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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.

Defective hydration of airway surface mucosa is associated with lung infection in cystic fibrosis (CF), partly caused by disruption of the epithelial barrier integrity. Although rehydration of the CF airway surface liquid (ASL) alleviates epithelium vulnerability to infection by junctional protein expression, the mechanisms linking ASL to barrier integrity are unknown. We show here the strong degradation of YAP1 and TAZ proteins in well-polarized CF human airway epithelial cells (HAECs), a process that was prevented by ASL rehydration. Conditional silencing of YAP1 in rehydrated CF HAECs indicated that YAP1 expression was necessary for the maintenance of junctional complexes. A higher plasma membrane tension in CF HAECs reduced endocytosis, concurrent with the maintenance of active β 1-integrin ectopically located at the apical membrane. Pharmacological inhibition of β 1-integrin accumulation restored YAP1 expression in CF HAECs. These results indicate that dehydration of the CF ASL affects epithelial plasma membrane tension, resulting in ectopic activation of a β 1-integrin/YAP1 signaling pathway associated with degradation of junctional proteins., (© 2024 Simonin et al.)

Cystic fibrosis (CF) is characterized by chronic bacterial infections leading to progressive bronchiectasis and respiratory failure. Pseudomonas aeruginosa (Pa) is the predominant opportunistic pathogen infecting the CF airways. The guanine nucleotide exchange factor Vav3 plays a critical role in Pa adhesion to the CF airways by inducing luminal fibronectin deposition that favors bacteria trapping. Here we report that Vav3 overexpression in CF is caused by upregulation of the mRNA-stabilizing protein HuR. We found that HuR accumulates in the cytoplasm of CF airway epithelial cells and that it binds to and stabilizes Vav3 mRNA. Interestingly, disruption of the HuR-Vav3 mRNA interaction improved the CF epithelial integrity, inhibited the formation of the fibronectin-made bacterial docking platforms, and prevented Pa adhesion to the CF airway epithelium. These findings indicate that targeting HuR represents a promising antiadhesive approach in CF that can prevent initial stages of Pa infection in a context of emergence of multidrug-resistant pathogens.

Background: Manifestations of cystic fibrosis, although well-characterized in the proximal airways, are understudied in the distal lung. Characterization of the cystic fibrosis lung 'matrisome' (matrix proteome) has not been previously described, and could help identify biomarkers and inform therapeutic strategies., Methods: We performed liquid chromatography-mass spectrometry, gene ontology analysis, and multi-modal imaging, including histology, immunofluorescence, and electron microscopy for a comprehensive evaluation of distal human lung extracellular matrix (matrix) structure and composition in end-stage cystic fibrosis., Results: Quantitative proteomic profiling identified sixty-eight (68) matrix constituents with significantly altered expression in end-stage cystic fibrosis. Over 90% of significantly different matrix peptides detected, including structural and basement membrane proteins, were expressed at lower levels in cystic fibrosis. However, the total abundance of matrix in cystic fibrosis lungs was not significantly different from control lungs, suggesting that cystic fibrosis leads to loss of diversity among lung matrix proteins rather than an absolute loss of matrix. Visualization of distal lung matrix via immunofluorescence and electron microscopy revealed pathological remodeling of distal lung tissue architecture and loss of alveolar basement membrane, consistent with significantly altered pathways identified by gene ontology analysis., Conclusions: Dysregulation of matrix organization and aberrant wound healing pathways are associated with loss of matrix protein diversity and obliteration of distal lung tissue structure in end-stage cystic fibrosis. While many therapeutics aim to functionally restore defective cystic fibrosis transmembrane conductance regulator (CFTR), drugs that target dysregulated matrix pathways may serve as adjunct interventions to support lung recovery., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest exists, (Copyright © 2022 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)

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.)

Pseudomonas aeruginosa (Pa) represents the leading cause of airway infection in cystic fibrosis (CF). Early airways colonization can be explained by enhanced adhesion of Pa to the respiratory epithelium. RNA sequencing (RNA-seq) on fully differentiated primary cultures of airway epithelial cells from CF and non-CF donors predict that VAV3, β1 INTEGRIN, and FIBRONECTIN genes are significantly enriched in CF. Indeed, Vav3 is apically overexpressed in CF, associates with active β1 integrin luminally exposed, and increases fibronectin deposition. These luminal microdomains, rich in fibronectin and β1 integrin and regulated by Vav3, mediate the increased Pa adhesion to the CF epithelium. Interestingly, Vav3 inhibition normalizes the CF-dependent fibronectin and β1-integrin ectopic expression, improves the CF epithelial integrity, and prevents the enhanced Pa trapping to the CF epithelium. Through its capacity to promote a luminal complex with active β1 integrin and fibronectin that favors bacteria trapping, Vav3 may represent a new target in CF., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. 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.)

The recovery of an intact epithelium following injury is critical for restoration of lung homeostasis, a process that may be altered in cystic fibrosis (CF). In response to injury, progenitor cells in the undamaged areas migrate, proliferate and re-differentiate to regenerate an intact airway epithelium. The mechanisms regulating this regenerative response are, however, not well understood. In a model of circular wound injury of well-differentiated human airway epithelial cell (HAEC) cultures, we identified the gap junction protein Cx26 as an important regulator of cell proliferation. We report that induction of Cx26 in repairing HAECs is associated with cell proliferation. We also show that Cx26 is expressed in a population of CK14-positive basal-like cells. Cx26 silencing in immortalized cell lines using siRNA and in primary HAECs using lentiviral-transduced shRNA enhanced Ki67-labeling index and Ki67 mRNA, indicating that Cx26 acts a negative regulator of HAEC proliferation. Cx26 silencing also markedly decreased the transcription of KLF4 in immortalized HAECs. We further show that CF HAECs exhibited deregulated expression of KLF4, Ki67 and Cx26 as well enhanced rate of wound closure in the early response to injury. These results point to an altered repair process of CF HAECs characterized by rapid but desynchronized initiation of HAEC activation and proliferation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)