Bila G. Involvement of neutrophil extracellular traps in the animal inflammatory processes under the action of nanoparticles

The aim of the dissertation work was to establish the involvement of neutrophil extracellular traps (NETs) in the induction of inflammatory processes in animals and humans following contact with endogenous and exogenous nanoparticles (NPs), the development of methods for using NPs to control the formation of NETs, the search for means of targeted influence on neutrophil granulocytes and their ability to form NETs, particularly for the use of NET-inducing NPs in the composition of next-generation vaccines. The action of two types of NPs was studied: endogenous ones, which are spontaneously formed in the hepatobiliary system as a result of a high-fat, highcholesterol diet and a fructose-rich diet, and exogenous ones, including allotrope modifications of carbon – nanodiamonds and graphene oxide. The latter do not have receptors in the body, but their ability to induce the formation of NETs is known. The work thoroughly examined the possibility of using formed conjugates of NPs and coronavirus peptides for the rapid generation of an immune response to SARS-CoV-2 antigens upon administration into the body. To conduct the study, a model of non-alcoholic steatohepatitis (NASH) was created in laboratory mice using a high-lipid high-cholesterol diet (HLHCD) and a high-fructose diet (HFD). Both diets effectively reflected the content of lipids, cholesterol, and fructose typical for a «Western diet» or a diet with a high content of fast food, fried food, and sweet drinks. At the high cholesterol content in the presence of cholic acid, they tends to crystallize in the bile ducts as a result of natural bile concentration processes, forming nano- and micro-particles. In the liver, fructose is metabolized by the enzyme fructokinase C, which acts rapidly and lacks a feedback mechanism. When consuming a large amount of fructose present in sweet drinks, it leads to an instant decrease in ATP levels in hepatocytes, converting it into AMP. The formed AMP induces the purine degradation pathway through the activation of AMP deaminase, resulting in the formation of uric acid and the generation of mitochondrial oxidants. Hepatocytes being deprived of energy due to ATP depletion and with elevated levels of mitochondrial oxidants are prone to cell death, and when their integrity is compromised, uric acid will enter the extracellular space, where it will come into contact with a high concentration of sodium ions, converting into monosodium urate. The latter forms needle-shaped nano- and microcrystals, which are inducers of gout through the activation of NETs. To investigate the mechanism of NETs formation, we used a special line of C57BL6/N mice expressing the functional nnt gene, responsible for increased mitochondrial NADPH/NADP+ content, and characterized by high levels of neutrophil granulocytes in the blood with a high ability to form reactive oxygen species (ROS). The animals exposed to HLHCD and HFD treatment exhibited typical signs of NASH within 4 – 6 weeks, which is significantly shorter compared to other described mouse models of NASH (12 – 18 weeks). Histologically, NASH in the liver of the animals was confirmed by the accumulation of lipids, the appearance of microvesicular steatosis, nuclear degradation, and collagen accumulation. We adapted quantitative approaches for the analysis of histological images to assess tissue damage in the mouse liver. We proposed a progressive approach for verifying NASH liver markers using the immunoenzyme method based on molecular markers of the ELF – Enhanced Liver Fibrosis, test developed by Siemens, adapted for mice, detecting the amino-terminal peptide of procollagen III, tissue inhibitor of metalloproteinases-1 and hyaluronic acid. Using the reproducible and convenient NASH models in laboratory mice that we developed, we investigated the involvement of neutrophil granulocytes in the development of these pathologies. Leukocyte infiltration and vessel and bile duct destruction were observed in histological preparations. We developed an effective method for detecting eosinophils in histological samples using fluorescence microscopy, which allowed differentiation between neutrophilic and eosinophilic granulocytes among the observed leukocytes. It was established that neutrophils predominate among the infiltrating granulocytes in liver tissues.