Functional Characterization Of Immune Microenvironment In Murine Hepatic Alveolar Echinococcosis Based On Single-cell RNA Sequencing

Objective:To investigate the cellular composition and transcriptional profiles of liver tissues in mice secondary to Echinococcus multilocularis infection using 10×Genomics single-cell RNA sequencing technology,and to provide a theoretical basis for understanding the role of various cell types in the immune microenvironment of alveolar echinococcosis(AE).Methods:Two female BALB/c mice were selected to construct a mouse model of alveolar echinococcosis by intraperitoneal inoculation of protoscoleces.After 15months,the perilesional liver tissues(PL group)and paired distal liver tissues(DL group)of two mice were collected for single-cell RNA sequencing.All data analysis was completed based on the 10×Genomics official software Cell Ranger and R software.The Seurat package was used to perform quality control,standardization,data scaling,cell clustering,and cell type annotation analysis on the obtained raw data.Subsequently,differentially expressed genes of different cell types were found by differential expression analysis,and the GO and KEGG enrichment analysis were performed.In addition,Cell Chat software package was used to predict the cell communication network between T cell subsets.Results:In this study,43,710 cells in the liver tissue of mice with alveolar echinococcosis were analyzed,and 11 main cell types and 20 different cell subsets were annotated.It was found that T cells were the highest proportion of ones in the immune microenvironment.The up-regulated differentially expressed genes in neutrophils from the PL group were significantly enriched in inflammatory response,positive regulation of response to external stimulus,positive regulation of apoptosis process,TNF signaling pathway,and NF-κb signaling pathway.There were three different functional macrophage subsets in the immune microenvironment,among which tumor associated macrophagrs and Spp1~+macrophages were highly expressed to promote tumor metastasis and fibrosis-related genes Mt2,Mt1,Fn1,Cd9,and Arg1.The up-regulated differentially expressed genes in Spp1~+macrophages derived from PL tissues were significantly enriched in positive regulation of cell migration,positive regulation of cell motility,NF-κB signaling pathway,and TGF-beta signaling pathway.The T cells in the immune microenvironment could be classified into eight cell subsets,among which the number and proportion of CD4~+cytotoxic T cells,CD4~+helper T cells and Th2 cells were different between the PL group and the DL group.The differentially expressed genes of Th2 cells derived from PL group were related to negative regulation of leukocyte activation,negative regulation of inflammatory response,MAPK signaling pathway and apoptosis.CD4~+cytotoxic T cells in the immune microenvironment can interact with CD4~+effector T cells,CD8~+cytotoxic T cells and CD8~+naive T cells,and induce the high expression of various chemokines and their receptor genes.Among them,CD4~+cytotoxic T cells and CD4~+effector T cells communicate with Fasl-Fas and Ccl5-Ccr5 through immunosuppressive ligand-receptor pairs.Conclusion:In this study,the single-cell RNA sequencing technology was used to successfully map the liver tissue of mice at the advanced stage of alveolar echinococcosis,and the role of macrophages and T cells in the immune microenvironment was explored,which provided a theoretical basis for further understanding the immune evasion mechanism of mice at the advanced stage of alveolar echinococcosis.