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、先天免疫和获得性免疫等方面发挥着关键作用。在……里面 巨噬细胞对激活刺激的反应是产生细胞因子和生长因子来调节这些 流程。巨噬细胞的激活被归类为“经典”(炎症，M1)和“替代”。 (血管生成或伤口愈合，M2)。M1的激活是由IFNG和/或TLR激动剂(如脂多糖)介导的，而 M2的激活被广泛定义为由IL4或IL13通过共同的IL4ra介导的。我们发现了一个 TLR激动剂与腺苷A2A/A2B受体共同介导的IL4Ra非依赖性新途径 (A2AR/A2BR)激动剂。该协同途径抑制TNFa和IL-12的表达并诱导血管内皮细胞生长因子 和IL-10的表达，将巨噬细胞转化为我们称之为M2d的M2样表型。这 新的途径需要依赖内毒素诱导A2AR和A2BR的表达，诱导HIF-1a 磷脂酶-CB2(PLCb2)在转录后水平的表达 其信使核糖核酸不稳定。我们最近发现，miRNAs的一个子集在 单用内毒素(miR-155、miR146a、miR-146b、miR-221和miR-222)或联合内毒素/ A2AR/A2BR挑战(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)。在这项提议中，我们将首先 分析受内毒素特异性调控的miRNAs亚群对PLCb2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报告质粒在RAW264.7细胞中表达。然后我们将确定具体的角色(S) 脂多糖处理的巨噬细胞中受A2AR/A2BR激动剂调节并导致“M2d”激活的miRNAs 巨噬细胞基因的表达。我们假设这个特定子集中的miRNAs在切换中起作用 巨噬细胞从M1表型转变为M2d表型。我们将研究这些化合物的模拟物和拮抗剂的作用 MiRNAs对巨噬细胞表达TNFa和IL-12(M1激活产生的旗舰细胞因子)的影响，以及 血管内皮生长因子和IL-10(M2d激活产生的旗舰因子)。最后，我们将对其机理进行分析。 脂多糖对巨噬细胞PLCb2基因表达的失稳作用我们假设，内毒素调节着 含有调节因子的PLCb2 3‘UTR，如miRNAs或RNA结合蛋白，参与了 稳定基因的表达。MiR-466L和RNA结合蛋白Hur和Hur的保守重叠位点 TTP存在于3‘非编码区。我们将分析这些因素在调节PLCb2 mRNA稳定性中的作用 过度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。这些研究应该为巨噬细胞的差异基因调控提供新的见解。 在诱导“M2d”激活的条件下。

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.