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）的感觉和响应吸入真菌。一个人吸入 10亿孢子/天，但真菌在肺中被感知和抑制的模式尚不清楚。我们的初步 数据显示EC调节粘膜对两种主要病原体芽生菌和组织胞浆菌的免疫。我们 发现EC NF-κB活化是必需的，IL-1 R、MyD 88和CARD 9的上游信号以及mobiliza- 产生IL-17和GM-CSF的先天淋巴细胞的作用。我们认为EC亚群，特别是俱乐部细胞， 真菌和调节先天免疫，EC内在信号通过IL-1 R和MyD 88驱动NF κ B调节 产物以触发产生IL-17和GM-CSF的先天性淋巴细胞，其募集/激活骨髓效应物。 肺包含6个EC亚群：肺泡EC、俱乐部EC（clara）、杯状EC、神经内分泌EC、基底EC和纤毛EC 柱状的我们将使用转基因小鼠来测试Club细胞在调节粘膜免疫中的作用， 一个谱系特异性GFP报告基因来跟踪和分析这个子集的行为， 携带cre重组酶，通过将小鼠与IKK 2fl/fl或CRISPR/Cas9小鼠杂交来测试其在抗性中的作用。 阻断NF-κB信号传导。为了了解EC如何调动对真菌的先天免疫，我们将使用两种真菌病原体 代表典型细胞外（芽生菌）和细胞内（组织胞浆菌）致病生活方式 与哺乳动物肺部上皮细胞相对抗的细胞。我们的目标是： 1.阐明俱乐部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， 先进的成像和流式细胞仪分析，我们将跟踪的时间和激活EC和血细胞的位置， 造血细胞对吸入真菌的反应。转基因小鼠将被用来剖析俱乐部细胞的作用， 它们的信号通路和下游产物在抗性中的作用，以及用于GFP标签的CRISPR/Cas9， 晚期、剖析和功能性地询问Club细胞在调节先天免疫中的特定功能。 2.定义Club细胞和产品如何调节γδ T细胞和nTh 17细胞以抑制吸入性真菌。我们将 定义了俱乐部细胞及其信号对抗真菌TCR γδ和nTh 17细胞的调节作用-例如活化， 细胞因子的分泌或募集以及细胞因子的产生。先天性T细胞IL-17 A，GM-CSF和IL-22的作用将被讨论。 促进杀死真菌和促进EC完整性。可溶性信号，如CCL 20、IL-1、IL-6、IL-10、IL-11、IL-12、IL-14、IL-16、IL-18、IL-19、IL- 18和IL-23激活小鼠和人类的先天性T细胞，并鉴定细胞来源。 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， 洞察力和强大的基因编辑工具，将推动该领域的发展。通过推测抗真菌防御机制， 我们将改善针对早期事件的治疗前景，旨在优化粘膜免疫， 真菌和肺部炎症。