Innate immune memory mediates increased susceptibility to Alzheimer’s disease-like pathology in sepsis surviving mice 2021 Virginia L. De Sousa.pdf

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Brain,Behavior,andImmunity95(2021)287–298
ContentslistsavailableatScienceDirect
BrainBehaviorandImmunity
journalhomepage:
InnateimmunememorymediatesincreasedsusceptibilitytoAlzheimer’s
disease-likepathologyinsepsissurvivingmice
,újoa,,MarianaSilva-Queiroza,
,CarolinaSoaresa,FernandaBarros-Aragao~a,b,-Araujoa,
Viní,RobsonCoutinho-Silvac,,,c,d,
RobsonDaCostaa,,b,*,,b,*
aSchoolofPharmacy,FederalUniversityofRiodeJaneiro,RiodeJaneiro,RJ21944-590,Brazil
bInstituteofBiomedicalSciences,FederalUniversityofRiodeJaneiro,RiodeJaneiro,RJ21944-590,Brazil
cInstituteofBiophysicsCarlosChagasFilho,FederalUniversityofRiodeJaneiro,RiodeJaneiro,RJ21944-590,Brazil
dInstituteofMedicalBiochemistryLeopoldodeMeis,FederalUniversityofRiodeJaneiro,RiodeJaneiro,RJ21944-590,Brazil
ARTICLEINFOABSTRACT
Keywords:Sepsissurvivorsshowlong-termimpairments,
MicrogliasuggeststhatsystemicinflammationcontributestotheprogressionofAlzheimerśdisease(AD),butthemech-
(trained)anddiminished(tolerant)innateimmune
,theoccurrenceoflong-terminnate
Synapselossimmunememoryinthepost-,wedemonstratethatsepsiscauseslong-lasting
Amyloid-β
Innateimmunememorytrainedinnateimmunememoryinthemousebrain,leadingtoanincreasedsusceptibilitytoAβoligomers(AβO),
-survivingmiceshifttoanamoeboid/
phagocyticmorphologicalprofilewhenexposedtolowamountsofAβO,andthiseventwasaccompaniedbythe
upregulationofseveralpro-inflammatoryproteins(IL-1β,IL-6,INF-γandP2X7receptor)inthemousehippo-
campus,

blockageofbrainphagocyticcellsormicroglialdepletion,usingminocyclineandcolonystimulatingfactor1
receptorinhibitor(PLX3397),respectively,preventscognitivedysfunctioninducedbyAβOinsepsis-surviving
,ourfindingssuggestthatsepsisinducesalong-lastingtrainedinnateimmunememoryinthe
mousebrain,leadingtoanincreasedsusceptibilitytoAβO-inducedneurotoxicityandcognitiveimpairment.
-clinicaldatahavesuggestedthatsystemicinflammation
associatedwithsepsismayaffecttheprogressionofAlzheimer’sdisease
Sepsisisacomplexinflammatorysyndromethatresultsfroma(AD)(Ehleretal.,2017;Gasparottoetal.,2018;Nevesetal.,2018;
dysregulatedhostimmuneresponsetoinfection;itisasignificantpublicWangetal.,2018).Indeed,inflammatoryconditionsinhumansmay
healthproblemindevelopedand,mostnotably,indevelopingcountriesinduceorworsenpre-existingbraindysfunctions,includingAD(Guerra
(Ruddetal.,2020).Survivingpatientsshowanincreasedriskofdeathinetal.,2012;Iwashynaetal.,2010;Kaoetal.,2015;PrescottandAngus,
thefollowingmonthsandyearspost-sepsisandfrequentlyexperience2018;Semmleretal.,2013),butthemechanismslinkingsystemic
long-lastingdebilitatingconsequences,includingcognitiveimpairmentinflammationandneurodegenerationarestillunclear.
(PrescottandAngus,2018).ManypatientswhosurvivesepsisneverEvidencesuggeststhattheoccurrenceandseverityoflateconse-
recovernormalmemoryfunction(Iwashynaetal.,2010;Prescottandquencesofsepsisareassociatedwithfunctionalreprogrammingof
Angus,2018;Semmleretal.,2013),andpresentanincreasedriskofinnateimmunecells(Bomansetal.,2018;Neteaetal.,2016).Innate
developingdementialaterinlife(Guerraetal.,2012;Kaoetal.,2015).immunememorymayleadtoeitheraboosted(trained)oradiminished
*Correspondingauthors.
E-mailaddresses:******@(),******@().
/
Received16September2020;Receivedinrevisedform12March2021;Accepted3April2021
Availableonline8April2021
0889-1591/©:.
(2021)287–298
(tolerant)immuneresponsetoasecondinflammatorystimulus(-amyloidpeptideoligomers(AβO)
etal.,2016).Moststudieshaveshowntheprevalenceofanimmune
tolerantenvironmentaftersepsis,providingapossibleexplanationforAt45daysafterCLPsurgery,micereceivedanintra-
thewell-knownsepsis-inducedimmunosuppressionandhighersuscep-cerebroventricular(.)infusionofamyloid-βpeptideoligomers
tibilityofpatientstosecondaryinfections(Hotchkissetal.,2013).In(AβO,1or10pmol/site)oranequalvolumeofvehicleaspreviously
contrast,arecentstudydescribedthatperipheralmonocytesmaypre-described(Figueiredoetal.,2013).AβOwerepreparedaspreviously
sentatrainedimmunityfollowingsepsis(Bomansetal.,2018),sug-described(DeFeliceetal.,2008).Briefly,humanAβ1-42(American
gestingthatbothimmunetoleranceandtraininghavearoleinthePeptide)wasfirstsolubilizedinhexafluoroisopropanol,andthesolvent
pathophysiologyofsepsis-inducedlong-,whichweresubsequentlydis-
Inthepresentstudy,wehypothesizedthatsepsiscauseslong-lastingsolvedinsterileanhydrousdimethylsulfoxidetomakea5mMsolution.
innateimmunememoryinthebrain,leadingtoanincreasedsuscepti-Thissolutionwasdilutedto100μMinice-coldPBSandincubatedfor16
bilitytoAβoligomers(AβO),themainneurotoxinsfoundinADbrainshat4,000×gfor10minat
(SelkoeandHardy,2016).Here,usingamousemodelofsepsisinduced4Ctoremoveinsolubleaggregates(protofibrilsandfibrils),andthe
bycecalligationandperforation(CLP),weshowthatpost-septicmicesupernantanscontainingsolubleAβoligomerswerestoredat4oC.
aremoresusceptibletosynapsedamageandcognitiveimpairmentProteinconcentrationwasdeterminedusingtheBCAassay(Thermo
-ScientificPierce).Preparationswereroutinelycharacterizedbysize-
pocampalphagocyticcellsfromsepsis-survivingmiceshifttoanexclusionhigh-performanceliquidchromatographyandoccasionally
amoeboidmorphologicalprofilewhenexposedtolowamountsofAβO,byimmunoblotsusinganti-Aβ6E10oranti-AβoligomersNU4(Lambert
.,2007).
morphologicalchangeswereaccompaniedbytheupregulationof
severalpro-inflammatoryproteins(IL-1β,IL-6,INF-γandP2X7receptor)
inthehippocampusofpost-,wedemonstratedthat
brainexposuretolowamountsofAβOincreasedthephagocyticabilityImmediatelyaftersurgery,animalsreceivedasubcutaneous(.)
ofIba-1positivecellsagainsthippocampalsynapses,andthattheinjectionofsterilesaline(1mL)(Benjamimetal.,2005)andwere
pharmacologicalblockadeofbrainphagocyticcellsactivationortreatedwithanintraperitoneal(.)injectionoftheantimicrobialagent
microglialdepletionpreventscognitivedysfunctioninducedbyAβOinmeropenem(10mg/kg)5,-
sepsis-,ourfindingssuggestthatsepsisin-cologicalblockadeofmicroglia/macrophageactivation,micereceived
ducesalong-lastingtrainedinnateimmunememoryinthebrain,lead-vehicle(saline)orminocyclinehydrochloride(Sigma-Aldrich)either
ingtoanincreasedsusceptibilitytoAβO-(50mg/kg,
)orintracerebroventricularly(50μg/site,
).For
microglialdepletion,PLX3397(PLX;MedKooBiosciences,Morrisville,
)wasdissolvedindimethylsulfoxideanddilutedwithphosphate-
bufferedsaline(PBS);micereceiveddailytreatmentwithvehicle
NaïvemaleSwissmiceaged6to8weeks,weighingbetween25and(PBS)orPLX(40mg/kg)byoralgavagefortwentyonedayspriorto
30g,.
groupsofthreetofivepercage,withfreeaccesstofoodandwater,under
a12-hourlight/

(NOR)test
InstitutionalAnimalCareandUseCommitteeoftheHealthSciencesThenovelobjectrecognitiontestwasperformedinanopenfield
Center(CCS)fromtheFederalUniversityofRiodeJaneiro(UFRJ),arenameasuring30×30×45cm,whereobjectswerefixedtothebox
protocolnumber174/(Figueiredoetal.,2019).Preliminarytestsshowedthatnone

,eachanimalwassubmittedtoa5-min-longhabituationsession,

Sepsisinductionwasperformedusingthececalligationandpuncturethishabituationsession,totaldistancetraveledbyeachmousewas
model(CLP)aspreviouslydescribed(Bakeretal.,1983;Nevesetal.,measuredusingAnymazesoftware(StoeltingCo,WoodDale,IL)to
2018;Wichtermanetal.,1980)Briefly,
Ketamine100mg/kgandXylazine20mg/kggivenintraperitoneally(-longsessionduringwhichanimalswereplacedat
p.).Aftertrichotomyintheabdominalregionandasepsiswith75%
ethanol,

,%ethanoltoeliminate
regionbelowtheileocecalvalve,piercedtwicewithasterile18-,animalswereagainplacedin
needle,and,finally,itwaslightlycompressedtoallowleakageoffecalthearenafora5min-longtestsession,inwhichoneoftheobjectsused
,andthelap-,thetimespent
arotomywasclosedwithasurgical9---
animalsweresubjectedtothesamesurgicalprocedures;however,thecentageoftimeexploringeachobjectduringthetrainingortestsessions
-sampleStudent’sttestcomparingthe
individuallytodeterminesurvivalandclinicalsignsofasystemicin-meanexplorationtimeforeachobjectwiththefixedvalueof50(50%,i.
flammatoryresponse,suchaspiloerection,tremors,prostration,musclee.,nodifferentiationbetweenobjects).Animalsthatrecognizethe
atony,(.,learnthetask)explorethenovelobject>
discomfortwereseeninsham-%ofthetotaltime.
288:.
(2021)287–298
(TST)diametersofacellbody,,the
Thistestisdesignedtoassessthedepressivebehaviorofmice(CryanFeretmax–mindifferencestendtozero.
etal.,2005).Fortesting,(NIH;
:SCR_003070)tocountthenumberofcolocalized,pre-(synapto-
definedastheamountoftimeduringwhichthemicehungpassively,physin),orpostsynaptic(Homer-1)
withoutactivelytryingtoreleasetheirtailsfromtape(Steruetal.,engulfmentbymicroglia,threeconfocalZ-stackimagesfromtheCA1
1985).andCA3hippocampalregionfromdifferentmicegroupswereacquired
–12
,atleastoneofwhichwereanalyzedinde-
pendentlyaspreviouslydescribed(Bellesietal.,2017).Briefly,red