Single-Cell RNA Sequencing Analysis in Murine Biliary Atresia Model Reveals Subpopulation of Biliary Epithelial Cells with Proinflammatory and Fibrogenic Traits

Abstract

INTRODUCTION: Understanding the pathogenesis of liver fibrosis is critical to impacting outcomes of infants with biliary atresia (BA). In BA, Prominin-1 (Prom1)-expressing hepatic progenitor cell (HPC) populations give rise to biliary epithelial cells (BECs) with ductular reactions (DR). Loss of Prom1 function significantly decreases DRs and fibrosis. Hypothesis: Prom1-expressing HPCs give rise to BECs manifesting activated fibrogenic pathways in BA. METHODS: Experimental BA was induced in neonatal Balb/c mice with intraperitoneal injection of rhesus rotavirus (RRV) vs saline on postnatal day (p) 3. Livers were collected at p3, 10, and 17. EPCAM+/CD31–/CD45–/TER119– live BECs were isolated by fluorescence-activated cell sorting for single-cell RNA sequencing. RESULTS: A total of 44,877 cells were sequenced (saline: 29,195 cells, n = 11; RRV: 15,682 cells, n = 6), graph-based clustering identified 19 clusters of transcriptionally distinct cell types/states. Four distinct BEC subtypes were identified: stem/progenitor, immature, hepatocyte-like, and mature (Figure). All demonstrated activation of inflammation and fibrogenic pathways with RRV treatment. UMAP clustering revealed a unique population of Prom1-expressing immature BECs in RRV p17 samples. Ingenuity pathway analysis (IPA) of this population showed upregulation of several pathways specific to innate immune response, ie natural killer cell and Toll-like receptor signaling. IPA also showed evidence of NFκB signaling with upregulation of proinflammatory cytokines, IL-1β, IL-6, and TNF, and profibrogenic cytokines, Tgfβ1 and Ccl2, in this population.FigureCONCLUSION: A subset of Prom1-expressing immature BECs upregulate proinflammatory and profibrogenic cytokines that likely contribute to fibrosis in BA. These cells and these signaling pathways represent potential therapeutic targets.