PU.1/Dectin-1在气道上皮细胞应对烟曲霉感染的作用和机制研究

Invasive aspergillosis (IA) has increased incidence rate and lead to high mortality. Aspergillus fumigates conidia are inhaled into respiratory tract and met bronchial epithelial cells first. Bronchial epithelial cells can regulate immune status and enhance immune defense through recognition and chemiotaxis. The regulation and mechanism of immune barrier consisted by bronchial epithelial cells is not clear. Our preliminary study found that bronchial epithelial cells can translocated transcription factor PU.1 to nuclear and induce Dectin-1 expression when stimulated with conidia. Therefore, we hypothesize that bronchial epithelial cells enhance its immune defense through activate PU.1 and modify pattern recognition receptors expression in order to strengthen innate immune defense through a direct or indirect way. Our project try to evaluate the immune function and investigate the regulation mechanism of bronchial epithelial cells during Aspergillus fumigates infection. The conditional knockout bronchial epithelial cells and mouse would be used to study on PU.1 function in innate immune regulation and Dectin-1 upexpression regulation mechanism. Through this project, we expect to expand the knowledge of regulation mechanism in immune defense, reveal the role of bronchial epithelial cells in innate immune and build the theoretical basis of immune intervention for IA control and prevention.

侵袭性曲霉病（IA）发病率不断升高且危害严重。烟曲霉吸入呼吸道后，首先接触气道上皮细胞，上皮细胞能够调节自身天然免疫状态，通过早期识别和免疫趋化等途径发挥重要防御作用，但目前对于气道上皮细胞防御屏障功能的调节及其机制尚不清楚。课题组前期研究发现：烟曲霉感染时气道上皮细胞通过激活转录因子PU.1，启动Dectin-1受体表达，增强了免疫防御功能。因此我们提出假说：气道上皮细胞通过核转位PU.1，可能在转录水平参与Dectin-1表达调控，并直接或间接促进气道上皮细胞天然免疫功能。本项目拟通过气道上皮细胞表达干预模型以及组织特异性表达干预小鼠模型，探究PU.1天然免疫调控功能，解析Dectin-1调控机制，阐明气道上皮细胞免疫防御功能及其调节机制。期望从PU.1这个新视点，拓展对宿主抗真菌防御调控机制的认识，揭示气道上皮细胞在抗感染天然免疫中的作用，为今后开展免疫调控防治IA奠定基础。

侵袭性曲霉病（IA）发病率不断升高且危害严重。烟曲霉感染时，呼吸道天然免疫状态至关重要，通过早期识别和免疫趋化等途径发挥重要防御作用，目前对气道天然免疫防御屏障功能的调节及其机制尚不清楚。项目前期研究发现：烟曲霉感染时气道上皮细胞通过激活转录因子PU.1，启动Dectin-1受体表达，增强了免疫防御功能。.课题通过报告基因实验，确认PU.1表达量的增加，hDectin-1基因启动子活性逐渐升高；利用生物信息学网站预测、染色质免疫共沉淀实验证实，PU.1可与hDectin-1基因启动子（-1053~ -1047）、（-719~ -713）和（-527~ -521）序列结合；凝胶电泳迁移率实验证实PU.1能够与hDectin-1基因启动子区（-1053~ -1047）、（-719~ -713）、（-527~ -521）位点特异性地结合。构建重组腺病毒Ad-PU.1，并将其感染THP-1细胞，48 h后细胞内PU.1与Dectin-1的蛋白表达明显升高；对PU.1表达沉默的THP-1给予烟曲霉刺激，12 h后细胞内Dectin-1的蛋白表达上调受抑制。.课题组成功构建了表达目的基因Dectin-1的重组腺病毒Ad-Dectin-1。Ad-Dectin-1体外转染小鼠肺泡巨噬细胞MH-S，转染后Dectin-1基因mRNA和蛋白表达显著增加，Dectin-1高表达的细胞模型构建成功。在烟曲霉感染过程中，Decetin-1高表达的MH-S炎症因子表达量增加，对烟曲霉的杀灭能力增强，提示Ad-Dectin-1可调节天然免疫反应，提高宿主抗曲霉感染的能力。.通过气管给予Ad-Dectin-1成功上调小鼠肺组织Dectin-1水平，Dectin-1主要表达于细支气管上皮细胞，表达高峰在第5d，可持续2w以上。Ad-mDectin-1能够通过上调气道Dectin-1表达提高烟曲霉感染小鼠肺组织验证趋化因子水平，募集中性粒细胞，减轻肺内真菌负荷，提高烟曲霉感染后的生存率。.本项目构了建腺病毒载体和组织特异性表达干预小鼠模型，明确了PU.1天然免疫调控功能和Dectin-1调控机制，阐明THP-1细胞免疫防御功能及其调节机制。从PU.1/Dectin-1这个视点，拓展对宿主抗真菌防御调控机制的认识，揭示PU.1/Dectin-1在抗感染天然免疫中的作用，为IA的免疫治疗奠定理论基础。