The common HAQ STING variant impairs cGAS-dependent antibacterial responses and is

22 Pages·2017·3.26 MB·English

RESEARCHARTICLE
The common HAQ STING variant impairs
cGAS-dependent antibacterial responses and
is associated with susceptibility to
Legionnaires’ disease in humans
JuanS.Ruiz-Moreno1,LutzHamann2,JaveedA.Shah3,4,AnneliesVerbon5,Frank
P.Mockenhaupt6,MonikaPuzianowska-Kuznicka7,8,JanNaujoks1,LeifE.Sander1,9,
MartinWitzenrath1,9,10,JohnC.Cambier11,NorbertSuttorp1,9,10,RalfR.Schumann2,
a1111111111 LeiJin12,ThomasR.Hawn3,BastianOpitz1,9*,CAPNETZStudyGroup¶
a1111111111
1 DepartmentofInternalMedicine/InfectiousDiseasesandPulmonaryMedicine,Charite´-
a1111111111
Universita¨tsmedizinBerlin,corporatememberofFreieUniversita¨tBerlin,Humboldt-Universita¨tzuBerlin,and
a1111111111
BerlinInstituteofHealth,Berlin,Germany,2 InstituteofMicrobiologyandHygiene,Charite´-
a1111111111
Universita¨tsmedizinBerlin,corporatememberofFreieUniversita¨tBerlin,Humboldt-Universita¨tzuBerlin,and
BerlinInstituteofHealthBerlin,Berlin,Germany,3 DepartmentofMedicine,UniversityofWashington,
Seattle,Washington,UnitedstatesofAmerica,4 VAPugetSoundHealthCareSystem,Seattle,Washington,
UnitedstatesofAmerica,5 DepartmentofMedicalMicrobiologyandInfectiousdiseases,ErasmusUniversity
MedicalCenter,Rotterdam,TheNetherlands,6 InstituteofTropicalMedicineandInternationalHealth,
Charite´-Universita¨tsmedizinBerlin,corporatememberofFreieUniversita¨tBerlin,Humboldt-Universita¨tzu
OPENACCESS
Berlin,andBerlinInstituteofHealth,Berlin,Germany,7 DepartmentofHumanEpigenetics,Mossakowski
Citation:Ruiz-MorenoJS,HamannL,ShahJA, MedicalResearchCentre,PolishAcademyofSciences,Warsaw,Poland,8 DepartmentofGeriatricsand
Gerontology,MedicalCentreofPostgraduateEducation,Warsaw,Poland,9 GermanCenterforLung
VerbonA,MockenhauptFP,Puzianowska-Kuznicka
Research(DZL),Germany,10 CAPNETZSTIFTUNG,Hannover,Germany,11 DepartmentofImmunology
M,etal.(2018)ThecommonHAQSTINGvariant
andMicrobiology,UniversityofColoradoSchoolofMedicine,Aurora,Colorado,UnitedStatesofAmerica,
impairscGAS-dependentantibacterialresponses
12 DepartmentofMedicine,DivisionofPulmonary,CriticalCareandSleepMedicine,UniversityofFlorida,
andisassociatedwithsusceptibilityto
Gainesville,Florida,UnitedStatesofAmerica
Legionnaires’diseaseinhumans.PLoSPathog
14(1):e1006829.https://doi.org/10.1371/journal. ¶MembershipoftheCAPNETZStudyGroupisprovidedintheAcknowledgments.
ppat.1006829 *[email protected]
Editor:DarioS.Zamboni,UniversityofSãoPaulo
FMRP/USP,BRAZIL
Abstract
Received:March8,2017
Accepted:December18,2017 ThecyclicGMP-AMPsynthase(cGAS)-STINGpathwayiscentralforinnateimmunesens-
ingofvariousbacterial,viralandprotozoalinfections.Recentstudiesidentifiedthecommon
Published:January3,2018
HAQandR232HallelesofTMEM173/STING,butthefunctionalconsequencesofthesevar-
Copyright:©2018Ruiz-Morenoetal.Thisisan
iantsforprimaryinfectionsareunknown.HerewedemonstratethatcGAS-andSTING-defi-
openaccessarticledistributedunderthetermsof
theCreativeCommonsAttributionLicense,which cientmurinemacrophagesaswellashumancellsofindividualscarryingHAQTMEM173/
permitsunrestricteduse,distribution,and STINGwereseverelyimpairedinproducingtypeIIFNsandpro-inflammatorycytokinesin
reproductioninanymedium,providedtheoriginal
responsetoLegionellapneumophila,bacterialDNAorcyclicdinucleotides(CDNs).Incon-
authorandsourcearecredited.
trast,R232HattenuatedcytokineproductiononlyfollowingstimulationwithbacterialCDN,
DataAvailabilityStatement:Allrelevantdataare
butnotinresponsetoL.pneumophilaorDNA.InamousemodelofLegionnaires’disease,
withinthepaperanditsSupportingInformation
files. cGAS-andSTING-deficientanimalsexhibitedhigherbacterialloadsascomparedtowild-
typemice.Moreover,thehaplotypefrequencyofHAQTMEM173/STING,butnotofR232H
Funding:Thisworkwassupportedbythe
DeutscheForschungsgemeinschaft(www.dfg.de) TMEM173/STING,wasincreasedintwoindependentcohortsofhumanLegionnaires’dis-
(GRK1673/B5toJSRMandBO;GRK1673/A5to easepatientsascomparedtohealthycontrols.OurstudyrevealsthatthecGAS-STING
RRS,SFB/TR84projectA1/A5toBO,SFB/TR84
cascadecontributestoantibacterialdefenseagainstL.pneumophilainmiceandmen,and
projectC3/C6toMW,andSFB/TR84projectB1to
NS;andSPP1580/OP86/10-1toBO),theGerman
PLOSPathogens|https://doi.org/10.1371/journal.ppat.1006829 January3,2018 1/22
HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
MinistryforEducationandResearch(www.bmbf. providesimportantinsightintohowthecommonHAQTMEM173/STINGvariantaffectsanti-
de)(CAPSyS;grantTP4toMW),theMinistryof microbialimmuneresponsesandsusceptibilitytoinfection.
ScienceandHigherEducation,Poland(MEIN;
www.bmbf.de)(PBZ-MEiN-9/2/2006–K143/P01/
Trialregistration
2007/1toMPK),andtheNationalInstitutesof
Health(www.nih.gov)(grantsR21AI099346and
ClinicalTrials.govDRKS00005274,GermanClinicalTrialsRegister
T32AI022295toJCC).Thefundershadnorolein
studydesign,datacollectionandanalysis,decision
topublish,orpreparationofthemanuscript.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist. Authorsummary
Interferons(IFNs)andpro-inflammatorycytokinesarekeyregulatorsofgeneexpression
andantibacterialdefenseduringLegionellapneumophilainfection.Herewedemonstrate
thatproductionofthesemediatorswaslargelyorpartlydependentonthecyclicGMP-
AMPsynthase(cGAS)-STINGpathwayinhumanandmurinecells.Cellsofindividuals
carryingthecommonHAQalleleofTMEM173/STINGwerestronglyimpairedintheir
abilitytorespondtoL.pneumophila,bacterialDNAorcyclicdinucleotides(CDNs),
whereastheR232HallelewasonlyattenuatedinsensingofexogenousCDNs.Impor-
tantly,cGASandSTINGcontributedtoantibacterialdefenseinmiceduringL.pneumo-
philalunginfection,andtheallelefrequencyofHAQTMEM173/STING,butnotof
R232HTMEM173/STING,wasincreasedintwoindependentcohortsofhumanLegion-
naires’diseasepatientsascomparedtohealthycontrols.Hence,sensingofbacterialDNA
bythecGAS/STINGpathwaycontributestoantibacterialdefenseagainstL.pneumophila
infection,andthehypomorphicvariantHAQTMEM173/STINGisassociatedwith
increasedsusceptibilitytoLegionnaires’diseaseinhumans.
Introduction
Legionellapneumophilaisincreasinglyrecognizedasasignificantcauseofpneumoniain
ambulatoryandhospitalizedpatients.Thisformofpneumonia,commonlyreferredtoas
Legionnaires’disease,isassociatedwithhighmortalityrates,rangingfrom8to34%depending
onthestudy,despiteavailabilityofefficientantibiotictherapies[1].Knownriskfactorsfor
Legionnaires’diseaseincludechronicrespiratoryandcardiovasculardiseases,diabetes,cancer,
andimmunosuppression,althoughindividualswithoutthesepredisposingconditionsarealso
affectedbyLegionnaires’disease[1,2].InfectionoccursfollowinginhalationofL.pneumo-
phila-contaminatedwaterdroplets.Onceinthealveolarcompartment,thebacteriumis
phagocytosedbyalveolarmacrophages,whereitestablishesanintracellularreplicationvacu-
ole.ThisprocessrequirestheDot/IcmtypeIVsecretionsystem(T4SS)whichinjectsapprox.
300bacterialeffectormoleculesintothehostcytosol[3].
TheimmuneresponsetoL.pneumophilainthelungislargelydependentonproductionof
pro-inflammatorycytokinesandinterferons(IFNs)[4–9].WhileIL-1βandTNFαstimulate
antibacterialdefensebye.g.promotingneutrophilrecruitment[10,11],typeIandIIIFNsacti-
vateanIRG1-anditaconicacid-dependentmacrophage-intrinsicresistancepathway[12].
InfectedandbystandermacrophagesarethemainproducersofIL-1β,typeIIFNsandTNFα,
respectively[4,13–15],whereastypeIIIFNisreleasedbyinnateandadaptivelymphoidcells
[16].TheL.pneumophila-inducedtypeIIFNproductionhaspreviouslybeenshowntodepend
ontheT4SSandcytosolicsensingofbacterialDNA[4,8,13],althoughdetectionofthebacterial
cyclicdinucleotides(CDNs)cyclic30-50diguanylate(c-diGMP)hasalsobeenimplicated
inthisresponse[17].Moreover,werecentlyshowedthatinhibitionoftheendoplasmic
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
reticulum-associatedproteinSTING(stimulatorofIFNgenes,alsoknownasMITA,ERIS,
MPYS)reducedtypeIIFNresponsestoL.pneumophilainmurinemacrophages[4].
STINGisencodedbytheTMEM173/STINGgeneandfunctionsasbothasignalingadaptor
inthecytosolicDNAsensingpathway[18,19]andasareceptorforbacterialandendogenous
CDNs[20].UpstreamofSTING,sensingofDNAinthecytosoladditionallyrequiresthecyclic
GMP-AMPsynthase(cGAS),whichbindsmicrobialandhostDNAandmediatesproduction
ofthesecondmessengercyclic2’3’-GMP-AMP(2’3’-cGAMP)[21,22].Recentstudieshave
shownthatproductionoftypeIIFNsduringinfectionswithseveralbacterialpathogens
requiresboth,cGASandSTING[23–26].
Interestingly,humanTMEM173/STINGexhibitssignificantheterogeneity[27,28].Thesec-
ondmostcommonallelebesidestheWTalleleisHAQ,whichcontainsahaplotypecomprised
ofthethreenon-synonymoussinglenucleotidepolymorphisms(SNPs)R71H-G230A-R293Q.
PreviousstudiesindicatedthatHAQSTINGisaloss-of-functionvariantexhibitinglargely
reducedcapacitytostimulatetypeIIFNresponses[27–29].Moreover,thethirdmostcommon
allele,R232H,hasbeenfoundtobedefectiveinsensingbacterialCDNsbutnotof2’3’-
cGAMPorDNA[28,30].However,thefunctionofendogenousHAQandR232HSTINGin
primaryinfectionsandtheeffectofthesevariantsonsusceptibilitytodiseasesinhumanshave
notyetbeenaddressed.
Herewetestedthehypothesesthati)thecGAS/STINGpathwaymediatesdefenseagainstL.
pneumophilainmiceandmen,ii)thatcarriageofHAQTMEM173/STINGimpairstheanti-
bacterialimmuneresponse,andiii)thatcarriageofHAQTMEM173/STINGpredisposesto
Legionnaires’Disease.
Results
L.pneumophilainfectionstimulatestypeIIFNresponsesinvitroandin
vivoviacGASandSTING
GiventhecriticalroleoftypeIIFNsduringL.pneumophilainfection,wefirstinvestigatedthe
roleofSTINGininducingtypeIIFNresponsesinresponsetoL.pneumophilaaswellasLegio-
nellaDNAinmurinebonemarrow-derivedmacrophages(BMDMs).AstrongIfnbinduction
wasobservedinWTbutnotinSTING-deficientcellsinresponsetotwodifferentL.pneumo-
philastrains,bacterialDNAaswellasourcontroltreatmentcGAMP(Fig1Aand1B).The
uptakeofL.pneumophilaintoTmem173-/-BMDMsoritsreplicationwas,however,notsignifi-
cantlydifferentascomparedtoWTcells(S1AFig).Aspreviouslyshown,Legionellalacking
theT4SSeffectorproteinsdhA(ΔsdhA)[31],whichisinvolvedinmaintainingtheintegrityof
theLegionella-containingvacuole,inducedastrongertypeIIFNresponse,whereasamutant
lackinganessentialcomponentoftheT4SS(ΔdotA)wasunabletoactivateSTING-dependent
Ifnbexpression(Fig1B).Moreover,L.pneumophila-inducedexpressionoftheIFN-stimulated
geneIrg1andproductionoftheIFN-stimulatedchemokineIP-10werealsodiminishedin
Tmem173-/-cells(Fig1C,S1BFig).
InordertoexaminetheeffectofcGAS,BMDMswerefirsttransfectedwithasiRNAtarget-
ingcGasoracontrolsiRNA.cGas-specificsiRNAreducedtheexpressionofitstargetgene
(S2AFig),andstronglydiminishedtheinductionofIfnbandtheIFN-inducedgeneIrg1in
responsetoL.pneumophilaorbacterialDNA(S2BandS2CFig).Asexpected,cGAMP-stimu-
latedIfnbexpressionwasnotinhibitedbycGassiRNA,sincecGAMPisthesecondmessenger
produceddownstreamofcGAS[21,22].TofurtherdemonstratetheimportanceofcGAS,
wechallengedmurinecGAS-deficientBMDMswithL.pneumophila,DNAandcGAMP.
Importantly,L.pneumophila-andDNA-induced(butnotcGAMP-stimulated)IfnbandIrg1
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
Fig1.TypeIIFNresponsesduringL.pneumophilainfectionaremediatedbythecGAS/STINGpathway.(A-C)WTandTmem173-/-
mouseBMDMswereleftuntreatedorstimulatedwith1ug/mlL.pneumophilaDNA(JR32DNA)or5ug/ml2´3-cGAMP(A)orwere
infectedwithL.pneumophilaJR32WTand130bWT,ormutantstrainsdeficientfordotAorsdhAatMOI10for6h(B,C).Expressionof
Ifnb(A,B)orIrg1(C)wasmeasuredbyqRT-PCR.(D-G)WTandcGAS-deficientBMDMswerestimulatedwithL.pneumophilaDNAor
2´3-cGAMPorinfectedwithL.pneumophilaJR32WT,andexpressionofIfnbandIrg1wasquantifiedbyqRT-PCR(D-F)orproductionof
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
IP-10wasmeasuredbyELISA(G).(H-N)WT,STING-andcGAS-deficientmicewereintranasallyinfectedwith1×106L.pneumophilaJR32
WTorinstilledwithPBSascontrol(H-J).IfnbandIrg1expressioninthelungswasassessed48(H,I)or144hp.i.(K-N)byqRT-PCR,orIP-
10productionwasmeasuredat48h(J).Dataarerepresentedastherelativequantification(RQ)ofspecifiedmRNAs.Dataareshownasthe
mean+SEMofthreetofourindependentexperiments,measuredintechnicalduplicates(Fig.1A-G)or6to7micepergroup(Fig.H-N).
AnalyseswereperformedthroughtheMann-WhitneyUTest.Comparisonswithap<0.05wereconsideredsignificant.
https://doi.org/10.1371/journal.ppat.1006829.g001
expressionaswellasIP-10productionwerereducedincGAS-deficientBMDMsascompared
toWTcells(Fig1D–1G).
ToinvestigatetherelevanceofthecGAS/STINGpathwayforthetypeIIFNresponsedur-
inglunginfection,weintranasallyinfectedWTandTmem173-/-animalswithL.pneumophila.
WeobservedastronglydecreasedinductionofIfnbandIrg1expressionaswellasIP-10pro-
ductioninthelungsofSTING-deficientmice48hp.i(Fig1H–1J).Similarly,wefoundsignifi-
cantlyreducedexpressionlevelsofIfnbandIrg1inlunghomogenatesfromcGAS-and
STING-deficientmice144hp.i.(Fig1K–1N).Inconclusion,ourresultsshowthatthecGAS/
STINGpathwayislargelyresponsiblefortypeIIFNresponsestoL.pneumophilainfectionin
mice.
Productionofpro-inflammatorycytokinesinresponsetoL.pneumophilais
partlydependentoncGAS/STING
NextweexaminedtheimpactofthecGAS-STINGpathwayontheproductionofotherpro-
inflammatorycytokinesinresponsetoL.pneumophila.DeficiencyofSTINGorcGASsignifi-
cantlyreducedproductionofIL-1βandIL-6andadditionallyshowedsomeminoreffectson
TNFα(Fig2A–2F,S3A–S3CFig).Moreover,STING-deficientanimalsproducedlessIL-1β
andIL-6aswellasIFNγinresponsetoL.pneumophilainfectionofthelung(Fig2G,2Hand
2J),whereastheeffectofSTINGonTNFαproductioninvivowasnotsignificant(Fig2I).
ThesedataindicatethatthecGAS/STINGpathwayalsocontributestotheproductionofpro-
inflammatorymediatorsduringLegionellainfection.
EndogenousHAQSTINGisstronglyimpaired,butnotdeficient,in
mediatingtypeIIFNandpro-inflammatorycytokineresponsesto
LegionellainfectionorstimulationwithDNAandcGAMP
RecentstudiesshowedthatHAQSTINGpoorlyactivatestypeIIFNresponseswhenectopi-
callyexpressedinHEK293cells[27,28].Inordertoexaminetheactivityofendogenous
humanHAQSTING,wescreenedabout564healthyvolunteersforthepresenceofHAQ
TMEM173/STING.Weidentified8individualswhowerehomozygousforHAQ(andR232),
isolatedperipheralbloodmononuclearcells(PBMCs)from4ofthem,culturedthecellsfor7
daystoletthemonocytesdifferentiateintomacrophage-likecells,andcomparedthemwith
cellsfrompersonscarryingWTTMEM173/STING.InlinewithourresultsfromSTING-defi-
cientmurinemacrophages(seeS1Fig),wefoundthatreplicationofL.pneumophilawasnot
differentincellsexpressingWTorHAQSTING(S4Fig).Interestingly,however,weobserved
astrongreductioninIfnbexpressionandinproductionoftheIFN-dependentcytokineIP-10
incellsfromhomozygousHAQTMEM173/STINGcarriersascomparedtocellsfromWT
allelecarriersinresponsetocGAMP,syntheticDNA,Legionellainfection,andbacterialDNA,
butnotfollowingstimulationwiththeTLR7/8agonistResiquimod(R848)(Fig3A,S5Aand
S6Figs).HAQPBMCswerealsopartlydefectiveinproducingpro-inflammatorycytokines
suchasIL-1β,IL-6andTNFα(Fig3B–3D,S5B–S5DFig).Moreover,heterozygouscarriageof
HAQTMEM173/STINGalsoleadtoapartialreductionoftypeIIFNandIL-1βexpression,
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
Fig2.ThecGAS/STINGaxiscontributestotheproductionofpro-inflammatorycytokinesduringL.pneumophilainfection.(A-F)WT,
Tmem173-/-andcGas-/-BMDMswereinfectedfor6hwithL.pneumophilaWTatMOI10andrelativecytokineexpressionwasdeterminedby
qRT-PCR.(G-J)CytokineproteinconcentrationsinwholelunghomogenatesfromL.pneumophila-infectedmicewerequantifiedbysandwich
ELISA.Dataareshownasmean±SEM.(A-F)Datarepresentativeof3to4independentexperimentscarriedoutinduplicates.(G-J)Data
representativeof6o7micepergroup.DatawereanalyzedthroughtheMann-WhitneyUTest.Comparisonswithap<0.05wereconsidered
significant.
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
Fig3.EndogenousHAQSTINGisstronglyimpairedinmountingatypeIIFNandproinflammatorycytokineresponsesagainstLegionellainfection
orstimulationwithDNAorCDNs.(A-D)PBMCsfromhealthyvolunteers(N=4forWTandN=4forHAQ)wereisolatedbydensitygradient
centrifugation.7dafterisolationcellswereinfectedfor6hwithL.pneumophilaatMOIs10and50orstimulatedforthesameperiodwith1and5ug/ml
2´-3´cGAMPoreitherbacterialorsyntheticDNAataconcentrationof0.2or1ug/ml.RNAwasisolatedandtheexpressionofIFNB(A),IL1B(B),IL6(C)
andTNFA(D)wasdeterminedbyqRT-PCR.DataareshownastheRQofspecifiedmRNAs.Datarepresentthemean±SEMof4independent
experimentscarriedoutintriplicates.DifferenceswereassessedwiththeMann-WhitneyUTest.Comparisonswithap<0.05wereconsideredsignificant.
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
whichhoweveronlyreachedstatisticalsignificanceforcGAMP-andL.pneumophila-induced
IFNBinduction(S7Fig).
HomozygousHAQPBMCswerestronglyimpairedbutnotbluntedininducingtypeI
IFNresponsestoL.pneumophilainfectionorstimulationwithDNAorcGAMP(Fig3A,
S5AFig),suggestingthatHAQSTINGpossesseslargelyreducedbutnotbluntedactivity.In
linewiththissuggestion,THP-1cells,whichhavepreviouslybeenshowntoexpressHAQ
STING[30,32]andwhichweconfirmedtocarrytheHAQTMEM173/STINGalleleinhomo-
zygosity,respondedonlyweaklytoDNA,cGAMPandL.pneumophilastimulation(2-
10-foldincreaseinIfnbexpression,seeFig4).ThesetypeIIFNresponsesinTHP-1cells
wereconsiderablylowerascomparedtoPBMCsexpressingWTSTING(100-1000-foldIfnb
induction,seeFig3A).Interestingly,however,deletionofcGASexpressionbyCRISPR/
Cas9-mediatedgenomeediting[33]furtherdecreasedthetypeIIFNresponsesinTHP-1
cells(Fig4).Takentogether,ourdatademonstratethatendogenousHAQSTINGisahypo-
morphicvariantthatisstronglyimpaired(butnotdeficient)inmediatingtypeIIFNand
pro-inflammatorycytokineresponsestocGAMP,syntheticDNA,bacterialDNAandLegio-
nellainfection.
EndogenousR232HSTINGispartlydefectiveinsensingbacterialCDNs
butfullyfunctionalinmediatingresponsestoDNA,cGAMPandL.
pneumophilainfection
HEK293cellsexpressingamutatedmurineSTINGwithanalanineinsteadofarginine231
(R231A)respondnormallytoDNAbutnottobacterialCDNs[20].Thecorresponding
humanR232HalleleisthethirdmostcommonTMEM173/STINGallele[29],andhasalso
beenshowntobedefectiveinsensingbacterialCDNswhenoverexpressed[28,30].Inorderto
examinethefunctionofendogenousR232HSTINGandtherelevanceofc-diGMPsensingfor
hostresponsestoL.pneumophila,wescreenedhealthyvolunteersforcarriageofthisallele,iso-
latedcellsfrom3individualsharboringtheR232Halleleinhomozygosity,andcompared
themwithcellsexpressingWTSTING.Inagreementwithpreviousstudies,wefoundthat
humancellsexpressingR232HSTINGwerepartlyimpairedinsensingcGMPandRp,Rp-c-
diAMPSS(aRp,Rp-isomerofthedi-thiophosphateanalogueofthebacterialsecondmessenger
c-diAMP)(Fig5A–5D,S8Fig).Incontrast,expressionoftheR232HalleledidnotaffecttypeI
IFNorpro-inflammatorycytokineresponsestoL.pneumophilainfectionorDNAorcGAMP
stimulation.Moreover,theR232HSNPdidnotaffectreplicationofL.pneumophilainhuman
cells(S9Fig).ThesedataindicatethatendogenoushumanR232HSTINGispartlydefectivein
sensingbacterialCDNsandthatrecognitionofc-diGMPisnotcriticallyinvolvedinhuman
cellinteractionswithL.pneumophila.
cGASandSTINGcontributetoanti-bacterialhostdefenseagainst
Legionellainfectioninmice
Next,weinvestigatedtherelevanceofthecGAS/STING-dependentpathwayforantibacterial
defenseinvivo.cGAS-andSTING-deficientmiceaswellasWTcontrolswereintranasally
infectedwithL.pneumophila.6daysafterinfection,weobservedenhanced(2–3fold)bacterial
loadsinthelungsofcGas-/-andTmem173-/-miceascomparedtoWTcontrols(Fig6),demon-
stratingthatthecGAS/STINGpathwaycontributestoantibacterialdefenseagainstL.pneumo-
philainvivo.
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
Fig4.L.pneumophilainfectionandstimulationwithDNAorcGAMPinduceweakcGAS-dependenttypeIIFNresponsesinTHP-1cells.WT
THP-1orcGAS-/-THP-1clonesA5andB5werealloweddifferentiationpriortostimulationwitheithercGAMPorsyntheticDNA(A)orinfection
withtwodifferentstrainsofL.pneumophila(B).IFNBexpressionwasdeterminedbyqRT-PCR.Datarepresentmean±SEMof2independent
experimentscarriedoutinduplicates.AnalyseswereperformedbyemployingtheMann-WhitneyUTest.Comparisonswithap<0.05were
consideredsignificant.
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CarriageofHAQTMEM173/STINGmightpredisposeindividualsto
infection
Finally,wetestedforapotentialassociationbetweenHAQandR232HTMEM173/STINGcar-
riagesandsusceptibilitytowardsL.pneumophilainfection.Inanexploratoryanalysis,allele
frequenciesandgenotypeswerecomparedbetween59Legionnaires´diseasepatientsand100
healthycontrolsofsimilarageandsexdistribution.ThefrequencyofHAQTMEM173/STING
(butnotR232HTMEM173/STING)wassignificantlyincreasedamongcases(0.18)ascom-
paredtocontrols(0.075)(Table1);anunadjustedanalysisshowedthatcarriageofthehaplo-
typealmosttripledtheoddsofbeingalegionellosispatientinthiscohort(p=0.028;OR2.69;
95%CI,1.16–6.27).TheHAQhaplotyperemainedassociatedwiththediseasewhentheanaly-
siswasperformedwithanadjustmentforageandgender(p=0.01;OR2.70,95%CI1.24–
5.86;logisticregressionwithdominantgeneticmodel).
Tovalidatethesefindings,weexaminedanothercasecontrolcohort(N=91Legionnaires´
diseasepatientsand88controls)fromaflowershowoutbreakintheNetherlandsin1999that
hasbeendescribedindetailpreviously(S1Table)[34–37].TheHAQhaplotypewaspresentin
23casesand12controlsandassociatedwithincreasedsusceptibilitytoLegionnaires´disease
inanunadjustedanalysis(OR2.24,95%CI1.03–5.31;logisticregressionwithdominant
geneticmodel).Inananalysisadjustedforageandgender,theHAQhaplotyperemainedasso-
ciatedwithLegionnaires´disease(p=0.013;OR2.29,95%CI1.04–5.24)(Table2).Incontrast,
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HAQSTINGandsusceptibilitytoLegionellapneumophilainfection
Fig5.EndogenousR232HSTINGispartlydeficientinsensingbacterialCDNbutrespondsnormallytoLegionellainfectionorstimulationwith
DNA.(A-D)PBMCsfromhealthyvolunteers(N=3forWTandN=3forR232H)wereisolatedbydensitygradientcentrifugation.7dafterisolation
cellswereinfectedfor6hwithL.pneumophilaatMOI10orstimulatedforthesameperiodwith1ug/ml2´-3´cGAMP,Rp-c-diAMPSS,cGMPoreither
bacterialDNAataconcentrationof1ug/ml.RNAwasisolatedandtheexpressionofIFNB(A),IL1B(B),andIL6(C)andTNFA(D)wasdeterminedby
qRT-PCR.DataareshownastheRQofspecifiedmRNAs.Datarepresentthemean±SEMof3independentexperimentscarriedoutintriplicates.
DifferenceswereassessedwiththeMann-WhitneyUTest.Comparisonswithap<0.05wereconsideredsignificant.
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