The inflammatory response has been implicated in the pathogenesis of many chronic diseases. Along these lines, the modulation of inflammation by consuming bioactive food compounds, such as ω−3 fatty acids or procyanidins, is a powerful tool to promote good health. In the present study, the administration of DHA (docosahexaenoic acid) and B1, B2 and C1 procyanidins, alone or in combination, prevented the inflammatory response induced by the LPS (lipopolysaccharide) endotoxin in human macrophages and brought them to the homoeostatic state. DHA and B1 were strong and selective negative regulators of cyclo-oxygenase 1 activity, with IC50 values of 13.5 μM and 8.0 μM respectively. Additionally, B2 and C1 were selective inhibitors of pro-inflammatory cyclo-oxygenase 2 activity, with IC50 values of 9.7 μM and 3.3 μM respectively. Moreover, DHA and procyanidins prevented the activation of the NF-κB (nuclear factor κB) cascade at both early and late stages with shared mechanisms. These included inhibiting IκBα (inhibitor of NF-κB α) phosphorylation, inducing the cytoplasmic retention of pro-inflammatory NF-κB proteins through p105 (NF-κB1) overexpression, favouring the nuclear translocation of the p50–p50 transcriptional repressor homodimer instead of the p50–p65 pro-inflammatory heterodimer, inhibiting binding of NF-κB DNA to κB sites and, finally, decreasing the release of NF-κB-regulated cytokines and prostaglandins. In conclusion, DHA and procyanidins are strong and selective inhibitors of cyclo-oxygenase activity and NF-κB activation through a p105/p50-dependent regulatory mechanism.
The inflammatory response is a body defence mechanism that is triggered by an external aggression, such as trauma or infection by external pathogens, and it represents a complex network of cellular and molecular interactions that is responsible for facilitating tissue repair and for returning the cell to physiological homoeostasis [1,2]. The cellular response is composed of local and systemic activation processes and is mediated by transcription factors, such as NF-κB (nuclear factor κB), that modulate the synthesis and secretion of cytokines such as IL (interleukin)-6 or prostanoids formed by the metabolism of arachidonic acid by the COX (cyclo-oxygenase) pathway .
Macrophages can be considered as the main co-ordinators of the innate immune response, as they are the first to recognize exogenous pathogens. Through a series of receptors based on pattern recognition, including TLR (Toll-like receptor) types and complement receptors, macrophages are able to identify the most common pathogens that trigger the inflammatory response [1,4].
IL-6 is one of the most important pro-inflammatory cytokines secreted by macrophages in response to an inflammatory stimulus. IL-6 is involved in the modulation of a broad range of cellular and physiological responses . COX, or PGHS (prostaglandin H synthase), is responsible for the metabolic conversion of ω−6 AA (arachidonic acid), the most abundant fatty acid present in the cell membrane, into PGE2 (prostaglandin E2) and TX (thromboxane) [5–7]. Additionally, prostanoids can also be synthesized from ω−3 fatty acids, such as DHA (docosahexaenoic acid). The ω−6 and ω−3 fatty acids lead to two different sets of eicosanoids, the E2 and E3 series respectively . These eicosanoids act as chemical messengers for the immune system, and their principal function involves regulating the inflammatory response. Three isoforms of COX (EC number 188.8.131.52) responsible for the biosynthesis of eicosanoids have been described, which include COX1 (or prostaglandin endoperoxide synthase 1), COX2 (or prostaglandin endoperoxide synthase 2) and COX3 (or prostaglandin endoperoxide synthase 3) [7,9]. COX1 is constitutively expressed in most cells and is therefore regarded as a housekeeping molecule and is involved in the mediation of different physiological responses (e.g. the protection of the gastric epithelium or platelet aggregation). COX2 remains undetectable in the majority of mammalian tissues under basal conditions, although its expression is inducible by several inflammatory stimuli [e.g. LPS (lipopolysaccharide) or inflammatory cytokines], leading to an increase in PGE2 synthesis. Additionally, the activation of COX2 is implicated in numerous inflammation-associated diseases and instances of tumorigenesis [6,9]. Therefore COX1 and COX2 are of particular interest because they are the major targets of NSAIDs (non-steroidal anti-inflammatory drugs). COX3 has been described as a splice variant of COX1; however, its biological role is still poorly studied .
The transcription factor NF-κB regulates the transcription of numerous genes that control the immune response [11,12]. Although NF-κB plays an essential role in normal physiology and in the inflammatory immune response, the constitutive activation of NF-κB is associated with different diseases [11–14]. There are two NF-κB signalling pathways: the classical, or canonical, pathway and the alternative route. The main difference between these two activation pathways includes the specific IKK [IκB (inhibitor of NF-κB) kinase] family members that are involved . In mammals, the NF-κB family consists of five proteins, including p65 (RelA), RelB, c-Rel, p105/p50 (NF-κB1) and p100/p52 (NF-κB2), that associate with each other to form transcriptionally distinct homo- and hetero-dimeric complexes , although the p65–p50 heterodimer represents the most abundant of the Rel dimers. NF-κB family proteins are characterized by the presence of the RHD (Rel homology domain), a 300 amino acid N-terminal segment responsible for DNA binding, dimerization, nuclear translocation, interaction with IκB family proteins and transcriptional regulation [12–14]. In resting cells, the NF-κB heterodimer is retained in the cytoplasm and is bound to its inhibitory protein, IκB, a member of the IκB family of proteins. In response to an inflammatory stimulus, such as LPS from the bacterial cell wall, the classical pathway of NF-κB activation leads to the activation of IKKβ, a member of the IKK complex, and triggers the phosphorylation of IκBα protein (pIκBα). pIκBα is recognized by the ubiquitin ligase machinery, causing its polyubiquitination and subsequent proteasomal degradation. After pIκBα degradation, the NF-κB heterodimer is able to translocate to the nucleus, where, bound to the κB motif found in the promoter or enhancer regions, it induces the expression of numerous pro-inflammatory genes . The alternative activation pathway of NF-κB is not activated by LPS, and it is independent of IKKβ and IKKγ [11–13,16].
Procyanidins are phenolic compounds that are present in a variety of vegetables, fruits and cereals, but are mainly found in cocoa, grapes and apples. Procyanidins are polymers that are composed of flavan-3-ol units, such as catechin and epicatechin. Procyanidins can be defined as bioactive food compounds, owing to their influence on physiological status and cellular homoeostasis, as well as their beneficial effects on health, such as inhibition of the release of pro-inflammatory mediators [17–20]. The mechanisms involved in the modulation of the inflammatory response by procyanidins are not yet well understood and are currently a subject of intense study.
PUFAs (polyunsaturated fatty acids) are important constituents of cells that are mainly involved in preserving the fluidity of the cell membrane, regulating cellular signals or modulating gene expression . ω−3 fatty acids that are present as bioactive food compounds can replace the ω−6 fatty acids located in the cell membranes of macrophages, triggering a competition between ω−3 and ω−6 fatty acids during prostanoid formation . In general, the eicosanoids from the 3 series that are derived from ω−3 fatty acids have less inflammatory activity than the 2 series that are derived from ω−6 fatty acids. In some cases, the eicosanoids derived from the ω−3 fatty acids do not have the biological activities of their counterparts . DHA is present in certain aquatic organisms, such as fish (fish oil) or marine algae. Studies have indicated that the supplementation of diets with ω−3 fatty acids, such as DHA, produces beneficial effects on symptoms in diseases characterized by chronic inflammation, such as atherosclerosis, rheumatoid arthritis or obesity [24,25]. The mechanisms by which ω−3 fatty acids, specifically DHA, exert anti-inflammatory effects remain unclear.
The present study is focused on the regulation of the inflammatory response by DHA and the procyanidins B1, B2 and C1, alone or in combination, in LPS-stimulated human macrophages. This modulation of the inflammatory response was based upon the secretion of inflammatory mediators, such as IL-6 and PGE2, as well as modulation of the activity of the COX pathway. Additionally, the modulation effects on the expression of NF-κB family proteins and their implications in the NF-κB signalling pathway were investigated.
MATERIAL AND METHODS
Procyanidin dimers B1 [epicatechin-(4β→8)-catechin] and B2 [epicatechin-(4β→8)-epicatechin] were obtained from ExtraSynthese. Procyanidin trimer C1 [epicatechin-(4β→8)-epicatechin-(4β→8)-epicatechin] was obtained from TransGmbH. cis-4-,7,10,13,16,19-DHA was obtained from Sigma–Aldrich. RPMI 1640 culture medium was purchased from Gibco. Cell culture reagents were provided by BioWhittaker, except for the differentiating agent PMA, which was purchased from InvivoGen, and LPS (Escherichia coli 0111:B4), which was purchased from Sigma–Aldrich. The anti-p65, anti-p50/p105, anti-pIκBα and FITC-conjugated goat anti-rabbit IgG were acquired from Santa Cruz Biotechnology, and the anti-COX2 polyclonal antibody was purchased from Bioworld. The HRP (horseradish peroxidase)-conjugated monoclonal anti-rabbit IgG antibody was obtained from GE Healthcare, and the advanced ECL (enhanced chemiluminescence) Western blotting detection kit was provided by GE Healthcare. The NSAIDs nimesulide, sc-560 and indomethacin were purchased from Cayman Chemicals. Bradford reagent and Histopaque®-1077 were obtained from Sigma–Aldrich.
Cell lines and cell culture
Human THP-1 monocytes were purchased from the European Collection of Animal Cell Cultures (ECACC number 88081201). Cells were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 μg/ml streptomycin, 100 units/ml penicillin, 25 mM Hepes and 10% FBS (fetal bovine serum) at 37°C in a humidified incubator with 5% CO2. The differentiation of THP-1 monocytes into macrophages was induced by growing the cells in fresh medium containing 0.5 μg/ml PMA for 24 h. After differentiation, the medium containing PMA was removed and adherent THP-1 macrophages were incubated in RPMI medium for 24 h. Later, macrophages were treated with 25 μM DHA, 17.3 μM B1, 17.3 μM B2 or 11.5 μM C1, or a combination of DHA with one of the procyanidins in serum-free RPMI 1640-supplemented medium. For all experiments, control cells treated with ethanol alone (vehicle of the DHA and procyanidins) were included (final concentration in the medium was ≤0.1%). Finally, all of the macrophages, except for the unstimulated control, were stimulated with 1 μg/ml LPS. The experimental design is shown in Figure 1. Cell viability was assessed as better than 97% under all experimental conditions using Trypan Blue staining (results not shown).
Schematic diagram of the experimental design
Isolation of primary human monocyte-derived macrophages
Five healthy donors were recruited from the University Hospital Joan XXIII. The study was approved by the Institutional Review Board. All participants gave written informed consent for participation in medical research. Human PBMCs (peripheral blood mononuclear cells) were isolated from healthy blood donors by density gradient centrifugation using Histopaque®-1077, according to the manufacturer's protocol (Sigma–Aldrich). Histopaque®-1077 is a solution of polysucrose (5.7 g/dl) and sodium diatrizoate (9 g/dl), adjusted to a density of 1.077±0.001 g/ml. Monocytes were purified by adherence to plastic in serum-free RPMI 1640-supplemented medium inducing monocyte activation. Briefly, 2×106 cells/ml were seeded into 12-well plates and, after 2 h, non-adherent cells were removed by several washes with warm PBS. Freshly isolated primary activated monocytes were treated with DHA and/or procyanidins and were stimulated with LPS following the same protocol as previously described for THP-1 macrophages, which is shown in Figure 1.
The effect of DHA/procyanidin pre-treatments on IL-6 concentration was measured with an ELISA kit following the manufacturer's protocol (BioLegend). The secretion of IL-6 was normalized according to the protein content measured using a Bradford assay.
The effect of the DHA/procyanidin pre-treatments on PGE2 in LPS-stimulated macrophage supernatants was determined by a specific competitive immunoassay (EIA) following the manufacturer's protocol (Cayman Chemicals). The secretion of PGE2 in culture supernatants was normalized according to the protein concentration, which was determined using a Bradford assay.
COX1 and COX2 cell-free assay
The kinetic effects of the treatments (DHA, B1, B2 and C1) on the activity of COX1 and COX2 enzymes were determined using a cell-free inhibition assay, on the basis of the fact that the rate-limiting step in prostaglandin synthesis is catalysed by COXs. Then, the modulation of the synthesis of PGE2 from AA by DHA, B1, B2 and C1 was determined. Briefly, the cell-free assay was performed using purified COX1 from ram seminal vesicles and COX2 from sheep placental cotyledons (Cayman Chemicals). The COX isoforms were incubated with various concentrations of AA (0.15–25 μM) for 20 min (pH 8.0), at 37°C in the presence of 18 mM adrenaline (epinephrine), 5 μM haematin and concentrations of DHA, B1, B2, and C1 that ranged from 0.01 to 500 μM, as well as commercial inhibitors (indomethacin, nimesulide and sc-560). The reactions were stopped by the addition of 10 μl of 10% formic acid. The PGE2 concentration was determined using a specific competitive immunoassay (EIA, Cayman Chemicals) . IC50 values were expressed as the concentration of compound required to inhibit 50% of the PGE2 biosynthesis. The kinetic constants were analysed from double-reciprocal plots of velocity against AA concentration using GraphPad Prism (version 5.0). Kinetic parameters were obtained by fitting the points to the Michaelis–Menten kinetic equation:
where V is the initial rate, Vmax represents the maximum enzyme rate at saturating substrate concentrations, [S] is the substrate concentration and Km is the substrate concentration at which the reaction rate is half that of Vmax. The turnover number, or catalytic constant (kcat), was calculated from the following equation:
where [E]t represents the total active enzyme concentration. Additionally, the dissociation constant for inhibitor binding (Ki) was obtained from the following equation:
where Km,obs represents the Km value specific for each inhibitor, and Km is the value obtained under control conditions.
Nuclear and cytoplasm extract preparation
Pre-treated LPS-stimulated THP-1 cytoplasmic and nuclear proteins were extracted using methods that have been described previously [25,27]. Briefly, 2×106 cells were washed twice with ice-cold PBS. Cells were lysed in ice-cold buffer A [10 mM Hepes (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM PMSF, 0.5 mM DTT (dithiothreitol), 0.1% Nonidet P40 and 10 μg/ml protease and phosphatase inhibitors]. Subsequently, cells were centrifuged, and the supernatant, i.e. the cytoplasmic fraction, was removed and stored at −80°C. The nuclear pellet was washed once with PBS and resuspended in ice-cold buffer B [20 mM Hepes (pH 7.9), 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 25% glycerol, 0.5 mM PMSF and 10 μg/ml protease and phosphatases inhibitors]. The nuclear extract was obtained by centrifugation, and the supernatant, i.e. the nuclear fraction, was removed and stored at −80°C. The protein concentrations in the cytoplasmic and nuclear fractions were quantified with a Bradford assay using BSA as the standard.
Western blot analysis
Cytoplasmic and nuclear proteins were electrophoresed by SDS/PAGE, and separated proteins were transferred on to a PVDF membrane. The blots were blocked and incubated overnight at 4°C with the appropriate primary antibody. After washing, the blots were incubated with HRP-conjugated secondary antibody, developed with a chemiluminescent reagent (GE Healthcare) and exposed using the Alpha Innotech FluorChem FC2 Imager. A semi-quantitative analysis of the proteins was performed using ImageJ software.
Immunofluorescence staining of NF-κB p65 and confocal microscopy
THP-1-derived macrophages were cultured on sterile commercial poly-L-lysine-treated coverslips (BD BioCoat, BD Biosciences) prior to pre-treatment with DHA and/or procyanidins for 48 h. Then, THP-1 macrophages were activated with LPS for 1 h. Cells were washed, fixed in 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. After incubation with blocking buffer, the cells were incubated with NF-κB p65 primary antibody overnight at 4°C and were then incubated with FITC-conjugated goat anti-mouse IgG. The coverslips were washed, and the samples were then counterstained with ethidium bromide and were mounted in Sigma–Aldrich 10979 mounting medium with polyvinyl alcohol and anti-fading reagent. Confocal images were acquired with a laser confocal-scanning microscope (Nikon Eclipse TE2000-E) using EZ-C1 3.40 software.
The effect of pre-treatment with DHA and/or procyanidins on NF-κB p65 binding to target DNA was determined with the TransAM NF-κB Chemi Assay (Active Motif), according to the manufacturer's protocol.
Results are expressed as the means±S.E.M. Effects of the pre-treatments were assessed using ANOVA. We used a Tukey test to make pairwise comparisons. Differences were considered significant when the P values were <0.05. All calculations were performed using SPSS 17.0 software.
DHA, B1, B2 and C1 inhibit cytokine production in human macrophages
The secretion of IL-6 was regulated by the pre-treatment of macrophages with DHA, B1, B2 or C1, or the combination of DHA with a procyanidin for 48 h. In both THP-1 macrophages (Figure 2A) and primary human macrophages (Figure 2B), the release of IL-6 was inhibited by the pre-treatments when compared with the LPS-stimulated control. In THP-1 macrophages, pre-treatment with the B1 dimer, C1 trimer or any combination of DHA and procyanidins was more efficient in inhibiting the release of IL-6. Additionally, the secretion of IL-6 in primary human macrophages was significantly inhibited by B1, B2 and C1, and any combination of DHA with procyanidin dimers.
Modulation of IL-6 secretion in LPS-stimulated human macrophages
Differential modulation of PGE2 secretion and COX2 protein expression in human macrophages by DHA, B1, B2 or C1
The effect of the pre-treatments on the modulation of PGE2 secretion differed depending on the macrophage cell source. As shown in Figure 3(A), the pre-treatment of THP-1 macrophages with DHA and/or procyanidins did not lead to a significant decrease in PGE2 secretion. Additionally, the post-transcriptional COX2 expression (Figure 3B) was not modulated by DHA or procyanidins. Nevertheless, in primary human macrophages, the secretion of PGE2 was significantly inhibited with all of the pre-treatments relative to the LPS-stimulated control group. In these cases, the pre-treatments with B2 and combinations of DHA with B2 and C1 were the most efficient in decreasing the secretion of PGE2 levels to near the basal state, which corresponded to the LPS-unstimulated control group.
Effect of pre-treatment with 25 μM DHA, 17.3 μM B1, 17.3 μM B2 or 11.5 μM C1, or a combination of DHA and one of the procyanidins for 48 h on the secretion of PGE2 and COX2 protein expression in LPS-stimulated human macrophages
DHA and procyanidins are modulators of COX activity
To carefully study the modulation of COX activity during prostaglandin biosynthesis by DHA and procyanidins, a cell-free assay was performed. The concentration of inhibitor that reduced prostaglandin production by half (IC50) was used to evaluate the effects of DHA and procyanidins in COX1 and COX2 activity analyses. Three commercial NSAIDs, sc-560 (selective COX1 inhibitor), nimesulide (selective COX2 inhibitor) and indomethacin (non-selective COX inhibitor) were used as controls (Table 1). DHA and procyanidins discriminated between the two COX isoforms. DHA and the B1 dimer were strong selective COX1 inhibitors (IC50 value of 13.5 μM and 8.0 μM respectively), whereas the B2 dimer and C1 trimer were strong selective COX2 inhibitors (IC50 value of 9.7 μM and 3.3 μM respectively). In an effort to characterize the mechanism of inhibition of COX1 and COX2 by DHA and procyanidins, the effects of the AA concentration on the rate of enzyme inactivation were studied by using a fixed concentration of inhibitor (15 μM for DHA, and 10 μM for B1, B2 and C1). Kinetic parameters were determined by constructing Michaelis–Menten curves (Figure 4) and Lineweaver–Burk transformations (shown as insets in Figure 4A for COX1 and Figure 4B for COX2 respectively). The kinetic parameters for the inhibition of COX1 by DHA and B1 as well as the inhibition of COX2 by B2 and C1 are reported in Tables 2 and 3 respectively. The modulation of COX1 by DHA was a result of a decrease in the maximal reaction rate (Vmax) and an increase in the Michaelis–Menten constant (Km); therefore DHA acted as a mixed inhibitor of COX1 activity with a dissociation constant (Ki) of 0.73±0.17 μM for the interaction of DHA with COX1. B1 down-regulated the biosynthesis of PGE2; additionally, it exhibited an increase in Km, yet Vmax was not affected. This indicated that B1 competed with AA in the biosynthesis of PGE2 by COX1 with a Ki value of 6.87±0.66 μM. This competitive inhibition behaviour, indicated by an increase in Km and an unaffected Vmax, were also observed for the B2 and C1 regulation of PGE2 biosynthesis by COX2. The dissociation constants for the interactions of B2 and C1 with COX2 were 7.75±0.68 μM and 11.69±1.25 μM respectively. The catalytic efficiencies (kcat/Km) of COX1 and COX2 were also significantly decreased by DHA and B1, and by B2 and C1 respectively.
Determination of Michaelis–Menten and Lineweaver–Burk (insets) curves for the COX conversion of AA into PGE2
|Sample||Vmax (μM/min)||Km (μM)||kcat (s−1)||kcat/Km (s−1·μM−1)||Ki (μM)|
|Sample||Vmax (μM/min)||Km (μM)||kcat (s−1)||kcat/Km (s−1·μM−1)||Ki (μM)|
|Sample||Vmax (μM/min)||Km (μM)||kcat (s−1)||kcat/Km (s−1·μM−1)||Ki (μM)|
|Sample||Vmax (μM/min)||Km (μM)||kcat (s−1)||kcat/Km (s−1·μM−1)||Ki (μM)|
DHA and/or B1, B2 and C1 procyanidins are down-regulators of NF-κB activation
The transcription factor NF-κB plays a major role in the induction of transcription of numerous pro-inflammatory genes. Thus the phosphorylation of IκBα is an essential step for the nuclear translocation of the NF-κB p65–p50 heterodimer. Figure 5(A) shows the effect of DHA and/or procyanidins on the phosphorylation of IκBα. The pre-treatment of THP-1 macrophages with DHA or any combination of DHA and procyanidins slightly decreased the phosphorylation of IκBα. The expression and intracellular localization of several members of the NF-κB family are also essential steps during the activation of the NF-κB pathway. Therefore the effects of DHA and/or procyanidins on the cytoplasmic and nuclear p105, p50 and p65 expression were studied (Figure 5). The cytoplasmic localization of p105 was induced by the B2 dimer and the combination of DHA/B2 pre-treatments on THP-1 macrophages. However, B1, B2 and DHA/B1 pre-treatments inhibited the translocation of p50 to the nucleus through the induction of cytoplasmic p105 expression and inhibition of p50 translocation. Furthermore, all of the DHA and/or procyanidin pre-treatments inhibited the nuclear translocation of p65 in THP-1 macrophages through p65 cytoplasmic retention. Moreover, confocal microscopy was used to visualize the effects of the pre-treatments on nuclear and cytoplasmic p65 expression after 1 h of LPS activation. Figure 6 confirms the dramatic inhibition of nuclear translocation, shown in Figure 5, and demonstrates the retention of p65 in the cytoplasm rather than in the nuclei in all of the pre-treatments.
Inhibition of NF-κB pathway activation by DHA and/or procyanidins in THP-1 macrophages
Inhibition of NF-κB p65 nuclear translocation by DHA and/or procyanidins (B1, B2 and C1) in THP-1 macrophages
DHA, B1, B2 and C1 decrease the NF-κB p65 DNA-binding activity
The functional effects of DHA and/or procyanidins on NF-κB–DNA binding was assessed based on the inhibition of NF-κB p65 binding to the κB consensus sequence, which is located in the promoter and enhancer regions of several pro-inflammatory genes. Figure 7 shows the inhibitory effects of all pre-treatments (DHA and/or procyanidins) on NF-κB–DNA binding activity.
Inhibition of NF-κB p65 DNA-binding activity by DHA and/or B1, B2, and C1 pre-treatments in THP-1 macrophages
Bacterial LPS stimulates immune cells, such as monocytes and macrophages, to trigger the inflammatory response. This response is characterized by the release of an array of pro-inflammatory mediators, such as IL-6 or PGE2, as well as the activation of TLR-4, which results in a signal transduction that activates the NF-κB pathway and, subsequently, the transcription of numerous pro-inflammatory genes. Therefore the modulation of the inflammatory response by the dietary intake of bioactive food compounds is a powerful tool for promoting a healthy and homoeostatic condition and for preventing disease development. The molecular mechanisms underlying the antiinflammatory activities of bioactive food compounds, such as procyanidins and PUFAs, are poorly understood and have been under intense study. Furthermore, their combined effects are even less understood. The present study investigated the anti-inflammatory effects of the ω−3 fatty acid DHA, the procyanidins B1, B2 and C1, and the combination of DHA with any of the procyanidins in human macrophages. To determine whether the modulation of the inflammatory response was conserved between the THP-1 cell line and primary human macrophages, we determined the IL-6 and PGE2 levels for both human macrophage models.
The concentrations of DHA and procyanidins applied to the macrophage pre-treatments mimicked levels that would be typical from a Mediterranean diet [28–31]. It has previously been determined in a pharmacokinetic study that subjects who consumed fish once or twice a month had detectable plasma DHA levels of approximately 182 μM [32,33]. This concentration is higher than the 25 μM DHA tested in the present study. However, pharmacokinetic results from the administration of plant extracts rich in polyphenols, such as cocoa or grape seed extracts, concluded that plasma concentrations of unmodified compounds depended on the polyphenol source. In this respect, the range of physiological concentrations of dimers and trimers detected was 2–10 μM in the plasma of mice models [28,29,34], although the range of dimer and trimer concentrations in human plasma can be greater. In the present study, the concentrations used were 17.3 μM for the B1 and B2 dimers, and 11.5 μM for the C1 trimer, although we observed the same anti-inflammatory behaviour using half the concentration for each procyanidin (results not shown).
IL-6 is a potent pro-inflammatory cytokine involved in the modulation of a broad range of cellular and physiological responses, including the activation of JAK (Janus kinase)/STAT (signal transducer and activator of transcription) and MAPK (mitogen-activated protein kinase) family members [35–39]. Therefore an increase in IL-6 secretion is a hallmark of the activation of an inflammatory response. The pre-treatment of THP-1 macrophages and primary human macrophages with DHA with or without procyanidins B1, B2 and C1 translated into a significant fall in IL-6 secretion.
COX1 and COX2 enzymes catalyse the first committed steps during the synthesis of prostaglandins from AA. Actually, both isoforms are of particular interest because they are catalogued as the pharmacological targets of NSAIDs .
Although the secretion of PGE2 and LPS-inducible COX2 protein expression was not down-regulated in THP-1 macrophages by pre-treatments of DHA, procyanidins B1, B2 or C1, or combinations of DHA with each of the procyanidins, all of the pre-treatments significantly inhibited PGE2 secretion in primary human macrophages. The discrepancy between the modulation of PGE2 secretion in THP-1 macrophages and primary human macrophages could be explained by the THP-1 differentiation process. To induce the differentiation of THP-1 monocytes into functional macrophages, mimicking the in vivo process, it is necessary to treat THP-1 monocytes with a differentiating agent, such as PMA [40,41]. The differentiation of cells with PMA involves the up-regulation of COX1 expression and the enhanced capacity to produce PGE2 [42,43]. Thus the PGE2 levels released in THP-1 macrophages are not only due to LPS induction; the PGE2 levels could also be concealed due to the induction of COX1 by PMA.
We set up a cell-free assay to elucidate the kinetic relationship between DHA and procyanidins with both constitutive and LPS-induced COX isoforms. Through the determination of the IC50 values (Table 1), we demonstrated the discriminatory capacity of DHA and procyanidins compared with the COX isoforms. The DHA and B1 dimers were strong and selective inhibitors of COX1 activity, and the B2 dimer and C1 trimers were selective inhibitors of COX2 activity. Some authors have determined that, although COX1 and COX2 share 60% primary sequence identity, there are differences between their active sites. These subtle differences could play key roles in the different affinities of COX1 and COX2 towards DHA and the procyanidins, including the procyanidin dimer isoforms B1 and B2. On the basis of our kinetic cell-free assay, we confirmed that DHA and procyanidins altered the substrate specificity of COX1 and COX2 to AA, as reflected by the modulation of the kinetic parameters. This change in the AA specificity was the result of the modulation of the maximal reaction rate (Vmax) and the Michaelis–Menten constant (Km). DHA exhibited mixed inhibitory behaviour towards COX1 through the down-regulation of Vmax and the up-regulation of Km; therefore DHA binds to a different location than the active site where AA binds. For the B1 inhibition, a decrease in Km and no change in Vmax demonstrated that B1 competes with AA in binding to the active site in COX1. This competitive behaviour, a decrease in Km and no change in Vmax, was also observed in the inhibition of COX2 activity by B2 and C1. The decrease in apparent Km values from DHA and B1 treatments for COX1 as well as from B2 and C1 treatments for COX2 was the reason for the decrease in the catalytic efficiency (kcat/Km) of PGE2 biosynthesis. The selective inhibition of COX catalysis of PGE2 synthesis by DHA and procyanidins in primary human macrophages was strongly supported by the kinetic parameters that were obtained from the cell-free assay. LPS is well described as a potent stimulus for PGE2 secretion, which is predominantly performed by COX2 . As shown in Figure 3(A), the inhibition of PGE2 was greater for B2 (a selective COX2 inhibitor) pre-treatment than DHA or B1 (selective COX1 inhibitors), and the combination of DHA (selective inhibitor of COX1) with B2 and C1 (competitive inhibitors of COX2) inhibited the secretion of PGE2 to nearly basal levels.
The transcription factor NF-κB is one of the most important inducible transcription factors in the regulation of the expression of most genes involved in the control of the inflammatory response, cellular proliferation and cell adhesion [12,13]. As an activator and modulator of many inflammatory processes, such as pro-inflammatory cytokines and prostaglandin secretion, the modulation of the NF-κB signalling pathway by DHA and procyanidins in THP-1 macrophages was evaluated. The present study demonstrated the capacity of DHA, alone or in combination with procyanidins, to down-regulate IKKβ activity, which was expressed as a decrease in the phosphorylation of pIκBα, a key modulator of NF-κB activation (Figure 5A).
However, p105, or NF-κB1, is an NF-κB family member that is associated with a dual function in the NF-κB signalling system. LPS-induced NF-κB activation leads to the proteolytic degradation of p105 to p50; therefore p105 can be considered as a p50 precursor. Furthermore, p105 possesses ankyrin repeats in its structure, as do IκB family members. These allow for association with other members of the NF-κB family proteins in the cytoplasm and subsequently cause the inhibition of nuclear translocation and DNA binding of the NF-κB complex [45,46]. The p105 is bound and retained in the cytoplasm by all NF-κB proteins, in contrast with IκBα, which only binds to NF-κB dimers that contain a p65 subunit . The degradation of p105 also liberates the p105-associated MAPK, which is responsible for the activation of the ERK (extracellular-signal-regulated kinase)/MAPK cascade . Thus the effects of the presence of p105 in the cytoplasm can be related to the decreased activation of the NF-κB transcription factor [46,47] and the pro-inflammatory ERK/MAPK cascade. Our findings showed that pre-treatments with the B2 dimer, C1 trimer and any of the combinations of DHA with B1, B2 or C1 led to an increase in the p105 cytoplasmic retention in LPS-induced THP-1 macrophages (Figure 5).
The classical and most abundant NF-κB heterodimer is composed of p50 and p65 subunits and is characterized as a potent activator of the expression of several pro-inflammatory genes. Moreover, p50 is able to form homodimers itself, and the p50–p50 homodimers can be found in the nucleus bound to DNA. Although p65 possesses a transactivation domain, p50 does not; therefore the presence of the p50–p50 homodimer in the nucleus represses the transcriptional activity of NF-κB [14,48,49]. Thus the nuclear p50 subunit could modulate the NF-κB signalling pathway, participating with the pro-inflammatory heterodimer p65–p50 or the repressor homodimer p50–p50. Therefore the B2 dimer is the most powerful compound during the induction of p50 cytoplasmic expression and, together with B1 and DHA/B1, is an inhibitor of p50 nuclear translocation. However, the pre-treatment of LPS-stimulated THP-1 macrophages with any of the compounds studied (DHA and/or procyanidins) down-regulated the nuclear translocation of p65, and it favoured the cytoplasmic retention of and dramatically inhibited the nuclear translocation of p65. The stronger capacity of DHA and procyanidins to inhibit the nuclear translocation of p65 was corroborated by p65 immunostaining and confocal microscopy visualization (Figure 6). A greater nuclear expression of p50 than that of p65 was observed in p50 and p65 blots, as shown in Figure 5, for all of the pre-treatments. In keeping with the conventional NF-κB complex structure, the NF-κB heterodimer is formed by one subunit of each dimer, p50 and p65 [13,14]. The fact that the pre-treatment induced a greater nuclear expression of the p50 subunit than p65, and the fact that the inhibition of p65 nuclear translocation was more dramatic than that of p50, led us to consider that the fraction of p50 detected in the nucleus was forming p50–p50 homodimers. Therefore our bioactive molecules, procyanidins and, more significantly, DHA, not only inhibited the translocation of the p50–p65 pro-inflammatory heterodimer, but also induced the translocation of the p50–p50 transcriptional repressor homodimers.
In addition, we demonstrated that the pre-treatment of THP-1 macrophages with DHA and/or procyanidins down-regulated the binding activity of NF-κB p65 to the corresponding κB consensus sequence located in the promoter and enhancer regions of several pro-inflammatory genes (Figure 7).
In conclusion, most LPS-stimulated inflammatory responses lead to the activation of the transcription factor NF-κB signalling pathway, the secretion of cytokines and the formation of prostaglandins (Figure 8). Therefore the selective inhibition of COX activity in cell-free assays, the inhibition of IL-6 and PGE2 secretion, and the down-regulation of the NF-κB activation signal pathway in human macrophages by food bioactive elements, such as DHA and procyanidins B1, B2 and C1, lead to great interest in their use as potential anti-inflammatory compounds. Along these lines, the conserved and, in certain cases such as in PGE2 secretion, the improvement in the anti-inflammatory response due to DHA, B1, B2 and C1 pre-treatments in the THP-1 cell line and primary human macrophages is relevant proof of the capacity of bioactive food compounds to promote good health through the modulation of inflammation with diet.
NF-κB activation and proposed mechanism for its inhibition by DHA and procyanidins
inhibitor of nuclear factor κB
mitogen-activated protein kinase
nuclear factor κB
non-steroidal anti-inflammatory drug
peripheral blood mononuclear cell
polyunsaturated fatty acid
Neus Martinez-Micaelo, Noemi González-Abuín, Anna Ardèvol, Montserrat Pinent and Mayte Blay designed the research. Neus Martinez-Micaelo, Ximena Terra and Cristobal Richart isolated human macrophages. Neus Martinez-Micaelo performed the experiments. Neus Martinez-Micaelo and Mayte Blay interpreted the data and wrote the paper. Mayte Blay directed the project.
This work was supported by the Universitat Rovira i Virgili (for Ph.D. students) and the Ministerio de Educación y Ciencia (MEYC) of the Spanish Government [grant number AGL2008-00387].