Microglia are resident immune cells of the central nervous system that continuously monitor environment and become activated upon detecting pathological stimuli. In their activated state, microglia can polarize into a pro-inflammatory M1 or an anti-inflammatory M2 phenotype. M1 activation, induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ), is characterized by the release of inflammatory mediators such as tumor necrosis factor-α (TNF-α) and nitric oxide (NO), as well as increased secretion of enzymes including cysteine cathepsins. These lysosomal enzymes play important roles in immune signaling, extracellular matrix degradation, and neurotoxic processes. In contrast, polarization towards the M2 phenotype, stimulated by interleukin (IL)-4 and IL-13, results in increased arginase-1 activity, promotes anti-inflammatory mechanisms, regeneration and maintenance of homeostasis. The aim of this master’s thesis was to investigate the role of cysteine cathepsins, namely cathepsins B, S, and X in microglial polarization and to evaluate the effect of cysteine cathepsin inhibitors on microglia polarization and the regulation of the inflammatory response. The murine BV2 microglial cell line was used and polarized into M1 and M2 phenotypes. Results showed that LPS and IFN-γ induced morphological changes and increased NO release, which is characteristic of the M1 phenotype, while IL-4 and IL-13 increased arginase-1 activity without affecting cell morphology, indicating polarization into the M2 phenotype. Assessment of enzymatic activity revealed that LPS, and to a lesser extent IFN-γ, decreased intracellular activity of cathepsins B, S, and X, whereas IL-4 and IL-13 had no significant effect. Evaluation of the effects of cysteine peptidase inhibitors demonstrated differential impacts on the expression and release of inflammatory mediators and on enzymatic activity. The most effective in reducing levels of inflammatory factors such as NO and TNF-α, as well as regulating the proteolytic activity of cysteine cathepsins, were the broad-spectrum cysteine cathepsin inhibitors and inhibitors of cathepsins B and X, while a lesser effect was observed by the inhibitors of cathepsins L and S. These findings highlight the importance of mutual regulation among cysteine cathepsins in inflammatory processes and suggest that cathepsin inhibition may represent a potential strategy for modulating neuroinflammation.
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