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: 10152363
• 负责人: Pradipta Ghosh
• 金额: $ 48.7万
• 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/10152363
• 关键词: 3-Dimensional; Acute; Address; Anti-Inflammatory Agents; Arthritis; Atherosclerosis; Binding; Biochemical; Bioinformatics; Biological Assay; Bone Marrow; CREB1 gene; Cardiovascular Diseases; Cells; Chemicals; 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; Immunoblotting; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Immunoprecipitation; Immunotherapy; In Vitro; 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; Pharmacology; Physiological Processes; Play; Process; Repression; Role; Salmonella; Sequence Homology; Shapes; Signal Pathway; Signal Transduction; Signaling Protein; T cell response; T cell therapy; TLR4 gene; TNF gene; Tissues; Up-Regulation; Work; base; cell injury; cytokine; dysbiosis; experimental study; gastrointestinal; gut microbiome; healing; inflammatory disease of the intestine; 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 疾病（IBD）是疾病，在全球范围内严重衰弱了数百万人。免疫失调，微生物 营养不良，遗传学和环境因素都导致驱动疾病的慢性感染。 更具体地说，慢性炎症性疾病是通过炎症细胞因子表达增加的促进 招募免疫力，包括功能失调的促进性巨噬细胞，吸引，吸引 并传播促炎的T细胞反应。为了开发新颖的免疫疗法，更深层次 需要了解免疫稳态的机制，包括解决巨噬细胞的机制 功能障碍在永久注射中起着不可或缺的作用。因为分子机制 控制巨噬细胞中的慢性炎症反应尚未被充分阐明或剥削为治疗 目标，这既需要紧急又不满足。使用生物信息学（序列同源性）我们首先确定 GIV（又称吉尔丁），一种用于三聚体G蛋白GαI的鸟嘌呤核苷酸交换调节剂（GEM），作为一种新颖 TLR4的绑定合作伙伴。随后的工作不仅验证并深入验证了这种互动，还可以 揭示GIV是TLR4信号传导的调节剂，也是巨噬细胞极化和 炎性细胞因子表达。 GIV表达在M1中被抑制，并在M2偏振原则中进行了更新 巨噬细胞和GIV-GEM依赖性G蛋白信号传导是抑制前和 抗炎细胞因子对TLR4，Lipopolysachharide（LPS）的配体的上调。这是 假设GIV→GαI级联反应可通过切换巨噬细胞减少感染并有利于治愈 从促炎的M1到抗炎M2极化状态。该建议旨在阐明如何 GIV-GEM调节这种“开关”，并揭示了放松管制的GIV→GαI信号轴的后果 肠道最大的免疫系统内巨噬细胞面对最大的LP储备 微生物）。我们的目的是：1）剖析GIV在塑造TLR4信号体动力学中的作用 使用体外蛋白质 - 蛋白质相互作用测定的组合，3D词素模型引导的炎症 诱变，免疫沉淀测定，免疫荧光显微镜和特定的单AA突变体； 2） 确定GIV使用RNA-Seq转录组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和评估细菌摄取和清除率的测定； 3）研究使用小鼠模型在肠道注射中GIV失调的后果 炎症，例如LPS挑战； DSS诱导的化学结肠炎和感染性结肠炎（沙门氏菌和 柑橘类），有或没有GIV-GEM信号传导的药理学调节剂。获得的见解将有助于建立 TLR4-GIV信号轴是巨噬细胞炎症反应中的决定性信号通路，并提供 靶向GIV-GEM在慢性炎症推动的疾病中的原则证明。