Abstract: Objective　To investigate the cell composition and the transcriptional characteristics in microenvironments of hepatic tissues in mice at late stage of Echinococcus multilocularis infection at a single⁃cell level. Methods　Peri⁃lesion and paired distal hepatic specimens were collected from two BALB/c mice (6 to 8 weeks old) infected with E. multilocularis for single⁃cell RNA sequencing. The Seurat package in the R software was employed for quality control of data, multi⁃sample integration and correction of batch effects, and uniform manifold approximation and projection (UMAP) algorithm was used for cell clustering. Cell types were annotated using classical marker genes. Differentially expressed genes were screened in each cell type through differential gene expression analysis, and the biological roles of cells were predicted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Results　A total of 43 710 cells from peri⁃lesion and distal hepatic tissues of E. multilocularis⁃infected mice were analyzed, and were classified into 11 cell types, including neutrophils, T cells, macrophages, granulocyte⁃monocyte progenitor cells, B cells, plasma cells, basophils, hepatic stellate cells, endothelial cells, hepatocytes, and platelets. T cells were the largest population of immune cells in the microenvironment of hepatic tissues, including five CD4+ T cell subsets, two CD8+ T cell subsets and phosphoantigen⁃reactive γδT cells. The proportions of CD4+ helper T cells and cytotoxic CD4+ T cells decreased and the proportion of T helper 2 (Th2) cells increased in peri⁃lesion tissues relative to distal hepatic tissues. In addition, the differentially expressed genes in Th2 cells were associated with negative regulation of the immune system, and the highly expressed genes in cytotoxic CD4+ T cells correlated with activation of the immune system. Conclusions　Single⁃cell RNA sequencing deciphers the cell composition and distribution in microenvironments of hepatic tissues from mice infected with E. multilocularis, and the increased proportion of Th2 cells in peri⁃lesion hepatic tissues may be associated with formation of immunosuppressive microenvironments.

Key words: Alveolar echinococcosis, Single?cell RNA sequencing, Tissue microenvironment, Transcriptional profile

摘要： 目的　从单细胞水平探究多房棘球蚴感染小鼠晚期阶段肝脏组织微环境细胞组成及其转录谱特征。方法　收集2只多房棘球蚴感染BALB/c小鼠（6～8周龄）肝脏病灶旁组织和配对远端肝组织进行单细胞转录组测序。利用R软件Seurat包对获得的数据进行质量控制、多样本整合和批次效应校正，应用统一流形逼近与投影（uniform manifold approximation and projection，UMAP）算法进行细胞聚类，根据经典标记基因注释细胞类型。通过差异基因表达分析筛选各细胞类型的差异表达基因，进行基因本体论（Gene Ontology，GO）和京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）功能富集分析，预测细胞生物学作用。结果　对来自多房棘球蚴感染小鼠肝脏病灶旁和远端肝组织的43 710个细胞进行了分析，归类为11种细胞类型：中性粒细胞、T细胞、巨噬细胞、粒细胞⁃单核细胞祖细胞、B细胞、浆细胞、嗜碱性粒细胞、肝星状细胞、内皮细胞、肝细胞、血小板。T细胞是组织微环境中占比最高的免疫细胞，包括5种CD4+ T细胞、2种CD8+ T细胞和磷酸抗原反应γδ T细胞。与病灶远端肝组织相比，病灶旁肝组织中的CD4+ 辅助性T细胞和CD4+ 细胞毒性T细胞比例降低、辅助性T细胞2（Th2细胞）比例明显增高。Th2细胞差异表达基因主要与免疫系统负调控过程相关，CD4+ 细胞毒性T细胞高表达基因与免疫系统激活相关。结论　通过单细胞转录组测序揭示了多房棘球蚴感染小鼠肝组织微环境中的细胞组成和分布差异，病灶旁肝组织中Th2细胞升高可能与免疫抑制性微环境形成有关。

关键词: 泡型棘球蚴病, 单细胞转录组测序, 组织微环境, 转录谱特征