Novel sterile inflammatory pathways in alcoholic hepatitis

酒精性肝炎的新型无菌炎症途径

• 批准号: 9890961
• 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/9890961
• 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; Estrogen receptor positive; 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/antagonist; 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)影响退伍军人的比例高得不成比例，而且死亡率并没有 在过去的十年里发生了巨大的变化。AS的发病机制具有鲜明的特点，其特点是 非常严重的肝细胞损伤、无菌炎症和中性粒细胞募集；我们的目标是确定分子 在这些过程中扮演重要角色的目标。我们发现，Src同源2结构域的失调 含有(Shc)胶原相关蛋白在钙网蛋白(CTR)暴露和免疫原性中发挥重要作用 急性期的细胞死亡，加剧了无菌炎症信号的级联。研究Shc的调节作用 在AH中，我们证明了在AH患者中诱导Shc，并抑制肝细胞Shc。 急性胰腺炎动物模型可显著减轻炎症和氧化应激。在肝细胞中 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潮湿因素 酒精引起的死亡。此外，在中性粒细胞中，Shc/NOX2依赖的信号诱导中性粒细胞 细胞外陷阱的形成，从而进一步加重急性脑出血的炎性损伤。基于这些，我们 假设Shc信号的激活导致免疫原性细胞死亡和促氧化剂不育 急性髓细胞白血病的炎症途径。我们提出了三个具体目标，以解决 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的分子机制。 在酒精性肝炎期间，肝细胞内的钙网蛋白(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。要评估机械性 在p52Sh信号方面，缺失突变体将通过定点突变产生，以描绘密钥 磷酸化位点。应激信号将与ROS产生和CTR一起进行评估 易位。B)我们将使用这两种生化方法定义从ER引发CTR退出的信号 以及新型实时活细胞共聚焦荧光显微镜。在我们的第二个目标中，我们将重点关注角色 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，评估 Shc/NOX2在网络形成中的关键作用，并询问下游信号级联。我们发现 P52Shc直接与p47Phox NOX2亚基结合，直接作用于酶的激活。这个 ROS的相互作用将通过生物层干涉测定法和光敏素/Amplex红测定法进行评估 制作。我们将研究p47Phox动员与p52Shc结合相关的机制方面， 脂筏中NOX2复合体的激活并与网络形成相关(通过FRET显微镜和通过 动态活细胞成像)。B)由于以英语为母语的英语教师持续存在会加剧炎症，我们将调查 急性酒精性肝炎患者网络吞噬功能缺陷的机制。我们的第三个目标是研究Shc信号的体内效应 酒精性肝炎的炎症、氧化损伤与网络中毒我们将检验这样的假设：减少 SHC通过限制免疫原性死亡和净形成改善酒精性肝炎，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-/-小鼠来补充这些研究 在网络形成和适应性中性粒细胞转移实验中存在缺陷。要提供翻译版本 与这些研究相关，我们建议单独使用Shc抑制剂或PAD4抑制剂，或联合使用 小鼠急性胰腺炎模型。