基于免疫细胞微环境分析不同心肌病的异质性

Identification of heterogeneity in different cardiomyopathies based on immune cell microenvironment

摘要:

目的通过心脏免疫细胞的单细胞转录组测序（single cell RNA-sequencing, scRNA-seq）和批量转录组测序（bulk RNA-sequencing, RNA-seq）数据联合分析，探究不同类型心肌病的细胞异质性和免疫细胞微环境变化，为心脏免疫治疗提供一定的依据。 方法从基因表达综合数据库（gene expression omnibus, GEO）获取人类扩张型心肌病（dilated cardiomyopathy, DCM）、肥厚型心肌病（hypertrophic cardiomyopathy, HCM）、缺血性心肌病（ischemic cardiomyopathy, ICM）和健康对照心脏样本的免疫细胞scRNA-seq和RNA-seq数据，使用Seurat、Monocle和clusterProfiler等R包进行单细胞数据分析： 数据整合、差异表达基因（differentially expressed genes, DEGs）筛选、细胞类型鉴定、通路富集和拟时序分析等，利用CIBERSORT进行RNA-seq数据的免疫浸润分析。 结果整合了健康对照和3种心肌病的心脏免疫细胞scRNA-seq数据，优化筛选后获得19 348个细胞，并分为7种免疫细胞类型。分析发现自然杀伤细胞（natural killer cell, NK）在ICM中占比较高，说明NK细胞与心脏炎症高度相关。通过对胸腺依赖淋巴细胞（thymus dependent lymphocyte, T）进行亚型鉴定和拟时序分析，识别了10种T细胞亚型，发现应激反应状态的T细胞仅在ICM中形成拟时序轨迹，并进一步筛选到12个在T细胞各亚型间差异表达并且随拟时序变化的特异性的基因，主要在炎症和免疫调节过程中发挥作用。RNA-seq数据的免疫浸润分析发现，单核细胞（monocyte, MO）在DCM和ICM患者中浸润率较低，CD4+ T细胞和M2巨噬细胞（macrophage, Mφ）在HCM患者中浸润率较低，肥大细胞（mast cell, MC）则在HCM患者中浸润率较高。 结论发现了3种心肌病的免疫细胞异质性和免疫微环境变化，为探索不同心肌病的机制提供了一定的参考依据。

Abstract:

ObjectiveTo investigate the cell heterogeneity of different types and the changes of immune cell microenvironment of cardiomyopathy by jointly analyzing single cell RNA-sequencing(scRNA-seq) and bulk RNA-sequencing(RNA-seq) so as to provide some basis for cardiac immunotherapy. MethodsHuman immune cell scRNA-seq and RNA-seq data of dilated cardiomyopathy(DCM), hypertrophic cardiomyopathy(HCM), ischemic cardiomyopathy(ICM) and healthy controls were obtained from the Gene Expression Omnibus(GEO) database. The scRNA-seq and RNA-seq data were analyzed using R packages such as Seurat, monocle and clusterProfiler： data integration, differentially expressed genes(DEGs), cell type identification, enrichment analysis and pseudotime-ordered analysis. CIBERSOR was used for immunoinfiltration analysis of RNA-seq data. ResultsThe scRNA-seq data of cardiac immune cells from normal control and three types of cardiomyopathy were integrated, and 19 348 cells were obtained after optimized screening, and were divided into 7 types of immune cells. The high proportion of NK cells in ICM indicated that NK cells were associated with high cardiac inflammation. The T cells including ten subtypes identified by subtype identification and pseudotime-ordered analysis. It was found that T cells in stress response state only seemed to form a pseudo-temporal locus in ICM, and 12 specific genes of DEGs among T cell subtypes with pseudo-temporal variation were screened, which played a role in the process of inflammation and immune regulation. Immunoinfiltration analysis of RNA-seq data showed that monocytes had a low infiltration rate in DCM and ICM patients, CD4+T cells and M2 macrophages had a low infiltration rate in HCM patients, and mast cells had a high infiltration rate in HCM patients. ConclusionThe heterogeneity of immune cells and the changes of immune microenvironment in three kinds of cardiomyopathy are found in this study, which provide a certain reference for exploring the mechanism of different cardiomyopathy.