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、先天免疫和获得性免疫中发挥关键作用。在对激活刺激的反应中，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激活由IFN介导？和/或TLR激动剂如LPS，而M2激活被广泛定义为通过常见的IL4Ra由IL4或IL13介导。我们发现了一种新的il4ra独立途径，该途径由TLR激动剂和腺苷A2A/A2B受体（A2AR/A2BR）激动剂介导。这种协同途径抑制TNF？和IL-12的表达，并诱导VEGF和IL-10的表达，将巨噬细胞转变为我们称之为“M2d”的m2样表型。这种新途径需要依赖lps诱导A2AR和A2BR表达，诱导HIF-1a表达，并通过破坏PLCb2 mRNA的不稳定在转录后水平抑制磷脂酶- cb2 （PLCb2）表达。我们最近发现，巨噬细胞中有一部分mirna被LPS单独调控（miR-155、miR146a、miR-146b、miR-221和miR-222），或被LPS/ A2AR/A2BR联合调控（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被强烈下调）。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报告质粒荧光素酶表达的影响，分析LPS特异性调节的mirna亚群对PLCb2 mRNA稳定性的影响。然后，我们将确定由A2AR/A2BR激动剂调节的特定mirna在lps处理的巨噬细胞中的作用，从而导致巨噬细胞基因表达的“M2d”激活。我们假设这个特定亚群中的mirna在巨噬细胞从M1表型转换为“M2d”表型中发挥作用。我们将研究这些mirna的模拟物和拮抗剂对巨噬细胞TNF？和IL-12 （M1激活产生的旗舰细胞因子），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稳定的机制。我们假设LPS调节PLCb2 3'UTR与调节因子（如参与稳定mRNA的mirna或RNA结合蛋白）的相互作用。miR-466L和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，以及通过位点定向诱变修饰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。

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.