Modulation of Macrophage Polarization by Heterotrimeric G proteins: Implications of Gastrointestinal Inflammation

异源三聚体 G 蛋白对巨噬细胞极化的调节：胃肠道炎症的影响

• 批准号: 10397537
• 负责人: Pradipta Ghosh
• 金额: $ 48.73万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-21 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10397537
• 关键词: 3-Dimensional; Acute; Address; Anti-Inflammatory Agents; Arthritis; Atherosclerosis; Binding; Biochemical; Bioinformatics; Biological Assay; Bone Marrow; CREB1 gene; Cardiovascular Diseases; Cells; Chronic; Citrobacter; Colitis; Complex; Couples; Data; Diabetes Mellitus; Disease; Disease model; Dose; Environmental Risk Factor; Equilibrium; Event; Face; Family member; Fibrosis; Functional disorder; GTP-Binding Proteins; Genetic; Goals; Guanine Nucleotides; Heterotrimeric GTP-Binding Proteins; Homeostasis; Homology Modeling; Immune; Immune system; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Immunoprecipitation; Immunotherapy; In Vitro; Infectious colitis; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Innate Immune Response; Innate Immune System; Interleukin-1; Interleukin-10; Interleukin-4; Interleukin-6; Knockout Mice; Left; Ligands; Link; Lipopolysaccharides; MAP Kinase Gene; Malignant Neoplasms; Microbe; Molecular; Mus; Mutagenesis; Nature; Non-Insulin-Dependent Diabetes Mellitus; Organ; Pathogenesis; Pathologic Processes; Persons; Pharmacology; Physiological Processes; Play; Process; Repression; Role; Salmonella; Sequence Homology; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; Spontaneous colitis; T cell response; T cell therapy; TLR4 gene; TNF gene; Tissues; Up-Regulation; Western Blotting; Work; antagonist; base; cell injury; chemically induced colitis; cytokine; dextran sulfate sodium induced colitis; dysbiosis; experimental study; gastrointestinal; gut inflammation; gut microbiome; healing; insight; knock-down; macrophage; microbial; mouse model; mutant; new therapeutic target; novel; novel therapeutics; pathogenic bacteria; programs; protein protein interaction; receptor; recruit; response; therapeutic target; therapeutically effective; tissue repair; transcriptome; transcriptome sequencing; treatment strategy; tumor progression; uptake

Abstract: Chronic inflammatory disorders, whether it is arthritis, atherosclerosis, type II diabetes, or inflammatory bowel disease (IBD) are diseases that severely debilitate millions of people worldwide. Immune deregulation, microbial dysbiosis, genetics, and environmental factors, all contribute to the chronic inflammation that drives the disease. More specifically, chronic inflammatory disorders are fueled by increased expression of inflammatory cytokines and the recruitment of immune cells, including dysfunctional proinflammatory macrophages, that recruit, incite and propagate pro-inflammatory T-cell responses. In order to develop novel immunologic therapies, a deeper understanding of the mechanisms of immune homeostasis is needed, including those that address macrophage dysfunction, which plays an integral role in perpetuating inflammation. Because the molecular mechanisms that govern chronic inflammatory responses in macrophages are yet to be fully elucidated or exploited as therapeutic targets, this is a need that is both urgent and unmet. Using bioinformatics (sequence homology) we first identified GIV (a.k.a Girdin), a guanine-nucleotide exchange modulator (GEM) for trimeric G protein, Gαi, as a novel binding partner of TLR4. Subsequent work not just validated and characterized this interaction in-depth, but also revealed that GIV is a regulator of TLR4 signaling and a key determinant of macrophage polarization and inflammatory cytokine expression. GIV expression is suppressed in M1 and upregulated in M2-polarized primary macrophages, and GIV-GEM-dependent G protein signaling is required for the suppression of pro- and upregulation of anti-inflammatory cytokines in response to the ligand for TLR4, lipopolysachharide (LPS). It is hypothesized that the GIV→Gαi cascade reduces inflammation and favors healing by switching macrophages from the pro-inflammatory M1 to the anti-inflammatory M2 polarized state. This proposal seeks to elucidate how GIV-GEM regulates such a "switch" and reveal the consequences of deregulated GIV→Gαi signaling axis in the gut where macrophages within the largest immune system face-off the largest reservoir of LPS (i.e., luminal microbes). Our aims are: 1) Dissect GIV's role in shaping the dynamics of TLR4 signalosome during inflammation using a combination of in vitro protein-protein interaction assays, 3D-homology model-guided mutagenesis, immunoprecipitation assays, immunofluorescence microscopy, and specific single-AA mutants; 2) Determine GIV's ability to modulate macrophage inflammatory programs using RNA-seq transcriptome analysis, cell-based macrophage polarization assays, and assays evaluating bacterial uptake and clearance; and 3) Investigate the consequences of GIV dysregulation in intestinal inflammation using mouse models of inflammation e.g., LPS challenge; DSS-induced chemical colitis and infectious colitis (Salmonella and Citrobacter), with or without pharmacologic modulators of GIV-GEM signaling. Insights gained will help establish the TLR4-GIV signaling axis as a decisive signaling pathway in macrophage inflammatory responses and provide proof-of-principle for targeting of GIV-GEM in diseases that are fueled by chronic inflammation.

摘要： 慢性炎症性疾病，无论是关节炎、动脉粥样硬化、II型糖尿病还是炎症性肠病 疾病(IBD)是使全世界数百万人严重虚弱的疾病。免疫放松管制，微生物 生物失调、遗传和环境因素都是导致这种疾病的慢性炎症的原因。 更具体地说，慢性炎症性疾病是由炎性细胞因子表达增加引起的。 免疫细胞的招募，包括功能失调的促炎巨噬细胞，招募、煽动 并促进促炎T细胞反应。为了开发新的免疫疗法，更深层次的 需要了解免疫内稳态的机制，包括处理巨噬细胞的机制。 功能障碍，这在持久的炎症中起着不可或缺的作用。因为它的分子机制 巨噬细胞慢性炎症反应的调控尚未完全阐明或作为治疗手段加以利用 目标，这是一种既紧迫又未得到满足的需要。利用生物信息学(序列同源性)，我们首先确定了 三聚体G蛋白的鸟核苷酸交换调节剂GIV(又名GYDIN)--GαI TLR4的结合伙伴。随后的工作不仅深入地验证和表征了这种交互作用，而且还 GIV是TLR4信号的调节者，也是巨噬细胞极化的关键决定因素 炎性细胞因子表达。GIV表达在M1中被抑制，在M2极化的初级中上调 巨噬细胞和GIV依赖的GEM依赖的G蛋白信号转导是抑制前和 TLR4的配体--脂多糖(LPS)可上调抗炎细胞因子。它是 假设GIV→GαI级联通过转换巨噬细胞减少炎症并促进愈合 从促炎的M1到抗炎的M2极化状态。这项提议试图阐明如何 GIV-GEM调控了这种“开关”，并揭示了解除调控的GIV→GαI信号轴在 肠道中最大的免疫系统内的巨噬细胞与最大的内毒素储存库(即肠腔)对峙 微生物)。我们的目标是：1)剖析GIV在形成TLR4信号体动力学过程中的作用 使用3D同源模型引导的体外蛋白质-蛋白质相互作用分析相结合的炎症 诱变、免疫沉淀分析、免疫荧光显微镜和特异的单一氨基酸突变体； 用RNA-seq转录组确定GIV调节巨噬细胞炎症程序的能力 分析、基于细胞的巨噬细胞极化分析和评估细菌摄取和清除的分析； 3)用小鼠模型研究GIV失调在肠道炎症中的后果 炎症，例如，内毒素攻击；DSS诱导的化学性结肠炎和感染性结肠炎(沙门氏菌和 柠檬酸杆菌)，加或不加GIV-GEM信号的药物调节剂。所获得的见解将有助于建立 TLR4-GIV信号轴作为巨噬细胞炎症反应中的决定性信号通路并提供 针对慢性炎症引发的疾病靶向GIV-GEM的原则证明。