Diminished ICAM-1 expression and impaired pulmonary clearance of nontypeable Haemophilus influenzae in a mouse model of chronic obstructive pulmonary disease/emphysema

Abstract

The airways of patients with chronic obstructive pulmonary disease (COPD) are continually colonized with bacterial opportunists like nontypeable Haemophilus influenzae (NTHi), and a wealth of evidence indicates that changes in bacterial populations within the lung can influence the severity of COPD. In this study, we used a murine model for COPD/emphysema to test the hypothesis that COPD affects pulmonary clearance. Mice were treated with a pulmonary bolus of elastase, and as reported previously, the lungs of these mice were pathologically similar to those with COPD/emphysema at approximately 1 month posttreatment. Pulmonary clearance of NTHi was significantly impaired in elastase-treated versus mock-treated mice. While histopathologic analysis revealed minimal differences in localized lung inflammation between the two groups, lower levels of intercellular adhesion molecule 1 (ICAM-1) were observed for the airway epithelial surface of elastase-treated mice than for those of control mice. Following infection, elastase-treated mice had lung pathology consistent with pneumonia for as long as 72 h postinfection, whereas at the same time point, mock-treated mice had cleared NTHi and showed little apparent pathology. Large aggregates of bacteria were observed within damaged lung tissue of the elastase-treated mice, whereas sparse individual bacteria were observed in lungs of mock-treated mice at the same time point postinfection. Additional infection studies showed that NTHi mutants with biofilm defects were less persistent in the elastase-treated mice than the parent strain. These findings establish a model for COPD-related infections and support the hypotheses that ICAM-1 promotes clearance of NTHi. Furthermore, the data indicate that NTHi may form biofilms within the context of COPD-related infections.

FIG. 1.

Elastase-treated mice have fibrotic lung damage consistent with COPD. Mice were treated by nonsurgical intratracheal instillation of elastase (see Materials and Methods) and allowed to recover for 21 days posttreatment. Lung tissue samples from euthanized mice receiving vehicle (PBS) (A), elastase (B), or heat-inactivated (HI) elastase (C) were compared by hematoxylin-eosin staining of paraffin sections. COPD-like pulmonary damage was observed for the elastase-treated group that was not observed for either of the control groups. Magnification, ×4 for left panels and ×40 for right panels.

FIG. 2.

Impaired pulmonary clearance of NTHi from elastase-treated mice. Mice (five/group) were infected via the intratracheal route with ∼10⁷ CFU of NTHi 2019 and euthanized at the indicated times postinfection (see Materials and Methods). Symbols are CFU counts obtained from homogenized lung tissue from mock-treated (filled circles) or elastase-treated mice (open circles). The dotted line indicates the lower limit of detection, and symbols below that line indicate a mouse from which no bacteria were recovered. Asterisks indicate groups in which bacterial numbers were significantly higher than those in controls, as assessed by nonparametric statistical analysis.

FIG. 3.

Histopathologic assessment of lung tissue from infected animals. Lung tissue was sectioned and stained for histopathology analysis as described in Materials and Methods. Each panel is a portion of a representative hematoxylin-eosin-stained section from the indicated group (Control or Elastase group, left margin) at a given time point (Mock, 6 h, 24 h, 48 h, or 72 h).

FIG. 4.

Histopathology scores. Hematoxylin-eosin-stained sections from mock-treated (open bars) and elastase-treated (filled bars) mice were examined as a blinded set and scored for markers of pulmonary inflammation (see Materials and Methods).

FIG. 5.

Experimental COPD reduces the level and kinetics of immunohistochemical staining for ICAM-1 expression within lung tissue during infection. ICAM-1 levels in paraffin sections of lung tissue were assessed by immunohistochemical staining with monoclonal antibody 3E2 (BD/Pharmingen) as described in Materials and Methods. Sections were counterstained with hematoxylin. Panels show representative sections from the groups indicated at left. Graphs depict total ICAM-1 staining in all sections examined in the group as a percentage of total pixels. Magnification, ×20 for left panels and ×40 for right panels.

FIG. 6.

Immunofluorescent staining reveals multicellular bacterial communities within lung tissue of elastase-treated animals. Lung tissue was cryosectioned (see Materials and Methods) and stained with rabbit anti-H. influenzae sera (53) and fluorescent antibody conjugate (Jackson Laboratories). Panels A and B show merged differential interference-contrast/fluorescent images from a mock-treated animal (A) and elastase-treated animal (B) 48 h postinfection. The graph depicts quantified bacterial staining as the percentage of total pixels and was obtained from sections of tissue from all animals. Panels in C show sequential sections from the same tissue block stained to show the distribution of bacteria (fluorescent image marked “NTHi”) and cellular infiltrate within the lung (light microscopy image marked “H & E”). Magnification, ×20 for left set of panels in C and ×100 for right set of panels.

FIG. 7.

Clearance of NTHi mutants from mock-treated and elastase-treated mice. Mice were treated with elastase and infected as described in the legend to Fig. 1. Symbols represent CFU counts recovered from homogenized lung tissue from mock-treated (filled circles) and elastase-treated (open circles) animals 48 h postinfection. The dotted line indicates the lower limit of detection, and symbols below that line indicate a mouse from which no bacteria were recovered. Horizontal bars represent median values, and error bars represent standard errors of the mean for each group. Asterisks indicate groups in which bacterial numbers were significantly higher than in controls, as assessed by nonparametric statistical analysis.