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Epithelial polarity is fundamental in maintaining barrier integrity and tissue protection. In cystic fibrosis (CF), apicobasal polarity of the airway epithelium is altered, resulting in increased apical fibronectin deposition and enhanced susceptibility to bacterial infections. Here, we evaluated the effect of highly effective modulator treatment (HEMT) on fibronectin apical deposition and investigated the intracellular mechanisms triggering the defect in polarity of the CF airway epithelium. To this end, primary cultures of CF (F508del variant) human airway epithelial cells (HAECs) and a HAEC line, Calu-3, knocked down for CFTR (CF transmembrane conductance regulator) were compared with control counterparts. We show that CFTR mutation in primary HAECs and CFTR knockdown cells promote the overexpression and oversecretion of TGF-β1 and DKK1 when cultured at an air-liquid interface. These dynamic changes result in hyperactivation of the TGF-β pathway and inhibition of the Wnt pathway through degradation of β-catenin leading to imbalanced proliferation and polarization. The abnormal interplay between TGF-β and Wnt signaling pathways is reinforced by aberrant Akt signaling. Pharmacological manipulation of TGF-β, Wnt, and Akt pathways restored polarization of the F508del CF epithelium, a correction that was not achieved by HEMT. Our data shed new insights into the signaling pathways that fine-tune apicobasal polarization in primary airway epithelial cells and may provide an explanation to the mitigated efficacy of HEMT on lung infection in people with CF.

Background: Cystic fibrosis (CF), a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is characterized by dysfunction of the immune response in the airway epithelium that leads to prolonged infection, colonization and exacerbated inflammation. In this study, we determined the gene expression profile of airway epithelial cells knockdown for CFTR (CFTR KD) in response to bacterial and viral challenges., Methods: In a first approach, polarized CFTR KD and their control counterpart (CFTR CTL) cells were stimulated with P. aeruginosa-derived virulence factor flagellin. Next, we developed a model of Influenza A virus (IAV) infection in CTL and CFTR KD polarized cells. mRNA was collected for transcriptome analysis., Results: Beside the expected pro-inflammatory response, Gene Set Enrichment Analysis highlighted key molecular pathways and players involved in IAV and anti-viral interferon signaling. Although IAV replication was similar in both cell types, multiplex gene expression analysis revealed changes of key immune genes dependent on time of infection that were found to be CFTR-dependent and/or IAV-dependent. Interferons are key signaling proteins/cytokines in the antibacterial and antiviral response. To evaluate their impact on the altered gene expression profile in CFTR responses to pathogens, we measured transcriptome changes after exposure to Type I-, Type II- and Type III-interferons., Conclusions: Our findings reveal target genes in understanding the defective immune response in the CF airway epithelium in the context of viral infection. Information provided in this study would be useful to understand the dysfunctional immune response of the CF airway epithelium during infection., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Pathological remodeling of the airway epithelium is commonly observed in Cystic Fibrosis (CF). The different cell types that constitute the airway epithelium are regenerated upon injury to restore integrity and maintenance of the epithelium barrier function. The molecular signature of tissue repair in CF airway epithelial cells has, however, not well been investigated in primary cultures. We therefore collected RNA-seq data from well-differentiated primary cultures of bronchial human airway epithelial cells (HAECs) of CF (F508del/F508del) and non-CF (NCF) origins before and after mechanical wounding, exposed or not to flagellin. We identified the expression changes with time of repair of genes, the products of which are markers of the different cell types that constitute the airway epithelium (basal, suprabasal, intermediate, secretory, goblet and ciliated cells as well as ionocytes). Researchers in the CF field may benefit from this transcriptomic profile, which covers the initial steps of wound repair and revealed differences in this process between CF and NCF cultures.

Peroxisome proliferator-activated receptor gamma (PPARγ) represents a potential target to treat airway mucus hypersecretion in cystic fibrosis (CF). We aimed to determine if PPARγ is altered in CF human airway epithelial cells (HAECs), if PPARγ contributes to mucin expression and HAEC differentiation, and if PPARγ ligand therapy corrects the CF phenotype. To this end, well-differentiated CF and NCF HAEC primary cultures were wounded to monitor the expression of key genes involved in PPARγ activation and mucus homeostasis, and to evaluate the effect of a PPARγ agonist, at different times of repair. Hydroxyprostaglandin dehydrogenase (HPGD) converts prostaglandin E2 to 15-keto PGE2 (15kPGE2), an endogenous PPARγ ligand. Interestingly, PPARγ and HPGD expression dramatically decreased in CF HAECs. These changes were accompanied by an increase in the expression of MUC5B. The correlation between PPARγ and MUC5B was confirmed in an airway epithelial cell line after CFTR knock-down. Exposure of HAECs to 15kPGE2 did not correct the CF phenotype but revealed a defect in the process of basal cell (BC) differentiation. The HPGD/PPARγ axis is deregulated in primary HAEC cultures from CF patients, which may impact the maturation of BCs to differentiated luminal cells. Importantly, PPARγ therapy was inefficient in correcting the CF defect., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cell-to-cell communication via gap junctions regulates airway epithelial cell homeostasis and maintains the epithelium host defense. Quorum-sensing molecules produced by Pseudomonas aeruginosa coordinate the expression of virulence factors by this respiratory pathogen. These bacterial signals may also incidentally modulate mammalian airway epithelial cell responses to the pathogen, a process called interkingdom signaling. We investigated the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). C12 degradation and its effects on cells were monitored in various airway epithelial cell models grown under nonpolarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. C12 rapidly altered epithelial integrity and decreased GJIC in nonpolarized airway epithelial cells, whereas other quorum-sensing molecules had no effect. The effects of C12 were dependent on [Ca(2+)]i and could be prevented by inhibitors of Src tyrosine family and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12 except when undergoing repair after wounding. In vivo during colonization of intubated patients, C12 did not accumulate, but it paralleled bacterial densities. In vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in nonpolarized cells, whereas PON2 expression was increased during epithelial polarization. The cytotoxicity of C12 on nonpolarized epithelial cells, combined with its impaired degradation allowing its accumulation, provides an additional pathogenic mechanism for P. aeruginosa infections.

Background: Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation., Methods: Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells., Results: shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion., Conclusion: We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

Chronic infection and inflammation of the airways is a hallmark of cystic fibrosis (CF), a disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. The response of the CF airway epithelium to the opportunistic pathogen Pseudomonas aeruginosa is characterized by altered inflammation and apoptosis. In this study, we examined innate immune recognition and epithelial responses at the level of the gap junction protein connexin43 (Cx43) in polarized human airway epithelial cells upon infection by PAO1. We report that PAO1 activates cell surface receptors to elicit an intracellular signaling cascade leading to enhancement of gap junctional communication. Expression of Cx43 involved an opposite regulation exerted by JNK and p38 MAPKs. PAO1-induced apoptosis was increased in the presence of a JNK inhibitor, but latter effect was prevented by lentiviral expression of a Cx43-specific short hairpin RNA. Moreover, we found that JNK activity was upregulated by pharmacological inhibition of CFTR in Calu-3 cells, whereas correction of a CF airway cell line (CF15 cells) by adenoviral expression of CFTR reduced the activation of this MAPK. Interestingly, CFTR inhibition in Calu-3 cells was associated with decreased Cx43 expression and reduced apoptosis. These results indicate that Cx43 expression is a component of the response of airway epithelial cells to innate immune activation by regulating the survival/apoptosis balance. Defective CFTR could alter this equilibrium with deleterious consequences on the CF epithelial response to P. aeruginosa.

One of the main functions of the airway mucosa is to maintain a mechanical barrier at the air-surface interface and to protect the respiratory tract from external injuries. Differentiation of human airway epithelial cells (hAECs) to polarized airway mucosa can be reproduced in vitro by culturing the cells on microporous membrane at the air-liquid interface. Here, we describe approaches to study differentiation as well as repair of the hAECs by using a commercially available airway cell culture model called MucilAir™.

Stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by protease-activated receptors (PARs) at the basolateral membranes and by adenosine receptors (ADO-Rs) at the apical membrane maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Both pathways involve the release of prostaglandin E₂ (PGE₂) and the stimulation of their basolateral receptors (EP-Rs). We sought to determine whether gap junctions contribute to the coordination of these pathways for modulating CFTR activity and mucus hydration. We used RT-PCR and Western blotting to determine connexin (Cx), CD73, and EP-R expression in a Calu-3 airway epithelial cell line grown on Transwell (Corning Costar, Cambridge, MA) inserts. We used dye coupling to evaluate gap junctional intercellular communication (GJIC). We used Ussing chamber studies and X-Z confocal microscopy to monitor Cl(-) secretion and ASL volume regulation. We found that connexin 43 (Cx43)-mediated GJIC was increased either by endogenous ADO after the hydrolysis of purine nucleotides by CD73 or by the direct activation of ADO-Rs. Inhibition of phospholipase A2 and cyclooxygenase prevented ADO-dependent increases in GJIC, suggesting the involvement of PGE₂. PGE₂ was found to increase GJIC markedly by stimulating EP4-Rs. The modulation of ADO signaling also affected the PAR-dependent activation of CFTR. The reduction of GJIC by CD73 or Cx43 inhibition prevented PAR-evoked CFTR currents in Ussing chambers. The inhibition of GJIC resulted in a failure of PGE₂ to increase ASL volume in Calu-3 cells and in primary cultures of well-differentiated human airway epithelial cells. Thus, gap junctions coordinate a signaling network comprising CFTR, ADO-Rs, PARs, and EP-Rs, and are required for ASL volume homeostasis.

Highlights: [•] Repair of human airway epithelial cells from control individuals and patients with cystic fibrosis (CF) in response to injury is investigated. [•] Connexin26, a gap junction protein, is transiently induced in a subpopulation of basal airway epithelial cells expressing keratin14. [•] Connexin26 is induced by cell division and acts as a negative regulator of proliferation. [•] Krüppel-like factor (KLF) 4, a known transcription factor controlling cell proliferation, is transiently up-regulated in response to injury but did not appear to regulate Connexin26 expression. [•] The up-regulation of KLF4 following injury is absent in CF airway epithelial cells, which also exhibit desynchronized induction of Connexin26 expression and proliferation. [ABSTRACT FROM AUTHOR]

Rehabilitation of injured donor lungs can reduce the shortage of organs suitable for transplantation. We previously established a platform for the maintenance and recovery of extracorporeal human lungs using cross-circulation (XC) with a porcine host. Here, we characterize the human-swine immunologic interactions in this xenogeneic system. Human donor lungs declined for clinical transplantation were placed on XC with immunosuppressed (methylprednisolone, mycophenolate, tacrolimus), complement-depleted (Cobra Venom Factor) porcine hosts. Longitudinal analyses of the extracorporeal lung and porcine host were performed over 24 hours. For one of these lung (n = 1), single-cell lung suspensions and paraffin-embedded slides were prepared from biopsies of the lung collected at pre-XC baseline, 1h, and 24h. Infiltration of swine CD45+ immune cells and deposition of swine IgG and IgM in the human lung were assessed by immunohistochemistry and flow cytometry. The extracorporeal lung demonstrated improved PaO2/FiO2 (602%), dynamic compliance (238%), and gas exchange (O2, 974%; CO2, 198%) over the course of XC. Populations of human and porcine CD45+ leukocytes were readily identified within the post-XC lung. Human immune cells represented 1.48% and porcine immune cells represented 0.99% of isolated live single cells (Figure 1A). Infiltration of CD45+ porcine leukocytes into the lung increased over time, such that porcine neutrophils were visualized in the alveolar spaces (Figure 1B). By flow cytometry, porcine IgG was localized to lung epithelial cells (Figure 1C). Diffuse deposition of IgG and IgM was also detected by IHC (Figure 1D). In the xenogeneic XC system, porcine immune cell and immunoglobulin infiltration of the extracorporeal human lung can be detected. Despite this, XC was sufficient to facilitate physiologic improvement in the human lung. Further work is needed to minimize cross-species immune responses during ex vivo organ support. [ABSTRACT FROM AUTHOR]

Objective To investigate the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular communication (GJIC). Methods C12 degradation and its effects on cells were monitored in various human airway epithelial cell models grown under non-polarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. Results C12 rapidly altered epithelial integrity and decreased GJIC in non-polarized cells while other quorum-sensing molecules had no effect. C12 increased [Ca 2+ ] i by promoting calcium release from the ER and calcium influx. C12 cytotoxic effects were dependent on [Ca 2+ ] i and could be prevented by inhibitors of Src and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12. In vivo during colonization of intubated patients, C12 did not accumulate, but paralleled bacterial densities. Interestingly, in vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in non-polarized cells whereas PON2 expression was increased during epithelial polarization. Finally, in a wound injury model of primary airway epithelial cells grown at the air-liquid interface, repeated exposure of C12 impairs airway epithelial cell repair. Conclusion The cytotoxicity of C12 on non-polarized epithelial cells, combined with its impaired degradation allowing its accumulation, provide an additional pathogenic mechanism for P. aeruginosa infections. [ABSTRACT FROM AUTHOR]