Src kinase inhibition reduces inflammatory and cytoskeletal changes in ΔF508 human cholangiocytes and improves CFTR correctors efficacy

Abstract

CFTR, the channel mutated in cystic fibrosis (CF), is expressed by the biliary epithelium (i.e cholangiocytes) of the liver. Progressive clinical liver disease (CFLD) occurs in about 10% of CF patients and represents the third leading cause of death. Impaired secretion and inflammation contribute to CFLD, however the lack of human-derived experimental models has hampered the understanding of CFLD pathophysiology and the search for a cure. We have investigated the cellular mechanisms altered in human CF cholangiocytes using induced pluripotent stem cells (iPSC) derived from healthy controls and a ΔF508 CFTR patient. We have devised a novel protocol for the differentiation of human iPSC into polarized monolayers of cholangiocytes. Our results show that iPSC-cholangiocytes reproduced the polarity and the secretory function of the biliary epithelium. PKA/c-AMP mediated fluid secretion was impaired in ΔF508 cholangiocytes and negligibly improved by VX-770 and VX-809, two small molecule drugs used to correct and potentiate ΔF508 CFTR. Moreover, ΔF508 cholangiocytes showed increased phosphorylation of Src kinase and TLR4 and pro-inflammatory changes including increased NF-kB activation, secretion of pro-inflammatory chemokines (i.e MCP-1 and IL8), as well as alterations of the F-actin cytoskeleton. Treatment with Src inhibitor (PP2) decreased the inflammatory changes and improved cytoskeletal defects. Inhibition of Src along with administration of VX-770 and VX-809 successfully restored fluid secretion to normal levels. Conclusions: Our findings have strong translational potential and indicate that targeting Src kinase and decreasing inflammation may increase the efficacy of pharmacological therapies aimed at correcting the basic ΔF508 defect in CF liver patients. These studies also demonstrate the promise of applying iPSC technology in modeling human cholangiopathies. This article is protected by copyright. All rights reserved.

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