Bulk RNA-seq in cholangiocytes from mouse model of primary biliary cholangitis. Bulk RNA-seq in cholangiocytes from mouse model of primary biliary cholangitis

Background & Aims: Late-stage primary biliary cholangitis (PBC) is defined by bile duct loss, ductular reaction, peribiliary inflammation and fibrosis. Loss of anion exchanger 2 (AE2), the ‘bicarbonate umbrella’ and ductulo-canalicular junctions (DCJ) are hypothesized to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis controls cystic fibrosis transmembrane receptor (CFTR) activation and AE2 opening, and controls choleresis. We aimed to define the impact of Sct/SR signaling on biliary secretory processes and subsequent injury in late-stage PBC mouse model and human samples. Methods: Female and male wild-type (WT) and dominant-negative transforming growth factor beta receptor II (dnTGFβRII, late-stage PBC model) at 32 wks of age were treated with Sct for 1 wk or 8 wks. Pathways mediated by Sct were identified by RNA-seq in isolated cholangiocytes. Sct/SR/CFTR/AE2 expression and MUC1 levels were evaluated in human control and late-stage PBC. Results: Biliary Sct, SR, CFTR and AE2 expression is reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation and fibrosis in late-stage PBC models, as well as hepatic bile acid release and DCJ formation. RNA-seq identified that Sct promoted mature epithelial cell marker expression, specifically anterior grade 2 (Agr2, expressed only in cholangiocytes). Late-stage PBC models and human samples had reduced biliary MUC1, which was enhanced with Sct treatment. Conclusion: Loss of the Sct/SR pathway in late-stage PBC results in a faulty ‘bicarbonate umbrella’ and reduced Agr2 and mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and healthy bile duct growth. The Sct/SR axis may be a therapeutic target for late-stage PBC patients. Overall design: intrahepatic cholangiocytes analyzed from WT, WT+secretin 8 wk, PBC model (dnTGFBRII), and PBC model + secretin 8 wk. WT cholangiocytes are the control sample. We analyzed male and female samples separately. 6-8 mice per treatment group were pooled to extract cholangiocytes, and total RNA from this pooled population of cholangiocytes was sequenced in triplicate at GeneWiz using an Illumina platform.

研究背景与目的：晚期原发性胆汁性胆管炎（primary biliary cholangitis, PBC）以胆管丢失、胆管反应、胆管周围炎症及纤维化为特征。阴离子交换蛋白2（anion exchanger 2, AE2）作为“碳酸氢盐保护伞”以及胆管-小管连接（ductulo-canalicular junctions, DCJ）的缺失被认为可促进PBC进展。促胰液素（secretin, Sct）/促胰液素受体（secretin receptor, SR）轴可调控囊性纤维化跨膜转运蛋白（cystic fibrosis transmembrane receptor, CFTR）的活化与AE2的开放，并调控胆汁分泌。本研究旨在明确Sct/SR信号通路对晚期PBC小鼠模型及人体样本中胆管分泌过程及后续损伤的影响。

研究方法：将32周龄的野生型（wild-type, WT）及显性负性转化生长因子β受体II（dominant-negative transforming growth factor beta receptor II, dnTGFβRII，晚期PBC模型）雌雄小鼠分别用Sct处理1周或8周。通过对分离的胆管上皮细胞（cholangiocytes）进行RNA测序（RNA-seq），鉴定Sct介导的信号通路。在人类对照样本及晚期PBC样本中，评估Sct/SR/CFTR/AE2的表达水平及黏蛋白1（MUC1）的表达量。

研究结果：晚期PBC小鼠模型及人体样本中，胆管组织的Sct、SR、CFTR及AE2表达水平均降低。Sct处理可减轻晚期PBC模型中的胆管丢失、胆管反应、炎症及纤维化程度，同时减少肝脏胆汁酸释放并促进DCJ形成。RNA测序结果显示，Sct可促进成熟上皮细胞标志物的表达，尤其是仅在胆管上皮细胞中表达的前梯度蛋白2（anterior grade 2, Agr2）。晚期PBC模型及人体样本中的胆管MUC1表达水平降低，而Sct处理可上调该蛋白的表达。

研究结论：晚期PBC患者体内Sct/SR通路的缺失会导致“碳酸氢盐保护伞”功能异常，并降低Agr2及黏蛋白的生成。Sct可通过增强成熟胆管上皮细胞表型及健康胆管生长，恢复胆管上皮细胞的分泌过程及DCJ形成。Sct/SR轴有望成为晚期PBC患者的治疗靶点。

总体实验设计：对四组样本的肝内胆管上皮细胞进行分析：野生型（WT）组、WT+促胰液素处理8周组、PBC模型（dnTGFBRII）组、PBC模型+促胰液素处理8周组，其中WT胆管上皮细胞作为对照样本。本研究分别对雌雄样本进行独立分析。每个处理组选取6-8只小鼠的胆管上皮细胞进行混合，提取总RNA，随后在GeneWiz公司采用Illumina平台进行三次重复的RNA测序。