Wang Na, Yuan Jiang, Karim Md Rezaul, Zhong Ping, Sun Yan-Peng, Zhang Hong-Yan, Wang Yun-Fu
Department of Neurology, Taihe Hospital of Hubei University of Medicine, Shiyan, China.
Biomedical Research Institute of Hubei University of Medicine, Shiyan, China.
Front Neurol. 2020 Apr 7;11:238. doi: 10.3389/fneur.2020.00238. eCollection 2020.
This study was conducted to determine whether regulatory T cells (CD4CD25T, Tregs) show abnormal mitophagy as well as the function of Tregs in patients with myasthenia gravis (MG). CD4T cells and CD4CD25Treg cells were obtained from 15 patients with MG (MG group) and 15 controls (N group). Tregs from the MG group were subjected to rapamycin-induced culture for 48 h (Rapa group) and 3-methyladenine-induced culture for 48 h (3-MA group). The levels of mitophagy in Tregs were then observed through electron and confocal microscopy. Expression of the autophagy-related protein LC3-II was detected by western blotting, and mitochondrial function in each group was evaluated by flow cytometry. Inhibition of Treg cell proliferation was detected by flow cytometry. Mitophagy in the MG group was lower than that in the N group; it was higher in the Rapa group compared to that in the MG group and lowered in the 3-MA group than in the MG group. Expression of the autophagy-related protein LC3-II was lower in the MG group than in the N group, higher in the Rapa group than in the MG group, and lower in the 3-MA group than in the MG group. The mitochondrial membrane potential was lower in the MG group compared to that in the N group; it was higher in the Rapa group than in the MG group and lowered in the 3-MA group than in the MG group. Inhibition of Treg proliferation was lower in the MG group than in the N group; it was higher in the Rapa group than in the MG group and lowered in the 3-MA group than in the MG group. The decreased mitochondrial membrane potential and mitophagy in Tregs in the MG group may be related to a decreased inhibition of Treg proliferation. The mitochondrial membrane potential was increased after adding the autophagy agent Rapa to enhance mitophagy, and the proliferation inhibition function of Tregs was also enhanced. The autophagy agent 3-MA down-regulated mitophagy, which decreased the mitochondrial membrane potential and inhibitory effect of Tregs. These results reveal the possible cellular immune mechanism of Treg dysfunction in MG.
本研究旨在确定重症肌无力（MG）患者的调节性T细胞（CD4⁺CD25⁺T，Tregs）是否表现出异常的线粒体自噬以及Tregs的功能。从15例MG患者（MG组）和15例对照（N组）中获取CD4⁺T细胞和CD4⁺CD25⁺Treg细胞。MG组的Tregs进行雷帕霉素诱导培养48小时（雷帕霉素组）和3-甲基腺嘌呤诱导培养48小时（3-MA组）。然后通过电子显微镜和共聚焦显微镜观察Tregs中线粒体自噬的水平。通过蛋白质免疫印迹法检测自噬相关蛋白LC3-II的表达，并通过流式细胞术评估每组的线粒体功能。通过流式细胞术检测Treg细胞增殖的抑制情况。MG组的线粒体自噬低于N组；与MG组相比，雷帕霉素组的线粒体自噬更高，而3-MA组低于MG组。MG组中自噬相关蛋白LC3-II的表达低于N组，雷帕霉素组高于MG组，3-MA组低于MG组。MG组的线粒体膜电位低于N组；与MG组相比，雷帕霉素组的线粒体膜电位更高，而3-MA组低于MG组。MG组对Treg增殖的抑制低于N组；与MG组相比，雷帕霉素组的抑制更高，而3-MA组低于MG组。MG组Tregs中线粒体膜电位降低和线粒体自噬减少可能与Treg增殖抑制降低有关。添加自噬剂雷帕霉素以增强线粒体自噬后，线粒体膜电位升高，Tregs的增殖抑制功能也增强。自噬剂3-MA下调线粒体自噬，降低了线粒体膜电位和Tregs的抑制作用。这些结果揭示了MG中Treg功能障碍可能的细胞免疫机制。
