The Polyunsaturated Fatty Acids Arachidonic Acid and Docosahexaenoic Acid Induce Mouse Dendritic Cells Maturation but Reduce T-Cell Responses In Vitro

Long-chain polyunsaturated fatty acids (PUFAs) might regulate T-cell activation and lineage commitment. Here, we measured the effects of omega-3 (n-3), n-6 and n-9 fatty acids on the interaction between dendritic cells (DCs) and naïve T cells. Spleen DCs from BALB/c mice were cultured in vitro with ovalbumin (OVA) with 50 μM fatty acids; α-linolenic acid, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid or oleic acid and thereafter OVA-specific DO11.10 T cells were added to the cultures. Fatty acids were taken up by the DCs, as shown by gas chromatography analysis. After culture with arachidonic acid or DHA CD11c+ CD11b+ and CD11c+ CD11bneg DCs expressed more CD40, CD80, CD83, CD86 and PDL-1, while IAd remained unchanged. However, fewer T cells co-cultured with these DCs proliferated (CellTrace Violetlow) and expressed CD69 or CD25, while more were necrotic (7AAD+). We noted an increased proportion of T cells with a regulatory T cell (Treg) phenotype, i.e., when gating on CD4+ FoxP3+ CTLA-4+, CD4+ FoxP3+ Helios+ or CD4+ FoxP3+ PD-1+, in co-cultures with arachidonic acid- or DHA-primed DCs relative to control cultures. The proportion of putative Tregs was inversely correlated to T-cell proliferation, indicating a suppressive function of these cells. With arachidonic acid DCs produced higher levels of prostaglandin E2 while T cells produced lower amounts of IL-10 and IFNγ. In conclusion arachidonic acid and DHA induced up-regulation of activation markers on DCs. However arachidonic acid- and DHA-primed DCs reduced T-cell proliferation and increased the proportion of T cells expressing FoxP3, indicating that these fatty acids can promote induction of regulatory T cells.


Introduction
Lymphoid organs are embedded in fat [1] and fatty acids, especially long-chain polyunsaturated fatty acids (PUFAs) have immunoregulatory functions via several mechanisms. They are

Dendritic cell: T cell co-culture
The experimental design is shown in S1 Fig. Spleen DCs from mice were cultured in medium supplemented with fatty acids, see below, and the model antigen OVA for 3 days. On day 3 DCs were analyzed by flow cytometry for cell surface molecules (CD11b, CD11c, CD40, CD83, CD86, MHC class II IA d and PDL-1). Alternatively CellTrace™-stained OVA-specific CD4 + T cells, isolated from DO11.10 mice, were added and co-cultured with the DCs for another 6 days. Thereafter T-cell proliferation and phenotype (expression of CD25, CD69, CTLA-4, FoxP3, Helios and PD-1) were determined by flow cytometry.
For DCs, eight mice were sacrificed and spleens removed and prepared into single cell suspension. CD11c + cells were isolated with the MACS cell separation system (N418, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and LS columns, according to the manufacturer's instructions. CD11c + cells (5x10 4 ) were cultured in 96-well plates (Zellkultur Testplatte 96U, TPP) in Iscove's modified Dulbecco's medium (Sigma-Aldrich Co., St Louis, MO) supplemented with 10% fetal bovine serum, 1% β-mercaptoethanol (4 mM solution), 1% L-glutamine (200 mM solution, Sigma-Aldrich) and 0.1% gentamicin (50 mg/ml solution, GIBCO/Invitrogen, Eugene, OR). Fatty acids were dissolved in ethanol and added to DC cultures in a final concentration of 50 μM, a physiological concentration in plasma [33,34], within a range of concentrations that have been used in previous in vitro experiments [5,8,12]. The final ethanol concentration in the cell cultures was max 1%, when ethanol alone was used as control. The following fatty acids were used; 18:1 n-9 oleic acid (OA), 18:2 n-6 linoleic acid (LA), 18:3 n-3 αlinolenic acid (ALA), 20:4 n-6 arachidonic acid (AA), 20:5 n-3 eicosapentaenoic acid (EPA) and 22:6 n-3 DHA were used (all from Sigma-Aldrich with purity !98%). Purity of stock solutions was tested by gas chromatography-mass spectrometry analysis, and found to be > 90% (S2 Fig). Stock solutions of fatty acids as well as cell culture medium were tested for presence of staphylococcal enterotoxin with the SET-RPLA kit (Oxoid Ltd, Basingstoke, UK) and found to be endotoxin free. OVA (grade III, Sigma-Aldrich) was added to a final concentration of 0.5 μg/μl in cell cultures later used for T-cell analysis. For blocking experiments purified antimouse CD83 (HB15, clone Michel-17) or PD-1 (CD279) (clone RMP1-14) antibodies were used in a final concentration of 10 μg/ml (both from eBioscience Inc., San Diego, CA). After 3 days the DCs were either harvested for FACS analysis of phenotype or further co-cultured with OVA-specific T cells (5x10 4 , giving a DC: T cell ratio of 1:1). Notably, DCs were washed and medium renewed before co-culture with T cells to avoid direct stimulation from fatty acids on T cells. Responder OVA-specific DO11.10 T cells were isolated and purified from spleens using the CD4 + T cell isolation kit II (Miltenyi Biotec GmbH) according to the manufacturer's instructions. Isolated T cells were stained with CellTrace™ Violet (CellTrace™ Violet Cell Proliferation Kit, Molecular Probes/Invitrogen) in a final concentration of 5 μM, prior to addition to the CD11c + cell cultures. After 6 days of co-culture T cells were harvested for flow cytometry analysis of proliferation and phenotype.

Gas chromatography analysis of fatty acid content in dendritic cells
Cultured DCs were pooled to 4-8 Ã 10 6 cells and washed four times with PBS. After complete evaporation of PBS, 4 μg of an internal fatty acid standard, 17:0 in phospholipid form (Larodan AB, Malmö, Sweden), dissolved in dichloromethane was added to the samples. Total lipids were extracted with 5 ml 2:1 chloroform-methanol solution for 1 h. 2 ml milliQ-H 2 O were added and phases separated by centrifugation. The non-polar phase was transferred to a new tube before liquid evaporation. Fatty acids were methylated for 1 h at 75°C using 1 ml 10% acetyl chloride in methanol and 1 ml toluene. 1 ml milliQ-H 2 O and 5 ml petroleum ether were added. The non-polar phase was transferred to a new tube before liquid evaporation. The fatty acid methyl esters (FAME) were dissolved in 150 μl iso-octane and separated on a Agilent Technologies 7890A GC with a 5975C MSD Triple-Axis detector (Agilent Technologies, Inc., Santa Clara, CA), using a VF-WAXms column (30 m x 0,25 mm, film 0,25 μm; Agilent Technologies). The fatty acids were identified with the MSD ChemStation software version E.02.01.1177 (Agilent Technologies) and peaks identified using the GLC-463 reference standard (Nu-Chek Prep, Inc., Elysian, MN).

Flow cytometry
In brief, the procedure for flow cytometry was as follows. Cell cultures were centrifuged and resuspended in FACS wash buffer (prepared in-house) with a 1:100 solution of Fc-receptor block (anti-Mouse CD16/CD32 Purified, clone 93, eBioscience) and appropriate antibodies, diluted 1:100. Samples were incubated at 4°C for 20 min and washed twice prior to flow cytometry analysis. For intracellular staining of FoxP3, Helios and CTLA-4, the Foxp3/Transcription Factor Staining Buffer Set (eBioscience) was used. The last wash was followed by addition of permeabilization solution and samples were incubated at 4°C for 30 min. After centrifugation and 2x wash, cells were resuspended in permeabilization buffer. Fc-receptor block and antibodies (1:50) were added. After incubation, cells were washed twice in permeabilization buffer, followed by addition of FACS wash buffer and flow cytometry analysis.

Statistical analysis
Data was tested for normality with the D'Agostino & Pearson omnibus normality test. Parametric one-way ANOVA or non-parametric Kruskal-Wallis test was used to test statistical mean difference compared to control, ethanol but no fatty acid, with correction for multiple comparisons. For correlation analysis the parametric Pearson correlation test or the nonparametric Spearman correlation test was used. In blocking experiments, the parametric t test or the non-parametric Mann Whiney U-test was used for comparison between blocking and no blocking. All tests were performed two-tailed. Statistical significance levels was set at p 0.05 ( Ã ), p 0.01 ( ÃÃ ) p 0.001 ( ÃÃÃ ) and p 0.0001 ( ÃÃÃÃ ). For tests with all seven stimuli and eight mice only p-values below 0.01 were considered statistically significant.

Results
The supplemented fatty acids were taken up by the dendritic cells CD11c + DCs, isolated from the spleen of BALB/c mice, were cultured for 3 days with different fatty acids in the cell culture medium. Supplementation of arachidonic acid, DHA or oleic acid in the culture medium was reflected in the fatty acid content of the DCs. When measured as proportion of the cells' total amounts of fatty acids, the levels of oleic acid increased, from mean 12.7% to 33.2%, (p< 0.0001, Fig 1A) when added to the cell culture medium. Arachidonic acid changed from mean 7.0% to 8.7%, (p = 0.5253, Fig 1B) and DHA from mean 0.9% to 2.4%, (p = 0.7716, Fig 1C) but these changes did not reach statistical significance.
No change in proportion of CD11c + CD11b +/neg DCs after fatty acid supplementation After 3 days of in vitro culture with fatty acids DCs were analyzed by flow cytometry for separation of DC subsets and examination of their costimulatory molecule expression (Experimental design, S1 Fig). The FACS analysis revealed a high background in both the CD11c and CD11b channel (Fig 2A), reflecting low purity and pronounced autofluorescence of in vitro cultured cells. CD11c + cells were gated, based on higher fluorescence than to the FMO control (Fig 2A) and divided into CD11c + CD11b + (upper gate) and CD11c + CD11b neg (lower gate) subpopulations, shown in Fig 2A. This was made because the relative level of expression for several cell surface molecules were higher in the CD11b + group, as seen in the histograms in Figs 3 and 4. The proportion of CD11c + cells (Fig 2B) or CD11c + CD11b + cells ( Fig 2C) did not differ between cultures supplemented with fatty acids, compared to control cultures with ethanol only.
Increase of costimulatory molecules on DCs after culture with arachidonic acid or DHA We investigated whether supplementation with fatty acids affected DC expression of MHC class II (IA d ) and costimulatory molecules, CD80, CD86 and CD40. As seen in Fig 3A, expression of MHC class II, i.e. IA d , was unaffected by fatty acids, only a non-significant reduction was observed with arachidonic acid. Supplementation with oleic acid or α-linolenic acid induced non-significantly higher CD80 expression ( Fig 3A). In contrast, CD86 was significantly up-regulated on CD11c + CD11b neg DCs in response to arachidonic acid and DHA, and also tended to increase on CD11c + CD11b + cells in response to DHA (Fig 3A). CD40 expression was up-regulated on CD11c + CD11b + in cultures with arachidonic acid or DHA, and on CD11c + CD11b neg DCs in cultures with DHA ( Fig 3A). Representative histograms of MFI values for CD80 and CD86 are shown in Fig 3B and for CD40 and IA d in Fig 3C. The effect of fatty acid supplementation on expression of the inhibitory molecule PDL-1 and the DC maturation marker CD83 is shown in Fig 4A. CD83 was significantly increased on DCs cultured with arachidonic acid or DHA, which correlated with up-regulation of CD86

Generation of prostaglandin E 2 in arachidonic acid-primed dendritic cell cultures
We also measured soluble factors in the supernatant of DC cultures stimulated with the fatty acids arachidonic acid, DHA or oleic acid for 72 hours. We observed high levels of PGE 2 , >2 pg/ml, in DC cultures stimulated with arachidonic acid whereas <0.2 pg/ml of PGE 2 was found with the other stimuli ( Fig 5B). Since we observed an upregulation of CD83 expression on DCs supplemented with arachidonic acid and DHA we also measured soluble CD83 (sCD83) in the supernatant. Only low levels of sCD83 was detected, and without differences between the groups ( Fig 5A). The levels of IL-10, IL-12 p70, and IFNγ were below detection limit while TGF-β1 levels were in pair with the levels in fresh complete IMDM (containing fetal bovine serum), with no differences between the groups.
In conclusion, MHC expression on DCs was unaffected by fatty acids, while arachidonic acid and DHA up-regulated the costimulatory molecules CD40, CD83, CD86 and the inhibitory molecule PDL-1. However, arachidonic acid distinguished itself from the other fatty acids in its ability to induce PGE 2 production.

Dendritic cells cultured with arachidonic acid or DHA reduced T-cell proliferation
Next, we investigated if the fatty acid exposure of DCs would alter the outcome of the antigenpresentation to naïve T cells. Therefore, DCs were cultured with fatty acid and the model antigen OVA for 3 days, thereafter OVA-specific DO11.10 + T cells were added to the DC cultures (S1 Fig). The gating strategy of T cells is shown in Fig 6A and 6B. Briefly, lymphocytes were gated (upper 3 panels, Fig 6A), followed by gating for DO11.10 expression (middle panel, Fig  6A). Proliferation is shown in the lower panel of Fig 6A. After culture with OVA, about 25% of the T cells had divided, only 2% had divided in the absence of antigen. The solvent, ethanol had an inhibitory effect on proliferation, i.e. about 20% of the T cells proliferated in control cultures ( Fig 6A). Arachidonic acid-or DHA-primed DCs reduced subsequent T-cell proliferation, compared to the control DCs, from % 20% to % 5% ( Fig 6C). Expression of the activation markers CD69 and CD25 was reduced in parallel ( Fig 6D and 6E). There was a correlation between proliferation and expression of CD69 (r = 0.8587, p < 0.0001) as well as proliferation and CD25 (r = 0.8935, p < 0.0001) (S4 Fig). None of the other investigated fatty acids (linoleic acid, α-linolenic acid, oleic acid or EPA) suppressed the capacity to activate T cells.

Cell death in T-cell cultures with arachidonic acid-or DHA-primed dendritic cells
We determined the levels of apoptosis and necrosis in the T cells by staining with Annexin V and 7AAD. The gating strategy is shown in Fig 7A and 7B. Briefly, to identify debris, live cells (Annexin V -7AAD -) were gated and on those cells we put a non-debris gate from which CD4 + T cells were gated for Annexin V and 7AAD staining. There was a non-significantly lower proportion of live T cells in the DC: T cell co-cultures with arachidonic acid-primed DCs   Fig 7C) and a corresponding increase in late stage apoptotic/necrotic cells (Annexin V + 7AAD + , % 20% compared to 5%, Fig 7D), but no differences in early stage apoptotic T cells (Annexin V + 7AAD -, Fig 7E).

Regulatory T cells in cultures with arachidonic acid-or DHA-primed dendritic cells
Since arachidonic acid and DHA were the only fatty acids that affected DC phenotype as well as the DCs' ability to induce T-cell proliferation and activation markers, we chose to focus on these and included oleic acid as an inert fatty acid control. To investigate if the low T-cell proliferation observed with arachidonic acid-and DHA-primed DCs could be linked to presence of Tregs in the cultures we stained the T cells with FoxP3, Helios, CTLA-4 and PD-1. For representative dot plots and gating strategy see Fig 8A. We found that the proportion of FoxP3 + DO11.10 + T cells, either co-expressing Helios, CTLA-4 or PD-1 or not, were higher in DC: T cell co-cultures in which the DCs had been primed with arachidonic acid, and to some extent also DHA, compared to control cultures ( Fig 8B). In contrast the proportion of FoxP3 neg DO11.10 + T cells co-expressing CTLA-4 or PD-1 was lower (Fig 8B). With oleic acid-primed DCs the proportion of putative Tregs was similar as in the control cultures. If proliferation mainly occurred among effector T cells, and not Tregs, the higher proportion of Tregs with arachidonic acid and DHA might be due to limited proliferation in these cultures and not because of regulatory activity. Expression of Helios, CTLA-4 and PD-1 on the whole T cell population is shown in S5 There was a negative correlation between proliferation (% divided cells) and proportion of Tregs assessed by the following populations; FoxP3 + Helios + (Spearman r-value -0.92, p<0.0001), FoxP3 + CTLA-4 + (r = -0.94, p<0.0001) and FoxP3 + PD-1 + (r = -0.63, p = 0.0034) (Fig 9). In summary, DCs supplemented with arachidonic acid and, to some extent, DHA inhibit T-cell proliferation, accompanied by a higher proportion of Tregs. These Tregs have a similar phenotype as Tregs found in oleic acid or control cultures.  Lower levels of IL-10 and IFNγ with arachidonic acid-primed DCs in DC: T cell co-cultures The DC: T cell co-culture supernatant from samples with arachidonic acid-primed DCs contained lower levels of IL-10 and IFNγ compared to control (Fig 10A and 10B), which supports the low T-cell stimulatory activity with arachidonic acid. No reduction of cytokines was shown with DHA-primed DCs, compared to controls, which suggests less T-cell suppression (Fig 10A  and 10B). IL-12 p70 was below limit of detection, TGF-β1 was in pair with the levels in fresh complete IMDM, with no differences between the groups, while sCD83 was found in higher levels than those found in DC culture supernatant, but still without differences between the groups (S7 Fig). The PD-1: PDL1 pathway is partly responsible for the low proliferation in DC: T cell co-cultures with arachidonic acid-or DHA-primed DCs DCs supplemented with arachidonic acid or DHA had increased expression of PDL-1 and CD83. To evaluate if these markers were involved in the reduced T-cell activation induced by such DCs we added either anti-CD83 or anti-PD-1 blocking antibodies to the DC: T-cell co- cultures. To achieve a more efficient blocking we blocked PD-1 instead of PDL-1, because PD-1 can also bind another molecule, PDL-2. As demonstrated in Fig 11 blocking of PD-1 led to increased proliferation in all DC: T-cell co-cultures. Blocking of PD-1 in arachidonic acid cultures did not restore proliferation to control levels, indicating that other pathways are involved in the low T-cell response. Blocking of CD83 led to lower proliferation in all DC: T-cell co-cultures. This supports the literature describing CD83 as an activation [35] and maturation marker [36].
The PD-1: PDL-1 pathway promotes induction of Tregs [37], so therefore we tested if PD-1 blocking changed the Treg phenotype or functionality. Indeed, the proportion of FoxP3 +     Fig). Hence, PD-1 blocking in fatty acid supplemented cultures decreased the proportion of FoxP3 + Helios + . In this experiment there was a negative correlation between FoxP3 + Helios + T cells and proliferation (r = -0.7161, p < 0.0001) without blocking. With PD-1 blocking this negative correlation was increased (r = -0.7680, p < 0.0001). Fisher r-to-z transformation revealed that this change in r-value between PD-1 blocking and no blocking was statistically non-significant (z = 0.43, p (two-tailed) = 0.6672). Hence, with PD-1 blocking, the suppressive capacity of the FoxP3 + Helios + T cells was unaffected, but the proliferation was increased, possibly due to a lower proportion of FoxP3 + Helios + T cells.

Discussion
In the present study we tested the effect of six fatty acids on expression of MHC class II and costimulatory molecules on DCs and on the ability of the DCs to induce proliferation of naïve T cells in the presence of antigen. Culture of DCs with the long-chain PUFAs arachidonic acid or DHA, but no other fatty acid, led to increased expression of CD40, CD83 and CD86 as well as PDL-1 on the DC. Strikingly, however, DCs exposed to arachidonic acid or DHA induced lower antigen-specific T-cell proliferation and activation compared to DCs cultured in control medium. In parallel, a lower proportion of naïve T cells co-cultured with arachidonic acid-or DHA-primed DCs expressed the activation markers CD25 and CD69. Thus, despite a mature phenotype, DCs supplemented with the n-6 PUFA arachidonic acid or the n-3 PUFA DHA, did not activate T cells.
Tregs, that express the lineage-specific transcription factor FOXP3 are known to prevent effector T-cell proliferation [38]. Immunosuppressive effects of fatty acids mediated by induction of Tregs have been obtained by EPA supplementation during transplantation [39] and by dietary intake of arachidonic acid and DHA, together but not one by one, in a mouse model of dermatitis [40]. DC expression of the costimulatory molecules CD40, CD80 and CD86, has been inversely associated with generation of FoxP3 + T cells from naïve CD4 + T cells [41] but in our study the number of FoxP3 + Helios + DO11.10 + T cells were higher after supplementation with arachidonic acid or DHA to DCs, despite up-regulated activation markers. CTLA-4 is an inhibitory molecule important for Tregs, stored intracellular, but mobilized to the surface upon activation [42] where it binds CD80 or CD86. It has been reported as necessary for generation of Tregs in vitro [43]. In our study CTLA-4, measured intracellular, was not up-regulated in the T-cell cultures with suppressed proliferation. However our DC: T-cell co-culture lasted for 6 days, and Linsley et al. [44] have reported that surface expression of CTLA-4 peaks 48 h after activation and returns to normal levels after additionally 48 hours. Altogether, if the decreased T-cell proliferation was due to induction of Tregs, and the higher proportion of Tregs was not the result of limited T-cell proliferation, we have shown that arachidonic acid and DHA can induce FoxP3 + Helios + Tregs in vitro via DCs.
PD-1: PDL-1 interaction can inhibit T-cell proliferation and activation [23,45] and could therefore be expected to be responsible for the T-cell unresponsiveness. Proposed mechanisms are induction of Tregs [37]. With arachidonic acid and DHA DCs had higher PDL-1 MFI than control DCs. However, the corresponding receptor PD-1 was not up-regulated on T cells. Our blocking experiment showed that PD-1 blocking decreased the proportion of FoxP3 + Helios + T cells, which negatively correlated with increased proliferation. This effect was not limited to any of the stimuli. The FoxP3 + Helios + subset is generally regarded as thymus derived so we question if these cells are generated in vitro or if they're decreased due to higher proliferation of effector T cells. If so, FoxP3 + Helios neg T cells has proliferated to the same extent as FoxP3 neg T cells, since PD-1 blocking did not change the proportion of these. Expression of PD-1 has also been associated with programmed cell death [46]. Therefore we analyzed T-cell viability with Annexin V and 7AAD. The level of apoptotic and necrotic cells was generally low, with reduced viability in cultures with arachidonic acid or DHA. This might be due to lack of proliferation, an induction of tolerance via cell death.
CD83 was up-regulated with arachidonic acid and DHA. To our knowledge no association between fatty acids and CD83 has been reported. The most established role for CD83 is involvement in maturation of CD4 + T cells in the thymus [47]. In different setups CD83 has been shown to support expansion of newly primed naïve CD8 + T cells [48], be required for lymphocyte longevity [35], enhance DCs' stimulatory capacity [49] and thereby induce allogeneic T-cell proliferation [50]. However, the lack of a known ligand have hampered mechanistic explanations of CD83. CD83 expression has been correlated to expression of MHC class II [47,51,52] and CD86 [47,52]. Here, up-regulation of CD83 correlated with up-regulation of CD86, and CD83 blocking resulted in decreased proliferation, suggesting a stimulatory role for CD83. CD83 is also found in soluble form, sCD83, formed via proteolytic cleavage of membrane-bound CD83 [53]. The extracellular soluble CD83 domain can inhibit DC-mediated T-cell proliferation [54] as well as inhibit T-cell proliferation and production of IL-2 and IFNγ in vitro [55,56]. We measured sCD83 but found no differences between the groups, so this could not explain the T-cell unresponsiveness observed in cultures stimulated with arachidonic acid and DHA.
Rather than having an effect on cell membrane fluidity the effects of arachidonic acid and DHA can be caused by fatty-acid specific mechanisms. As free fatty acids, arachidonic acid and DHA are metabolically active compounds with ability to regulate intracellular signaling pathways and gene transcription but also serve as precursors for lipid mediators involved in inflammation. However, the suppressive effect of PUFAs can be independent of lipid mediators, as blocking of conversion enzymes in DCs not necessarily limit suppression of T-cell activation [25]. Because of the involvement in intracellular signaling the levels of free fatty acids are regulated on several levels. Most fatty acids are esterified and bound in the membrane or in triglycerides. Carrier proteins, such as fatty acid binding proteins (FABPs), can increase the solubility and regulate the accessibility of the fatty acids to their target molecules. PUFAs can affect expression for a plethora of genes, including the nuclear receptor peroxisome proliferator-activating receptors (PPARs) [57]. PPARγ is up-regulated in DCs upon differentiation [58] and its expression can be induced by DHA [32]. PPARγ induction reduces DC expression of CD40, CD80 and CD86 and capacity to activate naïve CD4 + T cells in vitro, as shown by decreased IL-2 and IFNγ production [59].
Other studies have determined the lipid content of DCs and the association to antigen presenting capacity. However the results are contradictory, DCs with high lipid content, in comparison to DCs with low lipid content, have been shown to be both tolerogenic [60] but also pro-immunogenic [61]. In a study by Herber et al. MHC molecules, CD40, CD80, CD86 and DC-SIGN were unaffected by triglyceride content while in a study by Ibrahim et al. lipid-laden DCs had higher expression of CD1d, CD40, CD54, CD80 and CD86. Our DCs, stimulated with arachidonic acid or DHA, displayed an activated phenotype but yet seemed to act in a more tolerogenic fashion resulting in low T-cell responses. We did not measure total lipid content of our DCs but it might be that total fatty acid content is not the parameter that drives DCs to become more or less tolerogenic. Instead ratios between different fatty acids or content of individual fatty acids might be the immunomodulatory parameter. Arachidonic acid had a stronger suppressive capacity than DHA and lowered the levels of IL-10 and IFNγ, but gave higher DC culture supernatant levels of PGE 2 . Our result is in line with the notion that longchain PUFAs reduce T-cell proliferation, but we can show that this is true not only when the fatty acids are added to the T cells but also when supplemented to DCs in an earlier cell culture. The induction of FoxP3 + Tregs warrants further studies of the capability of these Tregs to suppress other T cells.