Cystic Fibrosis Airway Epithelial Cells Research Articles

Interaction with CREB binding protein modulates the activities of Nrf2 and NF-κB in cystic fibrosis airway epithelial cells

Cystic fibrosis (CF) is characterized by inflammatory lung disease that significantly contributes to morbidity and mortality. Airway epithelial cells play a role in the inflammatory signaling in CF and have been reported to exhibit a number of dysfunctions in signaling cascades that modulate inflammation. Previously, we reported that the activity of nuclear factor erythroid-derived-like 2 (Nrf2), a transcription factor that regulates antioxidant and cytoprotective protein expression, is diminished in CF epithelia (7). In this report, we examined the mechanism of Nrf2 dysregulation in vitro in human airway epithelial cell lines and primary cells and in vivo in nasal epithelia excised from ΔF508 CF mutant mice. We found that cAMP-mediated signaling markedly reduces Nrf2 activity in CF vs. non-CF cells. Rp-cAMPS, a cAMP competitor, significantly corrected Nrf2 activity in CF cells, predominantly by increasing the nuclear accumulation of the transcription factor. Furthermore, we found that Rp-cAMPS significantly decreased NF-κB activation following inflammatory stimulation of CF cells. Further investigation revealed that Nrf2 and NF-κB compete for the transcriptional coactivator cAMP responsive element-binding protein (CREB) binding protein (CBP) and that Rp-cAMPS shifts CBP association in favor of Nrf2. Thus our findings provide a link between feedback to CF transmembrane regulator dysfunction and dysregulation of an inflammatory signaling pathway that modulates the coordinated activities of Nrf2 and NF-κB. Furthermore, our studies suggest that strategies that shift CBP association away from NF-κB and toward Nrf2 could have potential therapeutic efficacy for reducing inflammation in patients with CF.

MudPIT analysis of released proteins inPseudomonas aeruginosalaboratory and clinical strains in relation to pro-inflammatory effects

Pseudomonas aeruginosa (Pa) is the most common virulent pathogen contributing to the pathogenesis of cystic fibrosis (CF). During bacterial lung colonization, the products of its metabolism are released in the extracellular space contributing to the pathogenic events associated with its presence. To gain insights on the mechanisms involved in the Pa pathogenesis we focused our attention on proteins released by Pa using a MudPIT approach combined with cell biology assays. Conditioned medium (CM) collected under aerobic and microaerobic conditions from Pa clinical strains (in early and late colonization), unlike the laboratory strain, induced expression of IL-8 mRNA in CF airway epithelial cells. We have identified proteins released by clinically relevant Pa strains, focusing on the pro-inflammatory effects as metalloproteases (MMPs). In fact, their expression pattern was associated with the highest pro-inflammatory activity measured in the early clinically isolated strain. The relation was further supported by the result of the analysis of a larger and independent set of Pa isolates derived from sporadically and chronically infected CF patients: 76% of sporadic samples expressed protease activity (n = 44), while only 27% scored positive in the chronically infected individuals (n = 38, p < 0.0001, Fisher's exact test). Finally, looking for a possible mechanism of action of bacterial MMPs, we found that CM from early clinical isolates can cleave CXCR1 on the surface of human neutrophils, suggesting a potential role for the bacterially released MMPs in the protection of the pathogen from the host's response.

Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells

This article has been retractedVareille M, Kieninger E, Alves MP, et al. Impaired type I and type III interferon induction and rhinovirus control in human cystic fibrosis airway epithelial cells....

Amniotic Mesenchymal Stem Cells: A New Source for Hepatocyte-Like Cells and Induction of CFTR Expression by Coculture with Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with lung and liver manifestations. Because of pitfalls of gene therapy, novel approaches for reconstitution of the airway epithelium and CFTR expression should be explored. In the present study, human amniotic mesenchymal stem cells (hAMSCs) were isolated from term placentas and characterized for expression of phenotypic and pluripotency markers, and for differentiation potential towards mesoderm (osteogenic and adipogenic) lineages. Moreover, hAMSCs were induced to differentiate into hepatocyte-like cells, as demonstrated by mixed function oxidase activity and expression of albumin, alpha1-antitrypsin, and CK19. We also investigated the CFTR expression in hAMSCs upon isolation and in coculture with CF airway epithelial cells. Freshly isolated hAMSCs displayed low levels of CFTR mRNA, which even decreased with culture passages. Following staining with the vital dye CM-DiI, hAMSCs were mixed with CFBE41o- respiratory epithelial cells and seeded onto permeable filters. Flow cytometry demonstrated that 33–50% of hAMSCs acquired a detectable CFTR expression on the apical membrane, a result confirmed by confocal microscopy. Our data show that amniotic MSCs have the potential to differentiate into epithelial cells of organs relevant in CF pathogenesis and may contribute to partial correction of the CF phenotype.

Synergism Between Interleukin (IL)-17 and Toll-like Receptor 2 and 4 Signals to Induce IL-8 Expression in Cystic Fibrosis Airway Epithelial Cells

Cystic fibrosis (CF) is the most common lethal inherited disorder and is caused by mutations in the gene encoding the CF transmembrane regulator (CFTR). The CF lung expresses a profound proinflammatory phenotype that appears to be related to a constitutive hypersecretion of interleukin (IL)-8 from airway epithelial cells in response to microbial infection. Since overproduction of IL-8 in CF contributes to massive bronchial infiltrates of neutrophils, identification of the pathways underlying IL-8 induction could provide novel drug targets for treatment of neutrophil-dominated inflammatory diseases such as CF. Here, we show that IL-17A synergistically increases IL-8 production induced by a toll-like receptor (TLR) 2 agonist, peptidoglycan (PGN), or TLR4 agonist, lipopolysaccharide (LPS), in a human CF bronchial epithelial cell line (CFBE41o-). A strong synergism was also observed in primary human CF bronchial epithelial cells, but not in human non-CF cell lines and primary cells. Notably, despite the induction of nuclear factor-κB and MAP kinases during TLR2 or TLR4 activation in CFBE41o-, IL-17A-dependent synergism appears to be the result of enhanced PGN- or LPS-induced phosphorylation of p38. Taken together, these studies provide evidence that IL-17A is a critical factor in increasing IL-8 expression in bacteria-infected CF airways via a pathway that regulates p38 phosphorylation.

Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

Interferon γ Stimulates Accumulation of Gas Phase Nitric Oxide in Differentiated Cultures of Normal and Cystic Fibrosis Airway Epithelial Cells

SERCA2 Regulates Non-CF and CF Airway Epithelial Cell Response to Ozone

Calcium mobilization can regulate a wide range of essential functions of respiratory epithelium, including ion transport, ciliary beat frequency, and secretion of mucus, all of which are modified in cystic fibrosis (CF). SERCA2, an important controller of calcium signaling, is deficient in CF epithelium. We conducted this study to determine whether SERCA2 deficiency can modulate airway epithelial responses to environmental oxidants such as ozone. This could contribute to the pathogenesis of pulmonary exacerbations, which are important and frequent clinical events in CF. To address this, we used air-liquid interface (ALI) cultures of non-CF and CF cell lines, as well as differentiated cultures of cells derived from non-CF and CF patients. We found that ozone exposure caused enhanced membrane damage, mitochondrial dysfunction and apoptotic cell death in CF airway epithelial cell lines relative to non-CF. Ozone exposure caused increased proinflammatory cytokine production in CF airway epithelial cell lines. Elevated proinflammatory cytokine production also was observed in shRNA-mediated SERCA2 knockdown cells. Overexpression of SERCA2 reversed ozone-induced proinflammatory cytokine production. Ozone-induced proinflammatory cytokine production was NF-κB- dependent. In a stable NF-κB reporter cell line, SERCA2 inhibition and knockdown both upregulated cytomix-induced NF-κB activity, indicating importance of SERCA2 in modulating NF-κB activity. In this system, increased NF-κB activity was also accompanied by increased IL-8 production. Ozone also induced NF-κB activity and IL-8 release, an effect that was greater in SERCA2-silenced NF-κB-reporter cells. SERCA2 overexpression reversed cytomix-induced increased IL-8 release and total nuclear p65 in CFTR-deficient (16HBE-AS) cells. These studies suggest that SERCA2 is an important regulator of the proinflammatory response of airway epithelial cells and could be a potential therapeutic target.

Azithromycin fails to reduce inflammation in cystic fibrosis airway epithelial cells

Cystic fibrosis is a hereditary disease caused by a mutation in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene that encodes a chloride (Cl-) channel. Cystic fibrosis pulmonary pathophysiology is characterised by chronic inflammation and bacterial infections. Azithromycin, a macrolide antibiotic, has shown promising anti-inflammatory properties in some inflammatory pulmonary diseases. Moreover, all clinical studies have presented an improvement of the respiratory condition of cystic fibrosis patients, but the molecular and cellular mechanisms remain unknown. The aim of this study was to investigate, in bronchial epithelial cells, the effects of azithromycin on inflammatory pathways involved in cystic fibrosis. We have analysed the effects of azithromycin on cystic fibrosis and non-cystic fibrosis bronchial epithelial cell lines but also in non-immortalized non-cystic fibrosis human glandular cells. To create an inflammatory context, cells were treated with Tumor Necrosis Factor (TNF)-α or Interleukin (IL)1-β. Activation of the NF-κB pathway was investigated by luciferase assay, western blotting, and by Förster Resonance Energy Transfer imaging, allowing the detection of the interaction between the transcription factor and its inhibitor in live cells. In all conditions tested, azithromycin did not have an anti-inflammatory effect on the cystic fibrosis human bronchial epithelial cells and on CFTR-inhibited primary human bronchial glandular cells. More, our data showed no effect of azithromycin on IL-1β– or TNF-α–induced IL-8 secretion and NF-κB pathway activation. Taken together, these data show that azithromycin is unable to decrease in vitro inflammation in cystic fibrosis cells from airways.

Interaction and Localization of Synthetic Nanoparticles in Healthy and Cystic Fibrosis Airway Epithelial Cells: Effect of Ozone Exposure

Nanoparticles (NPs) produced by nanotechnology processes have taken the field of medicine by storm. Concerns about safety of these NPs in humans, however, have recently been raised. Although studies of NP toxicity have focused on lung disease the mechanistic link between NP exposure and lung injury remained unclear. This is primarily due to a lack of availability of appropriate airway disease models and sophisticated microscopic techniques to study nano-sized particulate delivery and resulting responses. Air-liquid interface (ALI) cultures of non-cystic fibrosis (CF) and CF airway epithelial cells were exposed to the FITC-labeled NPs using a PennCentury microsprayer™. Uptake of NPs was assessed by FACS. Laser scanning microscopy (LSM) was performed and the images were analyzed by an advanced imaging software to study particle deposition and uptake. Flow cytometry data revealed that CF cells accumulated increased amounts of NPs. The increased NP uptake could be attributed to the reduced CF transmembrane conductance regulator (CFTR) function as a similar increased retention/uptake was observed in cells whose CFTR expression was downregulated by antisense oligonucleotide. NPs alone did not induce pro-inflammatory cytokine release or cell death. The cell culture system was sensitive to ozone but exposure to the uncoated synthetic NPs used in this study, did not cause any synergistic or suppressive effects. LSM imaging and subsequent image restoration further indicated particle uptake and intracellular localization. Exposure to ozone increased nuclear uptake in both non-CF and CF cells. Our findings demonstrate the uptake of NPs using ALI cultures of non-CF and CF airway epithelial cells. The NPs used here were useful in demonstrating uptake by airway epithelial cells without causing adverse effects in presence or absence of ozone. However, to totally exclude toxic effects, chronic studies under in vivo conditions using coated particulates are required.

Personalized medicine for cystic fibrosis: the next generation.

The field of personalized medicine, based on genetic information, represents a tremendous but largely unfulfilled opportunity for a future generation of therapies [1]. While better clinical outcomes have been realized using predictive biomarkers in some forms of cancer [2], the influence of genetic information on the treatment of most other human diseases has been limited. However, in the case of the inherited genetic disease cystic fibrosis (CF), pre-emptive possibilities have recently been uncovered. In particular, an investigational drug known as VX-770, has demonstrated great promise in clinical trials in patients with a specific cystic fibrosis transmembrane conductance regulator (CFTR) allele selected for clinical investigation based on data from in vitro studies [3,4]. This article highlights some of the opportunities and challenges associated with developing novel genotype-directed therapies for CF.

Enhanced Ca2+entry due to Orai1 plasma membrane insertion increases IL‐8 secretion by cystic fibrosis airways

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, causes retention of CFTR in the endoplasmic reticulum (ER). Some CF abnormalities can be explained by altered Ca(2+) homeostasis, although it remains unknown how CFTR influences calcium signaling. This study examined the novel hypothesis that store-operated calcium entry (SOCE) through Orai1 is abnormal in CF. The significance of Orai1-mediated SOCE for increased interleukin-8 (IL-8) expression in CF was also investigated. CF and non-CF human airway epithelial cell line and primary cells (obtained at lung transplantation) were used in Ca(2+) imaging, electrophysiology, and fluorescence imaging experiments to explore differences in Orai1 function in CF vs. non-CF cells. Protein expression and localization was assessed by Western blots, cell surface biotinylation, ELISA, and image correlation spectroscopy (ICS). We show here that store-operated Ca(2+) entry (SOCE) is elevated in CF human airway epithelial cells (hAECs; ≈ 1.8- and ≈ 2.5-fold for total Ca(2+)(i) increase and Ca(2+) influx rate, respectively, and ≈ 2-fold increase in the I(CRAC) current) and is caused by increased exocytotic insertion (≈ 2-fold) of Orai1 channels into the plasma membrane, which is normalized by rescue of ΔF508-CFTR trafficking to the cell surface. Augmented SOCE in CF cells is a major factor leading to increased IL-8 secretion (≈ 2-fold). CFTR normally down-regulates the Orai1/stromal interaction molecule 1 (STIM1) complex, and loss of this inhibition due to the absence of CFTR at the plasma membrane helps to explain the potentiated inflammatory response in CF cells.

Vitamin D receptor agonists inhibit pro-inflammatory cytokine production from the respiratory epithelium in cystic fibrosis

Background 1,25-Dihydroxycholecalciferol (1,25(OH) 2D 3) has been shown to mitigate epithelial inflammatory responses after antigen exposure. Patients with cystic fibrosis (CF) are at particular risk for vitamin D deficiency. This may contribute to the exaggerated inflammatory response to pulmonary infection in CF. Methods CF respiratory epithelial cell lines were exposed to Pseudomonas aeruginosa lipopolysaccharide (LPS) and Pseudomonas conditioned medium (PCM) in the presence or absence of 1,25(OH) 2D 3 or a range of vitamin D receptor (VDR) agonists. Levels of IL-6 and IL-8 were measured in cell supernatants, and cellular total and phosphorylated IκBα were determined. Levels of human cathelicidin antimicrobial peptide (hCAP18) mRNA and protein were measured in cells after treatment with 1,25(OH) 2D 3. Results Pretreatment with 1,25(OH) 2D 3 was associated with significant reductions in IL-6 and IL-8 protein secretion after antigen exposure, a finding reproduced with a range of low calcaemic VDR agonists. 1,25(OH) 2D 3 treatment led to a decrease in IκBα phosphorylation and increased total cellular IκBα. Treatment with 1,25(OH) 2D 3 was associated with an increase in hCAP18/LL-37 mRNA and protein levels. Conclusions Both 1,25(OH) 2D 3 and other VDR agonists significantly reduce the pro-inflammatory response to antigen challenge in CF airway epithelial cells. VDR agonists have significant therapeutic potential in CF.

A Novel Role of Protein Tyrosine Kinase2 in Mediating Chloride Secretion in Human Airway Epithelial Cells

Ca2+ activated Cl− channels (CaCC) are up-regulated in cystic fibrosis (CF) airway surface epithelia. The presence and functional properties of CaCC make it a possible therapeutic target to compensate for the deficiency of Cl− secretion in CF epithelia. CaCC is activated by an increase in cytosolic Ca2+, which not only activates epithelial CaCCs, but also inhibits epithelial Na+ hyperabsorption, which may also be beneficial in CF. Our previous study has shown that spiperone, a known antipsychotic drug, activates CaCCs and stimulates Cl− secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro, and in Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) knockout mice in vivo. Spiperone activates CaCC not by acting in its well-known role as an antagonist of either 5-HT2 or D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Moreover, spiperone independently activates CFTR through a novel mechanism. Herein, we performed a mass spectrometry analysis and identified the signaling molecule that mediates the spiperone effect in activating chloride secretion through CaCC and CFTR. Proline-rich tyrosine kinase 2 (PYK2) is a non-receptor protein tyrosine kinase, which belongs to the focal adhesion kinase family. The inhibition of PYK2 notably reduced the ability of spiperone to increase intracellular Ca2+ and Cl− secretion. In conclusion, we have identified the tyrosine kinase, PYK2, as the modulator, which plays a crucial role in the activation of CaCC and CFTR by spiperone. The identification of this novel role of PYK2 reveals a new signaling pathway in human airway epithelial cells.

Reduced expression of Tis7/IFRD1 protein in murine and human cystic fibrosis airway epithelial cell models homozygous for the F508del-CFTR mutation

12-O-tetradecanoyl phorbol-13-acetate-induced sequence 7/interferon related development regulator 1 ( Tis7/ IFRD1) has been recently identified as a modifier gene in lung inflammatory disease severity in patients with cystic fibrosis (CF), based upon its capacity to regulate inflammatory activities in neutrophils. In CF patients, the F508del mutation in the Cftr gene encoding a chloride channel, the CF transmembrane conductance regulator (CFTR) in airway epithelial cells results in an exaggerated inflammatory response of these cells. At present, it is unknown whether the Tis7/ IFRD1 gene product is expressed in airway epithelial cells. We therefore investigated the possibility there is an intrinsic alteration in Tis7/IFRD1 protein level in cells lacking CFTR function in tracheal homogenates of F508del-CFTR mice and in a F508del-CFTR human bronchial epithelial cell line (CFBE41o − cells). When Tis7/IFRD1 protein was detectable, trachea from F508del-CFTR mice showed a reduction in the level of Tis7/IFRD1 protein compared to wild-type control littermates. A significant reduction of IFRD1 protein level was found in CFBE41o − cells compared to normal bronchial epithelial cells 16HBE14o −. Surprisingly, messenger RNA level of IFRD1 in CFBE41o − cells was found elevated. Treating CFBE41o − cells with the antioxidant glutathione rescued the IFRD1 protein level closer to control level and also reduced the pro-inflammatory cytokine IL-8 release. This work provides evidence for the first time of reduced level of IFRD1 protein in murine and human F508del-CFTR airway epithelial cell models, possibly mediated in response to oxidative stress which might contribute to the exaggerated inflammatory airway response observed in CF patients homozygous for the F508del mutation.

Down-regulation of Cytokine-induced Interleukin-8 Requires Inhibition of p38 Mitogen-activated Protein Kinase (MAPK) via MAPK Phosphatase 1-dependent and -independent Mechanisms

Down-regulation of overabundant interleukin (IL)-8 present in cystic fibrosis (CF) airways could ease excessive neutrophil burden and its deleterious consequences for the lung. IL-8 production in airway epithelial cells, stimulated with e.g. inflammatory cytokines IL-1β and tumor necrosis factor (TNF)-α, is regulated by several signaling pathways including nuclear factor (NF)-κB and p38 mitogen-activated protein kinase (MAPK). We previously demonstrated that the anti-inflammatory drugs dexamethasone and ibuprofen suppress NF-κB; however, only dexamethasone down-regulates cytokine-induced IL-8, highlighting the importance of non-NF-κB mechanisms. Here, we tested the hypothesis that down-regulation of cytokine-induced IL-8 requires modulation of the MAPK phosphatase (MKP)-1/p38 MAPK/mRNA stability pathway. The effects of dexamethasone (5 nm) and ibuprofen (480 μm) on this pathway and IL-8 were studied in CF (CFTE29o-, CFBE41o-) and non-CF (1HAEo-) airway epithelial cells. We observed that dexamethasone, but not ibuprofen, destabilizes IL-8 mRNA and up-regulates MKP-1 mRNA. Further, siRNA silencing of MKP-1, via p38 MAPK, leads to IL-8 overproduction and diminishes the anti-IL-8 potential of dexamethasone. However, MKP-1 overexpression does not significantly alter IL-8 production. By contrast, direct inhibition of p38 MAPK (inhibitor SB203580) efficiently suppresses IL-8 with potency comparable with dexamethasone. Similar to dexamethasone, SB203580 decreases IL-8 mRNA stability. Dexamethasone does not affect p38 MAPK activation, which excludes its effects upstream of p38 MAPK. In conclusion, normal levels of MKP-1 are necessary for a full anti-IL-8 potential of pharmacological agents; however, efficient pharmacological down-regulation of cytokine-induced IL-8 also requires direct effects on p38 MAPK and mRNA stability independently of MKP-1.

Leucine enhances aerosol performance of Naringin dry powder and its activity on cystic fibrosis airway epithelial cells

The effect of different amino acids (AAs) on the aerosol performance of N spray-dried powders was studied. Morphology, size distribution, density, dissolution rate were evaluated and correlated to process parameters. The aerosol performance was analyzed by both Single Stage Glass Impinger and Andersen Cascade Impactor. Results indicated that powders containing 5% (w/w) of leucine, proline or histidine and dried from 3:7 ethanol/water feeds showed very satisfying aerodynamic properties with fine particle fraction > 60%. Both neat N (raw and spray-dried) and N-leu1 dry-powder showing good aerodynamic properties were tested in cystic fibrosis (CF) and normal bronchial epithelial cells. Cell proliferation and expression levels of the key enzymes of the NF-κB and MAPK/ERK pathways, overactivated in CF cell lines, were evaluated. N-leu1 was able to significantly inhibit the expression levels of IKKα, IKKβ, as well as of the direct NF-κB inhibitor, IκBα. In addition N-Leu1 inhibited phosphorylation of ERK1/2 kinase and did not reduce cell proliferation as observed for the neat raw drug. Leucine co-spray-dried with the drug improved both aerodynamic properties and in vitro pharmacological activity of Naringin. The optimized N-Leu formulation as dry powder is potentially able to reduce hyperinflammatory status associated to CF.

Impaired expression of hypoxia-inducible factor-1α in cystic fibrosis airway epithelial cells – A role for HIF-1 in the pathophysiology of CF?

The continuous infection–inflammation cycle plays a crucial role in the progression of cystic fibrosis (CF) disease. This noxious loop can be aggravated by a reduced partial pressure of oxygen in the blood, hypoxemia, present in CF patients. These interconnected factors, hypoxia, inflammation and infection, by stabilizing the hypoxia-inducible factor-1α (HIF-1α) protein subunit, are able to activate the transcription factor HIF-1. To date, data investigating the potential role of HIF-1 in CF are scarce. Our results demonstrated that HIF-1α protein expression was altered in CF-affected compared to CFTR-corrected airway epithelial cells in unsimulated and simulated hypoxic conditions. In contrast, when CF-affected cells were infected with Pseudomonas aeruginosa, HIF-1α was more stabilized compared to CFTR-corrected cells. As HIF-1 is linked with an efficient immune response and pulmonary complications in cystic fibrosis, this difference in HIF-1α protein levels could have an impact in the CF pathology and the persistence of P. aeruginosa infection.

Innate Inflammatory Responses of Pediatric Cystic Fibrosis Airway Epithelial Cells

There is controversy regarding whether cystic fibrosis (CF) airway epithelial cells (AECs) are intrinsically proinflammatory. The objective of the current study was to characterize the inflammatory profiles of AECs from children with CF compared with cells from healthy control subjects. We obtained AECs from healthy children (12) and children with CF (27). Biochemical and functional characteristics were assessed by stimulating cells with IFNγ, LPS, a cocktail referred to as cytomix, which consists of IFNγ, IL-1β, TNF-α, and LPS, or with human rhinovirus (HRV). Cytokine production was assessed using ELISA. Apoptotic responses to HRV infection were measured via production of single-stranded DNA. Our results indicated that CF and healthy cells exhibited similar morphology in monolayer culture. CF cells constitutively produced greater amounts of IL-6, IL-1β, and prostaglandin E(2), but similar levels of IL-8 and soluble intracellular adhesion molecule-1 compared with healthy cells, and this profile was maintained through repeated passage. Stimulation with LPS or cytomix elicited similar levels of IL-8 in CF and non-CF cells. In contrast, exposure to HRV1b resulted in a marked increase in IL-8 production from CF compared with non-CF cells. CF cells also exhibited reduced apoptosis and increased viral replication compared with non-CF cells after exposure to HRV1b. We conclude that CF and healthy AECs have similar basal and stimulated expression of IL-8 in response to proinflammatory stimuli, but elevated IL-8 release in response to HRV infection. The elevated IL-8, together with dampened apoptotic responses by CF cells to HRV, could contribute to augmented airway inflammation in the setting of recurrent viral infections early in life.

Rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in cystic fibrosis airway epithelial cells

BackgroundIntermittent viral exacerbations in patients with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa (PA) infection are associated with increased bacterial load. A few clinical studies suggest that rhinoviruses (RV) are...

Extracellular Alkalinization Stimulates Calcium-activated Chloride Conductance in Cystic Fibrosis Human Airway Epithelial Cells

Background: The pH of the airway surface liquid (ASL) plays a pivotal role in maintaining the proper function of the respiratory epithelium. In patients with cystic fibrosis (CF) acidic ASL has been observed. Thus, alkalinization of ASL itself might be beneficial in CF. The aim of this study was to investigate the role of extracellular pH (pH_o) on the alternative Ca²⁺-activated Cl^- channels (CaCCs) in CF airway epithelial cells. Methods: The [Ca²⁺]_i and viability of CF airway epithelial cells (IB3-1) were assessed using Fluo-3/AM and YO-PRO-1 fluorescent dyes, respectively. Ion currents were detected in whole-cell configuration using the patch clamp technique. Results: Extracellular alkalinization (pH_o 8.2) stimulated Ca²⁺ entry and inward currents in low Na⁺ containing medium. The inward currents were blocked by the removal of extracellular Ca²⁺, chelating cytosolic Ca²⁺, as well as by the application of niflumic acid and DIDS. While Zn²⁺ promoted sustained Ca²⁺ entry in pH_o-dependent manner, it inhibited the anion conductance. The low external Na⁺ concentrations and alkaline pH_o were well tolerated by the cells. Conclusions: Stimulation of CaCCs could be achieved by alkalinization of the extracellular environment in CF airway epithelial cells. Zn²⁺ directly blocked, however indirectly enhanced the activity of Cl^- conductance.