Interleukin 2 (il 2)

Naïve CD4 + T cells were isolated from the spleens of male C57BL/6J mice using Naïve CD4 + T cells isolation kit (Miltenyi, Germany) (Au-Flaherty et al. 2015 ; Duan et al. 2019 (link)) and then cultured in 1640 medium containing 10% fetal bovine serum with plate-bound anti-CD3 (2 μg/mL, #14-0031-86, eBioscience, USA) and anti-CD28 (1 μg/mL, #14-0281-85, eBioscience, USA) antibodies for 4 days. IL-2 (200 U/mL, Cusabio, China) and TGF-β (0.5 ng/mL, Cusabio, China) were added to the culture medium for Treg cell differentiation; while IL-6 (10 ng/mL, Cusabio, China), IL-23 (10 ng/mL, Sinobiological, China) and TGF-β (2.5 ng/mL, Cusabio, China) were added to the culture medium for Th17 cell differentiation. After replacement of culture medium, cells were cultured with 200 µM PA and/or 100 nM exenatide in the presence or absence of 10 µM Foxrkhead box protein O1 (FoxO1) inhibitor (AS1842856, MedChemExpress, USA) for 24 h.

Concentrations of IFN-γ, IL-2, and KYN (all from CUSABIO) were analyzed by ELISA, according to the manufacturers’ instructions. Absorbance at 450 nm was measured on a Molecular Devices Multifilter F5 plate reader.