Effects of Aggregatibacter actinomycetemcomitans leukotoxin on neutrophil migration and extracellular trap formation

摘要

Background:AggressiveperiodontitisisassociatedwiththepresenceofAggregatibacteractinomycetemcomitans,aleukotoxin(Ltx)-producingperiodontalpathogen.Ltxhastheabilitytolysewhitebloodcellsincludingneutrophils.Objectives:ThisstudywasaimedatinvestigatingtheinteractionsbetweenneutrophilsandLtxwithregardtothechemotacticpropertiesofLtxandthereleaseofneutrophilextracellulartraps(NETs).Methods:Neutrophilsfromhealthyblooddonorswereisolatedandincubatedfor30minand3hwithincreasingconcentrationsofLtx(1,10,and100ng/mL)aswellaswithA.actinomycetemcomitansstrains(NCTC9710andHK1651)producingdifferentlevelsofLtx.FormationofNETsandcelllysiswereassessedbymicroscopy,fluorescence-basedassays,andmeasurementofreleasedlactatedehydrogenase.NeutrophilmigrationinresponsetodifferentLtxgradientswasmonitoredbyreal-timevideomicroscopy,andimageanalysiswasperformedusingImageJsoftware.Results:AlthoughLtx(10and100ng/mL)andtheleukotoxicA.actinomycetemcomitansstrainHK1651lysedsomeneutrophils,othercellswerestillcapableofperformingNETosisinaconcentration-dependentmanner.LowdosesofLtxandtheweaklyleukotoxicstrainNCTC9710didnotleadtoneutrophillysis,butdidinducesomeNETosis.Furthermore,allthreeconcentrationsofLtxenhancedrandomneutrophilmovement;however,lowdirectionalaccuracywasobservedcomparedwiththepositivecontrol(fMLP).Conclusions:TheresultsindicatethatLtxactsbothasaneutrophilactivatorandalsocausescelldeath.Inaddition,LtxdirectlyinducesNETosisinneutrophilspriortocelllysis.Infuturestudies,theunderlyingpathwaysinvolvedinLtx-meditatedneutrophilactivationandNETosisneedtobeinvestigatedfurther.Keywords:leukotoxin;Aggregatibacteractinomycetemcomitans;neutrophils;NETs;migration;chemotaxis;aggressiveperiodontitis(Published:8November2016)Citation:JournalofOralMicrobiology2016,8:33070-http://dx.doi.org/10.3402/jom.v8.33070