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.

抽象的： 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）是导致全世界数百万人严重衰弱的疾病。 生态失调、遗传和环境因素都会导致导致疾病的慢性炎症。 更具体地说，慢性炎症性疾病是由炎症细胞因子表达增加引起的 以及免疫细胞的招募，包括功能失调的促炎巨噬细胞，这些细胞会招募、煽动 并传播促炎性 T 细胞反应，以开发新的免疫疗法。 需要了解免疫稳态机制，包括那些涉及巨噬细胞的机制 功能障碍，在炎症持续存在中发挥着不可或缺的作用，因为其分子机制。 控制巨噬细胞慢性炎症反应的机制尚未完全阐明或用于治疗 我们首先利用生物信息学（序列同源性）确定了这一需求。 GIV（又名 Girdin），一种三聚体 G 蛋白 Gαi 的鸟嘌呤核苷酸交换调节剂 (GEM)，作为一种新型 随后的工作不仅深入验证和表征了这种相互作用，而且还验证了 TLR4 的结合伴侣。 揭示 GIV 是 TLR4 信号传导的调节剂，也是巨噬细胞极化和 炎症细胞因子的表达在 M1 中受到抑制，在 M2 极化的原代细胞中表达上调。 巨噬细胞和 GIV-GEM 依赖性 G 蛋白信号传导是抑制 pro-和 TLR4 配体脂多糖 (LPS) 导致抗炎细胞因子上调。 保留了 GIV→Gαi 级联通过切换巨噬细胞来减少炎症并有利于愈合 该提案旨在阐明如何从促炎 M1 到抗炎 M2 极化状态。 GIV-GEM 调节这样的“开关”并揭示 GIV→Gαi 信号轴失调的后果 肠道中最大的免疫系统中的巨噬细胞与最大的 LPS 储存库（即腔内 我们的目标是：1) 剖析 GIV 在塑造 TLR4 信号体动态过程中的作用。 结合体外蛋白质-蛋白质相互作用测定、3D 同源模型引导的炎症 诱变、免疫沉淀分析、免疫荧光显微镜和特定单 AA 突变分析 2) 使用 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测定以及评估细菌摄取和清除的测定； 3) 使用小鼠模型研究 GIV 失调对肠道炎症的影响 炎症，例如 DSS 诱发的化学性结肠炎和感染性结肠炎（沙门氏菌和 柠檬酸杆菌），有或没有 GIV-GEM 信号传导的药理调节剂，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 GIV-GEM 靶向治疗慢性炎症引起的疾病的原理验证。