Epithelial tissues protecting organs from the environment are the first-line of defense against pathogens. Therefore, efficient repair mechanisms after injury are crucial to maintain epithelial integrity. However, these healing processes can be insufficient to restore epithelial integrity, notably in infectious conditions. Pseudomonas aeruginosa infections in cutaneous, corneal, and respiratory tract epithelia are of particular concern because they are the leading causes of hospitalizations, disabilities, and deaths worldwide. Pseudomonas aeruginosa has been shown to alter repair processes, leading to chronic wounds and infections. Because of the current increase in the incidence of multi-drug resistant isolates of P. aeruginosa, complementary approaches to decrease the negative impact of these bacteria on epithelia are urgently needed. Here, we review the recent advances in the understanding of the impact of P. aeruginosa infections on the integrity and repair mechanisms of alveolar, airway, cutaneous and corneal epithelia. Potential therapeutic avenues aimed at counteracting this deleterious impact of infection are also discussed.

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.

The seasonal nature of outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. Accordingly, temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2, the virus responsible for the COVID-19 pandemic. The receptor-binding domain (RBD) of the Spike glycoprotein is known to bind to its host receptor angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Using biochemical, biophysical, and functional assays to dissect the effect of temperature on the receptor–Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike glycoprotein with the ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide (including the B.1.1.7 (α) lineage), bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

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 Δ lasR 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

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.

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 ofPseudomonas aeruginosaon 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 toP. aeruginosadiffusible 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 byP. 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.