Émilie Maillé

EGF and K+ channel activity control normal and cystic fibrosis bronchial epithelia repair

Severe lesions of airway epithelia are observed in cystic fibrosis (CF) patients. The regulatory mechanisms of cell migration and proliferation processes, involved in the repair of injured epithelia, then need to be better understood. A model of mechanical wounding of non-CF (NuLi) and CF (CuFi) bronchial monolayers was employed to study the repair mechanisms. We first observed that wound repair, under paracrine and autocrine EGF control, was slower (up to 33%) in CuFi than in NuLi. Furthermore, EGF receptor (EGFR) activation, following wounding, was lower in CuFi than in NuLi monolayers. Cell proliferation and migration assays indicated a similar rate of proliferation in both cell lines but with reduced (by 25%) CuFi cell migration. In addition, cell migration experiments performed in the presence of conditioned medium, collected from NuLi and CuFi wounded bronchial monolayers, suggested a defect in EGF/EGFR signaling in CF cells. We (49) recently demonstrated coupling between the EGF response and K(+) channel function, which is crucial for EGF-stimulated alveolar repair. In CuFi cells, lower EGF/EGFR signaling was accompanied by a 40-70% reduction in K(+) currents and KvLQT1, ATP-sensitive potassium (K(ATP)), and Ca(2+)-activated K(+) (KCa3.1) channel expression. In addition, EGF-stimulated bronchial wound healing, cell migration, and proliferation were severely decreased by K(+) channel inhibitors. Finally, acute CFTR inhibition failed to reduce wound healing, EGF secretion, and K(+) channel expression in NuLi. In summary, the delay in CuFi wound healing could be due to diminished EGFR signaling coupled with lower K(+) channel function, which play a crucial role in bronchial repair.

Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue

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-&Dgr;F508 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.

Regulation of normal and cystic fibrosis airway epithelial repair processes by TNF-α after injury.

Chronic infection and inflammation have been associated with progressive airway epithelial damage in patients with cystic fibrosis (CF). However, the effect of inflammatory products on the repair capacity of respiratory epithelia is unclear. Our objective was to study the regulation of repair mechanisms by tumor necrosis factor-α (TNF-α), a major component of inflammation in CF, in a model of mechanical wounding, in two bronchial cell lines, non-CF NuLi and CF CuFi. We observed that TNF-α enhanced the NuLi and CuFi repair rates. Chronic exposure (24-48 h) to TNF-α augmented this stimulation as well as the migration rate during repair. The cellular mechanisms involved in this stimulation were then evaluated. First, we discerned that TNF-α induced metalloproteinase-9 release, epidermal growth factor (EGF) shedding, and subsequent EGF receptor transactivation. Second, TNF-α-induced stimulation of the NuLi and CuFi wound-closure rates was prevented by GM6001 (metalloproteinase inhibitor), EGF antibody (to titrate secreted EGF), and EGF receptor tyrosine kinase inhibitors. Furthermore, we recently reported a relationship between the EGF response and K(+) channel function, both controlling bronchial repair. We now show that TNF-α enhances KvLQT1 and K(ATP) currents, while their inhibition abolishes TNF-α-induced repair stimulation. These results indicate that the effect of TNF-α is mediated, at least in part, through EGF receptor transactivation and K(+) channel stimulation. In contrast, cell proliferation during repair was slowed by TNF-α, suggesting that TNF-α could exert contrasting actions on repair mechanisms of CF airway epithelia. Finally, the stimulatory effect of TNF-α on airway wound repair was confirmed on primary airway epithelial cells, from non-CF and CF patients.

Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair

BackgroundExtensive alveolar epithelial injury and remodelling is a common feature of acute lung injury and acute respiratory distress syndrome (ARDS) and it has been established that epithelial regeneration, and secondary lung oedema resorption, is crucial for ARDS resolution. Much evidence indicates that K+ channels are regulating epithelial repair processes; however, involvement of the KCa3.1 channels in alveolar repair has never been investigated before.ResultsWound-healing assays demonstrated that the repair rates were increased in primary rat alveolar cell monolayers grown on a fibronectin matrix compared to non-coated supports, whereas an anti-β1-integrin antibody reduced it. KCa3.1 inhibition/silencing impaired the fibronectin-stimulated wound-healing rates, as well as cell migration and proliferation, but had no effect in the absence of coating. We then evaluated a putative relationship between KCa3.1 channel and the migratory machinery protein β1-integrin, which is activated by fibronectin. Co-immunoprecipitation and immunofluorescence experiments indicated a link between the two proteins and revealed their cellular co-distribution. In addition, we demonstrated that KCa3.1 channel and β1-integrin membrane expressions were increased on a fibronectin matrix. We also showed increased intracellular calcium concentrations as well as enhanced expression of TRPC4, a voltage-independent calcium channel belonging to the large TRP channel family, on a fibronectin matrix. Finally, wound-healing assays showed additive effects of KCa3.1 and TRPC4 inhibitors on alveolar epithelial repair.ConclusionTaken together, our data demonstrate for the first time complementary roles of KCa3.1 and TRPC4 channels with extracellular matrix and β1-integrin in the regulation of alveolar repair processes.

Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells

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. Exposure of airway epithelial cell to P. aeruginosa impairs CFTR function, expression and rescue by correctors http://ow.ly/IleTw

Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair.

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 Pseudomon asaeruginosa on airway epithelial repair. FASEBJ. 30, 3011–3025 (2016). www.fasebj.org

Deleterious impact of hyperglycemia on cystic fibrosis airway ion transport and epithelial repair

BACKGROUND Cystic fibrosis (CF)-related diabetes (CFRD) is associated with faster pulmonary function decline. Thus, we evaluated the impact of hyperglycemia on airway epithelial repair and transepithelial ion transport, which are critical in maintaining lung integrity and function. METHODS Non-CF and CF airway epithelial cells were exposed to low (LG) or high (HG) glucose before ion current and wound repair rate measurements. RESULTS CFTR and K+ currents decreased after HG treatments. HG also reduced the wound healing rates of non-CF and CF cell monolayers. Although CFTR correction with VRT-325 accelerated the healing rates of CF cells monolayers under LG conditions, this improvement was significantly abrogated under HG conditions. CONCLUSIONS Our data highlights a deleterious impact of hyperglycemia on ion transport and epithelial repair functions, which could contribute to the deterioration in lung function in CFRD patients. HG may also interfere with the beneficial effects of CFTR rescue on airway epithelial repair.

Vx-809/Vx-770 treatment reduces inflammatory response to Pseudomonas aeruginosa in primary differentiated cystic fibrosis bronchial epithelial cells

Cystic fibrosis patients exhibit chronic Pseudomonas aeruginosa respiratory infections and sustained proinflammatory state favoring lung tissue damage and remodeling, ultimately leading to respiratory failure. Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function is associated with MAPK hyperactivation and increased cytokines expression, such as interleukin-8 [chemoattractant chemokine (C-X-C motif) ligand 8 (CXCL8)]. Recently, new therapeutic strategies directly targeting the basic CFTR defect have been developed, and ORKAMBI (Vx-809/Vx-770 combination) is the only Food and Drug Administration-approved treatment for CF patients homozygous for the F508del mutation. Here we aimed to determine the effect of the Vx-809/Vx-770 combination on the induction of the inflammatory response by fully differentiated primary bronchial epithelial cell cultures from CF patients carrying F508del mutations, following exposure to P. aeruginosa exoproducts. Our data unveiled that CFTR functional rescue with Vx-809/Vx-770 drastically reduces CXCL8 (as well as CXCL1 and CXCL2) transcripts and p38 MAPK phosphorylation in response to P. aeruginosa exposure through a CFTR-dependent mechanism. These results suggest that ORKAMBI has anti-inflammatory properties that could decrease lung inflammation and contribute to the observed beneficial impact of this treatment in CF patients.

Quorum Sensing Down-Regulation Counteracts the Negative Impact of Pseudomonas aeruginosa on CFTR Channel Expression, Function and Rescue in Human Airway Epithelial Cells

The function of cystic fibrosis transmembrane conductance regulator (CFTR) channels is crucial in human airways. However unfortunately, chronic Pseudomonas aeruginosa infection has been shown to impair CFTR proteins in non-CF airway epithelial cells (AEC) and to alter the efficiency of new treatments with CFTR modulators designed to correct the basic CFTR default in AEC from cystic fibrosis (CF) patients carrying the F508del mutation. Our aim was first to compare the effect of laboratory strains, clinical isolates, engineered and natural mutants to determine the role of the LasR quorum sensing system in CFTR impairment, and second, to test the efficiency of a quorum sensing inhibitor to counteract the deleterious impact of P. aeruginosa both on wt-CFTR and on the rescue of F508del-CFTR by correctors. We first report that exoproducts from either the laboratory PAO1 strain or a clinical ≪Early≫ isolate (from an early stage of infection) altered CFTR expression, localization and function in AEC expressing wt-CFTR. Genetic inactivation of the quorum-sensing LasR in PAO1 (PAO1ΔlasR) or in a natural clinical mutant (≪Late≫ CF-adapted clinical isolate) abolished wt-CFTR impairment. PAO1 exoproducts also dampened F508del-CFTR rescue by VRT-325 or Vx-809 correctors in CF cells, whereas PAO1ΔlasR had no impact. Importantly, treatment of P. aeruginosa cultures with a quorum sensing inhibitor (HDMF) prevented the negative effect of P. aeruginosa exoproducts on wt-CFTR and preserved CFTR rescue by correctors in CF AEC. These findings indicate that LasR-interfering strategies could be of benefits to counteract the deleterious effect of P. aeruginosa in infected patients.

CFTR rescue with VX-809 and VX-770 favors the repair of primary airway epithelial cell cultures from patients with class II mutations in the presence of Pseudomonas aeruginosa exoproducts

BACKGROUND Progressive airway damage due to bacterial infections, especially with Pseudomonas aeruginosa remains the first cause of morbidity and mortality in CF patients. Our previous work revealed a repair delay in CF airway epithelia compared to non-CF. This delay was partially prevented after CFTR correction (with VRT-325) in the absence of infection. Our goals were now to evaluate the effect of the Orkambi combination (CFTR VX-809 corrector + VX-770 potentiator) on the repair of CF primary airway epithelia, in infectious conditions. METHODS Primary airway epithelial cell cultures from patients with class II mutations were mechanically injured and wound healing rates and transepithelial resistances were monitored after CFTR rescue, in the absence and presence of P. aeruginosa exoproducts. RESULTS Our data revealed that combined treatment with VX-809 and VX-770 elicited a greater beneficial impact on airway epithelial repair than VX-809 alone, in the absence of infection. The treatment with Orkambi was effective not only in airway epithelial cell cultures from patients homozygous for the F508del mutation but also from heterozygous patients carrying F508del and another class II mutation (N1303 K, I507del). The stimulatory effect of the Orkambi treatment was prevented by CFTR inhibition with GlyH101. Finally, Orkambi combination elicited a slight but significant improvement in airway epithelial repair and transepithelial resistance, despite the presence of P. aeruginosa exoproducts. CONCLUSIONS Our findings indicate that Orkambi may favor airway epithelial integrity in CF patients with class II mutations. Complementary approaches would however be needed to further improve CFTR rescue and airway epithelial repair.