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

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.