scholarly journals Assembly of NADPH Oxidase in Human Neutrophils Is Modulated by the Opacity-Associated Protein Expression State of Neisseria gonorrhoeae

2013 ◽  
Vol 82 (3) ◽  
pp. 1036-1044 ◽  
Author(s):  
Asya Smirnov ◽  
Kylene P. Daily ◽  
Alison K. Criss
Keyword(s):  
Reactive Oxygen Species ◽  
Neisseria Gonorrhoeae ◽  
Nadph Oxidase ◽  
Plasma Membranes ◽  
Human Neutrophils ◽  
Ros Production ◽  
Oxygen Species ◽  
Content Type ◽  
The Difference ◽  
Strain Fa1090

ABSTRACTNeisseria 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 (gp91phox/p22phox) and the cytoplasm (p47phox, p67phox, and p40phox). 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. gp91phoxand p22phoxwere 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 p47phox“organizing” subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.

Involvement of Caspases in Neutrophil Apoptosis: Regulation by Reactive Oxygen Species

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4808-4818 ◽  
Author(s):  
Bengt Fadeel ◽  
Anders Åhlin ◽  
Jan-Inge Henter ◽  
Sten Orrenius ◽  
Mark B. Hampton
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Nadph Oxidase ◽  
Reactive Oxygen ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Dependent Manner ◽  
Caspase Activity ◽  
Oxygen Species ◽  
Spontaneous Apoptosis

Abstract Human neutrophils have a short half-life and are believed to die by apoptosis or programmed cell death both in vivo and in vitro. We found that caspases are activated in a time-dependent manner in neutrophils undergoing spontaneous apoptosis, concomitant with other characteristic features of apoptotic cell death such as morphologic changes, phosphatidylserine (PS) exposure, and DNA fragmentation. The treatment of neutrophils with agonistic anti-Fas monoclonal antibodies (MoAbs) significantly accelerated this process. However, in cells treated with the potent neutrophil activator phorbol 12-myristate 13-acetate (PMA), caspase activity was only evident after pharmacologic inhibition of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Similarily, inhibition of the NADPH oxidase in constitutive and Fas/APO-1–triggered apoptosis resulted in increased rather than suppressed levels of caspase activity, suggesting that reactive oxygen species may prevent caspases from functioning optimally in these cells. Moreover, oxidants generated via the NADPH oxidase were essential for PS exposure during PMA-induced cell death, but not for neutrophils undergoing spontaneous apoptosis. We conclude that caspases are an important component of constitutive and Fas/APO-1–triggered neutrophil apoptosis. However, these redox sensitive enzymes are suppressed in activated neutrophils, and an alternate oxidant-dependent pathway is used to mediate PS exposure and neutrophil clearance under these conditions.

183 ADDITION OF DIPHENYLENEIODONIUM OR DEHYDROEPIANDROSTERONE TO CULTURE MEDIA INHIBITS REACTIVE OXYGEN SPECIES PRODUCTION DURING THE EARLY CLEAVAGE STAGES OF IN VITRO-PRODUCED PORCINE EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 208
Author(s):  
N. W. K. Karja ◽  
K. Kikuchi ◽  
M. Ozawa ◽  
M. Fahrudin ◽  
T. Somfai ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Culture Media ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Ros Production ◽  
Oxygen Species

Nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase), an enzyme required to catalyze the oxidation of NADPH to NADP during the metabolism of glucose via the pentose phosphate pathway (PPP), was considered as contributing to intracellular reactive oxygen species (ROS) production. Production of superoxide anion and H2O2 via NADPH oxidase has been reported on a rabbit blastocyst surface (Manes and Lai 1995 J. Reprod. Fertil. 104, 69–75). The objective of this study was to examine the effects on in vitro development and intracellular ROS content after the addition of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, or dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PDH), to culture medium during the early embryonic development of in vitro-produced (IVP) porcine embryos. To confirm that these inhibitors lead to reduction in NADPH concentration in the embryo and hence likely to be inhibiting the PPP, a brilliant cresyl blue (BCB) test was performed on Day 2 (the day of insemination = Day 0) of culture. Porcine cumulus–oocyte complexes were matured and fertilized in vitro as described previously (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Prezumptive zygotes were then cultured in NCSU-37 supplemented with 5.5 mM glucose and DPI at concentrations of 0.5 or 1 nM or DHEA at concentrations of 10 or 100 �M (DPI-0.5, DPI-1, DHEA-10 and DHEA-100 groups, respectively) from Day 0 to Day 2 of culture. All of the embryos were cultured subsequently until Day 6 in NCSU-37 supplemented with only 5.5 mM glucose. Data were analyzed by ANOVA. On Day 6, the development to the blastocyst stage of embryos in DPI-0.5, DPI-1, DHEA-10, and DHEA-100 groups were 16.1, 17.6, 16.1, and 19.5%, respectively, which were not significantly different from that of the control group (17.5%) (n d 165 per group, 5 replicates). However, the mean cell number in blastocysts derived from DPI-1, DHEA-10, and DHEA-100 groups (40.8 � 2.3, 39.3 � 1.7, and 42.5 � 2.7, respectively) was significantly higher (P < 0.01) than those in the control (33.4 � 1.6) and DPI-0.5 (32.7 � 1.6) groups. At 20 min after an exposure to BCB, the percentage of BCB+ embryos in DPI-1, DHEA-10, and DHEA-100 groups (73.8, 79.9, and 77.8%, respectively) were significantly higher (P < 0.01) than those in the control and DPI-0.5 groups (42% and 53.9%, respectively) (n = 81-92 per group, 6 replicates), indicating that these two inhibitors effectively induce the reduction of NADPH concentration in the embryos. Moreover, the addition of DPI at 1 nM or DHEA at 10 or 100 �M significantly decreased the H2O2 content of Day 2 embryos as compared with control embryos (n = 48-53 per group, 7 replicates). These results suggest that the addition of either DPI or DHEA to the medium during the first 2 days of culture did not impair the development of the embryos to the blastocyst stage. Decrease of cellular ROS production in Day 2 embryos in this study is interpreted as a result of inhibition of the NADPH oxidase by DPI or of the G6PDH by DHEA.

scholarly journals β-Aminobutyric Acid Primes an NADPH Oxidase–Dependent Reactive Oxygen Species Production During Grapevine-Triggered Immunity

2010 ◽  
Vol 23 (8) ◽  
pp. 1012-1021 ◽  
Author(s):  
Carole Dubreuil-Maurizi ◽  
Sophie Trouvelot ◽  
Patrick Frettinger ◽  
Alain Pugin ◽  
David Wendehenne ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Molecular Mechanisms ◽  
Reactive Oxygen ◽  
Aminobutyric Acid ◽  
Ros Production ◽  
Oxygen Species ◽  
Defense Gene Expression ◽  
Diphenylene Iodonium ◽  
Signaling Events

The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by β-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase–dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem.

NAD(P)H oxidase-mediated reactive oxygen species production alters astrocyte membrane molecular order via phospholipase A2

2009 ◽  
Vol 421 (2) ◽  
pp. 201-210 ◽  
Author(s):  
Donghui Zhu ◽  
Chunhua Hu ◽  
Wenwen Sheng ◽  
Kevin S. Tan ◽  
Mark A. Haidekker ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Phospholipase A2 ◽  
Actin Polymerization ◽  
Plasma Membranes ◽  
Reactive Oxygen ◽  
Membrane Dynamics ◽  
Mitogen Activated Protein Kinase ◽  
Ros Production ◽  
Oxygen Species ◽  
Mitogen Activated Protein

ROS (reactive oxygen species) overproduction is an important underlying factor for the activation of astrocytes in various neuropathological conditions. In the present study, we examined ROS production in astrocytes and downstream effects leading to changes in the signalling cascade, morphology and membrane dynamics using menadione, a redox-active compound capable of inducing intracellular ROS. NAD(P)H oxidase-mediated menadione-induced ROS production, which then stimulated phosphorylation of p38 MAPK (mitogen-activated protein kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2), and increased actin polymerization and cytoskeletal protrusions. We also showed that astrocyte plasma membranes became more molecularly ordered under oxidative stress, which was abrogated by down-regulating cPLA2 (cytosolic phospholipase A2) either with a pharmacological inhibitor or by RNA interference. In addition, mild disruption of F-actin with cytochalasin D suppressed menadione-enhanced phosphorylation of cPLA2 and membrane alterations. Taken together, these results suggest an important role for ROS derived from NAD(P)H oxidase in activation of astrocytes to elicit biochemical, morphological and biophysical changes reminiscent of reactive astrocytes in pathological conditions.

scholarly journals Reactive oxygen species cause endothelial dysfunction in chronic flow overload

2011 ◽  
Vol 110 (2) ◽  
pp. 520-527 ◽  
Author(s):  
X. Lu ◽  
X. Guo ◽  
C. D. Wassall ◽  
M. D. Kemple ◽  
J. L. Unthank ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Shear Stress ◽  
Endothelial Dysfunction ◽  
Nadph Oxidase ◽  
Group Treatment ◽  
Carotid Arteries ◽  
Reactive Oxygen ◽  
Ros Production ◽  
Oxygen Species ◽  
Enos Uncoupling

Although elevation of shear stress increases production of vascular reactive oxygen species (ROS), the role of ROS in chronic flow overload (CFO) has not been well investigated. We hypothesize that CFO increases ROS production mediated in part by NADPH oxidase, which leads to endothelial dysfunction. In six swine, CFO in carotid arteries was induced by contralateral ligation for 1 wk. In an additional group, six swine received apocynin (NADPH oxidase blocker and anti-oxidant) treatment in conjunction with CFO for 1 wk. The blood flow in carotid arteries increased from 189.2 ± 25.3 ml/min (control) to 369.6 ± 61.9 ml/min (CFO), and the arterial diameter increased by 8.6%. The expressions of endothelial nitric oxide synthase (eNOS), p22/p47phox, and NOX2/NOX4 were upregulated. ROS production increased threefold in response to CFO. The endothelium-dependent vasorelaxation was compromised in the CFO group. Treatment with apocynin significantly reduced ROS production in the vessel wall, preserved endothelial function, and inhibited expressions of p22/p47phox and NOX2/NOX4. Although the process of CFO remodeling to restore the wall shear stress has been thought of as a physiological response, the present data implicate NADPH oxidase-produced ROS and eNOS uncoupling in endothelial dysfunction at 1 wk of CFO.

scholarly journals NADPH oxidase-mediated induction of reactive oxygen species and extracellular matrix deposition by insulin-like growth factor binding protein-5

2019 ◽  
Vol 316 (4) ◽  
pp. L644-L655 ◽  
Author(s):  
Hidekata Yasuoka ◽  
Sara M. Garrett ◽  
Xinh-Xinh Nguyen ◽  
Carol M. Artlett ◽  
Carol A. Feghali-Bostwick
Keyword(s):  
Reactive Oxygen Species ◽  
Extracellular Matrix ◽  
Growth Factor ◽  
Nadph Oxidase ◽  
Dependent Manner ◽  
Ros Production ◽  
Oxygen Species ◽  
Insulin Like Growth Factor ◽  
Factor Binding

Insulin-like growth factor binding protein-5 (IGFBP-5) induces production of the extracellular matrix (ECM) components collagen and fibronectin both in vitro and in vivo and is overexpressed in patients with fibrosing lung diseases, such as idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc). However, the mechanism by which IGFBP-5 exerts its fibrotic effect is incompletely understood. Recent reports have shown a substantial role of reactive oxygen species (ROS) in fibrosis; thus we hypothesized that IGFBP-5 induces production of ROS to mediate the profibrotic process. In vitro analyses revealed that ROS production was induced by recombinant and adenoviral vector-mediated IGFBP-5 (AdBP5) in a dose- and time-dependent manner, regulated through MEK/ERK and JNK signaling, and primarily mediated by NADPH oxidase (Nox). Silencing IGFBP-5 in SSc and IPF fibroblasts reduced ROS production. The antioxidants diphenyleneiodonium and N-acetylcysteine blocked IGFBP-5-stimulated ECM production in normal, SSc, and IPF human primary lung fibroblasts. In murine fibroblasts lacking critical components of the Nox machinery, AdBP5-stimulated ROS production and fibronectin expression were reduced compared with wild-type fibroblasts. IGFBP-5 stimulated transcriptional expression of Nox3 in human fibroblasts while selective knockdown of Nox3 reduced ROS production by IGFBP-5. Thus IGFBP-5 mediates fibrosis through production of ROS in a Nox-dependent manner.

scholarly journals Modulation of Neutrophil Extracellular Trap and Reactive Oxygen Species Release by Periodontal Bacteria

2017 ◽  
Vol 85 (12) ◽  
Author(s):  
Josefine Hirschfeld ◽  
Phillipa C. White ◽  
Michael R. Milward ◽  
Paul R. Cooper ◽  
Iain L. C. Chapple
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Reactive Oxygen ◽  
Oral Bacteria ◽  
Oxygen Species ◽  
Antimicrobial Proteins ◽  
Bacterial Killing ◽  
Content Type ◽  
Periodontal Bacteria ◽  
Net Release

ABSTRACT Oral bacteria are the main trigger for the development of periodontitis, and some species are known to modulate neutrophil function. This study aimed to explore the release of neutrophil extracellular traps (NETs), associated antimicrobial proteins, and reactive oxygen species (ROS) in response to periodontal bacteria, as well as the underlying pathways. Isolated peripheral blood neutrophils were stimulated with 19 periodontal bacteria. NET and ROS release, as well as the expression of NET-bound antimicrobial proteins, elastase, myeloperoxidase, and cathepsin G, in response to these species was measured using fluorescence-based assays. NET and ROS release was monitored after the addition of NADP (NADPH) oxidase pathway modulators and inhibitors of Toll-like receptors (TLRs). Moreover, bacterial entrapment by NETs was visualized microscopically, and bacterial killing was assessed by bacterial culture. Certain microorganisms, e.g., Veillonella parvula and Streptococcus gordonii, stimulated higher levels of ROS and NET release than others. NETs were found to entrap, but not kill, all periodontal bacteria tested. NADPH oxidase pathway modulators decreased ROS production but not NET production in response to the bacteria. Interestingly, TLR inhibitors did not impact ROS and NET release. These data suggest that the variability in the neutrophil response toward different bacteria may contribute to the pathogenesis of periodontal diseases by mechanisms such as bacterial avoidance of host responses and activation of neutrophils. Moreover, our results indicate that bacterium-stimulated NET release may arise in part via NADPH oxidase-independent mechanisms. The role of TLR signaling in bacterium-induced ROS and NET release needs to be further elucidated.

Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species

2006 ◽  
Vol 290 (1) ◽  
pp. C66-C76 ◽  
Author(s):  
Tatyana Milovanova ◽  
Shampa Chatterjee ◽  
Yefim Manevich ◽  
Irina Kotelnikova ◽  
Kris DeBolt ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Proliferation ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Nadph Oxidase ◽  
Reactive Oxygen ◽  
Proliferation Index ◽  
Ros Production ◽  
Oxygen Species ◽  
Wild Type

Acute cessation of flow (ischemia) leads to depolarization of the endothelial cell (EC) membrane mediated by KATP channels and followed by production of reactive oxygen species (ROS) from NADPH oxidase. We postulated that ROS are a signal for initiating EC proliferation associated with the loss of shear stress. Flow cytometry was used to identify proliferating CD31-positive pulmonary microvascular endothelial cells (mPMVECs) from wild-type, Kir6.2−/−, and gp91 phox−/− mice. mPMVECs were labeled with PKH26 and cultured in artificial capillaries for 72 h at 5 dyn/cm2 (flow adaptation), followed by 24 h of stop flow or continued flow. ROS production during the first hour of ischemia was markedly diminished compared with wild-type mice in both types of gene-targeted mPMVECs. Cell proliferation was defined as the proliferation index (PI). After 72 h of flow, >98% of PKH26-labeled wild-type mPMVECs were at a single peak (PI 1.0) and the proportion of cells in the S+G2/M phases were at 5.8% on the basis of cell cycle analysis. With ischemia (24 h), PI increased to 2.5 and the ratio of cells in S+G2/M phases were at 35%. Catalase, diphenyleneiodonium, and cromakalim markedly inhibited ROS production and cell proliferation in flow-adapted wild-type mPMVECs. Significant effects of ischemia were not observed in Kir6.2−/− and gp91 phox−/− cells. ANG II activation of NADPH oxidase was unaffected by KATP gene deletion. Thus loss of shear stress in flow-adapted mPMVECs results in cell division associated with ROS generated by NADPH oxidase. This effect requires a functioning cell membrane KATP channel.

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