Cellular Oxidative Stress Response Controls the Antiviral and Apoptotic Programs in Dengue Virus ...

18 Pages·2014·2.81 MB·English

Cellular Oxidative Stress Response Controls the Antiviral
and Apoptotic Programs in Dengue Virus-Infected
Dendritic Cells
David Olagnier1¤*, Suraj Peri2, Courtney Steel1, Nadine van Montfoort1, Cindy Chiang1,
Vladimir Beljanski1, Michael Slifker2, Zhong He1, Carmen N. Nichols1, Rongtuan Lin3,
Siddharth Balachandran2, John Hiscott1*
1Vaccine&GeneTherapyInstituteofFlorida,PortSt.Lucie,Florida,UnitedStatesofAmerica,2FoxChaseCancerCenter,Philadelphia,Pennsylvania,UnitedStatesof
America,3LadyDavisInstitute,JewishGeneralHospital,McGillUniversity,Montreal,Quebec,Canada
Abstract
Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide,
leadingto50,000deathsannually.Becausedendriticcells(DC)intheskinandbloodarethefirsttargetcellsforDENV,we
sought toinvestigatetheearly moleculareventsinvolvedinthehost responsetothe virusinprimary humanmonocyte-
deriveddendriticcells(Mo-DC).Usingagenome-widetranscriptomeanalysisofDENV2-infectedhumanMo-DC,threemajor
responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-kB-driven antiviral and
inflammatorynetworks,aswellasthestimulationofanoxidativestressresponsethatincludedthestimulationofanNrf2-
dependentantioxidantgenetranscriptionalprogram.DENV2infectionresultedintheintracellularaccumulationofreactive
oxygenspecies(ROS)thatwasdependentonNADPH-oxidase(NOX).AdecreaseinROSlevelsthroughchemicalorgenetic
inhibitionoftheNOX-complexdampenedtheinnateimmuneresponsestoDENVinfectionandfacilitatedDENVreplication;
ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune
responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation
markers andwere lesssusceptibleto viral replication.Wehave identifieda criticalroleforthe transcription factorNrf2 in
limitingbothantiviralandcelldeathresponsestothevirusbyfeedbackmodulationofoxidativestress.SilencingofNrf2by
RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate
thatthelevelofoxidativestressiscriticaltothecontrolofbothantiviralandapoptoticprogramsinDENV-infectedhuman
Mo-DCand highlight the importanceofredox homeostasis inthe outcomeofDENVinfection.
Citation:OlagnierD,PeriS,SteelC,vanMontfoortN,ChiangC,etal.(2014)CellularOxidativeStressResponseControlstheAntiviralandApoptoticProgramsin
DengueVirus-InfectedDendriticCells.PLoSPathog10(12):e1004566.doi:10.1371/journal.ppat.1004566
Editor:GlennRandall,TheUniversityofChicago,UnitedStatesofAmerica
ReceivedJuly14,2014;AcceptedNovember10,2014;PublishedDecember18,2014
Copyright:(cid:2)2014Olagnieretal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
DataAvailability:Theauthorsconfirmthatalldataunderlyingthefindingsarefullyavailablewithoutrestriction.Allrelevantdataarewithinthepaperandits
SupportingInformationfilesexceptforthearraydatawhichisavailableintheNCBIGeneExpressionOmnibus(GEOSeriesaccessionnumberGSE58278).
Funding:ThisprojectwassupportedbyfundingfromVGTIFloridaandtheCanadianInstituteofHealthandResearch.Thefundershadnoroleinstudydesign,
datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*Email:[email protected](DO);[email protected](JH)
¤Currentaddress:LadyDavisInstitute,JewishGeneralHospital,McGillUniversity,Montreal,Quebec,Canada
Introduction infection manifests as self-limiting dengue fever or progresses to
life-threatening illnessremains unanswered.
Dengue virus (DENV) is the leading arthropod-borne viral Dengue is an RNA virus of the Flaviviridae family with 4
infectionintheworld,andrepresentsamajorglobalhumanhealth closely related serotypes that exhibit inter- and intra-serotypic
concern.DENVisendemicinmorethan100countrieswithupto genetic diversity [5–9]. Innate recognition of DENV involves a
3billionpeopleintropicalregionsoftheworldatriskofinfection spectrum of pattern recognition receptors (PRR) that sense
[1–3].Recently,DENVhasexpandeditsglobalrange,withlong- conserved molecular components termed pathogen associated
term outbreaks in South America and reintroduction into North molecular patterns (PAMP), and together orchestrate antiviral
AmericathroughFloridaandTexas,witheachoftheseoutbreaks responses to the viral infection. The cytoplasmic helicases RIG-I
accompanied by increased disease severity. Of the estimated 50– andMDA-5haveacentralroleinthehostresponsetoDENVby
100 million annual cases, the majority of infected individuals contributing to DENV protection in hepatocytes [10]. Addition-
develop a self-limiting febrile illness, but approximately 500,000 ally,TLR3andTLR7recognizeDENVRNAandmountarapid
clinicalcasesresultinmoreseveremanifestations,suchasDENV- protective immune response in human monocytic cells and
induced hemorrhagic fever and shock syndrome [1], leading to plasmacytoid dendritic cells, respectively [11,12]. Signaling
25–50,000 deaths per year [4]. The pathogenesis of dengue is through these different cellular sensors leads to the activation of
incompletely understood and the factors that determine whether the interferon pathway that restricts viral proliferation and
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RedoxRegulationoftheHostResponsetoDENV
disease severity in DENV-infected patients, suggesting a possible
Author Summary
role for oxidative stress in DENV-induced pathogenesis [41–44].
Dengue virus (DENV), the leading arthropod-borne viral Interestingly, circulating monocytes from glucose-6-phosphate
infection in the world, represents a major human health dehydrogenase (G6PD)-deficient patients, displayed an increased
concern with a global at risk population of over 3 billion susceptibilitytoDENVinfectionandreplication[45].TheG6PD
people. Currently, there are no antivirals or vaccines deletion affects ROS production, thus linking cellular oxidative
available to treat patients with dengue fever, nor is it state and susceptibility to DENV infection. Altogether, these
possible to predict which patients will progress to life- observationsunderlinetheimportanceoftheredoxhomeostasisin
threatening severedengue fever.Markers associatedwith
DENV infection and suggest an important interplay between the
oxidative stressresponses have been reportedin patients
generationofoxidativestressandtheimmunopathologyofdengue
with severe DENV infection, suggesting a relationship
disease.
between oxidative stress and viral pathogenesis. In order
Initial contact between DENV and innate immune cells plays
to uncover biological processes that determine the
an essential role in the outcome of the infection. Indeed, DENV
outcome of disease in patients, we utilized human
infection pushes monocytes towards a CD16+ inflammatory
dendritic cells, the primary target of DENV infection, in
an in vitro model. Transcriptional analysis of pathways phenotypethatfacilitatesplasmablastdifferentiationandinduction
activated upon de novo DENV infection revealed a major of anti-DENV antibody responses [46]. Given the importance of
roleforcellularoxidativestressintheinductionofantiviral, DC in bridging the innate and adaptive immune response, and
inflammatory, and cell death responses. We also demon- sinceDCintheskinandperipheralbloodarethefirsttargetcells
strated that antioxidant mechanisms play a critical role in for DENV after transmission via a mosquito bite [47–49],
controlling antiviral and cell death responses to the virus, evaluation of the early molecular events in DC is crucial to the
acting as feedback regulators of the oxidative stress understanding of DENV pathogenesis. In the present study, we
response. This report highlights the importance of oxida- generatedin-depthtranscriptomeanalysis,coupledwithbiochem-
tive stress responses in the outcome of DENV infection, ical and functional analyses of the early host response to DENV
andidentifiesthispathwayasapotentialnewentry-point infection in primary Mo-DC. DENV infection triggered an
for treatingdengue-associated diseases. NADPH-oxidase (NOX)-dependent oxidativestress responsethat
was required for the activation of IRF3/7/STAT1 and NF-kB-
contributestotheestablishmentofadaptiveimmuneresponsesvia mediated antiviral responses and for mitochondrial-dependent
NF-kB-mediated cytokine and chemokine release [13–16]. Inter- apoptosis. Furthermore, we have identified a critical role for the
estingly, the host immune response, activated in response to transcription factor Nrf2 in regulating both antiviral and
DENVinfection,notonlymediatesprotectionagainstdisease,but inflammatory gene response to the virus by feedback modulation
alsocontributestodiseaseseverity[1].Forexample,highlevelsof ofoxidativestress.Overall,thesestudieshighlighttheimportance
circulatingpro-inflammatorycytokinessuchasIL-1borTNF-ain of redoxhomeostasis inthe outcomeof DENV infection.
DENV-infected patients correlates with severe dengue fever,
compared topatients suffering withmild denguefever [17]. Results
Reactive oxygen species (ROS) production, generated as a
consequenceofmicrobialinvasion,haslongbeenknowntoexert DENV2 highly infects Mo-DC and generates a broad
an antimicrobial effect in phagocytes [18]. The activation of the antiviral response
antiviral and inflammatory signaling pathways has also been An in vitro model of de novo DENV infection was established
linkedwiththeproductionofROS[19–23],whichincludeoxygen using primary human monocytes differentiated in vitro with Mo-
ions and peroxides that are produced as byproducts of aerobic DC-differentiation medium containing GM-CSF and IL-4.
metabolism.BecauseofthehighchemicalreactivityofROS,cells Primary CD14+ CD1a2 monocytes were less permissive to
possess scavenger antioxidant mechanisms that maintain redox DENV2 infection, whereas infectivity increased progressively as
homeostasis [24–26]. Signaling pathways downstream of ROS the cells differentiated toward the Mo-DC (CD142 CD1a+)
detectionactivatethetranscriptionfactornuclearfactor-erythroid phenotype(4.6660.45%ofDENV+cellsinmonocytesatday0vs
2-related factor 2 (Nrf2) [24–26], which binds antioxidant 79.660.47% in Mo-DC at day 7) (Fig.1A). A strong statistical
responseelements (ARE)withinthepromotersofgenesencoding correlation between a CD142CD1a+ phenotype and DENV
antioxidantanddetoxifyingenzymes.Nrf2-dependentantioxidant infection was confirmed by the nonparametric Spearman test
genes act synergistically to reduce oxidative stress by quenching (r=0.9829;p,0.0001;n=15).DENV2viralRNAaccumulation
ROS [24–26]. wasdetectedafteralagperiodof6 handincreasedexponentially
IncreasedgenerationofROSandchangesinredoxhomeostasis thereafter(Fig.1B),whichcorroboratesapreviousreportdemon-
have been described in the context of many viral infections strating release of infectious particles [50]. Prior to the onset of
[23,27–33] and the failure to maintain an appropriate redox detectable DENV replication, an antiviral response was mounted
balance contributes to viral pathogenesis through alterations of by the infected Mo-DC population, as demonstrated by the
biological structures and themassive induction of celldeath [34– increase in IFN-b, IFIT1 and CCL5 gene expression (Fig.1B).
36]. In the flavivirus family, hepatitis C virus (HCV) has been DENV infected Mo-DC in a dose dependent manner to a
shown to promote oxidative stress and manipulate antioxidant maximum of ,80% infectivity at a MOI of 20 (Fig.1C). As a
systems,leadingtochronicdisease[31,37,38].Aswell,DENVwas consequence of early virus sensing, a broad antiviral and
shown to stimulate oxidative stress in hepatocytes leading to inflammatoryresponsewasgeneratedasshownbythephosphor-
production of the chemokine CCL5 and to activation of the ylation of IRF3 and STAT1 (Fig.1D) and significant release of
transcriptional regulator C/EBP beta [39]. Furthermore, HepG2 IFN-a, TNF-a and IL-6 (Fig.1E) by the infected cells. Previous
xenografted SCID mice presented alterations in oxidative stress studies reported cleavage of the endoplasmic reticulum adaptor
status and increased inflammatory cytokines following DENV STING upon DENV infection in Mo-DC [51]. However, in our
infection [40]. More recently, oxidative stress-induced damage experimentalmodelandwiththeviralstrainused,amodest20%
andalterationsinredoxstatushavebeenassociatedwithincreased decreaseinSTINGexpressionwasobservedat48hafterinfection
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Fig.1.Mo-DCarehighlysusceptibletoDENVinfectionandgenerateabroadantiviralandinflammatoryresponse.(A)Thecorrelation
betweenthepercentageofinfectedcellsdeterminedbyintracellularstainingofDENVEproteinexpressionandthepercentageofCD142CD1a+Mo-
DC was calculated during the course of Mo-DC differentiation (n=15; Spearman test). The FACS panels on the left hand side represent the
progressionofMo-DCdifferentiationatdays0,3and7.(B)DENVRNAlevelsaswellasIFN-b,IFIT1,andCCL5geneexpressionlevelsweredetermined
byqPCRatvarioustimesofDENV2infection(MOI10).Pvaluesweredeterminedbasedonthecomparisonwithcellsattime3hafterinfection.Data
arefromoneexperimentperformedonthreeindividualdonors.(C)Mo-DCwereinfectedwithincreasingamountsofvirus(MOI0.04-MOI20)for24h.
PercentageofDENV2-infectedcellswasdeterminedbyintracellularstaining(ICS)ofDENVEproteinexpressionusingflowcytometry.Dataarethe
means 6 SEM from one experiment performed on three different donors. (D) Mo-DC were challenged with DENV2 (MOI 20) and antiviral and
inflammatoryresponseswereexaminedbyimmunoblotting.Dataarefromonerepresentativeexperiment.(E)Cytokinereleasewasevaluatedby
cytokine bead array (CBA) 24h after DENV2 challenge (MOI 10) in the supernatants of infected cells. P values were determined based on the
comparisonwithuninfectedcells.Dataarethemeans6SEMfromoneexperimentperformedonthreedifferentdonors.
doi:10.1371/journal.ppat.1004566.g001
(Fig.1D).AltogetherthesedatademonstratethatDENV-infected antiviral pathways as well as the NF-kB-dependent pro-inflam-
Mo-DC generate a broad host response and secrete an array of matory pathways were all highly enriched after de novo DENV2
antiviral and inflammatory cytokines inresponsetothevirus. infection (Fig.2B). We also noticed an enrichment of networks
associated with the generation of a pro- and anti-oxidant stress
Transcriptome kinetics of the host response to DENV2 response (Fig. 2B). Further gene analysis represents the top 50
infection DEG over time following DENV infection (Fig.2C); among the
Tocharacterizesignalingpathwaysinvolvedinthehostintrinsic topup-regulatedgenes,twosubclassespredominated–interferon-
responsetoDENV2infection,atranscriptomeanalysisofDENV- stimulated genes (ISGs) such as ISG15, IFIT1, IFIT2, IFIT3,
infected Mo-DC was performed; Fig.2A represents a waterfall OASL, OAS2, CCL5, HES4 (presented in black) and more
plotofdifferentiallyexpressedgenes(DEG;selectedbasedonfold surprisingly a large set of antioxidant genes belonging to the
change .61.3, p value ,0.05) after DENV2 infection. Most metallothionein familyincluding MT1A,MT2A, MT1E,MT1X,
changesingeneexpressionappearedearly,withover7000genes MT1G,MT1H,andMT1F(presentedinred)(Fig.2C).Basedon
either up- or down-regulated by 6h after infection (Fig.2A). the regulation of gene networks activated or repressed after
Pathway analysis identified multiple canonical networks coordi- DENV2infection,Fig.2Dillustratesawordcloudmapofpossibly
natelyregulatedatalltimesafterinfection;theexpectedIFN/IRF activated(red)orinhibited(green)transcriptionfactorscontrolling
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Fig. 2. Transcriptome analysis of the host response to DENV2 infection in Mo-DC. Mo-DC were infected with DENV2 (MOI 20) for
designatedperiodsoftime.SampleswereanalyzedbyIlluminageneexpressionarrayanddifferentiallyexpressedgenes(DEGs)thatsatisfiedap
value (,0.05) with $1.3 fold change (up or down) were selected. (A) Waterfall plot representing the total number of up-regulated and down-
regulatedgenesateachtimepoint.(B)Heatmapshowsstatisticallysignificantcanonicalpathways(IngenuityPathwayAnalysisSoftware)commonly
regulatedat6h,12h,18hand24hwhencomparedtobaseline.Genesthathadadjustedp-value,0.05ateachtimepointandfoldchange.1.3or
,21.3andassociatedwithacanonicalpathwayinIngenuity’sKnowledgeBasewereusedforpathwayanalysis.Heatmapcolorsrepresenttheratio
of regulated genes/pathway genes after dengue infection (red and blue correspond to over- and under-represented, respectively). The over-
representationtestwasperformedusingFisherExactTest.Statisticalsignificanceachievedatp,0.05.Thedataarerepresentativeofoneexperiment
performedonthreedifferentdonors.(C)Geneexpressionheatmapofthetop50differentiallyexpressedgenesinducedbydengueinfectioninMo-
DCatvarioustimeswhencomparedtobaseline.GenesareselectedasdifferentiallyexpressedinatleastonecomparisonfollowingANOVAFtestas
implementedintheLIMMApackage.Thescaleshowsthelevelofgeneexpressionwhereredandbluecorrespondtoup-anddown-regulation
respectively.Apanelofantiviral(black)andantioxidant(red)DEGisrepresentedontherighthandsideoftheheatmap.(D)Wordcloudsrepresenting
potentiallyactivated(red)/inhibited(green)transcriptionfactorsat6hand24hafterDENV2challenge.IPAUpstreamRegulatorAnalysiswasusedto
identifymoleculesupstreamofthegenesinthedatasetthatcouldexplainthedetectedexpressionchanges.Thep-valueofoverlap,whichmeasures
the enrichment of network-regulated genes in the data set, is represented by the size of the word. The activation z-score which predicts likely
regulatingmoleculeswasusedtocolorthepredictedactivationstate.
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Fig.3.FunctionalcharacterizationofgenesdifferentiallyexpressedbyDENV2infection.Differentiallyexpressedgenesweresubjectedto
IngenuityPathwayAnalysisforboth6h(A)and24h(B)timepointsbasedonp-values(,0.001)andfoldchange$61(log2).Redrepresentgenes
inducedbyDENV,andgreenindicatedgenesdownregulatedbythevirus;theintensityofthecolorisrepresentativeofthefoldchange.Largercircles
indicatetranscriptionfactors.Genesenrichedforapathwayarerepresentedasacloud.
doi:10.1371/journal.ppat.1004566.g003
genenetworksat6hand24hafterDENV2challenge(Fig.2D). (Fig.4C). Although ROS are generated intracellularly, the
At 6 h after infection, two subclasses of transcription factors primary sources of ROS are plasma membrane oxidases,
predominated: 1) transcription factors associated with cellular particularly NADPH oxidases. ROS were detected as early as
stress-responses including TP53 (p53), EPAS1, HIF1A and 3 h after infection, and ROS production was suppressed by pre-
NFE2L2 (Nrf2); and 2) transcriptional regulators associated with treatment with the antioxidant diphenyleneidonium chloride
the antiviral program including IRF1/3/7, STAT1/ISGF3 and (DPI), an NADPH-oxidase (NOX) inhibitor (Fig.4D). ROS
NF-kB complex (Fig.2D). By 24h post-infection, the activity of production was independently confirmed in Mo-DC by the use
stress-relatedtranscriptionfactorsdecreased,withtheexceptionof of pyocyanin (N-methyl-1-hydroxyphenazine), an oxidative stress
TP53, while transcription factors driving the antiviral response - inducer,asdenotedbythe1.8foldincreaseinROSgenerationat
predominantly IRF7andNF-kB-were highlyactive (Fig.2D). 3 h after stimulation (Fig.4D). The involvement of NOX in
AFluidigmBioMarkhighthroughputqPCRassayencompass- DENV-inducedROSaccumulationwasfurtherconfirmedbythe
ing a cross-section of genes identified inthe genomicanalysis(S1 increased phosphorylation of the p47 subunit of the NADPH-
Table)wasusedtovalidatethetranscriptomedata;thepatternof oxidase (p=0.0404) (Fig.4E). Interference with NADPH-oxidase
gene expression at various times after DENV2 infection was activityusingsiRNA-mediatedsilencingofthecatalyticgp91phox
similar for three different donors (S1A Figure). Computational subunitlimitedROSaccumulationinresponsetodenovoDENV
analysis identified different kinetics of IFN induction, as well as infection (p=0.0328) (Fig.4F). To examine whether cellular
sustained up-regulation of chemokines, Th1 cytokines, ISGs and oxidative stress impactedtheimmediatehost responsetoDENV,
antiviral transcription factors (S1B Figure). A strong statistical weevaluatedtheeffectofexogenousROSadditiononexpression
correlationbetweenthelogfoldchangeforthemicroarrayvalues of DENV-induced antiviral genes. Treatment with increasing
andthelogfoldchangefortheBioMarkvalueswasconfirmedbya concentrations of hydrogen peroxide (H O ) did not stimulate
2 2
Spearman correlation test (S1C Figure) (r=0.8399194; immune responses in Mo-DC; however addition of H O
2 2
p=4.576e-14; n=49). In order to gain systems-wide insight into moderately potentiated the elevation of DENV-induced antiviral
DENV-modulated transcriptome, a functional clustering (node geneexpression (Fig.4G).
analysis) (Fig.3), as well as gene-pathway checkerboard analysis Next, the role of ROS in triggering the early host response to
(S2 Figure) of DENV-induced DEGs was performed. This DENV2 was evaluated by treating infected Mo-DC with
functional clustering identified at 6 h (Fig.3A and S2A Figure) increasing concentrations of DPI, an NADPH-oxidase inhibitor.
and24h(Fig.3BandS2BFigure)avarietyoftranscriptionalsub- Strikingly,phosphorylationofIRF3,STAT1andIkBa,aswellas
networksandbiologicalprocessesregulatedbyDENV.TheNrf2- the induction of ISGs such as RIG-I and IFIT1 – all markers of
mediated oxidative stress response pathway, the top differentially the antiviral response – were inhibited in a dose-dependent
regulatedpathwayinDENV-infectedMo-DCat6 h(S2AFigure), manner by DPI (Fig.5A). The observation that NOX-inhibitor
wastriggeredpriortotheonsetofviralreplicationandintersected blocked DENV-induced immune response was further confirmed
with other pathways such as NF-kB, IRF and STAT signaling by quantitative intracellular measurement of STAT1 phosphory-
(Fig.3A and S2A Figure). At the same time, hypoxia pathway lation. Indeed, DPI prevented the increase in STAT1 phosphor-
controlled by the transcription factor HIF1-a was predominantly ylation detected by PhosFlow following DENV infection. Impor-
downregulated(Fig.3AandS2AFigure).By24htheactivityof tantly,IFNb-inducedSTAT1phosphorylationwasnotaffectedby
the Nrf2-driven pathway decreased, whereas the expansion and the DPI treatment (Fig.5B). Using a customized BioMark chip,
increasedinteractionamongtheantiviral,inflammatoryanddeath antiviral and inflammatory genes such as type I IFNs (IFNA2,
response networks predominated (Fig.3B and S2B Figure). IFNB1), pro-inflammatory cytokines and chemokines (IL1b,
Concomitantly, genes related to mitochondrial function were all CCL5) and ISGs (MX1, IFITM1/2/3, OASL, IDO1, OAS3,
significantly down regulated and presumably associated with an DDX58) were inhibited by DPI in a dose dependent manner in
increase intheapoptotic response (Fig.3BandS2B Figure). DENV-infected cells (Fig.5C, upper right box). Cytokine release
(IFN-a,TNF-aandIL-6)wasalsoimpairedinthepresenceofthe
DENV-induced NOX-derived ROS accumulation is antioxidant molecule (Fig.5D). The use of antioxidant molecules
essential for induction of innate immune responses withdifferentmodesofaction(S3AFigure)recapitulatedtheeffect
The role of reactive oxygen species (ROS) as specific second observed with DPI and impaired the induction of antiviral and
messengers in signaling cascades involved in cell proliferation, inflammatory gene expression (Fig.5E). Importantly, all antiox-
differentiation andimmune activation has been well documented idant moleculestested in this paneldid not affect cell survival, as
[52].InlightofthearraydataandtoevaluateifROSareinvolved shown in S3B Figure and S3C Figure. Inhibition of NADPH-
in the recognition of DENV, Mo-DC were infected and ROS oxidase activity using transient knock-down of the catalytic
formation was monitored by flow cytometry using the oxidant- gp91phox subunit also decreased IFIT1 protein expression
sensitive fluorescent detection probe CM-H2DCFDA. ROS following de novo DENV infection (Fig.5F). No increase in
production was induced in DENV-infected Mo-DC, as reflected DENV RNA accumulation was detected in the presence of the
in the 2-fold increase in DCF fluorescence detected by FACS at NOX-inhibitor (3mM) after 24h of infection (S4 Figure).
18h after infection (p=0.0405) (Fig.4A). Also, DENV infection However, pre-treatment of cells with a higher concentration of
increased intracellular ROS accumulation in a dose dependent DPI(30 mM)ledtoanincreaseinDENVviralRNAaccumulation
manner (Fig.4B). A strong statistical correlation between DENV in the same conditions (S4 Figure). Importantly, DPI treatment
infection and the accumulation of ROS was confirmed by the resulted in increased DENV infectivity and replication at 48h
nonparametric Spearman test (r=0.7635; p,0.0001; n=15) post-infection, as demonstrated by the increased number of
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Fig.4.DENV-infectedcellsaccumulateintracellularNOX-derivedROSwhichpotentiatetheimmuneresponse.(A)ROSgenerationin
DENV-infected Mo-DC (MOI 20) was monitored by FACS using CM-H2DCFDA (1mM) 18h after infection. Data represent the mean 6 SEM from
experimentsperformedonfourdifferentdonors.(B)AccumulationofROSwasmeasuredbyflowcytometryusingCM-H2DCFDA(1mM)24hafter
infectionwithincreasingamountofvirus.Datarepresentthemean6SEMfromoneexperimentsperformedonthreedifferentdonors.Pvalueswere
determinedbasedonthecomparisonwithuninfectedcells(C)Thecorrelationbetweenthepercentageofinfectedcellsdeterminedbyintracellular
stainingofDENVEproteinexpressionandtheintracellularaccumulationofROSmeasuredbyDCFDAstainingwascalculated(n=15;Spearmantest).
(D)OxidativestressgenerationwasdetectedbyflowcytometryusingtheCM-H2DCFDA(1mM)fluorescentprobeonMo-DCpretreatedwithDPI
(3mM) and infected with DENV2 (MOI 20) for 3h or treated with the stress-inducer Pyocyanin (100mM). Data represent the geometric mean
fluorescence6SEMfromanexperimentperformedontwodifferentdonors.(E)ExpressionlevelofthephosphorylatedNADPH-oxidasep47subunit
wasassessedbyimmunoblotting3hafterDENV2infection.Histogramsrepresentthefoldchangeratioofphosphorylated-p47overtotalp47.Data
arethemeans6SEMofindependentexperimentsperformedonthreedifferentdonors.Pvaluewasdeterminedbasedonthecomparisonwith
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uninfectedcells.(F) Mo-DCweretransfected withcontrol or gp91phox siRNAand 48h later were infected withDENV2 (MOI20) for 18h. ROS
accumulationwasmeasuredbyFACSusingtheCM-H2DCFDAfluorescentprobe.Dataarethemeans6SEMofthreeindependentexperiments
performedondifferentdonors.PvaluewasdeterminedbasedonthecomparisonwithcellsinfectedwithDENVandtransfectedwiththecontrol
siRNAsequences(G)Mo-DCweretreatedwithincreasingconcentrationsofhydrogenperoxide(10–50mM)inthepresenceorabsenceofDENV2
(MOI20).CCL5andCXCL10mRNAexpressionlevelsweremonitoredbyqPCR24hfollowingtreatmentandinfection.Datarepresentthemeans6
SEMfromoneexperimentperformedonthreeindividualdonors.Experimenthasbeenrepeatedtwice.Pvaluesweredeterminedbasedonthe
comparisonwithDENV2-infectedcells.
doi:10.1371/journal.ppat.1004566.g004
DENV-infectedcells(i–ii)andviraltiters(iii)(Fig.5G).TheROS- While infected cells displayed apoptotic markers as described
mediated induction of antiviral and inflammatory genes required above, uninfected bystander Mo-DC cells did not undergo
live and replicating virus, since formalin-inactivation and UV- apoptosis, but rather increased expression of the differentiation
inactivationofDENV2completelysuppressedtheinductionofthe and activation markers CD83 and CD86 (Fig.6G and S6A–C
immune response (S5A Figure and S5B Figure). Also, DPI Figure),CD40,CD80,CD86andPD-L1(S6DFigure).Whencells
inhibited antiviral and inflammatory responses induced by were pre-treated with DPI prior to DENV infection, the number
DENV2strain16681(S5CFigure),indicatingthatROS-mediated ofCD83-positivebystandercellsdecreasedby2.2fold,compared
antiviral induction is a common feature of the DENV2 serotype to non-treated cells (Fig. 6H). To determine if ROS production
and is not restricted to a specific strain. Collectively, these data altered the antiviral response in uninfected bystander cells via
demonstrate that DENV infection of Mo-DC triggers an cytokinerelease,conditionedmediafromDENV-infectedDCpre-
intracellular accumulation of NOX-derived ROS, which are treated or not with DPI was transferred to uninfected Mo-DC
essential for the induction of the antiviral and inflammatory (Fig.6I).Pre-treatmentwithconditionedmediafromDPI-treated
immune responsesandthecontrol of DENV infection. DCalteredthesusceptibilityofna¨ıvecellstoDENVinfection,as
shown by the ,2-fold increase in DENV E protein expression.
DENV-inducedROSdrivemitochondrialapoptosisinMo- Altogether, ROS contributes to mitochondria-dependent apopto-
DC and contribute to bystander cell maturation sis,andalsocontributestothematurationofuninfectedbystander
DC.
DENV-infectedDCwereclearlyapoptotic,basedonAnnexin-
Vstaining:2765.15%(infected)vs6.6961%(control)at24hand
Nrf2 transcription factor controls DENV infection and its
73.6564.2% (infected) vs 22.9663.88% (control) at 48h
(Fig.6A). Upregulation of mRNA levels for pro-apoptotic genes associated immune and apoptotic response
suchasBCLX,BIM,andCASP4uponDENVinfection(Fig.6B) Defense against sustained antioxidant production and the
was consistent with the transcriptome analysis that identified the inhibitionof ROSareimportantprotective mechanismsthat are
induction of apoptosis-associated pathways 24h after DENV regulated by the activation of Nrf2-transcription factor and
infection (Fig.2BandFig.3B). downstreamNrf2-targetgenes.Basedonthearraydata(Fig. 3A),
To assess therelease of mitochondrial ROS, cells were stained Nrf2targetgenessuchasHMOX-1,SOD2,NQO1,aswellasthe
with mitoSOX, a probe specific for mitochondria-derived ROS; metallothionein and ferritin families, were all rapidly stimulated
the number of mitoSOX-positive cells increased from bydenovoDENV2infection(Fig. 7A)andtransientinductionof
31.9612.3% (uninfected) to 56.867.9% (infected) at 48h after these genes was confirmed by qPCR (Fig. 7B). Levels of heme-
infection. DiOC was also used to determine the loss of oxygenase-1 (HMOX-1) and superoxide dismutase-2 (SOD-2)
6
mitochondrial potential upon DENV infection: only 7.960.2% mRNA were sensitive to the ROS scavenger DPI which
uninfected cells were positive, whereas 47.2610.2% of infected abrogatedtheincreaseinHMOX-1andSOD-2(Fig. 7C).When
cells were positive for mitochondrial depolarization. Consistent Nrf2 expression was silenced using Nrf2-specific siRNA (both at
with the release of mitochondrial ROS and mitochondrial the mRNA (Fig. 7D) and at the protein level (S7A Figure),
depolarization, intracellular levels of cleaved caspase-3 increased decreases in the mRNA levels of Nrf2-dependent antioxidant
from5.661.3%inuninfectedcellsto28.961.2%ininfectedcells genes were also observed (S7B Figure). Functionally, the redox
(Fig.6C). Regression analysis indicated that the percentage of homeostasis was critically affected in Nrf2-deleted Mo-DC, as
infected cells at 24h correlated with the percentage of apoptotic shown by the ,3 fold increase in ROS accumulation (S7C
cells at 48h after infection (Fig.6D (i)). Furthermore, both Figure). Although silencing of Nrf2 only slightly increased
mitochondrial ROS release and mitochondrial depolarization DENV2 RNA accumulation (Fig. 7E) and DENV infectivity
were statistically associated with apoptosis induction (Fig.6D (ii) (Fig. 7F) after 24 h of infection, the impairment of Nrf2
and (iii)), thus demonstrating DENV-infected Mo-DC undergo expression drastically potentiated oxidative stress response in
mitochondrial-dependent apoptosis. When DC were pre-treated DENV-infected cells (Fig. 7G). Indeed, a ,2 fold increase in
withtheNOX-inhibitorDPI,astatisticallysignificantdecreasein ROS generation was observed between DENV-infected control-
apoptosisofDENV-infectedcellswasobserved(,80%forDENV and siRNA-expressing, Nrf2-transfected cells (Fig. 7G) for the
only infection compared to ,52% for DENV+DPI infection), samenumberofinfectedcells(Fig. 7F).Finally,themRNAlevels
indicating that mitochondrial-dependent apoptosis was also of genes associatedwith the antiviral and inflammatoryresponse
dependent, at least in part, on NOX-generated ROS (Fig. 6E). such as IFIT1, RSAD2, DDX58, CXCL10 and IFNb (Fig. 7H),
Based on the array data, a key sensor of cellular stress, the as well as genes involved in the apoptotic response such as
transcription factor p53 was strongly activated following DENV NOXA, BCLX, and RIPK1 (Fig. 7I) were all significantly
infection (Fig.2D). Inhibition of p53, using the specific inhibitor increased. Altogether, these data demonstrate that the Nrf2-
pifithrin-awasabletopartiallysuppressDENV-inducedapoptosis regulated antioxidant pathway is stimulated as part of the stress
(Fig.6F),asdidthepan-caspaseinhibitorZ-VAD-fmkinMo-DC response after DENV infection; the Nrf2-dependent genes
(Fig.6F). Altogether, these results argue that NOX-dependent regulate the levels of ROS production and thus modulate the
induction of ROS stimulated p53-regulated mitochondrial and immune and apoptotic responses against DENV infection
caspase-dependent apoptosis. (Fig. 8).
PLOSPathogens | www.plospathogens.org 8 December2014 | Volume 10 | Issue 12 | e1004566
RedoxRegulationoftheHostResponsetoDENV
Fig.5.CellularoxidativestressresponseisrequiredtomediateDENV-inducedinnateimmuneresponses.Mo-DCwerepre-treated
withDPI(0.3or3mM)for1h,andsubsequentlyinfectedwithDENV2(MOI20)for24h.(A)Antiviralandinflammatoryresponsesweremonitoredby
immunoblotting. Dataare representative of at leasttwoindependentexperiments onseparatedonors. (B) Intracellularlevels of phosphorylated
PLOSPathogens | www.plospathogens.org 9 December2014 | Volume 10 | Issue 12 | e1004566
RedoxRegulationoftheHostResponsetoDENV
STAT1weredetectedbyPhosFlowinMo-DCinfectedfor24hbyDENV2(MOI20)orstimulatedfor30minwithIFN-b(1000IU/mL)andpre-treated
ornotwithDPI(1mM).Datarepresentthemeans6SEMfromtwoexperimentsperformedonseparatedonors.(C)Highthroughputanalysisofgene
expression evaluated by qPCR BioMark analysis. Gene expression levels were calculated using the DDCt method and gene-wise standardized
expression (z-score) were generated for each gene. The scale represents z-score values where red shows an up-regulation and blue a down-
regulationingeneexpression.Dataarerepresentativeofoneexperimentperformedonthreeindividualdonors.Eachboxoftheheatmaprepresents
onedonor.(D)Cytokinereleasewasevaluatedbycytokinebeadarray(CBA)onthesupernatantsofDENVinfectedcellspre-treatedornotwithDPI
(0.3–3mM).Datarepresentthemeans6SEMfromthreeindividualdonors.PvaluesweredeterminedbasedonthecomparisonwithDENV2-infected
cells.(E)Mo-DCwerepre-treatedwithDPI(3mM),Apocynin(3mM),TEMPOL(3mM),Ebselen(10mM),PDTC(40mM)andTrolox(5mM)for1h,and
subsequentlyinfectedwithDENV2(MOI20)for24h.AntiviralandinflammatorygeneexpressionwasdeterminedbyqPCR.Datarepresentthemeans
6SEMfromoneexperimentperformedonthreeindividualdonors.PvaluesweredeterminedbasedonthecomparisonwithDENV2-infectedcells.
(F) Mo-DC were transfected with control or gp91 phox siRNA and 48h later were infected with DENV2 (MOI 20). IFIT1 and gp91 phox protein
expressionlevelsweremeasuredbyimmunoblotanalysis.Resultisrepresentativeofoneexperiment.(G)Mo-DCwerepre-treatedwithDPI(0.1–
1mM)for1h,andsubsequentlyinfectedwithDENV2(MOI1)for48h.PercentageofinfectedcellswasdeterminedbyintracellularstainingofDENVE
protein(i–ii).DENVtitersweredeterminedbytransferringsupernatantsfromMo-DC-infectedcellsonA549cellsandstainingforDENVEprotein(iii).
DENVtiterswereexpressedasthenumberofinfectiousunits/mL.Datarepresentthemeans6SEMofexperimentsperformedonfour(i–ii)andtwo
differentdonors(iii).
doi:10.1371/journal.ppat.1004566.g005
Discussion syntheticdsRNAPoly(I:C)[65].Furthermore,exogenousaddition
of oxidative stress potentiated the TLR3 response to dsRNA in
Evaluation of the early host immune response to DENV airway epithelial cells [66]. Finally, the specific TLR7 agonist
infectionisessentialforacompleteunderstandingofthecomplex imiquimod also elevated basal superoxide production through
immunopathogenesis associated with the development of mild or enhancedNOX2activityinmacrophages[67].Furtherstudiesare
severedenguefeverinpatients.Previousstudieshavedemonstrat- now required to determine the exact mechanism(s) involved in
ed that DENV can trigger an innate immune response that DENV-inducedNOX-dependentROSproductioninhumanMo-
includes the release of antiviral and inflammatory cytokines DC.
[50,53,54], while other studies demonstrate the ability of DENV ROS were long considered as toxic, microbe-induced by-
toantagonizetheinductionofinnateresponsesviacleavageofthe products involved inthekillingofpathogens[18];however, their
endoplasmicreticulumadaptorSTING[51,55].Touncovernovel function as second messengers that regulate immune signaling
regulatory pathways involved in DENV infection of Mo-DC, we suggestsamuchbroaderroleinhostdefenseagainstviruses[19–
have for the first time used a transcriptome-wide expression 23]. ROS production was in fact required to trigger the antiviral
analysis, coupled with biochemical dissection, to investigate the and inflammatory responses to DENV infection in DC, and was
early host response to DENV infection in primary human confirmed by both chemical and genetic inhibition of the NOX
dendritic cells - an important pool of cells infected early in vivo complex. Blockade of NOX activation or ROS production
afterthebiteofthemosquitoAedesaegypti.Here,wedemonstrate inhibited antiviral and inflammatory responses, including the
that: 1) DENV preferentially infected myeloid cells as they IRF3/STAT1 antiviral axis and the NF-kB inflammatory
differentiated in vitro to mature Mo-DC; 2) DENV2 infection pathway (Fig.4). The IRF3 pathway has previously been
triggered antiviral, inflammatory, and oxidative stress pathways demonstrated to be regulated by oxidative stress variations.
with distinct kinetics; 3) DENV2 infection generated a NOX- Indeed,theexpressionlevelofthenon-canonicalIKK-likekinase,
dependentintracellularaccumulationofROS;4)ROSproduction IKKe, is itsef NOX-regulated and participated in the immune
mediated activation of the IRF3/STAT1- and NF-kB-mediated response induced by the respiratory syncytial virus (RSV) [68].
innate immune responses; 5) ROS production mediated p53 NOX-derived ROS were also shown to activate the RIG-I/
mitochondrial-dependent apoptosis and contributed to bystander MAVS/IRF3antiviralaxisinepithelialcells,andwererequiredto
Mo-DCmaturation/activation;and6)Nrf2-regulatedtargetgenes maintain the constitutive level of MAVS expression [22]. In
limited the oxidative stress response, and ultimately modulated contrast, statistical changes in MAVS or IKKe expression
ROS-induced immune and apoptotic responses. These results following NOX inhibition in primary DENV-infected DC were
highlight a requirement for the oxidative stress response in the notobservedinthisstudy(S8A–CFigure),suggestingthatDENV-
generation of the host innate immune response to DENV induced ROS may regulate host response via post-translational
infection. modification of proteins involved in antiviral signaling, as was
Activation of the NADPH-oxidase (NOX) complex and described previously for S-glutathionylation of TRAF3 and
generation of reactive oxygen species (ROS) has been described TRAF6 [19]. Other non-infectious biological processes such as
for several viral infections, including hepatitis C virus (HCV), impairment of autophagy also support the idea that oxidative
Rhinovirus, and HIV [56–58]. We demonstrate that DENV stress modulates the sensitivity to antiviral signaling. Indeed,
infectionalsoactivatesNOX-dependentROSproductioninMo- blocking of autophagy allows for oxidative stress accumulation
DC.Insomeinfectionmodels,viralproteinssuchasNefandTat through defective mitochondria and leads to the amplification of
for HIV and NS3 for HCV were shown to specifically stimulate RLR signaling [69]. Altogether, these studies cumulatively
the NOX complex [59–61]. NOX activity was also regulated by highlight the complexity of ROS involvement in the stimulation
spleen tyrosine kinase (Syk)-mediated phosphorylation of the ofantiviralresponsesandarguesthattheinnateimmuneresponse
NOX p47phox subunit [62]; Syk kinase is downstream of the integrates both viral RNA sensing and detection of homeostatic
surface receptor CLEC5A, which was shown to promote perturbations tocoordinatean appropriate host response.
inflammasome activation and inflammatory cytokine release in The Nrf2-mediated antioxidant response was one of the top
DENVinfection[63,64].Importantly,TLR3,areceptorcritically differentially regulated pathways early after DENV infection,
involvedinRNAsensing,wasrecentlyshowntostimulateNOX- resultingintheexpressionofmanycytoprotectiveenzymessuchas
dependent ROS production that was required for NF-kB, IRF3 HMOX-1, SOD2, NQO1, GCLC and GCLM, that function
andSTAT1activationinmurinemacrophagesinresponsetothe togethertomaintainanappropriateredoxstatus,andthusprotect
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