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

摘要 接触蟑螂变应原会导致过敏性过敏，增加过敏性哮喘的风险。 然而，其潜在的分子机制目前还不是很清楚。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，我们小组在揭开 蟑螂抗原与过敏性哮喘的发生发展。具体地说，我们对N-连接的多糖的分析来自 蟑螂变应原鉴定了几种与甘露糖受体MRC1/CD206具有高亲和力的主要多糖。 此外，我们已经确定了MRC1在过敏原清除中的一个关键但以前未被认识的作用，即作为一种 自然防御机制及其在限制蟑螂变应原过敏进展和严重程度中的作用 哮喘小鼠模型的炎症反应。这是通过巨噬细胞清除的改变而发生的。 吸入蟑螂变应原与M1/M2巨噬细胞极化平衡。这一开始令人费解。 由于MRC1缺乏任何已知的信号基序，因此，在过敏原诱导的过程中，MRC1的信号级联 呼吸道炎症和巨噬细胞极化仍不清楚。我们对更深层次理解的突破 MRC1信号通路的研究伴随着人们认识到一个关键的调节miR-511-3p，由两者编码 小鼠和人的MRC1基因与巨噬细胞中的MRC1基因在转录水平上共同调节。这些令人兴奋的东西 这些发现导致我们提出了一个新的假设，即MRC1作为一种天然的 防御机制，MRC1编码的miR-511-3p参与介导MRC1下游免疫 反应和预防过敏原引起的呼吸道炎症。这一假设得到了进一步的支持 我们最近的研究发现，哮喘患者的血浆miR-511-3p水平比对照组低得多，并且 腺相关病毒(AAV)介导的miR-511-3p过表达可改善变应原诱导的过敏反应 MRc1-/-小鼠的呼吸道炎症，但miR-511-3p基因敲除小鼠显示变应原诱导的呼吸道炎症增加 发炎。这些令人兴奋的数据为批判性地评估miR-511-3p在体内的功能意义奠定了基础。 过敏性哮喘及其潜在机制。提出了三个既相互独立又相互联系的具体目标。目标 1将通过定量检测血浆、痰中miR-511-3来确定miR-511-3p在过敏性哮喘中的意义。 过敏性哮喘患者血浆和痰中的胞外小泡及其在巨噬细胞中的作用 极化和功能。目标2将定义是否使用全球miR-511-3p预防哮喘 和巨噬细胞条件性基因敲除小鼠(例如，LysM-cre；miR-511-3pflx/flx)和甘露糖修饰的EV-miR- 511-3P组小鼠。AIM 3将通过整合基因图谱和我们独特的亲和力来识别miR-511-3p靶点- 基于miR-511-3P结合伙伴mRNAs/长非编码RNA的转录切割方法。总的来说，我们的 研究将提供一个概念框架，将过敏原、MRC1和miR-511-3p与哮喘的关键特征联系起来。 最终，这些研究可能允许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.