The etiology of BA is still poorly understood. Biliatresone, a plant isoflavonoid-related 1,2-diaryl-2-propenone has been implicated in BA-like syndrome outbreaks in Australian livestock also causes bile duct destruction in zebrafish13, mouse cholangiocyte spheroids and ex-vivo bile duct culture15, and in mouse neonates 16. This study investigated the effects of biliatresone toxin on human cholangiocytes. Using human liver organoids, we showed that biliatresone induced dysmorphogenesis; disturbed apical-basal and cytoskeleton organization; defective development; reduction of primary cilia and cilia mechano-sensory function of cholangiocytes. Our data provide evidence showing that biliatresone induces BA-like morphological and developmental changes in cholangiocytes, suggesting that environmental toxins could contribute to BA pathogenesis in human.
Biliatresone treatment induces developmental defects of human liver organoid similar to the defects of BA liver tissue derived liver organoids19,21, which include a retarded growth; disturbed apical-basal organization and a defective cholangiocyte development. We have observed a reduction of cholangiocyte MDR transport, a reduction of ciliated cholangiocyte and cilia sensory response in biliatrsone-treated organoids, which further corroborates a defective cholangiocyte development in biliatresone treatment. In line with our observations, disrupted apical-basal polarity15, and a reduced number of primary cilia13 of cholangiocytes were reported in biliatresone-treated mouse cholangiocyte organoids.
Cholangiocytes, like many other cells, present primary cilia extending from their apical plasma membranes into the bile duct lumen, acting as flow-stress sensors to detect changes in bile flow, composition and osmolality, and adjust the secretion of bile acid and CO32− accordingly26–28. Defects in ciliary function could cause developmental or degenerative disorders of a wide spectrum29. Mutations of genes encoding cholangiocyte ciliary-associated proteins polycystin-1 and − 2; fibrocystin cause biliary dysgenesis, hepatic fibrosis, and cystogenesis of liver diseases such as Autosomal Dominant Polycystic Kidney Disease (ADPKD) and Autosomal Recessive PKD (ARPKD). Our recent whole genome sequencing study detected rare, deleterious de novo or biallelic variants of liver-expressed ciliary genes, including PCNT, KIF3B and TTC17, in around 30% of non-syndromic BA patients30. Cholangiocyte cilia act as mechano-, chemo-, and osmo-sensors for bile flow and regulator of bile formation in bile ducts. Through the ciliary specific and associated receptors, ion channels and sensory signalling molecules on the ciliary membrane, bile content changes are detected and signals are transduced to regulate cholangiocyte function. It was found that bile acids induced hyper-proliferation of non-ciliated cholangiocyte, but not of ciliated cholangiocyte31. Besides bile acids, there are neuropeptides, neurotransmitters, and hormones that promote cholangiocyte hyper-proliferation during cholestasis, whereas cholangiocyte hyper-proliferation could promote hepatic fibrosis32. Shorter, abnormally oriented and less abundant cilia were observed in the intra-hepatic cholangiocytes of syndromic and non-syndromic BA patients24,25. In extra-hepatic cholangiocytes, cilia were almost completely absent in BA neonates23. Fibrocystin, a protein that localizes to cholangiocyte cilia and basal body of bile ducts, was reduced in BA livers33. Taken all the above indicated that abnormal cilia and dysregulated cilia functions of cholangiocytes are intimately associated with development of BA. Exposure of bile duct cells to toxin such as biliatresone may lead to a reduction of ciliated cholangiocyte and dysregulated sensory responses to bile acids or other regulators, which could then lead to cholangiocyte hyper-proliferation and hepatic fibrosis in BA.
Biliatresone decreased the cellular antioxidant glutathione (GSH) and SOX17 level of mouse cholangiocytes15. GSH and SOX17 are essential for the stabilization of cellular cytoskeleton, which plays a vital role in maintaining the cellular polarity and in ciliogenesis13,15. SOX17 is expressed in the endoderm from the onset of gastrulation and plays essential roles in cholangiocyte and bile duct development34,35. Sox17 heterozygous mice develop BA-like phenotype as the gallbladder epithelium becomes detached from the luminal wall, which indicates that Sox17 is required to maintain the epithelial architecture of the gallbladder and cystic duct34. A small but significant drop of SOX17 expression in day-2 biliatresone-treated organoids was detected (Figure S3), which was associated with a perturbation of cytoskeleton, apical-basal polarity and ciliogenesis defects, and loss of epithelial integrity in human cholangiocytes as revealed by abnormal F-actin, α-tubulin and ZO-1 immuno-fluorescence (Fig. 3, 4 & S1). We also observed a small but significant drop of GSH level in biliatresone-treated organoids at 3 hours, but the GSH level was restored in biliatresone-treated organoids at 6, 24 and 48 hours (Figure S3). Biliatresone and decreases in GSH indirectly downregulated Sox1736. GSH levels were shown to drop in the first few hours, but were increased and recovered to normal level at 24 hours of biliatresone treatment. However, Sox17 expression was still significantly lower in biliatresone-treated mouse cholangiocytes at 24 hours of treatment15. Taken all these together suggested that a transient drop of GSH level is sufficient to induce a downregulation of SOX17, and a later recovery of GSH level is unable to restore the normal SOX17 expression.
In sum, biliatresone induced cholangiocyte injury that includes decrease of cell-cell tight junctions, polarity changes, increased epithelial permeability, and loss of cilia and cilia function of human cholangiocytes. The biliatresone induced morphological and developmental changes may provide important insights into the BA pathogenesis in human, and may lead to the development of new preventive measures for environmental toxin or novel treatments. Although pregnant women are unlikely exposed to biliatresone itself, humans may be exposed to other GSH-depleting molecules/toxins, and that some of these may cross the placenta causing oxidative stress and injury to the fetal hepatobiliary system, immune dysregulation in BA development. Human liver derived organoids can serve as a human proxy for screening of these possible GSH-depleting molecules/toxins.