Department of Medical Oncology, National Center for Tumor Diseases, University Hospital Heidelberg, Heidelberg, Germany; Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg, Germany.
Liver Cancer Center Heidelberg, University Hospital Heidelberg, Heidelberg, Germany; Institute of Pathology, University Hospital Heidelberg, Heidelberg, Germany.
Gastroenterology. 2019 Mar;156(4):1190-1205.e14. doi: 10.1053/j.gastro.2018.11.018. Epub 2018 Nov 13.
BACKGROUND & AIMS: Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction.
We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immunohistochemistry for levels of RELB and lymphotoxin β (LTB). We studied mice with liver parenchymal cell (LPC)-specific disruption of the cylindromatosis (CYLD) lysine 63 deubiquitinase gene (Cyld), with or without disruption of Relb (Cyld mice and Cyld/Relb mice) and compared them with C57BL/6 mice (controls). Mice were fed 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) or standard chow diets to induce biliary injury or were given injections of CCl to induce non-cholestatic liver fibrosis. Liver tissues were analyzed by histology, immunohistochemistry, immunoblots, in situ hybridization, and quantitative real-time polymerase chain reaction. Cholangiocytes were isolated from normal human liver, incubated with LTB receptor agonist, and transfected with small interfering RNAs to knock down RELB.
In liver tissues from patients with primary sclerosing cholangitis, primary biliary cholangitis, chronic infection with hepatitis B or C virus, autoimmune hepatitis, or alcoholic liver disease, we detected increased nuclear translocation of RELB and increased levels of LTB in cholangiocytes that formed reactive bile ducts compared with control liver tissues. Human cholangiocytes, but not those with RELB knockdown, proliferated with exposure to LTB. The phenotype of Cyld mice, which included ductular reaction, oval cell activation, and biliary fibrosis, was completely lost from Cyld/Relb mice. Compared with livers from control mice, livers from Cyld mice (but not Cyld/Relb mice) had increased levels of mRNAs encoding cytokines (LTB; CD40; and tumor necrosis factor superfamily [TNFSF] members TNFSF11 [RANKL], TNFSF13B [BAFF], and TNFSF14 [LIGHT]) produced by reactive cholangiocytes. However, these strains of mice developed similar levels of liver fibrosis in response to CCl exposure. Cyld mice and Cyld/Relb mice had improved liver function on the DDC diet compared with control mice fed the DDC diet.
Reactive bile ducts in patients with chronic liver diseases have increased levels of LTB and nuclear translocation of RELB. RELB is required for the ductular reaction and development of biliary fibrosis in Cyld mice. Deletion of RELB and CYLD from LPCs protects mice from DDC-induced cholestatic liver fibrosis.
胆管细胞增殖和胆管反应是肝脏疾病发生和进展的原因。关于转录因子核因子-κB（NF-κB）在这一过程中的作用，我们知之甚少。我们研究了 RELB 原癌基因 NF-κB 亚单位在人类胆管细胞和以胆管反应为特征的肝脏疾病小鼠模型中的活性。
我们从德国的一个组织库中获得了原发性硬化性胆管炎、原发性胆汁性胆管炎、乙型或丙型肝炎病毒感染、自身免疫性肝炎、酒精性肝病或无这些疾病（对照组）患者的肝组织样本。通过免疫组织化学分析 RELB 和淋巴毒素 β（LTB）的水平。我们研究了肝实质细胞（LPC）特异性破坏胱抑素 D（CYLD）赖氨酸 63 去泛素酶基因（Cyld）的小鼠，有无 Relb 破坏（Cyld 小鼠和 Cyld/Relb 小鼠），并将其与 C57BL/6 小鼠（对照组）进行比较。用 5-二乙氧羰基-1,4-二氢-collidine（DDC）或标准 chow 饮食喂养小鼠诱导胆管损伤，或用 CCl 注射诱导非胆汁淤积性肝纤维化。通过组织学、免疫组织化学、免疫印迹、原位杂交和实时定量聚合酶链反应分析肝组织。从正常人肝中分离胆管细胞，用 LTB 受体激动剂孵育，并转染小干扰 RNA 敲低 RELB。
在原发性硬化性胆管炎、原发性胆汁性胆管炎、乙型或丙型肝炎慢性感染、自身免疫性肝炎或酒精性肝病患者的肝组织中，与对照组肝组织相比，我们检测到胆管细胞中 RELB 的核易位增加和反应性胆管形成的 LTB 水平增加。与人胆管细胞不同，暴露于 LTB 会使 RELB 敲低的胆管细胞增殖。包括胆管反应、卵圆细胞激活和胆管纤维化在内的 Cyld 小鼠表型完全从 Cyld/Relb 小鼠中消失。与对照小鼠的肝脏相比，Cyld 小鼠（但不是 Cyld/Relb 小鼠）的肝脏中细胞因子（LTB；CD40；和肿瘤坏死因子超家族[TNFSF]成员 TNFSF11[RANKL]、TNFSF13B[BAFF]和 TNFSF14[LIGHT]）编码的 mRNA 水平升高。然而，这些品系的小鼠在暴露于 CCl 时发展出相似水平的肝纤维化。与 DDC 饮食喂养的对照小鼠相比，Cyld 小鼠和 Cyld/Relb 小鼠在 DDC 饮食上具有改善的肝功能。
慢性肝病患者的反应性胆管中 LTB 水平升高，RELB 核易位增加。RELB 是 Cyld 小鼠胆管反应和胆管纤维化发展所必需的。从 LPC 中删除 RELB 和 CYLD 可保护小鼠免受 DDC 诱导的胆汁淤积性肝纤维化。
