Novel Role of EGFR in Virus-Induced Asthma Exacerbations

描述（由申请人提供）：大多数哮喘加重是由病毒感染引起的，儿童病毒感染有助于哮喘的发病机制。由于没有有效的抗病毒治疗方法，病毒引起的哮喘恶化仍然是造成严重发病率和死亡率的原因。在哮喘中，气道上皮干扰素-λ (IFN-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irf1依赖的IFN-λ，从而导致病毒感染增加。此外，我们发现抑制EGFR增加IRF1和IFN-λ抑制病毒感染。这一结果对哮喘有潜在的影响，因为EGFR信号在哮喘上皮中增加。然而，由于现有的EGFR抑制剂可能存在副作用，我们研究了IFN-λ抑制EGFR，发现IFN-λ抑制EGFR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运输到细胞核以影响关键的细胞功能，其中包括细胞核中的钙（Ca2+）信号。初步研究发现，核Ca2+信号的抑制减弱了EGFR对IRF1的抑制，这意味着核EGFR依赖的Ca2+信号抑制了IRF1。最近，我们发现痰中EGFR表达与哮喘严重程度相关。由于巨噬细胞是痰中主要的细胞类型，我们研究了巨噬细胞中的EGFR信号。初步实验显示，痰液和pbmc来源的巨噬细胞中存在功能性EGFR。此外，EGFR激活的增加抑制了IRF1，这与我们在气道上皮中的发现相似。因此，本提案的假设是EGFR信号是通过抑制气道上皮细胞和巨噬细胞中irf1依赖性IFN-λ对哮喘病毒感染易感性的组成部分。在Aim 1中，我们将使用病毒诱导哮喘加重的小鼠模型来研究EGFR抑制对炎症的影响。遗传方法将抑制气道上皮EGFR，我们将研究鼻内EGFR siRNA和IFN-λ。目的2将研究核EGFR依赖的Ca2+信号来抑制IRF1和IFN-λ，使用共聚焦显微镜，构建和siRNA靶向EGFR运输到细胞核并抑制核Ca2+信号。这将包括实验研究IFN-λ抑制气道上皮细胞核EGFR的作用。目的3将研究哮喘患者和健康人痰和血巨噬细胞中的EGFR信号，包括IFN-λ对巨噬细胞核EGFR的影响。最后，利用基因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加重中抑制炎症的有效性，这将为新疗法提供见解；2)核EGFR依赖的Ca2+信号影响抗病毒免疫的新机制；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.