Repair Process Impairment by Pseudomonas aeruginosa in Epithelial Tissues: Major Features and Potential Therapeutic Avenues

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

Activation of an ATP-dependent K+ conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules

AbstractIn rabbit proximal convoluted tubules, an ATP-sensitive K+ (KATP) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na+ transport and basolateral K+ conductance. This K+ conductance is inhibited by taurine. We sought to isolate this K+ channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K+ conductance, largely inhibited by extracellular Ba2+ and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K+ current which was Ba2+-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K+ channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K+ current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K+ current. The possible involvement of AK in the KATP channel’s regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations

K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

AbstractActive Na+ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and KATP K+ channel activities exerts sustained control in Na+ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K+ channels, and 2) to determine the physiological impact of K+ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and KATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to KATP activation by pinacidil. Conversely, pharmacological KvLQT1 and KATP inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K+ channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and KATP activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K+ channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K+ channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance

The Quebec Respiratory Health Network Biobank

The Quebec Respiratory Health Network (RHN) Biobank is a multi-site infrastructure located in the province of Quebec (Canada) to collect, store, and supply high-quality human biological specimens for research on respiratory diseases. The sample types are diverse (plasma, serum, buffy coat, primary lung cells, lung parenchyma, bronchial biopsies, polyps, others), disease-oriented, and mirror research activities conducted at each site. The biobank currently manages approximately 57,000 specimens from 8,000 research participants or patients treated by standard of care. Specimens’ inventory and corresponding clinical data from all sites are denominalized and linked to a centralized database with retrieval and querying capabilities. Archival samples from recent to nearly 20-year collections are available to academic and industry researchers studying respiratory diseases.   Funding statement: The infrastructure is supported by the Quebec Respiratory Health Network (rsr.chus.qc.ca) of the 'Fonds de la recherche du Québec – Santé' (FRQS), the research centers involved, local foundations and users of the biobank. Each biobank site is responsible to sustain their activities

Repair Process Impairment by Pseudomonas aeruginosa in Epithelial Tissues: Major Features and Potential Therapeutic Avenues

International audienceEpithelial 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

Canaux potassiques et physiologie de l’épithélium respiratoire

Plus de 30 canaux potassiques différents, appartenant aux 3 principales classes de canaux K+, sont exprimés au sein de l’épithélium respiratoire recouvrant les voies aériennes et les alvéoles. La signification physiologique de cette diversité est encore mal connue et relativement peu étudiée. Pourtant, plusieurs études ont démontré un rôle crucial des canaux KvLQT1, KCa et KATP dans le transport ionique et liquidien, participant ainsi à la régulation de la composition et du volume des fluides pulmonaires. D’autre part, ils participent à d’autres fonctions essentielles, telles que l’adaptation au niveau d’oxygène ou la capacité de l’épithélium à se défendre contre les agressions extérieures

Vx-809/Vx-770 treatment reduces inflammatory response to Pseudomonas aeruginosa

International audienceCystic 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