View Abstract

A3856 - Investigating the Effects of Cigarette Smoke on Innate Antiviral Responses to Human Rhinovirus
Author Block: M. Love1, S. Wiehler1, R. Leigh2, D. Proud1; 1Department of Physiology and Pharmacology, University of Calgary, Calgary, AB, Canada, 2Department of Medicine, University of Calgary, Calgary, AB, Canada.
RATIONALE: Human rhinovirus (HRV) is the most frequently detected viral pathogen in patients hospitalized with acute exacerbations of chronic obstructive pulmonary disease (COPD). Cigarette smoke, the primary etiological risk factor for COPD is associated with more frequent and severe respiratory viral infections, resulting in worse clinical outcomes in patients with COPD. We hypothesize that exposure to cigarette smoke alters the kinetics of antiviral and pro-inflammatory mediator induction in primary human bronchial epithelial cells (HBE) during an HRV infection, resulting in an altered pattern of viral replication. While the effects of cigarette smoke on epithelial production of antivirals have been studied at a specific time points, the kinetics of antiviral induction upon exposure to whole cigarette smoke have not previously been reported. Such studies are possible using highly differentiated HBE cells cultured at the air-liquid interface, as they permit collection of samples from a single culture over extended periods of time.
METHODS: HBE cells isolated from non-transplanted lungs were differentiated in PneumaCultTM medium at the air liquid interface for 4 weeks following expansion in bronchial epithelial growth media. Highly differentiated cultures were then exposed to whole cigarette smoke for over a period of days and subsequently infected with HRV apically. Serial collections of apical washes and basolateral media were performed every 24h over the course of infection. Cell lysates were also collected to evaluate levels of intracellular viral RNA and cellular RNA at 24h intervals. Viral replication and associated epithelial production of antivirals were quantitatively assessed using RT-PCR and ELISA.
RESULTS: Our study demonstrates that exposure to whole cigarette smoke alters HRV replication patterns in infected HBE cells. Maximal intracellular and shed viral levels were reached at 48h post infection in clean air control cells. However cigarette smoke exposure significantly lowered absolute levels of intracellular and shed HRV at this time point. Modest suppression of IFNλ, IFNβ, IFIT2, IP-10, IFI44 and ISG15 transcripts was also measured over the course of infection.
CONCLUSIONS: Suppression of both viral replication and antiviral production in differentiated HBE cells treated with whole cigarette smoke, suggests that cigarette smoke impairs cellular handling of HRV. We speculate that the discrepancy between peak viral levels and antiviral induction in cigarette smoke treated cultures is related to an imbalance in the production of pro-inflammatory mediators in these cells.