[Immune cell characteristics of the liver in a mouse model of fatty liver disease induced by high-ethanol-producing Klebsiella pneumoniae based on single-cell transcriptome sequencing].

Objective: To construct a single-cell transcriptomic atlas of the liver in a mouse model of fatty liver disease induced by high-ethanol-producing Klebsiella pneumoniae (HiAlc Kpn) and characterize the heterogeneity of hepatic immune cell subtypes in this model. Methods: Twelve six-week-old male C57BL/6 mice were randomly divided into a control group (n=6) and a model group (n=6). The model group was gavaged with a HiAlc Kpn TH1 strain (HiAlc Kpn, 1×107 CFU/ml, 200 μl in YEPD medium) every 2 days for 8 weeks to induce fatty liver disease, while controls received YEPD medium alone. After the model establishment, livers were harvested for single-cell RNA sequencing (10×Genomics). Sequencing data were processed in R for quality control, dimensionality reduction, clustering, and cell-type annotation, followed by pseudo-time trajectory analysis, pathway enrichment analysis, and ligand-receptor interaction network analysis. Results: The single-cell atlas revealed marked immunological shifts in the model livers compared to controls. T cells were less prevalent in the model (46.48% vs. 64.91%, χ2=569.14, P<0.001), whereas B cells were more abundant (29.97% vs. 15.19%,χ2=530.21, P<0.001). Within T cell subtypes, the model group had fewer effector CD4+T cells (15.55% vs. 29.50%, χ2=530.21, P<0.001), effector CD8+T cells (13.41% vs. 20.19%, χ2=66.96, P<0.001), and a cytotoxic CD8+T subset (1.18% vs. 4.89%, χ2=84.68, P<0.001). By contrast, naive CD4+T cells (17.96% vs. 11.32%, χ2=79.67, P<0.001), naive CD8+T cells (30.59% vs. 15.90%, χ2=277.63, P<0.001), and exhausted CD8+T cells (7.75% vs. 5.99%, χ2=10.44, P=0.001) were more frequent in the model. Notably, exhausted cytotoxic CD8+T cells in the model exhibited diminished expression of cytotoxic genes (Ifng, Gzma, t=2.74, P=0.006; t=1.97, P=0.048) alongside upregulation of inhibitory receptors (Lag3, Tigit, t=-3.09, P=0.002; t=-2.38, P=0.017), indicating functional exhaustion. B cell compartment changes were also pronounced. The model showed a corresponding reduction in germinal center B cells (5.67% vs. 56.53%, χ2=1157.50, P<0.001) and plasma cells (9.09% vs. 21.56%, χ2=109.81, P<0.001), with a dramatic expansion of naive B cells (42.71% vs. 6.81%, χ2=542.07, P<0.001) and proliferating B cells (33.16% vs. 7.02%, χ2=332.20, P<0.001). Pathway enrichment analysis indicated that B cell subtypes in the model group were enriched for pathways related to B cell activation, whereas B cells in the control group were enriched for pathways maintaining homeostasis. Additionally, ligand-receptor interaction analysis revealed an enhanced communication network between plasma B cells and T cell subtypes in the model group compared to controls. Conclusion: This study has constructed a comprehensive single-cell transcriptomic map of HiAlc Kpn-induced fatty liver disease under high glucose conditions in mice and provided new insights into the immune cell heterogeneity underlying this disease model.