O1118: Single Cell RNA Sequencing Analysis in Murine Biliary Atresia Model Reveals Subpopulation of Biliary Epithelial Cells with Pro-inflammatory and Fibrogenic Traits

Background: 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: 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. 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, i.e., natural killer cell and Toll-like receptor signaling. IPA also showed evidence of NFκB signaling with upregulation of pro-inflammatory cytokines, IL-1β, IL-6 and TNF and pro-fibrogenic cytokines, Tgfb1 and Ccl2 in this population.

Conclusion: A subset of Prom1-expressing immature BECs upregulate pro-inflammatory and pro-fibrogenic cytokines which likely contribute to fibrosis in BA. These cells and these signaling pathways represent potential therapeutic targets.