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. Keywords Autophagy • Neutrophil extracellular traps • Nitric oxide • Peroxynitrite • Phosphoinositide 3-kinases • Reactive nitrogen species Abbreviations 3-MA 3-Methyladenine ABAH Aminobenzoic acid hydrazide AKT Protein kinase B CGD Chronic granulomatous disease CI Calcium ionophore A23187 DHR 123 Dihydrorhodamine 123 DPI Diphenyleneiodonium ERK Extracellular signal-regulated kinases IL Interleukin LC3 protein Light chain 3 protein L-NAME N-nitroarginine methyl ester LPS Lipopolysaccharide MAPK Mitogen-activated protein kinases MPO Myeloperoxidase NAC N-acetylcysteine NE Neutrophil elastase NEi Neutrophil elastase inhibitor GW 311616A NETs Neutrophil extracellular traps NBT Nitroblue tetrazolium NO Nitric oxide NOS Nitric oxide synthase Cellular and Molecular Life Sciences Malgorzata Wachowska and Urszula Demkow have contributed equally.
Over a decade ago, the formation of neutrophil extracellular traps (NETs) was described as a novel mechanism employed by neutrophils to tackle infections. Currently applied methods for NETs release quantification are often limited by the use of unspecific dyes and technical difficulties. Therefore, we aimed to develop a fully automatic image processing method for the detection and quantification of NETs based on live imaging with the use of DNA-staining dyes. For this purpose, we adopted a recently proposed Convolutional Neural Network (CNN) model called Mask R-CNN. The adopted model detected objects with quality comparable to manual counting—Over 90% of detected cells were classified in the same manner as in manual labelling. Furthermore, the inhibitory effect of GW 311616A (neutrophil elastase inhibitor) on NETs release, observed microscopically, was confirmed with the use of the CNN model but not by extracellular DNA release measurement. We have demonstrated that a modern CNN model outperforms a widely used quantification method based on the measurement of DNA release and can be a valuable tool to quantitate the formation process of NETs.
Zinc plays an important physiological role in the entire body, especially in the immune system. It is one of the most abundant microelements in our organism and an essential component of enzymes and antibacterial proteins. Zinc levels were reported to be correlated with the intensity of innate immunity responses, especially those triggered by neutrophils. However, as the results are fragmentary, the phenomenon is still not fully understood and requires further research. In this study, we aimed to perform a comprehensive assessment and study the impact of zinc on several basic neutrophils’ functions in various experimental setups. Human and murine neutrophils were preincubated in vitro with zinc, and then phagocytosis, oxidative burst, degranulation and release of neutrophil extracellular traps (NETs) were analyzed. Moreover, a murine model of zinc deficiency and zinc supplementation was introduced in the study and the functions of isolated cells were thoroughly studied. We showed that zinc inhibits NETs release as well as degranulation in both human and murine neutrophils. Our study revealed that zinc decreases NETs release by inhibiting citrullination of histone H3. On the other hand, studies performed in zinc-deficient mice demonstrated that low zinc levels result in increased release of NETs and enhanced neutrophils degranulation. Overall, it was shown that zinc affects neutrophils’ functions in vivo and in vitro. Proper zinc level is necessary to maintain efficient functioning of the innate immune response.
Neutrophils apply several antimicrobial strategies including degranulation, phagocytosis, the generation of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs) to fight pathogens. Iron is considered to be an invaluable constituent of host immune defense and plays a dual role in immunity. It is a well-known component of antimicrobial proteins and is a necessary microelement for pathogen survival. The aim of this study was to broaden the knowledge regarding the impact of iron on the function of neutrophils. Neutrophils from healthy blood donors and patients with mild irondeficiency anemia and HL-60 cells differentiated toward granulocyte-like cells were incubated with Fe 2+ , Fe 3+ or holo-transferrin (holo-Tf). Moreover, we isolated murine neutrophils of HFE gene knockout (KO) mice and mice fed irondeficient, iron-equivalent and high-iron diets. We analyzed the release of NETs, phagocytosis, degranulation of azurophilic granules, ROS release, bactericidal activity of granulocytes against Escherichia coli and neutrophil elastase (NE) activity. We show that holo-Tf inhibits the release of NETs stimulated by phorbol 12-myristate 13-acetate by inhibiting NE activity. Studies performed in mice models reveal that iron overload inhibits the release of NETs and ROS production in neutrophils isolated from HFE KO mice and mice fed a high-iron diet. No impact of a low-iron diet on neutrophil phagocytosis, ROS production or release of NETs was observed. Our study underscores the physiological significance of iron in neutrophil function, specifically in the release of NETs.
Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related (ATG) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils’ antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. ATG5 gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.
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