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

描述（由申请人提供）：囊性纤维化（CF）是一种常见的临床严重遗传性疾病，由CFTR突变引起，CFTR是一种膜蛋白，介导许多分泌上皮细胞（包括胆管树）的Cl-和液体分泌。约占CF的30% 患者存在生化肝脏异常，并且其中约10%发展为临床上显著的肝脏疾病和肝胆并发症，其特征在于慢性胆管病，其最终可演变为硬化性胆管炎和局灶性胆汁性肝硬化。囊性纤维化肝病（CFLD）对CF患者的生活质量和生存率产生负面影响，可能需要肝移植，然而，这种疾病的发病机制尚未完全了解。在Cftr-KO小鼠中，胆汁流量和胆汁分泌减少;然而CFLD的自发发展是极其罕见的，这表明除胆汁淤积以外的遗传和/或获得性因素在起作用。已经表明，通过口服给予葡聚糖诱导的门静脉内毒素血症特异性地导致Cftr-KO小鼠的胆道损伤，但在其野生型同窝仔中没有4。使用这个实验模型，我们最近表明，胆损伤和炎症引起的治疗与DSS在Cftr-KO小鼠不能防止恢复胆汁分泌与nor-UDCA，而且，暴露培养的CFTR缺陷的胆管细胞在体外LPS，显着增加细胞因子分泌和NF-？B活性。NF-？通过抑制TLR 4来预防B。我们还发现，Src的活性，参与细胞对LPS的反应，上调CF细胞中的酪氨酸激酶，并导致TLR 4的磷酸化增加。我们认为Src是CFTR和TLR之间潜在的分子联系。事实上，治疗Src抑制剂PP 2阻断TLR 4磷酸化和NF-？响应于LPS的B活化。我们还发现参与Src调控的Csk和EBP-50的表达和分布在Cftr-KO胆管细胞中改变。在本申请中，我们将进一步研究这种新的范式转换假说，特别是我们将阐明1）不同TLR在CFLD中的发病作用，2）Src在连接CFTR和TLR介导的信号传导中的作用，以及3）PPAR？激动剂作为一种策略，以抑制TLR/NF-？CF胆管细胞中的B通路。这些研究将在Cftr缺陷小鼠和雪貂中进行，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CF-胆管病发病机制和治疗的认识。此外，更好地了解胆管上皮中TLR途径的调节，也将为研究TLR途径的作用机制提供重要的见解。 其他炎症介导的上皮疾病的发病机制，为今后在更广泛的上皮免疫学领域的研究提供了坚实的基础。

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 littermates4. 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-?B activity as compared to WT cells. The increased activation of NF-?B 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-?B 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 PPAR? agonist as a strategy to inhibit the TLR/NF-?B 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.