毒胡萝卜素诱导内质网应激对小鼠调节性T细胞免疫功能的影响

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

目的探讨内质网应激(ERS)特异性诱导剂毒胡萝卜素(TG)体外刺激对小鼠调节性T细胞(Treg)免疫功能及ERS相关信号通路的影响。方法采用免疫磁珠分离法分选BALB/c小鼠脾脏Treg,流式细胞术检测其分选纯度。采用流式细胞术检测Treg叉头翼状螺旋转录因子(Foxp3)及细胞毒性T细胞相关抗原4(CTLA-4)表达的时间及剂量效应关系。Western blotting检测细胞ERS信号通路相关分子葡萄糖调节蛋白78(GRP78)、真核细胞转录起始因子2α(e IF2α)、磷酸化e IF2α(p-e IF2α)、转录激活因子4(ATF4)表达/活化水平。将Treg单独或与效应T细胞(Teff)共培养,采用流式细胞术检测Teff的增殖活性,ELISA法检测Treg白细胞介素(IL)-10和转化生长因子(TGF)-β生成以及共培养上清中IL-2、IL-4、干扰素(IFN)-γ水平。结果磁珠分选Treg的纯度为91.0%。依据Treg Foxp3及CTLA-4表达的时效-量效关系,选定TG 0.1μmol/L和12h为实验刺激浓度和时间。0.1μmol/L TG刺激12h后,Treg中ERS信号通路相关分子GRP78、磷酸化e IF2α、ATF4等表达/活化水平明显上调,差异均有统计学意义(P<0.05)。与对照组相比,TG刺激组Treg IL-10和TGF-β分泌量明显增加,其对Teff增殖活性抑制能力明显增强,与Teff共培养上清中IL-4/IFN-γ比值明显升高,IL-2水平明显下降,差异均有统计学意义(P<0.05)。结论 TG能够有效地活化Treg中ERS相关信号通路,进而显著增强Treg介导的免疫抑制反应。

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 78 k D glucose-regulated protein（GRP78）, eukaryotic translation initiation factor 2α（e IF2α）, P-e IF2α 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. ResultsThe 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（P〈0.05）. In a dose-dependent response, the expression of Foxp3 was markedly enhanced in treatment with 0.1 and 0.2μmol/L TG（P〈0.05）. However, there was no significant difference in Foxp3 expression between 12 h and 24 h groups as well with 0.1 and 0.2μmol/L groups（P〉0.05）. CTLA-4 expression was not significantly changed in each time groups and dose groups（P〉0.05）. After 12 h stimulation with 0.1μmol/L TG, the expression or activation of ERS signaling pathway-related molecules such as GRP78, P-e IF2α and ATF4 were significantly up-regulated in Tregs（P〈0.05）. In addition, compared with control group, the levels of IL-10 and TGF-β significantly elevated in TG-stimulated Tregs（P〈0.05）, and inhibitory effect of Tregs on Teffs obviously enhanced（P〈0.05）, the ratio of IL-4/IFN-γ increased（P〈0.05）, and IL-2 release markedly decreased in the co-culture supernatants with Teffs（P〈0.05）. Conclusion TG stimulation can obviously activate the ERS-related signal transduction pathway of Tregs, thereby contributing to enhancement of Treg-mediated immunosuppressive response.