A role for miRNAs in adenosine-dependent alternative macrophage activation

摘要： 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。在 巨噬细胞对激活刺激作出反应，产生细胞因子和生长因子，介导这些反应。 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活化由IFNg和/或TLR激动剂如LPS介导，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 TLR激动剂与腺苷A2 A/A2 B受体共同介导的新的IL 4 Ra非依赖性途径 (A2AR/A2 BR）激动剂。这种协同途径抑制TNF α和IL-12表达，并诱导VEGF 和IL-10表达，将巨噬细胞转变为我们称为“M2 d”的M2样表型。这 新的途径需要LPS依赖性诱导A2 AR和A2 BR表达，诱导HIF-1a 表达，并通过在转录后水平抑制磷脂酶-Cb 2（PLCb 2）表达， 使其mRNA不稳定。我们最近发现一个miRNAs的子集在细胞内被特异性调控， 通过单独的LPS（miR-155、miR-146 a、miR-146 b、miR-221和miR-222）或通过组合的LPS/ A2 AR/A2 BR攻击（miR-483、miR-877、miR-337- 5 p、miR-546和miR-494上调，而miR-770- 547上调）。 5 p、miR-487 b、miR-220、miR-212和miR-712被强烈下调）。在本提案中，我们将首先 分析LPS特异性调控的miRNAs亚组对PLCb 2稳定性的影响 通过测定这些miRNAs的模拟物和拮抗剂对荧光素酶表达的影响， 我们的pPLC <$2 - 3 'UTR-Luc报告基因质粒在RAW 264.7细胞中的表达。然后，我们将确定具体的 在LPS处理的巨噬细胞中由A2 AR/A2 BR激动剂调节的导致“M2 d”活化的miRNA 巨噬细胞基因的表达。我们假设，在这个特定的小分子RNA的子集中发挥作用，在开关 巨噬细胞从M1到“M2 d”表型。我们将研究这些的模拟物和拮抗剂的作用， miRNA对TNF α和IL-12（由M1激活产生的旗舰细胞因子）的巨噬细胞表达的影响，以及 VEGF和IL-10（由M2 d激活产生的旗舰因子）。最后，我们将分析 LPS对巨噬细胞中PLCb 2 mRNA的不稳定作用。我们假设LPS调节了 PLCb 2 3 'UTR与调控因子如参与调控的miRNA或RNA结合蛋白， 稳定mRNA。miR-466 L和RNA结合蛋白HuR和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PLCb 2 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， 诱变这些研究将为巨噬细胞的差异基因调控提供新的见解 在诱导“M2 d”活化的条件下。

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.