MicroRNA directed pathway discovery in allergy and asthma

项目摘要 哮喘是一种高度流行的慢性炎症性气道疾病，与哮喘的发生有密切的遗传和机制联系。 过敏IgE同种型抗体的过度产生是变态反应和“Th 2高”的标志性特征。 变应性哮喘内型当B细胞经历类别转换重组（CSR）时产生IgE， 分化为分泌抗体的浆细胞。分泌的IgE武装肥大细胞和嗜碱性粒细胞释放 炎症介质对过敏原暴露的反应，刺激急性过敏反应和 慢性炎症靶向IgE的治疗性抗体可减轻哮喘症状和急性发作， 许多患者患有中度至重度的持续性疾病。理解分子程序 可能导致新的和更有效的方法来预防和治疗 过敏和哮喘。 微小RNA（miRNAs）是基因表达的微小调节因子，介导强大的生物学效应 通过它们对靶mRNA网络的协同作用。B中表达超过100种不同的miRNA 细胞，但关键的生理功能已分配给少数，到目前为止。几年前，我们开发了一个 用于揭示辅助性T细胞分化的miRNA调节剂的强大筛选平台。我们现在已经完全 将该系统用于原代B细胞，并使用它来揭示CSR和IgE的miRNA调节剂 包括miR-221/222、miR-155和其他几种强有力的新候选物，以供进一步研究。 该提案的中心目标是利用B细胞miRNA：靶向网络来发现基因， 参与过敏和哮喘的发展和发病机制的途径。以强有力的初步指导 数据，我们将严格地询问miR-221/222的功能，并选择其他与miR-221/222相关的miRNA家族。 使用体外培养系统和小鼠哮喘空气变应原暴露模型产生IgE。我们将 在B细胞上进行比较性Ago 2 HITS-CLIP，并将该生物化学方法与基因表达联合收割机结合 和计算分析，以生成实验确定的功能性miRNA结合位点的图谱 整个B细胞转录组。这些数据将促进miRNA指导的基因和途径的发现 参与IgE的产生。基于初步数据，我们选择了转录因子Foxp 1和两个 其他新的miR-221/222靶点将成为详细分析的第一个对象，利用现有的 突变小鼠、小分子抑制剂和小鼠和人类CRISPR/Cas9基因组编辑方案 原代B细胞。我们希望拟议的研究能够产生关于过敏性致敏的新见解， 过敏性哮喘的免疫发病机制。此外，我们的研究将促进我们的基本 了解miRNA调控基因表达和细胞行为的网络特性。

PROJECT SUMMARY Asthma is a highly prevalent chronic inflammatory airway disease with tight genetic and mechanistic links to allergy. Hyperproduction of antibodies of the IgE isotype is a hallmark feature of allergy and the “Th2 high” allergic asthma endotype. IgE is produced when B cells undergo class switch recombination (CSR) and differentiate into antibody secreting plasma cells. Secreted IgE arms mast cells and basophils to release inflammatory mediators in response to allergen exposure, fueling both acute anaphylactic responses and chronic inflammation. A therapeutic antibody targeting IgE reduces asthma symptoms and exacerbations in many patients with moderate to severe persistent disease. Understanding the molecular programming that underlies IgE production may lead to novel and more effective approaches for the prevention and treatment of allergy and asthma. MicroRNAs (miRNAs) are tiny regulators of gene expression that mediate powerful biological effects through their concerted action on networks of target mRNAs. Over 100 distinct miRNAs are expressed in B cells, but critical physiological functions have been assigned to few, so far. Several years ago, we developed a robust screening platform for uncovering miRNA regulators of helper T cell differentiation. We have now fully adapted this system for use in primary B cells and used it to uncover miRNA regulators of CSR and IgE production, including miR-221/222, miR-155 and several other strong novel candidates for further study. The central objective of this proposal is to leverage B cell miRNA:target networks to discover genes and pathways involved in the development and pathogenesis of allergy and asthma. Guided by strong preliminary data, we will rigorously interrogate the function of miR-221/222 and select other miRNA families implicated in IgE production using in vitro culture systems and mouse aeroallergen exposure models of asthma. We will perform comparative Ago2 HITS-CLIP on B cells and combine this biochemical approach with gene expression and computational analyses to generate a map of experimentally defined functional miRNA binding sites throughout the B cell transcriptome. These data will facilitate miRNA-directed discovery of genes and pathways involved in IgE production. Based on preliminary data, we have selected the transcription factor Foxp1 and two other novel miR-221/222 targets to be the first subjects of detailed analysis, taking advantage of existing mutant mice, small molecule inhibitors, and CRISPR/Cas9 genome editing protocols in mouse and human primary B cells. We expect the proposed research to generate novel insights about allergic sensitization and the immunopathogenesis of allergic asthma. In addition, our studies will advance our fundamental understanding of the network properties of miRNA regulation of gene expression and cell behavior.