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Quantitative Analysis of Mitochondrial Damage in T Lymphocytes from Patients with Autoimmune Diseases and Evaluation of Its Clinical Value
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摘要:
目的 评估自身免疫性疾病(autoimmune disease，AID)患者外周血T淋巴细胞线粒体损伤情况，以期为AID病因学研究提供思路。 方法 回顾性收集2023年3—4月北京协和医院诊治的AID患者及同期体检健康人群的临床资料，并依据外周血辅助性T淋巴细胞(T helper cell, Th)百分比/杀伤性T淋巴细胞(T cytotoxic cell, Tc)百分比比值，将AID患者分为免疫功能低下亚组和免疫功能正常亚组。采用流式细胞术评估AID患者T淋巴细胞线粒体损伤情况，以线粒体膜电位偏低细胞百分比(mitochondrial membrane potential low percentage，MMP-low%)表示线粒体功能障碍程度并分析其与AID的相关性。 结果 共入选符合纳入与排除标准的AID患者70例，健康人群20名。AID患者中，免疫功能低下者20例(Th/Tc比值＜0.70)，免疫功能正常者50例(Th/Tc比值≥0.70)；系统性红斑狼疮(systemic lupus erythematosus，SLE)患者33例，类风湿关节炎(rheumatoid arthritis，RA)患者19例，干燥综合征(Sjögren syndrome，SS)患者18例。SLE、RA、SS患者CD3+T淋巴细胞线粒体膜电位偏低细胞百分比(T MMP-low%)、CD3+CD4+T淋巴细胞线粒体膜电位偏低细胞百分比(Th MMP-low%)、CD3+CD8+T淋巴细胞线粒体膜电位偏低细胞百分比(Tc MMP-low%)均低于健康人群(P均＜0.05)。AID患者中，免疫功能低下亚组和免疫功能正常亚组T MMP-low%、Th MMP-low%、Tc MMP-low%均低于健康人群(P均＜0.05)；相较于免疫功能正常亚组，免疫功能低下亚组T MMP-low%、Th MMP-low%、Tc MMP-low%均呈降低趋势，但差异无统计学意义(P均＞0.05)。Spearman相关性分析显示，线粒体损伤指标中，仅发现Th MMP-low%/Tc MMP-low%比值与AID患者免疫功能(Th/Tc比值)具有相关性(r=-0.39，P=0.001)。受试者工作特征曲线显示，T MMP-low%、Tc MMP-low%、Th MMP-low%在AID的识别中均具有良好效能，曲线下面积分别为0.83(95% CI: 0.74~0.92)、0.82(95% CI: 0.73~0.92)、0.77(95% CI: 0.67~0.88)。 结论 AID患者存在一定程度的外周血T淋巴细胞线粒体损伤, 尤其在免疫功能低下者中此现象更显著。T淋巴细胞线粒体损伤相关指标或可作为AID辅助诊断的分子标志物。 Abstract:Objective To evaluate the mitochondrial damage of peripheral blood T lymphocytes in patients with autoimmune diseases (AID) and provide insights for etiological research. Methods Clinical data were retrospectively collected from the AID patients treated at the Peking Union Medical College Hospital from March 2023 to April 2023 and from a population that was physically healthy during the same period. Based on the ratio of peripheral blood helper T cells (Th) to cytotoxic T cells (Tc), the AID patients were divided into an immunodeficiency subgroup and an immunocompetent subgroup. Flow cytometry was used to assess the mitochondrial damage of T lymphocytes in the AID patients, with the percentage of cells showing low mitochondrial membrane potential (MMP-low%) as an indicator of mitochondrial dysfunction, and its correlation with AID was analyzed. Results A total of 70 AID patients and 20 healthy individuals who met the inclusion and exclusion criteria were included. Among the AID patients, there were 20 immunodeficient cases (Th/Tc ratio < 0.70) and 50 immunocompetent cases (Th/Tc ratio ≥0.70); 33 patients had systemic lupus erythematosus (SLE), 19 had rheumatoid arthritis (RA), and 18 had Sjögren syndrome (SS). The percentage of CD3+ T lymphocytes showing low mitochondrial membrane potential (T MMP-low%), CD3+CD4+ T lymphocytes showing low mitochondrial membrane potential (Th MMP-low%), and CD3+CD8+ T lymphocytes showing low mitochondrial membrane potential (Tc MMP-low%) in SLE, RA, and SS patients were all lower than those in healthy individuals (all P < 0.05). In the AID patients, the percentages of T MMP-low%, Th MMP-low%, and Tc MMP-low% in both the immunodeficient subgroup and immunocompetent subgroup were lower than those in healthy individuals (P < 0.05). Compared to the immunocompetent subgroup, the immunodeficient subgroup showed a decreasing trend in the percentages of T MMP-low%, Th MMP-low%, and Tc MMP-low%, but the differences were not statistically significant (all P > 0.05). Spearman correlation analysis showed that among the mitochondrial damage indicators, only the Th MMP-low%/Tc MMP-low% ratio was correlated with the immune function (Th/Tc ratio) of the AID patients (r=-0.39, P=0.001). The receiver operating characteristic curve showed that Tc MMP-low%, Tc MMP-low%, and Th MMP-low% all had good performance in identifying AID, with respective areas under the curve of 0.83(95% CI: 0.74-0.92), 0.82(95% CI: 0.73-0.92), and 0.77(95% CI: 0.67-0.88), respectively. Conclusions Peripheral blood T lymphocytes in AID patients have varying degrees of mitochondrial damage, especially in immunodeficient individuals. Mitochondrial damage-related indicators of T lymphocytes may serve as molecular markers for auxiliary diagnosis of AID. -
Key words:
- autoimmune disease /
- T lymphocytes /
- mitochondrial damage /
- immunocompromised
作者贡献：吴子燕负责论文撰写；李昊隆负责实验研究；奉福泰、徐洪琳负责数据分析；张蜀澜、李永哲负责研究设计。利益冲突：所有作者均声明不存在利益冲突 -
图 1 AID患者与健康人群CD3+T淋巴细胞、CD3+CD4+T淋巴细胞、CD3+CD8+T淋巴细胞线粒体质量比较
T MMP-low%、Th MMP-low%、Tc MMP-low%、AID、SLE、RA、SS：同表 1
图 2 AID患者Th MMP-low%/Tc MMP-low%比值与Th/Tc比值的相关性
Th MMP-low%、Tc MMP-low%、AID：同表 1；Th/Tc：Th百分比与Tc百分比的比值
表 1 AID患者与健康人群外周血T淋巴细胞及其亚群线粒体质量比较[M(P25, P75), %]
组别 T MMP-low% Th MMP-low% Tc MMP-low% SLE患者(n=33) 20.91(11.96，28.17)* 14.52(10.18，18.75)* 22.83(13.49，32.95)* RA患者(n=19) 25.51(21.59，36.07)*# 18.20(12.31，27.46)* 35.64(24.12，41.97)*# SS患者(n=18) 19.92(11.09，34.50)* 19.09(4.78，30.85)* 26.56(11.99，43.09)* 健康人群(n=20) 38.24(31.35，48.54) 25.05(23.47，33.32) 48.41(35.59，65.41) P值 ＜0.0001 0.0006 ＜0.0001 AID：自身免疫性疾病；SLE：系统性红斑狼疮；RA：类风湿关节炎；SS：干燥综合征；T MMP-low%：CD3+T淋巴细胞线粒体膜电位偏低细胞百分比；Th MMP-low%：CD3+CD4+T淋巴细胞线粒体膜电位偏低细胞百分比；Tc MMP-low%：CD3+CD8+T淋巴细胞线粒体膜电位偏低细胞百分比；与健康人群比较，* P＜0.05；与SLE患者比较，# P＜0.05 
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表 2 不同免疫状态的AID患者与健康人群T淋巴细胞及其亚群线粒体质量比较[M(P25, P75)]
组别 T MMP-low%(%) Th MMP-low%(%) Tc MMP-low%(%) Th MMP-low%/Tc MMP-low%比值 AID组(n=70) 免疫功能低下亚组(n=20) 17.15 (10.71，31.16)* 15.36 (10.62，25.46)* 18.07 (10.99，35.85)* 0.86(0.59，1.27) 免疫功能正常亚组(n=50) 23.88 (17.16，32.52)* 16.27 (10.30，22.67)* 30.61 (18.15，38.85)* 0.56(0.38，0.85) 健康人群(n=20) 38.24 (31.35，48.54) 25.05(23.47，33.32) 48.41(35.59，65.41) 0.60(0.43，0.72) P值 0.0002 0.005 0.0001 0.056 T MMP-low%、Th MMP-low%、Tc MMP-low%、AID：同表 1；与健康人群比较，* P＜0.05
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表 3 T MMP-low%、Tc MMP-low%、Th MMP-low% 识别AID的临床价值
诊断指标 T MMP-low% Th MMP-low% Tc MMP-low% AUC 0.83 0.77 0.82 最佳临界值(%) 24.07 21.95 42.57 灵敏度(%) 100 85.00 70.00 特异度(%) 57.14 72.86 82.86 阴性预测值(%) 100 94.44 90.63 阳性预测值(%) 40.00 47.22 53.85 假阳性率(%) 42.86 27.14 17.14 假阴性率(%) 0 15.00 30.00 准确度(%) 66.67 75.56 80.00 Youden指数 0.57 0.58 0.53 AUC：曲线下面积；T MMP-low%、Th MMP-low%、Tc MMP-low%、AID：同表 1
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