A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: its usefulness in studying efficiency of gene transfer

Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.     

High lysosomal activities in cystic fibrosis tracheal gland cells corrected by adenovirus-mediated CFTR gene transfer

Human tracheal gland serous (HTGS) cells are now believed to be a major target of cystic fibrosis (CF) gene therapy. To evaluate the efficiency of adenovirus-mediated gene transfer in these cells we tested the adenovirus construction containing β-galactosidase cDNA. We observed that the endogenous β-galactosidase activity in cultured CF-HTGS cells was too strong to allow us to detect any exogenous β-galactosidase activity. Immunohistological study on sections of human tracheal tissue confirmed the presence of β-galactosidase in the serous component of the submucosal glands. We then looked for other lysosomal activities in normal and CF-HTGS cells. We showed that normal cells already have elevated enzyme values and that CF-HTGS cells contained 2–4-fold more β-galactosidase, α-fucosidase, α-mannosidase and β-glucuronidase activities than normal cells. An analysis of their kinetic constants has shown that this difference could be attributed to a lower K_m of CF lysosomal enzymes. More importantly, these differences are eliminated after adenovirus-mediated CFTR gene transfer and not after β-galactosidase gene transfer.     

Calcium homeostasis is abnormal in cystic fibrosis airway epithelial cells but is normalized after rescue of F508del-CFTR

Retention of F508del-CFTR proteins in the endoplasmic reticulum (ER) is dependent upon chaperone proteins, many of which require Ca(2+) for optimal activity. Here, we show in human tracheal gland CF-KM4 cells, that after correction of F508del-CFTR trafficking by miglustat (N-butyldeoxynojirimycin) or low temperature (27 degrees C), the Ca(2+) mobilization is decreased compared to uncorrected cells and becomes identical to the Ca(2+) response observed in non-CF MM39 cells. In CF-KM4 and human nasal epithelial CF15 cells, we also show that inhibiting vesicular trafficking by nocodazole prevents not only the rescue of F508del-CFTR but also the Ca(2+) mobilization decrease. Finally, experiments using the CFTR inhibitor CFTR(inh)-172 showed that the presence but not the channel activity of F508del-CFTR at the plasma membrane is required to decrease the Ca(2+) mobilization in corrected CF cells. These findings show that correction of the abnormal trafficking of F508del-CFTR proteins might have profound consequences on cellular homeostasis such as the control of intracellular Ca(2+) level.     

Gene transduction and cell entry pathway of fiber-modified adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells

Monolayers of cystic fibrosis transmembrane conductance regulator (CFTR)-deficient human tracheal glandular cells (CF-KM4) were subjected to phage biopanning, and cell-internalized phages were isolated and sequenced, in order to identify CF-KM4-specific peptide ligands that would confer upon adenovirus type 5 (Ad5) vector a novel cell target specificity and/or higher efficiency of gene delivery into airway cells of patients with cystic fibrosis (CF). Three different ligands, corresponding to prototypes of the most represented families of phagotopes recovered from intracellular phages, were designed and individually inserted into Ad5-green fluorescent protein (GFP) (AdGFP) vectors at the extremities of short fiber shafts (seven repeats [R7]) terminated by scissile knobs. Only one vector, carrying the decapeptide GHPRQMSHVY (abbreviated as QM10), showed an enhanced gene transduction of CF-KM4 cells compared to control nonliganded vector with fibers of the same length (AdGFP-R7-knob). The enhancement in gene transfer efficiency was not specific to CF-KM4 cells but was observed in other mammalian cell lines tested. The QM10-liganded vector was referred to as AdGFP-QM10-knob in its knobbed version and as AdGFP-QM10 in its proteolytically deknobbed version. AdGFP-QM10 was found to transduce cells with a higher efficiency than its knob-bearing version, AdGFP-QM10-knob. Consistent with this, competition experiments indicated that the presence of knob domains was not an absolute requirement for cell attachment of the QM10-liganded vector and that the knobless AdGFP-QM10 used alternative cell-binding domains on its capsid, including penton base capsomer, via a site(s) different from its RGD motifs. The QM10-mediated effect on gene transduction seemed to take place at the step of endocytosis in both quantitative and qualitative manners. Virions of AdGFP-QM10 were endocytosed in higher numbers than virions of the control vector and were directed to a compartment different from the early endosomes targeted by members of species C Ad. AdGFP-QM10 was found to accumulate in late endosomal and low-pH compartments, suggesting that QM10 acted as an endocytic ligand of the lysosomal pathway. These results validated the concept of detargeting and retargeting Ad vectors via our deknobbing system and redirecting Ad vectors to an alternative endocytic pathway via a peptide ligand inserted in the fiber shaft domain.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

CFTR Delivery to 25% of Surface Epithelial Cells Restores Normal Rates of Mucus Transport to Human Cystic Fibrosis Airway Epithelium

Dysfunction of CFTR in cystic fibrosis (CF) airway epithelium perturbs the normal regulation of ion transport, leading to a reduced volume of airway surface liquid (ASL), mucus dehydration, decreased mucus transport, and mucus plugging of the airways. CFTR is normally expressed in ciliated epithelial cells of the surface and submucosal gland ductal epithelium and submucosal gland acinar cells. Critical questions for the development of gene transfer strategies for CF airway disease are what airway regions require CFTR function and how many epithelial cells require CFTR expression to restore normal ASL volume regulation and mucus transport to CF airway epithelium? An in vitro model of human CF ciliated surface airway epithelium (CF HAE) was used to test whether a human parainfluenza virus (PIV) vector engineered to express CFTR (PIVCFTR) could deliver sufficient CFTR to CF HAE to restore mucus transport, thus correcting the CF phenotype. PIVCFTR delivered CFTR to >60% of airway surface epithelial cells and expressed CFTR protein in CF HAE approximately 100-fold over endogenous levels in non-CF HAE. This efficiency of CFTR delivery fully corrected the basic bioelectric defects of Cl⁻ and Na⁺ epithelial ion transport and restored ASL volume regulation and mucus transport to levels approaching those of non-CF HAE. To determine the numbers of CF HAE surface epithelial cells required to express CFTR for restoration of mucus transport to normal levels, different amounts of PIVCFTR were used to express CFTR in 3%–65% of the surface epithelial cells of CF HAE and correlated to increasing ASL volumes and mucus transport rates. These data demonstrate for the first time, to our knowledge, that restoration of normal mucus transport rates in CF HAE was achieved after CFTR delivery to 25% of surface epithelial cells. In vivo experimentation in appropriate models will be required to determine what level of mucus transport will afford clinical benefit to CF patients, but we predict that a future goal for corrective gene transfer to the CF human airways in vivo would attempt to target at least 25% of surface epithelial cells to achieve mucus transport rates comparable to those in non-CF airways.     

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.     

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.     

CFTR inhibition mimics the cystic fibrosis inflammatory profile

Primary airway epithelial cells grown in air-liquid interface differentiate into cultures that resemble native epithelium morphologically, express ion transport similar to those in vivo, and secrete cytokines in response to stimuli. Comparisons of cultures derived from normal and cystic fibrosis (CF) individuals are difficult to interpret due to genetic differences besides CFTR. The recently discovered CFTR inhibitor, CFTR(inh)-172, was used to create a CF model with its own control to test if loss of CFTR-Cl(-) conductance alone was sufficient to initiate the CF inflammatory response. Continuous inhibition of CFTR-Cl(-) conductance for 3-5 days resulted in significant increase in IL-8 secretion at basal (P = 0.006) and in response to 10(9) Pseudomonas (P = 0.0001), a fourfold decrease in Smad3 expression (P = 0.02), a threefold increase in RhoA expression, and increased NF-kappaB nuclear translocation upon TNF-alpha/IL-1beta stimulation (P < 0.000001). CFTR inhibition by CFTR(inh)-172 over this period does not increase epithelial sodium channel activity, so lack of Cl(-) conductance alone can mimic the inflammatory CF phenotype. CFTR(inh)-172 does not affect IL-8, IL-6, or granulocyte/macrophage colony-stimulating factor secretion in two CF phenotype immortalized cell lines: 9/HTEo(-) pCEP-R and 16HBE14o(-) AS, or IL-8 secretion in primary CF cells, and inhibitor withdrawal abolishes the increased response, so CFTR(inh)-172 effects on cytokines are not direct. Five-day treatment with CFTR(inh)-172 does not affect cells deleteriously as evidenced by lactate dehydrogenase, trypan blue, ciliary activity, electron micrograph histology, and inhibition reversibility. Our results support the hypothesis that lack of CFTR activity is responsible for the onset of the inflammatory cascade in the CF lung.     

Increased NaCl-induced interleukin-8 production by human bronchial epithelial cells is enhanced by the DeltaF508/W1282X mutation of the cystic fibrosis transmembrane conductance regulator gene

A satisfactory model describing the airway surface fluid (ASF) in the airways of persons with cystic fibrosis (CF) remains to be established due to theoretical challenges to both the "Hydration Hypothesis" and the "Salt Hypothesis." Irrespective of these models, inhaled hypertonic saline is often used to facilitate clearance of inspissated secretions. Hypertonicity induces interleukin-8 (IL-8) expression, a potent chemokine for neutrophils. The objectives of this study were: (i) to determine the relative contribution of three potential cis-regulatory elements in the regulation of NaCl-induced IL-8 production in BEAS-2B human bronchial epithelial cells, (ii) to compare NaCl-induced IL-8 expression in IB3-1 bronchial epithelial cells, which have the DeltaF508/W1282X mutation of the CF transmembrane conductance regulator (CFTR) gene, with that in C38 cells, which are IB3-1 cells stably transfected with a truncated but functional CFTR gene, and (iii) to compare equal osmolar concentrations of NaCl and D-sorbitol in the induction of IL-8 in all three cell types. In human bronchial epithelial cells, binding sites for NFkappaB, AP-1, and NF-IL6 in the 5'-flanking region of the IL-8 promoter are necessary for optimal NaCl induction of IL-8. Human bronchial epithelial cells with the DeltaF508/W1282X CFTR mutation produce an exaggerated amount of basal and NaCl-induced IL-8.     

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.     

Effects of cystic fibrosis transmembrane conductance regulator and DeltaF508CFTR on inflammatory response, ER stress, and Ca2+ of airway epithelia

We tested whether cystic fibrosis (CF) airway epithelia have larger innate immune responses than non-CF or cystic fibrosis transmembrane conductance regulator (CFTR)-corrected cells, perhaps resulting from ER stress due to retention of DeltaF508CFTR in the endoplasmic reticulum (ER) and activation of cytosolic Ca(2+) (Ca(i)) and nuclear factor (NF)-kappaB signaling. Adenovirus infections of a human CF (DeltaF508/DeltaF508) nasal cell line (CF15) provided isogenic comparisons of wild-type (wt) CFTR and DeltaF508CFTR. In the absence of bacteria, there were no or only small differences among CF15, CF15-lacZ (beta-galactosidase-expressing), CF15-wtCFTR (wtCFTR-corrected), and CF15-DeltaF508CFTR (to test ER retention of DeltaF508CFTR) cells in NF-kappaB activity, interleukin (IL)-8 secretion, Ca(i) responses, and ER stress. Non-CF and CF primary cultures of human bronchial epithelial cells (HBE) secreted IL-8 equivalently. Upon infection with Pseudomonas aeruginosa (PA) or flagellin (key activator for airway epithelia), CF15, CF15-lacZ, CF15-wtCFTR, and CF15DeltaF508CFTR cells exhibited equal PA binding, NF-kappaB activity, and IL-8 secretion; cells also responded similarly to flagellin when both CFTR (forskolin) and Ca(i) signaling (ATP) were activated. CF and non-CF HBE responded similarly to flagellin + ATP. Thapsigargin (Tg, releases ER Ca(2+)) increased flagellin-stimulated NF-kappaB and ER stress similarly in all cells. We conclude that ER stress, Ca(i), and NF-kappaB signaling and IL-8 secretion were unaffected by wt- or DeltaF508CFTR in control and during exposure to PA, flagellin, flagellin + ATP, or flagellin + ATP + forskolin. Tg, but not wt- or DeltaF508CFTR, triggered ER stress. Previous measurements showing hyperinflammatory responses in CF airway epithelia may have resulted from cell-specific, rather than CFTR- or DeltaF508CFTR-specific effects.     

Role of the cystic fibrosis transmembrane conductance regulator in internalization of Pseudomonas aeruginosa by polarized respiratory epithelial cells

Pseudomonas aeruginosa is an important human pathogen, producing lung infection in individuals with cystic fibrosis (CF), patients who are ventilated and those who are neutropenic. The respiratory epithelium provides the initial barrier to infection. Pseudomonas aeruginosa can enter epithelial cells, although the mechanism of entry and the role of intracellular organisms in its life cycle are unclear. We devised a model of infection of polarized human respiratory epithelial cells with P. aeruginosa and investigated the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in adherence, uptake and IL-8 production by human respiratory epithelial cells. We found that a number of P. aeruginosa strains could invade and replicate within cells derived from a patient with CF. Intracellular bacteria did not produce host cell cytotoxicity over a period of 24 h. When these cells were transfected with wild-type CFTR, uptake of bacteria was significantly reduced and release of IL-8 following infection enhanced. We propose that internalized P. aeruginosa may play an important role in the pathogenesis of infection and that, by allowing greater internalization into epithelial cells, mutant CFTR results in an increased susceptibility of bronchial infection with this microbe.