Mannose Receptor, miR-511-3p, and Macrophage Polarization in Asthma

摘要 生命早期的过敏原暴露是过敏性哮喘发展的一个强有力的预测因素。特别是暴露 对蟑螂过敏原过敏会导致过敏性致敏，增加患过敏性哮喘的风险。期间 在这一建议的试点阶段，我们在认识一个重要环节方面取得了实质性突破 在蟑螂抗原致敏哮喘通路中。我们已经证明甘露糖受体 (MRC1/CD 206）在蟑螂变应原诱导的气道炎症中提供了令人印象深刻的保护功能。 我们最近的初步研究表明，小鼠中MRC 1的缺失加剧了蟑螂过敏原诱导的肺 炎症，沿着巨噬细胞向M1巨噬细胞极化的趋势（炎症） 表型这一发现起初令人困惑，因为MRC 1缺乏传统的信号基序，因此， MRC 1介导的巨噬细胞极化和变应原诱导的肺部炎症的机制 仍然模糊。我们对这一途径的更深入理解的突破来自于对一个 关键调控microRNA（miR-511- 3 p，miR-511的功能链）。miR-511- 3 p编码于 MRC 1基因和转录在巨噬细胞中共调节。这个提议的核心是我们的新假设 甘露糖受体对过敏原诱导的巨噬细胞极化的显著保护作用， 肺部炎症是由于miR-511- 3 p的调节影响。事实上，我们发现miR-511- 3 p 与非哮喘患者相比，哮喘患者血液中的miR-511- 3 p显著降低， 在蟑螂提取物攻击小鼠的肺巨噬细胞中，miR-511- 3 p显著降低， 缺乏MRC 1的骨髓源性巨噬细胞（BMDM）和过表达miR-511- 3 p的BMDM倾向于 向M2表型转化。这些令人兴奋的数据为批判性地评估功能性 miR-511- 3 p在MRC 1介导巨噬细胞极化和过敏原诱导的过敏反应中的意义 炎症目的1提出了检测血清和单核细胞来源的miR-511- 3 p水平的研究。 巨噬细胞，并分析其与过敏性哮喘的关系，特别是那些对蟑螂过敏的人。目的 2提出了通过以下方法确定miR-511- 3 p在MRC 1介导的巨噬细胞极化中的作用的实验： 在巨噬细胞和过敏原诱导的肺部炎症中过表达或敲低表达miR-511- 3 p， 用造血干/祖细胞（HS/PC）miR-511- 3 p产生骨髓嵌合小鼠， 表达或敲低以及使用miR-511模拟物处理。目标3提出了实验来识别 通过使用miR-511 - 3 p过表达或抑制实验，荧光素酶 报告基因和生物素下拉测定。总之，这项工作将提供一个框架，以了解miRNA的目标， 并将导致后续的研究，以探讨MRC 1/miR-511- 3 p调节的细胞凋亡的机制。 巨噬细胞活化和过敏性炎症，重点是这些确定的目标。这项研究可能 最终为发现过敏性哮喘的新治疗靶点提供了机会。

ABSTRACT Allergen exposure early in life is a strong predictor for the development of allergic asthma. Particularly, exposure to cockroach allergen can lead to allergic sensitization and increase the risk of developing allergic asthma. During the pilot stage for this proposal we have made a substantive breakthrough in understanding an important link within the cockroach antigen-sensitization-asthma pathway. We have demonstrated that the mannose receptor (MRC1/CD206) provides an impressive protective function in cockroach allergen induced airway inflammation. Our recent pilot study suggests that deletion of MRC1 in mice exacerbates cockroach allergen-induced lung inflammation, along with a tendency to polarize macrophages toward a M1 macrophage (inflammatory) phenotype. This finding was at first perplexing because MRC1 lacks a traditional signaling motif, therefore, the mechanisms underlying MRC1 mediated macrophage polarization and allergen induced lung inflammation remained obscure. Our breakthrough for a deeper understanding of this pathway came with the recognition of a key regulatory microRNA (miR-511-3p, the functional strand of miR-511). The miR-511-3p is encoded within the MRC1 gene and transcriptionally co-regulated in macrophages. This proposal centers on our novel hypothesis that the impressive protective effect of the mannose receptor on allergen-induced macrophage polarization and lung inflammation is due to the regulatory influences of miR-511-3p. Indeed, we have found that miR-511-3p was significantly lower in the blood of asthmatics compared to non-asthmatics, miR-511-3p is significantly increased in lung macrophages from cockroach extract-challenged mice, miR-511-3p is significantly reduced in bone marrow derived macrophages (BMDMs) lacking the MRC1, and BMDMs over-expressing miR-511-3p tend to polarize toward a M2 phenotype. These exciting data set the stage to critically evaluate the functional significance of miR-511-3p in MRC1-mediated macrophage polarization and allergen-induced allergic inflammation. Aim 1 proposes studies to detect the levels of miR-511-3p in serum and monocyte-derived macrophages and analyze their associations with allergic asthma, particularly those with cockroach allergy. Aim 2 proposes experiments to determine the role of miR-511-3p in MRC1-mediated macrophage polarization by over- or knockdown-expressing miR-511-3p in macrophages and in allergen-induced lung inflammation by generating bone-marrow chimeric mice with hematopoietic stem/progenitor cells (HS/PCs) miR-511-3p over- expression or knock-down and by using miR-511 mimic treatment. Aim 3 proposes experiments to identify the potential targets for miR-511-3p by using miR-511-3p overexpression or inhibition experiments, luciferase reporter and biotin pulldown assays. Together, this work will provide a framework to understand miRNA targets, and will lead to subsequent studies to probe the mechanisms underlying the MRC1/miR-511-3p-modulated macrophage activation and allergic inflammation with a focus on these identified targets. The study may ultimately offer an opportunity for the discovery of novel therapeutic targets for allergic asthma.