Regulation of T cell-derived cytokines in allergic airway inflammation

T 细胞衍生细胞因子在过敏性气道炎症中的调节

• 批准号: 10424606
• 负责人: Jennifer S Chen
• 金额: $ 5.18万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10424606
• 关键词: 3' Untranslated Regions; Affect; Affinity; Allergens; Allergic Disease; Alternaria; Antibodies; Antibody Affinity; Asthma; B-Lymphocytes; Basophils; Binding; Binding Sites; Cell Survival; Complementary DNA; Data; Development; Disease; Event; Extrinsic asthma; Genetic Transcription; Helper-Inducer T-Lymphocyte; HuR protein; IgE; Immune response; Immunization; Immunoglobulin Class Switching; Immunologics; Immunoprecipitation; Inflammation; Interleukin-4; Internal Ribosome Entry Site; Length; Lipopolysaccharides; Maps; Mediating; Mediator of activation protein; Messenger RNA; Modeling; Molecular; Mus; Mutate; Mutation; Pathogenesis; Pathogenicity; Patients; Play; Population; Post-Transcriptional Regulation; Prevalence; Production; Protein Isoforms; Proteins; Public Health; Pulmonary Inflammation; RNA-Binding Proteins; Regulation; Reporter; Role; Signal Transduction; Source; Structure of germinal center of lymph node; Surface; T cell regulation; T-Lymphocyte; Th2 Cells; Training; Transcript; Translations; airborne allergen; allergic airway disease; allergic airway inflammation; anti-IgE; asthma exacerbation; chemical release; crosslink; cytokine; improved; insight; mRNA Expression; mRNA Stability; mast cell; microbial; mouse model; prevent; response; targeted treatment; therapeutically effective

Project Summary The increasing prevalence of asthma over the past few decades signals an urgent need to understand disease pathogenesis and to develop more effective therapeutics. High affinity IgE plays a central role in the disease by mediating mast cell and basophil degranulation, which releases chemical mediators responsible for asthma exacerbation. T follicular helper (Tfh) cells promote the relevant high affinity antibodies for a given immune response through the cytokines they secrete. Tfh cell-derived IL-4 is required to induce high affinity IgE in response to allergens. However, IL-4 has also been considered a canonical Tfh cell cytokine, produced even during microbial immunizations that do not elicit IgE. Such studies largely rely on the use of an IL-4 cytokine reporter that indicates activation of Il4 transcription but not necessarily transcript stability or translation. Our lab has established murine models of Alternaria-induced allergic airway inflammation (AAI), which involves high affinity IgE production, as well as lipopolysaccharide (LPS)-induced lung inflammation, which does not. Using these two models, I observed Il4 mRNA expression in Tfh cells from both conditions; in contrast, I found that only Tfh cells in AAI produce IL-4 protein. My preliminary data suggest that Il4 mRNA in Tfh cells from AAI is more stable than that from LPS-induced inflammation, uncovering a post-transcriptional regulatory mechanism that governs IL-4 production in Tfh cells. My overall hypothesis is that post-transcriptional regulation of Il4 is a checkpoint dictating the high affinity IgE-promoting capacity of Tfh cells. My proposal therefore suggests a new paradigm for the regulation of IL-4 protein production in Tfh cells and the molecular basis of IgE-mediated AAI. My first aim is to identify the mRNA regulatory sequences responsible for Il4 post-transcriptional regulation in Tfh cells during AAI versus LPS-induced lung inflammation. To accomplish this, I will clone Il4 expression constructs mutated in regulatory sequences to determine the effect on Il4 mRNA stability, IL-4 protein production, and high affinity IgE responses. My second aim is to evaluate the role of an RNA-binding protein (RBP) on IL-4 production in Tfh cells during AAI. To do this, I will map RBP-Il4 binding sites, mutate these binding sites in Il4 expression constructs, and evaluate the effect of such mutations on Il4 mRNA stability, IL-4 protein production, and high affinity IgE responses. If successful, this project will define a previously undescribed mechanism of IgE regulation in asthma while also elucidating the immunologic rules that prevent the inappropriate induction of IgE in non-allergic responses. Such findings will offer valuable insight into new strategies for blocking IgE development in asthma and other allergic diseases.

项目概要 过去几十年来哮喘患病率不断上升，表明迫切需要了解疾病 发病机制并开发更有效的治疗方法。高亲和力 IgE 在该疾病中发挥着核心作用 介导肥大细胞和嗜碱性粒细胞脱粒，释放导致哮喘的化学介质 恶化。滤泡辅助 T (Tfh) 细胞促进特定免疫的相关高亲和力抗体 通过它们分泌的细胞因子做出反应。 Tfh 细胞来源的 IL-4 是诱导高亲和力 IgE 所必需的 对过敏原的反应。然而，IL-4 也被认为是一种典型的 Tfh 细胞因子，甚至产生 在不引发 IgE 的微生物免疫过程中。此类研究很大程度上依赖于 IL-4 细胞因子的使用 指示 Il4 转录激活但不一定是转录稳定性或翻译的报告基因。我们的实验室 已经建立了链格孢属引起的过敏性气道炎症（AAI）的小鼠模型，该模型涉及高 亲和力 IgE 的产生，以及脂多糖 (LPS) 诱导的肺部炎症，而后者则不然。使用 在这两个模型中，我观察了两种条件下 Tfh 细胞中 Il4 mRNA 的表达；相比之下，我发现 AAI 中只有 Tfh 细胞产生 IL-4 蛋白。我的初步数据表明，来自 AAI 的 Tfh 细胞中的 Il4 mRNA 是 比 LPS 诱导的炎症更稳定，揭示了转录后调节机制 控制 Tfh 细胞中 IL-4 的产生。我的总体假设是 Il4 的转录后调控是 检查点决定 Tfh 细胞的高亲和力 IgE 促进能力。因此，我的建议提出了一个新的 Tfh 细胞中 IL-4 蛋白产生的调节范例以及 IgE 介导的 AAI 的分子基础。 我的第一个目标是确定负责 Il4 转录后的 mRNA 调控序列 AAI 与 LPS 诱导的肺部炎症期间 Tfh 细胞的调节。为了实现这一点，我将克隆 Il4 表达构建体在调节序列中突变以确定对 Il4 mRNA 稳定性、IL-4 的影响 蛋白质生产和高亲和力 IgE 反应。我的第二个目标是评估 RNA 结合的作用 蛋白 (RBP) 对 AAI 期间 Tfh 细胞 IL-4 产生的影响。为此，我将映射 RBP-Il4 结合位点，进行突变 这些在 Il4 表达构建体中的结合位点，并评估此类突变对 Il4 mRNA 稳定性的影响， IL-4 蛋白生产和高亲和力 IgE 反应。如果成功，该项目将定义一个先前的 未描述的 IgE 调节哮喘的机制，同时也阐明了预防哮喘的免疫学规则 非过敏反应中 IgE 的不适当诱导。这些发现将为新的研究提供宝贵的见解 阻止哮喘和其他过敏性疾病中 IgE 发育的策略。