NCF4 attenuates colorectal cancer progression by modulating inflammasome activation and immune surveillance.

The spatiotemporal regulation of inflammasome activation remains unclear. To examine the mechanism underlying the assembly and regulation of the inflammasome response, here we perform an immunoprecipitation-mass spectrometry analysis of apoptosis-associated speck-like protein containing a CARD (ASC) and identify NCF4/1/2 as ASC-binding proteins. Reduced NCF4 expression is associated with colorectal cancer development and decreased five-year survival rate in patients with colorectal cancer. NCF4 cooperates with NCF1 and NCF2 to promote NLRP3 and AIM2 inflammasome activation. Mechanistically, NCF4 phosphorylation and puncta distribution switches from the NADPH complex to the perinuclear region, mediating ASC oligomerization, speck formation and inflammasome activation. NCF4 functions as a sensor of ROS levels, to establish a balance between ROS production and inflammasome activation. NCF4 deficiency causes severe colorectal cancer in mice, increases transit-amplifying and precancerous cells, reduces the frequency and activation of CD8+ T and NK cells, and impairs the inflammasome-IL-18-IFN-γ axis during the early phase of colorectal tumorigenesis. Our study implicates NCF4 in determining the spatial positioning of inflammasome assembly and contributing to inflammasome-mediated anti-tumor responses.

Orthologs of NOX5 and EC-SOD/SOD3: dNox and dSod3 Impact Egg Hardening Process and Egg Laying in Reproductive Function of Drosophila melanogaster.

The occurrence of ovarian dysfunction is often due to the imbalance between the formation of reactive oxygen species (ROS) and the ineffectiveness of the antioxidative defense mechanisms. Primary sources of ROS are respiratory electron transfer and the activity of NADPH oxidases (NOX) while superoxide dismutases (SOD) are the main key regulators that control the levels of ROS and reactive nitrogen species intra- and extracellularly. Because of their central role SODs are the subject of research on human ovarian dysfunction but sample acquisition is low. The high degree of cellular and molecular similarity between Drosophila melanogaster ovaries and human ovaries provides this model organism with the best conditions for analyzing the role of ROS during ovarian function. In this study we clarify the localization of the ROS-producing enzyme dNox within the ovaries of Drosophila melanogaster and by a tissue-specific knockdown we show that dNox-derived ROS are involved in the chorion hardening process. Furthermore, we analyze the dSod3 localization and show that reduced activity of dSod3 impacts egg-laying behavior but not the chorion hardening process.

The Characteristics and Expression Analysis of the Tomato SlRBOH Gene Family under Exogenous Phytohormone Treatments and Abiotic Stresses.

Respiratory burst oxidase homologs (RBOHs), also known as NADPH oxidases, contribute significantly to the production of ROS in plants, alongside other major sources such as photosynthesis and electron transport in chloroplasts. It has been shown that plant RBOHs play an active role in plant adversity response and electron transport. However, the phylogenetic analysis and characterization of the SlRBOH gene family in tomatoes have not been systematically studied. This study identified 11 SlRBOH genes in the tomato genome using a genome-wide search approach. The physicochemical properties, chromosomal localization, subcellular localization, secondary structure, conserved motifs, gene structure, phylogenetics, collinear relationships, cis-acting elements, evolutionary selection pressures, tissue expressions, and expression patterns under exogenous phytohormones (ABA and MeJA) and different abiotic stresses were also analyzed. We found that the SlRBOHs are distributed across seven chromosomes, collinearity reflecting their evolutionary relationships with corresponding genes in Arabidopsis thaliana and rice. Additionally, all the SlRBOH members have five conserved domains and 10 conserved motifs and have similar gene structures. In addition, the results of an evolutionary selection pressure analysis showed that SlRBOH family members evolved mainly by purifying selection, making them more structurally stable. Cis-acting element analyses showed that SlRBOHs were responsive to light, hormone, and abiotic stresses. Tissue expression analysis showed that SlRBOH family members were expressed in all tissues of tomato to varying degrees, and most of the SlRBOHs with the strongest expression were found in the roots. In addition, the expressions of tomato SlRBOH genes were changed by ABA, MeJA, dark period extension, NaCl, PEG, UV, cold, heat, and H2O2 treatments. Specifically, SlRBOH4 was highly expressed under NaCl, PEG, heat, and UV treatments, while SlRBOH2 was highly expressed under cold stress. These results provide a basis for further studies on the function of SlRBOHs in tomato.

First Report on Chronic Granulomatous Disease from Nepal and a Review of CYBC1 Deficiency.

Chronic granulomatous disease (CGD) primarily results from inherited defects in components of the nicotinamide adenine dinucleotide phosphate oxidase enzyme complex. These include gene defects in cytochrome B-245/558 subunit α/ß and neutrophil cytosolic factors 1, 2, and 4. Recently, homozygous loss-of-function variants in cytochrome B-245 chaperone 1 gene (CYBC1) have been discovered to cause CGD (CYBC1-CGD). Data on variant-proven CGD from low-income countries, the most underprivileged regions of the world, remain sparse due to numerous constraints. Herein, we report the first cohort of patients with CGD from Nepal, a low-income country in the Himalayas' challenging terrain. Our report includes a description of a new case of CYBC1 deficiency who was first diagnosed with CGD at our center. Only a dozen cases of CYBC1-CGD have been described in the literature thus far which have been reviewed comprehensively herein. Most of these patients have had significant infections and autoimmune/inflammatory manifestations. Pulmonary and invasive/disseminated bacterial/fungal infections were the most common followed by skin and soft-tissue infections. Inflammatory bowel disease (IBD) was the most common inflammatory manifestation (median age at diagnosis: 9 years) followed by episodes of recurrent/prolonged fever. Other autoimmune/inflammatory manifestations reported in CYBC1-CGD include acute pancreatitis, hemophagocytic lymphohistiocytosis, systemic granulomatosis, interstitial lung disease, arthritis, autoimmune hemolytic anemia, uveitis, nephritis, and eczema. Our analysis shows that patients with CYBC1-CGD are at a significantly higher risk of IBD-like illness as compared to other forms of CGD which merits further confirmatory studies in the future.

Immune Thrombocytopenic Purpura (ITP) and Chorioretinopathy in Chronic Granulomatous Disease: A Case Report.

BACKGROUND: Chronic Granulomatous Disease (CGD) is a rare immunodeficiency disorder characterized by impaired phagocytic function, leading to recurrent infections and granuloma formation. Genetic mutations in NADPH oxidase complex components, such as CYBB, NCF1, NCF2, and CYBA genes, contribute to the pathogenesis. This case report explores the possible ocular and hematologic complications associated with CGD. CASE PRESENTATION: A 6-year-old girl with a history of vitrectomy, membranotomy, and laser therapy due to congenital blindness (diagnosed with chorioretinopathy) was referred to the hospital with generalized ecchymosis and thrombocytopenia. Diagnostic workup initially suggested chronic immune thrombocytopenic purpura (ITP). Subsequent admissions revealed necrotic wounds, urinary tract infections, and recurrent thrombocytopenia. Suspecting immunodeficiency, tests for CGD, Nitroblue tetrazolium (NBT) and dihydrorhodamine (DHR) were performed. She had a low DHR (6.7), and her NBT test was negative (0.0%). Her whole exome sequencing results confirmed autosomal recessive CGD with a homozygous NCF1 mutation. CONCLUSION: This case underscores the diverse clinical manifestations of CGD, including recurrent thrombocytopenia and possible early-onset ocular involvement. The diagnostic challenges highlight the importance of a multidisciplinary approach involving hematologists, immunologists, and ophthalmologists for accurate diagnosis and management. The rare coexistence of ITP in CGD emphasizes the intricate link between immunodeficiency and autoimmunity, requiring tailored therapeutic strategies.

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.

LbSakA-mediated phosphorylation of the scaffolding protein LbNoxR in the ectomycorrhizal basidiomycete Laccaria bicolor regulates NADPH oxidase activity, ROS accumulation and symbiosis development.

Ectomycorrhizal symbiosis, which involves mutually beneficial interactions between soil fungi and tree roots, is essential for promoting tree growth. To establish this symbiotic relationship, fungal symbionts must initiate and sustain mutualistic interactions with host plants while avoiding host defense responses. This study investigated the role of reactive oxygen species (ROS) generated by fungal NADPH oxidase (Nox) in the development of Laccaria bicolor/Populus tremula × alba symbiosis. Our findings revealed that L. bicolor LbNox expression was significantly higher in ectomycorrhizal roots than in free-living mycelia. RNAi was used to silence LbNox, which resulted in decreased ROS signaling, limited formation of the Hartig net, and a lower mycorrhizal formation rate. Using Y2H library screening, BiFC and Co-IP, we demonstrated an interaction between the mitogen-activated protein kinase LbSakA and LbNoxR. LbSakA-mediated phosphorylation of LbNoxR at T409, T477 and T480 positively modulates LbNox activity, ROS accumulation and upregulation of symbiosis-related genes involved in dampening host defense reactions. These results demonstrate that regulation of fungal ROS metabolism is critical for maintaining the mutualistic interaction between L. bicolor and P. tremula × alba. Our findings also highlight a novel and complex regulatory mechanism governing the development of symbiosis, involving both transcriptional and posttranslational regulation of gene networks.

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.

Effects of Nox4 upregulation on PECAM-1 expression in a mouse model of diabetic retinopathy.

Diabetic Retinopathy (DR) is the leading cause of vision loss in working-age adults. The hallmark features of DR include vascular leakage, capillary loss, retinal ischemia, and aberrant neovascularization. Although the pathophysiology is not fully understood, accumulating evidence supports elevated reactive oxygen species associated with increased activity of NADPH oxidase 4 (Nox4) as major drivers of disease progression. Previously, we have shown that Nox4 upregulation in retinal endothelial cells by diabetes leads to increased vascular leakage by an unknown mechanism. Platelet endothelial cell adhesion molecule 1 (PECAM-1) is a cell surface molecule that is highly expressed in endothelial cells and regulates endothelial barrier function. In the present study, using endothelial cell-specific human Nox4 transgenic (TG) mice and endothelial cell-specific Nox4 conditional knockout (cKO) mice, we investigated the impact of Nox4 upregulation on PECAM-1 expression in mouse retinas and brain microvascular endothelial cells (BMECs). Additionally, cultured human retinal endothelial cells (HRECs) transduced with adenovirus overexpressing human Nox4 were used in the study. We found that overexpression of Nox4 increases PECAM-1 mRNA but has no effect on its protein expression in the mouse retina, BMECs, or HRECs. Furthermore, PECAM-1 mRNA and protein expression was unchanged in BMECs isolated from cKO mice compared to wild type (WT) mice with or without 2 months of diabetes. Together, these findings do not support a significant role of Nox4 in the regulation of PECAM-1 expression in the diabetic retina and endothelial cells. Further studies are warranted to elucidate the mechanism of Nox4-induced vascular leakage by investigating other intercellular junctional proteins in endothelial cells and their implications in the pathophysiology of diabetic retinopathy.

Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.

Salmonella Typhimurium employs spermidine to exert protection against ROS-mediated cytotoxicity and rewires host polyamine metabolism to ameliorate its survival in macrophages.

Salmonella infection entails a cascade of attacks and defence measures. After breaching the intestinal epithelial barrier, Salmonella is phagocytosed by macrophages, where the bacteria encounter multiple stresses, to which it employs relevant countermeasures. Our study shows that, in Salmonella, the polyamine spermidine activates a stress response mechanism by regulating critical antioxidant genes. Salmonella Typhimurium mutants for spermidine transport and synthesis cannot mount an antioxidative response, resulting in high intracellular ROS levels. These mutants are also compromised in their ability to be phagocytosed by macrophages. Furthermore, it regulates a novel enzyme in Salmonella, Glutathionyl-spermidine synthetase (GspSA), which prevents the oxidation of proteins in E. coli. Moreover, the spermidine mutants and the GspSA mutant show significantly reduced survival in the presence of hydrogen peroxide in vitro and reduced organ burden in the mouse model of Salmonella infection. Conversely, in macrophages isolated from gp91phox-/- mice, we observed a rescue in the attenuated fold proliferation previously observed upon infection. We found that Salmonella upregulates polyamine biosynthesis in the host through its effectors from SPI-1 and SPI-2, which addresses the attenuated proliferation observed in spermidine transport mutants. Thus, inhibition of this pathway in the host abrogates the proliferation of Salmonella Typhimurium in macrophages. From a therapeutic perspective, inhibiting host polyamine biosynthesis using an FDA-approved chemopreventive drug, D, L-α-difluoromethylornithine (DFMO), reduces Salmonella colonisation and tissue damage in the mouse model of infection while enhancing the survival of infected mice. Therefore, our work provides a mechanistic insight into the critical role of spermidine in stress resistance of Salmonella. It also reveals a bacterial strategy in modulating host metabolism to promote their intracellular survival and shows the potential of DFMO to curb Salmonella infection.

Proteasome inhibitors induce apoptosis by superoxide anion generation via NADPH oxidase 5 in human neuroblastoma SH-SY5Y cells.

The ubiquitin-proteasome system (UPS) is a major proteolytic system that plays an important role in the regulation of various cell processes, such as cell cycle, stress response, and transcriptional regulation, especially in neurons, and dysfunction of UPS is considered to be a cause of neuronal cell death in neurodegenerative diseases. However, the mechanism of neuronal cell death caused by UPS dysfunction has not yet been fully elucidated. In this study, we investigated the mechanism of neuronal cell death induced by proteasome inhibitors using human neuroblastoma SH-SY5Y cells. Z-Leu-D-Leu-Leu-al (MG132), a proteasome inhibitor, induced apoptosis in SH-SY5Y cells in a concentration- and time-dependent manner. Antioxidants N-acetylcysteine and EUK-8 attenuated MG132-induced apoptosis. Apocynin and diphenyleneiodonium, inhibitors of NADPH oxidase (NOX), an enzyme that produces superoxide anions, also attenuated MG132-induced apoptosis. It was also found that MG132 treatment increased the expression of NOX5, a NOX family member, and that siRNA-mediated silencing of NOX5 and BAPTA-AM, which inhibits NOX5 by chelating calcium, suppressed MG132-induced apoptosis and production of reactive oxygen species in SH-SY5Y cells. These results suggest that MG132 induces apoptosis in SH-SY5Y cells through the production of superoxide anion by NOX5.

NCF4 regulates antigen presentation of cysteine peptides by intracellular oxidative response and restricts activation of autoreactive and arthritogenic T cells.

Autoimmune diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematous, are regulated by polymorphisms in genes contributing to the NOX2 complex. Mutations in both Ncf1 and Ncf4 affect development of arthritis in experimental models of RA, but the different regulatory pathways mediated by NOX2-derived reactive oxygen species (ROS) have not yet been clarified. Here we address the possibility that intracellular ROS, regulated by the NCF4 protein (earlier often denoted p40phox) which interacts with endosomal membranes, could play an important role in the oxidation of cysteine peptides in mononuclear phagocytic cells, thereby regulating antigen presentation and activation of arthritogenic T cells. To study the role of NCF4 we used mice with an amino acid replacing mutation (NCF4R58A), which is known to affect interaction with endosomal membranes, leading to decreased intracellular ROS production. To study the impact of NCF4 on T cell activation, we used the glucose phosphate isomerase peptide GPI325-339, which contains two cysteine residues (325-339c-c). Macrophages from mice with the NCF458A mutation efficiently presented the peptide when the two cysteines were intact and not crosslinked, leading to a strong arthritogenic T cell response. T cell priming occurred in the draining lymph nodes (LNs) within 8 days after immunization. Clodronate treatment, which depletes antigen-presenting mononuclear phagocytes, ameliorated arthritis severity, whereas treatment with FYT720, which traps activated T cells in LNs, prohibited arthritis. We conclude that NCF4-dependent intracellular ROS maintains cysteine peptides in an oxidized crosslinked state, which prevents presentation of peptides recognized by non-tolerized T cells and thereby protects against autoimmune arthritis.

Reactive oxygen species augment contractile responses of saphenous artery in 10-15-day-old but not adult rats: Substantial role of NADPH oxidases.

Reactive oxygen species (ROS) produced by NADPH oxidases (NOX, a key source of ROS in vascular cells) are involved in the regulation of vascular tone, but this has been explored mainly for adult organisms. Importantly, the mechanisms of vascular tone regulation differ significantly in early postnatal ontogenesis and adulthood, while the vasomotor role of ROS in immature systemic arteries is poorly understood. We tested the hypothesis that the functional contribution of NADPH oxidase-derived ROS to the regulation of peripheral arterial tone is higher in the early postnatal period than in adulthood. We studied saphenous arteries from 10- to 15-day-old ("young") and 3- to 4-month-old ("adult") male rats using lucigenin-enhanced chemiluminescence, quantitative PCR, Western blotting, and isometric myography. We demonstrated that both basal and NADPH-stimulated superoxide anion radical (O2•-) production was significantly higher in the arteries from young in comparison to adult rats. Importantly, pan-inhibitor of NADPH oxidase VAS2870 (10 µM) reduced NADPH-induced O2•- production in arteries of young rats. Saphenous arteries of both young and adult rats demonstrated high levels of Nox2 and Nox4 mRNAs, while Nox1 and Nox3 mRNAs were not detected. The protein contents of NOX2 and NOX4 were significantly higher in arterial tissue of young compared to adult animals. Moreover, VAS2870 (10 µM) had no effect on methoxamine-induced contractile responses of adult arteries but decreased them significantly in young arteries; such effect of VAS2870 persisted after removal of the endothelium. Finally, NOX2 inhibitor GSK2795039 (10 µM), but not NOX1/4 inhibitor GKT137831 (10 µM) weakened methoxamine-induced contractile responses of arteries from young rats. Thus, ROS produced by NOX2 have a pronounced contractile influence in saphenous artery smooth muscle cells of young, but not adult rats, which is associated with the increased vascular content of NOX2 protein at this age.

Changed regulation of granulocyte NADPH oxidase activity in the mouse model of obesity-induced type 2 diabetes mellitus.

NADPH oxidase is a target of hyperglycemia in type 2 diabetes mellitus (T2DM), which causes dysregulation of enzyme. Alterations in regulation of NADPH oxidase activity mediated receptor and non-receptor signaling in bone marrow granulocytes of mice with obesity-induced T2DM were studied. The animals fed high fat diet (516 kcal/100 g) for 16 weeks. NADPH oxidase-related generation of reactive species (RS) at normo- and hyperthermia was estimated using chemiluminescent analysis. The redox status of the cells was assessed by Redox Sensor Red CC-1. Baseline biochemical indicators in blood (glucose, cholesterol, HDL and LDL levels) were significant higher in T2DM mice versus controls. Using specific inhibitors, signaling mediated by formyl peptide receptors (FPRs) to NADPH oxidase was shown to involve PLC, PKC, cytochrome p450 in both control and T2DM groups and PLA2 in controls. In T2DM regulation of NADPH oxidase activity via mFpr1, a high-affinity receptors, occurred with a significant increase of the role of PKC isoforms and suppression of PLA2 participation. Significant differences between this regulation via mFpr2, low-affinity receptors, were not found. Non-receptor activation of NADPH oxidase with ionomycin (Ca2+ ionophore) or phorbol ester (direct activator of PKC isoforms) did not revealed differences in the kinetic parameters between groups at 37 °C and 40 °C. When these agents were used together (synergistic effect), lower sensitivity of cells to ionophore was observed in T2DM at both temperatures. Redox status in responses to opsonized zymosan was higher in T2DM mice at 37 °C and similar to control levels at 40 °C. ROC-analysis identified Tmax, RS production and effect of opsonized zymosan as the most significant predictors for discriminating between groups. It was concluded that Ca2+-dependent/PKC-mediated regulation of NADPH oxidase activity was altered in BM granulocytes from diabetic mice.

Naringenin Inhibits Acid Sphingomyelinase-Mediated Membrane Raft Clustering to Reduce NADPH Oxidase Activation and Vascular Inflammation.

Macrophage inflammation and oxidative stress promote atherosclerosis progression. Naringenin is a naturally occurring flavonoid with antiatherosclerotic properties. Here, we elucidated the effects of naringenin on monocyte/macrophage endothelial infiltration and vascular inflammation. We found naringenin inhibited oxidized low-density lipoprotein (oxLDL)-induced pro-inflammatory cytokines such as IL-1ß, IL-6, and TNF-α toward an M2 macrophage phenotype and inhibited oxLDL-induced TLR4 (Toll-like receptor 4) membrane translocation and downstream NF-κB transcriptional activity. Results from flow cytometric analysis showed that naringenin reduced monocyte/macrophage infiltration in the aorta of high-fat-diet-treated ApoE-deficient mice. The aortic cytokine levels were also inhibited in naringenin-treated mice. Further, we found that naringenin reduced lipid raft clustering and acid sphingomyelinase (ASMase) membrane gathering and inhibited the TLR4 and NADPH oxidase subunit p47phox membrane recruitment, which reduced the inflammatory response. Recombinant ASMase treatment or overexpression of ASMase abolished the naringenin function and activated macrophage and vascular inflammation. We conclude that naringenin inhibits ASMase-mediated lipid raft redox signaling to attenuate macrophage activation and vascular inflammation.

Particulate matter stimulates the NADPH oxidase system via AhR-mediated epigenetic modifications.

Stimulation of human keratinocytes with particulate matter 2.5 (PM2.5) elicits complex signaling events, including a rise in the generation of reactive oxygen species (ROS). However, the mechanisms underlying PM2.5-induced ROS production remain unknown. Here, we show that PM2.5-induced ROS production in human keratinocytes is mediated via the NADPH oxidase (NOXs) system and the Ca2+ signaling pathway. PM2.5 treatment increased the expression of NOX1, NOX4, and a calcium-sensitive NOX, dual oxidase 1 (DUOX1), in human epidermal keratinocyte cell line. PM2.5 bound to aryl hydrocarbon receptor (AhR), and this complex bound to promoter regions of NOX1 and DUOX1, suggesting that AhR acted as a transcription factor of NOX1 and DUOX1. PM2.5 increased the transcription of DUOX1 via epigenetic modification. Moreover, a link between DNA demethylase and histone methyltransferase with the promoter regions of DUOX1 led to an elevation in the expression of DUOX1 mRNA. Interestingly, PM2.5 increased NOX4 expression and promoted the interaction of NOX4 and Ca2+ channels within the cytoplasmic membrane or endoplasmic reticulum, leading to Ca2+ release. The increase in intracellular Ca2+ concentration activated DUOX1, responsible for ROS production. Our findings provide evidence for a PM2.5-mediated ROS-generating system network, in which increased NOX1, NOX4, and DUOX1 expression serves as a ROS signal through AhR and Ca2+ activation.

Characterization of intestinal O-glycome in reactive oxygen species deficiency.

Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation resulting from an inappropriate inflammatory response to intestinal microbes in a genetically susceptible host. Reactive oxygen species (ROS) generated by NADPH oxidases (NOX) provide antimicrobial defense, redox signaling and gut barrier maintenance. NADPH oxidase mutations have been identified in IBD patients, and mucus layer disruption, a critical aspect in IBD pathogenesis, was connected to NOX inactivation. To gain insight into ROS-dependent modification of epithelial glycosylation the colonic and ileal mucin O-glycome of mice with genetic NOX inactivation (Cyba mutant) was analyzed. O-glycans were released from purified murine mucins and analyzed by hydrophilic interaction ultra-performance liquid chromatography in combination with exoglycosidase digestion and mass spectrometry. We identified five novel glycans in ileum and found minor changes in O-glycans in the colon and ileum of Cyba mutant mice. Changes included an increase in glycans with terminal HexNAc and in core 2 glycans with Fuc-Gal- on C3 branch, and a decrease in core 3 glycans in the colon, while the ileum showed increased sialylation and a decrease in sulfated glycans. Our data suggest that NADPH oxidase activity alters the intestinal mucin O-glycans that may contribute to intestinal dysbiosis and chronic inflammation.

Curcumin Modulates NOX Gene Expression and ROS Production via P-Smad3C in TGF-ß-Activated Hepatic Stellate Cells.

Background: Liver fibrosis, associated with hepatic stellate cells (HSCs), occurs when a healthy liver sustains damage, thereby impairing its function. NADPH oxidases (NOXs), specifically isoforms 1, 2, and 4, play a role in reactive oxygen species (ROS) production during hepatic injuries, resulting in fibrosis. Curcumin has shown strong potential in mitigating liver fibrosis. Our research aimed to investigate the effects of curcumin on lowering NOX and ROS levels. This compound was also studied for its effects on NOXs, ROS concentrations through the inhibition of Smad3 phosphorylation in transforming growth factor beta (TGF-ß)-activated human HSCs. Methods: MTT assay investigated the cytotoxic effects of curcumin on HSCs. The cells were activated by exposure to TGF-ß (2 ng/mL) for 24 hours. After activating, the cells were treated with curcumin at 25-150 µM concentrations. After administering curcumin to the cells, we employed RT-PCR and Western blot techniques to evaluate the related gene and protein expression levels. This evaluation was primarily focused on the mRNA expression levels of NOX1, NOX2, NOX4 and phosphorylated Smad3C. Results: The mRNA expression level of aforesaid NOXs as well as α-smooth muscle actin (α-SMA), collagen1-α, and ROS levels were significantly reduced following 100 µM curcumin treatment. Furthermore, curcumin significantly decreased the p-Smad3C protein level in TGF-ß-activated cells, with fold changes of 3 and 2 observed at 75 and 100 µM, respectively. Conclusion: Curcumin decreased the levels of ROS and NOX, as well as the expression of α-SMA and collagen1-α. The primary mechanism for this reduction could be linked to the level of p-Smad3C. Hence, curcumin could serve as an effective therapeutic agent for liver fibrosis.