Lung epithelial cell regulation of immunity to inhaled fungi

肺上皮细胞对吸入真菌免疫的调节

• 批准号: 10222492
• 负责人: BRUCE Steven KLEIN
• 金额: $ 46.17万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 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/10222492
• 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

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.

项目摘要 我们建议定义肺上皮细胞（EC）如何感知并对遗传的真菌做出反应。一个人继承一个 每天十亿个孢子，但是在肺中感知和限制真菌的模式尚不清楚。我们的初步 数据表明，EC调节粘膜免疫对两种主要病原体，胚泡和组织肿瘤。我们 发现EC NF-κB激活是必不可少的，IL-1R，MyD88和Card9和Mobiliza-的上游信号也是必不可少的。 使IL-17和GM-CSF产生的先天淋巴细胞的影响。我们肯定了ECES集，尤其是俱乐部细胞 真菌和调节先天免疫，以及通过IL-1R和MyD88驱动NFKB调节的EC内在信号 触发IL-17和GM-CSF产生的先天淋巴细胞的产品，从而募集/激活髓样影响。 肺包含6个EC集：肺泡，俱乐部（以前为Clara），Goblet，神经内分泌，基底和纤毛 柱子。我们将通过使用显示的转基因小鼠来测试俱乐部细胞在控制粘膜免疫中的作用 谱系特异性的GFP记者，用于跟踪和分析该子集的行为，并且由于我们的记者鼠标 Harbors CRE重组酶，通过将鼠标与IKK2FL/FL或CRISPR/CAS9小鼠交叉测试其在电阻中的作用 要了解EC如何动员对真菌的先天免疫，我们将使用两种真菌病原体 代表范式的细胞外（Blastomyces）和细胞内（组织刺）的致病生活方式 面对哺乳动物肺上皮。我们的目标是： 1。描述俱乐部细胞在感测遗传真菌中的作用和作用。通过使用NFκB-EGFP记者小鼠， 高级成像和FACS分析，我们将跟踪EC和HEMATO-激活的时间和位置 诗歌细胞响应遗传真菌。转基因小鼠将用于剖析俱乐部细胞的作用， 它们的信号通路和下游产品具有阻力，CRISPR/CAS9用于GFP标签，ISO- 迟到，轮廓和功能质疑俱乐部细胞控制先天免疫的特定功能。 2。定义俱乐部细胞和产物如何调节γδT细胞和NTH17细胞以限制吸入真菌。我们将 定义俱乐部细胞及其信号在抗真菌TCRγδ和NTH17细胞上的调节作用 - 例如活动 招募或招募和细胞因子产生。先天T细胞IL-17A，GM-CSF和IL-22的作用将是 首选以促进真菌杀死并促进EC的完整性。 CCL20，IL-1，IL-6，IL-等可溶性信号 将定义激活小鼠和人类先天T细胞的18＆IL-23，并确定细胞源。 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，从而提供新的新 洞察力和强大的基因编辑工具将推进整个领域。通过分配抗真菌防御机制， 我们将提高针对旨在优化粘膜免疫力的早期事件的热门目标的前景 真菌和肺部感染。