《英语国际会议》PPT课件.ppt

1 MacrophagesfrompatientswithatopicdermatitisshowareducedCXCL10expressioninresponsetostaphylococcala toxin CONTENTS Introduction Results MaterialsandMethods 2 Discussion 3 background Keywords atopicdermatitis 特应性皮炎 chemokines 趋化因子 humanmacrophages 人巨噬细胞 IP 10 CXCL10 MDC CCL22 psoriasis 牛皮癣 银屑病 Staphylococcusaureus 金黄色葡萄球菌 toxin 毒素 4 Background Patientswithatopicdermatitis AD andpsoriasisarefrequentlycolonizedwithStaphylococcusaureus S aureus one thirdofthemproducing toxin whichiscor relatedwiththeseverityofeczemainAD Distinctexpressionofchemokine C Cmotif ligand 配体CCL andchemokine C X Cmotif ligand 配体CXCL chemokineshasbeendocumentedinbothdiseases theeffectsofa toxinonhumanmacrophagesinpatientswithADandpsoriasis 5 Presentstudy 6 Recentstudiesdemonstratedthatinfiltrationofinflammatorycellsintotissuesisregulatedbychemokines Twomainsubfamilies recentlyrenamedchemokine C Cmotif ligand CCL andchemokine C X Cmotif ligand CXCL chemo kines havebeendistinguished SeveralstudieshavesuggestedacrucialinvolvementofCCLchemokinesininflammation ChemokinesandtheirreceptorsareimplicatedinthedevelopmentofsymptomsofADandpsoriasis CXCL10playsaroleinpathogenesisofpsoriasis Purpose 7 Theeffectsof toxinonhumanmacrophagesstillremainunclear Weshowhereforthefirsttimethat toxininducestheTh1 relatedchemokineCXCL10inhumanmacrophages westudiedtheeffectsof toxinonTh1 andTh2 relatedchemokinesinmacrophagesfrompatientswithADandpsoriasiswheretheintrinsicabnormalanddifferentchemokinesproductionprofileiswelldefined MaterialsandMethods 8 MaterialsandMethods Patients Cellisolationandculture Statisticalanalysis CytokineassessmentbyELISA Westernblot Methods 9 macrophage derivedchemokine MDC CCL22 Cellsurfacemarkers expressionandchemotaxis IFN c inducedproteinof10 kDa IP 10 CXCL10 qRT PCRorELISA flowcytometryBoydenchambertechnique 10 Patients healthydonorsandpatientswithADorpsoriasis Cellisolationandculture Peripheralbloodmononuclearcells PBMCs wereisolatedbyLymphoprepdensity gradientcentrifugationfromhealthydonorsandfrompatientswithADandpsoriasis Chemotaxisassay Thechemotacticactivityofsupernatantsfroma toxinstimulatedmacrophagesonlymphocytes CD4 Tcells wasdeterminedusingaBoydenchambertechnique 11 Westernblot macrophageswereprestimulatedwitha toxin 100ng ml IFN c 10ng ml TNF a 10ng ml orLPS CellextractsweresubjectedtoWesternblotanalysis CytokineassessmentbyELISA Cell freeculturesupernatantswereharvestedandanalysedforCXCL10orCCL22 usingacommerciallyavailableenzyme linkedimmunosorbentassay ELISA system Results 12 OneInductionofIP 10 CXCL10bystaphylococcala toxininhumanmacrophages ThreeLoweffectofa toxinonCXCL10induction Th1 relatedchemokine inmacrophagesfrompatientswithAD Twoa Toxin stimulatedmacrophagescouldinducethemigrationofhumanCD4 TcellsviaCXCR3 FourEffectofa toxinonMDCproduction Th2 relatedchemokine inmacrophagesfrompatientswithchronicin ammatoryskindiseases InductionofIP 10 CXCL10bystaphylococcala toxininhumanmacrophages 13 MicroarrayanalysisrevealedthatCXCL10wasthemoststronglyup regulatedgeneinmacrophages Anincreasedexpressionwasnotobservedforotherchemokines Themeanratioforallthesechemokineswasbetween0 2and2whencomparingnotstimulatedwitha toxin stimulatedmacrophages 14 Figure1Inductionofchemokine C X Cmotif ligand CXCL 10followinga toxinstimulationinhumanmacrophagesatthemRNAlevel Figure2Inductionofchemokine C X Cmotif ligand CXCL 10followinga toxinstimulationinhumanmacrophagesattheproteinlevel 15 Figure3Punchbiopsies 3mm fromhealthyindividualswerelefteitherunstimulated A orstimulatedwitha toxin 100ng ml B orIFN c 100ng ml C for24hat37C 5 lmparaf nsectionswerestainedforCXCL10alongwithappropriateisotypeaswellasCD68 a Toxin stimulatedmacrophagescouldinducethemigrationofhumanCD4 TcellsviaCXCR3 16 Bothinhibitors anti IP 10antibody anti CXCR3antibody abrogatedthemigrationinducedbya toxin treatedmacrophagesupernatants a ToxinincellculturemediumwithoutmacrophagesdidnotdirectlyinducethemigrationofhumanCD4 lymphocytes 17 Figure4Chemotacticactivityofa toxin stimulatedmacrophagesandinhibitionofchemokine C X Cmotif ligand CXCL 10 inducedchemotaxisusinganeutralizinganti CXCL10oranti CXCR3Ab Loweffectofa toxinonCXCL10induction Th1 relatedchemokine inmacrophagesfrompatientswithAD 18 a toxininducedsigni cantlylowerlevelsofCXCL10expressionorsecretioninpatientswithADcomparedwithhealthycontrolsaswellaspsoriasisatalltimepointsanddosestested 19 Figure5Macrophagesfrompatientswithatopicdermatitis AD andpsoriasis PSO aswellasfromhealthycontrols HC werelefteitherunstimulated mediumcontrol orstimulatedwitha toxinasindicatedorwithIFN c 10ng ml orTNF a 20ng ml respectively fortheindicatedperiodsoftime Effectofa toxinonMDCproduction Th2 relatedchemokine inmacrophagesfrompatientswithchronicinfammatoryskindiseases 20 MacrophagesfrompatientswithADaswellaspatientswithpsoriasisshowedanintrinsicallyhigherCCL22productioncomparedtohealthycontrolsbytrend Onthewhole a toxindidnotregulateCCL22secretion 21 Thankyou