Rhinovirus accelerates wound repair via surface signaling EGFR activation

描述(申请人提供)：呼吸道上皮表面代表一个“战场”，宿主在这里拦截来自病原体的信号，并激活先天防御系统以对抗感染。在慢性呼吸道疾病中，伤口修复是呼吸道上皮细胞对损伤做出反应的一项基本功能。鼻病毒(RV)感染是导致其中几种疾病恶化的原因。我们假设呼吸道上皮细胞对低浓度的RV有反应，以加速伤口修复。此外，我们预测高浓度的轮状病毒对呼吸道上皮细胞有毒性，并抑制伤口修复。由于表皮生长因子受体(EGFR)参与创面修复，我们推测RV通过激活EGFR的表面信号通路促进创面修复。我们的长期目标是研究病毒与呼吸道上皮细胞的相互作用，以加深我们对健康和疾病中先天免疫的理解。短期内，我们将重点关注三个具体目标。(1)研究RV感染对呼吸道上皮损伤修复的浓度依赖性影响，并对损伤上皮中的病毒感染进行定位。(2)我们将研究RV加速创面修复中激活EGFR的表面信号通路。(3)最后，我们将研究RV感染所需的分子是否参与了这一表面信号级联反应。在初步实验中，RV在划痕伤口模型中加速了伤口修复，这种效果在较高的病毒浓度下是看不到的。为了确定病毒浓度增加的影响，我们将测量伤口修复率和细胞坏死标记物。为了定位轮状病毒感染，我们将使用免疫组织化学和荧光显微镜。在初步实验中，一种选择性的EGFR酪氨酸激酶抑制剂阻止了RV加速的伤口修复，暗示着EGFR的激活。我们计划使用中和抗体、金属蛋白酶和氧化酶抑制剂、ROS清除剂和siRNA来评估激活EGFR的表面信号级联。此外，我们还将测量EGFR配体和ROS的释放。为了评估ICAM-1、神经酰胺和PKC在轮状病毒加速伤口修复中的作用，我们将使用一种抗体来防止病毒附着，神经酰胺和PKC抑制剂。综上所述，我们认为这个表面信号级联涉及：RV 16-&GT；ICAM-1-&GT；神经酰胺-PKC-&GT；Duox1-&GT；ROS-“TACE-”转化生长因子-α-&GT；EGFR磷酸化。轮状病毒感染具有临床重要性，因为该病毒是普通感冒的主要原因，并导致哮喘和慢性阻塞性肺病的恶化。由于目前还没有针对轮状病毒的治疗方法，因此研究病毒-上皮细胞的相互作用可能会为抗病毒治疗带来新的靶点，并可能增加我们对呼吸道防御损伤和感染的理解。

DESCRIPTION (provided by applicant): The surface of the airway epithelium represents a "battleground" where the host intercepts signals from pathogens and activates innate defenses to combat infection. Wound repair is an essential function of the airway epithelium in response to injury in chronic airway diseases. Rhinovirus (RV) infection is responsible for exacerbations of several of these diseases. We hypothesize that the airway epithelium responds to low concentrations of RV to accelerate wound repair. Further, we predict that high concentrations of RV are toxic to airway epithelial cells and inhibit wound repair. Because the epidermal growth factor receptor (EGFR) is involved in wound repair, we hypothesize that RV accelerates wound repair via a surface signaling pathway activating EGFR. Our long term goals are to investigate viral interactions with the airway epithelium to further our understanding of innate immunity in health and disease. In the short term we will focus on three specific aims. (1) We will study the concentration-dependent effects of RV infection on airway epithelial wound repair and localize viral infection in the wounded epithelium. (2) We will investigate the surface signaling pathway in RV-accelerated wound repair that activates EGFR. (3) Finally, we will examine whether molecules required for RV infection are involved in this surface signaling cascade. In preliminary experiments, RV accelerated wound repair in a scratch wound model, an effect that was not seen with a higher viral concentration. To determine the effects of increasing concentrations of the virus, we will measure the rate of wound repair and markers of cell necrosis. To localize RV infection we will use immunohistochemistry and fluorescent microscopy. In preliminary experiments an inhibitor selective for EGFR tyrosine kinase prevented RV-accelerated wound repair, implicating EGFR activation. We plan to evaluate the surface signaling cascade activating EGFR, using neutralizing antibodies, metalloprotease and oxidase inhibitors, ROS scavengers, and siRNA. In addition, we will measure the release of EGFR ligands and ROS. To evaluate the role of ICAM-1, ceramide and PKC in RV-accelerated wound repair, we will use an antibody to prevent viral attachment, and ceramide and PKC inhibitors. In summary, we suggest that this surface signaling cascade involves: RV 16-> ICAM-1 -> ceramide-PKC-> Duox1-> ROS-" TACE-" TGF- a-> EGFR phosphorylation. RV infection has clinical importance because the virus is the predominant cause of the common cold and causes exacerbations of asthma and COPD. Because therapies for RV are not available, investigating viral-epithelial interactions may lead to novel targets for anti-viral therapies and may increase our understanding of airway defenses against injury and infection.