Gene Expression and Prognostic Value of NADPH Oxidase Enzymes in Breast Cancer.

NADPH oxidase enzymes (NOX) are involved in all stages of carcinogenesis, but their expression levels and prognostic value in breast cancer (BC) remain unclear. Thus, we aimed to assess the expression and prognostic value of NOX enzymes in BC samples using online databases. For this, mRNA expression from 290 normal breast tissue samples and 1904 BC samples obtained from studies on cBioPortal, Kaplan-Meier Plotter, and The Human Protein Atlas were analyzed. We found higher levels of NOX2, NOX4, and Dual oxidase 1 (DUOX1) in normal breast tissue. NOX1, NOX2, and NOX4 exhibited higher expression in BC, except for the basal subtype, where NOX4 expression was lower. DUOX1 mRNA levels were lower in all BC subtypes. NOX2, NOX4, and NOX5 mRNA levels increased with tumor progression stages, while NOX1 and DUOX1 expression decreased in more advanced stages. Moreover, patients with low expression of NOX1, NOX4, and DUOX1 had lower survival rates than those with high expression of these enzymes. In conclusion, our data suggest an overexpression of NOX enzymes in breast cancer, with certain isoforms showing a positive correlation with tumor progression.

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.

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.

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.

ER-tethered RNA-binding protein controls NADPH oxidase translation for hydrogen peroxide homeostasis.

Hydrogen peroxide (H2O2) functions as a signaling molecule in diverse cellular processes. While cells have evolved the capability to detect and manage changes in H2O2 levels, the mechanisms regulating key H2O2-producing enzymes to maintain optimal levels, especially in pancreatic beta cells with notably weak antioxidative defense, remain unclear. We found that the protein EI24 responds to changes in H2O2 concentration and regulates the production of H2O2 by controlling the translation of NOX4, an enzyme that is constitutively active, achieved by recruiting an RNA-binding protein, RTRAF, to the 3'-UTR of Nox4. Depleting EI24 results in RTRAF relocating into the nucleus, releasing the brake on NOX4 translation. The excessive production of H2O2 by liberated NOX4 further suppresses the translation of the key transcription factor MafA, ultimately preventing its binding to the Ins2 gene promoter and subsequent transcription of insulin. Treatment with a specific NOX4 inhibitor or the antioxidant NAC reversed these effects and alleviated the diabetic symptoms in beta-cell specific Ei24-KO mice. This study revealed a new mechanism through which cells regulate oxidative stress at the translational level, involving an ER-tethered RNA-binding protein that controls the expression of the key H2O2-producing enzyme NOX4.

NADPH oxidase exerts a B cell-intrinsic contribution to lupus risk by modulating endosomal TLR signals.

Genome-wide association studies in systemic lupus erythematosus (SLE) have linked loss-of-function mutations in phagocytic NADPH oxidase complex (NOX2) genes, including NCF1 and NCF2, to disease pathogenesis. The prevailing model holds that reduced NOX2 activity promotes SLE via defective efferocytosis, the immunologically silent clearance of apoptotic cells. Here, we describe a parallel B cell-intrinsic mechanism contributing to breaks in tolerance. In keeping with an important role for B cell Toll-like receptor (TLR) pathways in lupus pathogenesis, NOX2-deficient B cells exhibit enhanced signaling downstream of endosomal TLRs, increased humoral responses to nucleic acid-containing antigens, and the propensity toward humoral autoimmunity. Mechanistically, TLR-dependent NOX2 activation promotes LC3-mediated maturation of TLR-containing endosomes, resulting in signal termination. CRISPR-mediated disruption of NCF1 confirmed a direct role for NOX2 in regulating endosomal TLR signaling in primary human B cells. Together, these data highlight a new B cell-specific mechanism contributing to autoimmune risk in NCF1 and NCF2 variant carriers.

Mycobacterium tuberculosis protein PPE15 (Rv1039c) possesses eukaryote-like SH3 domain that interferes with NADPH Oxidase assembly and Reactive Oxygen Species production.

Inhibition of Reactive Oxygen Species (ROS) is one of the strategies that Mycobacterium tuberculosis (Mtb) employs as its defence mechanism. In this study, the role of PPE15 (Rv1039c), a late-stage protein, has been investigated in modulating the cellular ROS. We discovered PPE15 to be a secretory protein that downregulates ROS generation in THP1 macrophages. Our in-silico analysis revealed the presence of a eukaryote-like SH3 (SH3e) domain in PPE15. The predicted SH3e-domain of PPE15 was found to interact with cytosolic components of NADPH Oxidase (NOX), p67phox and p47phox through molecular docking. In-vitro experiments using THP1 macrophages showed a diminished NADP/NADPH ratio, indicating reduced NOX activity. We also observed increased levels of p67phox and p47phox in the cytoplasmic fraction of PPE15 treated macrophages as compared to the plasma membrane fraction. To understand the role of the SH3e-domain in ROS modulation, this domain was deleted from the full-length PPE15 (PPE15-/-SH3). We observed an increase in cellular ROS and NADP/NADPH ratio in response to PPE15-/-SH3 protein. The interaction of PPE15-/-SH3 with p67phox or p47phox was also reduced in the cytoplasm, indicating migration of NOX subunits to the plasma membrane. Additionally, M. smegmatis expressing PPE15 was observed to be resistant to oxidative stress with significant intracellular survival in THP1 macrophages as compared to M. smegmatis expressing PPE15-/-SH3. These observations suggest that the SH3e-domain of PPE15 interferes with ROS generation by sequestering NOX components that inhibit NOX assembly at the cell membrane. Therefore, PPE15 acts like a molecular mimic of SH3-domain carrying eukaryotic proteins that can be employed by Mtb at late stages of infection for its survival. These findings give us new insights about the pathogen evading strategy of Mtb which may help in improving the therapeutics for TB treatment.

DGK5ß-derived phosphatidic acid regulates ROS production in plant immunity by stabilizing NADPH oxidase.

In plant immunity, phosphatidic acid (PA) regulates reactive oxygen species (ROS) by binding to respiratory burst oxidase homolog D (RBOHD), an NADPH oxidase responsible for ROS production. Here, we analyze the influence of PA binding on RBOHD activity and the mechanism of RBOHD-bound PA generation. PA binding enhances RBOHD protein stability by inhibiting vacuolar degradation, thereby increasing chitin-induced ROS production. Mutations in diacylglycerol kinase 5 (DGK5), which phosphorylates diacylglycerol to produce PA, impair chitin-induced PA and ROS production. The DGK5 transcript DGK5ß (but not DGK5α) complements reduced PA and ROS production in dgk5-1 mutants, as well as resistance to Botrytis cinerea. Phosphorylation of S506 residue in the C-terminal calmodulin-binding domain of DGK5ß contributes to the activation of DGK5ß to produce PA. These findings suggest that DGK5ß-derived PA regulates ROS production by inhibiting RBOHD protein degradation, elucidating the role of PA-ROS interplay in immune response regulation.

In adult X-CGD patients, regulatory T cells are expanded while activated T cells display a NOX2-independent ROS increase.

The X-linked chronic granulomatous disease (X-CGD), a rare genetic disease characterised by recurrent infections, is caused by mutations of NOX2. Significant proportions of X-CGD patients display signs of immune dysregulation. Regulatory T cells (Tregs) are CD4+T lymphocytes that expand in active inflammation and prevent autoimmune disorders. Here we asked whether X-CGD is associated to Treg dysfunctions in adult patients. To this aim, the frequency of Tregs was analysed through intracellular flow cytometry in a cohort of adult X-CGD patients, carriers and controls. We found that Tregs were significantly expanded and activated in blood of adult X-CGD patients, and this was associated with activation of conventional CD4+T cells (Tconvs). T cell activation was characterised by accumulation of intracellular ROS, not derived from NOX2 but likely produced by cellular metabolism. The higher TNF production by Tconvs in X-CGD patients might contribute to the expansion of Tregs through the TNFR2 receptor. In summary, our data indicate that Tregs expand in adult X-CGD in response to immune activation, and that the increase of NOX2-independent ROS content is a feature of activated T cells.

NADPH oxidase 1 promotes hepatic steatosis in obese mice and is abrogated by augmented skeletal muscle mass.

Exercise as a lifestyle modification is a frontline therapy for nonalcoholic fatty liver disease (NAFLD), but how components of exercise attenuate steatosis is unclear. To uncouple the effect of increased muscle mass from weight loss in obesity, myostatin knockout mice were bred on a lean and obese db/db background. Myostatin deletion increases gastrocnemius (Gastrocn.) mass and reduces hepatic steatosis and hepatic sterol regulatory element binding protein 1 (Srebp1) expression in obese mice, with no impact on adiposity or body weight. Interestingly, hypermuscularity reduces hepatic NADPH oxidase 1 (Nox1) expression but not NADPH oxidase 4 (Nox4) in db/db mice. To evaluate a deterministic function of Nox1 on steatosis, Nox1 knockout mice were bred on a lean and db/db background. NOX1 deletion significantly attenuates hepatic oxidant stress, steatosis, and Srebp1 programming in obese mice to parallel hypermuscularity, with no improvement in adiposity, glucose control, or hypertriglyceridemia to suggest off-target effects. Directly assessing the role of NOX1 on SREBP1, insulin (Ins)-mediated SREBP1 expression was significantly increased in either NOX1, NADPH oxidase organizer 1 (NOXO1), and NADPH oxidase activator 1 (NOXA1) or NOX5-transfected HepG2 cells versus ?-galactosidase control virus, indicating superoxide is the key mechanistic agent for the actions of NOX1 on SREBP1. Metabolic Nox1 regulators were evaluated using physiological, genetic, and diet-induced animal models that modulated upstream glucose and insulin signaling, identifying hyperinsulinemia as the key metabolic derangement explaining Nox1-induced steatosis in obesity. GEO data revealed that hepatic NOX1 predicts steatosis in obese humans with biopsy-proven NAFLD. Taken together, these data suggest that hypermuscularity attenuates Srebp1 expression in db/db mice through a NOX1-dependent mechanism.NEW & NOTEWORTHY This study documents a novel mechanism by which changes in body composition, notably increased muscle mass, protect against fatty liver disease. This mechanism involves NADPH oxidase 1 (NOX1), an enzyme that increases superoxide and increases insulin signaling, leading to increased fat accumulation in the liver. NOX1 may represent a new early target for preventing fatty liver to stave off later liver diseases such as cirrhosis or liver cancer.

Commensal lifestyle regulated by a negative feedback loop between Arabidopsis ROS and the bacterial T2SS.

Despite the plant health-promoting effects of plant microbiota, these assemblages also comprise potentially detrimental microbes. How plant immunity controls its microbiota to promote plant health under these conditions remains largely unknown. We find that commensal bacteria isolated from healthy Arabidopsis plants trigger diverse patterns of reactive oxygen species (ROS) production dependent on the immune receptors and completely on the NADPH oxidase RBOHD that selectively inhibited specific commensals, notably Xanthomonas L148. Through random mutagenesis, we find that L148 gspE, encoding a type II secretion system (T2SS) component, is required for the damaging effects of Xanthomonas L148 on rbohD mutant plants. In planta bacterial transcriptomics reveals that RBOHD suppresses most T2SS gene expression including gspE. L148 colonization protected plants against a bacterial pathogen, when gspE was inhibited by ROS or mutation. Thus, a negative feedback loop between Arabidopsis ROS and the bacterial T2SS tames a potentially detrimental leaf commensal and turns it into a microbe beneficial to the host.

[Pulmonary complications of Chronic Granulomatous Disease]. / Complications pulmonaires de la granulomatose septique chronique.

Chronic Granulomatosis Disease (CGD) is an inherited immune deficiency due to a mutation in the genes coding for the subunits of the NADPH oxidase enzyme that affects the oxidative capacity of phagocytic cells. It is characterized by increased susceptibility to bacterial and fungal infections, particularly Aspergillus, as well as complications associated with hyperinflammation and granulomatous tissue infiltration. There exist two types of frequently encountered pulmonary manifestations: (1) due to their being initially pauci-symptomatic, possibly life-threatening infectious complications are often discovered at a late stage. Though their incidence has decreased through systematic anti-bacterial and anti-fungal prophylaxis, they remain a major cause of morbidity and mortality; (2) inflammatory complications consist in persistent granulomatous mass or interstitial pneumoniae, eventually requiring immunosuppressive treatment. Pulmonary complications recurring since infancy generate parenchymal and bronchial sequelae that impact functional prognosis. Hematopoietic stem cell allograft is a curative treatment; it is arguably life-sustaining and may limit the morbidity of the disease. As a result of improved pediatric management, life expectancy has increased dramatically. That said, new challenges have appeared with regard to adults: difficulties of compliance, increased inflammatory manifestations, acquired resistance to anti-infectious therapies. These different developments underscore the importance of the transition period and the need for multidisciplinary management.

Aliskiren-Loaded Nanoparticles Downregulate (Pro)renin Receptor and ACE Gene Expression in the Heart of Spontaneously Hypertensive Rats: Effect on NADPH Oxidase.

We aimed to determine effects of aliskiren, a direct renin inhibitor, loaded onto polymeric nanoparticles on the (pro)renin receptor (Atp6ap2), angiotensin II type 1 receptor (Agtr1), and angiotensin-converting enzyme (ACE) gene expression in the heart of spontaneously hypertensive rats (SHR). Twelve-week-old male SHRs were divided into an untreated group and groups treated with powdered aliskiren or aliskiren-loaded nanoparticles (25 mg/kg/day). After three weeks, the accumulation of aliskiren, distribution of polymeric nanoparticles, gene expression of Atp6ap2 and Agtr1 receptors and ACE, and protein expression of NADPH oxidase along with the conjugated diene (CD) concentration were analyzed. The accumulation of aliskiren in the heart was higher in the aliskiren-loaded nanoparticle group than in the powdered group. The fluorescent signals of nanoparticles were visible in cardiomyocytes, vessel walls, and erythrocytes. Aliskiren-loaded nanoparticles decreased the gene expression of Atp6ap2 and ACE, while not affecting Agtr1. Both forms of aliskiren decreased the protein expression of NADPH oxidase, with a more pronounced effect observed in the aliskiren-loaded nanoparticle group. CD concentration was decreased only in the aliskiren-loaded nanoparticle group. We hypothesize that aliskiren-loaded nanoparticle-mediated downregulation of Atp6ap2 and ACE may contribute to a decrease in ROS generation with beneficial effects in the heart. Moreover, polymeric nanoparticles may represent a promising tool for targeted delivery of aliskiren.

Leiomyoma and the importance of genetic variation on genes related to the vasculature system - CßS, MTHFR, NOS3, CYBA, and ACE1.

OBJECTIVE: The link between the systemic vasculature system and tumor biology is here investigated by studying the contribution of CßS (844ins68), MTHFR (677C > T), NOS3 (4a/4b), CYBA (C242T), and ACE1 (I/D) genes to leiomyoma onset, uterus and leiomyoma volumes. METHODS: DNA samples from 130 women with leiomyomas and 527 from healthy women were genotyped by PCR or PCR-RFLP. Qui-square (χ2) or Fisher's exact test were used to test associations. All the mentioned tests were performed in IBM® SPSS® Statistics Version 28. Statistical significance was defined as a p-value < 0.05. RESULTS: Results revealed that CßS (in the codominant and allelic models, p = 0.044 and, p = 0.015, OR = 1.791 [1.114-2.879], respectively), MTHFR (in the codominant, allelic and dominant models, p = 0.009, p = 0.002, OR = 0.585 [0.416-0.824] and p = 0.003, OR = 0.527 [0.346-0.802], respectively) and ACE1 (dominant model, p = 0.045, OR = 0.639 [0.411-0.992]) genes are associated with leiomyoma onset. NOS3 4a4a genotype is associated with a lower uterus volume (p = 0.004). This study also uncovers intriguing epistatic interactions among some genes that further accentuate their roles in disease modulation. Indeed, the epistatic interactions between the CC genotype (MTHFR) and (+/+) (CßS; p = 0.003), 4b4b (NOS3; p = 0.006, OR = 2.050 [1.223-3.439]) or DD (ACE1; p < 0.001, OR = 2.362 [1.438-3.880]) were shown to be associated with the disease, while 4a presence (NOS3) in epistasis with I presence (ACE1), increased the effect protection having just the I allele presence (p = 0.029, OR = 0.446 [0.214-0.930]). CONCLUSIONS: We conclude that variation in genes related to the systemic vascular system can play a role in the onset and development of leiomyoma.

XYLEM CYSTEINE PEPTIDASE 1 and its inhibitor CYSTATIN 6 regulate pattern-triggered immunity by modulating the stability of the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D.

Plants produce a burst of reactive oxygen species (ROS) after pathogen infection to successfully activate immune responses. During pattern-triggered immunity (PTI), ROS are primarily generated by the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD). RBOHD is degraded in the resting state to avoid inappropriate ROS production; however, the enzyme mediating RBOHD degradation and how to prevent RBOHD degradation after pathogen infection is unclear. In this study, we identified an Arabidopsis (Arabidopsis thaliana) vacuole-localized papain-like cysteine protease, XYLEM CYSTEINE PEPTIDASE 1 (XCP1), and its inhibitor CYSTATIN 6 (CYS6). Pathogen-associated molecular pattern-induced ROS burst and resistance were enhanced in the xcp1 mutant but were compromised in the cys6 mutant, indicating that XCP1 and CYS6 oppositely regulate PTI responses. Genetic and biochemical analyses revealed that CYS6 interacts with XCP1 and depends on XCP1 to enhance PTI. Further experiments showed that XCP1 interacts with RBOHD and accelerates RBOHD degradation in a vacuole-mediated manner. CYS6 inhibited the protease activity of XCP1 toward RBOHD, which is critical for RBOHD accumulation upon pathogen infection. As CYS6, XCP1, and RBOHD are conserved in all plant species tested, our findings suggest the existence of a conserved strategy to precisely regulate ROS production under different conditions by modulating the stability of RBOHD.

Grass carp Il-2 promotes neutrophil extracellular traps formation via inducing ROS production and autophagy in vitro.

Interleukin (IL)-2 has been reported to regulate neutrophil functions in humans, mice, pigs and chicken although it is a key regulator of T cells. Consistently, we found that grass carp (Ctenopharyngodon idellus) interleukin-2 (gcIl-2) is capable of modulating the antimicrobial activities of neutrophils via regulating granzyme B- and perforin-like gene expression in our previous study. In the present study, stimulation of gcIl-2 on neutrophil extracellular traps (NETs) formation in grass carp neutrophils was demonstrated by detecting free DNA release, histone H3 citrullination and morphological changes of the cells. Further investigation revealed that reactive oxygen species (ROS) production from NADPH oxidase but not mitochondria was involved in NETosis induced by gcIl-2. Aside from ROS, autophagy was disclosed to be indispensable for NETosis induced by gcIl-2. These converging lines of evidence suggested that fish Il-2 could induce NETs formation via NADPH oxidase-derived ROS- and autophagy-dependent pathways in fish species which is evolutionarily conserved with that in mammals. It is noteworthy that these two pathways did not interplay with each other in Il-2-stimulated NETosis. The mechanisms governing autophagy induced by Il-2 were also explored in the present study, showing that Il-2 modulated the action of high mobility group box 1 (HMGB1) protein to stimulate autophagy, leading to NETs formation in fish neutrophils. These results provided a new insight to the function of Il-2 in fish neutrophils, and a clue about the regulation of NETosis in the lower vertebrates.

Hypoxia stabilizes the H2 O2 -producing oxidase Nox4 in cardiomyocytes via suppressing autophagy-related lysosomal degradation.

The hydrogen peroxide (H2 O2 )-producing NADPH oxidase Nox4, forming a heterodimer with p22phox , is expressed in a variety of cells including those in the heart to mediate adaptive responses to cellular stresses such as hypoxia. Since Nox4 is constitutively active, H2 O2 production is controlled by its protein abundance. Hypoxia-induced Nox4 expression is observed in various types of cells and generally thought to be regulated at the transcriptional level. Here we show that hypoxia upregulates the Nox4 protein level and Nox4-catalyzed H2 O2 production without increasing the Nox4 mRNA in rat H9c2 cardiomyocytes. In these cells, the Nox4 protein is stabilized under hypoxic conditions in a manner dependent on the presence of p22phox . Cell treatment with the proteasome inhibitor MG132 results in a marked decrease of the Nox4 protein under both normoxic and hypoxic conditions, indicating that the proteasome pathway does not play a major role in Nox4 degradation. The decrease is partially restored by the autophagy inhibitor 3-methyladenine. Furthermore, the Nox4 protein level is upregulated by the lysosome inhibitors bafilomycin A1 and chloroquine. Thus, in cardiomyocytes, Nox4 appears to be degraded via an autophagy-related pathway, and its suppression by hypoxia likely stabilizes Nox4, leading to upregulation of Nox4-catalyzed H2 O2 production.