AJCN Tufts Nutrition Symposium, Boston & Online Sept 2009
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American Journal of Clinical Nutrition, Vol. 87, No. 4, 939-948, April 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Comparison of cytokine modulation by natural peroxisome proliferator–activated receptor {gamma} ligands with synthetic ligands in intestinal-like Caco-2 cells and human dendritic cells—potential for dietary modulation of peroxisome proliferator–activated receptor {gamma} in intestinal inflammation 1,2,3

Rachel Marion-Letellier, Matt Butler, Pierre Déchelotte, Raymond J Playford and Subrata Ghosh

1 From the Gastroenterology Section, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, London, United Kingdom (RM-L, MB, RJP, and SG), and Appareil Digestif Environnement Nutrition, Faculté de Médecine-Pharmacie, Institut Fédératif de Recherches Multidisciplinaires sur les Peptides, Gambetta, France (PD)

2 Supported by Nutricia Research Foundation (RML).

3 Address reprint requests and correspondence to S Ghosh, Gastroenterology Section, Imperial College Faculty of Medicine, Hammersmith Hospital Campus, Ducane Road, London W12 0NN, United Kingdom. E-mail: s.ghosh{at}imperial.ac.uk.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) plays a role in the regulation of intestinal inflammation and is activated by both natural (polyunsaturated fatty acid; PUFAs) and synthetic (troglitazone) ligands. The fatty acid content of defined formula diets may play a role in mediating the antiinflammatory effect, but the mechanism is unclear.

Objective: We evaluated to what extent the effect of PUFAs on intestinal inflammation is mediated via PPAR{gamma}.

Design: The human enterocyte-like cell line Caco-2 and human dendritic cells were stimulated by interleukin (IL) 1β and lipoprotein polysaccharide, respectively, in the presence of PPAR{gamma} agonists (troglitazone or PUFAs) or antagonist (GW9662). Five PUFAs were tested: {alpha}-linolenic acid (ALA), conjugated linoleic acid (CLA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and {gamma}-linolenic acid (GLA). Cytokine production was measured by enzyme-linked immunosorbent assay and PPAR{gamma}, I-{kappa}B, and inducible nitric oxide synthase (iNOS) expression by Western blot.

Results: In Caco-2 cells, IL-6 secretion was significantly decreased by troglitazone, DHA, EPA, and GLA. IL-8 production was significantly decreased by troglitazone, ALA, DHA, EPA, and GLA. PPAR{gamma} expression was significantly increased by troglitazone, DHA, and EPA. iNOS expression was significantly decreased by troglitazone, DHA, and EPA. Troglitazone and PUFAs at 0.1 µmol/L tended to increase the expression of I-{kappa}B. Addition of GW9662 reversed the effect of troglitazone and PUFAs at 0.1 µmol/L on IL-8 production and decreased the expression of PPAR{gamma}. EPA and DHA also modulated the dendritic cell response to lipoprotein polysaccharide.

Conclusions: The tested PUFAs exerted an antiinflammatory effect in vitro in both models. This effect of PUFAs in Caco-2 cells is similar to that of troglitazone on intestinal inflammation mediated by PPAR{gamma}, and the potency of the antiinflammatory effect is linked to the number of double bonds.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the human intestine, sentinels cells including intestinal epithelial cells and mucosal dendritic cells (DCs), continuously sense the intestinal environment and coordinate responses that protect the intestinal mucosa. Recent data suggest that dietary lipid components may play a role in maintaining the gut in a state of controlled activation (1). Defined formula diets are anti-inflammatory in Crohn disease (CD) and have been shown to down-regulate proinflammatory cytokines such as interleukin (IL) 1β (2). In vivo and in vitro evidence suggests that the anti-inflammatory effects of defined formula diets in inflammatory bowel disease (IBD) may be dependent on the fatty acid composition of the diet (3-5). However, a linoleic acid–rich composition was more effective than an oleic acid–rich formula in inducing remission in CD (3), which challenges the notion that such anti-inflammatory effects are solely mediated via modulation of arachidonic acid precursors. We speculate that modulation of peroxisome proliferators–activated receptor {gamma} (PPAR{gamma}) may be involved.

PPAR{gamma} is a member of a family of nuclear receptors (6). PPAR{gamma} plays a role in the regulation of intestinal inflammation. Indeed, PPAR{gamma} ligands have beneficial effects in different models of rodent colitis (7-11), and a recent study involved the colonic epithelium in the protective effect of PPAR{gamma} against dextran sodium sulfate–induced colitis (12).

PPAR{gamma} is activated by both natural ligands, such as polyunsaturated fatty acids (PUFAs) (13, 14), or by synthetic ligands, such as thiazolidinediones—a class of antidiabetic drug (14). Although PPAR{gamma} is expressed in various tissues and cell types, including pancreas, liver, kidney, immune cells (eg, lymphocytes, monocytes, and macrophages), and DCs, PPAR{gamma} is abundant in adipose tissue and colon (15). The colon is a major tissue expressing PPAR{gamma} in epithelial cells and to a lesser degree macrophages and lymphocytes (15).

Administration of PPAR{gamma} ligands is able to reduce the production of inflammatory cytokines in DCs (16) and can inhibit several other proinflammatory pathways such as inducible nitric oxide synthase (iNOS) (17) or matrix metalloproteinase-9 (18) in Caco-2 cells. In addition, ulcerative colitis patients have been shown to have a reduced expression of PPAR{gamma}, particularly in colonic epithelial cells, without any mutation in the PPAR{gamma} gene (19), whereas in human CD, the gene encoding PPAR{gamma} (ppar{gamma}) was recently identified as a susceptibility gene (20). PPAR{gamma} is also a key receptor mediating the effect of 5-aminosalicylic acid in IBD (21). In small pilot studies, PPAR{gamma} agonists such as rosiglitazone have been shown to have antiinflammatory effects in ulcerative colitis (22).

The aim of the present study was to test the antiinflammatory effects of natural PPAR{gamma} ligands in 2 human sentinel cells—an intestinal epithelial cell line and monocyte-derived DCs—in response to a proinflammatory inducer.


   SUBJECTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The experimental design is schematized in Figure 1Go.


Figure 1
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  		FIGURE 1.. Experimental design of the study. A: The human enterocyte-like cell line Caco-2 was cultured in DMEM culture medium at 37 °C. Caco-2 cells were incubated with interleukin (IL) 1β (IL-1β; 1 ng/mL), vehicle, polyunsaturated fatty acids (PUFAs) (0.1–10 µmol/L), or troglitazone (0.01, 0.1, and 1 µmol/L), with or without GW9662 (0.1–1 µmol/L) for 18 h. Cell supernatant was collected until enzyme-linked immunosorbent assay (ELISA) analysis for IL-6 and IL-8. Cells were harvested and proteins extracted, and the expression of peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}), inducible nitric oxide synthase (iNOS), and I-{kappa}B was studied by Western blot. B: CD14+ monocytes were isolated from peripheral blood mononuclear cells in fresh peripheral blood (from healthy volunteers) by positive selection and differentiation in dendritic cells (DCs) by cytokine treatment (25 ng/mL of IL-4 and 50 ng/mL of granulocyte macrophage colony stimulating factor) on day 0 and day 4. DCs were incubated with lipopolysaccharide (LPS), vehicle, docosahexaenoic acid (DHA), eicsopentaenoic acid (EPA), troglitazone, or GW9662 for 24 h. Cell supernatant was collected until ELISA analysis for IL-10 and tumor necrosis factor-{alpha} (TNF-{alpha}). To evaluate the stimulatory capacity of DCs, DCs were also incubated with allogeneic CD4+ T cells, used as responders, for 5 d. At 18 h before harvesting, 1 µCi tritiated methyl thymidine was added to each well and [3H]thymidine incorporation was measured by liquid scintillation spectroscopy. Cell supernatant was collected until ELISA analysis for IL-13 and interferon {gamma} (IFN-{gamma}). FCS, fetal calf serum.

 
Materials
Bovine serum albumin, phosphate-buffered saline (PBS), protease inhibitor cocktail, and CelLytic-M were purchased from Sigma (St Louis, MO). The 4–12% Bis-Tris gels, Invitrolon PVDF membranes, the blue colloidal kit, and multimark multicolored standard were obtained from Invitrogen (Paisley, United Kingdom). The mouse monoclonal immunoglobulin G1 (IgG1) antibody anti-PPAR{gamma}, the mouse monoclonal IgG1 antibody anti- I{kappa}B-{alpha}, and the secondary antibody goat anti-mouse IgG1 horseradish conjugated were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The mouse IgG1 against iNOS was obtained from BD Biosciences (Oxford, United Kingdom). The BCA protein assay kit was purchased from Pierce (Rockford, IL). Fatty acids [{alpha}-linolenic acid (ALA), conjugated linoleic acid (CLA; 9Z, 11E), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and {gamma}-linolenic acid (GLA)], troglitazone, and GW9662 were purchased from Cayman Chemicals (Axxora, United Kingdom).

Preparation of fatty acids
The fatty acids, troglitazone, and GW9662 were dissolved in 100% ethanol, portioned, and stored at 0.01 mol/Lt –20 °C. The vehicle control for fatty acids, troglitazone, or GW9662 was culture media (DMEM or X-VIVO) containing the same volume of ethanol (0.1%). The PUFAs used in the present study are summarized in Table 1Go with their respective dietary origins.


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  		TABLE 1. List of the tested peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) modulators1

 
Cell culture and cytokine treatment of Caco-2 cells
The human intestinal Caco-2 cell line has been extensively used as an enterocyte-like model. Caco-2 cells have been previously used to document intestinal inflammatory response (18) and the modulating effect of nutrients on inflammatory response (17, 23, 24). The human intestinal epithelial cell line Caco-2 was grown at 37 °C in DMEM (Gibco, Paisley, United Kingdom) and supplemented with 10% (by vol) heat-inactivated fetal bovine serum (Biosera, United Kingdom) and 1% penicillin:streptomycin (Gibco). Cells were maintained with 5% CO2 in 75-cm2 flasks (T75; Corning, United Kingdom). Cells were plated at a density of 1 x 105 cells/well for 24-well plates and 5 x 105 cells/well for 6-well plates. The DMEM medium was refresh every second day. Cells were passaged twice a week. DMEM culture media without fetal calf serum was used for stimulation. Human recombinant interleukin-1β (Sigma) was added in DMEM at a concentration of 1 ng/mL. In preliminary experiments, we initially used PUFAs at concentrations ranging from 0.01 to 100 µmol/L (data not shown). With regard to initial data, we finally decided that the optimum concentrations were a range of concentrations from 0.1 to 10 µmol/L.

Monocyte purification and DC differentiation
Fresh peripheral blood (from healthy volunteers) was collected in 25-mL aliquots and then made up to 50 mL with sterile PBS containing 10 U heparin. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation on a Ficoll-Hypaque (Nycomed, United Kingdom) density gradient according to the manufacturer's instructions. The upper layer was collected and then washed with cold sterile PBS. CD14+ monocytes were isolated by positive selection using MACS beads (Miltenyi Biotech, Germany) as per the manufacturer's instructions. Cells were transferred to 6-well tissue culture plates. To induce differentiation, cells were given 25 ng/mL IL-4 and 50 ng/mL granulocyte macrophage colony stimulating factor (First Link Ltd, United Kingdom) on day 0 and day 4. DCs were exposed to lipopolysaccharide (LPS) because we previously showed the pivotal role of LPS in DC phenotype and function from DC experiments in CD (25). Indeed, LPS from bacteria provide a major signal for DC maturation and activation and doing fatty acid experiments without LPS would be artificial.

Cytokine production by enzyme-linked immunosorbent assay
After treatment, supernatants were harvested and frozen until analyzed. One hundred microliters of capture antibody solution at 1 µg/mL in PBS [anti-IL-8, anti-tumor necrosis factor-{alpha} (TNF-{alpha}), anti-interferon {gamma} (IFN-{gamma}), anti-IL13 from BD Pharmingen; anti-IL-6 from Immunotools (Germany); and anti IL-10 from R&D Systems] was added to the wells of an enhanced protein-binding enzyme-linked immunosorbent assay plate (Nunc Maxisorb) and incubated overnight. After 3 washes (PBS-Tween 0.1%), nonspecific binding was blocked by PBS-bovine serum albumin 1% for 3 h. After 3 washes, standards and neat supernatants were added and incubated overnight. After 3 washes, the biotinylated anti-IL8 (Invitrogen, UK) anti-TNF-{alpha}, anti-IFN-{gamma}, anti-IL13 (BD Pharmingen) or anti-IL-6 (Immunotools, Germany) detection antibody was added for 2–3 h. After 3 washes, the avidin-horseradish peroxidase conjugate was added for 45 min. After 3 washes, the substrate solution was added, and the color reaction was stopped by stop solution. The absorption was read with a microplate reader set to 450 nm.

PPAR{gamma}, iNOS, and I-{kappa}B expression by Western blot
After treatment, cells were washed twice in PBS and harvested by scraping in CellLytic buffer to which a protease inhibitor cocktail was added. Protein extracts were obtained after centrifugation of the lysates at 13 000 x g at 4 °C for 20 min. Protein concentrations were determined with a BCA protein assay kit, and cell treatment withy PUFAs, troglitazone or GW9662 did not significantly affect the protein concentration of the sample. Twenty micrograms of each total protein was separated on a 4–12% bis-Tris gel, and proteins were transferred to Invitrolon polyvinyl difluoride membrane and gel was stained with colloidal blue to verify the amount of protein per lane. The membrane was blocked for 2 h in blocking buffer and then incubated overnight with a mouse monoclonal IgG1 antibody anti-PPAR{gamma} (dilution 1:1000) or anti-iNOS (dilution 1:2000) or incubated 2 h with anti-I-{kappa}B{alpha} (dilution 1:1000) The secondary antibody, a horseradish peroxidase conjugated goat anti-mouse IgG (Santa Cruz Biotechnology) diluted at 1:1000 was incubated with the membrane for 90 min at room temperature. The detection of the reaction was performed with an enhanced chemiluminescence detection kit according to the supplier's protocol (Amersham, Les Ulis, France). The results were densitometrically analyzed by using GelDoc-It Imaging System (UVP).

Mixed lymphocyte reaction
DCs were harvested and were then plated in 96-well plates in 100 µL X-VIVO serum-free medium. Allogeneic CD4+ T cells (isolated from PBMCs of healthy volunteers by negative selection) were used as responders at a DC:T cell ratio of 1:100. Plates were incubated for 5 d. At 18 h before harvesting, 1 µCi tritiated methyl thymidine (Amersham Int, United Kingdom) was added to each well. Cells were harvested onto glass fiber filter mats (Wallac, United Kingdom), and [3H]thymidine incorporation was measured by liquid scintillation spectroscopy in a β counter (Wallac).

Statistical analysis
Statistical comparison was performed by using GRAPHPADPRISM 5 (GraphPad Software Inc, San Diego, CA). Data are expressed as means (± SEMs) from ≥3 independent experiments. Results were compared with one-factor analysis of variance (ANOVA) with Tukey's or Dunnett's post test or 2-factor ANOVA with Bonferroni's post test as appropriate. Values were considered significant at P < 0.05. In Caco-2 cells experiments, we used the condition IL-1β + vehicle as a control for Dunnett's post test, and the control condition was not re-used for each permutation of tested PUFA. Correlations of IL-6 and IL-8 production of Caco-2 cells with PPAR{gamma} expression were analyzed with Spearman's correlation test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PPAR{gamma} expression in Caco-2 cells
Western blot analysis showed that PPAR{gamma} was expressed by Caco-2 cells and was elevated by the addition of 1 ng/mL IL-1β (Figure 2AGo). Expression was also enhanced by the addition of the PPAR{gamma} agonist, troglitazone, in IL-1β–stimulated Caco-2 cells (Figure 2BGo). Addition of 0.1 µmol/L troglitazone was found to be the most effective concentration (239.1% ± 132.7 compared with IL-1 + vehicle; P < 0.05, Figure 2Go and Table 2Go), but activity was also seen at 0.01 µmol/L (Figure 2CGo).


Figure 2
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  		FIGURE 2.. Peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) expression in intestinal-like Caco-2 cells. Caco-2 cells were incubated alone, with interleukin (IL) 1β (IL-1; 1 ng/mL), vehicle, and troglitazone (TGZ; 0.01, 0.1, and 1 µmol/L) for 18 h. PPAR{gamma} expression was determined by Western blot analysis. The results were densitometrically analyzed. A: Effect of IL-1 alone and IL-1 + vehicle on PPAR{gamma} expression. B: Dose-response to troglitazone on PPAR{gamma} expression. C: Mean (±SEM) densitometric expression of PPAR{gamma}. n = 3–7. Bars under the line were compared by using one-factor ANOVA followed by Tukey's multiple-comparisons post test; the P value from the ANOVA is above the line. *Significantly different from IL-1, P < 0.05. #Significantly different from IL-1 + vehicle, P < 0.05.

 

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  		TABLE 2. Top-rank of peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) agonists in the present study1

 
Cytokine production by stimulated Caco-2 cells is modulated by the synthetic PPAR{gamma} agonist troglitazone
Inflammatory cytokine production by Caco-2 was effectively induced by the addition of 1 ng/mL IL-1β for 18 h. Addition of IL-1β increased significantly the secretion of IL-6 and IL-8 (P < 0.001 for both; Figure 3Go), whereas the simultaneous addition of vehicle (ethanol) had no effect. By contrast, responses to IL-1β were reduced in the presence of troglitazone (0.1 µmol/L), with significant reductions in IL-6 (P = 0.0262; Figure 3AGo) and IL-8 (P = 0.0008; Figure 3BGo) responses. Again, 0.1 µmol/L of troglitazone showed the most efficient inhibitory effect (P < 0.05 for IL-6 and P < 0.01 for IL-8 compared with IL-1; Figure 3Go). The inhibitory effects of troglitazone could be reversed by the addition of GW9662—a synthetic PPAR{gamma} antagonist. GW9662 significantly reversed the inhibitory effect of troglitazone at 0.1 µmol/L on IL-6 and IL-8 production (P = 0.0018 and P < 0.0001; Figure 3, C and DGo, respectively). These results show that inflammatory cytokine production in response to IL-1β can be regulated by PPAR{gamma} activity in Caco-2 intestinal epithelial cells.


Figure 3
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  		FIGURE 3.. Mean (±SEM) cytokine production was modulated by the synthetic peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) agonist troglitazone (TGZ) in intestinal-like Caco-2 cells. Caco-2 cells were incubated with interleukin (IL) 1β (IL-1; 1 ng/mL) and TGZ (0.1 and 1 µmol/L) and/or GW9662 (GW; 0.1–1 µmol/L) for 18 h. Cytokine production in the supernatant was determined by enzyme-linked immunosorbent assay. Bars under the line were compared by using one-factor ANOVA (P value above the line) with Dunnett's multiple-comparisons post test and the condition IL-1 + vehicle was used as a control. A and B: IL-6 and IL-8 production of Caco-2 cells in response to TGZ (n = 6–16 separate experiments in duplicate) *Significantly different from IL-1, P < 0.05. **Significantly different from IL-1 + vehicle, P < 0.01. C and D: IL-6 and IL-8 production of IL-1–treated Caco-2 cells in response to TGZ (0.1 µmol/L) and GW9662 (n = 5–16 separate experiments in duplicate). *, **, ***Significantly different from IL-1 + vehicle: *P < 0.05, **P < 0.01, ***P < 0.001.

 
Cytokine production by stimulated Caco-2 cells is modulated by natural PPAR{gamma} agonists: effect of PUFAs
We next analyzed a panel of PUFAs (Table 1Go) for their ability to inhibit IL-8 and IL-6 production in IL-1β–activated Caco-2 cells. DHA and EPA significantly decreased IL-6 secretion (P = 0.0054 and P = 0.028, respectively; Figure 4Go), whereas reductions seen in the presence of ALA, CLA, and GLA were not significant. Similarly, DHA and EPA significantly decreased IL-8 secretion (P = 0.0001 and P < 0.0001, respectively; Figure 4Go); however, in this case, a significant reduction was also seen with ALA and GLA (P = 0.0275 and P < 0.0001, respectively). As with IL-6, reductions in the production of IL-8 observed in the presence of CLA were not significant. We also correlated IL-6 and IL-8 secretion with PPAR{gamma} expression (Spearman's rank correlation test). We then found that IL-6 production was significantly correlated with PPAR{gamma} expression for PUFAs alone (r = –0.54, P = 0.0067) and for PUFAs and troglitazone (r = –0.57, P = 0.0011). A similar inverse correlation was observed for IL-8 production with PPAR{gamma} expression for PUFAs alone (r = –0.43, P = 0.0252) and for PUFAs and troglitazone (r = –0.45, P = 0.0087).


Figure 4
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  		FIGURE 4.. Means (±SEM) cytokine production by Caco-2 cells treated with interleukin (IL) 1β (IL-1) was modulated by natural polyunsaturated fatty acids. Caco-2 cells were incubated with IL-1 (1 ng/mL) and polyunsaturated fatty acids (0.1–10 µmol/L) for 18 h. Cytokine production in the supernatant was determined by enzyme-linked immunosorbent assay. n = 5–17 separate experiments in duplicate. One-factor ANOVA with Dunnett's multiple-comparisons post test was used for the statistical analysis, and IL-1 + vehicle was used as a control. *,**Significantly different from IL-1 + vehicle: *P < 0.05, **P < 0.01. ALA, {alpha}-linolenic acid; CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; GLA, {gamma}-linolenic acid.

 
The antiinflammatory effects of PUFAs can be reversed in the presence of a PPAR{gamma} antagonist, GW9662
We next tested whether the antiinflammatory effects of PUFAs could be reversed by the addition of the PPAR{gamma} antagonist, GW9662. As previously, Caco-2 cells were stimulated with IL-1β in the presence or absence of PUFAs. A concentration of 0.5 µmol/L GW9662 was found to be sufficient to reverse the effects of all PUFAs on IL-8 production in Caco-2 cells (P < 0.0001; Figure 5Go). Thus, it is likely that PUFAs mediate their antiinflammatory activity in Caco-2 cells via the effects on PPAR{gamma}.


Figure 5
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  		FIGURE 5.. Effects of polyunsaturated fatty acids (PUFAs) were dependent on peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) activation. Caco-2 cells were incubated with interleukin (IL) 1β (1 ng/mL) and PUFAs at 0.1 µmol/L for 18 h with or without 0.5 µmol/L GW9662. A: Mean (±SEM) IL-8 production by enzyme-linked immunosorbent assay. n = 5–17 separate experiments in duplicate. Two-factor ANOVA with a Bonferroni post test was used for the statistical analysis (treatment by inhibitor). B: PPAR{gamma} expression by Western blot. Below the immunoblot, the minus sign indicates no GW9662 treatment, and plus sign indicates treatment with 0.5 µmol/L GW9662. ALA, {alpha}-linolenic acid; CLA, conjugated linoleic acid; DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; GLA, {gamma}-linolenic acid.

 
Natural and synthetic PPAR{gamma} agonists regulate the expression of PPAR{gamma}, I-{kappa}B, and iNOS
Finally, we analyzed the effect of PUFAs on the expression levels of PPAR{gamma}, I-{kappa}B (an inhibitor of nuclear transcription factor {kappa}B; NF-{kappa}B), and the effector molecule, iNOS. Troglitazone, DHA, and EPA significantly increased PPAR{gamma} expression by 239.1%, 173.2%, and 173.1% (P < 0.05 for all; Figure 6Go and Table 2Go). Conversely, both troglitazone and the PUFAs tended to enhance the expression of I-{kappa}B although not significantly so (Figure 4BGo and Table 2Go). Last, troglitazone, DHA, EPA, and GLA significantly decreased iNOS expression by 77.2% (P < 0.001), 62.0% (P < 0.05), 66.1% (P < 0.05), and 64.3% (P < 0.05) (Figure 6Go).


Figure 6
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  		FIGURE 6.. Peroxisome proliferator–activated receptor {gamma} (PPAR{gamma}) agonists regulate the expression of PPAR{gamma}, I-{kappa}B, and inducible nitric oxide synthase (iNOS). Expression of PPAR{gamma} (A), I-{kappa}B (B), and iNOS (C) was determined by Western blot, and the results were densitometrically analyzed. For each factor, a graph of the densitometric data are above a representative immunoblot. Caco-2 cells were incubated for 18 h with 1 ng/mL IL-1β and 0.1 µmol/L of the polyunsaturated fatty acids {alpha}-linolenic acid (ALA), conjugated linoleic acid (CLA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and {gamma}-linolenic acid (GLA). Troglitazone (TGZ; 0.1 µmol/L) was used as a positive control and vehicle as a negative control. Values are means ± SEMs from 5 to 8 separate experiments. One-factor ANOVA with Dunnett's multiple-comparisons post test was used for statistical analysis, and IL-1β + vehicle was used as a control. *,**Significantly different from IL-1β + vehicle: *P < 0.05, **P < 0.01.

 
DHA and EPA inhibit cytokine production in LPS-stimulated DCs
We then tested the effect of the 2 most potent PUFAs in the first part of our study in a second human sentinel cell type: human monocyte-derived DCs. DCs are antigen-presenting cells that play a key role in immune response. Treatment with troglitazone, DHA, and EPA inhibited IL-10 production in a dose-dependent manner in LPS-stimulated DCs (2-factor ANOVA: treatment effect, P = 0.0218; concentration effect, P = 0.0037; Figure 7Go), whereas incubation with 5 µmol/L GW9662, a PPAR{gamma} antagonist, had no significant effect on IL-10 production (P = 0.5499; Figure 7BGo). At 5 µmol/L, only DHA and EPA significantly decreased TNF-{alpha} production (P < 0.001 for both; Figure 7CGo) in LPS-stimulated DCs.


Figure 7
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  		FIGURE 7.. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) inhibit cytokine production in human monocyte-derived dendritic cells (DCs). In the gut, lipopolysaccharide (LPS) from bacteria provide a major signal for DC maturation and activation. DCs were treated with troglitazone (TGZ), DHA, and EPA at doses of 1, 5, and 25 µmol/L for 24 h in the presence of LPS (10 ng/mL). Interleukin-10 (IL-10) and tumor necrosis factor-{alpha} (TNF-{alpha}) production were measured by capture enzyme-linked immunosorbent assay in the cell supernatant. Values are means ± SEMs of ≥4 separate experiments. A: Dose-dependent inhibition of the production of IL-10 by TGZ, DHA, and EPA in LPS-stimulated DCs. Data were analyzed by 2-factor ANOVA (treatment by concentration) and a Bonferroni post test. B: GW9662 reversed the inhibitory effect of TGZ on IL-10 production in LPS-stimulated DCs. Data were analyzed by 2-factor ANOVA (treatment by inhibitor) and a Bonferroni post test. C: DHA and EPA, but not TGZ, inhibited TNF-{alpha} production in LPS-stimulated DCs. Data were analyzed by 1-factor ANOVA with Dunnett's post test. ***Significantly different from vehicle, P < 0.001.

 
Troglitazone, DHA, and EPA impair the stimulatory capacity of DCs in mixed lymphocyte reaction
We therefore assessed DC functions after LPS stimulation in the presence of PPAR{gamma} agonists and antagonists to assess the importance of PPAR{gamma} pathway activity in regulating stimulatory responses induced via the Toll-like receptor (TLR)–NF-{kappa}B pathway. To evaluate the ability of DCs to induce T cell proliferation, DCs were cocultured with T cells in a mixed lymphocyte reaction. Troglitazone and DHA at 1 µmol/L slightly decreased T cell proliferation in nonstimulated DCs. (P < 0.01 and P < 0.05, respectively; Figure 8Go). EPA at 5 µmol/L decreased the T cell stimulatory capacity of DCs in response or not to LPS (P < 0.001 for both; Figure 8AGo). Troglitazone, EPA, and DHA significantly increased IL-13 production (P < 0.001 for all; Figure 8BGo), whereas decreased IFN-{gamma} production in mixed lymphocyte reaction in response to LPS (P < 0.001 for all; Figure 8CGo).


Figure 8
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  		FIGURE 8.. Eicosapentaenoic acid (EPA), but not docosahexaenoic acid (DHA) or troglitazone (TGZ), impairs dendritic cell (DC) stimulatory capacity in mixed lymphocyte reaction. In the gut, lipopolysaccharide (LPS) from bacteria provide a major signal for DC maturation and activation. To mimic inflammation, DCs were exposed ({blacksquare}) or not exposed ({square}) to LPS. DCs were plated in 96-well plates in 100 µL X-VIVO serum-free medium. Allogeneic CD4+ T cells (isolated from peripheral blood mononuclear cells of healthy volunteers by negative selection) were used as responders at a DC:T cell ratio of 1:100. Thymidine incorporation (A) and cytokine production (B and C) were measured on day 6. Values are means ± SEMs of ≥4 separate experiments, and data were analyzed with a 2-factor ANOVA with a Bonferroni post test. *,**,***Significantly different from vehicle alone: *P < 0.05, **P < 0.01, ***P < 0.001. $$$Significantly different from vehicle with LPS, P < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In our study, we used activated intestinal epithelial cells to investigate the antiinflammatory potential of PUFAs. While identifying specific PUFAs with strong antiinflammatory activity, we were also able to show that these PUFAs mediate their effect through PPAR{gamma} in Caco-2 cells. In particular, DHA and EPA reduced the release of proinflammatory cytokines IL-8 and IL-6 and abrogated iNOS expression. We next questioned whether the 2 most effective PUFAs in Caco-2 cells could also modulate the inflammatory response in a second sentinel cell type. We chose to evaluate their effects in human monocyte-derived DCs in response to LPS. DCs can be detected in close proximity to the intestinal epithelial cells overlying Peyer's patches in the gut. Gastrointestinal DC subsets are predominantly of myeloid origin, and isolated myeloid DCs from gut biopsy samples respond to bacterial pathogen-associated molecular patterns (PAMPs) in a manner very similar to in vitro generated DCs (cytokine responses, costimulatory molecule up-regulation) (25). Intestinal DCs isolated from the lamina propria have already been exposed to PAMPs in the intestinal environment and therefore may not represent the situation when DCs are first exposed to PAMPs. In addition, PPAR{gamma} has been shown to be highly expressed in gut DCs in situ and in isolated monocyte-derived DCs (16). For these reasons, we felt that monocyte-derived DCs would provide a better model for preliminary investigations into PPAR{gamma} activity in myeloid cells in vitro. DCs in the mucosa of the intestinal tract are likely to be repeatedly exposed to bacterial antigens via receptor groups, such as the TLR family. In our study, we used LPS recognition, via TLR4, as a simplified model for DC interactions with bacterial components. Activation of the TLR pathway leads to NF-{kappa}B activation, inflammatory cytokine secretion, and DC maturation (enhanced T cell stimulatory capacity). In our second model, EPA and DHA also exerted antiinflammatory properties in human DCs in response to LPS. This is consistent with studies that showed that dietary PUFAs reduced the production of proinflammatory cytokines such as TNF-{alpha} in different models of colitis (7, 8) and in peripheral blood mononuclear cells (26). Moreover, a recent study showed that transgenic mice rich in endogenous n–3 PUFAs are protected from experimental colitis (27). Crucially, we were able to show that the observed antiinflammatory effect of PUFAs is mediated through PPAR{gamma} because it is mimicked by troglitazone, which binds to the PPAR{gamma} ligand-binding domain and is reversed by GW9662, a synthetic antagonist for PPAR{gamma} in Caco-2 cells.

Moreover, EPA and DHA modulate DC responses to LPS. We observed a reduced production of the cytokines TNF-{alpha} and IL-10 in PUFA-treated DCs. We next analyzed the effect of DHA and EPA on DC function on the basis of their ability to induce the proliferation of T cells in a mixed lymphocyte reaction. The addition of EPA decreased the ability of DCs to reduce proliferation. Treatment with EPA also increased IL-13 production, but reduced IFN-{gamma} production in mixed lymphocyte reaction. This finding is consistent with that of studies that showed that PPAR{gamma} ligands reduce the secretion of IFN-{gamma} and chemokines involved in T helper cell 1 recruitment (16).

The role of PPAR{gamma} in the regulation of intestinal inflammation has been well demonstrated: thiazolidinediones given in mice colitis reduce dramatically the disease severity (9, 10, 28). Moreover, PPAR{gamma}+/– heterozygotous mice exhibit an increased susceptibility to develop colitis (29). The mechanism of action of PPAR{gamma} in colitis seems to involve the attenuation of NF-{kappa}B (30, 31)—a key transcription factor in inflammation. In IBD patients, NF-{kappa}B concentrations are elevated in intestinal mucosa (19). After binding to a ligand, PPAR{gamma} forms a heterodimer with the retinoid X receptor, recruits coactivators containing histone acetylase activity, and binds to the peroxisome proliferator response element gene promoter, which leads to the regulation of gene transcription.

We also observed an inverse link between the level of fatty acid saturation (Table 1Go) and their level of inhibitory activity on cytokine secretion and iNOS expression (Table 2Go). The 2 most potent PUFAs in our study were DHA and EPA, which have 6 and 5 double bonds and a chain length of 22 and 20 carbons, respectively. Both are n–3 PUFAs derived from fish oil. The effects of EPA could also be mediated by pathways other than PPAR{gamma}, such as inhibition of activation of extracellular signal-regulated kinase (32-34) or its antiinflammatory metabolites 15-lipoxygenase (35) or resolving E1 (36, 37).

GLA also reduced cytokine production in the present study and could exert antiinflammatory properties by modulating the production of IL-1β. In LPS-treated monocytes, dietary administration of GLA reduces the IL-1β production mainly by reducing autoinduction of IL-1β and by increasing the amount of IL-1 receptor antagonist (38). The antiinflammatory effects of PUFAs in our study are observed at low concentrations (from 0.1 to 10 µmol/L). The n–3 and n–6 PUFAs are the 2 main families of PUFAs. The n–3 PUFAs are derived from ALA. ALA is converted to stearidonic acid via the {Delta}6-desaturase, rate-limiting step of the pathway and is then elongated and desaturated by the action of {Delta}5-desaturase into EPA. Through a series of elongase, action by {Delta}6-desaturase and peroxisomal β-oxidation, EPA is converted to DHA. In retinoblastoma cells, a low concentration of ALA is more efficient than is a higher concentration in terminating the conversion of ALA to DHA (39). The authors of this study suggested that a high concentration of ALA could exert a down-regulating effect on the conversion of ALA to DHA (39). In addition, nitrolinoleic acid, a derivative of linoleic acid, has been found at concentrations of 0.5 µmol/L in the blood of healthy individuals and is a potent endogenous ligand for PPAR{gamma} at a Ki of 0.133 µmol/L (40). Choosing the right concentration of PUFA may be critical in mediating antiinflammatory effects in vivo.

In our study, in vitro CLA did not modulate the inflammatory response in Caco-2 cells, contrary to the demonstrated effect of CLA in murine colitis (7). In the colitis study (7), a mixture of 2 CLAs (9Z, 11E and 10E, 12Z) has been used, and we used a single isomer of this mixture (9Z, 11E)—the predominant isomer of CLA in animal tissue.

The mechanism of action of defined formula diets as antiinflammatory agents is unclear (41). Because both elemental and polymeric diets appear equally effective, we have shown that altering the composition of the elemental diet may alter the antiinflammatory effect of such a diet in vitro (42). Increasing evidence implicates the fat composition of a defined formula diet as being critically important in its antiinflammatory effect. A Japanese randomized controlled study of low-, medium-, and high-fat modified elemental diets in patients with CD showed the highest remission rate in the low-fat group, and the antiinflammatory effect was abolished in the high-fat group (4). In a further and larger European randomized controlled trial, rather surprisingly, the defined formula diet that contained a higher proportion of linoleic acid (45%) resulted in a higher remission rate than did a diet with a low linoleic acid composition (6.5%), contrary to expectations based on the arachidonic acid precursor hypothesis (3). Although the investigators failed to come up with a clear explanation of this unexpected result, we propose an alternative explanation based on fatty acid ligand signaling via PPAR{gamma}.

To the best of our knowledge, the present study is the first to evaluate the effects of a range of 5 PUFAs on inflammatory responses in IL-1–treated enterocyte-like Caco-2 cells and to directly compare the effects of natural PUFAs compared with troglitazone. In addition, our study established a link between the effect of PUFAs and PPAR{gamma} by reproducing the results of troglitazone, a synthetic PPAR{gamma} agonist, and by blocking their effects by GW9662, a PPAR{gamma} antagonist. Moreover, the present study established a link between the efficiency of the antiinflammatory effect of PUFAs and its structure, ie, saturation and carbon chain length. Use of PUFAs in intestinal inflammation could exert beneficial effects by reducing the production of proinflammatory mediators and attenuating the activation of NF-{kappa}B. It could mimic the effect of commensal bacteria to attenuate inflammation by regulating PPAR{gamma}, as shown by Kelly et al (43). The results of the present study suggest that normal daily intake and composition of fatty acids in our food interact with intestinal epithelium and modulate inflammation. This may offer further therapeutic avenues for designing diets beneficial in IBD.


   ACKNOWLEDGMENTS
 
The authors' responsibilities were as follows—RM-L: was responsible for the conduct, analysis, and data interpretation of the study and wrote the manuscript with help from all other authors; MB: participated in the design and the conduct of the study, particularly the culture of human DCs; PD and RJP: contributed to the design of the study and analysis of the data; and SG: supervised the study. None of the authors had any personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication June 27, 2007. Accepted for publication October 13, 2007.




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R Marion-Letellier, P Dechelotte, M Iacucci, and S Ghosh
Dietary modulation of peroxisome proliferator-activated receptor gamma
Gut, April 1, 2009; 58(4): 586 - 593.
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