RESUMEN

Trichomoniasis is caused by a sexually transmitted flagellate protozoan parasite Trichomonas vaginalis. T. vaginalis-derived secretory products (TvSP) contain lipid mediators such as leukotriene B4 (LTB4) and various cysteinyl leukotrienes (CysLTs) which included LTC4, LTD4, and LTE4. However, the signaling mechanisms by which T. vaginalis-induced CysLTs stimulate interleukin (IL)-8 production in human mast cells remain unclear. In this study, we investigated these mechanisms in human mast cells (HMC-1). Stimulation with TvSP resulted in increased intracellular reactive oxygen species (ROS) generation and NADPH oxidase 2 (NOX2) activation compared to unstimulated cells. Pre-treatment with NOX2 inhibitors such as diphenyleneiodonium chloride (DPI) or apocynin significantly reduced ROS production in TvSP-stimulated HMC-1 cells. Additionally, TvSP stimulation increased NOX2 protein expression and the translocation of p47phox from the cytosol to the membrane. Pretreatment of HMC-1 cells with PI3K or PKC inhibitors reduced TvSP-induced p47phox translocation and ROS generation. Furthermore, NOX2 inhibitors or NOX2 siRNA prevented CREB phosphorylation and IL-8 gene expression or protein secretion induced by TvSP. Pretreatment with a CysLTR antagonist significantly inhibited TvSP-induced ROS production, CREB phosphorylation, and IL-8 production. These results indicate that CysLT-mediated activation of NOX2 plays a crucial role in ROS-dependent IL-8 production in human mast cells stimulated by T. vaginalis-secreted CysLTs. These findings enhance our understanding of the inflammatory response in trichomoniasis and may inform the development of targeted therapies to mitigate this response.

Asunto(s)

Interleucina-8 , Mastocitos , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno , Receptores de Leucotrienos , Trichomonas vaginalis , Humanos , Trichomonas vaginalis/efectos de los fármacos , Trichomonas vaginalis/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Especies Reactivas de Oxígeno/metabolismo , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Mastocitos/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/parasitología , Mastocitos/inmunología , Línea Celular , Receptores de Leucotrienos/metabolismo , Receptores de Leucotrienos/genética , NADPH Oxidasas/metabolismo , Transducción de Señal/efectos de los fármacos , Leucotrienos/metabolismo

RESUMEN

M2-polarized, tumor-associated macrophages (TAMs) produce pro-tumorigenic and angiogenic mediators, such as interleukin-8 (IL-8) and IL-10. Leucine-rich repeat-containing protein 8 members (LRRC8s) form volume-regulated anion channels and play an important role in macrophage functions by regulating cytokine and chemokine production. We herein examined the role of LRRC8A in IL-8 and IL-10 expression in THP-1-differentiated M2-like macrophages (M2-MACs), which are a useful tool for investigating TAMs. In M2-MACs, the pharmacological inhibition of LRRC8A led to hyperpolarizing responses after a transient depolarization phase, followed by a slight elevation in the intracellular concentration of Ca2+. Both the small interfering RNA-mediated and pharmacological inhibition of LRRC8A repressed the transcriptional expression of IL-8 and IL-10, resulting in a significant reduction in their secretion. The inhibition of LRRC8A decreased the nuclear translocation of phosphorylated nuclear factor-erythroid 2-related factor 2 (Nrf2), while the activation of Nrf2 reversed the LRRC8A inhibition-induced transcriptional repression of IL-8 and IL-10 in M2-MACs. We identified the CCAAT/enhancer-binding protein isoform B, CEBPB, as a downstream target of Nrf2 signaling in M2-MACs. Moreover, among several upstream candidates, the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) suppressed the Nrf2-CEBPB transcriptional axis in M2-MACs. Collectively, the present results indicate that the inhibition of LRRC8A repressed IL-8 and IL-10 transcription in M2-MACs through the NOX2-Nrf2-CEBPB axis and suggest that LRRC8A inhibitors suppress the IL-10-mediated evasion of tumor immune surveillance and IL-8-mediated metastasis and neovascularization in TAMs.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT , Interleucina-10 , Interleucina-8 , Macrófagos , Proteínas de la Membrana , NADPH Oxidasa 2 , Factor 2 Relacionado con NF-E2 , Humanos , Interleucina-10/metabolismo , Interleucina-10/genética , Interleucina-8/metabolismo , Interleucina-8/genética , Factor 2 Relacionado con NF-E2/metabolismo , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Transducción de Señal , Regulación hacia Abajo , Células THP-1

RESUMEN

Arsenic (As) has been classified as a carcinogen for humans. There is abundant evidence indicating that arsenic increases the risk of bladder cancer among human populations. However, the underlying mechanisms have yet to be fully understood and elucidated. NADPH oxidases (NOXs) are the main enzymes for ROS production in the body. NADPH Oxidase 2 (NOX2), which is the most distinctive and ubiquitously expressed subunit of NOXs, can promote the formation and development of tumors. The utilization of NOX2 as a therapeutic target has been proposed to modulate diseases resulting from the activation of NOD-like receptor thermal protein domain associated protein 3 (NLRP3). Matrine has been reported to exhibit various pharmacological effects, including anti-inflammatory, antifibrotic, antitumor, and analgesic properties. However, it has not been reported whether matrine can inhibit malignant transformation induced by arsenic in uroepithelial cells through NOX2. We have conducted a series of experiments using both a sub-chronic NaAsO2 exposure rat model and a long-term NaAsO2 exposure cell model. Our findings indicate that arsenic significantly increases cell proliferation, migration, and angiogenesis in vivo and in vitro. Arsenic exposure resulted in an upregulation of reactive oxygen species (ROS), NOX2, and NLRP3 inflammasome expression. Remarkably, both in vivo and in vitro, the administration of matrine demonstrated a significant improvement in the detrimental impact of arsenic on bladder epithelial cells. This was evidenced by the downregulation of proliferation, migration, and angiogenesis, as well as the expression of the NOX2 and NLRP3 inflammasomes. Collectively, these findings indicate that matrine possesses the ability to reduce NOX2 levels and inhibit the transformation of bladder epithelial cells.

Asunto(s)

Alcaloides , Arsénico , Proliferación Celular , Transformación Celular Neoplásica , Matrinas , NADPH Oxidasa 2 , Quinolizinas , Especies Reactivas de Oxígeno , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Animales , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/inducido químicamente , Humanos , Arsénico/toxicidad , Arsénico/efectos adversos , Alcaloides/farmacología , Especies Reactivas de Oxígeno/metabolismo , Ratas , Quinolizinas/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/metabolismo , Movimiento Celular/efectos de los fármacos , Línea Celular , Masculino

RESUMEN

PURPOSE: Chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by pathogenic variants of genes encoding the enzyme complex NADPH oxidase. In countries where tuberculosis (TB) is endemic and the Bacillus Calmette-Guérin (BCG) vaccine is routinely administered, mycobacteria are major disease-causing pathogens in CGD. However, information on the clinical evolution and treatment of mycobacterial diseases in patients with CGD is limited. The present study describes the adverse reactions to BCG and TB in Mexican patients with CGD. METHODS: Patients with CGD who were evaluated at the Immunodeficiency Laboratory of the National Institute of Pediatrics between 2013 and 2024 were included. Medical records were reviewed to determine the clinical course and treatment of adverse reactions to BCG and TB disease. RESULTS: A total of 79 patients with CGD were included in this study. Adverse reactions to BCG were reported in 55 (72%) of 76 patients who received the vaccine. Tuberculosis was diagnosed in 19 (24%) patients. Relapse was documented in three (10%) of 31 patients with BGC-osis and six (32%) of 19 patients with TB, despite antituberculosis treatment. There was no difference in the frequency of BCG and TB disease between patients with pathogenic variants of the X-linked CYBB gene versus recessive variants. CONCLUSIONS: This report highlights the importance of considering TB in endemic areas and BCG complications in children with CGD to enable appropriate diagnostic and therapeutic approaches to improve prognosis and reduce the risk of relapse.

Asunto(s)

Vacuna BCG , Enfermedad Granulomatosa Crónica , NADPH Oxidasa 2 , Tuberculosis , Humanos , Enfermedad Granulomatosa Crónica/diagnóstico , Enfermedad Granulomatosa Crónica/epidemiología , Enfermedad Granulomatosa Crónica/complicaciones , Vacuna BCG/efectos adversos , Masculino , Femenino , Niño , Tuberculosis/epidemiología , Tuberculosis/inmunología , Preescolar , Lactante , Adolescente , NADPH Oxidasa 2/genética , Estudios de Cohortes , Mycobacterium bovis , México/epidemiología , Antituberculosos/uso terapéutico , NADPH Oxidasas/genética

RESUMEN

BACKGROUND: This study evaluated the effects of concurrent isolated training (T) or training combined with the antioxidant N-acetylcysteine (NAC) on cardiac remodeling and oxidative stress in spontaneously hypertensive rats (SHR). METHODS: Six-month-old male SHR were divided into sedentary (S, n = 12), concurrent training (T, n = 13), sedentary supplemented with NAC (SNAC, n = 13), and concurrent training with NAC supplementation (TNAC, n = 14) groups. T and TNAC rats were trained three times a week on a treadmill and ladder; NAC supplemented groups received 120 mg/kg/day NAC in rat chow for eight weeks. Myocardial antioxidant enzyme activity and lipid hydroperoxide concentration were assessed by spectrophotometry. Gene expression of NADPH oxidase subunits Nox2, Nox4, p22 phox, and p47 phox was evaluated by real time RT-PCR. Statistical analysis was performed using ANOVA and Bonferroni or Kruskal-Wallis and Dunn. RESULTS: Echocardiogram showed concentric remodeling in TNAC, characterized by increased relative wall thickness (S 0.40 ± 0.04; T 0.39 ± 0.03; SNAC 0.40 ± 0.04; TNAC 0.43 ± 0.04 *; * p < 0.05 vs T and SNAC) and diastolic posterior wall thickness (S 1.50 ± 0.12; T 1.52 ± 0.10; SNAC 1.56 ± 0.12; TNAC 1.62 ± 0.14 * mm; * p < 0.05 vs T), with improved contractile function (posterior wall shortening velocity: S 39.4 ± 5.01; T 36.4 ± 2.96; SNAC 39.7 ± 3.44; TNAC 41.6 ± 3.57 * mm/s; * p < 0.05 vs T). Myocardial lipid hydroperoxide concentration was lower in NAC treated groups (S 210 ± 48; T 182 ± 43; SNAC 159 ± 33 *; TNAC 110 ± 23 *# nmol/g tissue; * p < 0.05 vs S, # p < 0.05 vs T and SNAC). Nox 2 and p22 phox expression was higher and p47 phox lower in T than S [S 1.37 (0.66-1.66); T 0.78 (0.61-1.04) *; SNAC 1.07 (1.01-1.38); TNAC 1.06 (1.01-1.15) arbitrary units; * p < 0.05 vs S]. NADPH oxidase subunits did not differ between TNAC, SNAC, and S groups. CONCLUSION: N-acetylcysteine supplementation alone reduces oxidative stress in untreated spontaneously hypertensive rats. The combination of N-acetylcysteine and concurrent exercise further decreases oxidative stress. However, the lower oxidative stress does not translate into improved cardiac remodeling and function in untreated spontaneously hypertensive rats.

Asunto(s)

Acetilcisteína , Hipertensión , NADPH Oxidasas , Estrés Oxidativo , Ratas Endogámicas SHR , Remodelación Ventricular , Animales , Masculino , Estrés Oxidativo/efectos de los fármacos , Acetilcisteína/farmacología , Remodelación Ventricular/efectos de los fármacos , Hipertensión/fisiopatología , Hipertensión/tratamiento farmacológico , Hipertensión/metabolismo , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , Ratas , Antioxidantes/farmacología , Condicionamiento Físico Animal , Modelos Animales de Enfermedad , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Miocardio/metabolismo , Miocardio/patología , Peróxidos Lipídicos/metabolismo , Función Ventricular Izquierda/efectos de los fármacos , Suplementos Dietéticos , Hipertrofia Ventricular Izquierda/fisiopatología , Hipertrofia Ventricular Izquierda/prevención & control , Hipertrofia Ventricular Izquierda/metabolismo

RESUMEN

NADPH oxidase 2 (NOX2) is an enzyme responsible for generating reactive oxygen species, primarily found in phagocytes. Chronic Granulomatous Disease (CGD), along with bacterial infections such as Mycobacterium tuberculosis (Mtb), is a representative NOX2-deficient X-linked disease characterized by uncontrolled inflammation. However, the precise roles of host-derived factors that induce infection-mediated hyperinflammation in NOX2-deficient condition remain incompletely understood. To address this, we compared Mtb-induced pathogenesis in Nox2-/- and wild type (WT) mice in a sex-dependent manner. Among age- and sex-matched mice subjected to Mtb infection, male Nox2-/- mice exhibited a notable increase in bacterial burden and lung inflammation. This was characterized by significantly elevated pro-inflammatory cytokines such as G-CSF, TNF-α, IL-1α, IL-1ß, and IL-6, excessive neutrophil infiltration, and reduced pulmonary lymphocyte levels as tuberculosis (TB) progressed. Notably, lungs of male Nox2-/- mice were predominantly populated with CD11bintLy6GintCXCR2loCD62Llo immature neutrophils which featured mycobacterial permissiveness. By diminishing total lung neutrophils or reducing immature neutrophils, TB immunopathogenesis was notably abrogated in male Nox2-/- mice. Ultimately, we identified G-CSF as the pivotal trigger that exacerbates the generation of immature permissive neutrophils, leading to TB immunopathogenesis in male Nox2-/- mice. In contrast, neutralizing IL-1α and IL-1ß, which are previously known factors responsible for TB pathogenesis in Nox2-/- mice, aggravated TB immunopathogenesis. Our study revealed that G-CSF-driven immature and permissive pulmonary neutrophils are the primary cause of TB immunopathogenesis and lung hyperinflammation in male Nox2-/- mice. This highlights the importance of quantitative and qualitative control of pulmonary neutrophils to alleviate TB progression in a phagocyte oxidase-deficient condition.

Asunto(s)

Pulmón , Ratones Noqueados , Mycobacterium tuberculosis , NADPH Oxidasa 2 , NADPH Oxidasas , Neutrófilos , Animales , Masculino , Ratones , Neutrófilos/inmunología , NADPH Oxidasas/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/inmunología , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/inmunología , NADPH Oxidasa 2/metabolismo , Pulmón/inmunología , Pulmón/patología , Pulmón/microbiología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/patología , Tuberculosis Pulmonar/microbiología , Femenino , Ratones Endogámicos C57BL , Enfermedad Granulomatosa Crónica/inmunología , Enfermedad Granulomatosa Crónica/microbiología , Enfermedad Granulomatosa Crónica/patología , Fagocitos/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad

RESUMEN

Reactive oxygen species (ROS) serve vital physiological functions, but aberrant ROS production contributes to numerous diseases. Unfortunately, therapeutic progress targeting pathogenic ROS has been hindered by the limited understanding of whether the mechanisms driving pathogenic ROS differ from those governing physiological ROS generation. To address this knowledge gap, we utilised a cellular model of Parkinson's disease (PD), as an exemplar of ROS-associated diseases. We exposed SH-SY5Y neuroblastoma cells to the PD-toxin, MPP+ (1-methyl-4-phenylpyridinium) and studied ROS upregulation leading to cell death, the primary cause of PD. We demonstrate: (1) MPP+ stimulates ROS production by raising cytoplasmic Ca2+ levels, rather than acting directly on mitochondria. (2) To raise the Ca2+, MPP+ co-stimulates NADPH oxidase-2 (NOX2) and the Transient Receptor Potential Melastatin2 (TRPM2) channel that form a positive feedback loop to support each other's function. (3) Ca2+ exacerbates mitochondrial ROS (mtROS) production not directly, but via Zn2+. (4) Zn2+ promotes electron escape from respiratory complexes, predominantly from complex III, to generate mtROS. These conclusions are drawn from data, wherein inhibition of TRPM2 and NOX2, chelation of Ca2+ and Zn2+, and prevention of electron escape from complexes -all abolished the ability of MPP+ to induce mtROS production and the associated cell death. Furthermore, calcium ionophore mimicked the effects of MPP+, while Zn2+ ionophore replicated the effects of both MPP+ and Ca2+. Thus, we unveil a previously unrecognized signalling circuit involving NOX2, TRPM2, Ca2+, Zn2+, and complex III that drives cytotoxic ROS production. This circuit lies dormant in healthy cells but is triggered by pathogenic insults and could therefore represent a safe therapeutic target for PD and other ROS-linked diseases.

Asunto(s)

Calcio , Mitocondrias , NADPH Oxidasa 2 , Enfermedad de Parkinson , Especies Reactivas de Oxígeno , Canales Catiónicos TRPM , Zinc , Especies Reactivas de Oxígeno/metabolismo , Humanos , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Zinc/metabolismo , Canales Catiónicos TRPM/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Calcio/metabolismo , Línea Celular Tumoral , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , 1-Metil-4-fenilpiridinio

RESUMEN

Obesity is linked to an increased risk of atrial fibrillation (AF) via increased oxidative stress. While NADPH oxidase 2 (NOX2), a major source of oxidative stress and reactive oxygen species (ROS) in the heart, predisposes to AF, the underlying mechanisms remain unclear. Here, we studied NOX2-mediated ROS production in obesity-mediated AF using Nox2-knockout mice and mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs). Diet-induced obesity (DIO) mice and hiPSC-aCMs treated with palmitic acid (PA) were infused with a NOX blocker (apocynin) and a NOX2-specific inhibitor, respectively. We showed that NOX2 inhibition normalized atrial action potential duration and abrogated obesity-mediated ion channel remodeling with reduced AF burden. Unbiased transcriptomics analysis revealed that NOX2 mediates atrial remodeling in obesity-mediated AF in DIO mice, PA-treated hiPSC-aCMs, and human atrial tissue from obese individuals by upregulation of paired-like homeodomain transcription factor 2 (PITX2). Furthermore, hiPSC-aCMs treated with hydrogen peroxide, a NOX2 surrogate, displayed increased PITX2 expression, establishing a mechanistic link between increased NOX2-mediated ROS production and modulation of PITX2. Our findings offer insights into possible mechanisms through which obesity triggers AF and support NOX2 inhibition as a potential novel prophylactic or adjunctive therapy for patients with obesity-mediated AF.

Asunto(s)

Fibrilación Atrial , Ratones Noqueados , Miocitos Cardíacos , NADPH Oxidasa 2 , Obesidad , NADPH Oxidasa 2/genética , NADPH Oxidasa 2/metabolismo , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Fibrilación Atrial/etiología , Fibrilación Atrial/enzimología , Animales , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Ratones , Humanos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Miocitos Cardíacos/enzimología , Especies Reactivas de Oxígeno/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Estrés Oxidativo , Remodelación Atrial

Asunto(s)

Lupus Eritematoso Sistémico , NADPH Oxidasa 2 , Receptor Toll-Like 7 , Humanos , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Riñón , Animales

RESUMEN

Ca2+/calmodulin-dependent protein kinase II α (CaMKIIα) signaling in the brain plays a critical role in regulating neuronal Ca2+ homeostasis. Its dysfunctional activity is associated with various neurological and neurodegenerative disorders, including Parkinson's disease (PD). Using computational modeling analysis, we predicted that, two essential cysteine residues contained in CaMKIIα, Cys30 and Cys289, may undergo redox modifications impacting the proper functioning of the CaMKIIα docking site for Ca2+/CaM, thus impeding the formation of the CaMKIIα:Ca2+/CaM complex, essential for a proper modulation of CaMKIIα kinase activity. Our subsequent in vitro investigations confirmed the computational predictions, specifically implicating Cys30 and Cys289 residues in impairing CaMKIIα:Ca2+/CaM interaction. We observed CaMKIIα:Ca2+/CaM complex disruption in dopamine (DA) nigrostriatal neurons of post-mortem Parkinson's disease (PD) patients' specimens, addressing the high relevance of this event in the disease. CaMKIIα:Ca2+/CaM complex disruption was also observed in both in vitro and in vivo rotenone models of PD, where this phenomenon was associated with CaMKIIα kinase hyperactivity. Moreover, we observed that, NADPH oxidase 2 (NOX2), a major enzymatic generator of superoxide anion (O2●-) and hydrogen peroxide (H2O2) in the brain with implications in PD pathogenesis, is responsible for CaMKIIα:Ca2+/CaM complex disruption associated to a stable Ca2+CAM-independent CaMKIIα kinase activity and intracellular Ca2+ accumulation. The present study highlights the importance of oxidative stress, in disturbing the delicate balance of CaMKIIα signaling in calcium dysregulation, offering novel insights into PD pathogenesis.

Asunto(s)

Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Calmodulina , NADPH Oxidasa 2 , Oxidación-Reducción , Enfermedad de Parkinson , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Enfermedad de Parkinson/metabolismo , Humanos , Calmodulina/metabolismo , Animales , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Calcio/metabolismo , Cisteína/metabolismo , Ratones

RESUMEN

Loss of NADPH oxidase (NOX2) exacerbates systemic lupus erythematosus (SLE) in mice and humans, but the mechanisms underlying this effect remain unclear. To identify the cell lineages in which NOX2 deficiency drives SLE, we employed conditional KO and chimeric approaches to delete Cybb in several hematopoietic cell lineages of MRL.Faslpr SLE-prone mice. Deletion of Cybb in macrophages/monocytes exacerbated SLE nephritis, though not to the degree observed in the Cybb global KOs. Unexpectedly, the absence of Cybb in B cells resulted in profound glomerulonephritis and interstitial nephritis, rivaling that seen with global deletion. Furthermore, we identified that NOX2 is a key regulator of TLR7, a driver of SLE pathology, both globally and specifically in B cells. This is mediated in part through suppression of TLR7-mediated NF-κB signaling in B cells. Thus, NOX2's immunomodulatory effect in SLE is orchestrated not only by its function in the myeloid compartment, but through a pivotal role in B cells by selectively inhibiting TLR7 signaling.

Asunto(s)

Linfocitos B , Lupus Eritematoso Sistémico , Macrófagos , Ratones Noqueados , NADPH Oxidasa 2 , Receptor Toll-Like 7 , Animales , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 7/genética , Ratones , Linfocitos B/inmunología , Linfocitos B/metabolismo , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Transducción de Señal , Femenino , Modelos Animales de Enfermedad , FN-kappa B/metabolismo , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Nefritis Lúpica/metabolismo , Nefritis Lúpica/genética , Glicoproteínas de Membrana

RESUMEN

Hydrogen peroxide (H2O2) is one of the key signaling factors regulating skeletal muscle adaptation to muscle contractions. Eccentric (ECC) and concentric (CONC) contractions drive different muscle adaptations with ECC resulting in greater changes. The present investigation tested the hypothesis that ECC produces higher cytosolic and mitochondrial H2O2 concentrations [H2O2] and alters gene expression more than CONC. Cytosolic and mitochondrial H2O2-sensitive fluorescent proteins, HyPer7 and MLS-HyPer7, were expressed in the anterior tibialis muscle of C57BL6J male mice. Before and for 60 min after either CONC or ECC (100 Hz, 50 contractions), [H2O2]cyto and [H2O2]mito were measured by in vivo fluorescence microscopy. RNA sequencing was performed in control (noncontracted), CONC, and ECC muscles to identify genes impacted by the contractions. [H2O2]cyto immediately after ECC was greater than after CONC (CONC: +6%, ECC: +11% vs. rest, P < 0.05) and remained higher for at least 60 min into recovery. In contrast, the elevation of [H2O2]mito was independent of the contraction modes (time; P < 0.0042, contraction mode; P = 0.4965). The impact of ECC on [H2O2]cyto was abolished by NADPH oxidase 2 (Nox2) inhibition (GSK2795039). Differentially expressed genes were not present after CONC or ECC + GSK but were found after ECC and were enriched for vascular development and apoptosis-related genes, among others. In conclusion, in mouse anterior tibialis, ECC, but not CONC, evokes a pronounced cytosolic H2O2 response, caused by Nox2, that is mechanistically linked to gene expression modifications.NEW & NOTEWORTHY This in vivo model successfully characterized the effects of eccentric (ECC) and concentric (CONC) contractions on cytosolic and mitochondrial [H2O2] in mouse skeletal muscle. Compared with CONC, ECC induced higher and more sustained [H2O2]cyto-an effect that was abolished by Nox2 inhibition. ECC-induced [H2O2]cyto elevations were requisite for altered gene expression.

Asunto(s)

Peróxido de Hidrógeno , Ratones Endogámicos C57BL , Contracción Muscular , Músculo Esquelético , NADPH Oxidasa 2 , Animales , Masculino , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Ratones , Peróxido de Hidrógeno/metabolismo , Expresión Génica/genética , Mitocondrias/metabolismo

RESUMEN

Striated muscle cells, encompassing cardiac myocytes and skeletal muscle fibers, are fundamental to athletic performance, facilitating blood circulation and coordinated movement through contraction. Despite their distinct functional roles, these muscle types exhibit similarities in cytoarchitecture, protein expression, and excitation-contraction coupling. Both muscle types also undergo molecular remodeling in energy metabolism and cell size in response to acute and repeated exercise stimuli to enhance exercise performance. Reactive oxygen species (ROS) produced by NADPH oxidase (NOX) isoforms 2 and 4 have emerged as signaling molecules that regulate exercise adaptations. This review systematically compares NOX2 and NOX4 expression, regulation, and roles in cardiac and skeletal muscle responses across exercise modalities. We highlight the many gaps in our knowledge and opportunities to let future skeletal muscle research into NOX-dependent mechanisms be inspired by cardiac muscle studies and vice versa. Understanding these processes could enhance the development of exercise routines to optimize human performance and health strategies that capitalize on the advantages of physical activity.

Asunto(s)

Adaptación Fisiológica , Ejercicio Físico , Músculo Esquelético , Miocardio , NADPH Oxidasa 2 , Especies Reactivas de Oxígeno , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Músculo Esquelético/enzimología , Miocardio/metabolismo , Miocardio/enzimología , Ejercicio Físico/fisiología , Animales , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Especies Reactivas de Oxígeno/metabolismo , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Transducción de Señal

RESUMEN

Skeletal muscle wasting is a clinically proven pathology associated with Japanese encephalitis virus (JEV) infection; however, underlying factors that govern skeletal muscle damage are yet to be explored. The current study aims to investigate the pathobiology of skeletal muscle damage using a mouse model of JEV infection. Our study reveals a significant increment in viral copy number in skeletal muscle post-JEV infection, which is associated with enhanced skeletal muscle cell death. Molecular and biochemical analysis confirms NOX2-dependent generation of reactive oxygen species, leading to autophagy flux inhibition and cell apoptosis. Along with this, an alteration in mitochondrial dynamics (change in fusion and fission process) and a decrease in the total number of mitochondria copies were found during JEV disease progression. The study represents the initial evidence of skeletal muscle damage caused by JEV and provides insights into potential avenues for therapeutic advancement.

Asunto(s)

Virus de la Encefalitis Japonesa (Especie) , Encefalitis Japonesa , Dinámicas Mitocondriales , Músculo Esquelético , Especies Reactivas de Oxígeno , Animales , Especies Reactivas de Oxígeno/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/virología , Ratones , Encefalitis Japonesa/metabolismo , Dinámicas Mitocondriales/fisiología , Apoptosis/fisiología , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Autofagia/fisiología , Modelos Animales de Enfermedad

RESUMEN

Energy status is linked to the production of reactive oxygen species (ROS) in macrophages, which is elevated in obesity. However, it is unclear how ROS production is upregulated in macrophages in response to energy overload for mediating the development of obesity. Here, we show that the Rab-GTPase activating protein (RabGAP) TBC1D1, a substrate of the energy sensor AMP-activated protein kinase (AMPK), is a critical regulator of macrophage ROS production and consequent adipose inflammation for obesity development. TBC1D1 deletion decreases, whereas an energy overload-mimetic non-phosphorylatable TBC1D1S231A mutation increases, ROS production and M1-like polarization in macrophages. Mechanistically, TBC1D1 and its downstream target Rab8a form an energy-responsive complex with NOX2 for ROS generation. Transplantation of TBC1D1S231A bone marrow aggravates diet-induced obesity whereas treatment with an ultra-stable TtSOD for removal of ROS selectively in macrophages alleviates both TBC1D1S231A mutation- and diet-induced obesity. Our findings therefore have implications for drug discovery to combat obesity.

Asunto(s)

Proteínas Activadoras de GTPasa , Macrófagos , Obesidad , Especies Reactivas de Oxígeno , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Activadoras de GTPasa/genética , Especies Reactivas de Oxígeno/metabolismo , Obesidad/metabolismo , Obesidad/genética , Animales , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Metabolismo Energético , Masculino , Mutación , Dieta Alta en Grasa/efectos adversos , Ratones Noqueados , Humanos , Proteínas Quinasas Activadas por AMP/metabolismo , Inflamación/metabolismo

RESUMEN

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease mainly caused by mutations in the X-linked CYBB gene that abrogate reactive oxygen species (ROS) production in phagocytes and microbial defense. Gene repair using the CRISPR/Cas9 system in hematopoietic stem and progenitor cells (HSPCs) is a promising technology for therapy for CGD. To support the establishment of efficient and safe gene therapies for CGD, we generated a mouse model harboring a patient-derived mutation in the CYBB gene. Our CybbC517del mouse line shows the hallmarks of CGD and provides a source for Cybb-deficient HSPCs that can be used to evaluate gene-therapy approaches in vitro and in vivo. In a setup using Cas9 RNPs and an AAV repair vector in HSPCs, we show that the mutation can be repaired in 19% of treated cells and that treatment restores ROS production by macrophages. In conclusion, our CybbC517del mouse line provides a new platform for refining and evaluating novel gene therapies and studying X-CGD pathophysiology.

Asunto(s)

Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Terapia Genética , Enfermedad Granulomatosa Crónica , NADPH Oxidasa 2 , Enfermedad Granulomatosa Crónica/terapia , Enfermedad Granulomatosa Crónica/genética , Animales , Terapia Genética/métodos , Ratones , NADPH Oxidasa 2/genética , Especies Reactivas de Oxígeno/metabolismo , Células Madre Hematopoyéticas/metabolismo , Humanos , Macrófagos/metabolismo , Mutación

RESUMEN

Urban air pollution, a significant environmental hazard, is linked to adverse health outcomes and increased mortality across various diseases. This study investigates the neurotoxic effects of particulate matter (PM), specifically PM2.5 and PM10, by examining their role in inducing oxidative stress and subsequent neuronal cell death. We highlight the novel finding that PM increases mitochondrial ROS production via stimulating NOX4 activity, not through its expression level in Neuro-2A cells. Additionally, PMs provoke ROS production via increasing the expression and activity of NOX2 in SH-SY5Y human neuroblastoma cells, implying differential regulation of NOX proteins. This increase in mitochondrial ROS triggers the opening of the mitochondrial permeability transition pore (mPTP), leading to apoptosis through key mediators, including caspase3, BAX, and Bcl2. Notably, the voltage-dependent anion-selective channel 1 (VDAC1) increases at 1 µg/mL of PM2.5, while PM10 triggers an increase from 10 µg/mL. At the same concentration (100 µg/mL), PM2.5 causes 1.4 times higher ROS production and 2.4 times higher NOX4 activity than PM10. The cytotoxic effects induced by PMs were alleviated by NOX inhibitors GKT137831 and Apocynin. In SH-SY5Y cells, both PM types increase ROS and NOX2 levels, leading to cell death, which Apocynin rescues. Variability in NADPH oxidase sources underscores the complexity of PM-induced neurotoxicity. Our findings highlight NOX4-driven ROS and mitochondrial dysfunction, suggesting a potential therapeutic approach for mitigating PM-induced neurotoxicity.

Asunto(s)

Apoptosis , Mitocondrias , NADPH Oxidasa 4 , Neuronas , Material Particulado , Especies Reactivas de Oxígeno , Material Particulado/toxicidad , NADPH Oxidasa 4/metabolismo , NADPH Oxidasa 4/genética , Especies Reactivas de Oxígeno/metabolismo , Apoptosis/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Neuronas/patología , Línea Celular Tumoral , Estrés Oxidativo/efectos de los fármacos , Animales , Ratones , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética

RESUMEN

BACKGROUND: The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. We examined whether irradiation causes chronic vascular injury and whether short-term administration of statins during and after irradiation is sufficient to prevent chronic injury compared with long-term administration. METHODS AND RESULTS: C57Bl/6 mice were pretreated with pravastatin for 72 hours and then exposed to 12 Gy X-ray head-and-neck irradiation. Pravastatin was then administered either for an additional 24 hours or for 1 year. Carotid arteries were tested for vascular reactivity, altered gene expression, and collagen deposition 1 year after irradiation. Treatment with pravastatin for 24 hours after irradiation reduced the loss of endothelium-dependent vasorelaxation and protected against enhanced vasoconstriction. Expression of markers associated with inflammation (NFκB p65 [phospho-nuclear factor kappa B p65] and TNF-α [tumor necrosis factor alpha]) and with oxidative stress (NADPH oxidases 2 and 4) were lowered and subunits of the voltage and Ca2+ activated K+ BK channel (potassium calcium-activated channel subfamily M alpha 1 and potassium calcium-activated channel subfamily M regulatory beta subunit 1) in the carotid artery were modulated. Treatment with pravastatin for 1 year after irradiation completely reversed irradiation-induced changes. CONCLUSIONS: Short-term administration of pravastatin is sufficient to reduce chronic vascular injury at 1 year after irradiation. Long-term administration eliminates the effects of irradiation. These findings suggest that a prospective treatment strategy involving statins could be effective in patients undergoing radiation therapy. The optimal duration of treatment in humans has yet to be determined.

Asunto(s)

Inhibidores de Hidroximetilglutaril-CoA Reductasas , Ratones Endogámicos C57BL , Estrés Oxidativo , Pravastatina , Animales , Pravastatina/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/efectos de la radiación , Factores de Tiempo , Vasoconstricción/efectos de los fármacos , Vasoconstricción/efectos de la radiación , Vasodilatación/efectos de los fármacos , Vasodilatación/efectos de la radiación , Masculino , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Transcripción ReIA/metabolismo , NADPH Oxidasas/metabolismo , Ratones , Traumatismos Experimentales por Radiación/prevención & control , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/tratamiento farmacológico , Esquema de Medicación , Arterias Carótidas/efectos de la radiación , Arterias Carótidas/efectos de los fármacos , Enfermedad Crónica , Modelos Animales de Enfermedad , NADPH Oxidasa 4

RESUMEN

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

Asunto(s)

Macrófagos , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 2 , Animales , Ratones , Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/virología , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Encefalitis Japonesa/inmunología , Especies Reactivas de Oxígeno/metabolismo , Virus de la Encefalitis Japonesa (Especie) , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/inmunología , Enfermedades Neuroinflamatorias/virología , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología

RESUMEN

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

Asunto(s)

Ferroptosis , Trastornos de la Memoria , Microglía , NADPH Oxidasa 2 , Enfermedades Neuroinflamatorias , Rotenona , Animales , Ratones , NADPH Oxidasa 2/metabolismo , NADPH Oxidasa 2/genética , Microglía/metabolismo , Microglía/patología , Microglía/efectos de los fármacos , Rotenona/toxicidad , Ferroptosis/efectos de los fármacos , Enfermedades Neuroinflamatorias/metabolismo , Enfermedades Neuroinflamatorias/patología , Enfermedades Neuroinflamatorias/inducido químicamente , Enfermedades Neuroinflamatorias/genética , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/genética , Trastornos de la Memoria/patología , Masculino , Ratones Endogámicos C57BL , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/efectos de los fármacos , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/metabolismo , Disfunción Cognitiva/genética , Disfunción Cognitiva/patología , Neuronas/metabolismo , Neuronas/patología , Neuronas/efectos de los fármacos , Ratones Noqueados