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A2856 - Glucocorticoid Receptor Dysfunction in Response to Respiratory Syncytial Virus Infection in Primary Human Airway Smooth Muscle Cells
Author Block: T. J. Harford1, V. Bokun2, F. Rezaee3, G. Piedimonte4; 1Pediatric Research Center, Cleveland Clinic Childrens, Cleveland, OH, United States, 2Pathobiology Pediatric Research Center, Cleveland Clinic Lerner Research Institute, Cleveland, OH, United States, 3Pediatrics, Center for Pediatric Research, Cleveland, OH, United States, 4Pediatrics, The Cleveland Clinic, Cleveland, OH, United States.
RATIONALE: Airway epithelial cells are the primary target for respiratory syncytial virus (RSV) infection, however studies have shown human airway smooth muscle cells (HASMCs) can also be infected. The primary carepath in treating severe RSV-induced bronchiolitis includes the use of corticosteroids however, is not recommended in pediatric patients due to lack of effectiveness and serious side effects. A better understanding of the effects of RSV on glucocorticoid receptor (GR) signaling in HASMCs from pediatric donors is necessary to more effectively treat RSV bronchiolitis in children. We therefore hypothesized that RSV infection of hASMCs leads to disruption of the GR signaling pathway. METHODS Primary hASMCs isolated from the lungs of pediatric donors were infected with control medium or replicating RSV strain A2 expressing recombinant red protein (rrRSV) at a multiplicity of infection (MOI) of 1. Cells were treated with vehicle or dexamethasone (1µM) then analyzed for RSV mediated changes in GR expression, phosphorylation or subcellular localization by western blot analysis. Changes in GR function was assessed by quantitative PCR of glucocorticoid induced leucine zipper (GILZ) gene. RESULTS GR expression was assessed 24 hours post RSV infection (MOI of 1) and found a 20% reduction in protein expression. We then analyzed the effect of RSV infection on dexamethasone induced phosphorylation of GR. At 24 hours post infection, dexamethasone induced phosphorylation of GR was reduced by 30%. We then analyzed the effect of RSV infection on dexamethasone induced translocation of GR from the cytoplasm to the nucleus. Nuclear GR was reduced in response to RSV infection at 24 hours both in absence and presence of dexamethasone treatment by 50% and 30% respectively. Lastly, we analyzed function of nuclear GR by expression of GILZ gene. Dexamethasone induced GILZ gene expression was reduced by 20% at 24 hours post RSV infection. CONCLUSIONS: This study demonstrates GR expression, phosphorylation, translocation and functional alterations occur in response to RSV infection in primary human airway smooth muscle cells derived from pediatric donors. These changes could be responsible for steroid resistance in children suffering RSV induced bronchiolitis. These data provides important insight for the development of novel treatments for RSV induced bronchiolitis in children who are unresponsive to current therapies.