Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation文档格式.docx
- 文档编号:6639510
- 上传时间:2023-05-07
- 格式:DOCX
- 页数:24
- 大小:87.52KB
Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation文档格式.docx
《Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation文档格式.docx》由会员分享,可在线阅读,更多相关《Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation文档格式.docx(24页珍藏版)》请在冰点文库上搜索。
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
Goto:
KeyPoints
∙CD68-GFPreportermiceshowGFPtransgeneexpressioninbothmonocytesandtissueresidentmacrophagepopulations.
∙AdoptivelytransferredCD68-GFPmonocytesmaintainGFPexpressionafterrecruitmentinanongoinginflammatoryresponse.
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.
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.
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-µ
mcellstrainertoobtainasinglecellsuspension.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/6Jmicewereadministered500µ
Lof100µ
gzymosanAinPBS(Sigma-Aldrich)intraperitoneally30minutesbeforeIVadministrationof1.5×
106isolatedmonocytes(70%to80%GFP-positive,in200µ
L).After16or72hours,micewereeuthanizedandperitonealexudateswerecollectedbyperitoneallavagewith5mLoficecoldsterilePBS-2mMEDTA.Totalcellcountsandcellularcompositionofperitonealexudat
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation
链接地址:https://www.bingdoc.com/p-6639510.html