Toll-like receptor 3 mediates PROMININ-1 expressing cell expansion in biliary atresia via Transforming Growth Factor-Beta

Background: In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-beta (TGF beta) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGF beta pathway activation in vitro. Methods: PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I: C), +/- small molecule inhibitors nafamostat, or SB431542. Results: Poly I: C induced myofibroblastic-like morphologic changes, degradation of I kappa B-alpha consistent with TLR3-NF kappa B activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24 h (p < 0.05), and an 8.2-fold increase in Prom1 at 72 h (p < 0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1 alpha staining following Poly I: C treatment. Degradation of I kappa Ba was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1. Conclusions: TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGF beta pathway activation to promote BA-associated biliary fibrosis. (C) 2016 Elsevier Inc. All rights reserved.