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

游离 DNA 通过 TLR9 激活驱动非酒精性脂肪性肝炎

• 批准号: 9378394
• 负责人: WAJAHAT Zafar MEHAL
• 金额: --
• 依托单位: VA CONNECTICUT HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-10-01 至 2020-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9378394
• 关键词: Acute; Ankle; Award; Biochemical; Cell Line; Cells; Cessation of life; Characteristics; Cirrhosis; Clinical; Clinical Trials; DNA; DNA receptor; Data; Development; Etiology; Fatigue; Fatty Liver; Grant; Health; Healthcare; Hepatocyte; Human; Icterus; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Interleukin-1 beta; Investigation; Kupffer Cells; Ligands; Liver; Liver Failure; Liver diseases; Malignant Neoplasms; Malignant neoplasm of liver; Metabolic syndrome; Mitochondria; Mitochondrial DNA; Modeling; Molecular; Morbidity - disease rate; Mus; Obesity; Oxides; Pancreas; Pathway interactions; Patients; Phase; Phase II Clinical Trials; Plasma; Plasma Cells; Population; Primary carcinoma of the liver cells; Production; Pruritus; Publications; Reporter; Role; Safety; Source; Sterility; Swelling; TLR9 gene; Testing; Tissues; Up-Regulation; Veterans; Work; cell free DNA; cytokine; effective therapy; immune activation; inhibitor/antagonist; liver inflammation; liver injury; macrophage; monocyte; mortality; mouse model; nonalcoholic steatohepatitis; oxidation; public health relevance

 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.

 描述（由申请人提供）：非酒精性脂肪性肝炎（NASH）是退伍军人中最常见的肝脏疾病，其特征为肝脂肪变性、无菌性炎症和肝损伤[1]。NASH与代谢综合征相关，并且可以进展为肝硬化和癌症。NASH发展的机制尚不清楚，目前没有有效的治疗方法。我们已经表明，DNA受体TLR 9（TOLL样受体9）是肝脏和胰腺中急性无菌性炎症所必需的[2-4]。在这里，我们建议检查无细胞DNA（cfDNA）在TLR 9激活中的作用及其在小鼠和人类NASH中的重要性。我们的假设是来自肝细胞的线粒体DNA水平的增加通过Kupffer细胞、单核细胞和肝细胞上的TLR 9活化促进NASH的发展。 我们将专注于鉴定cfDNA的细胞来源和生物化学（氧化）状态，以及cfDNA在NASH中的功能作用。我们的数据将支持已经安全用于人类的TLR 9拮抗剂的临床试验进展。初步数据：i）在喂食HFD的小鼠和患有NASH的肥胖患者中，cfDNA的血浆水平较高，并且cfDNA是线粒体来源的。ii）来自NASH受试者的血浆具有完整线粒体（mt）的增加，并且大多数在微粒（MP）中。iii）来自患有NASH的小鼠的血浆DNA是TLR 9的有效配体。iv）当去除MP时，来自NASH患者的血浆激活TLR 9报告细胞系的能力增加显著降低。v）NASH患者血浆中mtDNA氧化态的增加。vi）TLR 9的全身缺失在NASH的HFD模型中导致较少的NASH。vii）巨噬细胞上TLR 9的选择性缺失导致更少的NASH。viii）来自喂食HFD的小鼠的肝细胞mtDNA具有更大程度的氧化和更大的刺激炎性途径而不是抗炎途径的能力。ix）TLR 9拮抗剂IRS 954保护小鼠免受NASH。目的1：确定NASH中负责TLR 9激活的DNA的细胞来源和生化特征。目的2：鉴定TLR 9活化对a）单核细胞和枯否细胞和B）肝细胞的细胞特异性作用。目标三：确定mtDNA氧化对促炎通路选择性激活的作用，并在NASH小鼠模型中测试临床可用的TLR 9拮抗剂。该基金将确定NASH中升高的TLR 9配体血浆cfDNA的细胞起源，并确定NASH的发展是否增加DNA作为TLR 9配体的能力。它将进一步确定KC、单核细胞和肝细胞上的TLR 9活化在HFD NASH发展中的作用。最后，临床上可用的TLR 9拮抗剂在NASH小鼠模型中的功效的证明将允许合理地进展到临床试验。