Pharmacological inhibition of sodium-calcium exchange activates NADPH oxidase and induces infection-independent NETotic cell death.
Ontology highlight
ABSTRACT:
SUBMITTER: Inoue M
PROVIDER: S-EPMC8105669 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:AimsPrior studies have focused on the role of the kidney and vasculature in salt-induced modulation of blood pressure; however, recent data indicate that sodium accumulates in tissues and can activate immune cells. We sought to examine mechanisms by which salt causes activation of human monocytes both in vivo and in vitro.Methods and resultsTo study the effect of salt in human monocytes, monocytes were isolated from volunteers to perform several in vitro experiments. Exposure of human monocytes to elevated Na+ex vivo caused a co-ordinated response involving isolevuglandin (IsoLG)-adduct formation, acquisition of a dendritic cell (DC)-like morphology, expression of activation markers CD83 and CD16, and increased production of pro-inflammatory cytokines tumour necrosis factor-α, interleukin (IL)-6, and IL-1β. High salt also caused a marked change in monocyte gene expression as detected by RNA sequencing and enhanced monocyte migration to the chemokine CC motif chemokine ligand 5. NADPH-oxidase inhibition attenuated monocyte activation and IsoLG-adduct formation. The increase in IsoLG-adducts correlated with risk factors including body mass index, pulse pressure. Monocytes exposed to high salt stimulated IL-17A production from autologous CD4+ and CD8+ T cells. In addition, to evaluate the effect of salt in vivo, monocytes and T cells isolated from humans were adoptively transferred to immunodeficient NSG mice. Salt feeding of humanized mice caused monocyte-dependent activation of human T cells reflected by proliferation and accumulation of T cells in the bone marrow. Moreover, we performed a cross-sectional study in 70 prehypertensive subjects. Blood was collected for flow cytometric analysis and 23Na magnetic resonance imaging was performed for tissue sodium measurements. Monocytes from humans with high skin Na+ exhibited increased IsoLG-adduct accumulation and CD83 expression.ConclusionHuman monocytes exhibit co-ordinated increases in parameters of activation, conversion to a DC-like phenotype and ability to activate T cells upon both in vitro and in vivo sodium exposure. The ability of monocytes to be activated by sodium is related to in vivo cardiovascular disease risk factors. We therefore propose that in addition to the kidney and vasculature, immune cells like monocytes convey salt-induced cardiovascular risk in humans.
| S-EPMC8064439 | biostudies-literature
Project description:Neutrophils cast neutrophil extracellular traps (NETs) to defend the host against invading pathogens. Although effective against microbial pathogens, a growing body of literature now suggests that NETs have negative impacts on many inflammatory and autoimmune diseases. Identifying mechanisms that regulate the process termed "NETosis" is important for treating these diseases. Although two major types of NETosis have been described to date, mechanisms regulating these forms of cell death are not clearly established. NADPH oxidase 2 (NOX2) generates large amounts of reactive oxygen species (ROS), which is essential for NOX-dependent NETosis. However, major regulators of NOX-independent NETosis are largely unknown. Here we show that calcium activated NOX-independent NETosis is fast and mediated by a calcium-activated small conductance potassium (SK) channel member SK3 and mitochondrial ROS. Although mitochondrial ROS is needed for NOX-independent NETosis, it is not important for NOX-dependent NETosis. We further demonstrate that the activation of the calcium-activated potassium channel is sufficient to induce NOX-independent NETosis. Unlike NOX-dependent NETosis, NOX-independent NETosis is accompanied by a substantially lower level of activation of ERK and moderate level of activation of Akt, whereas the activation of p38 is similar in both pathways. ERK activation is essential for the NOX-dependent pathway, whereas its activation is not essential for the NOX-independent pathway. Despite the differential activation, both NOX-dependent and -independent NETosis require Akt activity. Collectively, this study highlights key differences in these two major NETosis pathways and provides an insight into previously unknown mechanisms for NOX-independent NETosis.
| S-EPMC4352781 | biostudies-literature
Project description:The vascular endothelium plays an important role in the regulation of inflammatory responses after trauma and hemorrhage. Interactions of neutrophils with endothelial cells (ECs) contribute to the activation of specific EC responses involved in innate immunity. We have previously reported that oxidants derived from the neutrophil reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is a critical regulator to EC activation. Our objective was to test the role of neutrophil NADPH oxidase-derived oxidants in mediating and enhancing hemorrhagic shock (HS)-induced activation of lung endothelial NADPH oxidase. Mice were subjected to HS and neutrophil depletion. The mice were also replenished with the neutrophil from NADPH oxidase-deficient mice. The resultant activation of lung NADPH oxidase was analyzed. The in vivo studies were also recapitulated with in vitro neutrophil-EC coculture system. HS induces NADPH oxidase activation in neutrophils and lung through high-mobility group box 1/Toll-like receptor 4-dependent signaling. In neutropenic mice, shock-induced NADPH oxidase activation in the lung was reduced significantly, but was restored upon repletion with neutrophils obtained from wild-type mice subjected to shock, but not with neutrophils from shock mice lacking the gp91(phox) subunit of NADPH oxidase. The findings were recapitulated in mouse lung vascular ECs cocultured with neutrophils. The data further demonstrate that neutrophil-derived oxidants are key factors mediating augmented High mobility group box 1 (HMGB1)-induced endothelial NADPH oxidase activation through a Rac1-dependent, but p38 mitogen-activated protein kinase-independent, pathway. Oxidant signaling by neutrophil NADPH oxidase is an important determinant of activation of endothelial NADPH oxidase after HS.
| S-EPMC3095934 | biostudies-literature
Project description:Seizure activity has been proposed to result in the generation of reactive oxygen species (ROS), which then contribute to seizure-induced neuronal damage and eventually cell death. Although the mechanisms of seizure-induced ROS generation are unclear, mitochondria and cellular calcium overload have been proposed to have a crucial role. We aim to determine the sources of seizure-induced ROS and their contribution to seizure-induced cell death. Using live cell imaging techniques in glioneuronal cultures, we show that prolonged seizure-like activity increases ROS production in an NMDA receptor-dependent manner. Unexpectedly, however, mitochondria did not contribute to ROS production during seizure-like activity. ROS were generated primarily by NADPH oxidase and later by xanthine oxidase (XO) activity in a calcium-independent manner. This calcium-independent neuronal ROS production was accompanied by an increase in intracellular [Na(+)] through NMDA receptor activation. Inhibition of NADPH or XO markedly reduced seizure-like activity-induced neuronal apoptosis. These findings demonstrate a critical role for ROS in seizure-induced neuronal cell death and identify novel therapeutic targets.
| S-EPMC4649505 | biostudies-literature
Project description:Rotenone, a widely used pesticide, reproduces parkinsonism in rodents and associates with increased risk for Parkinson disease. We previously reported that rotenone increased superoxide production by stimulating the microglial phagocyte NADPH oxidase (PHOX). This study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91(phox), the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91(phox). Functional studies showed that both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91(phox)/p22(phox)) and cytosolic subunits (p67(phox) and p47(phox)). Rotenone-elicited extracellular superoxide release in p47(phox)-deficient macrophages suggested that rotenone enabled activation of PHOX through a p47(phox)-independent mechanism. Increased membrane translocation of p67(phox), elevated binding of p67(phox) to rotenone-treated membrane fractions, and coimmunoprecipitation of p67(phox) and gp91(phox) in rotenone-treated wild-type and p47(phox)-deficient macrophages indicated that p67(phox) played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91(phox). Rac1, a Rho-like small GTPase, enhanced p67(phox)-gp91(phox) interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91(phox); such an interaction triggered membrane translocation of p67(phox), leading to PHOX activation and superoxide production.
| S-EPMC3253173 | biostudies-literature
Project description:It is now accepted that reactive oxygen species (ROS) are not only dangerous oxidative agents but also chemical mediators of the redox cell signaling and innate immune response. A central role in ROS-controlled production is played by the NADPH oxidases (NOXs), a group of seven membrane-bound enzymes (NOX1-5 and DUOX1-2) whose unique function is to produce ROS. Here, we describe the regulation of NOX5, a widespread family member present in cyanobacteria, protists, plants, fungi, and the animal kingdom. We show that the calmodulin-like regulatory EF-domain of NOX5 is partially unfolded and detached from the rest of the protein in the absence of calcium. In the presence of calcium, the C-terminal lobe of the EF-domain acquires an ordered and more compact structure that enables its binding to the enzyme dehydrogenase (DH) domain. Our spectroscopic and mutagenesis studies further identified a set of conserved aspartate residues in the DH domain that are essential for NOX5 activation. Altogether, our work shows that calcium induces an unfolded-to-folded transition of the EF-domain that promotes direct interaction with a conserved regulatory region, resulting in NOX5 activation.
| S-EPMC7317449 | biostudies-literature
Project description:Neutrophil extracellular traps (NETs) are cytotoxic DNA-protein complexes that play positive and negative roles in combating infection, inflammation, organ damage, autoimmunity, sepsis and cancer. However, NETosis regulatory effects of most of the clinically used drugs are not clearly established. Several recent studies highlight the relevance of NETs in promoting both cancer cell death and metastasis. Here, we screened the NETosis regulatory ability of 126 compounds belonging to 39 classes of drugs commonly used for treating cancer, blood cell disorders and other diseases. Our studies show that anthracyclines (e.g., epirubicin, daunorubicin, doxorubicin, and idarubicin) consistently suppress both NADPH oxidase-dependent and -independent types of NETosis in human neutrophils, ex vivo. The intercalating property of anthracycline may be enough to alter the transcription initiation and lead NETosis inhibition. Notably, the inhibitory doses of anthracyclines neither suppress the production of reactive oxygen species that are necessary for antimicrobial functions nor induce apoptotic cell death in neutrophils. Therefore, anthracyclines are a major class of drug that suppresses NETosis. The dexrazoxane, a cardioprotective agent, used for limiting the side effects of anthracyclines, neither affect NETosis nor alter the ability of anthracyclines to suppress NETosis. Hence, at correct doses, anthracyclines together with dexrazoxane could be considered as a therapeutic candidate drug for suppressing unwanted NETosis in NET-related diseases.
| S-EPMC6770146 | biostudies-literature
Project description:Candida albicans is one of the top leading causes of healthcare-associated bloodstream infection. Neutrophil extracellular traps (NET) are known to capture and kill pathogens. It is reported that opsonized C. albicans-triggered NETosis is NADPH oxidase-dependent. We discovered a NADPH oxidase-independent NETosis pathway in neutrophil response to unopsonized C. albicans. While CR3 engagement with opsonized C. albicans triggered NET, dectin-2 recognized unopsonized C. albicans and mediated NET formation. Engagement of dectin-2 activated the downstream Syk-Ca2+-PKC?-protein arginine deiminase 4 (PAD4) signaling pathway which modulated nuclear translocation of neutrophil elastase (NE), histone citrullination and NETosis. In a C. albicans peritonitis model we observed Ki67+Ly6G+ NETotic cells in the peritoneal exudate and mesenteric tissues within 3 h of infection. Treatment with PAD4 inhibitor GSK484 or dectin-2 deficiency reduced % Ki67+Ly6G+ cells and the intensity of Ki67 in peritoneal neutrophils. Employing DNA digestion enzyme micrococcal nuclease, GSK484 as well as dectin-2-deficient mice, we further showed that dectin-2-mediated PAD4-dependent NET formation in vivo restrained the spread of C. albicans from the peritoneal cavity to kidney. Taken together, this study reveals that unopsonized C. albicans evokes NADPH oxidase-independent NETosis through dectin-2 and its downstream signaling pathway and dectin-2-mediated NET helps restrain fungal dissemination.
| S-EPMC6834254 | biostudies-literature
Project description:Chronic inhalation of silica particles causes lung fibrosis and silicosis. Silica taken up by alveolar macrophages causes phagolysosomal membrane damage and leakage of lysosomal material into the cytoplasm to initiate apoptosis. We investigated the role of reactive oxygen species (ROS) in this membrane damage by studying the spatiotemporal generation of ROS. In macrophages, ROS generated by NADPH oxidase 2 (NOX2) was detected in phagolysosomes containing either silica particles or nontoxic latex particles. ROS was only detected in the cytoplasm of cells treated with silica and appeared in parallel with an increase in phagosomal ROS, as well as several hours later associated with mitochondrial production of ROS late in apoptosis. Pharmacological inhibition of NOX activity did not prevent silica-induced phagolysosomal leakage but delayed it. In Cos7 cells, which do not express NOX2, ROS was detected in silica-containing phagolysosomes that leaked. ROS was not detected in phagolysosomes containing latex particles. Leakage of silica-containing phagolysosomes in both cell types was transient, and after resealing of the membrane, endolysosomal fusion continued. These results demonstrate that silica particles can generate phagosomal ROS independent of NOX activity, and we propose that this silica-generated ROS can cause phagolysosomal leakage to initiate apoptosis.
| S-EPMC4569308 | biostudies-literature
Project description:Glycated albumin, an early-glycation Amadori-modified protein, stimulates transforming growth factor-beta (TGF-beta) expression and increases the production of the extracellular matrix proteins in mesangial cells, contributing to the pathogenesis of diabetic nephropathy. Glycated albumin has been shown to increase NADPH oxidase-dependent superoxide formation in mesangial cells. However, the mechanisms are not well understood. Therefore, in the present studies, we determined the mechanisms by which glycated albumin activates NADPH oxidase in primary rat mesangial cells and its contribution to glycated albumin-induced TGF-beta expression and extracellular matrix protein production. Our data showed that glycated albumin treatment stimulated NADPH oxidase activity and increased the formation of superoxide formation in rat mesangial cells. Moreover, glycated albumin treatment stimulated the expression and phosphorylation of p47phox, one of the cytosolic regulatory subunits of the NADPH oxidase. However, the levels of other NADPH oxidase subunits including Nox1, Nox2, Nox4, p22phox, and p67phox were not altered by glycated albumin. Moreover, siRNA-mediated knockdown of p47phox inhibited glycated albumin-induced NADPH oxidase activity and superoxide formation. Glycated albumin-induced TGF-beta expression and extracellular matrix production (fibronectin) was also inhibited by p47phox knock down. Taken together, these data suggest that up-regulation of p47phox is involved in glycated albumin-mediated activation of NADPH oxidase, leading to glycated albumin-induced expression of TGF-beta and extracellular matrix proteins in mesangial cells and contributing to the development of diabetic nephropathy.
| S-EPMC2900544 | biostudies-literature