流感病毒感染引起气道液体生物物理学特性改变及相关机制的研究

Ciliated epithelial cells line the upper airway and function as the first defense against deleterious pathogens, allergens, and pollutants. The attachment to luminal surface and fusogenicity of these allergic and infectious particles are regulated by the surface charges, pH, and volume of the luminal fluid. It is unknown how the host airway epithelium controls these parameters post viral infection, and what takes place secondary to invasion of these airborne substances. The airway epithelial monolayer is covered by a thin periciliary fluid layer which enables the cilia beat to clear microbial-containing mucus. This watery layer is also the main battlefront for pulmonary immune responses. The volume, pH, ion component, osmolarity, and other biophysical features are predominantly controlled by the balance of transepithelial fluid turnover and re-absorption. Positively-charged Na+ cations are removed via the apical epithelial sodium channels (ENaC), while the cystic fibrosis transmembrane conductance regulator (CFTR) is a key pathway for delivering negative charges to the lumen (mainly Cl- and HCO3- anions). ENaC is located in the “brush-like” cilia while CFTR proteins are at the plasma membrane between cilia. Electrically, the periciliary fluid and cilia surface are negatively charged by removing cations via ENaC and secreting anions though CFTR. The mechanical beating is at least partially governed by electrostatic repelling force between every negatively-charged cilium. Meanwhile, HCO3- anions react with H+ ions to neutralize pH. By detecting the functional changes of ENaC and CFTR in mouse tracheal epithelial monolayers infected with a seasonal influenza H1N1 strain (A/PR/8/34), we found these two critical pathways are impaired. Our central hypotheses are that ENaC and CFTR coordinately maintain a negatively-charged, finely regulated fluid layer covering the normal airway, and that influenza-induced changes in the biophysical profile of luminal fluid facilitate subsequent attachment and endocytosis of pathogens, allergens, and pollutants. We will characterize the intermolecular regulation between ENaC and CFTR in primary mouse tracheal epithelial cell monolayers challenged by influenza. We believe that the data we gather will identify novel cellular and molecular mechanisms for the biophysical responses of host airway luminal fluid to pathogens, allergens, and pollutants.

位于呼吸道表面的纤毛上皮细胞，是机体防御有害病原体、过敏原和污染物的首要屏障。气道管腔液体的表面电荷数、pH值和粘性，直接调控着过敏性、感染性颗粒对气道管腔表面的吸附和融合作用。带有正电荷的阳离子通过上皮钠通道（epithelial sodium channels，ENaC）进入细胞，而囊性纤维跨膜电导调节因子（cystic fibrosis transmembrane conductance regulator，CFTR）将阴离子运至管腔。通过分析感染一种流感病毒株A/PR/8/34的小鼠气管上皮中CFTR和ENaC的功能，发现这两个关键性途径都受到破坏。本课题的核心论点是流感病毒通过干扰两种转运电荷的CFTR和ENaC蛋白而改变气道管腔液体的生物物理学特性，继发加剧病原体、过敏原和污染物的致病过程。此研究结果将为宿主气道管腔液体对上述物质的生物物理学反应提供新的细胞和分子水平的机制。

正常人的呼吸道中，大约80%的细胞是带有纤毛的上皮细胞，这些组成紧密气道上皮层的极化细胞不仅可以作为一种有效防御各种有害物质的物理屏障，同时通过主动维持气道表面流体层的正常生物物理学特性而构成一个高度复杂的宿主防御系统，其中包括净负电荷、中性pH和黏性等。带有正电荷的阳离子通过上皮钠通道（epithelial sodium channels，ENaC）进入细胞，而囊性纤维跨膜电导调节因子（cystic fibrosis transmembrane conductance regulator，CFTR）将阴离子运至管腔。流感是一种主要的致命性呼吸系统疾病，常伴有多系统并发症，关于气道管腔表层液体对流感病毒、过敏原和污染物的生物物理学特征极少被人们关注。通过体外培养的小鼠气管上皮细胞我们发现气道管腔顶膜侧的CFTR与纤毛上的ENaC活性相关联，证实了小鼠气管上皮细胞中Na+的定向转运对于维持CFTR活性是必须的；同时发现流感病毒感染以时间和病毒剂量依赖性方式明显降低CFTR和ENaC的转录和蛋白表达水平；首次证实流感病毒能够增加小鼠气道表面液体高度，而黏性不受影响。无赖氨酸激酶4（with-no-lysine-kinase-4, WNK4）作为蛋白激酶C下游可以调节许多离子通道的活性，包括ENaC。实验结果表明流感病毒感染的小鼠气管上皮细胞中WNK4表达明显升高。应用siRNA特异性敲减WNK4基因可以减轻流感病毒对ENaC的影响，表明流感病毒可能是通过细胞内WNK4影响ENaC活性。病毒和细菌表面通常带有负电荷，病毒和细菌入侵宿主细胞需要在酸性pH下进行。本项目研究结果表明流感病毒主要通过干扰ENaC蛋白进而破坏气道管腔液体生物物理学特性，导致气道表层液体去极化和酸化，最终使机体更易受到各种病原体、过敏原和污染物的危害。此研究结果将为宿主气道管腔液体对上述物质的生物物理学反应提供新的细胞和分子水平的机制。通过本项目组成员的共同努力和合作，项目已基本按计划顺利完成。在国内外著名期刊公开发表标注基金资助论文38篇，其中被Sci收录20篇。获得2020实用新型专利一项，参加国际会议四次，培养硕/博士研究生共16人。通过本项目的研究工作基础获得2018年辽宁省重点研发计划指导计划项目一项。