Cigarette smoke modulates expression of human rhinovirus-induced airway epithelial host defense genes

Abstract

Human rhinovirus (HRV) infections trigger acute exacerbations of chronic obstructive pulmonary disease (COPD) and asthma. The human airway epithelial cell is the primary site of HRV infection and responds to infection with altered expression of multiple genes, the products of which could regulate the outcome to infection. Cigarette smoking aggravates asthma symptoms, and is also the predominant risk factor for the development and progression of COPD. We, therefore, examined whether cigarette smoke extract (CSE) modulates viral responses by altering HRV-induced epithelial gene expression. Primary cultures of human bronchial epithelial cells were exposed to medium alone, CSE alone, purified HRV-16 alone or to HRV-16+ CSE. After 24 h, supernatants were collected and total cellular RNA was isolated. Gene array analysis was performed to examine mRNA expression. Additional experiments, using real-time RT-PCR, ELISA and/or western blotting, validated altered expression of selected gene products. CSE and HRV-16 each induced groups of genes that were largely independent of each other. When compared to gene expression in response to CSE alone, cells treated with HRV+CSE showed no obvious differences in CSE-induced gene expression. By contrast, compared to gene induction in response to HRV-16 alone, cells exposed to HRV+CSE showed marked suppression of expression of a number of HRV-induced genes associated with various functions, including antiviral defenses, inflammation, viral signaling and airway remodeling. These changes were not associated with altered expression of type I or type III interferons. Thus, CSE alters epithelial responses to HRV infection in a manner that may negatively impact antiviral and host defense outcomes.

Figure 1. CSE suppresses HRV-induced expression of chemokines.

HRV-induced expression of mRNA (Panel A) and protein (Panel B) for CXCL10, as well as mRNA (Panel C) and protein (Panel D) for CCL5 were significantly inhibited in the presence of CSE. Data are presented as mean ± SEM from 4–7 donors. Asterisks indicate significant inhibition for HRV+CSE compared to HRV alone.

Figure 2. CSE suppresses HRV-induced expression of viral signaling molecules.

The effects of CSE on HRV-induced expression of: A. mda-5, B. RIG-I and C. STAT-1, was examined. In each case, the left panel shows effects mRNA expression (mean ± SEM, n = 4–7). Asterisks indicate significant inhibition for HRV+CSE compared to HRV alone. The right panels show western blots (representative of n = 3 in each case) for each protein studied. In the case of STAT-1, phospho-STAT-1 was also examined. For all blots, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was assessed to ensure equal protein loading.

Figure 3. CSE suppresses HRV-induced expression of antiviral proteins.

The effects of CSE on HRV-induced expression of: A. ISG56, B. Viperin and C. ISG15, was examined. In each case, the left panel shows effects mRNA expression (mean ± SEM, n = 4–7). Asterisks indicate significant inhibition for HRV+CSE compared to HRV alone. The right panels show western blots (representative of n = 3 in each case) for each protein studied. For all blots, GAPDH was assessed to ensure equal protein loading.

Figure 4. CSE suppresses HRV-induced expression of EPSTI1.

The left panel shows effects mRNA expression (mean ± SEM, n = 5). The right panel shows a western blot (representative of n = 3) for EPSTI1 protein. GAPDH was assessed to ensure equal protein loading.

Figure 5. CSE does not alter HRV-induced expression of interferons.

CSE did not significantly inhibit HRV-induced mRNA expression for : A. IFNβ, B. IFN-λ2 (IL28A) and C. IFN-λ1 (IL29). In each case, data are expresses as fold increase above medium control treatment (n = 5 in each case). Panel D. Shows protein expression for IFN-λ1 (IL29) expressed as pg/ml (n = 5). Data are presented as mean ± SEM in each case.