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Curcumin is the active compound in the extract of Curcuma longa rhizomes with anti-inflammatory properties mediated by inhibition of intracellular signalling. SOCS and MAPKinases are involved in the signalling events controlling the expression of IL-6, TNF-α and PGE2, which have important roles on chronic inflammatory diseases. The aim was to assess if these pathways are involved in curcumin-mediated effects on LPS-induced expression of these cytokines in macrophages. RAW 264.7 murine macrophages were stimulated with Escherichia coli LPS in the presence and absence of non-cytotoxic concentrations of curcumin. Curcumin potently inhibited LPS-induced expression of IL-6, TNF-α and COX-2 mRNA and prevented LPS-induced inhibition of SOCS-1 and -3 expression and the inhibition of the activation of p38 MAPKinase by modulation of its nuclear translocation. In conclusion, curcumin potently inhibits expression of LPS-induced inflammatory cytokines in macrophages via mechanisms that involve modulation of expression and activity of SOCS-1 and SOCS-3 and of p38 MAPK.
Curcumin is a constituent of the spice turmeric, one of the principal ingredients in curry powder. Many studies have evaluated the effect in vivo of curcumin in the treatment of conditions associated with an inflammatory/immune response component, including cancer,1–3 rheumatoid arthritis,4 Crohn‘s disease,5 inflammatory bowel disease,6 colitis7 and psoriasis.8 Understanding of the biological mechanisms involved in the effects of curcumin is improved by in vitro studies demonstrating inhibition of inflammatory cytokines such as TNF-α, IL-8, IL-1, IL-6, MMP-9, NO, COX-2 by curcumin in various cell types9–14; however, information on the macrophage-specific effects of curcumin are relatively scarce. Macrophages are associated with innate immunity and are important for the initiation and modulation of adaptive immunity, having a central role in inflammatory diseases.
Among the intracellular signalling pathways involved in cytokine production, the transcription factor NF-kB and mitogen-activated protein kinase (MAPK) cascades were shown to play fundamental roles. NF-kB is one of the most important inducible transcription factors in mammals and has been shown to play a pivotal role in the mammalian innate immune response15 and chronic inflammatory conditions.16 MAPKs, which include JNK, ERK, and p38, are well known stress-activated kinases implicated in the regulation of many genes, particularly those of the inflammatory and immune response.17 NF-kB is considered the primary target of curcumin, whereas MAPKinase activation is also affected by curcumin in some cell types. Besides NF-kB and MAPK, curcumin has been shown to modulate other signalling pathways involving protein tyrosine kinases as well as serine/threonine kinases, such as JAK/STAT/SOCS and PKC.18–21
The suppressor of cytokine signalling (SOCS) family of proteins22 represent an important endogenous negative regulatory mechanism of cytokine signalling that also affects pro-inflammatory cytokine expression. As an endogenous mechanism, SOCS proteins are22 essential to normal immune physiology, but they are also involved in the development of immunological disorders including inflammatory diseases such as atherosclerosis, arthritis, airway inflammation, diabetes and cancer. This has encouraged studies on the control of cytokine-related pathological conditions by mimicking the functions or modulating the expression of SOCS proteins.23 SOCS-1, also called SSI-1 (STAT-induced STAT inhibitor-1) was initially identified as an intracellular negative-feedback molecule that inhibits the overactivation of the JAK-STAT-mediated signal cascade initiated by various stimuli, including IFN-γ, IL-6, IL-4.24–26 Recent reports indicate an important role for SOCS-1 on the downregulation of LPS signal transduction alongside with direct effects on negative feedback inhibition of NF-kB signalling.22,27
SOCS-3 may have profound effects on the regulation of immunity and inflammation by affecting the activation, development and homeostatic functions of all lineages involved in immune and inflammatory responses.28 SOCS-3 is strongly induced by interleukin-1, -6, -10 and IFN-γ, and its main role seems to be the attenuation of inflammatory cytokine signalling. Previous studies have demonstrated that SOCS-1 deficiency results in severe synovial inflammation and joint destruction29–31 while SOCS-3 has also been implicated in the modulation of arthritis pathogenesis, being able to suppress the induction and development of joint inflammation in mouse models.31,32
While numerous in vitro and in vivo studies report on the anti-inflammatory role of curcumin, particularly on NF-kB activation, this is the first study to evaluate the effect of curcumin on the kinetics of SOCS-1 and SOCS-3 expression and on the activation of MAPKinases in macrophages stimulated by LPS and the effect of this modulation on expression of IL-6, TNF- α and PGE2.
In this study, we assess the effects of curcumin on the modulation of macrophage response to bacterial LPS, which is a well characterized, widely used and efficient model of evaluation of inflammatory response in vitro.33–37 Curcumin suppressed LPS-induced expression of TNF-α, IL-6 and PGE2; and this effect was paralleled by preventing the LPS-induced inhibition of SOCS-1 and -3 expression and the inhibition of the activation of p38 MAPKinase by modulation of its nuclear translocation.
The murine macrophage cell line RAW 264.7 (ATCC #TIB-71) was cultured in alpha-MEM supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% heat-inactivated foetal bovine serum and maintained in a humidified atmosphere at 37 °C and 5% CO2. LPS from Escherichia coli (serotype O55:B8) and curcumin were purchased from Sigma. E. coli LPS was diluted in PBS (pH 7.4) at 1 mg/mL (stock concentration) and used at a final concentration of 1 μg/mL (1:1000 dilution from the stock LPS solution). Curcumin was diluted in DMSO and used at the indicated concentrations. The concentration of LPS used for the stimulation, the experimental periods and the cell density used were optimized in preliminary experiments aimed at obtaining the maximum cell response (data not shown). NE-PER Nuclear and Cytoplasmic Extraction Reagent was purchased from Pierce (Thermo-Fisher Scientific). Rabbit polyclonal antibodies against phosphorylated forms of p38 MAP kinases, p65, ERK1/2, as well as to beta-actin, Lamin A/C and GAPDH were from Cell Signalling, as well as the secondary HRP-conjugated antibodies. Rabbit polyclonal antibodies for SOCS-1 and SOCS-3 were from Santa Cruz Biotechnology.
The effect of curcumin on cell viability was determined using a commercial kit (Cell Titer 96 Aqueous; Promega Corp.) according to the manufacturer's instructions. This kit evaluates cell viability by the activity of mitochondrial dehydrogenase enzymes that reduce a tetrazolium containing substrate solution to formazan, generating a colorimetric reaction. Briefly, 1 × 105 cells were plated in each well of 96-well plates, allowed to attach for 18 h, washed once with phosphate-buffered saline (PBS) and then de-induced for 6 h in culture medium with reduced concentration (0.3%) of FBS. Curcumin was added to the culture medium in various concentrations for a dose-response experiment and the cells incubated for 24 h. Controls were represented by the same volume of the DMSO vehicle. 20 μL of reagent containing the tetrazolium salt (MTS) substrate was added to each well and incubated 2 h and the results obtained by measuring the absorbance at 490 nm on a microplate reader (Bio-Rad Inc., model 550). The relative number of viable cells in the wells treated with curcumin was estimated in relation to the vehicle-treated control wells.
A total of 3 × 105 cells were grown for 24 h in each well of six-well plates, de-induced by incubation for 6 h in culture medium containing 0.3% foetal bovine serum and stimulated with E. coli lipopolysaccharide (1 μg/mL) for 24 h, both with and without a 30 min pretreatment with 5 or 10 μM of curcumin. Control wells were treated with the same volume of the DMSO vehicle used to dilute these compounds. Total RNA was isolated from the cells with Trizol reagent (Invitrogen Corp.) according to the manufacturer's instructions. The RNA was quantitated by spectrophotometry and 500 ng were reverse-transcribed into cDNA. The relative abundance of the transcripts of the candidate inflammatory genes were determined by real-time reverse transcription-PCR (RT-PCR) using Taqman chemistry and pre-designed sets of primers and probes (TaqMan Gene Expression Assays, Applied Biosystems) on a StepOne Plus Real-Time PCR System (Applied Biosystems). The reactions were carried out in a 96-well plate on a final reaction volume of 30 μL that included Taqman Universal PCR Master Mix (Applied Biosystems), Taqman Gene Expression Assays (Applied Biosystems) for each target gene: TNF-α (tumour necrosis factor alpha), NM_013693; IL-6 (interleukin 6), NM031168; PTGS-2 (prostaglandin-endoperoxide synthase-2), NM011198; GAPDH (glyceraldehyde-3-phosphate dehydrogenase), NM008084; and cDNA template (corresponding to 300 ng of total RNA). Optimized thermal cycling conditions were: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. For each sample, analyses of gene expression were performed in duplicate. Three independent experiments were performed. To normalize the amount of total RNA present in each reaction, the expression of GAPDH, which was not altered by the experimental conditions, was used as a housekeeping gene. To compare the expression levels among different samples, the relative expression level of the genes was calculated using the comparative Δ(ΔCT) method using the thermocycler's software.
Cell culture supernatants were collected from the same wells from which total RNA was harvested immediately thereafter. In brief, cells were stimulated with E. coli lipopolysaccharide for 24 h, both with and without a 30 min pretreatment with 5 or 10 μM of curcumin. Control wells were treated with the same volume of the DMSO vehicle used to dilute these compounds. These culture supernatants were cleared by centrifugation (5 min, 12,000 rpm at 4 °C), aliquoted and immediately stored at –80 °C until used. These samples were thawed only once and used in ELISA assays to quantify the secreted PGE2, IL-6 and TNF-α. These assays were performed according to the manufacturer's instructions (R&D Systems) and the target protein concentration was normalized to the total protein content determined by the Lowry method (DC assay, Bio-Rad).
3 × 105 cells were grown for 24 h in each well of six-well plates, routinely deinduced for 6 h and subsequently treated with 5 or 10 μM of curcumin 30 min before the stimulation with bacterial LPS. Control experiments were performed with the addition of the same volume of DMSO vehicle 30 min before LPS stimulation. Samples (cell lysates) were harvested 10, 30 and 60 min for the short-term experiments (activation of signalling pathways) and for 12, 24 and 48 h in the experiments to evaluate SOCS-1 and -3 expression. Whole-cell lysates were harvested by scraping the cells in sodium dodecyl sulfate sample buffer (62.5 mM Tris HCl buffer, pH 6.8, 10% glycerol, 50 mM dithiothreitol, 2% sodium dodecyl sulfate, 0.01% bromophenol blue) on ice, followed by sonication for 10 s and heat-denaturation at 95 °C for 5 min. For preparation of nuclear and cytoplasmic extracts, the cells were washed and scraped into phosphate-buffered saline 10, 30 and 60 min after LPS stimulation, and pelleted by centrifugation. For cytoplasmic/nuclear fractions, 1 × 106 cells were lysed in NE-PER (Nuclear and Cytoplasmic Extraction Reagent, Pierce) according to the manufacturer's protocol. Total protein content was quantified by Lowry method (DC Assay, Bio-Rad). For western blotting, 30 μg of total protein were separated on 10% Tris–HCl polyacrylamide gels run at 100 V for 60 min and subsequently electrotransferred to nitrocellulose membranes for another 60 min in a semidry apparatus at 110 mA/gel. The membranes were blocked (Tris-buffered saline with 5% nonfat dry milk, 0.1% Tween-20) for 1 h at room temperature and then probed with the primary antibodies overnight at 4 °C. The presence of the primary antibodies was detected by using horseradish peroxidase-conjugated secondary antibodies and a chemiluminescence system (SuperSignal West Pico Chemiluminescent Substrate; Pierce, Rockford, IL, USA). Digital images of the radiographic films exposed to the membranes were obtained on a gel documentation system (GelDoc XT; Bio-Rad). Expression levels of beta-actin were determined to verify equal loading of proteins, GAPDH and Lamin A/C were used as cytoplasmic and nuclear protein controls, respectively.
Initially, we performed a dose-response evaluation of the cytotoxic effects of curcumin. Since the biological effects of curcumin may be related to its cytotoxic effects, we intended to determine and use the highest non-cytotoxic concentration of curcumin. RAW 264.7 cells were treated with the indicated concentrations of curcumin (5–100 μM) for 24 h. The results of the MTS-based assay of DMSO vehicle-treated cells were set to 100% and the results for cells treated with curcumin are presented as relative increase or decrease. Concentrations of curcumin up to 10 μM produced no change in cell viability, whereas higher concentrations of curcumin (≥25 μM) caused significant cytotoxicity (Fig. 1). We then used 10 μM of curcumin as the maximum concentration for the subsequent experiments. We also used 5 μM of curcumin to assess possible dose-related effects.
LPS stimulation induced a significant increase on the expression of PTGS-2 (the murine homolog to human cyclooxygenase-2) mRNA by the macrophages. This increase was inhibited by pre-treatment of the cells with curcumin in a concentration-dependent manner; but statistical significance was only observed with 10 μM of curcumin. In the absence of LPS stimulation, curcumin induced a slight increase of PTGS-2 mRNA (Fig. 2). Detectable levels of PGE2 in the cell culture supernatants were detected only upon LPS stimulation and were completely abrogated by curcumin (at 5 and 10 μM) (Fig. 3).
Curcumin induced TNF-α and IL-6 mRNA expression in the absence of LPS stimulation. However, in cells stimulated with LPS curcumin dose-dependently inhibited the expression of IL-6, whereas inhibition of TNF-α mRNA was observed only with 10 μM of curcumin (Fig. 2). IL-6 and TNF-α protein were undetectable in the absence of LPS stimulation; and pre-treatment of the cells with 10 μM of curcumin significantly inhibited LPS-induced secretion of IL-6 and TNF-α (Fig. 3).
Considering the relevance of SOCS and MAPKinase signalling pathways for LPS-induced cytokine gene expression, we next evaluated the modulation of these pathways by curcumin in macrophages. As a positive control, we assessed the effects of curcumin on phosphorylation of p65 as indicative of NF-kB activation status; which is documented as a major target of curcumin. In the vehicle control experiments, equivalent volumes of the DMSO vehicle replaced curcumin. Macrophages were treated with curcumin (5 and 10 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 10, 30 and 60 min. LPS induced activation of NF-kB, p38 and ERK within 10 min. This activation was sustained, in all signalling pathways for at least 60 min. Pre-treatment of the cells with curcumin inhibited LPS-induced activation of p38 MAPK as well as of p65 (NF-kB) in a concentration-dependent manner starting 30 min after stimulation (Fig. 4).
The experiments assessing subcellular localization of the signalling intermediates show that LPS induced the nuclear translocation of p38 within 10 min and this induction was maintained at 30 min. Pre-treatment with 10 μM of curcumin inhibited nuclear translocation of p38 at both 10 and 30 min after LPS stimulation, whereas when curcumin was used at 5 μM this inhibition was observed only at 30 min (Fig. 5). Curcumin pre-treatment did not affect subcellular localization of ERK, but was associated with increased phosphorylation of ERK in the cytoplasmic compartment.
In the experiments to evaluate the modulation of SOCS-1 and SOCS-3, macrophages were treated with curcumin (5 and 10 μM) for 30 min followed by stimulation with LPS (1 μg/mL) for 12, 24 and 48 h. Curcumin (10 μM) inhibited constitutive expression of SOCS-3 at 12 and 24 h, but prevented the LPS-induced inhibition at 24 h. When used at 5 μM, curcumin did not alter constitutive expression of SOCS-3, but this lower concentration also prevented the LPS-induced inhibition at 12 h. SOCS-1 expression was increased by both concentrations of curcumin at 24 h and this increase was sustained at 48 h when cells were treated with 10 μM of curcumin. Curcumin treatment also increased SOCS-1 expression 12 h after LPS stimulation and dose-dependently prevented the LPS-induced inhibition on SOCS-1 expression after 48 h (Fig. 6).
In this study, we show for the first time, that curcumin prevented the LPS-induced inhibition of SOCS-1 and -3 and blocked the phosphorylation and nuclear translocation of p38 MAPK in murine macrophages. The modulation of signalling pathways can be related to potent inhibitory effect of curcumin on the expression of PGE2, IL-6 and TNF-α induced by LPS and observed in this work.
Curcumin was recently shown to dose-dependently inhibit the expression of TNF-α and IL-1β by LPS-stimulated RAW 264.7 cells.38 In this study, cells were treated with similar (5 and 10 μM) and higher (20 and 30 μM) concentrations of curcumin in comparison to the present report. The authors used a 2 h pre-treatment with curcumin before stimulation with LPS. The nature of LPS used was also different from the LPS used in the present study: LPS from E. coli that we used is a known activator of TLR4; whereas LPS from the periodontopathogenic microorganism Porphyromonas gingivalis used in the study by Chen et al.38 activates preferentially TLR2. These differences on the curcumin treatment and source of LPS used can account for the weak inhibition of both TNF-α and IL-1β with lower concentrations of curcumin reported by Chen et al.38 In our study, concentrations above 20 μM of curcumin were toxic for this cell line; moreover even lower concentrations of curcumin (5 μM) markedly decreased TNF-α protein production by RAW 264.7 cells stimulated with TLR4-activating LPS from E. coli. The concentrations of curcumin used in the present study were similar to those of Abe et al.39 who showed a marked inhibition of IL-1β, TNF-α, IL-8 and MIP-1α in LPS-stimulated human monocytes and alveolar macrophages; and higher than the maximum of 1 μM used by Jain et al.40 who reported a significant but less potent inhibition of IL-6 and TNF-α in human monocytes cultured in high glucose.
Curcumin has been shown to inhibit AP-1 and MAPKinases besides its prime target, NF-kB.21,41 In human intestinal epithelial cell line HT29 stimulated with TNF-α and IL-1β, curcumin was able to inhibit NF-kB and MAPKinases, such as p38 and JNK, but there was no inhibition of ERK1/2.42 On the other hand, inhibition of all three MAPKinases was observed in human esophageal cells stimulated by low pH,21 as well as in an adipocyte cell line treated with palmitate43 and in human microvascular endothelial cells stimulated with VEGF.44 Our data indicates that curcumin inhibits LPS-induced p38 MAPK activation in macrophages by reducing both its phosphorylation and nuclear translocation. Interestingly, curcumin at 10 μM had contrasting effects on the kinetic of LPS-induced activation of ERK MAPKinases: an initial dose-dependent increased activation 10 min after stimulation, was followed by a very marked inhibitory effect 30 and 60 min after LPS stimulation. This delayed inhibition may be attributed to cross-talk between these pathways resulting in a shift of the LPS/TLR4 signal to other signalling pathways due to the inhibition of NF-kB and p38 MAPK, in a compensatory activation mechanism. The fact that curcumin inhibits p38 while simultaneously not affecting or even increasing the activation of ERK MAPKinases suggests that curcumin exerts its effects relatively downstream on the MAPK cascade, perhaps at the MAP2K level (MKK3/6).
There is paucity information regarding the modulation of SOCS proteins by curcumin. In one of these few studies, the effect of curcumin on SOCS-3 levels was analyzed in ovarian and endometrial cancer cells and compared to non-neoplasic cells.45 The level of SOCS-3 was elevated in cancer cells compared to normal cells and its expression was inhibited following curcumin treatment. The authors correlated this marked decrease in SOCS-3 expression following curcumin treatment of cancer cells to the decrease on STAT3 activation.45 Our data show that higher dose of curcumin inhibits SOCS-3 expression in unstimulated macrophages and dose-dependently prevents the LPS-induced inhibition, which correlates with the potent inhibition of IL-6 production observed. In the absence of LPS stimulation, curcumin, particularly at 10 μM, increased expression of SOCS-1, which is consistent with the inhibition of NF-kB signalling,22 a major effect of curcumin. On the other hand, expression of SOCS-3 was reduced by curcumin at 10 μM in macrophages not stimulated by LPS, which is consistent with the finding in cancer cells.
Importantly, the marked effect of curcumin on cytokine expression may be dependent on this simultaneous inhibition of multiple signalling pathways. A novel observation is the rescue of SOCS-1 expression in LPS-stimulated macrophages. Recent data suggest that SOCS-1 regulates NF-kB signalling.22,46 The NF-kB component p65 was identified as an interaction partner for SOCS-1 but not for other members of the SOCS family. SOCS-1 bind to p65 within nucleus leading to its polyubiquitination and proteasomal degradation. Thus, SOCS-1 limits prolonged p65 signalling and terminates expression of NF-kB inducible genes by regulating the duration of NF-kB signal within the cell nucleus.46
According to these data, it is plausible to speculate that the increase of SOCS-1 by curcumin may be an additional mechanism involved in the modulation of NF-kB activation and NF-kB-inducible genes such as those evaluated in this study and, therefore, can be related to the anti-inflammatory effects of curcumin.
It will be interesting to define the specific target(s) of curcumin in the TLR4 signalling pathway. This prominent role of curcumin on the activation of innate immunity may have potential to be explored in the modulation of host response in chronic conditions associated with infectious stimuli, such as periodontal diseases and lower digestive tract diseases.
In summary, we have shown that curcumin modulates TLR signalling in macrophages and effectively inhibits cytokine gene expression induced by TLR stimulation indicating its therapeutic potential as a host modulatory agent in chronic infectious conditions.
Financial support was provided by Brazilian Federal Government through the National Council for Scientific and Technological Development (CNPq) and Coordination for Improvement of Higher Education Personnel (CAPES, #4638-05), and by the National Institutes of Health–National Institute of Dental and Craniofacial Research (NIH, 1R01DE018290 and 5P30GM103331-02).
National Council for Scientific and Technological Development (CNPq), Brazilian Ministry of Science. Coordination for Improvement of Higher Education Personnel (CAPES, #4638-05), Brazilian Ministry of Education. National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH, 1R01DE018290 and P30GM103331-02).
This is an in vitro study using commercially-available cell lines. No animal or human participation. There are no ethical considerations.
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