Role of excessive inflammatory response to Stenotrophomonas maltophilia lung infection in DBA/2 mice and implications for cystic fibrosis

Abstract

Stenotrophomonas maltophilia is a pathogen that causes infections mainly in immunocompromised patients. Despite increased S. maltophilia isolation from respiratory specimens of patients with cystic fibrosis (CF), the real contribution of the microorganism to CF pathogenesis still needs to be clarified. The aim of the present study was to evaluate the pathogenic role of S. maltophilia in CF patients by using a model of acute respiratory infection in DBA/2 mice following a single exposure to aerosolized bacteria. The pulmonary bacterial load was stable until day 3 and then decreased significantly from day 3 through day 14, when the bacterial load became undetectable in all infected mice. Infection disseminated in most mice, although at a very low level. Severe effects (swollen lungs, large atelectasis, pleural adhesion, and hemorrhages) of lung pathology were observed on days 3, 7, and 14. The clearance of S. maltophilia observed in DBA/2 mouse lungs was clearly associated with an early and intense bronchial and alveolar inflammatory response, which is mediated primarily by neutrophils. Significantly higher levels of interleukin-1beta (IL-1beta), IL-6, IL-12, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), GROalpha/KC, MCP-1/JE, MCP-5, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-2, and TARC were observed in infected mice on day 1 with respect to controls. Excessive pulmonary infection and inflammation caused systemic effects, manifested by weight loss, and finally caused a high mortality rate. Taken together, our results show that S. maltophilia is not just a bystander in CF patients but has the potential to contribute to the inflammatory process that compromises respiratory function.

FIG. 1.

Aerosol delivery system. The system, housed in a biosafety cabinet, allowed us to simultaneously expose up to 4 mice to aerosol preparations. A small compressor transferred a bacterial suspension as an aerosol to each of the four inhalation chambers, each consisting of a 30-ml syringe. The standard aerosol exposure cycle was as follows: 60 min for nebulization (infection), 5 min for cloud decay, and UV irradiation for 5 min (decontamination).

FIG. 2.

Experimental design. Groups of mice were exposed to aerosol with sterile PBS alone (control group; 6 mice/group) or containing S. maltophilia OBGTC9 (infected group; 12 mice/group). On day 0 (1 h after exposure), the pulmonary bacterial load of DBA/2 mice (n = 8) was assessed. Mice were then sacrificed on days 1, 2, 3, 7, and 14 after exposure for microbiological analysis of the lung and spleen, macroscopic description and histopathology of lungs, and measurement of pulmonary cytokines/chemokines. All mice were further monitored daily for survival and general health.

FIG. 3.

(A) Weight monitoring during S. maltophilia lung infection. DBA/2 mice (n = 92) were exposed on day 0 to aerosolized S. maltophilia OBGTC9 (▴) or PBS only (▪) and were examined daily for weight loss during the course of infection. The dotted line shows a 10% weight loss with regard to mean body weight before infection. **, P < 0.01 for control versus infected mice (unpaired Student's t test). Error bars represent SD. (B) Survival of DBA/2 mice after pulmonary infection with S. maltophilia OBGTC9. Results are the combination of two independent experiments (n = 28 for uninfected mice; n = 56 for infected mice) monitored over 14 days. The difference in survival between infected and control mice was statistically significant (P < 0.01; log rank test).

FIG. 4.

Lung clearance and dissemination of S. maltophilia infection. (A) DBA/2 mouse (n = 92) lung clearance kinetics after respiratory exposure to S. maltophilia strain OBGTC9. Lungs were collected, homogenized, and cultured for bacterial counts at 0 (1 h), 1, 2, 3, 7, and 14 days postexposure. Results were normalized to the lung wet weight (CFU/g) and are shown as means + SD. *, P < 0.05; **, P < 0.01 versus day 0 (1 h) and day 1 (ANOVA [P < 0.0001] followed by Bonferroni's multiple comparison posttest). (B) Percentages of DBA/2 mice (n = 92) with spleens positive for S. maltophilia following lung infection. Spleens were collected, homogenized, and cultured for bacterial counts after 0 (1 h), 1, 2, 3, 7, and 14 days. Results are expressed as percentages of spleens positive for S. maltophilia at culture analysis. ∧∧, P < 0.001 versus day 1, day 2, day 3, and day 14; **, P < 0.001 versus each time point; ○○, P < 0.001 versus day 1, day 2, day 3, and day 14 (chi-square test).

FIG. 5.

Macroscopic pathology of DBA/2 mouse lungs infected with S. maltophilia strain OBGTC9. (A) Macroscopic lung pathology in DBA/2 mice (n = 92) assessed on day 0 (1 h), 1, 2, 3, 7, and 14 postexposure by use of a four-point scoring system proposed by Dubin and Kolls (19). Results are shown as follows: the line within each box is the median; the upper and lower lines of the box are the 75th and 25th percentiles, respectively; and the whiskers are the highest and lowest values. *, P < 0.05 versus day 1 (Kruskal-Wallis test [P < 0.01] followed by Dunn's multiple comparison posttest). (B) Photograph of uninfected mouse lung on day 3. (C) Photograph of S. maltophilia-infected mouse lung on day 3.

FIG. 6.

(A) Tissue histopathology of lungs of DBA/2 mice infected with S. maltophilia strain OBGTC9. Lung sections of DBA/2 mice (n = 92) were stained with Giemsa stain and are representative of eight (infected group) or four (control group) mice per group studied at each time point (1 h and 1, 2, 3, 7, and 14 days). Magnification, ×20 (bronchial sections) and ×10 (alveolar sections). (B) Microscopic DBA/2 mouse lung pathology following infection with S. maltophilia strain OBGTC9. Lung pathologies of DBA/2 mice (n = 92) were assessed by use of a five-point scoring system proposed by Johansen et al. (27). Results are shown as median values. *, P < 0.05; **, P < 0.01 for bronchial (▪) versus alveolar (▴) sections (Kruskal-Wallis test [P < 0.01] followed by Dunn's multiple comparison posttest).

FIG. 7.

Time course expression of cytokines in control and infected lungs. (A) Cytokine levels measured on days 1, 3, and 7 postexposure in lung homogenates from control (white bars) and infected (gray bars) mice (n = 54). Results were normalized to the lung wet weight (pg/mg) and are shown as means plus SDs. Statistically significant (*, P < 0.05; **, P < 0.01 [ANOVA followed by Bonferroni's multiple comparison posttest]) differences were observed in the levels of protein expression between infected and control mice for IL-1β, IL-4, IL-6, IL-12, IFN-γ, and TNF-α. (B) Hierarchical clustering expression plot. Different colors in the rectangles represent the average log ratios, defined as log₂ (infected value/control value) for each cytokine. The dendrogram illustrates the degrees of similarity in expression between the cytokines tested. The color bar beneath the dendrogram represents the logarithmic expression values.

FIG. 8.

Time course expression of chemokines in control and infected lungs. (A) Chemokine levels measured at 1, 3, and 7 days postexposure in the lung homogenates from control (white bars) and infected (gray bars) mice (n = 54). Results were normalized to the lung wet weight (pg/mg) and graphed as means plus SD. Statistically significant (**, P < 0.01 [ANOVA followed by Bonferroni's multiple comparison posttest]) differences were observed in the levels of protein expression between infected and control mice for GROα/KC, MCP-1/JE, MCP-5, MIP-1α, MIP-2, and TARC. (B) Hierarchical clustering expression plot. Different colors in the rectangles represent the average log ratios, defined as log₂ (infected value/control value) for each chemokine. The dendrogram illustrates the degrees of similarity in expression between the chemokines tested. The color bar beneath the dendrogram represents the logarithmic expression values.