Figure 5.

Clathrin-dependent endocytosis of circulating exosomes affects MSC therapy in colitis mice subjected to restraint stress. (A) MSCs were pretreated with Pitstop2, a novel clathrin inhibitor, for 15 min. Then MSCs were exposed to PKH67-labeled plasma exosomes for 2 h. The effects of Pitstop2 on the uptake of plasma exosomes by MSCs were determined using cytometric analysis. Pitstop2-treated MSCs were infused into colitis mice subjected to restraint stress 3 days after DSS treatment. (B) Body weight loss and (C) disease activity index (DAI) were monitored daily during the entire experiment (n = 3 for DSS and DSS+MSC group; n = 4 for DSS+Stress+MSC and DSS+Stress+Pitstop2-MSC group) (*P< 0.05, **P< 0.01, DSS+Stress+Pitstop2-MSC versus DSS+Stress+MSC). At 10 days after DSS induction, mice were euthanized; (D) colon length was recorded, and (E) colonic epithelial/crypt destruction and inflammatory cell infiltration were visualized using HE staining (yellow arrows: goblet cells; green arrows: destruction of epithelial layer; red arrows: inflammatory cell infiltration). (F) Colonic damage was analyzed using histological activity index (HAI). (G, H) Splenocytes were harvested and the ratios of CD4⁺ IL17A⁺ Th17 cells and CD4⁺ CD25⁺ FOXP3⁺ Tregs among CD4⁺ T cells in the spleen were determined using flow cytometry. All results were verified in three independent experiments. A two-tailed Student’s t test (A) and one-way ANOVA with Bonferroni’s comparison test (B, C, D, F, G and H) were used for statistical analysis. Error bars represent the mean ± s.d. ***P < 0.005; **P < 0.01; *P < 0.05