Functional role of miR-511-3p in allergic asthma and its underlying mechanisms

摘要 接触蟑螂过敏原可导致过敏性致敏和过敏性哮喘的风险增加。 然而，潜在的分子机制目前还没有很好地建立。我们的长期目标是 阐明过敏性哮喘的基本潜在机制并确定新的治疗靶点。 在试点研究期间，我们的小组为解开以下两个方面的重要联系做出了重大贡献： 蟑螂抗原与过敏性哮喘的发生具体来说，我们对来自 蟑螂过敏原鉴定了几种对甘露糖受体MRC 1/CD 206具有高亲和力的主要聚糖。 此外，我们已经确定了MRC 1在过敏原清除中的一个关键但以前未被认识的作用， 自然防御机制和限制蟑螂过敏原诱导的过敏性疾病的进展和严重程度 哮喘小鼠模型中的炎症。这是通过改变巨噬细胞清除 吸入蟑螂变应原和M1/M2巨噬细胞极化的平衡。起初这令人困惑 由于MRC 1缺乏任何已知的信号基序，因此，MRC 1在过敏原诱导的 气道炎症和巨噬细胞极化仍然不清楚。我们的突破，更深入地了解 MRC 1信号通路的研究发现，一个关键的调节miR-511- 3 p，由两个基因编码， 小鼠和人MRC 1基因，在巨噬细胞中与MRC 1转录共调节。这些令人兴奋 这些发现使我们提出了一个新的假设，即MRC 1作为一种天然的免疫抑制剂，在很大程度上参与了过敏原的清除。 MRC 1编码的miR-511- 3 p参与介导MRC 1下游免疫应答， 反应和防止过敏原诱导的气道炎症。这一假设得到了进一步的支持， 我们最近发现，与对照组相比，哮喘患者的miR-511- 3 p血浆水平要低得多， 腺相关病毒（AAV）介导的miR-511- 3 p过表达改善了过敏原诱导的 Mrc 1-/-小鼠的气道炎症，但miR-511- 3 p敲除小鼠显示过敏原诱导的气道炎症增加， 炎症这些令人兴奋的数据为严格评估miR-511- 3 p在肿瘤中的功能意义奠定了基础。 过敏性哮喘及其潜在机制提出了三个独立但相关的具体目标。目的 1将通过定量血浆、痰液和血浆中的miR-511-3来确定miR-511 - 3 p在过敏性哮喘中的意义。 从过敏性哮喘患者血浆和痰液中分离细胞外囊泡（EVs），并检测其在巨噬细胞中的作用 极化和功能。目的2将确定miR-511- 3 p是否可以通过miR-511- 3 p全局 和巨噬细胞条件性敲除小鼠（例如，LysM-cre; miR-511- 3 pflox/flox）和甘露糖修饰的EV-miR- 511- 3 p处理的小鼠。目标3将通过整合基因谱和我们独特的亲和力来识别miR-511- 3 p靶点， 基于转录组学方法的miR-511- 3 p结合伴侣mRNA/长非编码RNA。总体而言，我们 研究将提供一个概念框架，将过敏原、MRC 1和miR-511- 3 p与哮喘的关键特征联系起来。 最终，这些研究可能有助于开发新的哮喘诊断和治疗靶点。

ABSTRACT Exposure to cockroach allergen can lead to allergic sensitization and an increased risk of allergic asthma. However, the underlying molecular mechanisms are currently not well-established. Our long-term goals are to elucidate the fundamental underlying mechanisms and identify novel therapeutic targets for allergic asthma. During the pilot studies, our group has made significant contributions to unraveling an important link between cockroach antigen and development of allergic asthma. Specifically, our profiling of N-linked glycans from cockroach allergen identified several major glycans with high affinity to mannose receptor, MRC1/CD206. Furthermore, we have identified a critical but previously unrecognized role of MRC1 in allergen clearance as a natural defense mechanism and in limiting the progression and severity of cockroach allergen-induced allergic inflammation in a mouse model of asthma. This occurs through alterations in macrophage clearance of the inhaled cockroach allergens and balance of M1/M2 macrophage polarization. This was at first perplexing because MRC1 lacks any known signaling motif, therefore, the signaling cascades of MRC1 in allergen-induced airway inflammation and macrophage polarization remain obscure. Our breakthrough for a deeper understanding of the MRC1 signaling pathway came with the recognition that a key regulatory miR-511-3p, encoded by both mouse and human MRC1 gene, is transcriptionally co-regulated with MRC1 in macrophages. These exciting findings lead us to propose a novel hypothesis that MRC1 is largely involved in allergen clearance as a natural defense mechanism, and MRC1-encoded miR-511-3p is involved in mediating MRC1 downstream immune responses and protecting against allergen-induced airway inflammation. This hypothesis is further buttressed by our recent findings that plasma levels of miR-511-3p were much lower in asthmatics compared to controls, and that adeno-associated virus (AAV)-mediated miR-511-3p over-expression ameliorated the allergen-induced airway inflammation in Mrc1-/- mice, but miR-511-3p knockout mice showed increased allergen-induced airway inflammation. These exciting data set the stage to critically evaluate the functional significance of miR-511-3p in allergic asthma and its underlying mechanisms. Three independent yet related specific aims are proposed. Aim 1 will determine the significance of miR-511-3p in allergic asthma by quantifying miR-511-3 in plasma, sputum and extracellular vesicles (EVs) from plasma and sputum of allergic asthmatics and testing its role in macrophage polarization and function. Aim 2 will define whether miR-511-3p protects against asthma using miR-511-3p global and macrophage conditional knockout mice (e.g., LysM-cre; miR-511-3pflox/flox) and mannose-decorated EV-miR- 511-3p-treated mice. Aim 3 will identify miR-511-3p targets by integrating gene profiling and our unique affinity- based transcriptomic approach for miR-511-3p binding partner mRNAs/long non-coding RNAs. Collectively, our studies will provide a conceptual framework linking allergens, MRC1, and miR-511-3p to key features of asthma. Ultimately, these studies may allow for the development of new diagnostic and therapeutic targets for asthma.