Project 2: Cholesterol Regulation of Inflammatory Macrophages in Atherosclerosis

项目2：动脉粥样硬化中炎症巨噬细胞的胆固醇调节

• 批准号: 10334095
• 负责人: Yury Miller
• 金额: $ 4.3万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-01 至 2022-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10334095
• 关键词: ATAC-seq; ATP binding cassette transporter 1; Agonist; Animal Model; Antibodies; Apolipoprotein A-I; Atherosclerosis; Automobile Driving; B-Lymphocyte Subsets; B-Lymphocytes; Binding Proteins; Biological Assay; Cardiovascular Diseases; Cell Communication; Cell physiology; Cells; Cellular Membrane; ChIP-seq; Cholesterol; Cholesterol Homeostasis; Chronic; Clinical; Complex; Cultured Cells; Cyclodextrins; DNA Sequencing Facility; Data; Desmosterol; Development; Diagnostic; Dimerization; Epigenetic Process; Epitopes; Excision; Experimental Designs; Feedback; Flow Cytometry; Gatekeeping; Gene Expression; Genetic Transcription; Human; Image; Immune; Immunity; Inflammation; Inflammatory; Inflammatory Response; Intervention; Knock-out; Knockout Mice; Leukocytes; Ligands; Liquid substance; Longitudinal cohort; Macromolecular Complexes; Malondialdehyde; Mediating; Membrane; Membrane Microdomains; Metabolic; Morbidity - disease rate; Mus; Myocardial Infarction; Oxides; Patients; Peptide Sequence Determination; Peripheral Blood Mononuclear Cell; Phenotype; Phospholipids; Play; Proteins; Regulation; Resources; Role; Signal Transduction; Solid; Source; Stroke; T-Lymphocyte; TLR4 gene; Testing; Training; Transgenic Mice; beta-Cyclodextrins; cholesterol control; diagnostic value; epigenetic memory; hypercholesterolemia; macrophage; metabolomics; mevalonate; mimetics; monocyte; mortality; novel strategies; overexpression; oxidation; oxidized lipid; peripheral blood; prevent; prognostic; prognostic value; receptor; recruit; scaffold; single cell proteins; transcriptome sequencing

Project 2 Summary Atherosclerosis is the primary cause of cardiovascular disease (CVD), which manifests in myocardial infarction and stroke, a major source of mortality and morbidity in the US. Hypercholesterolemia and chronic inflammation are leading causative factors in the development of atherosclerosis. Many inflammatory receptors and other proteins involved in the inflammatory response localize and become activated in cholesterol-rich membrane microdomains, often designated as lipid rafts, which provide a solid platform for protein assembly within a liquid membrane. Here, we propose the concept of inflammarafts, enlarged lipid rafts hosting assembled inflammatory signaling complexes, to help formalize the lipid raft-centric view of inflammation and its control by cholesterol metabolism. Cholesterol depletion from inflammarafts in activated cells disrupts inflammatory signaling, and the approaches that allow for targeting cholesterol removal selectively to inflammatory cells may be used for atheroprotection. In the context of atherosclerosis, monocytes and macrophages develop long-term adaptation, or trained immunity, which results in sustained inflammatory phenotypes and includes epigenetic memory, as well as transcriptional and metabolic alterations leading to a chronic inflammatory state. In this application, we will test the hypotheses that inflammarafts serve as a signaling platform that mediates cell reprogramming and that trained immune cells sustain lipid rafts to maintain the inflammatory response. We propose that this positive feedback mechanism between inflammarafts and metabolic, gene expression and epigenetic alterations plays a major role in the development of atherosclerosis. Specifically, to test the hypothesis that inflammarafts serve as gatekeepers and effectors of trained immunity in atherosclerosis, we will use inflammaraft assays to image and quantify lipid rafts, accessible cholesterol, membrane order, TLR4 dimerization and the assembly of other receptor complexes. RNA-seq, ATAC-seq, ChIPseq and metabolomics will be used to characterize trained immunity. To modulate ligand-dependent inflammaraft assembly, we will neutralize oxidized lipid DAMPs, abundant in plaques, in transgenic mice that overexpress oxidation-specific antibodies. APOA1, AAV-AIBP (apoA-I binding protein) and cyclodextrin interventions will be used for systemic and targeted depletion of cholesterol. Conversely, cholesterol and inflammarafts will be increased in inducible, macrophage-specific ABCA1/ABCG1 knockout mice. Statins/mevalonate and desmosterol mimetics will be used to modulate macrophage reprogramming. Importantly, we will evaluate the diagnostic and prognostic power of inflammaraft phenotyping of blood leukocytes in CVD. By using the resources of PPG’s clinical and single cell protein and RNA sequencing cores, we will test the hypotheses that the abundance of inflammarafts and raft-dependent receptor assemblies in peripheral blood monocytes and T and B cells reflect their trained immunity and inflammatory gene expression phenotypes and can be used for diagnostic and prognostic purposes in patients with CVD.

项目2摘要 动脉粥样硬化是心血管疾病（CVD）的主要原因，表现为心肌梗死 和中风，这是美国死亡率和发病率的主要来源。高胆固醇血症和慢性 炎症是动脉粥样硬化发展的主要致病因素。许多炎症受体 和其他参与炎症反应的蛋白质在富含胆固醇的 膜微区，通常称为脂筏，为蛋白质组装提供了坚实的平台 在液体膜中。在这里，我们提出了炎症筏的概念，扩大的脂筏托管 组装炎症信号复合物，以帮助正式确定炎症的脂筏中心观点， 其控制由胆固醇代谢。活化细胞中炎症因子的胆固醇消耗破坏了 炎症信号，以及允许选择性靶向胆固醇清除的方法， 炎性细胞可用于动脉粥样硬化保护。在动脉粥样硬化的背景下，单核细胞和 巨噬细胞发展长期适应，或训练免疫，这导致持续的炎症反应。 表型，包括表观遗传记忆，以及转录和代谢改变，导致 慢性炎症状态。在这个应用程序中，我们将测试的假设，炎症筏作为一个 信号平台，介导细胞重编程和训练的免疫细胞维持脂筏， 维持炎症反应。我们认为，这种积极的反馈机制之间， 炎症因子和代谢、基因表达和表观遗传学改变在发展中起着重要作用。 动脉粥样硬化具体地说，为了检验炎症筏作为炎症的看门人和效应器的假设， 在动脉粥样硬化的免疫训练中，我们将使用inflammaraft测定来成像和量化脂筏， 可接近的胆固醇、膜秩序、TLR 4二聚化和其他受体复合物的组装。 RNA-seq、ATAC-seq、ChIPseq和代谢组学将用于表征训练的免疫力。以调制 配体依赖性炎症因子组装，我们将中和氧化脂质DAMPs，丰富的斑块， 过表达氧化特异性抗体的转基因小鼠。APOA 1，AAV-AIBP（apoA-I结合蛋白） 和环糊精干预将用于胆固醇的全身性和靶向消耗。相反地， 胆固醇和炎性因子在可诱导的巨噬细胞特异性ABCA 1/ABCG 1敲除中会增加， 小鼠他汀类药物/甲羟戊酸和桥甾醇模拟物将用于调节巨噬细胞重编程。 重要的是，我们将评估血液炎症表型的诊断和预后能力。 CVD中的白细胞。通过利用PPG的临床和单细胞蛋白质和RNA测序核心资源， 我们将检验以下假设： 外周血单核细胞以及T和B细胞反映了它们训练的免疫和炎症基因表达 表型，并可用于诊断和预后的目的，在患者的CVD。