Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation.docx

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation.docx

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HumanCD68promoterGFPtransgenicmiceallowanalysisofmonocytetomacrophagedifferentiation

Blood.Oct9,2014;124（15）:

e33–e44.

PrepublishedonlineJul16,2014.doi:

 10.1182/blood-2014-04-568691

PMCID:

PMC4192756

HumanCD68promoterGFPtransgenicmiceallowanalysisofmonocytetomacrophagedifferentiationinvivo

AsifJ.Iqbal,1EileenMcNeill,2,3TheodoreS.Kapellos,1DanielRegan-Komito,1SophieNorman,4SarahBurd,1NicolaSmart,4DanielE.W.Machemer,5ElenaStylianou,6HelenMcShane,6KeithM.Channon,2,3AjayChawla,7andDavidR.Greaves

1

1SirWilliamDunnSchoolofPathology,UniversityofOxford,UnitedKingdom;

2DivisionofCardiovascularMedicine,UniversityofOxford,JohnRadcliffeHospital,Oxford,UnitedKingdom;

3WellcomeTrustCentreforHumanGenetics,UniversityofOxford,Oxford,UnitedKingdom;

4DepartmentofPhysiology,AnatomyandGenetics,UniversityofOxford,UnitedKingdom;

5Genentech,SouthSanFrancisco,CA;

6JennerInstitute,UniversityofOxford,Oxford,UnitedKingdom;and

7CardiovascularResearchInstitute,DepartmentsofPhysiologyandMedicine,UniversityofCaliforniaSanFrancisco,CA

Correspondingauthor.

A.J.I.andE.M.contributedequallytothisstudy.

Authorinformation►Articlenotes►CopyrightandLicenseinformation►

ReceivedApril8,2014;AcceptedJune30,2014.

Copyright©2014byTheAmericanSocietyofHematology

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KeyPoints

∙CD68-GFPreportermiceshowGFPtransgeneexpressioninbothmonocytesandtissueresidentmacrophagepopulations.

∙AdoptivelytransferredCD68-GFPmonocytesmaintainGFPexpressionafterrecruitmentinanongoinginflammatoryresponse.

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Abstract

Therecruitmentofmonocytesandtheirdifferentiationintomacrophagesatsitesofinflammationarekeyeventsindeterminingtheoutcomeoftheinflammatoryresponseandinitiatingthereturntotissuehomeostasis.Tostudymonocytetraffickingandmacrophagedifferentiationinvivo,wehavegeneratedanoveltransgenicreportermouseexpressingagreenfluorescentprotein（GFP）underthecontrolofthehumanCD68promoter.CD68-GFPmiceexpresshighlevelsofGFPinbothmonocyteandembryo-derivedtissueresidentmacrophagesinadultanimals.ThehumanCD68promoterdrivesGFPexpressioninallCD115+monocytesofadultblood,spleen,andbonemarrow;wetookadvantageofthistodirectlycomparethetraffickingofbonemarrow–derivedCD68-GFPmonocytestothatofCX3CR1GFPmonocytesinvivousingasterilezymosanperitonitismodel.UnlikeCX3CR1GFPmonocytes,whichdownregulateGFPexpressionondifferentiationintomacrophagesinthismodel,CD68-GFPmonocytesretainhigh-levelGFPexpressionfor72hoursafterdifferentiationintomacrophages,allowingcontinuedcelltrackingduringresolutionofinflammation.Insummary,thisnovelCD68-GFPtransgenicreportermouselinerepresentsapowerfulresourceforanalyzingmonocytemobilizationandmonocytetraffickingaswellasstudyingthefateofrecruitedmonocytesinmodelsofacuteandchronicinflammation.

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Introduction

Immunecellsofthemononuclearphagocytesystem（MPS）playakeyroleinhostimmuneresponses.Tissue-residentmacrophagesplayasentinelroleininitiatingacuteinflammatoryresponsesand,togetherwithmonocyte-derivedmacrophagesthatdifferentiatefromrecruitedmonocytes,theyregulatelocalinflammatoryresponses.Macrophagesplayanimportantroleininitiatingwoundrepairandrestoringtissuehomeostasisafterinfectionortissueinjury.1,2Insomecases,tissuehomeostasisisnotrestored,andmacrophagescanpromotecontinuingtissuedamageandchronicinflammation,suchasinrheumatoidarthritisandatherosclerosis.3MonocytesarecirculatingCD115+myeloidcellsthatdevelopfromthecommonmonocyteprecursorinthebonemarrowdownstreamofthemacrophagedendriticcell（DC）precursorinthebonemarrowbeforebeingreleasedintothebloodstream.4-7Factorsthatenhancemonocytereleasefrombonemarrowintobloodincludechemokinesderivedfromsitesofinflammation,8-11andrecentstudieshaveidentifiedhyperglycemiaandhyperlipidemiaasimportantfactorsthatregulatemonocytosisbyactingonbonemarrowprogenitorcells.12-16Bloodmonocytesfallinto2populations—Ly6ChiandLy6ClosubsetsthathavebeenpredictedtohavedifferentrolesinvivobecauseofdifferentialexpressionofchemokinereceptorsanddifferentbehaviorsinintravitalmicroscopystudiesinwhichLy6Clomonocyteshaveavascularpatrollingbehavior.17-19

HumanCD68anditsmurinehomolog,macrosialin（Cd68）,arebothheavilyglycosylatedtypeItransmembraneproteinsthatbelongtothelysosomal/endosomal-associatedmembraneproteins.20TheexactfunctionofCD68hasyettobeconfirmed,butaroleinantigenprocessingandasascavengerreceptorhavebeenpreviouslyproposed.20,21Intermsofexpression,CD68hasbeenreportedtoberestrictedtocellsofmyeloidlineage,specificallymonocyte/macrophages.22CD68tissuestainingiscommonlyusedasamarkerforinfiltratingmonocyte-derivedmacrophagesinatheroscleroticlesionsandothersitesofchronicinflammation.23

Themajorityofstudiesthatinvolvemonitoringleukocytetraffickingandinflammatorycellrecruitmentusetransgenicanimalsinwhichspecificcelltypeshavebeenlabeledbygenetic“knock-in”strategiesthataddafluorescentproteingeneintoagenelocusactiveinaspecificcelltype.ThisstrategyisexemplifiedbytheCX3CR1GFPknock-inmouse24andmorerecentlytheCCR2RFPknock-inmouse.25Bothofthesereportermicehavebeenwidelyusedtostudytraffickingofdifferentmonocytesubsetsduringrestingandinflammatoryconditions.Bothofthesetransgenicreporterstrainsrelyonchemokinereceptorgeneregulatoryelementstolabelmonocytesubsetsandbothrelyoncontinuedchemokinereceptorexpressiontolabelmacrophagesthatdifferentiatefrommonocytesinvivo.Byusingthepowerfulgeneregulatoryelementsthatwehaveidentifiedinthepromoterandintron1ofthehumanCD68gene,wesoughttoefficientlylabelalltissueresidentmacrophagepopulationsaswellasallmonocyte-derivedmacrophagesfoundatsitesofinflammation.

InthisstudywedemonstratethatCD68-GFPmicehavehigh-levelGFPreportergeneexpressioninmonocytesinblood,bonemarrow,andspleenaswellastissue-residentmacrophages.WehaveusedCD68-GFPmicetoinvestigatemonocytetraffickingandmacrophagedifferentiationinvivo.Importantly,wedemonstratethatadoptivelytransferredCD68-GFPmonocytesthattraffictositesofinflammationretainhigh-levelGFPexpressionafterdifferentiationintomacrophagesinsitu.

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Methods

Materials

AllcellculturemediaandbufferswereobtainedfromPAAsystems（Yeovil,UnitedKingdom）unlessotherwisespecified.AlllaboratorychemicalswereobtainedfromSigma-Aldrich（Gilligham,Dorset,UnitedKingdom）.ChemokinesandotherchemoattractantswerepurchasedfromPeprotech（London,UnitedKingdom）andR&DSystems（Abingdon,Oxford,UnitedKingdom）.

MolecularcloningandgenerationofCD68-GFPmice

AcomplementaryDNAfragmentencodingenhancedgreenfluorescentprotein（EGFP）（frompEGFP-N1vector,Invitrogen）wassubclonedthroughpSL301tochangetherestrictionsitesandthenclonedintothe1265vectorcontaininghumanCD68promoter（−2.9kb）.26DNAwasexcisedfromthecloningvectorandinjectedintoC57BL/6Jmouseoocytes.Transgenicoffspringweregenotypedbypolymerasechainreaction（PCR）usingthefollowingprimers:

TY1:

5′TTCTCGGCTCTGTGAATGACA3′and5′CAGCCCTCTCTTGGAAAGGAGG3′.FoundermicewerebackcrossedwithC57BL/6Jmice（F1-F4progeny）providingequalnumbersoftransgenepositiveoffspringandlittermatecontrols（subsequentlyreferredtoasWT）.

Animals

AllanimalstudieswereconductedwithapprovalfromtheDunnSchoolofPathologyLocalEthicalReviewCommitteeandinaccordancewiththeUKHomeOfficeregulations（GuidanceontheOperationofAnimals,ScientificProceduresAct,1986）.Male（10to14weeks）C57BL/6JmicewerefromHarlanLaboratories,Oxfordshire,UnitedKingdom;CX3CR1GFPmice24werekindlyprovidedbyDavidJacksonandLouiseJohnson（WeatherallInstituteofMolecularMedicine,UniversityofOxford）.

AdoptivetransferGFPmonocytesintozymosan-inducedperitonitis

MonocyteswereisolatedfrombonemarrowusingEasySepMouseMonocyteEnrichmentkit（StemCellTechnologies）.Briefly,femursandtibiaswereharvestedandflushedthroughwithicecoldphosphate-bufferedsaline（PBS）.Bonemarrowcellsuspensionswerepassedthrougha70-µmcellstrainertoobtainasinglecellsuspension.Redbloodcellswereremovedbyhypotoniclysis（Pharmlyse,BD）accordingtothemanufacturer'sinstructions.ThebonemarrowcellsuspensionwastreatedwiththeEasySepreagentsandmonocytesisolatedbydepletionusinganEasyPlatemagnet（StemCellTechnologies）.Cellswereappliedtothemagnetina96-wellplatefor5minutes,followedbyasecondselectionforafurther2minutesbecausethiswasfoundtoobtainahigheryieldofcellsthanthesingle10-minuteisolationrecommendedbythemanufacturer.ThepurityoftheresultingpopulationswasconfirmedbyflowcytometryusingCD45（BD,Bioscience）,CD11b（BD,Bioscience）,7/4（Serotec）,andLy-6G（Biolegend）.Bonemarrowisolationsweretypicallyfoundtoyield1to2×106cellsatapurityof75%to85%monocytes.

C57BL/6Jmicewereadministered500µLof100µgzymosanAinPBS（Sigma-Aldrich）intraperitoneally30minutesbeforeIVadministrationof1.5×106isolatedmonocytes（70%to80%GFP-positive,in200µL）.After16or72hours,micewereeuthanizedandperitonealexudateswerecollectedbyperitoneallavagewith5mLoficecoldsterilePBS-2mMEDTA.Totalcellcountsandcellularcompositionofperitonealexudat