CFTR modulates innate immune response in the biliary epithelium. Role in the path

描述(申请人提供)：囊性纤维化(CF)是一种常见的临床严重的遗传性疾病，由CFTR突变引起，CFTR是一种膜蛋白，调节包括胆道树在内的许多分泌性上皮细胞中的氯离子和液体分泌。大约30%的CF 患者出现生化肝脏异常，其中约10%发展为临床显著的肝病和肝胆并发症，其特征是慢性胆管病，最终可演变为硬化性胆管炎和局灶性胆汁性肝硬变。囊性纤维化肝病(CFLD)影响患者的生活质量和生存，可能需要肝移植，但其发病机制尚不清楚。在CFTR-KO小鼠中，胆汁流量和胆汁分泌减少；然而，CFLD的自发发展极其罕见，这表明除了胆汁淤积之外，遗传和/或获得性因素在起作用。研究表明，口服右旋糖苷所致的门静脉内毒素血症对CFTR-KO小鼠有特异性的胆道损伤作用，但对其野生型仔鼠无明显影响。利用这个实验模型，我们最近发现，用Nor-UDCA恢复胆汁分泌并不能防止DSS治疗CFTRKO小鼠引起的胆道损伤和炎症，与WT细胞相比，体外培养的CFTRKO小鼠胆管细胞暴露于脂多糖后，细胞因子分泌和NF-κB活性显著增加。活性的增加 抑制TLR4后，可抑制NF-κB的表达。我们还发现，参与细胞对内毒素反应的酪氨酸激酶Src的活性在CF细胞中上调，并导致TLR4的磷酸化增加。我们认为，Src是cftr和TLRs之间潜在的分子联系。事实上，用Src抑制剂PP2处理可阻断TLR4的磷酸化和内毒素诱导的核因子-κB的激活。我们还发现参与Src调控的CSK和EBP-50在CFTR-KO胆管细胞中的表达和分布发生了变化。在这一应用中，我们将进一步研究这一新的范式转换假说，特别是我们将阐明1)不同TLR在CFLD中的致病作用，2)sRc在连接CFTR和TLR介导的信号转导中的作用，3)PPARγ激动剂作为一种策略抑制CF胆管细胞中TLR/NF-κB通路的治疗价值。这些研究将在CFTR缺陷小鼠和雪貂身上进行，雪貂是一种自发发展CFLD的新型CF模型。这些研究将改变我们目前对胆囊性胆管病发病机制和治疗的认识。此外，更好地了解TLR通路在胆管上皮细胞中的调控，也将为其他炎症介导的上皮疾病的发病机制提供重要的见解，为未来更广泛的上皮免疫学领域的研究提供坚实的基础。

DESCRIPTION (provided by applicant): Cystic Fibrosis (CF) is a common and clinically severe genetic disease, caused by mutations in CFTR, a membrane protein that mediates Cl- and fluid secretion in a number of secretory epithelia, including the biliary tree. About 30% of CF patients present biochemical liver abnormalities and about 10% of these develop clinically significant liver disease and hepatobiliary complications characterized by a chronic cholangiopathy that can eventually evolve into sclerosing cholangitis and focal biliary cirrhosis. Cystic Fibrosis liver disease (CFLD) negatively impacts the quality of life and survival of CF patients, and may require liver transplantation, however, the pathogenesis of this condition is not well understood. In Cftr-KO mice, bile flow and biliary secretion are reduced; however the spontaneous development of CFLD is extremely rare, suggesting that genetic and/or acquired factors other than cholestasis are at play. It has been shown that portal endotoxemia, induced by oral administration of dextrans specifically causes biliary damage in Cftr-KO mice, but not in their wild type littermates. Using this experimental model, we recently showed that biliary damage and inflammation caused by treatment with DSS in Cftr-KO mice were not prevented by restoring biliary secretion with nor-UDCA, and that, exposure of cultured CFTR-defective cholangiocytes to LPS in vitro, significantly increased cytokine secretion and NF-κB activity as compared to WT cells. The increased activation of NF-κB was prevented by inhibition of TLR4. We also found that the activity of Src, a tyrosine kinase involved in cell response to LPS, was upregulated in CF cells and resulted in an increased phosphorylation of TLR4. We propose that Src is the potential molecular link between CFTR and TLRs. In fact, treatment with Src inhibitor PP2 blocked TLR4 phosphorylation and NF-κB activation in response to LPS. We have also found that the expression and distribution of Csk and EBP-50, involved in Src regulation, were altered in Cftr-KO cholangiocytes. In this application, we will further investigate this novel paradigm shifting hypothesis, and in particular we will elucidate 1) the pathogenetic role of different TLRs in CFLD, 2) the role of Src in linking CFTR and TLR- mediated signaling and 3) the therapeutic value of PPARγ agonist as a strategy to inhibit the TLR/NF-κB pathway in CF cholangiocytes. These studies will be performed in Cftr-defective mice and in ferrets, a novel CF model that spontaneously develops CFLD. These studies will change our current understanding of the pathogenesis and treatment of CF-cholangiopathy. Furthermore, better knowledge of the regulation of TLR pathways in the biliary epithelium, will also provide important insights into the pathogenesis of other inflammation-mediated diseases of the epithelium, providing a firm foundation for future studies in the broader field of epithelial immunology.