AlteredColonicMucosalPolyunsaturatedFattyAcid(PUFA) Derived Lipid Mediators in Ulcerative Colitis: NewInsightintoRelationshipwithDiseaseActivityandPathophysiologyMojganMasoodi1,2,3*.,DanielS.Pearl4,5,6*.,MichaelEiden2,JanisK.Shute5,JamesF.Brown5,PhilipC.Calder7,8,TimothyM.Trebble41Nestle Instituteof HealthSciences, Lausanne, Switzerland, 2Medical ResearchCouncil, Cambridge, Cambridgeshire, UnitedKingdom, 3Department of NutritionalSciences, University of Toronto, Ontario, Canada, 4Department of Gastroenterology, Portsmouth Hospital NHS Trust, Portsmouth, Hampshire, United Kingdom, 5Instituteof Biomedical and Biomolecular Sciences, University of Portsmouth, Portsmouth, Hampshire, United Kingdom, 6Department of Gastroenterology, Taunton and SomersetNHSFoundationTrust, Taunton, Somerset, UnitedKingdom, 7HumanDevelopment andHealthAcademicUnit, Universityof Southampton, Southampton, UnitedKingdom, 8NIHR Biomedical Research Centre in Nutrition, University Hospitals Southampton NHS Foundation Trust and University of Southampton, Southampton, UnitedKingdomAbstractObjectives: Ulcerativecolitis (UC) is arelapsinginflammatorydisorder of unconfirmedaetiology, variableseverityandclinical course, characterisedbyprogressivehistological inflammationandwithelevationof eicosanoids whichhaveaknown pathophysiological role in inflammation. Therapeutic interventions targetting eicosanoids (5-aminosalicylates (ASA))areeffectivefirst lineandadjunctivetreatments inmild-moderateUCfor achievingandsustainingclinical remission.However, the variable clinical response to 5-ASA and frequent deterioration in response to cyclo-oxygenase (COX) inhibitors,has prompted an in depth simultaneous evaluation of multiple lipid mediators (including eicosanoids) within theinflammatory milieu in UC. We hypothesised that severity of inflammation is associated with alteration of lipid mediators, inrelapsingUC.Design: Studywascase-control design. Mucosal lipidmediatorsweredeterminedbyLC-MS/MSlipidomics analysisonmucosal biopsies takenfrompatients attendingoutpatients withrelapsingUC. Univariateandmultivariatestatisticalanalyseswereusedtoinvestigatetheassociationofmucosal lipidmediators, withthediseasestateandseveritygradedhistologically.Results:LevelsofPGE2, PGD2, TXB2, 5-HETE, 11-HETE, 12-HETEand15-HETEaresignificantlyelevatedininflamedmucosaandcorrelatewithseverityofinflammation, determinedusingvalidatedhistological scoringsystems.Conclusions: Our approach of capturing inflammatory mediator signature at different stages of UC by combiningcomprehensivelipidomics analysis andcomputational modellingcouldbeusedtoclassifyandpredict mild-moderateinflammation; however, predictiveindexis diminishedinsevereinflammation. This newtechnical approachcouldbedevelopedtotailordrugtreatmentstopatientswithactiveUC, basedonthemucosal lipidmediatorprofile.Citation: Masoodi M, Pearl DS, Eiden M, Shute JK, Brown JF, et al. (2013) Altered Colonic Mucosal Polyunsaturated Fatty Acid (PUFA) Derived Lipid Mediators inUlcerativeColitis: NewInsightintoRelationshipwithDiseaseActivityandPathophysiology. PLoSONE8(10): e76532. doi:10.1371/journal.pone.0076532Editor:HiroyasuNakano, JuntendoUniversitySchool ofMedicine, JapanReceivedMay17, 2013; AcceptedAugust31, 2013; PublishedOctober18, 2013Copyright: 2013Masoodi etal. Thisisanopen-accessarticledistributedunder thetermsof theCreativeCommonsAttributionLicense, whichpermitsunrestricteduse, distribution, andreproductioninanymedium, providedtheoriginal authorandsourcearecredited.Funding: Salary and research bursary provided by Portsmouth Hospitals National Health Service (NHS) Trust and University of Portsmouth, Portsmouth, UnitedKingdomandMedical ResearchCouncil. Thefunders hadnoroleinstudydesign, datacollectionandanalysis, decisiontopublish, or preparationof themanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail: mojgan.masoodi@rd.nestle.com(MM); daniel.pearl@porthosp.nhs.uk(DSP).Theseauthorscontributedequallytothiswork.IntroductionUlcerative colitis (UC) is a chronic, relapsing intestinalinflammatory disorder of the colonic mucosa, with variabledistribution but limited to the distal bowel (distal colitis andproctitis)in60%ofcases[1].Indistalcolitisthereiscommonlyaclear demarcation between inflamed and non-inflamed tissues thatdemonstrate contrasting patterns of immunomodulator release [2].Despite advances in medical treatments, including biologics thattarget cytokine-led inflammatory responses for severe disease, longterm control of UC is variable with available therapeuticinterventions[35],with57%ofallpatientsfollowingarelapsingandremittingclinical course[6,7]. Inmildtomoderaterelapsingdisease only, a limited therapeutic repertoire is available toPLOSONE | www.plosone.org 1 October2013| Volume8| Issue 10| e76532patients principally as oral or topical 5-aminosalicylic acid (5-ASA)andcorticosteroids.Mucosal inflammation in UC is characterised by an infiltrate ofneutrophils, plasma cells and eosinophils, which correlate withdiseaseseverityandarepredictorsof diseaserelapse[810]. It isproposed that lipid inflammatory mediators, including eicosanoids,which are rapidly and locally formed and degraded in-situ,promote neutrophil chemotaxis [11,12], a pivotal step in theinflammatory cascade. Furthermore, mucosal inflammation in UCresponds to therapeutic interventions that target eicosanoidproductionsuchas 5-ASA[13,14]. However, theunderstandingof both the pathophysiology of UCand pharmacotherapeuticeffects of 5-ASA is limited, which has inhibited the development ofnewtherapeuticinterventions.Eicosanoids are a family of lipid mediators, derived frompolyunsaturatedfattyacids(PUFA) enzymaticallyandoxidatively[15]. Previous studies have demonstrated up regulation of theeicosanoidbiosyntheticenzymescyclooxygenase(COX)-1,COX-2, and5-lipoxygenase(LOX) inactiveUC[16]andelevationofboth prostaglandin (PG)E2[17] and leukotriene (LT)B4[18]derived fromthe n-6 PUFA arachidonic acid (AA) in UC.Eicosanoidsmay alsoderive fromthe n-3 PUFAeicosapentaenoicacid(EPA); EPA-derivedeicosanoids include PGE3andLTB5.Frequentlythemediators producedfromAAandEPAdiffer intheirinflammatorypotency[19,20]. However, studiesattemptingto alter eicosanoid production through dietary modification ofmucosal lipidprofileinactiveUChavebeendisappointing, withonly modest effects onrelapse rates andcorticosteroidrequire-ment, despite sound experimental results in healthy volunteers[21,22].Theaimsof thisstudyweretoinvestigatethelevelsof PUFA-derivedlipidmediatorswithin inflamedcolonicmucosa inmildtomoderateUC, todeterminetheirrelationshiptoendoscopicandhistological changes, to identify mediators that have not previouslybeen considered possible contributors to the inflammatory cascadein UC, and, as a result, to identify potential targets forintervention. The investigation of patients with distal colitisenabled comparative assessment of endoscopically normal andinflamedtissuesasamodel, aspreviouslydescribed[2].MaterialsandMethods2.1. SubjectsandMucosal BiopsyThis was a case-control of UC patients identified duringattendance at gastroenterology outpatients clinics at the RoyalHaslarandStMarysHospital,PortsmouthHospitalsNHSTrustbetween November 2008 and July 2009. The diagnosis of UC wasbased on endoscopic and histological investigation [23,24]. Allrecruitedpatientsunderwentaquestionnaire-basedassessmentoftheir demographiccharacteristics, previous andpresentingmed-ical history, and UC history. Clinical disease activity wasdeterminedusingtheUCDAIscore[25].Patients with clinical evidence of active disease underwentunpreparedflexible sigmoidoscopyexaminationas part of theirroutine clinical management. An endoscopy assessment wasundertakenbytwoendoscopists blindedtothepatients clinicalpresentationusingavalidatedscoringtool tostratifypatientsintoactiveorquiescentUCwithphotographicevidenceobtained,andtoidentifydemarcationofinflamedwithapparentlynon-inflamedmucosa. Biopsies were takenaccording toa standardprotocol,specificallydevelopedtomeasuretargetanalytesinadjacentareasof inflamed and non-inflamed mucosa in patients with active distalUC. Flexiblesigmoidoscopyinpatients withactivedistal UCtoabove visualdemarcationof normal mucosawas followed bygrabbiopsy, obtainedusingflexiblebiopsyforceps, inendoscopicallyinflamed and non-inflamed areas. All mucosal biopsy samplesweresnap-frozenandstoredinliquidnitrogenuntil use.Exclusion criteria were age less than 16 or greater than 80 years;refusal toundergoendoscopic evaluation; diagnosis of colitis ofalternativeaetiology; concurrent useof non-steroidal anti-inflam-matorymedication; inabilitytoprovideconsent.ThestudywasapprovedbytheIsleofWight, PortsmouthandSouth East Hampshire researchethics committee (project number08/H0501/82). All subjectsprovidedwritteninformedconsent.2.2. AnalysisofLipidMediators2.2.1. Reagentsandstandards. PGE2, PGD2, thrombox-ane (TX)B2, 6-keto-PGF1a, PGB2-d4, 13,14-dihydro-15-keto-PGE2, 9-hydroxy-10(E),12(Z)-octadecadienoic acid (9-HODE),13-hydroxy-9(Z),11(E)-octadecadienoic acid (13-HODE), 5-hy-droxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoicacid(5-HETE), 8-hy-droxy-5(Z),9(E),11(Z),14(Z)-eicosatetraenoic acid (8-HETE), 11-hydroxy-5(Z),8(Z),12(E),14(Z)-eicosatetraenoic acid (11-HETE),12(S)-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic-5,6,8,9,11,12,14,15-d8acid (12-HETE-d8), 9-hydroxy-5(Z),7(E),11(Z),14(Z)-eicosatetraenoic acid (9-HETE), 15-hy-droxy-5(Z),8(Z),11(Z),13(E)-eicosatetraenoic acid (15-HETE), 12-hydroxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid (12-HETE)andLTB4standards were purchasedfromCaymanChemicals(AnnArbor, MI). HPLC-grade acetonitrile, ethanol, methanol,hexane, and hydrochloric acid were from Fisher Chemicals(Loughborough, UK), HPLC-grade acetic acid and methylformate fromSigma (Dorset, UK), and solid-phase extraction(SPE) cartridges (C18-E, 500mg, 6mL) from Phenomenex(Macclesfield, UK).2.2.2. Solidphaseextraction. Extractionoflipidmediatorswascarriedoutaccordingtoapreviouslydescribedmethod[26].In summary, mucosal biopsy samples (approximately 35mg)were weighed on thawing and immediately transferred to 15% ice-coldmethanol inwater(3mL) andhomogenisedusingWheatonTapered tissue grinders. Internal standards (PGE2-d4and 12-HETE-d8(20ng)) wereaddedtoeachsample. Thesampleswereacidifiedwith0.05MhydrochloricacidtopH3.0andimmedi-ately applied to SPE cartridges that had been preconditioned with20mLof methanol, followedby20mLof water. Thecartridgeswerethenwashedwith20mLof15%(v/v) methanol, 20mLofwater, and10mLof hexane. Finally, the lipidmediators wereelutedwith10mLof methyl formate. Theorganicsolvent wasevaporated using a fine stream of nitrogen, the solute reconstitutedinethanol (100 mL), andthefinal solutionstoredat220uCuntilanalysiswithin48hours.2.2.3. Lipidomics analysis. Data-dependent LC-MS/MSlipidomics analysis was performedusingAccelaUHPLCsystem(Thermo Scientific, Hemel Hempstead, UK) coupled to LTQVelos (Thermo, Hemel Hempstead, UK) linear iontrap(LIT)-orbitrap as described previously [26]. The analysis on the orbitrapinstrument was carried out using heated electrospray ionization (h-ESI) in negative ion mode at sheath, auxiliary and sweep gas flowsof24,2and1,respectively[26].Thecapillaryandsourceheatertemperatureswereset to275uCand50uC, respectively. Theionspray voltage was adjusted to 4000V. MS/MS spectra, along withretention times and isotope distribution patterns fromthe MSspectrawereusedtoidentifylipidmediators inmucosal biopsysamples.TheidentifiedmetaboliteswerequantifiedusingAcquityUPLC(Waters, Hertsfordshire, UK) systemscoupledtoQTRAP4000 (AB Sciex, Concord, ON) quadrupole2linear ion trap(QqLIT) mass spectrometers as described previously [26]. ThelipidmediatorswereseparatedonaC18reversed-phase(RP) LCEicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 2 October2013| Volume8| Issue 10| e76532column(PhenomenexLuna, 3 mmparticles, 15062mm) usingalinear mobile phase gradient (A, 0.02% glacial acetic acid in water;B, 0.02%glacial aceticacidinacetonitrile) at 0.5mL/min. Thestartingconditionsconsistedof30%Bandthenincreasedto90%Bover 14minandfinallyreturnedtotheinitial conditions for2mintoallowequilibration.2.3. Histological AnalysisAssessment was by a histopathologist blinded to patientsidentities andclinical dataonparaffinembeddedserial haema-toxylin and eosin (H&E) stained sections using a validatedhistopathological scoring tool [24] (0, normal; 1, mildoedemaandinflammationinlaminapropria; 2, crypt abscess formationand inflammation in lamina propria; 3, more severe inflammationwithdestructivecryptabscesses +/2granulomata;4, moresevereinflammationwithactiveulceration) (Figure1).2.4. Statistical AnalysisLipid mediator concentration is expressed as pg/mg tissue.Unblinded bioinformatics analysis was carried out followingcompletionoflipidmediatormeasurementonblindedsamples.2.4.1. Univariate statistical analysis. Wilcoxons SignedRank Test was used as a non-parametric pair-wise univariatetestingmethodat aconfidenceinterval of 97.5%. Analysis wascarriedout usingcustomworkflows writtenintheprogramminglanguageR. Metaboliteswithavalueof p,0.05were regardedassignificantly different. Subgroup analysis to investigate the effect ofno 5-ASA or corticosteroid (n18) use was undertaken andmetaboliteswithavalueofp,0.05wereregardedassignificantlydifferent.2.4.2. Multivariate statistical analysis. Since univariatestatistical testingisunabletocharacterisetheinterrelationshipofvariables, multivariate statistical analysis was performed toexamine association with disease state (inflamed/non-inflamed)aswell asdiseaseseverityassessedhistologicallyusingtheGomesscoringsystem. Similartothepairedunivariatetesting, wewereprimarilyinterestedinthewithin-subject variationandthereforeperformedavariationsplit priortoanalysis (toremovebetweensubjectvariation), atechniquedevelopedinthecontextof multi-level multivariatedataanalysis[27,28].Ontheremainingwithin-subject variationdataweperformedPrincipal Component Analysis (PCA) for unsupervisedanalysis,followedby(Orthogonal)Partial LeastSquares(PLS)Analysisforthe associationwithdisease severity assessedhistologically, and(Orthogonal) Partial Least Squares Discriminant Analysis (PLS-DA) for the investigation of the disease state (inflamed/non-inflamed). Variation splitting was implemented using the pro-gramming language R, Partial Least Squares Approaches wereperformedusing Ras well as SIMCA-P12.01 (Umetrics, AB,Sweden).Results3.1. SubjectsPatients with active UC (n 69) were recruited; biopsy data sets ofpaired macroscopicallyinflamedandnon-inflamedtissues wereavailable in 54 UC patients. Baseline characteristics of patients areshowninTable1.3.2. LipidMediators: UnivariateAnalysisMucosal biopsies from inflamed and non-inflamed mucosa werescreened for all PUFA-derived bioactive lipids and deactivatedmetabolites. Reported metabolites included eicosanoids derivedfromAA (TXB2, 6-keto-PGF1a, PGE2, PGD2, 5-HETE, 15-HETE, 12-HETEand 11-HETE) and linoleic acid (LA) (13-HODE, 13-oxo-ODE, 9-HODE). EPA-derived eicosanoids (5-hydroxy-eicosapentaenoic acid (HEPE), 12-HEPE, 15-HEPE)were excluded fromanalysis as their concentration was belowthe limit of quantification. Neither PGE3 nor LTB5 were detected.3.2.1. LOX-relatedlipidmediators. 5-HETE(p,0.001)and 15-HETE (p,0.001) concentrations in inflamed mucosa weresignificantly higher than in adjacent non-inflamed mucosa(Table 2, Figure 2). Other 5-LOXmetabolites of AAsuchasLTB4 and 5-oxo-ETE were inconsistently detected. There were nosignificant differences intheconcentrations of LA-derivedLOXmetabolitessuchas9-HODE, 13-HODEand13-oxo-ODE.3.2.2. COX-related lipid mediators. PGE2(p,1026),PGD2(p,0.01) andTXB2(p,1026) concentrationsininflamedmucosaweresignificantlyhigher thaninadjacent non-inflamedmucosa (Table 2; Figure 3). There were no other significantdifferences inconcentrationof COXmetabolites betweennon-inflamedandinflamedmucosa. PGF2aandthedeactivatedformof PGE2, 13,14-dihydro-15-keto-PGE2, were only detectable in50%ofthesamplesandthereforewereexcludedfromanalysis.3.3. LipidMediators: MultivariateAnalysisThe principal component analysis (PCA), an unsupervisedmultivariateanalysis technique, whichis drivenbythevarianceinherent inthe data set and has no prior assumptionof classmembership, revealed a clear differentiation between inflamedand endoscopically non-inflamed adjacent mucosal tissue inpatientswithdistal UC(Figure4A).Furthermore, O2-PLSDA, a supervised analysis, was performedto actively check for differences in the lipid mediator profilebetweeninflamedandnon-inflamedtissue. AsexpectedfromthePCA analysis, the O2PLS-DA model (R2X(cum) =79%,R2Y(cum) =60%andQ2=60%) wasclearlyabletodifferentiatethetwophenotypicgroups (Figure4B). UsingtheO2PLS-DAmodel, 87.5%of inflamed tissue was correctly identified asinflamedbaseduponthelipidmediator profile, while87.5%ofnon-inflamedtissuewascorrectlyidentifiedasnon-inflamed.In order to investigate which metabolites made the greatestcontribution to the separation observed in O2PLS-DAmodel(Figure4B), avariableimportanceplot wascreated; PGE2hadthehighestcontributionfollowedbyTXB2, 15-HETE, 5-HETE,12-HETEand11-HETE, respectivelyindiminishingmagnitude(Figure5). This is consistent withthe results of the univariatestatistical analysis (Table 2), and demonstrates contribution of 12-HETE and 11-HETE which were significantly higher (all p,0.05)ininflamedcomparedwithnon-inflamedcolonicmucosaintheunivariate analysis. PGD2was excluded fromthis multivariateanalysisasitwasdetectedin ,85%ofsamples.3.4. AssociationbetweenLipidMediatorsandClinicalGradingO2-PLS Analysis was performed in order to examine therelationship between lipid mediator concentration and histologicalgrading (Figure 6). The model (R2X(cum) =86%,R2Y(cum) =57%and Q2=51%) was able to separate non-inflamedfrominflamedtissue;howeverwithintheinflamedtissuethere was anoverlapbetweenhistological grades (Figure6A).Althoughit was possibletopredict mild-moderateinflammation(grade 1 and 2) from the lipid mediator profile (Figure 6B), highlyinflamedtissue(grade3),failedtofitintothismodel;howeverthislattergroupcontained5subjectsonly.In order to investigate which metabolites had the greatestcontributiontotheseparationobservedintheO2PLSmodel, avariableimportanceplot was created; PGE2showedthehighestEicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 3 October2013| Volume8| Issue 10| e76532impact followedby5-HETE, 15-HETE, TXB2, 12-HETEand11-HETE, respectivelyindiminishingmagnitude(Figure7).3.5. 5-ASAandCorticosteroidNaivePatientsSubgroupanalysis withinpaired inflamedandnon-inflamedmucosafrom18UCpatients (age45.3years; malen10[55%],femalen8[45%]) not recently(within3months) on5-ASAorcorticosteroid treatment was carried out. Analysis of lipidmediatorsdemonstratedthatPGE2,PGD2,TXB2and15-HETE(all p,0.05) werehigherininflamedthannon-inflamedmucosa;however, the other measured lipid mediators were not significantlydifferent (Table3). Theresultswithinthesubgroupanalysisarecomparabletothetotal groupunivariateanalysis.Figure 1. Gomes histological classification of UC. 1A: Gomes 0 (normal); 1B: Gomes 1 (mild oedema and inflammation in lamina propria withcryptitis); 1C: Gomes2(cryptabscessformationandinflammation); 1D: Gomes3(destructivecryptabscesses +/2granulomata); 1EF: Gomes4(activeulcerationandformationofgranulationtissuewithneoangiogenesis).doi:10.1371/journal.pone.0076532.g001EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 4 October2013| Volume8| Issue 10| e765323.6. ControlsLipid mediator levels (TXB2 38.8 [27.551.6]; 6-keto-PGF1a 36.1 [27.849.5]; PGE2 14.9 [7.124.1]; PGD2 9.9 [4.017.7];9-HODE 38.3 [20.055.6]; 5-HETE 6.2 [3.012.4]; 15-HETE19.1[10.732.4]; 13-oxo-ODE21.6[14.841.3]; 13-HODE 55.4 [29.884.2]; 12-HETE 9.1 [5.516.5]; 11-HETE 9.4 [7.015.7]) were measured in normal colorectal mucosaobtainedfromagroupof 42control patientsundergoingroutineflexiblesigmoidoscopyexamination.DiscussionIn the current study we demonstrate differences in theconcentrationof anumber of lipidmediators betweeninflamedandnon-inflamedareasofthecolonicmucosafrompatientswithUC. The findings suggest that inflammation of the colonic mucosainUCisassociatedwithsignificantelevationinconcentrationsofPGE2, PGD2, TXB2, 5-HETE, 15-HETE, 12-HETEand 11-HETE, but not of othermeasuredlipidmediators(Table2). Inaddition, theprofileofthesesamelipidmediatorscorrelateswithseverityof inflammationmeasuredhistologically. Toour knowl-edge this is the first study to simultaneously demonstratecomprehensive alterations in multiple lipid mediators, whichcorrelateproportionatelytothedegreeof histological inflamma-tioninpatientswithUC. Thisisalsoinagreementwithpreviousstudies that showedupregulationineicosanoids, whichcorrelateproportionately to the degree of histological inflammation inpatientswithUC;however, previousstudieshavebeenlimitedtoinvestigation of selected enzymatic pathways (COX-2 and 5-LOX)inUC[1618,2932].Several of theidentifiedinflammatorymediators haveimmu-nomodulatoryroles as demonstratedeitherincohorts of inflam-matorybowel disease(IBD) patients or inexperimental studies.Forexample, PGE2isproducedviaCOX-1andCOX-2withintheAAcascadeandhaspro-inflammatory(viacytokineinductionpathways) and anti-inflammatory (via lipoxin induction) effects[33,34]. PGE2has been consistently demonstrated in previousstudiestobeelevatedininflamedcolonicmucosainUC[17].12-HETEis producedvia12-LOXwithinthe AAcascade andisknown to exert chemotactic effects on neutrophils [35,36]. 5-HETEandLTB4are bothproducts of 5-LOXwithinthe AAcascade; neutrophils containlargequantities of 5-LOXandareable to produce abundant 5-HETE and LTB4 [37,38]. 5-HETE isa potent activator of eosinophils and neutrophils via 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE). 5-HETE and 12-HETEwerereadilydetectedininflamedmucosal samples.Table1.BaselinecharacteristicsofpatientswithactiveUC.Category Subcategory ResultSex[n(%)] Male 26(48)Female 28(52)Age[yr]44.461.8CRP[mg/l]7.061.1BMI[kg/m2]27.560.8DiseaseDistribution[n(%)] Proctitis 32(60)Distal 16(30)Leftsided 3(6)Extensive 3(6)DiseaseLongevity[yr(%)] 05 22(41)510 15(28)1015 6(11)1520 3(6).20 7(13)unknown 1(2)Currentdrughistory[n(%)] 5-ASA 32(60)Corticosteroids 14(26)Thiopurines 8(15)Methotrexate 1(2)Cyclosporine 1(2)Diseaseactivity-UCDAI*,4.860.4Diseaseactivity-endoscopicscore[n(%)]1 36(67)2 13(24)3 5(9)Diseaseactivity-histologicalscore[n(%)]J0 8(15)1 24(44)2 11(20)3 8(15)4 3(6)Smokinghistory[n(%)] yes 6(11)no 48(89)*UCDAI-ulcerativecolitisdiseaseactivityindex[59](Dailystool frequency[0 =normal; 1 =12abovenormal; 2 =34abovenormal; 3 =.4abovenormal]+Rectal bleeding[0 =none; 1 =streaksofblood; 2 =obviousblood;3 =mostlyblood]+Sutherlandscore[seebelow]+Physiciansglobal assessment[0 =normal; 1 =mild; 2 =moderate; 3 =severe]).Sutherlandendoscopicscoringtool [25](0 =normal; 1 =mildfriability;2 =moderatefriability, bleedingoncontact; 3 =exudation, spontaneoushaemorrhage).JGomes Histological score [24] (0 =normal, 1 =mildoedema and inflammationinlaminapropria; 2 =cryptabscessformationandinflammationinlaminapropria; 3 =moresevereinflammationwithdestructivecryptabscesses +/2granulomata; 4 =moresevereinflammationwithactiveulceration).mean6 SEM.doi:10.1371/journal.pone.0076532.t001Table2.Lipidmediatorconcentrations(pg/mgtissue)incolonicmucosa(inflamedandnon-inflamed)inUCpatients.PaireddataLipidmediator InflamedJNon-inflamedJp-value*TXB246.2(27.270.7) 32.2(20.641.7) ,10266-keto-PGF1a34.9(17.951.4) 29.5(14.848.4) 0.201PGE224.1(13.456.2) 9.0(4.815.3) ,1026PGD213.9(5.921.9) 12.1(2.516.4) 0.0019-HODE 27.4(20.145.7) 33.9(19.542.3) 0.3685-HETE 9.4(4.023.2) 5.3(3.211.5) ,102315-HETE 39.2(26.097.0) 33.4(23.448.5) ,102313-oxo-ODE 30.1(19.142.0) 31.0(21.642.2) 0.32013-HODE 57.1(30.991.4) 67.1(31.988.8) 0.65012-HETE 13.5(7.521.4) 12.7(7.116.6) 0.02111-HETE 11.7(10.218.1) 10.5(7.314.7) 0.028JDataaremedian6IQR.*Wilcoxonsignedrankpairanalysis.doi:10.1371/journal.pone.0076532.t002EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 5 October2013| Volume8| Issue 10| e76532LTB4is a potent chemoattractant for neutrophils andotherleukcoytes [39]; however, LTB4 was not consistently demonstratedin our samples. LTB4is reported to be elevated in inflamedmucosa in UC; however, critical reviewof published methodsreveals that levels were measured ininvivo rectal dialysates ormucosal explants, whichwouldhaveledtoneutrophil activationinadvertently or by design [4048]. The presented methodcontrols for altered lipid mediator production induced byexperimental methods. The lack of consistent demonstrationofLTB4in the context of elevated 5-HETE (both 5-HPETEderivatives) may suggest that the bioactive concentration ofLTB4 is below the detectable limit, or that it is rapidly metabolisedininflamedmucosal samples. Analternativeexplanation, that 5-HETEisthepredominant 5-LOXderivedchemoattractant lipidFigure2. MedianandinterquartilerangeofLOX-relatedlipidmediators(pg/mgtissue)ingroupednon-inflamedandadjacentinflamedmucosawithdifferenceplotsofpairedsamples.2A: 11-HETE; 2B: 12-HETE; 2C: 15-HETE; 2D: 5-HETE.doi:10.1371/journal.pone.0076532.g002EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 6 October2013| Volume8| Issue 10| e76532mediator, or that PGE2mediated inhibition of FLAP (5-lipoxygenase activating protein) abrogates LTB4production,should be considered [49]; however, this was not specificallytestedinourstudy.Thepro-inflammatorymediator TXB2, astable derivativeofTXA2andbothAAderivatives viaCOX-dependent conversionfromPGH2,iselevatedininflamedcomparedwithnon-inflamedmucosa, ashasbeendemonstratedpreviously[50].In contrast, PGD2and its metabolite 15-deoxy D12,14PGJ2exert anti-inflammatory effects [51], and several studies havesuggested a role for PGD2 in resolution of inflammation, reductionof leukocyteinfiltrationandhealingwithinthecolon[32,5254].Consistent with other studies, we observed a significant increase inFigure3. MedianandinterquartilerangeofCOX-relatedlipidmediators(pg/mgtissue)ingroupednon-inflamedandadjacentinflamedmucosawithdifferenceplotsofpairedsamples.3A: PGE2; 3B: PGD2; 3C: TXB2.doi:10.1371/journal.pone.0076532.g003EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 7 October2013| Volume8| Issue 10| e76532thelevelsofPGD2ininflamedtissue, althoughitsmetabolite15-deoxyD12,14PGJ2wasnotdetectableinmucosal biopsies.Sub-groupanalysis (n18) of agroupof patients not receivingcorticosteroids and 5-ASAconfirmed significant differences inFigure4.Dataseparationofinflamed(triangle)andnon-inflamed(circle)mucosabasedonlipidmediatorconcentration.4A: PCAscoreplot(unsupervisedanalysis); 4B: O2PLS-DAplot(supervisedanalysis).doi:10.1371/journal.pone.0076532.g004EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 8 October2013| Volume8| Issue 10| e76532PGE2, PGD2, TXB2 and 15-HETE in inflamed mucosa (Table 3),withnosignificant differences observedin5-HETE, 11-HETEand12-HETEconcentrations ininflamedcomparedwithnon-inflamed mucosa. This may indicate the diminished power todetectdifferencesinallmeasuredeicosanoidsinthisgroupduetosample size, or possibly a more benign clinical course in thisuntreatedgroup.Usingpredictivemathematicalmodelling,wehavedemonstrat-ed that the measured lipid mediator profile may be used to predictpresenceorabsenceof histological inflammationwithsensitivity,specificity, and positive predictive and negative predictive values of87.5%. It was also possible to grade severity of inflammation basedonblindedhistological assessment;however, predictivemodellingwaslessconsistentinpatientswithsevereinflammation.Thismaybeduetothesmall groupof patients(n5) inthisgroup, ormaysuggestthatalternativeinflammatorymediatorsbecomepredom-inantinseveremucosal inflammation.Eicosanoids and other lipid mediators are generated viaoxidativepathways(COXandLOX) atthecytosolicinterfaceofthecellmembrane. Theyhavepleiotropic effectsand biochemicalactivityisinfluencedbythespeciesofPUFAfromwhichtheyarederived. In excess of 40 different eicosanoids from PUFAsubstratesarerecognised.Thesearederivedfromobligatedietaryconstituents, LA, AA and EPA, but principally from AA due to thehighermembranecontent of AAandthelowerbioavailabilityofEPA. Provision of EPA orally can influence the levels of AA in cellmembranes and reduce pro-inflammatory AA derived lipidmediators in patients with IBD[55] and in healthy subjects[56]; although clinical therapeutic efficacy is inconsistentlydemonstrated[21,57].In the current study, although AA-derived lipid mediators(PGE2, PGD2, TXB2, 5-HETE, 15-HETE, 12-HETEand11-HETE) were detectable in inflamed mucosa, EPA-derivedmediators (PGE3, PGD3, TXB3, 5-HEPE, 15-HEPE, 12-HEPEand 11-HEPE) were not, or were detected at unquantifiable levels.Wepreviouslyreportedthat activelyinflamedmucosainUCisassociated with increased AA and reduced EPAin both thecomplexlipidandfreefattyacidpools,withaconsistentlyalteredAA/EPA ratio compared with adjacent non-inflamed mucosa, andthat this alteration is despite no significant difference in the level ofdietary intake of PUFA(Pearl etal) [42]. These findings areconsistentandsuggestthatmetabolicalterationofAAproductionininflamedtissuemayleadtoenhancedsubstrateavailabilityforeicosanoid biosynthetic enzymes which are constitutively upregulated in actively inflamed mucosa, with resulting up regulationinall classesofeicosanoids, whichwehavedetected.These findings may be relevant to the failure of dietaryinterventionstudieswithEPA. ThismaybebecauseEPAanditslipid mediators including eicosanoids are present at very low levelsininflamedmucosa, whereasAA anditslipidmediators includingeicosanoids arepresent at muchhigher levels. SupplementationwithEPAmaynotbringaboutsignificantrebalancingof theAA/EPAratio, whichwouldberequiredtoreducethelevels of AAderivedeicosanoidlipidmediatorsininflamedmucosa.Inaddition, eicosanoids derivedfrombothCOX- andLOX-related pathways are significantly elevated indicating co-activationof multiple eicosanoid biosynthetic pathways within the AAcascade ininflamedmucosa. The role of eicosanoids is furthersupportedbytheknowntherapeuticeffectof5-ASAdrugs,whichact bybindingtoperoxisomeproliferatoractivatedreceptorcinFigure5. Variableimportanceplot inO2PLS-DAanalysisdemonstratesrelativecontributionof lipidmediatorstoseparationbetweeninflamedandnon-inflamedmucosa.(Fig. 4).doi:10.1371/journal.pone.0076532.g005EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 9 October2013| Volume8| Issue 10| e76532the colonic epithelium with subsequent suppression of pro-inflammatorylipidmediatorproduction[18].Co-activation of multiple synthetic pathways may suggestredundancy withineicosanoidsignalling networks; however, analternative explanation is that candidate eicosanoid-receptorsignallingindependently stimulates initiation, propagation, reso-lution and/or suppression stages of inflammation. This issupportedbytheobservedlossofintestinal eicosanoidhomeosta-sis, as occurs with COXinhibition during non-steroidal anti-inflammatorydruguse,whichisassociatedwithanincreasedriskofUCrelapseinquiescentdisease[58].Thisisalsosupportedbydetection of pro-inflammatory (PGE2) and anti-inflammatory(PGD2) lipidmediators withinthesametissuematrix, as inthecurrentstudy[51].The role of detected eicosanoids (PGE2, PGD2, TXB2, 5-HETE,15-HETE,12-HETEand11-HETE)inUCisdifficulttoaddress precisely, as this study was not designedto investigatechangesoccurringasaresult of therapy, ortemporal changesinmucosal lipidmediatorsassociatedwithdiseaseactivity.Figure6. O2PLSmodelshowingcomputedpredictionofdiseaseseveritybasedonlipidmediatorprofile. 6A: PlotshowingO2PLSregressionscoreplots; 6B: plotshowingactualvs. predictedGOMESscorebasedonlipidmediatorprofile.doi:10.1371/journal.pone.0076532.g006EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 10 October2013| Volume8| Issue 10| e76532There is therefore emergent evidence that UC is associated withchangesinPUFAprofilewithelevatedAA/EPAratiosuggestingalteration of production of the eicosanoid substrate AA (Pearl etal)[42], and with associated elevation of AA-derived eicosanoidswhichcorrelatewithgradeofhistological inflammation.Further research should focus on the use of predictive modellingbasedonmucosal derivedlipidmediators todetermine if drugnon-responders can be predicted, and to target appropriatetherapy. This wouldbe aunique development inmanagementofchronicinflammatorydisorders.Additional research could focus on characterization of lipidmediators biosynthesis during initiation, propagation, resolutionandsuppressionstages of inflammation, and to relate these tometabolicactivitywithinthefattyacidbiosyntheticcascade. Thiswouldallowafocussedtime-courseassessmentoftheroleoflipidmediators during the evolution of inflammation using distal UC asa model and may inform new targets for development oftherapeuticinterventionsinUCandinflammatorydisorders.Inconclusion,developmentofnewapproachesandtreatments,based on selective lipid mediators, may offer new investigative andtherapeutic strategies totarget treatment inpatients withmild-moderatechronicactivesteroidandimmunomodulatorresistantUC in whom an organ sacrificing approach via surgical colectomyisbeingconsidered.AcknowledgmentsProfessor AnoopChauhan(Director of Research, PortsmouthHospitalsNHS Trust (PHT)), for formal approval of IBDbiomedical sciencesresearchproject,supportandmentorship.Surgeon Commander Mark Whittaker (Consultant Histopathologist,PHT) forunwaveringenthusiasm.EndoscopystaffandpatientsatPHTFigure 7. Variable importance plot in O2PLS analysis demonstrates relative contribution of lipid mediators to separation betweenhistologicalgradesofseverity.(Fig. 6).doi:10.1371/journal.pone.0076532.g007Table3.Lipidmediatorconcentrations(pg/mgtissue)incolonicmucosa(inflamedandnon-inflamed)intreatmentnaiveUC.PaireddataLipidmediator InflamedJNon-inflamedJp-value*TXB243.4(27.659.3) 28.7(15.948.1) 0.0026-keto-PGF1a34.9(25.251.5) 37.9(5.847.4) 0.776PGE227.1(16.942.8) 7.1(2.415.9) ,1023PGD210.3(5.423.4) 3.8(2.215.4) 0.0169-HODE 25.2(15.046.8) 30.6(17.655.1) 0.8795-HETE 14.7(7.023.4) 10.4(4.619.4) 0.19115-HETE 40.8(23.3103.6) 39.1(13.651.0) 0.00513-oxo-ODE 24.4(15.137.1) 31.0(13.743.6) 0.91313-HODE 60.8(28.496.0) 67.5(29.295.4) 0.81112-HETE 14.7(7.725.2) 13.9(5.320.6) 0.13311-HETE 10.0(6.713.6) 9.1(5.110.7) 0.053JDataaremedian 6IQR.*Wilcoxonsignedrankpairanalysis.doi:10.1371/journal.pone.0076532.t003EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 11 October2013| Volume8| Issue 10| e76532AuthorContributionsConceived and designed the experiments: MMDSP TMTJFBJKS.Performedtheexperiments:MMDSP.Analyzedthedata:MMMEDSPTMTPCC.Contributed reagents/materials/analysistools:DSPMMMETMT. Wrote the paper: MM DSP ME. Critical revision of the manuscriptfor important intellectual content: DSPMMMEJFBJKSPCCTMT.Obtainedfunding: DSPMMJFBJKSTMT. StudysupervisionJFBJKSPCCTMT.Acquisitionofdata:MMDSPTMT.Statisticalanalysis:MEMMDSP.Technical ormaterial support: DSPMMMETMT.References1. 1.StonningtonCM,PhillipsSF,MeltonLJIII,ZinsmeisterAR(1987)Chroniculcerativecolitis: incidenceandprevalenceinacommunity.Gut28: 402409.2. Pearl DS, ShahK, Whittaker MA, Nitch-SmithH, BrownJF, et al. (2012)Cytokine mucosal expression in ulcerative colitis, the relationship betweencytokinereleaseanddiseaseactivity. JCrohnsColitis. S1873-9946(12)00333-9[pii];10.1016/j.crohns.2012.07.022[doi].3. LawsonMM, ThomasAG, AkobengAK(2006) Tumournecrosisfactoralphablocking agents for induction of remission in ulcerative colitis. CochraneDatabaseSystRevCD005112. 10.1002/14651858.CD005112.pub2[doi].4. Peyrin-BirouletL,LemannM(2011)Reviewarticle:remissionratesachievablebycurrent therapiesforinflammatorybowel disease. AlimentPharmacol Ther33: 870879. 10.1111/j.1365-2036.2011.04599.x[doi].5. Jakobovits SL, Travis SP (2005) Management of acute severe colitis. Br Med Bull7576: 131144. 75-76/1/131[pii];10.1093/bmb/ldl001[doi].6. LangholzE, MunkholmP, DavidsenM, BinderV(1994) Courseof ulcerativecolitis: analysisof changesindiseaseactivityoveryears. Gastroenterology107:311. S0016508594002155[pii].7. LangholzE(2010) Current trends ininflammatorybowel disease: thenaturalhistory. Therap Adv Gastroenterol 3: 7786. 10.1177/1756283X10361304[doi].8. GeboesJK(2003) Histopathologyof Crohnsdiseaseandulcerativecolitis. In:Satsangi J, SutherlandLR, Colombel JF, Fiocci C, LofbergRet al., editors.Inflammatory Bowel Disease. Edinburgh, London, Melbourne: ChurchillLivingstone. 255276.9. GeboesK,RiddellR,OstA,JensfeltB,PerssonT,etal.(2000)Areproduciblegrading scale for histological assessment of inflammation in ulcerative colitis. Gut47: 404409.10. Azad S, Sood N, Sood A(2011) Biological and histological parameters aspredi ctors of relapse i n ul cerati ve col i ti s: a prospecti ve study.S a u d i J Ga s t r o e n t e r o l 1 7 : 1 9 4 1 9 8 . S a u d i J Ga s t r o e n -terol_2011_17_3_194_80383[pii];10.4103/1319-3767.80383[doi].11. Nielsen OH, Elmgreen J (1987) Activation of neutrophil chemotaxis byleukotriene B4 and 5-hydroxyeicosatetraenoic acid in chronic inflammatorybowel disease. ScandJClinLabInvest47: 605611.12. Palmblad J (1984) The role of granulocytes in inflammation. Scand J Rheumatol13: 163172.13. Marteau P, Probert CS, Lindgren S, Gassul M, Tan TG, et al. (2005) Combinedoral andenematreatmentwithPentasa(mesalazine) issuperiortooral therapyalone in patients with extensive mild/moderate active ulcerative colitis: arandomised, doubleblind, placebocontrolledstudy. Gut 54: 960965. 54/7/960[pii];10.1136/gut.2004.060103[doi].14. Sutherland L, Roth D, Beck P, May G, Makiyama K (2000) Oral 5-aminosalicylic acid for inducing remission in ulcerative colitis. CochraneDatabaseSystRevCD000543.CD000543[pii];10.1002/14651858.CD000543[doi].15. Funk CD (2001) Prostaglandins and leukotrienes: advances in eicosanoidbiology. Science 294: 18711875. 10.1126/science.294.5548.1871 [doi];294/5548/1871[pii].16. JuppJ, Hillier K, Elliott DH, Fine DR, BatemanAC, et al. (2007) Colonicexpression of leukotriene-pathway enzymes in inflammatory bowel diseases.InflammBowel Dis13: 537546. 10.1002/ibd.20094[doi].17. RamptonDS, SladenGE, YoultenLJ(1980) Rectal mucosal prostaglandinE2release andits relationtodisease activity, electrical potential difference, andtreatmentinulcerativecolitis. Gut21: 591596.18. SharonP, StensonWF(1984) EnhancedsynthesisofleukotrieneB4bycolonicmucosa in inflammatory bowel disease. Gastroenterology 86: 453460.S0016508584000500[pii].19. LeeTH,SethiT,CreaAE,PetersW,ArmJP,etal.(1988)CharacterizationofleukotrieneB3: comparisonof its biological activities withleukotrieneB4andleukotriene B5 in complement receptor enhancement, lysozyme release andchemotaxisofhumanneutrophils. ClinSci (Lond) 74: 467475.20. NeedlemanP, RazA, Minkes MS, Ferrendelli JA, Sprecher H(1979) Trieneprostaglandins: prostacyclinandthromboxanebiosynthesisanduniquebiolog-ical properties. ProcNatl AcadSci USA76: 944948.21. TurnerD, SteinhartAH,GriffithsAM(2007) Omega3fattyacids(fishoil) formaintenance of remissioninulcerative colitis. Cochrane Database Syst RevCD006443. 10.1002/14651858.CD006443.pub2[doi].22. Calder PC(2008) Polyunsaturated fatty acids, inflammatory processes andinflammatory bowel diseases. Mol Nutr Food Res 52: 885897. 10.1002/mnfr.200700289[doi].23. DHaensGR,PanaccioneR,HigginsPD,VermeireS,Gassull M,etal.(2011)TheLondonPositionStatementoftheWorldCongressofGastroenterologyonBiological Therapy for IBD with the European Crohns and ColitisOrganization: whentostart, whentostop, whichdrugtochoose, andhowtopredictresponse?AmJGastroenterol106:199212.ajg2010392[pii];10.1038/ajg.2010.392[doi].24. GomesP,duBC,SmithCL,HoldstockG(1986)Relationshipbetweendiseaseactivityindicesandcolonoscopicfindingsinpatientswithcolonicinflammatorybowel disease. Gut27: 9295.25. Sutherland LR, Martin F, Greer S, Robinson M, Greenberger N, et al. (1987) 5-Aminosalicylic acid enema in the treatment of distal ulcerative colitis,proctosigmoiditis, and proctitis. Gastroenterology 92: 18941898.S0016508587001811[pii].26. Masoodi M, Eiden M, Koulman A, Spaner D, Volmer DA (2010)Comprehensive lipidomics analysis of bioactive lipids incomplex regulatorynetworks. Anal Chem82: 81768185. 10.1021/ac1015563[doi].27. JansenJJ,HoefslootHC,vanderGreefJ, TimmermanME,WesterhuisJA,etal. (2006) ASCA: analysis of multivariatedataobtainedfromanexperimentaldesign. JChemometrics19: 469481.28. vanVelzenEJ, WesterhuisJA,vanDuynhovenJP, vanDorstenFA, HoefslootHC, et al. (2008) Multilevel data analysis of a crossover designed humannutritional intervention study. J Proteome Res 7: 44834491. 10.1021/pr800145j[doi].29. Boughton-SmithNK,HawkeyCJ,WhittleBJ(1983) Biosynthesisoflipoxygen-ase and cyclo-oxygenase products from[14C]-arachidonic acid by humancolonicmucosa. Gut24: 11761182.30. Gould SR(1976) Assay of prostaglandin-like substances in faeces and theirmeasurementinulcerativecolitis. Prostaglandins11: 489497.31. Rampton DS, Sladen GE, Youlten LJ (1980) Prostaglandins and ulcerativecolitis. Gastroenterology78: 192193.32. Vong L, Ferraz JG, Panaccione R, Beck PL, Wallace JL (2010) A pro-resolutionmediator, prostaglandinD(2),isspecificallyup-regulatedinindividualsinlong-termremissionfromulcerativecolitis. ProcNatl AcadSci USA107: 1202312027. 1004982107[pii];10.1073/pnas.1004982107[doi].33. LevyBD, ClishCB,SchmidtB, GronertK,SerhanCN(2001) Lipidmediatorclass switching during acute inflammation: signals in resolution. Nat Immunol 2:612619. 10.1038/89759[doi];89759[pii].34. Vachier I, Chanez P, Bonnans C, Godard P, Bousquet J, et al. (2002)Endogenous anti-inflammatory mediators from arachidonate in humanneutrophils. Biochem Biophys Res Commun 290: 219224. 10.1006/bbrc.2001.6155[doi];S0006291X01961558[pii].35. PalmerRM, StepneyRJ, HiggsGA, Eakins KE (1980) Chemokinetic activity ofarachidonic and lipoxygenase products on leuocyctes of different species.Prostaglandins20: 411418.36. Turner SR, Tainer JA, Lynn WS (1975) Biogenesis of chemotactic molecules bythearachidonatelipoxygenasesystemofplatelets. Nature257: 680681.37. Borgeat P, SamuelssonB(1979) Arachidonicacidmetabolisminpolymorpho-nuclearleukocytes:unstableintermediateinformationofdihydroxyacids.ProcNatl AcadSci USA76: 32133217.38. Borgeat P, Samuelsson B (1979) Metabolism of arachidonic acid inpolymorphonuclear leukocytes. Structural analysis of novel hydroxylatedcompounds.JBiol Chem254: 78657869.39. Ford-HutchinsonAW, Bray MA, Doig MV, Shipley ME, SmithMJ (1980)LeukotrieneB,apotentchemokineticandaggregatingsubstancereleasedfrompolymorphonuclearleukocytes. Nature286: 264265.40. ShimizuT, Fujii T, Suzuki R, Igarashi J, OhtsukaY, et al. (2003) Effects ofhighlypurifiedeicosapentaenoicacidonerythrocytefattyacidcompositionandleukocyte and colonic mucosa leukotriene B4 production in children withulcerativecolitis. JPediatrGastroenterol Nutr37: 581585.41. Rask-MadsenJ, BukhaveK, LaursenLS, LauritsenK(1992) 5-Lipoxygenaseinhibitorsforthe treatmentofinflammatory boweldisease.Agents Actions SpecNo: C37C46.42. Pearl DS, Masoodi M, EidenM, BrummerJ, GullickD, et al. (2013) Alteredcolonic mucosal availability of n-3 and n-6 polyunsaturated fatty acids inulcerative colitis and the relationship to disease activity. J Crohns Colitis. S1873-9946(13)00137-2[pii];10.1016/j.crohns.2013.03.013[doi].43. LobosEA,SharonP,StensonWF(1987)Chemotacticactivityininflammatorybowel disease. RoleofleukotrieneB4. DigDisSci 32: 13801388.44. IkehataA,HiwatashiN,KinouchiY,Yamazaki H,ItoK,etal.(1995) AlteredleukotrieneB4metabolismincolonicmucosawithinflammatoryboweldisease.ScandJGastroenterol 30: 4449.45. GertnerDJ, RamptonDS, MaddenMV, TalbotIC, NichollsRJ, etal. (1994)IncreasedleukotrieneB4releasefromileal pouchmucosainulcerativecolitiscomparedwithfamilial adenomatouspolyposis. Gut35: 14291432.46. EliakimR, Karmeli F, ChorevM, OkonE, RachmilewitzD(1992) Effect ofdrugs on colonic eicosanoid accumulation in active ulcerative colitis.ScandJGastroenterol 27: 968972.EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 12 October2013| Volume8| Issue 10| e7653247. Cole AT, Pilkington BJ, McLaughlan J, Smith C, Balsitis M, et al. (1996)Mucosal factorsinducingneutrophil movementinulcerativecolitis: theroleofinterleukin8andleukotrieneB4. Gut39: 248254.48. CasellasF,PapoM,GuarnerF,AntolinM,SeguraRM,etal.(1995)Effectsofthromboxanesynthaseinhibitiononinvivoreleaseof inflammatorymediatorsinchroniculcerativecolitis. EurJGastroenterol Hepatol 7: 221226.49. Harizi H, JuzanM, MoreauJF, Gualde N(2003) Prostaglandins inhibit 5-lipoxygenase-activatingproteinexpressionandleukotrieneB4productionfromdendriticcellsviaanIL-10-dependentmechanism.JImmunol 170: 139146.50. CartyE, DeBM, Feakins RM, RamptonDS(2000) Measurement of invivorectal mucosal cytokine andeicosanoidproductioninulcerative colitis usingfilterpaper. Gut46: 487492.51. GilroyDW,Colville-NashPR,WillisD,ChiversJ,Paul-ClarkMJ,etal.(1999)Induciblecyclooxygenasemayhaveanti-inflammatoryproperties. Nat Med5:698701. 10.1038/9550[doi].52. Ajuebor MN, SinghA, Wallace JL(2000) Cyclooxygenase-2-derivedprosta-glandin D(2) is an early anti-inflammatory signal in experimental colitis.AmJPhysiol GastrointestLiverPhysiol 279: G238G244.53. RajakariarR,HilliardM,LawrenceT,TrivediS,Colville-NashP,etal.(2007)HematopoieticprostaglandinD2synthasecontrols theonset andresolutionofacuteinflammationthroughPGD2and15-deoxyDelta1214PGJ2. ProcNatlAcad Sci U S A 104: 2097920984. 0707394104 [pii];10.1073/pnas.0707394104[doi].54. Zamuner SR, Warrier N, Buret AG, MacNaughtonWK, WallaceJL(2003)Cyclooxygenase 2 mediates post-inflammatory colonic secretory and barrierdysfunction. Gut52: 17141720.55. Trebble TM, Arden NK, Wootton SA, Calder PC, Mullee MA, et al. (2004) Fishoil and antioxidants alter the composition and function of circulatingmononuclear cells inCrohndisease. AmJ ClinNutr 80: 11371144. 80/5/1137[pii].56. Trebble TM, Wootton SA, Miles EA, Mullee M, Arden NK, et al. (2003)ProstaglandinE2productionandTcell functionafterfish-oil supplementation:responsetoantioxidantcosupplementation. AmJClinNutr78: 376382.57. Hawthorne AB, DaneshmendTK, HawkeyCJ, Belluzzi A, Everitt SJ, et al.(1992) Treatment of ulcerative colitis with fish oil supplementation: a prospective12monthrandomisedcontrolledtrial. Gut33: 922928.58. AnanthakrishnanAN, Higuchi LM, HuangES, Khalili H, RichterJM, et al.(2012) Aspirin, nonsteroidal anti-inflammatory drug use, andrisk for Crohndiseaseandulcerativecolitis: acohort study. AnnInternMed156: 350359.156/5/350[pii];10.1059/0003-4819-156-5-201203060-00007[doi].59. DHaensG, SandbornWJ, FeaganBG, GeboesK, HanauerSB, etal.(2007) Areviewof activityindices andefficacyendpoints for clinical trials of medicaltherapyin adults with ulcerativecolitis.Gastroenterology 132: 763786.S0016-5085(06)02688-6[pii];10.1053/j.gastro.2006.12.038[doi].EicosanoidsandLipidMediatorsinUCPLOSONE | www.plosone.org 13 October2013| Volume8| Issue 10| e76532