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:
[email protected](MM);
[email protected](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.
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