Characterizing the role of WISP1 in liver pathophysiology

在急性或慢性暴露于肝毒物时，在肝脏中引起强烈的炎症反应。这一复杂反应的作用尚未得到充分理解。在这项提案中，我们的目标是解开基质细胞蛋白WISP 1（Wnt诱导的分泌蛋白-1）在急性和慢性肝病中的作用。我们的初步数据表明，急性和慢性CCl 4给药后，小鼠肝脏中WISP 1 mRNA被强烈诱导，TGF β可诱导原代小鼠肝细胞中WISP 1表达和分泌。此外，与野生型小鼠相比，WISP 1基因敲除小鼠在急性CCl 4中毒时表现出增强的肝损伤，伴随着较低的细胞因子（TNF α、IL-6、CCl 2）诱导和减少的中性粒细胞向小叶中心死细胞区域的再结合。这些结果表明，WISP 1可能是肝脏疾病中炎症反应的未被认识的调节剂。我们的建议将集中在四个方面。首先，我们将确定WISP 1的细胞来源在小鼠肝脏急性损伤后，由四氯化碳，和控制其表达的信号转导机制。将使用野生型和WISP 1 KO小鼠的肝组织，通过原位杂交和免疫染色检测肝组织中的WISP 1。其次，我们将确定WISP 1在全身炎症反应综合征，T细胞介导的肝炎，纤维化和肝细胞癌的小鼠模型中的作用，使用完善的小鼠模型为每种疾病。该模型将应用于野生型和WISP 1 KO小鼠。此外，我们将确定WISP 1在人类肝病样本（组织和血清）中的表达，并将其表达与疾病状态相关联。第三，我们将研究急性肝损伤时WISP 1产生白细胞化学引诱反应的机制。我们将通过FACS分析确定野生型和WISP 1 KO小鼠中哪些白细胞在肝损伤后差异募集。此外，我们将使用来自人类供体的原代白细胞在体外评估WISP 1的趋化潜力，并分析野生型和WISP 1 KO小鼠肝脏中趋化因子、细胞因子和粘附分子的表达。此外，我们将使用双光子显微镜来跟踪荧光标记的中性粒细胞和巨噬细胞的运动，以评估在WISP 1 KO小鼠中诱导的迁移改变。第四，我们将确定的信号和转录途径介导的WISP 1对靶细胞的影响，通过多重分析或免疫印迹磷酸化的信号转导蛋白，和钙离子成像与重组WISP 1在体外处理的细胞。我们希望我们的项目将揭示WISP 1在肝脏病理生理学中影响免疫反应的贡献和机制，为肝脏疾病中这种新型分子参与者的诊断和治疗应用奠定基础。

Upon acute or chronic exposure to hepatotoxicants, a strong inflammatory response is elicited in the liver. The role of this complex response is not fully understood. In this proposal, we aim to unravel the role of the matricellular protein WISP1 (Wnt-induced secreted protein-1) in acute and chronic liver disease. Our preliminary data indicates that WISP1 mRNA is strongly induced in mouse liver after acute and chronic CCl4 administration, and that WISP1 expression and secretion can be induced in primary mouse hepatocytes by TGFbeta. Furthermore, WISP1 knock out mice showed enhanced liver damage upon acute CCl4 intoxication compared to wild type counterparts, acompanied by lower induction of cytokines (TNFalpha, IL-6, Ccl2) and reduced recriutment of neutrophils to the centrilobular dead cell areas. These results suggest that WISP1 may be an unrecognized modulator of inflammatory responses in liver disease. Our proposal will be focused on four aspects. First, we will determine the cell source of WISP1 on mouse liver after acute injury by CCl4, and the signaling mechanisms controlling its expression. The detection of WISP1 in liver tissue will be performed by in situ hybridization and immunostaining using liver tissue from wild type and WISP1 KO mice. Second, we will determine the role of WISP1 in mouse models of sistemic inflammatory response syndrome, T-cell-mediated hepatitis, fibrosis and hepatocellular carcinoma, using well-established mouse models for each disease. The models will be applied in wild type and WISP1 KO mice. In addition, we will determine the expression of WISP1 in human liver disease samples (tissue and serum) and correlate its expression to disease state. Third, we will investigate the mechanisms by which WISP1 generates a chemoattractant response for leukocytes upon acute liver injury. We will establish which leukocytes become differentially recruited upon liver damage in wild type and WISP1 KO mice by FACS analysis. In addition, we will assess the chemoattractant potential of WISP1 in vitro using primary leukocytes from human donors and analyze the expression of chemokines, cytokines and adhesion molecules in wild type and WISP1 KO mouse liver. Also, we will use 2-photon microscopy to track the movement of fluorescently-labeled neutrophils and macrophages to assess the migration alterations induced in WISP1 KO mice. Fourth, we will identify the signaling and transcriptional pathways mediating WISP1 effects on its target cells, by muliplex assays or immunoblot for phosphorylated signal transducer proteins, and calcium imaging on cells treated with recombinant WISP1 in vitro. We expect that our project will unveil the contribution and the mechanisms by which WISP1 influences immune responses in liver pathophysiology, setting the basis for diagnostic and therapeutic applications focused on this novel molecular player in liver disease.