Microglia, the resident immune cells of the central nervous system (CNS), play a crucial role in maintaining brain homeostasis and immune response. These cells can be activated in response to various signals and possess a remarkable ability to adopt different phenotypes: the classic, M1 phenotype, or the alternative, M2 phenotype. The M1 polarized microglia is primarily associated with immune defense and the release of pro-inflammatory cytokines, contributing to neuroinflammation and tissue damage. The M2 polarized microglia is involved in tissue repair and trophic support, secreting anti-inflammatory factors. In addition to pro-inflammatory factors, M1 polarized microglia secretes an increased amount of lysosomal peptidases, including cysteine cathepsins. The latter are implicated in various pathological disorders, including neurodegeneration. Therefore, the regulation of their activity is essential for maintaining cellular homeostasis and preventing adverse outcomes. In our work, we studied the effect of autophagy regulation on the activity of cysteine cathepsins in microglia, focusing on cathepsins S and X. We set up a model for activated microglia using BV2 cell culture. First, we showed that TNF-a and LPS activate BV2 cells, whereas LPS induces a shift towards the M1 phenotype, which was confirmed by an increased amount of released NO. Moreover, we demonstrated that stimulation of BV2 cells with TNF-a increases the intracellular activity of cathepsin X, while it does not affect cathepsin S. In contrast, LPS has shown a slight decrease in the activity of both cathepsins. Furthermore, we pretreated BV2 cells with autophagy modulators. We demonstrated that stimulation of autophagy causes a slight decrease in the secretion of NO in activated BV2 cells, indicating the reduced shift towards the M1 phenotype. Meanwhile, the inhibition of autophagy did not affect microglia polarization. BV2 cells pretreated with autophagy inducers also showed increased intracellular activity of cathepsins S and X, which was demonstrated by measuring enzyme kinetics in cell lysates. Inhibition of autophagy, however, resulted in reduced activity of cysteine cathepsins in stimulated BV2 cells, which may be the result of increased secretion from the cell. The results have shown that the activation of autophagy in microglia reduces the polarization towards inflammatory phenotype, and affects the activity of cathepsins S and X. Thus, the regulation of autophagy represents a potential therapeutic target for treating neurodegenerative disorders associated with neuroinflammation.
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