Roscovitine enhances the bactericidal activity of the airway surface liquid of the cystic fibrosis bronchial epithelium but does not protect against Pseudomonas aeruginosa infection

Abstract

Cystic fibrosis (CF) is the most common genetic diseases in the Caucasian population. CFTR defects, the most common being F508del, lead to abnormal mucus accumulation. Respiratory failure caused by the resulting chronic infections is the leading cause of death in people with cystic fibrosis (pwCF). Pseudomonas aeruginosa is a major pathogen in CF and is responsible for a deterioration of respiratory function in pwCF. The increase of antibiotic-resistant P. aeruginosa strains encourages the search for alternative therapeutics for treating P. aeruginosa infection. In vitro studies have shown an interest in (R)-roscovitine (roscovitine) in the fight against bacterial infection in pwCF. Here we show a nuanced effect of roscovitine on ASL bactericidal activity and CF bronchial epithelium protection against P. aeruginosa. Using a 3D model of fully differentiated and functional F508del-CFTR human bronchial epithelium, we evidenced (i) an enhancement of the bactericidal activity of the airway surface liquid for 25 μM roscovitine but (ii) no limitation of the dynamic of the epithelium destruction upon roscovitine treatment whatever the concentrations. Our findings shed light on reasons for the lack of beneficial effects to prevent P. aeruginosa infection in pwCF treated with roscovitine in the ROSCO-CF clinical trial. We anticipate that our findings would have significant therapeutic implications in seeking to optimize roscovitine analogs.

Fig 1. Viability of CF bronchial epithelia treated with roscovitine.

(A) Resazurin based toxicity test in CF reconstructed bronchial epithelia treated with roscovitine (3, 6, 12, 25, 50 µM) for 9 days. The DMSO carrier solution was used for control 0 μM (CTRL) and the negative control (Neg CTRL) represents wells without epithelium. The Optical Density (OD_600nm) was measured 2 hours after incubation of epithelia with resazurin. Higher OD values indicate greater cell viability. Data represent mean values (2 replicates, n = 3 independent cultures from different pwCF). *P < 0.05 unpaired Student t-test. (B) Phase-contrast bright field micrograph of CF bronchial epithelia showing areas of destruction and detachment (arrows) after 9 days of treatment with 50μM of roscovitine. Bars = 50 µm.

Fig 2. Bactericidal activity against P. aeruginosa. of airway surface liquids from reconstructed CF bronchial epithelia treated with roscovitine.

The bactericidal activity of ASL from ALI cultures of CF bronchial cells from 6 independent pwCF (P1 to P6) is represented as bacterial survival (expressed as the ratio of CFU/mL_{ASL roscovitine-treated} over CFU/mL _{ASL non-treated}). Data represent mean values of 5 replicates for each cultures. The data were analysed using a Wilcoxon Signed Rank Test.

Fig 3. Infection with P. aeruginosa of CF bronchial epithelia treated with roscovitine.

(A) Time lapse images of dynamic cell death of CF bronchial epithelia treated with roscovitine, then infected with PA14. In the panel, the damaged airway epithelia appear in red. (B) Time of destruction of reconstructed CF bronchial epithelia generated with cells from 4 pwCF (P1 to P4) treated with roscovitine (3, 6, 12, 25  µM) for 10 days, then infected with PA14. The DMSO carrier solution was used for control (CTRL) and represents the untreated epithelium. Data represent mean values of 2 replicates for each culture. Data were analysed using a one-way ANOVA with a P value = 0.1129 and a comparison of the mean rank of each concentration with the mean rank of the CTRL using a Dunn’s multiple comparisons test.

Fig 4. Evaluation of the anti-proliferative effect of roscovitine on A549 cell line.

Cell proliferation was analysed using MTT assay after a treatment of (A) 2 or (B) 3 days with roscovitine at 12, 25 and 50 µM, purchased from Abcam or graciously gifted by Perha Therapeuticals (previously ManRos Therapeutics). The data represent mean values of 4 replicates (n = 3) and error bars indicates SD. One-sample t-test, *P < 0.05; **P < 0.01; ***P < 0.001; **** P < 0.0001. (C) Phase-contrast bright field micrographs showing A549 cells before and after a treatment of 3 days with roscovitine at 12 and 50 μM. Bars = 50 µm.