Fig. 2.

SSc fibroblast-derived exosomes upregulate surface marker expression characteristic of the SSc macrophage immunophenotype

Monocyte-derived human macrophages were stimulated for 48 h with 2 x 10⁹ exosomes purified from CM of healthy control or SSc patient fibroblasts that had been activated with TGF-beta for 24 h prior to harvest. Following incubation, macrophages were washed, and surface marker expression was analysed using flow cytometry. (A) Flow cytometry gating strategy of CD1a^-CD163⁺CD206⁺ macrophage selection. (B–E) Analysis of CD163, CD206, HLA-DR and CD16 macrophage surface marker expression. Surface levels were quantified using multi-colour flow cytometry and are presented in mean fluorescence intensity (MFI) units. *P < 0.05, **P < 0.01, macrophages treated with SSc patient fibroblast-derived exosomes vs macrophages treated with healthy donor fibroblast-derived exosomes. Data are representative of results obtained from analysis of 4 healthy donors and 5 SSc patients. Three separate experiments were performed for comparison. Error bars represent s.d.