PMA-induced Neutrophil Extracellular Traps Formation Research Articles

NET Formation Was Reduced via Exposure to Extremely Low-Frequency Pulsed Electromagnetic Fields.

Fracture-healing is a highly complex and timely orchestrated process. Non-healing fractures are still a major clinical problem and treatment remains difficult. A 16 Hz extremely low-frequency pulsed electromagnetic field (ELF-PEMF) was identified as non-invasive adjunct therapy supporting bone-healing by inducing reactive oxygen species (ROS) and Ca2+-influx. However, ROS and Ca2+-influx may stimulate neutrophils, the first cells arriving at the wounded site, to excessively form neutrophil extracellular traps (NETs), which negatively affects the healing process. Thus, this study aimed to evaluate the effect of this 16 Hz ELF-PEMF on NET formation. Neutrophils were isolated from healthy volunteers and exposed to different NET-stimuli and the 16 Hz ELF-PEMF. NETs were quantified using Sytox Green Assay and immunofluorescence, Ca2+-influx and ROS with fluorescence probes. In contrast to mesenchymal cells, ELF-PEMF exposure did not induce ROS and Ca2+-influx in neutrophils. ELF-PEMF exposure did not result in basal or enhanced PMA-induced NET formation but did reduce the amount of DNA released. Similarly, NET formation induced by LPS and H2O2 was reduced through exposure to ELF-PEMF. As ELF-PEMF exposure did not induce NET release or negatively affect neutrophils, the ELF-PEMF exposure can be started immediately after fracture treatment.

Insulin Can Delay Neutrophil Extracellular Trap Formation In Vitro-Implication for Diabetic Wound Care?

Diabetes is a worldwide evolving disease with many associated complications, one of which is delayed or impaired wound healing. Appropriate wound healing strongly relies on the inflammatory reaction directly after injury, which is often altered in diabetic wound healing. After an injury, neutrophils are the first cells to enter the wound site. They have a special defense mechanism, neutrophil extracellular traps (NETs), consisting of released DNA coated with antimicrobial proteins and histones. Despite being a powerful weapon against pathogens, NETs were shown to contribute to impaired wound healing in diabetic mice and are associated with amputations in diabetic foot ulcer patients. The anti-diabetic drugs metformin and liraglutide have already been shown to regulate NET formation. In this study, the effect of insulin was investigated. NET formation after stimulation with PMA (phorbol myristate acetate), LPS (lipopolysaccharide), or calcium ionophore (CI) in the presence/absence of insulin was analyzed. Insulin led to a robust delay of LPS- and PMA-induced NET formation but had no effect on CI-induced NET formation. Mechanistically, insulin induced reactive oxygen species, phosphorylated p38, and ERK, but reduced citrullination of histone H3. Instead, bacterial killing was induced. Insulin might therefore be a new tool for the regulation of NET formation during diabetic wound healing, either in a systemic or topical application.

Lipofundin mediates major inhibition of intravenous propofol on phorbol myristate acetate and Escherichia coli-induced neutrophil extracellular traps.

Neutrophil extracellular traps (NETs) consist of chromatin DNA networks that are studded with cytosolic and granular antimicrobial proteins to trap or kill an infected microorganism. A lipid emulsion, the solvent of pure propofol for intravenous application, is given to clinical patients who require intravenous feeding of fatty acids and fat for energy. Intravenous propofol is widely used to sedate critically ill patients. Both intravenous propofol and its lipid emulsion have immunomodulatory activity. However, the role of lipid emulsion of intravenous propofol on NET induction remains unclear. In this study, neutrophils were stimulated with phorbol myristate acetate (PMA) or Escherichia coli (E. coli) in the absence or presence of intravenous propofol (Propofol-Lipuro®), its solvent lipid emulsion (Lipofundin) or pure propofol, and NETs were stained with SYTOX Green for visualization and quantification. Total HOCl was determined by measuring the taurine-chloramine complex, and intracellular HOCl was evaluated with BioTracker™ TP-HOCl 1 dye. PMA-induced NETs were not efficiently inhibited whenPropofol-Lipuro® was addedafter PMA stimulation. Clinically relevant concentrations of Lipofundin exerted a significant reduction in PMA-induced NETs and total reactive oxidative species (ROS), which was comparable to that observed for Propofol-Lipuro®. Lipofundin transiently reduced intracellular HOCl production and thephosphorylation level of extracellular regulated kinase (p-ERK) but did not scavenge HOCl. Moreover, Lipofundin decreased E. coli-induced NETs in a ROS-independent pathway, similar to Propofol-Lipuro®. All data agree that Lipofundin, the major component of Propofol-Lipuro®, inhibits intracellular HOCl and p-ERK to suppress PMA-induced NET formation but reduces E.coli-induced NETs in a ROS-independent pathway.

Effect of Vitamin D3 in combination with Omega-3 Polyunsaturated Fatty Acids on NETosis in Type 2 Diabetes Mellitus Patients.

An understanding of the consequences of oxidative/halogenative stress triggered by neutrophil activation is impossible without considering NETosis. NETosis, formation of neutrophil extracellular traps (NETs), is known to promote microthrombus formation and impair wound healing in type 2 diabetes mellitus (T2DM) patients. Therefore, there is a need to search for drugs and treatment approaches that could prevent excessive NET formation. We aimed to evaluate the effect of vitamin D3 in combination with omega-3 polyunsaturated fatty acids (vitamin D3/omega-3 PUFAs) on NETosis in T2DM patients with purulent necrotizing lesions of the lower extremities. Patients and healthy subjects had vitamin D3 deficiency. Patients received, beyond standard treatment, 6000 IU of vitamin D3 and 480 mg of omega-3 PUFAs, and healthy subjects 1000 IU of vitamin D3 and 240 mg of omega-3 PUFAs daily for seven days. Neutrophil activation in ex vivo blood by phorbol-12-myristate-13-acetate (PMA) was used as a NETosis model. The percentage of blood NETs relative to leukocytes (NETbackground) before vitamin D3/omega-3 PUFA supplementation was 3.2%-4.9% in healthy subjects and 1.7%-10.8% in patients. These values rose, respectively, to 7.7%-9.1% and 4.0%-17.9% upon PMA-induced NETosis. In addition, the leukocyte count decreased by 700-1300 per 1 μL in healthy subjects and 700-4000 per 1 μL in patients. For both patients and healthy subjects, taking vitamin D3/omega-3 PUFAs had no effect on NETbackground but completely inhibited PMA-induced NET formation, though neutrophils exhibited morphological features of activation. Also, leukocyte loss was reduced (to 500 per 1 μL). For patients on standard treatment alone, changes occurred neither in background NETs and leukocytes nor in their amount after PMA stimulation. The decreased ability of neutrophils to generate NETs, which can be achieved by vitamin D3/omega-3 PUFA supplementation, could have a positive effect on wound healing in T2DM patients and reduce the incidence and severity of complications.

Protective effect of ethyl pyruvate on gut barrier function through regulations of ROS-related NETs formation during sepsis

BackgroundSepsis impairs the function of the intestinal barrier through neutrophil extracellular traps (NETs). Reactive oxygen species (ROS)-induced activation of mitogen-activated protein kinase (MAPK) is involved in NET formation. Ethyl pyruvate (EP), a potent and effective ROS scavenger, ameliorates sepsis-associated intestinal barrier dysfunction, but the detailed mechanism is unknown. The current study aimed to explore the eff ;ects of EP on sepsis-induced intestinal barrier dysfunction and whether ROS and NETs were involved. MethodsA sepsis model was induced in mice by intraperitoneal injection of LPS (10 mg/kg). The mice were divided into 4 groups: (1) sham group; (2) LPS group; (3) DNase-1 + LPS group; and (4) EP + LPS group. EP or DNase-1 was intraperitoneally injected after the LPS model was established. After 24 h, the small intestine and blood were collected for analysis. Human neutrophils were harvested and incubated with phorbol-12-myristate-13-acetate (PMA) or PMA + EP, and ROS and NET generation was measured. ResultsEP significantly decreased proinflammatory cytokines and MPO-DNA in the LPS model. In addition, EP suppressed NET formation in the intestines of endotoxemic mice. The decrease in NETs induced by EP or DNase-1 alleviated histopathological damage, intestinal cell apoptosis and increased tight junction expression. In vitro, the treatment of EP abolished PMA-induced ROS production and NET formation which could be reversed by H2O2 treatment. Meanwhile, EP also abolished MAPK ERK1/2 and p38 activation during PMA-induced NET formation. ConclusionThis study was the first to demonstrate that EP alleviated NET formation and sepsis-induced intestinal damage through blockage of ROS mediated MAPK ERK1/2 and p38 activation.

Propofol specifically reduces PMA-induced neutrophil extracellular trap formation through inhibition of p-ERK and HOCl

Neutrophil extracellular traps (NETs) are net-like chromatin fibers that can trap and kill microorganisms. Although several anti-inflammatory effects of intravenous anesthetics have been reported, it has not been investigated whether intravenous anesthetics influence NET formation. AimsTo compare the effects of four intravenous anesthetics (propofol, thiamylal sodium, midazolam, and ketamine) on phorbol myristate acetate (PMA)-induced NET formation and analyze the associated signaling pathways. Materials and methodsPMA-stimulated NETs formed in the absence or presence of intravenous anesthetics were stained with SYTOX Green and then quantified. Inhibitors were applied to investigate the related mechanism, which was confirmed by western blotting, and ROS were detected. Key findingsThe neutrophils incubated with propofol showed the lowest degree of NET formation compared with those incubated with the other intravenous anesthetics. Propofol significantly reduced the level of myeloperoxidase (MPO)-derived HOCl but not that of superoxide. Aminopyrine, an MPO inhibitor, markedly decreased the number of PMA-induced NETs, indicating the involvement of HOCl in the inhibitory effect of propofol on NET formation. According to western blotting results, the level of p-ERK was reduced by propofol during PMA-induced NET formation. The ERK inhibitor PD98059 decreased NET formation but did not inhibit PMA-induced HOCl generation, and aminopyrine did not reduce ERK phosphorylation. SignificanceThrough this study, we define a new anti-inflammatory effect of intravenous anesthetics. Of the four intravenous anesthetics tested, propofol was the most potent inhibitor of NET formation. Moreover, propofol resulted in a decrease in PMA-induced NET formation by two independent mechanisms: inhibition of HOCl and p-ERK.

Pathogenic Bacterium Acinetobacter baumannii Inhibits the Formation of Neutrophil Extracellular Traps by Suppressing Neutrophil Adhesion

Hospital-acquired infections caused by Acinetobacter baumannii have become problematic because of high rates of drug resistance. A. baumannii is usually harmless, but it may cause infectious diseases in an immunocompromised host. Although neutrophils are the key players of the initial immune response against bacterial infection, their interactions with A. baumannii remain largely unknown. A new biological defense mechanism, termed neutrophil extracellular traps (NETs), has been attracting attention. NETs play a critical role in bacterial killing by bacterial trapping and inactivation. Many pathogenic bacteria have been reported to induce NET formation, while an inhibitory effect on NET formation is rarely reported. In the present study, to assess the inhibition of NET formation by A. baumannii, bacteria and human neutrophils were cocultured in the presence of phorbol 12-myristate 13-acetate (PMA), and NET formation was evaluated. NETs were rarely observed during the coculture despite neutrophil PMA stimulation. Furthermore, A. baumannii prolonged the lifespan of neutrophils by inhibiting NET formation. The inhibition of NET formation by other bacteria was also investigated. The inhibitory effect was only apparent with live A. baumannii cells. Finally, to elucidate the mechanism of this inhibition, neutrophil adhesion was examined. A. baumannii suppressed the adhesion ability of neutrophils, thereby inhibiting PMA-induced NET formation. This suppression of cell adhesion was partly due to suppression of the surface expression of CD11a in neutrophils. The current study constitutes the first report on the inhibition of NET formation by a pathogenic bacterium, A. baumannii, and prolonging the neutrophil lifespan. This novel pathogenicity to inhibit NET formation, thereby escaping host immune responses might contribute to a development of new treatment strategies for A. baumannii infections.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen–antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Abstract LB-078: The role of CEACAM1 in neutrophil extracellular Trap mediated cancer metastasis

Abstract We have previously identified that Neutrophil Extracellular Traps (NETs) are an important contributing factor to the metastatic process. However, the underlying molecular mechanisms by which NETs facilitate metastasis remain unclear. Using mass spectrometry, amongst 583 distinct proteins, we identified CEACAM1 (CC1) in isolated human NETs and we sought to determine whether CC1 has a role in NET-mediated cancer metastasis. The presence of CC1 on NETs was confirmed by immunofluorescence. In vitro static adhesion of human colon cancer HT29 cells to isolated purified human NETs was reduced by 50% using a function blocking antibody against human CC1, but not isotype control, an effect equal to NET degradation with DNAse. Adhesion of murine colon cancer MC38-CC1L cells to C57BL/6 mouse neutrophils stimulated to produce NETs with phorbol myristate acetate (PMA) was increased 3-fold compared to untreated neutrophils, an effect that was completely attenuated with DNAse. PMA stimulation of neutrophils isolated from Ceacam1−/− knockout (KO) mice did not increase adhesion to MC38-CC1L cells. Using a parallel plate flow chamber, a physiologically relevant adhesion model under the shear conditions encountered in liver sinusoids, we flowed Lewis lung carcinoma (C10) cancer cells over neutrophils isolated from C57BL/6 or Ceacam1−/− KO mice. PMA-induced NET formation in C57BL/6 mouse neutrophils increased cancer cell adhesion 5-fold, an effect again completely attenuated by DNAse or use of neutrophils from Ceacam1−/− KO mice. Using a transwell chamber, MC38-CC1L cancer cells exposed to NET-stimulated Ceacam1−/− KO neutrophils had a 50% decrease in migration, compared to those exposed to NETs from C57BL/6 neutrophils. In order to delineate the role of CC1 in NET-related in vivo adhesion of cancer cells to liver sinusoids, we performed a series of neutrophil depletion and re-infusion experiments. C57BL/6 mice, depleted of neutrophils with anti-GR1 24 hrs prior, were re-infused with neutrophils isolated from C57BL/6 or Ceacam1−/− KO mice. This was followed by injection of MC38-CFSE labelled cells and hepatic intravital microscopy was used to quantify in vivo cancer cell adhesion and migration. PMA-stimulated C57BL/6 neutrophils prior to re-infusion was associated with a two-fold increase in adhesion compared to PMA-stimulated Ceacam1−/− KO neutrophils, an effect that was completely attenuated using DNAse. Our data support the notion that CEACAM1 is, at least in part, responsible for the increased cancer cell migration mediated by Neutrophil Extracellular Traps. We have thereby identified NET-associated CEACAM1 as a putative therapeutic target to prevent the metastatic progression of cancer cells. Citation Format: Phil Vourtzoumis, Rashmi Seth, Roni Rayes, Sara Najmeh, Jonathan Cools-Lartigue, Betty Giannias, France Bourdeau, Nicole Beauchemin, Simon Rousseau, Richard Blumberg, Jonathan D. Spicer, Lorrenzo Edwin Ferri. The role of CEACAM1 in neutrophil extracellular Trap mediated cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-078.

Prostaglandin E2 inhibits neutrophil extracellular trap formation through production of cyclic AMP

Upon stimulation, neutrophils release their nuclear contents called neutrophil extracellular traps (NETs), which contain unfolded chromatin and lysosomal enzymes. NETs have been demonstrated to play a critical role in host defence, although the role of PGE2 , a bioactive substance generated in inflammatory tissues, in the formation of NETs remains unclear. The effects of PGE2 , agonists and antagonists of its receptors, and modulators of the cAMP-PKA pathway on the formation of NETs were examined in vitro in isolated neutrophils and in vivo in a newly established mouse model. PGE2 inhibited PMA-induced NET formation in vitro through EP2 and EP4 Gαs-coupled receptors. Incubation with a cell-permeable cAMP analogue, dibutyryl cAMP, or various inhibitors of a cAMP-degrading enzyme, PDE, also suppressed NET formation. In the assay established here, where an agarose gel was s.c. implanted in mice and NET formation was detected on the surface of the gel, the extent of the NET formed was inhibited in agarose gels containing rolipram, a PDE4 inhibitor, and butaprost, an EP2 receptor agonist. PGE2 inhibits NET formation through the production of cAMP. These findings will contribute to the development of novel treatments for NETosis-related diseases.

Neutrophil Extracellular Trap Mitochondrial DNA and Its Autoantibody in Systemic Lupus Erythematosus and a Proof-of-Concept Trial of Metformin.

Neutrophil extracellular traps (NETs) have been shown to play an important role in systemic lupus erythematosus (SLE) pathogenesis by activating plasmacytoid dendritic cells (PDCs) and the type I interferon (IFN) pathway. NETs composed of self-DNA are considered to be of nuclear origin and are a major source of anti-DNA autoantibody generation. This study was undertaken to evaluate whether mitochondrial DNA (mtDNA) resides in NETs, to evaluate whether mtDNA and anti-mtDNA antibodies cause dysregulation of the PDC-IFNα pathway, and to investigate the clinical implication in SLE. Patients with SLE (n = 102), patients with rheumatoid arthritis (n = 30), and healthy donors (n = 40) were enrolled in in vitro studies. NETs were generated from phorbol 12-myristate 13-acetate (PMA)-stimulated peripheral neutrophils. Immunofluorescence staining was used to detect NET formation ex vivo and in lupus nephritis renal biopsy samples. The mtDNA levels and type I IFN-inducible gene scores were measured by quantitative polymerase chain reaction. Anti-mtDNA antibodies, anti-double-stranded DNA (anti-dsDNA) antibodies, and IFNα were detected by enzyme-linked immunosorbent assay. Purified PDCs were stimulated by isolated NETs, mtDNA, or dsDNA, combined with anti-mtDNA or dsDNA IgG, or other culture conditions. Additional patients with SLE (n = 113) were enrolled in a proof-of-concept trial. We evaluated the efficacy and safety of metformin on a background of corticosteroids and conventional immunosuppressive agents in patients with mild or moderate lupus. The primary end point was the efficacy of metformin for reducing disease flare. We detected mtDNA in NETs, and anti-mtDNA antibody levels were elevated in SLE patients compared with controls and significantly correlated with IFN scores and the disease activity index. The presence of anti-mtDNA antibodies was disproportionately associated with lupus nephritis, and correlated better than anti-dsDNA antibody levels with the lupus nephritis activity index. Mitochondrial DNA was deposited in NETs in lupus nephritis renal biopsy specimens. In addition, mtDNA/anti-mtDNA were greater inducers of PDC IFNα production via Toll-like receptor engagement than dsDNA/anti-dsDNA. We assessed the effect of metformin on down-regulating the NET mtDNA-PDC-IFNα pathway. Metformin decreased PMA-induced NET formation and CpG-stimulated PDC IFNα generation. A proof-of-concept trial of metformin add-on treatment of mild or moderate SLE resulted in decreases in clinical flares, prednisone exposure, and body weight. Our findings establish a link between mtDNA in NETs, anti-mtDNA antibodies, and PDC IFNα pathogenesis in SLE, and highlight that specific strategies to down-regulate this pathway, such as treatment with metformin, may be new approaches to treat SLE.

The Role of Rac and Pak in Neutrophil Histone Hypercitrullination and Neutrophil Extracellular Traps Formation

Under specific activating conditions, polymorphonuclear neutrophils (PMNs) release neutrophil extracellular traps (NETs) composed of decondensed chromatin lined with microbicidal protein such as neutrophil elastase and myeloperoxidase. NETs contribute to innate immunity but can also foster autoimmune diseases and thrombus formation. NET formation (NETosis) requires reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), allowing for chromatin decondensation. Rac GTPases are expressed in three isoforms: Rac1 is ubiquitously expressed and plays a role in PMN migration and oxidase function; Rac2 is hematopoietic-specific and the major isoform in PMNs and Rac3 is mostly neuronal. Rac1 and Rac2 regulate the cytoskeleton in PMNs, controlling actin polymerization, cell shape, adhesion and migration and are essential components of the NADPH oxidase complex. The present study aimed to explore the role of the Rac pathway on NETosis in PMNs, including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector of Rac, p21 activated kinase, Pak. We developed a flow cytometry-based quantification of H3 hypercitrullination (H3Cit). In response to phorbol myristate ester (PMA) stimulation, H3Cit is increased to 136% of basal in WT cells, compared with 103% in Rac2-/- (P<0.01) (Table). H3Cit levels observed by flow were confirmed in a NET formation assay. Rac2-/- PMNs formed significantly fewer NETs both spontaneously and after PMA stimulation (WT unstimulated 2.79%, Rac2-/- unstimulated 0.72%, WT+PMA 10.84%, Rac2-/-+PMA 1.39%, P< 0.05 for all pair comparisons). Furthermore, Rac2-/- mice demonstrated a trend towards reduced frequency of provoked thrombosis in an in vivo vena cava stenosis model (WT 78% and Rac2-/- 56% of mice with thrombus). Deletion of floxed Rac1 sequences in a Rac2-/- background in vivoallows generation and purification of PMNs lacking both Rac isoforms. Rac1Δ/Δ,Rac2-/- PMNs, which are defective in actin polymerization, had reduced basal H3Cit and a nearly complete lack of PMA-induced increase in H3Cit (136% vs 69%, WT vs Rac1Δ/Δ,Rac2-/-, P<0.01) (Table). Null knockouts of the GEFs Vav1 (hematopoietic-specific), Vav2, Vav3 or both Vav1 and 3 did not impair H3Cit response to PMA (Table). We next studied downstream effectors of Rac. Group A Paks include Pak1, 2 and 3 isoforms. Pretreatment of wild-type PMNs with either PF3758309 or IPA-3, two group A Pak inhibitors with distinct mechanisms of action, led to reduced H3Cit after PMA stimulation (induction reduced from 36% to 11% for both PF3758309 and IPA-3 treated (Table). To validate this in a genetic model, we studied Pak2Δ/Δ PMNs, since we have recently demonstrated the dependence of hematopoietic stem cell migration on Pak2. Pak2Δ/Δ demonstrated a reduced basal level of H3Cit and a significantly reduced PMA-induced increase in H3Cit (136% vs 94%, WT vs Pak2Δ/Δ, P<0.05, Table). In summary, we describe a flow-based assay that quantitates the early processes of NET formation and validated that this assay reliably predicts agonist-induced NET formation in a genetic model. The results establish that both Rac1 and Rac2, and the downstream effector Pak2, regulate histone H3 hypercitrullination and NET formation in PMNs, while suggesting that Vav does not activate the Rac pathway in PMA-induced NET formation. These data further delineate the role of the Rac pathway in NETosis, linking cytoskeleton and oxidase functions. Furthermore, these data indicate Pak could represent a therapeutic target for a wide array of pathological processes related to NETosis such as thrombosis and numerous autoimmune diseases.TableIntensity of H3Cit staining as determined by flow cytometry-based assay.Basal H3Cit levelPMA-induced H3CitPMA-induced changeWT PMN100±2%136±5%36%Rac2-/-82±9% ns103±15.3%**21%Rac1Δ/Δ, Rac2-/-64±10%***69±10%**5%Vav1-/-86±9% ns145±15% ns59%Vav2-/-91±4% ns134±18% ns43%Vav3-/-153±30%*171±28% ns18%Vav1,3-/-125±20% ns144±24% ns19%WT+PF 5nM76±17% ns87±8%*11%WT+ IPA 5µM100±4% ns111±10% ns11%Pak2Δ/Δ75±7%*94±11%*19%Results are expressed as mean±SEM % of the untreated WT control of each experiment. Results are from ≥3 independent experiments. * P<0.05, **P<0.01. P<0.001, ns non-significant, by two-tailed t-test. DisclosuresNo relevant conflicts of interest to declare.

Rab27a Is Essential for the Formation of Neutrophil Extracellular Traps (NETs) in Neutrophil-Like Differentiated HL60 Cells

Neutrophils play a crucial role in host defence. In response to a variety of inflammatory stimulation, they form neutrophil extracellular traps (NETs). NETs are extracellular structures composed of chromatin fibers decorated with antimicrobial proteins and developing studies indicate that NETs contribute to extracellular microbial killing. While the intracellular signaling pathways that regulate NET formation remain largely unknown, there is growing evidence that generation of reactive oxygen species (ROS) is a key event for NET formation. The Rab family small GTPase Rab27a is an important component of the secretory machinery of azurophilic granules in neutrophils. However, the precise mechanism of NET formation and whether or not Rab27a contributes to this process are unknown. Using neutrophil-like differentiated HL60 cells, we show here that Rab27a plays an essential role in both phorbol myristate acetate (PMA)- and Candida albicans-induced NET formation by regulating ROS production. Rab27a-knockdown inhibited ROS-positive phagosome formation during complement-mediated phagocytosis. To investigate the role of Rab27a in neutrophil function in detail, both primary human neutrophils and neutrophil-like differentiated HL60 cells were treated with PMA, and NET formation process was assessed by measurement of release of histone H3 into the medium, citrullination of the arginine in position 3 of histone H4 and chase of the nuclear change of the living cells in the co-existence of both cell-permeable and -impermeable nuclear indicators. PMA-induced NET formation occured sequentially in both neutrophil-like differentiated HL60 cells and primary neutrophils, and Rab27a-knockdown clearly inhibited NET formation in association with reduced ROS production. We also found that serum-treated Candida albicans triggers NET formation in a ROS-dependent manner, and that Rab27a-knockdown inhibits this process as well. Our findings demonstrate that Rab27a plays an important role in NET formation induced by both Candida albicans infection and PMA treatment by regulating ROS production.

Uric Acid Induces NADPH Oxidase–Independent Neutrophil Extracellular Trap Formation

IntroductionNeutrophils play a critical role in the innate immune response against various kinds of microbes. In addition to phagocytosis, neutrophil extracellular traps (NETs) have been identified as a novel killing mechanism, capturing microbes with extracellular structures consisting of DNA fibers and antimicrobial granule proteins. On the other hand, aberrant NET formation has been associated with the development of autoimmune and cardiovascular diseases. Various kinds of stimuli, including phorbol myristate acetate (PMA), are known to induce NETs by stimulating the production of reactive oxygen species (ROS), especially singlet oxygen (1O2), through the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) in neutrophils.Uric acid (UA), a product of purine metabolism, is a scavenger of 1O2 and regulates oxidative stress in humans. UA is believed to suppress Nox-dependent NET formation. By contrast, monosodium urate crystals, an endogenous danger signal, stimulate NET formation in patients with gout, suggesting that a high concentration of UA itself causes NET formation. At present, there are no data explaining the dual action of UA. In this study, we examined the direct effects of UA on NET formation. Materials and MethodsHuman neutrophils were isolated from the peripheral blood of healthy volunteers and two patients with chronic granulomatous disease (CGD), who provided written informed consent. Detection of 1O2 from the stimulated neutrophils was carried out by chemiluminescence assay using trans-1-(2'-methoxyvinyl)pyrene (MVP) as a 1O2-specific probe. NET formation was analyzed by scanning electron microscope (SEM) or confocal laser microscopy after staining with Sytox Green (dsDNA), and quantified by measuring the Sytox-positive area per cell. Involvement of the NF-κB pathway in NET formation was examined by Western blotting using anti-phospho-NF-κB p65 antibody. Data were compared with Student's t-test, and p values < 0.05 were considered significant. ResultsFirst, we examined the effects of UA on 1O2 production and NET formation by PMA-stimulated neutrophils. The addition of UA (0.05 – 5.0 mg/dL) significantly suppressed 1O2 production by PMA-stimulated neutrophils in a dose-dependent manner. At lower concentrations of UA (≤ 0.5 mg/dL), PMA-induced NET formation was suppressed, as predicted, while a high concentration of UA (5.0 mg/dL) failed to suppress PMA-induced NET formation.Next, we explored the direct effects of UA on 1O2 production and NET formation. UA treatment alone did not produce any detectable levels of 1O2 or other types of ROS. However, unexpectedly, UA-stimulated neutrophils formed NETs in a dose-dependent manner (1.0 – 8.0 mg/dL of UA). Nox inhibitors (diphenyleneiodonium [DPI] or apocynin) and a 1O2 scavenger (α-phenyl-N-tert-butyl nitrone [PBN]) did not suppress UA-induced NET formation. Moreover, neutrophils in CGD patients produced NETs after treatment with UA, although they had a defect in ROS formation and failed to make NETs after PMA stimulation. These data demonstrate that UA induces NET formation in a Nox-independent manner.Finally, we explored the signaling pathway in UA-stimulated neutrophils for inducing NET formation. Treatment with NF-κB inhibitor significantly suppressed UA-stimulated NET formation. In addition, using Western blotting, we detected phospho-NF-κB p65 in UA-activated neutrophils. These results indicate that NF-κB activation is indispensable for UA-induced NET formation. ConclusionUA suppressed PMA-induced NET formation at low concentrations (≤ 0.5 mg/dL), working as a 1O2 scavenger. By contrast, high concentrations of UA (≥ 5.0 mg/dL) induced NET formation in a Nox- or ROS-independent manner through NF-κB activation. To our knowledge, this is the first study to report on UA-induced NETs. Our findings are clinically significant in that a high level of serum UA may promote the chronic formation of NETs in circulating neutrophils, thereby contributing to vascular damage. Our data may explain one of the missing links between hyperuricemia and the risk of cardiovascular diseases. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Role of p38 MAPK in PMA‐induced NETs release from human neutrophils

Neutrophil extracellular traps (NETs) are produced by the neutrophils to bind and kill the extracellular microorganisms. It is composed of chromatin decorated with granular proteins such as elastase, myeloperoxidase, cathepsin G, pentraxin, lactoferrin etc. In the present study we have investigated the role of p38 MAPK in PMA induced reactive oxygen species (ROS) generation and NETs formation. Human neutrophils treated with PMA induced free radical generation and NETs release in a time (1–3 hrs.) and concentration (10 – 50 nM) dependent manner as demonstrated by DCF response and elastase/DNA release respectively. Since NETs release is dependent on free radicals effect of SB 202190 (50 μM, p38MAPK inhibitor) was investigated on PMA induced free radical formation as assessed by DCF and NBT, which was not significantly reduced. NETs formation was however inhibited by SB 202190 or DPI (10 μM, NADPH oxidase inhibitor), suggesting that p38 MAPK play a role downstream to the NADPH oxidase in PMA induced NETs release. NETs are made up of DNA and elastase, their release was also inhibited by DPI and SB 202190. PMA treatment induced the phosphorylation of p38 MAPK, which was inhibited by DPI. The results of the present study suggest that PMA induced NETs formation was mediated through the activation of NADPH oxidase and p38 MAPK.