Abstract
Background: Biliary atresia (BA) is the most prevalent serious neonatal biliary obstructive disorder and is a complex multifactorial liver disorder. Genome-wide association studies have identified Adducin 3 (ADD3) as a BA susceptibility gene but the mechanisms involved in disease causation and progression remain unclear. Methods: ADD3 knockout human pluripotent stem cells were differentiated into cholangiocyte organoids to assess the effect of ADD3 deletion on biliary development in vitro. Add3 deletion in rhesus rotavirus (RRV)-induced experimental BA mice were employed as the in vivo model to address the impact of reduced Add3 expression on BA pathogenesis. Findings: ADD3 knockout organoids displayed defective cholangiocyte differentiation, failure in the recruitment of βII-spectrin to the cell membrane, abnormal primary cilia development, reduced expression of tight junction proteins, lower transepithelial electrical resistance (TEER) and increased paracellular permeability. Statistical significantly reduced tight junction (TJ) proteins expression and lower TEER in Add3+/- and Add3-/- liver tissue-derived cholangiocytes were observed. Reduced number of TJs and enlarged paracellular spaces without any detectable TJ were detected in the intra-hepatic bile ducts of Add3+/- and Add3-/- livers. A statistical significantly higher incidence and a more advanced form of BA with statistical significantly higher serum bilirubin, liver necrosis and fibrosis, and accumulation of macrophages and activated hepatic stellate cells were observed in Add3 knockout BA mice as compared to wild-type BA mice. Interpretation: Dysregulated ADD3 expression caused an abnormal development and impaired barrier function of cholangiocytes, and the resultant increase in bile duct permeability rendered the foetus/neonate susceptible to a more severe injury response to an external insult. The findings support the hypothetical pathogenic model of genetic susceptibility genes being involved in hepatobiliary development/structure, and the perturbed embryogenesis of the biliary tree and its disrupt integrity increase the host susceptibility to biliary injury and BA. Funding: Theme-based Research Scheme 2021 (T12-712/21-R); Health and Medical Research Fund (06172096; 03143476); RGC CERG Grant 2019/20 (17105119) to VCHL; Commissioned HMRF (PR-HKU-1) to PKHT.