Immunomodulatory Effects of Curcumin

Curcumin (diferuloylmethane), found in the spice turmeric, exhibits anti-inflammatory, antioxidant, and chemopreventive activities. However, the effect of curcumin on the immunological responses largely remains unknown. In this study we have investigated the effect of curcumin on mitogen (phytohaemagglutinin; PHA) stimulated T-cell proliferation, natural killer (NK) cell cytotoxicity, production of cytokines by human peripheral blood mononuclear cells (PBMCs), nitric oxide (NO) production in mouse macrophage cells, RAW-264.7. Furthermore, we have carried out an electromobility shift assay to elucidate the mechanism of action of curcumin at DNA protein interaction level. We observed that curcumin inhibits PHA-induced T-cell proliferation, interleukin-2 production, NO generation, and lipopolysachharide-induced nuclear factor-κB (NF-κB) and augments NK cell cytotoxicity. Our results suggest that curcumin most likely inhibits cell proliferation and cytokine production by inhibiting NF-κB target genes involved in the induction of these immune parameters.     

Curcumin inhibits cell proliferation and promotes apoptosis in human osteoclastoma cell through MMP-9, NF-κB and JNK signaling pathways

Curcumin is a polyphenol compound extracted from ginger plant, turmeric, commonly used in a variety of food coloring and flavoring additives. Curcumin has many effects such as anti-inflammatory, anti-tumor, antioxidant and anti-microbial effects. However, the mechanism underlying the anti-cancer effect of curcumin on human osteoclastoma (Giant cell tumor, GCT) cells remains unclear. The objectives of this study were to determine the efficacy of curcumin on proliferation and apoptosis of GCT cells and its related mechanisms. In our study, cell viability, cellular apoptosis and caspase-3 activity of GCT cells were analyzed using 3.3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FCM) assay and commercial kits, respectively. Next, MMP-9 gene expression quantity, NF-κB activity and JNK protein expression of GCT cells were tested with real-time polymerase chain reaction (RT-PCR), commercial kits and western blotting assay, respectively. Firstly, MMP-9, NF-κB and JNK inhibitors were added into GCT cells and which was researched the mechanism of curcumin on human GCT cells. In this study, the efficacy of curcumin reduced cell viability, induced cellular apoptosis and increased caspase-3 activity of GCT cells. Furthermore, curcumin inhibited the MMP-9 gene expression quantity and NF-κB activity, and activated JNK protein expression in GCT cells. Meanwhile, down-regulation of MMP-9 gene expression quantity and NF-κB activity could promote the anti-cancer effect of curcumin on cell viability of GCT cells. Interesting, down-regulation of JNK protein expression could also reversed the anti-cancer effect of curcumin on cell viability of GCT cells. Taken together, our results suggest that curcumin inhibits cell proliferation and promotes apoptosis in osteoclastoma cell through suppression of MMP-9 and NF-κB, and activation JNK signaling pathways.     

Nitric oxide regulates clonal expansion and activation-induced cell death triggered by staphylococcal enterotoxin B.

Increased interest has recently been focused on nitric oxide (NO) due to its several biological roles. Apart from being a potential antimicrobial defense and a mediator of autoimmune diseases, NO also appears to be a strong mediator of T-cell responses. In this report, we have characterized the effect of NO on T-cell function. For this purpose, we analyzed in vivo T-cell responses to the bacterial superantigen produced by Staphylococcus aureus, staphylococcal enterotoxin B (SEB), in mice treated with an NO donor (isosorbide dinitrate [ISO]). We show that ISO partially prevents SEB-triggered activation-induced cell death of spleen and lymph node CD4Vbeta8+ T cells but not of CD8Vbeta8+ T cells. SEB-promoted thymic deletion is not abolished by ISO; however, a rapid recovery of thymocyte numbers due to increased double-positive (DP) CD4+ CD8+ thymocyte proliferation was clearly observed in ISO-treated, SEB-injected mice but not in controls (untreated SEB-injected mice). It was also found that ISO inhibits the early SEB-induced cell proliferation (i.e., that found 12 h after SEB injection), accelerating the clonal anergy usually observed 3 days after SEB injection. Inhibition of T-cell proliferation by the NO donor does not appear to be due to inhibition of cytokine production. These results show that NO interferes with apoptosis and facilitates thymic proliferation of DP thymocytes, although it inhibits peripheral T-cell proliferation.     

The effect of proteasome inhibitor MG132 on experimental inflammatory bowel disease

Immunoproteasome up-regulation enhances the processing of nuclear factor-κB (NF-κB) and degradation of IκBα, which correlates with increased amounts of NF-κB in the various cells. Aberrant activation of NF-κB is involved in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to elucidate the effect of proteasome inhibitor MG132 on experimental IBD. We investigated the effects of MG132 on intestinal inflammation and epithelial regeneration in both interleukin-10-deficient (IL-10^−/−) mice and mice with dextran sulphate sodium (DSS)-induced colitis. Body weight, histological findings and tumour necrosis factor (TNF)-α mRNA expression, epithelial cell proliferation and NF-κB p65 activity in colonic tissues were examined. The effects of MG132 on cell proliferation, migration and multiple drug resistance 1 (MDR1) gene expression were determined in vitro. MG132 ameliorated intestinal inflammation of IL-10^−/− mice by decreasing TNF-α mRNA expression in the colonic tissues, which was associated with suppression of NF-κB activation, and reduced significantly the number of Ki-67-positive intestinal epithelial cells. On the other hand, MG132 did not reduce intestinal inflammation in mice with DSS-induced colitis, and delayed significantly the recovery of body weight and epithelial regeneration. MG132 also suppressed significantly epithelial cell proliferation, cell migration and MDR1 gene expression in vitro. Proteasome inhibition reduces T cell-mediated intestinal inflammation, but may interrupt both epithelial regeneration and barrier function of colonic mucosa. Optimal use of proteasome inhibitor should be kept in mind when we consider its clinical application for patients with IBD.     

Inhibition of interleukin-12 production in mouse macrophages via suppression of nuclear factor-kappaB binding activity by Phyllostachys nigra var. henonis

Pharmacological inhibition of interleukin-12 (IL-12) production may be a therapeutic strategy for preventing development and progression of disease in experimental models of autoimmunity. The acetone fraction prepared from bamboo, Phyllostachys nigra var. henonis, potently inhibited the Lipo polysaccharide (LPS)-induced IL-12 production from RAW264.7 monocytic cell-line in a dose-dependent manner. The repressive effect mapped to a region in the IL-12 gene promoter containing a binding site for NF-κB. Furthermore, activation of macrophages by LPS resulted in markedly enhanced binding activity to the NF-κB site, which significantly decreased upon addition of the acetone fraction of Phyllostachys nigra var. henonis. This indicated that the acetone fraction inhibited IL-12 production in LPS-activated macrophages via inhibition of NF-κB binding activity.     

Inhibition of natural killer (NK) cell activity against varicella-zoster virus (VZV)-infected fibroblasts and lymphocyte activation in response to VZV antigen by nitric oxide-releasing agents

The addition of nitric oxide (NO)-releasing agents, S-nitroso-N-acetyl-DL-penicillamine (SNAP), 1-hydroxy-2-oxo-2,3-bis(2-aminoethyl)-1-triazene (NOC18), 30{(±)-(E)-ethyl-2′-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl}-pyridine (NOR4) significantly inhibited NK cell activity against VZV-infected cells, while antibody-dependent cell-mediated cytotoxicity (ADCC) against VZV-infected cells was unaffected. Interferon-alpha (IFN-α) production by non-adherent peripheral blood mononuclear cells (NPBMC) cultured with VZV-infected cells was decreased by the addition of NO-releasing agents. Lymphocyte proliferation and the expression IL-2 receptor (CD25) in response to VZV antigen were also inhibited by the addition of NO-releasing agents. These results suggest that the production of NO by an inflammatory process may lead to inhibition of NK cell- and T cell-mediated immunity to VZV infection.     

Inhibition of phosphoantigen-mediated gammadelta T-cell proliferation by CD4+ CD25+ FoxP3+ regulatory T cells

Tumour growth promotes the expansion of CD4(+) CD25(+) FoxP3(+) regulatory T cells (Tregs) which suppress various arms of immune responses and might therefore contribute to tumour immunosurveillance. In this study, we found an inverse correlation between circulating Treg frequencies and phosphoantigen-induced gammadelta T-cell proliferation in cancer patients, which prompted us to address the role of Tregs in controlling the gammadelta T-cell arm of innate immune responses. In vitro, human Treg-peripheral blood mononuclear cell (PBMC) co-cultures strongly inhibited phosphoantigen-induced proliferation of gammadelta T cells and depletion of Tregs restored the impaired phosphoantigen-induced gammadelta T-cell proliferation of cancer patients. Tregs did not suppress other effector functions of gammadelta T cells such as cytokine production or cytotoxicity. Our experiments indicate that Tregs do not mediate their suppressive activity via a cell-cell contact-dependent mechanism, but rather secrete a soluble non-proteinaceous factor, which is independent of known soluble factors interacting with amino acid depletion (e.g. arginase-diminished arginine and indolamine 2,3-dioxygenase-diminished tryptophan) or nitric oxide (NO) production. However, the proliferative activity of alphabeta T cells was not affected by this cell-cell contact-independent suppressive activity induced by Tregs. In conclusion, these findings indicate a potential new mechanism by which Tregs can specifically suppress gammadelta T cells and highlight the strategy of combining Treg inhibition with subsequent gammadelta T-cell activation to enhance gammadelta T cell-mediated immunotherapy.     

KUNBI-BOSHIN-HANGAM-TANG STIMULATES NITRIC OXIDE PRODUCTION THROUGH ACTIVATION OF NUCLEAR FACTOR-κB

The objective of the currently study was to determine the effect of Kunbi-Boshin-Hangam-Tang (KBH-Tang) on the production of nitric oxide (NO). Stimulation of RAW 264.7 cells with KBH-Tang after the treatment of recombinant interferon-γ (rIFN-γ) resulted in increased NO synthesis. KBH-Tang partially increased NO synthesis by itself. When KBH-Tang was used in combination with rIFN-γ, there was a marked cooperative induction of NO synthesis in a dose-dependent manner. This increase in NO synthesis was reflected as increased amount of inducible NO synthase (iNOS) protein. NO production was inhibited by N^G-monomethyl-_L-arginine (N^GMMA). Furthermore, activation of nuclear factor (NF)-κB was increased by KBH-Tang. These results suggest that KBH-Tang may stimulate the NO production through the activation of the NF-κB.     

Vγ2Vδ2 T-cell co-stimulation increases natural killer cell killing of monocyte-derived dendritic cells

Interactions between natural killer (NK) cells and dendritic cells (DC) affect maturation and function of both cell populations, including NK cell killing of DC (editing), which is important for controlling the quality of immune responses. We also know that antigen-stimulated Vγ2Vδ2 T cells co-stimulate NK cells via 4-1BB to enhance the killing of tumour cell lines but we do not know what regulates 4-1BB expression or whether other NK effector functions including DC killing, might also be influenced by NK–γδ T-cell cross-talk. Here we show that antigen-stimulated γδ T cells co-stimulate NK cells through inducible T-cell co-stimulator (ICOS)– ICOS ligand (ICOSL) and this signal increases NK cell killing of autologous DC. Effects of NK–γδ T-cell co-culture, which could be reproduced with soluble ICOS-Fc fusion protein, included increased CD69 and 4-1BB expression, interferon-γ, tumour necrosis factor-α, macrophage inflammatory protein-1β, I-309, RANTES and sFas ligand production, as well as elevated mRNA levels for co-stimulatory receptors OX40 (TNFRSF4) and GITR (TNFRSF18). Hence, ICOS–ICOSL co-stimulation of NK by Vγ2Vδ2 T cells had broad effects on NK phenotype and effector functions. The NK–γδ T-cell cross-talk links innate and antigen-specific lymphocyte responses in the control of cytotoxic effector function and DC killing.     

Lipopolysaccharide stimulates Epac1-mediated Rap1/NF-kappaB pathway in Raw 264.7 murine macrophages

Nuclear factor-kappa B (NF-κB) is regulated by various stimulants to show many physiological results. Lipopolysaccharide (LPS) activates NF-κB through toll-like receptor 4 (TLR4)-dependent signal transduction. LPS-treatment also produces cyclic AMP (cAMP) in Raw 264.7 murine macrophages. Two principal effector proteins for cAMP are protein kinase A (PKA) and cAMP-responsive guanine nucleotide exchange factor (Epac), a Rap GDP exchange factor. Here, we investigated whether NF-κB can be activated by cAMP production through Epac1-mediated Rap1 activation by using Epac-specific cAMP analogue, 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT). NF-κB activity was increased by the treatment with CPT but it was reduced by co-transfection with dominant negative of Rap1 (Rap1N17). In conclusion, NF-κB activation should be regulated through Epac1-mediated Rap1 stimulation for LPS-induced inflammatory responses in murine macrophages. It suggests that Epac1-mediated Rap1/NF-κB pathway could be helpful for interpretation on various cAMP-mediated physiological responses and it could be used as a target to control their pathological abnormalities.     

Divergent effects of FcγRIIIA ligands on the functional activities of human natural killer cells in vitro

The Fcγ receptor (R)IIIA (CD16) plays an important role in regulating the cytotoxic and non-cytotoxic functions of human natural killer (NK) cells. Some anti-CD 16 monoclonal antibodies (mAb) have been shown to stimulate NK activity, while human monomeric (m) IgG induces dose-dependent inhibition of NK activity. To explore further these interactions mediated via FcγRIIIA, purified NK cells were cultured for 2–3 days in the presence of mIgG, 3G8 mAb, interleukin-2 (IL-2) or a combination of mIgG or 3G8 with IL-2. Binding of mIgG or 3G8 to FcγRIIIA induced divergent effects of functions of cultured NK cells: 3G8 mAb + IL-2 induced dose-dependent inhibition of proliferation attributable to apoptosis; in contrast, mIgG + IL-2 significantly increased NK cell proliferation. Incubation of NK cells in the presence of mIgG up-regulated expression of surface activation markers (CD69, IL-2Rα, ICAM-1), cytotoxicity, cytokine production (IL-1β, IFN-γ and TNF-α) and release of soluble IL-2R. Thus, mIgG binding to FcγRIIIA induced stimulatory signals in human NK cells, leading to up-regulation of IL-2Rα expression, cell proliferation and cytokine release.     

Glycogen synthetase kinase 3 inhibition drives MIC-A/B to promote cytokine production by human natural killer cells in Dengue virus type 2 infection

Dengue virus (DENV) is the most widespread arbovirus worldwide and is responsible for major outbreaks. The host's immune response plays a crucial role in controlling this infection but might also contribute to the promotion of viral spread and immunopathology. In response to DENV infection, NK cells preferentially produce cytokines and are cytotoxic in the presence of specific antibodies. Here, we identified that DENV-2 inhibits glycogen synthase kinase 3 (GSK-3) activity to subsequently induce MHC class-1-related chain (MIC) A and MIC-B expression and IL-12 production in monocyte-derived DCs, independently of the STAT-3 pathway. The inhibition of GSK-3 by DENV-2 or small molecules induced MIC-A/B expression on monocyte-derived DCs, resulting in autologous NK cells of a specific increase in IFN-γ and TNF-α production, in the absence of direct cytotoxicity. Together, these findings identified GSK-3 as a regulator of MIC-A/B expression and suggested its role in DENV-2 infection to specifically induce cytokine production by NK cells.     

Role of IFN-alpha/beta and IL-12 in the activation of natural killer cells and interferon-gamma production during experimental infection with Trypanosoma cruzi

Control of Trypanosoma cruzi infection depends largely upon the production of interferon (IFN)-gamma. During experimental infection this cytokine is produced early, mainly by natural killer (NK) cells and later by T cells. As NK cells have been reported to participate in defence against T. cruzi, it is of importance to study the regulation of NK cell functions during infection with the parasite. Several innate cytokines regulate NK cell activity, among them being interferon (IFN)-alpha and IFN-beta (type 1 IFNs) and interleukin (IL)-12, which have all been reported to be involved in protection against T. cruzi. The role of these cytokines in regulation of NK cell functions and disease outcome were studied by infection of mutant mice lacking the IFN-alpha/beta receptor (IFNalpha/betaR-/-) or IL-12 (IL-12-/-) with T. cruzi. IFNalpha/betaR-/- mice were unable to activate the cytotoxic response but produced IFN-gamma, and were not more susceptible than controls. IL-12-/- mice were extremely susceptible and failed to produce T cell-derived IFN-gamma and nitric oxide (NO), although NK cytotoxicity was induced. The results indicate that IL-12 protects against T. cruzi by initiating T cell-mediated production of IFN-gamma, but that endogenous IFN-alpha/beta and NK cell cytotoxicity are not of major importance in defence.     

Differential modulating effect of natural killer (NK) T cells on interferon-γ production and cytotoxic function of NK cells and its relationship with NK subsets in Chlamydia muridarum infection

Natural killer T (NKT) cells are a newly identified T-cell population with potential immunomodulatory functions. Several studies have shown modulating effects of NKT cells activated by α-galactosylceramide, a model antigen, on NK cell function. We here report a differential modulating effect of NKT cells on the interferon-γ (IFN-γ) production and cytolytic function of NK cells in a chlamydial infection model, using NKT-cell-deficient mice and antibody blocking (anti-CD1d monoclonal antibody) approaches. Our results showed that both NKT and NK cells became activated and produced IFN-γ following Chlamydia muridarum infection in vitro and in vivo. The NK cells in NKT-cell-deficient mice and CD1d-blocked mice showed decreased CD69 expression, cellular expansion and IFN-γ production but surprisingly showed increased cytolytic activity (degranulation) of immature and more mature NK cell subsets, suggesting an inhibitory role of NKT cells on NK cell killing activity. The results suggest that NKT cells preferentially promote IFN-γ production but are inhibitory for the cytotoxic function of NK cells in this infection model. Furthermore, the differential modulating effect of NKT cells on the IFN-γ production and cytotoxicity of NK cells was observed in immature and mature NK cell subsets, although it was more dramatic in the relatively mature CD11b(high) CD27(high) NK cell subset. This finding demonstrates the complexity of innate cell interactions in infection and the possible differential impact of NKT cells on the variable functional aspects of other cell(s) even in one infection setting.