Distinct and overlapping functions of glutathione peroxidases 1 and 2 in limiting NF-κB-driven inflammation through redox-active mechanisms.

Glutathione peroxidase 2 (GPx2) is one of the five selenoprotein GPxs having a selenocysteine in the active center. GPx2 is strongly expressed in the gastrointestinal epithelium, as is another isoform, GPx1, though with a different localization pattern. Both GPxs are redox-active enzymes that are important for the reduction of hydroperoxides. Studies on GPx2-deficient mice and human HT-29 cells with a stable knockdown (kd) of GPx2 revealed higher basal and IL-1ß-induced expression of NF-κB target genes in vivo and in vitro. The activation of the IKK-IκBα-NF-κB pathway was increased in cultured GPx2 kd cells. Basal signaling was only restored by re-expressing active GPx2 in GPx2 kd cells but not by redox-inactive GPx2. As it is still not clear if the two isoforms GPx1 and GPx2 have different functions, kd cell lines for either GPx1 or GPx2 were studied in parallel. The inhibitory effect of GPx2 on NF-κB signaling and its target gene expression was stronger than that of GPx1, whereas cyclooxygenase (COX)- and lipoxygenase (LOX)-derived lipid mediator levels increased more strongly in GPx1 kd than in GPx2 kd cells. Under unstimulated conditions, the levels of the COX-derived prostaglandins PGE2 and PGD2 were enhanced in GPx2 as well as in GPx1 kd compared to control cells. Specifically, in GPx1 kd cells IL-1ß stimulation led to a dramatic shift of the PGE2/PGD2 ratio towards pro-inflammatory PGE2. Taken together, GPx2 and GPx1 have overlapping functions in controlling inflammatory lipid mediator synthesis and, most probably, exert their anti-inflammatory effects by preventing excessive PGE2 production. In view of the high activity of COX and LOX pathways during inflammatory bowel disease our data therefore provide new insights into the mechanisms of the protective function of GPx1 and GPx2 during colitis as well as inflammation-driven carcinogenesis.

Alkamides from Tropaeolum tuberosum inhibit inflammatory response induced by TNF-α and NF-κB.

ETHNO-PHARMACOLOGICAL RELEVANCE: Tropaeolum tuberosum, commonly known as "Mashua", is one of the plants most frequently used by Andean (Peruvian-Bolivian) people as food and medicine. It is used as a remedy against a wide range of diseases, especially those related with inflammation. OBJECTIVES: This study aims to identify compounds active against inflammatory related conditions. MATERIALS AND METHODS: A bioassay-guided isolation of anti-inflammatory compounds from black and purple tubers of T. tuberosum was performed measuring TNF-α and NF-κB production in THP-1 monocytic cells. RESULTS: The bioassay-guided isolation led to one active compound from purple T. tuberosum, N-oleoyldopamine (1), and another active compound from black T. tuberosum, N-(2-Hydroxyethyl)-7Z,10Z,13Z,16Z-docosatetraenamide (2). Both compounds displayed anti-TNF-α activity with IC50 values of 3.12 ±â€¯0.19 µM and 1.56 ±â€¯0.15 µM, respectively. Also, both compounds suppressed NF-κB with IC50 of 3.54 ±â€¯0.02 µM and 1.77 ±â€¯0.07 µM, respectively. CONCLUSIONS: We identified bioactive compounds from purple and black Tropaeolum tuberosum responsible for their anti-inflammatory activity: N-oleoyldopamine (1) and N-(2-Hydroxyethyl)-7Z,10Z,13Z,16Z-docosatetraenamide (2). This is the first report which isolates these compounds from T. tuberosum and describes their anti-inflammatory activities.

Efficient delivery of NF-κB siRNA to human retinal pigment epithelial cells with hyperbranched cationic polysaccharide derivative-based nanoparticles.

A hyperbranched cationic polysaccharide derivative-mediated small interfering (si)RNA interference strategy was proposed to inhibit nuclear transcription factor-kappa B (NF-κB) activation in human retinal pigment epithelial (hRPE) cells for the gene therapy of diabetic retinopathy. Two hyperbranched cationic polysaccharide derivatives containing the same amount of cationic residues, but with different branching structures and molecular weights, including 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) and amylopectin (DMAPA-Amp) derivatives, were developed for the efficient delivery of NF-κB siRNA into hRPE cells. The DMAPA-Glyp derivative showed lower toxicity against hRPE cells. Furthermore, the DMAPA-Glyp derivative more readily condensed siRNA and then formed the nanoparticles attributed to its higher branching architecture when compared to the DMAPA-Amp derivative. Both DMAPA-Glyp/siRNA and DMAPA-Amp/siRNA nanoparticles were able to protect siRNA from degradation by nuclease in 25% fetal bovine serum. The particle sizes of the DMAPA-Glyp/siRNA nanoparticles (70-120 nm) were smaller than those of the DMAPA-Amp/siRNA nanoparticles (130-180 nm) due to the higher branching architecture and lower molecular weight of the DMAPA-Glyp derivative. In addition, the zeta potentials of the DMAPA-Glyp/siRNA nanoparticles were higher than those of the DMAPA-Glyp/siRNA nanoparticles. As a result, siRNA was much more efficiently transferred into hRPE cells using the DMAPA-Glyp/siRNA nanoparticles rather than the DMAPA-Amp/siRNA nanoparticles. This led to significantly high levels of suppression on the expression levels of NF-κB p65 messenger RNA and protein in the cells transfected with DMAPA-Glyp/siRNA nanoparticles. This work provides a potential approach to promote hyperbranched polysaccharide derivatives as nonviral siRNA vectors for the inhibition of NF-κB activation in hRPE cells.

Oral nuclear factor-κB decoy oligonucleotides delivery system with chitosan modified poly(D,L-lactide-co-glycolide) nanospheres for inflammatory bowel disease.

Chitosan (CS)-modified poly(D,L-lactide-co-glycolide) (PLGA) nanospheres (NS) were developed and evaluated for use with a nuclear factor kappa B (NF-κB) decoy oligonucleotide (ODN) oral delivery system in an experimental model of ulcerative colitis (UC). Decoy ODN-loaded PLGA NS were prepared by an emulsion solvent diffusion method, and the physicochemical properties of NS were investigated. CS-modified PLGA NS (CS-PLGA NS) showed positive zeta potential, while unmodified PLGA NS (plain-PLGA NS) were negatively charged. Decoy ODN uptake studies with Caco-2 cells using confocal laser scanning microscopy (CLSM) indicated that CS-PLGA NS were more effectively taken up by the cells than plain-PLGA NS. Decoy ODN-loaded CS-PLGA NS were able to improve the stability of ODN against DNase I or an acidic medium, such as gastric juice. Daily oral administration of CS-PLGA NS in a rat model significantly improved dextran sulfate sodium-induced diarrhea, bloody feces, shortening of colon length, and myeloperoxidase activity. Furthermore, decoy ODN-loaded CS-PLGA NS were specifically deposited and adsorbed on the inflamed mucosal tissue of the UC model rat. These results suggested that CS-PLGA NS provide an effective means of colon-specific oral decoy ODN delivery in UC.

Síndrome de Sjögren primario: expresión del factor NF-kB en glándula salival menor / Primary Sjögren's syndrome: expression of NF-kB in minor salivary glands

Objetivo. Evaluar la translocación nuclear del factor NF-kB en las glándulas salivales menores de pacientes con síndrome de Sjögren primario (SSp). Métodos. Se realizaron biopsias de glándulas salivares menores en 24 pacientes mujeres con diagnóstico de SSp del Servicio de Reumatología del Hospital Rivadavia. Las glándulas fueron teñidas con H&E y la inmunohistoquímica para NF-kB, fueron clasificados de acuerdo con la puntuación de Chisholm y Masson. Resultados. Las biopsias de pacientes con SSp (H&E) mostraron infiltrados linfoplasmocitarios, formando focos periacinares y periductales cuyo número depende del estadio de la enfermedad. En las fases III y IV se observa la destrucción de los acinos y, en algunos casos, fibrosis. En las biopsias con diagnóstico de sialoadenitis observamos elementos linfoplasmocitarios intersticiales dispersos y también neutrófilos polimorfonucleares. Las biopsias de labio de pacientes con diagnóstico clínico serológico de SSp mostraron la translocación nuclear de NF-kB en los linfocitos de infiltración focal y en el epitelio de los acinos adyacentes a los infiltrados. En acinos y en las estructuras ductales alejadas de los infiltrados, no observamos translocación nuclear. Sin embargo, en pacientes con sialoadenitis se observaron linfocitos intersticiales con translocación nuclear pero no en acinos y ductos. Los pacientes con SSp con glándula normal no mostraron translocación nuclear del factor NF-kB, ni en acinos ni en el conducto. Conclusiones. Estos resultados nos permiten inferir la importancia de la interacción linfocitosepitelio y, la activación del factor NF-kB en pacientes con diagnóstico de SSp (AU)

Purpose. To evaluate nuclear NF-kB translocation in minor salivary glands (mSG) of human primary Sjögren Syndrome (pSS). Methods. Lip biopsies’ mSG were done in 24 female patients with pSS from the Rheumatology Service of Rivadavia Hospital. Glands were stained with H&E and immunostained for NF-kB. Specimens were classified according to the Chisholm and Masson score. Results. The biopsies (H&E staining) showed lymphoplasmocitic infiltrates, forming periacini and periductal focuses which number depending on the stage of the disease. In stages III and IV there was acini destruction and, in some cases, fibrosis. In the biopsies with a diagnosis of sialadenitis we observed interstitially-dispersed lymphoplasmocitic elements and also polimorphonuclear neutrophils. The lip biopsies’ mSG of patients with clinical-serological diagnosis of pSS showed nuclear translocation of NF-kB in lymphocytes of focal infiltrates and in the acini epithelium adjacent to the infiltrates. In distal acini and ductal structures from the infitrates we did not observe nuclear translocation. However, in SSp patients with sialadenitis interstitial lymphocytes with nuclear translocation were observed but neither in the acini or the ducts. SSp patients with normal glands did not show nuclear translocation of NF-kB factor either in the acini or in the ducts. Conclusions. These results allow us to infer the importance of lymphocyte-epithelium interaction on the activation of NF-kB in human pSS (AU)

NF-kappaB and AP-1 are key signaling pathways in the modulation of NAD(P)H:quinone oxidoreductase 1 gene by mercury, lead, and copper.

We have previously shown that Hg(2+), Pb(2+), and Cu(2+), significantly modulate the expression of NAD(P):quinone oxidoreductase 1 (Nqo1) in Hepa 1c1c7 cells through oxidative stress-dependent mechanisms. In the current study, we examined the role of redox-sensitive transcription factors, NF-kappaB and AP-1 signaling pathways in the modulation of Nqo1 by heavy metals. Our results show that the depletion of cellular GSH using L-buthionine-(S,R)-sulfoximine further potentiated the heavy metal-mediated induction of Nqo1 at the mRNA and activity levels. The NF-kappaB activator, PMA, significantly abolished the metal-mediated effects on Nqo1 mRNA and activity. In parallel, the NF-kappaB inhibitor, PDTC, further potentiated the Pb(2+)- and Hg(2+)-mediated induction of Nqo1 mRNA and activity levels, respectively. Inhibition of AP-1 upstream signaling pathway such as JNK by SP600125 significantly suppressed heavy metal-mediated induction of Nqo1 mRNA and activity levels. In contrast, inhibition of ERK by U0126 further potentiated heavy metal-mediated effects on Nqo1 mRNA, while only potentiated Hg(2+)-mediated induction of Nqo1 activity. Furthermore, p38 MAPK inhibitor, SB203580 further potentiated Pb(2+)- and Cu(2+)-mediated effects at the mRNA levels, whereas did not alter the activity levels. These results clearly demonstrate that activation of NF-kappaB negatively regulates the expression of Nqo1 by heavy metals, whereas AP-1 signaling pathways differentially modulates the heavy metal-mediated effects.

Injection of nuclear factor-kappa B decoy into the sciatic nerve suppresses mechanical allodynia and thermal hyperalgesia in a rat inflammatory pain model.

STUDY DESIGN: In vitro and in vivo study of a rat inflammatory pain model using nuclear factor-kappa B decoy. OBJECTIVES: To investigate transduction efficiency of nuclear factor-kappa B decoy into dorsal root ganglion, both in vivo and in vitro, and to assess the suppression of inflammatory pain by nuclear factor-kappa B decoy. SUMMARY OF BACKGROUND DATA: Transcription factor nuclear factor-kappa B is reported to play a crucial role in regulating pro-inflammatory cytokine gene expression. We hypothesized that inhibiting nuclear factor-kappa B gene expression with nuclear factor-kappa B decoy may suppress inflammatory pain. METHODS: Nuclear factor-kappa B decoy-fluorescein isothiocyanate (FITC) was induced in explant culture, endoneurally injected into the sciatic nerve, and its transduction efficiency into dorsal root ganglion measured. For behavioral testing, 12 rats received plantar injections of complete Freund's adjuvant and were divided into 3 groups: decoy group, single endoneural injection of 10 microL of nuclear factor-kappa B decoy (n = 4); saline group, single endoneural injection of 10 microL of saline (n = 4); and naïve group, untreated (n = 4). Behavioral testing was performed using von Frey filaments and a Hargreaves device with a heat source. RESULTS: Total transduction efficiency of nuclear factor-kappa B decoy-FITC was 53.6% in vitro and 20.5% in vivo. No statistical differences were observed with respect to types of cell size distributions of all FITC-positive neurons. In behavioral testing, withdrawal latencies or thresholds significantly differed between the decoy group and the saline group from 2 to 14 days after surgery in the mechanical allodynia experiments, and from 2 to 3 days after surgery in the thermal hyperalgesia experiments. CONCLUSIONS: Nuclear factor-kappa B decoy was conveyed and transduced into dorsal root ganglion both in vivo and in vitro. Additionally, nuclear factor-kappa B decoy reduced mechanical allodynia and thermal hyperalgesia in the rat inflammatory pain model, suggesting that inhibition of nuclear factor-kappa B with nuclear factor-kappa B decoy may represent a key mechanism for mediating inflammation or reducing inflammatory pain.

Intravenous administration of mannosylated cationic liposome/NFkappaB decoy complexes effectively prevent LPS-induced cytokine production in a murine liver failure model.

The purpose of this study was to inhibit endotoxin induced cytokines production and liver injury by liver non-parenchymal cell (NPC) selective delivery of nuclear factor kappaB (NFkappaB) decoy using mannosylated cationic liposomes (Man-liposomes). In this study, we examined the distribution, inhibitory effect on cytokines production and ALT/AST of intravenously injected Man-liposome/NFkappaB decoy complex. Man-liposome/[(32)P] NFkappaB decoy complexes mostly accumulated in the liver, preferentially in NPC. In a murine lipopolysaccharide-induced liver failure model, the production of tumor necrosis factor-alpha (TNFalpha), IFNgamma, IL1-beta, ALT and AST were effectively reduced by Man-liposome complexes. However, cationic or galactosylated cationic liposome complexes could not inhibit TNFalpha production.

Sequestration of serum response factor in the hippocampus impairs long-term spatial memory.

The formation of long-term memory has been shown to require protein kinase-mediated gene expression. One such kinase, mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), can lead to the phosphorylation of serum response factor (SRF) and Elk-1, enhancing the expression of target genes. However, a direct involvement of these transcription factors in memory storage has not been demonstrated. We have employed an oligonucleotide decoy technique to interrogate SRF and Elk-1. Previously, it has been shown that intra-amygdalal infusion of small double-stranded decoy oligonucleotides for nuclear factor-kappaB (NFkappaB) can impair long-term memory for fear-potentiated startle. Using this approach, we found that intra-hippocampal infusion of NFkappaB decoy oligonucleotides also impairs long-term spatial memory, consistent with a role for this factor in long-term memory storage. Decoy oligonucleotides containing the binding site for SRF, as confirmed by shift-western, did not influence memory acquisition but impaired long-term spatial memory. Analysis of search behavior during the transfer test revealed deficits consistent with a loss of precise platform location information. In contrast, oligonucleotides with a binding site for either Elk-1 or another target of ERK activity, SMAD3/SMAD4, did not interfere with memory formation or storage. These findings suggest that SRF-mediated gene expression is required for long-term spatial memory.

Selective blockade of NF-kappa B activity in airway immune cells inhibits the effector phase of experimental asthma.

Knockout mice studies have revealed that NF-kappaB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-kappaB also plays a role in the effector phase of airway allergy and whether inhibiting NF-kappaB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-kappaB activity in the lungs during OVA challenge. Intratracheal administration of NF-kappaB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-kappaB activity in the airways upon OVA provocation. NF-kappaB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-kappaB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-kappaB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.

Controlled release of NFkappaB decoy oligonucleotides from biodegradable polymer microparticles.

The objective of this study was to evaluate a poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) delivery system for nuclear factor-kappa B (NFkappaB) decoy phosphorothioated oligonucleotides (ODNs). PLGA/PEG microparticles loaded with ODNs were fabricated with entrapment efficiencies up to 70%. The effects of PEG contents (0, 5, and l0 wt%), ODN loading densities (0.4, 4, and 40 microg/mg), and pH of the incubation medium (pH 5, 7.4. and 10) on ODN release kinetics from the PLGA/PEG microparticles were investigated in vitro for up to 28 days. The release profiles in pH 7.4 phosphate buffered saline (PBS) were characterized by an initial burst during the first 2 days, a linear release phase until day 18, and a final release phase for the rest of the period. Up to 85% of the ODNs were released after 28 days in pH 7.4 PBS regardless of the ODN loading density and PEG content. Higher ODN loading densities resulted in lower entrapment efficiencies and greater initial burst effects. The bulk degradation of PLGA was not significantly affected by the PEG content and ODN loading density, but significantly accelerated at acidic buffer pH. Under acidic and basic conditions, the aggregation of microparticles resulted in significantly lower cumulative mass of released ODNs than that released at neutral pH. The effects of pH were reduced by the incorporation of PEG into PLGA microparticles. Since the PLGA degradation products are acidic, PLGA/PEG microparticles might provide a better ODN delivery vehicle than PLGA microparticles. These results suggest that PLGA/PEG microparticles are useful as delivery vehicles for controlled release of ODNs and merit further investigation in cell culture and animal models of glioblastoma.

Suppressed severity of collagen-induced arthritis by in vivo transfection of nuclear factor kappaB decoy oligodeoxynucleotides as a gene therapy.

OBJECTIVE: In both rheumatoid arthritis and collagen-induced arthritis (CIA), the nuclear factor kappaB (NF-kappaB) transcription factor plays a pivotal role in the coordinated transactivation of many cytokines related to pathogenesis. This study investigated whether synthetic double-stranded DNA that show a high affinity for NF-kappaB could be introduced in vivo as "decoy" cis elements to bind the transcription factor and block the activation of such proinflammatory cytokine genes as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNFalpha), and thus suppress the severity of joint destruction. METHODS: NF-kappaB decoy oligonucleotides (ODN) were introduced by an intraarticular injection into the bilateral hind ankle joints of CIA rats using the hemagglutinating virus of Japan (HVJ)-liposome method. Joint destruction was evaluated by histology and radiography. IL-1 and TNFalpha levels were assessed by enzyme-linked immunosorbent assay and Northern blot analysis. RESULTS: Using the HVJ-liposome method, the presence of fluorescein isothiocyanate-labeled ODN in the synovium was confirmed until 28 days after intraarticular injection. In vivo transfection of NF-kappaB decoy ODN by an intraarticular injection into CIA rats decreased the severity of hind-paw swelling. Histologic and radiographic studies showed a marked suppression of joint destruction treated by NF-kappaB decoy ODN transfection. This treatment method also suppressed the production of IL-1 and TNFalpha in the synovium of arthritic joints. CONCLUSION: The present results demonstrate that administration of NF-kappaB decoy ODN in arthritic joints of rats with CIA led to an amelioration of arthritis. These findings suggest that intraarticular transfection of NF-kappaB decoy ODN may provide a useful therapeutic approach for the treatment of inflammatory arthritis.

Synergistic transcriptional activation of the IL-8 gene by NF-kappa B p65 (RelA) and NF-IL-6.

Transcriptional activation of the IL-8 gene by several inflammatory mediators, including the cytokines IL-1 and TNF-alpha, is mediated through sequences located between nucleotide -94 and -71 of the IL-8 promoter. Because adjacent binding sites for the inducible transcription factors NF-kappa B and NF-IL-6 are located within this region, we examined the functional interaction of these two transcription factor families in IL-8 gene regulation. Maximal transcriptional activation by PMA in Jurkat T lymphocytes was shown to require intact binding sites for both NF-kappa B and NF-IL-6. Electrophoretic mobility shift analysis indicates that NF-IL-6, as well as other related members of this family, bind specifically to the NF-IL-6 site in the IL-8 promoter. In addition, NF-kappa B p65 (RelA), but not NF-kappa B p50 (NFKB1), binds specifically to the NF-kappa B site. When incubated together, RelA and NF-IL-6/C/EBP form a ternary complex with this region of the IL-8 promoter; this binding is dependent on intact binding sites for both NF-IL-6 and RelA. Transient cotransfection analyses indicate that the cooperative association of NF-IL-6 and RelA with the IL-8 promoter results in synergistic transcriptional activation. Mutational analyses of RelA demonstrate that the C-terminal transactivation domain and the DNA binding domain are required for synergistic activation with NF-IL-6. In addition, overexpression of the NF-kappa B inhibitor molecule, I kappa B, abolished the RelA- and RelA/NF-IL-6-dependent synergistic activation. These data demonstrate that RelA and members of the C/EBP/NF-IL-6 family can functionally cooperate in transcriptional activation of the IL-8 gene and suggest a common mechanism for inducible regulation of cytokine gene expression.