Reactive oxygen species and mitochondria: A nexus of cellular homeostasis

Downregulation of p22phox in Retinal Pigment Epithelial Cells Inhibits Choroidal Neovascularization in Mice

Relative contributions of mitochondria and NADPH oxidase to deoxycorticosterone acetate-salt hypertension in mice

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Cisplatin Fails To Stimulate Production Of Reactive Oxygen Species and Release Of Neutrophil Extracellular Traps By Human Neutrophils In Vitro

Trypanosoma evansi triggered neutrophil extracellular traps formation dependent on myeloperoxidase, neutrophil elastase, and extracellular signal-regulated kinase 1/2 signaling pathways

Impaired Formation of Neutrophil Extracellular Traps (NETs) in Patients with Myelodysplastic Syndrome (MDS)

Simple SummaryPolymorphonuclear leukocytes (PMN) are the first line and most abundant immune cells against infection. They kill bacteria via phagocytosis, degranulation, and the formation of neutrophil extracellular traps (NETs). The formation of NETs is always accompanied by the production of reactive oxygen species (ROS). We aimed to research the NETs and ROS formation capacity of PMN in different lactational stages of Holstein cows. We also validated a model to mimic infection and inflammation to study the NETs and ROS formation capacity in vitro. Results show that the basal NETs and ROS values of PMN harvested from peripartum and lactating cows were higher than those in nulliparous heifers. The in vitro stimulation, using inflammatory products, of PMN derived from nulliparous heifers increased the production of ROS and NETs. PMN isolated from peripartum and lactating cows are primed to produce NETs and ROS, and this potentially contributes to the cows’ unfavorable inflammatory and immune status at these critical periods in their lactation cycle. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic and study fundamental aspects of the production of NETs and ROS in vitro.We aimed to research the neutrophil extracellular traps (NETs) and reactive oxygen species (ROS) formation capacity of polymorphonuclear cells (PMN) during different lactational stages of Holstein cows. We also aimed to validate a model which could mimic infection and inflammation in vitro by adding increasing concentrations of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) to PMN suspensions isolated from nulliparous heifers and evaluate their capacity to produce NETs and ROS. In 3 replicates, we collected blood from nulliparous heifers (n = 3), cows at the end of gestation (n = 3), early postpartum (n = 3) and in mid-lactation (n = 3) in which PMN were isolated. The production of ROS in PMN were assessed using the 2’,7’-Dichlorofluorescein diacetate method, while the SYTOX Orange and Quant-iT™ PicoGreen dsDNA ultra-sensitive nucleic fluorescent acid staining methods were applied in order to quantitatively analyze the formation of NETs. Statistical analyses were performed via linear regression models using the replicate as a random. ROS values of PMN harvested from peripartum cows were 1.3 times increased compared with those in nulliparous heifers (p < 0.01). Compared with nulliparous heifers, the production of NETs by PMN isolated from mid-lactation and postpartum cows was 2.1 and 2.5 times higher (p < 0.01), respectively. In 3 replicates, in vitro stimulation of PMN isolated from nulliparous heifers (n = 3) with LPS linearly increased the production of ROS and NETs (R2 = 0.96 and 0.86, respectively). Similarly, when PMN isolated from nulliparous heifers were stimulated with PMA, a linear increase in the production of ROS (R2 = 0.99) and NETs (R2 = 0.78) was observed. The basal NETs and ROS production is lower in nulliparous heifers. Thus, they are an excellent model to mimic inflammation and study fundamental aspects of the production of NETs and ROS in vitro.

Autophagy is a key regulator of cellular homeostasis that can be activated by pathogen-associated molecules and recently has been shown to influence IL-1β secretion by macrophages. However, the mechanisms behind this are unclear. Here, we describe a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with Toll-like receptor ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further activation of autophagy with rapamycin induced the degradation of pro-IL-1β and blocked secretion of the mature cytokine. Inhibition of autophagy promoted the processing and secretion of IL-1β by antigen-presenting cells in an NLRP3- and TRIF-dependent manner. This effect was reduced by inhibition of reactive oxygen species but was independent of NOX2. Induction of autophagy in mice in vivo with rapamycin reduced serum levels of IL-1β in response to challenge with LPS. These data demonstrate that autophagy controls the production of IL-1β through at least two separate mechanisms: by targeting pro-IL-1β for lysosomal degradation and by regulating activation of the NLRP3 inflammasome.

BackgroundPrevious studies indicated that infection with Haemonchus contortus is host-specific (goat: susceptible host; gerbil: paratenic host; mouse: resistant host). Neutrophils play an essential role in host defense against parasitic infection through phagocytic engulfment, reactive oxygen species (ROS) generation, and neutrophil extracellular traps (NETs) formation. NETs are large web-like complexes consisting of a DNA scaffold decorated with various proteins components, including histones, myeloperoxidase, and elastase. They are released through both ROS-dependent and ROS-independent pathways. Previous studies have demonstrated both constraints and effectiveness of NETs in helminths. However, the roles of NETs in anti-infection of H. contortus in different hosts are still unclear.MethodsTo assess host-specific variations in NETs release, neutrophils isolated from goats, gerbils, and mice were co-cultured with Haemonchus contortus third-stage larvae (HcL3), followed by quantitative analysis of NETs formation using the PicoGreen® fluorescence assay. Subsequently, H. contortus excretory–secretory proteins (HcESPs) were co-cultured with neutrophils isolated from each host species. NETs release and ROS production were then quantitatively assessed using PicoGreen® fluorescence intensity and oxidation-sensitive dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. In addition, the neutrophil’s phagocytic ability for FITC-dextran was evaluated by flow cytometric analysis. Finally, to elucidate the signaling pathways involved in HcESP-induced NETs release in goat neutrophils, four specific inhibitors were employed for pretreatment prior to stimulation.ResultsOur results demonstrate that in vitro stimulation with HcL3 triggers NETs formation. The release of NETs exhibits significant host-specific variation, specifically, neutrophils from mice showed the highest NETs release, followed by gerbils, and a minimal response in goats. Moreover, HcESP treatment markedly inhibited ROS generation and phagocytic capacity in neutrophils from all three host species. Intriguingly, HcESPs exerted host-specific modulation of NETs release, with inhibition observed in goats, enhancement in mice, and context-dependent modulation in gerbils. Mechanistic investigations revealed that the NETs suppression in goats neutrophils involved both nicotinamide adenine dinucleotide phosphate (NADPH) oxidase- and neutrophil elastase-dependent pathways.ConclusionsOur results demonstrate that HcESPs significantly inhibit NETs formation in goat neutrophils through dual modulation of NADPH oxidase and neutrophil elastase activity. This finding highlights these two enzymes as promising molecular targets for anti-helminthic vaccine development.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-025-06956-z.

Uric Acid Induces NADPH Oxidase–Independent Neutrophil Extracellular Trap Formation

Background: Excess reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) promotes apoptotic cell death following ischemic/reperfusion injury. Effect of chlorpromazine and promethazine (C+P) on brain activity was reported to induce neuroprotection. The current study was designed to evaluate the inhibitory function of C+P on oxidative injury after stroke. Methods: Adult male Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) followed by 6 or 24 h of reperfusion. At the onset of reperfusion, rats received C+P, or apocynin (NOX inhibitor), or rottlerin [protein kinase C (PKC) δ inhibitor]. Brain damage was evaluated using infarct volumes and neurological deficits as well as apoptotic cell death (TUNEL). The enzymatic activity of NOX and ROS production as well as protein expressions of NOX subunits (gp91 phox , p67 phox , p47 phox , and p22 phox ), phosphorylation of PKC δ (p-PKC δ)/PKC δ and manganese superoxide dismutase (MnSOD) was examined. Neural SHSY5Y cells were used under 2 h of oxygen-glucose deprivation (OGD) followed by reoxygenation for 6 and 24 h with or without C+P treatment. ROS and protein levels of NOX subunits, p-PKC δ/PKC δ and MnSOD were detected. Moreover, measurement of PKC δ membrane translocation and detection of the interaction of p47 phox and PKC δ through co-immunoprecipitation were performed. Results: C+P reduced cerebral infarct volumes, neurological deficits, and apoptotic cell death in the ischemic rats, as well observed in the presence of NOX and PKC δ inhibitors. ROS production, NOX activity, expression of NOX subunits, p-PKC δ/PKC δ and MnSOD were significantly reduced by C+P. In ischemic rats administered with NOX and PKC δ inhibitors, ROS, activity of NOX and the NOX subunits protein levels were all decreased. In the OGD/R model, C+P decreased ROS and protein levels of NOX subunits, p-PKC δ/PKC δ and MnSOD. Furthermore, C+P reduced the PKC δ membrane translocation and the interaction of p47 phox and PKC δ. Conclusion: C+P treatments confers neuroprotection in severe stroke by suppressing oxidative stress and ROS production. PKC δ/NOX/MnSOD may be the vital regulators and the potential targets for an efficacious therapy following ischemic stroke.

Skin, our first interface to the external environment, is subjected to oxidative stress caused by a variety of factors such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particulate matters, and psychological stress. Excessive reactive species, including reactive oxygen species and reactive nitrogen species, exacerbate skin pigmentation and aging, which further lead to skin tone unevenness, pigmentary disorder, skin roughness and wrinkles. Besides these, skin microbiota are also a very important factor ensuring the proper functions of skin. While environmental factors such as UV and pollutants impact skin microbiota compositions, skin dysbiosis results in various skin conditions. In this review, we summarize the generation of oxidative stress from exogenous and endogenous sources. We further introduce current knowledge on the possible roles of oxidative stress in skin pigmentation and aging, specifically with emphasis on oxidative stress and skin pigmentation. Meanwhile, we summarize the science and rationale of using three well-known antioxidants, namely vitamin C, resveratrol and ferulic acid, in the treatment of hyperpigmentation. Finally, we discuss the strategy for preventing oxidative stress-induced skin pigmentation and aging.

Chronic granulomatous disease (CGD) is an inherited immunodeficiency characterized by impaired phagocytic cells due to mutations in genes encoding the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme complex, leading to recurrent severe infections. In the innate immune system, NETosis, which is a program for the formation of neutrophil extracellular traps (NETs) has been highlighted. Over the past years, NETs have been recognized as being involved in the pathogenesis of immune-mediated inflammatory renal diseases. On the mechanism underlying usual NETosis, reactive oxygen species (ROS) activation by NADPH oxidase (NOX) has been considered to be essential. CGD patients are theoretically unable to form such usual NETosis because of a deficiency of NOX-dependent ROS activation. However, according to previous reports, stimulated neutrophils derived from CGD patients showed NOX-independent unusual NETosis enriched in mitochondrial DNA. The mitochondrial ROS activation on NETosis in CGD was reported to induce an elevated inflammatory response, instead of the usual ROS generation, which might cause autoimmune diseases. We are interested in the renal autoimmune disorder in CGD, especially the inflamed renal disorder caused by unusual mitochondria-rich NETosis in CGD. Indeed, in vivo analysis has revealed severe renal disorder in MRL/lpr lupus-prone mice lacking NOX activity compared to wild-type of MRL/lpr. A few clinical reports showed aggravation of inflamed glomerulonephritis in patients with CGD. Taken together, we presumed that a defect of usual NOX-dependent NETosis might be a clue for aggravated renal disorder in CGD. In the present review, we summarize the features of NETosis in CGD and discuss the aggravation mechanism for renal disorder in CGD by referring to previous reports.

Inflammatory Bowel Disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), results from dysregulated immune responses that drive chronic intestinal inflammation. Neutrophils, as key effectors of the innate immune system, contribute to IBD through multiple mechanisms, including the release of reactive oxygen species (ROS), pro-inflammatory cytokines, and neutrophil extracellular traps (NETs). NETs are web-like structures composed of DNA, histones, and associated proteins including proteolytic enzymes and antimicrobial peptides. NET formation is increased in IBD and has a context-dependent role; under controlled conditions, NETs support antimicrobial defense and tissue repair, whereas excessive or dysregulated NETosis contributes to epithelial injury, barrier disruption, microbial imbalance, and thrombotic risk. This review examines the roles of neutrophils and NETs in IBD. We summarize recent single-cell and spatial-omics studies that reveal extensive neutrophil heterogeneity in the inflamed gut. We then address the dual role of neutrophils in promoting tissue damage-through cytokine release, immune cell recruitment, ROS production, and NET formation-and in supporting microbial clearance and mucosal healing. We also analyze the molecular mechanisms regulating NETosis, as well as the pathways involved in NET degradation and clearance. Focus is given to the ways in which NETs disrupt the epithelial barrier, remodel the extracellular matrix, contribute to thrombosis, and influence the gut microbiota. Finally, we discuss emerging therapeutic strategies aimed at restoring NET homeostasis-such as PAD4 inhibitors, NADPH oxidase and ROS pathway modulators, and DNase I-while emphasizing the need to preserve antimicrobial host defenses. Understanding neutrophil heterogeneity and NET-related functions may facilitate the development of new therapies and biomarkers for IBD, requiring improved detection tools and integrated multi-omics and clinical data.

Neutrophils participate in the regulation of pathogens by phagocytosis as well as by generating neutrophil extracellular traps (NETs). Antiphospholipid antibodies, particularly those targeting beta‐2‐glycoprotein I (β2GPI), stimulate monocytes, platelets, and endothelial cells with prothrombotic participation. This study aimed to explore NET generation in response to anti‐β2GPI/β2GPI. A series of experiments involving the separation of primary human leukocytes, NETosis quantification using propidium iodide, exploration of NETosis by fluorescence microscopy, western blotting, examination of free Zn2+ using FluoZin‐3, and reactive oxygen species (ROS) examination with dihydrorhodamine 123 were performed in this study. We found that anti‐β2GPI/β2GPI triggered NETosis, resembling phorbol 12‐myristate 13‐acetate (PMA)‐induced NETosis in magnitude and morphology. The anti‐β2GPI/β2GPI complex in isolation stimulated NETs without relying on p38, protein kinase B (AKT), extracellular signal‐related kinase (ERK) 1/2, and zinc signals. NET generation was unaffected by the NADPH oxidase suppressor DP1. The anti‐β2GPI/β2GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti‐β2GPI/β2GPI complex. In summary, our results indicate that the anti‐β2GPI/β2GPI complex reinforced NET generation by relying on ROS.The significance of the paper in the context of current knowledgeNeutrophils as one of the first lines of defence and essential in the response to pathogen invasion. They eradicate bacteria via phagocytosis or by releasing antimicrobial proteins in degranulation. In this study, we explored the capability of anti‐β2GPI/β2GPI to stimulate NETosis, demonstrating that anti‐β2GPI/β2GPI is a promising method for triggering NET. Anti‐β2GPI/β2GPI induced ROS generation without relying on NADPH oxidase, which contributes to NETosis independently of ERK1/2, Zn2+, or AKT. Our results showed that anti‐β2GPI/β2GPI triggered NETosis, resembling PMA‐induced NETosis in magnitude as well as morphology. The anti‐β2GPI/β2GPI complex in isolation stimulated NETs without relying on p38, AKT, ERK1/2, or zinc signals. The anti‐β2GPI/β2GPI complex stimulated ROS generation without relying on NADPH oxidase, which may participate in NET generation triggered via the anti‐β2GPI/β2GPI complex.