Induction of IFN-beta and the innate antiviral response in myeloid cells occurs through an IPS-1

18 Pages·2017·0.99 MB·English

WWaasshhiinnggttoonn UUnniivveerrssiittyy SScchhooooll ooff MMeeddiicciinnee
DDiiggiittaall CCoommmmoonnss@@BBeecckkeerr
Open Access Publications
2009
IInndduuccttiioonn ooff IIFFNN--bbeettaa aanndd tthhee iinnnnaattee aannttiivviirraall rreessppoonnssee iinn mmyyeellooiidd
cceellllss ooccccuurrss tthhrroouugghh aann IIPPSS--11--ddeeppeennddeenntt ssiiggnnaall tthhaatt ddooeess nnoott
rreeqquuiirree IIRRFF--33 aanndd IIRRFF--77
Stephane Daffis
Washington University School of Medicine in St. Louis
Mehul S. Suthar
University of Washington - Seattle Campus
Kristy J. Szretter
Washington University School of Medicine in St. Louis
Michael Gale Jr.
University of Washington - Seattle Campus
Michael S. Diamond
Washington University School of Medicine in St. Louis
Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs
Part of the Medicine and Health Sciences Commons
RReeccoommmmeennddeedd CCiittaattiioonn
Daffis, Stephane; Suthar, Mehul S.; Szretter, Kristy J.; Gale, Michael Jr.; and Diamond, Michael S.,
,"Induction of IFN-beta and the innate antiviral response in myeloid cells occurs through an
IPS-1-dependent signal that does not require IRF-3 and IRF-7." PLoS Pathogens. 5,10. e1000607. (2009).
https://digitalcommons.wustl.edu/open_access_pubs/754
This Open Access Publication is brought to you for free and open access by Digital Commons@Becker. It has been
accepted for inclusion in Open Access Publications by an authorized administrator of Digital Commons@Becker.
For more information, please contact [email protected].
Induction of IFN-b and the Innate Antiviral Response in
Myeloid Cells Occurs through an IPS-1-Dependent Signal
That Does Not Require IRF-3 and IRF-7
Stephane Daffis1, Mehul S. Suthar2, Kristy J. Szretter1, Michael Gale, Jr.2, Michael S. Diamond1,3,4*
1Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, United States of America, 2Department of Immunology, University of
WashingtonSchoolofMedicine,Seattle,Washington,UnitedStatesofAmerica,3DepartmentofMolecularMicrobiology,WashingtonUniversitySchoolofMedicine,St.
Louis,Missouri,UnitedStatesofAmerica,4DepartmentofPathology&Immunology,WashingtonUniversitySchoolofMedicine,St.Louis,Missouri,UnitedStatesof
America
Abstract
Interferon regulatory factors (IRF)-3 and IRF-7 are master transcriptional factors that regulate type I IFN gene (IFN-a/b)
inductionandinnateimmunedefensesaftervirusinfection.PriorstudiesinmicewithsingledeletionsoftheIRF-3orIRF-7
genes showed increased vulnerability to West Nile virus (WNV) infection. Whereas mice and cells lacking IRF-7 showed
reduced IFN-a levels after WNV infection, those lacking IRF-3 or IRF-7 had relatively normal IFN-b production. Here, we
generatedIRF-32/26IRF-72/2doubleknockout(DKO)mice,analyzedWNVpathogenesis,IFNresponses,andsignalingof
innatedefenses.Comparedtowildtypemice,theDKOmiceexhibitedabluntedbutnotabrogatedsystemicIFNresponse
and sustaineduncontrolledWNV replication leadingto rapidmortality. Exvivo analysis showed completeablation ofthe
IFN-aresponseinDKOfibroblasts,macrophages,dendriticcells,andcorticalneuronsandasubstantialdecreaseoftheIFN-b
response in DKO fibroblasts and cortical neurons. In contrast, the IFN-b response was minimally diminished in DKO
macrophages and dendritic cells. However, pharmacological inhibition of NF-kB and ATF-2/c-Jun, the two other known
components of the IFN-b enhanceosome, strongly reduced IFN-b gene transcription in the DKO dendritic cells. Finally, a
geneticdeficiencyofIPS-1,anadaptorinvolvedinRIG-I-andMDA5-mediatedantiviralsignaling,completelyabolishedthe
IFN-b response after WNV infection. Overall, our experiments suggest that, unlike fibroblasts and cortical neurons, IFN-b
generegulationafterWNVinfectioninmyeloidcellsisIPS-1-dependent butdoesnotrequirefulloccupancyoftheIFN-b
enhanceosomeby canonicalconstituent transcriptional factors.
Citation:DaffisS,SutharMS,SzretterKJ,GaleM,Jr.,DiamondMS(2009)InductionofIFN-bandtheInnateAntiviralResponseinMyeloidCellsOccursthroughan
IPS-1-DependentSignalThatDoesNotRequireIRF-3andIRF-7.PLoSPathog5(10):e1000607.doi:10.1371/journal.ppat.1000607
Editor:GanesSen,ClevelandClinic,UnitedStatesofAmerica
ReceivedMarch11,2009;AcceptedSeptember8,2009;PublishedOctober2,2009
Copyright: (cid:1) 2009 Daffis et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisstudywassupportedbyNIHgrantsAI057568,U54AI081680,U19AI083019,andAI074973.Thefundershadnoroleinstudydesign,datacollection
andanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
Introduction downstream of specific PRR (e.g., TLR3, MDA5, and RIG-I) to
inhibit RNA viruses, IRF-7 is both an ISG and a transcriptional
The rapid production of type I interferon (IFN-a/b) and the activator downstream of distinct PRR (e.g., TLR7 and TLR8);
IFN-induced antiviral response serve as primary host defense IRF-7participatesinanIFNamplificationloopbyinducingIFN-b
mechanismsagainstinfectionbymanyviruses(reviewedin[1–3]). andmanysubtypes of IFN-a[14].
IFN-a/b gene transcription is induced after host pattern Recognition of West Nile virus (WNV), a neurotropic virus of
recognition receptors (PRR) bind pathogen-associated molecular the Flaviviridae family of RNA viruses, by the intrinsic cellular
patterns(PAMP),suchasviralnucleicacids(reviewedin[4–6]).A immuneresponseisbelievedtooccurthroughconcertedsignalsby
current paradigm for type I IFN production after RNA virus several PRR (TLR3, TLR7, TLR8, RIG-I, and MDA5) that
infection describes a two-step or positive feedback model that is recognizesingleordouble-strandedRNAandsignalthroughtheir
modulated by the master transcription factors interferon regula- constituentadaptormolecules(TRIF,MyD88,andIPS-1).Tissue
toryfactors(IRF)-3and-7[7–9].Intheinitialphase,viralsensing culture experiments with murine embryonic fibroblasts (MEF)
by PRR induces nuclear localization of IRF-3, which stimulates suggested that RIG-I is an initial and primary PRR for WNV as
gene transcription and production of IFN-b and IFN-a4 by geneticallydeficientcellshadanabrogatedISGresponseatearly
infectedcells.Inthesecondphase,theseIFNsbindtoacommon time points after infection [15]. The late ISG response appears
IFN-a/breceptorinaparacrineandautocrinemannerandsignal moredependentonMDA5,suggestingadualrequirementofboth
through the JAK-STAT pathway resulting in the induced RIG-I and MDA5 for activation of an effective cellular antiviral
expression of hundreds of interferon stimulated genes (ISG) (e.g., response against WNV, at least in MEF [16]. In contrast, a
PKR, RNAse L, viperin, ISG15, and ISG20), which limit viral deficiencyofTLR3inMEF,macrophages,anddendriticcellsdid
replicationthroughmultiplemechanisms[10–13].WhereasIRF-3 not alter IFN-a/b production or viral burden, indicating a more
is constitutively expressed throughout many tissues and functions limitedroleofthissensorinrecognizingWNVinthesecelltypes
PLoSPathogens | www.plospathogens.org 1 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
experiments with myeloid dendritic cells (mDC) ex vivo, the
Author Summary
IFN-bresponseafterWNVinfectionwasabolishedbytheabsence
WestNilevirus(WNV)isamosquito-transmittedvirusthat of IPS-1 but was largely unaffected by a combined deficiency or
infects birds, horses, and humans and has become an the transcription factors IRF-3 and IRF-7 or individual deficien-
emerging infectious disease threat in the Western cies of IRF-1, IRF-5, or IRF-8. However, pharmacological
Hemisphere. In humans, WNV can invade into the brain inhibition of signaling by both NF-kB and ATF-2/c-Jun, the
and spinal cord and destroy neurons, causing severe two other factors, beside IRF-3 and IRF-7, that form the IFN-b
neurological disease, particularly in the immunocompro- gene transcriptional complex enhanceosome [23–25], strongly
mised and elderly. A better understanding of how the reducedtheIFN-bresponseinDKObutnotwildtypemDCafter
immune system controls WNV infection is critical for
WNV infection. These studies define cell type-specific molecular
developing new treatments and vaccines. In this study,
mechanisms and roles for IRFs in antiviral defense, and reveal a
using a mouse model of WNV infection, we evaluate the
differential requirement for components of the enhanceosome in
combinedroleoftwokeytranscriptionfactors,interferon-
inducingtheIFN-bgeneinresponse toRNAviruses.
regulatory factor-3 (IRF-3) and IRF-7, that orchestrate
antiviral and interferon (IFN) responses after infection.
MicethatlackbothIRF-3andIRF-7werehighlyvulnerable Results
to lethal infection and cells lacking IRF-3 and IRF-7 had a
IncreasedlethalityofDKOmiceafterinfectionwithWNV
markedly attenuated IFN-a response. Surprisingly, macro-
phagesanddendriticcellslackingIRF-3andIRF-7showed Because of the possible functional redundancy of IRF-3 and
arelativelynormalIFN-bresponse.Furthermore,agenetic IRF-7,andtofullyevaluatetheirnetcontributiontotheregulation
deficiency of IPS-1, a protein that signals downstream of of the IFN-b transcriptional response after WNV infection, we
the RIG-I- and MDA5 cytoplasmic viral RNA sensors, generated IRF-32/26 IRF-72/2 (DKO) mice. These animals
completely abolished IFN-b production. Our experiments wereinfectedsubcutaneouslywith102PFUofahighlypathogenic
suggestthatinspecificcelltypesinfectedwithWNV,IFN-b New York strain of WNV. Whereas wild type C57BL/6 mice
can be induced through an IPS-1-dependent transcrip- exhibiteda,60%survivalrate,congenicDKOmicedisplayeda
tional signal that does not require the master transcrip- severephenotypewith100%mortalityandameantimetodeath
tional regulatorsIRF-3 andIRF-7. of6.0days(Fig1A).Incomparison,IRF-32/2orIRF-72/2single
knockout mice infected with the same dose of WNV also had a
[17]. To date, no experiments have been published on thedirect 100%mortalityratebutwithmeantimetodeathsof9.3and7.4
role of TLR7 and TLR8 in the priming of IFN responses after days, respectively [21,22].
WNVinfectionincells.Arecentstudydemonstratednodecrease As IRF-3 and IRF-7 are key regulators of the type I IFN
in systemic production of type I IFN in TLR72/2 mice infected responsetoviralinfection,wealsocomparedtheDKOphenotype
withWNV[18].However,investigationswithotherRNAviruses with congenic IFN-abR2/2 mice. Similar to what we previously
suggest that the TLR7/MyD88/IRF-7 axis regulates type I IFN observed [13,26], IFN-abR2/2 mice were vulnerable to WNV
responses inspecific subsets ofdendritic cells [19,20]. infectionwith100%mortalityby4daysandameantimetodeath
Studies with genetically deficient mice have established an of3.5days(Fig1A,P,0.001ofaveragesurvival timecompared
importantroleofIRF-3inprotectionagainstlethalWNVinfection to DKO mice). Thus, although a combined deficiency of IRF-3
bycontrollingviralburdeninperipheralandcentralnervoussystem andIRF-7hasamoreseverephenotypeafterWNVinfectionthan
(CNS) tissues without affecting the systemic type I IFN response theirrespectivesingledeficiencies,thesurvivalpatterndidnotfully
[21]. Experiments performed with primary cells established that recapitulatethatobservedinIFN-abR2/2mice,suggestingtypeI
IRF-3 restricts WNV replication in cortical neurons and MEF IFN induction and/or regulation after WNV infection may
throughtypeIIFN-dependentmechanismswhereasinmyeloidcells require additional transcriptional regulators.
IRF-3 limits WNV infection independently of IFN by regulating
basalorWNV-inducedexpressionofhostdefensemolecules[21]. DKO mice show uncontrolled WNV replication in tissues
MorerecentstudiesdemonstratedapivotalroleofIRF-7invivoin TomorecompletelyevaluatetheimpactoftheIRF-3andIRF-
controlling WNV infection [22]. IRF-72/2 mice developed 7 deficiency on WNV pathogenesis in vivo, wild type and DKO
markedly elevated WNV burdens in multiple tissues and had a mice were infected subcutaneously with 102 PFU of WNV and
blunted systemic type I IFN response. Studies with primary cells viral burden was measured by fluorogenic quantitative RT-PCR
showedthatIRF-7controlsWNVinfectionlargelythroughanIFN- or viral plaque assay at days 1, 2, 3, 4, 5, 6 and 8 in blood,
a-dependent mechanism. Surprisingly, the IFN-b response re- peripheral organs (draining lymph nodes,spleen andkidney) and
mained intact suggesting possible redundant effects of IRF-3 and theCNS tissues (brainandspinal cord)(Fig1B–G).
IRF-7inregulatingIFN-bgeneexpression.Theseresultsagreewith Elevated viremia was observed in DKO mice when compared
anindependentstudyinwhichIRF-72/2MEFinfectedwithherpes towildtypemice(Fig1B).Byonedayafterinfection,,101.5-fold
simplex(HSV),vesicularstomatitis(VSV),orencephalomyocarditis higherlevelsofviralRNA(P,0.0001)weredetectedintheDKO
(EMCV)virusshowedresidualIFN-bresponsesthatwereabolished mice.Markedlyenhanced(104.6to106.2-foldincrease,P,0.0001)
inIRF-32/26IRF-72/2MEF[14]. WNVRNAlevelswereobservedinserumofDKOmiceondays2
TounderstandthecombinedrolesofIRF-3andIRF-7ininnate through 5,after whichmostanimals succumbed toinfection.
immuneprogramsfollowingWNVinfectioninvivo,wegenerated Inthedraininglymphnodes,similarlyhighlevels(102.8to105.0-
IRF-32/26IRF-72/2doubleknockout(DKO)mice.Anabsence fold increase, P,0.0001) of WNV RNA were observed in the
ofbothIRF-3andIRF-7ledtouncontrolledWNVreplicationin DKOmicethroughoutinfection(Fig1C).Inthespleen,infectious
tissues and more rapid death than either of the single gene WNVwasnotdetectedinwildtypemiceuntilday3.Incontrast,
deletions. Although severe, the DKO phenotype did not allDKOmice(10of10)hadmarkedlyelevatedviraltitersbyday
recapitulatethatobservedincongenicIFN-abR2/2mice.Despite 2(meantiterof107.7PFU/g)(Fig1D).AlthoughWNVinfection
a complete absence of both IRF-3 and IRF-7, IFN-b induction gradually increased in the spleens of wild type mice at days 3
remained largely intact in some cell types. Remarkably, in through 5, significantly higher (104.5 to 104.9-fold) viral burdens
PLoSPathogens | www.plospathogens.org 2 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
Figure1.Survivalandviralburdenanalysisinmice.A.Eightto12week-oldC57BL/6micewereinoculatedwith102PFUofWNVbyfootpad
injection and followed for mortality for 21 days. Survival differences were statistically significant between immunodeficient and wild type mice
(n=11,IFN-abR2/2;n=20,DKO;andn=20,wildtypemice,P,0.0001).AveragesurvivaltimebetweenIFN-abR2/2(3.5days)andDKO(6days)mice
wasalsostatisticallydifferent(P,0.001).B–G.ViralburdeninperipheralandCNStissuesafterWNVinfection.WNVRNAin(B)serumand(C)draining
lymphnode,andinfectiousvirusin(D)spleen,(E)kidney,(F)brainand(G)spinalcordweredeterminedfromsamplesharvestedontheindicated
daysusingqRT-PCR(BandC)orviralplaqueassay(D–G).DataisshownasviralRNAequivalentsorPFUpergramoftissuefor10to12micepertime
point.Forallviralloaddata,thesolidlinerepresentsthemedianPFUpergramattheindicatedtimepoint,andthedottedlinerepresentsthelimitof
sensitivityoftheassay.
doi:10.1371/journal.ppat.1000607.g001
were observed in DKO mice. Altered tissue tropism was also ments including spread toandpropagation withinnormally non-
observed in DKO mice with significant infection of the kidneys permissive organs.
(e.g. 105 PFU/g, by day 2 and 107.5 PFU/g, by day 5, Fig 1E), Analysisofviralburdeninthebrainandspinalcordshoweda
whereas wild type mice showed no productive infection of the rapid entry of WNV into the CNS of DKO mice. WNV was
kidneys.Thus,acombineddeficiencyofIRF-3andIRF-7results detectedinallbrainsfromDKOmiceatday2comparedtowild
in sustained and elevated WNV infection in peripheral compart- type mice, where WNV was not observed until day 5 (Fig 1F).
PLoSPathogens | www.plospathogens.org 3 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
Notably,atday5,DKOmicehad,105.2-foldhigherviraltiters IRF-3 and IRF-7 are required for the type I IFN and ISG
in the brain compared to wild type mice. A similar pattern was response in MEF after WNV infection
observedinthespinalcordofDKOmicewithallanimalsshowing
To better understand the net effect of IRF-3 and IRF-7 on
markedlyelevatedviralloadsafterday2(Fig1G).Incontrast,in
WNV infection and induction of a protective IFN response, we
wildtypemice,infectiousWNVwasnotdetectedinthemajority
infected primary cells from DKO and wild type mice. Because
ofanimalsuntilday8afterinfection.Thus,acombinedabsenceof
MEFhavebeenstudiedextensivelyinvirusinfection-hostimmune
IRF-3 and IRF-7 results inearly neuroinvasionand uncontrolled
response assays [16,29,30], we initially evaluated the effect of an
WNV replication. IRF-36IRF-7deficiencyinthesecells.Previousexperimentshad
shownabluntedIFN-aresponseandanormalIFN-bresponsein
ThesystemictypeIIFNresponseinDKOmiceisblunted IRF-72/2 MEF infected with WNV [22]. In contrast, IRF-32/2
but not abolished MEFhadadiminishedIFN-aand-bresponseearlyafterinfection
PreviousstudieshavereportedthatanabsenceofIRF-3invivo butdevelopednormallevelsatlatertimepoints(S.DaffisandM.
doesnotprofoundlyreducethelevelsoftypeIIFNinserumafter Diamond, unpublished data). In DKO MEF, IFN-a mRNA and
WNV[21,27]orotherviralinfections[14].Incontrast,IRF-72/2 protein secretion were completely abolished after WNV infection
mice infected with WNV had a reduced but not abrogated (Fig3Aand3B).Similarly,levelsofIFN-bmRNAwerestrongly
systemicIFNresponse[22].WethereforeusedtheDKOmiceto reduced at 24h (,70-fold decrease, P,0.0001) and 48h after
definewhethersignalingthroughIRF-3couldexplaintheresidual infection (,130-fold decrease, P,0.0001) but not entirely
systemic type I IFN response after WNV infection in IRF-72/2 abolished (Fig 3C). Measurement of secreted IFN-b in the cell
mice.UsingahighlysensitiveL929cellbioassay,wecomparedthe supernatants corroborated these findings as a ,4 and 20-fold
serum IFN levels in DKO and wild type mice after WNV reduction (P,0.0001) was observed in DKO MEF at 24 and
infection.Asobservedpreviously[21,22],inwildtypemicetypeI 48hours,respectively(Fig3D).Thus,normalinductionofIFN-a
IFNwasdetectedinserumbyday1withpeaklevelsmeasuredat and IFN-b in MEF after WNV infection primarily requires
days3and4afterinfection(Fig2).Incontrast,intheDKOmice, transcriptional activation byIRF-3 andIRF-7.
serumIFNlevelsatday1werebelowthedetectionlevel(0.01 IU/ We next assessed the expression pattern of selected ISG
ml) of the assay. Despite the higher viremia, serum IFN levels in including ISG49, ISG54 and RIG-I by Western blot analysis. In
theDKOmicewerereducedatdays2,3and4whencomparedto wild type MEF, these proteins become rapidly induced at 24h
wild type animals (,5 to 14-fold lower, P,0.005), but were not after infection and were sustained at 48h. In contrast, induction
abolished. This antiviral activity in the serum of infected DKO was absent in DKO MEF at 24h after infection and levels were
mice was confirmed as type I IFN-dependent by depletion comparably reduced at 48h (Fig 3E). To assess how this
experimentswithaneutralizingmAbagainsttheIFN-a/bR(data phenotype affected WNV replication, multi-step viral growth
not shown). Thus, a combined deficiency of IRF-3 and IRF-7 in analysis was performed (Fig 3F). Although no difference in viral
vivo delays and diminishes the accumulation of type I IFN in titers was observed at 24hours, enhanced WNV replication was
serum.However,overtime,typeIIFNaccumulatesintheserum seen at 48h (13-fold, P=0.004) and 72h (58-fold, P,0.0001) in
of DKO mice after WNV infection. Thus, additional regulatory DKOMEF.Moreover,thelevelsofWNVat48and72hinthe
factors must contribute to the systemic IFN response. Notably, DKOMEFweregreaterthanthosepreviouslyobservedwiththe
these results agree with a recent study in which independently- single IRF-32/2 or IRF-72/2 MEF (S. Daffis and M. Diamond,
generated DKO mice infected with mouse cytomegalovirus unpublished results and [22]). As IRF-3 and IRF-7 appear to
(MCMV) showed a residual systemic IFN response that was primarily regulate the IFN and ISG responses in MEF, we
entirely specific forIFN-b[28]. predictedthattheWNVreplicationphenotypeintheDKOMEF
should not differ substantially fromcongenic IFN-abR2/2MEF.
Indeed, when directly compared, only small differences in viral
growthwereobservedbetweenDKOandIFN-abR2/2MEF(2.7
fold, P,0.03 at 48h and2.3 fold,P,0.002at 72h) (Fig3F).
IRF-3 and IRF-7 regulate the type I IFN and ISG response
in cortical neurons after WNV infection
ToevaluatethecombinedrolesofIRF-3andIRF-7incontrol
of viral replication and the IFN response in neuronal cells, we
assessedWNVinfectionofprimarycorticalneuronsisolatedfrom
DKOmice.AnalysisofviralgrowthkineticsconfirmedthatIRF-3
and IRF-7 restrict WNV replication as a ,5 to 6-fold increase
(P,0.0001)inviraltiterwasobservedat24hand48hcompared
to wild type cells (Fig 4A). Somewhat surprisingly, the DKO
neuronsdidnotshowincreasedreplicationrelativetocellslacking
Figure2.LevelsoftypeIIFNlevelsinserumofwildtypeand
eitherIRF-3orIRF-7(datanotshownand[21,22]).Therelatively
DKOmiceinfectedwithWNV.Micewereinoculatedwith102PFUof
WNVbyfootpadinjectionandsacrificedattheindicatedtimes.TypeI modestreplicationphenotypeintheabsenceofIRF-3andIRF-7
IFNlevelsweredeterminedfromserumcollectedondays1to4after is consistent with only a small IFN-dependent antiviral effect in
WNVinfectionbyanEMCVbioassayinL929cells.Datareflectaverages these cells: IFN-a or -b pre-treatment inhibits WNV infection in
ofserumsamplesfrom5to10micepertimepointandthedataare cortical neurons a maximum of 5 to 8-fold [13]. Analysis of the
expressedasinternationalunits(IU)ofIFN-aperml.Thespecificityof
IFNresponseofWNV-infectedDKOneuronsshowedacomplete
the assay was confirmed with an anti-IFN-abR neutralizing antibody
ablation of the IFN-a gene induction confirming results with the
(data not shown). Asterisks indicate values that are statistically
significant(**,P,0.005,*,P,0.05). IRF-72/2 corticalneurons[22](Fig4B).Incontrast,andunlike
doi:10.1371/journal.ppat.1000607.g002 that observed with MEF or other primary myeloid cells (see
PLoSPathogens | www.plospathogens.org 4 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
Figure3.IRF-3andIRF-7controltheIFN-a/bgeneinductionandISGexpressioninMEF.A–D.MEFgeneratedfromwildtypeorDKOmice
wereinfectedatanMOIof0.1andanalyzedforIFN-a/bgeneinduction.TotalRNAfromuninfectedandWNV-infectedMEFwasharvestedatthe
indicatedtimesafterinfectionandlevelsof(A)IFN-aand(C)IFN-bmRNAweremeasuredbyqRT-PCR.Dataarenormalizedto18SrRNAandare
expressed as the relative fold increase over normalized RNA from uninfected controls. Accumulation of (B) IFN-a and (D) IFN-b protein in
supernatantswasevaluatedbyELISA.E.WholecelllysatesweregeneratedattheindicatedtimesfromwildtypeorDKOMEFthatwereuninfected
(Un)orinfectedwithWNV(W).ProteinlevelsofISG49,ISG54,RIG-I,WNVandtubulinwereexaminedbyimmunoblotanalysis.F.MEFgeneratedfrom
wildtype,IFN-abR2/2andDKOmicewereinfectedatanMOIof0.001andvirusproductionwasevaluatedbyplaqueassay.Thedataistheaverageof
atleastthreeindependentexperimentsperformedinquadruplicate,(***,P,0.0001).
doi:10.1371/journal.ppat.1000607.g003
below),inductionofIFN-bmRNAinDKOcorticalneuronswas increased WNV replication (,35, 250 and 310-fold, P,0.0001
also entirely abolished (Fig 4C). Consistent with this, analysis of at24,48and72hafterinfection, respectively)comparedtowild
ISG in DKO neurons showed a complete loss of induction of typeorevenIRF-32/2orIRF-72/2singlydeficientcells(Fig5A
ISG54, RIG-I and MDA5 following WNV infection (Fig 4D). and [21,22]). In contrast to that observed with MEF (see Fig 3),
Thus, in cortical neurons coordinate signals through the the DKO Mw produced ,14-fold less (P,0.05) WNV at 72h
transcriptional regulators IRF-3 and IRF-7 are required for compared to IFN-abR2/2 Mw infected in parallel. Thus, an
IFN-aandIFN-bgeneinduction afterWNV infection. absenceofbothIRF-3andIRF-7inMwdidnotrecapitulatethe
replicationphenotypeofIFN-abR2/2cells.Assuchadiscrepancy
IFN-bresponseinMwafterWNVinfectionispartiallyIRF- could be related to differences in the type I IFN and/or ISG
3 and IRF-7-dependent response,weanalyzedtheseinDKOMwafterWNVinfection.As
As previous studies suggested cell type-specific differences in expected, levels of IFN-a mRNA were completely abolished
typeIIFNinduction[21],weevaluatedtheeffectofthecombined (Fig 5B), consistent with results from IRF-72/2 Mw [22].
IRF-3andIRF-7deficiencyinmacrophages(Mw),acelltypethat WhereasIFN-bmRNAlevelswerereducedat24hafterinfection
ispermissive toWNV invivo [26].Priorexperiments established inDKOMw,theyaccumulatedtonormallevelsby48h(Fig5C).
that Mw lacking either IRF-3 or IRF-7 were more susceptible to WesternblotanalysisofISGexpressionconfirmedthispattern,as
WNV infection [21,22]. Analogously, DKO Mw supported theearlyinductionofseveralISGwasalteredat24hbutnot48h
PLoSPathogens | www.plospathogens.org 5 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
Figure4.IRF-3andIRF-7restrictWNVinfectionbyregulatingtheIFN-a/bresponseandISGexpressioninprimarycorticalneurons.
A.PrimarycorticalneuronsgeneratedfromwildtypeorDKOmicewereinfectedatanMOIof0.001andvirusproductionwasevaluatedatthe
indicatedtimesbyplaqueassay.Valuesareanaverageoftriplicatesamplesgeneratedfromthreeindependentexperiments.Asterisksindicatevalues
thatarestatisticallysignificant(***,P,0.0001).BandC.Levelsof(B)IFN-aand(C)IFN-bmRNAinWNV-infectedcorticalneuronsweremeasuredby
qRT-PCRasdescribedinthelegendofFigure3.D.WholecelllysatesweregeneratedattheindicatedtimesfromwildtypeorDKOMEFthatwere
uninfected(Un)orinfectedwithWNV(W).ProteinlevelsofISG54,RIG-I,andMDA5wereexaminedbyimmunoblotanalysis.Thedataistheaverageof
atleastthreeindependentexperimentsperformedinquadruplicate(***,P,0.0001).
doi:10.1371/journal.ppat.1000607.g004
followingWNVinfection(Fig5D).Thus,afterWNVinfectionof infection(Fig6F).ThesedatasuggestthattheexpressionofISGis
Mw, IRF-3 and IRF-7 coordinately regulate the early but are primarily IFN-b-dependent or that the IFN-a and -b have
dispensable for thelaterIFN-bandISGresponses. redundant effects in these cells. Nonetheless, as higher viral
replication was sustained in DKO mDC despite a relatively
normal IFN-b response and ISG expression profile, it remains
IRF-3 and IRF-7 are also dispensable for the IFN-b
possiblethatIRF-3directlyregulatesexpressionofakeysubsetof
response in mDC after WNV infection
ISGthat accounts foranti-WNV activityinthis celltype.
Because mDC are likely early targets for WNV infection in To determine whether the IFN-b induction response in DKO
animals[31–33]andhelporchestrateinnateandadaptiveantiviral mDC was specific for WNV, we performed experiments with
immune responses [34], we evaluated IFN and ISG responses in agonists for the TLR3 and TLR4 pathways and with unrelated
bone marrow-derived mDC. Prior studies showed that mDC viruses (Chikungunya virus (CHIK), an emerging human alpha-
lacking either IRF-3 or IRF-7 support enhanced WNV replica- virus,andEMCV,amodelrodentpicornavirus).Asexpected,wild
tion,IRF-32/2mDCdevelopednormalIFN-a/bresponsesafter typemDCthatweretreatedextracellularlywithTLR3(polyI:C)
WNV infection, and IRF-72/2 mDC had reduced IFN-a but or TLR4 (LPS) agonists rapidly induced IFN-b mRNA (Fig 7A).
relatively intact IFN-b responses after WNV infection (S. Daffis In DKO cells, and in contrast to that observed with WNV, the
and M. Diamond, unpublished results and [22]). Multi-step IFN-b response downstream of TLR treatment was effectively
growthcurveanalysisofDKOmDCinfectedwithWNVshowed abolished. Thus, activation of the IFN-b response in mDC after
a higher viral burden compared to wild type cells (16 to 95-fold, TLR3andTLR4stimulationrequiresbothIRF-3andIRF-7.To
P,0.0001) and IRF-32/2 or IRF-72/2 mDC (Fig 6A, S. Daffis definewhethertheIRF-3/IRF-7-independentIFN-bresponsewas
and M. Diamond, unpublished results, and [22]). The viral titers specific to WNV, we infected mDC with additional RNA viruses
in DKO mDC were similar to those obtained in IFN-abR2/2 (Fig 7B). In wild type mDC, EMCV infection induced a robust
mDC,suggestingadefectintypeIIFNsignalingintheDKOcells. IFN-bresponsesimilartoWNV,andhighlevelsofIFN-bmRNA
Surprisingly, whereas levels of IFN-a mRNA and protein were were detected by 24hours post infection (,100-fold increase).
abolished(Fig6Band6C),inductionofIFN-bgeneandprotein Infection with CHIK at a higher MOI also resulted in a similar
production was not significantly affected (P.0.2) after WNV IFN-b gene induction at 24hours post infection. In DKO cells,
infectionofDKOmDC(Fig6Dand6E).Thus,inmDC,IRF-3 theIFN-bresponseafterinfectionwithWNV,EMCV,andCHIK
andIRF-7regulatetheIFN-aresponsebutarelargelydispensable wasequivalenttothatobservedinwildtypecells.Thus,inmDC,
forinducingIFN-bafterWNVinfection.Westernblotanalysisof IRF-3 and IRF-7 are dispensable for activation of IFN-b gene
ISG corroborated these findings as similar levels of ISG were transcription, not only in response to WNV but also to other
observedinwildtypeandDKOmDCat24and48hafterWNV positive strand RNAvirusesthat aregenetically unrelated.
PLoSPathogens | www.plospathogens.org 6 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
Figure5.IRF-3andIRF-7partiallymodulatetheIFN-bresponseandISGexpressioninprimaryMw.A.Mwgeneratedfromwildtype,IFN-
abR2/2andDKOmicewereinfectedatanMOIof0.01andvirusproductionwasevaluatedattheindicatedtimespostinfectionbyplaqueassay.
Valuesareanaverageofquadruplicatesamplesgeneratedfromatleastthreeindependentexperiments.B.Wholecelllysatesweregeneratedatthe
indicatedtimesfromwildtypeandDKOMwthatwereuninfected(Un)orinfectedwithWNV(W).ProteinlevelsofISG49,ISG54,PKR,STAT1,RIG-I,
MDA5 and tubulinwere examinedby immunoblot analysis. C and D. Theinduction of (C) IFN-a and (D) IFN-b mRNA in WNV-infectedMw was
analyzedbyqRT-PCRasdescribedinFigure3.Asterisksindicatevaluesthatarestatisticallysignificant(***,P,0.0001,**,P,0.005,*,P,0.05).
doi:10.1371/journal.ppat.1000607.g005
TheIFN-bresponseinmDCafterWNVinfectiondoesnot whether the transcriptional signal after WNV infection of mDC
rely on type I IFN signaling and IRF-1 or IRF-8 cells was dependent on IRF-5. IRF-52/2 mDC showed only a
To begin to investigate the mechanism(s) that regulate IFN-b modest yet reproducible reduction (2.5-fold, P,0.05) in IFN-b
gene induction in mDC, we infected IRF-12/2 and IRF-82/2 genetranscriptionwithin24hoursofWNVinfection(Fig8D).By
48hours, however, these deficits were no longer apparent as
mDC with WNV. These transcription factors have been
equivalentorevengreaterlevelsofIFN-bmRNAweredetectedin
implicated in type I IFN gene transcription in DC after TLR
IRF-52/2 cells. Taken together, our experiments suggest that
stimulation or viral infection [35,36]. Notably, no difference in
IFN-aandIFN-bmRNAlevelswasobservedinIRF-12/2mDC IRF-1 and IRF-8 are dispensable for induction of IFN-b gene
transcriptioninmDC,whereasanIRF-5hasasmallandtransient
(Fig8Aand8B).Subsequently,weassessedwhethertheregulator
of IFN-b transcription was IFN-inducible, as has been suggested regulatory effect inthese cells after WNVinfection.
for IRF-8 in the context of infection by MCMV [36] or
paramyxovirus [37]. IFN-abR2/2 mDC were infected with A combined inhibition ofNF-kB and ATF-2/c-Jun in DKO
WNVandthelevelsofIFN-aandIFN-bmRNAweremeasured. mDC reduces the IFN-b response after WNV infection
Whereas the IFN-a response was diminished in these cells, likely The transcriptional activation of the IFN-b gene requires full
because of the abolition of the type I IFN positive feedback loop occupancy of an enhancer complex known as the ‘‘enhanceo-
(Fig 8C), the IFN-b gene response in IFN-abR2/2 mDC was some’’[23–25].Invitro,theactiveIFN-benhanceosomeisformed
greater(5to10-fold,P,0.05)comparedtowildtypecells(Fig8D). afterassociationofthetranscriptionfactorsIRF-3,IRF-7,ATF-2/
These results suggest that, unlike the regulation of the IFN-a c-Jun,andNF-kB.AlthoughsynergyintranscriptionoftheIFN-b
response, activation of the IFN-b gene after WNV infection in geneoccursaftercoordinatebindingoftheseregulatoryfactorsin
mDC is independent of the type I IFN positive feedback loop. severalcelltypes[25],themechanisticbasisforfulloccupancyof
Consistentwiththis,nodifferenceinIFN-aandIFN-binduction the enhanceosome remains incompletely understood. Since a
was observed after WNV infection of IRF-82/2 mDC (Fig 8A combined deficiency of IRF-3 and IRF-7 only modestly reduced
and 8B). the IFN-b gene induction in mDC after WNV infection, we
Recent studies have suggested that IRF-5 can activate hypothesizedthattheresidualIFN-bactivitycouldoccurthrough
transcription ofIFNandotherinflammatorycytokinegenesafter aNF-kB-dependentand/orATF-2/c-Jun-dependentsignalthatis
viral infection or TLR stimulation [38–41]. As IRF-5 also has mediated directly through PRR signaling. To test this possibility,
been implicated in IFN production and protection in vivo after we used the highly specific and validated pharmacological
infectionbynegativestrandRNAviruses(NewcastleDiseasevirus inhibitorsofNF-kB(BAY11-7082)[43,44]andp38MAPkinase
(NDV) and VSV) and DNA viruses (HSV) [38,42], we evaluated (SB202190),whichisessentialforphosphorylatingandactivating
PLoSPathogens | www.plospathogens.org 7 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
Figure6.TheWNV-inducedIFN-bresponseandISGexpressionisprimarilyIRF-3andIRF-7-independentinmDC.A.mDCgenerated
fromwildtype,IFN-abR2/2andDKOmicewereinfectedatanMOIof0.001andvirusproductionwasevaluatedattheindicatedtimespostinfection
byplaqueassay.Valuesareanaverageofquadruplicatesamplesgeneratedfromatleastthreeindependentexperiments(***,P,0.0001).B–E.Levels
of(B)IFN-aand(D)IFN-bmRNAaswellas(C)IFN-aand(E)IFN-bproteininWNV-infectedmDCweremeasuredbyqRT-PCRorELISAasdescribedin
thelegendofFigure3.F.WholecelllysatesweregeneratedattheindicatedtimesfromwildtypeandDKOmDCthatwereuninfected(Un)or
infectedwithWNV(W).ProteinlevelsofISG49,ISG54,PKR,STAT1,RIG-I,MDA5andtubulinwereexaminedbyimmunoblotanalysis.
doi:10.1371/journal.ppat.1000607.g006
the ATF-2/c-Jun complex [45]. Treatment of DKO mDC with confirmed that the difference in effect by the inhibitors was not
increasing concentrations of BAY 11-7082, the NF-kB inhibitor, due to differential cytotoxicity between wild type and DKO cells
yielded only a small reduction of WNV-induced IFN-b response (Fig 9A). These experiments suggest that, in mDC, efficient
(maximum of 3.9 fold decrease, P=0.006) (Fig 9A). In contrast, activation of the IFN-b gene does not require the full enhanceo-
theLPS-inducedTNF-aresponse,whichisdominantlyregulated someoccupancy,asacombinedfunctionalabsenceofIRF-3,IRF-
by NF-kB [46,47], was dose-dependently inhibited in wild type 7, NF-kB and ATF-2/c-Jun is necessary to strongly inhibit the
and DKO mDC treated with BAY 11-7082 (Fig 9C). Analo- IFN-bgene activationafter WNVinfection.
gously, treatment of DKO mDC with SB 202190 had a limited Since the activation of the IFN-b response in MEF was
effect on the WNV-induced IFN-b response (3.5 fold decrease, markedly reduced in the absence of both IRF-3 and IRF-7, we
P=0.02) (Fig 9A). Thus, in mDC lacking IRF-3 and IRF-7, hypothesized that, in contrast to that observed in DC, the
inhibitionofNF-kBorATF-2/c-Junaloneonlymodestlyreduced regulationoftheIFN-bgeneinMEFwoulddependmorestrongly
the IFN-b gene transcription after WNV infection. However, a on the full occupancy of the enhanceosome with canonical
combined inhibition of both NF-kB and ATF-2/c-Jun in DKO constituents.Toassessthis,wildtypeMEFweretreatedwithBAY
mDC strongly diminished the IFN-b response (29-fold decrease, 11-7082 and/or SB 202190, infected with WNV, and levels of
P,0.0001).Incontrast,amoresubtledecrease(2.5-folddecrease, IFN-b mRNA were measured. In contrast to that observed with
P=0.006) of IFN-b transcription after WNV infection was wild type mDC, inhibition of NF-kB in wild type MEF slightly
observed in wild type mDC that retained expression of IRF-3 decreased the IFN-b response (,2-fold decrease, P=0.01)
and IRF-7. Parallel experiments using an ATP metabolism assay (Fig 9B). Similarly, inhibition of ATF-2/c-Jun in wild type MEF
PLoSPathogens | www.plospathogens.org 8 October2009 | Volume 5 | Issue 10 | e1000607
IFNInductioninIRF3/7DKOMiceandCells
understand the signaling pathway between PRR and IFN gene
inductionafterexposuretoWNV,weinfectedIPS-12/2MEFand
mDC.WhereaslevelsofIFN-aand-bmRNAincreaseovertimein
wildtypemDCandMEF,inductionofbothwasvirtuallyabolished
in IPS-12/2 cells (Fig 10A–D). Consistent with this, we did not
detect any secreted IFN-a and b from IPS-12/2 DC or MEF
supernatants (data not shown). Thus, the induction of type I IFN
genesinmDCorMEFafterWNVinfectionisentirelydependent
on IPS-1 signaling. Since IRF-5 mayplay a partialrole in IFN-b
gene regulation in mDC (Fig 8D) and since MyD88 has been
suggested to mediate the TLR- and IRF-5-dependent cell type-
specificinductionoftypeIIFNafterNDVinfection[38],wealso
assessed the role of MyD88 in triggering the IFN-b response in
mDC.Asshown(Fig10EandF),theIFN-aandbtranscription
inducedbyWNVinfectioninmDCwasessentiallyindependentof
MyD88. Thus, an optimal IFN-b transcriptional signal in mDC
afterWNVinfectionisgeneratedthroughapathwaythatrequires
IPS-1,asubsetofthefour(IRF-3,IRF-7,NF-kBandATF-2/c-Jun)
components of the canonical enhanceosome and possibly, IRF-5,
afteraMyD88-independentactivationsignal.
TRAF3andTBK1regulatetheearlyIPS-1-dependenttype
I IFN response whereas TRAF6 modulates a later phase
AlthoughourexperimentssuggestedthatIFN-binductionafter
WNV infection required IPS-1 yet was mediated by a cell type-
specific complex of transcriptional regulators, the signaling
adaptors that connected these pathways remained uncertain. As
IPS-1-dependentregulationofthetypeIIFNgenesinresponseto
VSV and Sendai virus requires activation of TRAF3 [48], we
infectedTRAF32/2MEFwithWNVandmeasuredthelevelsof
IFN-bmRNAandsecretedcytokine.TRAF32/2MEFshoweda
significant reduction of IFN-b mRNA levels at 24hours and
48hourspostinfection(7.8-fold,P,0.0001and2.4-fold,P,0.05,
respectively),whichwasconfirmedbymeasuringIFN-binthecell
supernatant (3.7- and 1.4-fold decrease, respectively, P,0.0001)
Figure7.TheIFN-bresponseinmDCafterTLRstimulationand (Fig 11B and 11D). These data suggest that initial IPS-1-
viral infection.mDC generated from wild typeand DKOmice were dependent trigger of the IFN-b response after WNV infection is
(A)stimulatedwith50mg/mlofpoly(I:C)or2mg/mlLPSfor24hor(B) mediated at least partially by TRAF3. Consistent with this, the
infectedwithEMCV(MOI0.1),WNV(MOI0.1)andCHIK(MOI1)for24h. levels of IFN-a mRNA and protein, which reflect the type I IFN
LevelsofIFN-bweremeasuredeitherbyELISAorqRT-PCR.Valuesare positive feedback, also were decreased in TRAF32/2 MEF
an average of triplicate samples generated from three independent
(mRNA: 32-fold and 36- fold decreases, P,0.0001; protein: 1.3-
experiments. Asterisks indicate values that are statistically significant
(***, P,0.0001, ** P,0.0005) from wild type cells; n.s. indicates fold, P,0.0001) (Fig 11A and 11C). As TBK1 is a kinase that
differencesthatwerenotstatisticallysignificant. reportedly mediates the signal to induce IFN production
doi:10.1371/journal.ppat.1000607.g007 downstream of the IPS-1/TRAF3 axis [49], we assayed IFN-a/
b induction after WNV infection in TBK12/2 MEF. Similar to
also reduced the IFN-b response (,5-fold decrease, P=0.007). thatobservedinTRAF32/2MEF,thelevelsofmRNAandmore
More strikingly, inhibition of both NF-kB and ATF-2/c-Jun in importantly, secreted IFN-b were decreased in TBK12/2 MEF
wild type MEF strongly attenuated the IFN-b gene transcription (3.7- and 1.5-fold decrease, respectively, P,0.0001) (Fig 11D).
(,20-fold decrease, P,0.0001). Thus, disruption of function of However, levels of IFN-a mRNA and secreted cytokine in
singlecomponentsoftheenhanceosomeissufficienttoreducethe TBK12/2 MEF were not different compared to wild type MEF
IFN-b transcriptional response in MEF. Consistent with this, (P$0.2)(Fig11Aand11C).Thus,TBK1appearstomediatethe
inhibition of NF-kB and/or ATF-2/c-Jun in DKO MEF earlyIFN-bresponsedownstreamofTRAF3afterWNVinfection
essentiallyabolishedtheresidualIFN-bresponse(Fig9B).Taken
but appears dispensable for the later activation of IFN amplifica-
together, these data suggest that optimal regulation of the IFN-b
tionloop.Collectively, these datasuggestthat,inMEF,theearly
genetranscriptioninmDC,incontrasttoMEF,doesnotrequire IFN-b response depends on a signaling pathway involving IPS-1,
completeoccupancyfourcanonicalcomponentsoftheenhanceo- TRAF3, and TBK1 whereas the late IFN-b response may also
some. partiallyinvolveTRAF3.Unfortunately,becauseTRAF32/2and
TBK12/2micearelethalshortlyafterbirth[50,51],wecouldnot
AdeficiencyofIPS-1abolishestheIFN-bresponseinMEF confirmthissignaling pathway inmDC.
and mDC Recent studies with VSV-infected cells have suggested that
Immune detection of WNV by mDC likely occurs through the TRAF6regulatestheIPS-1-dependentsignaltoinducetypeIIFN
cytosolicPRR,RIG-IandMDA5andrequisitesignalingthrough [52]. To evaluate the contribution of TRAF6 to induction of the
the IPS-1 adaptor protein; this leads to activation of downstream IFN response after WNV infection, levels of mRNA and IFN-b
IRF family proteins and IFN gene transcription [16]. To better secretedproteinwereassayedinTRAF62/2MEF.Incontrastto
PLoSPathogens | www.plospathogens.org 9 October2009 | Volume 5 | Issue 10 | e1000607