Table_2_The Expression of ephrinA1/ephA2 Receptor Increases in Chronic Rhinosinusitis and ephrinA1/ephA2 Signaling Affects Rhinovirus-Induced Innate Immunity in Human Sinonasal Epithelial Cells.docx

EphA2 receptor and its ephrin ligands are involved in virus infection, epithelial permeability, and chemokine secretion. We hypothesized that ephrinA1/ephA2 signaling participates in rhinovirus (RV)-induced antiviral immune response in sinonasal mucosa of patients with chronic rhinosinusitis (CRS). Therefore, we investigated the expression of ephrinA1/ephA2 in normal and inflamed sinonasal mucosa and evaluated whether they regulate chemokine secretion and the production of antiviral immune mediators including interferons (IFNs) in RV-infected human primary sinonasal epithelial cells. For this purpose, the expression and distribution of ephrinA1/ephA2 in sinonasal mucosa were evaluated with RT-qPCR, immunofluorescence, and western blot. Their roles in chemokine secretion and the production of antiviral immune mediators such as type I and III IFNs, and interferon stimulated genes were evaluated by stimulating ephA2 with ephrinA1 and inactivating ephA2 with ephA2 siRNA or inhibitor in cells exposed to RV and poly(I:C). We found that ephrinA1/ephA2 were expressed in normal mucosa and their levels increased in inflamed sinonasal mucosa of CRS patients. RV infection or poly(I:C) treatment induced chemokine secretion which were attenuated by blocking the action of ephA2 with ephA2 siRNA or inhibitor. The production of antiviral immune mediators enhanced by rhinovirus or poly (I:C) is increased by blocking ephA2 compared with that of cells stimulated by either rhinovirus or poly(I:C) alone. In addition, blocking ephA2 attenuated RV replication in cultured cells. Taken together, these results describe a novel role of ephrinA1/ephA2 signaling in antiviral innate immune response in sinonasal epithelium, suggesting their participation in RV-induced development and exacerbations of CRS.EphA2受体（EphA2 receptor）及其ephrin配体（ephrin ligands）参与病毒感染、上皮通透性调控及趋化因子分泌过程。我们假设ephrinA1/EphA2信号通路参与慢性鼻窦炎（CRS）患者鼻窦黏膜内鼻病毒（RV）诱导的抗病毒免疫应答。为此，我们检测了正常与炎性鼻窦黏膜中ephrinA1/EphA2的表达水平，并评估二者是否可在RV感染的人原代鼻窦上皮细胞中调控趋化因子分泌，以及包括干扰素（IFNs）在内的抗病毒免疫介质的产生。为达成上述研究目的，我们采用实时荧光定量聚合酶链式反应（RT-qPCR）、免疫荧光及蛋白质免疫印迹（Western Blot）技术，检测了鼻窦黏膜中ephrinA1/EphA2的表达与分布情况。通过在RV与聚肌胞苷酸（poly(I:C)）处理的细胞中，用ephrinA1激活EphA2，或利用EphA2小干扰RNA（siRNA）与抑制剂阻断EphA2功能，我们评估了二者在趋化因子分泌，以及I型、III型干扰素与干扰素刺激基因等抗病毒免疫介质产生过程中的作用。研究结果显示，正常黏膜组织中即可检测到ephrinA1/EphA2的表达，且CRS患者的炎性鼻窦黏膜中二者的表达水平显著升高。RV感染或poly(I:C)处理可诱导趋化因子分泌，而使用EphA2 siRNA或抑制剂阻断EphA2功能可削弱这一过程。相较于单独使用RV或poly(I:C)刺激的细胞，阻断EphA2可增强RV或poly(I:C)诱导的抗病毒免疫介质生成。此外，阻断EphA2可抑制培养细胞内的RV复制。综上，本研究揭示了ephrinA1/EphA2信号通路在鼻窦上皮抗病毒天然免疫应答中的全新作用，提示其参与了RV诱导的CRS发生与病情加重过程。