SnRNA-seq reveals heterogeneity of mouse cholangiocyte in liver Mallory-Denk bodies formation

Mallory-Denk bodies (MDBs) are misfolded protein aggregates that include hepatocytes present in liver diseases, and are considered a typical feature of chronic liver diseases. Cholangiocytes are an epithelial cell type constituting the hepatic parenchyma, and play a role in liver regeneration if hepatocyte regeneration is compromised. UbD, which is overexpressed in hepatocellular carcinoma (HCC) plays an important role in immune regulation and MDB pathogenesis. In this study, we conducted the first comprehensive study on cholangiocyte heterogeneity to demonstrate the pathogenesis of MDBs formation using single-nucleus RNA sequencing (snRNA-seq) from fractionations of normal and DDC-treated mouse livers. We found that cholangiocytes in the MDB-forming groups were significantly expanded, and the enrichment pathways of differentially expressed genes (DEGs) were markedly involved in metabolic processes, neurodegenerative diseases, autophagy, and ubiquitin-mediated proteolysis. We proposed a cellular-state landscape for cholangiocytes using uniform manifold approximation and projection (UMAP) analysis, which includes numerous cell subpopulations, referred to as Clusters0 through 7, with Cluster1 being the sole subpopulation made up entirely of DDC-treated cells. UbD was highly expressed in Cluster4 and colocalized with the cholangiocyte marker gene cytokeratin 19 (Krt19), implying that cholangiocytes function as facultative liver progenitor cells (LPCs) to supply damaged hepatocytes to promote MDBs formation. In summary, we revealed, for the first time, the heterogeneity of cholangiocytes and provided a novel viewpoint for understanding the mechanism of MDBs formation as well as chronic liver disease progression. Overall design: SnRNA-seq was performed on two groups (Control vs. DDC), and each group independent sample consisted of liver tissues from three mice. Biological functions of cholangiocyte differentially expressed genes (DEGs) were well identified by the comparison between Control group and DDC group at the single-cell level. Gene ontology (GO) term, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathway and Gene set enrichment analysis (GSEA) software were used to reveal cholangiocytes heterogeneity. qRT-PCR and immunohistochemistry staining were used to evaluate the expression of signature genes in cholangiocytes. Finally, FISH analysis was used to assess the colocalization.