Respiratory viruses augment the adhesion of bacterial pathogens to respiratory epithelium in a viral species- and cell type-dependent manner

Abstract

Secondary bacterial infections often complicate respiratory viral infections, but the mechanisms whereby viruses predispose to bacterial disease are not completely understood. We determined the effects of infection with respiratory syncytial virus (RSV), human parainfluenza virus 3 (HPIV-3), and influenza virus on the abilities of nontypeable Haemophilus influenzae and Streptococcus pneumoniae to adhere to respiratory epithelial cells and how these viruses alter the expression of known receptors for these bacteria. All viruses enhanced bacterial adhesion to primary and immortalized cell lines. RSV and HPIV-3 infection increased the expression of several known receptors for pathogenic bacteria by primary bronchial epithelial cells and A549 cells but not by primary small airway epithelial cells. Influenza virus infection did not alter receptor expression. Paramyxoviruses augmented bacterial adherence to primary bronchial epithelial cells and immortalized cell lines by up-regulating eukaryotic cell receptors for these pathogens, whereas this mechanism was less significant in primary small airway epithelial cells and in influenza virus infections. Respiratory viruses promote bacterial adhesion to respiratory epithelial cells, a process that may increase bacterial colonization and contribute to disease. These studies highlight the distinct responses of different cell types to viral infection and the need to consider this variation when interpreting studies of the interactions between respiratory cells and viral pathogens.

FIG. 1.

Adhesion of NTHi and S. pneumoniae to respiratory epithelial cells is increased following infection with RSV. Respiratory epithelial cells were infected with RSV and at 24-h intervals incubated with NTHi-778 or S. pneumoniae. Adhesion is expressed as a percentage of inoculum. Antecedent infection of all cells with RSV resulted in a statistically significant increase in bacterial adhesion (*, P < 0.005; **, P < 0.05) compared to control cells not infected by RSV. (A) Adhesion of NTHi to respiratory epithelial cells. (B) Adhesion of pneumococci to respiratory epithelial cells. Data represent the mean ± standard error of the mean for six separate experiments.

FIG. 2.

Adhesion of NTHI and S. pneumoniae to respiratory epithelial cells is increased following infection with HPIV-3 and influenza virus. Respiratory epithelial cells were infected with HPIV-3 or influenza virus and at 24-h intervals incubated with NTHi-778 or S. pneumoniae. Adhesion is expressed as a percentage of inoculum. Bacterial adhesion was significantly increased to virally infected cells compared to adhesion to control cells not infected by viruses (*, P < 0.005; **, P < 0.01; ***, P < 0.05). (A) Adhesion of NTHi to respiratory epithelial cells. (B) Adhesion of pneumococci to respiratory epithelial cells. Data represents the means ± standard errors of the means of six separate experiments.

FIG. 3.

ICAM-1, CEACAM1, and PAF-r expression by epithelial cells after RSV infection. (A) Cells were infected by RSV for 24, 48, or 72 h, and receptor expression was quantified by FACS. Histograms illustrate the fluorescence intensities of control cells (not infected with virus) compared to those of infected cells at 24, 48, and 72 h. RSV infection up-regulated A549 cell ICAM-1 and CEACAM-1 expression at 48 to 72 h, BEAS-2B ICAM-1 and PAF-r expression at 48 to 72 h, and NHBE cell ICAM-1 and CEACAM-1 expression at 48 and 72 h. (B) Up-regulation of receptors by RSV infection of A549 cells was confirmed by Western blot analysis. Data are representative of three independent experiments.

FIG. 4.

Blocking ICAM-1 and PAF-r reduces NTHi adhesion to RSV-infected respiratory epithelial cells. A549 cells were infected with RSV for 24, 48, or 72 h and then incubated with anti-LFA-3/CD58, anti-ICAM-1, anti-PAF-r, or both anti-ICAM-1 and anti-PAFr Ab, and numbers of NTHi bacteria adhering to cells were determined. Shown is the inhibition observed after incubation of A549 cells with 25 μg/ml of Ab. (A) Adhesion of NTHi-778 at 24 h. (B) Adhesion after 48 h. (C) Adhesion after 72 h. Treatment with both anti-ICAM-1 and PAF-r antibodies but not control anti-LFA-3/CD58 Ab resulted in a significant inhibition of NTHi adhesion. (*, P ≤ 0.005; **, P = 0.01). Data represent the means ± standard errors for three independent experiments.

FIG. 5.

HPIV-3 infection increases ICAM-1 and CEACAM1 expression by respiratory epithelial cells. (A) Cells were infected by HPIV-3 for 24, 48, or 72 h, and receptor expression was quantified by FACS. Histograms illustrate the fluorescence intensities of control cells (not infected with virus) compared to those of infected cells at 24, 48, and 72 h. HPIV-3 infection significantly increased expression of ICAM-1 and modestly increased expression of CEACAM1 by A549 cells and significantly increased ICAM-1 and CEACAM1 expression by NHBE cells at 48 to 72 h after infection. (B) The increased expression of receptors by A549 cells following HPIV-3 infection was confirmed by Western blot analysis. Data are representative of three independent experiments.