mice was associated with increased numbers of neutrophils in the lung, lower rates of neutrophil apoptosis, and enhanced activation. Marked alterations in pulmonary cytokine/chemokine expression were also noted in the lungs of gp91(phox)(-/-) mice, characterized by elevated levels of tumor necrosis factor-alpha, KC, macrophage inflammatory protein-2, monocyte chemotactic protein-1, and IL-6. The greater numbers of neutrophils in gp91(phox)(-/-) mice were not associated with increased lung injury. Levels of neutrophil elastase in bronchoalveolar lavage were not decreased in gp91(phox)(-/-) mice.<h4>Conclusions</h4>During pneumococcal pneumonia, NADPH oxidase-derived reactive oxygen species are redundant for host defense but limit neutrophil recruitment and survival. Decreased NADPH oxidase-dependent reactive oxygen species production is well tolerated and improves disease outcome during pneumococcal pneumonia by removing neutrophils from the tight constraints of reactive oxygen species-mediated regulation.