Neutrophil NET Formation with Microbial Stimuli Requires Late Stage NADPH Oxidase Activity

Neutrophils respond to a range of stimuli by releasing extracellular traps (NETs), a mesh consisting of chromatin plus granule and cytoplasmic proteins. We have investigated NET release in response to phorbol myristate acetate (PMA), Pseudomonas aeruginosa (PAO1), Staphylococcus aureus and Candida albicans, and the involvement of NADPH oxidase (NOX2) and myeloperoxidase (MPO) activities. An oxidative mechanism was involved with each stimulus, and the NOX2 inhibitor diphenylene iodonium (DPI) gave almost total inhibition. Notably, DPI added up to 60–90 min after stimulation still gave significant inhibition of subsequent NET formation. As most of the NOX2 activity had already occurred by that time, this indicates a requirement for late-stage low-level oxidant production. Inhibition of histone citrullination did not suppress NET formation, indicating that this was not the essential oxidant-dependent step. With PMA and P. aeruginosa PAO1, MPO activity played an important role in the induction of NETs and MPO inhibitors added up to 30–90 min after stimulation suppressed NET formation. NET formation with S. aureus and C. albicans was insensitive to MPO inhibition. Thus, MPO products are important with some stimuli but not others. Our results extend earlier observations with PMA and show that induction of NETs by microbial stimuli requires late stage oxidant production. Others have shown that NET formation involves NOX2-dependent elastase release from granules. As this is an early event, we conclude from our results that there is more than one oxidant-dependent step.

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Carbon monoxide differentially inhibits TLR signaling pathways by regulating ROS-induced trafficking of TLRs to lipid rafts

Journal of Experimental Medicine ◽

10.1084/jem.20060845 ◽

2006 ◽

Vol 203 (10) ◽

pp. 2377-2389 ◽

Cited By ~ 267

Author(s):

Kiichi Nakahira ◽

Hong Pyo Kim ◽

Xue Hui Geng ◽

Atsunori Nakao ◽

Xue Wang ◽

...

Keyword(s):

Carbon Monoxide ◽

Nadph Oxidase ◽

Signaling Pathways ◽

Lipid Rafts ◽

Poly I:C ◽

Early Event ◽

Ros Production ◽

Tlr Signaling ◽

Polycytidylic Acid ◽

Diphenylene Iodonium

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 gp91phox-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 gp91phox-deficient macrophages, also indicating a role for NADPH oxidase. CO attenuated LPS-induced NADPH oxidase activity in vitro, potentially by binding to gp91phox. Thus, CO negatively controlled TLR signaling pathways by inhibiting translocation of TLR to lipid rafts through suppression of NADPH oxidase–dependent ROS generation.

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NADPH oxidase promotes NF-κB activation and proliferation in human airway smooth muscle

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00206.2001 ◽

2002 ◽

Vol 282 (4) ◽

pp. L782-L795 ◽

Cited By ~ 72

Author(s):

Sukhdev S. Brar ◽

Thomas P. Kennedy ◽

Anne B. Sturrock ◽

Thomas P. Huecksteadt ◽

Mark T. Quinn ◽

...

Keyword(s):

Smooth Muscle ◽

Nadph Oxidase ◽

Airway Smooth Muscle ◽

Nicotinamide Adenine Dinucleotide ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Nuclear Factor Κb ◽

Nicotinamide Adenine ◽

Phagocytic Cells ◽

Diphenylene Iodonium ◽

Reduced Nicotinamide Adenine Dinucleotide

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O[Formula: see text]) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O[Formula: see text] as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22phox. Transfection with p22phoxantisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-κB (NF-κB), and overexpression of a superrepressor form of the NF-κB inhibitor IκBα significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22phoxregulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-κB.

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Dextromethorphan Attenuates NADPH Oxidase-Regulated Glycogen Synthase Kinase 3β and NF-κB Activation and Reduces Nitric Oxide Production in Group A Streptococcal Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02045-17 ◽

2018 ◽

Vol 62 (6) ◽

pp. e02045-17 ◽

Cited By ~ 4

Author(s):

Chia-Ling Chen ◽

Miao-Huei Cheng ◽

Chih-Feng Kuo ◽

Yi-Lin Cheng ◽

Ming-Han Li ◽

...

Keyword(s):

Nitric Oxide ◽

Nadph Oxidase ◽

Nuclear Translocation ◽

Glycogen Synthase ◽

Glycogen Synthase Kinase ◽

Streptococcal Toxic Shock Syndrome ◽

Glycogen Synthase Kinase 3Β ◽

Diphenylene Iodonium ◽

Group A ◽

Gsk 3Β

ABSTRACTGroup AStreptococcus(GAS) is an important human pathogen that causes a wide spectrum of diseases, including necrotizing fasciitis and streptococcal toxic shock syndrome. Dextromethorphan (DM), an antitussive drug, has been demonstrated to efficiently reduce inflammatory responses, thereby contributing to an increased survival rate of GAS-infected mice. However, the anti-inflammatory mechanisms underlying DM treatment in GAS infection remain unclear. DM is known to exert neuroprotective effects through an NADPH oxidase-dependent regulated process. In the present study, membrane translocation of NADPH oxidase subunit p47phoxand subsequent reactive oxygen species (ROS) generation induced by GAS infection were significantly inhibited via DM treatment in RAW264.7 murine macrophage cells. Further determination of proinflammatory mediators revealed that DM effectively suppressed inducible nitric oxide synthase (iNOS) expression and NO, tumor necrosis factor alpha, and interleukin-6 generation in GAS-infected RAW264.7 cells as well as in air-pouch-infiltrating cells from GAS/DM-treated mice. GAS infection caused AKT dephosphorylation, glycogen synthase kinase-3β (GSK-3β) activation, and subsequent NF-κB nuclear translocation, which were also markedly inhibited by treatment with DM and an NADPH oxidase inhibitor, diphenylene iodonium. These results suggest that DM attenuates GAS infection-induced overactive inflammation by inhibiting NADPH oxidase-mediated ROS production that leads to downregulation of the GSK-3β/NF-κB/NO signaling pathway.

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Apoptotic cascade initiated by angiotensin II in neonatal cardiomyocytes: role of DNA damage

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00408.2003 ◽

2003 ◽

Vol 285 (6) ◽

pp. H2364-H2372 ◽

Cited By ~ 51

Author(s):

Valentina Grishko ◽

Viktor Pastukh ◽

Viktoriya Solodushko ◽

Mark Gillespie ◽

Junichi Azuma ◽

...

Keyword(s):

Nitric Oxide ◽

Dna Damage ◽

Angiotensin Ii ◽

Nadph Oxidase ◽

Ventricular Remodeling ◽

Oxidase Inhibitor ◽

Neonatal Cardiomyocytes ◽

Diphenylene Iodonium ◽

Nitric Oxide Inhibitor

Angiotensin II contributes to ventricular remodeling by promoting both cardiac hypertrophy and apoptosis; however, the mechanism underlying the latter phenomenon is poorly understood. One possibility that has been advanced is that angiotensin II activates NADPH oxidase, generating free radicals that trigger apoptosis. In apparent support of this notion, it was found that angiotensin II-mediated apoptosis in the cardiomyocyte is blocked by the NADPH oxidase inhibitor diphenylene iodonium. However, three lines of evidence suggest that peroxynitrite, rather than superoxide, is responsible for angiotensin II-mediated DNA damage and apoptosis. First, the inducible nitric oxide inhibitor aminoguanidine prevents angiotensin II-induced DNA damage and apoptosis. Second, based on ligation-mediated PCR, the pattern of angiotensin II-induced DNA damage resembles peroxynitritemediated damage rather than damage caused by either superoxide or nitric oxide. Third, angiotensin II activates p53 through the phosphorylation of Ser15 and Ser20, residues that are commonly phosphorylated in response to DNA damage. It is proposed that angiotensin II promotes the oxidation of DNA, which in turn activates p53 to mediate apoptosis.

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Exaggerated vasomotor response to ANG II in rats with fetal programming of hypertension associated with exposure to a low-protein diet during gestation

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00798.2005 ◽

2006 ◽

Vol 291 (4) ◽

pp. R1060-R1068 ◽

Cited By ~ 41

Author(s):

C. Yzydorczyk ◽

F. Gobeil ◽

G. Cambonie ◽

I. Lahaie ◽

N. L. O. Lê ◽

...

Keyword(s):

Nadph Oxidase ◽

Renin Angiotensin System ◽

Underlying Mechanism ◽

Maximal Response ◽

Ang Ii ◽

Vasomotor Response ◽

Enhanced Chemiluminescence ◽

Low Protein ◽

Diphenylene Iodonium ◽

Nadph Oxidase Complex

The renin-angiotensin system plays a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. The current studies were undertaken to verify whether vascular response to ANG II is increased in adult offspring of low-protein fed dams (LP) compared with control (CTRL) and if so, to examine underlying mechanism(s). ANG II-induced contraction of carotid rings was increased in LP (Emax, the maximum asymptote of the curve, relative to maximal response to KCl 80 mM: 230 ± 3% LP vs. 201 ± 2% CTRL, P < 0.05). In both groups, contraction to ANG II was mediated solely by AT1R. Responses to thromboxane A2 analog U-46619 and to KCl 80 mM under step increases in tension were similar between groups. Endothelium depletion enhanced contraction to ANG II in both groups, more so in LP. Blockade of endothelin formation had no effect on response to ANG II, and ANG-(1–7) did not elicit vasomotor response in either group. Superoxide dismutase (SOD) analog Tempol normalized LP without modifying CTRL response to ANG II. Basal levels of superoxide (aortic segments, lucigenin-enhanced chemiluminescence and fluorescent dye hydroethidine) were higher in LP. ANG II further increased superoxide production in LP only, and this was inhibited by coincubation with diphenylene iodonium or apocynin (inhibitor of NADPH oxidase complex). AT1R expression in carotid arteries was increased in LP, whereas SOD expression was unchanged. In conclusion, vasoconstriction to ANG II is exaggerated in this model of developmental programming of hypertension, secondary to enhanced vascular production of superoxide anion by NADPH oxidase with concomitant increase of AT1R expression.

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Telmisartan Attenuates Uric Acid-Induced Epithelial-Mesenchymal Transition in Renal Tubular Cells

BioMed Research International ◽

10.1155/2019/3851718 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12 ◽

Cited By ~ 5

Author(s):

Dongqing Zha ◽

Saiqun Wu ◽

Ping Gao ◽

Xiaoyan Wu

Keyword(s):

Uric Acid ◽

Epithelial Cells ◽

Nadph Oxidase ◽

Dependent Manner ◽

Tubular Epithelial Cells ◽

Mesenchymal Transition ◽

Tubular Cells ◽

Renal Tubular Cells ◽

Diphenylene Iodonium ◽

Renal Tubular

We examined whether and how uric acid induces epithelial to mesenchymal transition (EMT) in renal tubular cells, along with the mechanism by which telmisartan acts on uric acid-induced renal injury. Rat renal proximal tubular epithelial cells (NRK-52E) were exposed to various concentrations of uric acid in the presence or absence of telmisartan. Treatment with uric acid increased the expression of α-SMA, decreased the expression of E-cadherin, and promoted EMT in NRK-52E cells. Uric acid treatment also led to increased endothelin-1 (ET-1) production, activation of extracellular-regulated protein kinase 1/2 (ERK1/2), and the upregulation of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4). Use of ET-1 receptor inhibitor (BQ123 or BQ788) could inhibit uric acid-induced EMT in NRK-52E cells. Pretreatment with the ERK inhibitor (U0126 or PD98059) suppressed the release of ET-1 and EMT induced by uric acid. Additionally, pretreatment with a traditional antioxidant (diphenylene iodonium or apocynin) inhibited the activation of ERK1/2, release of ET-1, and uric acid-induced EMT in NRK-52E cells. These findings suggested that uric acid-induced EMT in renal tubular epithelial cells occurs through NADPH oxidase-mediated ERK1/2 activation and the subsequent release of ET-1. Furthermore, telmisartan (102 nmol/L to 104 nmol/L) inhibited the expression of NOX4, intracellular reactive oxygen species (ROS), activation of ERK1/2, and the release of ET-1 in a dose-dependent manner, thereby preventing uric acid-induced EMT in NRK-52E. In conclusion, telmisartan could ameliorate uric acid-induced EMT in NRK-52E cells likely through inhibition of the NADPH oxidase/ERK1/2/ET-1 pathway.

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Alpha-Synuclein and LRRK2 in Synaptic Autophagy: Linking Early Dysfunction to Late-Stage Pathology in Parkinson’s Disease

Cells ◽

10.3390/cells9051115 ◽

2020 ◽

Vol 9 (5) ◽

pp. 1115 ◽

Cited By ~ 1

Author(s):

Giulia Lamonaca ◽

Mattia Volta

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Late Stage ◽

Disease Onset ◽

Disease Process ◽

Alpha Synuclein ◽

Synaptic Activity ◽

Early Event ◽

Cellular Targets ◽

Disease Modifying

The lack of effective disease-modifying strategies is the major unmet clinical need in Parkinson’s disease. Several experimental approaches have attempted to validate cellular targets and processes. Of these, autophagy has received considerable attention in the last 20 years due to its involvement in the clearance of pathologic protein aggregates and maintenance of neuronal homeostasis. However, this strategy mainly addresses a very late stage of the disease, when neuropathology and neurodegeneration have likely “tipped over the edge” and disease modification is extremely difficult. Very recently, autophagy has been demonstrated to modulate synaptic activity, a process distinct from its catabolic function. Abnormalities in synaptic transmission are an early event in neurodegeneration with Leucine-Rich Repeat Kinase 2 (LRRK2) and alpha-synuclein strongly implicated. In this review, we analyzed these processes separately and then discussed the unification of these biomolecular fields with the aim of reconstructing a potential “molecular timeline” of disease onset and progression. We postulate that the elucidation of these pathogenic mechanisms will form a critical basis for the design of novel, effective disease-modifying therapies that could be applied early in the disease process.

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Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

AJP Cell Physiology ◽

10.1152/ajpcell.00571.2007 ◽

2008 ◽

Vol 294 (6) ◽

pp. C1552-C1565 ◽

Cited By ~ 23

Author(s):

Martin Barfred Friis ◽

Katrine Gribel Vorum ◽

Ian Henry Lambert

Keyword(s):

Nadph Oxidase ◽

Oxidase Activity ◽

Ros Production ◽

Taurine Release ◽

Nitric Oxide Synthase Inhibitor ◽

Nih3t3 Cells ◽

Hypotonic Stress ◽

Diphenylene Iodonium ◽

Synthase Inhibitor ◽

Si Rna

Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H2O2 potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19–32, 2003). The increase in ROS production [5-(and-6)-carboxy-2′, 7′-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 μM) but is unaffected by the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22phox, a NOX4 isotype; p47phox; and p67phox (real-time PCR). Exposure to the Ca2+-mobilizing agonist ATP (10 μM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47phox or p47phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca2+.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-23-8-1012 ◽

2010 ◽

Vol 23 (8) ◽

pp. 1012-1021 ◽

Cited By ~ 43

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.

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A newly synthesized molecule derived from ruthenium cation, with antitumour activity, activates NADPH oxidase in human neutrophils

Biochemical Journal ◽

10.1042/bj3280559 ◽

1997 ◽

Vol 328 (2) ◽

pp. 559-564 ◽

Cited By ~ 15

Author(s):

Modesto CARBALLO ◽

Rosario VILAPLANA ◽

Gracia MÁRQUEZ ◽

Manuel CONDE ◽

J. Francisco BEDOYA ◽

...

Keyword(s):

Nadph Oxidase ◽

Antitumour Activity ◽

Human Neutrophils ◽

Phagocytic Cells ◽

Diphenylene Iodonium ◽

Ru Complexes ◽

O2 Production ◽

Cytosolic Factors ◽

Oxygen Metabolites ◽

Antitumour Effects

To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-•) production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1,2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-• production. This increase was associated with the translocation of cytosolic factors p47phox and p67phox of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-• production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.

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