Redox-sensitive pathways Mediating RSV Pathogenesis

呼吸道上皮是环境与气道内环境之间的主要细胞屏障。在吸入感染剂后，上皮能够分泌参与炎症和免疫应答的多种分子，如细胞因子和趋化因子。呼吸道合胞病毒（RSV）是细胞因子的有效刺激物， 气道上皮细胞趋化因子基因表达。我们已经表明，在病毒脱壳，转录和病毒复制的过程中，RSV诱导几种细胞内信号，导致转录因子的一个子集，包括NF κ B，AP-1，STAT和IRF，这是必要的RSV诱导的趋化因子基因表达的激活。RSV感染后，NF-κ B、STAT和IRF的活化通过氧化还原敏感性途径发生，因为其被气道上皮细胞的抗氧化剂处理抑制。RSV感染是活性氧（ROS）的强诱导剂，活性氧是普遍存在的、高度扩散的和反应性的分子，其作为细胞内信号传导的重要调节剂起作用。在这个项目中，我们将研究ROS在调节细胞内蛋白磷酸酶（PTP）活性，以及激活IKK激酶，最近克隆的IKK样激酶，作为新的信号通路，导致STAT，IRF和NF-κ B诱导气道上皮细胞，呼吸道合胞病毒感染后的作用。我们将测试ROS产生与IKK β、PTP和下游转录因子激活的调节之间的关系，具体目标如下：研究ROS在蛋白酪氨酸磷酸酶活性中的作用及其与RSV诱导的STAT激活的关系。我们将研究在存在或不存在抗氧化剂的情况下感染RSV的细胞中PTP1B和SHP-2的酶活性，然后我们将通过过表达无催化活性的蛋白质或通过小干扰RNA（siRNA）下调其表达来确定它们在STAT激活中的作用。目标2.确定ROS在RSV诱导的IKK β激活中的作用。我们将研究IKK β基因表达，蛋白质合成和激酶活性在呼吸道合胞病毒感染的气道上皮细胞在存在或不存在抗氧化剂。我们将克隆IKK β启动子，以确定IKK β诱导是否是转录介导的，以及是否依赖于病毒诱导的ROS产生。目标3。确定调节NF-κ B驱动转录的ROS依赖性机制。我们将分析病毒诱导的NF-κ B磷酸化水平在气道上皮细胞处理或不抗氧化剂。类似的实验将在IKK β信号转导的细胞中进行。 被打乱了我们将确定IKKepsilon介导的NF-κ B转录激活是否需要特定的丝氨酸磷酸受体位点。最后，我们将使用野生型IKK激酶，在有或无抗氧化剂的情况下进行IKK激酶测定。 或丝氨酸突变的NF-κ B作为底物，以确定IKK β直接磷酸化NF-κ B的能力以及激酶活性是否是氧化还原依赖性的。从这些研究中获得的见解将有助于制定新的治疗方法， 治疗病毒引起的肺部炎症和感染后哮喘。

The respiratory epithelium represents the principal cellular barrier between the environment and the intemal milieu of the airways. Following inhalation of infectious agents, the epithelium is able to secrete a variety of molecules involved in inflammatory and immune responses, like cytokines and chemokines. Respiratory syncytial virus (RSV) is a potent stimulus of cytokines and chemokines gene expression in airway epithelial cells. We have shown that during the process of viral uncoating, transcription and viral replication, RSV induces several intracellular signals leading to activation of a subset of transcription factors, including NFkappaB, AP-1, STAT and IRF, which are necessary for RSV-induced chemokine gene expression. Activation of NF-kappaB, STAT and IRF, following RSV infection, occurs through redox-sensitive pathways since it is inhibited by antioxidant treatment of airway epithelial cells. RSV infection is a strong inducer of reactive oxygen species (ROS), ubiquitous, highly diffusable and reactive molecules which function as important regulators of intracellular signaling. In this project, we will investigate the role of ROS in modulating intracellular protein phosphatase (PTP) activity, as well as in activation of lKKepsilon, a recently cloned IKK-like kinase, as novel signaling pathways leading to STAT, IRF and NF-kappaB induction in airway epithelial cell, following RSV infection. We will test the relationship between ROS production and modulation of IKKepsilon, PTPs and downstream transcription factor activation in the following specific aims: Aim 1. To investigate the role of ROS in protein tyrosine phosphatase activity and its relationship to RSV-induced STAT activation. We will investigate PTP1B and SHP-2 enzymatic activity in cells infected with RSV in the presence or absence of antioxidants, and we will then determine their role in STAT activation by over-expressing catalytically-inactive proteins or down-regulating their expression by small interfering RNA (siRNA). Aim 2. To determine the role of ROS in RSV-induced IKKepsilon activation. We will investigate IKKepsilon gene expression, protein synthesis and kinase activity in airway epithelial cells infected with RSV in the absence or presence of antioxidants. We will clone the IKKepsilon promoter to determine if IKKepsilon induction is transcriptionally mediated and if it is dependent on viral-induced ROS generation. Aim 3. To identify ROS-dependent mechanisms regulating NF-kappaB driven transcription. We will analyze viral-induced levels of NF-kappaB phosphorylation in airway epithelial cells treated with or without antioxidant. Similar experiments will be performed in cells where IKKepsilon signaling has been disrupted. We will determine whether specific serine phosphoacceptor sites are required for IKKepsilon-mediated NF-kappaB transcriptional activation. Finally, we will perform IKKepsilon kinase assays, in the absence or presence of antioxidants, using wild type or serine mutated NF-kappaB as substrate, to determine the ability of IKKepsilon to directly phosphorylate NF-kappaB and if the kinase activity is redox-dependent. Insights gained from these studies will be instrumental in formulating novel therapeutic approaches for the treatment of viral-induced lung inflammation and post-infectious asthma.