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1MacrophagesfrompatientswithatopicdermatitisshowareducedCXCL10expressioninresponsetostaphylococcala-toxin.IntroductionResultsMaterialsandMethods2Discussion2020/10/313Introductionkeywordspresentstudypurposebackground2020/10/31Keywords•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）•α-toxin.（α-毒素）42020/10/31Background:•Patientswithatopicdermatitis(AD)andpsoriasisarefrequentlycolonizedwithStaphylococcusaureus(S.aureus).one-thirdofthemproducingα-toxin,whichiscor-relatedwiththeseverityofeczemainAD.•Distinctexpressionofchemokine(C-Cmotif)ligand(配体CCL)andchemokine(C-X-Cmotif)ligand(配体CXCL)chemokineshasbeendocumentedinbothdiseases.•theeffectsofa-toxinonhumanmacrophagesinpatientswithADandpsoriasis?52020/10/31Presentstudy6Recentstudiesdemonstratedthatinfiltrationofinflammatorycellsintotissuesisregulatedbychemokines.Twomainsubfamilies,recentlyrenamedchemokine(C-Cmotif)ligand(CCL)andchemokine(C-X-Cmotif)ligand(CXCL)chemo-kines,havebeendistinguished.SeveralstudieshavesuggestedacrucialinvolvementofCCLchemokinesininflammation.ChemokinesandtheirreceptorsareimplicatedinthedevelopmentofsymptomsofADandpsoriasis.CXCL10playsaroleinpathogenesisofpsoriasis.2020/10/31Purpose7Theeffectsofα-toxinonhumanmacrophagesstillremainunclear.Weshowhereforthefirsttimethatα-toxininducestheTh1-relatedchemokineCXCL10inhumanmacrophages.westudiedtheeffectsofα-toxinonTh1-andTh2-relatedchemokinesinmacrophagesfrompatientswithADandpsoriasiswheretheintrinsicabnormalanddifferentchemokinesproductionprofileiswelldefined.2020/10/31MaterialsandMethods8MaterialsandMethodsPatientsCellisolationandcultureStatisticalanalysisCytokineassessmentbyELISAWesternblot2020/10/31Methods9macrophage-derivedchemokine(MDC)/CCL22Cellsurfacemarkers’expressionandchemotaxisIFN-c-inducedproteinof10-kDa(IP-10)/CXCL10qRT–PCRorELISAflowcytometryBoydenchambertechnique2020/10/3110PatientshealthydonorsandpatientswithADorpsoriasisCellisolationandculturePeripheralbloodmononuclearcells(PBMCs)wereisolatedbyLymphoprepdensity-gradientcentrifugationfromhealthydonorsandfrompatientswithADandpsoriasisChemotaxisassayThechemotacticactivityofsupernatantsfroma-toxinstimulatedmacrophagesonlymphocytes(CD4+Tcells)wasdeterminedusingaBoydenchambertechnique2020/10/3111Westernblotmacrophageswereprestimulatedwitha-toxin(100ng/ml),IFN-c(10ng/ml),TNF-a(10ng/ml)orLPS.CellextractsweresubjectedtoWesternblotanalysisCytokineassessmentbyELISACell-freeculturesupernatantswereharvestedandanalysedforCXCL10orCCL22,usingacommerciallyavailableenzyme-linkedimmunosorbentassay(ELISA)system.2020/10/31Results12OneInductionofIP-10/CXCL10bystaphylococcala-toxininhumanmacrophagesThreeLoweffectofa-toxinonCXCL10induction(Th1-relatedchemokine)inmacrophagesfrompatientswithADTwoa-Toxin-stimulatedmacrophagescouldinducethemigrationofhumanCD4+TcellsviaCXCR3FourEffectofa-toxinonMDCproduction(Th2-relatedchemokine)inmacrophagesfrompatientswithchronicinflammatoryskindiseases2020/10/31InductionofIP-10/CXCL10bystaphylococcala-toxininhumanmacrophages13MicroarrayanalysisrevealedthatCXCL10wasthemoststronglyup-regulatedgeneinmacrophages.Anincreasedexpressionwasnotobservedforotherchemokines.Themeanratioforallthesechemokineswasbetween0.2and2whencomparingnotstimulatedwitha-toxin-stimulatedmacrophages.2020/10/3114Figure1Inductionofchemokine(C-X-Cmotif)ligand(CXCL)10followinga-toxinstimulationinhumanmacrophagesatthemRNAlevel.Figure2Inductionofchemokine(C-X-Cmotif)ligand(CXCL)10followinga-toxinstimulationinhumanmacrophagesattheproteinlevel.2020/10/3115Figure3Punchbiopsies(3mm)fromhealthyindividualswerelefteitherunstimulated(A)orstimulatedwitha-toxin(100ng/ml)(B)orIFN-c(100ng/ml)(C)for24hat37C.5-lmparaffinsectionswerestainedforCXCL10alongwithappropriateisotypeaswellasCD68.2020/10/31a-Toxin-stimulatedmacrophagescouldinducethemigrationofhumanCD4+TcellsviaCXCR316Bothinhibitors(anti-IP-10antibody&anti-CXCR3antibody)abrogatedthemigrationinducedbya-toxin-treatedmacrophagesupernatants.a-ToxinincellculturemediumwithoutmacrophagesdidnotdirectlyinducethemigrationofhumanCD4+lymphocytes.2020/10/3117Figure4Chemotacticactivityofa-toxin-stimulatedmacrophagesandinhibitionofchemokine(C-X-Cmotif)ligand(CXCL)10-inducedchemotaxisusinganeutralizinganti-CXCL10oranti-CXCR3Ab.2020/10/31Loweffectofa-toxinonCXCL10induction(Th1-relatedchemokine)inmacrophagesfrompatientswithAD18a-toxininducedsignificantlylowerlevelsofCXCL10expressionorsecretioninpatientswithADcomparedwithhealthycontrolsaswellaspsoriasisatalltimepointsanddosestested.2020/10/3119Figure5Macrophagesfrompatientswithatopicdermatitis(AD)andpsoriasis(PSO)aswellasfromhealthycontrols(HC)werelefteitherunstimulated(mediumcontrol)orstimulatedwitha-toxinasindicatedorwithIFN-c(10ng/ml)orTNF-a(20ng/ml),respectively,fortheindicatedperiodsoftime.2020/10/31Effectofa-toxinonMDCproduction(Th2-relatedchemokine)inmacrophagesfrompatientswithchronicinfammatoryskindiseases20MacrophagesfrompatientswithADaswellaspatientswithpsoriasisshowedanin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