Cell Surface Parameters for Accessing Neutrophil Activation Level with Atomic Force Microscopy.

In this study, we examine the topography and adhesion images of the cell surface of neutrophils during the activation process. Our analysis of cell surface parameters indicates that the most significant changes in neutrophils occur within the first 30 min of activation, suggesting that reactive oxygen species may require approximately this amount of time to activate the cells. Interestingly, we observed surface granular structure as early as 10 min after neutrophil activation when examining atomic force microscopy images. This finding aligns with the reorganization observed within the cells under confocal laser scanning microscopy. By analyzing the cell surface images of adhesion, we identified three spatial surface parameters that correlate with the activation time. This finding enables us to estimate the degree of activation by using atomic force microscopy maps of the cell surface.

Morphology of Neutrophils during Their Activation and NETosis: Atomic Force Microscopy Study.

Confocal microscopy and fluorescence staining of cellular structures are commonly used to study neutrophil activation and NETosis. However, they do not reveal the specific characteristics of the neutrophil membrane surface, its nanostructure, and morphology. The aim of this study was to reveal the topography and nanosurface characteristics of neutrophils during activation and NETosis using atomic force microscopy (AFM). We showed the main stages of neutrophil activation and NETosis, which include control cell spreading, cell fragment formation, fusion of nuclear segments, membrane disruption, release of neutrophil extracellular traps (NETs), and final cell disintegration. Changes in neutrophil membrane nanosurface parameters during activation and NETosis were quantified. It was shown that with increasing activation time there was a decrease in the spectral intensity of the spatial periods. Exposure to the activator A23187 resulted in an increase in the number and average size of cell fragments over time. Exposure to the activators A23187 and PMA (phorbol 12-myristate 13-acetate) caused the same pattern of cell transformation from spherical cells with segmented nuclei to disrupted cells with NET release. A23187 induced NETosis earlier than PMA, but PMA resulted in more cells with NETosis at the end of the specified time interval (180 min). In our study, we used AFM as the main research tool. Confocal laser-scanning microscopy (CLSM) images are provided for identification and detailed analysis of the phenomena studied. In this way, we exploited the advantages of both techniques.

Role of Mitochondria in the Regulation of Effector Functions of Granulocytes.

Granulocytes (neutrophils, eosinophils, and basophils) are the most abundant circulating cells in the innate immune system. Circulating granulocytes, primarily neutrophils, can cross the endothelial barrier and activate various effector mechanisms to combat invasive pathogens. Eosinophils and basophils also play an important role in allergic reactions and antiparasitic defense. Granulocytes also regulate the immune response, wound healing, and tissue repair by releasing of various cytokines and lipid mediators. The effector mechanisms of granulocytes include the production of reactive oxygen species (ROS), degranulation, phagocytosis, and the formation of DNA-containing extracellular traps. Although all granulocytes are primarily glycolytic and have only a small number of mitochondria, a growing body of evidence suggests that mitochondria are involved in all effector functions as well as in the production of cytokines and lipid mediators and in apoptosis. It has been shown that the production of mitochondrial ROS controls signaling pathways that mediate the activation of granulocytes by various stimuli. In this review, we will briefly discuss the data on the role of mitochondria in the regulation of effector and other functions of granulocytes.

Stages of NETosis Development upon Stimulation of Neutrophils with Activators of Different Types.

Before NETs are released, the neutrophil undergoes structural changes. First, it flattens, accompanied by a change in cell shape and rearrangement of the cytoskeleton. Then, nuclear swelling begins, which ends with the ejection of NETs into the extracellular space. We used widefield and confocal fluorescence microscopy to register morphological and structural changes in neutrophils during activation and NETosis. Different types of activators were used, such as NOX-dependent PMA and calcium ionophore A23187. The measurements were performed in a series of sequential stages. In the first stage (30 s after addition of activators and immediately after stimulation of neutrophils), the response of neutrophils to A23187 and PMA exposure was studied. Subsequently, the characteristics of neutrophils in different phases of activation were examined over a longer period of time (30, 60, 120, 180, and 240 min). The specific features of NETosis development were analyzed separately. During the first 30 s, neutrophils appeared to be heterogeneous in shape and structure of the actin cytoskeleton. Characteristic cell shapes included 30″ type 1 cells, similar in shape to the control, with F-actin concentrated in the center of the cytoplasm, and 30″ type 2 cells, which had flattened (spread) shapes with increased frontal dimensions and F-actin distributed throughout the cell. Later, the development of nuclear swelling, the corresponding changes in neutrophil membranes, and NET release into the extracellular space were evaluated. The conditions determining the initiation of chromatin ejection and two characteristic types of decondensed chromatin ejection were revealed. The results obtained contribute to a better understanding of the biophysical mechanisms of neutrophil activation and NETosis development.

Radiation from UV-A to Red Light Induces ROS-Dependent Release of Neutrophil Extracellular Traps.

Neutrophils release decondensed chromatin or extracellular traps (NETs) in response to various physiological and pharmacological stimuli. Apart from host defensive functions, NETs play an essential role in the pathogenesis of various autoimmune, inflammatory, and malignant diseases. In recent years, studies have been performed on photo-induced NET formation, mainly activated by UV radiation. Understanding the mechanisms of NET release under the influence of UV and visible light is important to control the consequences of the damaging effects of electromagnetic radiation. Raman spectroscopy was applied to record characteristic Raman frequencies of various reactive oxygen species (ROS) and low-frequency lattice vibrational modes for citrulline. NETosis was induced by irradiation with wavelength-switchable LED sources. Fluorescence microscopy was used to visualize and quantify NET release. The ability of five wavelengths of radiation, from UV-A to red light, to induce NETosis was investigated at three different energy doses. We demonstrated, for the first time, that NET formation is activated not only by UV-A but also by three spectra of visible light: blue, green, and orange, in a dose-dependent manner. Using inhibitory analysis, we established that light-induced NETosis proceeds through NADPH oxidase and PAD4. The development of new drugs designed to suppress NETosis, especially when induced by exposure to intense UV and visible light, can help to mitigate light-induced photoaging and other damaging effects of electromagnetic radiation.

Protein kinase C isoforms mediate the formation of neutrophil extracellular traps.

Neutrophils release extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune, inflammatory and malignant diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating the aberrant or excessive NET release. Protein kinase C (PKC) is a serine/threonine kinase which is involved in various neutrophil functions, however, little is known about its implication in NETosis activated by various physiological and pharmacological stimuli. Since there are conventional, novel and atypical PKC isoforms (α, ßI, ßII, δ, and ζ) found in human neutrophils, we investigated their impact in NETosis, oxidative burst and spreading applying pharmacological approach. Using specific inhibitors of PKC isoforms, we showed that PKCß, PKCδ, and PKCζ are involved in the oxidative burst, spreading and NETosis activated by calcium ionophore A23187, while only PKCß is implicated in these functions activated by phorbol 12-myristate 13-acetate (PMA). The data obtained in our study might help in the development of new drugs useful for the treatment of autoimmune and inflammatory diseases associated with NETs.

Mitochondrial permeability transition pore is involved in oxidative burst and NETosis of human neutrophils.

Neutrophils release neutrophil extracellular traps (NETs) in response to numerous pathogenic microbes as the last suicidal resource (NETosis) in the fight against infection. Apart from the host defense function, NETs play an essential role in the pathogenesis of various autoimmune and inflammatory diseases. Therefore, understanding the molecular mechanisms of NETosis is important for regulating aberrant NET release. The initiation of NETosis after the recognition of pathogens by specific receptors is mediated by an increase in intracellular Ca2+ concentration, therefore, the use of Ca2+ ionophore A23187 can be considered a semi-physiological model of NETosis. Induction of NETosis by various stimuli depends on reactive oxygen species (ROS) produced by NADPH oxidase, however, NETosis induced by Ca2+ ionophores was suggested to be mediated by ROS produced in mitochondria (mtROS). Using the mitochondria-targeted antioxidant SkQ1 and specific inhibitors of NADPH oxidase, we showed that both sources of ROS, mitochondria and NADPH oxidase, are involved in NETosis induced by A23187 in human neutrophils. In support of the critical role of mtROS, SkQ1-sensitive NETosis was demonstrated to be induced by A23187 in neutrophils from patients with chronic granulomatous disease (CGD). We assume that Ca2+-triggered mtROS production contributes to NETosis either directly (CGD neutrophils) or by stimulating NADPH oxidase. The opening of the mitochondrial permeability transition pore (mPTP) in neutrophils treated by A23187 was revealed using the electron transmission microscopy as a swelling of the mitochondrial matrix. Using specific inhibitors, we demonstrated that the mPTP is involved in mtROS production, NETosis, and the oxidative burst induced by A23187.

The role of mitochondrial ROS in antibacterial immunity.

Reactive oxygen species (ROS) are essential participants of various innate immune cell responses against microorganisms and are also involved in many cellular regulatory pathways. It was believed that the main pool of ROS in the innate immune cells is generated by the NADPH oxidase enzymatic complex. However, it was discovered recently that mitochondrial ROS (mtROS) are equally important for the functioning of the immune system. mtROS play an important role in the development of the antimicrobial innate immune responses. The present mini-review summarizes the most recent data on the role of mtROS in the antibacterial immunity. The principles of mtROS formation and possible mechanisms of their generation under the activation of innate immunity are highlighted in this review. We also speculate on the possibilities of using activators of mtROS production in clinical practice.

Mitochondrial reactive oxygen species are involved in chemoattractant-induced oxidative burst and degranulation of human neutrophils in vitro.

Activation of neutrophils is accompanied by the oxidative burst, exocytosis of various granule types (degranulation) and a delay in spontaneous apoptosis. The major source of reactive oxygen species (ROS) in human neutrophils is NADPH oxidase (NOX2), however, other sources of ROS also exist. Although the function of ROS is mainly defensive, they can also play a regulatory role in cell signaling. However, the contribution of various sources of ROS in these processes is not clear. We investigated a possible role of mitochondria-derived ROS (mtROS) in the regulation of neutrophil activation induced by chemoattractant fMLP in vitro. Using the mitochondria-targeted antioxidant SkQ1, we demonstrated that mtROS are implicated in the oxidative burst caused by NOX2 activation as well as in the exocytosis of primary (azurophil) and secondary (specific) granules. Scavenging of mtROS with SkQ1 slightly accelerated spontaneous apoptosis and significantly stimulated apoptosis of fMLP-activated neutrophils. These data indicate that mtROS play a critical role in signal transduction that mediates the major neutrophil functional responses in the process of activation.

Effects of the antioxidants Trolox, Tiron and Tempol on neutrophil extracellular trap formation.

Neutrophils can entrap and kill pathogens by releasing of neutrophil extracellular traps (NETs), in addition to their routine functions such as phagocytosis and degranulation. NETs consist of a DNA backbone supplemented by multiple bactericidal proteins from the nucleus, the cytoplasm and the granules. Neutrophils release NETs after their activation by a number of physiological and pharmacological stimuli. In addition to the antimicrobial function, NETs are involved in the pathogenesis of various autoimmune and inflammatory diseases. Since NET formation predominantly depends on the generation of reactive oxygen species (ROS), all substances that are capable of scavenging ROS or inhibiting the enzymes responsible for their synthesis should prevent ROS-associated NET release. The aim of this study was to test substances with an antioxidant activity, such as Trolox, Tiron, and Tempol, for their capacity to inhibit NET formation by primary human neutrophils in vitro. We revealed for the first time an inhibitory effect of Trolox on ROS-dependent NET release. We also established a suppressive effect of Tempol on NET formation that manifested itself in a wide range of concentrations. In this study, no inhibitory influence of Tiron on NET release was revealed. All tested substances exerted a significant dose-dependent antioxidative effect on ROS generation induced by phorbol 12-myristate 13-acetate (PMA). We suggest that the antioxidants Trolox and Tempol should be recommended for treating autoimmune and inflammatory diseases that implicate ROS-dependent NET release.

Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity.

The pathogenesis of many autoimmune diseases is initially based on a redundant or prolonged activation of the innate immune system. It was suggested that an excessive activation of the innate immunity is often the result of a chronic inflammatory process in the organism. This inflammation can be induced by exogenous and endogenous alarm factors, or alarmins. We believe that the recently discovered neutrophil extracellular traps, or NETs, completely meet the criteria of alarmins. This review summarizes current knowledge concerning the general characteristics of NETs, their antimicrobial properties, and their role in the development of chronic inflammatory processes that underlie the pathogenesis of psoriasis and atherosclerosis. Studies on the NETosis can provide the foundation for developing new diagnostic methods and effective treatment of chronic inflammatory and autoimmune diseases.

The bactericidal effects of electrolyzed oxidizing water on bacterial strains involved in hospital infections.

The study is designed to investigate bactericidal actions of electrolyzed oxidizing water on hospital infections. Ten of the most common opportunistic pathogens are used for this study. Cultures are inoculated in 4.5 mL of electrolyzed oxidizing (EO) water or 4.5 mL of sterile deionized water (control), and incubated for 0, 0.5, and 5 min at room temperature. At the exposure time of 30 s the EO water completely inactivates all of the bacterial strains, with the exception of vegetative cells and spores of bacilli which need 5 min to be killed. The results indicate that electrolyzed oxidizing water may be a useful disinfectant for hospital infections, but its clinical application has still to be evaluated.