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A role for miRNAs in adenosine-dependent alternative macrophage activation

miRNA 在腺苷依赖性巨噬细胞激活中的作用

基本信息

批准号：
8717565
负责人：
Samuel Joseph Leibovich
金额：
$ 22.42万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8717565
关键词：
3&apos Untranslated Regions Adenosine Agonist Binding Sites Bioinformatics Biological Assay Blood Cell surface Cells Exhibits Gene Expression Gene Expression Regulation Genes Growth Factor Health Human IL4 gene Inflammation Inflammatory Interleukin-10 Interleukin-12 Interleukin-13 Ligation Luciferases Macrophage Activation Mediating Messenger RNA MicroRNAs Modification Mus Natural Immunity Organism Pathway interactions Phagocytes Phenotype Phospholipase Plasmids Play Process Purinergic P1 Receptors RNA RNA Stability RNA-Binding Proteins Reading Reporter Role Site Site-Directed Mutagenesis Stimulus Subgroup Tissues Vascular Endothelial Growth Factors Wound Healing abstracting acquired immunity angiogenesis cytokine cytosolic receptor insight mRNA Expression mRNA Stability macrophage monocyte novel protein expression receptor research study response

项目摘要

Abstract: Macrophages play key roles in regulating inflammation, wound healing and innate and acquired immunity. In response to activational stimuli, macrophages produce cytokines and growth factors that mediate these processes. Activation of macrophages has been classified as "classical" (inflammatory, M1) and "alternative" (angiogenic or wound healing, M2). M1 activation is mediated by IFNg and/or TLR agonists such as LPS, while M2 activation is broadly defined as mediated by IL4 or IL13 through the common IL4Ra. We have discovered a novel IL4Ra-independent pathway that is mediated by TLR agonists together with adenosine A2A/A2B receptor (A2AR/A2BR) agonists. This synergistic pathway suppresses TNFa and IL-12 expression and induces VEGF and IL-10 expression, switching macrophages into an M2-like phenotype that we have termed "M2d". This novel pathway requires the LPS-dependent induction of A2AR and A2BR expression, induction of HIF-1a expression, and suppression of phospholipase-Cb2 (PLCb2) expression at the post-transcriptional level by destabilization of its mRNA. We have found recently that a subset of miRNAs are specifically regulated in macrophages by LPS alone (miR-155, miR146a, miR-146b, miR-221 and miR-222), or by combined LPS/ A2AR/A2BR challenge (miR-483, miR-877, miR-337-5p, miR-546 and miR-494 are up-regulated, while miR-770- 5p, miR-487b, miR-220, miR-212 and miR-712 are strongly down-regulated). In this proposal we will first analyze the effects of the subgroup of miRNAs that are specifically regulated by LPS on the stability of PLCb2 mRNA by determining the effects of mimics and antagonists of these miRNAs on luciferase expression from our pPLC¿2-3'UTR-Luc reporter plasmid in RAW264.7 cells. We will then determine the role(s) of the specific miRNAs that are regulated by A2AR/A2BR agonists in LPS-treated macrophages that result in "M2d" activation of macrophage gene expression. We hypothesize that miRNAs in this specific subset play a role in switching macrophages from an M1 to an "M2d" phenotype. We will study the effects of mimics and antagonists of these miRNAs on macrophage expression of TNFa and IL-12 (flagship cytokines produced by M1 activation), and VEGF and IL-10 (flagship factors produced by M2d activation). Finally, we will analyze the mechanism of destabilization of PLCb2 mRNA by LPS in macrophages. We hypothesize that LPS regulates the interaction of the PLCb2 3'UTR with regulatory factors such as miRNAs or RNA binding proteins that are involved in stabilizing the mRNA. Conserved overlapping sites for miR-466L and for the RNA binding proteins HuR and TTP are present in the 3'UTR. We will analyze the role of these factors in regulating PLCb2 mRNA stability by over-expressing or inhibiting these factors, and by modifying the binding sites in the 3'UTR by site-directed mutagenesis. These studies should provide novel insights into differential gene regulation in macrophages under conditions that induce "M2d" activation.

抽象的：巨噬细胞在调节炎症、伤口愈合以及先天和获得性免疫方面发挥着关键作用。在巨噬细胞对激活刺激作出反应，产生介导这些作用的细胞因子和生长因子流程。巨噬细胞的激活被分为“经典”（炎症，M1）和“替代” （血管生成或伤口愈合，M2）。 M1 激活由 IFNg 和/或 TLR 激动剂（例如 LPS）介导，而 M2 激活广义上定义为由 IL4 或 IL13 通过常见的 IL4Ra 介导。我们发现了一个由 TLR 激动剂与腺苷 A2A/A2B 受体共同介导的新型 IL4Ra 独立途径 (A2AR/A2BR) 激动剂。该协同途径抑制 TNFa 和 IL-12 表达并诱导 VEGF 和 IL-10 表达，将巨噬细胞转变为 M2 样表型，我们称之为“M2d”。这新途径需要 LPS 依赖性诱导 A2AR 和 A2BR 表达，诱导 HIF-1a 表达，并在转录后水平抑制磷脂酶-Cb2 (PLCb2) 表达其 mRNA 不稳定。我们最近发现 miRNA 的一个子集在单独使用 LPS（miR-155、miR146a、miR-146b、miR-221 和 miR-222）或组合使用 LPS/ A2AR/A2BR 挑战（miR-483、miR-877、miR-337-5p、miR-546 和 miR-494 上调，而 miR-770- (5p，miR-487b、miR-220、miR-212 和 miR-712 强烈下调)。在这个提案中，我们首先将分析LPS特异性调控的miRNA亚群对PLCb2稳定性的影响通过确定这些 miRNA 的模拟物和拮抗剂对荧光素酶表达的影响来分析 mRNA 我们的 pPLC¿2-3'UTR-Luc 报告质粒在 RAW264.7 细胞中。然后我们将确定具体的角色 LPS 处理的巨噬细胞中受 A2AR/A2BR 激动剂调节的 miRNA，导致“M2d”激活巨噬细胞基因表达。我们假设这个特定子集中的 miRNA 在转换中发挥作用巨噬细胞从 M1 到“M2d”表型。我们将研究这些的模拟物和拮抗剂的影响 miRNA 对巨噬细胞表达 TNFa 和 IL-12（M1 激活产生的旗舰细胞因子）的影响 VEGF 和 IL-10（M2d 激活产生的旗舰因子）。最后我们来分析一下其机制 LPS 在巨噬细胞中使 PLCb2 mRNA 不稳定。我们假设 LPS 调节以下相互作用： PLCb2 3'UTR 具有调节因子，例如 miRNA 或 RNA 结合蛋白，这些因子参与稳定 mRNA。 miR-466L 和 RNA 结合蛋白 HuR 的保守重叠位点 TTP 存在于 3'UTR 中。我们将分析这些因素在调节 PLCb2 mRNA 稳定性中的作用过表达或抑制这些因子，并通过定点修饰 3'UTR 中的结合位点诱变。这些研究应该为巨噬细胞差异基因调控提供新的见解在诱导“M2d”激活的条件下。