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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

基本信息

批准号：
10628032
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
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-05-21 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10628032
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-10 Interleukin-4 Interleukin-6 Knockout Mice Left Ligands Link Lipopolysaccharides MAP Kinase Gene Macrophage Malignant Neoplasms Microbe Molecular Mus Mutagenesis Nature Non-Insulin-Dependent Diabetes Mellitus Organ Pathogenesis Pathologic Processes Persons 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 Tissues Up-Regulation Western Blotting Work antagonist cell injury chemically induced colitis comparison control cytokine dextran sulfate sodium induced colitis dysbiosis experimental study gastrointestinal gut inflammation gut microbiome healing insight knock-down microbial mouse model mutant new therapeutic target novel novel therapeutics pathogenic bacteria pharmacologic programs protein protein interaction receptor recruit response therapeutic target therapeutically effective tissue repair transcriptome transcriptome sequencing treatment strategy tumor progression uptake

项目摘要

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 IBD是使全世界数百万人严重衰弱的疾病。免疫失调，微生物生态失调、遗传和环境因素，都有助于驱动疾病的慢性炎症。更具体地说，慢性炎性疾病是由炎性细胞因子的表达增加引起的以及免疫细胞的募集，包括功能失调的促炎巨噬细胞，并传播促炎性T细胞反应。为了开发新的免疫疗法，更深入的需要了解免疫稳态的机制，包括那些解决巨噬细胞功能障碍，这在使炎症持续存在中起着不可或缺的作用。因为分子机制在巨噬细胞中控制慢性炎症反应的机制尚未完全阐明或开发为治疗方法目标，这是一个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 GIV（a.k.a Girdin）是一种三聚体G蛋白Gαi的鸟嘌呤-核苷酸交换调节剂（GEM）， TLR 4的结合伴侣。随后的工作不仅深入验证和表征了这种相互作用，揭示了GIV是TLR 4信号传导的调节剂和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中受到抑制，在M2极化的原发性巨噬细胞，GIV-GEM依赖性G蛋白信号传导是抑制前和抗炎细胞因子响应于TLR 4的配体脂多糖（LPS）的上调。是假设GIV→Gαi级联通过转换巨噬细胞减少炎症并有利于愈合，从促炎M1到抗炎M2极化状态。本建议旨在阐明如何 GIV-GEM调节这样的“开关”，并揭示了GIV→Gαi信号轴失调的后果，最大免疫系统内的巨噬细胞面对最大的LPS储库的肠道（即，管腔微生物）。我们的目标是：1）解剖GIV在形成TLR 4信号体动力学过程中的作用，使用体外蛋白质-蛋白质相互作用测定、3D同源性模型引导的诱变、免疫沉淀试验、免疫荧光显微术和特异性单AA突变体; 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和评价细菌摄取和清除的测定; 和3）使用小鼠模型研究肠道炎症中GIV失调的后果炎症，例如，LPS激发; 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属），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 TLR 4-GIV信号传导轴作为巨噬细胞炎症反应中的决定性信号传导途径， GIV-GEM靶向治疗慢性炎症引发的疾病的原理验证。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Regulation of DNA damage response by trimeric G-proteins.

调节三聚体G蛋白的DNA损伤反应。

DOI：
10.1016/j.isci.2023.105973
发表时间：
2023-02-17
期刊：
ISCIENCE
影响因子：
5.8
作者：
Abd El-Hafeez, Amer Ali;Sun, Nina;Chakraborty, Anirban;Ear, Jason;Roy, Suchismita;Chamarthi, Pranavi;Rajapakse, Navin;Das, Soumita;Luker, Kathryn E.;Hazra, Tapas K.;Luker, Gary D.;Ghosh, Pradipta
通讯作者：
Ghosh, Pradipta

Deletion of intestinal epithelial AMP-activated protein kinase alters distal colon permeability but not glucose homeostasis.

DOI：
10.1016/j.molmet.2021.101183
发表时间：
2021-05
期刊：
Molecular metabolism
影响因子：
8.1
作者：
Olivier S;Pochard C;Diounou H;Castillo V;Divoux J;Alcantara J;Leclerc J;Guilmeau S;Huet C;Charifi W;Varin TV;Daniel N;Foretz M;Neunlist M;Salomon BL;Ghosh P;Marette A;Rolli-Derkinderen M;Viollet B
通讯作者：
Viollet B

Building unconventional G protein-coupled receptors, one block at a time.