Microglia cells are glial cells of the central nervous system (CNS), similar to macrophages, which maintain brain homeostasis and are among the most important cells of the innate immune system in the CNS. Under the influence of pathological and immunological stimuli, the polarization of microglia occurs, either into the pro-inflammatory phenotype M1, which promotes the processes of neuroinflammation, or the anti-inflammatory phenotype M2, which exhibits a protective function in the brain. Cysteine cathepsins released by M1 polarized microglia are increasingly recognized as important inflammatory factors that trigger signaling pathways, which in turn lead to aggravation of neuroinflammation. Among them is the cysteine peptidase cathepsin X, which has already been recognized as a pathogenic factor in inflammation-induced neurodegeneration. As part of the master's thesis, we evaluated the role of cathepsin X in the polarization of microglia cells. First, we established a cellular model of polarized microglia using the BV2 cell line, where we demonstrated the effect of stimulation with lipopolysaccharide (LPS), interferon-γ (IFN-γ), interleukin-4 (IL-4) and IL-13 on the course of polarization. Here, LPS and IFN-γ induced polarization of microglia to the M1 phenotype, which we demonstrated with increased levels of pro-inflammatory factors. On the other hand, the cytokines IL-4 and IL-13 induced polarization towards the M2 phenotype, which was confirmed by markers of the anti-inflammatory state. We further characterized the expression, activity, and vesicular localization of cathepsin X in stimulated BV2 cells. Stimulation with LPS and IFN-γ increased the intracellular expression of cathepsin X, which was demonstrated by western blotting, and at the same time decreased its activity due to the release of the mature form of cathepsin X from microglial cells and reduced its intracellular localization in lysosomes, which was determined by confocal microscopy. IL-4 and IL-13 had no effect on cathepsin X expression pattern and localization. In the last part of the master's thesis, we also evaluated the influence of the irreversible cathepsin X inhibitor, AMS36, on the polarization of microglia. We showed that AMS36 prevented the polarization of microglia into the M1 phenotype by inhibiting cathepsin X after stimulation with LPS and IFN-γ, as is also indicated by the reduction of the pro-inflammatory factor NO, whereas stimulation of BV2 cells with IL-4 and IL- 13 in the presence of AMS36 enhanced polarization of microglia into the M2 phenotype, which was demonstrated by increased arginase-1 activity and the expression of the marker CD206, which are characteristic of M2 polarized microglia. The obtained results suggest that cathepsin X plays an important role in the polarization of microglia into the pro-inflammatory M1 and the anti-inflammatory M2 phenotype; as such, it represents a potential therapeutic target for the prevention and treatment of neurodegenerative diseases associated with excessive inflammation.
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