Table 1_Integrative single-cell and bulk RNA-seq analyses identify CD4+ T-cell subpopulation infiltration and biomarkers of regulatory T cells involved in mediating the progression of atherosclerotic plaque.xlsx

BackgroundAtherosclerosis (AS) is a chronic inflammatory disease with a significant contributor to mortality worldwide. Regulatory T cells (Tregs) are atheroprotective. However, the potential pathways and genes associated with atherosclerotic plaque progression in Tregs remain largely unknown. Therefore, this study aimed to identify critical target genes and pathways of Tregs associated with the progression of AS.
MethodsThe gene expression data and single cell RNA-seq data of AS were downloaded from the Gene Expression Omnibus (GEO) database. Initially, we quantified CD4+ T cell proportions in non-plaque and plaque tissues using cell infiltration by estimation of RNA sequences (CIBERSORT) analysis, identifying pivotal transcription factors regulating the number of Tregs in atherosclerotic plaque. Subsequently, we identified significantly differential expressed genes of Tregs during the progression of atherosclerotic plaque and investigated the key pathways and transcription factors for these differentially expressed genes using gene ontology (GO) analysis and transcription factor enrichment analysis (TFEA), respectively. We also employed high dimensional weighted gene co-expression network analysis (hdWGCNA) and cell-cell communication analysis to elucidate the modules and cascade reaction of Tregs in the progression of AS. The key genes diagnostic potential was assessed via receiver operating characteristic (ROC) curve analysis. Finally, the target genes were validated in AS model using Ldlr−/− mice.
ResultsWe found that the proportion of Tregs significantly decreased, and Th2 cells showed a significant increase in atherosclerotic plaque compared to that in non-plaque arterial tissues. The five transcription factors (TEFC, IRF8, ZNF267, KLF2, and JUNB), identified as key targets associated with the function and the number of Tregs driving the progression of AS, primarily regulate immune response, ubiquitination, cytokine production, and T-cell differentiation pathways. ZNF267 may mainly involve in regulating ubiquitination, TGF-beta, and MAPK pathways of Tregs to regulate the function and the number of Tregs during the progress of AS. Interestingly, we found that IRF8 and ZNF267 as potential biomarkers were upregulated in circulating CD4+ T cells in patients with atherosclerotic coronary artery disease. Moreover, we also found that the changes of the function and the number of Tregs could modulate endothelial cell and smooth muscle cell functions to counteract AS through ligand–receptor pairs such as the MIF signaling pathway. Finally, we validated that two of the five transcription factors were also upregulated in mice atherosclerotic plaque through AS model using Ldlr−/− mice.
ConclusionOur results indicate that the transcription factors TEFC, IRF8, ZNF267, KLF2, and JUNB in Tregs could be potential targets for the clinical management of AS.

研究背景：动脉粥样硬化（Atherosclerosis, AS）是一种慢性炎症性疾病，是全球范围内致死的重要诱因之一。调节性T细胞（Regulatory T cells, Tregs）具有抗动脉粥样硬化的保护作用。然而，Tregs中与动脉粥样硬化斑块进展相关的潜在通路及基因仍尚未完全明确。因此，本研究旨在筛选与AS进展相关的Tregs关键靶基因及通路。
研究方法：本研究从基因表达综合数据库（Gene Expression Omnibus, GEO）下载了AS相关的基因表达数据与单细胞RNA测序（single cell RNA-seq）数据。首先，本研究通过基于RNA序列估算细胞浸润的CIBERSORT分析，量化了非斑块组织与斑块组织中CD4+ T细胞的占比，筛选出调控动脉粥样硬化斑块中Tregs数量的关键转录因子。随后，本研究筛选出动脉粥样硬化斑块进展过程中Tregs的差异表达基因，并分别通过基因本体论（gene ontology, GO）分析与转录因子富集分析（transcription factor enrichment analysis, TFEA）探究了这些差异基因的关键通路与调控转录因子。本研究还采用高维加权基因共表达网络分析（high dimensional weighted gene co-expression network analysis, hdWGCNA）与细胞间通讯分析，阐明了AS进展过程中Tregs的共表达模块与级联反应。通过受试者工作特征（receiver operating characteristic, ROC）曲线分析评估了关键基因的诊断潜力。最终，本研究在Ldlr基因敲除（Ldlr−/−）小鼠构建的AS模型中对靶基因进行了验证。
研究结果：本研究发现，与非斑块动脉组织相比，动脉粥样硬化斑块中Tregs的占比显著降低，而Th2细胞的占比则显著升高。本研究筛选出5个与驱动AS进展的Tregs功能及数量相关的关键转录因子（TEFC、IRF8、ZNF267、KLF2及JUNB），这些因子主要调控免疫应答、泛素化、细胞因子产生以及T细胞分化通路。ZNF267可能主要通过调控Tregs的泛素化、转化生长因子-β（TGF-β）及丝裂原活化蛋白激酶（MAPK）通路，进而在AS进展过程中调控Tregs的功能与数量。值得注意的是，本研究发现动脉粥样硬化性冠心病患者外周循环CD4+ T细胞中，IRF8与ZNF267作为潜在生物标志物呈上调表达。此外，本研究还发现Tregs的功能与数量变化可通过巨噬细胞迁移抑制因子（MIF）信号通路等配体-受体对，调控内皮细胞与平滑肌细胞功能，从而对抗AS的发生发展。最终，本研究通过Ldlr−/−小鼠构建的AS模型验证发现，5个转录因子中有2个在小鼠动脉粥样硬化斑块中同样呈上调表达。
研究结论：本研究结果表明，Tregs中的转录因子TEFC、IRF8、ZNF267、KLF2及JUNB可作为AS临床管理的潜在靶标。