Type 2 Immunity Elicited Through an LTE4/GPR99-Dependent Pathway

项目摘要 LTE4是稳定的半胱氨酰白三烯（cysLT），在哮喘患者的生物体液中检测到 由过敏原刺激阿司匹林和呼吸道病毒引发LTE4在正常人中引起皮肤水肿 哮喘患者肺内嗜碱性粒细胞和嗜酸性粒细胞的募集。我们组最近 确定CysLT3R（也称为Oxgr1或GPR99）为LTE4的高亲和力受体，并证明它 在缺乏常规cysLT受体CysLT 1R和CysLT 1R的小鼠中介导LTE4引起的皮肤水肿， CysLT2R。然而，LTE4诱导肺病理学的机制仍然知之甚少 CysLT3R的作用尚未阐明。我们已经发现CysLT3R在两种小鼠上都表达， 和人呼吸道上皮细胞（EpCs），并控制它们的激活和发育。作为CysLT3R 对目前可用的cysLT受体抑制剂具有耐药性，该提案的发现将决定 CysLT3R是否是减少粘液产生、气流阻塞或2型糖尿病的合理治疗靶点 哮喘中的炎症 使用单细胞RNA-Seq和我们已经开发的新型CysLT3R floxed菌株， 几个谱系报告基因和无效菌株，Aim 1将定义CysLT3R 调节鼠肺EpC活化和发育。在目标2中，我们将描述候选效应器 CysLT3R-扩增的气道EpCs中的信号通路，我们已经在RNA-seq数据集中定义。这一目标提出， 为了扩大我们在纯化的罕见EpC亚群上的RNA-seq数据，并使用细胞特异性敲除来表征 目的EpC效应蛋白。在目标3中，我们将利用不断增长的单细胞RNA-seq数据库 我们是从鼻息肉患者发展而来的。我们将利用巨大的转录差异， 在不同的疾病状态（阿司匹林加重的呼吸道疾病，阿司匹林耐受患者， 鼻息肉病和对照样品），以寻找独特的EpC效应程序，并确定那些可能是 由CysLT3R调节。最后，我们将在正常人支气管上皮细胞培养中使用体外系统 为了确定CysLT3R调节人气道EpC分化的程度，并验证CysLT3R对人气道EpC分化的影响， 这是通过什么途径发生的。

Project Summary LTE4 is the stable cysteinyl leukotriene (cysLT), detected in the biologic fluids of patients with asthma triggered by allergen challenge, aspirin, and respiratory viruses. LTE4 elicits cutaneous edema in normal controls and elicits basophil and eosinophil recruitment to the lung in patients with asthma. Our group recently identified CysLT3R (also called Oxgr1 or GPR99) as the high affinity receptor for LTE4 and demonstrated that it mediates LTE4-elicited cutaneous edema in mice lacking the conventional cysLT receptors, CysLT1R and CysLT2R. However, the mechanism(s) by which LTE4 induces lung pathobiology remains poorly understood and the role of CysLT3R has not been elucidated. We have found that CysLT3R is expressed on both murine and human respiratory epithelial cells (EpCs) and controls both their activation and development. As CysLT3R is resistant to currently available cysLT receptor inhibitors, the findings from this proposal will determine whether CysLT3R is a logical therapeutic target to reduce mucus production, airflow obstruction, or type 2 inflammation in asthma. Using single cell RNA-Seq and a novel CysLT3R floxed strain that we have developed, in addition to several lineage reporter and null strains, Aim 1 will define the cellular mechanisms by which CysLT3R regulates murine lung EpC activation and development. In Aim 2, we will characterize candidate effector pathways in CysLT3R-expanded airway EpCs that we have defined in an RNA-seq dataset. This aim proposes to expand our RNA-seq data on a purified rare EpC subset, and use a cell-specific knockout to characterize an EpC effector protein of interest. In Aim 3 we will take advantage of a growing single cell RNA-seq data base we are developing from patients with nasal polyps. We will exploit the tremendous transcriptional distinction we see across different disease states (aspirin-exacerbated respiratory disease, aspirin-tolerant patients with nasal polyposis, and control samples) to look for unique EpC effector programs and define those which may be regulated by CysLT3R. Finally, we will use an in vitro system in normal human bronchial epithelial cell culture to determine the extent to which CysLT3R regulates human airway EpC differentiation and validate the pathways through which this occurs.