Innate Immune Response to Hepatocyte Death

 描述(申请人提供)：肝脏损伤由病毒性肝炎感染、登革热等其他感染、乙醇等化学物质和生物毒素引起。原发性肝细胞损伤是由先天免疫系统介导的免疫病理过程中并发的，但其机制尚不清楚。这在一定程度上是因为实验模型，如缺血-再灌注和CCl4给药，同时影响多种类型的肝细胞。为了阐明肝脏免疫病理的机制，我们建立了一种新的模型来研究肝细胞死亡的后果。利用基因治疗载体在小鼠肝细胞中特异性表达白喉毒素受体，然后给予白喉毒素导致肝损伤。在血清丙氨酸氨基转移酶(ALT)急剧升高的同时，趋化因子基因迅速上调，混合髓系细胞大量涌入。我们做了两个引人注目的观察。首先，病理是通过Toll样受体-3(TLR-3)介导的，这表明RNA是重要的分子触发因素。其次，当我们分离和分离不同的肝细胞亚群时，到目前为止，肝细胞的先天免疫反应最显著。这给了我们第二个假设，即肝细胞是感知肝细胞损伤和协调先天免疫反应的主要推动者。在具体目标1中，我们通过实验直接解决核糖核酸的抑制，正式证明核糖核酸是信号，并确定什么形式的核糖核酸(S)可能是相关的。在特定的目标2中，我们解剖肝损伤以确定不同形式的肝细胞死亡的作用，测试肝细胞是否直接识别TLR-3介导的信号，然后检验肝细胞通过AsialoGylcoProtein受体与死亡的邻居结合的能力对这种识别至关重要的假设。到目前为止，我们的初步工作主要集中在肝细胞特异性白喉毒素模型上，但在特定的目标3中，我们使用乙肝病毒诱导的免疫病理学的实验模型来验证这一假设，即RNA和TLR3依赖的肝损伤发生在更生理的背景下。这些实验将深入了解TLR3介导的未被探索的肝损伤机制，并测试其更广泛的适用性。拟议工作的两个方面加强了其全球意义。首先，我们使用白喉毒素引起伤害，这是一种潜在的生物威胁。改善白喉毒素引起的细胞损伤的新方法将在发生生物攻击时保护种群方面具有重要价值其次，在特定目标1和3的新实验中使用了一种重组核糖核酸酶融合蛋白，该蛋白经过基因工程后可用作人类治疗剂，并已进入第二阶段临床试验。这意味着，如果体内RNA的降解确实改善了肝脏的免疫病理学，那么就有一条直接的途径将这一发现转化为临床医学。

 DESCRIPTION (provided by applicant): Liver injury results from viral hepatitis infection, from other infections such as Dengue Fever, from chemicals such as ethanol, and from biological toxins. Primary hepatocellular injury in complicated by immunopathology mediated by the innate immune system, but the mechanisms are poorly understood. This is partly because experimental models such as ischemia-reperfusion and CCl4 administration impact on many liver cell types simultaneously. To clarify the mechanisms of liver immunopathology we have created a novel model to study the consequences of hepatocyte death. A gene therapy vector is used to express the Diphtheria Toxin Receptor specifically in mouse hepatocytes, then administration of Diphtheria Toxin results in liver injury. There is rapid up-regulation of chemokine genes and a mixed myeloid influx, coinciding with a sharp elevation in serum Alanine Aminotransaminase (ALT). We made two striking observations. First, the pathology is mediated via Toll-Like Receptor-3 (TLR-3), suggesting the hypothesis that RNA is the important molecular trigger. Second, when we dissociate and isolate different subsets of liver cells, the innate immune response is by far most dramatic in hepatocytes. This gives us a second hypothesis that the hepatocytes are the prime movers in sensing hepatocellular injury and orchestrating the innate immune response. In Specific Aim 1 we directly address the RNA DAMP through experiments to formally prove that RNA is the signal, and to identify what form(s) of RNA are likely to be relevant. In Specific Aim 2, we dissect the liver injury to determine the role of different forms of hepatocyte death, test whether hepatocytes directly recognize the TLR-3 mediated signal, and then test the hypothesis that the capacity of hepatocytes to take up their dead neighbors through the AsialoGylcoprotein Receptor is critical for such recognition. So far we have focused our preliminary work on the hepatocyte-specific Diphtheria Toxin model, but in Specific Aim 3 we test the hypothesis that RNA- and TLR3-dependent liver injury occurs in a more physiological context, using an experimental model of the immunopathology induced by Hepatitis B Virus. These experiments will give insight into the mechanism of an under-explored mechanism of liver injury, mediated through TLR3, and test its wider applicability. Two aspects of the proposed work enhance its global significance. First, we provoke injury using Diphtheria Toxin, which is a potential biological threat. New ways to ameliorate Diphtheria Toxin-induced cell damage will be of great value in protecting the population in the event of a biological attack Secondly, new experiments in Specific Aims 1 and 3 use a recombinant RNase fusion protein that is genetically engineered for use as a human therapeutic agent, and is already in Phase II clinical trials. This means that if the in vivo degradation of RNA in fact ameliorates liver immunopathology, there is an immediate pathway to translate this discovery to clinical medicine.