Cross talk between epithelial, inflammatory and mesenchymal cells in the development of portal fibrosis

 描述（由申请人提供）：人们对了解胆道损伤与门脉纤维化之间的机制关系非常感兴趣，这是慢性胆管疾病进展的主要机制。先天性肝纤维化（CHF）和Caroli病（CD）是由PKHD 1基因突变引起的遗传性胆管病，PKHD 1基因编码纤维囊蛋白，其特征在于胆管发育不全、节段性导管扩张和进行性门静脉纤维化伴门静脉高压症。在CHF和CD中，胆管细胞功能障碍和门静脉纤维化是由胆管上皮中的遗传缺陷引起的，而不是由坏死性炎症损伤引起的，因此，代表了用于阐明胆管细胞在胆道疾病的门静脉纤维化中的作用的模型疾病。我们建议在CHF/CD小鼠模型中研究这些机制，该模型在Pkhd 1中携带缺失突变（Pkhd 1del 4/del 4小鼠）。 我们的初步数据突出了Pkhd 1del 4/del 4小鼠的几个有趣的特征。包括：a）胆管纤维化和门静脉高压症在12个月内缓慢发展，沿着胆管发育不良恶化; B）在早期疾病阶段，存在胆管周围细胞浸润的进行性积累，所述胆管周围细胞浸润包含约60%的巨噬细胞和高达20%的aSMA阴性胶原蛋白阳性细胞（纤维细胞），而α-SMA阳性门静脉肌成纤维细胞的积累在前六个月后变得显著; c）Pkhd 1del 4/del 4分离的胆管细胞分泌增加量的CXCL 1和CXCL 10，并显示在Ser 675处β-连环蛋白的PKA依赖性磷酸化增加; d）在Pkhd 1del 4/del 4胆管细胞中，CXCL 1和CXCL 10的分泌是β-连环蛋白介导的。 基于这些观察，我们提出，由于pSer-675-β-catenin的转录激活增加，Pkhd 1del 4/del 4胆管细胞分泌能够募集巨噬细胞的趋化因子巨噬细胞协调纤维化的沉积和从早期缓慢纤维化阶段到后期更活跃阶段的过渡，其特征在于巨噬细胞M2极化的增加。因此，在Pkhd 1del 4/del 4小鼠中门静脉纤维化的建立是由β-连环蛋白依赖性胆管细胞从胆管上皮分泌趋化因子启动的。这一系列事件是“副炎症”的典型实例，即对持续细胞功能障碍的适应性反应，产生低级炎症反应并导致纤维化机制的激活。为了证明这一新的假设，我们将追求以下具体目标：目标#1：阐明增加的pSer 675-β-连环蛋白信号传导如何刺激CXCL 1和CXCL 10的分泌。目的#2：检查巨噬细胞在Pkhd 1del 4/del 4小鼠纤维化形成中的作用。目标3：调查 Pkhd 1del 4/del 4小鼠中肌成纤维细胞的组织发生，以及纤维细胞的作用。 这些研究将为胆管细胞功能障碍和副炎症在门静脉纤维化中的作用提供一个新的模型。调控信号的知识可能会导致新的治疗策略。

 DESCRIPTION (provided by applicant): There is considerable interest in understanding the mechanistic relationships between biliary damage and portal fibrosis, the main mechanism of progression in chronic cholangiopathies. Congenital Hepatic Fibrosis (CHF) and Caroli disease (CD) are genetic cholangiopathies caused by mutations in PKHD1, the gene encoding for fibrocystin, characterized by biliary dysgenesis, segmental ductal dilations and progressive portal fibrosis with portal hypertension. In CHF and CD, cholangiocyte dysfunction and portal fibrosis are caused by a genetic defect in the biliary epithelium, rather than by necroinflammatory damage and thus, represent a model disease for elucidating the role of cholangiocytes in portal fibrosis of biliary diseases. We propose to study these mechanisms in a mouse model of CHF/CD, harboring a deleting mutation in Pkhd1 (Pkhd1del4/del4 mice). Our preliminary data highlights several interesting features of Pkhd1del4/del4 mice. Including: a) sevee biliary fibrosis and portal hypertension develop slowly over 12 months, along with worsening biliary dysgenesis; b) during early disease phases there is progressive accumulation of a peribiliary cell infiltrate that is comprised about 60% macrophages and up to 20% by aSMA-negative, collagen positive cells (fibrocytes), whereas the accumulation of a-SMA-positive portal myofibroblasts becomes significant after the first sixth months; c) Pkhd1del4/del4 isolated cholangiocytes secrete increased amount of CXCL1 and CXCL10 and show an increased PKA-dependent phosphorylation of ß-catenin at Ser675; d) secretion of CXCL1 and CXCL10 is ß-catenin- mediated in Pkhd1del4/del4 cholangiocytes. Based on these observations, we propose that, as a consequence of the increased transcriptional activation of pSer-675-ß-catenin, Pkhd1del4/del4 cholangiocytes secrete chemokines able to recruit macrophages (and possibly fibrocytes) to the pericystic area; macrophages orchestrate the deposition of fibrosis and the transition from the early slow fibrosis phase to the later more brisk phase marked by an increase in macrophage M2 polarization. Thus, establishment of portal fibrosis in Pkhd1del4/del4 mice is initiated by a ß-catenin-dependent cholangiocyte secretion of chemokines from the biliary epithelium. This sequence of events is a paradigmatic example of "para-inflammation" i.e. an adaptive response to a persistent cellular dysfunction, generating a low grade inflammatory response and resulting in activation of fibrogenic mechanisms. To demonstrate this novel hypothesis, we will pursue the following Specific Aims: Aim#1: Elucidate how increased pSer675-ß-catenin signaling stimulates the secretion of CXCL1 and CXCL10. Aim#2: Examine the role of macrophages in the establishment of fibrosis in Pkhd1del4/del4 mice. Aim#3: Investigate the histogenesis of myofibroblasts in Pkhd1del4/del4 mice, and the role of fibrocytes. These studies will provide a new model for role of cholangiocyte dysfunction and para-inflammation in portal fibrosis. Knowledge of the regulatory signaling could lead to new therapeutic strategies.