Lung epithelial cell regulation of immunity to inhaled fungi

肺上皮细胞对吸入真菌免疫的调节

基本信息

批准号：
10451274
负责人：
BRUCE Steven KLEIN
金额：
$ 8.18万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10451274
关键词：
Address Adult Air Alveolar Antifungal Agents Behavior Blastomyces C Type Lectin Receptors CCL20 gene CRISPR/Cas technology Cell Line Cell Lineage Cells Confocal Microscopy Data Defense Mechanisms Ecosystem Elements Enterobacteria phage P1 Cre recombinase Epithelial Epithelial Cells Event Fostering Fungal Spores Genes Granulocyte-Macrophage Colony-Stimulating Factor Hematopoietic Histoplasma Host Defense Human Image Immunity Individual Infection Inflammatory Inhalation Interleukin-1 Interleukin-17 Interleukin-18 Interleukin-6 Knowledge Learning Leukocytes Life Style Link Location Lung Lung Inflammation Lymphocyte Mammalian Cell Molecular Mucosal Immunity Mucous Membrane Mus Myelogenous Myeloid Cells Natural Immunity Neurosecretory Systems Outcome Pathogenicity Persons Production Public Health Regulation Reporter Reproduction spores Resistance Respiratory Mucosa Role Signal Pathway Signal Transduction Source T-Lymphocyte Testing Transgenic Mice Work climate change cytokine design extracellular fungus genome editing improved in vivo insight interleukin-22 interleukin-23 lymphocyte product particle pathogen pathogenic fungus promoter receptor recruit response restraint therapeutic target tissue repair tool γδ T cells

项目摘要

PROJECT SUMMARY We propose to define how lung epithelial cells (EC) sense and respond to inhaled fungi. A person inhales a billion spores/day, but the modes by which fungi are sensed and restrained in lung are unclear. Our preliminary data reveal that EC regulate mucosal immunity to two primary pathogens, Blastomyces and Histoplasma. We find that EC NF-κB activation is essential, as are upstream signals of IL-1R, MyD88 and CARD9 and mobiliza- tion of innate lymphocytes that make IL-17 and GM-CSF. We posit that EC subsets, notably Club cells, sense fungi and regulate innate immunity, and that EC intrinsic signals via IL-1R and MyD88 drive NFkB-regulated products to trigger IL-17- and GM-CSF-producing innate lymphocytes, which recruit/activate myeloid effectors. The lung contains 6 EC subsets: alveolar, club (formerly clara), goblet, neuroendocrine, basal and ciliated columnar. We'll test the role of Club cells in regulating mucosal immunity by using transgenic mice that display a lineage-specific GFP reporter to track and analyze the behavior of this subset and, since our reporter mouse harbors cre recombinase, test its role in resistance by crossing the mouse with IKK2fl/fl or CRISPR/Cas9 mice to block NF-κB signaling. To learn how EC mobilize innate immunity to fungi, we will use two fungal pathogens that represent paradigmatic extracellular (Blastomyces) and intracellular (Histoplasma) pathogenic lifestyles that confront the epithelium of mammalian lungs. Our aims are to: 1. Delineate the role and action of Club cells in sensing inhaled fungi. By using NFκB-eGFP reporter mice, advanced imaging and FACS analysis, we will track the timing and location of activation of EC and hemato- poietic cells in response to inhaled fungi. Transgenic mice will be used to dissect the roles of Club cells and their signaling pathways and downstream products in resistance, and CRISPR/Cas9 used to GFP tag, iso- late, profile and functionally interrogate specific functions of Club cells in regulating innate immunity. 2. Define how Club cells and products regulate γδ T cells and nTh17 cells to restrain inhaled fungi. We will define the regulatory role of Club cells and their signals on anti-fungal TCR γδ and nTh17 cells – e.g. activa- tion or recruitment and cytokine production. The role of innate T cell IL-17A, GM-CSF and IL-22 will be dis- sected in promoting killing of fungi and fostering EC integrity. Soluble signals such as CCL20, IL-1, IL-6, IL- 18 & IL-23 that activate innate T cells in mice and humans will be defined, and the cell source(s) identified. Our work will reveal how lung Club cells regulate innate mucosal immunity to fungi offering conceptually new insight and powerful gene editing tools that will advance the field. By divining antifungal defense mechanisms, we will improve prospects for therapeutically targeting early events designed to optimize mucosal immunity to fungi and lung inflammation.

项目概要我们建议定义肺上皮细胞（EC）如何感知和响应人吸入的真菌。十亿个孢子/天，但肺部感知和抑制真菌的方式尚不清楚。数据显示，EC 调节对两种主要病原体（芽生菌和组织胞浆菌）的粘膜免疫。发现 EC NF-κB 激活至关重要，IL-1R、MyD88 和 CARD9 的上游信号也是如此，并且动员我们假设 EC 亚群，尤其是 Club 细胞，具有感知能力。真菌并调节先天免疫，EC 内在信号通过 IL-1R 和 MyD88 驱动 NFkB 调节产品可触发产生 IL-17 和 GM-CSF 的先天淋巴细胞，从而招募/激活骨髓效应细胞。肺包含 6 个 EC 子集：肺泡、杵状细胞（以前称为克拉拉）、杯状细胞、神经内分泌细胞、基底细胞和纤毛细胞我们将通过使用表现出柱状的转基因小鼠来测试俱乐部细胞在调节粘膜免疫中的作用。谱系特异性 GFP 报告基因用于跟踪和分析该子集的行为，并且由于我们的报告基因小鼠含有 cre 重组酶，通过将小鼠与 IKK2fl/fl 或 CRISPR/Cas9 小鼠杂交来测试其在耐药性中的作用为了了解 EC 如何调动针对真菌的先天免疫，我们将使用两种真菌病原体。代表典型的细胞外（芽生菌）和细胞内（组织胞浆菌）致病生活方式面对哺乳动物肺部的上皮细胞，我们的目标是： 1. 描述 Club 细胞在感知吸入真菌中的作用和作用通过使用 NFκB-eGFP 报告小鼠，先进的成像和 FACS 分析，我们将跟踪 EC 和血液激活的时间和位置转基因小鼠对吸入真菌的反应将被用来剖析俱乐部细胞和细胞的作用。它们的信号通路和下游产物具有抗性，并且 CRISPR/Cas9 用于 GFP 标签、iso- 后期、剖析和功能性询问俱乐部细胞在调节先天免疫方面的特定功能。 2. 定义Club细胞和产品如何调节γδ T细胞和nTh17细胞以抑制吸入真菌。定义 Club 细胞及其信号对抗真菌 TCR γδ 和 nTh17 细胞的调节作用 - 例如先天性 T 细胞 IL-17A、GM-CSF 和 IL-22 的作用将被揭示。有助于杀死真菌和促进可溶性信号，如 CCL20、IL-1、IL-6、IL-。将定义激活小鼠和人类先天 T 细胞的 18 和 IL-23，并确定细胞来源。我们的工作将揭示肺俱乐部细胞如何调节对真菌的先天粘膜免疫，提供新的概念洞察力和强大的基因编辑工具将推动该领域的发展。我们将改善针对旨在优化粘膜免疫的早期事件的治疗前景真菌和肺部炎症。