DTIC ADA622592: Acute and Chronic Plasma Metabalomic and Liver Transcriptomic Stress Effects in a Mouse Model with Features of Post-Traumatic Stress Disorder

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RESEARCHARTICLE
Acute and Chronic Plasma Metabolomic and
Liver Transcriptomic Stress Effects in a Mouse
Model with Features of Post-Traumatic Stress
Disorder
AartiGautam1,2‡,PeterD’Arpa1,2‡,DuncanE.Donohue1,2,SeidMuhie3,
NabarunChakraborty1,2,BrianT.Luke3,DmitryGrapov4,EricaE.Carroll5,James
L.Meyerhoff1,2,RashaHammamieh1,MartiJett1*
1 USArmyCenterforEnvironmentalHealthResearch,FortDetrick,MD,UnitedStatesofAmerica,2 The
GenevaFoundation,Tacoma,WA98402,UnitedStatesofAmerica,3 AdvancedBiomedicalComputing
Center,FrederickNationalLaboratoryforCancerResearch,Frederick,MD,UnitedStatesofAmerica,4 NIH
WestCoastMetabolomicsCenter,UniversityofCaliforniaDavis,Davis,CA,UnitedStatesofAmerica,5
ArmyInstituteforPublicHealth,AberdeenProvingGround,Aberdeen,MD21010–5403,UnitedStatesof
America
‡Theseauthorscontributedequallytothiswork.
OPENACCESS
* [email protected]
Citation:GautamA,D’ArpaP,DonohueDE,Muhie
S,ChakrabortyN,LukeBT,etal.(2015)Acuteand
ChronicPlasmaMetabolomicandLiver Abstract
TranscriptomicStressEffectsinaMouseModelwith
FeaturesofPostTraumaticStressDisorder.PLoS
Acuteresponsestointensestressorscangiverisetopost-traumaticstressdisorder
ONE10(1):e0117092.doi:10.1371/journal.
pone.0117092 (PTSD).PTSDdiagnosticcriteriaincludetraumaexposurehistoryandself-reportedsymp-
toms.IndividualswhomeetPTSDdiagnosticcriteriaoftenmeetcriteriaforadditionalpsy-
AcademicEditor:AlexandraKavushansky,Technion
IsraelInstituteofTechnology,ISRAEL chiatricdiagnoses.BiomarkerspromisetocontributetoreliablephenotypesofPTSDand
comorbiditiesbylinkingbiologicalsystemalterationstobehavioralsymptoms.Herewe
Received:September29,2014
haveanalyzedunbiasedplasmametabolomicsandotherstresseffectsinamousemodel
Accepted:December18,2014
withbehavioralfeaturesofPTSD.Inthismodel,C57BL/6micearerepeatedlyexposedtoa
Published:January28,2015
trainedaggressormouse(albinoSJL)usingamodified,resident-intruder,socialdefeatpar-
Copyright:Thisisanopenaccessarticle,freeofall adigm.Ourrecentstudiesusingthismodelfoundthataggressor-exposedmiceexhibited
copyright,andmaybefreelyreproduced,distributed,
acutestresseffectsincludingchangedbehaviors,bodyweightgain,increasedbodytem-
transmitted,modified,builtupon,orotherwiseused
perature,aswellasinflammatoryandfibrotichistopathologiesandtranscriptomicchanges
byanyoneforanylawfulpurpose.Theworkismade
availableundertheCreativeCommonsCC0public ofhearttissue.Someoftheseacutestresseffectspersisted,reminiscentofPTSD.Herewe
domaindedication. reportelevatedproteinsinplasmathatfunctionininflammationandresponsestooxidative
DataAvailabilityStatement:Livertranscriptomics stressanddamagedtissueat24hrspost-stressor.Additionallyatthisacutetimepoint,
data mayviewGSE58275studyat:http://www.ncbi. transcriptomicanalysisindicatedliverinflammation.Theunbiasedmetabolomicsanalysis
nlm.nih.gov/geo/query/acc.cgi?acc=GSE58275.The
showedalteredmetabolitesinplasmaat24hrsthatonlypartiallynormalizedtowardcontrol
metabolomicsdatafileissubmittedasS1File.The
levelsafterstress-withdrawalfor1.5or4wks.Inparticular,gut-derivedmetaboliteswereal-
ProteindatafileissubmittedasFileS6.
teredat24hrspost-stressorandremainedalteredupto4wksafterstress-withdrawal.Also
Funding:ThisprojectissupportedbytheOfficeof
atthe4wktimepoint,hyperlipidemiaandsuppressedmetabolitesofaminoacidsandcar-
theAssistantSecretaryofDefenseforHealthAffairs
andtheMilitaryOperationalMedicineResearch bohydratesinplasmacoincidedwithtranscriptomicindicatorsofalteredlivermetabolism
Program(MOMRP),andthesupportofgrant (activatedxenobioticandlipidmetabolism).Collectively,thesesystem-widesequelaetore-
#USAMRMCNO:09284002ishighlyacknowledged.
peatedintensestresssuggestthatthesimultaneousperturbedfunctioningofmultipleorgan
DGwassupportedbyNIH1U24DK097154.The
PLOSONE|DOI:10.1371/journal.pone.0117092 January28,2015 1/24
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1. REPORT DATE 2. REPORT TYPE 3. DATES COVERED
26 AUG 2015 Research 00-01-2013 to 00-08-2014
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Acute and Chronic Plasma Metabalomic and liver transcriptomic stress
5b. GRANT NUMBER
effects in a mouse model with features of post-tramatic stress disorder
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
Aarti Gautam; Peter D’Arpa; Duncan Donohue; Seid Muhie; Nabarun
5e. TASK NUMBER
Chakraborty
4.0
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
U.S.Army Center for Environmental Health Research,568 Doughten REPORT NUMBER
14-16
Drive,Fort Detrick,MD,21702-5010
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
U.S. Army Medical Research and Materiel Command, 504 Scott Street, USAMRMC
Fort Detrick, MD, 21702-5010
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release; distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
Acute responses to intense stressors can give rise to post-traumatic stress disorder (PTSD). PTSD
diagnostic criteria include trauma exposure history and self-reported symp- toms. Individuals who meet
PTSD diagnostic criteria often meet criteria for additional psychiatric diagnoses. Biomarkers promise to
contribute to reliable phenotypes of PTSD and comorbidities by linking biological system alterations to
behavioral symptoms. Here we have analyzed unbiased plasma metabolomics and other stress effects in a
mouse model with behavioral features of PTSD. In this model, C57BL/6 mice are repeatedly exposed to a
trained aggressor mouse (albino SJL) using a modified, resident-intruder, social defeat paradigm. Our
recent studies using this model found that aggressor-exposed mice exhibited acute stress effects including
changed behaviors, body weight gain, increased body tem- perature, as well as inflammatory and fibrotic
histopathologies and transcriptomic changes of heart tissue. Some of these acute stress effects persisted,
reminiscent of PTSD. Here we report elevated proteins in plasma that function in inflammation and
responses to oxidative stress and damaged tissue at 24 hrs post-stressor. Additionally at this acute time
point, transcriptomic analysis indicated liver inflammation. The unbiased metabolomics analysis showed
altered metabolites in plasma at 24 hrs that only partially normalized toward control levels after
stress-withdrawal for 1.5 or 4 wks. In particular, gut-derived metabolites were al- tered at 24 hrs
post-stressor and remained altered up to 4 wks after stress-withdrawal. Also at the 4 wk time point,
hyperlipidemia and suppressed metabolites of amino acids and car- bohydrates in plasma coincided with
transcriptomic indicators of altered liver metabolism (activated xenobiotic and lipid metabolism).
Collectively, these system-wide sequelae to re- peated intense stress suggest that the simultaneous
perturbed functioning of multiple organ systems (e.g., brain, heart, intestine and liver) can interact to
produce injuries that lead to chronic metabolic changes and disorders that have been associated with
PTSD.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a REPORT b ABSTRACT c THIS PAGE Same as 24
unclassified unclassified unclassified Report (SAR)
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
fundershadnoroleinstudydesign,datacollection systems(e.g.,brain,heart,intestineandliver)caninteracttoproduceinjuriesthatleadto
andanalysis,decisiontopublish,orpreparationof chronicmetabolicchangesanddisordersthathavebeenassociatedwithPTSD.
themanuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Post-traumaticstressdisorder(PTSD)isacommonlyoccurring,debilitatinganxietydisorder
thatoftenpersistsformanyyearsandisfrequentlyassociatedwithexposuretomultipletrau-
mas[1].SignificantcontroversysurroundsthediagnosisofPTSD[2]thatisbasedonhistory
ofexposuretoextremetraumaticevent(s)andreportedsymptomsincludingpersistentre-
experiencingoftheevent(s),persistentavoidanceandnumbingtotrauma-relatedstimuli,in-
creasedarousal,andclinicallysignificantdistressthatimpairssocial,occupational,orotherim-
portantareasoffunctioningformorethanonemonth[3].ThePTSDlifetimeprevalencerate
fromtheNationalComorbiditySurveywasestimatedat7.8%amongthegeneralpopulationof
Americanmenandwomen[4].PTSDprevalenceratesarehigherinindividualsexposedtose-
veretraumas,andinpost-conflictsettingsrangedfrom16to37%[5,6]with22%of289,328
veteransoftheIraqandAfghanistanconflictsenteringVAhealthcarefrom2002to2008after
havingreceivedthediagnosisofPTSD[7].
Neurological/physiologicalalterationsassociatedwithPTSDcomprisebothcentralandau-
tonomicnervoussystems,andincludehyperarousalofthesympatheticnervoussystemwith
sensitizedandaugmentedacoustic-startleeyeblinkreflexandsleepabnormalities,aswellas
changesinnoradrenergic,hypothalamic-pituitary-adrenocortical,serotonergic,glutamatergic,
thyroid,endogenousopioid,andothersystems[6].Brainimaginghasrevealedreducedvolume
ofthehippocampusandanteriorcingulatecortex,excessiveamygdalaactivity,andreducedac-
tivationoftheprefrontalcortex.
PsychometricandpsychophysiologictechniqueshavebeendevelopedtoassessPTSD[8].
Self-reportingcomprisesalargepartofthediagnosisofPTSD,butitispronetocontroversial
diagnoses.AdditionalcontroversyinthediagnosisofPTSDrelatestothehighrateofcomorbid
conditions;i.e.,individualswhomeetthePTSDdiagnosticcriteriaarelikelytomeetcriteriafor
oneormoreadditionaldiagnoses[6,9].Assuch,PTSDmaybecategorizedintomultipledi-
mensionswithtraitssuchascognition,mood,andsocialinteractionsexistingonacontinuum
fromnormaltoextreme[10].Ongoingresearchaimstocontinuetolinkthesebehavioraldi-
mensionswithunderlyingmolecularpathologiesinordertoconstructvalid,measurabletraits
forPTSDaswellastoclarifytheboundariesandoverlapwithothermentaldisorders,toward
improvingPTSDprevention,diagnosisandtreatment[10].
Animalmodelsofanxietydisordersvarywidely.Inthecurrentstudy,weusedapreviously
validatedC57BL/6JmousemodelwithPTSD-likefeatures[11].Micearestressedbyexposures
totrainedaggressormicefor5or106-hrsessionsdailyinacage-in-cageenvironmentwherein
thesubjectmouseisremovedfromthesmallercageforuptothreerandomtimesduringeach
6hrsessiontobeattackedbytheaggressorforoneminuteortenstrikes,whichevercomes
first.Inpreviousstudieswiththismodel,subjectmiceexperiencedacutestresseffects,includ-
ingbodyweightgain,increasedbodytemperature,cardiacinflammationandtranscriptomic
changes,andbehaviorsindicativeoffearandanxiety.Someoftheseacutestresseffectsper-
sistedfollowing1.5to6weeksofstress-withdrawal,reminiscentofPTSD.Herewehaveevalu-
atedadditionalacuteandchronicstresseffectsinthismodel,includingchangesinthe
transcriptomeofliverandproteinsandmetabolitesinplasma.Inparticular,transcriptomics
indicatedliverinflammationat24hrspost-stressor.Unbiasedmetabolomicsidentifiedaltered
metabolitesderivedfromthegutmicrobiotaasearlyas24hrspost-stressorthatremainedal-
teredupto4wks.Also,atthischronictimepoint,aggressor-exposedmicedisplayed
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MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
Figure1.Studydesign.Micewereexposedtoatrainedaggressormousefor6hrsdailyfor5or10days
(blackrectangles).Controlmicewereexposedonthesamescheduleandunderthesameconditionsbutwith
noaggressormousepresent.Terminalbleedsandtissuesamples(redrectangles)wereobtained24hafter
the5-and10-dayregimensaswellas1.5weeksafterthe5-dayand4weeksafterthe10-daystressor
regimens.Miceweresinglyhousedthroughouttheexperiment.
doi:10.1371/journal.pone.0117092.g001
metabolomicmarkersofhyperlipidemiaandtranscriptomicindicatorsofalteredxenobiotic
andlipidmetabolisminliver.Theseobservationscontributetodefiningacutestresseffectsand
theirchronicsequelaefollowingrepeatedexposurestointensestressors.
ResultsandDiscussion
StudyDesign
C57BL/6Jmicewereanalyzedforunbiasedplasmametabolomics,serumproteins,histopathol-
ogy,andlivertranscriptomicsfollowingcontrol(Ctrl)oraggressorexposure(AggE)for6hrs
dailyforeither5or10days.Twenty-fourhoursafterthesestressorregimens,aswellas1.5
weeksafterthe5-dayregimen,and4weeksafterthe10-dayregimen,histopathologywasper-
formedandterminalbleedsandtissueswereobtainedforanalysis.Onehourpriortosample
collection,duringapartitiontest[11],micewereexposedtothepotentsituationalcueassociat-
edwiththeaggressorexposures.TheexperimentaldesignisshowninFig.1.
PlasmametabolitealterationsofAggEmiceat24hrspost-stressor
partiallynormalizedafterstress-withdrawalfor1.5or4wks
Theunbiasedplasmametabolomicsdetected330namedmetabolitesand166unidentified
compounds(S1File).Forthenamedmetabolites,thepairwisecomparisonsoftheirlevelsbe-
tweenCtrlandAggEwereassociatedwithhighq-values,likelyduetothesmallsamplesizeof
5or6micepergroupandtheknownhighlyvariableresponseofeveninbredmicetostress.
However,analysisofunadjustedp-valuesshowedtrendsinthedata.Clearertrendswereiden-
tifiedbyanalyzingsuperpathwaysandsubpathwaysofmetabolitesconstructedbasedon
KEGG,HMDBandtheinternalknowledgebaseofMetabolon,Inc.(Durham,NC).Within
manyofthesepathways,numbersofelevatedorsuppressedmetabolitelevelsinAggEvs.Ctrl
differedfromthenullhypothesisofequalnumbers(binomialtest).
Inpairwiseanalysis,AggE-alteredmetabolitesweremorenumerousat24hrsafterthelast
stresssessionascomparedtoafter1.5or4wksofstress-withdrawal(Welch’st-testand2-way
ANOVA):40and14metaboliteswerealteredat24hrsand1.5wks,respectively(p(cid:1)0.05),
afterthe5-daystressregimen;and,37and20metaboliteswerealteredat24hrsand4wks,re-
spectively(p(cid:1)0.05),afterthe10-daystressregimen.Thistrendsuggeststhattheacutemeta-
bolicperturbationofAggEmicepartiallynormalizedtowardtheCtrllevelafterthemicehad
beenwithdrawnfromthestressorfor1.5and4wks.
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MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
Evidenceforhabituationtothe10-dayvs.the5-daystressorregimen
Time-dependentchangesinplasmametaboliteswerealsoevidentinbothCtrlandAggE
mice.InAggEmice,103(5-dayregimen)and30(10-dayregimen)metabolitelevelswere
significantlydifferentbetweentheacute(24hrs)andchronictimepoints(1.5or4wks).
Similarly,incontrolmice,90(5-dayregimen)and76metabolitesdiffered(10-dayregimen)
betweentheacuteandchronictimepoints(S1Table).Thus,greaternumbersofmetabolites
werealteredwithtimeafterthe5-dayregimenascomparedtothe10-dayregimen,despitethe
10-day-regimenmicehavingreceivedfiveadditionalAggEsessionsandhavingbeenwithdrawn
fromthestressfor2.5wkslongerthanthe5-day-regimenmice.Thismayindicatethatthemice
receivingthe10-dayregimenhabituatedtothestressorbetweenthe5thand10thdailyexposure
(i.e.,metaboliteshadalreadynormalized24hrsafterthe10thstressorsession).Consistentwith
thisinterpretation,thetreatmenteffect(Ctrlvs.AggE,acuteandchronictimepointscombined),
thetimeeffect(acutevs.chronic;CtrlandAggEcombined),andthetreatmentandtimeinterac-
tion(two-wayANOVA),wereallgreaterafterthe5-daycomparedtothe10-daystressregimen
(S1Table).Thus,therewerefewermetabolicchanges24hrsafterthe10-daystressregimenas
comparedtothe5-daystressregimen,suggestinghabituationtothemetaboliceffectof
thestressor.
Consistentwithhabituation,thepercentofmicethatjumpedinresponsetotheaggressor
orfoughtbackagainsttheaggressordroppedbetweenthe4thand10thdaysofthestress
regimen(unpublisheddata).Additionally,intheheart,anorderofmagnitudefewerAggE-
vs.-Ctrltranscriptswereevidentat24hrsafterthe10-daythanafterthe5-dayregimen,and
inflammationtranscriptspeakedonthethirddayoftheAggEregimenandthenfellastissue
remodelingtranscriptsrose,suggestinghealingdespitethe6hrdailyAggEsessionsondays4
to10[12].
Asafurtherindicationofhabituation,inthelivertherewere~3-foldmoreAggE-vs.-Ctrl
differentiallyexpressedgenes(DEGs,>2-fold)at24hrsafterthe5-daystressorthanat24hrs
afterthe10-daystressor(2169vs.668for5-and10-daymice,respectively;Table1).Geneset
enrichmentanalysisoftheseDEGsshowedthedownstreamfunctions‘biosynthesisofhydro-
genperoxide’,‘accumulationofleukocytes’,and‘activationofphagocytes’asthetopthreeand
onlyactivatedfunctionsat24hrspost-5-daystressor(category‘PhysiologicalSystemDevelop-
mentandFunction’,(S2Table).Conversely,at24hrsafterthe10-daystressor,thetopactivat-
edfunctionwas‘bindingoffibroblastcelllines’,andthetoptwoinhibitedfunctionswere
‘quantityofneutrophils’and‘quantityofphagocytes’.Theseresultsareconsistentwiththose
fromhearttissue(belowand[12])andsuggestthatinflammationofliversofAggEmiceat24
hrspost-5-daystressormayhavetransitionedtofibroblastinfiltrationat24hrspost-10-
daystressor.
Table1.SummaryofdifferentiallyexpressedgenesbetweenliversofControlandAggEmice.
5-DayStressRegimen 10-DayStressRegimen
24hr 1.5wk 24hr 4wk
Up-regulatedgenes* 842 530 383 193
Down-regulatedgenes* 1327 522 285 139
Totals 2169 1052 668 332
*Basedon(cid:3)2foldchangeofprobeintensitywithp-value(cid:1)0.05.
doi:10.1371/journal.pone.0117092.t001
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MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
HearthistopathologywasuniquetoAggEmice;lymphoplasmacytic
infiltratesofotherorgansweresimilarinCtrlandAggEmicebutvaried
withstressordurationandpost-stresstime
Similartothehearttranscriptomics,histopathologyofhearttissueofAggEmiceat24hrsafter
the5-daystressregimenappearedtobeahighergradethanafterthe10-dayregimen[11].
Heartinflammatoryhistopathologiesofthe5-day-AggEmiceat24hrshaddisappearedby
1.5wkswhenfibrosisorfibroplasiaandmyocardialdegenerationwerepresent.In10-day
AggEmiceatthe24hrpoint,muchmorefibrosis,fibroplasiaanddegenerationwerepresent
comparedtoat24hrsafterthe5-dayAggE,indicatingtheinjuryhadstartedtohealbythe11th
dayafterthestartofthe10-dayregimen.Alargerpercentageof5-dayAggEmiceat1.5wks
thanthe10-dayAggEmiceat4wksshowedfibrosisorfibroplasiaandmyocardialdegenera-
tion,whicharelikelyremnantsoftheinjurythatpersistedat1.5wksbutwasevidentlyhealed
by4wks(S1Fig.).
Acute,intensepsychologicalstressorssuchasinter-maleaggressiveencountersorcatechol-
amineadministrationhavebeenshowntoinduceorgantissueinjury[13,14].Uponfighting,
EpidermalGrowthFactor(EGF)releasedfromsubmandibularsalivaryglandsprotected
againstheartinjury,butnotagainstliverinjury[15].However,twicedailyadrenergicstimula-
tiondecreasedtheEGFcontentofsubmandibularglandsbyalmost90%,andfurtheradrener-
gicstimulationdidnotincreaseplasmaEGFlevels[15].Thisresultsuggestsprotective
mechanismsagainstacutestresscanbecomeexhaustedinresponsetochronicstress.Inour
model,therepeated,intense,5-and10-dayAggEsessionslikelyexhaustedmechanismsfor
protectinghearttissuefromintenseadrenergicstimulation.
IncontrasttothecardiaclesionsfoundonlyinAggEmice,lymphoplasmacyticinfiltratesof
liver,gallbladder,kidney,andstomachwereofsimilargradesinCtrlandAggEmice.Thissug-
geststhepossibilitythattheCtrlcondition,whichreproducedtheAggEconditionwiththeex-
ceptionthattheaggressorwasabsent,wasasignificantstressor,likelyinvolvingsocialisolation
stress.AlthoughsimilarinCtrlandAggEmice,immunecellinfiltratesofliver,gallbladder,
kidney,andstomachdifferedfromeachotherintrendsofrateofdevelopmentandtheimpact
ofthestressorduration(S2Fig.).Kidneylymphoplasmacyticinterstitialinfiltratesapparently
worsenedwithtime,whileliverlymphoplasmacyticinfiltrateslessenedduringstress-withdraw-
al.Fororganswithinfiltratesthatincreasedduringstress-withdrawal,thetrendssuggestthe
possibilitythatthe10-dayregimenmighthavebeenmoredamagingthanthe5-dayregimen,
and/orthealterationscouldhavebeencumulativeordevelopedmorefullyby4wksthanby
1.5wks.
Elevatedplasmaproteinsat24hrspost-stressorindicatedamaged
tissueinAggEmice
Inplasma,wedetected41proteinsintheRodentMulti-AnalyteProfilev.2.0Antigens(Myri-
ad-RBM)panelat24hrsafterthe5-and10-daystressors.Thisanalysisshowedthathaptoglo-
binandmyeloperoxidaseandserumamyloidP-component(FalseDiscoveryRatep(cid:1)0.05
[16])weresignificantlyelevatedinplasmaofAggEoverCtrlmiceat24hrsforthe10-day-
exposedmiceaswellasforthecombinedgroupof5-day-and10-day-exposedmice(S2File).
Theseproteinsareinvolvedinresponsestooxidativestress,damagedtissue,andinflammation.
Myeloperoxidaseisstoredinazurophilicgranulesofneutrophils,oxidizeschlorideionstothe
potentbactericidaloxidanthypochlorousacid[17],andhasbeenwidelyusedasmarkerofin-
flammationinacuteandchronicconditions,includingcardiovasculardisease[18].Haptoglo-
binisanacutephaseprotein,increasedduringinflammation[19],whichhasbeenobservedto
riseinresponsetoanumberofpsychologicalstressesaswellasdepression[20,21].Serum
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MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
amyloidP-component(SAP)isamemberofthehighlyconservedpentraxinsuperfamilyalong
withtheclassicalacute-phasereactant,C-reactiveprotein(CRP);bothSAPandCRPtranscrip-
tionareactivatedinresponsetoendoplasmicreticulumstress[22].SAPhasanti-fibroticeffects
[23]andbindsmoleculararrayssuchasDNA,chromatin,histones,andphosphoethanolamine-
containingmembranes,suggestingitfunctionsintheclearanceoflateapoptoticcells[24].
Additionally,tissueinhibitorofmetalloproteinases1(TIMP-1)waselevatedin10-day
AggEoverCtrlmice(FDRcorrected,p=0.038),andmatrixmetalloproteinase-9(MMP-9)was
elevatedinthe5-day-exposedoverthe10-day-exposedmice(combinedCtrlandAggE
groups).MMP-9andmatrixmetalloproteinasefamilymembersareinvolvedinthedegrada-
tionofextracellularmatrixinprocessessuchaswoundhealing,andTIMP-1isnaturalinhibi-
torofMMP-9,andthetwoproteinsaremarkersconsistentlyindictedincardiovasculardisease
developmentandprognosis[25,26].MMP-9levelshavebeenassociatedwithdirecteffectsof
cortisolandnorepinephrine(referencesin[27]),andindependentlypredictednewcardiac
eventsinpatientswithstablecoronarydisease[28],aswellashavingbeenindependentlyasso-
ciatedwithpsychosocialfactorsinanormalmiddle-agedpopulation[27].Additionally,inour
model,inhearttissues,MMP-2andTIMP-1transcriptswereelevatedinAggEmiceat24hrs
afterthe5-dayand10-daystressregimens.
Incontrast,granulocytechemotacticprotein-2(GCP-2)andmacrophage-derivedchemo-
kine(MDC)wereelevatedinCtrlrelativetoAggEmiceat24hrs,whichmayindicateadiffer-
enttrajectoryofinflammationresultingfromthestressoftheCtrlcondition.
Gutmicrobiota-derivedmetabolitesinplasmaofAggEmicewerealtered
atbothacute(24hrs)andchronic(1.5&4wks)timepoints
Toexploremetabolicalterationsoccurringat24hrsthatpotentiallypersistedoutto1.5and4
wks,weusedrandomforests(RF)toclassifyAggEvs.Ctrlat24hrsand1.5&4wks(combined
5-dayand10-daygroups).Theresultinglistsofthetop10%ofmetaboliteswiththegreatest
‘meandecreaseaccuracy’(MDA)attheacuteandchronictimepointswereanalyzedforover-
laps[29].Ninemetabolitesoverlappedbetweenthetwolists(Table2,p=0.002,Fisher’sexact
test).Fiveofthe9overlappingmetabolitesweregut-derivedmetabolites:phenylpropionylgly-
cine,phenolsulfate,hippurate,3-phenylpropionate,andp-cresolsulfate.Threeofthese—3-
phenylpropionate,phenylpropionylglycine,andhippurate—wereelevatedinAggEmiceat
bothacuteandchronictimepointspost-10-dayregimen(t-test,p(cid:1)0.05),andtheacuteand
chronictimepointsofthecombined5-and10-daygroup(t-test,p(cid:1)0.05).Thesethreemetab-
olitesareamongthemostelevatedmetabolitesinAggEmice,beingonaverage6.7-foldelevat-
edat24hrsand2.8-foldelevatedat1.5&4wks.
Themetabolite3-phenylpropionateisaknownmetabolicproductofanaerobicbacteria
[30].Bothhippurateandphenylpropionylglycinearederivedfrom3-phenylpropionate.
Phenylpropionylglycineistheglycineconjugateof3-phenylpropionate.Hippurateisaglycine
conjugateofbenzoate,derivedfrom3-phenylpropionateviacatalysisbyMedium-chain
acyl-CoAdehydrogenase(MCAD)[31,32].Hippuratehasbeenfoundtobe17-foldlowerin
plasmaofgerm-freemicethanconventionalmice,andphenylpropionylglycinewasdetected
exclusivelyinconventionalsamples[30].Phenolsulfateandp-cresolsulfatehavebeenshown
tobereducedseveralhundredto1000-foldingerm-freemice[30].
TheremainingfourmetabolitesidentifiedbyRFthatoverlappedbetween24hrsand1.5&4
wkswereapparentlydiverse.Notably,2’-deoxycytidinewasthebestsingleclassifierofAggE
vs.Ctrlmiceinthecombined5-and10-daygroupsat24hrsusingeitherdistance-dependent
K-nearestneighboranalysis(sensitivity=90.9%,specificity=81.8%)orrule-basedmachine
learning.
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MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
Table2.MetabolitesthatclassifyAggEvs.Ctrlmiceatbothacuteandchronictimepoints.
24hrs 1.5&4wks
MDA FC p-value MDA FC p-value
Phenylpropionylglycine* 14.3 10 0.005 3.5 3.95 0.024
phenolsulfate* 14.2 1.27 0.090 5.9 0.77 0.143
Hippurate* 12.6 8.07 0.008 3 2.52 0.025
3-phenylpropionate(hydrocinnamate)* 7.9 2.16 0.021 7.3 1.97 0.015
p-cresolsulfate* 7.1 0.69 0.244 5.2 0.77 0.054
2'-deoxycytidine 22 1.64 0.001 2.1 1.13 0.152
palmitoylsphingomyelin(d18:1/16:0) 13.5 1.26 0.007 15 1.11 0.015
creatinine 7.8 0.76 0.022 8.2 0.88 0.159
2-linoleoylglycerophosphocholine 6.8 0.83 0.183 6.1 0.91 0.277
MDA—‘meandecreaseaccuracy’fromrandomforestsanalysis
FC—foldchange
*metabolitesderivedfromgutmicrobiota
doi:10.1371/journal.pone.0117092.t002
Gutmicrobiota-derivedandothermetabolitesinplasmaalteredateither
acuteorchronictimepoints
Inadditiontothegut-derivedmetabolitesidentifiedbyRF,pairwiseandcorrelationanalyses
revealedadditionalgut-derivedmetabolitesthatwereelevatedinAggEat24hrs.Twoofsix
metabolitesthatwere“perfectclassifiers”ofAggEvs.Ctrlmice(i.e.,levelswereallhigheror
lowerinAggEmice)at24hrspost-5-daystressoraremetabolitesofthegutmicrobiota;2-(4-
hydroxyphenyl)propionateandindolelactate.BothwereelevatedinAggEvs.Ctrlmice(com-
bined5-and10-daygroups).2-4-hydroxyphenyl)propionateisafecalbacteriafermentation
productoftyrosine[33,34],whichincreasedinfecesofantibiotic-treatedmice[35],anditalso
positivelycorrelatedinourdatasetwithindolelactate(r=0.86),whichistheotherelevatedper-
fectclassifier.IndolelactateisatryptophanmetaboliteproducedbyBifidobacteriumspecies
[36],andwasfoundtobeatleast10-foldmoreabundantinurineofgerm-freemicecolonized
withBifidobacteriumlongumthaningerm-freemiceormicecolonizedwithBacteroidesthe-
taiotaomicron[37].Itwasalso7-foldreducedinseruminamodelofinflammatoryboweldis-
ease[38].Additionally,2-(4-hydroxyphenyl)propionatecorrelatedwithindolepropionate(r=
0.71).Indolepropionatehasbeenidentifiedintheplasmaofconventionalmicebutnotin
germ-freemice[30].
Ofthetop10%,or33,highestMDAmetabolites,24didnotoverlapbetweenthe24hrand
1.5&4wktimepoints.Uniquetothe24hrtimepointsweretheadditionalgut-derivedmetabo-
litesindolelactateand2-(4-hydroxyphenyl)-propionate(alsoidentifiedasperfectclassifiers),
andoneoftheircorrelatinggut-derivedmetabolites,indolepropionate.Anothergut-derived
metabolitealteredat24hrswasphenyllactate[39].Thus,thesegut-derivedmetabolitesappear
alteredattheacutetime,inadditiontothegut-derivedmetabolitesalteredatboththeacute
andchronictimes.Additionally,twofibrinogencleavagepeptideswerehighlyrankedunique
RFclassifiersofAggEmiceatthe24hrtimepoint.Fibrinogencleavageproductshavebeenas-
sociatedwithinflammation[40].
TheRF-classifyingmetabolitesuniqueto1.5&4wksincludedthreeplant-derivedbiochemi-
cals,campesterol[41],piperidine[42]anddaidzein[43].Additionally,sevenlysophospholipids
wereamongtheRFtop-scoringmetabolitesat1.5&4wksandtheywereallelevated.Thesere-
sultssuggestthatAggEstressmayhavedamagedtheGItract[44].Inadditiontoalikely
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MetabolomicsandLiverTranscriptomicsinPTSDMouseModel
alteredgutmicrobiota,otherchangescouldpossiblyincludealteredintestinalabsorptionand
alteredlivermetabolism.TheGItractrespondstostressandmediatorsofstress(includingglu-
cocorticoidsandcatecholamines)withdisturbedmotoractivity,ulcerations,enteritis,andal-
teredtransepithelialiontransport[45–47],allofwhichcanaffectthecompositionofthe
microbiota[48].Also,gutbacteriacanresponddirectlytocatecholamineswithchangesin
growthandvirulencegeneexpression[45].Alterationsinintestinalbacterial-hostinteractions
havebeensuggestedtocauseliverinjury[49].
Metabolitesofthemajormetabolicfuelsinplasmawerealteredatacute
andchronictimepoints
Themajormetabolicfuels,carbohydrates,aminoacids,andlipidsdifferedinplasmalevelsde-
pendentuponthestressordurationandthelengthofthestress-withdrawalperiod(Fig.2).At
24hrsafterthe5-and10-daystressors,81%and76%ofthe21carbohydratemetaboliteswere
higherinplasmaofAggEmice(binomialtest,p=0.003,post-5-daystressor,andp=0.013,
post-10-daystressor).Butafterwithdrawalfromthestressfor1.5and4wks,only57%and
29%ofplasmacarbohydrateswereelevated,fewerthanat24hrs(testofequalproportions:p=
0.182andp=0.005,respectively;andp=0.002forthecombined5-dayand10-daygroup).
AcutelyelevatedcarbohydratemetabolitesinplasmaofAggEmicecouldpossiblyreflectthe
actionoftheprimarystressmediators,epinephrineandcorticosterone,onfuelstorageand
availability.Corticosteronemobilizesaminoacidsmainlyfrommuscle,makingmoreavailable
intheplasmatoentergluconeogenesisinthelivertopromoteglucoseandglycogensynthesis
[50].Thus,moreglycogenismadeavailableforglycogenolysisstimulatedbyepinephrine,
whichcanreleasewithinminuteslargeamountsofglucoseintotheblood[50].Therisein
bloodglucoseinturnstimulatesinsulinsecretion,buthighlevelsofglucocorticoidmakeskele-
talmuscleandadiposetissueresistanttoinsulin’sstimulatoryeffectsonglucoseuptakeand
utilization[50].Consistentwiththisscenario,attheacutetimepoint,glucoselevelshoweda
trendtobeelevated1.2-foldinthe5-day24hrgroup(p(cid:1)0.1)and1.18-foldinthecombined
5-dayand10-day24hrgroup(p(cid:1)0.1).Alsopossiblyconsistentwiththistrend,2-hydroxybu-
tyrate,anindicatorofinsulinresistanceandimpairedglucoseregulation[51–53],was1.74-
foldelevatedinthecombinedgroupof5-and10-dayAggEmiceat24hrs(p=0.007).Howev-
er,changesin2-hydroxybutyratehavealsobeenrelatedtotheoxidativestressresponseinthe
liverandincreasedglutathionesynthesis.
Conversely,thenumberofaminoacidmetaboliteselevatedinAggEmiceat24hrspost-5-
daystressorwasnotgreater,butmoreofthemwereelevatedat24hrsafterthe10-daystressor
Figure2.PercentofmetabolitesofmetabolicfuelshigherinAggEvs.Ctrlmice.TheCarbohydrate,
AminoacidandLipidsuperpathwaysarecomprisedof21,98,and134metabolites,respectively.Asterisks
indicatep(cid:1)0.05(*)andp(cid:1)0.01(**)forobtainingthepercentelevatedmetabolitesshown(exactbinomial
test).
doi:10.1371/journal.pone.0117092.g002
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