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Published ahead of print on February 7, 2008, doi:10.1165/rcmb.2007-0176OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 86-96, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0176OC

Transcription Factor Oligodeoxynucleotides to NF-{kappa}B Inhibit Transcription of IL-8 in Bronchial Cells

Valentino Bezzerri1,*, Monica Borgatti2,*, Elena Nicolis1, Ilaria Lampronti2, Maria Cristina Dechecchi1, Irene Mancini2, Paolo Rizzotti1, Roberto Gambari2,3 and Giulio Cabrini1

1 Laboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Haematology, University-Hospital, Verona, Italy; 2 ER-GenTech, Department of Biochemistry and Molecular Biology, 3 Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Ferrara, Italy

Correspondence and requests for reprints should be addressed to Roberto Gambari, PhD, Department of Biochemistry and Molecular Biology, Via L. Borsari 46, I-44100 Ferrara, Italy. E-mail: gam{at}unife.it or to Giulio Cabrini, MD, Laboratory of Molecular Pathology, Laboratory of Clinical Chemistry and Haematology, University Hospital of Verona, Piazzale Stefani 1, I-37126 Verona, Italy. Email:giulio.cabrini{at}azosp.vr.it


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Chronic pulmonary inflammation in patients affected by cystic fibrosis (CF) is characterized by massive bronchial infiltrates of neutrophils, which is sustained by the interaction of pathogens (e.g., Pseudomonas aeruginosa) with surface bronchial cells. To explore new treatment options focused on the reduction of neutrophil chemotaxis, we applied the transcription factor (TF) decoy approach, based on the intracellular delivery of double-stranded oligodeoxynucleotides (ODNs) causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. In CF bronchial IB3-1 cells, P. aeruginosa induced transcription of the neutrophil chemokines IL-8 and GRO-{gamma}, of the adhesion molecule intercellular adhesion molecule (ICAM)-1, and of the cytokines IL-1β and IL-6. Since consensus sequences for the TF, NF-{kappa}B, are contained in the promoters of all these genes, IB3-1, CuFi-1, Beas-2B, and CaLu-3 cells were transfected with double-stranded TF "decoy" ODNs mimicking different NF-{kappa}B consensus sequences. IL-8 NF-{kappa}B decoy ODN partially inhibited the P. aeruginosa–dependent transcription of IL-8, GRO-{gamma}, and IL-6, whereas decoy ODNs to both HIV-1 long terminal repeat and Igk produced a strong, 80 to 85% inhibition of transcription of IL-8, without reducing that of GRO-{gamma}, ICAM-1, IL-1β, and IL-6. In conclusion, intracellular delivery of "decoy" molecules aimed to compete with the TF, NF-{kappa}B, is a promising strategy to obtain inhibition of IL-8 gene transcription.

Key Words: inflammation • lung • chemokines • cytokines • gene regulation



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Decoy oligonucleotides targeted to transcription factors are a promising tool to intervene in modulating critical genes involved in the immune response in the lungs of patients with cystic fibrosis.

 
Cystic fibrosis (CF) is a genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes for a chloride channel expressed in several epithelia. Defective CFTR causes chronic pathology in the lungs, pancreas, liver, and reproductive system, with the airway tract disease being the most important cause of morbidity and mortality in CF. Leading hypotheses consider CF lung disease to be a result of reduced periciliary fluid and increased viscosity of submucosal gland secretions, which reduce the mucociliary clearance of entrapped pathogens (1, 2). The hallmarks in CF airway pathology are a chronic infection sustained by strikingly few pathogens, the most common isolate being Pseudomonas aeruginosa, and a neutrophil (PMN)-dominated chronic inflammation (3). Whether P. aeruginosa and bacterial pathogens activate the innate immune response or just aggravate an exaggerated underlying inflammatory background characteristic of CF airway epithelia has not been completely elucidated (4, 5).

Elevated concentrations of pro-inflammatory cytokines and chemokines have been found in the bronchoalveolar fluid of patients with CF (6–8). Consistent with these findings, experimental models support the concept that interaction of P. aeruginosa with CF respiratory epithelial cells promotes PMN recruitment, mainly by releasing the chemokine interleukin 8 (IL-8) (9). Within the lumen of CF airways, nucleic acids derived from PMNs further reduce mucociliary clearance, and proteases released from PMNs mediate epithelium injury, progressive bronchial wall damage, bronchiectasis, and peribronchiolar fibrosis. Hence, the exaggerated, PMN-dominated inflammatory process is thought to critically contribute to the gradual decline in CF lung function (10). To circumvent this process, clinical trials testing anti-inflammatory molecules, such as corticosteroids and ibuprofen, as well as new leukotriene B4 (LTB4) receptor antagonists, have been performed in patients with CF (11, 12). Enthusiasm has been diminished either by the limited efficacy or the occurrence of undesired effects, thus suggesting the need to widen the investigation to both alternative therapeutic targets and novel anti-inflammatory molecules (10, 12).

At the basis of the PMN-dominated lung inflammation in CF, P. aeruginosa is known to stimulate surface respiratory epithelial cells to express and release IL-8 through activation of the transcription factor NF-{kappa}B (13, 14). Besides IL-8, a large number of other genes contain {kappa}B sites in their 5'-upstream untranslated regions, including genes encoding cytokines, chemokines, and adhesion molecules that orchestrate PMN transbronchial migration, such as TNF-{alpha}, IL-1, GRO-{gamma}, and intercellular adhesion molecule (ICAM)-1, together with different other genes regulating innate immunity (15). Thus, NF-{kappa}B has been proposed as one target to down-modulate the immune processes involved in different pulmonary diseases, including CF (16).

The transcription factor (TF) decoy approach has been proposed to modulate gene expression in vitro. This approach is based on the intracellular delivery of double-stranded oligodeoxynucleotides causing inhibition of the binding of TF-related proteins to the different consensus sequences in the promoter of specific genes. Several decoy oligodeoxynucleotides have been studied to pursue the modulation of expression of genes relevant to human diseases (17, 18). Importantly, novel peptide nucleic acid (PNA)-DNA chimeras have been developed to improve delivery and stability into different living tissues and move toward their pharmaceutical application in vivo (19, 20). Due to its central role in immune response and cell cycle regulation, TF decoy oligodeoxynucleotides targeted to NF-{kappa}B have been studied in other and our laboratories (17, 18, 21–23). Regarding IL-8, limited success has been previously obtained by testing NF-{kappa}B decoy oligodeoxynucleotides to reduce IL-1β– and TNF-{alpha}–stimulated IL-8 expression in monocytes and respiratory epithelial cells (24, 25). Moreover, the application of the same oligodeoxynucleotide in murine lungs was completely inefficient in inhibiting bleomycin-induced release of IL-6 (26). Thus, to date, the inhibition of IL-8 expression by NF-{kappa}B decoy molecules has been limited both conceptually, by the potential lack of specificity related to the wide role of NF-{kappa}B in regulating expression of several genes, and practically, by the limited efficiency obtained in the preliminary experiments (24–26). Here we addressed the issues of efficiency and specificity by testing the effect of novel NF-{kappa}B decoy sequences on the expression of different genes induced by P. aeruginosa in human bronchial epithelial cells carrying either the wild type or the mutant CFTR gene.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Cultures
IB3-1 cells were obtained from LGC Promochem (Teddington, UK). Cells were grown in LHC-8 basal medium (Biofluids, Rockville, MD), supplemented with 5% FBS in the absence of gentamycin. All culture flasks and plates were coated with a solution containing 35 µg/ml bovine collagen (Becton-Dickinson, Franklin Lakes, NY), 1 µg/ml bovine serum albumin (Sigma, St. Louis, MO), and 1 µg/ml human fibronectin (Becton-Dickinson) as described (27). CuFi-1 cells, a generous gift of A. Klingelhutz, P. Karp, and J. Zabner (University of Iowa, Iowa City, IA), were derived from human bronchial epithelium from a patient with CF (CFTR {Delta}F-508/{Delta}F-508 mutant genotype) and had been transformed by reverse transcriptase component of telomerase, hTERT, and human Papillomavirus type 16 (HPV-16), E6, and E7 genes (28). This cell line was grown on human placental collagen type IV (Sigma)-coated flasks in bronchial epithelial growth medium (BEGM) (Cambrex BioScience, Walkersville, MD) medium, as previously described (28). Beas-2B cells, a kind gift of Curtis C. Harris (NCI, NIH, Bethesda, MD) was grown in BEGM (Cambrex BioScience). All culture flasks and plates were coated with human placental collagen type IV (Sigma). Calu-3 cells, obtained from a human lung adenocarcinoma and derived from serous cells of proximal bronchial airways (29), were cultured in culture flasks in Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose, supplemented with 10% FBS in a humidified atmosphere of 5% CO2 in air. For the experiments, Calu-3 cells were seeded onto cell culture inserts (pore size of 0.4 µm) for Falcon 24-well multitrays (BD Biosciences, Franklin Lakes, NJ) at a density of 70 x 103 cells/insert. Cells reached confluence after 12 to 15 days of culture. Transepithelial electrical resistance was measured with an epithelial voltometer (EVOM; World Precision Instruments, Sarasota, FL). The cell inserts were used for experiments when the resistance exceeded 2,000 {Omega}/cm2.

Decoy Oligodeoxynucleotides
Decoy oligodeoxynucleotides (ODNs) mimicking the NF-{kappa}B consensus sequences identified in the promoter of different genes were used: HIV-1 long terminal repeat (LTR) NF-{kappa}B decoy ODN, from the LTR of the promoter region of HIV-1 gene (sense: 5'-CGC TGG GGA CTT TCC ACG G-3'); Igk NF-{kappa}B decoy ODN, from the enhancer region in the promoter of the kappa light chain of immunoglobulin gene (sense: 5'-GGG GAT TCC CCT-3'); IL-8 NF-{kappa}B decoy ODN, from the NF-{kappa}B consensus sequence identified in the promoter of IL-8 gene (sense: 5'-AAT CGT GGA ATT TCC TCT-3'); and ICAM-1 site A/B/C NF-{kappa}B decoy ODNs, from the three consensus sequences identified in the promoter region of ICAM-1 gene (site A, –642 to –633, sense 5'-CGC CGG GAG GTG CCT GGC-3'; site B, –565 to –556, sense: 5'-GGG AGG GGC ATC CCT CAG-3'; site C, –278 to –269, sense: 5'-GCC GGG AGC AGC CCC CGG-3'). A "scrambled" ODN sequence (sense: 5'-CAC AAA GTG TAA CAG TCT-3') was used in each experiment. Synthesis and high-performance liquid chromatography–grade purification was obtained from Sigma. Sense and anti-sense freeze-dried ODNs were resuspended in DNAse-free sterile water at the stock concentration 5 µg/µl. Annealing to obtain double-stranded ODNs was performed by mixing equimolar concentrations of both forward and reverse strands (1 µg/µl in 150 mM NaCl, final concentration), incubating the solution at 100°C for 5 minutes, then leaving it overnight at room temperature.

Transfection of IB3-1 Cells with Decoy ODNs
IB3-1 cells have been transfected with ODNs by mixing the double-stranded ODNs with the cationic liposome Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Lipofectamine 2000 (4 µl) was diluted in 1 ml LHC-8 serum-free cell culture medium (Biofluids), according to the manufacturer's instructions. Double-stranded decoy or scrambled ODNs (2 µg) were added and incubated for 10 minutes. Liposome:DNA complexes in LHC-8 serum-free medium, or in BEGM (500 µl), were added to IB3-1cells or CuFi-1 and Beas-2B cells, respectively. Liposome:DNA complexes in serum-free DMEM were added to CaLu-3 cells grown in cell culture inserts (200 µl to apical, 700 µl to basolateral sides, respectively) and incubated at 37°C/5% CO2 for 6 hours. All cell lines were washed twice with culture serum-free medium and left at 37°C/5% CO2 for a further 18 hours before infection with P. aeruginosa.

Cell Infection with P. aeruginosa
PAO1, a well-characterized, nonmucoid motile laboratory strain of P. aeruginosa, kindly donated by Alice Prince (Columbia University, New York, New York), was grown in trypticase soy broth (TSB) or agar (TSA) (Difco, Detroit, MI) (30). Bacteria colonies from overnight cultures on TSA plates were grown with shaking in 20 ml TSB broth at 37°C until an optical density (at 660 nm) corresponding to 1 x 109 colony-forming units (CFU)/ml was reached, as determined by dilution plating. Bacteria were washed twice in PBS and finally diluted in each specific serum-free culture medium before infection. All cell lines were infected with ranging doses of PAO1 at 37°C for 4 hours.

Preparation of Nuclear Extracts from IB3-1 Cells
Nuclear extracts were prepared from IB3-1 cells as described (31). Cell were washed twice with PBS and detached by trypsinization. Nuclear proteins were obtained by hypotonic lysis, followed by high-salt extraction treatment of nuclei (31). Protein concentration was determined using Bio-Rad protein assay. Nuclear extracts were brought to a concentration of 0.5 µg/µl for electrophoretic mobility shift assay (EMSA) experiments.

Electrophoretic Mobility Shift Assay
EMSA was perfomed as previously described (21). Briefly, double-stranded synthetic oligodeoxynucleotides mimicking the NF-{kappa}B–binding site present in the promoter of the IL-8 gene (IL-8 NF-{kappa}B, sense: 5'-AATCGTGGAATTTCCTCT-3') were used. Oligodeoxynucleotides were labeled with {gamma}32-P-ATP using 10 Units of T4-polinucleotide-kinase (MBI Fermentas, St. Leon-Rot, Germany) in 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, 50 mM DTT, 1 mM spermidine, 1 mM EDTA in the presence of 50 µCi {gamma}32-P-ATP) in a volume of 20 µl for 45 minutes at 37°C. Reaction was brought to 150 mM NaCl, and 150 ng complementary oligodeoxynucleotide was added. Reaction temperature was increased to 100°C for 5 minutes and left diminishing to room temperature overnight. Nuclear extracts from IB3-1 cells or purified NF-{kappa}B p50 dimer protein (Promega, Madison, WI) were used at the specified concentrations and poly(dI:dC) (1 µg per reaction) was also added to abolish nonspecific binding. After 30 minutes of binding at room temperature, the samples were run at constant voltage (200 V) under low ionic strength conditions (0.25x TBE buffer: 22 mM Tris-borate, 0.4 mM EDTA) on 6% polyacrylamide gels. Gels were dried and subjected to standard autoradiographic procedures.

Quantitation of Transcripts of Inflammatory Genes
Total RNA from IB3-1 cells was isolated using High Pure RNA Isolation Kit (Roche, Mannheim, Germany). Total RNA (2.5 µg) was reverse-transcribed to cDNA using the High Capacity cDNA Archive Kit and random primers (Applied Biosystems, Foster City, CA) in a 100-µl reaction. The cDNA (2 µl) was then amplified for 50 PCR cycles using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) in an ABI Prism 5700 sequence detection system (Applied Biosystems). The real-time PCR reactions were performed in duplicates for both target and normalizer genes. Primer sequences and concentration are shown in Table 1. Primer sets were purchased from Sigma-Genosys (The Woodlands, TX). Results were collected with Sequence Detection Software (version 1.3; Applied Biosystems). Relative quantification of gene expression was performed using the comparative threshold (CT) method as described by the manufacturer (Applied Biosystems User Bulletin 2). Changes in mRNA expression level were calculated after normalization to calibrator gene. The ratios obtained after normalization are expressed as -fold change over untreated samples.


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  		TABLE 1. PRIMERS FOR QUANTIFICATION OF GENE TRANSCRIPTS

 

   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Induction of Pro-Inflammatory Genes by P. aeruginosa in IB3-1 Cells
Respiratory epithelial cells paving the conductive airways are known to play a key role as early sensors of the presence of pathogens by expressing a series of soluble molecules aimed at recruiting and activating immune cells. P. aeruginosa, a critical pathogen in CF chronic lung pathology, is known to induce a pro-inflammatory response in respiratory epithelial cells, mainly through interaction with Toll-like receptor (TLR)5, TLR2, and asialo-GM1 receptors (32, 33). Therefore, we exposed the bronchial epithelial cell line IB3-1, which expresses the {Delta}F508/W1282X-mutated CFTR protein, to the P. aeruginosa laboratory strain PAO1 for 4 hours, and total RNA was extracted to quantify transcripts. As shown in Figure 1A, PAO1 induced a very strong up-modulation of (1) IL-8 and GRO-{gamma}, which recruit polymorphonuclear neutrophils (PMNs); (2) of ICAM-1, which cooperates as an adhesion molecule in driving the migration of leukocytes through both endothelial and epithelial monolayers; and (3) of the pro-inflammatory cytokines IL-1β and IL-6 in IB3-1 cells, These data confirm the role of bronchial epithelial cells in the early induction of pro-inflammatory stimuli upon exposure to P. aeruginosa.


Figure 1
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  		Figure 1. Effects of P. aeruginosa–dependent cytokine transcription on IB3-1 cells and transcription factors consensus sequences. (A) Induction of cytokine transcription. IB3-1 cells (100,000/cm2) were seeded, serum starved for 18 hours, and incubated with P. aeruginosa laboratory strain PAO1 (100 colony-forming units [CFU]/cell) for 4 hours and cytokine transcript levels measured as described in MATERIALS AND METHODS. (B) Schematic representation of the position of the consensus sequences of the major transcription factors identified in the 5'-untranslated regions of IL-8, GRO{gamma}, ICAM-1, IL-1β and IL-6 genes (35, 44–47). Transcription factor consensus sequences have been completed by the free-access TF-search software (http://www.cbrc.jp/research/db/TFSEARCH.html). The positions of the TATA box and of the consensus sequences identified have been reported relatively to the translation start sites of each gene.

 
Search for Transcription Factors Signals within the Promoters of IL-8, GRO-{gamma}, ICAM-1, IL-1β, and IL-6
Binding of P. aeruginosa to TLR5, TLR2, and asialoGM1 receptors on bronchial epithelial cells is known to induce expression of genes of the innate immunity by activating different TFs, such as NF-{kappa}B, AP-1, Elk-1, and NF–IL-6 (14, 33, 34). To propose a TF decoy approach to down-modulate gene expression, the analysis of the promoter sequences of the target genes is very important. To this end, we evaluated the presence of TF signals within the genes modulated by PAO1 treatment of IB3 cells. The analysis reported in Figure 1B describes the presence of several sequences that are putative targets of transcription factors. Among these sequences, NF-{kappa}B–binding sites are present in all the promoters analyzed. Since NF-{kappa}B plays a well-known pivotal role in inflammatory processes, we choose it as the first molecular target for the TF decoy approach.

Effect of NF-{kappa}B Decoy ODNs in Nuclear Extracts
Due to its role in recruiting PMNs in the bronchial wall and lumen of the patients, down-modulation of IL-8 is considered a key target to reduce the damage of the CF airway tract. Thus the CF bronchial IB3-1 cells were transfected with TF decoy ODNs that have been previously designed to mimic the DNA-binding site of NF-{kappa}B and tested in their capacity to interfere with the NF-{kappa}B–dependent transcription (24). A double-stranded ODN homologous to the NF-{kappa}B–binding site present in the LTR of the immunodeficiency type I virus (HIV-1 LTR NF-{kappa}B decoy ODN) has been previously demonstrated by our research group to efficiently interact with NF-{kappa}B p52 transcription factor (21). Second, a decoy ODN mimicking the NF-{kappa}B sequence contained in the promoter of the gene encoding the immunoglobulin light kappa chain (Igk NF-{kappa}B decoy ODN) has been employed (35). Third, a new decoy ODN homologous to the NF-{kappa}B consensus sequence identified in the promoter of the IL-8 gene (IL-8 NF-{kappa}B decoy ODN) was designed and prepared for this purpose. We exposed our model system of cells to the P. aeruginosa laboratory strain PAO1 and studied the activation of NF-{kappa}B by EMSA. As shown in Figure 2A, P. aeruginosa increases NF-{kappa}B TF, as expected from previously reported data. To test the ability of the decoy ODNs to compete for the binding of NF-{kappa}B TF with the sequence contained in the promoter of IL-8, all the decoy ODNs were incubated with nuclear extracts from IB3-1 cells in the presence of a radiolabeled probe 100% homologous with the IL-8 NF-{kappa}B sequence, and EMSA was performed. Complete inhibition of interaction of the 32P-labeled IL-8 NF-{kappa}B probe with NF-{kappa}B proteins present in the nuclear extracts (NF-{kappa}B:DNA complex) was obtained with very low concentrations of the IL-8 NF-{kappa}B cold probe and both HIV-1 LTR and IgK NF-{kappa}B ODNs (5 and 10 ng), as shown in Figure 2B. To further validate the specificity of the competition, the same ODNs were used in the binding experiments of the 32P-labeled IL-8 NF-{kappa}B probe with purified TF NF-{kappa}B subunits. Pre-incubation of the HIV-1 LTR or IgK NF-{kappa}B ODNs with purified NF-{kappa}B p50 dimer completely abolish the binding to the 32P-labeled IL-8 NF-{kappa}B probe, as reported in Figure 2C. Interestingly, the competition of the HIV-1 LTR or IgK NF-{kappa}B ODNs was even more effective than that obtained with the IL-8 NF-{kappa}B decoy ODN, with a core sequence 100% homologous to the consensus sequence contained in the promoter of IL-8, that shows the presence of a residual, albeit very small, p50:DNA complex (Figure 2C). Altogether, these EMSAs provide proof of principle of the competition of these NF-{kappa}B decoy ODNs for the DNA consensus sequence contained in the promoter of the IL-8 gene.


Figure 2
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  		Figure 2. Effect of decoy ODNs on molecular interactions between nuclear proteins and the 32P-labeled IL-8 NF-{kappa}B DNA target molecule. (A) IB3-1 cells (2 x 106 cells/Petri dish) were infected with P. aeruginosa laboratory strain PAO1 (20 cfu/cell) or PAO1 suspension medium (not infected) for 2 hours. Nuclear extracts (from 0.5 to 4 µg) were incubated for 20 minutes in the presence of 32P-labeled IL-8 NF-{kappa}B 18-mer probe. Protein:DNA complexes were separated by polyacrilamide gel electrophoresis. The arrow indicates the protein:DNA complexes. The asterisk indicates the free 32P-labeled 18-mer probe; (–) indicates 32P-labeled 18-mer probe without nuclear extracts. (B) Nuclear extracts from IB3-1 cells (3 µg) were incubated for 20 minutes in the absence (–) or presence of the indicated amounts (5 or 10 ng) of the HIV-1 LTR NF-{kappa}B, Igk NF-{kappa}B, or IL-8 NF-{kappa}B cold probe DNA/DNA molecules. After this incubation period, a further 20-minute incubation step was performed in the presence of 32P-labeled IL-8 NF-{kappa}B 18-mer probe. (C) Purified NF-{kappa}B p50 dimer protein (10 ng) was incubated for 20 minutes in the absence (–) or presence of 100 ng of the HIV-1 LTR NF-{kappa}B, Igk NF-{kappa}B, or IL-8 NF-{kappa}B cold probe DNA/DNA molecules.

 
Effect of NF-{kappa}B Decoy ODNs in Intact Cells
To test decoy ODNs in intact cells, IB3-1 cells were transfected with HIV-1 LTR NF-{kappa}B, Igk NF-{kappa}B, and IL-8 NF-{kappa}B decoy ODNs complexed with cationic liposomes. The efficiency of intracellular uptake was tested by incubating the three fluorescein-labeled NF-{kappa}B decoy ODNs with IB3-1 cells and analyzing the fluorescent signal by fluorescence microscopy. All the three NF-{kappa}B decoy ODNs showed similar intracellular uptake efficiency, as reported in Figure 3, indicating uptake of the decoy ODNs in a population of the cells in culture. To test the effect of ODNs on gene transcription, complexes of cationic liposomes with NF-{kappa}B or scrambled ODNs were pre-incubated with IB3-1 cells 24 hours before exposure to the PAO1 laboratory strain of P. aeruginosa for a further 4 hours. Both HIV-1 LTR NF-{kappa}B and Igk NF-{kappa}B decoy ODNs strongly inhibited transcription of IL-8 mRNA, whereas, as reported in the dose–response experiment shown in Figure 4A, the IL-8 NF-{kappa}B decoy ODN resulted in a less potent effect with respect to bacteria-treated cells, either when compared with untreated or scrambled ODN-treated IB3-1 cells. In view of the strong inhibition observed for both HIV-1 LTR NF-{kappa}B and Igk NF-{kappa}B decoy ODNs, we tested whether one of the two decoy ODNs could be more effective at lower doses in IB3-1 cells. The dose–response experiments reported in Figures 4B and 4C suggest that the HIV-1 LTR NF-{kappa}B exhibits its maximal inhibitory effect at a lower concentration with respect to the Igk NF-{kappa}B decoy ODN. Since DNA-binding sequences for the TF NF-{kappa}B have been reported also in the promoters of the other four genes that we found induced by P. aeruginosa in IB3-1 cells, as described in Figure 1, the question arose if whether, in addition to IL-8, the NF-{kappa}B decoy ODNs inhibit the transcription of other pro-inflammatory genes induced by PAO1. HIV-1 LTR NF-{kappa}B decoy ODN did not show inhibitory effects on the P. aeruginosa–dependent transcription of GRO-{gamma}, ICAM-1, and IL-1β and only minor and not significant effects were observed of the expression of IL-6 gene, as shown in Figure 5A. To understand whether the specific effect of HIV-1 LTR NF-{kappa}B decoy ODN on IL-8 transcription is restricted to the IB3-1 cell line, we extended the analysis to the CuFi-1 CF bronchial epithelial cell line carrying the {Delta}F508 mutation on both the CF gene alleles. As reported in Figure 5B, the same specific effect of HIV-1 LTR NF-{kappa}B decoy ODN on IL-8 transcription was observed also in CuFi-1 cells infected with P. aeruginosa. The newly designed IL-8 NF-{kappa}B decoy ODN was also tested on the transcription of the different genes induced by P. aeruginosa. As shown in Figure 5C, IL-8 NF-{kappa}B decoy ODN inhibits IL-8 transcription induced by P. aeruginosa, although with lower efficiency than that observed with HIV-1 LTR NF-{kappa}B, together with a partial inhibition of P. aeruginosa–dependent transcription of GRO-{gamma} and IL-6. So far, the main message from these results indicates that newly designed decoy ODNs directed against the transcription factor NF-{kappa}B can inhibit P. aeruginosa–dependent transcription of IL-8. To test possible differential effects of the decoy ODNs between wild-type and CF mutated bronchial cell lines, the analysis was extended to other bronchial cell lines. As reported in Figure 6A, all three NF-{kappa}B decoy ODNs inhibited very effectively P. aeruginosa–dependent transcription of IL-8 mRNA on CuFi cells. Interestingly, significant, albeit less strong, inhibition was observed in the Beas-2B bronchial epithelial cells carrying the wild-type CF gene (Figure 6B). Finally, the HIV-1 LTR NF-{kappa}B decoy ODN significantly inhibited IL-8 mRNA transcription also in the bronchial serous submucosal gland–derived wild-type cell line CaLu-3 grown polarized on collagen-coated inserts and exposed to P. aeruginosa from the apical side, as reported in Figure 6C. Altogether, the results shown in Figures 4 to 6GoGo suggest that the inhibitory effect of these NF-{kappa}B decoy ODNs is not restricted to CFTR-mutated bronchial cells, although the overall potency seems higher with respect to that observed in cells carrying the wild-type CF gene. Since different NF-{kappa}B decoy ODNs have been previously tested in respiratory cells stimulated exposed to the pro-inflammatory cytokine TNF-{alpha}, obtaining a much lower degree of inhibition (25), we performed further experiments, exposing IB3-1 cells to either TNF-{alpha} and IL-1β, with the aim to gain preliminary information on whether the difference was more likely related to the ODN sequences or the pro-inflammatory challenge used. Both pro-inflammatory stimuli induced transcription of the same pattern of genes induced by P. aeruginosa, as shown in Figures 7A and 7B. However, when cells were challenged with both stimuli and P. aeruginosa, the most effective HIV-1 LTR NF-{kappa}B decoy ODN inhibited significantly IL-8 mRNA induced by PAO1 and IL-1β but had a much more limited effect on TNF-{alpha}–dependent transcription of this chemokine, as reported in Figure 7C.


Figure 3
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  		Figure 3. Uptake of decoy ODNs in IB3-1 cells. IB3-1 cells were seeded and cultured in 8-wells chamber slides for 2 days in LHC-8 medium. Fluorescein-labeled decoy ODNs were mixed with Lipofectamine 2000 and diluted with medium as described in MATERIALS AND METHODS. Liposome:DNA complexes (2 µl:1 µg in 500 µl volume) were added to IB3-1 cells. Fluorescence was collected at excitation and emission wavelengths of 490 nm and 520 to 560 nm, respectively, with a Nikon TMD inverted fluorescence microscope. Representative fluorescence field at 4 hours after incubation: (A) IB3-1 cells exposed to Lipofectamine/fluorescein-labeled HIV-1 LTR NF-{kappa}B decoy ODN complex; (B) IB3-1 cells exposed to Lipofectamine/fluorescein-labeled Ig{kappa} NF-{kappa}B decoy ODN complex; (C) IB3-1 cells exposed to Lipofectamine/fluorescein-labeled IL-8 NF-{kappa}B decoy ODN complex; (D) IB3-1 cells exposed to Lipofectamine alone. Right side shows the bright-field of each panel.

 

Figure 4
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  		Figure 4. Effect of NF-{kappa}B decoy ODNs on P. aeruginosa–dependent induction of IL-8 mRNA in IB3–1 cells. (A) Cells were pre-incubated for 24 hours with HIV-1 LTR, Igk NF-{kappa}B, IL-8 NF-{kappa}B decoys, scrambled ODNs, or medium alone before infection with P. aeruginosa, PAO1 strain. Total RNA was extracted 4 hours after infection and IL-8 mRNA was quantified as described in MATERIALS AND METHODS. Effect of (B) HIV-1 LTR NF-{kappa}B decoy ODN and (C) Ig{kappa} NF-{kappa}B decoy ODN on IL-8 transcription after infection with PAO1 (100 CFU/cell) for 4 hours. Data are mean ± SEM and are representative of three separate experiments.

 

Figure 5
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  		Figure 5. Effect of NF-{kappa}B decoy ODNs on induction of different cytokines. HIV-1 LTR NF-{kappa}B decoy ODN was tested on transcription of ICAM-1, IL-8, GRO{gamma}, IL-1β, and IL-6 genes in (A) IB3-1 and (B) CuFi-1 bronchial cells derived from patients with CF after infection with P. aeruginosa. (C) Effect of IL-8 NF-{kappa}B ODN decoy on cytokine transcripts induced by infection with P. aeruginosa in IB3-1 cells. Decoy or scrambled ODNs were pre-incubated with IB3-1 or CuFi-1 cells for 24 hours before infection with PAO1 (150 cfu/cell) for 4 hours. Total RNA was extracted and processed for quantification of transcripts as described in MATERIALS AND METHODS. Values are mean ± SEM of four separate experiments. Significance in Student's t test between each scrambled and decoy ODNs: *P < 0.05 or **P < 0.01.

 

Figure 6
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  		Figure 6. Effect of NF-{kappa}B decoy ODNs on P. aeruginosa–dependent induction of IL-8 mRNA in different bronchial cell lines. (A) CuFi-1 cells were pre-incubated for 24 hours with ODNs before infection with PAO1 (100 CFU/cell). (B) Beas-2B cells were pre-incubated with ODNs for 24 hours before infection with PAO1 (100 CFU/cell). (C) Polarized CaLu-3 cells were pre-incubated with decoy or scrambled ODNs or medium alone for 24 hours before infection with PAO1 (100 CFU/cell). Total RNA was extracted and processed for quantitation of transcripts as described in MATERIALS AND METHODS. Values are mean ± SEM of three separate experiments. Significance in Student's t test between each scrambled and decoy ODNs: *P < 0.05, **P < 0.01, or ***P < 0.001.

 

Figure 7
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  		Figure 7. Effect of different pro-inflammatory stimuli on transcription of IL-8 mRNA. Induction of cytokine transcription by (A) TNF-{alpha} (50 ng/ml) and (B) IL-1β (10 ng/ml) in IB3-1 cells. Cells were seeded, serum starved for 18 hours, and incubated with the different stimuli for 4 hours. (C) Effect of HIV-1 LTR NF-{kappa}B decoy ODN on IL-8 transcription in IB3-1 cells treated with different stimuli. Decoy or scrambled ODNs were pre-incubated with IB3-1 cells for 24 hours before stimulation for 4 hours by IL-1β (50 ng/ml), TNF-{alpha} (10 ng/ml), or infection with PAO1 (100 CFU/cell) for 4 hours. Total RNA was extracted and processed for quantitation of transcripts as described in MATERIALS AND METHODS. Values are mean ± SEM of three separate experiments. Significance in Student's t test between each scrambled and decoy ODNs: *P < 0.05 or **P < 0.01. (D) Effect of ICAM-1 NF-{kappa}B decoy ODNs on ICAM-1 transcription. NF-{kappa}B decoy ODNs, designed on the three consensus sequences identified in the promoter of ICAM-1 gene reported in Figures 1 and 8, were pre-incubated separately or together in IB3-1 cells 24 hours before infection with PAO1 (100 CFU/cell). ICAM-1 mRNA was quantified as described. Values are mean ± SEM of four separate experiments. The difference between each decoy and the scrambled ODN was not significant as measured by Student's t test.

 
Effect of Decoy ODNs Designed on NF-{kappa}B Sites of ICAM-1 Gene
The partial or absent inhibitory effect of HIV-1 LTR and IL-8 NF-{kappa}B decoy ODNs that we observed in P. aeruginosa–dependent transcription of ICAM-1, GRO-{gamma}, IL-1β, and IL-6 genes could be explained in different ways. The most straightforward hypothesis takes into consideration the presence of different degrees of homology between the sequences of the decoy ODN and the NF-{kappa}B–binding sites in the different promoters. As reported in Table 2, the sequences of the seven NF-{kappa}B sites in the promoters of these genes are not completely homologous to one another. As reported in Figure 8, 8 out of 10 nucleotides of the HIV-1 LTR NF-{kappa}B decoy ODN are homologous with the site contained in the IL-8 promoter, whereas the degree of homology in the sites of other promoters is lower than in the IL-8 one. In particular, three different NF-{kappa}B sites have been identified in the promoter of ICAM-1, each with low sequence homology for the HIV-1 LTR NF-{kappa}B decoy ODN (7/10, 7/10, and 5/10 nucleotides, respectively), as reported in Figure 8. To test this hypothesis, three decoy ODNs 100% homologous with the NF-{kappa}B–binding sites identified in the promoter of ICAM-1 gene (35) were designed and tested. The three decoy ODNs have been transfected separately or together in IB3-1 cells and their effect on P. aeruginosa–dependent ICAM-1 induction was checked. As shown in Figure 7D, neither the separate nor the combined pre-incubation with the ICAM-1 NF-{kappa}B decoy ODNs produced inhibition of PAO1-dependent up-modulation of the ICAM-1 transcript, suggesting that other, so far unidentified, TFs could be more relevant than NF-{kappa}B in transcription of the ICAM-1 gene and could be studied as a molecular target for an affective decoy approach to shut down the P. aeruginosa–dependent transcription of this gene.


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  		TABLE 2. CONSENSUS SEQUENCES FOR THE TRANSCRIPTION FACTOR NF-{kappa}B

 

Figure 8
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  		Figure 8. Sequence homologies between LTR HIV-1 NF-{kappa}B decoy ODN (central nucleotides) and the promoter regions of IL-8, GRO-{gamma}, ICAM-1, IL-1β, and IL-6. The sequence of the LTR HIV1 NF-{kappa}B decoy ODN is reported above that of NF-{kappa}B identified in the promoter regions of the genes of interest. Sequence homologies have been indicated by short vertical bars between the decoy ODN (upper sequence) and each gene of interest (lower sequence).

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
The transcription factor decoy approach has been, in the past few years, applied to modulate gene expression both in vitro (19–26) and in vivo (17, 18), suggesting that this approach has potential value in nonviral gene therapy (41). In this study, we applied a TF decoy strategy aimed at down-modulating the expression of pro-inflammatory genes in wild-type and CF bronchial epithelial cells infected with P. aeruginosa. Transfection of bronchial cells with decoy ODNs mimicking the NF-{kappa}B sequences contained in the HIV-1 LTR, Igk chain, or IL-8 genes clearly inhibited P. aeruginosa–dependent expression of IL-8. Second, no inhibitory effect was observed on other genes containing NF-{kappa}B sites in their promoters, such as ICAM-1 and IL-1β. Third, minor differences in the sequence of the decoy ODNs, such as between the HIV LTR and IL-8 NF-{kappa}B decoy ODNs, may produce partial inhibition in the transcription of other genes, such as GRO-{gamma} and IL-6.

A TF decoy strategy to down-modulate IL-8 expression was first proposed by Cooper and colleagues in human monocytes stimulated with IL-1β (24). In that experimental model, transfection of a 10-nucleotide decoy ODN 100% homologous to the core sequence of NF-{kappa}B identified in IL-8 promoter produced an average 60% inhibition of the IL-1β–dependent expression of IL-8 gene. Application of TF decoy ODNs has been tried by Griesenbach and coworkers with a different 20-nucleotide NF-{kappa}B decoy ODN in CF tracheal epithelial cells stimulated with TNF-{alpha}, as they obtained only an average 40% inhibition of IL-8 secretion (25). The same decoy ODN was unable to inhibit IL-6 secretion after bleomycin-induced pneumonia in murine lungs (26). How can we reconcile these previous results with the highly efficient inhibition of IL-8 expression we report in the present work? Different decoy ODN sequences have been used in our work with respect to all the previous studies, which could, by itself, explain the discrepancies. In addition, Cooper and colleagues (24) used decoy ODNs without flanking regions, which could be more easily degraded by nucleases cleaving at the 5' or 3' ends of the ODNs, thus reducing their effectiveness in competing for active NF-{kappa}B. Also, the application of different pro-inflammatory stimuli could have impact on the efficiency of the NF-{kappa}B decoy ODNs, a stimulus that we chose considering this pathogen particularly relevant to study anti-inflammatory approaches for CF lung pathology. From this point of view, we report here that the HIV-1 LTR NF-{kappa}B decoy ODN has a lower potency of inhibition when IB3-1 cells have been stimulated with TNF-{alpha} instead of IL-1β and P. aeruginosa (Figure 7C). This is in agreement with the weak inhibition that Griesenbach and coworkers (25) observed after using the same stimulus. This can be partly explained considering that TNF-{alpha} activates a panel of receptors, adapters, and kinases (36), which is quite different than those induced by P. aeruginosa, thus suggesting that transcription factors other than NF-{kappa}B could have a predominant role. Thus differences in the pro-inflammatory stimuli used could have an important role besides that played by the choice of the sequences of the decoy ODNs.

NF-{kappa}B proteins comprise a family of transcription factors involved in the control of a large number of genes related to cellular growth, developmental processes, apoptosis, and immune and inflammatory responses (for synopsis see http://www.NF-{kappa}B.org). As far as our bronchial epithelial model system is concerned, we found that P. aeruginosa induces transcription of IL-8, GRO{gamma}, ICAM-1 IL-1β, and IL-6 genes, all of them containing NF-{kappa}B consensus sequences in their promoters, as summarized also in Figure 1. How can we possibly explain that the HIV-1 LTR NF-{kappa}B decoy ODN strongly inhibits the P. aeruginosa–dependent transcription of IL-8, but not that of the other four genes induced in parallel both in IB3-1 and CuFi-1 cells? We initially considered that the consensus sequences for NF-{kappa}B are not 100% homologous in the genes regulated by this transcription factor. Many nucleotide variations could be present, as summarized in the general NF-{kappa}B consensus usually reported (5'-GGG[A/G]NN[C/T][C/T]CC-3'), which in principle accounts for the possibility of 27 different variants. In our specific case, none of the seven NF-{kappa}B consensus sequences identified in the promoters of the five genes that we found induced by P. aeruginosa are perfectly homologous with at least one of the others (see Table 2). Consequently, different degrees of homology between the HIV-1 LTR NF-{kappa}B decoy ODN and the consensus sequences in the promoters can be observed (see Figure 8). Thus we hypothesized that an important parameter modulating the efficacy the NF-{kappa}B decoy ODNs in inhibiting transcription of IL-8, GRO{gamma}, ICAM-1, IL-1β, and IL-6 should be the degree of homology between decoy ODN and consensus sequences. For instance, the three consensus sequences identified within the promoter of ICAM-1 gene present the highest number of mismatches with the HIV-1 LTR NF-{kappa}B decoy ODN that prompted us to test the effect of new decoy ODNs designed with 100% homology. Disappointingly, none of the three ICAM-1 NF-{kappa}B decoy ODNs inhibited P. aeruginosa–dependent ICAM-1 transcription, even after transfecting the three decoy ODNs together (see Figure 7D). Although this unsuccessful attempt could be related to different parameters (e.g., length of the ODN, choice of the 5'- and 3'-flanking nucleotides, secondary structure, etc.), overall these negative results seem to basically disprove the hypothesis. Second, it should be noted that the HIV-1 LTR NF-{kappa}B decoy ODN is even more homologous to the consensus sequences reported in the GRO-{gamma} and IL-6 than in the IL-8 promoter (see Figure 8), whereas we proved the decoy ODN being effective in inhibiting transcription of the latter but not of the former genes (see Figure 5). Third, the IL-8 NF-{kappa}B decoy ODN, which is much more homologous than the HIV-1 LTR NF-{kappa}B decoy ODN with the consensus sequence in the promoter of IL-8, is less efficient in inhibiting IL-8 expression than the HIV-1 LTR NF-{kappa}B decoy ODN and inhibits partially also the transcription of GRO-{gamma} and IL-6. Therefore it becomes apparent that the extent of homology between decoy ODN and consensus sequences is important but not sufficient to explain all the differences in effectiveness of each NF-{kappa}B decoy ODN in inhibiting the transcription of the five genes that we observed induced by P. aeruginosa in bronchial epithelial cells. Extensive analyses of a large series of ODNs mutated in their core consensus and/or flanking regions need future investigation to thoroughly answer these questions.

A further explanation of the different efficiency of NF-{kappa}B decoy ODNs could be related to the complex interplay of transcription factors in the expression of the different genes of interest in the present study. Surface structures of P. aeruginosa, like flagellum and pili, interact with different receptors expressed on respiratory epithelial cells (e.g., TLR5, TLR4, and Asialo GM-1 receptor), leading eventually to activation of transcription factors such as NF-{kappa}B, AP-1, Elk-1, and NF–IL-6, as already reported (14, 33, 34). Interestingly, all the five genes we found induced by P. aeruginosa contain in their promoters one or more consensus sequences for NF-{kappa}B and some of them also for the transcription factors AP-1, NF–IL-6, and Sp-1 (see Figure 1). Therefore, we hypothesize that the competition of the decoy ODN for the binding of NF-{kappa}B to its consensus sequence is not sufficient to inhibit drastically GRO{gamma}, ICAM-1, IL-1β, and IL-6 transcription also because of the hierarchical roles of AP-1, NF–IL-6, and Sp-1 in respect to NF-{kappa}B for the transcription of some of those genes.

As far as the mechanism of action of the decoy ODNs is concerned, we can infer various suggestions. The degree of homology between specific decoy ODNs and the consensus sequences for the corresponding TF is important but not sufficient to predict the efficiency in inhibiting gene transcription. Efficiency of each decoy ODN could depend on the secondary structure and the kind of flanking regions of the ODNs, and on the hierarchy of the different TFs in regulating expression of specific genes. Thus, from a theoretical point of view, our results strengthen the utility of the TF decoy strategy to provide useful insights on the issue of regulation of gene transcription. Conversely, as a practical consequence useful to applications in human diseases, decoy ODNs appear to present the advantage of a much higher gene specificity than previously expected. For instance, one may have anticipated a broad inhibitory effect of our NF-{kappa}B decoy ODNs on all the five genes containing NF-{kappa}B consensus sequences, whereas experimental results showed that only IL-8 is strongly inhibited with selected decoy ODNs.

Considering the therapeutic applications of TF decoy ODNs to lung disease of patients with CF and/or to other human lung diseases in which the innate immunity is involved, we propose here a hint for inhibiting master genes in CF pulmonary pathology. This means that novel anti-inflammatory approaches focused on specific target genes relevant to specific diseases, without having too broad and undesired effects on the innate immune response, could be seriously taken into consideration. On the opposite side, knowing the redundancy of this response, a disease-oriented intervention should be tailored, focusing on the inhibition of the relevant pro-inflammatory genes with complementary TF decoy molecules. Different molecular approaches can be investigated to obtain a novel anti-inflammatory intervention. Strictly single-gene–related inhibitory approaches could be devised considering the application of anti-sense and Silencing inhibitory RNA (SiRNA) nucleotides. However, due to the redundancy of the inflammatory response to P. aeruginosa observed in CF lungs, inhibition of one chemokine (e.g., IL-8) could be bypassed by other potent chemoattractants (e.g., Gro-{gamma}). A much broader inhibitory approach such as the modulation of the receptors for pathogens (e.g., TLRs [37]) would present the disadvantage of reducing the wide array of genes induced by pathogen-associated molecular patterns that are critical to maintain the proper anti-infective defenses against pathogens. In terms of receptor blockade, those for chemokines could also be a theoretically interesting target (38). However, since chemokine receptors are expressed on circulating immune cells, the systemic delivery of antagonist or competing molecules could raise safety concerns. All this considered, TF decoy ODNs directed toward NF-{kappa}B regulating pro-inflammatory genes transcribed in CF respiratory epithelial cells exposed to P. aeruginosa could present the advantage of extending or reducing the range of target genes by changing the ODN sequence, as observed also in the results presented here. In the perspective of application in the respiratory tract, NF-{kappa}B decoy ODNs conjugated with novel lipoplexes have been successfully utilized in murine lungs, obtaining effective inhibition of endotoxic shock (39). However, we are perfectly aware that the delivery of decoy ODNs in the conductive airways still requires extensive research and development, in particular to ensure sufficient stability in respect to degradation in the biological fluids. Recent progress obtained in other and our laboratories with chimeric TF decoy molecules that are significantly more permeable to the plasma membrane and resistant to the nucleases contained in serum and other biological fluids (21, 23, 40–43) are rendering closer the possibility of extending the TF decoy strategy to in vivo pre-clinical experiments.


   Acknowledgments
 
The authors are grateful to Alice Prince for donating the P.aeruginosa strain PAO1, to Curtis C. Harris (NCI, NIH, Bethesda, MD) for donating BEAS-2B cells, to A. Klingelhutz, P. Karp and J. Zabner (University of Iowa, Iowa city, IA) for donating the CuFi-1 cell line, to Stephan J. Reshkin for helpful discussion and critical revising of the manuscript, to Anna Tamanini for helpful discussions, to Maria Grazia Giri for help in statistical analysis, to Baroukh M. Assael for support, and to Federica Quiri for excellent technical assistance.


   Footnotes
 
This work was supported by grants from the Italian Cystic Fibrosis Research Foundation (grants # 15/2004 and # 13/2007 to R.G. and G.C.) and Fondazione Cariverona–Bando 2005—Malattie rare e della povertà (to G.C.). R.G. received grants from Fondazione Cariparo and MUR COFIN-2005. V.B. is a fellow of the "Fondazione Cariverona." E.N. is a fellow of the "Azienda Ospedaliera di Verona."

* These authors contributed equally to this work. Back

Originally Published in Press as DOI: 10.1165/rcmb.2007-0176OC on February 7, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form May 17, 2007

Accepted in final form November 27, 2007


   References
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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