Cell-free DNA is a driver of non-alcoholic steatohepatitis via TLR9 activation

 描述(申请人提供)：非酒精性脂肪性肝炎(NASH)是退伍军人中最常见的肝脏疾病，其特征是肝脏脂肪变性、无菌炎症和肝脏损伤[1]。NASH与代谢综合征有关，并可进展为肝硬化和癌症。控制NASH发展的机制尚不清楚，目前还没有有效的治疗方法。我们已经证明，DNA受体TLR9(Toll样受体9)是肝脏和胰腺急性无菌炎症所必需的[2-4]。在这里，我们建议研究无细胞DNA(CfDNA)在TLR9激活中的作用及其在小鼠和人类NASH中的重要性。我们的假设是，肝细胞线粒体DNA水平的增加通过激活Kupffer细胞、单核细胞和肝细胞上的TLR9而促进NASH的发生。我们将着重于鉴定cfDNA的细胞来源和生化(氧化)状态，以及cfDNA在NASH中的功能作用。我们的数据将支持TLR9拮抗剂的临床试验进展，该拮抗剂已经在人体上安全使用。初步数据：i)服用HFD的小鼠和患有NASH的肥胖患者的血浆cfDNA水平较高，cfDNA来自线粒体。2)NASH患者血浆中完整线粒体(MT)增多，且多数为微粒(MP)。Iii)NASH小鼠的血浆DNA是TLR9的有效配体。IV)去除MP后，NASH患者血浆激活TLR9报告细胞系的能力显著降低。(5)NASH患者血浆线粒体DNA氧化态增加。VI)在NASH的HFD模型中，TLR9的整体缺失导致较少的NASH。(Vii)TLR9在巨噬细胞上的选择性缺失导致NASH减少。(Viii)喂饲HFD的小鼠肝细胞mtDNA氧化程度更高，有更强的刺激炎症途径的能力，但不能抗炎。9)TLR9拮抗剂IRS954保护小鼠免受NASH的伤害。目的1：确定NASH中TLR9激活相关DNA的细胞来源和生化特性。目的：研究TLR9激活对单核细胞、库普弗细胞和肝细胞的细胞特异性影响。目的3：确定线粒体DNA氧化在促炎途径选择性激活中的作用，并在NASH小鼠模型中测试临床可用的TLR9拮抗剂。这笔赠款将确定NASH中TLR9配体血浆cfDNA升高的细胞来源，并确定NASH的发展是否增加了DNA作为TLR9配体的能力。这将进一步确定TLR9在KC、单核细胞和肝细胞上的激活在HFD NASH发生中的作用。最后，临床可用的TLR9拮抗剂在NASH小鼠模型中的有效性的证明将使临床试验的合理进展成为可能。

 DESCRIPTION (provided by applicant): Nonalcoholic steatohepatitis (NASH) is the most common liver disease among veterans and is characterized by hepatic steatosis, sterile inflammation and liver damage [1]. NASH is associated with the metabolic syndrome, and can progress to cirrhosis and cancer. The mechanisms governing NASH development are not known, and currently there is no effective therapy. We have shown that the DNA receptor TLR9 (TOLL like receptor 9) is required for acute sterile inflammation in the liver and pancreas [2-4]. Here we propose to examine role of cell-free DNA (cfDNA) in TLR9 activation and its importance in murine and human NASH. Our hypothesis is that increased levels of mitochondrial DNA from hepatocytes contributes to the development of NASH via TLR9 activation on Kupffer cells, monocytes and hepatocytes. We will focus on identifying the cellular source and biochemical (oxidation) status of cfDNA, and the functional role of cfDNA in NASH. Our data will support the progression to clinical trials of a TLR9 antagonist that has already been safely used in humans. Preliminary data: i) Plasma levels of cfDNA are higher in mice fed a HFD and in obese patients with NASH, and the cfDNA is of mitochondrial origin. ii) Plasma from NASH subjects has an increase in intact mitochondria (mt), and the majority are in microparticles (MP). iii) Plasma DNA from mice with NASH is a potent ligand for TLR9. iv) Increased ability of plasma from NASH patients to activate a TLR9 reporter cell line is significantly reduced when MP are removed. v) Increase in oxidation state of mtDNA in plasma from patients with NASH. vi) Total body deletion of TLR9 results in less NASH in a HFD model of NASH. vii) Selective deletion of TLR9 on macrophages results in less NASH. viii) Hepatocytes mtDNA from mice fed a HFD has a greater degree of oxidation and a greater ability to stimulate inflammatory, but not anti-inflammatory pathway. ix) The TLR9 antagonist IRS954 protects mice against NASH. Aim 1: Identify the cellular origin and biochemical characteristics of DNA responsible for TLR9 activation in NASH. Aim 2: Identify the cell specific effects of TLR9 activation on a) monocytes and Kupffer cells and b) hepatocytes. Aim 3: Identify the role of mtDNA oxidation on the selective activation of pro-inflammatory pathways, and test a clinically available TLR9 antagonist in a mouse model of NASH. This grant will identify the cellular origins of the elevated the TLR9 ligand plasma cfDNA in NASH, and identify if the development of NASH increases the ability of DNA to function as a ligand for TLR9. It will further establish the role of TLR9 activation on KC, monocytes, and hepatocytes in the development of HFD NASH. Finally demonstration of the efficacy of a clinically available TLR9 antagonist in a mouse model of NASH will allow for the rational progression to clinical trials.