A role for miRNAs in adenosine-dependent alternative macrophage activation

描述(申请人提供)：巨噬细胞在调节炎症、伤口愈合以及先天和后天免疫方面发挥关键作用。作为对激活刺激的反应，巨噬细胞产生细胞因子和生长因子来调节这些过程。巨噬细胞的激活被归类为“经典”(炎症，M1)和“替代”(血管生成或创伤愈合，M2)。M1激活是由干扰素介导的。和/或TLR激动剂，如脂多糖，而M2的激活被广泛定义为由IL4或IL13通过共同的IL4ra介导。我们发现了一种由TLR激动剂和腺苷A2A/A2B受体(A2AR/A2BR)激动剂共同介导的IL4Ra非依赖性通路。这种协同作用途径抑制肿瘤坏死因子？和IL-12的表达，并诱导血管内皮生长因子和IL-10的表达，将巨噬细胞转变为M2样表型，我们称之为M2d。这一新的途径需要依赖内毒素诱导A2AR和A2BR的表达，诱导HIF-1a的表达，并通过其mRNA的失稳在转录后水平抑制磷脂酶-CB2(PLCb2)的表达。我们最近发现，在巨噬细胞中，miRNAs的一个子集被脂多糖(miR-155、miR146a、miR-146b、miR-221和miR-222)或脂多糖/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)在巨噬细胞中特异性调节。在本提案中，我们将首先通过确定这些miRNAs的模拟物和拮抗剂对我们的pPLCb2-3‘UTR-Luc报告质粒在RAW264.7细胞中荧光素酶表达的影响，来分析脂多糖特异性调控的miRNAs亚群对PLCb2 mRNA稳定性的影响。然后，我们将确定由A2AR/A2BR激动剂调控的特定miRNAs在脂多糖处理的巨噬细胞中的作用(S)，这将导致巨噬细胞基因表达的M2d激活。我们假设这一特定亚群中的miRNAs在将巨噬细胞从M1表型转换为“M2d”表型中发挥作用。我们将研究这些miRNAs的模拟物和拮抗剂对巨噬细胞表达肿瘤坏死因子？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，我们将分析内毒素引起巨噬细胞PLCb2mRNA失稳的机制。我们假设脂多糖调节PLCb2 3‘UTR与调节因子的相互作用，如miRNAs或RNA结合蛋白，这些调节因子参与稳定mRNA。在3‘非编码区中存在miR-466L以及RNA结合蛋白Hur和TTP的保守重叠位点。我们将通过过度表达或抑制这些因素，以及通过定点突变改变3‘UTR中的结合位点，来分析这些因素在调节PLCb2 mRNA稳定性中的作用。这些研究应该为巨噬细胞在诱导“M2d”激活的条件下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方面发挥关键作用。巨噬细胞对微环境刺激表现出极高的敏感性，并可被激活以表达一系列调控炎症或伤口愈合反应的基因。我们建议研究miRNAs在激活的巨噬细胞基因表达调控中的作用，以及RNA的稳定性和周转可能如何影响基因表达。

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.