Cigarette smoke exposure impairs pulmonary bacterial clearance and alveolar macrophage complement-mediated phagocytosis of Streptococcus pneumoniae

Abstract

Cigarette smoke exposure increases the risk of pulmonary and invasive infections caused by Streptococcus pneumoniae, the most commonly isolated organism from patients with community-acquired pneumonia. Despite this association, the mechanisms by which cigarette smoke exposure diminishes host defense against S. pneumoniae infections are poorly understood. In this study, we compared the responses of BALB/c mice following an intratracheal challenge with S. pneumoniae after 5 weeks of exposure to room air or cigarette smoke in a whole-body exposure chamber in vivo and the effects of cigarette smoke on alveolar macrophage phagocytosis of S. pneumoniae in vitro. Bacterial burdens in cigarette smoke-exposed mice were increased at 24 and 48 h postinfection, and this was accompanied by a more pronounced clinical appearance of illness, hypothermia, and increased lung homogenate cytokines interleukin-1beta (IL-1beta), IL-6, IL-10, and tumor necrosis factor alpha (TNF-alpha). We also found greater numbers of neutrophils in bronchoalveolar lavage fluid recovered from cigarette smoke-exposed mice following a challenge with heat-killed S. pneumoniae. Interestingly, overnight culture of alveolar macrophages with 1% cigarette smoke extract, a level that did not affect alveolar macrophage viability, reduced complement-mediated phagocytosis of S. pneumoniae, while the ingestion of unopsonized bacteria or IgG-coated microspheres was not affected. This murine model provides robust additional support to the hypothesis that cigarette smoke exposure increases the risk of pneumococcal pneumonia and defines a novel cellular mechanism to help explain this immunosuppressive effect.

FIG. 1.

Cigarette smoke exposure increases pulmonary bacterial burden and worsens clinical signs of pneumococcal pneumonia. Female BALB/c mice were exposed to room air (open bars) or cigarette smoke (CS) (solid bars) for 5 weeks, followed by an intratracheal challenge with live S. pneumoniae (10⁴ CFU). (A) Lung homogenates were assessed for bacterial burdens at 24 and 48 h postinfection. (B) The clinical appearance was evaluated 6 h after infection and every 12 h thereafter, as described in Materials and Methods. (C) Core temperatures were taken at the time of euthanasia, and data are presented as changes from baseline (core temperature of mice that were not infected). Bars represent means ± standard errors of the means (n = 7 or 8 mice per group). *, P < 0.05 compared with air-exposed mice at the same time point, by ANOVA (A and C) and the Kruskal-Wallis test (B).

FIG. 2.

Cytokine levels in lung homogenates obtained from mice following an intratracheal challenge with live S. pneumoniae. Female BALB/c mice were exposed to room air (open bars) or cigarette smoke (CS) (solid bars) for 5 weeks, followed by an intratracheal challenge with live S. pneumoniae (10⁴ CFU). Homogenates were prepared from lungs removed from euthanized mice at 24 and 48 h postinfection and were assessed for IL-1β, IL-6, IL-10, MIP-2, and TNF-α as described in Materials and Methods. *, P < 0.05 versus air-exposed mice, by t test.

FIG. 3.

Elevated neutrophil counts in cigarette smoke-exposed mice following intratracheal challenge with heat-killed S. pneumoniae. Leukocytes were obtained by bronchoalveolar lavage from mice exposed to room air (open bars) or cigarette smoke (CS) (solid bars) for 5 weeks, with no subsequent challenge (A) or 24 h (B) or 48 h (C) following an intratracheal challenge with heat-killed S. pneumoniae (10⁶ CFU-equivalent dose). Differential counts were calculated by multiplying total cell counts by the percentages of lymphocytes (Lymph), neutrophils (PMN), and monocytes/macrophages (Mono/Mac) following differential staining. Bars represent means ± standard errors of the means (n = 4 to 6 mice per group for panel A and 7 or 8 mice per group for panels B and C). *, P < 0.05 by paired t test.

FIG. 4.

Cytokine levels in lung homogenates obtained from mice following intratracheal challenge with heat-killed S. pneumoniae. Female BALB/c mice were exposed to room air (open bars) or cigarette smoke (CS) (solid bars) for 5 weeks, followed by an intratracheal challenge with S. pneumoniae (10⁶ CFU). Lung homogenates were assessed for IL-1β, IL-6, IL-10, MIP-2, and TNF-α as described in Materials and Methods. *, P < 0.05 versus air-exposed mice, by t test.

FIG. 5.

Cigarette smoke extract impairs complement-mediated phagocytosis of S. pneumoniae by AMs in vitro. (A) AMs were cultured overnight with medium alone (control) (open bar) or with medium containing 1% CSE (solid bars), and phagocytosis of S. pneumoniae opsonized with normal rat serum (NS), heat-inactivated rat serum (HIS), C3-deficient serum (C3-DS), or no serum (UNOP) or of IgG-coated microspheres (IgG-MS) was assessed as described in Materials and Methods. (B) Female BALB/c mice were exposed to room air (open bars) or cigarette smoke (CS) (solid bars) for 4 h, and AMs were recovered by lavage and cultured overnight. On the following day, phagocytosis of S. pneumoniae opsonized with normal rat serum (NS) or C3-deficient serum (C3-DS) was assessed. Data were normalized to their respective controls. Bars represent the means ± SEM for three to five experiments. *, P < 0.05 compared with control AMs, by ANOVA.