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Nuclear Factor-κB (NF-κB) is a transcription factor regulating several genes involved in important physiological and pathological processes. NF-κB has been found constitutively activated in many inflammatory/immune diseases. In addition, a positive correlation between persistent activation of NF-κB and tumor promotion has been demonstrated. Since the IKK (IκB kinase) activation is an indispensable component of all pro-inflammatory signaling pathways leading to NF-κB activation, considerable efforts have been done in order to develop novel anti-inflammatory therapeutics targeting IKK. Association of the IKK complex relies on critical interactions between the C-terminus NBD (NEMO binding domain) of the catalytic subunits IKKα and IKKβ, and the regulatory subunit NEMO (NF-κB Essential Modulator). Thus, this IKK/NEMO interacting region provides an attractive target to prevent the IKK complex formation and NF-κB activation. In this regard, we have identified non-peptide small molecule disruptors of IKKβ/NEMO complex through a structure-based virtual screening (SBVS) of the NCI chemical library. Phenothiazine 22 and its close analogues (22.2, 22.4 and 22.10) were able to reduce nitrite production and iNOS mRNA expression in J774 murine macrophages stimulated with LPS for 24h. These effects were associated with a reduced NF-κB/DNA binding activity as well as a decreased expression of phosphorylated IKKβ, IκBα and NF-κB/p65 in these cells. These observations suggest that compound 22 and its three structural analogues by inhibiting IKKβ/NEMO association mediate the blockage of NF-κB signaling pathway and may prove effective in treatment of diseases in which the IKK/NF-κB pathway is dysregulated., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Macrophages play crucial roles in atherosclerotic immune responses. Recent investigation into macrophage autophagy (AP) in atherosclerosis has demonstrated a novel pathway through which these cells contribute to vascular inflammation. AP is a cellular catabolic process involving the delivery of cytoplasmic contents to the lysosomal machinery for ultimate degradation and recycling. Basal levels of macrophage AP play an essential role in atheroprotection during early atherosclerosis. However, AP becomes dysfunctional in the more advanced stages of the pathology and its deficiency promotes vascular inflammation, oxidative stress, and plaque necrosis. In this paper, we will discuss the role of macrophages and AP in atherosclerosis and the emerging evidence demonstrating the contribution of macrophage AP to vascular pathology. Finally, we will discuss how AP could be targeted for therapeutic utility.

Nuclear factor-kappaB (NF-kappaB) is involved in several pathological processes, such as inflammation. Pro-inflammatory genes expression can be down-regulated by using an oligonucleotide (ODN) decoy to NF-kappaB. Cationic liposomes are largely used to improve ODN uptake into cells, although a higher transfection efficiency and a lower toxicity are required to use them in therapy. In this work, we investigated the potential of a novel liposome formulation, based on the recently synthesised cationic lipid (2,3-didodecyloxypropyl) (2-hydroxyethyl) dimethylammonium bromide (DE), as the delivery system for a double stranded ODN decoy to NF-kappaB. Liposomes composed of DE or DE mixed with 1,2-dioleyl-sn-glycero-3-phosphoethanolamine or cholesterol as helper lipids were complexed with ODN at different +/- charge ratios. In vitro uptake and the effect of ODN, naked or complexed with DE-containing liposomes, were evaluated in lipopolysaccharide-stimulated RAW 264.7 macrophages. The use of helper lipids increased liposome physical stability up to 1 year at 4 degrees C. ODN complexed with DE/cholesterol liposomes, at the +/- charge ratio of 8, showed a limited cytotoxicity and the highest inhibition of nitrite production, inducible nitric oxide synthase protein expression and NF-kappaB/DNA binding activity. Confocal microscopy confirmed a high ODN cell uptake obtained with DE/cholesterol liposomes at the highest +/- charge ratio.

We investigated the effect of PS ( 1) on nitrite and reactive oxygen species (ROS) production in J774 macrophages stimulated with bacterial lipopolysaccharide (LPS) for 24 h. PS ( 1) inhibited in a concentration-dependent manner nitrite and ROS production as well as inducible nitric oxide synthase (iNOS) protein expression induced by LPS. Incubation of cells with PS ( 1) determined a significant decrease of nuclear factor-kappaB (NF-kappaB)/DNA binding activity which was correlated with a marked reduction of iNOS mRNA levels. These results show that PS ( 1) inhibits NF-kappaB activation and iNOS gene expression by preventing the reactive species production and suggest a role for this compound in controlling oxidative stress and/or inflammation.

Inducible nitric oxide synthase (iNOS) is an homodimeric enzyme which produces large amounts of nitric oxide (NO) in response to inflammatory stimuli. Several factors affect the synthesis and catalytic activity of iNOS. Particularly, dimerization of NOS monomers is promoted by heme, whereas an intracellular depletion of heme and/or L-arginine considerably decreases NOS resistance to proteolysis. In this study, we found that oxalomalate (OMA, oxalomalic acid, alpha-hydroxy-beta-oxalosuccinic acid), an inhibitor of both aconitase and NADP-dependent isocitrate dehydrogenase, inhibited nitrite production and iNOS protein expression in lipopolysaccharide (LPS)-activated J774 macrophages, without affecting iNOS mRNA content. Furthermore, injection of OMA precursors to LPS-stimulated rats also decreased nitrite production and iNOS expression in isolated peritoneal macrophages. Interestingly, alpha-ketoglutarate or succinyl-CoA administration reversed OMA effect on NO production, thus correlating NO biosynthesis with the anabolic capacity of Krebs cycle. When protein synthesis was blocked by cycloheximide in LPS-activated J774 cells treated with OMA, iNOS protein levels, evaluated by Western blot analysis and (35)S-metabolic labelling, were decreased, suggesting that OMA reduces iNOS biosynthesis and induces an increase in the degradation rate of iNOS protein. Moreover, we showed that OMA inhibits the activity of the iNOS from lung of LPS-treated rats by enzymatic assay. Our results, demonstrating that OMA acts regulating synthesis, catalytic activity and degradation of iNOS, suggest that this compound might have a potential role in reducing the NO overproduction occurring in some pathological conditions.

Nitric oxide (NO) plays an important role in the pathogenesis of celiac disease. We have examined the involvement of nuclear factor-kappaB (NF-kappaB), interferon regulatory factor-1 (IRF-1), and signal transducer and activator of transcription-1alpha (STAT-1alpha) on the synergistic induction of inducible nitric oxide synthase (iNOS) gene expression by gliadin (G) in association with interferon-gamma (IFN-gamma) in RAW 264.7 macrophages. We found that IFN-gamma was efficient in enhancing the basal transcription of the iNOS promoter at 1, 6, and 24 h, whereas G had no effect. The G plus IFN-gamma association caused an increase in iNOS promoter activity which was inhibited by pyrrolidine dithiocarbammate (PDTC) at 6 and 24 h as well as by genistein (Gen) and tyrphostine B42 (TB42) at 1 h, inhibitors of NF-kappaB, IRF-1, and STAT-1alpha activation, respectively. Similarly, the IFN-gamma and G combination treatment led to a higher increase in iNOS mRNA levels at 1, 6, and 24 h compared with IFN-gamma alone. Gen and TB42 inhibited iNOS mRNA levels at 1 h, whereas PDTC inhibited iNOS mRNA levels at 6 and 24 h. In addition, the synergistic induction of iNOS gene expression by G plus IFN-gamma correlated with the induction of NF-kappaB, IRF-1, and STAT-1alpha/DNA binding activity and mRNA expression. In conclusion, our study, which provides evidence that the effect of G on iNOS gene transcription in IFN-gamma-stimulated RAW 264.7 cells can be ascribed to all three transcription factors, may contribute to lead to new insights into the molecular mechanisms governing the inflammatory process in celiac disease.

We investigated the role of constitutive transcription factor nuclear factor kappaB (NF-kappaB) in nitric oxide (NO)-mediated apoptosis in J774 macrophages. Our results show that NF-kappaB is present in untreated J774 cells in a form constitutively active. Incubation of cells with sodium nitroprusside (SNP) and S-nitroso-glutathione (GSNO), two NO-generating compounds, caused: (a) inhibition of constitutive NF-kappaB/DNA binding activity; (b) decrease of cell viability; (c) DNA fragmentation; (d) ApopTag positivity. Pyrrolidine dithiocarbamate (PDTC) and N-alpha-para-tosyl-L-lysine chloromethyl ketone (TLCK), two inhibitors of NF-kappaB activation, showed the same effects of both NO-generating compounds. Furthermore, SNP and GSNO as well as PDTC and TLCK significantly increased the cytoplasmic level of IkappaBalpha. All together these results demonstrate that constitutive NF-kappaB protects J774 macrophages from NO-induced apoptosis. Moreover, these findings show, for the first time, that NO-generating compounds may induce apoptosis in J774 macrophages by down-regulating constitutive NF-kappaB/DNA binding activity and suggest a novel mechanism by which NO induces apoptosis.

We investigated the effect of cyclolinteinone, a sesterterpene from Caribbean sponge Cacospongia linteiformis, on inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2) protein expression in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microgram/ml) caused an increase of both iNOS and COX-2 protein expression, which was prevented in a concentration-dependent fashion by cyclolinteinone (12.5, 25 and 50 microM). Electrophoretic mobility-shift assay indicated that cyclolinteinone blocked the activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for either iNOS or COX-2 induction. Cyclolinteinone also blocked disappearance of I(kappa)B-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show that cyclolinteinone down-regulates iNOS and COX-2 protein expression by inhibiting NF-kappaB activation and suggest that it may represent a novel anti-inflammatory compound capable of controlling the excessive production of prostaglandins and nitric oxide occurring in several inflammatory diseases.

We investigated the effect of PGE2 and iloprost (a prostacyclin analogue) on inducible nitric oxide synthase (iNOS) protein expression and nuclear factor-kappaB (NF-kappaB) activation in lipopolysaccharide (LPS)-stimulated J774 macrophages. Incubation of J774 cells with LPS (10 microg/ml) caused an increase of iNOS protein expression which was prevented in a concentration-dependent fashion by PGE2 (0.1, 1, 10 microM) and iloprost (0.01, 0.1, 1 microM). Electrophoretic mobility shift assay indicated that both prostanoids blocked the activation of NF-kappaB, a transcription factor necessary for NO synthase induction. PGE2 and iloprost also blocked disappearance of I kappaB-alpha from cytosolic fraction and nuclear translocation of NF-kappaB subunits p50 and p65. These results show for the first time that PGE2 and iloprost down-regulate iNOS protein expression by inhibiting NF-kappaB activation and suggest a negative feed-back mechanism that may be important for limiting excessive or prolonged NO production in pathological events.

We investigated the involvement of NF-kappaB in the regulation of COX-2 protein expression and prostaglandin production in LPS-stimulated J774 macrophages. Incubation of J774 cells with LPS (1 microg/ml) for 24 h caused an increase of COX-2 protein expression and accumulation of both PGE2 and 6-keto-PGF1alpha in the cell culture medium. Ammonium pyrrolidinedithiocarbamate (APDC, 0.1, 1, 10 microM) and N-alpha-p-tosyl-L-lysine chloromethylketone (TLCK, 1, 10, 100 microM), two inhibitors of NF-kappaB activation, suppressed in a concentration-dependent manner both LPS-induced COX-2 protein expression and prostanoid generation. Moreover, APDC and TLCK both inhibited the LPS-induced increase of NF-kappaB DNA binding activity and prevented IkappaB-alpha degradation. Our results show for the first time that NF-kappaB is involved in COX-2 protein expression in LPS-stimulated J774 macrophages and suggest that inhibitors of NF-kappaB activation may represent a useful tool for the pharmacological control of inflammation.

Glucocorticoids inhibit inducible-type NO synthase activity in a variety of cell types. We report here that proteins recovered from the medium of dexamethasone-treated J774 macrophages (1, 10, 100 microg/ml) inhibited lipopolysaccharide-stimulated nitrite generation by 10.0 +/- 3.0%, 32.3 +/- 5.3% and 55.0 +/- 6.0%, respectively, and inducible NO synthase mRNA expression in these cells. Immunoblotting analysis of crude and partially purified glucocorticoid-induced proteins with an anti-lipocortin-1 polyclonal antiserum revealed the presence of lipocortin-1-like immunoreactive species with a molecular mass of 35-37 kDa. Furthermore, inhibition of lipopolysaccharide-induced nitrite production by glucocorticoid-induced proteins in J774 cells was reversed by addition of anti-lipocortin-1 neutralizing polyclonal antibody (1:60 dilution; 4 h before lipopolysaccharide). Comparison of glucocorticoid-induced proteins inhibition of both nitrite production and inducible NO synthase mRNA expression suggests that these effects result mainly from inhibition of lipopolysaccharide-mediated inducible NO synthase gene expression. These results indicate that negative regulation of inducible NO synthase by glucocorticoids is, at least in part, mediated by glucocorticoid-induced proteins that involve also members of the lipocortin-like superfamily.

Pre-incubation of J774 murine macrophages with 5,6-dihydroxyindole-2-carboxylic acid (DHICA), a diffusible intermediate in the biosynthesis of eumelanins, leads to a marked increase in the levels of nitric oxide (NO) produced by lipopolysaccharide (LPS)-induced NO-synthase (iNOS). The effect varies with DHICA concentration being maximum at a concentration of 1 x 10(-6)M, and is suppressed by the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA). No stimulation is observed when macrophages are exposed to DHICA after activation with LPS, indicating that the indole does not affect the catalytic activity of iNOS. These results point to a hitherto unrecognized role of DHICA as a chemical messenger mediating interaction between active melanocytes and macrophages in epidermal inflammatory and immune responses.

The effect of opioids on NO production by LPS-stimulated murine macrophages J774 has been investigated. Morphine (mu and k opioid receptor agonist), DAGO (selective mu receptor agonist) and U50-488H (selective k receptor agonist), added (10(-10)-10(-6) M) to the cells 0.5 h before activation with LPS, significantly inhibited NO production. This effect was reverted by naloxone (10(-12)-10(-8) M), an opioid specific antagonist. In contrast, DPDPE and deltorphin II respectively delta 1 and delta 2 receptor agonists (10(-10)-10(-6) M) did not affect NO generation. Morphine was not able to inhibit NO production when added after LPS challenge. The results of the present study indicate that opioids are able to inhibit NO formation in LPS-activated macrophages through the involvement of specific opioid receptors. Moreover, the ability of morphine to inhibit NO production only when given before LPS challenge suggests that the opiate inhibits the induction but not the activity of the inducible NO synthase.

The effect of dexamethasone on the ability of activated J774 macrophages to inhibit platelet aggregation was investigated. After 24h incubation with lipopolysaccharide and gamma-interferon, J774 cells generated large amounts of nitric oxide and inhibited platelet aggregation. Incubation of the cells with dexamethasone (0.01-10 microM) caused a concentration-dependent inhibition of both the NO2- production and the anti-aggregating activity of the cells. The inhibitory effect of dexamethasone was blocked by the glucocorticoid antagonist RU 38486. These data suggested that the protective effect of glucocorticoids in endotoxin-induced hypotension and their immunosuppressive action may both depend, at least in part, on the inhibition by these steroids of the induction of the nitric oxide synthase.

The effect of glucocorticoids on the production of NO2- and NO by the macrophage cell line J774 was investigated. Stimulation of the cells with lipopolysaccharide (LPS) resulted in a time-dependent accumulation of NO2- in the medium, reaching a plateau after 48h. Concomitant incubation of the cells for 24h with dexamethasone (0.001-1.0 microM) or hydrocortisone (0.01-10.0 microM) caused a concentration-dependent inhibition of NO2- formation. The cytosol of J774 cells stimulated with LPS and IFN-gamma produced a time-dependent increase in the release of NO. This was blocked in a concentration-dependent manner by dexamethasone and hydrocortisone, but not progesterone, administered concomitantly with the immunological stimulus. None of these compounds had any effect on the release of NO once the enzyme had been induced. The inhibitory effect of hydrocortisone on NO formation was blocked by cortexolone. These data suggest that part of the anti-inflammatory and immunosuppressive actions of glucocorticoids is due to their inhibition of the induction of the NO synthase.