Influenza virus infection decreases tracheal mucociliary velocity and clearance of Streptococcus pneumoniae

Abstract

Influenza virus infections increase susceptibility to secondary bacterial infections, such as pneumococcal pneumonia, resulting in increased morbidity and mortality. Influenza-induced tissue damage is hypothesized to increase susceptibility to Streptococcus pneumoniae infection by increasing adherence to the respiratory epithelium. Using a mouse model of influenza infection followed by S. pneumoniae infection, we found that an influenza infection does not increase the number of pneumococci initially present within the trachea, but does inhibit pneumococcal clearance by 2 hours after infection. To determine whether influenza damage increases pneumococcal adherence, we developed a novel murine tracheal explant system to determine influenza-induced tissue damage and subsequent pneumococcal adherence. Murine tracheas were kept viable ex vivo as shown by microscopic examination of ciliary beating and cellular morphology using continuous media flow for up to 8 days. Tracheas were infected with influenza virus for 0.5-5 days ex vivo, and influenza-induced tissue damage and the early stages of repair to the epithelium were assessed histologically. A prior influenza infection did not increase pneumococcal adherence, even when the basement membrane was maximally denuded or during the repopulation of the basement membrane with undifferentiated epithelial cells. We measured mucociliary clearance in vivo and found it was decreased in influenza-infected mice. Together, our results indicate that exposure of the tracheal basement membrane contributes minimally to pneumococcal adherence. Instead, an influenza infection results in decreased tracheal mucociliary velocity and initial clearance of pneumococci, leading to an increased pneumococcal burden as early as 2 hours after pneumococcal infection.

Figure 1.

Effects of influenza infection on pneumococcal clearance. (A) Influenza plaque-forming units (PFUs) in tracheas from uninfected mice or mice infected with influenza virus for 3 or 6 days. Line represents median value for each infection. (B) Initial adherence and clearance of Streptococcus pneumoniae in tracheas from uninfected mice or mice infected with influenza for 3 or 6 days. ***P < 0.001, significant differences between mice infected with S. pneumoniae only and mice coinfected with influenza and S. pneumoniae; *P < 0.05 significant differences between mice coinfected with influenza for 3 or 6 days followed by S. pneumoniae. Data expressed as means (±SD). Data analyzed using one-way ANOVA followed by the Bonferroni post test. Results represent 5–6 mice for 0- and 30-minute time points, and 8–11 mice for 60- and 120-minute time points.

Figure 2.

Diagram of tracheal explant system showing media reservoir connected to pump and flask with tracheal explant. Tracheas were suspended from blunt-ended 18-gauge needles, which were connected to silicone tubing through a pump with a constant media flow rate of 0.1 ml/min. Media (cRPMI) and explants were incubated at 37°C in 5% CO₂. Inset: picture of tracheal explant and portion of lung connected to needle inside flask. The arrow indicates trachea attached to needle.

Figure 3.

Uninfected tracheal explants at 0, 4, 6, and 8 days after ex vivo culture stained with hemotoxylin and eosin (H&E) or for β-tubulin and Clara cell secretory protein (CCSP). Intact cilia are present at all time points. Tracheal respiratory epithelium is on right-hand side of images. Magnification, 400× for H&E staining, 600× for β-tubulin and CCSP staining.

Figure 4.

Influenza PR8 virus plaque-forming units (PFUs) in tracheal explants at various time points after ex vivo influenza infection. Data are expressed as means (±SD). Results represent 12–15 tracheas per day of influenza infection.

Figure 5.

Uninfected tracheal explants and tracheal explants infected ex vivo with influenza virus for 1–5 days. Hemotoxylin and eosin (H&E) staining and corresponding influenza immunofluorescence staining of serial sections from tracheal explants at various time points after ex vivo influenza infection. Magnification, 600×. Tracheal respiratory epithelium is on right-hand side of images.

Figure 6.

Influenza-infected epithelium of tracheal explant. Colocalization of immunofluorescence staining for influenza virus (red) and β-tubulin (green) in tracheal explant infected with influenza virus for 1 day. Uninfected tracheal explant shows β-tubulin staining only. Magnification, 600×. Tracheal respiratory epithelium is on right-hand side of images.

Figure 7.

Effect of Streptococcus pneumoniae inoculum dose on S. pneumoniae adherence to uninfected and influenza-infected (1 d) tracheal explants. Tracheas were inoculated with 5 × 10³, 5 × 10⁵, 5 × 10⁶, 5 × 10⁷, or 5 × 10⁸ S. pneumoniae CFUs/ml for 1 hour to allow for adherence. Data are expressed as means (±SD). Data were analyzed using one-way ANOVA followed by the Bonferroni post test. Results represent four to five tracheas per dose, except eight tracheas were used for the 5 × 10⁷ dose.

Figure 8.

Effect of influenza infection on Streptococcus pneumoniae adherence to tracheal explants at various times after influenza infection (0.5–5 d). Data are expressed as means (±SD). Data were analyzed using one-way ANOVA followed by the Bonferroni post test. Results represent 8–10 tracheas per time point, except 0 and 0.5 days with and without influenza, which had 14 and 15 tracheas each, respectively.

Figure 9.

Effects of influenza infection on tracheal mucociliary velocity. (A) Tracheal mucociliary velocities of uninfected mice or mice infected with influenza virus for 3 or 6 days. Line represents median value for each infection. ***P < 0.001, significant differences between uninfected mice and influenza-infected mice. Data were analyzed using one-way ANOVA followed by the Bonferroni post test. (B) Lung influenza plaque-forming units (PFUs) from mice used for tracheal mucociliary velocity measurements. Line represents median value for each infection. ***P < 0.001, significant differences between mice infected with influenza for 3 or 6 days. Data were analyzed using one-way ANOVA followed by the Bonferroni post test.