Characterizing the role of WISP1 in liver pathophysiology

急性或慢性暴露于肝毒物后，肝脏会引起强烈的炎症反应。这种复杂反应的作用尚不完全清楚。在这项提议中，我们的目标是揭示基质细胞蛋白WISP1 （wnt诱导分泌蛋白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CCl4给药后，小鼠肝脏强烈诱导WISP1 mRNA表达，tgf - β可诱导小鼠原代肝细胞中WISP1的表达和分泌。此外，与野生型小鼠相比，WISP1敲除小鼠在急性CCl4中毒时表现出更严重的肝损伤，同时细胞因子（TNFalpha, IL-6, Ccl2）的诱导降低，中性粒细胞向小叶中心死细胞区域的募集减少。这些结果表明，WISP1可能是肝脏疾病中未被识别的炎症反应调节剂。我们的建议将集中在四个方面。首先，我们将确定CCl4急性损伤小鼠肝脏中WISP1的细胞来源，以及控制其表达的信号机制。采用野生型和WISP1 KO小鼠的肝组织进行原位杂交和免疫染色，检测肝组织中WISP1的表达。其次，我们将确定WISP1在系统性炎症反应综合征、t细胞介导的肝炎、纤维化和肝细胞癌小鼠模型中的作用，使用成熟的每种疾病小鼠模型。该模型将应用于野生型和WISP1 KO小鼠。此外，我们将测定WISP1在人类肝脏疾病样本（组织和血清）中的表达，并将其表达与疾病状态联系起来。第三，我们将研究急性肝损伤时WISP1对白细胞产生化学引诱反应的机制。我们将通过FACS分析确定野生型和WISP1小鼠肝损伤时哪些白细胞被不同地募集。此外，我们将利用人供体原代白细胞在体外评估WISP1的趋化潜能，并分析野生型和WISP1 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跟踪荧光标记的中性粒细胞和巨噬细胞的运动，以评估WISP1 KO小鼠诱导的迁移改变。第四，我们将通过多重试验或磷酸化信号换能器蛋白的免疫印迹，以及体外重组WISP1处理细胞的钙成像，确定介导WISP1对靶细胞作用的信号传导和转录途径。我们期望我们的项目将揭示WISP1在肝脏病理生理中影响免疫反应的贡献和机制，为这一新的肝脏疾病分子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.