Lung epithelial cell regulation of immunity to inhaled fungi

项目总结 我们建议定义肺上皮细胞(EC)如何感知和对吸入真菌做出反应。一个人吸入了一种 数十亿个孢子/天，但真菌在肺部的感受和抑制模式尚不清楚。我们的预赛 资料显示，EC调节粘膜对两种主要病原体--芽囊霉菌和组织胞浆菌的免疫。我们 发现EC NF-κB的激活是必不可少的，IL-1R、MyD88和CARD9的上游信号和激动剂也是必不可少的。 产生IL-17和GM-CSF的天然淋巴细胞的激活。我们假设EC子集，特别是俱乐部细胞，感觉 真菌和调节先天免疫，EC通过IL-1R和MyD88内在信号驱动NFkB调节 触发产生IL-17和GM-CSF的固有淋巴细胞的产品，这些淋巴细胞招募/激活髓系效应器。 肺含有6个EC亚群：肺泡型、棒状型、杯状型、神经内分泌型、基底型和纤毛型。 专栏。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 一个特定于血统的GFP记者来跟踪和分析这个子集的行为，自从我们的记者鼠标 携带cre重组酶，用IKK2fl/fl或CRISPR/Cas9小鼠与小鼠杂交，检测其在耐药中的作用 阻断核因子-κB信号转导。为了了解EC如何调动对真菌的先天免疫力，我们将使用两种真菌病原体 代表聚合的胞外(芽生霉菌)和胞内(组织胞浆)致病的生活方式 它们与哺乳动物肺部的上皮细胞对峙。我们的目标是： 1.阐明Club细胞在感知吸入性真菌中的作用和作用。通过使用NFκB-绿色荧光蛋白报告小鼠， 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- 细胞对吸入真菌的反应。转基因小鼠将被用来剖析Club细胞和 它们的信号通路和下游产物的抗性，以及用于GFP标签的CRISPR/Cas9，iso- 后期，概述和功能询问俱乐部细胞在调节先天免疫中的特定功能。 2.明确俱乐部细胞及其产物如何调节γδT细胞和nTh17细胞以抑制吸入真菌。我们会 明确俱乐部细胞及其信号对抗真菌TcRγδ和nTh17细胞的调节作用。 招聘或招募以及细胞因子的产生。天然T细胞IL-17A、GM-CSF和IL-22的作用将不同 致力于促进杀灭真菌和培养欧共体的诚信。CCL20、IL-1、IL-6、IL-6等可溶性信号 18&IL-23激活小鼠和人的固有T细胞，并确定细胞来源(S)。 我们的工作将揭示肺俱乐部细胞如何调节对真菌的天然粘膜免疫，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。通过预测抗真菌防御机制， 我们将改善针对早期事件的治疗前景，旨在优化黏膜免疫 真菌和肺部炎症。

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.