Rhinovirus and Airway Epithelial Cell Responses

鼻病毒(RV)感染在哮喘加重中占很大比例。气道中性粒细胞与IL-8 在RV诱导的恶化中，水平增加，这表明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。 在前期研究中，我们发现RV39可诱导原代、小鼠和小鼠的IL-8、ENA-78和Gro-ct的表达。 球状分化的人气管上皮细胞。在16HBE140-细胞中，RV39感染激活了Src、Pi 3-激酶、Akt和ERK在感染后几分钟内被激活，这些激酶的激活是IL-8表达所必需的。 RV可增加IL-13和TNFa两种促哮喘细胞因子诱导的C-X-C趋化因子表达。菲-- 总之，C57/BL6小鼠RV1B感染增加了呼吸道中性粒细胞和MIP-2的水平，这是一种小鼠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，为轮状病毒引起的哮喘加重提供了机制。 具体目标1：鉴定PI3-激酶的上游激活子和下游效应子 RV诱导ELR()C-X-C趋化因子表达。我们假设：1)RV与Src，Pi3- 脂筏中的蛋白激酶、Akt和Grb2；2)PI3-激酶/Akt通路的激活需要Src；3)Class 在RV诱导的IL-8、ENA-78和GROOT的最大表达中，需要IA、II和IIIPI 3-激酶； 4)最大的核因子-KB激活需要依赖PI3-激酶的NADPH氧化酶的激活。 具体目标2：确定负责协同效应的生化信号机制 RV和促哮喘细胞因子对呼吸道上皮细胞IL-8表达的影响我们假设：1)ERKand JNK通过激活AP-1启动子位点来调节IL-8的表达，而AP-1启动子位点是一种基础水平的EN-2。 2)RV39和TNFa的相加效应是通过增加p65relA的磷酸化和NF-α来实现的。 3)RV39和IL-13的协同作用是通过增加AP-1的反式激活来实现的。 具体目标3：确定轮状病毒诱导信号所需或足够的病毒生命周期中的步骤 和趋化因子的反应，反过来，决定宿主细胞信号转导的需求 病毒感染。我们假设：1)ICAM1是激活Src，PI 3-所必需的且是充分的。 激酶、Akt、ERK和JNK；2)激活这些信号中间产物不需要病毒复制； (3)RV39的内化需要PI-3-激酶的激活。 特定目标4：确定PI3-激酶信号和ELR()C-X-C趋化因子对 RV诱导的体内反应。我们假设：1)RV1B感染足以引起呼吸道炎症 2)PI3K在RV1B诱导的体内气道炎症中起重要作用； 3)C-X-C趋化因子受体(CXCR)-2在体内调节RV1B诱导的气道炎症。 了解轮状病毒引起的哮喘恶化将导致这种疾病治疗的改进。

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.