A nox2/cybb zebrafish mutant with defective myeloid cell reactive oxygen species production displays normal initial neutrophil recruitment to sterile tail injuries.

Reactive oxygen species are important effectors and modifiers of the acute inflammatory response, recruiting phagocytes including neutrophils to sites of tissue injury. In turn, phagocytes such as neutrophils are both consumers and producers of reactive oxygen species. Phagocytes including neutrophils generate reactive oxygen species in an oxidative burst through the activity of a multimeric phagocytic nicotinamide adenine dinucleotide phosphate oxidase complex. Mutations in the NOX2/CYBB (previously gp91phox) nicotinamide adenine dinucleotide phosphate oxidase subunit are the commonest cause of chronic granulomatous disease, a disease characterized by infection susceptibility and an inflammatory phenotype. To model chronic granulomatous disease, we made a nox2/cybb zebrafish (Danio rerio) mutant and demonstrated it to have severely impaired myeloid cell reactive oxygen species production. Reduced early survival of nox2 mutant embryos indicated an essential requirement for nox2 during early development. In nox2/cybb zebrafish mutants, the dynamics of initial neutrophil recruitment to both mild and severe surgical tailfin wounds was normal, suggesting that excessive neutrophil recruitment at the initiation of inflammation is not the primary cause of the "sterile" inflammatory phenotype of chronic granulomatous disease patients. This nox2 zebrafish mutant adds to existing in vivo models for studying reactive oxygen species function in myeloid cells including neutrophils in development and disease.

Structural basis for EROS binding to human phagocyte NADPH oxidase NOX2.

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.

Microglial gp91phox-mediated neuroinflammation and ferroptosis contributes to learning and memory deficits in rotenone-treated mice.

Apart from dopaminergic neurotoxicity, exposure to rotenone, a commonly used insecticide in agriculture, also adversely affects hippocampal and cortical neurons, resulting in cognitive impairments in mice. We recently established a role of microglia-mediated neuroinflammation in rotenone-elicited deficits of cognition, yet the mechanisms remain elusive. Here, we investigated the involvement of NADPH oxidase 2 (NOX2) catalytic subunit gp91phox in rotenone-induced cognitive deficits and the associated mechanisms. Our study demonstrated that rotenone exposure elevated expression of gp91phox and phosphorylation of the NOX2 cytosolic subunit p47phox, along with NADPH depletion in the hippocampus and cortex of mice, indicating NOX2 activation. Specific knockdown of gp91phox in microglia via adeno-associated virus delivery resulted in reduced microglial activation, proinflammatory gene expression and improved learning and memory capacity in rotenone-intoxicated mice. Genetic deletion of gp91phox also reversed rotenone-elicited cognitive dysfunction in mice. Furthermore, microglial gp91phox knockdown attenuated neuronal damage and synaptic loss in mice. This intervention also suppressed iron accumulation, disruption of iron-metabolism proteins and iron-dependent lipid peroxidation and restored the balance of ferroptosis-related parameters, including GPX4, SLC711, PTGS2, and ACSL4 in rotenone-lesioned mice. Intriguingly, pharmacological inhibition of ferroptosis with liproxstatin-1 conferred protection against rotenone-induced neurodegeneration and cognitive dysfunction in mice. In summary, our findings underscored the contribution of microglial gp91phox-dependent neuroinflammation and ferroptosis to learning and memory dysfunction in rotenone-lesioned mice. These results provided valuable insights into the pathogenesis of cognitive deficits associated with pesticide-induced Parkinsonism, suggesting potential therapeutic avenues for intervention.

Atorvastatin combined with imipenem alleviates lung injury in sepsis by inhibiting neutrophil extracellular trap formation via the ERK/NOX2 signaling pathway.

Sepsis is a systemic inflammatory response syndrome caused by the invasion of pathogenic microorganisms. Despite major advances in diagnosis and technology, morbidity and mortality remain high. The level of neutrophil extracellular traps (NETs) is closely associated with the progression and prognosis of sepsis, suggesting the regulation of NET formation as a new strategy in sepsis treatment. Owing to its pleiotropic effects, atorvastatin, a clinical lipid-lowering drug, affects various aspects of sepsis-related inflammation and immune responses. To align closely with clinical practice, we combined it with imipenem for the treatment of sepsis. In this study, we used a cecum ligation and puncture-induced lung injury mouse model and employed techniques including western blot, immunofluorescence, and enzyme-linked immunosorbent assay to measure the levels of NETs and other sepsis-related lung injury indicators. Our findings indicate that atorvastatin effectively inhibited the formation of NETs. When combined with imipenem, it significantly alleviated lung injury, reduced systemic inflammation, and improved the 7-day survival rate of septic mice. Additionally, we explored the inhibitory mechanism of atorvastatin on NET formation in vitro, revealing its potential action through the ERK/NOX2 pathway. Therefore, atorvastatin is a potential immunomodulatory agent that may offer new treatment strategies for patients with sepsis in clinical settings.

Inhibition of NADPH oxidase 2 enhances resistance to viral neuroinflammation by facilitating M1-polarization of macrophages at the extraneural tissues.

BACKGROUND: Macrophages play a pivotal role in the regulation of Japanese encephalitis (JE), a severe neuroinflammation in the central nervous system (CNS) following infection with JE virus (JEV). Macrophages are known for their heterogeneity, polarizing into M1 or M2 phenotypes in the context of various immunopathological diseases. A comprehensive understanding of macrophage polarization and its relevance to JE progression holds significant promise for advancing JE control and therapeutic strategies. METHODS: To elucidate the role of NADPH oxidase-derived reactive oxygen species (ROS) in JE progression, we assessed viral load, M1 macrophage accumulation, and cytokine production in WT and NADPH oxidase 2 (NOX2)-deficient mice using murine JE model. Additionally, we employed bone marrow (BM) cell-derived macrophages to delineate ROS-mediated regulation of macrophage polarization by ROS following JEV infection. RESULTS: NOX2-deficient mice exhibited increased resistance to JE progression rather than heightened susceptibility, driven by the regulation of macrophage polarization. These mice displayed reduced viral loads in peripheral lymphoid tissues and the CNS, along with diminished infiltration of inflammatory cells into the CNS, thereby resulting in attenuated neuroinflammation. Additionally, NOX2-deficient mice exhibited enhanced JEV-specific Th1 CD4 + and CD8 + T cell responses and increased accumulation of M1 macrophages producing IL-12p40 and iNOS in peripheral lymphoid and inflamed extraneural tissues. Mechanistic investigations revealed that NOX2-deficient macrophages displayed a more pronounced differentiation into M1 phenotypes in response to JEV infection, thereby leading to the suppression of viral replication. Importantly, the administration of H2O2 generated by NOX2 was shown to inhibit M1 macrophage polarization. Finally, oral administration of the ROS scavenger, butylated hydroxyanisole (BHA), bolstered resistance to JE progression and reduced viral loads in both extraneural tissues and the CNS, along with facilitated accumulation of M1 macrophages. CONCLUSION: In light of our results, it is suggested that ROS generated by NOX2 play a role in undermining the control of JEV replication within peripheral extraneural tissues, primarily by suppressing M1 macrophage polarization. Subsequently, this leads to an augmentation in the viral load invading the CNS, thereby facilitating JE progression. Hence, our findings ultimately underscore the significance of ROS-mediated macrophage polarization in the context of JE progression initiated JEV infection.

Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

Impact of Surgery-Induced Myeloid-derived Suppressor Cells and the NOX2/ROS Axis on Postoperative Survival in Human Pancreatic Cancer.

Preclinical studies imply that surgery triggers inflammation that may entail tumor outgrowth and metastasis. The potential impact of surgery-induced inflammation in human pancreatic cancer is insufficiently explored. This study included 17 patients with periampullary cancer [pancreatic ductal adenocarcinoma (PDAC) n = 14, ampullary carcinoma n = 2, cholangiocarcinoma n = 1] undergoing major pancreatic cancer surgery with curative intent. We analyzed the potential impact of preoperative and postoperative immune phenotypes and function on postoperative survival with >30 months follow-up. The surgery entailed prompt expansion of monocytic myeloid-derived suppressor cells (M-MDSC) that generated NOX2-derived reactive oxygen species (ROS). Strong induction of immunosuppressive M-MDSC after surgery predicted poor postoperative survival and coincided with reduced functionality of circulating natural killer (NK) cells. The negative impact of surgery-induced M-MDSC on survival remained significant in separate analysis of patients with PDAC. M-MDSC-like cells isolated from patients after surgery significantly suppressed NK cell function ex vivo, which was reversed by inhibition of NOX2-derived ROS. High NOX2 subunit expression within resected tumors from patients with PDAC correlated with poor survival whereas high expression of markers of cytotoxic cells associated with longer survival. The surgery-induced myeloid inflammation was recapitulated in vivo in a murine model of NK cell-dependent metastasis. Surgical stress thus induced systemic accumulation of M-MDSC-like cells and promoted metastasis of NK cell-sensitive tumor cells. Genetic or pharmacologic suppression of NOX2 reduced surgery-induced inflammation and distant metastasis in this model. We propose that NOX2-derived ROS generated by surgery-induced M-MDSC may be targeted for improved outcome after pancreatic cancer surgery. SIGNIFICANCE: Pancreatic cancer surgery triggered pronounced accumulation of NOX2+ myeloid-derived suppressor cells that inhibited NK cell function and negatively prognosticated postoperative patient survival. We propose the targeting of M-MDSC as a conceivable strategy to reduce postoperative immunosuppression in pancreatic cancer.

NADPH oxidase 2 mediates cardiac sympathetic denervation and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced cardiomyopathy.

Doxorubicin has been used extensively as a potent anticancer agent, but its clinical use is limited by its cardiotoxicity. However, the underlying mechanisms remain to be fully elucidated. In this study, we tested whether NADPH oxidase 2 (Nox2) mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy, resulting in cardiac atrophy and dysfunction in doxorubicin-induced heart failure. Nox2 knockout (KO) and wild-type (WT) mice were randomly assigned to receive a single injection of doxorubicin (15 mg/kg, i.p.) or saline. WT doxorubicin mice exhibited the decreases in survival rate, left ventricular (LV) wall thickness and LV fractional shortening and the increase in the lung wet-to-dry weight ratio 1 week after the injections. These alterations were attenuated in Nox2 KO doxorubicin mice. In WT doxorubicin mice, myocardial oxidative stress was increased, myocardial noradrenergic nerve fibers were reduced, myocardial expression of PGP9.5, GAP43, tyrosine hydroxylase and norepinephrine transporter was decreased, and these changes were prevented in Nox2 KO doxorubicin mice. Myocyte autophagy was increased and myocyte size was decreased in WT doxorubicin mice, but not in Nox2 KO doxorubicin mice. Nox2 mediates cardiac sympathetic nerve terminal abnormalities and myocyte autophagy-both of which contribute to cardiac atrophy and failure after doxorubicin treatment.

The NADPH oxidase 2 subunit p47phox binds to the WAVE regulatory complex and p22phox in a mutually exclusive manner.

The actin cytoskeleton and reactive oxygen species (ROS) both play crucial roles in various cellular processes. Previous research indicated a direct interaction between two key components of these systems: the WAVE1 subunit of the WAVE regulatory complex (WRC), which promotes actin polymerization and the p47phox subunit of the NADPH oxidase 2 complex (NOX2), which produces ROS. Here, using carefully characterized recombinant proteins, we find that activated p47phox uses its dual Src homology 3 domains to bind to multiple regions within the WAVE1 and Abi2 subunits of the WRC, without altering WRC's activity in promoting Arp2/3-mediated actin polymerization. Notably, contrary to previous findings, p47phox uses the same binding pocket to interact with both the WRC and the p22phox subunit of NOX2, albeit in a mutually exclusive manner. This observation suggests that when activated, p47phox may separately participate in two distinct processes: assembling into NOX2 to promote ROS production and engaging with WRC to regulate the actin cytoskeleton.

Food therapy of scutellarein ameliorates pirarubicin­induced cardiotoxicity in rats by inhibiting apoptosis and ferroptosis through regulation of NOX2­induced oxidative stress.

Pirarubicin (THP) is one of the most commonly used antineoplastic drugs in clinical practice. However, its clinical application is limited due to its toxic and heart­related side effects. It has been reported that oxidative stress, inflammation and apoptosis are closely associated with cardiotoxicity caused by pirarubicin (CTP). Additionally, it has also been reported that scutellarein (Sc) exerts anti­inflammatory, antioxidant, cardio­cerebral vascular protective and anti­apoptotic properties. Therefore, the present study aimed to investigate the effect of food therapy with Sc on CTP and its underlying molecular mechanism using echocardiography, immunofluorescence, western blot, ROS staining, and TUNEL staining. The in vivo results demonstrated that THP was associated with cardiotoxicity. Additionally, abnormal changes in the expression of indicators associated with oxidative stress, ferroptosis and apoptosis were observed, which were restored by Sc. Therefore, it was hypothesized that CTP could be associated with oxidative stress, ferroptosis and apoptosis. Furthermore, the in vitro experiments showed that Sc and the NADPH oxidase 2 (NOX2) inhibitor, GSK2795039 (GSK), upregulated glutathione peroxidase 4 (GPX4) and inhibited THP­induced oxidative stress, apoptosis and ferroptosis. However, cell treatment with the ferroptosis inhibitor, ferrostatin­1, or inducer, erastin, could not significantly reduce or promote, respectively, the expression of NOX2. However, GSK significantly affected ferroptosis and GPX4 expression. Overall, the results of the present study indicated that food therapy with Sc ameliorated CTP via inhibition of apoptosis and ferroptosis through regulation of NOX2­induced oxidative stress, thus suggesting that Sc may be a potential therapeutic drug against CTP.

Preimplantation genetic testing for X-linked chronic granulomatous disease induced by a CYBB gene variant: A case report.

INTRODUCTION: X-linked recessive chronic granulomatous disease (XR-CGD) is a severe primary immunodeficiency principally caused by a CYBB (OMIM: 300481) gene variant. Recurrent fatal bacterial or fungal infections are the main clinical manifestations of XR-CGD. PATIENT CONCERNS: In the current case, in vitro fertilization (IVF) associated with preimplantation genetic testing for monogenic disorder (PGT-M) was applied for a Chinese couple who had given birth to a boy with XR-CGD. DIAGNOSIS: Next-generation sequencing-based SNP haplotyping and Sanger-sequencing were used to detect the CYBB gene variant (c.804 + 2T>C, splicing) in this family. INTERVENTIONS: The patient was treated with IVF and PGT-M successively. OUTCOMES: In this IVF cycle, 7 embryos were obtained, and 2 of them were euploid and lacked the CYBB gene variant (c.804 + 2T>C). The PGT results were verified by prenatal diagnosis after successful pregnancy, and a healthy girl was eventually born. CONCLUSION: PGT-M is an effective method for helping families with these fatal and rare inherited diseases to have healthy offspring. It can availably block the transmission of disease-causing loci to descendant.

Rebalancing NOX2/Nrf2 to limit inflammation and oxidative stress across gut-brain axis in migraine.

Migraine is one of the most common neurological illnesses, and it is characterized by complicated neurobiology. It was confirmed the influence of inflammation and oxidative stress in migraines and also in distal organs such as the intestine. Indeed, the constant bidirectional communication between the Central Nervous System (CNS) and the gastrointestinal (GI) tract, known as the gut-brain axis, has become an attractive target involved in different human disorders. Herein, we explored the role of NADPH oxidase 2 (NOX2) in nitroglycerin (NTG)-induced migraine in mice models to discover the mechanism by which, during migraine attack, oxidative stress is sustained within trigeminal neurons and GI. Considering the inverse relationship between NOX2 and Nrf2, Nrf2 upregulation seems to be a promising approach to decrease NOX2 expression and consequently limit oxidative stress and inflammation spread in neurological and non-neurological diseases. With this aim, we exploited tempol's Nrf2-inducer ability to better understand the involvement of Nrf2/NOX2 axis in migraine and associated GI comorbidities. Behavioral tests confirmed that tempol, in a dose-dependent manner, moderated clinical signs of migraine and abdominal pain. Moreover, we demonstrated that the decrease in migraine-related symptomatology was strongly linked to the modulation of Nrf2/NOX2 signaling pathway in the brain and colon. In the brain, the rebalancing of Nrf2/NOX2 prevented neuronal loss, decreased glia reactivity while inhibiting NF-κB and NLRP3 inflammasome activation. In the colon, Nrf2 upregulation and consequent NOX2 decrease reduced the histological damage, mast cells infiltration as well as tumor necrosis factor (TNF)-α and interleukin (IL)-1ß release. Furthermore, the attenuation of inflammation and oxidative stress led to the restoration of the intestinal barrier through TJs replacement. Taken as a whole, data suggested that the regulation of Nrf2/NOX2 balance is a successful way to reduce neurological and related intestinal impairments during migraine and could be of relevance for migraine-like attacks in humans.

Machine learning-based identification of CYBB and FCAR as potential neutrophil extracellular trap-related treatment targets in sepsis.

Objective: Sepsis related injury has gradually become the main cause of death in non-cardiac patients in intensive care units, but the underlying pathological and physiological mechanisms remain unclear. The Third International Consensus Definitions for Sepsis and Septic Shock (SEPSIS-3) definition emphasized organ dysfunction caused by infection. Neutrophil extracellular traps (NETs) can cause inflammation and have key roles in sepsis organ failure; however, the role of NETs-related genes in sepsis is unknown. Here, we sought to identify key NETs-related genes associate with sepsis. Methods: Datasets GSE65682 and GSE145227, including data from 770 patients with sepsis and 54 healthy controls, were downloaded from the GEO database and split into training and validation sets. Differentially expressed genes (DEGs) were identified and weighted gene co-expression network analysis (WGCNA) performed. A machine learning approach was applied to identify key genes, which were used to construct functional networks. Key genes associated with diagnosis and survival of sepsis were screened out. Finally, mouse and human blood samples were collected for RT-qPCR verification and flow cytometry analysis. Multiple organs injury, apoptosis and NETs expression were measured to evaluated effects of sulforaphane (SFN). Results: Analysis of the obtained DEGs and WGCNA screened a total of 3396 genes in 3 modules, and intersection of the results of both analyses with 69 NETs-related genes, screened out seven genes (S100A12, SLC22A4, FCAR, CYBB, PADI4, DNASE1, MMP9) using machine learning algorithms. Of these, CYBB and FCAR were independent predictors of poor survival in patients with sepsis. Administration of SFN significantly alleviated murine lung NETs expression and injury, accompanied by whole blood CYBB mRNA level. Conclusion: CYBB and FCAR may be reliable biomarkers of survival in patients with sepsis, as well as potential targets for sepsis treatment. SFN significantly alleviated NETs-related organs injury, suggesting the therapeutic potential by targeting CYBB in the future.

NOX2 control over energy metabolism plays a role in acute myeloid leukaemia prognosis and survival.

Acute myeloid leukaemia (AML) is a highly heterogeneous disease, however the therapeutic approaches have hardly changed in the last decades. Metabolism rewiring and the enhanced production of reactive oxygen species (ROS) are hallmarks of cancer. A deeper understanding of these features could be instrumental for the development of specific AML-subtypes treatments. NADPH oxidases (NOX), the only cellular system specialised in ROS production, are also involved in leukemic metabolism control. NOX2 shows a variable expression in AML patients, so patients can be classified based on such difference. Here we have analysed whether NOX2 levels are important for AML metabolism control. The lack of NOX2 in AML cells slowdowns basal glycolysis and oxidative phosphorylation (OXPHOS), along with the accumulation of metabolites that feed such routes, and a sharp decrease of glutathione. In addition, we found changes in the expression of 725 genes. Among them, we have discovered a panel of 30 differentially expressed metabolic genes, whose relevance was validated in patients. This panel can segregate AML patients according to CYBB expression, and it can predict patient prognosis and survival. In summary, our data strongly support the relevance of NOX2 for AML metabolism, and highlights the potential of our discoveries in AML prognosis.

Myelodysplasia after clonal hematopoiesis with APOBEC3-mediated CYBB inactivation in retroviral gene therapy for X-CGD.

Stem cell gene therapy using the MFGS-gp91phox retroviral vector was performed on a 27-year-old patient with X-linked chronic granulomatous disease (X-CGD) in 2014. The patient's refractory infections were resolved, whereas the oxidase-positive neutrophils disappeared within 6 months. Thirty-two months after gene therapy, the patient developed myelodysplastic syndrome (MDS), and vector integration into the MECOM locus was identified in blast cells. The vector integration into MECOM was detectable in most myeloid cells at 12 months after gene therapy. However, the patient exhibited normal hematopoiesis until the onset of MDS, suggesting that MECOM transactivation contributed to clonal hematopoiesis, and the blast transformation likely arose after the acquisition of additional genetic lesions. In whole-genome sequencing, the biallelic loss of the WT1 tumor suppressor gene, which occurred immediately before tumorigenesis, was identified as a potential candidate genetic alteration. The provirus CYBB cDNA in the blasts contained 108 G-to-A mutations exclusively in the coding strand, suggesting the occurrence of APOBEC3-mediated hypermutations during the transduction of CD34-positive cells. A hypermutation-mediated loss of oxidase activity may have facilitated the survival and proliferation of the clone with MECOM transactivation. Our data provide valuable insights into the complex mechanisms underlying the development of leukemia in X-CGD gene therapy.

Novel mutations in CYBB Gene Cause X-linked chronic Granulomatous Disease in Pakistani patients.

BACKGROUND: Chronic Granulomatous Disease (CGD) is a primary immunodeficiency that causes susceptibility to recurrent fungal and bacterial infections. The CYBB gene encodes gp91phox component of the Phagocytic Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and specifically, X-linked CGD is caused by mutations in the CYBB gene, located on the X chromosome. The aim of the study was to characterize functional and genetic mutations in X-linked CGD. METHODS: Functional analysis was conducted on the whole blood of seventeen male individuals who were suspected to have X-linked chronic granulomatous disease (CGD). Flow cytometry was employed to assess the capacity of NADPH oxidase, measuring both H2O2 production and gp91phox protein expression in neutrophils. Additionally, DNA Sanger sequencing was performed for genetic analysis. The pathogenicity of novel mutations was assessed by pathogenicity prediction tools. RESULT: Among the seventeen patients evaluated, five patients (P1, P2, P3, P4, and P5) displayed impaired H2O2 production by their neutrophils upon stimulation with Phorbol myristate acetate (PMA), accompanied by abnormal gp91phox expression. DNA sequencing of the CYBB gene identified specific mutations in each patient. In P1 and P2 (previously reported cases), a hemizygous missense mutation, c.925G > A/p.E309K was identified. In P3 and P4 (novel cases), hemizygous nonsense mutations, c.216T > A/p.C72X were found. Lastly, in P5 (also a novel case), a hemizygous missense mutation, c.732T > G/p.C244W was detected. These mutations reside in exons 9,3 and 7 of the CYBB gene, respectively. CONCLUSIONS: The current study contributes to the understanding of the clinical and genetic spectrum associated with X-linked chronic granulomatous disease (CGD). It highlights the significance of early diagnosis in CGD and emphasizes the importance of lifelong prophylaxis to prevent severe infections.

Hematopoietic stem cell transplantation for CYBB heterozygous mutation resulting in very early onset inflammatory bowel disease in children: a case report.

BACKGROUND: Inflammatory bowel disease (IBD) is a heterogeneous group of disorders associated with environmental triggers and dysregulated immune responses resulting in chronic, recurrent intestinal inflammation. Very early-onset IBD (VEO-IBD) refers to patients with symptoms or diagnosis before the age of 6 years and is widely thought to be associated with monogenic mutations. Traditional drug therapy is often ineffective in this patient population, while hematopoietic stem cell transplantation (HSCT) represents the definitive cure for patients with gene mutations. CASE PRESENTATION: We report a case of VEO-IBD associated with a monogenic mutation in a 2-year-old girl presenting mainly with gastrointestinal symptoms, including recurrent hematochezia and abdominal pain for more than 3 months. A gastroscopy revealed erosive gastritis and bulbar duodenitis, while a colonoscopy indicated erosive colitis. Abnormal results were obtained from the dihydrohodamine (DHR) assay and immunoglobulin testing. Whole-exome sequencing identified a heterozygous and de novo nonsense mutation (c.388 C > T; p.R130X) in the CYBB gene leading to deficiency of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) (encoded by CYBB), a critical component of phagocytes. HSCT was performed successfully, and the DHR assay showed that normal neutrophil function was restored. Six months after HSCT, clinical remission was observed, and a repeat colonoscopy revealed intestinal mucosal healing was attained. CONCLUSIONS: Patients with CYBB mutations often develop recurrent or severe bacterial or fungal infections, mostly in the lungs, skin, lymph nodes, and liver. Here, we report on a young female child with CYBB mutations presenting predominantly with gastrointestinal symptoms. This study explores the mechanisms of inflammatory bowel disease caused by a monogenic mutation in CYBB to improve early diagnosis and effective treatment rates of this patient population.

Functional NADPH oxidase 2 in T cells amplifies salt-sensitive hypertension and associated renal damage.

Infiltrating T cells in the kidney amplify salt-sensitive (SS) hypertension and renal damage, but the mechanisms are not known. Genetic deletion of T cells (SSCD247-/-) or of the p67phox subunit of NADPH oxidase 2 (NOX2; SSp67phox-/-) attenuates SS hypertension in the Dahl SS rat. We hypothesized that reactive oxygen species produced by NOX2 in T cells drive the SS phenotype and renal damage. T cells were reconstituted by adoptively transferring splenocytes (∼10 million) from the Dahl SS (SS→CD247) rat, the SSp67phox-/- rat (p67phox→CD247), or only PBS (PBS→CD247) into the SSCD247-/- rat on postnatal day 5. Animals were instrumented with radiotelemeters and studied at 8 wk of age. There were no detectable differences in mean arterial pressure (MAP) or albuminuria between groups when rats were maintained on a low-salt (0.4% NaCl) diet. After 21 days of high-salt diet (4.0% NaCl), MAP and albuminuria were significantly greater in SS→CD247 rats compared with p67phox→CD247 and PBS→CD247 rats. Interestingly, there was no difference between p67phox→CD247 and PBS→CD247 rats in albuminuria or MAP after 21 days. The lack of CD3+ cells in PBS→CD247 rats and the presence of CD3+ cells in rats that received the T cell transfer demonstrated the effectiveness of the adoptive transfer. No differences in the number of CD3+, CD4+, or CD8+ cells were observed in the kidneys of SS→CD247 and p67phox→CD247 rats. These results indicate that reactive oxygen species produced by NOX2 in T cells participates in the amplification of SS hypertension and renal damage.NEW & NOTEWORTHY Our current work used the adoptive transfer of T cells that lack functional NADPH oxidase 2 into a genetically T cell-deficient Dahl salt-sensitive (SS) rat model. The results demonstrated that reactive oxygen species produced by NADPH oxidase 2 in T cells participate in the amplification of SS hypertension and associated renal damage and identifies a potential mechanism that exacerbates the salt-sensitive phenotype.

The Expression and Role of NADPH Oxidase 2 in Colon Cancer.

BACKGROUND/AIM: Recent studies have reported that nicotinamide adenine dinucleotide phosphate oxidases (NOXs) are expressed in various cancers and play important roles in tumor progression. However, no studies have examined the expression and role of NOX2 in colon cancer. The aim of this study is to investigate the pathophysiological roles of NOX2 in colon cancer patients and cell lines. PATIENTS AND METHODS: One-hundred and sixteen primary colon cancer samples of patients who underwent radical resection for locally advanced colon cancer were used for immunohistochemistry of NOX2 protein. The relationship between NOX2 expression and clinicopathological factors was assessed and the prognostic significance of NOX2 expression was evaluated in colon cancer patients. NOX2 siRNA transfection experiments were performed using two colon cancer cell lines (HCT116 and RKO) to analyze the impact of NOX2 expression on cellular physiological functions. RESULTS: The expression of NOX2 protein in noncancerous tissue was scarcely observed, and 45 samples (38.8%) showed positively stained NOX2 expression in cancer tissue. There were no clinicopathological factors significantly associated with NOX2 expression. The 5-year recurrence-free survival rate of the NOX2 positive group was significantly lower than that of the NOX2 negative group (61.1% vs. 79.3%, p=0.029). NOX2 depletion significantly inhibited cell proliferation with G1 arrest, and motility in the two cell lines. CONCLUSION: NOX2 expression level has a close association with the prognosis of colon cancer patients and physiological functions of colon cancer cells. NOX2 may be a useful prognostic biomarker for colon cancer patients.

[A case report of chronic granulomatous disease with CYBB gene mutation in an adult].

We reported a 28-year-old male patient who had been admitted to a local hospital for several times in the past four years because of recurrent fever and cough. Each chest CT scan during hospitalization showed consolidation accompanied by exudation and mild pleural effusion. After treatment, the consolidation apparently absorbed, but similar symptoms recurred within half a year, and the new consolidation appeared. For this reason, he was diagnosed with tuberculosis or bacterial pneumonia several times in other hospitals, and was hospitalized two to three times a year. Finally, he was diagnosed with chronic granulomatous disease (CGD) with CYBB gene mutation through whole-exome sequencing.