Gp91 Phox Research Articles

Prodigiosin inhibits gp91 phox and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia–ischemia

This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen–glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100 μg/kg, i.v.) at 1 h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91 phox), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood–brain barrier (BBB) by activation of nuclear factor-kappa B (NF-κB). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91 phox and iNOS via activation of the NF-κB pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91 phox and iNOS expression possibly by impairing NF-κB activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice.

NF-κB in the paraventricular nucleus modulates neurotransmitters and contributes to sympathoexcitation in heart failure.

Findings from our laboratory indicate that proinflammatory cytokines and their transcription factor, nuclear factor-kappaB (NF-κB), are increased in the hypothalamic paraventricular nucleus (PVN) and contribute towards the progression of heart failure. In this study, we determined whether NF-κB activation within the PVN contributes to sympathoexcitation via interaction with neurotransmitters in the PVN during the pathogenesis of heart failure. Heart failure was induced in rats by left anterior descending coronary artery ligation. Sham-operated control (SHAM) or heart failure rats were treated for 4weeks through bilateral PVN infusion with SN50, SN50M or vehicle via osmotic minipump. Rats with heart failure treated with PVN vehicle or SN50M (inactive peptide for SN50) had increased levels of glutamate, norepinephrine (NE), tyrosine hydroxylase (TH), superoxide, gp91(phox) (a subunit of NAD(P)H oxidase), phosphorylated IKKβ and NF-κB p65 activity, and lower levels of gamma-aminobutyric acid (GABA) and the 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN compared with those of SHAM rats. Plasma levels of cytokines, norepinephrine, epinephrine and angiotensin II, and renal sympathetic nerve activity (RSNA) were increased in heart failure rats. Bilateral PVN infusion of SN50 prevented the decreases in PVN GABA and GAD67, and the increases in RSNA and PVN glutamate, norepinephrine, TH, superoxide, gp91(phox), phosphorylated IKKβ and NF-κB p65 activity observed in vehicle or SN50M-treated heart failure rats. A same dose of SN50 given intraperitoneally did not affect neurotransmitters concentration in the PVN and was similar to vehicle-treated heart failure rats. These findings suggest that NF-κB activation in the PVN modulates neurotransmitters and contributes to sympathoexcitation in rats with ischemia-induced heart failure.

Gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis

Despite the fundamental function of neutrophils (PMNs) in innate immunity, their role in Escherichia coli K1 (EC-K1) induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by down regulating rac1, rac2 and gp91phox transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis.

Ultraviolet B radiation to the eye induces pigmentation in the epidermis via the activation of the subunit gp91 phox of reduced nicotinamide adenine dinucleotide phosphate oxidase

Irradiation by ultraviolet (UV)B is known to increase the number of dopamine (Dopa)-positive melanocytes in the skin. In this study, a 2.5-kJ/m(2) dose of UVB radiation was delivered by a sunlamp to the ear or the eye of wild-type C57BL/6j mice and of gp91 phox(-/-) C57BL/6j mice that had a knockout mutation of the gp91 phox subunit of reduced nicotinamide adenine dinucleotide phosphate oxidase (NADPH). The degree of change in the Dopa-positive melanocyte expression in was reduced in gp91 phox(-/-) mice given UVB irradiation to the eye, but not in those given irradiation to the ear. The plasma level of α-melanocyte-stimulating hormone (α-MSH) in the blood increased in the C57BL/6j mice after irradiation to either the eye or the ear, but it did not increase in the gp91 phox(-/-) mice given UVB irradiation to the eye. Both gp91 phox and α-MSH in the central nervous system seem to contribute to pigmentation after UVB irradiation of the eye in mice.

Long-term methylglyoxal treatment impairs smooth muscle contractility in organ-cultured rat mesenteric artery

Methylglyoxal (MGO), a metabolite of glucose accumulates in vascular tissues of hypertensive rats. We recently showed that short-term (30 min) treatment with MGO inhibits noradrenaline (NA)-induced smooth muscle contraction in rat aorta and mesenteric artery. In the present study, long-term effect of MGO was examined using organ culture method. The contractility, morphology, and protein expression of rat mesenteric artery after organ culture with MGO for 3 days were examined. MGO (4 and 42 μM) inhibited NA (0.1 nM to 3 μM) or KCl (72.7 mM)-induced contraction. The inhibitory effect was higher in endothelium-denuded than endothelium-intact artery. An anti-oxidant drug, N-acetyl- l-cysteine (NAC; 1 mM) or an inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX), gp91ds-tat (0.1 μM) prevented the inhibitory effect of MGO. MGO increased superoxide production as detected by lucigenin assay. In the medial layer of the arteries cultured with MGO, apoptotic morphological change was observed, and NAC or gp91ds-tat prevented it. MGO significantly increased expression of a homolog of gp91 phox, NOX1 but not gp91 phox as determined by Western blotting. An NF-κB inhibitor, pyrrolidine dithiocarbamate prevented the MGO-induced NOX1 expression. MGO had no effect on protein expression of p22 phox, p67 phox, p47 phox, as well as superoxide dismutase (SOD)-1, SOD-2 and SOD-3. Present results indicate that long-term MGO treatment has an inhibitory effect on contractility of isolated blood vessel, which is likely mediated via increased NOX1-derived superoxide production and subsequent apoptosis.

Role of Putative Second Transmembrane Region of Nox2 Protein in the Structural Stability and Electron Transfer of the Phagocytic NADPH Oxidase

Flavocytochrome b(558) (cytb) of phagocytes is a heterodimeric integral membrane protein composed of two subunits, p22(phox) and gp91(phox). The latter subunit, also known as Nox2, has a cytosolic C-terminal "dehydrogenase domain" containing FAD/NADPH-binding sites. The N-terminal half of Nox2 contains six predicted transmembrane α-helices coordinating two hemes. We studied the role of the second transmembrane α-helix, which contains a "hot spot" for mutations found in rare X(+) and X(-) chronic granulomatous disease. By site-directed mutagenesis and transfection in X-CGD PLB-985 cells, we examined the functional and structural impact of seven missense mutations affecting five residues. P56L and C59F mutations drastically influence the level of Nox2 expression indicating that these residues are important for the structural stability of Nox2. A53D, R54G, R54M, and R54S mutations do not affect spectral properties of oxidized/reduced cytb, oxidase complex assembly, FAD binding, nor iodonitrotetrazolium (INT) reductase (diaphorase) activity but inhibit superoxide production. This suggests that Ala-53 and Arg-54 are essential in control of electron transfer from FAD. Surprisingly, the A57E mutation partially inhibits FAD binding, diaphorase activity, and oxidase assembly and affects the affinity of immunopurified A57E cytochrome b(558) for p67(phox). By competition experiments, we demonstrated that the second transmembrane helix impacts on the function of the first intracytosolic B-loop in the control of diaphorase activity of Nox2. Finally, by comparing INT reductase activity of immunopurified mutated and wild type cytb under aerobiosis versus anaerobiosis, we showed that INT reduction reflects the electron transfer from NADPH to FAD only in the absence of superoxide production.

Impact of N-Acetylcysteine on Neonatal Cardiomyocyte Ischemia-Reperfusion Injury

Reactive oxygen species (ROS) are hypothesized to play a key role in myocardial ischemia-reperfusion (IR) injury after cardiopulmonary bypass in children. Clinical studies in adults and several animal models suggest that myocardial IR injury involves cardiomyocyte apoptosis and necrosis. This study investigated a potential relationship between IR-induced ROS production and neonatal cardiomyocyte apoptosis using both in vitro and ex vivo techniques. For in vitro experiments, embryonic rat cardiomyocytes (H9c2 cells) exposed to hypoxia-reoxygenation (HR) showed a time-dependent increase in gp91 phox (a marker for ROS production by NADPH oxidases), caspase-3 (a key mediator of apoptosis) expression, and a decrease in the glutathione redox ratio. N-acetylcysteine (NAC; 0.25-2 mM), a potent antioxidant, decreased gp91 phox and caspase-3 expression, inhibited apoptosis and restored the glutathione redox ratio. For ex vivo study, IR injury significantly reduced left ventricular (LV) function and increased the expression of gp91 phox and caspase-3 in Langendorff-perfused neonatal (7-14 d) rabbit hearts. NAC (0.4 mM) treatment completely attenuated LV dysfunction after IR. In summary, neonatal myocardial IR injury is associated with an increase in cardiomyocyte oxidative stress and apoptosis. NAC attenuates apoptosis in an in vitro embryonic rat cardiomyocyte model of HR, and myocardial dysfunction in an ex vivo neonatal rabbit model of myocardial IR injury.

Depot-Specific Overexpression of Proinflammatory, Redox, Endothelial Cell, and Angiogenic Genes in Epicardial Fat Adjacent to Severe Stable Coronary Atherosclerosis

Pro- and antiinflammatory genes are expressed in epicardial adipose tissue (EAT). Our objectives were to characterize genes in EAT that may contribute specifically to coronary atherogenesis and to measure circulating adipokines matched to their messenger RNAs (mRNAs) in EAT. We hypothesized that severe coronary atherosclerosis (CAD) would preferentially affect gene expression in EAT as compared to substernal fat or subcutaneous thoracic adipose tissue (SAT), as well as circulating levels of adipokines. Fat mRNA was quantified using reverse transcription polymerase chain reaction (RT-PCR), and circulating adipokines were measured by enzyme-linked immunosorbent assays (ELISAs) in patients with severe stable CAD and controls without severe CAD undergoing open heart surgery. A total of 39 of 70 mRNAs in EAT were significantly increased in CAD. Only 4 and 3 of these mRNAs were increased in substernal fat and SAT, respectively. Of the mRNAs increased in EAT, 17 were either inflammatory adipokines or proteins known to be involved in inflammatory processes, 7 were involved in oxidative stress and or oxygen species regulation, whereas 15 were proteins involved in metabolism and regulation of gene transcription or proteins unique to fat cells. The largest increases, over three-fold, were seen in GPX3, gp91 phox, p47phox, heme oxygenase, and interleukin-8 (IL-8). Tpl2 mRNA was uniquely elevated in all three fat depots from CAD patients, and its expression in SAT, but not in EAT or substernal fat, was directly correlated with homeostasis model assessment of insulin resistance (HOMA-IR) values. Compared to controls, there were no associations between circulating levels of IL-8, lipocalin-2, nerve growth factor (NGF), RANTES, CD-163, GPX-3, monocyte chemotactic protein-1 (MCP-1)/CCL2, leptin, soluble vascular endothelial growth factor receptor-1 (sFLT1), fatty acid binding protein-4 (FABP-4), and plasminogen activator inhibitor-1 (PAI-1) and increases in their gene expression in EAT adjacent to CAD. Expression of proinflammatory, redox, endothelial cell, and angiogenic genes in EAT is depot specific and supports the hypothesis that pathophysiologically EAT contributes locally to CAD. CAD links with these fat depots might involve Tpl2 as a primary response indicator.

Two X-Linked Chronic Granulomatous Disease Patients with Unusual NADPH Oxidase Properties

Chronic granulomatous disease (CGD) is an immune deficiency syndrome caused by defects in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme that generates reactive oxygen species (ROS) in phagocytizing leukocytes. This study evaluates the NADPH oxidase capacity in two X-linked CGD patients with mutations in gp91(phox) that alter the regions in this membrane-bound NADPH oxidase component involved in docking of the cytosolic component p47(phox). Hydrogen peroxide and superoxide generation, bactericidal activity, and NADPH oxidase protein expression by the patients' neutrophils were measured, and genetic analysis was performed. We report two patients, each with a novel missense mutation in CYBB, the gene that encodes gp91(phox). Surprisingly, neutrophils from these patients showed total absence of superoxide production, although they retained 13-30% of the hydrogen peroxide production capability. We speculate that this is due to direct electron transfer from flavin adenine dinucleotide (FAD) in gp91(phox) to oxygen, leading to inefficient hydrogen peroxide formation instead of efficient superoxide production. X-linked CGD patients with mutations that alter the gp91(phox) protein in regions involved in docking of the cytosolic NADPH oxidase component p47(phox) may have higher than expected hydrogen peroxide generation capability.

Rosuvastatin attenuates the elevation in blood pressure induced by overexpression of human C-reactive protein

C-reactive protein (CRP) has been shown to function as an inflammatory factor to induce endothelial dysfunction and hypertension in rats. The anti-inflammatory effects of statins suggest that they may attenuate CRP-induced endothelial dysfunction and hypertension in Sprague-Dawley rats. Male Sprague-Dawley rats were injected with an adeno-associated virus (AAV) to induce overexpression of human CRP (AAV-hCRP) or green fluorescent protein (GFP) control (AAV-GFP). At 2 months after injection, rats were administered rosuvastatin by daily oral gavage (10 mg kg(-1)) for 2 additional months. Rosuvastatin administration attenuated the increased blood pressure and loss of vascular endothelial nitric oxide synthase expression in AAV-hCRP-treated rats, and N-nitro-L-arginine methyl ester blocked its hypotensive effect. Rosuvastatin also activated phosphoinositide 3-kinases/Akt, and inhibited Rho kinase activity in aorta. Rosuvastatin reduced the production of reactive oxygen species through downregulation of nicotinamide adenine dinucleotide phosphate oxidase subunits, p22 phox and gp91 phox, and upregulation of superoxide dismutase 1 expression. Rosuvastatin attenuated the increase in blood pressure in AAV-hCRP-treated rats through endothelial protection and antioxidant effects. Our data reveals a novel mechanism through which statins may lower blood pressure.

Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

A Selective Phosphodiesterase-4 Inhibitor Reduces Leukocyte Infiltration, Oxidative Processes, and Tissue Damage after Spinal Cord Injury

We tested the hypothesis that a selective phosphodiesterase type 4 inhibitor (PDE4-I; IC486051) would attenuate early inflammatory and oxidative processes following spinal cord injury (SCI) when delivered during the first 3 days after injury. Rats receiving a moderately severe thoracic-clip-compression SCI were treated with the PDE4-I (0.5, 1.0, and 3.0 mg/kg IV) in bolus doses from 2-60 h post-injury. Doses at 0.5 mg/kg and 1.0 mg/kg significantly decreased myeloperoxidase (MPO) enzymatic activity (neutrophils), expression of a neutrophil-associated protein and of ED-1 (macrophages), and estimates of lipid peroxidation in cord lesion homogenates at 24 h and 72 h post-injury by 25-40%. The 3.0 mg/kg dose had small or no effects on these measures. The PDE4-I treatment (0.5 or 1.0 mg/kg) reduced expression of the oxidative enzymes gp91(phox), inducible nitric oxide synthase, and cyclooxygenase-2, and diminished free radical generation by up to 40%. Treatment with 0.5 mg/kg PDE4-I improved motor function (as assessed by the Basso-Beattie-Bresnahan scale) significantly from 4-8 weeks after SCI (average difference 1.3 points). Mechanical allodynia elicited from the hindpaw decreased by up to 25%. The PDE4-I treatment also increased white matter volume near the lesion at 8 weeks after SCI. In conclusion, the PDE4-I reduced key markers of oxidative stress and leukocyte infiltration, producing cellular protection, locomotor improvements, and a reduction in neuropathic pain. Early inhibition of PDE4 is neuroprotective after SCI when given acutely and briefly at sufficient doses.

Eplerenone potentiates protective effects of amlodipine against cardiovascular injury in salt-sensitive hypertensive rats

The clinical value of the combination of amlodipine and eplerenone is unclear. This study was undertaken to test whether eplerenone potentiates the protective effects of amlodipine against hypertensive cardiovascular injury. Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were given (1) vehicle, (2) an antihypertensive dose of amlodipine, (3) a non-antihypertensive dose of eplerenone or (4) combined amlodipine and eplerenone for 6 weeks, and the effects on cardiovascular injuries were compared. There was no significant difference among the four groups regarding plasma aldosterone, urine volume or urinary electrolytes. A subpressor dose of eplerenone markedly ameliorated vascular endothelial dysfunction, cardiac inflammation and fibrosis in DS rats to a similar degree as an antihypertensive dose of amlodipine. Addition of eplerenone to amlodipine, without affecting blood pressure, enhanced the improvement by amlodipine of vascular endothelial function, cardiac inflammation, fibrosis and diastolic dysfunction in DS rats. Additive beneficial effects of eplerenone were attributed to additive potentiation of eNOS and Akt phosphorylation and additive reduction of oxidative stress. Eplerenone significantly attenuated cardiovascular NADPH oxidase activity by reducing gp91(phox) upregulation and attenuated the upregulation of cardiovascular AT1 receptor, but amlodipine failed to affect them. Thus, the normalization by eplerenone of gp91(phox) and AT1 receptor upregulation seems to be at least partially responsible for the additive benefits of eplerenone in the prevention of hypertensive cardiovascular injury. The combination of amlodipine and eplerenone may be a promising therapeutic strategy for cardiovascular disease in salt-sensitive hypertension.

Role for NADPH oxidase in macrophage clearance of apoptotic cells (54.2)

Abstract Ingestion of apoptotic neutrophils by macrophages (efferocytosis) is important for both neutrophil (PMN) homeostasis and resolution of tissue inflammation following infection or injury. In this study, we demonstrated that apoptotic PMN triggered activation of the NADPH oxidase in engulfing mouse macrophages, leading to oxidant production in phagosomes containing PMNs (also called efferosomes). Consistently, immunofluorescent staining revealed accumulation of gp91phox, a membrane-associated subunit of the oxidase, on the efferosome membrane. Examination of MyD88-/- macrophages illustrated a partial requirement for MyD88 in apoptotic PMN-mediated activation of the oxidase. To delineate the role of the NADPH oxidase in degradation of PMNs by macrophages, we observed that in macrophages from gp91 phox -/- mice, which do not generate oxidants during efferocytosis, lysosome-efferosome fusion occurred at a slower rate, thus implicating the importance of oxidants in efferosome maturation. Degradation of ingested PMNs within macrophages was further monitored by staining PMN myeloperoxidase (MPO), which was much more prominent in gp91phox-/- macrophages 6 hours after efferocytosis compared to WT macrophages, suggesting slower degradation in the absence of oxidants. Taken together, our data support that, in addition to its well-characterized anti-microbial function, macrophage NADPH oxidase may be an integral player in PMN clearance to facilitate the resolution of inflammation.

Selective activation of NADPH oxidase subunit 2 (NOX2) by A3 adenosine receptor in mouse aorta

The NADPH oxidase (NOX) subunits 1, 2 and 4 are the major sources for reactive oxygen species (ROS) in vascular tissue. We have shown earlier that activation of A3 adenosine receptor (A3AR) leads to contraction of aorta as well a decrease in coronary flow (Talukder, et al., 2002; Ansari, et al., 2007). In conditions such as ischemia‐reperfusion and hypoxia, both ROS and adenosine are released suggesting a possible interaction. Our aim in this study was to examine the relationship between A3AR and NOX1, NOX2 (gp91 phox) and NOX4 in mouse aorta. The protein and mRNA expression of the NOX1, NOX2 and NOX4 were measured in aortic rings from WT and A3AR knockout (A3KO) mice. A3AR agonist Cl‐IBMECA (10−7 M) was used in the presence or absence of the NOX inhibitor (apocynin, 10−5M). Cl‐IBMECA selectively increased protein expression of NOX2 by 150±15% in WT but not in A3KO mouse aortas and this increase was inhibited by apocynin. Neither NOX1 nor NOX4 protein expressions were affected by Cl‐IBMECA. Interestingly, the mRNA of NOX2 was not changed by Cl‐IBMECA in either WT or A3KO aortas, suggesting that NOX2 protein increase by Cl‐IBMECA may be through either a change in protein translation or turnover. In conclusion, A3AR selectively activates NOX2 without affecting NOX1 or NOX4 in mouse aorta, suggesting a role for NOX2 in adenosine mediated control of vascular tone through A3AR (Supported by NIH grants HL 027339, HL 094447, HL071802).

G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction.

Glucose-6-phosphatase, an enzyme localized in the endoplasmic reticulum (ER), catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. In humans, there are three differentially expressed glucose-6-phosphatase catabolic genes (G6PC1-3). Recently, it has been shown that mutations in the G6PC3 gene result in a syndrome associating congenital neutropenia and various organ malformations. The enzymatic function of G6PC3 is dependent on G6P transport into the ER, mediated by G6P translocase (G6PT). Mutations in the gene encoding G6PT result in glycogen storage disease type-1b (GSD-1b). Interestingly, GSD-1b patients exhibit a similar neutrophil dysfunction to that observed in G6PC3-deficient patients. To better understand the causes of neutrophil dysfunction in both diseases, we have studied the neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of patients with G6PC3 and G6PT syndromes. Unexpectedly, sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments indicated hypo-glycosylation of gp91(phox), the electron-transporting component of the NADPH oxidase, in all of these patients. Rigorous mass spectrometric glycomic profiling showed that most of the complex-type antennae which characterize the neutrophil N-glycome of healthy individuals were severely truncated in the patients' neutrophils. A comparable truncation of the core 2 antenna of the O-glycans was also observed. This aberrant neutrophil glycosylation is predicted to have profound effects on the neutrophil function and merit designation of both syndromes as a new class of congenital disorders of glycosylation.

Gp91phox contributes to the development of experimental inflammatory bowel disease

Inflammatory bowel disease (IBD) is related to dysfunction of intestinal immunity. Neutrophils have an important role in innate immunity via the oxidative burst, using the p47phox- and gp91(phox)-containing NAD(P)H oxidase known as Nox2. In dextran sulphate sodium (DSS)-induced colitis, no significant difference in inflammation between p47(phox-/-) and wild-type (WT) mice was reported, but there was improved endothelium-dependent arteriolar dilation in gp91(phox-/-) mice, compared with that in WT mice. Gp91(phox) and p47 (phox) are not only essential components of phagocyte Nox2, but also have roles in other enzymes. Thus the differences in response of their respective gene knockout mice to DSS challenge are not completely unexpected, but need further investigation. The clinicopathological changes and immunological responses to DSS challenge have not been fully described in gp91(phox-/-) mice. Thus we treated WT and gp91(phox-/-) mice with 2.5% DSS for 7 days. The gp91(phox-/-) mice developed less severe colitis than WT mice following DSS treatment, reflected by a smaller body weight loss, less rectal bleeding and fewer histopathological changes. Less colonic myeloperoxidase was observed in gp91(phox-/-), compared with WT mice, following DSS challenge, correlating with interleukin (IL)-6 production. IL-10 was upregulated in both gp91(phox-/-) and WT mice, but was significantly higher in the latter, following 7 days DSS challenge. These results suggest that gp91(phox-/-) mice are less susceptible to acute DSS-induced colitis, possibly because of a reduced oxidative burst in the intestine and, consequently, less tissue damage.

Attenuated Allergic Airway Inflammation in gp91 phox KO Mice is Due to Altered Dendritic Cell Function

RATIONALE: NADPH oxidase is a membrane bound enzyme complex comprised of five phagocytic oxidase subunits, including the membrane bound, catalytic subunit gp91phox. This complex is integral to the production of reactive oxygen species, which have been associated with inflammatory lung diseases such as asthma.

Germline CYBB mutations that selectively affect macrophages in kindreds with X-linked predisposition to tuberculous mycobacterial disease

Germline mutations in CYBB, the human gene encoding the gp91(phox) subunit of the phagocyte NADPH oxidase, impair the respiratory burst of all types of phagocytes and result in X-linked chronic granulomatous disease (CGD). We report here two kindreds in which otherwise healthy male adults developed X-linked recessive Mendelian susceptibility to mycobacterial disease (MSMD) syndromes. These patients had previously unknown mutations in CYBB that resulted in an impaired respiratory burst in monocyte-derived macrophages but not in monocytes or granulocytes. The macrophage-specific functional consequences of the germline mutation resulted from cell-specific impairment in the assembly of the NADPH oxidase. This 'experiment of nature' indicates that CYBB is associated with MSMD and demonstrates that the respiratory burst in human macrophages is a crucial mechanism for protective immunity to tuberculous mycobacteria.

Role of neutrophils and NADPH phagocyte oxidase in host defense against respiratory infection with virulent Francisella tularensis in mice

Francisella tularensis subspecies (subsp.) tularensis is a CDC Category A biological warfare agent and inhalation of as few as 15 bacilli can initiate severe disease. Relatively little is known about the cellular and molecular mechanisms of host defense against respiratory infection with subsp. tularensis. In this study, we examined the role of neutrophils and NADPH phagocyte oxidase in host resistance to pulmonary infection in a mouse intranasal infection model. We found that despite neutrophil recruitment to the lungs and increased concentrations of neutrophil-chemotactic chemokines (KC, MIP-2 and RANTES) in the bronchoalveolar lavage fluid following intranasal inoculation of the pathogen, neither depletion of neutrophils nor enhancement of their recruitment into the lungs had any impact on bacterial burdens or survival rate/time. Nevertheless, mice deficient in NADPH phagocyte oxidase (gp91 phox–/– ) did exhibit higher tissue and blood bacterial burdens and succumbed to infection one day earlier than wild-type C57BL/6 mice. These results imply that although neutrophils are not a major effector cell in defense against subsp. tularensis infection, NADPH phagocyte oxidase does play a marginal role.