NF-κB Decoy Oligodeoxynucleotides Research Articles

NF-κB Decoy Oligodeoxynucleotide-Loaded Poly Lactic-co-glycolic Acid Nanospheres Facilitate Socket Healing in Orthodontic Tooth Movement.

Orthodontic space closure following tooth extraction is often hindered by alveolar bone deficiency. This study investigates the therapeutic use of nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides loaded with polylactic-co-glycolic acid nanospheres (PLGA-NfDs) to mitigate alveolar bone loss during orthodontic tooth movement (OTM) following the bilateral extraction of maxillary first molars in a controlled experiment involving forty rats of OTM model with ethics approved. The decreased tendency of the OTM distance and inclination angle with increased bone volume and improved trabecular bone structure indicated minimized alveolar bone destruction. Reverse transcription-quantitative polymerase chain reaction and histomorphometric analysis demonstrated the suppression of inflammation and bone resorption by downregulating the expression of tartrate-resistant acid phosphatase, tumor necrosis factor-α, interleukin-1β, cathepsin K, NF-κB p65, and receptor activator of NF-κB ligand while provoking periodontal regeneration by upregulating the expression of alkaline phosphatase, transforming growth factor-β1, osteopontin, and fibroblast growth factor-2. Importantly, relative gene expression over the maxillary second molar compression side in proximity to the alveolus highlighted the pharmacological effect of intra-socket PLGA-NfD administration, as evidenced by elevated osteocalcin expression, indicative of enhanced osteocytogenesis. These findings emphasize that locally administered PLGA-NfD serves as an effective inflammatory suppressor and yields periodontal regenerative responses following tooth extraction.

Sex differences of NF-κB-targeted therapy for mitigating osteoporosis associated with chronic inflammation of bone.

Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR). Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN. We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males.

Nuclear Factor-κB Decoy Oligodeoxynucleotide Attenuates Cartilage Resorption In Vitro.

Cartilage harvest and transplantation is a common surgery using costal, auricular, and septal cartilage for craniofacial reconstruction. However, absorption and warping of the cartilage grafts can occur due to inflammatory factors associated with wound healing. Transcription factor nuclear factor-κB (NF-κB) is activated by the various stimulation such as interleukin-1 (IL-1), and plays a central role in the transactivation of this inflammatory cytokine gene. Inhibition of NF-κB may have anti-inflammatory effects. The aim of this study was to explore the potential of an NF-κB decoy oligodeoxynucleotide (Decoy) as a chondroprotective agent. Safe and efficacious concentrations of Decoy were assessed using rabbit nasal septal chondrocytes (rNSChs) and assays for cytotoxicity, proteoglycan (PG) synthesis, and PG turnover were carried out. The efficacious concentration of Decoy determined from the rNSChs was then applied to human nasal septal cartilage (hNSC) in vitro and analyzed for PG turnover, the levels of inflammatory markers, and catabolic enzymes in explant-conditioned culture medium. Over the range of Decoy conditions and concentrations, no inhibition of PG synthesis or cytotoxicity was observed. Decoy at 10 μM effectively inhibited PG degradation in the hNSC explant, prolonging PG half-life by 63% and decreasing matrix metalloprotease 3 (MMP-3) by 70.7% (p = 0.027). Decoy may be considered a novel chondroprotective therapeutic agent in cartilage transplantation due to its ability to inhibit cartilage degradation due to inflammation cytokines.

NF-κB Decoy ODN-Loaded Poly(Lactic-co-glycolic Acid) Nanospheres Inhibit Alveolar Ridge Resorption.

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-κB) decoy oligodeoxynucleotides (ODNs) are double-stranded DNA sequences capable of downregulating the expression of downstream genes of the NF-κB pathway, which is recognized for regulating prototypical proinflammatory signals, physiological bone metabolism, pathologic bone destruction, and bone regeneration. The aim of this study was to investigate the therapeutic effect of NF-κB decoy ODNs on the extraction sockets of Wistar/ST rats when delivered by poly(lactic-co-glycolic acid) (PLGA) nanospheres. Microcomputed tomography and trabecular bone analysis following treatment with NF-κB decoy ODN-loaded PLGA nanospheres (PLGA-NfDs) demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1β, tumor necrosis factor-α, receptor activator of NF-κB ligand, turnover rate, and increased transforming growth factor-β1 immunopositive reactions and relative gene expression. These data demonstrate that local NF-κB decoy ODN transfection via PLGA-NfD can be used to effectively suppress inflammation in a tooth-extraction socket during the healing process, with the potential to accelerate new bone formation.

Overcoming thickened pathological skin in psoriasis via iontophoresis combined with tight junction-opening peptide AT1002 for intradermal delivery of NF-κB decoy oligodeoxynucleotide

Transdermal delivery of nucleic acid therapeutics has been demonstrated to be effective for psoriasis treatment. We previously reported the utility of iontophoresis (IP) using weak electric current (0.3–0.5 mA/cm2) for intradermal delivery of nucleic acid therapeutics via weak electricity-mediated intercellular junction cleavage, and subsequent exertion of nucleic acid function. However, the thickened pathological skin in psoriasis hampers permeation of IP-administered macromolecules. Thus, approaches are needed to more strongly cleave intercellular spaces and overcome the psoriatic skin barrier. Herein, we applied a combination of tight junction-opening peptide AT1002 with IP, as synergistic effects of weak electricity-mediated intercellular junction cleavage and the tight junction-opening ability of AT1002 may help overcome thickened psoriatic skin and facilitate macromolecule delivery. Pretreatment with IP of an AT1002 analog exhibiting positively-charged moieties before fluorescence-labeled oligodeoxynucleotide IP resulted in the oligodeoxynucleotide permeation into psoriatic skin, whereas IP of the oligodeoxynucleotide alone did not. Moreover, psoriasis-induced upregulation of inflammatory cytokine mRNA levels was significantly suppressed by NF-κB decoy oligodeoxynucleotide IP combined with the AT1002 analog, resulting in amelioration of epidermis hyperplasia. These results suggest that synergistic effects of IP and an AT1002 analog can overcome thickened psoriatic skin and enable intradermal delivery of NF-κB decoy oligodeoxynucleotide for psoriasis treatment.

Suppression of NF-κB-induced chronic inflammation mitigates inflammatory osteolysis in the murine continuous polyethylene particle infusion model.

Wear particle-associated bone loss (periprosthetic osteolysis) constrains the longevity of total joint arthroplasty (TJA). Wear particles induce a prolonged upregulation of nuclear factor kappa B (NF-κB) signaling in macrophages and osteoclasts. Synthetic double-stranded oligodeoxynucleotides (ODNs) can prevent the binding of NF-κB to the promoter regions of targeted genes and inhibit genetic activation. We tested the hypothesis that polyethylene-particle induced chronic inflammatory bone loss could be suppressed by local delivery of NF-κB decoy ODNs in murine in vivo model. Polyethylene particles were continuously infused into the medullary cavity of the distal femur for 6 weeks to induce chronic inflammation, and micro-computational tomography and immunohistochemical analysis were performed. Particle-induced chronic inflammation resulted in lower BMD values, an increase in osteoclastogenesis and nuclear translocation of p65, a prolonged M1 pro-inflammatory macrophage phenotype, and a decrease of M2 anti-inflammatory macrophage phenotype. Delayed timing of local infusion of NF-κB decoy ODN for the last 3 weeks reversed polyethylene-particle associated chronic inflammatory bone loss and facilitated bone healing. This study demonstrated that polyethylene-particle associated chronic inflammatory osteolysis can be effectively modulated via interference with the NF-κB pathway; this minimally invasive intervention could potentially be an efficacious therapeutic strategy for periprosthetic osteolysis after TJA.

Abstract 5356: Targeted in vivo delivery of NF-κB decoy oligodeoxynucleotide augments efficacy of radiation therapy against B-cell lymphomas

Abstract Despite recent advances in the treatment of non-Hodgkin B-cell lymphoma (BCL), significant numbers of patients relapse or remain refractory to current therapies. Treatment resistance in BCL is associated with survival signaling via NF-κB transcription factor. While NF-κB is an attractive molecular target in BCL and many other human cancers, as a transcription factor it remains undruggable. Here, we describe a B-cell-selective NF-κB inhibitor consisting of a NF-κB-specific decoy oligodeoxynucleotide (dODN) conjugated to a type B CpG ODN sequence targeting Toll-like receptor-9 (TLR9)-expressing B-cell lymphoma cells. Bc-NFκBdODN showed efficient uptake by human diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL), such as U2932, OCI-Ly3 and Jeko1, respectively. In addition, we confirmed that Bc-NFκBdODN inhibited nuclear translocation and DNA-binding activity of NF-κB together with the expression of CCND2 and MYC target genes. In vitro Bc-NFκBdODN enhanced direct and ionizing radiation-induced cytotoxicity in lymphoma cells. In xenotransplanted models of human U2932 and Ly3 lymphomas, local injections of Bc-NFκBdODN reduced NF-κB activity in whole tumors and effectively arrested lymphoma progression when combined with a single, local 3Gy or 10Gy dose of radiation. In immune-competent mice, intratumoral treatment with Bc-NFκBdODN abrogated growth of directly treated as well as distant A20 lymphomas and triggered CD8+ T cell-mediated anti-tumor immune responses. Bc-NFκBdODN but not control Bc-scrambled oligonucleotides downregulated PD-1 expression on CD8+ and CD4+ T cells, while reducing Treg percentage in tumor draining lymph nodes (TDLN). Bc-NFκBdODN treatment also downregulated PD-L1 on tumor-associated myeloid cells and reduced percentage of tumor-infiltrating M-MDSCs. Our results underscore clinical relevance of NF-κB-specific decoy strategy, which can restore efficacy of standard radiation therapy to benefit patients with resistant or relapsed BCL. Citation Format: Zhuoran zhang, Xingli Zhao, Dayson Moreira, Yu-Lin Su, Piotr Swiderski, Stephen Forman, Larry Kwak, Marcin Kortylewski. Targeted in vivo delivery of NF-κB decoy oligodeoxynucleotide augments efficacy of radiation therapy against B-cell lymphomas [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5356.

Targeted and triple therapy-based liposomes for enhanced treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that is currently incurable. Clinical practice has shown significant benefits of combined therapies for RA treatment. This study aims to develop and demonstrate an efficient triple therapy for RA in vitro and in vivo. Three anti-inflammatory agents, NF-κB decoy oligodeoxynucleotides (ODNs), gold nanorods (GNRs), and dexamethasone (DEX), were encapsulated into folate (FA) modified liposomes (FA-lip(DEX+GNRs/ODNs)). The FA-lip(DEX+GNRs/ODNs) showed favorable physicochemical properties and efficient intracellular uptake by inflamed macrophages. Combined with laser irradiation, FA-lip(DEX+GNRs/ODNs) greatly reduced the secretion of proinflammatory proteins and oxidative factors in vitro. In adjuvant-induced arthritis (AIA) mice, FA-lip(DEX+GNRs/ODNs) achieved prolonged and enhanced accumulation at inflamed paws. FA-lip(DEX+GNRs/ODNs)+laser treatment reduced clinical arthritis scores and serum cytokine levels and protected cartilage. In summary, the triple therapy demonstrated significantly enhanced anti-inflammatory efficacy and is a promising strategy to treat RA via combined anti-inflammatory mechanisms.

Crosstalk between dental pulp stem cells and endothelial cells augments angiogenic factor expression.

We aimed to investigate whether the mesenchymal stem cell-endothelial cell crosstalk enhances angiogenic factor expression via nuclear factor-kappa B (NF-κB)-dependent mechanisms. Human dermal microvascular endothelial cells (HDMECs) and stem cells from human exfoliated deciduous teeth (SHEDs) were cocultured for 96hr, in the presence of NF-κB decoy oligodeoxynucleotides (ODNs) or scramble (control). Vascular endothelial cell growth factor (VEGF) and phospho-NF-κB p65 were measured with enzyme-linked immunosorbent assay. Angiogenesis-related gene expression was analyzed with microarray analysis followed by real-time polymerase chain reaction. Tube formation assay was conducted in the presence of NF-κB decoy. The VEGF and phospho-NF-κB p65 levels were significantly higher in the coculture with NF-κB decoy scramble than in single culture and coculture with NF-κB decoy ODN. Microarray analysis of SHEDs and HDMECs with NF-κB decoy scramble showed higher expression of proangiogenic genes, Bcl-2, NF-κB1, VEGFA, CXCL8, and CXCR1, and lower expression of proapoptotic genes, Bax and Caspase 9, compared to cells with NF-κB decoy ODN. Real-time PCR results for Bcl-2 and CXCL8 showed a similar trend. Tube formation assay showed more tube development in the presence of NF-κB decoy scramble. The SHED-HDMEC crosstalk enhanced proangiogenic factor expression via NF-κB-dependent pathways.

Inhibition of Nuclear Factor Kappa B Prevents the Development of Experimental Periapical Lesions

IntroductionNuclear factor kappa B (NF-κB) is an important transcriptional regulator of angiogenesis involving B-cell lymphoma 2 (Bcl-2) and Bcl-2–associated X protein (Bax) signaling pathways. Thus, inhibition of NF-κB may suppress the development of periapical lesions via blockage of angiogenesis. Accordingly, we examined the effects of NF-κB decoy oligodeoxynucleotide (ODN) treatment on experimentally induced periapical lesions. MethodsPeriapical lesions were induced in the mandibular first molars of 5-week-old male Wistar rats by the application of lipopolysaccharide to the pulp. NF-κB decoy ODN or NF-κB decoy scramble (control) was injected intraperitoneally every 7 days, starting 1 day before pulp exposure. After 28 days, the samples were retrieved, and digital radiographs were taken for radiomorphometry. Samples were processed for (1) immunohistochemistry of CD31, Bcl-2, and Bax; (2) laser capture microdissection to analyze Bcl-2, Bax, chemokine (C-X-C motif) ligand 1 (CXCL1), CXC receptor 2 (CXCR2), and vascular endothelial cell growth factor receptor 2 (VEGFR2) messenger RNA (mRNA) expression in CD31+ endothelial cells; (3) enzyme-linked immunosorbent assay to determine NF-κB/p65 activity; and (4) Western blotting for vascular endothelial growth factor expression. ResultsNF-κB decoy ODN treatment significantly reduced lesion size, NF-κB/p65 activity, and the density of CD31+ endothelial cells in the lesion. NF-κB decoy ODNs also down-regulated CXCL1, CXCR2, and VEGFR2 mRNAs and up-regulated Bax mRNA in endothelial cells but did not affect Bcl2 mRNA in endothelial cells. Vascular endothelial growth factor protein expression in the lesions was significantly decreased. ConclusionsThe inhibition of NF-κB activity by decoy ODN treatment suppressed the development of experimentally induced periapical lesions with a concomitant reduction in angiogenic responses in endothelial cells.

NF-κB Decoy Oligodeoxynucleotide-Coated Balloon Catheter for Arteriovenous Fistula in Hemodialysis

IntroductionNew treatments to inhibit neointimal formation after percutaneous transluminal angioplasty (PTA) are needed for patients undergoing chronic hemodialysis (HD). We compared the efficacy and safety of AMG0102, a balloon catheter containing nuclear factor κB (NF-κB) decoy oligodeoxynucleotide (ODN) with the PTA balloon catheter (control group) for arteriovenous fistula (AVF) stenosis.MethodsIn total, 175 patients (age ≥20 years, undergoing HD, with venous stenosis at the anastomotic region) were registered in this prospective open-label, randomized study. Patients were followed postoperatively for 36 weeks. The duration of primary patency on the targeted venous stenosis site (primary endpoint) was estimated by the Kaplan–Meier method.ResultsA lower restenosis risk was observed for the AMG0102 group, but it was not statistically significant (stratified log-rank test P = 0.250, hazard ratio [HR] 0.774; 95% confidence interval [CI]: 0.500–1.198). The median duration of primary patency was 245 days and 172 days in the AMG0102 and control groups, respectively. After stratification based on the status of diabetes complications, the HR was 0.666 (95% CI: 0.366–1.212; P = 0.183) and the median duration of primary patency was prolonged by 108 days in the AMG0102 group with diabetes complications (245 days) compared with the control group (137 days). Adverse event (AE) incidence up to 36 postoperative weeks did not differ between groups. Four device failures occurred in 3 patients (AMG0102 group), but none resulted in AEs.ConclusionFurther modifications to enhance NF-κB decoy ODN uptake and efficacy are necessary to show its clinical utility for AVF stenosis in chronic HD.

Prevention of Asthma Exacerbation in a Mouse Model by Simultaneous Inhibition of NF-κB and STAT6 Activation Using a Chimeric Decoy Strategy.

Transactivation of inflammatory and immune mediators in asthma is tightly regulated by nuclear factor κB (NF-κB) and signal transducer and activator of transcription 6 (STAT6). Therefore, we investigated the efficacy of simultaneous inhibition of NF-κB and STAT6 using a chimeric decoy strategy to prevent asthma exacerbation. The effects of decoy oligodeoxynucleotides were evaluated using an ovalbumin-induced mouse asthma model. Ovalbumin-sensitized mice received intratracheal administration of decoy oligodeoxynucleotides 3 days before ovalbumin challenge. Fluorescent-dye-labeled decoy oligodeoxynucleotides could be detected in lymphocytes and macrophages in the lung, and activation of NF-κB and STAT6 was inhibited by chimeric decoy oligodeoxynucleotide transfer. Consequently, treatment with chimeric or NF-κB decoy oligodeoxynucleotides protected against methacholine-induced airway hyperresponsiveness, whereas the effect of chimeric decoy oligodeoxynucleotides was significantly greater than that of NF-κB decoy oligodeoxynucleotides. Treatment with chimeric decoy oligodeoxynucleotides suppressed airway inflammation through inhibition of overexpression of interleukin-4 (IL-4), IL-5, and IL-13 and inflammatory infiltrates. Histamine levels in the lung were reduced via suppression of mast cell accumulation. A significant reduction in mucin secretion was observed due to suppression of MUC5AC gene expression. Interestingly, the inhibitory effects on IL-5, IL-13, and histamine secretion were achieved by transfer of chimeric decoy oligodeoxynucleotides only. This novel therapeutic approach could be useful to treat patients with various types of asthma.

Ultrasound microbubble-mediated transfection of NF-κB decoy oligodeoxynucleotide into gingival tissues inhibits periodontitis in rats in vivo.

Periodontitis is a chronic infectious disease for which the fundamental treatment is to reduce the load of subgingival pathogenic bacteria by debridement. However, previous investigators attempted to implement a nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN) as a suppressor of periodontitis progression. Although we recently reported the effectiveness of the ultrasound-microbubble method as a tool for transfecting the NF-κB decoy ODN into healthy rodent gingival tissue, this technique has not yet been applied to the pathological gingiva of periodontitis animal models. Therefore, the aim of this study was to investigate the effectiveness of the technique in transfecting the NF-κB decoy ODN into rats with ligature-induced periodontitis. Micro computed tomography (micro-CT) analysis demonstrated a significant reduction in alveolar bone loss following treatment with the NF-κB decoy ODN in the experimental group. RT-PCR showed that NF-κB decoy ODN treatment resulted in significantly reduced expression of inflammatory cytokine transcripts within rat gingival tissues. Thus, we established a transcutaneous transfection model of NF-κB decoy ODN treatment of periodontal tissues using the ultrasound-microbubble technique. Our findings suggest that the NF-κB decoy ODN could be used as a significant suppressor of gingival inflammation and periodontal disease progression.

Amelioration of collagen-induced arthritis using antigen-loaded dendritic cells modified with NF-κB decoy oligodeoxynucleotides.

Dendritic cells (DCs) play an important role in the initiation of autoimmunity in rheumatoid arthritis (RA); therefore, the use of DCs needs to be explored to develop new therapeutic approaches for RA. Here, we investigated the therapeutic effect of bovine type II collagen (BIIC)-loaded DCs modified with NF-κB decoy oligodeoxynucleotides (ODNs) on collagen-induced arthritis (CIA) in rats and explored the underlying mechanisms. DCs treated with BIIC and NF-κB decoy ODNs exhibited features of immature DCs with low levels of costimulatory molecule (CD80 and CD86) expression. The development of arthritis in rats with CIA injected with BIIC + NF-κB decoy ODN-propagated DCs (BIIC–decoy DCs) was significantly ameliorated compared to that in rats injected with BIIC-propagated DCs or phosphate-buffered saline. We also found that the BIIC–decoy DCs exerted antiarthritis effects by inhibiting self-lymphocyte proliferative response and suppressing IFN-γ and anti-BIIC antibody production and inducing IL-10 antibody production. Additionally, antihuman serum antibodies were successfully produced in the rats treated with BIIC–decoy DCs but not in those treated with NF-κB decoy ODN-propagated DCs; moreover, the BIIC–decoy DCs did not affect immune function in the normal rats. These findings suggested that NF-κB decoy ODN-modified DCs loaded with a specific antigen might offer a practical method for the treatment of human RA.

Orthopaedic wear particle-induced bone loss and exogenous macrophage infiltration is mitigated by local infusion of NF-κB decoy oligodeoxynucleotide.

Excessive production of wear particles from total joint replacements induces chronic inflammation, macrophage infiltration, and consequent bone loss (periprosthetic osteolysis). This inflammation and bone remodeling are critically regulated by the transcription factor NF-κB. We previously demonstrated that inhibition of NF-κB signaling by using the decoy oligodeoxynucleotide (ODN) mitigates polyethylene wear particle-induced bone loss using in vitro and in vivo models. However, the mechanisms of NF-κB decoy ODN action, and in particular its impact on systemic macrophage recruitment, remain unknown. In the current study, this systemic macrophage infiltration was examined in our established murine femoral continuous particle infusion model. RAW264.7 murine macrophages expressing a luciferase reporter gene were injected into the systemic circulation. Quantification of bioluminescence showed that NF-κB decoy ODN reduced the homing of these reporter macrophages into the distal femurs exposed to continuous particle delivery. Particle-induced reduction in bone mineral density at the distal diaphysis of the femur was also mitigated by infusion of decoy ODN. Histological staining showed that the decoy ODN infusion decreased osteoclast and macrophage numbers, but had no significant effects on osteoblasts. Local infusion of NF-κB decoy ODN reduced systemic macrophage infiltration and mitigated particle-induced bone loss, thus providing a potential strategy to treat periprosthetic osteolysis. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3169-3175, 2017.

Inhibition of Allergic Response by Intranasal Selective NF-κB Decoy Oligodeoxynucleotides in a Murine Model of Allergic Rhinitis.

PurposeIt remains unknown whether local inhibition of Nuclear factor-kappa B (NF-κB) could have therapeutic value in the treatment of allergic rhinitis (AR). This study aimed to evaluate the effect of selective NF-κB inhibition using NF-κB decoy oligodeoxynucleotides (ODNs) for the local treatment of AR in ovalbumin (OVA)-sensitized wild-type mice.MethodsBALB/c mice were sensitized with OVA and alum, and then challenged intranasally with OVA. NF-κB decoy ODNs were given intranasally to the treatment group, and NF-κB scrambled ODNs were given to the sham treatment group. Allergic symptom scores, eosinophil infiltration, cytokine levels in the nasal mucosa, nasal lavage fluid, and spleen cell culture, serum total and OVA-specific immunoglobulins, as well as intercellular adhesion molecure-1 (ICAM-1) in the nasal mucosa, were analyzed.ResultsNF-κB decoy ODNs significantly reduced allergic symptoms and eosinophil infiltration in the nasal mucosa. They also suppressed serum levels of total IgE, OVA-specific IgE, and IgG1. IL-5 and TNF-α levels and the expression of ICAM-1 were decreased in the nasal mucosa of the treatment group compared to the positive control and sham treatment groups. In addition, IL-6 levels were significantly decreased in the nasal lavage fluid of the treatment group. Furthermore, NF-κB decoy ODNs significantly reduced expression of the systemic Th2 cytokines, IL-4 and IL-5 in spleen cell culture.ConclusionsThis study demonstrates for the first time that local NF-κB inhibition using NF-κB decoy ODNs suppressed the allergic response in a murine AR model. This shows the therapeutic potential of local NF-κB inhibition in the control of AR.

NF-κB decoy oligodeoxynucleotide mitigates wear particle-associated bone loss in the murine continuous infusion model

Total joint replacement is a cost-effective surgical procedure for patients with end-stage arthritis. Wear particle-induced chronic inflammation is associated with the development of periprosthetic osteolysis. Modulation of NF-κB signaling in macrophages, osteoclasts, and mesenchymal stem cells could potentially mitigate this disease. In the current study, we examined the effects of local delivery of decoy NF-κB oligo-deoxynucleotide (ODN) on wear particle-induced bone loss in a murine continuous femoral particle infusion model. Ultra-high molecular weight polyethylene particles (UHMWPE) with or without lipopolysaccharide (LPS) were infused via osmotic pumps into hollow titanium rods placed in the distal femur of mice for 4weeks. Particle-induced bone loss was evaluated by μCT, and immunohistochemical analysis of sections from the femur. Particle infusion alone resulted in reduced bone mineral density and trabecular bone volume fraction in the distal femur. The decoy ODN reversed the particle-associated bone volume fraction loss around the implant, irrespective of the presence of LPS. Particle-infusion with LPS increased bone mineral density in the distal femur compared with particle-infusion alone. NF-κB decoy ODN reversed or further increased the bone mineral density in the femur (3–6mm from the distal end) exposed to particles alone or particles plus LPS. NF-κB decoy ODN also inhibited macrophage infiltration and osteoclast number, but had no significant effects on osteoblast numbers in femurs exposed to wear particles and LPS. Our study suggests that targeting NF-κB activity via local delivery of decoy ODN has great potential to mitigate wear particle-induced osteolysis. Statement of SignificanceTotal joint replacement is a cost-effective surgical procedure for patients with end-stage arthritis. Chronic inflammation is crucial for the development of wear particle-associated bone loss. Modulation of NF-κB signaling in macrophages (pro-inflammatory cells), osteoclasts (bone-resorbing cells), and osteoblasts (bone-forming cells) could potentially mitigate this disease. Here we demonstrated that local delivery of decoy NF-κB oligo-deoxynucleotide (ODN) mitigated ultra-high molecular weight polyethylene (UHMWPE) wear particle induced bone loss in a clinically relevant murine model. The protective effects of decoy ODN was associated with reduced macrophage infiltration and osteoclast activation, but had no significant effects on osteoblast numbers. Our study suggests that targeting NF-κB activity via local delivery of decoy ODN has great potential to mitigate wear particle-induced bone loss.

NF-κB decoy polyplexes decrease P-glycoprotein-mediated multidrug resistance in colorectal cancer cells.

Multidrug resistance (MDR), a major cause for chemotherapy failure, has been linked to upregulation of ATP-dependent membrane efflux systems that limit intracellular accumulation of cytotoxic anticancer agents. P-glycoprotein (P-gp) encoded by the human ABCB1 gene was the first efflux transporter identified to contribute to MDR. ABCB1 gene expression is correlated with constitutive activation of the NF-κB signaling pathway in tumor cells. The objective of this research is to modulate P-gp activity in colon cancer cells using NF-κB decoy oligodeoxynucleotides (ODNs) that are effectively delivered into the nucleus of colorectal cancer cells by self-assembling nonviral nanoparticles comprising the novel poly[N-(2-hydroxypropyl)methacrylamide]-poly(N,N-dimethylaminoethylmethacrylate) diblock copolymer (pHPMA-b-pDMAEMA). Ethidium bromide intercalation and gel retardation assays demonstrated high DNA condensation capacity of pHPMA-b-pDMAEMA. Nanoparticles prepared with and without decoy ODNs did not significantly compromise cellular safety at N/P ratios ⩽4. Transfection efficiency of pHPMA-b-pDMAEMA polyplexes (N/P=4) in Caco-2 cells was comparable to TurboFect transfection standard, resulting in a 98% reduction in P-gp protein levels. As a pharmacodynamic consequence, intracellular accumulation of the P-gp substrate Rhodamine123 significantly increased by almost twofold. In conclusion, NF-κB ODN polyplexes fabricated with pHPMA-b-pDMAEMA polymer effectively reduced P-gp-mediated efflux activity in Caco-2 cells, suggesting successful interference with NF-κB-binding sites in the promoter region of the ABCB1 gene.

NF-κB Decoy Oligodeoxynucleotides Inhibits Kupffer Cell Activation

Objective: To investigate the inhibitory effects and the mechanism of NF-κB decoy oligodeoxynucleotides (ODN) on Kupffer cells (KCs) activation. Methods: KCs were isolated and randomly divided into three groups: (1) a control group; (2) an LPS stimu1ation group and (3) an NF-κB decoy group, in which the KCs were transduced with an NF-κB decoy ODN prior to LPS stimulation. Following 6 hours of LPS stimulation, the NF-κB activity was assayed by an electrophoretic mobility shift assay (EMSA). The CD80 mRNA expression in the KCs was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the production of TNF-α and IL-6 in the supernatant was measured by an enzyme-linked immune sorbent assay (ELISA). Results: The NF-κB decoy ODN could efficiently inhibit KCs activation by LPS stimulus. The NF-κB activity was significantly decreased to 0.53 fold as compared with the LPS group. The CD80 mRNA expression, TNF-α production, and IL-6 level were significantly decreased to 0.46, 0.37, and 0.60 fold, respectively. Conclusion: The NF-κB decoy ODN could efficiently suppress transcription activity of NF-κB and inhibit co-stimulatory molecules and cytokines expression by KCs, which afford reliable experimental data for the in vivo application of NF-κB decoy ODN. Keywords: Cytokines, Kupffer cells, LPS, NF-κB, oligodeoxynucleotides, TNF-α.

NF-κB decoy oligodeoxynucleotide inhibits wear particle-induced inflammation in a murine calvarial model.

Wear particles induce periprosthetic inflammation and osteolysis through activation of nuclear factor kappa B (NF-κB), which up-regulates the downstream target gene expression for proinflammatory cytokines in macrophages. It was hypothesized that direct suppression of NF-κB activity in the early phases of this disorder could be a therapeutic strategy for preventing the inflammatory response to wear particles, potentially mitigating osteolysis. NF-κB activity can be suppressed via competitive binding with double stranded NF-κB decoy oligodeoxynucleotides (ODNs) that blocks this transcription factor from binding to the promoter regions of targeted genes. In this murine calvarial study, clinically relevant polyethylene particles (PEs) with/without ODN were subcutaneously injected over the calvarial bone. In the presence of PE particles, macrophages migrated to the inflammatory site and induced tumor necrosis factor alpha (TNF-α) and receptor activator of nuclear factor kappa B ligand (RANKL) expression, resulting in an increase in the number of osteoclasts. Local injections of ODN mitigated the expression of TNF-α, RANKL, and induced the expression of two anti-inflammatory, antiresorptive cytokines: interleukin-1 receptor antagonist and osteoprotegerin. Local intervention with NF-κB decoy ODN in early cases of particle-induced inflammation in which the prosthesis is still salvageable may potentially preserve periprosthetic bone stock.