Cystic Fibrosis Airway Epithelial Cells Research Articles

Inhibition of mucus secretion by niclosamide and benzbromarone in airways and intestine

The Ca2+ activated Cl− channel TMEM16A (anoctamin 1; ANO1) is expressed in secretory epithelial cells of airways and intestine. Previous studies provided evidence for a role of ANO1 in mucus secretion. In the present study we investigated the effects of the two ANO1-inhibitors niclosamide (Niclo) and benzbromarone (Benz) in vitro and in vivo in mouse models for cystic fibrosis (CF) and asthma. In human CF airway epithelial cells (CFBE), Ca2+ increase and activation of ANO1 by adenosine triphosphate (ATP) or ionomycin was strongly inhibited by 200 nM Niclo and 1 µM Benz. In asthmatic mice airway mucus secretion was inhibited by intratracheal instillation of Niclo or Benz. In homozygous F508del-cftr mice, intestinal mucus secretion and infiltration by CD45-positive cells was inhibited by intraperitoneal injection of Niclo (13 mg/kg/day for 7 days). In homozygous F508del-cftr rats intestinal mucus secretion was inhibited by oral application of Benz (5 mg/kg/day for 60 days). Taken together, well tolerated therapeutic concentrations of niclosamide and benzbromarone corresponding to plasma levels of treated patients, inhibit ANO1 and intracellular Ca2+ signals and may therefore be useful in inhibiting mucus hypersecretion and mucus obstruction in airways and intestine of patients suffering from asthma and CF, respectively.

18 CFTR modulators increase viral mobility in healthy and cystic fibrosis airway epithelial cells

18 CFTR modulators increase viral mobility in healthy and cystic fibrosis airway epithelial cells

Olive Leaf Extract (OLE) as a Novel Antioxidant That Ameliorates the Inflammatory Response in Cystic Fibrosis.

The deletion of phenylalanine at position 508 (F508del) produces a misfolded CFTR protein that is retained in the ER and degraded. The lack of normal CFTR channel activity is associated with chronic infection and inflammation which are the primary causes of declining lung function in Cystic Fibrosis (CF) patients. Moreover, LPS-dependent oxidative stress downregulates CFTR function in airway epithelial cells. Olive leaf extract (OLE) is used in traditional medicine for its effects, including anti-oxidant and anti-inflammatory ones. We found that OLE decreased the intracellular ROS levels in a dose-response manner in CFBE cells. Moreover, OLE attenuates the inflammatory response to LPS or IL-1β/TNFα stimulation, mimicking the infection and inflammatory status of CF patients, in CFBE and primary nasal epithelial (HNE) cells. Furthermore, we demonstrated that OLE restored the LPS-mediated decrease of TrikfaftaTM-dependent F508del-CFTR function in CFBE and HNE cultures. These findings provide strong evidence of OLE to prevent redox imbalance and inflammation that can cause chronic lung damage by enhancing the antioxidant activity and attenuating inflammation in CF airway epithelial cells. Additionally, OLE might be used in combination with CFTR modulators therapy to improve their efficacy in CF patients.

Targeting HuR-Vav3 mRNA interaction prevents Pseudomonas aeruginosa adhesion to the cystic fibrosis airway epithelium

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.

426 Metformin improves high mobility group box protein 1–induced mucociliary dysfunction in cystic fibrosis airway epithelial cells

426 Metformin improves high mobility group box protein 1–induced mucociliary dysfunction in cystic fibrosis airway epithelial cells

Elexacaftor/Tezacaftor/Ivacaftor Accelerates Wound Repair in Cystic Fibrosis Airway Epithelium.

Background: Cystic fibrosis (CF) airway epithelium shows alterations in repair following damage. In vitro studies showed that lumacaftor/ivacaftor (Orkambi) may favor airway epithelial integrity in CF patients. Our aim was to evaluate the effect of the novel triple combination elexacaftor/tezacaftor/ivacaftor (ETI) on wound repair in CF airway epithelial cells. Methods: A tip-based scratch assay was employed to study wound repair in monolayers of CFBE14o- cells overexpressing the F508del mutation. ETI was added during wound repair. Results: ETI efficiently rescued CFTR F508del maturation and activity, accelerated wound closure and increased wound healing rates of the injured CF cell monolayers. Conclusions: The triple corrector/potentiator combination ETI shows promise in ameliorating wound healing of the airway epithelium in F508del patients.

Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough.

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors’ clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.

A Splice Switch in SIGIRR Causes a Defect of IL-37-Dependent Anti-Inflammatory Activity in Cystic Fibrosis Airway Epithelial Cells.

Cystic fibrosis (CF) is a hereditary disease typically characterized by infection-associated chronic lung inflammation. The persistent activation of toll-like receptor (TLR) signals is considered one of the mechanisms for the CF hyperinflammatory phenotype; however, how negative regulatory signals of TLRs associate with CF inflammation is still elusive. Here, we showed that the cell surface expression of a single immunoglobulin interleukin-1 receptor (IL-1R)-related molecule (SIGIRR), a membrane protein essential for suppressing TLRs- and IL-1R-dependent signals, was remarkably decreased in CF airway epithelial cells compared to non-CF cells. Notably, CF airway epithelial cells specifically and highly expressed a unique, alternative splice isoform of the SIGIRR that lacks exon 8 (Δ8-SIGIRR), which results in the production of a C-terminal truncated form of the SIGIRR. Δ8-SIGIRR was expressed intracellularly, and its over-expression abolished the cell surface expression and function of the full-length SIGIRR (WT-SIGIRR), indicating its dominant-negative effect leading to the deficiency of anti-inflammatory activity in CF cells. Consistently, IL-37, a ligand for the SIGIRR, failed to suppress viral dsRNA analogue poly(I:C)-dependent JNK activation and IL-8 production, confirming the reduction in the functional WT-SIGIRR expression in the CF cells. Together, our studies reveal that SIGIRR-dependent anti-inflammatory activity is defective in CF airway epithelial cells due to the unique splicing switch of the SIGIRR gene and provides the first evidence of IL-37-SIGIRR signaling as a target of CF airway inflammation.

EPS6.04 Specialised pro-resolving mediators’ biosynthesis by cystic fibrosis airway epithelial cells and their impact on mucociliary clearance

ePS6.04 Specialised pro-resolving mediators’ biosynthesis by cystic fibrosis airway epithelial cells and their impact on mucociliary clearance

Human Amniotic Mesenchymal Stem Cells and Fibroblasts Accelerate Wound Repair of Cystic Fibrosis Epithelium.

Cystic fibrosis (CF) airways are affected by a deranged repair of the damaged epithelium resulting in altered regeneration and differentiation. Previously, we showed that human amniotic mesenchymal stem cells (hAMSCs) corrected base defects of CF airway epithelial cells via connexin (CX)43-intercellular gap junction formation. In this scenario, it is unknown whether hAMSCs, or fibroblasts sharing some common characteristics with MSCs, can operate a faster repair of a damaged airway epithelium. A tip-based scratch assay was employed to study wound repair in monolayers of CFBE14o- cells (CFBE, homozygous for the F508del mutation). hAMSCs were either co-cultured with CFBE cells before the wound or added to the wounded monolayers. NIH-3T3 fibroblasts (CX43+) were added to wounded cells. HeLa cells (CX43-) were used as controls. γ-irradiation was optimized to block CFBE cell proliferation. A specific siRNA was employed to downregulate CX43 expression in CFBE cells. CFBE cells showed a delayed repair as compared with wt-CFTR cells (16HBE41o-). hAMSCs enhanced the wound repair rate of wounded CFBE cell monolayers, especially when added post wounding. hAMSCs and NIH-3T3 fibroblasts, but not HeLa cells, increased wound closure of irradiated CFBE monolayers. CX43 downregulation accelerated CFBE wound repair rate without affecting cell proliferation. We conclude that hAMSCs and fibroblasts enhance the repair of a wounded CF airway epithelium, likely through a CX43-mediated mechanism mainly involving cell migration.

Genomic characterization of lytic bacteriophages targeting genetically diverse Pseudomonas aeruginosa clinical isolates

SummaryPseudomonas aeruginosa infections can be difficult to treat and new therapeutics are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and used them to screen and isolate over a dozen P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. We evolved bacterial mutants that were resistant to phage infection for four different phages, and used genome sequencing and functional analysis to study them further. We also tested phages for their ability to kill P. aeruginosa grown in biofilms in vitro and ex vivo on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

Cilia Stimulatory and Antibacterial Activities of T2R Bitter Taste Receptor Agonist Diphenhydramine: Insights into Repurposing Bitter Drugs for Nasal Infections.

T2R bitter taste receptors in airway motile cilia increase ciliary beat frequency (CBF) and nitric oxide (NO) production. Polymorphisms in some T2Rs are linked to disease outcomes in chronic rhinosinusitis (CRS) and cystic fibrosis (CF). We examined the expression of cilia T2Rs during the differentiation of human nasal epithelial cells grown at air–liquid interface (ALI). The T2R expression increased with differentiation but did not vary between CF and non-CF cultures. Treatment with Pseudomonas aeruginosa flagellin decreased the expression of diphenhydramine-responsive T2R14 and 40, among others. Diphenhydramine increased both NO production, measured by fluorescent dye DAF-FM, and CBF, measured via high-speed imaging. Increases in CBF were disrupted after flagellin treatment. Diphenhydramine impaired the growth of lab and clinical strains of P. aeruginosa, a major pathogen in CF and CF-related CRS. Diphenhydramine impaired biofilm formation of P. aeruginosa, measured via crystal violet staining, as well as the surface attachment of P. aeruginosa to CF airway epithelial cells, measured using colony-forming unit counting. Because the T2R agonist diphenhydramine increases NO production and CBF while also decreasing bacterial growth and biofilm production, diphenhydramine-derived compounds may have potential clinical usefulness in CF-related CRS as a topical therapy. However, utilizing T2R agonists as therapeutics within the context of P. aeruginosa infection may require co-treatment with anti-inflammatories to enhance T2R expression.

Genome-Wide RNAi Screening Identifies Novel Pathways/Genes Involved in Oxidative Stress and Repurposable Drugs to Preserve Cystic Fibrosis Airway Epithelial Cell Integrity.

Recurrent infection-inflammation cycles in cystic fibrosis (CF) patients generate a highly oxidative environment, leading to progressive destruction of the airway epithelia. The identification of novel modifier genes involved in oxidative stress susceptibility in the CF airways might contribute to devise new therapeutic approaches. We performed an unbiased genome-wide RNAi screen using a randomized siRNA library to identify oxidative stress modulators in CF airway epithelial cells. We monitored changes in cell viability after a lethal dose of hydrogen peroxide. Local similarity and protein-protein interaction network analyses uncovered siRNA target genes/pathways involved in oxidative stress. Further mining against public drug databases allowed identifying and validating commercially available drugs conferring oxidative stress resistance. Accordingly, a catalog of 167 siRNAs able to confer oxidative stress resistance in CF submucosal gland cells targeted 444 host genes and multiple circuitries involved in oxidative stress. The most significant processes were related to alternative splicing and cell communication, motility, and remodeling (impacting cilia structure/function, and cell guidance complexes). Other relevant pathways included DNA repair and PI3K/AKT/mTOR signaling. The mTOR inhibitor everolimus, the α1-adrenergic receptor antagonist doxazosin, and the Syk inhibitor fostamatinib significantly increased the viability of CF submucosal gland cells under strong oxidative stress pressure. Thus, novel therapeutic strategies to preserve airway cell integrity from the harsh oxidative milieu of CF airways could stem from a deep understanding of the complex consequences of oxidative stress at the molecular level, followed by a rational repurposing of existing “protective” drugs. This approach could also prove useful to other respiratory pathologies.

Increased susceptibility of cystic fibrosis airway epithelial cells to ferroptosis

BackgroundDefective chloride transport in airway epithelial cells (AECs) and the associated lung disease are the main causes of morbidity and early mortality in cystic fibrosis (CF). Abnormal airway iron homeostasis and the presence of lipid peroxidation products, indicative of oxidative stress, are features of CF lung disease.ResultsHere, we report that CF AECs (IB3-1) are susceptible to ferroptosis, a type of cell death associated with iron accumulation and lipid peroxidation. Compared to isogenic CFTR corrected cells (C38), the IB3-1 cells showed increased susceptibility to cell death upon exposure to iron in the form of ferric ammonium citrate (FAC) and the ferroptosis inducer, erastin. This phenotype was accompanied by accumulation of intracellular ferrous iron and lipid peroxides and the extracellular release of malondialdehyde, all indicative of redox stress, and increased levels of lactate dehydrogenase in the culture supernatant, indicating enhanced cell injury. The ferric iron chelator deferoxamine (DFO) and the lipophilic antioxidant ferrostatin-1 inhibited FAC and erastin induced ferroptosis in IB3-1 cells. Glutathione peroxidase 4 (GPX4) expression was decreased in IB3-1 cells treated with FAC and erastin, but was unchanged in C38 AECs. Necroptosis appeared to be involved in the enhanced susceptibility of IB3-1 AECs to ferroptosis, as evidenced by partial cell death rescue with necroptosis inhibitors and enhanced mixed lineage kinase domain-like (MLKL) localisation to the plasma membrane.ConclusionThese studies suggest that the increased susceptibility of CF AECs to ferroptosis is linked to abnormal intracellular ferrous iron accumulation and reduced antioxidant defences. In addition, the process of ferroptotic cell death in CF AECs does not appear to be a single entity and for the first time we describe necroptosis as a potential contributory factor. Iron chelation and antioxidant treatments may be promising therapeutic interventions in cystic fibrosis.Graphical

The sodium/glucose cotransporters as potential therapeutic targets for CF lung diseases revealed by human lung organoid swelling assay

Cystic fibrosis (CF) is a lethal autosomal-recessive inherited disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In the present work, we derived human proximal lung organoids (HLOs) from patient-derived pluripotent stem cells (PSCs) carrying disease-causing CFTR mutations. We evaluated the forskolin (Fsk)-stimulated swellings of these HLOs in the presence of CFTR modulators (VX-770 and/or VX-809) and demonstrated that HLOs respond to CFTR modulators in a mutation-dependent manner. Using this assay, we examined the effects of the sodium-dependent glucose cotransporter 1/2 (SGLT1/2) inhibitor drugs phlorizin and sotagliflozin on the basis of our findings that SGLT1 expression is upregulated in CF HLOs and airway epithelial cells compared with their wild-type counterparts. Unexpectedly, both drugs promoted dF/dF HLO swelling. These results reveal SGLTs, especially SGLT1, as potential therapeutic targets for treating CF lung diseases and demonstrate the use of PSC-derived HLOs as a preclinical tool in CF drug development.

TLR5 signalling is hyper-responsive in porcine cystic fibrosis airways epithelium

Excessive lung inflammation and airway epithelium damage are hallmarks of cystic fibrosis (CF) disease. It is unclear whether lung inflammation is related to an intrinsic defect in the immune response or to chronic infection. We aimed to determine whether TLR5-mediated response is defective in the CF airway epithelium. We used a newborn CF pig model to study intrinsic alterations in CF airway epithelium innate immune response. Airway epithelial cells (AECs) were stimulated with flagellin or lipopolysaccharide to determine responses specific for TLR5 and TLR4, respectively. We observed a significant increase in cytokine secretion when CF AECs were stimulated with flagellin compared to wild type (WT) AECs. These results were recapitulated when AECs were treated with an inhibitor of CFTR channel activity. We show that TLR5-signalling is altered in CF lung epithelium at birth. Modulation of TLR5 signalling could contribute to better control the excessive inflammatory response observed in CF lungs.

Microbial interaction: Prevotella spp. reduce P. aeruginosa induced inflammation in cystic fibrosis bronchial epithelial cells

BackgroundIn Cystic Fibrosis (CF) airways, the dehydrated, thick mucus promotes the establishment of persistent polymicrobial infections and drives chronic airways inflammation. This also predisposes the airways to further infections, the vicious, self-perpetuating cycle causing lung damage and progressive lung function decline. The airways are a poly-microbial environment, containing both aerobic and anaerobic bacterial species. Pseudomonas aeruginosa (P. aeruginosa) infections contribute to the excessive inflammatory response in CF, but the role of anaerobic Prevotella spp., frequently found in CF airways, is not known. MaterialsWe assessed innate immune signalling in CF airway epithelial cells in response to clinical strains of P. histicola, P. nigresens and P. aeruginosa. CFBE41o- cells were infected with P. aeruginosa (MOI 100, 2h) followed by infection with P. histicola or P. nigrescens (MOI 100, 2h). Cells were incubated under anaerobic conditions for the duration of the experiments. ResultsOur study shows that P. histicola and P. nigresens can reduce the growth of P. aeruginosa and dampen the inflammatory response in airway epithelial cells. We specifically illustrate that the presence of the investigated Prevotella spp. reduces Toll-like-receptor (TLR)-4, MAPK, NF-κB(p65) signalling and cytokine release (Interleukin (IL)-6, IL-8) in mixed infections. ConclusionOur work, for the first time, strongly indicates a relationship between P. aeruginosa and anaerobic Prevotella spp.. The observed modified NF-κB and MAPK signalling indicates some mechanisms underlying this interaction that could offer a novel therapeutic approach to combat chronic P. aeruginosa infection in people with CF.

Extracellular phosphate enhances the function of F508del-CFTR rescued by CFTR correctors

Background: The clinical response to cystic fibrosis transmembrane conductance regulator (CFTR) modulators varies between people with cystic fibrosis (CF) of the same genotype, in part through the action of solute carriers encoded by modifier genes. Here, we investigate whether phosphate transport by SLC34A2 modulates the function of F508del-CFTR after its rescue by CFTR correctors.Methods: With Fischer rat thyroid (FRT) cells heterologously expressing wild-type and F508del-CFTR and fully-differentiated CF and non-CF human airway epithelial cells, we studied SLC34A2 expression and the effects of phosphate on CFTR-mediated transepithelial ion transport. F508del-CFTR was trafficked to the plasma membrane by incubation with different CFTR correctors (alone or in combination) or by low temperature.Results: Quantitative RT-PCR demonstrated that both FRT and primary airway epithelial cells express SLC34A2 mRNA and no differences were found between cells expressing wild-type and F508del-CFTR. For both heterologously expressed and native F508del-CFTR rescued by either VX-809 or C18, the magnitude of CFTR-mediated Cl− currents was dependent on the presence of extracellular phosphate. However, this effect of phosphate was not detected with wild-type and low temperature-rescued F508del-CFTR Cl− currents. Importantly, the modulatory effect of phosphate was observed in native CF airway cells exposed to VX-445, VX-661 and VX-770 (Trikafta) and was dependent on the presence of both sodium and phosphate.Conclusions: Extracellular phosphate modulates the magnitude of CFTR-mediated Cl− currents after F508del-CFTR rescue by clinically-approved CFTR correctors. This effect likely involves electrogenic phosphate transport by SLC34A2. It might contribute to inter-individual variability in the clinical response to CFTR correctors.

The Anti-Infectious Role of Sphingosine in Microbial Diseases.

Sphingolipids are important structural membrane components and, together with cholesterol, are often organized in lipid rafts, where they act as signaling molecules in many cellular functions. They play crucial roles in regulating pathobiological processes, such as cancer, inflammation, and infectious diseases. The bioactive metabolites ceramide, sphingosine-1-phosphate, and sphingosine have been shown to be involved in the pathogenesis of several microbes. In contrast to ceramide, which often promotes bacterial and viral infections (for instance, by mediating adhesion and internalization), sphingosine, which is released from ceramide by the activity of ceramidases, kills many bacterial, viral, and fungal pathogens. In particular, sphingosine is an important natural component of the defense against bacterial pathogens in the respiratory tract. Pathologically reduced sphingosine levels in cystic fibrosis airway epithelial cells are normalized by inhalation of sphingosine, and coating plastic implants with sphingosine prevents bacterial infections. Pretreatment of cells with exogenous sphingosine also prevents the viral spike protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from interacting with host cell receptors and inhibits the propagation of herpes simplex virus type 1 (HSV-1) in macrophages. Recent examinations reveal that the bactericidal effect of sphingosine might be due to bacterial membrane permeabilization and the subsequent death of the bacteria.

CF monocyte-derived macrophages have an attenuated response to extracellular vesicles secreted by airway epithelial cells.

Mutations in CFTR alter macrophage responses, for example, by reducing their ability to phagocytose and kill bacteria. Altered macrophage responses may facilitate bacterial infection and inflammation in the lungs, contributing to morbidity and mortality in cystic fibrosis (CF). Extracellular vesicles (EVs) are secreted by multiple cell types in the lungs and participate in the host immune response to bacterial infection, but the effect of EVs secreted by CF airway epithelial cells (AEC) on CF macrophages is unknown. This report examines the effect of EVs secreted by primary AEC on monocyte-derived macrophages (MDM) and contrasts responses of CF and wild type (WT) MDM. We found that EVs generally increase pro-inflammatory cytokine secretion and expression of innate immune genes in MDM, especially when EVs are derived from AEC exposed to Pseudomonas aeruginosa and that this effect is attenuated in CF MDM. Specifically, EVs secreted by P. aeruginosa exposed AEC (EV-PA) induced immune response genes and increased secretion of proinflammatory cytokines, chemoattractants, and chemokines involved in tissue repair by WT MDM, but these effects were less robust in CF MDM. We attribute attenuated responses by CF MDM to differences between CF and WT macrophages because EVs secreted by CF AEC or WT AEC elicited similar responses in CF MDM. Our findings demonstrate the importance of AEC EVs in macrophage responses and show that the Phe508del mutation in CFTR attenuates the innate immune response of MDM to EVs.