Angela Sandri

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.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

20 In vivo monitoring of lung inflammation in CFTR–/– mice

and pulmonary disease management (ECFS; Sermet-Gaudelus et al. 2010). Results: There are currently 9 children diagnosed with CF following NBS enrolled at the paediatric CF specialized centre (14.8% of all patients), with a mean age of 11 months (range, 3–25). The mean age for confirmatory sweat test was 27 days (range, 11–48). There was a 97.1% compliance rate to ECFS guidelines (33 out of 34 recommendations), especially in the areas of nutritional and pulmonary disease management, with 100% compliance. The only area of no compliance was the lack of communication with primary care providers. Conclusions: We have identified an excellent conformity level between the care provided by the specialized CF centre and ECFS guidelines. Measures to improve communication with primary care providers are currently underway.

WS10.3 Monitoring the pro-inflammatory effect of Pseudomonas aeruginosa culture supernatants and the inhibitory effect of azithromycin by in vivo imaging in IL-8 transiently transgenized mice

Objectives Airway inflammation is frequently associated with chronic bacterial infections. The IL-8 mediated lung inflammation induced by virulence factors, specifically metalloproteases (MP), secreted by Pseudomonas aeruginosa has been monitored in vivo in a mouse model transiently expressing the luciferase reporter gene under the control of an IL-8 bovine promoter. The model was also used to test the possible anti-inflammatory activity of AZMthromycin (AZM) mediated by its effect on Pseudomonas MP expression. Methods Culture supernatants (SNs) from two P. aeruginosa clinical strains, VR1 and VR2 respectively, were obtained after growth in the absence or presence of a sub-lethal dose of AZM. The MP activity was detectable in the SN from VR1 grown without AZM but was greatly reduced when cells grew with the antibiotic. Results The pro-inflammatory activity of VR1 SN was clearly visible in vivo in the transgenized mice while a significant decrease of the inflammation response was observed with the SN grown in the presence of AZM. Further, VR1 SN stimulated the recruitment of WBC and neutrophils and the expression of a number of cytokines. This later effect was clearly decreased when SN from VR1 grown with AZM was used. Contrarily, SN from VR2 showed neither protease activity or detectable inflammation in the mouse lung. Conclusion The present animal model has revealed useful to the in vivo, long term monitoring of the effect of bacterial virulence factors in the lung tissue and the possible beneficial, anti-inflammatory effect of molecules for therapeutic use. This study was supported by Lega Italiana Fibrosi Cistica and Italian CF Research Foundation (grant #18/2013).