Characterization of degranulation regulators in human mast cells

项目摘要/摘要 该项目的长期目标是了解人类肥大细胞(MC)功能的调节，并 阐明MC在人类健康和疾病中的作用。单核细胞是免疫球蛋白E相关的主要效应细胞 反应(如哮喘、过敏、寄生虫免疫)。这些细胞也可能对某些自身免疫起作用。 在疾病和启动对病原体和其他病原体的反应方面。激活后，MC会经历 脱颗粒，导致分泌许多介质(包括储存的产品，如组胺、肝素 以及脂类介体的产生，例如LTC4和PGD2，以及许多细胞因子和 增长因素。根据环境的不同，MC可以具有效应器和/或免疫调节作用。多数 对MC发育和功能的研究采用了各种小鼠模型。然而，我们 对调节人类巨噬细胞激活的机制知之甚少。同样，相对地， 关于人类MC中信号通路的扰动如何促成MC-Mc-1，我们知之甚少。 相关病理学。这反过来又阻碍了用于治疗和/或治疗的治疗剂的设计 预防人类受试者的过敏和其他肥大细胞相关疾病。我们最近开发了一种 采用功能基因组学，结合高分辨率单细胞共聚焦成像的技术平台， 它可以快速识别人类肥大细胞脱颗粒的调节因子。我们现在建议使用这个 在培养的供体中识别IgE/FceRI依赖的信号转导通路的关键调节因子的平台- 衍生的原代人类肥大细胞。具体地说，我们将尝试在原代人类肥大细胞中验证 选定以前在啮齿动物肥大细胞中发现的主要脱颗粒调节剂。我们还将使用目标 排列的人CRISPR-Cas9文库以识别新的(即以前未识别的)IgE/FceRI调节因子- 原代人类肥大细胞中依赖的信号通路。此外，我们将调查功能 CRISPR-Cas9法检测肥大细胞增多症患者Kit D816V突变的意义 基因编辑技术在人HMC-1细胞系和原代人类中诱导/纠正相同突变 肥大细胞。为了实现我们的目标，我们提出了两个目标。在目标1中，我们将鉴定新的人类肥大细胞 脱颗粒调节剂和系统地定义依赖于IgE/FceRI的信号通路 人类肥大细胞。在目标2中，我们将使用CRISPR-Cas9“PRIME EDITING”进行HMC的突变研究。 1人肥大细胞系和原代人肥大细胞。总之，这项工作将有助于创建一个详细的模型 并将开始分析KIT D816V突变如何可能 影响人类肥大细胞生物学。

PROJECT SUMMARY/ABSTRACT This project’s long-term goals are to understand the regulation of human mast cell (MC) function and to elucidate the roles of MCs in human health and disease. MCs are major effector cells in IgE-associated responses (e.g., asthma, allergy, parasite immunity). These cells also may contribute to certain autoimmune disorders and in the initiation of responses to pathogens and other agents. Upon activation, MCs undergo degranulation, leading to the secretion of many mediators (including stored products, e.g., histamine, heparin and proteases), as well as the production of lipid mediators, e.g., LTC4 and PGD2, and many cytokines and growth factors. Depending on the setting, MCs can have effector and/or immunoregulatory roles. Most research investigating MC development and function has employed various mouse models. However, we know considerably less about the mechanisms that regulate the activation of human MCs. Similarly, relatively little is known about how the perturbation of signaling pathways in human MCs can contribute to MC- associated pathology. This, in turn, has hampered the design of therapeutic agents for the treatment and/or prevention of allergies and other mast cell-associated diseases in human subjects. We recently developed a technology platform employing functional genomics, coupled with high-resolution single-cell confocal imaging, which can rapidly identify regulators of degranulation in human mast cells. We now propose to use this platform to identify key regulators of IgE/FceRI-dependent signal transduction pathways in cultured, donor- derived, primary human mast cells. Specifically, we will attempt to validate, in primary human mast cells, selected major degranulation regulators previously identified in rodent mast cells. We also will use targeted arrayed human CRISPR-Cas9 libraries to identify novel (i.e., previously unidentified) regulators of IgE/FceRI- dependent signaling pathways in primary human mast cells. In addition, we will investigate the functional significance of the KIT D816V mutation detected in mastocytosis patients by using the CRISPR-Cas9-based gene editing technologies to induce/correct the same mutation in human HMC-1 cell lines and primary human mast cells. To achieve our goals, we propose two aims. In Aim 1, we will identify novel human mast cell degranulation regulators and systematically define IgE/FceRI-dependent signaling pathways in primary human mast cells. In Aim 2, we will use CRISPR-Cas9 “prime editing” to perform mutational studies of HMC- 1 human mast cell lines and primary human mast cells. Together, this work will help create a detailed model of mast cell degranulation in human mast cells and will begin to analyze how the KIT D816V mutation may influence human mast cell biology.