Neutrophil Extracellular Traps Formation In Neutrophils Research Articles

Tumor-Derived Exosomes Induce the Formation of Neutrophil Extracellular Traps: Implications For The Establishment of Cancer-Associated Thrombosis

Cancer patients are at an increased risk of developing thromboembolic complications. Several mechanisms have been proposed to explain cancer-associated thrombosis including the release of tumor-derived extracellular vesicles and the activation of host vascular cells. It was proposed that neutrophil extracellular traps (NETs) contribute to the prothrombotic phenotype in cancer. In this study, we evaluated the possible cooperation between tumor-derived exosomes and NETs in cancer-associated thrombosis. Female BALB/c mice were orthotopically injected with 4T1 breast cancer cells. The tumor-bearing animals exhibited increased levels of plasma DNA and myeloperoxidase in addition to significantly increased numbers of circulating neutrophils. Mice were subjected to either Rose Bengal/laser-induced venous thrombosis or ferric chloride-induced arterial thrombosis models. The tumor-bearing mice exhibited accelerated thrombus formation in both models compared to tumor-free animals. Treatment with recombinant human DNase 1 reversed the prothrombotic phenotype of tumor-bearing mice in both models. Remarkably, 4T1-derived exosomes induced NET formation in neutrophils from mice treated with granulocyte colony-stimulating factor (G-CSF). In addition, tumor-derived exosomes interacted with NETs under static conditions. Accordingly, the intravenous administration of 4T1-derived exosomes into G-CSF-treated mice significantly accelerated venous thrombosis in vivo. Taken together, our observations suggest that tumor-derived exosomes and neutrophils may act cooperatively in the establishment of cancer-associated thrombosis.

The interrelationship between phagocytosis, autophagy and formation of neutrophil extracellular traps following infection of human neutrophils by Streptococcus pneumoniae.

Neutrophils play an important role in the innate immune response to infection with Streptococcus pneumoniae, the pneumococcus. Pneumococci are phagocytosed by neutrophils and undergo killing after ingestion. Other cellular processes may also be induced, including autophagy and the formation of neutrophil extracellular traps (NETs), which may play a role in bacterial eradication. We set out to determine how these different processes interacted following pneumococcal infection of neutrophils, and the role of the major pneumococcal toxin pneumolysin in these various pathways. We found that pneumococci induced autophagy in neutrophils in a type III phosphatidylinositol-3 kinase dependent fashion that also required the autophagy gene Atg5. Pneumolysin did not affect this process. Phagocytosis was inhibited by pneumolysin but enhanced by autophagy, while killing was accelerated by pneumolysin but inhibited by autophagy. Pneumococci induced extensive NET formation in neutrophils that was not influenced by pneumolysin but was critically dependent on autophagy. While pneumolysin did not affect NET formation, it had a potent inhibitory effect on bacterial trapping within NETs. These findings show a complex interaction between phagocytosis, killing, autophagy and NET formation in neutrophils following pneumococcal infection that contribute to host defence against this pathogen.

Iron-chelating agent, deferasirox, inhibits neutrophil activation and extracellular trap formation.

Iron-chelating agents, which are frequently prescribed to transfusion-dependent patients, have various useful biological effects in addition to chelation. Reactive oxygen species (ROS) produced by neutrophils can cause pulmonary endothelial cell damage, which can lead to acute lung injury (ALI). We previously reported that deferasirox (DFS), an iron-chelating agent, inhibits phorbol myristate acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP)-induced ROS production in neutrophils, in vitro. Here, we investigate whether DFS inhibits vacuolization in neutrophils and neutrophil extracellular trap (NET) formation. Human neutrophils were incubated with DFS and stimulated with PMA or fMLP. Human neutrophils were separated from heparinized peripheral blood using density gradient centrifugation, and subsequently incubated with DFS. After 10minutes, neutrophils were stimulated by PMA or fMLP. Vacuole formation was observed by electron microscopy. For observing NET formations using microscopes, immunohistological analyses using citrullinated histone H3 and myeloperoxidase antibodies, and SYTOX Green (an impermeable DNA detection dye) staining, were conducted. NET formation was measured as the quantity of double-stranded DNA (dsDNA), using the AccuBlue Broad Range dsDNA Quantitation Kit. DFS (50μmol/L) inhibited vacuole formation in the cytoplasm and NET formation. Additionally, 5-100μmol/L concentration of DFS inhibited the release of dsDNA in a dose-independent manner. We demonstrate that DFS inhibits not only ROS production but also vacuolization and NET formation in neutrophils. These results suggest the possibility of protective effects of DFS against NET-related adverse effects, including ALI and thrombosis.

Neutrophil extracellular traps as a potential source of autoantigen in cocaine-associated autoimmunity.

Exposure to illicit cocaine and its frequent adulterant, levamisole, is associated with ANCAs targeting neutrophil elastase (NE), neutropenia and vasculitic/thrombotic skin purpura. The mechanisms of cocaine/levamisole-associated autoimmunity (CLAA) are unknown. The aim of this study was to assess the ability of cocaine and levamisole to induce the release of neutrophil extracellular traps (NETs), a potential source of autoantigen and tissue injury. We performed quantitative and qualitative assessment of NET formation in neutrophils from healthy donors exposed to either drug in vitro . In addition, IgG from sera of individuals with CLAA (CLAA-IgG) was assessed for its ability to enhance formation of, and to bind to, drug-induced NETs. Both cocaine and levamisole could induce formation of NETs enriched in NE and, potentially, inflammatory mitochondrial DNA. Both drugs could also augment simultaneous release of B cell-activating factor belonging to the TNF family (BAFF). CLAA-IgG, but not IgG from healthy individuals, could potentiate drug-induced NETosis. Furthermore, CLAA-IgG, but not ANCA + control IgG, bound to drug-induced NETs in a pattern consistent with NE targeting. Both cocaine and levamisole may contribute to the development of ANCAs by inducing release of potentially inflammatory NETs in association with NE autoantigen and BAFF. Enhancement of drug-induced NET release by CLAA-IgG provides a potential mechanism linking vasculitis/pupuric skin disease to acute drug exposure in patients with CLAA. Further study of this under-recognized form of autoimmunity will be likely to provide mechanistic insight into ANCA-associated vasculitis and other diseases associated with NETosis.

Inhibition of Neutrophil Extracellular Trap Formation after Stem Cell Transplant by Prostaglandin E2.

Autologous and allogeneic hematopoietic stem cell transplant (HSCT) patients are susceptible to pulmonary infections, including bacterial pathogens, even after hematopoietic reconstitution. We previously reported that murine bone marrow transplant (BMT) neutrophils overexpress cyclooxygenase-2, overproduce prostaglandin E2 (PGE2), and exhibit defective intracellular bacterial killing. Neutrophil extracellular traps (NETs) are DNA structures that capture and kill extracellular bacteria and other pathogens. To determine whether NETosis was defective after transplant and if so, whether this was regulated by PGE2 signaling. Neutrophils isolated from mice and humans (both control and HSCT subjects) were analyzed for NETosis in response to various stimuli in the presence or absence of PGE2 signaling modifiers. NETs were visualized by immunofluorescence or quantified by Sytox Green fluorescence. Treatment of BMT or HSCT neutrophils with phorbol 12-myristate 13-acetate or rapamycin resulted in reduced NET formation relative to control cells. NET formation after BMT was rescued both in vitro and in vivo with cyclooxygenase inhibitors. Additionally, the EP2 receptor antagonist (PF-04418948) or the EP4 antagonist (AE3-208) restored NET formation in neutrophils isolated from BMT mice or HSCT patients. Exogenous PGE2 treatment limited NETosis of neutrophils collected from normal human volunteers and naive mice in an exchange protein activated by cAMP- and protein kinase A-dependent manner. Our results suggest blockade of the PGE2-EP2 or EP4 signaling pathway restores NETosis after transplantation. Furthermore, these data provide the first description of a physiologic inhibitor of NETosis.

Super-low Dose Endotoxin Pre-conditioning Exacerbates Sepsis Mortality

Sepsis mortality varies dramatically in individuals of variable immune conditions, with poorly defined mechanisms. This phenomenon complements the hypothesis that innate immunity may adopt rudimentary memory, as demonstrated in vitro with endotoxin priming and tolerance in cultured monocytes. However, previous in vivo studies only examined the protective effect of endotoxin tolerance in the context of sepsis. In sharp contrast, we report herein that pre-conditioning with super-low or low dose endotoxin lipopolysaccharide (LPS) cause strikingly opposite survival outcomes. Mice pre-conditioned with super-low dose LPS experienced severe tissue damage, inflammation, increased bacterial load in circulation, and elevated mortality when they were subjected to cecal-ligation and puncture (CLP). This is in contrast to the well-reported protective phenomenon with CLP mice pre-conditioned with low dose LPS. Mechanistically, we demonstrated that super-low and low dose LPS differentially modulate the formation of neutrophil extracellular trap (NET) in neutrophils. Instead of increased ERK activation and NET formation in neutrophils pre-conditioned with low dose LPS, we observed significantly reduced ERK activation and compromised NET generation in neutrophils pre-conditioned with super-low dose LPS. Collectively, our findings reveal a mechanism potentially responsible for the dynamic programming of innate immunity in vivo as it relates to sepsis risks.

Characterization of rapid neutrophil extracellular trap formation and its cooperation with phagocytosis in human neutrophils

Neutrophils, as the first cellular line of innate host defense, employ phagocytosis and formation of neutrophil extracellular traps (NETs) to combat infections. Classical NET formation induced by phorbol myristate acetate requires several hours to complete. However, recent studies demonstrated rapid NET formation in neutrophils upon stimulation by platelets, Staphylococcus aureus or fungal products. Here we describe that antibody- or complement-induced phagocytosis triggers rapid NET formation. In contrast to classical NETosis, chemical inhibition of NADPH oxidase as well as using NADPH oxidase-deficient patient neutrophils did not affect rapid NET formation. Although phagocytosis and rapid NET formation may not be the prerequisite of each other, cooperation of phagocytosis and rapid NET formation may be essential to improve the efficiency of defense mechanisms in combating disseminating bacteria. Dissecting the differential mechanisms of NET formation is crucial to develop novel therapeutic strategies for infectious and auto-immune diseases where NETs play an essential role.

Effective NET formation in neutrophils from individuals with G6PD Taiwan-Hakka is associated with enhanced NADP+ biosynthesis

In response to infection, neutrophils employ various strategies to defend against the invading microbes. One of such defense mechanisms is the formation of neutrophil extracellular traps (NETs). Recent studies suggest that reactive oxygen species is a signal critical to NET formation. This prompts us to examine whether neutrophils from individuals with glucose-6-phosphate dehydrogenase (G6PD) Taiwan-Hakka variant, which are prone to oxidative stress generation, have altered ability to form NET. We adopted an image-based method to study the NET formation potential in neutrophils from G6PD-deficient patients. Neutrophils from either normal or G6PD-deficient individuals underwent NETosis in response to phorbol 12-myristate 13-acetate (PMA). The extent of NETosis in the former did not significantly differ from that of the latter. Diphenyleneiodonium sulfate (DPI) and 3-methyladenine (MA) inhibited PMA-stimulated NET formation in these cells, suggesting the involvement of NADPH oxidase and autophagy in the process. Glucose oxidase (GO) and xanthine oxidase/xanthine (XO/X) could induce a similar extent of NET formation in normal and G6PD-deficient neutrophils. GO- or XO-induced NETosis was not inhibitable by MA, implying that reactive oxygen species (ROS) can act as an independent signal for activation of NETosis. Mechanistically, enhanced superoxide production in neutrophils was associated with increases in levels of NAD+ and NADP+, as well as activation of NAD+ kinase. Taken together, these findings suggest that G6PD-deficient neutrophils are as equally efficient as normal cells in NET formation, and their deficiency in G6PD-associated NADPH regeneration capacity is largely compensated for by nicotinamide nucleotide biosynthesis.

Deoxyribonuclease Is a Potential Counter Regulator of Aberrant Neutrophil Extracellular Traps Formation after Major Trauma

Introduction. Neutrophil extracellular traps (NET) consist of a DNA scaffold that can be destroyed by Deoxyribonuclease (DNase). Thus DNases are potential prerequisites for natural counter regulation of NETs formation. In the present study, we determined the relationship of NETs and DNase after major trauma. Methods. Thirty-nine major trauma patients, 14 with and 25 without sepsis development were enrolled in this prospective study. Levels of cell-free (cf)-DNA/NETs and DNase were quantified daily from admission until day 9 after admission. Results. Levels of cf-DNA/NETs in patients who developed sepsis were significantly increased after trauma. In the early septic phase, DNase values in septic patients were significantly increased compared to patients without sepsis (P < 0.05). cf-DNA/NETs values correlated to values of DNase in all trauma patients and patients with uneventful recovery (P < 0.01) but not in septic patients. Recombinant DNase efficiently degraded NETs released by stimulated neutrophils in a concentration-dependent manner in vitro. Conclusions. DNase degrades NETs in a concentration-dependent manner and therefore could have a potential regulatory effect on NET formation in neutrophils. This may inhibit the antibacterial effects of NETs or protect the tissue from autodestruction in inadequate NETs release in septic patients.

Neutrophil Extracellular Traps (NETs) in Patients with Congenital Neutropenia

Abstract 15Activated neutrophils kill microbes by phagocytosis and extracellular mechanisms, including neutrophil extracellular traps (NETs), which are composed of decondensed chromatin and granular proteins such as neutrophil elastase (NE) and cathepsin G. Enzymatic generation of reactive oxygen species (ROS) by NADPH oxidase and the release of serine proteases such as NE have been shown to be essential factors for NET formation. Patients with chronic granulmatous disease (CGD), who lack a normal generation of ROS, show a defective NET formation.Since myeloid cells of patients with severe congenital neutropenia (CN) show an aberrant expression pattern of granular proteases such as neutrophil elastase (NE) or myeloperoxidase, we aimed to analyse NET formation in activated neutrophils of these patients. CN is a heterogeneous hematological disorder, characterized by peripheral blood neutrophil counts below 0,2×109/l and a maturation arrest of myelopoiesis at the promyelocytic/myelocytic stage. 60% of CN patients harbor autosomal dominant mutations within the ELA2 gene encoding for NE, but also mutations in other genes (e.g. HAX1, G6PC3, WAS, GFI1, p14) have been found to be disease-causing. Previously, we described severely diminished levels of NE in myeloid cells of CN patients. Here, we aimed to explore NET formation in neutrophils of CN patients. Moreover, we intended to analyze the effects of a reduced ELA2 expression and gene mutations as seen in CN patients on NET formation in vitro.Granulocytes of CN patients undergoing G-CSF therapy were extracted by density centrifugation, stimulated with phorbol myristate acetate (PMA, 50 nM, up to 240 min) and then tested for NET formation. NETs were stained with an extracellular DNA dye or DAPI. Our analyses showed normal NET formation in peripheral blood granulocytes of two patients with HAX1-related neutropenia, whereas there was a significantly lower amount of NETs in two patients with ELA2 mutations. One further patient out of three CN patients with unknown mutations showed a reduced amount of NETs in bone marrow PMNs.To further evaluate the possible effect of downregulated ELA2 expression on NET formation, we transduced primary human CD34+ cells with a lentiviral-based shRNA construct downregulating the expression of NE. Subsequently, these cells were differentiated into granulocytes with a cytokine cocktail containing G-CSF and tested for their ability to form NETs. We found an almost completely abolished NET formation in cells transduced with ELA2 shRNA as compared to control cells.Hitherto, CGD is the only immunodeficiency with a clearly defective NET formation. Our results point to an impaired formation of NETs also in CN patients carrying ELA2 mutations. This supports the recent finding of a central role for NE in NET formation. Two patients with HAX1 related CN showed a normal ability to form NETs. Our further work will aim to better define the subgroup of CN patients defective in NET formation. Disclosures:No relevant conflicts of interest to declare.