Respiratory tract infection with Mycoplasma pneumoniae in interleukin-12 knockout mice results in improved bacterial clearance and reduced pulmonary inflammation

Abstract

Mycoplasma pneumoniae is a leading cause of pneumonia and is associated with asthma. Evidence links M. pneumoniae respiratory disease severity with interleukin-12 (IL-12) concentration in respiratory secretions. We evaluated the microbiologic, inflammatory, and pulmonary function indices of M. pneumoniae pneumonia in IL-12 (p35) knockout (KO) mice and wild-type (WT) mice to determine the role of IL-12 in M. pneumoniae respiratory disease. Eight-week-old wild-type BALB/c mice and 8-week-old IL-12 (p35) KO BALB/c mice were inoculated once intranasally with 10(7) CFU of M. pneumoniae. Mice were evaluated at days 2, 4, and 7 after inoculation. Outcome variables included quantitative bronchoalveolar lavage (BAL) M. pneumoniae culture, lung histopathologic scores (HPS), BAL cytokine concentrations determined by enzyme-linked immunosorbent assay (tumor necrosis factor alpha [TNF-alpha], gamma interferon [IFN-gamma], IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-10, and granulocyte-macrophage colony-stimulating factor) and plethysmography, before and after methacholine, to assess airway obstruction (AO) and airway hyperreactivity (AHR). IL-12 (p35) KO mice infected with M. pneumoniae were found to have significantly lower BAL M. pneumoniae concentrations compared with M. pneumoniae-infected WT mice. Lung HPS and the parenchymal pneumonia subscores (neutrophilic alveolar infiltrate), as well as AO, were significantly lower in infected KO mice. No difference was found for AHR. Infected KO mice had significantly lower BAL concentrations of IFN-gamma than WT mice; a trend toward lower BAL concentrations was observed for IL-10 (P = 0.065) and TNF-alpha (P = 0.078). No differences were found for IL-1beta, IL-2, IL-4, IL-5, or IL-6. The lack of IL-12 in experimental M. pneumoniae pneumonia was associated with less severe pulmonary disease and more rapid microbiologic and histologic resolution.

FIG. 1.

Quantitative M. pneumoniae (Mp) cultures of BAL fluid samples of WT and IL-12 KO mice inoculated with M. pneumoniae. Values shown are the means ± standard deviations (error bars). Data represent results from two separate experiments with eight mice in each group per time point. *, P = 0.002 between WT and IL-12 KO mice inoculated with M. pneumoniae (two-way ANOVA).

FIG. 2.

Lung HPS and pneumonia subscores of WT and IL-12 KO mice inoculated with M. pneumoniae (Mp) or sterile SP4 broth (controls). Values shown are the means ± standard deviations (error bars). Data represent results from two separate experiments with eight mice in each group per time point. *, P < 0.001 for HPS and P = 0.002 for the pneumonia subscore between WT and IL-12 KO mice inoculated with M. pneumoniae (two-way ANOVA).

FIG. 3.

Comparative histopathological appearance of the lungs of uninfected wild type mice (a; HPS = 0), IL-12 KO mice (b; HPS = 0), M. pneumoniae-infected WT mice (c; HPS = 16) and M. pneumoniae-infected IL-12 KO mice (d; HPS = 9) on day 4 postinoculation. Magnification, ×20.

FIG. 4.

AO and AHR were assessed by whole-body plethysmography by measuring Penh in WT and IL-12 KO mice inoculated with M. pneumoniae (Mp) or sterile SP4 broth (controls). Values shown are the means ± standard deviations (error bars). Data represent results from two separate experiments with eight mice in each group per time point. *, P = 0.022 between WT and IL-12 KO mice inoculated with M. pneumoniae (two-way ANOVA). +, P < 0.001 between WT and IL-12 KO mice inoculated with SP4 broth (controls) (two-way ANOVA).

FIG. 5.

Cytokine concentrations in BAL fluid specimens from WT and IL-12 KO mice inoculated with M. pneumoniae (Mp) or sterile SP4 broth (controls). Values shown are the medians and the 25th to 75th percentile (error bars). Data represent results from two separate experiments with eight mice in each group per time point. *, P < 0.001 between WT and IL-12 KO mice inoculated with M. pneumoniae (two-way ANOVA).