Inhibition of Pseudomonas aeruginosa secreted virulence factors reduces lung inflammation in CF mice

Abstract

Background: Cystic fibrosis (CF) lung infection is a complex condition where opportunistic pathogens and defective immune system cooperate in developing a constant cycle of infection and inflammation. The major pathogen, Pseudomonas aeruginosa, secretes a multitude of virulence factors involved in host immune response and lung tissue damage. In this study, we examined the possible anti-inflammatory effects of molecules inhibiting P. aeruginosa virulence factors.

Methods: Pyocyanin, pyoverdine and proteases were measured in bacterial culture supernatant from different P. aeruginosa strains. Inhibition of virulence factors by sub-inhibitory concentrations of clarithromycin and by protease inhibitors was evaluated. Lung inflammatory response was monitored by in vivo bioluminescence imaging in wild-type and CFTR-knockout mice expressing a luciferase gene under the control of a bovine IL-8 promoter.

Results: The amount of proteases, pyocyanin and pyoverdine secreted by P. aeruginosa strains was reduced after growth in the presence of a sub-inhibitory dose of clarithromycin. Intratracheal challenge with culture supernatant containing bacteria-released products induced a strong IL-8-mediated response in mouse lungs while lack of virulence factors corresponded to a reduction in bioluminescence emission. Particularly, sole inactivation of proteases by inhibitors Ilomastat and Marimastat also resulted in decreased lung inflammation.

Conclusions: Our data support the assumption that virulence factors are involved in P. aeruginosa pro-inflammatory action in CF lungs; particularly, proteases seem to play an important role. Inhibition of virulence factors production and activity resulted in decreased lung inflammation; thus, clarithromycin and protease inhibitors potentially represent additional therapeutic therapies for P. aeruginosa-infected patients.

Keywords: Cystic fibrosis; Pseudomonas aeruginosa; clarithromycin; in vivo imaging; lung inflammation; protease inhibitors; virulence factors.

Figure 1.

Proteases (A), pyocyanin (B) and pyoverdine (C) measured in culture supernatant collected from P. aeruginosa strains grown in absence/presence of 45 ug/ml CLM. Each value represents the mean ± SEM of 3 experiments. Statistical analysis was performed by Mann-Whitney test; *p < 0.05, **p < 0.01, ***p < 0.001 and **** p < 0.0001.

Figure 2.

Growth curves (A), growth rate (B) and CFU count (C) measured from VR1 growth in absence/presence of 45 ug/ml CLM. Absorbance at 600 nm was measured every hour for 6 hours (A) and growth rate per minute was calculated from exponential phase of growth curves (B). CFU were counted on TSA after plating serial dilutions of inocula (C). Each value represents the mean ± SEM of 3 experiments.

Figure 3.

Representative images (upper panels) and photon emission measurement (lower panels) of bioluminescence emission in WT (A, C) and CF (B, D) mice challenged with concentrated culture supernatant from VR1 grown in absence/presence of 45 ug/ml CLM. Control mice were treated with TSB. Photon emission is expressed as folds of increase (FOI) vs. baseline. Each value represents the mean ± SEM of 6 animals (biological replicates) per group. Statistical analysis was performed by 2way ANOVA followed by Dunnett’s multiple comparisons test; *p < 0.05, ***p < 0.001, **** p < 0.0001.

Figure 4.

Protease activity in VR1 concentrated culture supernatant was measured in presence of MMPIs Marimastat (MAM), Batimastat (BAM) and Ilomastat (ILM). Each value represents the mean ± SD of 3 experiments. Statistical analysis was performed by Kruskal-Wallis test and Dunn’s multiple comparisons test; * p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 vs. non-treated supernatant (0 µM MMPIs).

Figure 5.

Representative images (upper panels) and photon emission measurement (lower panels) of bioluminescence emission in WT (A, C) and CF (B, D) mice challenged with VR1 concentrated culture supernatant pre-treated with 50 and 150 µM Ilomastat (ILM) and 150 µM Marimastat (MAM). VR1 supernatant was used as positive control (black), TSB was used as negative control. Photons emission is expressed as folds of increase (FOI) vs. baseline. Each value represents the mean ± SEM of 6 animals (biological replicates) per group. Statistical analysis was performed by 2way ANOVA followed by Dunnett’s multiple comparisons test; *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.