Novel sterile inflammatory pathways in alcoholic hepatitis

酒精性肝炎的新型无菌炎症途径

• 批准号: 10554317
• 负责人: Natalie J. Torok
• 金额: --
• 依托单位: VETERANS ADMIN PALO ALTO HEALTH CARE SYS
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2024-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10554317
• 关键词: Address; Affect; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcohols; Animal Model; Area; Attenuated; Binding; Biochemical; Biological Assay; Cell Death; Cells; Cessation of life; Collagen; Complement; Complex; Data; Diet; Disease; Enzyme Activation; Fatty acid glycerol esters; Fibrosis; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Goals; Heavy Drinking; Hepatocyte; Hospitalization; Inflammation; Inflammatory; Inflammatory Response; Injury; Interferometry; Label; Link; Liver; Liver diseases; Lucigenin; Mediating; Membrane; Membrane Microdomains; Microscopy; Modeling; Molecular; Molecular Target; Mus; NADPH Oxidase; National Institute on Alcohol Abuse and Alcoholism; Neutrophil Infiltration; Oxidation-Reduction; Oxidative Stress; Pathogenesis; Pathway interactions; Patients; Phagocytosis; Phosphorylation Site; Play; Process; Production; Protein Tyrosine Phosphatase; Proteins; Reactive Oxygen Species; Role; Signal Induction; Signal Transduction; Site-Directed Mutagenesis; Sterility; Stress; T-cell protein tyrosine phosphatase; Testing; Therapeutic; Time; Treatment Protocols; United States Department of Veterans Affairs; Veterans; calreticulin; cell type; endoplasmic reticulum stress; experimental study; extracellular; hepatocyte injury; immunogenic; immunogenic cell death; immunogenicity; improved; in vivo; inhibitor; live cell imaging; mortality; mouse model; mutant; neutrophil; novel; novel therapeutic intervention; oxidative damage; targeted treatment; therapeutic target

Alcoholic hepatitis (AH) affects disproportionally high numbers of veterans, and has a mortality rate that has not changed substantially over the last decade. As AH pathogenesis has distinct features and is characterized by very severe hepatocyte injury, sterile inflammation and neutrophil recruitment; our goal is to identify molecular targets that have significant roles in these processes. We found that dysregulation of the Src homology 2 domain containing (Shc) collagen-related proteins play important roles in calreticulin (CTR) exposure, and immunogenic cell death during AH, exacerbating sterile inflammatory signalling cascades. To study the regulatory role of Shc proteins in AH, we demonstrated that Shc was induced in patients with AH, and inhibition of hepatocyte Shc in an animal model with AH resulted in significantly attenuated inflammation and oxidative stress. In hepatocytes Shc played a role in CTR translocation and exposure, a critical DAMP that determines immunogenicity of cell death elicited by alcohol. Furthermore in neutrophils Shc/NOX2-dependent signals induced neutrophil extracellular trap formation thereby further augmenting inflammatory injury in AH. Based on these, we hypothesize that activation of Shc signals leads to immunogenic cell death and pro-oxidant sterile inflammatory pathways in AH. We propose three SPECIFIC AIMS to address the key areas generated by the main hypothesis: Our first aim is to determine the molecular mechanism by which Shc proteins are involved in redox signaling and calreticulin (CTR) exposure as DAMP in hepatocytes during alcoholic hepatitis. a) We propose to study the mechanism of redox-mediated p52Shc activation. To evaluate the mechanistic aspects of p52Sh signals, deletion mutants will be generated by site-directed mutagenesis to delineate the key phosphorylation sites. Stress signaling will be evaluated in conjunction with ROS production, and CTR translocation. b) We will define the signals eliciting CTR exit from the ER using both biochemical approaches and the novel real-time, live-cell confocal fluorescence microscopy. In our second aim we will focus on the role of neutrophil extracellular trap (NET) formation in alcoholic hepatitis. a) We propose studies that evaluate the key role of Shc/NOX2 in NET formation, and interrogate downstream signaling cascades. We discovered that p52Shc directly binds to the p47phox NOX2 subunit and directly plays a role in enzyme activation. The interaction will be assessed by biolayer interferometry assays and lucigenin/Amplex red assays for ROS production. We will study the mechanistic aspects of p47phox mobilization in correlation to p52Shc binding, activation of the NOX2 complex in lipid rafts and correlate to NET formation (by FRET microscopy, and by dynamic live cell imaging). b) As persistence of NETs can fuel further inflammation, we will investigate the mechanism of defective NET phagocytosis in AH. Our third aim is to study the in vivo effects of Shc signaling on inflammation, oxidative injury and NETosis in alcoholic hepatitis. We will test the hypothesis that reducing Shc improves alcoholic hepatitis by limiting immunogenic death and NET formation, by using conditional cell- specific ShcKO mice (ShchepKO and Shcneutko). We will complement these studies by using the PAD4-/- mice that are defective in NET formation, and adaptive neutrophil transfer experiments. To provide translational relevance to these studies, we propose to use Shc inhibitors or PAD4 inhibitors alone, or in combination in the mouse models of AH.

酒精性肝炎（AH）会影响不成比例的退伍军人，并且死亡率尚未 在过去的十年中发生了很大的变化。由于AH发病机理具有独特的特征，并以此为特征 非常严重的肝细胞损伤，无菌炎症和中性粒细胞募集；我们的目标是识别分子 在这些过程中具有重要作用的目标。我们发现SRC同源性2域的失调 含有（SHC）胶原蛋白相关蛋白在钙网蛋白（CTR）暴露和免疫原性中起重要作用 AH期间的细胞死亡，加剧无菌炎症信号级联。研究SHC的调节作用 AH中的蛋白质，我们证明了AH患者诱导SHC，并抑制肝细胞SHC 具有AH的动物模型导致明显减弱的炎症和氧化应激。在肝细胞中 SHC在CTR易位和暴露中发挥了作用，这是一种确定细胞免疫原性的严重潮湿 酒精引起的死亡。此外，中性粒细胞SHC/NOX2依赖性信号诱导中性粒细胞 细胞外陷阱形成，从而进一步增加了AH的炎症损伤。基于这些，我们 假设SHC信号的激活导致免疫原性死亡和促氧化无菌性 AH的炎症途径。我们提出了三个具体目标，以解决由 主要假设： 我们的第一个目的是确定SHC蛋白参与氧化还原信号的分子机制 在酒精性肝炎期间，肝细胞中的炎症（CTR）暴露为潮湿。 a）我们建议研究氧化还原介导的p52SHC激活的机制。评估机械 p52SH信号的各个方面，缺失突变体将通过定点诱变产生，以描绘钥匙 磷酸化位点。应力信号将与ROS的产生一起评估，并且CTR 易位。 b）我们将使用两种生化方法来定义从ER启发CTR退出的信号 以及新型的实时活细胞共聚焦荧光显微镜。在我们的第二个目标中，我们将专注于角色 酒精性肝炎中嗜中性粒细胞外陷阱（净）形成。 a）我们提出了评估的研究 SHC/NOX2在净形成中的关键作用，并询问下游信号级联。我们发现了这一点 p52SHC直接与p47phox NOX2亚基结合，并直接在酶活性中起作用。这 相互作用将通过Biolayer干涉法测定法和ROS的Lucigenin/Amplex红色测定法进行评估 生产。我们将研究p47phox动员与p52SHC结合相关的机械方面， 脂质筏中NOX2复合物的激活，并与净形成相关（通过FRET显微镜和通过 动态活细胞成像）。 b）由于网的持续性会加剧进一步的炎症，我们将调查 AH中有缺陷的净吞噬作用机理。我们的第三个目的是研究SHC信号的体内效应 关于酒精性肝炎的炎症，氧化损伤和肠病。我们将测试减少的假设 SHC通过使用条件细胞 - 特定的Shcko小鼠（Shchepko和Shcneutko）。我们将通过使用PAD4 - / - 鼠标来补充这些研究 在净形成和自适应嗜中性粒细胞转移实验方面有缺陷。提供翻译 与这些研究相关，我们建议仅使用SHC抑制剂或PAD4抑制剂，或者在 AH的鼠标模型。

项目成果

Strategies and endpoints of antifibrotic drug trials: Summary and recommendations from the AASLD Emerging Trends Conference, Chicago, June 2014.

• DOI: 10.1002/hep.27720
• 发表时间: 2015-08
• 期刊: Hepatology (Baltimore, Md.)
• 作者: Torok NJ;Dranoff JA;Schuppan D;Friedman SL
• 通讯作者: Friedman SL

NADPH oxidase 4 (Nox4) deletion accelerates liver regeneration in mice.

• DOI: 10.1016/j.redox.2020.101841
• 发表时间: 2021-04
• 期刊: Redox biology
• 影响因子: 11.4
• 作者: Herranz-Itúrbide M;López-Luque J;Gonzalez-Sanchez E;Caballero-Díaz D;Crosas-Molist E;Martín-Mur B;Gut M;Esteve-Codina A;Jaquet V;Jiang JX;Török NJ;Fabregat I
• 通讯作者: Fabregat I

Extracellular vesicles and ceramide: new mediators for macrophage chemotaxis?

• DOI: 10.1194/jlr.c066191
• 发表时间: 2016-02-01
• 期刊: JOURNAL OF LIPID RESEARCH
• 影响因子: 6.5
• 作者: Toeroek, Natalie J.

Editorial: Noninvasive Fibrosis Biomarkers in Patients With NASH With Diabetes.

• DOI: 10.1002/hep4.1662
• 发表时间: 2021-04
• 期刊: Hepatology communications
• 影响因子: 5.1
• 作者: Fan W;Torok NJ
• 通讯作者: Torok NJ

P300, A New Player in Mechanosensitivity and Activation of Cancer-Associated Fibroblasts.

• DOI: 10.1053/j.gastro.2018.05.002
• 发表时间: 2018-06
• 期刊: Gastroenterology
• 影响因子: 29.4
• 作者: Torok NJ