Effect of thapsigargin-induced endoplasmic reticulum stress on immune function of regulatory T cells

Objective To investigate the effect of endoplasmic reticulum stress (ERS) induced by thapsigargin (TG) on the immune function and ERS-related signaling pathways in splenic regulatory T cells (Tregs) of mice. Methods Magnetic activated cell sorting (MACS) kit was used to segregate Tregs in the spleens of BALB/c mice, and the purity of Tregs was determined by flow cytometry. Tregs were cultured with or without TG stimulation (cultured with 0.1μmol/L TG for 0, 6, 12 and 48 hours, or cultured with TG for 12 hours at different concentrations of 0, 0.05, 0.1 and 0.2μmol/L, respectively), n=4. The time and dose-effect relationships of the expression of forkhead or winged helix transcription factor (Foxp3) and cytotoxic T cell-associated antigen 4 (CTLA-4) in Tregs were measured by flow cytometry. After 12-h stimulation with 0.1μmol/L TG, the expressions of ERS-related markers 78kD glucose-regulated protein (GRP78), eukaryotic translation initiation factor 2α (eIF2α), P-eIF2α and activating transcription factor 4 (ATF4) were analyzed by Western blotting. Tregs were cultured or co-cultured with effector T cells (Teffs), the proliferative activity of Teffs were measured by flow cytometry. Enzyme linked immunosorbent assay (ELISA) was used to determine the cytokine levels including interleukin (IL)-10 and transforming growth factor (TGF)-β in Tregs and the levels of IL-4, interferon (IFN)-γ and IL-2 in co-culture supernatants. Results The purity of Treg was 91.0%. Compared to the control group, the expression of Foxp3 was obviously up-regulated after TG (0.1μmol/L) stimulation for 12 and 24 hours (P0.05). CTLA-4 expression was not significantly changed in each time groups and dose groups (P>0.05). After 12h stimulation with 0.1μmol/L TG, the expression or activation of ERS signaling pathway-related molecules such as GRP78, P-eIF2α and ATF4 were significantly up-regulated in Tregs (P