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

• 批准号: 8385795
• 负责人: Samuel Joseph Leibovich
• 金额: $ 17.59万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8385795
• 关键词: 3' 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; Interferons; 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; TNF gene; Tissues; Vascular Endothelial Growth Factors; Wound Healing; acquired immunity; angiogenesis; cytokine; cytosolic receptor; insight; mRNA Expression; mRNA Stability; macrophage; monocyte; novel; protein expression; receptor; research study; response

DESCRIPTION (provided by applicant): 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 IFN? 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 TNF? 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 pPLCb2-3'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 TNF? 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. PUBLIC HEALTH RELEVANCE: Macrophages play a key role in regulating inflammation, wound healing and angiogenesis. Macrophages exhibit exquisite sensitivity to micro-environmental stimuli, and can be activated to express sets of genes that regulate either inflammatory or wound healing responses. We propose to investigate the role of miRNAs in the regulation of gene expression in activated macrophages, as well as to investigate how RNA stability and turnover may influence gene expression.

描述（由申请人提供）：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。为了响应激活刺激，巨噬细胞产生介导这些过程的细胞因子和生长因子。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 激活是由 IFN 介导的吗？和/或 TLR 激动剂，如 LPS，而 M2 激活广泛定义为由 IL4 或 IL13 通过常见的 IL4Ra 介导。我们发现了一种新的不依赖于 IL4Ra 的途径，该途径由 TLR 激动剂和腺苷 A2A/A2B 受体 (A2AR/A2BR) 激动剂共同介导。这种协同途径抑制 TNF？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”。这一新途径需要 LPS 依赖性诱导 A2AR 和 A2BR 表达，诱导 HIF-1a 表达，并通过使其 mRNA 不稳定来在转录后水平抑制磷脂酶-Cb2 (PLCb2) 表达。我们最近发现，巨噬细胞中的一部分 miRNA 在单独的 LPS（miR-155、miR146a、miR-146b、miR-221 和 miR-222）或 LPS/A2AR/A2BR 联合攻击（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)。在本提案中，我们将首先通过确定这些 miRNA 的模拟物和拮抗剂对 RAW264.7 细胞中 pPLCb2-3'UTR-Luc 报告质粒的荧光素酶表达的影响，分析受 LPS 特异性调节的 miRNA 亚组对 PLCb2 mRNA 稳定性的影响。然后，我们将确定 LPS 处理的巨噬细胞中受 A2AR/A2BR 激动剂调节的特定 miRNA 的作用，从而导致巨噬细胞基因表达的“M2d”激活。我们假设这个特定子集中的 miRNA 在巨噬细胞从 M1 表型转变为“M2d”表型中发挥作用。我们将研究这些 miRNA 的模拟物和拮抗剂对巨噬细胞 TNF 表达的影响？和 IL-12（M1 激活产生的旗舰细胞因子），以及 VEGF 和 IL-10（M2d 激活产生的旗舰因子）。最后，我们将分析LPS在巨噬细胞中破坏PLCb2 mRNA稳定性的机制。我们假设 LPS 调节 PLCb2 3'UTR 与参与稳定 mRNA 的调节因子（例如 miRNA 或 RNA 结合蛋白）的相互作用。 miR-466L 以及 RNA 结合蛋白 HuR 和 TTP 的保守重叠位点存在于 3'UTR 中。我们将通过过表达或抑制这些因子以及通过定点诱变修饰 3'UTR 中的结合位点来分析这些因子在调节 PLCb2 mRNA 稳定性中的作用。这些研究应该为诱导“M2d”激活的条件下巨噬细胞的差异基因调控提供新的见解。 公共卫生相关性：巨噬细胞在调节炎症、伤口愈合和血管生成中发挥着关键作用。巨噬细胞对微环境刺激表现出敏锐的敏感性，并且可以被激活以表达调节炎症或伤口愈合反应的基因组。我们建议研究 miRNA 在调节活化巨噬细胞基因表达中的作用，以及研究 RNA 稳定性和周转如何影响基因表达。