Interleukin 4 (il 4)

BMDCs from the mice were extracted and cultured as previously described 26 (link). Briefly, 5×10⁵ BMDCs were stimulated using lipopolysaccharides (LPS) (1 μg/mL, Sigma-Aldrich) and bile acids (10 µM, Sigma-Aldrich) or INT-747(100 µM, MedChem Express, USA) or INT-777 (100 µM, MedChem Express) for 24 h.
Mice CD4+ T cells were isolated using mouse CD4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cocultured with BMDCs pretreated with LCA or INT-777 at a ratio of 5:1 (CD4+ T cells: BMDCs) for 5 days.
Human CD14+ monocytes in PBMCs were isolated using Human CD14 microbeads (Miltenyi Biotec). CD14+ monocytes were incubated with 50 ng/mL IL-4 (AcroBiosystems, Newark, NJ, USA) and 100 ng/mL GM-CSF (AcroBiosystems) to induce DC maturation. 7 days later, 5×10⁵ MD-DCs were treated with 100 ng/ml LPS and 10 µM LCA or 100 µM INT-777 for 24 h.

CD14 and CD4 mAb-conjugated magnetic microbeads (Miltenyi Biotec, Germany) were used to isolate CD14⁺ monocytes and CD4⁺ T cells from VKH and the healthy subjects (purity >90%). The RPMI 1640 medium with 10% fetal bovine serum and 100 U/mL penicillin/streptomycin was used to culture the cells. To generate the monocyte-derived DCs, CD14⁺ monocytes were stimulated with 100 ng/ml granulocyte macrophage colony stimulating factor (GM-CSF, Acro Biosystems, Newark, DE, USA) and 50 ng/ml interleukin-4 (IL-4, Acro Biosystems, Newark, DE, USA) for 6 days. Half of the medium was then refreshed at the 4^th day. Mature DCs were subsequently generated by stimulation with 100 ng/ml lipopolysaccharide (LPS) for 24 hours. The co-culture experiment was performed as previously described^{32 (link)}. Briefly, the DCs were co-cultured with CD4⁺ T cells (DC: T cell ratio = 1:5) with or without DAC for 5 days. The flow cytometry tests were performed to detect the intracellular IFN-γ and IL-17. The culture supernatants were used for ELISA assay.

RAW264.7 macrophages were seeded at a density of 1.5 × 10⁴ cells per well in a 6-well plate and cultured for 24 h. After cells adhered to the wall, they were washed once with PBS and induced as follows: (a) Osteoclast differentiation: cells were induced with 100 ng/mL RANKL (Sino Biological, China) and 30 ng/mL M-CSF (Sino Biological, China) for 6 days with medium changed every 2 days; (b) M1 macrophage polarization: cells were induced with 100 ng/mL LPS (Solarbio Science & Technology, China) and 20 ng/mL IFN-γ (Sino Biological, China) for 24 h; (c) M2 macrophage polarization: cells were induced with 20 ng/mL IL-4 (ACRO Biosystems, USA) for 48 h. All treatments were performed in complete medium containing 10% exosome-free FBS. To obtain exosome-free FBS, FBS was ultracentrifuged at 130, 000×g for 4 h, at 4 °C in advance.