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. 2024 May 16;22:255. doi: 10.1186/s12951-024-02509-1

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© The Author(s) 2024, corrected publication 2024

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Fig. 5 — Senescent ASC-EVs induce a pro-inflammatory phenotype in synovial macrophages and alter the phenotype of synovial fibroblasts. Primary human synoviocytes were pretreated with LPS or IFNγ and TNFα (I/T; 20 ng/mL and 10 ng/mL, respectively) for 24 h and EVs (Medium dose: 500 ng) from healthy or senescent ASCs (H-EV or S-EV) were added for 48 days. (A) Representative gating strategy of macrophages (CD11b⁺ cells in the CD45⁺ cells) in the synovial cell population and picture of the synovial cells at P0. (B) The average ratio of CD163⁺/CD80⁺ and CD163⁺/CD86⁺ macrophages after LPS stimulation (n = 12). (C) The average ratio of CD163⁺/CD80⁺ and CD163⁺/CD86⁺ macrophages after I/T stimulation (n = 10). (D) Expression of macrophage markers as expressed as fold change (n = 10). (E) Quantification of cytokines in the supernatants of I/T-stimulated macrophages by ELISA (n = 8). (F) Representative gating strategy of synovial fibroblasts (CD73⁺ and CD45⁻ cells) in the synovial cell population and picture of the synovial cells at P4. (G) Expression of markers in fibroblasts as expressed as fold change (n = 9). Data are shown as mean ± SEM. Statistical analysis used the one-sample Wilcoxon test comparing the treated sample to the IL1β control (*p < 0.05, **p < 0.01) or the Mann-Whitney test for comparing H-EV and S-EV samples and in panel F (# p < 0.05)