|Home | About | Journals | Submit | Contact Us | Français|
One of the clinical features of cystic fibrosis (CF) is a deep inflammatory process, which is characterized by production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against CF to reduce the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. In order to demonstrate that TFD against NF-kappaB interferes with the NF-kappaB pathway we proved, by chromatin immunoprecipitation (ChIP) that treatment with TFD oligodeoxyribonucleotides of cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa leads to a decrease occupancy of the Il-8 gene promoter by NF-kappaB factors. In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. In this respect PNA-DNA-PNA (PDP) chimeras are molecules of great interest from several points of view: (1) they can be complexed with liposomes and microspheres; (2) they are resistant to DNases, serum and cytoplasmic extracts; (3) they are potent decoy molecules. By using electrophoretic mobility shift assay and RT-PCR analysis we have demonstrated that (1) the effects of PDP/PDP NF-kappaB decoy chimera on accumulation of pro-inflammatory mRNAs in P.aeruginosa infected IB3–1 cells reproduce that of decoy oligonucleotides; in particular (2) the PDP/PDP chimera is a strong inhibitor of IL-8 gene expression; (3) the effect of PDP/PDP chimeras, unlike those of ODN-based decoys, are observed even in the absence of protection with lipofectamine. These informations are of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy of cystic fibrosis.
Cystic fibrosis (CF) is a severe inherited disease caused by mutations of a gene encoding a chloride channel termed Cystic fibrosis transmembrane conductance regulator (CFTR).1 Although most of the CF patients are affected by multiple organ pathologies, lung disease is the major cause of morbidity and mortality in CF. In the lung of CF patients a hyper-inflammatory condition is established, which is characterized by predominant infiltrates of polymorphonuclear neutrophils (PMNs) in bronchial lumina and increased expression of pro-inflammatory cytokines and chemokines, in particular Interleukin-8 (IL-8).2-8 Recently, it has been established that excessive IL-8 release from lung epithelial cells may also play a role in bacterial proliferation and adhesion.9 Accordingly, IL-8 is presently considered a critical pharmacological target to reduce the excessive inflammation in CF lungs.10 We recently described an analysis of the transcription machinery of the IL-8 gene in human bronchial epithelial cells, and found NF-kappaB pathway very important.11,12 These results are in line with several reports indicating lung inflammation in CF related to an increased NF-kappaB signaling causing the induction of IL-8 gene expression.13
One of the possible strategies to inhibit IL-8 gene transcription is the so called “transcription factor decoy” (TFD) approach,14-17 based on oligodeoxynucleotides (ODNs) mimicking consensus sequences identified within the proximal promoter region of the IL-8 gene. In order to verify whether the TFD against NF-kappaB interferes with the NF-kappaB pathway we employed in this study chromatin immunoprecipitation (ChIP) on cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa and treated with TFD ODNs against NF-kappaB factors.
In consideration of the fact that an important drawback of the decoy approach designed for the modulation of gene expression is the presence of intracellular and extracellular DNases,18 therefore, large amounts of DNA must be loaded into target cells in order to obtain biological responses leading to alteration of gene expression. On the other hand, modified oligonucleotides (either methylphosphonate or phosphorothioate) have the advantage in that they are resistant to DNase cleavage; unfortunately, these molecules are highly toxic.18
In this respect, in recent reports we presented the possible use of peptide nucleic acids (PNAs)19-21 as alternative reagents in experiments aimed at the control of gene expression involving the TFD approach. In PNAs, the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units.19 PNAs hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices21 and are resistant to both nucleases and proteases.22 PNAs were found to be excellent candidates for antisense and antigéne therapies.23-26 Among PNA-based molecules, we found that PNA-DNA chimeras are of great potential in gene therapy being active as decoy molecules against the NF-kappaB and Sp1 transcription factors.23,27-29
Following these considerations, we determined the effect of PNA-DNA-PNA (PDP) chimeras mimicking the binding sites of NF-kappaB on the transcription of IL-8 gene in cystic fibrosis IB3–1 cells infected by Pseudomonas aeruginosa. We have described this system in several reports and reviews, pointing out that after Pseudomonas aeruginosa exposure, IB3–1 are induced to increase accumulation of IL-8 mRNA in respect to basal levels of uninfected cells. In addition to IL-8 mRNA, other sequences induced by PAO1 are GRO-γ, GRO-α, IL-6, IL-1β, ICAM-1.11,12 Therefore, IB3–1 cells infected by P.aeruginosa are an excellent system to verify (1) whether decoy molecules against NF-kappaB inhibit IL-8 gene transcription and (2) whether this effect is restricted to the IL-8 gene, or affects other genes containing NF-kappaB binding sites, such as GRO-γ, IL-6, IL-1β and ICAM-1.
Figure 1 shows the experimental strategy followed in our study. Complexes of cationic liposomes with NF-kappaB ODNs or PDP/PDP chimeras have been pre-incubated with cystic fibrosis IB3–1 cells 24 h before exposure to the PAO1 laboratory strain of P.aeruginosa (100 CFU/cell) for a further four hour time period. A scrambled ODN was used as negative control (Table 1). After the treatment RNA was extracted and real-time quantitative RT-PCR performed; at the same time chromatin was purified from IB3–1 cells for chromatin immunoprecipitation assay (ChIP). In the first set of experiments, described in Figure 2, we provided evidences that the TF decoy approach interfere(s) with the NF-kappaB activity in vitro and in the IB3–1 cellular system. In order to verify the ability of this decoy ODNs to compete for the binding of NF-kappaB to the sequences contained in the promoter of IL-8 gene, the NF-kappaB decoy ODNs was incubated with nuclear extracts from IB3–1 cells in the presence of a radiolabeled probe 100% homologous with the NF-kappaB binding sequences and performed electrophoretic mobility shift assays (EMSA). Complete inhibition of interaction of the 32P-labeled probes with specific transcription factor proteins (NF-kappaB/DNA complexes) has been obtained, providing the proof of principle of the competition of this NF-kappaB decoy ODNs for the DNA consensus sequence contained in the promoter of the IL-8 gene. On the contrary, other ODN containing the binding sites for other transcription factors are inactive. ODN for CREB, NF-IL6, AP-1 and CHOP were selected because these transcription factors play a crucial role in the control of transcription of the IL-8 gene.11
The results reported in Figure 3A show that treatment with 0.5 μg/ml of ODN NF-kappaB decoy is sufficient to obtain decreased expression of the IL-8 gene. However also the other signals from transcription factors present within the IL-8 gene promoter (Fig. 1) might play an important transcriptional role; this is supported by the observation that decoys for CREB, AP-1 and CHOP-1 also induce to some extent inhibition of IL-8 mRNA content in parallel experiments (data not shown). In any case, some decoy oligonucleotides are not active in inhibiting P. aeruginosa induced IL-8 gene increased expression, as outlined in Figure 3B, showing that treatment of PAO1 infected IB3–1 CF cells with NF-IL6 decoy does not lead to significant reduction of IL-8 mRNA. In order to clarify the effects of decoy molecules on the NF-kappaB intracellular dynamics, we studied by chromatin immunoprecipitation (ChIP) the possible effects of the treatment with decoy ODNs on the recruitment of NF-kappaB to the IL-8 gene promoters. Chromatin was isolated from uninfected IB3–1 cells, from cells infected with PAO1, and from cells infected with PAO1 and treated with 2 μg/ml of ODN NF-kappaB decoy. After immunoprecipitation (Fig. 1B), amplification of the IL-8 gene promoter segment containing the NF-kappaB binding sites was performed; as a negative control a parallel PCR was also performed using primers amplifying an upstream sequence of the IL-8 gene (located at - 5 Kb from the transcription start site) lacking NF-kappaB consensus elements. Figure 3B shows one important feature of our experimental cell systems, i.e., that NF-kappaB is recruited with high efficiency to the IL-8 gene promoter following infection with PAO1; second, the data clearly indicate that the NF-kappaB decoy ODN interferes with this recruitment. No changes in occupancy by NF-kappaB were detected when ChIP-DNA was amplified using PCR primers specific for the upstream sequence of the IL-8 gene lacking NF-kappaB consensus elements.
While it is firmly established that the entry of oligonucleotides into eukaryotic cells can occur through a nucleic acid channel,30 the complexation with lipofectamine (or other delivery systems) is required in vitro, since the degradation of ODNs exposed to fetal calf serum is an important drawback of these transfection approaches.31,32 On the contrary, we have elsewhere reported that, unlike ODN-based decoys, PDP/PDP chimeras are fully resistant to serum and cytoplasmic extracts.18,28 This information is of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy. Therefore, we compared the effects of ODN-based and PDP-based NF-kappaB decoys on P. aeruginosa infected cells in the presence or in the absence of the lipofectamine transfection reagents. In the first set of experiments we confirmed that the NF-kappB decoy PNA-DNA-PNA chimera (see Fig. 4A for molecular structure) reproduces the activity of the NF-kappaB decoy ODN on P.aeruginosa induced genes. In fact, the NF-kappaB PDP/PDP chimera exhibits differential effects on expression of PAO1 activated genes (Fig. 4B), such as ICAM-1, GRO-γ, IL-1β, IL-6, IL-8. The major inhibitory effect was found on IL-8 gene expression, confirming on one hand elsewhere reported studies on the role of NF-kappaB for IL-8 gene transcription and, on the other hand, that decoy for NF-kB might retain differential effects of genes regulated by promoters containing NF-kB signal sequences, such as ICAM-1, GRO-γ, IL-1β, IL-6. This suggest that other transcription factors are the master regulators of these genes (such as Sp1 for IL-6 gene).33 Despite the fact that this specific issue should be object of future investigations, the effects of the NF-kappaB PNA-DNA-PNA chimeras, as shown in the insert of Figure 4B, reproduce those obtained using NF-kappB ODNs. The results shown in Figure 5, while confirming that the NF-kappaB decoy ODN exhibit lipofectamine-dependent effects, provide evidence that the NF-kappaB decoy PDP interferes with the NF-kappaB activity without the need of lipofectamine.
Recent studies have indicated that the transcription factor decoy (TFD) strategy targeting NF-kappaB might be of interest to develop anti-inflammatory approaches for cystic fibrosis.12,13 The first set of data of the present study demonstrate that decoy ODNs against NF-kappaB interfere with the NF-kappaB pathway in cystic fibrosis cells infected with Pseudomonas aeruginosa. This was demonstrated by chromatin immunoprecipitation (ChIP) using as a model system cystic fibrosis IB3–1 cells infected with Pseudomonas aeruginosa. Treatment with ODN decoys against NF-kappaB led to a decreased occupancy of the IL-8 gene promoter by NF-kappaB factors.
In order to develop more stable therapeutic molecules, peptide nucleic acids (PNAs) based agents were considered. PNAs are DNA mimicking molecules in which the pseudopeptide backbone is composed of N-(2-aminoethyl)glycine units.19-21 PNAs are resistant to both nucleases and proteases22 and, more importantly, hybridize with high affinity to complementary sequences of single-stranded RNA and DNA, forming Watson-Crick double helices.21 For these reasons, PNAs were found to be excellent candidates for antisense and antigéne therapies.23-26 In recent studies, PNA-DNA chimeras have been described as reagents for the transcription factor decoy approach.27-29 PNA-DNA chimeras are PNA-DNA covalently bonded hybrids and were designed on one hand to improve the poor cellular uptake and solubility of PNAs, on the other hand to exhibit biological properties typical of DNA, such as the ability to stimulate RNaseH activity and to act as substrate for cellular enzymes (for instance DNA polymerases). The results published by Romanelli et al.29 Borgatti et al.27,28 and Moggio et al.34 firmly demonstrate that decoy molecules based on PNA-DNA chimeras are powerful decoy molecules. In respect to experiments aimed at pharmacological modification of gene expression, PNA-DNA-PNA chimeras are molecules of interest for several points of view: (1) unlike PNAs, they can be complexed with liposomes and microspheres;28 (2) unlike ODNs, they are resistant to DNases, serum and cytoplasmic extracts;22 (3) unlike PNA/PNA and PNA/DNA hybrids,35 they are potent decoy molecules.27-29
The second set of results presented in this paper (Figs. Four and 5) demonstrated that PDP/PDP chimeras targeting NF-kappaB are strong inhibitors of IL-8 gene expression even in the absence of protection with lipofectamine (Fig. 5) and mimick the biological activity of ODN-based lipofectamine-delivered decoys (insert of Fig. 4B). The extent of inhibition of IL-8 gene expression using naked NF-kappaB decoy ODN (Fig. 5C) approaches that found in lipofectamine-delivered NF-kappaB decoy ODN (Fig. 5B). This is the first research article from our group reporting this information, which is of great impact, in our opinion, for the development of stable molecules to be used in non-viral gene therapy. Despite the fact that these results should be considered as a proof-of-principle that PNA-DNA chimeras targeting NF-kB retain selective biological functions, we like to remark that no translation to clinical settings can be proposed based only on data reported in the present paper. On the other hand, we like to underline that IL-8 is one of the master genes in pro-inflammatory processes affecting cystic fibrosis. Accordingly, inhibition of its functions might have a clinical relevance and research efforts on this issue deserve great attention.
The synthetic oligonucleotides used in this study were purchased from Pharmacia.
Chimeras were assembled on solid phase, by sequential elongation of the PNA fragment, to whom DNA first and PNA were attached. The chimeras were obtained using Mmt(Bz)PNA monomers, as reported in previously published papers.29,36 ESI-MS for gcg-ACC CCT GAA AGG T-gcc: [M+4H]4+: 1438.2, [M+5H]5+ 1150.5, calculated. for C193H245N90O95P13 5748.26. ESI-MS for cgc-TGG GGA CTT TCC A-cgg: [M+4H]4+: 1443.6, [M+5H]5+: 1154.9, calculated for C194H247N86O99P13 5770.26.
IB3–1 cells (LGC Promochem) are human bronchial epithelial cells immortalized with adeno12/SV40, derived from a CF patient with a mutant F508del/W1282X genotype.37 Cells were grown in LHC-8 basal medium (Biofluids) supplemented with 5% fetal bovine serum (FBS). All culture flasks and plates were coated with a solution containing 35 μg/ml bovine collagen (Becton-Dickinson), 1μg/ml BSA (Sigma-Aldrich) and 1 μg/ml human fibronectin (Becton-Dickinson). P.aeruginosa, PAO1 laboratory strain, was kindly provided by A. Prince (Columbia University). Bacteria were grown in trypticase soy broth (TSB) or agar (TSA) (Difco).
The double-stranded oligonucleotides (ODN) used in the EMSA are reported in Table 1. ODN (3 pmol each) were 32P-labeled using 10 U T4 polinucleotide kinase (MBI Fermentas) annealed to an excess of complementary ODN and purified from [γ-32P]ATP (Perkin Elmer). Binding reactions were performed by incubating 2.5 µg of nuclear extract, obtained from IB3 cells as previously described,12 and 16 fmol of 32P-labeled double-stranded ODN, with or without competitor in a final volume of 20 µL of binding buffer (20 mM TRIS-HCl, pH 7.5, 50 mM KCl, 1 mM MgCl2, 0.2 mM EDTA, 5% glycerol, 1 mM dithiothreitol, 0.01% TritonX100, 0.05 µg•µL-1 of poly dI-dC, 0.05 µg•µL-1 of a single-stranded ODN). Competitor (100-fold excess of unlabeled ODNs) and nuclear extract mixture were incubated for 15 min and then probe was added to the reaction. After a further incubation of 30 min at room temperature samples were immediately loaded onto a 6% nondenaturing polyacrylamide gel containing 0.25 x Tris/borate/EDTA (22.5 mM Tris, 22.5 mM boric acid, 0.5 mM EDTA, pH 8) buffer. Electrophoresis was performed at 200 V. Gels were vacuum heat-dried and subjected to autoradiography.
IB3–1 cells were seeded in 24-wells plates at a density of 30,000 cells/cm2 and transfected with ODNs using cationic liposome vector Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Lipofectamine 2000 (4 μl) was diluted in 1 ml of serum-free LHC-8 basal medium (Biofluids) and double-stranded decoy or PDP/PDP chimeras were added and incubated for 10 min to generate Liposome:DNA complexes. In parallel, a scrambled ODN was employed as negative control (Table 1). Then, the complexes were added to IB3–1 cells and incubated for 6 h. After this time of incubation cells were washed twice with serum-free culture medium and left at 37°C and 5% CO2 for 20–24 h before pro-inflammatory challenge with P.aeruginosa (100 CFU/ml), IL-1β (10 ng/ml) or TNF-α (50 ng/ml) for a further 4 h.
Total RNA from IB3–1 cells was purified using High Pure RNA Isolation Kit (Roche) and 2.5 μg RNA were reverse-transcribed to cDNA using the High Capacity cDNA Archive Kit and random primers (Applied Biosystems) in a final reaction volume of 100 μl. cDNA (5 μl) and primers (15 nM each) (Table 1) were mixed with Fast SYBR Green Master Mix (Applied Biosystems). GAPDH gene expression was determined as normalizer gene. Primer sets were purchased from Sigma-Genosys. Real Time PCR was performed in duplicate for both target and normalizer genes using the ABI PRISM 7900 HT Fast Real Time PCR System (Applied Biosystems) as follows: 50°C for 2 min, 95°C for 20 sec and 40 two-step cycles: 95°C for 1 sec and 60°C for 20 sec. Results were collected with SDS 2.3 Software (Applied Biosystems) and relative quantification was performed using the comparative threshold (Ct) method. Data were analyzed with RQ Manager Software 1.2 (Applied Biosystems). Changes in mRNA expression levels were calculated after normalization to calibrator gene. The ratios obtained after normalization are expressed as -fold change over untreated samples.
Chromatin immunoprecipitation assays were performed by using the Chromatin Immunoprecipitation Assay Kit (Upstate Biotechnology). A total of 5 x 106 IB3 cells were treated, for 10 min at room temperature, with 1% formaldehyde culture medium. Cells were washed in phosphate-buffered saline, and then glycine was added to a final concentration of 0.125 M. The cells were then suspended in 0.5 ml of lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris-Cl, pH 8.1) plus protease inhibitors (1 μg/ml pepstatin A, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) and the chromatin subjected to sonication (using a Sonics Vibracell VC130 sonicator with a 2 mm probe). Fifteen 15 sec sonication pulses at 30% amplitude were required to shear chromatin to 200–1000 bp fragments. 0.2 ml aliquots of chromatin were diluted to 2 ml in ChIP dilution buffer containing protease inhibitors and then cleared with 75 μl of salmon sperm DNA/protein A-agarose 50% gel slurry (Upstate Biotechnology) for 1 h at 4°C before incubation on a rocking platform with either 10 μg of NF-kappaB p65 specific antiserum (sc-372X, Santa Cruz Biotechnology) or normal rabbit serum as negative control IgG (Upstate Biotechnology). Twenty μl of diluted chromatin was saved and stored for later PCR analysis as 1% of the input extract. Incubations occurred overnight at 4°C and continued an additional 1 h after the addition of 60 μl protein A-agarose slurry. Thereafter the agarose pellets were washed consecutively with low salt, high salt and LiCl buffers. DNA/protein complexes were recovered from the pellets with elution buffer (0.1 M NaHCO3 with 1% SDS), and cross-links were reversed by incubating overnight at 65°C with 0.2 M NaCl. The samples were treated with RNase A and proteinase K, extracted with phenol/chloroform and ethanol-precipitated. The pelletted DNAs were washed with 70% ethanol and dissolved in 40 μl of Tris/EDTA. Two μl aliquots were used for each real-time PCR reaction to quantitate immunoprecipitated promoter fragments. For quantitative real-time PCR reaction, a pair of primers that amplify a 301 bp region on the IL-8 promoter, containing the NF-kappaB binding site, was designed (IL-8 ChIP f, IL-8 ChIP r, Table 1). PCR reactions were also performed using negative control primers that amplify a 255 bp genomic region about 5 kb upstream of the IL-8 promoter, lacking NF-kappaB binding sites (Neg ChIP f, Neg ChIP r, Table 1). Each Real-time PCR reactions were performed in 25 μl of final volume, using 2 μl of template DNA (from chromatin immunoprecipitations), 10 pmol of primers and 1 x iQ™ SYBR® Green Supermix (Bio-Rad) for a total of 45 cycles (96°C for 15 sec, 66°C for 30 sec, and 72°C for 20 sec) using an iCycler IQ® (Bio-Rad). The relative proportions of immunoprecipitated promoter fragments were determined based on the threshold cycle (Tc) value for each PCR reaction. Real time PCR data analysis were obtained using the comparative cycle threshold method.38 The data represent the ratios between the IL-8 promoter Q-PCR performed on NF-kappaB specific ChIP and negative IgG (preimmune rabbit serum) ChIP. Each value was derived employing chromatin isolated from untreated IB3–1 cells or cells infected with PAO-1 in the absence or in the presence of pre-treatment with the NF-kappaB decoy ODN. Each sample was performed in duplicate on at least three separate experiments.
Results are expressed as mean ± standard error of the mean (SEM). Comparisons between groups were made by using paired Student's t test and a one-way analysis of variance (ANOVA). Statistical significance was defined with p < 0.05 (*, significant) and p < 0.01 (**, highly significant).
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.). R.G. is granted by Fondazione Cariparo, by MUR COFIN-2005, by TELETHON (GGP10124), by C.I.B. and by Associazione Veneta per la Lotta alla Talassemia. V.B. was fellow of the “Fondazione Cariverona.”
No potential conflicts of interest were disclosed.
Previously published online: www.landesbioscience.com/journals/artificialdna/article/21061