Spontaneous Neutrophil Extracellular Traps Formation Research Articles

Neutrophil extracellular traps in the anti-infectious defense of human organism

Background. Neutrophil extracellular traps (NETs) are net-like structures that have been investigated in inflammatory diseases. However, the presence of NETs in infected persons without clinical symptoms has not been yet studied.Aim. To reveal NETs in healthy persons during and after the H1N1 influenza pandemic as well as to study the functional activity of NETs.Materials and methods. The study included two groups of volunteers (n = 10 in each group) aged 20–25 years. The first group of volunteers was examined in the absence of acute diseases during one month before the study and in the absence of chronic diseases in the medical history. Volunteers of the second group were in contact with patients with influenza, but did not get sick. The comparative study also included patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, abscess; 12 patients) and 9 patients with non-specific ulcerative colitis. Neutrophils were isolated from the blood by the traditional method of Ficoll density centrifugation. The number, morphology, and functional activity of NETs were determined (by capture of Klebsiella pneumoniae). SYBR Green I-based fluorescence microscopy was used to visualize and quantify NETs.Results. In healthy volunteers who were not in contact with infected patients, spontaneous NETs formation did not occur. Neutrophils of persons who were in contact with infected patients spontaneously formed NETs. In this case the number of NETs reached 8.58 ± 0.51%, and the size of NETs amounted to 39.68 ± 3.52 µm. NETs effectively captured cells of the tested microorganism, which was accompanied by retraction of network fibers and transformation of the network structure into a cloud-like one, which retained 89.38 ± 5.86 microbial cells. For comparison, the NETs in patients with acute inflammation in the abdominal cavity captured and bound 20.2 ± 1 .67 microbial cells and with non-specific ulcerative colitis – 5.53 ± 0.34 cells.Conclusion. High binding capacity of NETs is a factor contributing to effective defense of the body against the development of an infectious disease with manifested clinical symptoms.

Primary sarcopenia is associated with elevated spontaneous NET formation.

Introduction: Sarcopenia is a frequent complication of liver cirrhosis, but it can also occur independently as a result of any underlying cause. The immune system plays an important role in the pathogenesis of both liver cirrhosis and sarcopenia. Neutrophil function, including neutrophil extracellular trap (NET) formation, is linked to chronic inflammation; however, it has not been extensively studied in patients with sarcopenia. Here, we aim to study if main neutrophil functions, such as phagocytosis, reactive oxygen species (ROS) production, and NET formation, are altered in patients with sarcopenia with or without liver cirrhosis. Methods: Neutrophils from 92 patients (52 patients with liver cirrhosis and sarcopenia, 25 patients with liver cirrhosis without sarcopenia, and 15 patients with sarcopenia without liver cirrhosis) and 10 healthy controls were isolated and stimulated with heat-inactivated E. coli (250 bacteria/cell), phorbol 12-myristate 13-acetate (PMA) (100nM), or incubation medium in duplicates for 2h at 37°C. Cells were fixed with paraformaldehyde and stained with 4',6-diamidino-2-phenylindole (DAPI). Pictures of 10 random fields of vision per slide were taken with an Olympus BX51 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) at 600x total magnification. The DNA Area and NETosis Analysis (DANA) algorithm was used to quantify the percentage of NET formation per patient. Phagocytosis and ROS production were assessed with the PhagotestTM kit and PhagoburstTM kit (Glycotope, Heidelberg, Germany) in 92 patients and 21 healthy controls, respectively. Results: Spontaneous NET formation was significantly elevated in patients with only sarcopenia compared to patients with cirrhosis and sarcopenia (p = 0.008) and healthy controls (p = 0.039). NET formation in response to PMA was significantly decreased in patients with cirrhosis (p = 0.007), cirrhosis and sarcopenia (p < 0.001), and sarcopenia (p = 0.002) compared to healthy controls. There was no significant difference in NET formation in response to E. coli between the groups. The DANA algorithm was successfully optimized and validated for assessment of clinical samples. There were no significant changes in neutrophil phagocytosis between patients' groups compared to healthy controls. A significantly lower percentage of neutrophils produced ROS in response to N-formylmethionine-leucyl-phenylalanine (fMLF) in patients compared to healthy controls. Discussion: Spontaneous NET formation might contribute to chronic inflammation and sarcopenia pathogenesis. This, however, does not result in the impairment of the NET formation function of neutrophils in response to a bacterial stimulus and, therefore, cannot be not linked with the increased risk of bacterial infections neither in sarcopenia nor in cirrhosis. The semi-automated NET formation analysis can be successfully implemented to analyze the vast amount of data generated within clinical studies. This approach opens up the possibilities to develop an NET formation-based biomarker in different diseases including sarcopenia and implement NET formation analysis into clinical settings. Phagocytosis and ROS production were not affected in patients with sarcopenia. Further research is needed to explore the mechanism of NET formation in patients with sarcopenia and its potential as a biomarker in sarcopenia.

Immune Checkpoint Inhibitors (ICI) Promote Neutrophil-Platelet Aggregate and NET Formation in Tumor-Bearing Mice

Introduction: Immune checkpoint inhibitor (ICI) therapy has revolutionized cancer treatment. Despite remarkable clinical benefit, there are numerous immune-related adverse events (irAEs) associated with ICI therapy. Cancer-associated thrombosis (CAT) is a common complication of cancer, and ICI-associated thrombosis (ICI-CAT) is an underappreciated irAE. There is no data available concerning the mechanisms underlying ICI-CAT. To address this issue, we have established a murine model of ICI-CAT in which tumor-bearing mice treated with ICI develop larger thrombi than those treated with control IgG. We have observed that these ICI-treated mice display greater numbers of neutrophils in thrombi, and increased levels of circulating neutrophil extracellular traps (NETs). Methods: ICI (anti-PD-1 and anti-CTLA-4) was infused into Balb/c mice bearing subcutaneous CT26 colorectal tumors. Neutrophils (CD11b+ Ly6G+) as a percentage of total WBC was assessed using an automated hematology analyzer. Neutrophil-platelet doublets and aggregates were determined by flow cytometry analysis in using antibodies for CD41 (GPIIb) and CD62P (P-selectin). Circulating neutrophils were isolated from tumor-bearing mice treated with IgG and ICI 48h after IVC occlusion using an EasySep mouse neutrophil enrichment kit (STEMCELL technologies). For NET staining, isolated neutrophils (5x105 cells/well) were seeded on poly-L lysine coated coverslips, and treated with or without phorbol myristate acetate (PMA, 100 nM) for 4 h in 37°C. Neutrophils were then blocked using 5% FBS and stained, without permeabilization, using rabbit monoclonal anti-histone H3 (citrulline R2+R8+R17) antibody (ab5103, Abcam). Extracellular CitH3 was detected using anti-rabbit Alexa Fluor 488 secondary antibody. Results: Thrombi in ICI-treated mice were larger and contained greater numbers of neutrophils than thrombi from IgG-treated mice. Complete blood counts demonstrated that neutrophil numbers increased 3-fold in tumor bearing (38.2%) vs non-tumor bearing mice (12.8%) (Data not shown). However, neutrophils from ICI-treated tumor-bearing mice demonstrated significantly more spontaneous NET formation than those isolated from mice treated with control IgG (66.7% vs 18.2%). Smaller differences were also noted in response to PMA (Fig. A). Since binding of platelets to neutrophils is one cause of NET release, we also determined the frequency of neutrophil-platelet aggregates circulating in tumor-bearing mice treated with ICI or control IgG. Increased levels of circulating neutrophil-platelet aggregate in ICI-treated mice were present in a doublet CD41+Ly6G+ population (44.8% vs 22.3%); similar results were noted when platelets were stained with the activation marker CD62 (P-selectin) (8.5% vs 1%) (Fig. B, Left). The frequency of larger neutrophil-platelet aggregates followed a similar trend (ICI vs IgG; 75.8% vs 30% for CD41; 48.1% vs 7.7% for CD62P) (Fig. B, Right). Conclusions: Circulating neutrophils from ICI-treated, tumor bearing mice demonstrate increased spontaneous NET release. Moreover, elevated levels of circulating platelet-neutrophil doublets and aggregates are present in ICI-treated mice compared to those treated with control IgG. These results suggest a potential mechanism of ICI-induced thrombosis, and that platelet-neutrophil interactions may stimulate NET release in ICI-treated mice. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 123: Genetic Deficiency Of The Ectoenzyme CD73 Increases Neutrophil Extracellular Trap Formation In Patients With ACDC

Background/Purpose: CD73 is a ecto-5′-nucleotidase located on the plasma membrane that generates adenosine from AMP in the extracellular space, and functions as a checkpoint against immune and vascular activation. CD73 inhibitors that eliminate this immune checkpoint blockade are being studied in phase 2/3 cancer clinical trials. We recently identified that lack of CD73 promotes vascular thromboinflammation and neutrophil extracellular trap (NET) formation in mice. The role of CD73 in human NETosis, however, remains to be studied. Arterial calcification due to deficiency of CD73 (ACDC) is a rare, autosomal recessive condition (~20 patients globally) that results in occlusive peripheral vascular disease. We hypothesized that the congenital absence of CD73 and its function as an immune checkpoint would heighten innate immune activation in patients with ACDC. Methods: To test this hypothesis, six patients with molecularly confirmed ACDC were recruited to the NIH for mechanistic studies. Using purified neutrophils from ACDC patients and healthy controls, spontaneous and LPS-induced NET formation was quantified by both Sytox extracellular DNA (eDNA) labeling and NET-associated MPO activity. Results: Absence of surface CD73 was confirmed by flow cytometry. Relative to healthy controls, four of six (4/6) patients with ACDC demonstrated increased ex vivo spontaneous NET formation, but not LPS-triggered NETosis. The four ACDC patients with increased spontaneous NETosis had a mean 1.7-fold increase in Sytox-stained eDNA and 2-fold increase in MPO activity compared with contemporary healthy controls. Conclusion: These data reveal marked, spontaneous NETosis in patients deficient in the ectoenzyme CD73. Comparison with LPS-stimulated neutrophils demonstrates near maximal NETosis at baseline in patients with ACDC, consistent with a model where CD73 tonically suppresses neutrophil activation to maintain vascular and immune homeostasis. Ongoing studies are exploring the mechanism of spontaneous NETosis, thrombin formation, and fibrinolysis in patients with ACDC. Taken together, these data will have relevance to patients with ACDC, and inform the thromboinflammatory risk of cancer patients receiving CD73 inhibitors.

Внеклеточные ловушки нейтрофилов: динамика образования, ассоциированная с переходом от ремиссии к активному ревматоидному артриту

Tne aim of the research. To evaluate the main parameters of neutrophil extracellular traps formation (NETosis) and their dynamics in rheumatoid arthritis (RA) patients associated with the transition from remission to active autoimmune infl ammation. Material and methods. A total of 37 patients (6 males and 31 females) with verifi ed RA according to the ACR/EULAR 2010 criteria were enrolled in the study and formed the main group. Th e comparison group was constituted by 30 healthy volunteers. Circulating neutrophils were isolated with one-step density gradient centrifugation using double layers of iohexol gradient in the original modifi cation. Th e quantitative composition of neutrophils was assessed through microscopy of smears stained according to May-Grunwald. Cell viability as well as non-specifi c activation were evaluated microscopically using trypan blue exclusion assay and nitroblue tetrazolium test, respectively. Neutrophil extracellular trap (NETs) formation was induced by 50 nM phorbol-12-myristate-13- acetate and assessed using fl uorescence microscopy. Results. Neutrophils isolated from RA patients in clinical remission have been revealed to have a higher ability for spontaneous and induced NETs formation than neutrophils from the comparison group. Th e transition from remission to active RA was characterised by a signifi cant increase in induced and, especially, spontaneous formation of NETs. Th e growth rate of spontaneous NET formation was 3.9-fold higher than the induced NETs formation. Spontaneous formation of NETs above 16.4 % may serve as a possible RA activation indicator. Neutrophils from ACPA-positive RA patients were found to have increased spontaneous and induced NETs formation compared to ACPA-negative RA patients. Conclusion. We have studied the main parameters of NETs formation and their dynamics associated with the transition from remission to active autoimmune infl ammation in RA patients. Th e activity of RA has a more pronounced eff ect on the intensity of NETs formation than the presence of ACPA. NETs may be considered a new potential diagnostic and prognostic biomarker

Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity.

Pregnancy is associated with elevated maternal levels of cell-free DNA of neutrophil extracellular trap (NET) origin, as circulatory neutrophils exhibit increased spontaneous NET formation, mainly driven by G-CSF and finely modulated by sex hormones. The postpartum period, on the other hand, involves physiological alterations consistent with the need for protection against infections and fatal haemorrhage. Our findings indicate that all relevant serum markers of neutrophil degranulation and NET release are substantially augmented postpartum. Neutrophil pro-NETotic activity in vitro is also upregulated particularly in post-delivery neutrophils. Moreover, maternal puerperal neutrophils exhibit a strong pro-NETotic phenotype, associated with increased levels of all key players in the generation of NETs, namely citH3, MPO, NE, and ROS, compared to non-pregnant and pregnant controls. Intriguingly, post-delivery NET formation is independent of G-CSF in contrast to late gestation and complemented by the presence of TF on the NETs, alterations in the platelet activity status, and activation of the coagulation cascade, triggered by circulating microparticles. Taken together, our results reveal the highly pro-NETotic and potentially procoagulant nature of postpartum neutrophils, bridging an overt immune activation with possible harmful thrombotic incidence.

Neutrophil extracellular traps contribute to immune dysregulation in bullous pemphigoid via inducing B-cell differentiation and antibody production.

Bullous pemphigoid (BP), an autoimmune skin disease, is characterized by autoantibodies against hemidesmosomal proteins in the skin and mucous membranes. Neutrophils infiltrate BP skin lesions, however, their role in immune dysregulation remains unclear. We investigated whether BP involves aberrant neutrophil extracellular traps (NETs) formation in skin lesions and circulation; and examined the triggers and deleterious immuno-inflammatory consequences. In the present study, we found that circulating NET-related biomarker levels increased in serum and blister fluid of BP patients and significantly correlated with disease severity. Additionally, circulating neutrophils from BP patients displayed enhanced spontaneous NETs formation than healthy controls. In vitro, BP180-NC16A immune complexes-induced NETosis in neutrophils from BP patients, which was abrogated by Fcγ receptor and/or NADPH pathway blockade. Furthermore, the elevated levels of NETs from BP patients boosted autoantibody production by inducing B-cell differentiation into plasma cells, mediated by MAPK P38 cascade activation. Together, our findings provide strong evidence that NETs are involved in a pathogenic loop, causing excessive differentiation of B cells and promotion of autoantibody production. Hence, targeting aberrant neutrophil responses will provide novel potential targets for the treatment of BP.

Peptidylarginine Deiminase Inhibition Prevents Diabetes Development in NOD Mice.

Protein citrullination plays a role in several autoimmune diseases. Its involvement in murine and human type 1 diabetes has recently been recognized through the discovery of antibodies and T-cell reactivity against citrullinated peptides. In the current study, we demonstrate that systemic inhibition of peptidylarginine deiminases (PADs), the enzymes mediating citrullination, through BB-Cl-amidine treatment, prevents diabetes development in NOD mice. This prevention was associated with reduced levels of citrullination in the pancreas, decreased circulating autoantibody titers against citrullinated glucose-regulated protein 78, and reduced spontaneous neutrophil extracellular trap formation of bone marrow–derived neutrophils. Moreover, BB-Cl-amidine treatment induced a shift from Th1 to Th2 cytokines in the serum and an increase in the frequency of regulatory T cells in the blood and spleen. In the pancreas, BB-Cl-amidine treatment preserved insulin production and was associated with a less destructive immune infiltrate characterized by reduced frequencies of effector memory CD4+ T cells and a modest reduction in the frequency of interferon-γ–producing CD4+ and CD8+ T cells. Our results point to a role of citrullination in the pathogenesis of autoimmune diabetes, with PAD inhibition leading to disease prevention through modulation of immune pathways. These findings provide insight in the potential of PAD inhibition for treating autoimmune diseases like type 1 diabetes.

Defective Neutrophil Function in Patients with Sepsis Is Mostly Restored by ex vivo Ascorbate Incubation.

BackgroundNeutrophil function is essential for effective defence against bacterial infections but is defective in patients with sepsis. Ascorbate or vitamin C, which is low in the plasma of patients with sepsis, is stored inside human neutrophils and is essential for their normal function.ObjectiveThis study aimed to determine if ascorbate treatment ex vivo improved neutrophil function in patients with sepsis.Patients and MethodsHuman blood neutrophils were isolated from 20 patients with sepsis and 20 healthy age-matched controls. Neutrophils were incubated with or without ascorbate (1, 5, 10, 20 and 40 mM) for periods up to 2h. Chemotaxis was evaluated using a chemotactic chamber in response to the chemoattractant, fMLP. Phagocytosis (uptake of pHrodo red stained S. aureus) and apoptosis (annexin-V/propidium iodide staining) were measured by flow cytometry. Neutrophil extracellular trap (NET) formation was detected and quantified using DAPI, anti-myeloperoxidase and anti-neutrophil elastase immuno-fluorescence staining. Quantifluor detected the amount of dsDNA in NET supernatants, while quantitative PCR identified changes in expression of PADI4 gene.ResultsChemotactic and phagocytic activities were decreased in patients with sepsis but increased after treatment with the high concentrations of ascorbate. Apoptosis was increased in the sepsis patients but not altered by ascorbate treatment. Spontaneous NET formation was observed in patients with sepsis. A quantity of 1mM ascorbate decreased spontaneous NETosis to that of normal, healthy neutrophils, while high concentrations of ascorbate (>10mM) further promoted NET formation.ConclusionDysregulated neutrophil function was observed in patients with sepsis which could contribute to disease pathology and outcomes. Exposure to ascorbate could reverse some of these changes in function. These novel discoveries raise the possibility that ascorbate treatment could be used as an adjunctive therapy that could result in improved neutrophil function during sepsis.

Enhanced NETosis generation in radiographic axial spondyloarthritis: utility as biomarker for disease activity and anti-TNF-\u03b1 therapy effectiveness

BackgroundRadiographic axial spondyloarthritis (r-axSpA) is a chronic inflammatory form of arthritis in which tumor necrosis factor (TNF)-α, a potent inducer of inflammatory response and a key regulator of innate immunity and of Th1 immune responses, plays a central role. NETosis is a mechanism of innate immune defense that is involved in diverse rheumatology diseases. Nevertheless, spontaneous NETosis generation in r-axSpA, its association to disease pathogenesis, and the NETosis involvement on anti-TNF-α therapy’s effects has never been explored.MethodsThirty r-axSpA patients and 32 healthy donors (HDs) were evaluated. Neutrophil extracellular trap (NET) formation, mediators of signal-transduction cascade required for NETosis induction and cell-free NETosis-derived products were quantified. An additional cohort of 15 r-axSpA patients treated with infliximab (IFX) for six months were further analyzed. In vitro studies were designed to assess the effects of IFX in NETosis generation and the inflammatory profile triggered.ResultsCompared to HDs, neutrophils from r-axSpA patients displayed augmented spontaneous NET formation, elevated expression of NET-associated signaling components, nuclear peptidylarginine deiminase 4 translocation and increased citrullinated histone H3. Furthermore, patients exhibited altered circulating levels of cell-free NETosis-derived products (DNA, nucleosomes and elastase). Additional studies revealed that cell-free NETosis-derived products could be suitable biomarkers for distinguish r-axSpA patients from HDs. Correlation studies showed association between cell-free NETosis-derived products and clinical inflammatory parameters. Besides, nucleosomes displayed potential as a biomarker for discriminate patients according to disease activity.IFX therapy promoted a reduction in both NETosis generation and disease activity in r-axSpA patients. Mechanistic in vitro studies further unveiled the relevance of IFX in reducing NET release and normalizing the augmented inflammatory activities promoted by NETs in mononuclear cells.ConclusionsThis study reveals that NETosis is enhanced in r-axSpA patients and identifies the NETosis-derived products as potential disease activity biomarkers. In addition, the data suggests the potential role of NET generation analysis for assessment of therapeutic effectiveness in r-axSpA.

P6396LDL cholesterol promotes neutrophil extracellular trap formation

Abstract Background Pro-protein convertase subtilisin/kexin 9 (PCSK9) is a regulator of low density lipoprotein (LDL) receptor (LDLR) expression and has gained attention in the treatment of hyperlipidemia. Serum levels of LDL are correlated with numbers of activated neutrophils in the circulation of hyperlipidemic patients. Activated neutrophils can form neutrophil extracellular traps (NETs) by expelling their chromatin, and NETs have been recognized as important risk factors for acute myocardial infarction (AMI) and stroke. Purpose We analyzed the influence of serum LDL levels on neutrophil activation, their propensity to form NETs, and the correlation of NET surrogate markers with systemic inflammatory responses in AMI. Methods We recruited 249 consecutive patients with AMI (mean age 58 years, 20% females) and assessed laboratory parameters, inflammatory markers, and serum lipid profiles 72 h after primary percutaneous coronary intervention. Double-stranded DNA (dsDNA) and citrullinated histone H3 were determined from plasma as markers of in vivo NET formation. In a subset of patients (n=25), ex vivo NET formation in response to PCSK9 and ionomycin was analyzed, neutrophils were stained for CD11b, LDLR, and lectin-like oxidized LDLR (LOX-1) using flow cytometry at baseline and after activation with phorbol myristate acetate. PCSK9 levels were measured by ELISA. Results Patients with serum LDL levels above the median [median (IQR) LDL 111 mg/dl (87–141), mean ± SD LDL 115±41 mg/dl] had significantly higher concentrations of circulating dsDNA. Levels of dsDNA [median 121 ng/ml, IQR 101–156] were linked with levels of the specific NET marker citH3 [median (IQR) 252 ng/ml (655–1600), rs=0.157]. Plasma concentrations of dsDNA and citH3 correlated significantly with CRP (dsDNA rs=0.328; citH3 rs=0.209), proBNP (dsDNA rs=0.286; citH3 rs=0.192), troponin T (dsDNA rs=0.282; citH3 rs=0.197), and IL-6 (dsDNA rs=0.242; citH3 rs=0.216). In the subset of patients in whom neutrophils were characterized, pre-treatment of neutrophils with PCSK9 could significantly decrease ionomycin induced NET release in a dose-dependent manner. Levels of serum LDL were associated with spontaneous NET formation (r=0.504) ex vivo and activated CD11b on neutrophils (r=0.495). LOX-1 expression correlated significantly with LDLR expression (rs=0.670) and PCSK9 levels (r=0.520). Patient neutrophil stimulation in whole blood led to a significant decrease of the LDLR positive neutrophil population. This effect was greater when PCSK9 levels were higher. Conclusion Our data indicate a role for LDL in boosting neutrophil activation depending on PCSK9.

Neutrophil extracellular traps, B cells, and type I interferons contribute to immune dysregulation in hidradenitis suppurativa.

Hidradenitis suppurativa (HS), also known as acne inversa, is an incapacitating skin disorder of unknown etiology manifested as abscess-like nodules and boils resulting in fistulas and tissue scarring as it progresses. Given that neutrophils are the predominant leukocyte infiltrate in HS lesions, the role of neutrophil extracellular traps (NETs) in the induction of local and systemic immune dysregulation in this disease was examined. Immunofluorescence microscopy was performed in HS lesions and detected the prominent presence of NETs. NET complexes correlated with disease severity, as measured by Hurley staging. Neutrophils from the peripheral blood of patients with HS peripheral also displayed enhanced spontaneous NET formation when compared to healthy control neutrophils. Sera from patients recognized antigens present in NETs and harbored increased antibodies reactive to citrullinated peptides. B cell dysregulation, as evidenced by elevated plasma cells and IgG, was observed in the circulation and skin from patients with HS. Peptidylarginine deiminases (PADs) 1 to 4, enzymes involved in citrullination, were differentially expressed in HS skin, when compared to controls, in association with enhanced tissue citrullination. NETs in HS skin coexisted with plasmacytoid dendritic cells, in association with a type I interferon (IFN) gene signature. Enhanced NET formation and immune responses to neutrophil and NET-related antigens may promote immune dysregulation and contribute to inflammation. This, along with evidence of up-regulation of the type I IFN pathway in HS skin, suggests that the innate immune system may play important pathogenic roles in this disease.

Deficiency of adenosine deaminase 2 triggers adenosine-mediated NETosis and TNF production in patients with DADA2

AbstractReduction of adenosine deaminase 2 (ADA2) activity due to autosomal-recessive loss-of-function mutations in the ADA2 gene (previously known as CECR1) results in a systemic vasculitis known as deficiency of ADA2 (DADA2). Neutrophils and a subset of neutrophils known as low-density granulocytes (LDGs) have been implicated in the pathogenesis of vasculitis, at least in part, through the formation of neutrophil extracellular traps (NETs). The study objective was to determine whether neutrophils and NETs play a pathogenic role in DADA2. In vivo evidence demonstrated NETs and macrophages in affected gastrointestinal tissue from patients with DADA2. An abundance of circulating LDGs prone to spontaneous NET formation was observed during active disease in DADA2 and were significantly reduced after remission induction by anti–tumor necrosis factor (TNF) therapy. Increased circulating LDGs were identified in unaffected family members with monoallelic ADA2 mutations. Adenosine triggered NET formation, particularly in neutrophils from female patients, by engaging A1 and A3 adenosine receptors (ARs) and through reactive oxygen species– and peptidylarginine deiminase–dependent pathways. Adenosine-induced NET formation was inhibited by recombinant ADA2, A1/A3 AR antagonists, or by an A2A agonist. M1 macrophages incubated with NETs derived from patients with DADA2 released significantly greater amounts of TNF-α. Treatment with an A2AAR agonist decreased nuclear translocation of NF-κB and subsequent production of inflammatory cytokines in DADA2 monocyte-derived macrophages. These results suggest that neutrophils may play a pathogenic role in DADA2. Modulation of adenosine-mediated NET formation may contribute a novel and directed therapeutic approach in the treatment of DADA2 and potentially other inflammatory diseases.

SPARC Is a New Myeloid-Derived Suppressor Cell Marker Licensing Suppressive Activities.

Myeloid-derived suppressor cells (MDSC) are well-known key negative regulators of the immune response during tumor growth, however scattered is the knowledge of their capacity to influence and adapt to the different tumor microenvironments and of the markers that identify those capacities. Here we show that the secreted protein acidic and rich in cysteine (SPARC) identifies in both human and mouse MDSC with immune suppressive capacity and pro-tumoral activities including the induction of epithelial-to-mesenchymal transition (EMT) and angiogenesis. In mice the genetic deletion of SPARC reduced MDSC immune suppression and reverted EMT. Sparc−/− MDSC were less suppressive overall and the granulocytic fraction was more prone to extrude neutrophil extracellular traps (NET). Surprisingly, arginase-I and NOS2, whose expression can be controlled by STAT3, were not down-regulated in Sparc−/− MDSC, although less suppressive than wild type (WT) counterpart. Flow cytometry analysis showed equal phosphorylation of STAT3 but reduced ROS production that was associated with reduced nuclear translocation of the NF-kB p50 subunit in Sparc−/− than WT MDSC. The limited p50 in nuclei reduce the formation of the immunosuppressive p50:p50 homodimers in favor of the p65:p50 inflammatory heterodimers. Supporting this hypothesis, the production of TNF by Sparc−/− MDSC was significantly higher than by WT MDSC. Although associated with tumor-induced chronic inflammation, TNF, if produced at high doses, becomes a key factor in mediating tumor rejection. Therefore, it is foreseeable that an unbalance in TNF production could skew MDSC toward an inflammatory, anti-tumor phenotype. Notably, TNF is also required for inflammation-driven NETosis. The high level of TNF in Sparc−/− MDSC might explain their increased spontaneous NET formation as that we detected both in vitro and in vivo, in association with signs of endothelial damage. We propose SPARC as a new potential marker of MDSC, in both human and mouse, with the additional feature of controlling MDSC suppressive activity while preventing an excessive inflammatory state through the control of NF-kB signaling pathway.

Neutrophils contribute to vasculitis by increased release of neutrophil extracellular traps in Behçet's disease

Background and objectivesBehçet’s disease (BD) is a multi-system inflammatory disorder that can cause vasculitis. Here we questioned whether Neutrophils in BD cause vasculitis via releasing Neutrophil Extracellular Traps (NETs), a process called NETosis. MethodsCirculating neutrophils were isolated from a cohort of Middle Eastern BD patients with an active disease and healthy volunteers. The percentage of NETs release was monitored in neutrophils stimulated or not with BD serum, and treated or not with Colchicine, Dexamethasone, Cl-amidine or N-Acetyl Cysteine (NAC). The mRNA expression levels of PAD4 (a key enzyme in NETosis) was also assessed. The effect of NETs on the proliferation and cell death of endothelial cells was investigated using an in vitro co-culture model. The presence of NETs in skin tissues of BD patients was examined using immunolabeling of NETs associated proteins. ResultsCirculating Neutrophils from BD patients were more prone to release NETs in vitro and expressed higher levels of PAD4 compared to healthy volunteers. Spontaneous NETs formation in BD neutrophils was inhibited by Colchicine and Dexamethasone, two drugs used to treat BD. NETs formation was also inhibited by Cl-amidine, a specific PAD4 inhibitor, and by NAC, a ROS inhibitor. Interestingly, serum from BD patients stimulated circulating neutrophils from healthy volunteers to release more NETs and increased their mRNA PAD4 expression. Moreover, endothelial cells cultured in the presence of NETs from BD patients showed a decrease in proliferation and an increase in apoptosis and cell death. Finally, NETosis was predominantly identified around affected blood vessels in biopsies of vasculitis from BD patients. ConclusionOur results provide evidence on the implication of NETosis in the pathophysiology of BD especially in inducing vasculitis.

Experimental lupus is aggravated in mouse strains with impaired induction of neutrophil extracellular traps.

Many effector mechanisms of neutrophils have been implicated in the pathogenesis of systemic lupus erythematosus (SLE). Neutrophil extracellular traps (NETs) have been assigned a particularly detrimental role. Here we investigated the functional impact of neutrophils and NETs on a mouse model of lupus triggered by intraperitoneal injection of the cell death-inducing alkane pristane. Pristane-induced lupus (PIL) was aggravated in 2 mouse strains with impaired induction of NET formation, i.e., NOX2-deficient (Ncf1-mutated) and peptidyl arginine deiminase 4-deficient (PAD4-deficient) mice, as seen from elevated levels of antinuclear autoantibodies (ANAs) and exacerbated glomerulonephritis. We observed a dramatically reduced ability to form pristane-induced NETs in vivo in both Ncf1-mutated and PAD4-deficient mice, accompanied by higher levels of inflammatory mediators in the peritoneum. Similarly, neutropenic Mcl-1ΔMyelo mice exhibited higher levels of ANAs, which indicates a regulatory function in lupus of NETs and neutrophils. Blood neutrophils from Ncf1-mutated and human individuals with SLE exhibited exuberant spontaneous NET formation. Treatment with specific chemical NOX2 activators induced NET formation and ameliorated PIL. Our findings suggest that aberrant NET is one of the factors promoting experimental lupus-like autoimmunity by uncontrolled release of inflammatory mediators.

Formation of Neutrophil Extracellular Traps under Low Oxygen Level.

Since their discovery, neutrophil extracellular traps (NETs) have been characterized as a fundamental host innate immune defense mechanism. Conversely, excessive NET-release may have a variety of detrimental consequences for the host. A fine balance between NET formation and elimination is necessary to sustain a protective effect during an infectious challenge. Our own recently published data revealed that stabilization of hypoxia-inducible factor 1α (HIF-1α) by the iron chelating HIF-1α-agonist desferoxamine or AKB-4924 enhanced the release of phagocyte extracellular traps. Since HIF-1α is a global regulator of the cellular response to low oxygen, we hypothesized that NET formation may be similarly increased under low oxygen conditions. Hypoxia occurs in tissues during infection or inflammation, mostly due to overconsumption of oxygen by pathogens and recruited immune cells. Therefore, experiments were performed to characterize the formation of NETs under hypoxic oxygen conditions compared to normoxia. Human blood-derived neutrophils were isolated and incubated under normoxic (21%) oxygen level and compared to hypoxic (1%) conditions. Dissolved oxygen levels were monitored in the primary cell culture using a Fibox4-PSt3 measurement system. The formation of NETs was quantified by fluorescence microscopy in response to the known NET-inducer phorbol 12-myristate 13-acetate (PMA) or Staphylococcus (S.) aureus wild-type and a nuclease-deficient mutant. In contrast to our hypothesis, spontaneous NET formation of neutrophils incubated under hypoxia was distinctly reduced compared to control neutrophils incubated under normoxia. Furthermore, neutrophils incubated under hypoxia showed significantly reduced formation of NETs in response to PMA. Gene expression analysis revealed that mRNA level of hif-1α as well as hif-1α target genes was not altered. However, in good correlation to the decreased NET formation under hypoxia, the cholesterol content of the neutrophils was significantly increased under hypoxia. Interestingly, NET formation in response to viable S. aureus wild-type or nuclease-deficient strain was retained under hypoxia. Our results lead to the conclusion that hypoxia is not the ideal tool to analyze HIF-1α in neutrophils. However, the data clearly suggest that neutrophils react differently under hypoxia compared to normoxia and thereby highlight the importance of the usage of physiological relevant oxygen level when studying neutrophil functions.

A novel image-based quantitative method for the characterization of NETosis

NETosis is a newly recognized mechanism of programmed neutrophil death. It is characterized by a stepwise progression of chromatin decondensation, membrane rupture, and release of bactericidal DNA-based structures called neutrophil extracellular traps (NETs). Conventional ‘suicidal’ NETosis has been described in pathogenic models of systemic autoimmune disorders. Recent in vivo studies suggest that a process of ‘vital’ NETosis also exists, in which chromatin is condensed and membrane integrity is preserved. Techniques to assess ‘suicidal’ or ‘vital’ NET formation in a specific, quantitative, rapid and semiautomated way have been lacking, hindering the characterization of this process. Here we have developed a new method to simultaneously assess both ‘suicidal’ and ‘vital’ NETosis, using high-speed multi-spectral imaging coupled to morphometric image analysis, to quantify spontaneous NET formation observed ex-vivo or stimulus-induced NET formation triggered in vitro. The use of imaging flow cytometry allows automated, quantitative and rapid analysis of subcellular morphology and texture, and introduces the potential for further investigation using NETosis as a biomarker in pre-clinical and clinical studies.

Enhanced neutrophil extracellular trap generation in rheumatoid arthritis: analysis of underlying signal transduction pathways and potential diagnostic utility.

IntroductionNeutrophil extracellular traps (NETs) have recently been implicated in a number of autoimmune conditions, including rheumatoid arthritis (RA). We examined the underlying signaling pathways triggering enhanced NETosis in RA and ascertained whether the products of NETosis had diagnostic implications or usefulness.MethodsNeutrophils were isolated from RA patients with active disease and from controls. Spontaneous NET formation from RA and control neutrophils was assessed in vitro with microscopy and enzyme-linked immunosorbent assay (ELISA) for NETosis-derived products. The analysis of the signal-transduction cascade included reactive oxygen species (ROS) production, myeloperoxidase (MPO), neutrophil elastase (NE), peptidyl arginine deiminase 4 (PAD4), and citrullinated histone 3 (citH3). NET formation was studied in response to serum and synovial fluid and immunoglobulin G (IgG) depleted and reconstituted serum. Serum was analyzed for NETosis-derived products, for which receiver operator characteristic (ROC) curves were calculated.ResultsNeutrophils from RA cases exhibited increased spontaneous NET formation in vitro, associated with elevated ROS production, enhanced NE and MPO expression, nuclear translocation of PAD4, PAD4-mediated citrullination of H3, and altered nuclear morphology. NET formation in both anti-citrullinated peptide antibody (ACPA)-positive and -negative RA was abolished by IgG depletion, but restored only with ACPA-positive IgG. NETosis-derived products in RA serum demonstrated diagnostic potential, the ROC area under the curve for cell-free nucleosomes being >97%, with a sensitivity of 91% and a specificity of 92%. No significant difference was observed between ACPA-positive and -negative cases.ConclusionsSignaling elements associated with the extrusion of NETs are significantly enhanced to promote NETosis in RA compared with healthy controls. NETosis depended on the presence of ACPA in ACPA-positive RA serum. The quantitation of NETosis-derived products, such as cell-free nucleosomes in serum, may be a useful complementary tool to discriminate between healthy controls and RA cases.