Abstract

Background: Biliary atresia (BA) is the most common reason for liver transplantation in infants, but its etiopathology is still not completely understood. A nationwide genomic study identified mutations in PKD1L1 as a BA candidate gene. Liver-specific deletion of Pkd1l1 in mice results in liver pathology analogous to changes seen in BA, but how Pkd1l1-deficient cholangiocytes differ from wild type cells is unknown. The aim of this study was to elucidate the role of Pkd1l1 in cell homeostasis using isolated biliary epithelial cells (BECs) and intrahepatic biliary organoids (ICOs).

Methods: Intrahepatic BECs were isolated from 6–8-week-old male Pkd1l1^Fl/Fl (control) and Pkd1l1-deficient (KO) mice by fluorescence-activated cell sorting. Two models were used: BEC Transwell cultures grown to polarized monolayers; and isolated BECs to generate ICOs in 3-D cultures. Cells were incubated with DMSO, CDCA (Chenodeoxycholic Acid, 100mM), UDCA (Ursodeoxycholic Acid, 100mM) and Lipopolysaccharides (LPS, 100 IU/ml) from 1 to 24 hours. Organoid formation was visualized using light microscopy. BECs and organoids were characterized by RT-qPCR for known reactive ductular genes and immunofluorescence (IF) to explore cell structure and tight junction integrity.

Results: ICOs derived from KO mice were significantly smaller in size compared to control (average size 60204 mm² vs 2912 mm², 20-fold decrease; p<0.0001). Tight junctions, as indicated by IF of markers such as beta-catenin, were disorganized in KO compared to control cells in both systems. After challenge with CDCA and LPS to induce a reactive biliary phenotype, ICOs demonstrated aberrant mRNA expression of several proinflammatory cytokines in KO versus control (e.g., Ccl5 and Cxcl2 with 5-fold and 4-fold higher expression in KO compared to control cells respectively). A similar, pro-inflammatory phenotype has been observed in Transwell cultures with a basal increase of several markers (e.g, Cxcl1, with 9-fold increase in KO as compared to control).

Conclusion: Our studies demonstrate that Pkd1l1 is essential for the maintenance of normal BEC epithelial tight junctional structure and regulation of inflammatory response. This enhanced reactive ductular phenotype of KO cells mirrors features of human BA livers. These findings support the relevance of Pkd1l1 KO as a mouse model of the cholangiopathy leading to the BA phenotype and a valuable tool for evaluation of possible therapies of this devastating disease.