Nuclear factor-kappaB Decoy Research Articles

580. Prevention of Airway Inflammation by Simultaneous Inhibition of NFkB and STST6 Using Chimeric Decoy Oligonucleotides in a Mouse Model of Asthma

Allergic asthma is a common inflammatory disease of the airways characterized by reversible airflow obstruction and airway hyperresponsiveness (AHR) to bronchoconstrictor stimuli. The pathophysiology in asthma mainly occurs as a consequence of overexpression of the TH2 cytokines. These inflammatory factors are tightly regulated by a transcriptional network system. Nuclear factor kappaB (NFkB) and STAT6 are well known to regulate a set of gene associated with inflammatory and immune responses, and is thought to play an important role in the induction of allergic asthma. Therefore, we focused on the simultaneous inhibition of these important transcription factors using a decoy strategy to develop a novel therapeutic approach for treating asthma. For increased efficacy of decoy oligodeoxynucleotides (ODN) in vivo, we employed chimeric decoy ODN containing consensus sequences of both NFkB and STAT6 binding sites. In addition, two strands of decoy ODN were combined by the chemical spacer to increase its resistance to endonuclease for intratracheal administration. The therapeutic effect of chimeric decoy ODN was investigated using OVA-induced experimental asthma in mice. Mice were sensitized by intraperitoneal injections of 20 ug of ovalbumin (OVA) and 2 mg aluminum hydroxide constituted in 0.1 ml of saline on days 0 and 14. On days 21, 22 and 23, mice were challenged with 1% OVA aerosol for 20 min. At 24 hours after the last challenge, AHR was measured by methacholine-induced airflow obstruction. Intratracheal administration of decoy ODN was performed in OVA-sensitized mice at 3 days before the first challenge with aerosolized OVA. FITC-labeled chimeric decoy ODN could be detected in macrophages and monocytes migrating into the lung and airway, and NFkB and STAT6 activity were simultaneously inhibited by chimeric decoy ODN. Twenty-four hours after the last OVA challenge, treatment with chimeric or single transfection of NFkB decoy ODN was protected from methacholine-induced AHR, while mice treated with scrambled decoy ODN or saline developed a significantly increase in airway reactivity to methacholine. Importantly, this inhibitory effect of chimeric decoy ODN on airway hyperresponsiveness was significantly greater than that of NFkB decoy ODN. Treatment with chimeric decoy ODN markedly suppressed airway inflammation after OVA sensitization and challenge as compared with control and scrambled decoy ODN treatment. Inflammatory infiltrate, such as macrophage, was significantly inhibited by chimeric decoy ODN through suppression of ICAM-1 and eotaxin expression. In addition, secretion of Th2 cytokines including IL-4, IL-5 and IL-13 in BALF, and histamine in the whole lung were reduced by chimeric decoy ODN. Furthermore, a significant reduction of mucin secretion was observed by chimeric decoy ODN treatment accompanied by a suppression of MUC5AC gene expression. However, intratracheal administration of chimeric decoy ODN did not affected IgE synthesis. The present study provides a novel strategy for treating bronchial asthma by the simultaneous inhibition of both NFkB and STAT6 using chimeric decoy ODN. Further modification of chimeric decoy ODN would be useful to treat asthma as a decoy-based therapy.

Eight-Year Follow-Up of an Initial Case with NF-?B Decoy Oligodeoxynucleotide Transfection After Coronary Stent Implantation

Background: Nuclear factor–kappa B (NF-kB) plays a pivotal role in vascular remodeling after percutaneous coronary intervention (PCI) with a stent implantation. Methods and Results: This is the first clinical report to demonstrate the effect of NF-kB decoy oligodeoxynucleotide (ODN) on the prevention of restenosis after an eight-year observation period. We revealed that decoy ODN treatment suppressed neointimal formation in comparison to that without ODN transfected stent lesion in the same artery. Conclusion: We showed the long-term usefulness of NF-kB decoy ODN transfer after PCI. Keywords: CT, coronary intervention, gene therapy, nuclear factor–kappa B, oligodeoxynucleotide, restenosis, stents

Nuclear factor-κB decoy oligodeoxynucleotides ameliorate impaired glucose tolerance and insulin resistance in mice with cecal ligation and puncture-induced sepsis*

Insulin-resistant hyperglycemia is commonly observed in septic patients and may actually lead to some of adverse outcomes. We examined the changes in insulin signaling and glucose uptake regulation in sepsis and the involvement of the nuclear factor-kappaB pathway. Controlled animal study. University research laboratory. One hundred fifty-four BALB/c mice (8-12 wks of age). The following four experimental groups were studied: sham-operated control, cecal ligation and puncture-induced sepsis, sepsis + nuclear factor-kappaB decoy oligodeoxynucleotide treatment, and sepsis + scrambled decoy oligodeoxynucleotide treatment. Septic mice were markedly hyperinsulinemic with apparently normal blood glucose levels in the fasted state, suggesting they are insulin-resistant. In fact, glucose clearance in response to insulin was markedly impaired in septic mice. They had impaired GLUT4 membrane translocation resulting from impaired insulin signaling as indicated by the decreased amount of insulin receptor substrate protein and the reduced activation of phosphatidylinositol 3-kinase and Akt. Interestingly, injection of nuclear factor-kappaB decoy oligodeoxynucleotide into the skeletal muscle dramatically improved all of the changes, including glucose clearance and insulin signaling. We also found that the Cbl-associated protein to TC10 pathway, another pathway regulating GLUT4 translocation, was up-regulated in septic mice in a nuclear factor-kappaB-dependent manner. This pathway may be one of the compensatory mechanisms to translocate GLUT4 because silencing of the individual components of the pathway with small interfering RNAs further reduced GLUT4 translocation in muscles of septic mice. In sepsis, skeletal muscle GLUT4 translocation is impaired as a result of the reduced phosphatidylinositol 3-kinase/Akt pathway associated with insulin receptor substrate down-regulation through nuclear factor-kappaB activation.

Effects of intratracheal administration of nuclear factor-kappaB decoy oligodeoxynucleotides on long-term cigarette smoke-induced lung inflammation and pathology in mice

To determine if nuclear factor-κB (NF-κB) activation may be a key factor in lung inflammation and respiratory dysfunction, we investigated whether NF-κB can be blocked by intratracheal administration of NF-κB decoy oligodeoxynucleotides (ODNs), and whether decoy ODN-mediated NF-κB inhibition can prevent smoke-induced lung inflammation, respiratory dysfunction, and improve pathological alteration in the small airways and lung parenchyma in the long-term smoke-induced mouse model system. We also detected changes in transcriptional factors. In vivo, the transfection efficiency of NF-κB decoy ODNs to alveolar macrophages in BALF was measured by fluorescein isothiocyanate (FITC)-labeled NF-κB decoy ODNs and flow cytometry post intratracheal ODN administration. Pulmonary function was measured by pressure sensors, and pathological changes were assessed using histology and the pathological Mias software. NF-κB and activator protein 1(AP-1) activity was detected by the electrophoretic motility shift assay (EMSA). Mouse cytokine and chemokine pulmonary expression profiles were investigated by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and lung tissue homogenates, respectively, after repeated exposure to cigarette smoke. After 24 h, the percentage of transfected alveolar macrophages was 30.00 ± 3.30%. Analysis of respiratory function indicated that transfection of NF-κB decoy ODNs significantly impacted peak expiratory flow (PEF), and bronchoalveolar lavage cytology displayed evidence of decreased macrophage infiltration in airways compared to normal saline-treated or scramble NF-κB decoy ODNs smoke exposed mice. NF-κB decoy ODNs inhibited significantly level of macrophage inflammatory protein (MIP) 1α and monocyte chemoattractant protein 1(MCP-1) in lung homogenates compared to normal saline-treated smoke exposed mice. In contrast, these NF-κB decoy ODNs-treated mice showed significant increase in the level of tumor necrosis factor-α(TNF-α) and pro-MMP-9(pro-matrix metalloproteinase-9) in mice BALF. Further measurement revealed administration of NF-κB decoy ODNs did not prevent pathological changes. These findings indicate that NF-κB activation play an important role on the recruitment of macrophages and pulmonary dysfunction in smoke-induced chronic lung inflammation, and with the exception of NF-κB pathway, there might be complex mechanism governing molecular dynamics of pro-inflammatory cytokines expression and structural changes in small airways and pulmonary parenchyma in vivo.

Effects of nuclear factor-kappa B decoy oligonucleotide on the apoptosis of HepG2 cells

Objective To investigate the apoptosis of HepG2 cells and their sensitivities to the ciglitazone after inhibiting the activity of nuclear factor-kappa B (NF-kB) by NF-kB decoy oligonucleotide. Methods After transfecting HepG2 cells with NF-kB decoy oligonucleotide, the activity of NF-kB was observed by electrophonetic mobility shift assay and the protein expression of Bcl-2 and Fas by Western blot. The transfected and untransfected HepG2 cells were processed with 100 umol/L of ciglitazone for 1 to 4 days, and the growth curve and cell cycle of HepG2 cells were observed. Results After transfecting NF-kB decoy oligonucleotide to HepG2 cells, the activity of the NF-kB was inhibited, the Bcl-2 protein expression decreased and the Fas protein expression increased. The inhibition effect of the ciglitazone on the growth of HepG2 ceils was strengthened and more HepG2 cells were arrested at G1/G0 phase. Conclusions NF-kB decoy oligonucleotide could accelerate the apoptosis of HepG2 cells and enhance the inhibition effect of ciglitazone on HepG2 proliferation, the mechanism of which might be attributable to the increased expression of Fas protein and the decreased expression of Bcl-2 protein after NF-kB decoy oligonucleotide inhibiting the activity of NF-kB. Key words: Nuclear factor-kappa B decoy oligonucleotide; Liver cancer; HepG2 cell; Apoptosis

Transgene Persistence and Cell Turnover in the Diarthrodial Joint: Implications for Gene Therapy of Chronic Joint Diseases

Local gene therapy for chronic joint diseases requires prolonged transgenic expression, but this has not been reliably achieved in animal models. Using normal and immunocompromised animals, we examined the capacity of various cell types in joint tissues to maintain and express exogenous transgenes after direct intra-articular gene delivery. We found that transgenic expression could persist for the lifetime of the animal but required precise immunological compatibility between the vector, transgene product, and host. It was not dependent on vector integration or promoter origin. We identified two phenotypically distinct sub-populations of genetically modified cells within the joint: (i) transient cells, with a half-life of a few weeks, and (ii) stable cells that reside in the joint tissues indefinitely. Contrary to the prevailing assumption, the transient sub-population was composed almost exclusively of synovial fibroblasts, indicating that the synovium is not an appropriate tissue upon which to base a long-term therapy. Instead, fibroblasts in the ligaments, tendons, and capsule emerged as the primary cell types capable of sustained therapeutic transgene expression. This study sheds new light on the cellular dynamics of articular tissues and suggests that cell turnover and immune reactivity are the key determinants in achieving sustained transgenic expression intra-articularly.

The effects of nuclear factor-kappaB decoy oligonucleotides on dextran sulphate sodium-induced colitis: experiment with mice

To investigate the effects of nuclear factor-kappaB (NF-kappaB) decoy oligonucleotide (ODN) on dextran sulphate sodium (DSS)-induced colitis. Nine female BABL/C mice underwent infusion of 0.15 ml normal saline into the distant colon and used as controls (Group 1). Twenty-seven female BABL/C mice were made into DSS-induced colitis models and then randomly divided into 3 groups: Group 2 (underwent infusion of 0.15 ml normal saline into the distant colon), Group 3 (infused with NF-kappaB decoy ODN 25 nmol solved in 0.15 ml), and Group 4 (infused with NF-kappaB scrambled decoy ODN 25 nmol solved in 0.15 ml). Disease active index (DAI) was observed every day. Nine days later the mice were killed and their colons were taken out to undergo histological examination. The tumor necrosis factor (TNF)-alpha level of the colon mucosa was measured by enzyme linked immunosorbent assay (ELISA). NF-kappaB expression was determined by immunohistochemical staining. The distribution of NF-kappaB decoy ODN was investigated by confocal laser microscopy. (1) The DAI scores, histological scores and TNF-a level in the colon mucosa of Groups 2 - 4 were all significantly higher than those of Group 1 (all P < 0.05). The DAI scores, histological scores and TNF-a level in the colon mucosa of Group 3 were all significantly lower than those of Groups 2 and 4 (all P < 0.01). (2) In the tissue sections NF-kappaB p65 was positive mainly in the nucleus in the 3 DSS-treated groups without significant differences among these 3 groups, and was mainly positive in the cytoplasm in the control group. (3) Confocal laser microscopy showed that NF-kappaB decoy ODN could be ingested efficiently into the mucosa and submucous layer of colon. (4) There were no significant differences in the liver function, kidney function, and blood glucose among all groups. NF-kappaB pathway is associated with the pathogenesis of DSS-induced colitis which is very similar to human UC. Blockade of NF-kappaB pathway by NF-kappaB decoy ODN shows protective effect on the mice with DSS-induced colitis.

Spinal NF-kB activation induces COX-2 upregulation and contributes to inflammatory pain hypersensitivity.

Cyclooxygenase-2 (COX-2) is a major contributor to the elevation of spinal prostaglandin E2, which augments the processing of nociceptive stimuli following peripheral inflammation, and dynorphin has been shown to have an important role in acute and chronic pain states. Moreover, the transcription factor, nuclear factor-kappa B (NF-kB), regulates the expressions of both COX-2 and dynorphin. To elucidate the role of spinal NF-kB in the induction of inflammatory pain hypersensitivity, we examined whether activated NF-kB affects pain behavior and the expressions of the mRNAs of COX-2 and prodynorphin following peripheral inflammation. Intrathecal pretreatment with different NF-kB inhibitors, namely, NF-kB decoy or pyrrolidine dithiocarbamate, significantly reduced mechanical allodynia and thermal hyperalgesia following unilateral hindpaw inflammation evoked by complete Freund's adjuvant (CFA). These NF-kB inhibitors also suppressed the activation of spinal NF-kB and the subsequent remarkable elevation of spinal COX-2 mRNA, but not that of prodynorphin mRNA. In addition, the activation of spinal NF-kB following CFA injection was inhibited by intrathecal pretreatments with interleukin-1 beta receptor antagonist or caspase-1 inhibitor. In view of the fact that interleukin-1 beta (IL-1 beta) is the major inducer of spinal COX-2 upregulation following CFA injection, our results suggest that IL-1 beta-induced spinal COX-2 upregulation and pain hypersensitivity following peripheral inflammation are mediated through the activation of the NF-kB-associated pathways.

Augmented regeneration of partial liver allograft induced by nuclear factor-kappaB decoy oligodeoxynucleotides-modified dendritic cells.

To investigate the effect of NF-kappaB decoy oligodeoxynuleotides (ODNs) - modified dendritic cells (DCs) on regeneration of partial liver allograft. Bone marrow (BM)- derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs, respectively. GM-CSF-propagated DCs were treated with double-strand NF-kappaB decoy ODNs containing two NF-kappaB binding sites or scrambled ODNs. Allogeneic (SD rat to LEW rat) 50% partial liver transplantation was performed. Normal saline (group A), GM-CSF -propagated DCs (group B), GM-CSF+IL-4 - propagated DCs (group C), and GM-CSF+NF-kappaB decoy ODNs (group D) or scrambled ODNs -propagated DCs (group E) were injected intravenously into recipient LEW rats 7 days prior to liver transplantation and immediately after transplantation. DNA synthesis (BrdU labeling) and apoptosis of hepatocytes were detected with immunostaining and TUNEL staining postoperative 24 h, 48 h, 72 h and 84 h, respectively. Liver graft-resident NK cell activity, hepatic IFN-gamma mRNA expression and recipient serum IFN-gamma level at the time of the maximal liver allograft regeneration were measured with (51)Cr release assay, semiquantitative RT-PCR and ELISA, respectively. Regeneration of liver allograft was markedly promoted by NF-kappaB decoy ODNs-modified immature DCs but was significantly suppressed by mature DCs, the DNA synthesis of hepatocytes peaked at postoperative 72 h in group A, group B and group E rats, whereas the DNA synthesis of hepatocytes peaked at postoperative 84 h in group C rats and 48 h in group D rats, respectively. The maximal BrdU labeling index of hepatocytes in group D rats was significantly higher than that in the other groups rats. NF-kappaB decoy ODNs-modified immature DCs markedly suppressed but mature DCs markedly promoted apoptosis of hepatocytes, liver-resident NK cell activity, hepatic IFN-gamma mRNA expression and recipient serum IFN-gamma production. At the time of the maximal regeneration of liver allograft, the minimal apoptosis of hepatocytes, the minimal activity of liver-resident NK cells, the minimal hepatic IFN-gamma mRNA expression and serum IFN-gamma production were detected in group D rats. The apoptotic index of hepatocytes, the activity of liver- resident NK cells, the hepatic IFN-gamma mRNA expression level and the serum IFN-gamma level in group D rats were significantly lower than that in the other groups rats at the time of the maximal regeneration of liver allograft. The data suggest that the augmented regeneration of partial liver allograft induced by NF-kappaB decoy ODNs-modified DCs may be attributable to the reduced apoptotic hepatocytes, the suppressed activity of liver-resident NK cells and the reduced IFN-gamma production.

Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1.

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O(2)(-)). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [(3)H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O(2)(-) and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH(2)F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappaB, and inhibition of this activity by nuclear factor-kappaB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O(2)(-) produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.

Transcription factor decoy for nuclear factor-kappaB inhibits tumor necrosis factor-alpha-induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.

Several cytokines and adhesion molecules released from endothelium play an important role in inflammation, immune responses, and probably atherogenesis. To determine whether the transcription factor nuclear factor-kappaB mediated expression of these genes involved in the inflammatory response of endothelial cells to tumor necrosis factor-alpha, by using transcription factor decoy oligodeoxynucleotides. We first transfected fluorescein isothiocyanate (FITC)-labeled double-stranded oligodeoxynucleotides into endothelial cells by a cationic liposome-mediated method of gene transfer. We then confirmed that the decoy oligodeoxynucleotides could block binding of nuclear factor-kappaB to its specific cis element effectively. In addition, we transfected the reporter gene chloramphenicol acetyltransferase driven by three repeated nuclear factor-kappaB binding sequences in the promoter and enhancer region. FITC-labeled oligodeoxynucleotides were detected in the nuclei of approximately 70% of the total cells. Tumor necrosis factor--stimulated expression of chloramphenicol acetyltransferase was partially inhibited by transfection of nuclear factor-kappaB decoy oligodeoxynucleotides, but not by transfection of scrambled oligodeoxynucleotides. Also nuclear factor-kappaB decoy oligodeoxynucleotides but not scrambled oligodeoxynucleotides inhibited tumor necrosis factor-induced expression of interleukin-6 and intracellular adhesion molecule-1 both at the messenger RNA and at protein level (assessed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay). Our results demonstrate that nuclear factor-kappaB decoy oligodeoxynucleotides transfected by cationic liposome method inhibited tumor necrosis factor--induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.