Urban dust particles are a major pathogenic factor in respiratory diseases such as asthma and chronic obstructive pulmonary disease, and also increase the risk of cardiovascular diseases, and lung cancer. Nanoparticles (NPs) of various origins are an important component of urban dust, but their effects on the human body are barely studied. In the present work, the effect of urban dust NPs on innate immune cells, neutrophils and macrophages was investigated in vitro. The study used urban dust NPs isolated from urban dust samples using the coiled tube field-flow fractionation technique. Urban dust NPs were shown to induce a significant increase in the production of reactive oxygen species in human neutrophils. Preincubation of neutrophils with dust NPs resulted in a fourfold increase in production of reactive oxygen species in response to the chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), which indicates an effect of neutrophil priming with nanoparticles. On macrophages differentiated from the monocytic line THP-1, urban dust NPs stimulated the secretion of pro-inflammatory cytokines, tumor necrosis factor, and interleukin-6. The inflammatory activation of neutrophils and macrophages was reduced by antibiotic polymyxin B, which is able to bind bacterial wall lipopolysaccharide. The results suggest that the pro-inflammatory effect of urban dust NPs on neutrophils and macrophages is, at least in part, due to the presence of bacterial wall lipopolysaccharide.

Neutrophils are the first line of defense of the human immune system against pathogens. Photobiomodulation, mediated by mitochondrial photoacceptors such as cytochrome c oxidase, has emerged as a method to modulate neutrophil function through targeted light exposure. Despite the extensive characterization of neutrophil extracellular traps (NETs) formation (NETosis), the wavelength-specific modulation of neutrophil photoactivation and the involvement of redox pathways remain poorly defined. In this study, the effects of monochromatic (365 nm, 415 nm, 437 nm, and 625 nm) and dichromatic LED-light irradiation on NETs formation were systematically examined. The highest netotic responses were elicited by UV-A (365 nm) and violet-blue light (415 nm), whereas 437 nm showed the lowest induction and 625 nm stimulated a moderate netotic response. The pharmacological inhibition of NETosis induced by 365 nm and 415 nm irradiation with specific NADPH oxidase inhibitor, apocynin, and mitochondrial reactive oxygen species (mtROS) scavenger, MitoTEMPO, attenuated NETs formation by engaging both enzymatic and mitochondrial oxidative sources. Notably, mtROS played a dominant role under 415 nm stimulation in contrast to 365 nm-induced NETosis as demonstrated by higher sensitivity to MitoTEMPO. Importantly, combined simultaneous irradiation with 415 nm and 625 nm LEDs resulted in a significant suppression of NETs formation by more than 50%, highlighting a potent inhibitory synergy observed for the first time and suggesting a new approach of wavelength pairing to modulate neutrophil activation. These results were further supported by measurements of ROS production using a luminol-amplified chemiluminescence assay. Collectively, these findings delineate a wavelength- and ROS-dependent framework for light-induced neutrophil activation, with mitochondrial pathways exerting central control particularly under short-wavelength irradiation.

Multiple sclerosis (MS) is among the most common neurological diseases. The number of MS affected people is constantly growing worldwide. Untreated MS leads to disability of the most capable part of the population of young age, and in recent years it has been diagnosed more often in elderly patients. The second part of our review is focused on the prospects of MS therapies under development. Mitochondria and the use of mitochodria-targeted antioxidants, neutrophils, as well as immune cells affected by pathology and other specialized cells, which can be reprogrammed and replaced by healthy cells using stem cells, pre-oligodendrocytes able to accelerate maturation and remyelinating ability on the antihistamine action, are considered as targets in MS treatment. Helminth therapy, accompanied by a shift in the composition of the microbiota of MS patients and the release of antioxidants in the tissues of humans and model animals, may lead to immunomodulation and reduction of oxidative stress, providing significant mitigation of the disease. Approaches to the treatment of elderly MS patients are discussed.

Multiple sclerosis (MS) is among the most common diseases of the central nervous system. The disease leads to pathological demyelination of axons in the white matter of the brain, followed by demyelination of gray matter, and is accompanied by progressive neurodegeneration in patients. The etiology of the disease is not fully understood. However, a number of external and internal factors that increase the likelihood of MS among the active capable part of the population have been established. The characteristics of age patients exacerbating the course of MS have been identified. The review discusses the mechanism of inflammation activation at MS involving NLRP3 inflammasome and neutrophils identified in recent years, the effect of inflammation on damage to the blood-brain barrier and MS progression, as well as reactive oxygen species-mediated participation of mitochondria in MS pathology development.

Neutrophil extracellular traps (NETs) are decondensed nuclear chromatin, decorated with bactericidal proteins of various cell organelles and performing an eff ector function aimed to combat pathogens at the site of infl ammation. At the same time, NETs play an important role in the pathogenesis of many autoimmune and infl ammatory diseases as well as malignancies. Rabbits are one of the most commonly used species of laboratory animals in medical and biological research. A large number of models of various diseases of the cardiovascular, immune and other human systems have been developed in rabbits. However, there is no information in the scientifi c literature about the ability of rabbit neutrophils to undergo NETosis in response to well-known pharmacological stimuli. The purpose of the present work was to study in in vitro system the ability of neutrophils of Soviet chinchilla rabbit to form NETs in response to mimetic of diacylglycerol phorbol 12-myristate 13-acetate (PMA) and calcium ionophore A23187. To isolate rabbit neutrophils, the one-step density gradient centrifugation on Ficoll-Hypaque method with modifi cations was used. Oxidative burst was assessed with luminol-amplifi ed chemiluminescence method, and NET formation was assessed with immunofl uorescence analysis. The work shows for the fi rst time that neutrophils of Soviet chinchilla rabbit do not form NETs in response to PMA, but form traps in response to A23187, as well as have a low level of oxidative burst in response to PMA, A23187 and chemoattractant N-formyl-methionylleucyl-phenylalanine.

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.

Neutrophils release decondensed nuclear chromatin or Neutrophil Extracellular Traps (NETs) in response to a great number of physiological stimuli to protect the host from pathogens. However, NETs have recently been shown to play an important role in the pathogenesis of autoimmune, infl ammatory, and malignant diseases. Therefore, understanding the molecular mechanisms underlying NETs formation, usually leading to the neutrophil death (NETosis), is extremely important to control the aberrant release of chromatin. Mitogen-activated protein kinases (MAP-kinases) are involved in various cellular functions such as oxidative burst, chemotaxis, degranulation, adhesion, and apoptosis, but their role in NETosis is not well understood. Three families of MAP-kinases, p38, ERK1/2, and JNK, have been described in human neutrophils, and we investigated the contribution of p38, ERK1/2, and protein kinase B Akt1/2 in oxidative burst and NETosis using inhibitory analysis. We have shown that MAPkinase p38 as well as protein kinase B Akt1/2 are activated upon stimulation of oxidative burst and NETosis with calcium ionophore ionomycin. However, these kinases are not involved in the oxidative burst induced by diacylglycerol mimetic phorbol 12-myristate 13-acetate (PMA) but are involved in PMA-induced NETosis.

Background. Chronic sinusitis occurs in the Russian Federation in 16.4±10.89% of the population and has a great impact on the quality of life of patients. The inflammatory process underlying this pathology is often resistant to conservative treatment and causes surgical intervention. The study of the role of neutrophil extracellular traps as an important part of the immune response, as well as the capabilities of drugs capable of influencing the processes of netosis, is an important and relevant area of modern research. Aim. Studying the role of NETs in the pathogenesis of CRS without polyps, assessing the effect of azoximer bromide on the metabolism of NETs in patients with CRS. Materials and methods. The study included 82 patients diagnosed with chronic rhinosinusitis (average age 37±12 years), and 40 healthy volunteers (average age 34±10 years). Patients with CRS were treated with surgery and a course of azoximer bromide, the severity of the disease did not differ in patients. Nasal secretions and venous blood were analyzed in all study participants with the determination of surrogate markers of neutrophil extracellular traps – myeloperoxidase complexes with DNA and the detection of double-stranded DNA (Quant Pico Green dsDNA kit). In patients who received azoximer bromide, the material was taken twice – before the start of treatment and 10 days after the course of treatment. Results. In patients with CRS, the amount of NETs in nasal secretions and venous blood is higher compared to the control group (p0.05). The use of azoximer bromide in CRS outside of exacerbation reduces the activity of NETosis processes with intranasal use of the drug not only in the area of inflammation (reduction of NET in nasal secretions); p0.05, but also at the general level (decrease NETs in venous blood); p0.05. Conclusion. An increase in the amount of NETs in nasal flushes and venous blood in patients with CRS without exacerbation compared with the control group may indicate a likely pathological role of NETosis processes, and an increase in the amount of NETs in the blood of patients with CRS without exacerbation compared with the control group indicates the systemic effect of a local inflammatory process in the mucous membrane of the nasal cavity and paranasal sinuses.

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.

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.

Neutrophils are the most numerous blood leukocytes and are the “first line” of defense against pathogens in the focus of inflammation, where they perform effector functions of phagocytosis, degranulation, generation of reactive oxygen species, and the formation of neutrophil extracellular traps. It was believed for a long time that neutrophils are short-lived terminally differentiated phagocytes. However, this view changed after it was discovered that neutrophils are able to interact with other populations of leukocytes as well as being responsible for the relationship between innate and adaptive immunity. A lot of data indicating the ability of neutrophils to acquire the function of antigen-presenting cells in pathological and inflammatory conditions has accumulated in recent years. In addition, neutrophils can express major histocompatibility complex class II molecules and costimulatory molecules when exposed to specific cytokines in the in vitro system and activate T lymphocytes. The review summarizes recent data on the antigen-presenting function of neutrophils, the proposed mechanisms of regulation of this process, and its significance in normal and pathological conditions.

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.

In this study, we aimed to investigate the impact of radiation across a wide range of wavelengths, from UV-A to red visible light, on the role of neutrophils in inflammatory, autoimmune, and oncological diseases.Our focus was on understanding the photoacceptance process involving two cytochromes: cytochrome_b558 and cytochrome_c oxidase.Through the utilization of Raman spectroscopy, we recorded characteristic Raman frequencies corresponding to various reactive oxygen species (ROS) and low-frequency lattice vibrational modes for citrulline.By employing selective inhibitors of NADPH oxidase (apocynin) and PAD4 (GSK484), we were able to establish that when neutrophils are exposed to light of different wavelengths, it activates signaling pathways that lead to the formation of NETs (neutrophil extracellular traps) through the involvement of NADPH oxidase and PAD4.During the irradiation of neutrophils, we observed distinct peaks indicating the presence of ROS and citrulline, suggesting the participation of intracellular ROS during light exposure.Development of novel drugs aimed at suppressing NETs formation could potentially inhibit NET formation at sites exposed to UV and visible light.This could result in a reduction in symptoms related to UV-induced photoaging and other forms of organ damage.

Neutrophils release decondensed nuclear chromatin or Neutrophil Extracellular Traps (NETs) in response to a great number of physiological and pharmacological stimuli. However, apart from the host defensive function, NETs play an essential role in the pathogenesis of various autoimmune, inflammatory, and malignant diseases. Therefore, understanding the molecular mechanisms of NETs formation, usually leading to the neutrophil death (NETosis), is important to control the probable aberrant or excessive NETs release. The Src-family kinases (Src-kinases) are non-receptor tyrosine kinases that are involved in a variety of human functions. However, their role in NETosis and oxidative burst has not been sufficiently studied. Since three representatives of Src-kinases (Hck, Fgr, and Lyn) have been described in human neutrophils, we studied their contribution to NETosis and oxidative burst using inhibitory analysis. We have shown that Src-kinases are involved in the oxidative burst and NETosis induced by the calcium ionophore A23187 but not the mimetic of diacylglycerol phorbol-12-myristate-13-acetate (PMA).

Neutrophils release decondensed nuclear chromatin or neutrophil extracellular trap (NETs) in response to a large number of different physiological stimuli in order to protect the host from the pathogens. However, as it has been recently established, NETs play an important role in the pathogenesis of autoimmune, inflammatory, and oncological diseases. In this regard, understanding molecular mechanisms underlying the formation of NETs and leading, as a rule, to the death of neutrophils (NETosis) is extremely important to provide a control of aberrant chromatin release. Mitogen-activated protein kinases (MAP kinases) are involved in diverse cellular functions, such as oxidative burst, chemotaxis, degranulation, adhesion, and apoptosis; however, their role in NETosis was not sufficiently studied. Three families of MAP kinases were described in human neutrophils, including p38, ERK1/2, and JNK. In our work, the involvement of p38, ERK1/2, as well as protein kinase B Akt1/2, in the oxidative burst and NETosis was studied using an inhibitory analysis. We demonstrated that p38 MAP kinase and protein kinase B Akt1/2 are activated upon stimulation of the oxidative burst and NETosis by calcium ionophore ionomycin. At the same time, these kinases are not involved in the oxidative burst induced by diacylglycerol mimetic phorbol 12-myristate 13-acetate (PMA), but are involved in PMA-induced NETosis.

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.

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, βI, βII, 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 PKCβ, PKCδ, and PKCζ are involved in the oxidative burst, spreading and NETosis activated by calcium ionophore A23187, while only PKCβ 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.

Neutrophils release decondensed nuclear chromatin or neutrophil extracellular traps (NET) in response to a great number of physiological and pharmacological stimuli. However, apart from the host defensive function, NETs play an essential role in the pathogenesis of various autoimmune, inflammatory, and malignant diseases. Therefore, understanding the molecular mechanisms of NET formation, usually leading to the neutrophil death (NETosis), is important to control the consequences of aberrant or excessive NET release. Protein kinase C (PKC) is a serine/threonine kinase that is involved in a variety of neutrophil functions, but its role in NETosis is not well understood. Since five PKC isoforms (α, βI, βII, δ, and ζ) have been described in human neutrophils, we studied their contribution to NETosis and oxidative burst using inhibitory analysis. Using specific PKC isoform inhibitors, we have shown that PKCβ, PKCδ, and PKCζ are involved in the oxidative burst and NETosis activated by calcium ionophore A23187, while PKCβ is involved in the oxidative burst and NETosis upon cell activation by diacylglycerol mimetic phorbol 12‑myristate 13-acetate.

Chronic rhinosinusitis is one of the most common diseases in the structure of ENT pathology. A combination of several factors often leads to the chronicity of this disease, which significantly complicates the choice of treatment tactics while maintaining persistent inflammation in the mucous membrane of the nasal cavity and paranasal sinuses. The emergence of new data on changes occurring at the cellular-molecular level significantly affects the understanding of the processes occurring during the pathogenesis of chronic rhinosinusitis and other diseases of the ENT organs. In recent years, more and more information has been accumulating on the role of neutrophils and eosinophils, as well as on such an immune response mechanism as the formation of extracellular traps in the structure of diseases of the upper and lower respiratory tract. The review presents current information on the effect of neutrophilic and eosinophilic extracellular traps on the chronic inflammatory process in the mucous membrane of the respiratory tract using the example of chronic rhinosinusitis.