Fig. 4: Eid3 from MSC-sEVs destabilized RORγt in Th17 cells.

A Volcano plot of the differentially expressed mRNAs in MSC-sEVs-treated Th17 cells compared with untreated control Th17 cells. The dashed horizontal line indicates a p value of 0.05. The Eid3 gene, proteasome-related genes, and the other genes are colored red, blue, and gray, respectively. B The expression level of Eid3 in MSC-sEVs-treated T cells. The protein level of Eid3 was increased in MSC-sEVs-treated T cells. A representative image from three independent immunoblots is shown. The eid3/actin ratio is shown as a bar graph. No sti: without CD3/CD28 stimulation; Sti: with CD3/CD28 stimulation. ***p ≤ 0.001. C The protein level of Eid3 is expressed in each cell lysate. The relative expression of Eid3 in MSC-sEVs was the highest, and a negligible amount of Eid3 was observed in T cells and splenocytes, meaning that Eid3 was not intrinsic to activated T cells but came from MSC-sEVs. A representative image from three independent immunoblots is shown. The Eid3/actin ratio is shown as a bar graph. hADSC: human adipose-derived stem cell. ***p ≤ 0.001; ****p ≤ 0.0001. D mRNA level of Eid3 by real-time PCR (normalized by GAPDH). MSCs and MSC-sEVs both contained Eid3 mRNA. NSC: neural stem cell; MSC: mesenchymal stem cell. ***p ≤ 0.001. E Expression of RORγt after transduction of lentiviral Eid3-ORF (GFP-tagged) into Th17 cells (5 MOI). A lentiviral control-ORF particle (GFP-tagged) was used as a control (Lenti-control, 5 MOI). The downregulation of RORγt expression was also found in the lentiviral Eid3-ORF-transduced group. F The expression level of Eid3 in MSCs, Eid3-knockdown MSCs, MSC-derived sEVs, and Eid3-knockdown MSC-derived sEVs. Knockdown of Eid3 was performed by transducing the lentiviral Eid3-shRNA construct and was confirmed by western blotting. G Expression of RORγt in naive CD4+T cells, Th17 cells, MSC-sEVs-treated Th17 cells, Eid3-knockdown MSC-sEVs-treated Th17 cells, and C646-treated Th17 cells. The expression level of RORγt was assessed by immunoprecipitation with an anti-RORγt antibody. Eid3-knockdown MSC-derived sEVs failed to reduce the expression of RORγt. H Expression of p300 and RORγt in Th17 cells and MSC-sEVs-treated Th17 cells detected by western blotting. The expression levels of both p300 and RORγt were significantly decreased in MSC-sEVs-treated Th17 cells. A representative image from three independent immunoblots is shown. The p300/GAPDH and RORγt/GAPDH ratios are shown as bar graphs. *p ≤ 0.05; **p ≤ 0.01. I Regulation of p300 and RORγt by Eid3. Immunoprecipitation was conducted using an anti-p300 antibody, and immunoblotting was conducted using anti-p300, Eid3, and RORγt antibodies. The upregulated expression of Eid3 by lentiviral transduction or delivery of Eid3 through MSC-sEVs downregulated both p300 and RORγt. J Assessment of the polyubiquitination of K63 and the acetylation of RORγt by coimmunoprecipitation. Thymocytes treated with MSC-sEVs or C646 were immunoprecipitated using an anti-RORγt antibody. Immunoblotting was conducted using anti-p300, K63 polyubiquitin, acetyl-K, and RORγt antibodies. Lysate refers to thymocyte lysate without immunoprecipitation used as a positive control. Eid3 derived from MSC-sEVs destabilized RORγt by suppressing K63 polyubiquitination and acetylation through the inhibition of p300.