Published online before print July 16, 2008
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:RXR heterodimers: comparison with other polyunsaturated fatty acids
* Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain;
Laboratory of Oncological Immunology, Department of Medical and Molecular Genetics, IDIBELL-Cancer Research Institute, Barcelona, Spain;
Laboratory 123, Institut de Recerca, Hospital Universitari Vall d'Hebron, Barcelona, Spain;
Infectious Diseases Unit, Internal Medicine Department, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain; and
|| CIBER Fisiopatologia de la Obesidad y Nutrición, Instituto de Salud Carlos III, Barcelona, Spain
1 Correspondence: Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona. Diagonal 645, Edifici Nou, Planta-1, 08028 Barcelona, Spain. E-mail: jcdomingo{at}ub.edu
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(PPAR) activator, and the retinoid 9-cis-retinoic acid (9cRA), a retinoid X receptor (RXR) activator. Moreover, DHA induced the expression of PPAR target genes pyruvate dehydrogenase kinase-4 and aP-2 in immature DCs. The combination of DHA with Rosi or 9cRA produced additive effects. Furthermore, when DCs were cultured in the presence of a specific PPAR inhibitor, all of the changes induced by DHA were blocked. Together, these results strongly suggest that the PPAR:RXR heterodimer is the pathway component activated by DHA to induce its immunomodulatory effect on DCs.
Key Words: lymphocyte proliferation • immunological regulation • nuclear receptors • dentritic cell maturation
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Dendritic cells (DCs) are professional APCs capable of migrating to lymph nodes and inducing potent primary responses [14 15 16 ]. DCs play a pivotal role in the immune response, balancing between tolerance and immunity and connecting innate and adaptive responses [14 , 16 17 18 ].
Despite the large number of studies already describing the effects of fish oils and omega-3 PUFAs on many immune cell types, there has recently been increasing interest in the action of DHA on DCs. In this sense, some of the effects of DHA, as well as EPA and arachidonic acid, on DCs have been reported [19 20 21 ]. However, the precise mechanism of the action of DHA and related FAs on the regulation of DC activities remains unknown.
One of the important regulators in lipid metabolism is peroxisome proliferator-activated receptor (PPAR), which regulates genes responsible for lipid uptake, accumulation, and storage [22
]. Besides its role in adipocytes, it was also reported that PPAR is expressed in cells of monocytic origin such as macrophages and DCs, where it plays an anti-inflammatory role by blocking the induction of several proinflammatory cytokines [23
24
25
] and regulates lipid metabolism. PPAR forms a heterodimer with retinoic X receptors (RXR), subsequently regulating many target genes upon activation. PPAR are expressed at high levels in DCs, and DHA derivatives have been found to act as potent PPAR agonists [26
]. Furthermore, DHA itself has been reported to be a natural ligand of RXR [27
, 28
]. This fact together with the similar effects of the specific PPAR and RXR activators [i.e., Rosiglitazone (Rosi) and 9cis Retinoic acid (9cRA)] on DCs to those reported here suggest to us the possibility that these nuclear receptors are involved in the mechanism of action of DHA on DCs.
In this paper, we show that DHA acts on DCs by inhibiting the expression of some costimulatory markers and the secretion of important regulatory cytokines, as well as by limiting the lymphocyte stimulatory capacity of DCs, and we confirm an impairment of DC maturation described previously [21
]. Furthermore, additive effects of DHA with other specific activators of PPAR or RXR are also described. The effects induced by DHA action on DCs are more intense than those induced by any other monounsaturated FA or PUFA studied.
The DHA-induced expression of PPAR target genes in DCs together with the phenotypic and functional alterations and the blocking effects of specific PPAR:RXR inhibitors on the DHA-induced activity suggest a mechanism of action for DHA involving activation of the heterodimer PPAR:RXR. These results point toward the potential implications for immunological function of a DHA-rich diet and the possible usefulness of these FAs in reducing or complementing treatments with agonists of PPAR:RXR receptors and suggest a possible participation of this FA and its putative receptors in the physiological mechanisms of control of DC differentiation and maturation.
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When required, a negative selection purification method using monocyte-negative isolation kit II, according to the instructions of the manufacturer, was also used (Dynal Biotech ASA, Oslo, Norway). After magnetic retention of Dynabead-attached cells, the supernatant fraction contained more than 85% CD14+ cells.
Monocytes were seeded (1x106 cells/well) in six-well plates (Costar, Cambridge, MA, USA) and differentiated to immature DCs by adding IL-4 (750 U/ml; Sigma-Aldrich, St. Louis, MO, USA) and GM-CSF (100 ng/ml; Leucomax 400, Novartis, Barcelona, Spain) in RPMI medium (Sigma-Aldrich). Cultures were fed with cytokines every 2–3 days. At Day 6, DCs showed phenotypic characteristics of immature DCs (CD14neg, CD83low, CD1ahigh, HLA-DRhigh, CD80low, CD86low). When mature DCs were needed, LPS (500 ng/ml, Sigma-Aldrich, EC 055:B5), TNF-
(Sigma-Aldrich), or polyinosinic:polycytidylic acid (Sigma-Aldrich) was added at Day 6, and DCs were cultured for 2 additional days.
DC treatment
Prior to addition of DC-differentiating cytokines, the indicated concentrations of DHA, EPA, oleic acid (OA), linoleic acid (LA), 9cRA (Sigma-Aldrich), and Rosi (GlaxoSmithKline, Mississauga, Ontario, Canada) were added alone or in combinations. When necessary, 10 µM GW9662 (Sigma-Aldrich) was added to cultures and incubated for 90 min at 37°C, 5% CO2, prior to any other treatment. The GW9662 addition was repeated on Day 3.
Flow cytometry
DCs were collected, and mAb, directly conjugated to FITC or PE, were subsequently used. The FITC-conjugated mouse mAb were anti-CD1a, anti-CD83, anti-HLA-DR, and anti-CD36, and the PE-conjugated mouse mAb were anti-CD14, anti-CD80, and anti-CD86 (BD PharMingen, San Diego, CA, USA). Data for at least 5 x 103 DC regions/sample were acquired and analyzed on a FACScan flow cytometer using CellQuest (BD Biosciences, Mountain View, CA, USA), FlowJo (Tree Star, Inc., Ashland, OR, USA), and WinMDI software.
Chemotaxis assay
Immature DCs (5x105) were seeded into a transwell chamber (5 µm, Millipore, Billerica, MA, USA) in a 12-well plate, and migration to MIP-1 (10 ng/ml; R&D Systems, Wiesbaden, Germany) was analyzed after 1 h, counting the migrated cells by gating DCs in a FACSCalibur cytometer for 4 min. After DC maturation with LPS, as described above, migration to MIP-3β (10 ng/ml; PeproTech, Rocky Hill, NJ, USA) was performed under similar conditions as for MIP-1.
Analysis of endocytic capacity
For the analysis of endocytic activity, 2 x 105 DCs were incubated with FITC-Dextran (Sigma-Aldrich) for 30 min at 37°C. As a control, 2 x 105 DCs were precooled to 4°C prior to incubation with dextran at 4°C for 30 min. The cells were washed four times and analyzed on a FACSCalibur cytometer.
Carboxyfluorescein diacetate-succinimidyl ester (CFDA-SE) staining
To determine proliferation, lymphocytes obtained as described above were thawed and washed immediately with PBS to eliminate DMSO. Cells were incubated for 15 min at 4–8°C with DNase and MgCl2 to avoid DNA debris and stained with 5 µM CFDA-SE (Molecular Probes, Leiden, Netherlands) for 10 min at 37°C, followed by two steps of incubation (10 min at 37°C) and washing.
DC autologous stimulation of lymphocytes
Primary DCs were pulsed with Staphylococcal enterotoxin B (SEB; Sigma-Aldrich) antigen (1 µg/mL) for 4 h at 37°C in a humid atmosphere of 5% CO2 and then irradiated prior to coincubation with lymphocytes. Autologous stimulation was conducted in 96-well culture plates in 200 µL RPMI 1640 containing 10% inactivated FCS and antibiotics. DCs were mixed with 1 x 105 autologous CFSE-labeled lymphocytes at a DC:lymphocyte ratio between 1:10 and 1:160. Cells were allowed to proliferate for 96 h and were then collected and analyzed in a FACScan flow cytometer using appropriate software. The percent divided cells (%DvC; the initial number of lymphocytes that become activated by DCs) and the proliferation index (PI; the average number of divisions in the lymphocyte population) were calculated using the FlowJo proliferation platform. Comparison between %DvC from samples and controls at different ratios of DC:lymphocyte permits an approximated calculation of the stimulatory capacity of the DCs.
RNA isolation and quantitative real-time RT-PCR analysis of PPAR target genes
Total RNA was prepared from 
106 DCs, differentiated and subjected to the treatments described above. After extraction with a guanidinium HCl/phenol mixture and isopropanol precipitation following the Roche Tripure isolation reagent protocol [30
], RNA was dissolved in diethyl pyrocarbonate-treated water, and the integrity of the RNA samples was checked by electrophoresis in agarose gel and the concentrations estimated spectrophotometrically. Typically, 6–10 µg total RNA was obtained from one extraction. RNA was treated with RNase-free DNase for 1 h at 37°C. Quantitative mRNA expression analysis was performed using TaqMan real-time RT-PCR (Applied Biosystems, Foster City, CA, USA). The RT reaction was performed on 0.5 µg RNA using TaqMan standardized reagents, and the real-time PCR reaction was performed using TaqMan Universal PCR Master Mix and the following standardized gene expression primer probes ("Assay-on-Demand"): FA-binding protein 4 (FABP4/aP2; Hs99699791_m1) and pyruvate dehydrogenase kinase-4 (PDK4; Hs00176875_m1). Samples were run in duplicate on the ABI-PRISM 7700HT sequence detection system (Applied Biosystems), and the mean value of the duplicate was used to calculate the mRNA expression of the gene of interest, which was normalized to that of the reference control [hypoxanthine guanine phosphoribosyl transferase (HPRT); Hs99999909_m1] using the 2-
-comparative threshold method, following the manufacturer’s instructions.
Measurement of cytokines
On Day 7, LPS was added to the DC cultures, and supernatants were harvested 24 h later. Samples were then frozen at –80°C until use. The human IL-10 and IL-12 levels were determined using the human IL-12 (p70) subunit and IL-10 BD OptEIA ELISA set, according to the instructions of the manufacturer (Becton Dickinson, Franklin Lakes, NJ, USA). IL-6 gene expression was analyzed following the method described in the previous section. The primers and probe used were an Hs00174131_m1, Assay-on-Demand combination from Applied Biosystems. Results were normalized to that of the reference control (HPRT, Hs99999909_m1)
Statistics
Statistical analysis was performed using the SPSS 12.0 statistical package (SPSS Inc., Chicago, IL, USA), and the nonparametric Wilcoxon signed rank test was used for comparisons between control and experimental groups [31
]. The significance level was placed at P < 0.05.
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Figure 1. Changes in the differentiation process from monocyte to immature and mature DCs in the presence or absence of 50 µM DHA. Adherent monocytes were differentiated to DCs by 6 days culture in the presence of GM-CSF and IL-4. Maturation of DCs was induced by 2 additional days of culture with 500 ng/mL LPS. The surface expression level is indicated as mean fluorescence intensity (MFI) in each histogram.
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Table 1. Impact of DHA Concentration on the Immature and LPS-Matured DC Phenotypea
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Effect of DHA on chemotaxis and uptake capacity in DCs
As phenotypic changes induced by DHA suggested differences in the immature and mature characteristics of the treated DCs, the migration to MIP-1 in immature DCs and to MIP-3β in mature DCs was examined. As can be observed in Figure 2
, DHA increases MIP-1 chemotaxis in immature DCs, whereas GW9662 blocks the process (P<0.05). Contrarily, for MIP-3β, the chemotactic capacity of the DHA-treated, mature DCs was decreased significantly (P<0.05) and the effect inhibited by GW9662 (Fig. 2)
.
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Figure 2. Chemotaxis induced by MIP-1 and MIP-3β on immature and mature DCs, respectively. Mean of 10 different assays. Results are expressed as mean of the MFIs of the different flow cytometry assays. Statistical analysis was performed using the Wilcoxon signed rank test; *, P < 0.05 (different from untreated).
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DHA effects are partially reproduced by other FAs
To determine whether the effects observed with DHA on DCs are restricted to this FA or are a general feature of PUFAs, the activities of other PUFAs: EPA, LA, and OA, belonging to the omega-3, omega-6, and omega-9 FA families, respectively—were also tested. Immature DCs treated with the different PUFAs (Fig. 3A
) displayed similar, although less intense, phenotypic alterations to those obtained with DHA, except for EPA and LA, which in contrast to DHA, reduced CD36 expression. OA induced no phenotypic changes except the up-regulation of CD80. The weak effects shown for LA and OA could be incremented with higher concentrations of these FAs but without reaching the intensities shown for DHA and EPA. When the other FAs were tested on mature DCs, the results paralleled those obtained with DHA but again, to a lesser degree (Fig. 3B)
. The only difference was again in the expression of CD36, which was not up-regulated with these FAs. OA once again produced no effects in the mature stage, in comparison with those using DHA.
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Figure 3. Changes in the differentiation process from monocytes cultured with GM-CSF and IL-4 to produce immature DCs (A) and LPS-matured DCs (B) induced by the presence 50 µM DHA, EPA, LA, or OA.
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Figure 4. Cytokine secretion of LPS-matured DCs in the presence of PUFAs. ELISA displaying IL-12 (A) and IL-10 (B) secretion at different FA concentrations normalized to control (100%). (C) Effect of DHA on the expression of the IL-6 gene studied by real-time RT-PCR. Statistical analysis was performed using the Wilcoxon signed rank test; *, P < 0.05 (relative to untreated). C+, positive control (matured DCs without any additional treatment).
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DHA down-regulates DC stimulation of autologous lymphocytes
DCs, differentiated in the presence of 50 µM of the following FAs: DHA, EPA, LA, or OA, were loaded with the SEB antigen, irradiated, and cocultured with CFSE-labeled lymphocytes at a ratio of 1:10 DC:lymphocyte. Analysis of proliferation using the FlowJo proliferation platform confirmed the results observed in the histograms, being 43.4 ± 11.1%DvC (for untreated DCs) and 16.3 ± 6.8% for DHA-treated DCs (P<0.05; Table 2
), which indicates a proliferation decrease of 69% with respect to the untreated DCs. EPA- and LA-treated DCs induced a less-pronounced decrease in lymphoproliferation. Finally, OA-treated DCs did not reduce lymphoproliferation (46.2±9.2; Table 2
). DHA did not induce changes in CD4, CD8 T cell subpopulations under the culture conditions of this work nor were there significant variations in CD62 ligand and CD25 high regulatory T cell (Treg)-related markers.
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Table 2. Impact of FA Treatment on Lymphocyte Activation
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activators, and additionally, this FA is able to bind to RA receptor RXR [27
, 28
]. Treatment of DCs with DHA yielded similar results to those reported by Nencioni et al. [32
] and us [29
], obtained using the PPAR and RXR ligands Rosi and 9cRA, respectively. In this sense, Rosi, 9cRA, and DHA induced the same phenotypic variations (Fig. 5
). Rosi and DHA down-regulated IL-10 and IL-12 secretion in mature DCs, and all three compounds also inhibited autologous lymphocyte proliferation stimulated by DCs. Furthermore, PPAR and RXR ligands produced additive or synergic effects when combined in cultures with complementary activators of both sides of the PPAR:RXR heterodimer. These additive effects have been demonstrated in many different cell types [33
34
35
], including DCs [29
]. To test whether DHA could mimic these effects, combination studies of DHA with the previously mentioned, specific, strong agonists for PPAR and RXR were performed. At concentrations for which agonists alone produced scarcely any effect in DCs, additive effects were seen in combination with DHA. Thus, in immature DCs, additive effects of DHA with Rosi or 9cRA coincubation were observed for CD86, CD36, HLA-DR, CD1a, and CD80 (Fig. 5A)
. In mature DCs, once again, the additive effects observed in the combination of DHA with Rosi or 9cRA were the down-regulation of CD86, CD80, CD83, CD1a, and HLA-DR and the up-regulation of CD36 (Fig. 5B)
. When using higher concentrations of these ligands, the additive effects were increased in immature and mature DCs (Fig. 5 A and B)
.
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Figure 5. Phenotypic, additive effects of DHA when combined with 9cRA or Rosi. MFI from immature (A) and LPS-matured (B) DCs belonging to four healthy individuals. In low concentrations (LC), DCs were incubated with 5 µM DHA and/or 100 nM Rosi or 1 nM 9cRA. In high concentrations (HC), DCs were incubated with 25 µM DHA and/or 1 µM Rosi or 10 nM 9cRA.
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target genes in DC cells [25
], FABP4 (aP2) and PDK4, was studied by real-time RT-PCR. When DCs were treated with DHA, an increase in the expression of these two genes (12-fold for FABP4; 18-fold for PDK4) also induced by Rosi (53-fold for FABP4; sixfold for PDK4) was observed (Fig. 6
). When DCs were treated with combinations of DHA and Rosi, additive effects in the expression of both genes were observed (Fig. 6)
. Additive effects, but in a lower range, were also observed when DCs were treated with combinations of DHA plus HX630 (a RXR-specific agonist) or 9cRA (Fig. 6)
.
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Figure 6. Effect of DHA on the expression of PPAR target genes FABP4 (aP2; A) and PDK4 (B) studied by real-time RT-PCR. DCs were treated with combinations of DHA and/or PPAR or RXR agonists or GW9662, a specific inhibitor of PPAR. Statistical analysis was performed using the Wilcoxon signed rank test; *, P < 0.05 (relative to untreated). C–, positive control (matured DCs without any additional treatment).
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inhibitor, blocks DC changes induced by DHA activation but not that of PPAR or -β and also blocks heterodimer formation [37
]. Therefore, in the presence of GW9662, no activation is possible through the RXR or the PPAR component of the heterodimer. To further determine if changes observed in DHA-treated DCs were produced through the PPAR:RXR heterodimer pathway, GW9662 was added to the monocyte cultures 90 min prior to treatment with DHA and the addition of the differentiating cytokines. In GW9662-treated, immature DCs as well as in mature DCs, all of the markers studied were inhibited, except CD36, which was up-regulated, blocking the effects of DHA and strongly suggesting that this FA is acting through the PPAR:RXR heterodimer (Fig. 7 A and B
). In the same way, GW9662 was able to inhibit the DHA induction of PPAR target gene expression (Fig. 6)
. Two particular cases were CD1a, with GW9662 partially blocking DHA inhibition, and CD36, which is up-regulated by DHA and surprisingly, GW9662 in an additive manner (Fig. 7 A and B)
. When cytokine production was studied, GW9662 blocked the effects of DHA-treated DCs on the secretion of IL-12 (Fig. 8C
), but production of IL-10 was inhibited, whether or not DHA was present (Fig. 8D)
. In the same way, GW9662 blocks the effects of DHA-treated DCs on the stimulation of lymphoproliferation. Control DCs loaded with 1 µg SEB induced a lymphocyte proliferation of 43% of %DvC and a PI of 4.71 (Fig. 8A
, shaded) in comparison with the cells treated with GW9662, which induced a slight increase (%DvC, 55%; PI, 6.41; Fig. 8A
, dashed line). Previous treatment of DCs with 50 µM DHA reduced induction of lymphocyte proliferation (%DvC, 15%; PI, 2.02; Fig. 8B
, shaded), whereas GW9662 treatment reversed the DHA-induced inhibition (%DvC, 36%; PI, 4.26; Fig. 8B
, dashed line).
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Figure 7. Modulation of DHA effects on DC phenotype by GW9662. MFI from eight healthy individuals in immature DCs at Day 6 (A) and in LPS-matured DCs (B). DCs were incubated with or without 25 µM DHA in the presence or not of 10 µM GW9662. All of these compounds were present in culture medium from the 1st day of differentiation from monocytes to DCs. GW9662 was added to cultures, and incubation was for 90 min at 37°C, 5% CO2, before any other treatment; this addition was repeated on Day 3 and only in LPS maturation on Day 6.
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Figure 8. Lymphoproliferation and cytokine secretion in the presence of GW9662. Representative lymphoproliferations from three different, healthy individuals triggered by DCs loaded with 1 µg SEB, with (A, dashed line) or without (A, shaded histogram) GW9662. The same experiment was performed in the presence of 50 µM DHA alone (B, shaded histogram) or with GW9662 (B, dashed line). FL1-H, Fluorescence 1-height. The DC:lymphocyte ratio was 1:10. ELISA displaying IL-12 (C) and IL-10 (D) secretion from 10 µM GW9662-treated DCs and untreated in the presence or not of 50 µM DHA. GW9662 was present in culture medium from the 1st day of differentiation from monocyte to DC. The results are shown normalized to the controls. Statistical analysis was performed using the Wilcoxon signed rank test; *, P < 0.05 (relative to untreated).
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:RXR in the physiological maturation process of these cells [29
], which could also be modulated by dietary PUFAs. |
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In the present report, we have addressed the effects that DHA and other unsaturated FAs exert on human monocyte-derived DCs. DHA, as well as EPA and LA, although to a lesser degree, lead to induction or inhibition in the expression of some costimulation and antigen-presentation markers, depending on the DC maturation stage. CD36, which was always up-regulated, is implicated in noninflammatory antigen apoptotic cell capture [57 ]. These FAs also diminished secretion of the cytokines IL-10 and IL-12 and the DC stimulatory capacity for T cells. IL-10 and IL-12 play a central role in the regulation of the lymphocyte activity induced by DCs; IL-10 is able to commit T lymphocytes to a TH2 activity and in some circumstances, to Treg, whereas IL-12 directs lymphocytes to TH1 activity. OA, the fourth FA studied, did not alter the DC phenotype, except for an unexpected increase in CD80 surface expression.
The results obtained here could help to explain the abundant benefits reported for DHA and EPA in chronic inflammatory and autoimmune diseases [58 ]. In any case, the potent down-regulation of CD1a expression in DCs treated with DHA and EPA is particularly interesting. CD1a is implicated in the activation of the subpopulation of cytolytic T cells restricted for CD1 that can be stimulated by immature and mature DCs through CD1 presentation. It is known that at least one parasite, Leishmania donovani, is able to down-regulate CD1 in DCs to avoid activation of these T cells [59 ]. Moreover, it has been demonstrated recently that group 1 CD1 (CD1a,b,c) but not group 2 (CD1d) is crucial for the presentation of Mycobacterium tuberculosis antigens [60 ]. As a consequence, a diet primarily based on omega-3 FAs could potentially inhibit group 1 CD1 presentation by APCs. It could be worthwhile to study its possible relation to the high incidence of the infectious tuberculosis disease observed in the Eskimo population (who follow omega-3-enriched diets and present a 10–20 times higher tuberculosis incidence than other populations, despite their low population density [61 ]).
PPAR is an important regulator of many immunogenic functions in monocytes, macrophages, lymphocytes, and DCs [36
, 62
63
64
65
66
67
68
]. Many reports have independently described the same effects in these cells and NK cells for DHA treatment [2
, 3
, 6
, 69
70
71
72
73
74
] and PPAR activators [32
, 36
, 62
63
64
65
66
67
68
, 75
]. The actions of DHA and PPAR ligands are also significant in diabetes and cardiovascular diseases such as atherosclerosis and hypertension [75
76
77
]. The PPAR:RXR heterodimer can be activated independently by RXR or PPAR ligands [35
, 78
, 79
]. In fact, DHA derivatives have been found to act as potent PPAR agonists [26
], and DHA itself has been reported to be a natural ligand of RXR [27
, 28
]. All of these lines of evidence suggest a likely relationship between this nuclear receptor and the mechanism of action of DHA, which despite years of study, remains obscure. On the other hand, it has been reported that combinations of RXR and PPAR activators exert additive or synergistic effects on glucose and lipid metabolism and also on the expression of some genes, through the simultaneous activation of both heterodimer partners [33
, 80
81
82
]. We have already demonstrated these additive effects in DCs combining Rosi and 9cRA [29
]. As expected, if the DHA mechanism of action was dependent on PPAR, when DHA was combined with Rosi, 9cRA, or HX630 (a RXR-specific agonist), it induced additive effects on the DC phenotype (Fig. 5)
and on the expression of the PPAR target genes FABP4 (aP2) and PDK4 (Fig. 6)
. Both combinations showed similar effects, although that of DHA with Rosi was more pronounced, suggesting that although DHA is a promiscuous activator of both sides of the PPAR:RXR heterodimer, it is more prone to RXR.
To further investigate this mechanism, we added the selective PPAR inhibitor GW9662 to the DC cultures, prior to addition of DHA. In the case whereby RXR:RXR homodimers or other heterodimers were responsible for the effects induced by DHA, then the inhibitor would not interfere with DHA function. However, our results indicated that GW9662 blocked DHA effects, suggesting the PPAR:RXR heterodimer pathway as the main site of action.
The lack of toxicity in humans of DHA treatment and the additive effects observed in this work with other PPAR or RXR activators, which could show toxicity or side-effects at the therapeutically effective dosage, point to DHA as a potential candidate for therapeutic use. DHA used as a complementary adjuvant with other PPAR and RXR agonists could increase their activity and/or reduce their required dose without loss of effectiveness. This could have important implications in the treatment of diabetes, inflammatory disorders, and cardiovascular diseases such as atherosclerosis and hypertension. Finally, the effects of fish oil-rich diets on CD1 presentation in vivo require more intensive research to clarify a possible role in the spread of M. tuberculosis in certain populations.
Received October 10, 2007; revised May 30, 2008; accepted June 17, 2008.
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  S. Kapoor Immunomodulatory properties of omega-3 fatty acids: a possible explanation for their systemic, anti-carcinogenic effects J. Leukoc. Biol., January 1, 2009; 85(1): 2 - 3. [Full Text] [PDF]
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