RESUMEN
Chronic Pseudomonas aeruginosa lung infections are associated with progressive epithelial damage and lung function decline. In addition to its role in tissue injury, the persistent presence of P. aeruginosa-secreted products may also affect epithelial repair ability, raising the need for new antivirulence therapies. The purpose of our study was to better understand the outcomes of P. aeruginosa exoproducts exposure on airway epithelial repair processes to identify a strategy to counteract their deleterious effect. We found that P. aeruginosa exoproducts significantly decreased wound healing, migration, and proliferation rates, and impaired the ability of directional migration of primary non-cystic fibrosis (CF) human airway epithelial cells. Impact of exoproducts was inhibited after mutations in P. aeruginosa genes that encoded for the quorum-sensing (QS) transcriptional regulator, LasR, and the elastase, LasB, whereas impact was restored by LasB induction in ΔlasR mutants. P. aeruginosa purified elastase also induced a significant decrease in non-CF epithelial repair, whereas protease inhibition with phosphoramidon prevented the effect of P. aeruginosa exoproducts. Furthermore, treatment of P. aeruginosa cultures with 4-hydroxy-2,5-dimethyl-3(2H)-furanone, a QS inhibitor, abrogated the negative impact of P. aeruginosa exoproducts on airway epithelial repair. Finally, we confirmed our findings in human airway epithelial cells from patients with CF, a disease featuring P. aeruginosa chronic respiratory infection. These data demonstrate that secreted proteases under the control of the LasR QS system impair airway epithelial repair and that QS inhibitors could be of benefit to counteract the deleterious effect of P. aeruginosa in infected patients.-Ruffin, M., Bilodeau, C., Maillé, É., LaFayette, S. L., McKay, G. A., Trinh, N. T. N., Beaudoin, T., Desrosiers, M.-Y., Rousseau, S., Nguyen, D., Brochiero, E. Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.
Asunto(s)
Proteínas Bacterianas/metabolismo , Células Epiteliales/microbiología , Pseudomonas aeruginosa/fisiología , Movimiento Celular , Proliferación Celular , Células Cultivadas , Regulación Bacteriana de la Expresión Génica/fisiología , Humanos , Mutación , Mucosa Respiratoria/citología , Sistema RespiratorioRESUMEN
The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells. CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements. A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells. These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Infecciones por Pseudomonas/metabolismo , ARN Mensajero/metabolismo , Mucosa Respiratoria/metabolismo , Células Cultivadas , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/microbiología , Humanos , Piperazinas/farmacología , Infecciones por Pseudomonas/complicaciones , Pseudomonas aeruginosa , Quinazolinas/farmacología , Mucosa Respiratoria/citología , Mucosa Respiratoria/microbiología , Adulto JovenRESUMEN
Airway damage and remodelling are important components of lung pathology progression in cystic fibrosis (CF). Although repair mechanisms are engaged to restore the epithelial integrity, these processes are obviously insufficient to maintain lung function in CF airways. Our aims were therefore to study how the basic cystic fibrosis transmembrane conductance regulator (CFTR) defect could impact epithelial wound healing and to determine if CFTR correction could improve it. Wound-healing experiments, as well as cell migration and proliferation assays, were performed to study the early phases of epithelial repair in human CF and non-CF airway cells. CFTR function was evaluated using CFTR small interferring (si)RNA and inhibitor GlyH101 in non-CF cells, and conversely after CFTR rescue with the CFTR corrector VRT-325 in CF cells. Wound-healing experiments first showed that airway cells from CF patients repaired slower than non-CF cells. CFTR inhibition or silencing in non-CF primary airway cells significantly inhibited wound closure. GlyH101 also decreased cell migration and proliferation. Interestingly, wild-type CFTR transduction in CF airway cell lines or CFTR correction with VRT-325 in CFBE-ΔF508 and primary CF bronchial monolayers significantly improved wound healing. Altogether our results demonstrated that functional CFTR plays a critical role in wound repair, and CFTR correction may represent a novel strategy to promote the airway repair processes in CF.