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Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis ...

Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM)

Université de Montréal

K(+) channels play a crucial role in epithelia by repolarizing cells and maintaining electrochemical gradient for Na(+) absorption and Cl(-) secretion. In the airway epithelium, the most frequently studied K(+) channels are KvLQT1 and K(Ca). A functional role for K(ATP) channels has been also suggested in the lung, where K(ATP) channel openers activate alveolar clearance and attenuate ischemia-reperfusion injury. However, the molecular identity of this channel is unknown in airway and alveolar epithelial cells (AEC). We adopted an RT-PCR strategy to identify, in AEC, cDNA transcripts for Kir channels (Kir6.1 or 6.2) and sulfonylurea receptors (SUR1, 2A, or 2B) forming K(ATP) channels. Only Kir6.1 and SUR2B were detected in freshly isolated and cultured alveolar cells. To determine the physiological role of K(+) channels in the transepithelial transport of alveolar monolayers, we studied the effect, on total short-circuit currents (I(sc)), of basolateral application of glibenclamide, an inhibitor of K(ATP) channels, as well as clofilium, charybdotoxin, clotrimazole, and iberiotoxin, inhibitors of KvLQT1 and K(Ca) channels, respectively. Interestingly, activity of the three types of K(+) channels was detected, since all tested inhibitors decreased I(sc). Furthermore, these K(+) channel inhibitors reduced amiloride-sensitive Na(+) currents (mediated by ENaC) and completely abolished stimulation of Cl(-) currents by forskolin. Conversely, pinacidil, an activator of K(ATP) channels, increased Na(+) and Cl(-) transepithelial transport by 33-35%. These results suggest the presence, in AEC, of a K(ATP) channel, formed from Kir6.1 and SUR2B subunits, which plays a physiological role, with KvLQT1 and K(Ca) channels, in Na(+) and Cl(-) transepithelial transport.

Molecular identity and function in transepithelial transport of K(ATP) channels in alveolar 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. 

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

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.

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

Extensive 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.

Complementary roles of KCa3.1 channels and β1-integrin during alveolar 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 Pseudomonas aeruginosa on airway epithelial repair.

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

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.

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.

Article	Year
Molecular identity and function in transepithelial transport of K(ATP) channels in alveolar epithelial cells. American journal of physiology. Lung cellular and molecular physiology\| PubMed ID: 14729507	2004
Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells. The European respiratory journal\| PubMed ID: 25792634	2015
Deleterious impact of hyperglycemia on cystic fibrosis airway ion transport and epithelial repair. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 25920899	2016
Complementary roles of KCa3.1 channels and β1-integrin during alveolar epithelial repair. Respiratory research\| PubMed ID: 26335442	2015
Quorum-sensing inhibition abrogates the deleterious impact of Pseudomonas aeruginosa on airway epithelial repair. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 27178322	2016
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. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society\| PubMed ID: 29661510	2018

Emmanuelle Brochiero

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