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Clinical Nutrition 30 (2011) 678e687

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Clinical Nutrition

journal homepage: http: / /www.elsevier .com/locate/clnu

Original article

Anti-inflammatory and anti-angiogenic effect of long chain n-3 polyunsaturatedfatty acids in intestinal microvascular endothelium

Ayman Ibrahim a, Khaly Mbodji a, Aktham Hassan a, Moutaz Aziz b, Nabile Boukhettala a, Moïse Coëffier a,c,Guillaume Savoye a,d, Pierre Déchelotte a,c, Rachel Marion-Letellier a,*aAppareil Digestif Environnement Nutrition (ADEN EA 4311), Medicine, University of Rouen, I.F.R. 23, Institute of Biomedical Research, 22 boulevard Gambetta,76183 Rouen cedex, Franceb Laboratory of Anatomo-Pathology, Rouen University Hospital, FrancecNutrition Unit, Rouen University Hospital, 1 rue de Germont, 76031 Rouen cedex, FrancedDepartment of Gastroenterology, Rouen University Hospital, 1 rue de Germont, 76031 Rouen cedex, France

a r t i c l e i n f o

Article history:Received 3 January 2011Accepted 2 May 2011

Keywords:ColitisEndotheliumHIMECInflammationn-3 PUFA

Abbreviations: ALA, a-linolenic acid; CD, Crohoxygenase-2; DHA, docosahexaenoic acid; EC, endpentaenoic acid; GM-CSF, Granulocyte macrophagHIMEC, human intestinal microvascular endotheliaadhesion molecule-1; IBD, inflammatory bowel diseapolysaccharides; LTB4, leukotrienes B4; NF-kB, nucleaPBMC, peripheral blood mononuclear cells; PGE2, prisome proliferator-activated receptor; PUFA, polyunsat6-trinitrobenzen sulfonic acid; TLR, Toll-like receptor;VCAM1, vascular cell adhesion molecule-1; VEGFR2,factor receptor 2.* Corresponding author. Tel.: þ33 (0) 2 35 14 82 45

E-mail address: marion_rachel@hotmail.com (R. M

0261-5614/$ e see front matter � 2011 Elsevier Ltd adoi:10.1016/j.clnu.2011.05.002

s u m m a r y

Background & aims: The role of endothelial cells in inflammatory bowel disease has been recentlyemphasized. Endothelial activation and expression of adhesion molecules are critical for leukocytesrecruitment into the inflammatory wall. Compelling evidence demonstrated anti-inflammatory effects oflong chain n-3 PUFA in inflammatory models. We previously showed that long chain n-3 PUFA (EPA andDHA) inhibited inflammatory response in epithelial and dendritic cells. As long chain n-3 PUFA treatmentled to a decreased expression of adhesion molecules in endothelial cells from other organs, we have nowinvestigated their effect on intestinal endothelial cells in vitro and in colitic rats.Methods: In vitro study: Primary culture of human intestinal microvascular endothelial cells (HIMEC)were pre-treated with DHA and then incubated with IL-1b. In vivo study: Colitis was induced in 2 groupsat day0 by intrarectal injection of 2-4-6-trinitrobenzen sulfonic acid (TNBS). Rats received by gavageeither fish oil, rich in EPA and DHA (TNBSþn-3) or an isocaloric isolipidic oil formula for 14 days.Results: DHA led to a decreased VCAM-1, TLR4, cyclooxygenase-2 and VEGFR2 expression anda decreased production of IL-6, IL-8 and GM-CSF and a reduced production of PGE2 and LTB4 (p < 0.001)in IL-1b-induced HIMEC. Similarly, dietary intervention with fish oil rich in EPA and DHA significantlydecreased colon production of PGE2 and LTB4, endothelial VCAM-1 and VEGFR2 in rats with colitis.Conclusions: Data obtained from in vitro and in vivo studies reveal a potential anti-angiogenic role of longchain n-3 PUFA in intestinal endothelial cells. This protective effect of long chain n-3 PUFA may partlyexplain the observed benefit of dietary intake of long chain n-3 PUFA in IBD development.

� 2011 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

n’s Disease; COX-2, cyclo-othelial cells; EPA, eicosa-e colony-stimulating factor;l cells; ICAM-1, intercellularse; IL, interleukin; LPS, lipo-r factor-kB; PA, palmitic acid;ostaglandin E2; PPAR, perox-urated fatty acids; TNBS, 2-4-TNFa, Tumor necrosis factor-a;Vascular endothelial growth

.arion-Letellier).

nd European Society for Clinical N

1. Introduction

The etiology of inflammatory bowel disease (IBD) remainsdebated but seems to result mainly from an aberrant immuneresponse in genetically predisposed subjects. IBD studies aremainly focused on mechanisms involving immune cells but othercell types such as epithelial or endothelial cells also play a key rolein the inflammatory process.1 In fact, microvascular endothelialcells (EC) regulate the migration of leukocytes from the intravas-cular compartment into the inflammatory tissue. Migration ofleucocytes is a key process in inflammation that involves adhesionmolecules such as ICAM-1 and VCAM-1. Primary cultures of humanintestinal microvascular endothelial cells (HIMEC) have beendeveloped to evaluate the role of EC in IBD.1,2 Under inflammatoryconditions, mimicked in vitro by pro-inflammatory stimuli,

utrition and Metabolism. All rights reserved.

Fig. 1. Experimental design. Colitis was induced in 2 groups at day0 by intrarectalinjection of TNBS while control rats received the vehicle. Rats received by gavage eitherfish oil, rich in EPA and DHA (TNBSþn-3) or an isocaloric isolipidic oil formula for 14days (control, TNBS).

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687 679

adhesion molecules such as ICAM-1 and VCAM-1 are up-regulatedin HIMEC.2 Thus, HIMEC may be a cellular target for a specializedpharmacological or nutritional modulation.

The key role of angiogenesis during IBD1 has been recentlyemphasized: increased vascularisation in IBDmucosa, up-regulationof angiogenic factors and angiogenic activity1 shown by IBDmucosalextracts-induced HIMEC migration. In chronic inflammatory disor-ders, angiogenesis appears deleterious by promoting tissue damage.Inhibition of angiogenesis may contribute to decrease immune cellsrecruitment and production of pro-inflammatory mediators.

Docosahexaenoic acid (DHA, 22:6(n-3)) and eicosapentaenoicacid (EPA, 20:5(n-3)) are fish oil-derived (n-3) PUFA with anti-inflammatory properties in several experimental models.3e5 Inaddition, incubation with fish oil, containing DHA and EPAincreases the ratio of Interleukin-1 receptor antagonist (IL-1ra) toIL-1b in biopsies from IBD patients.6 We have previously shownthat DHA was the most potent in a range of 5 PUFA to inhibit pro-inflammatory cytokine production in human intestinal epithelialcells in response to interleukin-1b (IL-1b).4 We also tested DHA andEPA in LPS-stimulated human dendritic cells and have shown thatthey down-regulate production of TNFa, a key cytokine in IBDpathogenesis.4 DHA also inhibits adhesion molecules expression inretinal EC3 as well as in human umbilical vein EC.7 The effects oflong chain n-3 PUFA (EPA and DHA) on intestinal microvascularendothelial cells, which play a key role in IBD, have not yet beendefined.

The aim of the present study was to evaluate the effect of longchain n-3 PUFA on intestinal endothelial cells in vitro and in ratswith TNBS-induced colitis.

2. Materials and methods

2.1. Material

BSA, PBS, 2, 4, 6-trinitrobenzen sulfonic acid (TNBS), proteaseinhibitor cocktail, antibody against b-actin and CelLytic-M werepurchased from Sigma Aldrich-Company (Saint-Quentin Fallavier,France). The 4e12% Bis-Tris gels, nitrocellulose membranes, the bluecolloidal kit, multimark multi-colored standard were obtained fromInvitrogen (Cergy Pontoise, France). Themonoclonal antibodies anti-VCAM-1, anti-ICAM-1, anti-VEGFR2, anti-PPARg, anti- IkBa, goatpolyclonal antibody anti COX-2 (sc-1747), rabbit polyclonal antibodyanti-phospho-p38 (sc-7975), mouse monoclonal antibody anti-phospho-JNK (sc-6254) and secondary antibodies IgG1 horseradishconjugated were obtained from Santa Cruz biotechnology (Tebu, LePerray-en-Yvelines, France). DHA and palmitic acid (PA) werepurchased from Cayman Chemicals Company (Ann Arbor, MI, USA).

2.2. Cell culture and cytokine treatment of HIMEC

Human intestinal microvascular endothelial cells (HIMEC) wereobtained from ScienCell Research Laboratories (Carlsbad, CA, USA)and grown at 37 �C in endothelial cell medium supplemented with5% of fetal bovine serum, 1% of endothelial cell growth supplementand 1% ml of penicillin/streptomycin solution (all from ScienCellResearch Laboratories). Cells were maintained with 5% CO2 in25 cm2 flasks (BD Primaria, Dominique Dutscher, Brumath, France).Cells were plated at a density of 105 cells/well for 6 well plates and2.5 � 105 cells per 25 cm2 flasks. The culture medium wasrefreshed every second day. Cells were passaged twice a week.Passages 3 to 6 were used in the experiments. Human recombinantInterleukin-1b (Sigma) was added to culture medium at 1 ng/mL.

In our preliminary experiments, a range of fatty acids concen-tration from 5 to 125 mM (data not shown) was used and in regardsto our first data. However, we subsequently decided that the

optimum concentrations were 5 and 25 mM. The fatty acids werenot complexed with BSA for addition to cell culture media. Cellswere firstly pre-treated with either DHA or PA (5 or 25 mM) for 24 hand then incubated with IL-1b for 8 h or 24 h. After cell treatment,supernatants were harvested and stored at �80 �C until analysis.After three washes with PBS, cells were lysed in presence ofCellytic� buffer. Preparation of fatty acids for cell culture: the fattyacids were dissolved in 100% ethanol, aliquoted and stored at0.01 M at �20 �C. The vehicle control for fatty acids was culturemedia containing the same volume of ethanol (0.025%). For IL-1binduction, control incubations received the same amount of PBS.

2.2.1. Choice of DHAIn our previous study, EPA and DHA had similar anti-

inflammatory response.4 in the present study, we chose to testinvitroonly theeffect ofDHAbecause its inhibitoryeffecton cytokineproduction was more marked.4 in addition, we reported that theanti-inflammatory potency was inversely linked to the chain lengthof the PUFA and the degree of saturation4: DHA is 22:6n-3while EPAis 20:5n-3. in addition, De Caterina et al. have compared therespective effects of DHA and EPA on VCAM-1 expression in endo-thelial cells. Only, DHAwas able to inhibit VCAM-1 expressionwhileEPAdid not 5. Similarly, inhibitory effects on COX-2, NF-kB and TLR-4activation was more pronounced with DHA compared to EPA inmacrophages.8

2.2.2. Choice of pre-treatmentCells were pre-treated with DHA or PA and then incubated with

IL-1b. Collie-Duguid et al. have compared the effect of n-3 treat-ment with pre-treatment on endothelial cells and they demon-strated that n-3 PUFA treatment prior to IL-1b induction modulatedadhesion molecules expression while n-3 treatment concomitantlyto IL-1b did not [4]. We also thought that n-3 PUFA pre-treatmentwas more relevant to IBD pathophysiology. Indeed, nutritionalintervention with n-3 PUFA may participate to maintain IBDpatients in remission. in addition, recent epidemiological studieshighlighted the role of DHA consumption to minor IBD risk [5].

2.3. Rats and study design

The experimental design is schematized in Fig. 1.Animal care and experimentation complied with both French

regulations andEuropean Community regulations (Official Journal of the Euro-

pean Community L 358, 18/12/1986) and RML and MC are autho-rized by the French government to use this rat model(Authorization n�76e106 and n� 76e107, respectively). Spra-gueeDawley male rats weighing 200e250 g were obtained fromJanvier (Le Genest St Isle, France). Each rat was kept individually ina metabolic cage to record daily food intake. Rats were weighed

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687680

each day. Water and food were provided ad libitum. The rats wererandomized into three groups (n ¼ 8 per group): Control, TNBS andTNBSþn-3. The rats were killed using an overdose of anesthesia.

2.4. Diet

Rats received 450 mg kg�1.day�1 per body weight of n-3 PUFAby gastric gavage because we have recently shown that this dosewas efficient to inhibit inflammatory response and oxidative stressin rats with TNBS-induced colitis.9 Rats were supplemented withfish oil rich formula (DHA/EPA: 1:1) instead of DHA alone because,to our point of view, it enables an easier extrapolation to humanstudies (Table 1). To our knowledge, IBD clinical trials or casereports investigating the effects of long chain n-3 PUFA wereadministered in the form of fish oil10 or DHA þ EPA mix.11,12 Thetested formula is already available for human studies. In contrary,we cannot determine whether the observed effect of the formula isdue to DHA or EPA. Rats received once a day a fish oil formula(Omegaven�, Fresenius-Kabi, TNBSþn-3 group) while the othercolitic group (TNBS) and the control group received an isocaloricisolipidic formula (intralipides 10%�, Fresenius-Kabi) for 14 d (fromd-7 to d7). Rats were food- deprived for 24 h prior to induction ofcolitis and were allowed free access to tap water throughout thestudy.

2.5. Induction of colitis

Colitis was induced at day 0 by administration of TNBS aspreviously described.9 Colitis was induced in 2 groups at day 0 byintrarectal injection of TNBS (TNBS and TNBSþn-3) whereas thecontrol group received the vehicle (0.25 mL of 50% ethanol). Briefly,rats were anesthetized with an intraperitoneal injection of ket-amine and chlorpromazine following 24 h food deprivation. TNBSdissolved in 50% (v/v) ethanol was instilled into the colon througha canula (25 mg in a volume of 0.25 mL) to induce chronic colitis.Following the instillation of the hapten, the rats weremaintained ina head-down position for a few minutes to prevent leakage of theintracolon instillate. Control groups received the vehicle using thesame volume.

Table 1

Ingredient (g/100 ml) Omegaven� Intralipide�

Fish oil 10 e

Soybean oil e 10Glycerol 2.50 2.20Egg purified phospholipids 1.20 1.20Energy (Kcal/100 ml) 112 110Fatty acids composition

of the fish oil (g/100 ml)14:0 0.1e0.616:0 0.25e1.016:1(n-9) 0.3e0.918:0 0.05e0.218:1(n-9) 0.6e1.318:2(n-6) 0.1e0.718:3(n-3) � 0.218:4(n-3) 0.05e0.420:1(n-9) 0.05e0.320:4(n-6) 0.1e0.420:5(n-3) 1.25e2.8222:1(n-9) � 0.1522:5(n-6) 0.15e0.4522:6(n-3) 1.44e3.09P

(n-3) fatty acids 2.94e6.51P

(n-6) fatty acids 0.35e1.55P

(n-9) fatty acids 1.1e2.65

2.6. Immunohistochemistry

Fixed intestinal tissues were embedded in paraffin wax blocksand 5 mm sections were stained with hematoxylin-eosin. Immuno-histochemistry was performed as previously described.9 Briefly,slides were incubated with the primary antibodies anti-rat ICAM-1at 1/50 (202403, Biolgend), rabbit anti-rat VCAM-1 at 1/100 (sc-8304, Santa Cruz) and rabbit VEGFR2 (ab2349, Abcam). The numberof immunostained leukocytes was determined by an observerblinded to treatment allocation on five visual fields within themucosa (magnification � 400). The staining of endothelial cells wasdetermined semiquantitatively using a four grade scale (0-negative;1-slight staining; 2-moderate staining; 3-strong staining).

2.7. Determination of cytokine production

Concentrations of GM-CSF, MCP-1, IL-1a, IL-1Ra, IL-6, IL-8, IL-10and TNFa were measured in duplicate by the Multiplex assay (R&Dsystems, Abington, UK). This assay relied on the use of polystyrenebeads, each with a unique signature mix of fluorescent dyes thatcan be discriminated by a laser-based detection instrument (Bio-plex 2200). Each bead type was coated with a specific antibody tothe cytokine of interest. The cytokine antibody pairs in this multi-plex assay do not cross react with other analytes in the panel.

2.8. Determination of PGE2 and LTB4 production by ELISA

Culture supernatants were centrifuged and filtered before theassay. PGE2 and LTB4 were quantified using commercial humanELISA kit according to the manufacturer’s instructions (R&Dsystems).

2.9. Western blot

At the end of treatment, cells were harvested in Cellytic reagentwith 0.1% of protease inhibitor cocktail (Sigma) and 1% of phos-phatase inhibitor cocktail (Sigma). Homogenates were centrifuged(12,000 g, 15 min, 4 �C) and the supernatants were collected andstored at �80 �C. Protein concentration was determined followingBradford’s colorimetric method. Aliquots of supernatants contain-ing equal amounts of protein (25 mg) were separated on 4e12%NuPAGE gel (Invitrogen). In the next step, the proteins were elec-trophoretically transferred to a nitrocellulose membrane (Hybond,GE Healthcare, Orsay, France). After blocking in 5% nonfat dry milk(for VEGFR2, PPARg, ICAM-1, VCAM-1, COX-2, b-actin and IkBa) or5% nonfat dry milk plus 0.5% of NP40 (for P-JNK and P-p38),membranes were incubated with specific primary antibodies. Eachfilter was washed three times for 5 min and incubated with thesecondary horseradish peroxidase. Immunodetection was per-formed using enhanced chemiluminiscence light-detecting kit (GEHealthcare) and autoradiography (ECL and Hyperfilm ECL; GEHealthcare). Protein bands were quantified by densitometry usingImageScanner III and ImageQuant TL software (GE Healthcare).ImageQuant TL software analyses 1 D gels image captured bya scanner. Lane creation mode is used to define the positions andareas occupied by the lanes on the gel image. Lanes is created for allchannels in a multi-channel image. Background substraction isperformed on the images to account for the background intensity ofthe gel material on the image.

2.10. Determination of nitric oxide (NO) production

NO production is estimated based on the sum of nitrites andnitrates (total NO) determined by using the total NO kit accordingto manufacturer’s instructions (R&D systems). Briefly, in the first

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687 681

stage, nitrate is converted to nitrite by the enzyme nitrate reduc-tase. The reaction was followed by a colorimetric detection ofnitrite as an azodye product of the Griess reaction. The detectionlimit is less than 1.35 mmol/l. Then, data were expressed in mmol/lper total protein mg.

2.11. Proteolytic pathway activities

Proteolytic pathway activities such as caspase-like andchymotrypsin-like were evaluated as previously described.13

Briefly, evaluation of their activities was performed by spectro-fluorimetry on a microtiter plate fluorometer (Mithras LB 940,Berthold Technologies) using fluorogenic proteasome substrate inthe presence or absence of specific proteasome inhibitors.

2.12. Statistical analysis

Statistical comparisons were performed using GraphPadPrism 5(version 5.01). Data are expressed as mean � S.E.M. Body weightchanges and food intake were analyzed with 2-way ANOVA forrepeatedmeasures with Bonferroni’s post hoc tests. Other variableswere analyzed by Student’s t test, 1-way ANOVA with Tukey’s posthoc test or KruskaleWallis test as appropriate. Normality waschecked by ShapiroeWilk normality test. Values were consideredsignificant at P < 0.05.

3. Results

3.1. Effects of IL-1b stimulation on adhesion molecules expression inHIMEC cells (Fig. 2)

ICAM-1 and VCAM-1 expression were significantly increased byincubationwith IL-1b for8h (p<0.05andp<0.001 respectively) and24 h (p < 0.01 and p < 0.05 respectively) in HIMEC cells (Fig. 2A, B).

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Fig. 2. DHA pre-treatment modulates IL-1b-induced adhesion molecules expression in HIME1b for 8h or 24h. Expression of ICAM-1 and VCAM were studied by western blot and the resuICAM-1 (A) and VCAM-1 (B) expression. Effect of pre-treatment with DHA or palmitic acid onseparate experiments. Values were compared by using one-factor ANOVA (P value abovedifferent from no IL-1, #P < 0.05, ##P < 0.01, ###P < 0.001. *,*** means significantly differentconcentration, V P < 0.05.

3.2. Effects of DHA on adhesion molecules expression in HIMEC cells

ICAM-1 expression was significantly affected by our cultureconditions at 24 h (one-way ANOVA P¼ 0.0432) but the nutritionalpre-treatments did not significantly modify its expression (P> 0.05for all, post-test, Fig. 2C). DHA pre-treatment significantlydecreased VCAM-1 expression in response to IL-1b (p < 0.05 at5 mM and p < 0.001 at 25 mM) whereas PA had no effect on VCAM-1expression (p > 0.05 versus IL-1 and p < 0.05 versus DHA 5 mM,Fig. 2D).

3.3. Effects of IL-1b and DHA on cytokine production in HIMEC cells(Fig. 3)

In response to IL-1b, IL-6, IL-8 and GM-CSF production wassignificantly increased (p < 0.001 for all). DHA pre-treatmentreduced IL-6 production (p < 0.001 for 5 and 25 mM, Fig. 3A). Thisinhibitory effect was specific for DHA and PA did not influence IL-6production (p > 0.05 versus IL-1b and p < 0.001 versus DHA at thesame concentration). IL-8 production was significantly decreasedby DHA or PA at 25 mM (p < 0.05 for both, Fig. 3B). DHA tended toincrease IL-10 production but this did not reach statistical signifi-cance (Fig. 3C). GM-CSF production was significantly decreased byDHA at 25 mM whereas PA had no effect (p > 0.05 versus IL-1 andp < 0.01 versus DHA 25 mM, Fig. 3D).

3.4. Effects of IL-1b and DHA on lipid mediators in HIMEC cells(Fig. 4)

IL-1b significantly increased COX-2 expression (p< 0.001 versuscontrol, Fig. 4A) whereas DHA significantly reversed this enhancedexpression (p < 0.01 for both). IL-1b significantly increased LTB4and PGE2 production (p < 0.001 and p < 0.01 respectively, Fig. 4B,C). DHA and PA significantly decreased IL-1b-induced LTB4production (p < 0.001 for both). DHA at 25 mM significantly

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C. HIMEC cells were incubated by DHA or palmitic acid and then treated by interleukin-lts were densitometrically analyzed and expressed as a ratio of b-actin. Effect of IL-1b onICAM-1 (C) and VCAM-1 (D) expression. Values are expressed as means � SEM from 5the bars) with Tukey’s multiple-comparisons post test. #,##,### means significantly

from IL-1, *P < 0.05, ***P < 0.001. V means significantly different from DHA at the same

Fig. 3. Cytokine production is modulated by DHA in IL-1b-treated HIMEC cells. HIMEC cells were incubated by DHA or palmitic acid and then treated by interleukin-1b for 24h.Cytokine production was evaluated by multiplex technology. A: IL-6, B:IL-8, C:IL-10, D:GM-CSF. Values are expressed as means � SEM from 5 separate experiments. Values werecompared by using one-factor ANOVA (P value above the bars) with Tukey’s multiple-comparisons post test. *,*** means significantly different from IL-1, *P < 0.05, ***P < 0.001.VV,VVV means significantly different from DHA at the same concentration, VV P < 0.01, VVV P < 0.001.

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687682

decreased PGE2 production whereas PA had no effect (P > 0.05versus IL-1).

Several signal transduction proteins were modulated by IL-1b andDHA in HIMEC cells. Expression of IkB, the inhibitor protein ofnuclear factor NF-kB, was significantly increased by DHA at 25 mM(P¼ 0.0388, Fig. 5A). Expression of signalling proteins phospho-p38was decreased by DHA at 25 mM (P ¼ 0.0145) while phospho-JNKwas also not altered by DHA pre-treatment (one way ANOVA,P ¼ 0.9720). We also investigated whether DHA modulates theexpression of pro-inflammatory signalling pathways involved ininnate immunity: Toll-like receptor-2 (TLR2) and TLR4 (Fig. 6). TLR4expression was significantly decreased by DHA at 5 mM whereasTLR2 was not affected. As DHA is a natural ligand of PPARg,14 westudied the expression of PPARg in response to DHA. In the presentstudy, PPARg expression was markedly enhanced by IL-1b but notmodulated by DHA. Expression of VEGFR2 was up-regulated byIL-1b and DHA pre-treatment decreased IL-1b-induced increase ofVEGFR2 expression. Under our conditions, NO production in HIMECcells was not significantly altered (1way ANOVA, P ¼ 0.2774, datanot shown).

3.5. Effects of dietary fish oil, rich in EPA and DHA on body weightand food intake in rats with TNBS-induced colitis (Fig. 7)

Supplementation with n-3 PUFA did not significantly modifyfood intake (Fig. 7A) or body weight (Fig. 7B) in colitic rats.

3.6. Effects of dietary fish oil, rich in EPA and DHA on adhesionmolecules and VEGFR2 in rats with TNBS-induced colitis (Fig. 8)

Endothelial VCAM-1 staining (Fig. 8A) was higher in the TNBSgroup than in the control group (P< 0.001) and were normalized inthe TNBSþn-3 group (P < 0.05). TNBS administration increasedendothelial VEGFR2 (Fig. 8B, P < 0.01) staining and fish oil-supplementation normalized this effect (P < 0.05). ICAM-1 stain-ing did not differ among the groups (data not shown).

3.7. Effects of dietary fish oil, rich in EPA and DHA on eicosanoids(PGE2 and LTB4,) and proteolytic activities in rats with TNBS-induced colitis (Fig. 9)

Colon production of LTB4 was significantly higher in the TNBSgroup (P < 0.001) than in the control group and fish oil-supplementation normalized this effect (P < 0.001, Fig. 9A). Colonproduction of PGE2 did not significantly differ among groups (1wayANOVA, P ¼ 0.07, Fig. 9C) but colon production of PGE2 wassignificantly lower in the TNBSþn-3 group than in the TNBS group(P ¼ 0.0236).

Caspase-like proteolytic activity did not differ among groups(Fig. 9B). Chymotrypsin-like activity was higher in the TNBS groupthan in the control group (P < 0.05, Fig. 9D) and was intermediateand did not differ from either group in the TNBSþn-3 group.

4. Discussion

The present study reports to our knowledge for the first time theanti-inflammatory effects of DHA in HIMEC. Indeed, DHA decreasedthe IL-1b-induced expression of VCAM-1, and the production of thepro-inflammatory cytokines IL-6, IL-8 and GM-CSF. Moreover, DHAalso decreased COX-2 expression and production of the pro-inflammatory lipid mediators LTB4 and PGE2. Finally, several sig-nalling pathways regulating the inflammatory cascade were alsomodulated by DHA.

In concordance with the literature, the present study demon-strates an up-regulation of adhesion molecules2 such as ICAM-1and VCAM-1 and an increased production of pro-inflammatorycytokines IL-6, IL-8 and GM-CSF in response to the pro-inflammatory inducer IL-1b. The down-regulatory effect of DHAon VCAM-1 expression was similar to that obtained with culturedEC from other organs. Furthermore, DHA at 100 mM inhibitedcytokine-induced expression of VCAM-1 in human retinal vascularEC.3 DHA also inhibited VCAM-1 expression in a dose-dependentmanner5 and decreased VCAM-1 mRNA levels in IL-1-treatedhuman saphenous vein EC.7 The effect of DHA on TNFa-treatedhuman umbilical vein EC seems to be dependent on its

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Fig. 4. Lipid inflammatory mediators are modulated by DHA in IL-1b-treated HIMECcells. HIMEC cells were incubated by DHA or palmitic acid and then treated by inter-leukin-1b for 24h. Expression of COX-2 (A) was assessed by western blot and theresults were densitometrically analyzed and expressed as a ratio of b-actin. LTB4 (B)and PGE2 (C) productionwas evaluated by ELISA. Values are expressed as means � SEMfrom 5 separate experiments. Values were compared by using one-factor ANOVA (Pvalue above the bars) with Tukey’s multiple-comparisons post test. *,**,*** meanssignificantly different from IL-1, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. DHA at 25 mM modulates expression of IkB and p-p38 in IL-1b-treated HIMECcells. HIMEC cells were incubated by DHA and then treated by interleukin-1b for 24h.Expression of IkB, p-p38, p-JNK and b-actin was assessed by western blot and theresults were densitometrically analyzed and expressed as a ratio of b-actin. * meansP < 0.05.

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687 683

concentration: DHA at 50 mM decreased VCAM-1 expression15

whereas DHA at 10 mM had no effect.16 In the previous study,DHA at 10 mM did not affect TNFa-induced adhesion molecules butsignificantly reduced monocyte rolling and adhesion to activatedHUVEC.16 In our study, we also confirmed the inhibitory effect oflong chain n-3 PUFAs on VCAM-1 in vivo in rats with TNBS-inducedcolitis.

We have also observed an inhibitory effect of DHA on IL-6 andIL-8 cytokine production in IL-1b treated HIMEC. This is in accor-dance with our previous study where DHA was the most potent ina range of 5 PUFAs to reduce the release of IL-6 and IL-8 by IL-1b-treated enterocyte-like Caco-2 4. Similarly, pre-incubation withDHA from 0 to 100 mM inhibited IL-1b, IL-6 and TNFa release in LPS-stimulated monocytic THP-1 cells. In the present study, DHAtreatment decreased IL-1b-induced GM-CSF production. Accord-ingly, an inhibitory effect of n-3 PUFAs on LPS-induced GM-CSFproduction has been previously reported in peripheral bloodmononuclear cells isolated from Alzheimer’s disease patientsreceiving a daily supplementation with DHA and EPA compared

with placebo for 6 months.17 In vivo studies have shown beneficialeffects of n-3 PUFA in colitis models.18e21 Shoda et al. have shownthe inhibitory effect of n-3 PUFA supplementation on ulcer indexand plasma LTB4 in rats with TNBS-induced colitis. We alsoconfirmed this inhibitory effect of n-3 PUFAs on colon production ofLTB4 and PGE2. Moreover, transgenicmicewith high endogenous n-3 PUFAs are protected from experimental colitis.20 In addition,dietary administration of olive oil supplementedwith fish oil rich inEPA and DHA improves inflammation in chemically-inducedcolitis18,19 but these studies did not permit to determine thespecific effect of DHA.

Anti-inflammatory effects of DHA may be mediated by compe-tition with the n-6 PUFAs. In addition, DHA is an n-3 PUFA that actsas a competitive substrate for the n-6 PUFA metabolism thatconverts arachidonic acid into inflammatory mediators such asprostaglandins and leukotrienes.22 COX-2 is responsible for theoverproduction of PGE2 from arachidonic acid during the inflam-matory processes and its expression23 and LTB4 production areincreased during IBD. Four-hour incubationwith fish oil, the dietary

Fig. 6. DHA pre-treatment modulates expression of signalling pathways involved in intestinal inflammation in IL-1b-treated HIMEC cells. HIMEC cells were incubated by DHA andthen treated by interleukin-1b for 24h. Expression of TLR2, TLR4, PPARg, VEGFR2 and b-actin was assessed by western blot and the results were densitometrically analyzed andexpressed as a ratio of b-actin. Representative immunoblot. * means P < 0.05.

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687684

source of DHA, is also able to down-regulate COX-2 expression inboth intestinal epithelial cell lines Caco-2 and HT-29.24 Inhibition ofCOX-2 leads to an inhibition of angiogenesis and an alteredproduction of angiogenic factors and its effect is probably mediatedthrough PGE2.25 In the present study, DHA pre-treatment led to analtered expression of COX-2 and an inhibition of LTB4 and PGE2release. We also demonstrated this inhibitory effect of n-3 PUFA onproduction of LTB4 and PGE2 in rats with TNBS-induced colitis.

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Fig. 7. Food intake (A) or body weight (B) of rats with TNBS-induced colitis. Colitis wasinduced in 2 groups at day0 by intrarectal injection of TNBS while control rats receivedthe vehicle. Rats received by gavage either fish oil, rich in EPA and DHA (TNBSþn-3) oran isocaloric isolipidic oil formula for 14 days (control, TNBS).

Similarly, inhibition of plasma LTB4 by elemental diet supple-mented with 2% of n-3 PUFA has been previously reported in colitismodel.21 Inhibition of COX-2 expression and PGE2 production byDHA suggests that DHA may possess anti-angiogenic properties.Similarly, a 24 h-incubationwith DHA attenuates COX-2 expressionand activity in human saphenous vein endothelial cells in responseto IL-1b.26 Very recently, it has been shown that curcumin,a pigment from turmeric, is also able to inhibit angiogenesis inHIMEC via COX-2 pathway.27 In our study, COX-2 inhibition may bemediated by TLR4. Indeed, it has been shown that DHA is able todown-regulate COX-2 in a monocytic cell line through the modu-lation of TLR4-mediated signalling pathways.8 Nutritional inter-vention with PUFA in a necrotizing enterocolitis rat model also ledto a down-regulation of TLR4.28

We also investigated several signalling pathways to betterunderstand how DHA can down regulate the inflammatoryresponse in HIMEC. Since adhesion molecules and cytokinesexpression are regulated by NF-kB signalling pathway, we investi-gated the effect of DHA on IkB expression. IkB expression wasincreased by DHA at 25 mM. In contrast, pre-treatment with DHAdid not modify TNFa-induced phosphorylation of IkB in HUVEC,a human endothelial cell line.15 We have previously shown thatDHA induced PPARg expression in enterocyte-like Caco-2 cells inresponse to IL-1b4 while its expression was not significantlyaffected by DHA in IL-1b-treated HIMEC. This discrepancy could bedue to the cell type. Indeed, we previously showed that anti-inflammatory effect of DHA in LPS-treated human dendritic cellswas notmediated by PPARg.4 In addition, PPARg is highly expressedin intestinal epithelial cells and plays a key role in this cell type toregulate intestinal inflammation.29 We also speculated thatdifferent signalling pathways might be regulated in regards to thedose of DHA (Fig. 10). Lower dose of DHA may inhibit TLR4 sig-nalling and induced PPARg pathway while higher dose of DHA mayincrease IkB expression and decrease p38MAPK. Both pathways ledto an altered activation of NF-kB and an inhibition of intestinalinflammation.

In the present study, p-JNK expression was not affected by DHAand or by IL-1b while it has been previously reported that DHA at20 mM inhibits TNF-a-induced p-JNK expression in endothelial

Fig. 8. Endothelial VCAM-1 (A) and VEGFR2 (B) staining in rats with TNBS-induced colitis. Results are expressed as means � SEM and were compared by one-way ANOVA followedby Tukey’s post test. *, **, *** means P < 0.05, P < 0.01, P < 0.001.

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687 685

cells.30 Similarly, n-3 PUFA prevent inflammation induced by high-fat diet in obese diabetic mice by inhibiting JNK pathway.31 Wehypothesized that the absence of effect on p-JNK could be due toour experimental design: IL-1b and 24h cell treatment because IL-1b-induced activation of p-JNK was transient and was not inhibitedby MAPK inhibitors in brain endothelial cells.32

In addition, molecular targets of PUFA are numerous and otherpathways might explain the beneficial effect of n-3 PUFA in intes-tinal inflammation. Nuclear receptors such as PPARa are dietary lipidsensors and synthetic agonists of PPARa have demonstrated theiranti-inflammatory properties in experimental models of IBD.33

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Fig. 9. Colon production of eicosanoids (LTB4, PGE2) and proteolytic activities (caspase-like,rats. Values are means � SEM, n ¼ 7e8. A: LTB4, B: caspase-like, C: PGE2, D: Chymotrypsin

In agreement with both studies in endothelial cells,34,35 DHAtreatment did not affect NO production by IL-1b-induced HIMEC.Currently, the role of VEGFA, a key angiogenic mediator has beendemonstrated in IBD.36 Levels of VEGFA and its receptor VEGFR2 areincreased in IBD patients and in colitis mice.36 In our study, we alsoobserved an up-regulation of VEGFR2 in response to a pro-inflammatory stimulus (IL-1b in HIMEC) and in colitic rats. Inter-estingly, DHA-pretreatment leads to a down-regulation of VEGFR2in HIMEC in response to IL-1b. We also confirmed this finding incolitic rats where dietary intervention with fish oil rich in EPA andDHA normalized endothelial VEGFR2 levels.

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chymotrypsin-like) on day 7 after induction of colitis in control, TNBS, and TNBSþn-3-like. * means P < 0.05, *** means P < 0.001.

Fig. 10. Hypothetical diagram of the mechanism through which DHA regulates IL-1b-induced inflammatory response in HIMEC. Our results suggest that mechanismsbehind the effects of DHA differ in regards to the dose of DHA. At low concentration,DHA down-regulates TLR4 expression and increased PPARg. At high dose, DHAdecreased p38MAPK pathway while it decreased IkB expression. All these mechanismscontribute to an inhibition of NF-kB pathway and subsequent down-regulatespro-inflammatory genes. That leads to a decreased production of cytokines, a down-regulated expression of adhesion molecules and an inhibited expression of cyclo-oxygenase-2.

A. Ibrahim et al. / Clinical Nutrition 30 (2011) 678e687686

Palmitic acid is a saturated fatty acid (16:0) and was chosen asa lipid control. The choice of an adequate control lipid is a matter ofdebate. We and others have demonstrated the ability of unsatu-rated fatty acids4,9,37e39 (ALA, DHA, EPA, CLA, GLA, LA) to modulateintestinal inflammatory response. Oleic acid, an n-9 MUFA, isgenerally considered as a good candidate to be used as a lipidcontrol because it did not modulate eicosanoids generation.40 Asoleic acid consumption has been associated with decreased risk ofUC development, we did not consider it as an appropriate neutrallipid in regards to intestinal inflammation.41 We thus chose to usePA because it had no effect on adhesion molecule expression inboth cytokine-stimulated human EC.3,7 However, PA had an anti-inflammatory effect on IL-8 release and on LTB4 production inHIMEC in response to IL-1b. Our results regarding PA differed fromseveral in vitro studies showing no anti-inflammatory effect of PA.In monocytic THP-1 cells, DHA effect was compared to PA on LPS-stimulated cytokine production.42 Whereas DHA had an inhibi-tory effect on IL-6, IL-1b and TNFa production and mRNA levelscompared to LPS alone, PA had no effect on the studied parametersexcept a stimulatory effect on IL-6 mRNA levels.42 In addition,incubation of adipocytes with PA resulted in an increased produc-tion of TNFa compared to untreated cells whereas DHA treatmenthad no effect.43 However, the effect of PA on inflammatory medi-ators seems to be dependent on the concentration: in adipocytes,PA at 10 mM tended to decrease MCP-1 release whereas at 100 mM itsignificantly increased its production.44

Compelling evidences suggests that proteolysis is a majorcomponent of inflammation regulation. Under inflammatoryconditions, the ubiquitin-proteasome proteolytic pathway is up-regulated and increased chymotrypsin-like activity has beendescribed in CD.45 We previously described in a rat colitis model anincreased colon chymotrypsin-like activity by TNBS that was

attenuated by nutritional intervention with ALA. In the presentstudy, we confirmed this up-regulated chymotrypsin-like activityby TNBS and we also observed that fish oil formula attenuated thiseffect. Down-regulation of chymotrypsin-like activities by EPA hasbeen reported in murine models of muscle proteolysis.46,47

Environment, in particular dietary habits, appears to influenceIBD development. Recently, dietary intake of long chain n-3 PUFAs,in particular, DHA have been shown to be protective from ulcerativecolitis in a UK cohort.48 This protective effect of n-3 PUFAs may bemediated, at least in part, by an inhibitory effect on endothelialVCAM-1 or VEGFR2.

In conclusion, the role of endothelial cells in the pathogenesis ofIBD certainly warrants further investigations. We have previouslyshown that long chain PUFA such as DHA or EPA are able to down-regulate inflammatory response in sentinel cells such as intestinalepithelial cells and dendritic cells.4 We have now extended thesefindings by showing in the present study the anti-inflammatory (IL-8, VCAM-1, TLR4, IL-6, IL-8, GM-CSF) and anti-angiogenic (COX-2,VEGFR2) effects of long chain n-3 PUFA on intestinal endothelialcells in vitro and in a TNBS-induced colitis model. This supportsfurther evaluation of the mechanisms involved in the protectivepotential of long chain n-3 PUFA in IBD development.

5. Conflict of interest statement & Statement of authorshipNone of the authors had any personal or financial conflict of

interest.AI & KM contributed equally to this work. AI participated in the

conduct and analysis of the data and performed all cell cultureexperiments. AH, KM and NB participated in multiplex or westernblot experiments. MC, GS and PD contributed to the analysis anddata interpretation of the study. KM & RML performed animalstudies. RML was responsible for the conduct, analysis, data inter-pretation and supervised the study. RML wrote the manuscriptwith help from all other authors.

Acknowledgments

We thank Richard Medeiros (Medical Editor - Rouen UniversityHospital) for editing the manuscript.

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