USP15-modified ADMSCs-Exo alleviates chondrocyte damage and effectively relieved osteoarthritis by inducing M2 polarization of macrophages through deubiquitinating FOXC1.

BACKGROUND: The damage to chondrocytes and inflammatory responses are considered the key factors in the pathogenesis of osteoarthritis (OA). Ubiquitin-specific protease 15 (USP15) has been shown to be involved in OA. This study aimed to explore the mechanism of USP15-modified adipose-derived mesenchymal stem cells (ADMSCs) exosome (Exo) in alleviating OA. METHODS: ADMSC-Exo with USP15 overexpression was isolated by magnetic beads method, and the Exo marker proteins were identified by western blot assay. M1 and M2 phenotypic markers of THP1-M0 cells were analyzed by flow cytometry. ELISA was used to detect the expression of inflammatory factors in cells. CCK-8, EdU, Transwell, and flow cytometry were used to detect the cell activity, proliferation, apoptosis and migration ability. The interaction between forkhead box C1 (FOXC1) and USP15 was verified by Glutathione-S-transferase (GST) pull-down and Co-immunoprecipitation (Co-IP) experiments. The stability of FOXC1 was measured by cycloheximide (CHX), and its ubiquitination level was analyzed by exogenous ubiquitination assay. RESULTS: The Exos from ADMSCs overexpressing USP15 (oe-USP15/Exos) were successfully isolated. It was confirmed that oe-USP15/Exo inhibited the M1 polarization of THP1-M0 cells caused by lipopolysaccharide (LPS) but induced the M2 polarization and the release of inflammatory inhibitory factors. Meanwhile, the damage of chondrocytes caused by LPS was also prevented by oe-USP15/Exo. Besides, USP15 was validated to exert a deubiquitination effect by binding to FOXC1 and positively regulate FOXC1 expression. And the effects of oe-USP15/Exo were abolished after FOXC1 silencing. CONCLUSION: USP15-modified ADMSC-derived Exos facilitated M2 polarization of macrophages and improved chondrocyte injury by deubiquitination of FOXC1.