Three common polymorphisms in the CYBA gene form a haplotype associated with decreased ROS generation

NOX enzymes are reactive oxygen species (ROS)‐generating NADPH oxidases. Several members of the NOX family depend on the p22^phox subunit, encoded by the CYBA gene. CYBA is highly polymorphic, and has been widely studied as a potential risk factor for various diseases, with conflicting results. In the present study, we used Epstein‐Barr (EBV)‐transformed B‐lymphocytes from 50 healthy unrelated individuals to analyze their CYBA mRNA sequence and NOX2‐dependent ROS generation. Seven single‐nucleotide polymorphisms (SNPs) were identified (five previously described, two novel). The combination of these SNPs yielded 11 distinct haplotypes, which could be grouped into seven haplogroups (A–G). Haplogroup C (c.214T>C, c.521T>C, and c.^*24G>A) showed a significantly lower ROS generation, as compared to the most frequent haplogroup, A. CYBA variants from the seven haplogroups were transduced into p22^phox‐deficient B‐lymphocytes. The haplogroup C variant showed significantly lower ROS production. c.214T>C and c.521T>C lead to nonsynonymous codon changes, while c.^*24G>A lies within the 3′UTR. Using a luciferase/3′UTR construct, we showed that the ^*24A allele led to decreased reporter gene activity. These results help to unravel the complex nature of how genetic variations in CYBA influence NOX2 activity, and indicate that haplotypes, rather than individual SNPs, define the effect on ROS generation. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47^phox, p67^phox, p40^phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA^11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal–regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47^phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the “MAPK–p47^phox phosphorylation–NOX2 activation” axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.     

NADPH oxidases and MMP-9/TIMP-1 balance in human glomeruli: putative links to diabetic nephropathy

Study aim Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H₂O₂ and angiotensin‐2. Material and methods NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival. Results NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40^phox, p67^phox and p47^phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H₂O₂ in glomerular extracts, while addition of 25 mm glucose blunted these findings. Conclusion Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H₂O₂ from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.     

HDL抑制人脐静脉内皮细胞NADPH氧化酶4表达和活性氧生成

目的分析HDL对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)内NAD-PH氧化酶4(NADPH oxidase4,NOX4)及其亚组分p22phox、p67phox、Rac1与活性氧(reactive oxygen species,ROS)生成的影响,研究HDL对LDL的拮抗作用。方法用Ⅱ型胶原酶消化法分离培养HUVEC;用RT-PCR、Western印迹观察NOX4、p22phox、p67phox、Rac1表达的变化;用流式细胞仪检测细胞内ROS的变化。结果HDL处理HUVEC能下调NOX4和p67phox mRNA和蛋白水平,但不影响p22phox和Rac1的表达。高浓度LDL刺激HUVEC时,NOX4表达上调,细胞内ROS生成增加,但p22phox、p67phox和Rac1的表达没有明显变化。用HDL预处理再加高浓度LDL刺激时,NOX4表达下调,ROS生成量减少;结论HDL可通过下调HUVEC NOX4表达降低ROS水平。     

Macrophage‐specific NOX2 contributes to the development of lung emphysema through modulation of SIRT1/MMP‐9 pathways

Reactive oxygen species (ROS) participate in the pathogenesis of emphysema. Among ROS‐producing enzymes, NOX NADPH oxidases are thought to be responsible for tissue injury associated with several lung pathologies. To determine whether NOX2 and/or NOX1 participate in the development of emphysema, their expression patterns were first studied by immunohistochemistry in the lungs of emphysematous patients. Subsequently, we investigated their contribution to elastase‐induced emphysema using NOX2‐ and NOX1‐deficient mice. In human lung, NOX2 was mainly detected in macrophages of control and emphysematous lungs, while NOX1 was expressed in alveolar epithelium and bronchial cells. We observed an elevated number of NOX2‐positive cells in human emphysematous lungs, as well as increased NOX2 and NOX1 mRNA expression in mouse lungs following elastase exposure. Elastase‐induced alveolar airspace enlargement and elastin degradation were prevented in NOX2‐deficient mice, but not in NOX1‐deficient mice. This protection was independent of inflammation and correlated with reduced ROS production. Concomitantly, an elevation of sirtuin 1 (SIRT1) level and a decrease of matrix metalloproteinase‐9 (MMP‐9) expression and activity were observed in alveolar macrophages and neutrophils. We addressed the specific role of macrophage‐restricted functional NOX2 in elastase‐induced lung emphysema using Ncf1 mutant mice and Ncf1 macrophage rescue mice (Ncf1 mutant mice with transgenic expression of Ncf1 only in CD68‐positive mononuclear phagocytes; the MN mouse). Compared to WT mice, the lack of functional NOX2 led to decreased elastase‐induced ROS production and protected against emphysema. In contrast, ROS production was restored specifically in macrophages from Ncf1 rescue mice and contributes to emphysema. Taken together, our results demonstrate that NOX2 is involved in the pathogenesis of human emphysema and macrophage‐specific NOX2 participates in elastase‐induced emphysema through the involvement of SIRT1/MMP‐9 pathways in mice. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Glucose‐6‐phosphate dehydrogenase inhibition attenuates acute lung injury through reduction in NADPH oxidase‐derived reactive oxygen species

Acute lung injury (ALI) is a heterogeneous disease with the hallmarks of alveolar capillary membrane injury, increased pulmonary oedema and pulmonary inflammation. The most common direct aetiological factor for ALI is usually parenchymal lung infection or haemorrhage. Reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX2) are thought to play an important role in the pathophysiology of ALI. Glucose‐6‐phosphate dehydrogenase (G6PD) plays an important role both in production of ROS as well as their removal through the supply of NADPH. However, how G6PD modulation affects NOX2‐mediated ROS in the airway epithelial cells (AECs) during acute lung injury has not been explored previously. Therefore, we investigated the effect of G6PD inhibitor, 6‐aminonicotinamide on G6PD activity, NOX2 expression, ROS production and enzymatic anti‐oxidants in AECs in a mouse model of ALI induced by lipopolysaccharide (LPS). ALI led to increased G6PD activity in the AECs with concomitant elevation of NOX2, ROS, SOD1 and nitrotyrosine. G6PD inhibitor led to reduction of LPS‐induced airway inflammation, bronchoalveolar lavage fluid protein concentration as well as NOX2‐derived ROS and subsequent oxidative stress. Conversely, ALI led to decreased glutathione reductase activity in AECs, which was normalized by G6PD inhibitor. These data show that activation of G6PD is associated with enhancement of oxidative inflammation in during ALI. Therefore, inhibition of G6PD might be a beneficial strategy during ALI to limit oxidative damage and ameliorate airway inflammation.     

Colitis susceptibility in mice with reactive oxygen species deficiency is mediated by mucus barrier and immune defense defects

Reactive oxygen species (ROS) generated by NADPH oxidases (NOX/DUOX) provide antimicrobial defense, redox signaling, and gut barrier maintenance. Inactivating NOX variants are associated with comorbid intestinal inflammation in chronic granulomatous disease (CGD; NOX2) and pediatric inflammatory bowel disease (IBD; NOX1); however Nox-deficient mice do not reflect human disease susceptibility. Here we assessed if a hypomorphic patient-relevant CGD mutation will increase the risk for intestinal inflammation in mice. Cyba (p22^phox) mutant mice generated low intestinal ROS, while maintaining Nox4 function. The Cyba variant caused profound mucus layer disruption with bacterial penetration into crypts, dysbiosis, and a compromised innate immune response to invading microbes, leading to mortality. Approaches used in treatment-resistant CGD or pediatric IBD such as bone marrow transplantation or oral antibiotic treatment ameliorated or prevented disease in mice. The Cyba mutant mouse phenotype implicates loss of both mucus barrier and efficient innate immune defense in the pathogenesis of intestinal inflammation due to ROS deficiency, supporting a combined-hit model where a single disease variant compromises different cellular functions in interdependent compartments.     

NOX1-derived ROS drive the expression of Lipocalin-2 in colonic epithelial cells in inflammatory conditions

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22^PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.     

Dexamethasone increases ROS production and T cell suppressive capacity by anti-inflammatory macrophages

Macrophages have been demonstrated to suppress T cell responses by producing reactive oxygen species (ROS) leading to the subsequent induction of T regulatory cells in a ROS-dependent manner. Macrophages may therefore be instrumental in downregulating T cell responses in situations of exacerbated immune responses. Here we investigated the effect of immunosuppressive drugs on ROS production by macrophage subsets and the subsequent effects on T cell activation. Macrophage types 1 and 2 were differentiated with GM-CSF or M-CSF, in presence or absence of dexamethasone, cyclosporine A, FK506, rapamycin, or mycophenolic acid. The ROS producing capacity of fully differentiated Mph was highest in anti-inflammatory Mph2 and not affected by exposure to immunosuppressive drugs. However, presence of rapamycin during Mph2 differentiation decreased the ROS production of these cells. In contrast, other immunosuppressive drugs, with dexamethasone being the most potent, increased the ROS producing capacity of Mph2. Intriguingly although the ROS producing ability of Mph1 was unaffected, dexamethasone strongly increased the ROS producing capabilities of dendritic cells. Both at the mRNA and protein level we found that dexamethasone enhanced the expression of NOX2 protein p47^phox. Functionally, dexamethasone further enhanced the capacity of Mph2 to suppress T cell mediated IFN-γ and IL-4 production. In vivo, only in rats with normal ROS production (congenic DA.Ncf1^E3/E3) it was observed that dexamethasone injection resulted in long-lasting upregulation of ROS production by macrophages and induced higher levels of Treg in a ROS-dependent manner. In conclusion, we show that the anti-inflammatory drug dexamethasone increases the ROS producing capacity of macrophages.     

BCR‐induced superoxide negatively regulates B‐cell proliferation and T‐cell‐independent type 2 Ab responses

Superoxide and its derivatives have been implicated as secondary messenger molecules that influence signaling cascades in non‐phagocytes. B lymphocytes produce superoxide after BCR ligation. We found that these ROS regulate B‐cell signaling and entry into the cell cycle. B cells from mice deficient in the gp91^phox subunit of the NADPH oxidase complex are unable to generate ROS after BCR ligation. However, after BCR stimulation, more gp91^phox KO B cells enter the G1 stage of the cell cycle and proliferate than WT B cells. BCR ligation leads to a more rapid decrease in p27^Kip1 levels in gp91^phox KO B cells. Gp91^phox KO mice display enhanced T‐cell‐independent type 2, but normal T‐dependent Ab responses. ROS‐dependent regulation of BCR‐induced proliferation may help modulate the size of the humoral response to T‐cell‐independent type 2 Ag immunization.     

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

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells'' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91^phox/p22^phox) and the cytoplasm (p47^phox, p67^phox, and p40^phox). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa⁻) Gc does not stimulate neutrophil ROS production. Using constitutively Opa⁻ and OpaD-positive (OpaD⁺) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa⁻ Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD⁺ Gc. gp91^phox and p22^phox were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa⁻ Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47^phox “organizing” subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.     

Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides

Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2^−/− knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2^−/− mice died earlier than the WT strain. Immunization of the NOX2^−/− and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2^−/− mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis.     

Three novel mutations in CYBA among 22 Iranians with Chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by defect in one of the components of nicotinamide adenine dinucleotide phosphate (NADPH)‐oxidase enzyme. The enzyme is at least composed of membrane‐bound subunits gp91‐phox and p22‐phox (also named cytochrome b₅₅₈), and cytosolic ones p40‐phox, p47‐phox and p67‐phox. A defect in the enzyme activity leads to impaired intracellular killing of phagocytic cells. The CYBA gene encoding p22‐phox is located on chromosome 16q24. In this study, new genetic changes of CYBA gene in 22 Iranian patients with autosomal recessive‐CGD (AR‐CGD) were identified. Twenty‐two patients with CGD were referred to Immunology, Asthma and Allergy Research Institute (IAARI) and enrolled in this study based on defect in NADPH oxidase activity, demographic data and clinical histories. All patients had p22‐phox deficiency based on Western blotting. Genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs), and PCR followed by direct sequencing was performed to find p22‐phox mutations. Mutation analysis of CYBA revealed 12 different mutations, including three novel mutations: one was deletion of exon 1, and two were point mutations in exon 3 (c.136G>A (p.Gly46Ser)), and exon 6 (c.388C>T (p.Gln130X)). Three new mutations of CYBA gene in four of 22 Iranian patients with AR‐CGD were found. These three novel mutations can partly complete the database of Human Gene Mutation Database (HGMD) and other related ones. It can also be helpful for further prenatal diagnosis in the affected families. Given that currently bone marrow transplantation is considered to be the curative treatment for patients with CGD, finding mutations will also be useful for timely decision‐making in bone marrow transplantation.     

Regulatory T cell features in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (T_regs) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in T_regs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated T_regs from gp91^phox-, p47^phox- and p40^phox-deficient CGD patients separately. Results show that T_reg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91^phox-deficiency effector T_reg (eT_reg) numbers are decreased. Expression of T_reg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in T_reg functionality or activation state. No correlation was seen between eT_reg numbers and patients' clinical phenotype. To conclude, the only difference between T_regs from CGD patients and healthy controls is a decrease in circulating eT_regs in gp91^phox-deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived T_regs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for T_regs in CGD-related autoinflammation/autoimmunity.     

Expression and localization of NOX2 and NOX4 in primary human endothelial cells

Reactive oxygen species (ROS) control the integrity of the vascular endothelium. Our laboratory has recently shown that transduction of human umbilical vein endothelial cells (HUVECs) with an active variant of the small GTPase Rac promotes the production of ROS, ROS-dependent activation of p38 mitogen-activated protein kinase, and loss of vascular/endothelial-cadherin-mediated cell-cell adhesion. Here we show that HUVECs express mRNAs for NOX2 as well as NOX4 mRNA, but not for NOX1 or NOX3. Interestingly, NOX4 was expressed at 100-fold higher levels compared with NOX2. NOX4-green fluorescent protein largely localizes to an intracellular compartment that costained with a marker for the endoplasmic reticulum, and its distribution did not overlap with lysosomes, Weibel-Palade bodies, or mitochondria. The NOX2-regulatory proteins p47(phox) and p67(phox) associated with the actin cytoskeleton and were found in cell protrusions and membrane ruffles, colocalizing with Rac1. This translocation to the cell periphery was promoted by tumor necrosis factor (TNF)-alpha. Finally, scavenging of ROS was found to impair TNF-alpha-induced cytoskeletal rearrangements and the formation of a confluent endothelial monolayer. Together, these data prove the differential mRNA expression of NOX family members in human endothelium and indicate that these NOX proteins and their regulators may be involved in the control of endothelial cell spreading, motility, and cell-cell adhesion.     

Impaired p53/CEP‐1 is associated with lifespan extension through an age‐related imbalance in the energy metabolism of C. elegans

In the nematode Caenorhabditis elegans, the mammalian tumor suppressor p53 ortholog CEP‐1 mediates the stress response, activates germ line apoptosis and regulates meiotic chromosome segregation. A reduction in its expression, which frequently occurs in mammalian cancer cells, extends lifespan and induces an adaptive response in C. elegans. However, these effects do not involve an increase in oxidative stress resistance. Here, we showed that intermittent exposure to hyperoxia, which induces oxidative stress resistance and lowers the production of ROS derived from mitochondrial respiration in C. elegans, slightly improved the lifespan extension of cep‐1 mutant. Interestingly, ATP levels were increased without an increase in oxygen consumption in cep‐1 mutant during aging. In the wild‐type, lactate levels and consequentially the lactate/pyruvate ratio decreased during aging in adults. Furthermore, the expression levels of mitochondrial respiration‐related sco‐1, which is a target of p53/CEP‐1, as well as those of gluconeogenesis regulation and mammalian sirtuin ortholog genes, were also increased in the aged and adaptive conditioned wild‐type animals. In contrast, the lactate/pyruvate ratio increased in cells of the cep‐1 mutant and was amplified by intermittent hyperoxia. These results suggest that impaired p53/CEP‐1 leads to an imbalance in the age‐related energy metabolic alteration between mitochondrial oxidative phosphorylation and aerobic glycolysis and plays an important role in the extension of both intact and adaptive lifespans.     

Defining the pathogenicity of creatine deficiency syndrome

This work examined nine patients with creatine deficiency syndrome (CDS): six with a creatine transport (CRTR) defect and three with a GAMT defect. Eleven nucleotide variations were detected: six in SLC6A8 and five in GAMT. These changes were analyzed at the mRNA level and specific alleles (most of which bore premature stop codons) were selected as nulls because they provoked nonsense‐mediated decay activation. The impact of these CDS mutations on metabolic stress (ROS production, p38MAPK activation, aberrant proliferation and apoptosis) was analyzed in patient fibroblast cultures. Oxidative stress contributed toward the severe form of CDS, with increases seen in the intracellular ROS content and the percentage of apoptotic cells. An altered cell cycle was also seen in a number of CRTR and GAMT fibroblast cell lines (mostly those carrying null alleles). p38MAPK activation only correlated with oxidative stress in the CRTR cells. Based on intracellular creatine levels, the contribution of energy depletion toward metabolic stress was demonstrable only in selected CRTR cells. Together, these findings suggest that the apoptotic response to genotoxic damage in the present CDS cells may have been triggered by different cell signaling pathways. They also suggest that reducing oxidative stress could be helpful in treating CDS. Hum Mutat 32:1–10, 2011. © 2011 Wiley‐Liss, Inc.     

Gp91<Superscript>phox</Superscript> (NOX2) in classically activated microglia exacerbates traumatic brain injury

Background  

We hypothesized that gp91^phox (NOX2), a subunit of NADPH oxidase, generates superoxide anion (O₂ ^-) and has a major causative role in traumatic brain injury (TBI). To evaluate the functional role of gp91^phox and reactive oxygen species (ROS) on TBI, we carried out controlled cortical impact in gp91^phox knockout mice (gp91^phox-/-). We also used a microglial cell line to determine the activated cell phenotype that contributes to gp91^phox generation.     

Macrophage‐derived reactive oxygen species protects against autoimmune priming with a defined polymeric adjuvant

Understanding the nature of adjuvants and the immune priming events in autoimmune diseases, such as rheumatoid arthritis, is a key challenge to identify their aetiology. Adjuvants are, however, complex structures with inflammatory and immune priming properties. Synthetic polymers provide a possibility to separate these functions and allow studies of the priming mechanisms in vivo. A well‐balanced polymer, poly‐N‐isopropyl acrylamide (PNiPAAm) mixed with collagen type II (CII) induced relatively stronger autoimmunity and arthritis compared with more hydrophilic (polyacrylamide) or hydrophobic (poly‐N‐isopropylacrylamide‐co‐poly‐N‐tertbutylacrylamide and poly‐N‐tertbutylacrylamide) polymers. Clearly, all the synthesized polymers except the more hydrophobic poly‐N‐tertbutylacrylamide induced arthritis, especially in Ncf1‐deficient mice, which are deficient in reactive oxygen species (ROS) production. We identified macrophages as the major infiltrating cells present at PNiPAAm‐CII injection sites and demonstrate that ROS produced by the macrophages attenuated the immune response and the development of arthritis. Our results reveal that thermo‐responsive polymers with high immune priming capacity could trigger an autoimmune response to CII and the subsequent arthritis development, in particular in the absence of NOX2 derived ROS. Importantly, ROS from macrophages protected against the autoimmune priming, demonstrating a critical regulatory role of macrophages in immune priming events.