Redox-sensitive pathways Mediating RSV Pathogenesis

呼吸道上皮是环境和呼吸道内环境之间的主要细胞屏障。吸入感染剂后，上皮细胞能够分泌多种参与炎症和免疫反应的分子，如细胞因子和趋化因子。呼吸道合胞病毒(RSV)是一种强有力的细胞因子和 趋化因子基因在呼吸道上皮细胞中的表达我们发现，在病毒去壳、转录和病毒复制的过程中，RSV诱导多种细胞内信号，导致转录因子亚群的激活，包括NFkappaB、AP-1、STAT和IRF，这些转录因子是RSV诱导的趋化因子基因表达所必需的。呼吸道合胞病毒感染后，NF-kappaB、STAT和IRF的激活通过氧化还原敏感的途径发生，因为它被呼吸道上皮细胞的抗氧化处理所抑制。呼吸道合胞病毒(RSV)感染是一种强烈的活性氧(ROS)诱导物，ROS是一种普遍存在的、高度可扩散的反应性分子，是细胞内信号转导的重要调节因子。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lKKepsilon中的作用。lKKepsilon是最近克隆的一种新的信号通路，在RSV感染后导致呼吸道上皮细胞中STAT、IRF和NF-kappaB的诱导。我们将从以下几个方面探讨ROS的产生与IKKepsilon、PTPs的调控以及下游转录因子激活的关系：目的1.探讨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，然后我们将通过过度表达催化失活的蛋白质或通过小干扰RNA(SiRNA)下调它们的表达来确定它们在STAT激活中的作用。目的2.探讨ROS在RSV诱导的IKKepsilon激活中的作用。我们将研究在没有或存在抗氧化剂的情况下，呼吸道上皮细胞感染RSV后IKKepsilon基因的表达、蛋白质合成和激酶活性。我们将克隆IKKepsilon启动子，以确定IKKepsilon的诱导是否是转录介导的，以及它是否依赖于病毒诱导的ROS产生。目的3.确定ROS依赖的调控核因子-kappaB转录的机制。我们将分析病毒诱导的含或不含抗氧化剂的呼吸道上皮细胞中核因子-kappaB的磷酸化水平。类似的实验将在IKKepsilon信号传递的细胞中进行 已经被破坏了。我们将确定IKKepsilon介导的NF-kappaB转录激活是否需要特定的丝氨酸磷酸受体位点。最后，我们将在没有或存在抗氧化剂的情况下，使用野生型进行IKKepsilon激酶检测 或丝氨酸突变的核因子-kappaB作为底物，以确定IKKepsilon直接磷酸化核因子-kappaB的能力以及该激酶的活性是否依赖于氧化还原。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.