Rhinovirus and Airway Epithelial Cell Responses

鼻病毒（RV）感染占哮喘急性发作的很大一部分。气道中性粒细胞和IL-8 在RV诱导的急性加重中水平升高，表明RV通过诱导 上皮细胞表达（El_R）+ C-X-C趋化因子，导致过度的炎症反应。 在初步研究中，我们已经表明，RV 39诱导IL-8，ENA-78和GRO-α表达的主要，μ- 纤毛分化的人气管上皮细胞。在16 HBE 140-细胞中，RV 39感染激活Src，PI 3-激酶、Akt和ERK，并且这些激酶的活化是IL-8表达所需的。 RV增加由两种促哮喘细胞因子IL-13和TNF α诱导的C-X-C趋化因子表达。菲- 总之，RV 1B感染C57/BL 6小鼠增加气道中性粒细胞和MIP-2水平，MIP-2是一种小鼠ELR（+）C- X-C趋化因子因此，我们假设RV足以激活生化信号通路 参与哮喘反应，为RV诱导的哮喘加重提供了机制。 具体目标1：表征PI 3-激酶的上游激活物和下游效应物， RV诱导的ELR（+）C-X-C趋化因子表达。我们假设：1）RV与Src，PI 3共定位， 2）Src是PI 3-激酶/Akt通路激活所必需的; 3）类 IA、II和III PI 3-激酶是最大RV诱导的IL-8、ENA-78和GROot表达所需的;和 4)最大的NF-κ B活化需要NADPH氧化酶的PI 3-激酶依赖性活化。 具体目标2：确定负责协同效应的生化信号传导机制， RV和促哮喘细胞因子对气道上皮细胞IL-8表达的影响。我们假设：1）ERKand JNK通过激活AP-1启动子位点调节IL-8的表达，AP-1启动子位点作为一个基础水平， 增强; 2）RV 39和TNF α的累加效应由增加的p65 RelA磷酸化和NF-κ B介导。 3）RV 39和IL-13的协同作用由增加的AP-1反式激活介导。 具体目标3：确定RV诱导信号传导所需或足够的病毒生命周期步骤 和趋化因子反应，相反，决定了宿主细胞信号转导的需要， 病毒感染我们假设：1）ICAM 1连接是Src，PI 3- 1激活所必需的，并且是充分的。 激酶、Akt、ERK和JNK; 2）这些信号传导中间体的激活不需要病毒复制; 和3）PI 3-激酶活化是RV 39内化所必需的。 具体目标4：确定PI 3-激酶信号传导和ELR（+）C-X-C趋化因子的需求， RV诱导的体内反应。我们假设：1）RV 1B感染足以导致气道炎症 2）PI 3-激酶是RV 1B诱导的气道炎症所必需的; C-X-C趋化因子受体（CXCR）-2在体内调节RV 1B诱导的气道炎症。 了解RV引起的哮喘急性发作将导致这种疾病治疗的改善。

Rhinovirus (RV) infection accounts for a large fraction of asthma exacerbations. Airway neutrophils and IL-8 levels are increased in RV-induced exacerbations, suggesting that RV stimulates exacerbations by inducing epithelial cell expression of (El_R)+ C-X-C chemokines, leading to an exaggerated inflammatory response. In pilot studies, we have shown that RV39 induces IL-8, ENA-78 and GRO-ct expression in primary, mu- cociliary-differentiated human tracheal epithelial cells. In 16HBE14o- cells, RV39 infection activates Src, PI 3-kinase, Akt and ERK minutes after infection, and activation of these kinases is required for IL-8 expression. RV increases C-X-C chemokine expression induced by two pro-asthmatic cytokines, IL-13 and TNFa. Fi- nally, RV1B infection of C57/BL6 mice increases airway neutrophils and levels of MIP-2, a murine ELR(+) C- X-C chemokine. Wetherefore hypothesize that RV is sufficient to activate biochemical signalingpathways involved in the asthmatic response, providing a mechanism for RV-induced asthma exacerbations. Specific Aim 1: Characterize upstream activators and downstream effectors of PI 3-kinase required for RV-induced ELR(+) C-X-C chemokine expression. We hypothesize that: 1) RV colocalizes with Src, PI 3- kinase, Akt and Grb2 in lipid rafts; 2) Src is required for activation of the PI 3-kinase/Akt pathway; 3) Class IA, II and III PI 3-kinases are required for maximal RV-induced expression of IL-8, ENA-78 and GROot; and 4) maximal NF-KB activation requires PI 3-kinase-dependent activation of NADPH oxidase. Specific Aim 2: Determine the biochemical signaling mechanisms responsible for cooperative effects of RV and pro-asthmatic cytokines on airway epithelial cell IL-8 expression. We hypothesize that: 1) ERKand JNK regulate IL-8 expression via activation of the AP-1 promoter site, which functions as a basal level en- hancer; 2) additive effects of RV39 and TNFa are mediated by increased p65 RelA phosphorylation and NF- transactivation; 3) synergistic effects of RV39 and IL-13 are mediated by increased AP-1 transactivation. Specific Aim 3: Determine the steps in the viral life cycle required or sufficient for RV-induced signaling and chemokine responses and, conversely, determine the requirement of host cell signal transduction for viralinfection. We hypothesize that: 1) ICAM1 ligation is required and sufficient for activation of Src, PI 3- kinase, Akt, ERK and JNK; 2) viral replication is not required for activation of these signaling intermediates; and 3) PI 3-kinase activation is required for RV39 internalization. Specific Aim 4: Determine the requirements of PI 3-kinase signaling and ELR(+) C-X-C chemokines for RV-inducedresponses in vivo. We hypothesize that: 1) RV1B infection is sufficient for airway inflammation and epithelial cell signaling in vivo; 2) PI 3-kinase is required for RV1B-induced airway inflammation in vivo; and 3) C-X-C chemokine receptor (CXCR)-2 regulates RV1B-induced airway inflammation in vivo. Understandina RV-induced asthma exacerbations will lead to improvements in the treatment of this disease.