系统性红斑狼疮患者肠道菌群分析与CD4^(+)T细胞亚群关系的研究

Study on the relationship between intestinal flora analysis and CD4^(+)T lymphocyte subsets in patients withsystemic lupus erythematosus

探索SLE患者的肠道菌群特征,并进一步挖掘SLE的菌群与CD4^(+)T细胞亚群和疾病活动情况的关系。方法收集96例SLE患者和96例性别和年龄匹配的健康对照(HC)的粪便样本。采用16s rRNA测序法检测肠道菌群,流式细胞仪检测外周血Th1、Th2、Th17、Treg等CD4T细胞亚群。同时记录患者的ESR、CRP、血红蛋白(Hb)、补体C3和C4等疾病活动指标,并记录每位患者的SLEDAI。利用edgeR算法进行差异丰度分析。采用Wilcoxon秩和检验比较各组间的alpha多样性指数、细菌丰度和F/B比值。采用R软件4.0.1进行统计学分析,采用Pearson相关分析评估样品中细菌属相对丰度与血清ESR、CRP、C3和C4水平、CD4^(+)T细胞亚群之间的相关性。结果与HC相比,SLE肠道菌群的丰度(ACE和Chao1)和多样性(Shannon和Simpson)指数显著降低。通过edgeR算法,发现SLE和HC之间的肠道菌群差异有统计学意义。与HC相比,SLE患者在属水平上有24种细菌差异有统计学意义(P<0.05)。相关性分析提示:CRP与粪球菌属(Coprococcus)有显著正相关性(r=0.30,P=0.014);C4与棒状杆菌(Corynebacterium)(r=0.31,P=0.013)和粪杆菌属(Faecalibacterium)(r=0.25,P=0.048)呈正相关,血红蛋白与摩根菌属(Morganella)(r=0.41,P=0.001)呈正相关;而疾病整体活动度与棒状杆菌属呈正相关关系(r=0.25,P=0.047)。B细胞(r=0.52,P=8.45×10^(-6))、Treg细胞(r=0.26,P=0.036)与挑剔真杆菌(Eubacterium eligens group)呈正相关关系,CD8+T细胞(r=0.20,P-=0.034)CD4+T(r=0.32,P=0.010)细胞、NK细胞(r=0.56,P<0.001)与棒状杆菌呈正相关关系,除此之外,Th1与大肠志贺杆菌(Escherichia Shigella)(r=0.33,P=0.008)呈正相关关系,Th2与Dielma(r=0.51,P<0.001)呈正相关。结论SLE患者肠道菌群的丰度和多样性显著降低,多种差异表达的菌与患者的CD4+T淋巴细胞亚群和疾病活动指标密切相关。

Objective To explore the characteristics of intestinal microbiota in patients with systemic lupus erythematosus(SLE),and further explore the relationship between microbiota and CD4^(+)T lymphocyte subsets and disease activity.Methods Fecal samples were collected from 96 patients with SLE,and 96 sexand age-matched healthy controls(HCs).The gut microbiota were investigated via 16s rRNA sequencing.Flow cytometry was used to detect peripheral CD4^(+)T lymphocyte subsets of Th1,Th2,Th17 and Treg cells.Indicators of disease activity such as erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),complement C3 and C4,Systemic lupus erythematosus disease activity index(SLEDAI)for each patient were recorded.Differential abundance analysis was carried out using the edgeR algorithm.The Wilcoxon rank-sum test was used to compare alpha diversity indices,bacterial abundances,and the F/B ratio between groups.R(version 4.0.1)was used for comparative statistics,and Pearson's correlation analysis was used to assess the correlations between the relative abundances of bacterial genera and serum levels of ESR,CRP,C3 and C4 in the samples.Results The alpha estimators of richness(ACE and Chao I)were significantly reduced in SLE feces samples compared with those of HCs(P<0.01).Bacterial diversity estimators,including the Shannon(P<0.01)and Simpson's(P<0.01)indices,were also significantly lower in SLE.Significant differences in gut microbiota composition between SLE and HCs were found using the edgeR algorithm.Compared with HC,24 species of bacteria were significantly different in SLE patients at the genus level(P<0.05).Moreover,there was a significant positive correlation between CRP and Coprococcus(r=0.30,P=0.014),C4 and Corynebacterium(r=0.31,P=0.013)and Faecalibacterium(r=0.25,P=0.048),Hemoglobin and Morganella(r=0.41,P=0.001),as well as SLIDA and Corynebacterium(r=0.25,P-0.047).In terms of lymphocyte subsets,there was significant positive correlation between B cells,Treg cells and Eubacterium eligens group,as well as CD8+T,CD4^(+)T,NK cells and Corynebacterium.In additional,Th1 was positively correlated with Shigella Escherichia coli(r=0.52,P=0.008),and Th2 was positively correlated with Dielma(r=0.51,P<0.001).Conclusion The abundance and diversity of intestinal flora in SLE patients were significantly reduced,and the differentially expressed bacteria were closely related to the CD4^(+)T lymphocyte subsets and disease activity indicators of patients.