MPO-DNA Complexes Research Articles

Neutrophil Extracellular Traps Induced By Activated Platelets Contribute to Hypercoagulable State in Patients with Colorectal Cancer

Background: Patients with colorectal cancer (CRC) are at increased risk of venous thromboembolism (VTE), but the precise mechanisms of hypercoagulability in CRC remain largely unknown. Neutrophil extracellular traps (NETs) are web-like chromatin structures decorated with cytoplasmic, granular and nuclear components of neutrophils, which can participate in both antimicrobial responses and contribute to a number of autoimmune and thrombotic diseases. However, a definitive role of NETs in the hypercoagulable state in CRC patients is still unclear. The aims of this study were to identify the novel role of NET in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs).Methods: Ninety-two CRC patients and 30 healthy controls were included. The presence of NETs was assessed using immunofluorescence microscopy. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit, myeloperoxidase (MPO)-DNA complex was measured using a capture enzyme linked immunosorbent assay (ELISA). Thrombin-antithrombin (TAT) complex of NETs was evaluated by ELISA. Coagulation time of NETs, platelets and ECs was assessed by coagulation time (CT) using one-stage recalcification time assays, purified coagulation complex and fibrin turbidity were measured using ELISA. PS exposure on platelets and ECs, and fibrin formation on ECs were detected with flow cytometry and confocal microscopy.Results: We showed that the levels of cf-DNA and MPO-DNA complexes in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage III and IV patients compared to healthy subjects (all P<0.01). In addition, NETs released by CRC patients shortened coagulation time (CT), significantly enhanced the generation of TAT complexes and the formation of fibrin fibrils compared to healthy controls (all P<0.05). Moreover, DNase1-mediated degradation of NETs resulted in decreased PCA in patients with CRC (P<0.001). Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1 (P<0.01). Conversely, NETs from CRC patients could also induce the exposure of PS on platelets and the release of platelet MPs (PMPs), leading to markedly enhanced intrinsic/extrinsic FXa and FIIa, as well as shortened CT (all P<0.05). Importantly, endothelial cells (ECs) were converted to a procoagulant phenotype when exposed to NETs from CRC patients. The PCA of NETs-activated platelets or ECs could be inhibited either by the cleavage of NETs with DNase1 or the blockage of histone with activated protein C (APC) (all P<0.05). Our study also showed that the levels of NETs in CRC patients was positively correlated with TAT complexes and D-dimer (all P<0.05).Conclusion: Our results suggest that activated platelets promote NETs formation through the release of HMGB1 and result in an elevated PCA in CRC patients. In turn, NETs induce platelet PS exposure and PMPs release, forming a vicious cycle. In addition, NETs could also induce a procoagulant phenotype of ECs, indicating the complex relationship among these cellular constituents and highlighting the procoagulant role and cytotoxic effects of NETs in CRC. We propose that the rapid developments in the field of NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antibody Stabilization of Neutrophil Extracellular Trap-Platelet Factor 4 Complexes Is Therapeutic in a Murine Model of Endotoxemia

Sepsis is characterized by life-threatening organ dysfunction caused by a dysregulated response to infection. Neutrophils play a crucial role in sepsis during which they release neutrophil extracellular traps (NETs), webs of negatively charged cell-free DNA (cfDNA) complexed with positively-charged histones that kill pathogens but also damage host tissue. While it has been proposed that NET digestion may be beneficial in the treatment of sepsis, it is also possible that this strategy leads to the release of harmful NET degradation products (NDPs), such as cfDNA, histones, and myeloperoxidase (MPO) that cause endothelial damage. An alternative approach would be treatment strategies that prevent NET lysis and the release of NDPs. We posited that NET-bound platelet factor 4 (PF4, CXCL4) may have this effect. PF4 is a highly-positively charged, platelet-specific chemokine released in high concentrations following platelet activation and aggregates polyanionic molecules like heparin, polyphosphates, and DNA. We have found that PF4 causes physical compaction of NETs without inducing histone release. We studied the effect of this compaction using an in vitro microfluidic assay in which NETs released from neutrophils stimulated by tissue-necrosis factor (TNF) α were infused over a layer of cultured human umbilical vein endothelial cells (HUVECs) ± PF4 (25 μg/ml) and incubated for 12 hours. Channels exposed to PF4 were “heathier” with significantly more residual attached endothelial cells (44±24 vs. 150±29 cells/hpf, n=6, p=0.0002). We have previously shown that mice that overexpress PF4 are protected from mortality in lipopolysaccharide (LPS) endotoxemia. Compared to wildtype animals, PF4 null mice have increased circulating levels of NET markers including cfDNA, citrullinated histones, and MPO. Treatment with exogenous PF4 leads to a decrease in plasma levels of NET components and a reduction in mortality. In a microfluidic assay in which channel-adherent NETs were treated with increasing concentrations of PF4 (0-25 µg/ml) and then infused with DNase I (100 U/ml), we found that compacted NET-PF4 complexes become resistant to DNase I digestion. Prior studies have revealed that when PF4 binds to polyanionic molecules, it exposes HIT-like antigenic sites. We confirmed that the NET-PF4 complexes similarly bind HIT IgG isolated from clinical samples and the HIT-like monoclonal antibody KKO. Interestingly, KKO binding further enhanced DNase I resistance, a phenomenon not seen with a polyclonal anti-PF4 antibody (abcam). We hypothesized that KKO which causes PF4 oligomerization unlike the polyclonal antibody, further stabilized the crosslinked NET-PF4 complexes, providing additional protection from DNase I digestion and preventing the release of NDPs. We then asked whether KKO may serve as a targeted therapy in the treatment of sepsis, enhancing NET-PF4 complex resistance to nuclease digestion and leading to the sequestration of NDPs. As KKO is an IgG2bk antibody, we were unable to use pepsin digestion to remove the Fc region that causes platelet activation. We therefore used an IgG-specific endoglycosidase to develop a deglycosylated version of KKO (DG-KKO) that retains the ability to bind to NET-PF4 complexes (data not shown), but has a reduced capacity to interact with hematopoietic cell Fc receptors. DG-KKO has a markedly decreased ability to activate human platelets compared to KKO in the presence of added PF4 as measured by P-selectin level (3710±140 control vs. 1580±300 treated MFI, n = 2). Compared to control, DG-KKO infusion in the murine endotoxemia model prevented thrombocytopenia (mean platelet count 286±16 control vs. 537±69 treated X103/µl, n=4-5, p=0.016), the release of cfDNA (8.5±0.3 vs. 3.7±0.7 µg/ml, n=4-5, p=0.016), and the emission of MPO-DNA complexes (234±14 vs. 99±5 % increase from non-LPS injected controls, n=4-5, p=0.016) As depicted in Figure 1, we propose that in sepsis, NETosis occurs (Step 1) causing the release of harmful NDPs (Step 2). PF4 expelled from activated platelets stabilizes PF4-NET complexes (Step 3). Infused DG-KKO enhances NET stability and decreases release of NDPs following DNase I digestion (Step 4). These studies provide mechanistic insights into the release of NDPs during endotoxemia and offer a targeted, novel therapeutic to prevent their contribution to inflammatory states such as sepsis. [Display omitted] DisclosuresPoncz:Incyte Corporation: Consultancy, Research Funding.

Neutrophil Extracellular Traps Promote Hypercoagulability in ST-Elevated Myocardial Infarction Following Fibrinolytic Administration

Introduction:Fibrinolysis plays an important role in the treatment of ST-elevated myocardial infarction (STEMI) when percutaneous coronary intervention is not readily available. Early and successful myocardial reperfusion with thrombolytic therapy effectively reduces the infarct size and improves the clinical outcome. However, the process of restoring blood flow to the ischemic myocardium can induce injury and reduce the beneficial effects of myocardial reperfusion. Previous studies had shown that platelets, leukocytes and TF play important role in thrombotic complications after fibrinolysis in AMI. However, there are still 10-15% patients who have risk for re-occlusion after antiplatelet and anticoagulant therapies. Thus, we speculate that there may be other mechanisms involved in the hypercoagulability after STEMI fibrinolysis. Neutrophil extracellular traps (NETs) are double-edge swords that could ensnare and kill microbial pathogens but also contribute to thrombosis. However, the role of NETs during STEMI fibrinolysis-induced re-occlusion is largely unknown. Our aims were to determine the procoagulant role of NETs after successful thrombolysis, and to elucidate its interaction with endothelial cells (ECs).Methods:31 STEMI patients with successfully fibrinolysis and 12 healthy controls were enrolled. Patient blood samples were collected at 0 h, 2 h, 6 h, 12 h and 24 h after fibrinolysis. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes and TAT (thrombin-antithrombin) complexes. Wright-Giemsa and immunofluorescence confocal microscope were used to analyze and quantify NETs formation in neutrophil cells. ECs were incubated in growth media containing 20% pooled serum obtained from healthy donors in the presence or absence of 20-fold concentrated neutrophil extracellular chromatin. The procoagulant activity (PCA) of neutrophils and ECs was measured by clotting time and purified coagulation complex assays. DNase I or anti-TF were included in the inhibition assays.Results: We found that cf-DNA, MPO-DNA and TAT are significantly reduced at 2 hours in STEMI patients with successful fibrinolysis. Their levels then increased and peaked at 6 hours (Figure 1A, B, E). Interestingly, the level of cf-DNA at 6 hours in STEMI thrombotic patients was positively correlated with TAT (r=0.959; p<0.01; Figure 1G). Wright-Giemsa and immunofluorescence staining showed that NETs were released by STEMI reperfusion neutrophils or by control neutrophils treated with plasma obtained from STEMI patients with fibrinolysis (Figure 1D,F), and the percentage of NETs-releasing PMNs was about 30% (Figure 1C). Isolated neutrophils from fibrinolytic patients in vitro demonstrated significantly shortened coagulation time and increased fibrin formation after 2 hours fibrinolysis, and peaked at 6 hours. DNase I but not anti-tissue factor antibody could inhibit these effects. Co-incubation assays revealed that NETs triggered PS exposure on ECs, converting them to a procoagulant phenotype. Confocal imaging of NETs-treated ECs illustrated that bound FVa and FXa colocalized within PS-enriched areas of ECs to form prothrombinase, and further supported fibrin formation. Moreover, patients with recurrent ischemia showed significantly higher NETs release and thrombin generation than non-recurrent ischemia.Conclusions: Our study reveals that the PCA of STEMI following fibrinolytic administration decrease after 2 hours, then increase and peak at 6 hours, which is at least partly due to the release of NETs induced by activated PMNs. Additionally, NETs partly contribute to ECs injury after myocardial reperfusion. DNase I can disconnect NETs and may therefore serve as a promising therapeutic target in STEMI reinfarction and recurrent ischemia. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neutrophil Extracellular Traps Enhance Procoagulant Activity in Patients with Oral Squamous Cell Carcinoma

Background: Cancer patients are considered to be prothrombotic with major disturbances in hemostasis that are associated with an increased risk of venous thromboembolism, especially in patients with advanced cancer. Recently reports show that the high levels of circulating microparticles (MPs) have procoagulant activity (PCA) in oral squamous cell carcinoma (OSCC). However, this study did not address the question of what specific mechanism might underlie the PCA in OSCC. Neutrophil extracellular traps (NETs) are activated neutrophil-derived web-like structures, which have emerged as important mediators in cancer progression, metastasis and cancer-associated thrombosis. Additionally, the cytokines and neutrophils were known to become aggregated in cancers and are usually present in high numbers in OSCC patients and are associated with poor outcomes. The exact molecular mechanisms responsible for modulation of neutrophils procoagulant functions in OSCC are, however, poorly understood. Thus, we hypothesized that cytokines might activate neutrophils to release NETs, thereby predisposing OSCC patients to a hypercoagulative state. Moreover, we evaluated NETs interaction with human umbilical vein endothelial cells (HUVECs) and their association with pathological lesions in this disease.Methods: OSCC patients (n = 58) were divided into four stages according to the 2009 guidelines of the American Joint Committee on Cancer staging classification, and compared to healthy controls (n = 25). Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes, TAT (thrombin-antithrombin) complexes, neutrophil elastase, nucleosomes, and cytokines. PCA of NETs was evaluated using coagulation time and purified coagulation complex and fibrin production assays. Phosphatidylserine (PS) exposure, fibrin strands, and FVa/Xa binding on cells were observed using confocal microscopy.Results:Plasma levels of NET markers in patients with stage III/IV OSCC were significantly higher than those in stage I/II patients or controls (all p<0.05), and positively correlated with thrombin-antithrombin (TAT) complex and fibrinogen levels. Interestingly, neutrophils from OSCC patients with stage III/IV were more prone to release NETs compared to those from stage I/II patients and controls. Additionally, we found that plasma from patients with stage III/IV OSCC was able to prime neutrophils to generate higher amounts of NETs than from stage I/II patients and controls. Depleting IL-8, IL-6 and TNF-a reduced plasma-enhance NETs release. In addition, NETs released by stage III/IV OSCC neutrophils significantly increased the potency of control plasma to generate thrombin and fibrin, greatly shortened the coagulation time (all p<0.05). These effects were attenuated by DNase I. Finally, isolated NETs induced ECs to lose normal morphology and retract from their cell-cell junctions, converting them to a pro-coagulant phenotype. DNase I attenuated this cytotoxicity.Conclusions:These results suggest that OSCC creates a systemic inflammation environment that primes neutrophils to release procoagulant NETs in patients with stage III/IV OSCC. The NETs formation correlated positively with the parameters of disease severity. The information that results from these investigations may serve as a rational basis for the design of future drug intervention trials that target coagulation reactions, mechanisms and/or interactions relevant to OSCC. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Quantification of NETs-associated markers by flow cytometry and serum assays in patients with thrombosis and sepsis.

Neutrophil extracellular traps (NETs) are networks of extracellular fibres produced from neutrophil DNA with a pathogenic role in infection, thrombosis and other conditions. Reliable assays for measuring NETs are desirable as novel treatments targeting NETs are being explored for the treatment of these conditions. We compare a whole blood flow cytometry method with serum assays to measure NETs-associated markers in patients with sepsis and thrombosis. Patients with deep venous thrombosis (n=25), sepsis (n=21) and healthy controls (n=23) were included in the study. Neutrophil surface NETs markers were determined by flow cytometry on whole blood samples by gating of neutrophils stained for surface citrullinated histone (H3cit) and myeloperoxidase (MPO). Serum double-stranded (ds) DNA, MPO, myeloid-related protein, nucleosomes, DNAse, elastase, human high-mobility group box 1 and MPO-DNA complexes were quantified as circulating markers of NETs. Neutrophil NETs markers by flow cytometry and serum NETs markers were significantly higher in patients with thrombosis and sepsis compared with healthy controls. Neutrophil NETs markers significantly correlated with the serum marker dsDNA. Flow cytometry detection of neutrophil NETs markers is feasible in whole blood and correlates with serum markers of NETs. We propose the flow cytometry detection of MPO/H3cit positive neutrophils and serum dsDNA as simple methods to quantify cellular and extracellular NET markers in patients with thrombosis and sepsis.

Neutrophil Extracellular Traps Exacerbate Inflammatory Responses and Thrombotic Tendency in Both a Murine Colitis Model and Patients with Inflammatory Bowel Disease

Introduction: Inflammatory bowel disease (IBD) arises from a combination of genetic susceptibility and environmental factors, which trigger inappropriate mucosal inflammatory responses, the pathogenesis of which still remains exclusive. Therapeutic strategies for IBD mainly focus on controlling the active inflammation, intestinal epithelial barrier destruction and thrombotic tendency. Our group has recently demonstrated, for the first time, that patients with active IBD exhibited enhanced neutrophil extracellular traps (NETs) release, leading to a hypercoagulable state (He Z et al, Thromb Haemost 2016). However, the mechanisms by which NETs drive the pathogenesis of IBD remain unclear. The aim of this study was to further identify the novel role of NETs in the initiation and progression of IBD.Methods: 51 consecutive patients with IBD were studied. Disease activity was assessed by using the Mayo Score (MS) for patients with (ulcerative colitis) UC and (Crohn's disease) CD. Acute disease was induced by the treatment of C57BL/6 mice with 3.5% DSS in drinking water for 6 days. For an inhibition assay, DNase I perfusion or neutrophil depletion with anti-Ly6G antibody (1A8 clone) was performed. Cell-free DNA (cf-DNA) was quantified using the Quant-iT PicoGreen dsDNA Assay Kit. ELISA was used to detect MPO-DNA complexes, TAT (thrombin-antithrombin) complexes, nucleosomes, chemokines, and cytokines.Results: Compared to subjects with inactive UC or CD, patients with active UC or CD had significantly increased levels of cf-DNA, nucleosomes and NETs formation (MPO-DNA complexes). Neutrophils from active CD and UC demonstrated more spontaneous NET release as compared to inactive patients and controls. In DSS-induced colitis, significantly higher levels of serum cf-DNA and NETs formation were found in mice on day 4 and day 6 after DSS initiation. Western blot analysis and immunofluorescent staining of colonic tissues from mice with DSS-induced colitis showed increased NETs release and deposition. Mice treated with DNase I were protected from DSS-induced colitis, showing slighter weight loss, lower disease activity index, improved survival rate, diminished colon length shortening and decreased histologic signs of inflammation compared with controls. Furthermore, DNase I perfusion also decreased MPO levels by 62% on day 4 and by 58% on day 6, indicating DNase down-regulated neutrophil infiltration during DSS-induced colitis. The expression of Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, CXCL2, CXCL10 and monocyte chemotactic protein-1(MCP-1) messenger RNA in colonic extracts were lower in DNase I perfused DSS-induced mice compared with saline perfused DSS-induced mice. Incubation of normal platelets with NETs from active IBD patients, but not inactive IBD patients, significantly enhanced their procoagulant activity by 32% and the ability to support fibrin formation by 42%. This effect was blocked by DNase I treatment.Conclusions: We have extended our previous study and demonstrated that NETs constitute a central component in the initiation and progression of colitis through mediating inflammation cell infiltration, driving cytokines release and thrombotic tendency. NET degradation through DNase I perfusion protected mice from severe DSS-induced colitis. Thus, strategies focusing on the application of DNase I to accelerate the degradation of excessive NET release and deposition may offer potential therapeutic benefits for patients with inflammatory bowel disease.Disclosure of Interest: None declared. DisclosuresNo relevant conflicts of interest to declare.

Increased levels of neutrophil extracellular trap remnants in the serum of patients with rheumatoid arthritis.

Neutrophil extracellular traps (NETs) comprise a unique form of non-apoptotic cell death exhibited by neutrophils, which occurs in a stepwise process termed NETosis. It has been postulated that NETosis plays an important role in the pathogenesis of autoimmune disorders. The aim of this study was to evaluate serum levels of NET remnants in patients with rheumatoid arthritis (RA), as well as potential associations between NET remnants and indicators of RA. Serum levels of myeloperoxidase (MPO)-DNA complexes (NET remnants) were examined in 74 RA patients and 50 healthy controls using a modified enzyme-linked immunosorbent assay. Associations between the levels of these complexes and indicators of RA were then statistically evaluated. RA patients exhibited significantly higher levels of MPO-DNA complexes than the healthy controls, and these levels were associated with increased neutrophil counts and positivity for rheumatoid factor (RF) and anti-citrullinated protein/peptide antibodies (ACPA). Among the 63 ACPA-positive RA patients examined, those with ACPA titers > 1600 U/mL showed significantly increased MPO-DNA levels. Receiver operating characteristic analysis determined that the area under the curve for all 74 RA patients was 0.798, with a sensitivity of 91.9% and a specificity of 56.0%, while that for the ACPA-negative patients was 0.891, with a sensitivity and specificity of 81.8% and 84.0%, respectively. The results of this study suggest that the disease status of RA is associated with increased NETosis. In particular, evaluation of serum MPO-DNA levels may comprise a useful complementary tool for discriminating RA patients from healthy individuals.

IL-33 exacerbates liver sterile inflammation by amplifying neutrophil extracellular trap formation

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. 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. 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. 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. 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.

Neutrophil Extracellular Traps Accelerate Cholestatic Liver Injury through Bile Acids in Bile Duct Ligation Mice

Introduction: The mechanisms involved in cholestatic liver injury remain elusive although emerging evidence indicate neutrophils, rather than bile acids (BAs), are the predominant source of damage. Neutrophil extracellular traps (NETs) are double-edge swords that serve to ensnare and kill microbial pathogens but also contribute to excessive inflammation and tissue damage. However, the role of NETs during bile duct ligation (BDL)-induced cholestatic liver injury is largely unknown. Moreover, whether neutrophils and BAs cooperatively participate in this process also needs to be investigated. Our objectives were to localize and determine the magnitude of NETs in the hepatic microvasculature, define the role of NETs on hematological and histological changes, and characterize the role of BAs in the induction of NETs in BDL mice.Methods: BDL was induced in C57BL/6 mice by ligation of the common bile duct. After 3 days, liver tissue was collected and NETs were detected by immunostaining and western blot. Serum myeloperoxidase (MPO)-DNA complexes were measured by ELISA. Cholestatic liver injury was characterized by hematological and histological assessment. NETs were dismantled by DNase I or depleted with peptidylarginine deiminase 4 (PAD4) inhibitor Cl-amidine. The apoptosis of hepatocytes was measured by TUNEL and the induction of chemokines on Kupffer cells was analyzed by quantitative polymerase chain reaction (qPCR). BAs and inflammatory cytokines were utilized collaboratively to induce NETs formation both in vitro and in vivo. Diphenylene iodonium (DPI) was used to inhibit reactive oxygen species (ROS) formation. Plasma was obtained from healthy controls and patients with entrahepatic cholestasis.Results: We found that NETs formed in the sinusoids of cholestatic liver lobes in vivo, which was associated with significantly increased serum MPO-DNA complexes (P<0.01) and tissue levels of citrullinated-histone 3 (P<0.05) in BDL mice compared with sham operated mice. The formation of NETs exacerbated BDL-induced liver injury as evidenced by markedly increased levels of serum alamine transaminase (ALT) and percentage of necrotic tissue area (both P<0.05 vs. sham group, Figure 1). Depleting of NETs with DNase I or Cl-amidine decreased NETs formation, protected hepatocytes and dramatically alleviated cholestatic liver injury, indicating the pathophysiological role of NETs in BDL mice (Figure 1). In addition, NETs promoted the apoptosis of hepatocytes and induced the release of chemokines from Kupffer cells in BDL mice compared to sham group. Both effects could be inhibited by DNase I or Cl-amidine. Moreover, BAs induced mouse neutrophils, primed with inflammatory cytokines, to release NETs in vitro in a dose-dependent manner and mediated by PAD4 and ROS. Intravenously injection of BAs and inflammatory cytokines to healthy mice lead to the formation of NETs and exacerbated liver injury, suggesting that depleting NETs can prevent organ damage. Furthermore, we found NETs markers were significantly increased in the serum of cholestatic patients, correlating positively with increased levels of ALT and BAs.Conclusions: Our results suggest that BAs promote NETs formation via PAD4 and ROS. Development of NETs subsequently initiates inflammatory responses and exacerbates organ damage during BDL-induced cholestatic liver injury, and may therefore serve as a promising therapeutic target in cholestasis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Arsenic Trioxide Potentiates Extracellular Traps-Formation Cell Death in Acute Promyelocytic Leukemia through mTOR-Regulated Autophagy

Background:Emerging clinical data shows that arsenic trioxide (ATO) exerts selective cytotoxicity against acute promyelocytic leukemia (APL) without severe side effects that mainly ascribed to nonspecific induction of apoptosis. It is attractive to speculate whether other uncovered APL cell death exists which can be specifically sparked by ATO administration. We have recently demonstrated that APL cells can undergo a previously unrecognized pathway for death by releasing extracellular DNA traps (ETs), termed ETosis (Ma R et al, Cell Death Dis 2016). However, besides apoptosis, whether ATO induces this APL-specific ETotic cell death remains to be explored. We wereto investigated the effects of a wide range of concentrations of ATO on ETotic death in APL cells and elucidate the underlying molecular mechanisms.Methods: Bone marrow samples were obtained from sixteen APL patients before and after the continuous administration of ATO for two weeks. APL cells were isolated and cultured in the presence and absence of ATRA for 3 days. We used confocal microscopy to image ET formation by APL cells and the percentage of ETotic cells was simultaneously quantified. ELISA was used to measure the concentration of myeloperoxidase (MPO)-DNA complexes in the supernant. We also assessed the effects of a wide range of concentrations (0.1, 0.25, 0.5, 0.75, 1.0, 2.0 μM) of ATO treatment for 24, 48 and 72 hours on ETosis in APL-cell line NB4 cells in vitro. Autophagy activation and leukemia-initiating cell (LIC) activity were evaluated by immunoblotting and imaged by immunostaining. LICs were analyzed using colony-forming unit (CFU) assays, and identified and quantified by flow cytometry.Results: APL cells isolated from ATO-treated APL patients underwent significantly increased spontaneous (P = 0.005) and ATRA-stimulated (P = 0.002) ETosis compared to those from newly diagnosed patients (n = 16). In vitro ATO treatment showed that the percentage of ETotic NB4 cells dramatically increased at 0.5 μM (8 ± 1.6%), peaked at 0.75 μM (15 ± 2.4%) and was gradually suppressed at higher concentrations. The concentration of MPO-DNA complexes, an indirect marker of ETosis, parallelled the dose-dependent change in the percentage of ETotic cells. Interestingly, inhibition experiments indicated that ATO caused concentration-dependent APL cell death: ATO primarily triggered ETosis at moderate concentrations (0.5 to 0.75 μM) and switched to apoptosis at relatively high doses (1.0 to 2.0 μM). Furthermore, We found that ATO induced ETosis through mammalian target of rapamycin (mTOR)-regulated autophagy. Surprisingly, inhibition of ETosis spared LIC activity from ATO reduction, while combined treatment with ATO and rapamycin further increased ETosis-mediated LIC loss (~3.5-fold).Conclusions: This is the first study to show that ATO potentiates ETotic death in APL cells through mTOR-regulated autophagy. Importantly, further investigation suggests that ATO specifically targets the APL LICs to ETosis. This implies that, in combination with ATO, therapy-triggered ETosis by targeting the corresponding signaling pathways could be a novel and effective strategy to improve a long relapse-free survival through LIC clearance, avoid lethal apoptosis-related complications and overcome apoptosis resistance.Conflict of interest statement: None. DisclosuresNo relevant conflicts of interest to declare.

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils&lt;em&gt; In Vitro&lt;/em&gt;

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils developed several antimicrobial mechanisms including phagocytosis, degranulation and formation of neutrophil extracellular traps (NETs). NETs consist of a DNA scaffold decorated with histones and several granule markers including myeloperoxidase (MPO) and human neutrophil elastase (HNE). NET release is an active process involving characteristic morphological changes of neutrophils leading to expulsion of their DNA into the extracellular space. NETs are essential to fight microbes, but uncontrolled release of NETs has been associated with several disorders. To learn more about the clinical relevance and the mechanism of NET formation, there is a need to have reliable tools capable of NET quantitation. Here three methods are presented that can assess NET release from human neutrophils in vitro. The first one is a high throughput assay to measure extracellular DNA release from human neutrophils using a membrane impermeable DNA-binding dye. In addition, two other methods are described capable of quantitating NET formation by measuring levels of NET-specific MPO-DNA and HNE-DNA complexes. These microplate-based methods in combination provide great tools to efficiently study the mechanism and regulation of NET formation of human neutrophils.

Superoxide induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-dependent mechanism.

Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil Extracellular Traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and non-parenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH Oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination, and increased levels of MPO-DNA complexes occurring in a TLR-4 dependent manner. Inhibition of superoxide generation by Allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT, but not TLR-4 KO mice. To reduce circulating superoxide we pretreated mice undergoing I/R with Allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings.

Periodontal bacteria in human carotid atherothrombosis as a potential trigger for neutrophil activation

ObjectiveEpidemiological, biological and clinical links between periodontal and cardiovascular diseases are now well established. Several human studies have detected bacterial DNA corresponding to periodontal pathogens in cardiovascular samples. Intraplaque hemorrhage has been associated with a higher risk of atherosclerotic plaque rupture, potentially mediated by neutrophil activation. In this study, we hypothesized that plaque composition may be related to periodontal pathogens. MethodsCarotid culprit plaque samples were collected from 157 patients. Macroscopic characterization was performed at the time of collection: presence of blood, lipid core, calcification and fibrosis. Markers of neutrophil activation released by carotid samples were quantified (myeloperoxidase or MPO, cell-free DNA and DNA-MPO complexes). PCR analysis using specific primers for Porphyromonas gingivalis, Aggregatibacter actinomycetemcommitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia was used to detect DNA from periodontal pathogens in carotid tissues. In addition, bacterial lipopolysaccharide (LPS) and Immunoglobulins G against T. forsythia were quantified in atherosclerotic carotid conditioned medium. ResultsIntraplaque hemorrhage was present in 73/157 carotid samples and was associated with neutrophil activation, reflected by the release of MPO, cell-free DNA and MPO-DNA complexes. LPS levels were also linked to intraplaque hemorrhage but not with the neutrophil activation markers. Seventy-three percent of the carotid samples were positive for periodontal bacterial DNA. Furthermore, hemoglobin levels were associated with the detection of T. forsythia and neutrophil activation/inflammation markers. ConclusionThis study suggests a potential role of periodontal microorganisms, especially T. forsythia, in neutrophil activation within hemorrhagic atherosclerotic carotid plaques.

Netting neutrophils in autoimmune small-vessel vasculitis

Small-vessel vasculitis (SVV) is a chronic autoinflammatory condition linked to antineutrophil cytoplasm autoantibodies (ANCAs). Here we show that chromatin fibers, so-called neutrophil extracellular traps (NETs), are released by ANCA-stimulated neutrophils and contain the targeted autoantigens proteinase-3 (PR3) and myeloperoxidase (MPO). Deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes suggest that NET formation triggers vasculitis and promotes the autoimmune response against neutrophil components in individuals with SVV.