Abstract
Despite great interest, the mechanism of neutrophil extracellular traps (NETs) release is not fully understood and some aspects of this process, e.g. the role of reactive nitrogen species (RNS), still remain unclear. Therefore, our aim was to investigate the mechanisms underlying RNS-induced formation of NETs and contribution of RNS to NETs release triggered by various physiological and synthetic stimuli. The involvement of RNS in NETs formation was studied in primary human neutrophils and differentiated human promyelocytic leukemia cells (HL-60 cells). RNS (peroxynitrite and nitric oxide) efficiently induced NETs release and potentiated NETs-inducing properties of platelet activating factor and lipopolysaccharide. RNS-induced NETs formation was independent of autophagy and histone citrullination, but dependent on the activity of phosphoinositide 3-kinases (PI3K) and myeloperoxidase, as well as selective degradation of histones H2A and H2B by neutrophil elastase. Additionally, NADPH oxidase activity was required to release NETs upon stimulation with NO, as shown in NADPH-deficient neutrophils isolated from patients with chronic granulomatous disease. The role of RNS was further supported by increased RNS synthesis upon stimulation of NETs release with phorbol 12-myristate 13-acetate and calcium ionophore A23187. Scavenging or inhibition of RNS formation diminished NETs release triggered by these stimuli while scavenging of peroxynitrite inhibited NO-induced NETs formation. Our data suggest that RNS may act as mediators and inducers of NETs release. These processes are PI3K-dependent and ROS-dependent. Since inflammatory reactions are often accompanied by nitrosative stress and NETs formation, our studies shed a new light on possible mechanisms engaged in various immune-mediated conditions.
Highlights
Neutrophils are effector cells of the innate immune system responsible for the defense against invading pathogens upon recruitment to the site of infection, injury or inflammation
We observed that exogenously added peroxynitrite induced neutrophil extracellular traps (NETs) formation in a concentrationdependent manner (Fig. 1a, b) with statistical significance for concentrations, 100 and 200 μM
NETs formation was assessed fluorometrically (b) and by fluorescent microscopy (c). b one-way ANOVA, n = 6 kinase-activation patterns induced by various NETs-inducers and we found that stimulation with phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187 (CI), strong synthetic NETs inducers, led to phosphorylation of p38 and extracellular signal–regulated kinase (ERK) kinases (Fig. 4a)
Summary
Neutrophils are effector cells of the innate immune system responsible for the defense against invading pathogens upon recruitment to the site of infection, injury or inflammation. After migration to the tissue, neutrophils are able to destroy pathogens through several mechanisms, including the release of neutrophil extracellular traps (NETs) [1]. These antimicrobial structures are composed of DNA complexed with histones as well as granular and cytoplasmic proteins, such as neutrophil elastase (NE), myeloperoxidase (MPO), and cathepsin G. Most studies point to nucleus as the source of NETs-bound DNA, the release of mitochondrial DNA has been described [2] In physiological conditions, these DNA-containing traps immobilize and kill microorganisms, serving as an important mechanism to control bacterial, fungal, parasitic, and viral infections. Imbalance between NETs formation and clearance can contribute to multiple pathological conditions such as systemic lupus erythematosus, thrombosis, cystic fibrosis or even formation of cancer metastases, being “a double-edged sword” [2, 3]
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