Proinflammatory cytokine secretion is suppressed by TMEM16A or CFTR channel activity in human cystic fibrosis bronchial epithelia

Cystic fibrosis (CF) is caused by the functional expression defect of the CF transmembrane conductance regulator (CFTR) chloride channel at the apical plasma membrane. Impaired bacterial clearance and hyperactive innate immune response are hallmarks of the CF lung disease, yet the existence of and mechanism accounting for the innate immune defect that occurs before infection remain controversial. Inducible expression of either CFTR or the calcium-activated chloride channel TMEM16A attenuated the proinflammatory cytokines interleukin-6 (IL-6), IL-8, and CXCL1/2 in two human respiratory epithelial models under air–liquid but not liquid–liquid interface culture. Expression of wild-type but not the inactive G551D-CFTR indicates that secretion of the chemoattractant IL-8 is inversely proportional to CFTR channel activity in cftr^{∆F508/∆F508} immortalized and primary human bronchial epithelia. Similarly, direct but not P2Y receptor–mediated activation of TMEM16A attenuates IL-8 secretion in respiratory epithelia. Thus augmented proinflammatory cytokine secretion caused by defective anion transport at the apical membrane may contribute to the excessive and persistent lung inflammation in CF and perhaps in other respiratory diseases associated with documented down-regulation of CFTR (e.g., chronic obstructive pulmonary disease). Direct pharmacological activation of TMEM16A offers a potential therapeutic strategy to reduce the inflammation of CF airway epithelia.     

Involvement of CFTR in uterine bicarbonate secretion and the fertilizing capacity of sperm

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed in a wide variety of epithelial cells, mutations of which are responsible for the hallmark defective chloride secretion observed in cystic fibrosis (CF). Although CFTR has been implicated in bicarbonate secretion, its ability to directly mediate bicarbonate secretion of any physiological significance has not been shown. We demonstrate here that endometrial epithelial cells possess a CFTR-mediated bicarbonate transport mechanism. Co-culture of sperm with endometrial cells treated with antisense oligonucleotide against CFTR, or with bicarbonate secretion-defective CF epithelial cells, resulted in lower sperm capacitation and egg-fertilizing ability. These results are consistent with a critical role of CFTR in controlling uterine bicarbonate secretion and the fertilizing capacity of sperm, providing a link between defective CFTR and lower female fertility in CF.     

Complement yourself: transcomplementation rescues partially folded mutant proteins

Cystic fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing ΔF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the F508 deletion mutant. Transcomplementation rescues the processing and channel activity of ΔF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.     

The expression of the mitochondrial gene MT-ND4 is downregulated in cystic fibrosis

Cystic fibrosis (CF) is a disease produced by mutations in the CFTR channel. We have previously reported that the CFTR chloride transport activity indirectly regulates the differential expression of several genes, including SRC and MUC1. Here we report that MT-ND4, a mitochondrial gene encoding a subunit of the mitochondrial Complex I (mtCx-I), is also a CFTR-dependent gene. A reduced expression of MT-ND4 was observed in CFDE cells (derived from a CF patient) when compared to CFDE cells ectopically expressing wild-type CFTR. The differential expression of MT-ND4 in CF was confirmed by RT-PCR. In situ hybridizations of deparaffinized human lung tissue slices derived from wt-CFTR or CF patients also showed downregulation of ND4 in CF. In addition, the CFTR chloride transport inhibitors glibenclamide and CFTR(inh)-172 also reduced MT-ND4 expression in CFDE cells ectopically expressing wt CFTR. These results suggest that the CFTR chloride transport activity indirectly up-regulates MT-ND4 expression.     

Estrogen and the cystic fibrosis gender gap

Cystic fibrosis (CF) is the most frequent inherited disease in Caucasian populations and is due to a defect in the expression or activity of a chloride channel encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Mutations in this gene affect organs with exocrine functions and the main cause of morbidity and mortality for CF patients is the lung pathology in which the defect in CFTR decreases chloride secretion, lowering the airway surface liquid height and increasing mucus viscosity. The compromised ASL dynamics leads to a favorable environment for bacterial proliferation and sustained inflammation resulting in epithelial lung tissue injury, fibrosis and remodeling. In CF, there exist a difference in lung pathology between men and women that is termed the “CF gender gap”. Recent studies have shown the prominent role of the most potent form of estrogen, 17β-estradiol in exacerbating lung function in CF females and here, we review the role of this hormone in the CF gender dichotomy.     

Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.     

Cystic fibrosis: a disease of vulnerability to airway surface dehydration

Cystic fibrosis (CF) lung disease involves chronic bacterial infection of retained airway secretions (mucus). Recent data suggest that CF lung disease pathogenesis reflects the vulnerability of airway surfaces to dehydration and collapse of mucus clearance. This predisposition is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, resulting in (i) the absence of CFTR-mediated Cl- secretion and regulation of epithelial Na+ channel (ENaC) function; and (ii) the sole dependence on extracellular ATP to rebalance these ion transport processes through P2 purinoceptor signaling. Recent clinical studies indicate that inhalation of hypertonic saline osmotically draws sufficient water onto CF airway surfaces to provide clinical benefit.     

Control of the proinflammatory state in cystic fibrosis lung epithelial cells by genes from the TNF-alphaR/NFkappaB pathway

BACKGROUND: Cystic fibrosis (CF) is the most common, lethal autosomal recessive disease affecting children in the United States and Europe. Extensive work is being performed to develop both gene and drug therapies. The principal mutation causing CF is in the CFTR gene ([Delta F508]CFTR). This mutation causes the mutant protein to traffic poorly to the plasma membrane, and degrades CFTR chloride channel activity. CPX, a candidate drug for CF, binds to mutant CFTR and corrects the trafficking deficit. CPX also activates mutant CFTR chloride channel activity. CF airways are phenotypically inundated by inflammatory signals, primarily contributed by sustained secretion of the proinflammatory cytokine interleukin 8 (IL-8) from mutant CFTR airway epithelial cells. IL-8 production is controlled by genes from the TNF-alphaR/NFkappaB pathway, and it is possible that the CF phenotype is due to dysfunction of genes from this pathway. In addition, because drug therapy with CPX and gene therapy with CFTR have the same common endpoint of raising the levels of CFTR, we have hypothesized that either approach should have a common genomic endpoint. MATERIALS AND METHODS: To test this hypothesis, we studied IL-8 secretion and global gene expression in IB-3 CF lung epithelial cells. The cells were treated by either gene therapy with wild-type CFTR, or by pharmacotherapy with the CFTR-surrogate drug CPX. CF cells, treated with either CFTR or CPX, were also exposed to Pseudomonas aeruginosa, a common chronic pathogen in CF patients. cDNA microarrays were used to assess global gene expression under the different conditions. A novel bioinformatic algorithm (GENESAVER) was developed to identify genes whose expression paralleled secretion of IL-8. RESULTS: We report here that IB3 CF cells secrete massive levels of IL-8. However, both gene therapy with CFTR and drug therapy with CPX substantially suppress IL-8 secretion. Nonetheless, both gene and drug therapy allow the CF cells to respond with physiologic secretion of IL-8 when the cells are exposed to P. aeruginosa. Thus, neither CFTR nor CPX acts as a nonspecific suppressor of IL-8 secretion from CF cells. Consistently, pharmacogenomic analysis indicates that CF cells treated with CPX greatly resemble CF cells treated with CFTR by gene therapy. Additionally, the same result obtains in the presence of P. aeruginosa. Classical hierarchical cluster analysis, based on similarity of global gene expression, also supports this conclusion. The GENESAVER algorithm, using the IL-8 secretion level as a physiologic variable, identifies a subset of genes from the TNF-alphaR/NFkappaB pathway that is expressed in phase with IL-8 secretion from CF epithelial cells. Certain other genes, previously known to be positively associated with CF, also fall into this category. Identified genes known to code for known inhibitors are expressed inversely, out of phase with IL-8 secretion. CONCLUSIONS: Wild-type CFTR and CPX both suppress proinflammatory IL-8 secretion from CF epithelial cells. The mechanism, as defined by pharmacogenomic analysis, involves identified genes from the TNF-alphaR/NFkappaB pathway. The close relationship between IL-8 secretion and genes from the TNF-alphaR/NFkappaB pathway suggests that molecular or pharmaceutical targeting of these novel genes may have strategic use in the development of new therapies for CF. From the perspective of global gene expression, both gene and drug therapy have similar genomic consequences. This is the first example showing equivalence of gene and drug therapy in CF, and suggests that a gene therapy-defined endpoint may prove to be a powerful paradigm for CF drug discovery. Finally, because the GENESAVER algorithm is capable of isolating disease-relevant genes in a hypothesis-driven manner without recourse to any a priori knowledge about the system, this new algorithm may also prove useful in applications to other genetic diseases.     

Cross-linking of ΔF508-CFTR promotes its trafficking to the plasma membrane

CFTR is a cAMP-activated chloride channel responsible for agonist stimulated chloride and fluid transport across epithelial surfaces.¹ Mutations in the CFTR gene lead to cystic fibrosis (CF) which affects the function of secretory organs like the intestine, the pancreas, the airways and the sweat glands. Most of the morbidity and mortality in CF has been linked to a decrease in airway function.² The ΔF508 mutation is the most common CF-related mutation in the Caucasian population and represents 90% of CF alleles. Homozygote carriers of this mutation present with a severe CF phenotype.³ The ΔF508 mutation causes misfolding of the nascent CFTR polypeptide, which leads to inefficient export from the endoplasmic reticulum (ER) and rapid degradation by the proteasome.⁴     

Neutrophil-Mediated Phagocytic Host Defense Defect in Myeloid Cftr-Inactivated Mice

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.     

Airway epithelial cell inflammatory signalling in cystic fibrosis

Cystic fibrosis (CF) is the most common lethal monogenic disorder in Caucasians, estimated to affect one out of 2500-4000 new-borns. In patients with CF, lack of CF transmembrane conductance regulator (CFTR) Cl(-) channel function leads to progressive pulmonary damage and ultimately to death. Severe and persistent polymorphonuclear neutrophil-dominated endobronchial inflammation and chronic bacterial infection are characteristic hallmarks of CF lung disease. Whether CFTR dysfunction results directly in an increased predisposition to infection and whether inflammation arises independent of infection remains to be established. The loss of functional CFTR in airway epithelial cells promotes depletion and increased oxidation of the airway surface liquid. Activated neutrophils present in airways produce large amounts of proteases and reactive oxygen species (ROS). Together these changes are associated with diminished mucociliary clearance of bacteria, activation of epithelial cell signalling through multiple pathways, and subsequent hyperinflammatory responses in CF airways. The NF-kappaB pathway and Ca(2+) mobilization in airway epithelial cells are believed to be of key importance for control of lung inflammation through regulated production of mediators such as interleukin-8 that participate in recruitment and activation of neutrophils, modulation of apoptosis, and control of epithelial barrier integrity. In this review, the current understanding of the molecular mechanisms by which airway epithelial cells contribute to abnormal lung inflammation in CF, as well as the anti-inflammatory strategies that can be proposed are discussed.     

Aspergillus fumigatus generates an enhanced Th2-biased immune response in mice with defective cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) lung disease is characterized by persistent airway inflammation and airway infection that ultimately leads to respiratory failure. Aspergillus sp. are present in the airways of 20-40% of CF patients and are of unclear clinical significance. In this study, we demonstrate that CF transmembrane conductance regulator (CFTR)-deficient (CFTR knockout, Cftr(tm1Unc-)TgN(fatty acid-binding protein)CFTR) and mutant (DeltaF508) mice develop profound lung inflammation in response to Aspergillus fumigatus hyphal Ag exposure. CFTR-deficient mice also develop an enhanced Th2 inflammatory response to A. fumigatus, characterized by elevated IL-4 in the lung and IgE and IgG1 in serum. In contrast, CFTR deficiency does not promote a Th1 immune response. Furthermore, we demonstrate that CD4+ T cells from naive CFTR-deficient mice produce higher levels of IL-4 in response to TCR ligation than wild-type CD4+ T cells. The Th2 bias of CD4+ T cells in the absence of functional CFTR correlates with elevated nuclear levels of NFAT. Thus, CFTR is important to maintain the Th1/Th2 balance in CD4+ T cells.     

Terminal glycosylation in cystic fibrosis

Cystic fibrosis (CF) is a common genetic disease for which the gene was identified within the last decade. Pulmonary disease predominates in this ultimately fatal disease and current therapy only slows the progression. CF transmembrane regulator (CFTR), the gene product, is an integral membrane glycoprotein that normally functions as a chloride channel in epithelial cells. The most common mutation, deltaF508, results in mislocalization and altered glycosylation of CFTR. Altered fucosylation and sialylation are hallmarks of both membrane and secreted glycoproteins in CF and the focus here is on these investigations. Oligosaccharides from CF membrane glycoproteins have the Lewis x, selectin ligand in terminal positions. In addition, two major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins, which recognize fucose in alpha1,3 linkage and asialoglycoconjugates. We speculate that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to the chronic infection and robust inflammatory response in the CF lung. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as therapy for CF.     

Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies

Cystic fibrosis (CF) is caused by a defect in the transmembrane conductance regulator (CFTR) protein that functions as a chloride channel. Dysfunction of the CFTR protein results in salty sweat, pancreatic insufficiency, intestinal obstruction, male infertility and severe pulmonary disease. In most patients with CF life expectancy is limited due to a progressive loss of functional lung tissue. Early in life a persistent neutrophylic inflammation can be demonstrated in the airways. The cause of this inflammation, the role of CFTR and the cause of lung morbidity by different CF-specific bacteria, mostly Pseudomonas aeruginosa, are not well understood. The lack of an appropriate animal model with multi-organ pathology having the characteristics of the human form of CF has hampered our understanding of the pathobiology and chronic lung infections of the disease for many years. This review summarizes the main characteristics of CF and focuses on several available animal models that have been frequently used in CF research. A better understanding of the chronic lung infection caused particularly by P. aeruginosa, the pathophysiology of lung inflammation and the pathogenesis of lung disease necessitates animal models to understand CF, and to develop and improve treatment.     

Cytokine pattern in cystic fibrosis patients during antibiotic therapy and gene therapy using adenoviral vector

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite improvements in treatment, pulmonary disease still remains the primary cause of death among these patients. In order to introduce a normal CFTR gene copy into airway epithelial cells, adenoviral vectors (AV) have been developed. AV are known to induce an inflammatory reaction that limits transgene expression, and can be potentially harmful. No human study has clearly monitored simultaneously, systemic and local inflammatory reaction, during AV administration. We report here the levels of C-reactive protein (CRP), interleukin (IL)-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 receptor antagonist (IL-1Ra) in plasma and bronchoalveolar lavage fluid (BALF) from six cystic fibrosis patients receiving AV encoding CFTR (AdCFTR). AdCFTR was administered to three cohorts of two patients into the nose on day 0, at doses ranging from 105 to 4 x 108 plaque-forming units (pfu), followed, on day 1, by aerosolization of 107 to 5.4 x 108 pfu. In order to ensure that patients were in the best clinical condition, and to further attenuate the broncho-pulmonary inflammation secondary to bacterial infection, they received antibiotic therapy, two weeks prior to AdCFTR administration, until 9 to 11 days after. We found that antibiotics markedly decreased CRP, TNF-alpha, IL-6, IL-1Ra levels in blood. In BALF, antibiotics slightly decreased TNF-alpha levels but had no effect on IL-8 and IL-1Ra, while IL-6 levels increased. AdCFTR administration did not induce any systemic or local cytokine release. In both blood and BALF, CRP, IL-8, IL-1Ra, TNF-alpha decreased, while IL-6 levels increased between day -7 and day 3. One patient presented an asymptomatic increase of all parameters in the BALF on day 7. Twenty one days later, he displayed a clinical deterioration suggestive of an exacerbation. In conclusion, this study demonstrates that antibiotic administration tends to attenuate systemic but not local broncho-pulmonary inflammation in CF patients. In the setting of our study, AdCFTR administration did not induce cytokine release. Further studies are necessary to investigate other inflammatory markers and the mechanisms involved during AV-mediated gene transfer for a better understanding of the immune reaction, which continues to hamper the development of gene therapy for CF patients.     

Spectrum of mutations in cystic fibrosis

Cystic fibrosis (CF) is a disorder characterized by elevated sweat electrolytes and thick mucous secretions due to abnormal chloride permeability in epithelial tissues. The gene responsible for this disease, the CF transmembrane conductance regulator (CFTR) was identified by a positional cloning approach 3 years ago. Since that time, over two hundred mutations have been found in CFTR genes from affected individuals. Analysis of these disease-associated mutations has provided new insight into the etiology of this disease and into the mechanisms of epithelial electrolyte secretion.