Neutrophils Extracellular Traps Formation Research Articles

Heterozygous Variants in the Clpbgene Associated with Neutropenia Affect Neutrophil Function

Introduction The CLPB gene encodes a caseinolytic peptidase belonging to the ATPase family and acts as a mitochondrial chaperone. While biallelic mutations in CLPB are associated with the 3-methylglutaconic aciduria and severe neutropenia, (MIM number: 616271 3), some heterozygous variants seem to induce neutropenia exclusively. In the current study, we evaluated for the first time a function of neutrophils among neutropenic carriers of CLPB mutation. Methods Among 282 individuals with neutropenia studied with targeted Next-Generation Sequencing (panel of 725 genes related to hematological disorders), three neutropenic probands with heterozygous CLPB amino acid substitutions were identified. Neutrophils from patients and control healthy donors were isolated from peripheral blood with a Pancoll density gradient. The percentages of freshly isolated neutrophils undergoing apoptosis or necrosis were determined by flow cytometry using Annexin V - FITC and Propidium Iodide (PI) staining. Phagocytic capacity was evaluated using pHrodo Green S. aureus BioParticles Conjugates. A Boyden's chamber model was used to determine the level of the neutrophil's mobility and chemotaxis response to 10% human serum. Formation of Neutrophils Extracellular Traps (NETs) and induction of the oxidative burst were studied upon phorbol 12-myristate 13-acetate (PMA) stimulation (25 nM) with SYTOX™ Green Nucleic Acid and flow cytometry with CellROX Green assay, respectively. The intracellular and circulating concentrations of anti-microbial compounds (Myeloperoxidase MPO, Lactotransferrin LTF, Cathelicidin-LL37, and defensins) were measured using commercially available ELISA assays. The results were evaluated with an unpaired t-test using GraphPad Prism 9. Quantitative data presented as medians (25%-75%). Results Genetic analysis identified three heterozygous variants of CLPB gene p.Arg327Trp, p.Arg628Cys, p .Arg629Cys in three different families. Two of these mutations were novel p.Arg327Trp and p .Arg629Cys. Interestingly, the p.Arg327Trp variant was located outside the ATPase domain. In total 7 family members who suffered from neutropenia and were carriers of the CLPB variants were available for neutrophil functional assays (Figure 1). Neutrophils collected from CLPB-deficient individuals have a higher rate of apoptosis as compared to healthy neutrophils: 57(52-66)% vs 23(20-27)%, P<0.0001, and necrosis: 0.56(0.31-1.07)% vs 0.13(0.10-0.21)%, P=0.02. CLPB-deficient neutrophils display reduced mobility in the absence of stimulation: 298(191-413) vs 1011(936-1201) migrating neutrophils, P<0.0001, and after 10% human serum treatment: 2090(1746-2458) vs 3736(3071-4828) migrating neutrophils, P=0.0014. Similarly, reduced phagocytosis of S. aureus bioparticles was observed in CLPB-deficient neutrophils: 1890(1679-2063) vs 3352(2472-4440) Fluorescence Units, respectively, P=0.0029. Upon PMA stimulation, neutrophils from CLPB patients produced less reactive oxygen species compared to controls: 311(175-542)FMI vs 775(766-1150)FMI, P=0.08, and their ability to produce NETs is almost entirely abolished: 0.5(0.21-0.79)ng/µl vs 1.3(1.08-3.05)ng/µl, P=0.0075. Moreover, CLPB-deficient neutrophils have reduced intracellular concentration of myeloperoxidase: 35(31-39)ng/mg vs 55(41-63)ng/mg, P=0.0043 and cathelicidin LL-37: 5.5(5-6.3)ng/µg vs 19.22(14.21-23.28)ng/µg, P=0.0003. The concertation of the LL-37 is also reduced in patients' plasma 1.34(0.84-1.78)ng/ml vs 7.17(5.38-8.03)ng/ml, P<0.0001. Both intracellular and circulating concentrations of LTF and defensins did not differ between study groups. Conclusion Neutrophils from neutropenic carriers of CLPB mutations are functionally impaired, especially concerning their ability for NETs formation and cathelicidin LL-37 production.

Neutrophils extracellular traps formation may serve as a biomarker for disease activity in oligoarticular juvenile idiopathic arthritis: a pilot study

BackgroundJuvenile idiopathic arthritis (JIA) is the most common chronic rheumatic disease in children, causing significant morbidity. Despite the dramatic improvement in treatment, many patients do not achieve complete remission, and biomarkers for subclinical disease, flares, and response to treatment are lacking. Neutrophils and neutrophil extracellular traps (NETs) play key roles in the pathogenesis of autoimmune and inflammatory conditions. In this study, we characterized neutrophil enzyme activity and NETs formation in oligoarticular and polyarticular JIA and explored their association with disease activity.MethodsNeutrophils from 6 healthy controls and 7 patients with oligoarticular and polyarticular JIA were freshly isolated at time of diagnosis and after glucocorticoid intra-articular injection. Enzymatic activity of neutrophil granular enzymes was monitored by colorimetry and PMA-activated NETs formation was assessed using fluorescent microscopy.ResultsIn this pilot and feasibility study, we revealed that NETs were significantly increased in oligoarticular JIA patients at time of diagnosis compared to healthy controls. Anti-inflammatory treatment using intra-articular steroid injection normalized NETs formation in these patients. Correlation between NETs formation and clinical Juvenile Activity Disease Activity Score-10 (cJADAS-10) was linear and significant (P = 0.007) in oligo but not in poly JIA patients.ConclusionsThis is the first study exploring the link of NETs formation with oligo and poly JIA activity. We demonstrated a statistically significant linear correlation between cJADAS-10 and NETs formation in oligo but not in poly JIA patients. Hence, we suggest that NETs may reflect clinical disease activity in JIA, and may serve as a putative biomarker. Further work is needed to validate these initial results and determine the dynamics of NETs formation in JIA.

Targeting neutrophils extracellular traps (NETs) reduces multiple organ injury in a COVID-19 mouse model

BackgroundCOVID-19 is characterized by severe acute lung injury, which is associated with neutrophil infiltration and the release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment.MethodsHere, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. SARS-CoV-2-infected K18-hACE2 mice were performed for clinical sickness scores and lung pathology. Moreover, the levels of NETs were assessed and lung injuries were by histopathology and TUNEL assay. Finally, the injury in the heart and kidney was assessed by histopathology and biochemical-specific markers.ResultsDNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potentially deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I.ConclusionsTogether, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

Inhibition of Netosis with PAD Inhibitor Attenuates Endotoxin Shock Induced Systemic Inflammation.

Neutrophils play a pivotal role in innate immunity by releasing neutrophils extracellular traps (NETs). Excessive NETs are detrimental to the local tissue and further exacerbate inflammation. Protein arginine deiminases (PAD) mediate histone citrullination and NET formation that, in turn, exacerbate endotoxin shock damages. In this study, we further investigated the molecular mechanism underlying PAD and NETs in endotoxic stress in mice. The control group mice were injected with solvent, the LPS endotoxic shock group mice were intraperitoneally injected with LPS at 35 mg/kg only, while the LPS and PAD inhibitor YW3-56 treatment group mice were injected with YW3-56 at 10 mg/kg prior to the LPS injection. YW3-56 significantly prolonged the survival time of the LPS-treated mice. NETs, cfDNA, and inflammatory factors were detected by ELISA in serum, paitoneal cavity, and lung at 24 h after LPS administration. Lung injuries were detected by immunostaining, and lung tissue transcriptomes were analyzed by RNA-seq at 24 h after LPS administration. We found that YW3-56 altered neutrophil tissue homeostasis, inhibited NET formation, and significantly decreased cytokines (IL-6, TNFα and IL-1β) levels, cytokines gene expression, and lung tissue injury. In summary, NET formation inhibition offers a new avenue to manage inflammatory damages under endotoxic stress.

Colchicine Inhibits NETs and Alleviates Cardiac Remodeling after Acute Myocardial Infarction.

Colchicine, a multipotent anti-inflammatory drug, has been reported to alleviate cardiac remodeling and improve cardiac function after acute myocardial infarction (AMI). However, the underlying mechanism remains incompletely understood. Because neutrophils extracellular traps (NETs) enhance inflammation and participate in myocardial ischemia injury, and colchicine can inhibit NETosis, we thus aimed to determine whether colchicine exerts cardioprotective effects on AMI via suppressing NETs. Adult C57BL/6 mice were subjected to permanent ligation of the left anterior descending coronary artery and treated with colchicine (0.1mg/kg/day) or Cl-amidine (10mg/kg/day) for 7 or 28days after AMI. Cardiac function was evaluated by echocardiography, and NETs detected by immunofluorescence. ROS production was detected using 2',7'-dichlorodihydrofluorescein diacetates (DCFH-DA) fluorometry. Intracellular Ca2+ concentration was assessed by a fluorometric ratio technique. We found that colchicine treatment significantly increased mice survival (89.8% in the colchicine group versus 67.9% in control, n = 32 per group; log-rank test, p < 0.05) and improved cardiac function at day 7 (left ventricular ejection fraction (LVEF): 28.0 ± 9.2% versus 12.6 ± 3.9%, n = 8 per group; p < 0.001) and at day 28 (LVEF: 26.2 ± 7.2% versus 14.8 ± 6.7%, n = 8 per group; p < 0.001) post-AMI. In addition, the administration of colchicine inhibited NETs formation and inflammation. Furthermore, colchicine inhibited NETs formation by reducing NOX2/ROS production and Ca2+ influx. Moreover, prevention of NETs formation with Cl-amidine significantly alleviated AMI-induced cardiac remodeling. Colchicine inhibited NETs and cardiac inflammation, and alleviated cardiac remodeling after acute myocardial infarction.

Investigating the Link between Alpha-1 Antitrypsin and Human Neutrophil Elastase in Bronchoalveolar Lavage Fluid of COVID-19 Patients.

Neutrophils play a pathogenic role in COVID-19 by releasing Neutrophils Extracellular Traps (NETs) or human neutrophil elastase (HNE). Given that HNE is inhibited by α1-antitrypsin (AAT), we aimed to assess the content of HNE, α1-antitrypsin (AAT) and HNE–AAT complexes (the AAT/HNE balance) in 33 bronchoalveolar lavage fluid (BALf) samples from COVID-19 patients. These samples were submitted for Gel-Electrophoresis, Western Blot and ELISA, and proteins (bound to AAT or HNE) were identified by Liquid Chromatography-Mass Spectrometry. NETs’ release was analyzed by confocal microscopy. Both HNE and AAT were clearly detectable in BALf at high levels. Contrary to what was previously observed in other settings, the formation of HNE–AAT complex was not detected in COVID-19. Rather, HNE was found to be bound to acute phase proteins, histones and C3. Due to the relevant role of NETs, we assessed the ability of free AAT to bind to histones. While confirming this binding, AAT was not able to inhibit NET formation. In conclusion, despite the finding of a high burden of free and bound HNE, the lack of the HNE–AAT inhibitory complex in COVID-19 BALf demonstrates that AAT is not able to block HNE activity. Furthermore, while binding to histones, AAT does not prevent NET formation nor their noxious activity.

Activated Platelets Autocrine 5-Hydroxytryptophan Aggravates Sepsis-Induced Acute Lung Injury by Promoting Neutrophils Extracellular Traps Formation.

Excessive neutrophil extracellular trap (NET) formation is an important contributor to sepsis-induced acute lung injury (ALI). Recent reports indicate that platelets can induce neutrophil extracellular trap formation. However, the specific mechanism remains unclear. Tph1 gene, which encodes the rate-limiting enzyme for peripheral 5-hydroxytryptophan (5-HT) synthesis, was knocked out in mice to simulate peripheral 5-HT deficiency. Cecal ligation and puncture (CLP) surgery was performed to induce sepsis. We found that peripheral 5-HT deficiency reduced NET formation in lung tissues, alleviated sepsis-induced lung inflammatory injury, and reduced the mortality rate of CLP mice. In addition, peripheral 5-HT deficiency was shown to reduce the accumulation of platelets and NETs in the lung of septic mice. We found that platelets from wild-type (WT), but not Tph1 knockout (Tph1 −/− ), mice promote lipopolysaccharide (LPS)-induced NET formation. Exogenous 5-HT intervention increased LPS-induced NET formation when Tph1 −/− platelets were co-cultured with WT neutrophils. Therefore, our study uncovers a mechanism by which peripheral 5-HT aggravated sepsis-induced ALI by promoting NET formation in the lung of septic mice.

Neutrophils Extracellular Traps Formation and Reactive Oxygen Species (ROS) Production by Milk Immune Cells from Camels with Subclinical Mastitis

Subclinical mammary gland infections are within the most important infectious diseases in dromedary camels with high impact on milk production and animal health. Using flow cytometry, the present study analysed the capacity of milk phagocytes to form neutrophil extracellular traps (NETs) and to produce reactive oxygen species (ROS) in vitro. Based on the California mastitis test, clinically healthy camels were divided into animals with subclinical mastitis (n = 5) and camels with healthy mammary gland (n = 5). The ex vivo ROS production and the NETs formation activity of milk phagocytes were compared between healthy and affected animals. A basic fraction of phagocytes (10.0 ± 1.7% of total cells) with positive staining with the NETs-sensitive dye SYTOX™ Green was detected in milk samples from healthy camels. The NETs-positive fraction was significantly lower in milk from camels with subclinical mastitis (4.8 ± 1.5 % of total cells) compared to milk samples from healthy camels. Stimulation of milk cells with the gram-negative bacteria E. coli resulted in enhanced ROS production in milk phagocytes from both healthy and affected camels. The two groups, however, did not differ in the ROS level in their unstimulated or stimulated phagocytes. In conclusion, the present study identified basic levels of NETs formation by milk phagocytes separated from healthy camels. The reduced NETs formation by cells from infected camels may play a role in the pathogenesis of subclinical mammary gland infections in camels.

Neutrophils Extracellular Traps Induced By Lymphoma-Derived IL-8 Could Promote Diffuse Large B Cell Lymphoma Progression

Background: Neutrophils extracellular traps (NETs) are extracellular strands of decondensed DNA in complex with citrullinated histones (H3cit) and granule proteins including myeloperoxidase (MPO) (Sørensen O E, et al. 2016). Although this structure was initially described as an antimicrobial defense mechanism (Brinkmann V, et al. 2007), recent evidence has linked NETs to tumor metastases, and a previous study has demonstrated that the interaction of NETs and CD5+ B cells favored the transition from autoimmunity to lymphoma (Sangaletti S, et al. 2014). But it remains unclear whether NETs promote diffuse large B cell lymphoma (DLBCL) progression and can be a therapeutic candidate. Thus, our objectives were to detect NETs in the DLBCL samples, define the role of NETs in progression of DLBCL and reveal the underlying mechanism.Methods: We collected 123 tissue samples from DLBCL patients. NETs in tissues were detected by immunofluroscent staining. In cocultured model, we tested the effect of NETs on cell proliferation and migration of DLBCL cell lines. In vivo, murine A20 cells were inoculated in BALB/C mice (n = 5 mice/group), tumor growth and axillary lymph nodes metastasis were monitored by computer tomography. Tumor tissues and plasma were collected and NETs were detected by immunofluroscence and ELISA.Results: We detected more NETs structures in advanced stage samples than early stage samples (P= 0.035). Higher NETs expression was significantly associated with more B symptoms, extranodal involvement, higher IPI, elevated LDH level and lower overall response rate. In a univariate analysis, we found high NETs expression was associated with significant inferior survival (OS, HR: 3.250, 95%CI: 1.451-7.278, P= 0.002; progression free survival, HR: 2.678, 95%CI: 1.271-5.642, P= 0.007). In a multivariate analysis, high NETs expression was also found to be an independent predictor of OS and PFS (OS, HR: 2.824, 95%CI: 1.242-6.423, P= 0.013; PFS, HR: 2.398, 95%CI: 1.125-5.113, P= 0.024).Next, we wanted to determine whether DLBCL could induce NETs formation and how tumor-induced NETs were formed. As previous study proved that IL-8 could prime neutrophils for NETosis (Alfaro C, et al.2016), we detected IL-8 expression in the same tissue samples by immunostaining, and found advanced stage presented with high IL-8 expression and IL-8 also predicted inferior OS, suggesting that IL-8 may interact with NETs. Using a transwell chamber assay, we found only the media of human Su-dhl2 and murine A20, not human Su-dhl4 or Su-dhl6, potentiated species-matched neutrophils to generate extensive NETs. The level of IL-8 was highest in the supernatant of Su-dhl2 (3181.00 ± 386.60 pg/ml), nearly 10.63 and 8.57 folds higher than Su-dhl4 and Su-dhl6 (P= 0.002); and blocking IL-8 significantly reduced the ability of Su-dhl2 to induce NETs. CXCL1 and CXCL2, IL-8 murine homologues, were also high in the media of A20 cells, and NETosis induced by A20 cells was attenuated by murine CXCL1 or/and CXCL2 neutralizing antibody. We also found IL-8 mainly bound to CXCR2, not CXCR1, to induce NETs via the activation of MAPK and Src pathway.Then we sought to investigate whether NETs formation affected the growth and dissemination of DLBCL. In vitro, we treated Su-dhl2 cells and A20 cells with PMA-stimulating neutrophils media, and we observed that the treatment of NETs increased tumor proliferation at 72 hours and promoted cell migration, which were attenuated by administration of DNase I, a known inhibitor of NETs formation. In vivo, we found that NETs formed in the tumor tissues, which was associated with significantly increased serum MPO-DNA complexes in untreated mice. However, the systemic administration of DNase I or CXCR2 neutralizing antibody significantly decreased NETs formation, and retarded heterotopic tumor growth and axillary lymph nodes metastases compared with untreated group.Conclusion: In the present study, we firstly demonstrated the value of NETs to predict DLBCL inferior survival, and provided clear evidence that tumor-derived IL-8 bound to its receptor CXCR2, not CXCR1, to induce NETs formation via the activation of Src and MAPK pathway. And we also proved that this structure could promote DLBCL progression. Most importantly, our findings provided preliminary evidence that the use of DNase I or CXCR2 neutralizing antibody could reduce DLBCL progression and will hopefully pave the way for future clinical trials. DisclosuresNo relevant conflicts of interest to declare.

The Antibody Receptor Fc Gamma Receptor IIIb Induces Calcium Entry via Transient Receptor Potential Melastatin 2 in Human Neutrophils.

Human neutrophils express two unique antibody receptors for IgG, the FcγRIIa and the FcγRIIIb. FcγRIIa contains an immunoreceptor tyrosine-based activation motif (ITAM) sequence within its cytoplasmic tail, which is important for initiating signaling. In contrast, FcγRIIIb is a glycosylphosphatidylinositol (GPI)-linked receptor with no cytoplasmic tail. Although, the initial signaling mechanism for FcγRIIIb remains unknown, it is clear that both receptors are capable of initiating distinct neutrophil cellular functions. For example, FcγRIIa is known to induce an increase in L-selectin expression and efficient phagocytosis, while FcγRIIIb does not promote these responses. In contrast, FcγRIIIb has been reported to induce actin polymerization, activation of β1 integrins, and formation of neutrophils extracellular traps (NET) much more efficiently than FcγRIIa. Another function where these receptors seem to act differently is the increase of cytoplasmic calcium concentration. It has been known for a long time that FcγRIIa induces production of inositol triphosphate (IP3) to release calcium from intracellular stores, while FcγRIIIb does not use this phospholipid. Thus, the mechanism for FcγRIIIb-mediated calcium rise remains unknown. Transient Receptor Potential Melastatin 2 (TRPM2) is a calcium permeable channel expressed in many cell types including vascular smooth cells, endothelial cells and leukocytes. TRPM2 can be activated by protein kinase C (PKC) and by oxidative stress. Because we previously found that FcγRIIIb stimulation leading to NET formation involves PKC activation and reactive oxygen species (ROS) production, in this report we explored whether TRPM2 is activated via FcγRIIIb and mediates calcium rise in human neutrophils. Calcium rise was monitored after Fcγ receptors were stimulated by specific monoclonal antibodies in Fura-2-loaded neutrophils. The bacterial peptide fMLF and FcγRIIa induced a calcium rise coming initially from internal pools. In contrast, FcγRIIIb caused a calcium rise by inducing calcium entry from the extracellular medium. In addition, in the presence of 2-aminoethoxydiphenyl borate (2-APB) or of clotrimazole, two inhibitors of TRPM2, FcγRIIIb-induced calcium rise was blocked. fMLF- or FcγRIIa-induced calcium rise was not affected by these inhibitors. These data suggest for the first time that FcγRIIIb aggregation activates TRPM2, to induce an increase in cytoplasmic calcium concentration through calcium internalization in human neutrophils.

The Crosstalk Between Cancer Cells and Neutrophils Enhances Hepatocellular Carcinoma Metastasis via Neutrophil Extracellular Traps-Associated Cathepsin G Component: A Potential Therapeutic Target.

BackgroundEmerging evidences have highlighted the roles of neutrophils, as the major host microenvironment component, in the development of hepatocellular carcinoma (HCC). Neutrophils extracellular traps (NETs) produced in the infection can strengthen the behavior of cancer metastasis. Here, we investigated the roles of NETs in HCC metastasis and further explore the underlying mechanism of how NETs interact with cancer.MethodsThe neutrophils were isolated from whole blood of HCC patients and used to evaluate the formation of NETs. NET markers were detected in tissue samples, plasma and cell climbing slice. Mouse models were used to evaluate the roles of NETs in HCC metastasis in vivo, and the corresponding mechanisms were explored using in vivo and in vitro assays.ResultsAn increase in the release of NETs in patients with HCC, particularly those with portal vein tumor thrombosis (PVTT). The presence of NETs in HCC tumor tissues closely correlated with a poor prognosis. Functionally, the invasion ability of HCC cells was enhanced by co-culture with HCC neutrophils, through NETs formation, while the neutrophils from a healthy donor (HD) exhibited the inhibition of the invasion ability. Furthermore, we observed an enhanced ability of forming NETs in neutrophils from HCC patients in vitro, especially patients with PVTT or extra-hepatic metastasis. An in-vivo animal study demonstrated that neutrophils of HCC facilitated the metastatic behavior towards the lung. The further mechanistic investigation unveiled that HCC cells-derived cytokine IL-8 triggered NETs formation in an NADPH oxidase-dependent manner, and NETs-associated cathepsin G (cG) promoted HCC metastasis in vitro as well as vivo. Clinically, the expression of the cG protein in tumor tissues displayed a close correlation with the disease prognosis of HCC patients.ConclusionOur findings implicated that the induction of NETs by HCC cells is a critical metastasis-supporting cancer–host interaction and that NETs may serve as an immune-based potential therapeutic target against HCC progression.

Exogenous hydrogen sulfide inhibits neutrophils extracellular traps formation via the HMGB1/TLR4/p-38 MAPK/ROS axis in hyperhomocysteinemia rats

Hydrogen sulfide (H2S) prevents platelet activation and neutrophils extracellular traps (NETs) formation. However, the mechanism of sodium hydrosulfide (NaHS, a donor that produces H2S) inhibits the formation of NETs in hyperhomocysteinemia (HHcy) rats has not been previously investigated. In the experiment, the expressions of HMGB1 of platelets, the expressions of TLR4, PAD4 and the phosphor-p38 of neutrophils were measured. The NETs formations, the concentration of DNA in the serum and the culture solution of cultured neutrophils which was stimulated by platelet-rich plasma (PRP) were tested. Additionally, the cellular ROS level and SOD activity were detected. The platelets were activated and the expression of HMGB1 of platelets and NETs formation, the concentration of DNA, and the expressions of TLR4, phosphor-p38 and PAD4, the ROS level were all increased while the activity of SOD decreased in the HHcy group compared to the control group. NaHS significantly inhibited the activation of platelets, the production of ROS and the formation of NETs in neutrophils, reversed the expressions of HMGB1, TLR4, phosphor-p38, PAD4 and decreased concentration of DNA which was caused by high homocysteine. Our results demonstrate that the donor of H2S inhibits NETs formation of neutrophils via the HMGB1/TLR4/p38 MAPK/ROS pathway in hyperhomocysteinemia.

G-CSF Infusion for Stem Cell Mobilization Transiently Increases Serum Cell-Free DNA and Protease Concentrations.

G-CSF for stem cell mobilization increases circulating levels of myeloid cells at different stages of maturation. Polymorphonuclear cells (PMNs) are also mobilized in high numbers. It was previously reported that G-CSF primes PMNs toward the release of neutrophils extracellular traps (NETs). Since NETs are often involved in thrombotic events, we hypothesized that high G-CSF blood concentrations could enhance PMN priming toward NET formation in healthy hematopoietic stem cell donors, predisposing them to thrombotic events. However, we found that G-CSF does not prime PMNs toward NETs formation, but increases the serum concentration of cell-free DNA, proteases like neutrophils elastase and myeloperoxidase, and reactive oxygen species. This could possibly create an environment disposed to induce thrombotic events in the presence of additional predisposing factors.

Direct Visualization of Circulating Neutrophil Extracellular Traps Using Cell Fluorescence during Human Septic Shock-Induced Disseminated Intravascular Coagulation

Septic shock is the most severe form of infection, defined as a subset of sepsis in which circulatory, cellular and metabolic abnormalities are profound and responsible for multiple organ failure and a high mortality-rate. Septic shock is characterized by a broad coagulation activation that can lead to uncontrolled thrombin and fibrin generation, which may evolve to disseminated intravascular coagulation (DIC). DIC increases the risk of death, thus representing a therapeutic target of interest. However, the pathophysiological mechanisms of DIC are not fully understood. Polymorphonuclear neutrophils (PMNs) have recently been identified as potential players of the response to infection by releasing their content, including DNA, histones and granules enzymes. These structures, called neutrophils extracellular traps (NETs), form a large net-like structure in which pathogens get trapped. NETs capture invading pathogens, but also represent a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. In this study, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC using a simple and robust method. Blood samples were obtained from healthy human volunteers (n=3), patients with septic shock without DIC (n=3) and with DIC (n=3). PMNs were immediately purified immediately after sampling using negative immune-magnetic sorting method. 5x104 purified PMNs were subsequently spotted on microscope slides using cytocentrifugation at 35g for 5 minutes, thus preserving cell integrity and morphology. Positive controls for NET formation were obtained using PMNs isolated from healthy volunteers that were stimulated in-vitro with 4 μM ionomycin. PMNs were stained with mouse anti-human FITC anti-myeloperoxidase (MPO) antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DAPI stained DNA fibers associated to MPO detected by immunofluorescence microscopy. The degree of NETs formation was blindly quantified into 5 categories according to the proportion of neutrophils forming NETs per microscopic field: absence (0% of neutrophils forming NETs), scarce (1-25%), moderate (26% to 50%), high (50% to 75%) or very high degree of NETS formation (76% to 100%). PMNs from healthy donors stimulated by ionomycin released NETs, evidenced as double-stained DAPI-positive chromatin structures decorated with MPO in the extracellular space using fluorescence microscopy. NET structures similar to those observed after ionomycin stimulation were unambiguously visualized in PMNs from septic shock patients with DIC, but neither in PMNs from septic shock patients without DIC, nor in unstimulated PMNs from healthy donors. DIC patients were stratified into "high" or "very high" degree of NETs formation groups (&amp;gt;75 % of PMNs displaying DNA release), not different from ionomycin-stimulated PMNs (50 to 75%). Conversely, PMNs from patients devoid of DIC as well as unstimulated normal PMNs from healthy volunteers were stratified into "scarce" or "absence" of PMNs forming NET categories. Blind review of May-Giemsa-Grunwald (MGG) stained slides showed the correlation of the detection of NETs with nuclear decondensation and vacuolation of the cytoplasm of PMNs, features that are associated with neutrophil activation. Accordingly, neutrophils' side-fluorescence (NEUT-SFL), a Sysmex™ CBC analyzers parameter previously associated to NETosis (Stiel et al., 2016, Delabranche et al., 2017) was increased during septic shock induced DIC as well as following in-vitro ionomycin stimulation of normal PMNs. In this study, we showed for the first time direct evidence of circulating NETs in peripheral blood of patients with septic shock-induced DIC using immunofluorescence. These NETs are undistinguishable from NETs features observed following in-vitro ionomycin stimulation of normal PMNs. NETs images were furthermore associated with modifications of PMN morphology that could be detected using MGG staining. This observation may be of great interest in order to stratify patients eligible for future treatment protocols with molecules targeting NETs. Figure Disclosures Meziani: STAGO: Research Funding.

First visualization of circulating neutrophil extracellular traps using cell fluorescence during human septic shock-induced disseminated intravascular coagulation

Disseminated intravascular coagulation (DIC) is a severe complication of septic shock. Polymorphonuclear neutrophils (PMNs) may play a key role in septic shock-induced DIC via the release of neutrophils extracellular traps (NETs). NETs capture invading pathogens, but also act as a pro-coagulant surface at the interface between immunity and thrombosis. During septic shock-induced DIC, neutrophil activation may result in excessive NET formation. Herein, we originally report the presence of circulating NETs in human blood during septic shock-induced DIC.To investigate NET formation during shock-induced DIC neutrophils were isolated from patients in septic shock associated with (n = 3) or without (n = 3) DIC. Neutrophils from healthy donors (n = 3) were stimulated in vitro with ionomycin as NET formation positive controls. PMNs smears were stained with mouse anti-human FITC anti-myeloperoxidase antibody and the blue-fluorescent DAPI nucleic acid stain. NETs were identified as elongated extracellular DNA fibers associated to myeloperoxidase detected by immunofluorescence.NETs were unambiguously observed in PMNs from septic shock patients with DIC but not from patients without DIC. NETs features in DIC+ patients were undistinguishable from those observed in ionomycin-induced PMNs from healthy donors. Fluorescence images of NETs were associated to extracellular cytoplasmic expansions.Our data report for the first time the direct visualization of circulating NETs in patients with septic shock-induced DIC. The in vivo relevance of previously reported indirect markers of NETosis (neutrophil side fluorescence) is confirmed.

Exogenous hydrogen sulfide protects from endothelial cell damage, platelet activation, and neutrophils extracellular traps formation in hyperhomocysteinemia rats

Hydrogen sulfide (H2S) prevents endothelial cells damage and P-selectin of platelets promotes neutrophils extracellular traps (NETs) formation. However, how sodium hydrosulfide (NaHS), a donor that produces H2S regulates the activation of platelets and whether H2S inhibits the formation of neutrophils extracellular traps in hyperhomocysteinemia rats have not been previously investigated. The morphological and ultrastructural alterations of endothelial cells (ECs) and platelets were tested by transmission electron microscopy. The expressions of P-selectin of platelets were determined by flow cytometry. Additionally, the cellular ROS and the H2S level were detected by DCFH-DA staining and H2S probe, the expressions of Bax and Bcl-2 in arteries and cultured ECs from rat thoracic aortas and the phosphor-p38 mitogen-activated protein kinase (MAPK), CSE and CBS of platelets were measured by western blotting. The NETs formations, the concentration of DNA in serum and supernatant of cultured neutrophils stimulated with platelet-rich plasma (PRP) were tested by Sytox Green and PicoGreen commercial Kits. The vascular ECs damaged, the expression of P-selectin of platelets and NETs formation increased; the concentration of DNA in serum and supernatant of cultured neutrophils stimulated with PRP also increased; the expression of Bax increased while Bcl-2 decreased in arteries, the phosphor-p38 MAPK of platelets increased while CSE and CBS have no statistically significant changes in the HHcy group compared to the control group. In the cultured ECs, the ROS level increased while the H2S level decreased after 48 and 72 h treatment by HHcy; the expression of Bcl-2 decreased while Bax increased after 72 h treatment by HHcy. NaHS significantly inhibited the ECs injured, cellular ROS production, platelet activation and NETs formation, reversed the expressions of Bax, Bcl-2, phosphor-p38 MAPK, P-selectin and the increased concentration of DNA in serum and supernatant of cultured neutrophils which caused by high homocysteine. Our results demonstrate that the donor of H2S inhibits the platelets activation and NETs formation, which concerts the protection of ECs in hyperhomocysteinemia.

Nickel (II) nitrate hexahydrate triggered canine neutrophil extracellular traps release in vitro

Nickel (II) nitrate hexahydrate (Ni) is a common heavy metal material in battery manufacturing, electroplating alloy parts and ceramic staining, therefore we frequently contact with Ni-related products in daily life. In this study, we aimed to investigate the effects of Ni on neutrophils extracellular traps (NETs) release by canine polymorphonuclear neutrophils (PMNs). The structure of Ni-induced NETs was observed by fluorescence confocal microscopy. Ni-triggered NETs release was quantified by Pico Green® and fluorescence microplate reader. In addition, the inhibitors of NADPH oxidase, ERK1/2-, p38 - signaling pathways were used for preliminary inquiry into the potential mechanism of this process. The results showed that Ni markedly triggered the formation of NETs-like structures, and these structures were mainly consisted of DNA decorated with NE and MPO. Furthermore, quantification experiments showed that Ni significantly increased NETs formation compared to control groups. These results forcefully confirmed that nickel nitrate possesses the ability to induce NETs formation. However, inhibiting the NADPH oxidase, ERK1/2- and p38 MAPK-signaling pathways did not significantly change the quantitation of Ni-induced NETs release. To our knowledge, this study is the first report of Ni-triggered NETs release in vitro, which might provide an entirely new mechanism of several diseases and health issues induced by nickel overexposure.

D(-) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression.

Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(−) lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(−) lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(−) lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET) production (NETosis) in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(−) lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(−) lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1). d(−) lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(−) lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(−) lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

Canine Neutrophil Extracellular Traps Release Induced by the Apicomplexan Parasite Neospora caninum In Vitro.

Neosporosis is considered as one of the main causes of abortion and severe economic losses in dairy industry. The Canis genus serving as one of the confirmed definitive hosts of the apicomplexan parasite Neospora caninum (N. caninum) plays a critical role in its life cycle. However, the effects of N. caninum on its definitive hosts of neutrophils extracellular traps (NETs) formation remain unclear. In the present study, N. caninum tachyzoite-induced canine NETs formation was observed by scanning electron microscopy (SEM). Visualization of DNA decorated with H3, neutrophil elastase (NE), and myeloperoxidase (MPO) within N. caninum tachyzoite-induced NETs were examined using fluorescence confocal microscopy analyses. Furthermore, the formation of canine NETs was quantified using Sytox Green staining, and the LDH levels in supernatants were examined by an LDH Cytotoxicity Assay® kit. The results clearly showed that NETs-like structures were induced by N. caninum tachyzoites, and the major components within these structures induced by N. caninum tachyzoite were further confirmed by fluorescence confocal microscopy visualization. These results suggest that N. caninum tachyzoites strongly induced NETs formation in canine polymorphonuclear neutrophils (PMN). In functional inhibition assays, the blockings of NADPH oxidase, NE, MPO, SOCE, ERK 1/2, and p38 MAPK signaling pathways significantly inhibited N. caninum tachyzoite-induced NETs formation. To our knowledge, this study is the first to report the formation of NETs in canine PMN against N. caninum infection.

NETosis is induced by high glucose and associated with type 2 diabetes.

The role of neutrophils in diabetes and its complications is unclear. Upon challenge with microbes and inflammatory triggers, neutrophils release enzymes and nuclear material, forming neutrophils extracellular traps (NETs) and thereby dying by NETosis. We herein tested NET formation and NETosis products in high glucose and in the setting of type 2 diabetes (T2D). NETosis was assessed in vitro in cells exposed to 0, 5, 25mM glucose and 25mM mannitol, DMSO and PMA using immunofluorescence staining for elastase, DNA and chromatin. Single-cell morphometric analysis was used to detect enter of elastase in the nucleus and extrusion of nuclear material. Release of NETs was quantified by staining with Hoechst 33342. In 38 T2D and 38 age- and sex-matched non-diabetic individuals, we determined plasma elastase, mono- and oligonucleosomes and double-strand (ds) DNA, as circulating NETosis products. NETosis was accurately reproduced in vitro: high (25mM) glucose increased NETosis rate and release of NETs compared with 5mM glucose and 25mM mannitol. T2D patients showed increased plasma elastase, mono- and oligonucleosomes and dsDNA compared with non-diabetic control individuals. A positive correlation was found between HbA1c and mono- and oligonucleosomes, whereas dsDNA was correlated with the presence of nephropathy and cardiovascular disease. Serum IL-6 concentrations were higher in T2D compared with CTRL and correlated with serum dsDNA levels. High glucose and hyperglycemia increase release of NETs and circulating markers of NETosis, respectively. This finding provides a link among neutrophils, inflammation and tissue damage in diabetes.