Elucidation of pericholangitis and periductal fibrosis in cholestatic liver diseases via extracellular vesicles released by polarized biliary epithelial cells.

Cholestatic liver diseases causes inflammation and fibrosis around bile ducts. However, the pathological mechanism has not been elucidated. Extracellular vesicles (EVs) are released from both the basolateral and apical sides of polarized biliary epithelial cells. We aimed to investigate the possibility that EVs released from the basolateral sides of biliary epithelial cells by bile acid stimulation induce inflammatory cells and fibrosis around bile ducts, and they may be involved in the pathogenesis of cholestatic liver disease. Human biliary epithelial cells (H69) were grown on cell culture inserts and stimulated with chenodeoxycholic acid + IFN-γ. Human THP-1-derived M1-macrophages, LX-2 cells, and KMST-6 cells were treated with the extracted basolateral EVs, and inflammatory cytokines and fibrosis markers were detected by RT-PCR. Highly expressed proteins from stimulated EVs were identified, and M1-macrophages, LX-2, KMST-6 were treated with these recombinant proteins. Stimulated EVs increased the expression of TNF, IL-1ß, and IL-6 in M1-macrophages, TGF-ß in LX-2 and KMST-6 compared with the corresponding expression levels in unstimulated EVs. Nucleophosmin, nucleolin, and midkine levels were increased in EVs from stimulated cells compared with protein expression in EVs from unstimulated cells. Leukocyte cell-derived chemotaxin-2 (LECT2) is highly expressed only in EVs from stimulated cells. Stimulation of M1-macrophages with recombinant nucleophosmin, nucleolin, and midkine significantly increased the expression of inflammatory cytokines. Stimulation of LX-2 and KMST-6 with recombinant LECT2 significantly increased the expression of fibrotic markers. These results suggest that basolateral EVs are related to the development of pericholangitis and periductal fibrosis in cholestatic liver diseases.NEW & NOTEWORTHY Our research elucidated that the composition of basolateral EVs from the biliary epithelial cells changed under bile acid exposure and the basolateral EVs contained the novel inflammation-inducing proteins NPM, NCL, and MK and the fibrosis-inducing protein LECT2. We report that these new results are possible to lead to the potential therapeutic target of cholestatic liver diseases in the future.

Role of Bile-Derived Extracellular Vesicles in Hepatocellular Proliferation after Partial Hepatectomy in Rats.

Although liver regeneration has been extensively studied, the effects of bile-derived extracellular vesicles (bile EVs) on hepatocytes has not been elucidated. We examined the influence of bile EVs, collected from a rat model of 70% partial hepatectomy (PH), on hepatocytes. We produced bile-duct-cannulated rats. Bile was collected over time through an extracorporeal bile duct cannulation tube. Bile EVs were extracted via size exclusion chromatography. The number of EVs released into the bile per liver weight 12 h after PH significantly increased. Bile EVs collected 12 and 24 h post-PH, and after sham surgery (PH12-EVs, PH24-EVs, sham-EVs) were added to the rat hepatocyte cell line, and 24 h later, RNA was extracted and transcriptome analysis performed. The analysis revealed that more upregulated/downregulated genes were observed in the group with PH24-EVs. Moreover, the gene ontology (GO) analysis focusing on the cell cycle revealed an upregulation of 28 types of genes in the PH-24 group, including genes that promote cell cycle progression, compared to the sham group. PH24-EVs induced hepatocyte proliferation in a dose-dependent manner in vitro, whereas sham-Evs showed no significant difference compared to the controls. This study revealed that post-PH bile Evs promote the proliferation of the hepatocytes, and genes promoting cell cycles are upregulated in hepatocytes.