Acetaminophen Toxicity in Mice Lacking NADPH Oxidase Activity: Role of Peroxynitrite Formation and Mitochondrial Oxidant Stress

Previous data have indicated that activated macrophages may play a role in the mediation of acetaminophen toxicity. In the present study, we examined the significance of superoxide produced by macrophages by comparing the toxicity of acetaminophen in wild-type mice to mice deficient in gp91phox, a critical subunit of NADPH oxidase that is the primary source of phagocytic superoxide. Both groups of mice were dosed with 300?mg/kg of acetaminophen or saline and sacrificed at 1, 2, 4 or 24?h. Glutathione in total liver and in mitochondria was depleted by approximately 90% at 1?h in wild-type and knock out mice. No significant differences in toxicity (serum transaminase levels or histopathology) were observed between wild-type and mice deficient in gp91phox. Mitochondrial glutathione disulfide, as a percent of total glutathione, was determined as a measure of oxidant stress produced by increased superoxide, leading to hydrogen peroxide and/or peroxynitrite. The percent mitochondrial glutathione disulfide increased to approximately 60% at 1?h and 70% at 2?h in both groups of mice. Immunohistochemical staining for nitrotyrosine was present in vascular endothelial cells at 1?h in both groups of mice. Acetaminophen protein adducts were present in hepatocytes at 1?h in both wild-type and knock out animals. These data indicate that superoxide from activated macrophages is not critical to the development of acetaminophen toxicity and provide further support for the role of mitochondrial oxidant stress in acetaminophen toxicity.     

NADPH oxidase but not myeloperoxidase protects lymphopenic mice from spontaneous infections

The adaptive immune system plays an important role in host defense against invading micro-organisms. Yet, mice deficient in T- and B-cells are surprisingly healthy and develop few spontaneous infections when raised under specific pathogen-free conditions (SPF). The objective of this study was to ascertain what role phagocyte-associated NADPH oxidase or myeloperoxidase (MPO) plays in host defense in mice lacking both T- and B-cells. To do this, we generated lymphopenic mice deficient in either NADPH oxidase or MPO by crossing gp91(phox)-deficient (gp91 ko) or MPO ko mice with mice deficient in recombinase activating gene-1 (RAG ko). We found that neither gp91 ko, MPO ko mice nor lymphocyte-deficient RAG ko mice developed spontaneous infections when raised under SPF conditions and all mice had life spans similar to wild-type (WT) animals. In contrast, gp91xRAG double-deficient (DKO) but not MPOxRAG DKO mice developed spontaneous multi-organ bacterial and fungal infections early in life and lived only a few months. Infections in the gp91xRAG DKO mice were characterized by granulomatous inflammation of the skin, liver, heart, brain, kidney, and lung. Addition of antibiotics to the drinking water attenuated the spontaneous infections and increased survival of the mice. Oyster glycogen-elicited polymorphonuclear neutrophils (PMNs) and macrophages obtained from gp91 ko and gp91xRAG DKO mice had no detectable NADPH oxidase activity whereas WT, RAG ko, and MPOxRAG DKO PMNs and macrophages produced large and similar amounts of superoxide in response to phorbol myristate acetate. The enhanced mortality of the gp91xRAG DKO mice was not due to defects in inflammatory cell recruitment or NO synthase activity (iNOS) as total numbers of elicited PMNs and macrophages as well as PMN- and macrophage-derived production of nitric oxide-derived metabolites in these mice were similar and not reduced when compared to that of WT mice. Taken together, our data suggest that that NADPH oxidase but not MPO (nor iNOS) is required for host defense in lymphopenic mice and that lymphocytes and NADPH oxidase may compensate for each other's deficiency in providing resistance to spontaneous bacterial infections.     

Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst

Reactive nitrogen species (RNS) play an essential role in host defence against Mycobacterium tuberculosis (MTB) in the mouse model of tuberculosis (TB), as evidenced by the increased susceptibility of mice deficient in the inducible isoform of nitric oxide synthase (NOS2). In contrast, the role of reactive oxygen species (ROS) in protection against MTB is less clear, and mice defective in the ROS-generating phagocyte NADPH oxidase (Phox) are relatively resistant. This suggests that MTB might possess efficient mechanisms to evade or counter the phagocyte oxidative burst, effectively masking the impact of this host defence mechanism. In order to assess the role of ROS detoxification pathways in MTB virulence, we generated a katG null mutant of MTB, deficient in the KatG catalase-peroxidase-peroxynitritase, and evaluated the mutant's ability to replicate and persist in macrophages and mice. Although markedly attenuated in wild-type C57Bl/6 mice and NOS2(-/-) mice, the DeltakatG MTB strain was indistinguishable from wild-type MTB in its ability to replicate and persist in gp91(Phox-/-) mice lacking the gp91 subunit of NADPH oxidase. Similar observations were made with murine bone marrow macrophages infected ex vivo: growth of the DeltakatG MTB strain was impaired in macrophages from C57Bl/6 and NOS2(-/-) mice, but indistinguishable from wild-type MTB in gp91(Phox-/-) macrophages. These results indicate that the major role of KatG in MTB pathogenesis is to catabolize the peroxides generated by the phagocyte NADPH oxidase; in the absence of this host antimicrobial mechanism, KatG is apparently dispensable.     

CD4+ T lymphocytes mediate hypercholesterolemia-induced endothelial dysfunction via a NAD(P)H oxidase-dependent mechanism

Although hypercholesterolemia is known to impair endothelium-dependent vasodilation (EDV) long before the appearance of atherosclerotic plaques, it remains unclear whether the immune mechanisms that have been implicated in atherogenesis also contribute to the early oxidative stress and endothelial cell dysfunction elicited by hypercholesterolemia. EDV (wire myography), superoxide generation (cytochrome c reduction), and NAD(P)H oxidase mRNA expression were monitored in aortic rings from wild-type (WT) and mutant mice placed on either a normal diet or a cholesterol-enriched diet (HC) for 2 wk. WT mice on HC exhibited impaired EDV, enhanced superoxide generation, and increased expression of NAD(P)H oxidase subunit Nox-2 mRNA. The impaired EDV and increased superoxide generation induced by HC were significantly blunted in severe combined immunodeficient (SCID) mice and CD4+ T lymphocyte-deficient mice. These responses were also attenuated in HC mice genetically deficient in IFN-gamma; however, adoptive transfer of WT-HC CD4+ T lymphocytes to IFN-gamma-deficient recipients restored HC-induced responses. The HC-induced impaired EDV and oxidative stress were also attenuated in HC mice genetically deficient in Nox-2 (gp91(phox-/-)) and in WT-->gp91(phox-/-)-HC chimeras. HC-induced gp91(phox) mRNA expression was significantly blunted in mice deficient in CD4+ T cells or IFN-gamma and was restored with adoptive transfer of WT-HC CD4+ T cells to IFN-gamma-deficient recipients. These findings implicate the immune system in the early endothelial cell dysfunction associated with hypercholesterolemia and are consistent with a mechanism of impaired EDV that is mediated by CD4+ T cells and IFN-gamma, acting through the generation of superoxide from vascular NAD(P)H oxidase.     

Role of NADPH oxidase in the brain injury of intracerebral hemorrhage

The major risk factors for intracerebral hemorrhage (ICH) are hypertension and aging. A fundamental mechanism for hypertension- and aging-induced vascular injury is oxidative stress. We hypothesize that oxidative stress has a crucial role in ICH. To test our hypothesis, we used bacterial collagenase to produce ICH in wild-type C57BL/6 and gp91phox knockout (gp91phox KO) mice (deficient in gp91phox subunit of the superoxide-producing enzyme NADPH oxidase). All animals were studied at 20-35 weeks of age, resembling an older patient population. We found that collagenase produced less bleeding in gp91phox KO mice than wild-type mice. Total oxidative product was lower in gp91phox KO mice than in wild-type mice, both under basal conditions and after ICH. Consistent with the ICH volume, brain edema formation, neurological deficit and a high mortality rate was noted in wild-type but not in gp91phox KO mice. This ICH-induced brain injury in wild-type mice is associated with enhanced expression of the gp91phox subunit of NADPH oxidase. In conclusion, the oxidative stress resulting from activation of NADPH oxidase contributes to ICH induced by collagenase and promotes brain injury.     

NADPH oxidase restrains the matrix metalloproteinase activity of macrophages

Matrix metalloproteinases (MMPs) regulate numerous functions in normal and disease processes; thus, irreversibly blocking their activity is a key step in regulating MMP catalysis. We previously showed in vitro that oxidizing intermediates generated by phagocytes inactivate MMPs by modifying specific amino acids. To assess whether this mechanism operates in vivo, we focused on MMP-12, a macrophage-specific MMP known to mediate emphysema in mouse models. We found that mice lacking gp91(phox), a phagocyte-specific component of the NADPH oxidase, developed extensive, spontaneous emphysematous destruction of their peripheral air spaces, whereas mice deficient in both NADPH oxidase and MMP-12 were protected from spontaneous emphysema. Although gp91(phox)-null and wild-type macrophages produced equivalent levels of MMP-12 protein, the oxidant-deficient cells had greater MMP-12 activity than wild-type macrophages. These findings indicate that reactive intermediates provide a physiological mechanism to protect tissues from excessive macrophage-mediated damage during inflammation.     

Intermittent hypoxia-induced cognitive deficits are mediated by NADPH oxidase activity in a murine model of sleep apnea

Background

In rodents, exposure to intermittent hypoxia (IH), a hallmark of obstructive sleep apnea (OSA), is associated with neurobehavioral impairments, increased apoptosis in the hippocampus and cortex, as well as increased oxidant stress and inflammation. Excessive NADPH oxidase activity may play a role in IH-induced CNS dysfunction.

Methods and Findings

The effect of IH during light period on two forms of spatial learning in the water maze and well as markers of oxidative stress was assessed in mice lacking NADPH oxidase activity (gp91phox ^_/Y) and wild-type littermates. On a standard place training task, gp91phox ^_/Y displayed normal learning, and were protected from the spatial learning deficits observed in wild-type littermates exposed to IH. Moreover, anxiety levels were increased in wild-type mice exposed to IH as compared to room air (RA) controls, while no changes emerged in gp91phox ^_/Y mice. Additionally, wild-type mice, but not gp91phox ^_/Y mice had significantly elevated levels of NADPH oxidase expression and activity, as well as MDA and 8-OHDG in cortical and hippocampal lysates following IH exposures.

Conclusions

The oxidative stress responses and neurobehavioral impairments induced by IH during sleep are mediated, at least in part, by excessive NADPH oxidase activity, and thus pharmacological agents targeting NADPH oxidase may provide a therapeutic strategy in sleep-disordered breathing.     

Role of gp91phox-containing NADPH oxidase in left ventricular remodeling induced by intermittent hypoxic stress

Intermittent hypoxia due to sleep apnea syndrome is associated with cardiovascular diseases. However, the precise mechanisms by which intermittent hypoxic stress accelerates cardiovascular diseases are largely unclear. The aim of this study was to investigate the role of gp91(phox)-containing NADPH oxidase in the development of left ventricular (LV) remodeling induced by intermittent hypoxic stress in mice. Male gp91(phox)-deficient (gp91(-/-)) mice (n = 26) and wild-type (n = 39) mice at 7-12 wk of age were exposed to intermittent hypoxia (30 s of 4.5-5.5% O(2) followed by 30 s of 21% O(2) for 8 h/day during daytime) or normoxia for 10 days. Mean blood pressure and LV systolic and diastolic function were not changed by intermittent hypoxia in wild-type or gp91(-/-) mice, although right ventricular systolic pressure tended to be increased. In wild-type mice, intermittent hypoxic stress significantly increased the diameter of cardiomyocytes and interstitial fibrosis in LV myocardium. Furthermore, intermittent hypoxic stress increased superoxide production, 4-hydroxy-2-nonenal protein, TNF-alpha and transforming growth factor-beta mRNA, and NF-kappaB binding activity in wild-type, but not gp91(-/-), mice. These results suggest that gp91(phox)-containing NADPH oxidase plays a crucial role in the pathophysiology of intermittent hypoxia-induced LV remodeling through an increase of oxidative stress.     

Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox

Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.     

Acetaminophen toxicity. Opposite effects of two forms of glutathione peroxidase

Acetaminophen is one of the most extensively used analgesics/antipyretics worldwide, and overdose or idiopathic reaction causes major morbidity and mortality in its victims. Research into the mechanisms of toxicity and possible therapeutic intervention is therefore essential. In this study, the response of transgenic mice overexpressing human antioxidant enzymes to acute acetaminophen overdose was investigated. Animals overexpressing superoxide dismutase or plasma glutathione peroxidase demonstrated dramatic resistance to acetaminophen toxicity. Intravenous injection of glutathione peroxidase provided normal mice with nearly complete protection against a lethal dose of acetaminophen. Surprisingly, animals overexpressing intracellular glutathione peroxidase in the liver were significantly more sensitive to acetaminophen toxicity compared with nontransgenic littermates. This sensitivity appears to be due to the inability of these animals to efficiently recover glutathione depleted as a result of acetaminophen metabolism. Finally, the results suggest that glutathione peroxidase overexpression modulates the synthesis of several acetaminophen metabolites. Our results demonstrate the ability of glutathione peroxidase levels to influence the outcome of acetaminophen toxicity.     

Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 μM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.     

Phenotypic analysis of the ccp1Delta and ccp1Delta-ccp1W191F mutant strains of Saccharomyces cerevisiae indicates that cytochrome c peroxidase functions in oxidative-stress signaling

Yeast cytochrome c peroxidase (CCP) efficiently catalyzes the reduction of H₂O₂ to H₂O by ferrocytochrome c in vitro. The physiological function of CCP, a heme peroxidase that is targeted to the mitochondrial intermembrane space of Saccharomyces cerevisiae, is not known. CCP1-null-mutant cells in the W303-1B genetic background (ccp1Δ) grew as well as wild-type cells with glucose, ethanol, glycerol or lactate as carbon sources but with a shorter initial doubling time. Monitoring growth over 10 days demonstrated that CCP1 does not enhance mitochondrial function in unstressed cells. No role for CCP1 was apparent in cells exposed to heat stress under aerobic or anaerobic conditions. However, the detoxification function of CCP protected respiring mitochondria when cells were challenged with H₂O₂. Transformation of ccp1Δ with ccp1^W191F, which encodes the CCP^W191F mutant enzyme lacking CCP activity, significantly increased the sensitivity to H₂O₂ of exponential-phase fermenting cells. In contrast, stationary-phase (7-day) ccp1Δ-ccp1^W191F exhibited wild-type tolerance to H₂O₂, which exceeded that of ccp1Δ. Challenge with H₂O₂ caused increased CCP, superoxide dismutase and catalase antioxidant enzyme activities (but not glutathione reductase activity) in exponentially growing cells and decreased antioxidant activities in stationary-phase cells. Although unstressed stationary-phase ccp1Δ exhibited the highest catalase and glutathione reductase activities, a greater loss of these antioxidant activities was observed on H₂O₂ exposure in ccp1Δ than in ccp1Δ-ccp1^W191F and wild-type cells. The phenotypic differences reported here between the ccp1Δ and ccp1Δ-ccp1^W191F strains lacking CCP activity provide strong evidence that CCP has separate antioxidant and signaling functions in yeast.     

Gp91phox contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide (O2-*) levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in O2-* levels between adventitial cells and medial SMC in aorta from gp91phox-deficient (gp91phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91phox KO and wild-type FB to account for these observations. Because gp91phox is associated with membranes, we examined NADPH-stimulated O2-. production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91phox in FB, antisense to gp91phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91phox did not alter expression of the gp91phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91phox contributes to NADPH oxidase function in vascular FB, but not SMC.     

Multiple deficiencies in antioxidant enzymes in mice result in a compound increase in sensitivity to oxidative stress

To examine the effect of compound deficiencies in antioxidant defense, we have generated mice (Sod2^+/−/Gpx1^−/−) that are deficient in Mn superoxide dismutase (MnSOD) and glutathione peroxidase 1 (Gpx1) by breeding Sod2^+/− and Gpx1^−/− mice together. Although Sod2^+/−/Gpx1^−/− mice showed a 50% reduction in MnSOD and no detectable Gpx1 activity in either mitochondria or cytosol in all tissues, they were viable and appeared normal. Fibroblasts isolated from Sod2^+/−/Gpx1^−/− mice were more sensitive (4- to 6-fold) to oxidative stress (t-butyl hydroperoxide or γ irradiation) than fibroblasts from wild-type mice, and were twice as sensitive as cells from Sod2^+/− or Gpx1^−/− mice. Whole-animal studies demonstrated that survival of the Sod2^+/−/Gpx1^−/− mice in response to whole body γ irradiation or paraquat administration was also reduced compared with that of wild-type, Sod2^+/−, or Gpx1^−/− mice. Similarly, endogenous oxidative stress induced by cardiac ischemia/reperfusion injury led to greater apoptosis in heart tissue from the Sod2^+/−/Gpx1^−/− mice than in that from mice deficient in either MnSOD or Gpx1 alone. These data show that Sod2^+/−/Gpx1^−/− mice, deficient in two mitochondrial antioxidant enzymes, have significantly enhanced sensitivity to oxidative stress induced by exogenous insults and to endogenous oxidative stress compared with either wild-type mice or mice deficient in either MnSOD or Gpx1 alone.     

Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.     

Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33 μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6 μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

Female X-chromosome mosaicism for NOX2 deficiency presents unique inflammatory phenotype and improves outcome in polymicrobial sepsis

Cellular X-chromosome mosaicism, which is unique to females, may be advantageous during pathophysiological challenges compared with the single X-chromosome machinery of males, and it may contribute to gender dimorphism in the inflammatory response. We tested the hypothesis of whether cellular mosaicism for the X-linked gp91phox (NOX2) deficiency, the catalytic component of the superoxide anion-generating NADPH oxidase complex, is advantageous during polymicrobial sepsis. Deficient, wild-type (WT), and heterozygous/mosaic mice were compared following polymicrobial sepsis initiated by cecal ligation and puncture. Compared with WT littermates, sepsis-induced mortality was improved in deficient mice, as well as in mosaic animals carrying both deficient and WT phagocyte subpopulations. In contrast, blood bacterial counts were greatest in deficient mice. Consistent with poor survival, WT mice also showed the most severe organ damage following sepsis. In mosaic animals, the deficient neutrophil subpopulations displayed increased organ recruitment and elevated CD11b membrane expression compared with WT neutrophil subpopulations within the same animal. The dynamics of sepsis-induced blood and organ cytokine content and WBC composition changes, including lymphocyte subsets in blood and bone marrow, showed differences among WT, deficient, and mosaic subjects, indicating that mosaic mice are not simply the average of the deficient and WT responses. Upon oxidative burst, interchange of oxidants between WT and deficient neutrophil subpopulations occurred in mosaic mice. This study suggests that mice mosaic for gp91phox expression have multiple advantages compared with WT and deficient mice during the septic course.     

Tissue-specific oxidative imbalance and mitochondrial dysfunction during Trypanosoma cruzi infection in mice

In this study, we examined the tissue specificity of inflammatory and oxidative responses and mitochondrial dysfunction in mice infected by Trypanosoma cruzi. In acute mice, parasite burden and associated inflammatory infiltrate was detected in all tissues (skeletal muscle>heart>stomach>colon). The extent of oxidative damage and mitochondrial decay was in the order of heart>stomach>skeletal muscle>colon. In chronic mice, a low level of parasite burden and inflammation continued in all tissues; however, oxidant overload and mitochondrial inefficiency mainly persisted in the heart tissue (also detectable in stomach). Further, we noted an unvaryingly high degree of oxidative stress, compromised antioxidant status, and decreased mitochondrial respiratory complex activities in peripheral blood of infected mice. A pair-wise log analysis showed a strong positive correlation in the heart-versus-blood (but not other tissues) levels of oxidative stress markers (malonyldialdehyde, glutathione disulfide), antioxidants (superoxide dismutase, MnSOD, catalase), and mitochondrial inhibition of respiratory complexes (CI/CIII) in infected mice. T. cruzi-induced acute inflammatory and oxidative responses are widespread in different muscle tissues. Antioxidant/oxidant status and mitochondrial function are consistently attenuated in the heart, and reflected in the peripheral-blood of T. cruzi-infected mice. Our results provide an impetus to investigate the peripheral-blood oxidative responses in relation to clinical severity of heart disease in chagasic human patients.     

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection

^•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91^{phox −/−} or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with ^•NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.