A role for miRNAs in adenosine-dependent alternative macrophage activation

描述（由申请人提供）：巨噬细胞在调节炎症、伤口愈合以及先天性和获得性免疫中起关键作用。巨噬细胞对激活刺激作出反应，产生细胞因子和生长因子，介导这些过程。巨噬细胞的活化被分类为“经典”（炎症，M1）和“替代”（血管生成或伤口愈合，M2）。M1的激活是由IFN？和/或TLR激动剂如LPS，而M2活化被广义地定义为通过共同的IL 4 Ra由IL 4或IL 13介导。我们已经发现了一种新的IL 4 Ra非依赖性途径，其由TLR激动剂与腺苷A2 A/A2 B受体（A2 AR/A2 BR）激动剂一起介导。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为“M2 d”的M2样表型。这种新途径需要LPS依赖性诱导A2 AR和A2 BR表达，诱导HIF-1 α表达，并通过使其mRNA不稳定来抑制磷脂酶-Cb 2（PLCb 2）在转录后水平的表达。我们最近发现巨噬细胞中的一个miRNAs亚组仅受LPS的特异性调节（miR-155、miR-146 a、miR-146 b、miR-221和miR-222）或通过组合的LPS/A2 AR/A2 BR激发（miR-483、miR-877、miR-337- 5 p、miR-546和miR-494上调，而miR-770- 5 p、miR-487 b、miR-220、miR-212和miR-712强烈下调）。在这个提议中，我们将首先分析由LPS特异性调节的miRNA亚组对PLCb 2 mRNA稳定性的影响，通过确定这些miRNA的模拟物和拮抗剂对来自我们的pPLCb 2 - 3 'UTR-Luc报告质粒的荧光素酶表达的影响在RAW 264.7细胞中。然后，我们将确定由A2 AR/A2 BR激动剂在LPS处理的巨噬细胞中调节的特定miRNA的作用，其导致巨噬细胞基因表达的“M2 d”活化。我们假设，在这个特定的子集中的miRNA在将巨噬细胞从M1表型转换为“M2 d”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TNF？和IL-12（由M1活化产生的旗舰细胞因子），以及VEGF和IL-10（由M2 d活化产生的旗舰因子）。最后，我们将分析LPS在巨噬细胞中使PLCb 2 mRNA不稳定的机制。我们假设LPS调节PLCb 2 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-466 L和RNA结合蛋白HuR和TTP的保守重叠位点存在于3 'UTR中。我们将分析这些因子在调控PLCb 2 mRNA稳定性中的作用，通过过表达或抑制这些因子，并通过定点突变修饰3 'UTR中的结合位点。这些研究应提供新的见解差异基因调控巨噬细胞的条件下，诱导“M2 d”激活。 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。我们打算研究miRNAs在活化巨噬细胞基因表达调控中的作用，以及研究RNA稳定性和周转率如何影响基因表达。

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.