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A3848 - Cytoskeletal Alterations of Rhinovirus-Infected Airway Epithelial Cells
Author Block: A. N. Michi, D. Proud; Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada.
RATIONALE: Asthmatic airways show characteristic structural characteristics collectively referred to as airway remodeling (AR). These structural changes include impaired barrier function and increased epithelial junctional fragility. Bronchial biopsy studies reveal evidence of AR in wheezing pre-school age children even before the formal diagnosis of asthma is made. Interestingly, such features are not observed in symptomatic infants with variable airflow limitation, suggesting that AR occurs in response to a stimulus in early life. Human rhinovirus (HRV)-induced wheezing illnesses are a major risk factor for the subsequent development of asthma in children. The human airway epithelial cell is the major site of HRV infection and replication. Based on studies of other picornaviruses, we hypothesize that:
HRV-infection of normal human bronchial epithelial cells (HBE) causes cytoskeletal alterations during viral replication which result in transient intercellular junctional changes; and we speculate that asthmatic HBE cells have a diminished capacity to recover from cytoskeletal changes, leading to a permanently altered barrier.
METHODS: Highly differentiated air-liquid interface (ALI) cultures of HBE, isolated from normal human lungs, were infected with three different HRV serotypes: HRV-16, HRV-1A, and HRV-C15 for 24, 48, 72, 96, 120, 144 hours, and the junctional protein, ZO-1, was examined. Cytoskeletal studies were conducted using HBEs grown in submersion culture. Other stimuli include replication-deficient HRV, poly(I:C) (dsRNA mimic), cytochalasin D (actin disruptor), and paclitaxel (microtubule stabilizer). Cells were fixed for immunofluorescence imaging using a resonant scanning confocal microscope. HRV replication kinetics were determined by serially washing ALI cultures every 24 hours to determine viral release by RT-qPCR. Barrier function studies were conducted in Ussing chambers using FITC-dextran (permeability) and real-time measurements of transepithelial electrical resistance (TEER).
RESULTS: HRV-1A and HRV-C15, but not HRV-16 induce dramatic remodeling of ALI cellular architecture, including the co-localization of nuclei with apically expressed ZO-1. This response is HRV replication-dependent as replication-deficient HRV does not induce these changes. Additionally, the cells associated with remodeled architecture are positive for HRV infection, as assessed by expression of virus replication intermediates. Preliminary data suggests that cytoskeletal inhibitors diminish the ability of HRV replication to occur, indicating a critical role for the actin cytoskeleton in HRV replication.
CONCLUSION: We conclude that some, but not all, HRV serotypes induce altered junctional architecture in HBE that is associated with disruption of the actin cytoskeleton. We speculate that more prolonged and extensive changes will be observed in cells from patients with asthma.