Novel Role of EGFR in Virus-Induced Asthma Exacerbations

描述(申请人提供)：大多数哮喘恶化是由病毒感染引起的，儿童病毒感染有助于哮喘的发病。由于缺乏有效的抗病毒治疗，病毒引起的哮喘加重仍然是发病率和死亡率的重要原因。在哮喘中，呼吸道上皮细胞干扰素-λ缺乏，导致抗病毒免疫反应受损。我们已经证明，病毒激活表皮生长因子受体(EGFR)来抑制依赖IRF1的干扰素-λ，从而导致病毒感染的增加。此外，我们还发现抑制表皮生长因子受体可以增加IRF1和干扰素-λ的表达，从而抑制病毒感染。这一结果对哮喘有潜在的意义，因为哮喘上皮细胞中的EGFR信号增加。然而，由于现有的表皮生长因子受体抑制剂可能存在副作用，我们研究了干扰素-λ抑制表皮生长因子受体的作用，发现干扰素-λ抑制了表皮生长因子受体的激活。EGFR生物学的一个新兴领域是这种细胞表面酪氨酸激酶受体向细胞核传递，以影响关键的细胞功能，包括细胞核中的钙(Ca2+)信号。初步研究发现，抑制核内钙信号可减弱EGFR对IRF1的抑制作用，这意味着核内依赖于EGFR的钙信号抑制了IRF1。最近，我们发现痰中EGFR的表达与哮喘的严重程度相关。因为巨噬细胞是痰中的主要细胞类型，所以我们研究了巨噬细胞中的EGFR信号。初步实验表明，在痰和PBMC来源的巨噬细胞中具有功能的EGFR。此外，增加的EGFR激活抑制了IRF1，这与我们在呼吸道上皮细胞中的发现相似。因此，这一建议的假设是，通过抑制呼吸道上皮细胞和巨噬细胞中依赖于IRF1的干扰素-λ，EGFR信号转导与哮喘病毒感染的易感性是不可或缺的。在目标1中，我们将使用病毒诱导的哮喘加重的小鼠模型来研究EGFR抑制对炎症的影响。基因方法将抑制呼吸道上皮细胞表皮生长因子受体，我们将研究鼻腔内表皮生长因子受体siRNA和干扰素-λ。目的2利用共聚焦显微镜技术，构建靶向EGFR转运到细胞核并抑制核内钙信号转导的小干扰RNA，研究其抑制iRF1和干扰素-λ的作用。这将包括研究干扰素-λ抑制呼吸道上皮细胞核表皮生长因子受体的作用的实验。目的研究哮喘患者和健康人痰和血巨噬细胞的表皮生长因子受体信号，包括干扰素-λ对巨噬细胞胞核表皮生长因子受体的影响。最后，使用遗传模型去除巨噬细胞中的EGFR，我们将研究EGFR信号在病毒诱导的哮喘加重小鼠模型中的细胞特异性贡献。这些研究将阐明：1)EGFR抑制在病毒诱导的哮喘发作中抑制炎症的有效性，这将为新的治疗方法提供见解；2)核EGFR依赖的钙信号影响抗病毒免疫的新机制；以及3)巨噬细胞EGFR信号在哮喘中的作用，这将扩展我们目前的EGFR生物学模型。

DESCRIPTION (provided by applicant): The majority of asthma exacerbations are caused by viral infections, and childhood viral infections contribute to asthma pathogenesis. Virus-induced asthma exacerbations continue to account for significant morbidity and mortality, because effective antiviral therapies are not available. In asthma, airway epithelial Interferon-lambda (IFN-λ) is deficient, which results in impaired antiviral immune responses. We have shown that viruses activate epidermal growth factor receptor (EGFR) to suppress IRF1-dependent IFN-λ, which results in increased viral infection. In addition, we found that inhibiting EGFR increased IRF1 and IFN-λ to suppress viral infection. This result has potential implications in asthma because EGFR signaling is increased in asthmatic epithelium. However, because of possible side effects with existing EGFR inhibitors, we investigated IFN-λ to suppress EGFR, and found that IFN-λ inhibits EGFR activation. An emerging area of EGFR biology is that this cell surface tyrosine kinase receptor traffics to the nucleus to influence key cell functions, which include calcium (Ca2+) signaling in the nucleus. Preliminary work found that inhibition of nuclear Ca2+ signaling attenuates EGFR suppression of IRF1, which implicates nuclear EGFR-dependent Ca2+ signaling to suppress IRF1. More recently, we found that sputum EGFR expression correlates with asthma severity. Because macrophages are the predominant cell type in sputum, we investigated EGFR signaling in macrophages. Preliminary experiments showed functional EGFR in sputum and PBMC-derived macrophages. In addition, increased EGFR activation suppressed IRF1, which parallels our findings in airway epithelium. Therefore, the hypothesis of this proposal is that EGFR signaling is integral to susceptibility to viral infectionin asthma via suppression of IRF1-dependent IFN-λ in both airway epithelial cells and macrophages. In Aim 1, we will use a murine model of virus-induced asthma exacerbations to study the effect of EGFR inhibition on inflammation. A genetic approach will suppress airway epithelial EGFR, and we will study intranasal EGFR siRNA and IFN-λ. Aim 2 will investigate nuclear EGFR-dependent Ca2+ signaling to suppress IRF1 and IFN-λ using confocal microscopy, constructs, and siRNA that target EGFR trafficking to the nucleus and inhibit nuclear Ca2+ signaling. This will include experiments to study the effect of IFN-λ to inhibit nuclear EGFR in airway epithelium. Aim 3 will investigate EGFR signaling in sputum and blood macrophages from asthmatics and healthy individuals, which will include the effect of IFN-λ on nuclear EGFR in macrophages. Finally, using a genetic model to remove EGFR in macrophages, we will study the cell-specific contribution of EGFR signaling in the mouse model of virus-induced asthma exacerbations. These studies will elucidate: 1) the effectiveness of EGFR inhibition to suppress inflammation in virus-induced asthma exacerbations, which will provide insights for novel therapies, 2) a new mechanism for nuclear EGFR-dependent Ca2+ signaling to influence antiviral immunity, and 3) the role of macrophage EGFR signaling in asthma, which will expand our current model of EGFR biology.