NOX2-Deficient Neutrophils Facilitate Joint Inflammation Through Higher Pro-Inflammatory and Weakened Immune Checkpoint Activities.

Immune-mediated arthritis is an important chronic inflammatory disease of joints causing debilitating morbidity in affected patients. The mechanisms underlying immune-mediated arthritis have been intensively investigated, however the cellular and molecular factors contributing to the joint inflammation in different redox conditions have not been clearly elucidated. Previous research showed that phagocyte-produced reactive oxygen species (ROS) plays an anti-inflammatory role in K/BxN serum-transfer arthritis and NOX2-deficient mice tend to have more severe arthritis. Although many leukocytes play critical roles in the development of immune-mediated arthritis, the role of neutrophils, which are the main producers of ROS in inflammation, is still controversial. We hence assessed the immunomodulatory function of neutrophils from arthritic joints of NOX2-deficient and wild type mice in this study. We found more neutrophils accumulation in NOX2-deficient inflamed joints. RNA-sequencing and quantitative PCR revealed significantly increased expression of acute inflammation genes including IL1b, Cxcl2, Cxcl3, Cxcl10 and Mmp3 in activated neutrophils from the inflamed joints of NOX2-deficient mice. Moreover, gene set enrichment analysis (GSEA) showed enriched gene signatures in type I and II IFN responses, IL-6-JAK-STAT3 signaling pathway and TNF-α signaling pathway via NF-κB in NOX2-deficient neutrophils. In addition, we found that NOX2-deficient neutrophils expressed lower levels of PD-L1 and were less suppressive than WT neutrophils. Moreover, treatment of PD-L1-Fc decreased cytokine expression and ameliorated the severity of inflammatory arthritis. Our results suggest that NOX2-derived ROS is critical for regulating the function and gene expression in arthritic neutrophils. Both the strong pro-inflammatory and weakened anti-inflammatory functions of neutrophils due to abnormal redox regulation may be targets of treatment for immune-mediated arthritis.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

Mice Deficient in NOX2 Display Severe Thymic Atrophy, Lymphopenia, and Reduced Lymphopoiesis in a Zymosan-Induced Model of Systemic Inflammation.

Patients with chronic granulomatous disease (CGD) who have mutated phagocyte NADPH oxidase are susceptible to infections due to reduced reactive oxygen species production and exhibit autoimmune and inflammatory diseases in the absence of evident infection. Neutrophils and macrophages have been extensively studied since phagocyte NADPH oxidase is mainly found only in them, while the impact of its deficiency on lymphocyte cellularity is less well characterized. We showed herein a zymosan-induced systemic inflammation model that CGD mice deficient in the phagocyte NADPH oxidase gp91phox subunit (NOX2) exhibited more severe thymic atrophy associated with peripheral blood and splenic lymphopenia and reduced lymphopoiesis in the bone marrow in comparison with the wild-type mice. Conversely, the zymosan-exposed CGD mice suffered from more remarkable neutrophilic lung inflammation, circulating and splenic neutrophilia, and enhanced granulopoiesis compared with those in zymosan-exposed wild-type mice. Overall, this study provided evidence that NOX2 deficiency exhibits severe thymic atrophy and lymphopenia concomitant with enhanced neutrophilic inflammation in a zymosan-induced systemic inflammation model.

Toll-like receptor 5 deficiency diminishes doxorubicin-induced acute cardiotoxicity in mice.

Rationale: Clinical application of doxorubicin (DOX) is limited by its toxic cardiovascular side effects. Our previous study found that toll-like receptor (TLR) 5 deficiency attenuated cardiac fibrosis in mice. However, the role of TLR5 in DOX-induced cardiotoxicity remains unclear. Methods: To further investigate this, TLR5-deficient mice were subjected to a single intraperitoneal injection of DOX to mimic an acute model. Results: Here, we reported that TLR5 expression was markedly increased in response to DOX injection. Moreover, TLR5 deficiency exerted potent protective effects against DOX-related cardiac injury, whereas activation of TLR5 by flagellin exacerbated DOX injection-induced cardiotoxicity. Mechanistically, the effects of TLR5 were largely attributed to direct interaction with spleen tyrosine kinase to activate NADPH oxidase (NOX) 2, increasing the production of superoxide and subsequent activation of p38. The toxic effects of TLR5 activation in DOX-related acute cardiac injury were abolished by NOX2 deficiency in mice. Our further study showed that neutralizing antibody-mediated TLR5 depletion also attenuated DOX-induced acute cardiotoxicity. Conclusion: These findings suggest that TLR5 deficiency attenuates DOX-induced cardiotoxicity in mice, and targeting TLR5 may provide feasible therapies for DOX-induced acute cardiotoxicity.

NOX2 Is Critical to Endocardial to Mesenchymal Transition and Heart Development.

NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfß2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.

Nox2 dependent redox-regulation of Akt and ERK1/2 to promote left ventricular hypertrophy in dietary obesity of mice.

BACKGROUND: A Nox2 containing NADPH oxidase (Nox2) is involved in the global oxidative stress found in dietary obesity and metabolic disorders. However, the effects of high fat diet (HFD) on cardiac Nox2 activation and signaling in left ventricular hypertrophy (LVH) remain unknown. METHODS: Left ventricular (LV) tissues isolated from C57BL/6J wild-type (WT) and Nox2 knockout (Nox2KO) mice (11 months old, n = 6 per group) after 4 months of HFD treatment were used. Cardiomyocyte sizes were measured digitally on LV cross-sections. The levels of cardiac reactive oxygen species (ROS) production was determined using lucigenin-chemiluminescence and in situ dihydroethidium (DHE) fluorescence. The levels of Nox subunit expression and redox signaling were examined by immunoblotting and immunofluorescence. RESULTS: In comparison to WT normal chow diet control hearts, WT HFD hearts had 1.8-fold increases in cardiomyocyte size, a sign of cardiac hypertrophy, and this was accompanied with ≥2-fold increase in the levels of ROS production, Nox2 expression and the phosphorylation of Akt and ERK1/2. Increased ROS production measured in HFD heart homogenates was inhibited to control levels by Tiron (a cell membrane permeable O2•-scavenger), diphenyleneiodonium (DPI, a flavohaemoprotein inhibitor) and Nox2 ds-tat (a Nox2 assembly inhibitor). However, all of these abnormalities were significantly reduced or absent in Nox2KO hearts under the same HFD. CONCLUSIONS: Nox2 activation in response to dietary obesity and metabolic disorders plays a key role in cardiac oxidative stress, aberrant redox signaling and cardiomyocyte hypertrophy. Knockout of Nox2 protects hearts from oxidative damage associated with obesity and metabolic disorders.

Innate Effector Systems in Primary Human Macrophages Sensitize Multidrug-Resistant Klebsiella pneumoniae to Antibiotics.

Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are difficult to treat with conventional antibiotics. Thus, alternative strategies to control the growth of MDR Klebsiella are warranted. We hypothesized that activation of innate effector systems could sensitize MDR K. pneumoniae to conventional antibiotics. Thus, human primary macrophages were stimulated with compounds known to activate innate immunity (vitamin D3, phenylbutyrate [PBA], and the aroylated phenylenediamine HO53) and then infected with MDR Klebsiella in the presence or absence of antibiotics. Antibiotics alone were ineffective against MDR Klebsiella in the cellular model, whereas vitamin D3, PBA, and HO53 reduced intracellular growth by up to 70%. The effect was further improved when the innate activators were combined with antibiotics. Vitamin D3- and PBA-induced bacterial killing was dependent on CAMP gene expression, whereas HO53 needed the production of reactive oxygen species (ROS), as shown in cells where the CYBB gene was silenced and in cells from a patient with reduced ROS production due to a deletion in the CYBB gene and skewed lyonization. The combination of innate effector activation by vitamin D3, PBA, and HO53 was effective in sensitizing MDR Klebsiella to conventional antibiotics in a primary human macrophage model. This study provides new evidence for future treatment options for K. pneumoniae.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

RATIONALE: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.

The Emerging Roles of Nicotinamide Adenine Dinucleotide Phosphate Oxidase 2 in Skeletal Muscle Redox Signaling and Metabolism.

Significance: Skeletal muscle is a crucial tissue to whole-body locomotion and metabolic health. Reactive oxygen species (ROS) have emerged as intracellular messengers participating in both physiological and pathological adaptations in skeletal muscle. A complex interplay between ROS-producing enzymes and antioxidant networks exists in different subcellular compartments of mature skeletal muscle. Recent evidence suggests that nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are a major source of contraction- and insulin-stimulated oxidants production, but they may paradoxically also contribute to muscle insulin resistance and atrophy. Recent Advances: Pharmacological and molecular biological tools, including redox-sensitive probes and transgenic mouse models, have generated novel insights into compartmentalized redox signaling and suggested that NOX2 contributes to redox control of skeletal muscle metabolism. Critical Issues: Major outstanding questions in skeletal muscle include where NOX2 activation occurs under different conditions in health and disease, how NOX2 activation is regulated, how superoxide/hydrogen peroxide generated by NOX2 reaches the cytosol, what the signaling mediators are downstream of NOX2, and the role of NOX2 for different physiological and pathophysiological processes. Future Directions: Future research should utilize and expand the current redox-signaling toolbox to clarify the NOX2-dependent mechanisms in skeletal muscle and determine whether the proposed functions of NOX2 in cells and animal models are conserved into humans.

Increased ILC3s associated with higher levels of IL-1ß aggravates inflammatory arthritis in mice lacking phagocytic NADPH oxidase.

The role of redox regulation in immune-mediated arthritis has been previously described. However, the relationship between innate immune cells, including innate lymphoid cells (ILCs) and phagocyte-derived ROS, in this process remains unclear. Here, we characterize ILCs and measure the IL-1 family cytokines along with other cytokines relevant to ILC functions and development in serum-induced arthritic joints in wild type and phagocytic NADPH oxidase (NOX2)-deficient Ncf1-/- mice. We found more severe serum-induced joint inflammation and increased NCR+ ILC3s in inflamed joints of Ncf1-/- mice. Furthermore, in vitro stimulation with IL-1ß on Tbet+ ILC1s from joints facilitated their differentiation into ROR-γt+ ILC3s. Moreover, treatment with IL-1 antagonists effectively lowered the proportions of NCR+ ILC3s and IL-17A producing ILC3s in Ncf1-/- arthritic mice and ameliorated the joint inflammation. These results suggest that NOX2 is an essential regulator of ILC transdifferentiation and may mediate this process in a redox-dependent manner through IL-1ß production in the inflammatory joint. Our findings shed important light on the role of ILCs in the initiation and progression in tissue inflammation and delineate a novel innate immune cell-mediated pathogenic mechanism through which redox regulation may determine the direction of immune responses in joints.

Coordination between NADPH oxidase and vascular peroxidase 1 promotes dysfunctions of endothelial progenitor cells in hypoxia-induced pulmonary hypertensive rats.

Previous studies have demonstrated that NADPH oxidase (NOX)/vascular peroxidase (VPO1) pathway - mediated oxidative stress plays an important role in the pathogenesis of multiple cardiovascular diseases. This study aims to evaluate the correlation between NOX/VPO1 pathway and endothelial progenitor cells (EPCs) dysfunctions in hypoxia-induced pulmonary hypertension (PH). The rats were exposed to 10% hypoxia for 3 weeks to establish a PH model, which showed increases in right ventricle systolic pressure, right ventricular and pulmonary vascular remodeling, acceleration in apoptosis and impairment in functions of the peripheral blood derived - EPCs (the reduced abilities in adhesion, migration and tube formation), accompanied by up-regulation of NOX (NOX2 and NOX4) and VPO1. Next, normal EPCs were cultured under hypoxia to induce apoptosis in vitro. Consistent with the in vivo findings, hypoxia enhanced the apoptosis and dysfunctions of EPCs concomitant with an increase in NOX and VPO1 expression, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) production; these phenomena were attenuated by NOX2 or NOX4 siRNA. Knockdown of VPO1 showed similar results to that of NOX siRNA except no effect on NOX expression and H2O2 production. Based on these observations, we conclude that NOX/VPO1 pathway-derived reactive oxygen species promote the oxidative injury and dysfunctions of EPCs in PH, which may contribute to endothelial dysfunctions in PH.

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.

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 (Tregs ) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in Tregs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated Tregs from gp91phox -, p47phox - and p40phox -deficient CGD patients separately. Results show that Treg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91phox -deficiency effector Treg (eTreg ) numbers are decreased. Expression of Treg 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 Treg functionality or activation state. No correlation was seen between eTreg numbers and patients' clinical phenotype. To conclude, the only difference between Tregs from CGD patients and healthy controls is a decrease in circulating eTregs in gp91phox -deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived Tregs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for Tregs in CGD-related autoinflammation/autoimmunity.

Alveolar Macrophage Chemokine Secretion Mediates Neutrophilic Lung Injury in Nox2-Deficient Mice.

Acute lung injury (ALI), developing as a component of the systemic inflammatory response syndrome (SIRS), leads to significant morbidity and mortality. Reactive oxygen species (ROS), produced in part by the neutrophil NADPH oxidase 2 (Nox2), have been implicated in the pathogenesis of ALI. Previous studies in our laboratory demonstrated the development of pulmonary inflammation in Nox2-deficient (gp91phox-/y) mice that was absent in WT mice in a murine model of SIRS. Given this finding, we hypothesized that Nox2 in a resident cell in the lung, specifically the alveolar macrophage, has an essential anti-inflammatory role. Using a murine model of SIRS, we examined whole-lung digests and bronchoalveolar lavage fluid (BALf) from WT and gp91phox-/y mice. Both genotypes demonstrated neutrophil sequestration in the lung during SIRS, but neutrophil migration into the alveolar space was only present in the gp91phox-/y mice. Macrophage inflammatory protein (MIP)-1α gene expression and protein secretion were higher in whole-lung digest from uninjected gp91phox-/y mice compared to the WT mice. Gene expression of MIP-1α, MCP-1, and MIP-2 was upregulated in alveolar macrophages obtained from gp91phox-/y mice at baseline compared with WT mice. Further, ex vivo analysis of alveolar macrophages, but not bone marrow-derived macrophages or peritoneal macrophages, demonstrated higher gene expression of MIP-1α and MIP-2. Moreover, isolated lung polymorphonuclear neutrophils migrate to BALf obtained from gp91phox-/y mice, further providing evidence of a cell-specific anti-inflammatory role for Nox2 in alveolar macrophages. We speculate that Nox2 represses the development of inflammatory lung injury by modulating chemokine expression by the alveolar macrophage.

Pulmonary metastatic colonisation and granulomas in NOX2-deficient mice.

Metastasis is the leading cause of death in cancer patients, and successful colonisation of a secondary organ by circulating tumour cells (CTCs) is the rate-limiting step of this process. We used tail-vein injection of B16-F10 melanoma cells into mice to mimic the presence of CTCs and to allow for the assessment of host (microenvironmental) factors that regulate pulmonary metastatic colonisation. We found that mice deficient for the individual subunits of the NADPH oxidase of myeloid cells, NOX2 (encoded by Cyba, Cybb, Ncf1, Ncf2, and Ncf4), all showed decreased pulmonary metastatic colonisation. To understand the role of NOX2 in controlling tumour cell survival in the pulmonary microenvironment, we focused on Cyba-deficient (Cybatm1a ) mice, which showed the most significant decrease in metastatic colonisation. Interestingly, histological assessment of pulmonary metastatic colonisation was not possible in Cybatm1a mice, owing to the presence of large granulomas composed of galectin-3 (Mac-2)-positive macrophages and eosinophilic deposits; granulomas of variable penetrance and severity were also found in Cybatm1a mice that were not injected with melanoma cells, and these contributed to their decreased survival. The decreased pulmonary metastatic colonisation of Cybatm1a mice was not due to any overt defects in vascular permeability, and bone marrow chimaeras confirmed a role for the haematological system in the reduced metastatic colonisation phenotype. Examination of the lymphocyte populations, which are known key regulators of metastatic colonisation, revealed an enhanced proportion of activated T and natural killer cells in the lungs of Cybatm1a mice, relative to controls. The reduced metastatic colonisation, presence of granulomas and altered immune cell populations observed in Cybatm1a lungs were mirrored in Ncf2-deficient (Ncf2tm1a ) mice. Thus, we show that NOX2 deficiency results in both granulomas and the accumulation of antitumoural immune cells in the lungs that probably mediate the decreased pulmonary metastatic colonisation. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

[Viewing sepsis and autoimmune disease in relation with infection and NETs-formation].

Neutrophil has been widely recognized as body's first line of defence against pathogens. NETosis was first reported in 2004 as a programmed cell death of neutrophil and distinguished from apoptosis and necrosis. This phenomenon has been already observed in both basic and clinical research. NETosis is induced by various stimulants such as PMA, IL-8, DAMPs/PAMPs, bacteria, and antigen-antibody complex including self-antibody such as ANCA. It is known that there are two types of NETosis following bacterial infections. Although both of them have the ability to capture and kill bacteria, they also damage the host tissues. The inhibition of the NETs-related enzymes prevents the NETs formation at that time. The production of O2- from respiratory burst of neutrophils triggers NETs formation. In the first type of NETosis, neutrophils are completely collapsed, while in the second type, they maintain the morphology and the ability of phagocytosis. However, bacteria can escape from NETs by degrading NETs with their secreting nucleases. Thus the animal models of infection, using these bacteria, oftentimes suffer from severe infectious diseases. Human CGD (Chronic Granulomatosis Disease) patients who do not have Nox2 are immunocompromised, and highly susceptible to infection due to the defect of NETs formation. On the other hand, SLE patients are unable to break down the NETs as their serum inhibits the DNase1 activity, which results in autoantibody generation against NETs as well as self-DNA. It is getting clear that there is a relationship between inflammatory diseases, including infectious diseases, Sepsis and autoimmune diseases, and NETs. Therefore, it is important to re-evaluate the inflammatory disorders from NETs' perspective, and to incorporate the emerging concepts for better understanding the mechanisms involved.

Nox2 in regulatory T cells promotes angiotensin II-induced cardiovascular remodeling.

The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II-induced (Ang II-induced) pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline, whereas Ang II-induced effector T cell (Teff) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of Ang II-induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 antibody depletion of Tregs. Mechanistically, Nox2-/y Tregs showed higher in vitro suppression of Teff proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on Ang II-induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling.

Reactive oxygen species regulate axonal regeneration through the release of exosomal NADPH oxidase 2 complexes into injured axons.

Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-ß1-dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K-phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2-PI3K-p-Akt signalling pathway.

A combination antioxidant therapy to inhibit NOX2 and activate Nrf2 decreases secondary brain damage and improves functional recovery after traumatic brain injury.

Uncontrolled oxidative stress contributes to the secondary neuronal death that promotes long-term neurological dysfunction following traumatic brain injury (TBI). Surprisingly, both NADPH oxidase 2 (NOX2) that increases and transcription factor Nrf2 that decreases reactive oxygen species (ROS) are induced after TBI. As the post-injury functional outcome depends on the balance of these opposing molecular pathways, we evaluated the effect of TBI on the motor and cognitive deficits and cortical contusion volume in NOX2 and Nrf2 knockout mice. Genetic deletion of NOX2 improved, while Nrf2 worsened the post-TBI motor function recovery and lesion volume indicating that decreasing ROS levels might be beneficial after TBI. Treatment with either apocynin (NOX2 inhibitor) or TBHQ (Nrf2 activator) alone significantly improved the motor function after TBI, but had no effect on the lesion volume, compared to vehicle control. Whereas, the combo therapy (apocynin + TBHQ) given at either 5 min/24 h or 2 h/24 h improved motor and cognitive function and decreased cortical contusion volume compared to vehicle group. Thus, both the generation and disposal of ROS are important modulators of oxidative stress, and a combo therapy that prevents ROS formation and potentiates ROS disposal concurrently is efficacious after TBI.