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 (ELR)+ 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-a expression in primary, mucociliary-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. Finally, RV1B infection of C57/BL6 mice increases airway neutrophils and levels of MIP-2, a murine ELR(+) C- X-C chemokine. We therefore hypothesize that RV is sufficient to activate biochemical signaling pathways 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 GRO-a; and 4) maximal NF-kappaB 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) ERK and JNK regulate IL-8 expression via activation of the AP-1 promoter site, which functions as a basal level enhancer; 2) additive effects of RV39 and TNFa are mediated by increased p65 RelA phosphorylation and NF-kappaB 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 viral infection. 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-induced responses 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. Understanding RV-induced asthma exacerbations will lead to improvements in the treatment of this disease.
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