STING and IRF3 activation by cyclic dinucleotides in early alcoholic liver disease

项目总结/摘要 在这里，我们试图研究直接参与干扰素基因刺激因子激活的成分 （STING），一种内质网跨膜蛋白，负责细胞内的关键事件， 早期酒精性肝病免疫发病机制STING的活化导致磷酸化 干扰素调节因子3（IRF3）是一种转录因子，在细胞因子的激活中起重要作用。 先天免疫和乙醇给药后肝细胞中促凋亡信号的激活。在 在免疫细胞中，STING被环状二核苷酸激活。2'3 '-cGAMP是哺乳动物环状二核苷酸， 由环GMP-AMP合酶（cGAS）（细胞内dsDNA传感器）合成。c-di-GMP，细菌 在90%以上的细菌中，二核苷酸由二鸟苷酸环化酶（DGC）合成。最近，循环 二核苷酸已显示通过间隙连接从细胞传递到细胞。之前我们已经报道 STING或IRF3缺陷的小鼠可以免受酒精性肝病的影响。在这里，我们展示了老鼠 保护cGAS缺乏的人免受急性或慢性加急性乙醇诱导的肝损伤， IRF3的磷酸化。我们还表明，药理学抑制肝脏缝隙连接， 急性或慢性加急性乙醇诱导的肝损伤。我们发现哺乳动物和细菌 环状二核苷酸能够激活原代肝细胞中的STING和IRF 3。最后，我们表明， 间隙连接在体外将乙醇诱导的IRF3磷酸化传播到邻近细胞。基于这些 根据这些发现，我们假设来自宿主动物和肠道微生物组的环状二核苷酸会聚在一起， STING和IRF3在早期酒精性肝病中的激活。 作为这项研究计划的一部分，提出的目标包括基础分子生物学，分析化学， 以及使用小分子抑制剂和病毒载体介导的基因转移技术， 在两种小鼠早期酒精性肝病模型中进行治疗干预。主要目标可以是 摘要如下： 1.检测和定量哺乳动物和细菌的环二核苷酸的血清，全肝裂解物后， 酒精性肝损伤 2.研究间隙连接介导的环状二核苷酸的时空动力学，导致乙醇- 诱导肝脏中的STING和IRF3活化。 3.表征细菌二鸟苷酸环化酶及其相关环二核苷酸作为 使用小分子药物治疗早期酒精性肝病的可行治疗靶点 抑制剂和/或腺相关病毒相关基因治疗。

PROJECT SUMMARY/ABSTRACT Here we seek to investigate components directly involved in activation of Stimulator of Interferon Genes (STING), an endoplasmic reticulum trans-membrane protein that is responsible for key events in immunopathogenesis of early alcoholic liver disease. Activation of STING leads to phosphorylation of Interferon Regulatory Factor 3 (IRF3), a transcription factor that plays an important role in the activation of innate immunity and in activation pro-apoptotic signaling in hepatocytes after ethanol administration. In immune cells, STING is activated by cyclic dinucleotides. 2'3'-cGAMP is a mammalian cyclic dinucleotide synthesized by cyclic GMP-AMP synthase (cGAS), an intracellular dsDNA sensor. c-di-GMP, the bacterial dinucleotide, is synthesized by diguanylate cyclase (DGC) in over 90% of bacteria. Recently, cyclic dinucleotides have been shown to pass from cell to cell through gap junctions. We have previously reported that mice deficient in STING or IRF3 are protected from alcoholic liver disease. Here, we show that mice deficient in cGAS are protected from acute or acute-on-chronic ethanol-induced liver injury and phosphorylation of IRF3. We also show that pharmacological inhibition of hepatic gap junctions protects from acute or acute-on-chronic ethanol-induced liver injury. We show evidence that both mammalian and bacterial cyclic dinucleotides are capable of activation STING and IRF3 in primary hepatocytes. Lastly, we show that gap junctions propagate ethanol-induced phosphorylation of IRF3 to neighboring cells in vitro. Based on these findings, we hypothesize that cyclic dinucleotides from the host animal and gut microbiome converge in activation of STING and IRF3 in early alcoholic liver disease. The aims proposed as part of this research proposal encompass basic molecular biology, analytical chemistry, as well as the use of small-molecule inhibitors and viral vector-mediated gene transfer technology as therapeutic interventions in two models of early alcoholic liver disease in mice. The main objectives can be summarized as follows: 1. Detect and quantify mammalian and bacterial cyclic dinucleotides in serum, whole liver lysate after ethanol-induced liver injury. 2. Study the spatiotemporal dynamics of gap junction-mediated cyclic dinucleotides that lead to ethanol- induced STING and IRF3 activation in the liver. 3. Characterize the potential of bacterial diguanylate cyclase and its associated cyclic dinucleotides as viable therapeutic targets in the treatment of early alcoholic liver disease using small-molecule inhibitors and/or adeno-associated virus associated gene therapy.