P47 Phox Knockout Mice Research Articles

Lpp of Escherichia coli K1 inhibits host ROS production to counteract neutrophil-mediated elimination

Escherichia coli (E. coli) is the most common Gram-negative bacterial organism causing neonatal meningitis. The pathogenesis of E. coli meningitis, especially how E. coli escape the host immune defenses, remains to be clarified. Here we show that deletion of bacterial Lpp encoding lipoprotein significantly reduces the pathogenicity of E. coli K1 to induce high-degree of bacteremia necessary for meningitis. The Lpp-deleted E. coli K1 is found to be susceptible to the intracellular bactericidal activity of neutrophils, without affecting the release of neutrophil extracellular traps. The production of reactive oxygen species (ROS), representing the primary antimicrobial mechanism in neutrophils, is significantly increased in response to Lpp-deleted E. coli. We find this enhanced ROS response is associated with the membrane translocation of NADPH oxidase p47phox and p67phox in neutrophils. Then we constructed p47phox knockout mice and we found the incidence of bacteremia and meningitis in neonatal mice induced by Lpp-deleted E. coli is significantly recovered by p47phox knockout. Proteomic profile analysis show that Lpp deficiency induces upregulation of flagellar protein FliC in E. coli. We further demonstrate that FliC is required for the ROS induction in neutrophils by Lpp-deleted E. coli. Taken together, these data uncover the novel role of Lpp in facilitating intracellular survival of E. coli K1 within neutrophils. It can be inferred that Lpp of E. coli K1 is able to suppress FliC expression to restrain the activation of NADPH oxidase in neutrophils resulting in diminished bactericidal activity, thus protecting E. coli K1 from the elimination by neutrophils.

Serum and cecal metabolic profile of the insulin resistant and dyslipidemic p47phox knockout mice

Involvement of NOX-dependent oxidative stress in the pathophysiology of metabolic disorders as well as in the maintenance of metabolic homeostasis has been demonstrated previously. In the present study, the metabolic profile in p47phox–/– and WT mice fed on a chow diet was evaluated to assess the role of metabolites in glucose intolerance and dyslipidemia under altered oxidative stress conditions. p47phox–/– mice displayed glucose intolerance, dyslipidemia, hyperglycemia, insulin resistance (IR), hyperinsulinemia, and altered energy homeostasis without any significant change in gluconeogenesis. The expression of genes involved in lipid synthesis and uptake was enhanced in the liver, adipose tissue, and intestine tissues. Similarly, the expression of genes associated with lipid efflux in the liver and intestine was also enhanced. Enhanced gut permeability, inflammation, and shortening of the gut was evident in p47phox–/– mice. Circulating levels of pyrimidines, phosphatidylglycerol lipids, and 3-methyl-2-oxindole were augmented, while level of purine was reduced in the serum. Moreover, the cecal metabolome was also altered, as was evident with the increase in indole-3-acetamide, N-acetyl galactosamine, glycocholate, and a decrease in hippurate, indoxyl sulfate, and indigestible sugars (raffinose and melezitose). Treatment of p47phox–/– mice with pioglitazone, marginally improved glucose intolerance, and dyslipidemia, with an increase in PUFAs (linoleate, docosahexaenoic acid, and arachidonic acid). Overall, the results obtained in p47phox–/– mice indicate an association of IR and dyslipidemia with altered serum and cecal metabolites (both host and bacterial-derived), implying a critical role of NOX-derived ROS in metabolic homeostasis.

Inhibition of endothelial Nox2 activation by LMH001 protects mice from angiotensin II-induced vascular oxidative stress, hypertension and aortic aneurysm

Endothelial oxidative stress and inflammation attributable to the activation of a Nox2-NADPH oxidase are key features of many cardiovascular diseases. Here, we report a novel small chemical compound (LMH001, MW = 290.079), by blocking phosphorylated p47phox interaction with p22phox, inhibited effectively angiotensin II (AngII)-induced endothelial Nox2 activation and superoxide production at a small dose (IC50 = 0.25 μM) without effect on peripheral leucocyte oxidative response to pathogens. The therapeutic potential of LMH001 was tested using a mouse model (C57BL/6J, 7-month-old) of AngII infusion (0.8 mg/kg/d, 14 days)-induced vascular oxidative stress, hypertension and aortic aneurysm. Age-matched littermates of p47phox knockout mice were used as controls of Nox2 inhibition. LMH001 (2.5 mg/kg/d, ip. once) showed no effect on control mice, but inhibited completely AngII infusion-induced excess ROS production in vital organs, hypertension, aortic walls inflammation and reduced incidences of aortic aneurysm. LMH001 effects on reducing vascular oxidative stress was due to its inhibition of Nox2 activation and was abrogated by knockout of p47phox. LMH001 has the potential to be developed as a novel drug candidate to treat oxidative stress-related cardiovascular diseases.

P47phox-Dependent Oxidant Signalling through ASK1, MKK3/6 and MAPKs in Angiotensin II-Induced Cardiac Hypertrophy and Apoptosis.

The p47phox is a key regulatory subunit of Nox2-containing NADPH oxidase (Nox2) that by generating reactive oxygen species (ROS) plays an important role in Angiotensin II (AngII)-induced cardiac hypertrophy and heart failure. However, the signalling pathways of p47phox in the heart remains unclear. In this study, we used wild-type (WT) and p47phox knockout (KO) mice (C57BL/6, male, 7-month-old, n = 9) to investigate p47phox-dependent oxidant-signalling in AngII infusion (0.8 mg/kg/day, 14 days)-induced cardiac hypertrophy and cardiomyocyte apoptosis. AngII infusion resulted in remarkable high blood pressure and cardiac hypertrophy in WT mice. However, these AngII-induced pathological changes were significantly reduced in p47phox KO mice. In WT hearts, AngII infusion increased significantly the levels of superoxide production, the expressions of Nox subunits, the expression of PKCα and C-Src and the activation of ASK1 (apoptosis signal-regulating kinase 1), MKK3/6, ERK1/2, p38 MAPK and JNK signalling pathways together with an elevated expression of apoptotic markers, i.e., γH2AX and p53 in the cardiomyocytes. However, in the absence of p47phox, although PKCα expression was increased in the hearts after AngII infusion, there was no significant activation of ASK1, MKK3/6 and MAPKs signalling pathways and no increase in apoptosis biomarker expression in cardiomyocytes. In conclusion, p47phox-dependent redox-signalling through ASK1, MKK3/6 and MAPKs plays a crucial role in AngII-induced cardiac hypertrophy and cardiomyocyte apoptosis.

285 - Protein phosphatase 2A (PP2A) inhibition in NAFLD leads to an underlying intestinal fibrotic pathology that is NOX2 dependent

Increased oxidative stress exacerbates the condition of Non-Alcoholic Fatty Liver Disease (NAFLD) which is a global pandemic, leading to its progression to the state of chronic inflammation and fibrosis (NASH). Protein phosphatase 2A (PP2A) is an enzyme that plays an important role in fibrosis, but its role in controlling the production of reactive oxygen species is unclear. Our studies show that the environmental toxin microcystin, an inhibitor of PP2A, specifically inhibits the enzyme and generates higher oxidative stress. The above process activated TGF-β fibrotic signaling pathway in the intestines that was mediated by NOX2. Results using mouse NAFLD models and transformed rat intestinal epithelial cells showed that inhibiting PP2A by administration of microcystin led to increased NOX2 activation, increased SMAD2/3-SMAD4 co-localization and increased alpha smooth muscle actin immuno-reactivity. This process was greatly attenuated in p47 phox knockout mice signifying an important role of NOX2. Mechanistically, rat intestinal epithelial cells were treated with apocynin, DMPO and phenylboronic acid which showed significantly decreased NOX2 activation, SMAD co-localization and α-SMA immuno-reactivity. A significant decrease of NOX2 and TGF-β signaling in NOX4 inhibitor treated samples indicated that there might be a possibility of dependency of NOX isoforms on each other. This study gives us hope to explore novel therapeutic strategies by increasing PP2A in situ by administration of PP2A agonists for the treatment of NASH.

Clozapine attenuates mitochondrial burdens and abnormal behaviors elicited by phencyclidine in mice via inhibition of p47 phox; Possible involvements of phosphoinositide 3-kinase/Akt signaling.

Oxidative stress and mitochondrial dysfunction have been implicated in the pathophysiology of schizophrenia. We investigated whether antipsychotic clozapine modulates nicotinamide adenine dinucleotide phosphate oxidase and mitochondrial burdens induced by phencyclidine in mice. We examined the effect of clozapine on nicotinamide adenine dinucleotide phosphate oxidase activation, mitochondrial burdens (i.e. oxidative stress and mitochondrial dysfunction), and activities of enzymatic antioxidant in the prefrontal cortex, and subsequent abnormal behaviors induced by repeated treatment with phencyclidine. p47 phox Knockout mice and LY294002, a phosphoinositide 3-kinase inhibitor, were employed to elucidate the pharmacological mechanism of clozapine. Phencyclidine treatment resulted in an early increase nicotinamide adenine dinucleotide phosphate oxidase activity, membrane translocation of p47 phox, interaction between p-Akt and p47 phox, and mitochondrial burdens in wild-type mice. Although these increases returned to near control level four days post-phencyclidine, mitochondrial superoxide dismutase and glutathione peroxidase activities were decreased at that time. Clozapine, LY294002, or p47 phox knockout significantly ameliorated social withdrawal and recognition memory deficits produced by phencyclidine. Importantly, LY294002 did not significantly alter the effects of clozapine against abnormal behaviors and the interaction between p-Akt and p47 phox induced by phencyclidine. Furthermore, neither LY294002 nor clozapine exhibited any additive effects to the protection afforded by p47 phox knockout against phencyclidine insult. Our results suggest that p47 phox gene mediates phencyclidine-induced mitochondrial burdens and abnormal behaviors, and that the interactive modulation between p47 phox and phosphoinositide 3-kinase/Akt is important for the understanding on the pharmacological mechanism of clozapine.

High circulatory leptin mediated NOX-2-peroxynitrite-miR21 axis activate mesangial cells and promotes renal inflammatory pathology in nonalcoholic fatty liver disease

High circulatory insulin and leptin followed by underlying inflammation are often ascribed to the ectopic manifestations in non-alcoholic fatty liver disease (NAFLD) but the exact molecular pathways remain unclear. We have shown previously that CYP2E1-mediated oxidative stress and circulating leptin in NAFLD is associated with renal disease severity. Extending the studies, we hypothesized that high circulatory leptin in NAFLD causes renal mesangial cell activation and tubular inflammation via a NOX2 dependent pathway that upregulates proinflammatory miR21. High-fat diet (60% kcal) was used to induce fatty liver phenotype with parallel insulin and leptin resistance. The kidneys were probed for mesangial cell activation and tubular inflammation that showed accelerated NASH phenotype and oxidative stress in the liver. Results showed that NAFLD kidneys had significant increases in α-SMA, a marker of mesangial cell activation, miR21 levels, tyrosine nitration and renal inflammation while they were significantly decreased in leptin and p47 phox knockout mice. Micro RNA21 knockout mice showed decreased tubular immunotoxicity and proinflammatory mediator release. Mechanistically, use of NOX2 siRNA or apocynin,phenyl boronic acid (FBA), DMPO or miR21 antagomir inhibited leptin primed-miR21-mediated mesangial cell activation in vitro suggesting a direct role of leptin-mediated NOX-2 in miR21-mediated mesangial cell activation. Finally, JAK-STAT inhibitor completely abrogated the mesangial cell activation in leptin-primed cells suggesting that leptin signaling in the mesangial cells depended on the JAK-STAT pathway. Taken together the study reports a novel mechanistic pathway of leptin-mediated renal inflammation that is dependent on NOX-2-miR21 axis in ectopic manifestations underlying NAFLD-induced co-morbidities.

Glucose and lipid metabolism alterations in liver and adipose tissue pre-dispose p47phox knockout mice to systemic insulin resistance

Oxidative stress due to enhanced production or reduced scavenging of reactive oxygen species (ROS) has been associated with diet (dyslipidemia) induced obesity and insulin resistance (IR). The present study was undertaken to assess the role of p47phox in IR using wild type (WT) and p47phox−/− mice, fed with different diets (HFD, LFD or Chow). Augmented body weight, glucose intolerance and reduced insulin sensitivity were observed in p47phox−/− mice fed with 45% HFD and 10% LFD. Further, body fat and circulating lipids were increased significantly with 5 weeks LFD feeding in p47phox−/− mice, while parameters of energy homeostasis were reduced as compared with WT mice. LFD fed knockout (KO) mice showed an enhanced hepatic glycogenolysis, and reduced insulin signalling in liver and adipose tissue, while skeletal muscle tissue remained unaffected. A significant increase in hepatic lipids, adiposity, as well as expression of genes regulating lipid synthesis, breakdown and efflux were observed in LFD fed p47phox−/− mice after 5 weeks. On the other hand, mice lacking p47phox demonstrated altered glucose tolerance and tissue insulin sensitivity after 5 weeks chow feeding, while changes in body weight, respiratory exchange ratio (RER) and heat production are non-significant. Our data demonstrate that lack of p47phox is sufficient to induce IR through altered glucose and lipid utilization by the liver and adipose tissue.

218 Knockout p47phox reduces angiotensin ii-induced cardiac oxidative stress and hypertrophy

Oxidative stress due to the activation of a Nox2-containing NADPH oxidase is involved in Angiotensin II (AngII)-induced cardiovascular dysfunction. p47phox is a key regulatory subunit of Nox2. However, the role...

Adipokine Leptin mediated NOX-2 promotes kidney inflammation in nonalcoholic fatty liver disease via miR21-dependent mesangial cell immune activation

Abstract High insulin and leptin resistance followed by underlying inflammation is often ascribed to the ectopic manifestations in nonalcoholic fatty liver disease (NAFLD) but the exact molecular pathways remain unclear. We have shown previously that CYP2E1-mediated oxidative stress and circulating leptin in NAFLD is associated with renal disease severity. Extending the studies, we hypothesized that high circulatory leptin in NAFLD causes renal mesangial cell activation and tubular inflammation via a NOX2 dependent pathway that upregulates proinflammatorymiR21. High fat diet (60%kcal) was used to induce fatty liver phenotype with parallel insulin and leptin resistance in WT, Leptin knockout (ob/ob) and p47 phox knockout mice. Kidneys were collected after pathology showed NASH-like phenotype and accelerated oxidative stress in the liver. The kidneys were probed for mesangial cell activation and tubular inflammation. Results showed that NAFLD kidneys had significant increases in α-SMA, a marker of mesangial cell activation, miR21 levels, tyrosine nitration and renal inflammation while they were significantly decreased in leptin and p47 phox knockout mice. Micro RNA21 knockout mice showed decreased tubular immunotoxicity and proinflammatory mediator release. Mechanistically, use of apocynin or phenyl boronic acid (FBA) or DMPO or miR21 antagomir inhibited leptin primed-miR21-mediated mesangial cell activation in vitro suggesting a direct role of leptin-mediated NOX-2 in miR21-mediated mesangial cell activation. Finally JAK-STAT inhibitor completely abrogated the mesangial cell activation in leptin-primed cells suggesting that leptin signaling in the mesangial cells depended on the JAK-STAT pathway.

NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

NADPH oxidase-generated reactive oxygen species (ROS) from immune cells are well known to be important for pathogen killing in response to TLR ligands. Here, we investigated a new aspect of NADPH oxidase in the TLR2/6-induced release of the immunologically relevant GM-CSF by endothelial cells. Stimulation of human endothelial cells with TLR2/6 agonist, MALP-2 (macrophage-activating lipopeptide of 2 kDa), induced NADPH oxidase activation and ROS formation. Inhibition by ROS scavengers and NADPH oxidase inhibitors blocked MALP-2-induced GM-CSF release. NADPH oxidase activators or ROS donors alone did not result in GM-CSF secretion; however, additional superoxide supply augmented MALP-2-induced GM-CSF secretion and restored GM-CSF levels after NADPH oxidase inhibition. MALP-2-dependent NF-ĸB activation was suppressed by NADPH oxidase inhibition, and inhibition of NF-κB completely blunted MALP-2-induced GM-CSF release. Vascular explants from mice that were deficient for the NADPH oxidase subunit p47 phox showed diminished intimal superoxide production and GM-CSF release after ex vivo stimulation with MALP-2. Moreover, an increase in circulating progenitor cells after MALP-2 injection was completely abolished in p47phox-knockout mice. Finally, MALP-2 stimulation increased mRNA expression of the major subunit NADPH oxidase, (Nox)2, in endothelial cells, and Nox2 inhibition prevented MALP-2-induced GM-CSF release. Our findings identify a Nox2-containing NADPH oxidase as a crucial regulator of the immunologic important growth factor GM-CSF after TLR2/6 stimulation in endothelial cells.-Schuett, J., Schuett, H., Oberoi, R., Koch, A.-K., Pretzer, S., Luchtefeld, M., Schieffer, B., Grote, K. NADPH oxidase NOX2 mediates TLR2/6-dependent release of GM-CSF from endothelial cells.

90 - High Circulatory Leptin Mediated NOX-2 Promotes Kidney Inflammation in Nonalcoholic Fatty Liver Disease Via MiR21-Dependent Mesangial Cell Activation

High insulin and leptin resistance followed by underlying inflammation is often ascribed to the ectopic manifestations in non-alcoholic fatty liver disease (NAFLD) but the exact molecular pathways remain unclear. We have shown previously that CYP2E1-mediated oxidative stress and circulating leptin in NAFLD is associated with renal disease severity. Extending the studies, we hypothesized that high circulatory leptin in NAFLD causes renal mesangial cell activation and tubular inflammation via a NOX2 dependent pathway that upregulates proinflammatory miR21. High fat diet (60%kcal) was used to induce fatty liver phenotype with parallel insulin and leptin resistance in WT, Leptin knockout (ob/ob) and p47 phox knockout mice. Kidneys were collected after pathology showed NASH-like phenotype and accelerated oxidative stress in the liver. The kidneys were probed for mesangial cell activation and tubular inflammation. Results showed that NAFLD kidneys had significant increases in α-SMA, a marker of mesangial cell activation, miR21 levels, tyrosine nitration and renal inflammation while they were significantly decreased in leptin and p47 phox knockout mice. Micro RNA21 knockout mice showed decreased tubular immunotoxicity and proinflammatory mediator release. Mechanistically, use of apocynin or phenyl boronic acid (FBA) or DMPO or miR21 antagomir inhibited leptin primed-miR21-mediated mesangial cell activation in vitro suggesting a direct role of leptin-mediated NOX-2 in miR21-mediated mesangial cell activation. Finally JAK-STAT inhibitor completely abrogated the mesangial cell activation in leptin-primed cells suggesting that leptin signaling in the mesangial cells depended on the JAK-STAT pathway. Taken together the study reports a novel mechanistic pathway of leptin-mediated renal inflammation in NAFLD. Grant support R00ES019875 to SC

Mechanosensitive Regulation of Cortactin by p47 phox

p47phox is 1 of the 5 subunits that makes up the multisubunit complex of the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2), a major source of cellular reactive oxygen species (ROS).1 The prototype Nox2 comprises 2 transmembrane subunits (gp91phox [Nox2] and p22phox) and 3 cytosolic subunits (p47phox, p67phox, and p40phox).2,3 Nox2 is characteristically found in phagocytic cells, where it has a critical antimicrobial function through the production of superoxide anion.2 Nox2, as well as the other Nox homologues, including Nox1, Nox4, and Nox5, is expressed in many cell types, including those of the cardiovascular system.3 Although the function of phagocytic Nox2 is clearly defined, the exact pathophysiological significance of cardiovascular Nox2 is still unclear. Article, see p 1542 p47phox is a major regulator of Nox2.1 In the resting state, it localizes in the cytoplasm where its activity is inhibited through an autoinhibitory phosphorylation. However, on cell stimulation, p47phox interacts with cytosolic subunits p67phox and p40phox and the complex translocates to the cell membrane to assemble the active oxidase, which generates superoxide anion.2 This process is triggered by phosphorylation of p47phox, which is the key for activation of Nox and consequent ROS production. Upregulation of Nox and associated increased ROS generation (oxidative stress) have been implicated in many cardiovascular pathologies, processes that are p47phox dependent because deletion of this subunit reduces oxidative stress and ameliorates pathological cardiac and vascular inflammation and remodeling.4–7 On the basis of this paradigm, in this issue of Circulation Research , Patel et al8 questioned the putative protective effect of p47phox deletion/downregulation in a mouse model of transverse aortic constriction–induced pressure overload and heart failure. Intriguingly and …

NADPH Oxidase Mediates Depressive Behavior Induced by Chronic Stress in Mice

Stress is a potent risk factor for depression, yet the underlying mechanism is not clearly understood. In the present study, we explored the mechanism of development and maintenance of depression in a stress-induced animal model. Mice restrained for 2 h daily for 14 d showed distinct depressive behavior, and the altered behavior persisted for >3 months in the absence of intervention. Acute restraint induced a surge of oxidative stress in the brain, and stress-induced oxidative stress progressively increased with repetition of stress. In vitro, the stress hormone glucocorticoid generated superoxide via upregulation of NADPH oxidase. Consistently, repeated restraints increased the expression of the key subunits of NADPH oxidase, p47phox and p67phox, in the brain. Moreover, stressed brains markedly upregulated the expression of p47phox to weak restress evoked in the poststress period, and this molecular response was reminiscent of amplified ROS surge to restress. Pharmacological inhibition of NADPH oxidase by the NADPH oxidase inhibitor apocynin during the stress or poststress period completely blocked depressive behavior. Consistently, heterozygous p47phox knock-out mice (p47phox(+/-)) or molecular inhibition of p47phox with Lenti shRNA-p47phox in the hippocampus suppressed depressive behavior. These results suggest that repeated stress promotes depressive behavior through the upregulation of NADPH oxidase and the resultant metabolic oxidative stress, and that the inhibition of NADPH oxidase provides beneficial antidepression effects.

Abstract 8711: Double Serine Phosphorylation of P47 Phox At 303-304 is Critical for Acute Tnfα-Induced Ros Production but Not for Mapk Activation in Endothelial Cells

p47 phox is a major regulatory subunit of Nox2 enzyme, which by generating superoxide plays important role in TNFα-induced endothelial dysfunction. The p47 phox has several serines clustered within the carboxyl terminus , and their phosphorylation has been reported to be critical in Nox2 activation and endothelial reactive oxygen species (ROS) production in response to stimulation. To understand the mechanisms of p47 phox phosphorylation in mediating acute TNFα redox-signaling, we replaced serines (303-4, 310, 315, 320, 328, 345, 348, 359, 370 and 379) to alanines and transfected mouse microvascular endothelial cells (SVEC4-10) with these constructs. Compared to vector controls, S303-4A (double mutation), S310A, S315A, S328A, S345A, S370A and S379A reduced significantly the TNFα (100 U/mL for 30 min)-induced ROS production (P<0.05), whereas only S328A, S345A, S370A and S379A (but not S303-4A, S310A and S315A) inhibited TNFα-induced ERK1/2 phosphorylation. The different effects of S303-304A on TNFα-induced ROS production and TNFα signaling were further investigated using coronary microvascular endothelial cells isolated from p47 phox knockout mice. Compared to the cells transfected with a wild-type p47 phox cDNA, S303-4A mutation significantly inhibited (~51+2%) TNFα-induced ROS production, but showed no significant effect on TNFα-induced MAPK activation, p47 phox association with TRAF4, p47 phox binding to p22 phox and p47 phox membrane translocation. Knockdown of TRAF4 with siRNA abolished TNFα-induced ERK1/2 phosphorylation in S303-4A transfected p47 phox knockout cells. In conclusion, phosphorylation of p47 phox at different serines plays distinct roles in endothelial cell response to acute TNFα stimulation. Double serine phosphorylation (S303 and S304) of p47 phox is crucial in acute TNFα-induced ROS production, but not involved in TNFα-signaling through TRAF4 and MAPK in endothelial cells.

Genetic ablation of phagocytic NADPH oxidase in mice limits TNFα-induced inflammation in the lungs but not other tissues

In vitro and limited in vivo evidence suggests that reactive oxygen species derived from NADPH oxidases (NOX-ROS) play an important role in inflammatory responses by enhancing the activity of redox-sensitive cell signaling pathways and transcription factors. Here, we investigated the role of NOX-ROS in TNFα-induced acute inflammatory responses in vivo, using mice deficient in the gp91phox (NOX2) or p47phox subunits of NADPH oxidase. Age- and body weight-matched C57BL/6J wild-type (WT) and gp91phox or p47phox knockout mice were injected intraperitoneally with 50μg TNFα/kg bw or saline vehicle control and sacrificed at various time points up to 24h. Compared to WT mice, gp91phox −/− mice exhibited significantly diminished (P<0.05) TNFα-induced acute inflammatory responses in the lungs but not other tissues, including heart, liver, and kidney, as evidenced by decreased activation of the redox-sensitive transcription factor NF-κB, and decreased gene expression of interleukin (IL)-1β, IL-6, TNFα, E-selectin, and other cellular adhesion molecules. Similar results were observed in p47phox −/− mice. Interestingly, decreased lung inflammation in knockout mice was accompanied by increased leukocyte infiltration into the lungs compared to other tissues. Our data suggest that phagocytic NOX-ROS signaling plays a critical role in promoting TNFα-induced, NF-κB-dependent acute inflammatory responses and tissue injury specifically in the lungs, which is effected by preferential leukocyte infiltration.

Francisella Acid Phosphatases Inactivate the NADPH Oxidase in Human Phagocytes

Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [DeltaABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the DeltaABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida DeltaABCH strain stimulated a significant level of reactive oxygen species. The DeltaABCH mutant, but not the wild-type strain, strongly colocalized with p47(phox) and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47(phox) knockout mice. Finally, purified AcpA strongly dephosphorylated p47(phox) and p40(phox), but not p67(phox), in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.

Knockout of p47 phox Uncovers a Critical Role of p40 phox in Reactive Oxygen Species Production in Microvascular Endothelial Cells

p40(phox) is an important regulatory subunit of NADPH oxidase, but its role in endothelial reactive oxygen species (ROS) production remains unknown. Using coronary microvascular endothelial cells isolated from wild-type and p47(phox) knockout mice, we found that knockout of p47(phox) increased the level of p40(phox) expression, whereas depletion of p40(phox) in wild-type cells increased p47(phox) expression. In both cases, the basal ROS production (without agonist stimulation) was well preserved. Double knockout of p40(phox) and p47(phox) dramatically reduced (approximately 65%) ROS production and cells started to die. The transcriptional regulation of p40(phox) and p47(phox) expressions involves HBP1. p40(phox) was prephosphorylated in resting cells. PMA stimulation induced p40(phox) swift dephosphorylation (within 1 minute) in parallel with the start of p47(phox) phosphorylation. p40(phox) was then rephosphorylated, and this was accompanied with an increase in ROS production. Depletion of p40(phox) resulted in approximately 67% loss in agonist-induced ROS production despite the presence of p47(phox). These were further supported by experiments on mouse aortas stimulated with angiotensin II. p40(phox) is prephosphorylated in resting endothelial cells and can compensate p47(phox) in keeping basal ROS production. Dephosphorylation of p40(phox) is a prerequisite for agonist-induced p47(phox) phosphorylation, and p40(phox) through its dynamic dephosphorylation and rephosphorylation is involved in the regulation of agonist-induced ROS production.

Roles of Endothelial Oxidases in Endothelium-derived Hyperpolarizing Factor Responses in Mice

The endothelium synthesizes and releases several vasodilator substances, including prostacyclin, nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an EDHF in animals and humans and that superoxide anions derived from endothelial nitric oxide synthases (NOSs) system are an important precursor for EDHF/H2O2 in mice. There are several intracellular sources of superoxide anions other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, and mitochondrial electron transport chain. In this study, we examined the possible role of endothelial oxidases other than NOSs in the EDHF-mediated responses. In angiotensin II-infused mice, both EDHF-mediated relaxations and hyperpolarizations to acetylcholine were significantly reduced, nitric oxide-mediated relaxations were rather enhanced, and vascular smooth muscle responses were preserved. Antihypertensive treatment normalized blood pressure but failed to improve EDHF-mediated responses in those mice. Acute inhibition of endothelial oxidases other than NOSs, including NAD(P)H oxidase, xanthine oxidase, lipoxygenase, or mitochondrial electron transport chain, had no inhibitory effects on EDHF-mediated responses. Furthermore, in p47phox-knockout mice, EDHF-mediated responses were unaltered. These results suggest that endothelial oxidases other than NOSs are not involved in EDHF/H2O2 responses in mice, suggesting a specific link between endothelial NOSs system and EDHF responses under physiological conditions.

Neurofurans, Novel Indices of Oxidant Stress Derived from Docosahexaenoic Acid

Isoeicosanoids are free radical-catalyzed isomers of the enzymatic products of arachidonic acid. They are formed in situ in cell membranes, are cleaved, circulate, and are excreted in urine. Isomers of prostaglandin F(2alpha), the F(2)-isoprostanes, have emerged as sensitive indices of lipid peroxidation in vivo. Analogous compounds formed from docosahexaenoic acid (DHA) are termed neuroprostanes and are more abundant than isoprostanes (iPs) in brain. Isofurans are another class of isoeicosanoids characterized by a substituted tetrahydrofuran ring. They are preferentially formed, relative to iPs, under conditions of elevated oxygen tension. Here, we report the discovery of neurofurans (nFs), the analogous family of compounds formed from DHA. Formation of nFs is characterized by mass spectrometry and confirmed by oxidation of DHA in vitro and following CCl(4) administration in liver in vivo. It is demonstrated that the levels of nFs are elevated in the brain cortex of a mouse model of Alzheimer disease and are depressed in mouse brain cortex by deletion of p47(phox), an essential component of the phagocyte NADPH oxidase. Measurement of the nFs may ultimately prove useful in diagnosis, timing, and selection of dose in the treatment and chemoprevention of neurodegenerative disease.