Novel sterile inflammatory pathways in alcoholic hepatitis

酒精性肝炎的新型无菌炎症途径

• 批准号: 10427122
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10427122
• 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; Phosphoric Monoester Hydrolases; Phosphorylation Site; Play; Process; Production; Proteins; Reactive Oxygen Species; Role; Signal Transduction; Site-Directed Mutagenesis; Sterility; Stress; T-cell protein tyrosine phosphatase; Testing; Therapeutic; Time; Treatment Protocols; United States Department of Veterans Affairs; Veterans; base; 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）影响了大量退伍军人，并且死亡率没有 在过去的十年里发生了巨大的变化。由于AH的发病机制具有鲜明的特点， 非常严重的肝细胞损伤，无菌性炎症和中性粒细胞募集;我们的目标是确定分子 在这些过程中发挥重要作用的目标。我们发现Src同源2结构域的失调 含（Shc）胶原相关蛋白在钙网蛋白（CTR）暴露中起重要作用， AH期间细胞死亡，加剧无菌炎症信号级联反应。研究Shc的调节作用 我们证明了AH患者的Shc是诱导的，并且在AH患者中抑制肝细胞Shc。 具有AH的动物模型导致炎症和氧化应激显著减弱。肝细胞中 Shc在CTR易位和暴露中发挥作用，CTR是决定细胞免疫原性的关键DAMP 酒精导致死亡。此外，在中性粒细胞中，Shc/NOX 2依赖性信号诱导中性粒细胞 细胞外陷阱形成，从而进一步增强AH中的炎性损伤。基于这些，我们 假设Shc信号激活导致免疫原性细胞死亡和促氧化不育 AH中的炎症通路。我们提出了三个具体目标，以解决由 主要假设： 我们的第一个目标是确定Shc蛋白参与氧化还原信号传导的分子机制 以及酒精性肝炎期间肝细胞中钙网蛋白（CTR）作为DAMP的暴露。 a）我们建议研究氧化还原介导的p52 Shc激活的机制。为了评估 在p52 Sh信号方面，将通过定点诱变产生缺失突变体，以描绘p52 Sh信号的关键 磷酸化位点。将结合ROS产生和CTR评估应激信号传导。 易位B）我们将使用两种生物化学方法定义引发CTR从ER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，我们将重点关注 酒精性肝炎中性粒细胞胞外陷阱（NET）的形成。a）我们建议进行研究， Shc/NOX 2在NET形成中的关键作用，并询问下游信号级联。我们发现 p52 Shc直接结合p47 phox NOX 2亚基，并直接在酶活化中起作用。的 将通过生物层干涉测定法和光泽精/Amplex红测定法评估相互作用 生产我们将研究与p52 Shc结合相关的p47 phox动员机制， 脂筏中的NOX 2复合物的活化，并与NET形成相关（通过FRET显微镜， 动态活细胞成像）。B）由于NET的持续存在会进一步引发炎症，我们将研究 AH中有缺陷的NET吞噬机制。我们的第三个目标是研究Shc信号转导的体内作用 对酒精性肝炎的炎症、氧化损伤和NETosis的影响。我们将检验减少的假设 Shc通过限制免疫原性死亡和NET形成改善酒精性肝炎，通过使用条件性细胞- 特异性ShcKO小鼠（ShchepKO和Shcneutko）。我们将通过使用PAD 4-/-小鼠来补充这些研究， 在NET形成和适应性中性粒细胞转移实验中有缺陷。提供翻译 与这些研究相关，我们建议单独使用Shc抑制剂或PAD 4抑制剂，或联合使用， AH小鼠模型。