Neutrophil extracellular trap induced by HMGB1 exacerbates damages in the ischemic brain.
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ABSTRACT:
SUBMITTER: Kim SW
PROVIDER: S-EPMC6556959 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cardiovascular disease (CVD) is the leading cause of death in patients with chronic kidney disease (CKD). Endothelial cell (EC) dysfunction is a key CKD-specific risk factor; however, the mechanisms by which uremia harms the endothelium are still unclear. We report a role for excessive neutrophil extracellular trap (NET) formation induced by uremic serum on EC injury. Level of plasma nucleosome and myeloperoxidase-DNA, established in vivo markers of NETs, as well as intracellular adhesion molecule (ICAM)-1 were measured in hemodialysis (HD) patients and healthy volunteers (HV) and their prognostic role evaluated. For in vitro studies, HV-derived neutrophils and differentiated HL-60 cells by retinoic acid were used to determine the effect of uremic serum-induced NETs on human umbilical vein EC (HUVEC). The level of in vivo NETs was significantly higher in incident HD patients compared to HV, and these markers were strongly associated with ICAM-1. Specifically, nucleosome and ICAM-1 levels were independent predictors of a composite endpoint, all-cause mortality, or vascular access failure. In vitro, HD-derived uremic serum significantly increased NET formation both in dHL-60 and isolated neutrophils compared to control serum, and these NETs decreased EC viability and induced their apoptosis. In addition, the level of ICAM-1, E-selectin and von Willebrand factor in HUVEC supernatant was significantly increased by uremic serum-induced NETs compared to control serum-induced NETs. Dysregulated neutrophil activities in the uremic milieu may play a key role in vascular inflammatory responses. The high mortality and CVD rates in ESRD may be explained in part by excessive NET formation leading to EC damage and dysfunction.
| S-EPMC8563801 | biostudies-literature
Project description:BACKGROUND & AIMS:Neutrophils and liver sinusoidal endothelial cells (LSECs) both contribute to sterile inflammatory injury during ischemia/reperfusion (I/R), a well-known liver surgical stress. Interleukin-33 (IL-33) has been shown to drive neutrophil infiltration during inflammatory responses through its receptor ST2. We recently reported that infiltrating neutrophils form neutrophil extracellular traps (NETs), which exacerbate sterile inflammatory injury in liver I/R. Here, we sought to determine the role of IL-33 in NET formation during liver sterile inflammation. METHODS:Evaluation of IL-33 forming NETs was investigated using a partial liver I/R model to generate sterile injury in healthy WT, IL-33 and ST2 knockouts. Serum levels of IL-33 and myeloperoxidase (MPO)-DNA complex were measured in both humans and mice after the first surgery. Liver damage was assessed. Mouse neutrophil depletion was performed by intraperitoneal injection of anti-Ly6G antibody before I/R. RESULTS:Patients undergoing liver resection showed a significant increase in serum IL-33 compared to healthy volunteers. This coincided with higher serum MPO-DNA complexes. NET formation was decreased in IL-33 and ST2 knockout mice compared with control mice, after liver I/R. IL-33 or ST2 deficiency protected livers from I/R injury, whereas rIL-33 administration during I/R exacerbated hepatotoxicity and systemic inflammation. In vitro, IL-33 is released from LSECs to promote NET formation. IL-33 deficient LSECs failed to induce NETs. ST2 deficient neutrophils limited their capacity to form NETs in vitro and adoptive transfer of ST2 knockout neutrophils to neutrophil-depleted WT mice significantly decreased NET formation. CONCLUSIONS:Data establish that IL-33, mainly released from LSECs, causes excessive sterile inflammation after hepatic I/R by inducing NET formation. Therapeutic targeting of IL-33/ST2 might extend novel strategies to minimize organ damage in various clinical settings associated with sterile inflammation. LAY SUMMARY:Liver ischemia and reperfusion injury results in the formation of neutrophil extracellular traps, which contribute to organ damage in liver surgeries. Herein, we show that IL-33 is released from liver sinusoidal endothelial cells to promote NET formation during liver I/R, which exacerbates inflammatory cascades and sterile inflammation.
| S-EPMC5862757 | biostudies-literature
Project description:UnlabelledInnate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage-associated molecular patterns, which trigger an innate immune and inflammatory cascade through pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to organ damage and innate immune and inflammatory responses. Formation of neutrophil extracellular traps (NETs) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase-DNA complexes, and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. Damage-associated molecular patterns, such as High Mobility Group Box 1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout or TLR9 knockout neutrophils confers significant protection from liver I/R injury with a significant decrease in NET formation. In addition, we found inhibition of NET formation by the peptidyl-arginine-deiminase 4 inhibitor and that DNase I reduces High Mobility Group Box 1 and histone-mediated liver I/R injury.ConclusionDamage-associated molecular patterns released during liver I/R promote NET formation through the TLR signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R.
| S-EPMC4515210 | biostudies-literature
Project description:Histones, the major protein components of chromatin, are released into the extracellular space during sepsis, trauma, and ischemia-reperfusion injury, and subsequently mediate organ failure. Extracellular histones can promote endothelial damage and platelet aggregation, which can be suppressed by administration of recombinant thrombomodulin (rTM). The present study aimed to clarify whether histones can activate neutrophils to induce NET formation and whether rTM can prevent histone-induced NET formation. NET formation was analyzed in vitro by stimulating human neutrophils with histones in the absence or presence of rTM. NET formation was further analyzed in vivo by intravenous infusion of histones into rats with or without rTM. Histones induced NET release in a dose-dependent manner in vitro and NET release was induced as early as 1 h after stimulation. Histone-induced NET release was independent of NADPH oxidase. rTM suppressed histone-induced NET release in vitro as well as in vivo. The suppression might be mediated by rTM binding to histones, as suggested by analysis using a quartz crystal microbalance system. The present findings suggest that histones can activate neutrophils to form NETs and that rTM can inhibit histone-induced NET formation.
| S-EPMC6828967 | biostudies-literature
Project description:ObjectiveThe release of neutrophil extracellular traps (NETs) by hyperactive neutrophils has recently been recognized to play an important role in antiphospholipid syndrome (APS). This study was undertaken to evaluate autoantibodies targeting NETs in patients with primary APS, and to determine their potential functions and clinical associations.MethodsWe measured global anti-NET activity in 76 patients with primary APS, 23 patients with systemic lupus erythematosus without antiphospholipid antibodies (aPL), 11 patients with a history of unprovoked venous thrombosis without aPL, and 44 healthy controls. The ability of APS sera to degrade NETs was also assessed.ResultsWe found markedly elevated levels of anti-NET IgG and IgM in patients with primary APS compared with healthy controls (for IgG, mean ± SD optical density 0.55 ± 0.34 versus 0.33 ± 0.17; for IgM, mean ± SD optical density 0.76 ± 0.51 versus 0.26 ± 0.23). This anti-NET activity did not correlate with levels of traditional aPL and was relatively stable over time. Mechanistically, anti-NET antibodies (especially of the IgG isotype) impaired the ability of patient sera to degrade NETs (r = 0.4, P = 0.003). Levels of anti-NET IgM inversely correlated with complement C4 (r = 0.4, P = 0.019). Clinically, anti-NET antibodies associated with certain APS clinical manifestations, and in particular recurrent venous thrombosis (odds ratio 4.3; P = 0.002). Interestingly, anti-NET antibody levels also appeared to be associated with unprovoked venous thrombosis in the general population (for IgM, mean ± SD optical density 0.67 ± 0.34 versus 0.26 ± 0.23).ConclusionOur data indicate high levels of anti-NET antibodies in patients with primary APS, which may impair NET clearance and activate the complement cascade. These findings may ultimately enable more effective risk stratification.
| S-EPMC7722115 | biostudies-literature
Project description:ObjectiveThe role of neutrophil extracellular traps (NETs) in acute heart failure is unknown. We recently showed that interleukin 8, a putative NETs stimulator, was associated with myocardial recovery in acute heart failure complicating ST-elevation myocardial infarction (STEMI). In this exploratory post-hoc study, we aimed to investigate the role of NETs components in relation to myocardial function and interleukin 8 in STEMI patients with symptomatic acute heart failure.MethodsIn 61 STEMI patients developing acute heart failure within 48 hours of successful revascularization, wall motion score index (WMSI), global longitudinal strain (GLS) and left ventricular ejection fraction (LVEF) were assessed by echocardiography at baseline and on day 5. Blood drawn at baseline and days 1, 2 and 5 was used to quantify double-stranded DNA (dsDNA), myeloperoxidase-DNA complexes (MPO-DNA) and citrullinated histone 3 (CitH3). The area under the curve (AUC) of each NETs marker and interleukin 8 was approximated for the first 5 days.ResultsdsDNAAUC and MPO-DNAAUC correlated significantly with change in WMSI from baseline to day 5 (rs = 0.28 for both, p≤0.05), whereas NETs AUCs did not correlate with changes in GLS and LVEF. dsDNAAUC was significantly correlated with interleukin 8AUC (r = 0.40, p = 0.003). However, mixed model regression could not identify a significant effect of the NETs components on myocardial function parameters.ConclusionsIn this cohort with acute heart failure complicating STEMI, NETs components were partly correlated with myocardial function and interleukin 8 levels, yet no causal relationship between NETs components and myocardial recovery could be established.Clinical trial registrationClinicalTrials.gov, identifier: NCT00324766.
| S-EPMC7595325 | biostudies-literature
Project description:BACKGROUND AND PURPOSE:Recent data suggest that neutrophil extracellular traps (NETs) form aggregates with microparticles (MPs) upon activation of neutrophils although the functional role of NET-MP complexes remain elusive. The objective of this study was to examine the role of NET-MP aggregates in leukocyte recruitment in vivo. EXPERIMENTAL APPROACH:PMA stimulation of murine bone marrow neutrophils generated NET-MP complexes and pretreatment with caspase and calpain inhibitors resulted in the formation of NETs depleted of MPs. Leukocyte-endothelium interactions were studied by using intravital microscopy of the mouse cremaster microcirculation. KEY RESULTS:Intrascrotal injection of NET-MP aggregates dose-dependently increased leukocyte recruitment. In contrast, leukocyte responses were markedly reduced after administration of NETs depleted of MPs. Neutrophil depletion abolished intravascular and extravascular leukocytes in response to challenge with NET-MP complexes. Electron microscopy revealed that NET-associated MPs express HMGB1. Notably, immunoneutralization of HMGB1 markedly decreased NET-MP complex-induced neutrophil accumulation. Moreover, inhibition of TLR2 and TLR4 significantly reduced neutrophil recruitment in response to NET-MP aggregates. CONCLUSIONS AND IMPLICATIONS:These data show that NET-MP complexes are potent inducers of neutrophil recruitment, which is dependent on HMGB1 expressed on MPs and mediated via TLR2 and TLR4. Blocking MP binding to NETs or downstream inhibition of the HMGB1-TLR2/TLR4 axis might provide useful targets to attenuating NET-dependent tissue damage in acute inflammation.
| S-EPMC6692579 | biostudies-literature
Project description:Ischemic stroke is major cause of disability and mortality worldwide, and aging is strong risk factor for poor post-stroke outcome. Neutrophils traffic rapidly to the brain following ischemic stroke, and recent evidence has suggested that aging may alter neutrophil function after tissue injury. In this study, we hypothesize that aging enhances the pro-inflammatory function of neutrophils, directly contributing to the poorer outcomes seen in aging patients. We utilized demographic data and biological specimens from ischemic stroke patients and an experimental mouse model to determine the correlation between age, neutrophil function and stroke outcomes. In ischemic stroke patients, age was associated with increased mortality and morbidity and higher levels of neutrophil-activating cytokines. In mice, aged animals had higher stroke mortality and morbidity, higher levels of neutrophil-activating cytokines and enhanced generation of neutrophil reactive oxygen species compared to young mice. Finally, depletion of neutrophils via a specific monoclonal antibody after ischemic stroke led to long-term benefits in functional outcome in aged male and female animals, with no benefit observed in young. These results demonstrate that aging is associated with augmented neutrophil pathogenicity in ischemic stroke, and that neutrophil-targeted therapies may confer greater benefit in aged subjects.
| S-EPMC6977697 | biostudies-literature
Project description:BackgroundNeutrophil-induced tissue damage contributes to the rejection in xenotransplantation. Therefore, suppressing neutrophil function could be effective in suppressing xenogeneic rejection. In a previous study, we demonstrated that the ectopic expression of human cluster of differentiation 31 (CD31) on porcine endothelial cells (PEC) significantly suppressed neutrophil-mediated cytotoxicity through the homophilic binding of CD31. Cluster of differentiation 177 (CD177) was recently reported to be a high-affinity heterophilic binding partner for CD31 on endothelial cells. Thus, we hypothesized that human CD177 on PEC might induce a stronger suppression in neutrophil-mediated cytotoxicity compared with CD31. In this study, the inhibitory function of human CD177 on PEC in neutrophil-mediated cytotoxicity was investigated.MethodsPEC were transfected with a cloning plasmid containing cDNA inserts that encoded for hCD177 and hCD31 genes. Neutrophil-induced cytotoxicity was evaluated by flow cytometry after coculturing with PEC or PEC/CD177 in the presence of phorbol 12-myristate 13-acetate. To elucidate the mechanisms responsible for hCD177-induced suppression, the phosphorylation of src homology region 2 domain containing phosphatase 1 was measured by immunoblot analysis.ResultsHuman CD177 on PEC induced a significant reduction in neutrophil-induced cytotoxicity. In addition, CD177 on PEC induced a significant increase in the phosphorylation of src homology region 2 domain-containing phosphatase 1 in neutrophils and suppressed NETosis.ConclusionsThese findings suggest that human CD177 suppresses neutrophil-mediated cytotoxicity through the inhibition of NETosis.
| S-EPMC8439991 | biostudies-literature
Project description:Neutrophils undergo a unique form of cell death to generate neutrophil extracellular traps (NETs). It is well established that citrullination of histones (e.g., CitH3) facilitates chromatin decondensation during NET formation (NETosis), particularly during calcium-induced NETosis that is independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activation. However, the importance of other forms of histone modifications in NETosis has not been established. We considered that acetylation of histones would also facilitate NETosis. To test this hypothesis, we induced NOX-dependent NETosis in human neutrophils with phorbol myristate acetate or lipopolysaccharide (from Escherichia coli 0128), and NOX-independent NETosis with calcium ionophores A23187 or ionomycin (from Streptomyces conglobatus) in the presence or absence of two pan histone deacetylase inhibitors (HDACis), belinostat and panobinostat (within their half maximal inhibitory concentration (IC50) range). The presence of these inhibitors increased histone acetylation (e.g., AcH4) in neutrophils. Histone acetylation was sufficient to cause a significant increase (~20%) in NETosis in resting neutrophils above baseline values. When acetylation was promoted during NOX-dependent or -independent NETosis, the degree of NETosis additively increased (~15?30%). Reactive oxygen species (ROS) production is essential for baseline NETosis (mediated either by NOX or mitochondria); however, HDACis did not promote ROS production. The chromatin decondensation step requires promoter melting and transcriptional firing in both types of NETosis; consistent with this point, suppression of transcription prevented the NETosis induced by the acetylation of histones. Collectively, this study establishes that histone acetylation (e.g., AcH4) promotes NETosis at baseline, and when induced by both NOX-dependent or -independent pathway agonists, in human neutrophils. Therefore, we propose that acetylation of histone is a key component of NETosis.
| S-EPMC6359456 | biostudies-literature