Vav Proteins Are Key Regulators of Card9 Signaling for Innate Antifungal Immunity

13 Pages·2016·2.87 MB·English

by  Susanne Roth

Article
Vav Proteins Are Key Regulators of Card9 Signaling
for Innate Antifungal Immunity
Graphical Abstract Authors
SusanneRoth,HannaBergmann,
MartinJaeger,...,MihaiNetea,
Xose´ R.Bustelo,Ju€rgenRuland
Correspondence
[email protected]
In Brief
CLR/Card9signalingisessentialfor
antifungalimmunity.Rothetal.have
identifiedtheVavfamilyofproteinsaskey
activatorsoftheCard9/NF-kBpathway,
essentialforCLR-inducedinflammatory
responsesandhostdefenseagainst
fungi.
Highlights Accession Numbers
d VavproteinscontrolCLR-mediatedinflammatoryresponses GSE83736
d CLR-inducedNF-kBactivationisregulatedbyVavproteins
d Vav/Card9signalingiscriticalforantifungalhostdefense
Rothetal.,2016,CellReports17,2572–2583
December6,2016CrownCopyrightª2016
http://dx.doi.org/10.1016/j.celrep.2016.11.018
Cell Reports
Article
Vav Proteins Are Key Regulators of Card9 Signaling
for Innate Antifungal Immunity
SusanneRoth,1,2HannaBergmann,1MartinJaeger,3,4AssaYeroslaviz,5KonstantinNeumann,1Paul-AlbertKoenig,1
ClarissaPrazeresdaCosta,6LesleyVanes,7VinodKumar,8MelissaJohnson,9MauricioMenacho-Ma´rquez,10
BiancaHabermann,5VictorL.Tybulewicz,7,11MihaiNetea,3Xose´ R.Bustelo,10,12andJu€rgenRuland1,13,14,15,*
1Institutfu€rKlinischeChemieundPathobiochemie,KlinikumrechtsderIsar,TechnischeUniversita¨tMu€nchen,81675Mu€nchen,Germany
2ChirurgischeKlinik,Universita¨tsklinikumHeidelberg,Ruprecht-Karls-Universita¨t,69120Heidelberg,Germany
3DepartmentofMedicine,RadboudUniversity,MedicalCentre,6500HBNijmegen,theNetherlands
4RadboudCenterforInfectiousDiseases,6500HBNijmegen,theNetherlands
5MaxPlanckInstituteofBiochemistry,ResearchGroupComputationalBiology,82152Martinsried,Germany
6Institutfu€rMedizinischeMikrobiologie,Immunologie,undHygiene,KlinikumrechtsderIsar,TechnischeUniversita¨tMu€nchen,
81675Mu€nchen,Germany
7FrancisCrickInstitute,LondonNW11AT,UK
8DepartmentofGenetics,UniversityMedicalCenterGroningen,UniversityofGroningen,Groningen,9700RB,theNetherlands
9DukeUniversityMedicalCenter,DukeBox102359,Durham,NC27710,USA
10CentrodeInvestigacio´ndelCa´ncer,CSIC-UniversityofSalamanca,37007Salamanca,Spain
11DepartmentofMedicine,ImperialCollege,LondonW120NN,UK
12CentrodeInvestigacionBiomedicaenRed-Oncologia,CarlosIIIHealthInstitute,Spain
13GermanCancerConsortium(DKTK),69120Heidelberg,Germany
14GermanCenterforInfectionResearch(DZIF),PartnerSiteMunich,Munich,Germany
15LeadContact
*Correspondence:[email protected]
http://dx.doi.org/10.1016/j.celrep.2016.11.018
SUMMARY for host defense and tissue homeostasis (Takeuchi and Akira,
2010).OneimportantfamilyofsignalingPRRsonmyeloidcells
Fungalinfectionsaremajorcausesofmorbidityand are the Syk-coupled C-type lectin receptors (Kerrigan and
mortality, especially in immunocompromised indi- Brown, 2011; Sancho and Reis e Sousa, 2012). These PRRs
viduals. The innate immune system senses fungal playabroadroleininnateimmunityandareparticularlyimpor-
pathogensthroughSyk-coupledC-typelectinrecep- tantforhostdefenseagainstfungalinfections(Ferwerdaetal.,
tors(CLRs),whichsignalthroughtheconservedim- 2009; Robinson et al., 2009; Saijo et al., 2007, 2010; Sato
etal.,2006;Tayloretal.,2007;Wellsetal.,2008),whichconsti-
mune adaptor Card9. Although Card9 is essential
tuteanincreasinghealththreatbecauseofgrowingnumbersof
for antifungal defense, the mechanisms that couple
patientsatriskmainlybecauseofimmunosuppressivemedical
CLR-proximal events to Card9 control are not well
interventions and AIDS. In the context of antifungal immunity,
defined.Here,weidentifyVavproteinsaskeyactiva-
theC-typelectinreceptor(CLR)familymemberDectin-1senses
tors of the Card9 pathway. Vav1, Vav2, and Vav3
b-glucans in fungal cell walls (Brown and Gordon, 2001),
cooperate downstream of Dectin-1, Dectin-2, and whereas Mincle and Dectin-2 detect a-mannose, glycolipids,
MincletoengageCard9forNF-kBcontrolandproin- and a-mannans, respectively (Sancho and Reis e Sousa,
flammatory gene transcription. Although Vav family 2012).AgonistbindingbyDectin-1leadstothephosphorylation
members show functional redundancy, Vav1/2/3(cid:1)/(cid:1) of immunoreceptor tyrosine-based activation motif (ITAM)-like
mice phenocopy Card9(cid:1)/(cid:1) animals with extreme motifsin its cytoplasmictail bySrc family kinases,resulting in
susceptibilitytofungi.Inthiscontext,Vav3isthesin- activation of the tyrosine kinase Syk. Likewise, Dectin-2 and
Mincle also activate Syk, indicating that these CLR signals
glemostimportantVavinmice,andapolymorphism
in human VAV3 is associated with susceptibility to engagecommoneffectormechanisms(Mo´csaietal.,2010;San-
choandReiseSousa,2012).Theinnateimmuneadaptorprotein
candidemiainpatients.Ourresultsrevealamolecu-
Card9iscriticalforCLRsignaling.Itassemblessignalingcom-
lar mechanism for CLR-mediated Card9 regulation
plexes that also contain Bcl10 and Malt1 (Card9-BCl10-Malt1
thatcontrolsinnateimmunitytofungalinfections.
[CBM]complexes)andthatserveasscaffoldsforactivationof
thecanonicalnuclearfactorkB(NF-kB)pathway(RothandRu-
INTRODUCTION land,2013).ThismechanismactivatestheinhibitorofkappaB
(IkB) kinase (IKK) complex, which phosphorylates inhibitory
Cellsoftheinnateimmunesystemsensemicrobialcomponents IkBs,leadingtotheirproteasomaldegradationandtherelease
or cell damage-associated structures via germline-encoded of NF-kB dimers to the nucleus to activate gene transcription
pattern recognition receptors (PRRs) that subsequently signal (VallabhapurapuandKarin,2009).Malt1canalsofunctionasa
2572 CellReports17,2572–2583,December6,2016CrownCopyrightª2016
ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
protease upon CBM complex assembly that cleaves a set of bySrcandSykkinases(Bustelo,2014).Todeterminetheimpor-
NF-kB regulators, including RelB, to fine-tune immune gene tanceoftheCLR-proximalSrcandSykkinasesinVavphosphor-
expression(Hailfingeretal.,2011;Jaworskietal.,2014).These ylation,wepretreatedBMDCswiththeSrcinhibitorPP2orthe
Card9 signaling complexes operate downstream of all tested SykinhibitorR406priortotheadditionofC.albicans.BothSrc
Syk-coupled CLRs (Roth and Ruland, 2013) and are essential and Syk inhibition blocked Vav1-Tyr174 phosphorylation in
for innate antifungal immunity. Indeed, Card9-deficient mice adose-dependentmanner,whereaspretreatmentwithPP3,an
are highly susceptible to infection with Candida (Gross et al., inactiveanalogofPP2,didnotaffectC.albicans-inducedphos-
2006; Jia et al., 2014), Aspergillus (Jhingran et al., 2012), and phorylationofVav1atTyr174(Figure1C).Together,theseresults
Cryptococcus (Yamamoto et al., 2014) species. Moreover, indicate that Vav1 is activated in myeloid antigen-presenting
loss-of-function mutationsinhuman CARD9havebeenidenti- cells specifically after fungal sensing via an Src and Syk ki-
fiedascausesofmucocutaneousandinvasivefungalinfections nase-dependentmechanism.
(Glockeretal.,2009;Pe´rezdeDiegoetal.,2015).Nevertheless,
despite the critical role for CLR-triggered Card9 signaling in CLR-MediatedInflammatoryResponsesAreCritically
innate immunity and mammalian host defense, the molecular DependentonVavProteins
mechanismsthatlinkCLRligationtoCard9-dependenteffector Next, we studied the functional relevance of Vav proteins in
mechanismsarenotwellunderstood. C.albicans-inducedinflammatoryresponses.Becausedendritic
Here, we used a mass spectrometry-based proteomic cellsexpressVav2(Spurrelletal.,2009)inadditiontoVav1and
approach and identified Vav proteins as regulators of Card9 Vav3,whichhavepartiallyredundantfunctionsinimmunecells
signaling. Vav1, Vav2, and Vav3 cooperate downstream of (Fujikawa et al., 2003; Tybulewicz, 2005), we investigated
Dectin-1,Dectin-2,andMincletoengageCard9complexesfor BMDCs from mice that lack each individual Vav isoform, two
NF-kB control and proinflammatory gene transcription. Like Vav isoforms, or all three Vav family members. Notably, the
Card9-deficientmice,Vav1/2/3triple-deficientmiceareseverely lackofanyoneorallVavmoleculesdidnotimpairBMDCdiffer-
impairedininflammatoryresponsestoCandidaalbicansinfec- entiation (data not shown; Graham et al., 2007; Spurrell et al.,
tionandhostdefenseagainstthefungus.Moreover,wereport 2009). However, BMDCs from Vav1/2/3 triple-deficient mice
ahumanpolymorphisminVAV3thatisassociatedwithsuscep- wereseverelydefectiveinC.albicans-inducedTNFproduction,
tibilitytocandidemia.Thus,ourresultsestablishVavproteinsas whereas tumor necrosis factor (TNF) secretion in response to
essential regulators of CLR-mediated Card9 control in innate Toll-like receptor (TLR) stimulation with LPS or Pam3CSK4
antifungalimmunity. wasunaffectedinthesecells(Figure2A).Moreover,theproduc-
tionofinterleukin-2(IL-2)andIL-10wasalsodefectiveinBMDCs
RESULTS fromVav1/2/3(cid:1)/(cid:1)mice(Figure2A).ThefindingthatVav1,Vav2,
orVav3single-deficientorVav2/3(cid:1)/(cid:1)myeloidantigen-present-
FungalInfectionInducesTyrosinePhosphorylationof ingcellsareonlypartiallyimpairedinC.albicans-inducedTNF,
VavinMyeloidCells IL-2,andIL-10generation(Figure2B)indicatesredundancyfor
ToinvestigatethemechanismsofSyk-coupledCLRsignaling,we Vavproteinsinfungus-inducedcytokineresponses.
stimulatedwild-typemurinebonemarrow-deriveddendriticcells Oneimportantinflammatorycytokineforantifungalimmunity
(BMDCs), comprisingconventional DCs and monocyte-derived isIL-1b(Vonketal.,2006),thegenerationofwhichiscontrolled
macrophages(Helftetal.,2015),for10minwithzymosan,ayeast by NF-kB-dependent pro-IL-1b gene transcription followed by
cellwallpreparationthatishighlyenrichedinDectin-1andDec- NLRP3 inflammasome-mediated Caspase-1-dependent pro-
tin-2agonists,andsubsequentlyaffinity-purifiedtyrosine-phos- IL-1bprocessing(Grossetal.,2009;Hiseetal.,2009).Stimula-
phorylated proteins for mass spectrometric analysis (Strasser tion of wild-type BMDCs with C. albicans induced the robust
etal.,2012).Undertheseconditions,weobservedsignal-induced secretionofmatureIL-1bintotheculturesupernatant(Figure2C).
tyrosinephosphorylationofVav1andVav3,whicharecytosolic In contrast, Vav1/2/3-deficient cells were almost completely
signaling scaffolds and guanine nucleotide exchange factors defective in Candida-induced IL-1b production (Figure 2C).
thatcanplaycontext-specificrolesinimmunereceptorpathways Thisdefectwascausedbytheselectiveimpairmentofpro-IL-
(Bustelo,2014).Tovalidatethesefindings,westimulatedBMDCs 1bgenetranscriptioninVav1/2/3(cid:1)/(cid:1)cells(Figure2D),whereas
withC.albicanshyphaeandspecificallyanalyzedVav1phosphor- thedeficiencyof Vavproteins didnotaffectNLRP3inflamma-
ylationbywesternblotanalysis.Indeed,Vav1wastyrosine-phos- some-dependent Caspase-1 activation (Figure 2E). Together,
phorylatedafterC.albicansinfection(Figure1A).Thesedataarein these experiments demonstrate that Vav proteins are critical
linewithpreviouslypublishedresultsthatdemonstratedthatstim- forcytokineproductionbymyeloidcellsfollowingthedetection
ulationwithb-glucanorzymosantriggerstyrosinephosphoryla- ofwholefungalcells.
tionofVav1alsoinmicroglialcellsorneutrophils(Lietal.,2011; Next,westudiedthespecificrequirementofVavproteinsfor
Shah et al., 2009). Further analysis with a phosphospecific Dectin-1-, Dectin-2-, and Mincle-induced cytokine responses
antibody raised against the regulatory Tyr174 residue of Vav1 usingselectiveagonistsforeachindividualreceptor.Stimulation
additionallyrevealedthatthisspecificresidueisphosphorylated of Dectin-1 with curdlan, a purified particulate b-glucan (Lei-
uponC.albicansdetection,indicatingVav1activation(Aghazadeh bundGut-Landmannetal.,2007),inducedtherobustproduction
etal.,2000),butnotuponLPSstimulation(Figure1B). of TNF in wild-type BMDCs, whereas Vav1/2/3-deficient
Recent work has demonstrated that Vav phosphorylation cells failed to produce TNF in response to Dectin-1 triggering
downstreamofdifferentITAM-containingreceptorsismediated (Figure 2F). Likewise, stimulation of BMDCs with agonistic
CellReports17,2572–2583,December6,2016 2573
A B
C
Figure1. Vav1IsTyrosine-PhosphorylatedinResponsetoCandida
(A)BMDCswereuntreated((cid:1))orstimulatedwithC.albicanshyphae(MOI5)for15min.Proteinsfromcelllysateswereimmunopurifiedwithanti-phospho-
tyrosineantibodiesandanalyzedbyimmunoblotwithaVav1antibody(left).AlsoshownisquantificationofVav1bydensitometry(right).
(B)ImmunoblotanalysisofBMDCsunstimulated((cid:1)),treatedwithC.albicanshyphae(MOI0.5)fortheindicatedtimes,orstimulatedwithLPS(100ng/ml)for
45minandprobedwithantibodiesagainstphospho-Vav1(Tyr174)orVav1(left).Alsoshownisdensitometricalquantificationofphospho-Vav1relativetototal
Vav1,normalizedtoBMDCstreatedwithC.albicansfor45min(right).
(C)BMDCswereuntreated((cid:1))orpreincubatedwiththeSykinhibitorR406(0.5,1,and2mM)ortheSrc-kinaseinhibitorPP2(1.5,3,and6mM)oritsinactiveanalog
PP3(1.5,3,and6mM)andthenstimulatedwithC.albicanshyphae(MOI1)for30min.Lysateswereimmunoblottedwithanti-phospho-Vav1(Tyr174)oranti-Vav1
antibodies(left).Alsoshownisdensitometricalquantificationofphospho-Vav1relativetototalVav1,normalizedtoBMDCstreatedwithC.albicanswithoutany
inhibitors(right).Dataofatleastthreeindependentexperimentsareshownasmean±SEM.
*p<0.05,**p<0.01,***p<0.001;Student’sttest(A)andone-wayANOVAandposthocTukey-Kramertest(C).n.s.,notsignificant.
antibodies against Dectin-2 or with trehalose-6,6-dibehenate Mincle ligand TDM (Ishikawa et al., 2009; Miyake et al., 2013)
(TDB),asyntheticanalogofthemycobacterialcordfactortreha- intowild-typeandVav1/2/3(cid:1)/(cid:1)mice.Consistentwithpreviously
lose6,60-dimycolate(TDM),whichspecificallyactivatesMincle publishedresults(Ishikawaetal.,2009),TDMinjectionresulted
(Lobato-Pascual et al., 2013; Miyake et al., 2013; Schoenen in the strong systemic production of TNF, IL-1b, and IL-10 in
etal.,2010),inducedTNFinwild-typecells,butthisresponse wild-type mice (Figure 2H). However, these responses were
wasseverelyimpairedinVav1/2/3-deficientBMDCs(Figure2F). defectiveinVav1/2/3triple-deficientanimals(Figure2H).Next,
Therefore,althoughVavsingle-ordouble-mutantcellsexhibited westudiedtheroleofVavproteinsininflammatorycytokinere-
apartiallyimpairedcytokineresponseuponselectiveDectin-1, sponses after fungal infection in vivo. To this end, we intrave-
Dectin-2,orMinclestimulation(Figure2G),theVavfamilyasa nouslyinjectedwild-typeorVav1/2/3tripleknockoutmicewith
whole is essential for cytokine production after Syk-coupled liveC.albicansandmeasuredserumTNFandIL-6levelsafter
CLRligationinvitro. 6 hr. Notably, the production of both inflammatory cytokines
was almost completely abolished in Vav1/2/3(cid:1)/(cid:1) mice (Fig-
VavProteinsRegulateCLR-InducedInflammatory ure3A).Subsequently,westudiedtheimportanceofindividual
ResponsesInVivoandAreEssentialforAntifungal Vavproteins in hostprotection against fungi byinfecting wild-
Defense type or Vav single-, double-, or triple-deficient mice with 1 3
To investigate the function of Vav proteins in CLR-mediated 105colony-formingunits(CFUs)ofC.albicans.Fourdayslater,
inflammatory responses in vivo, we intravenously injected the we assessed intravital fungal growth in the kidneys, the main
2574 CellReports17,2572–2583,December6,2016
A B
C D E
F G
H
Figure2. VavProteinsAreEssentialforSyk-CoupledCLR-InducedProinflammatoryResponses
(AandB)TNF,IL-2,andIL-10levelsinthesupernatantsofwild-type(WT)andVav1/2/3-/-BMDCs(A),orVavsingle-ordoublemutantBMDCsoftheindicated
genotypes(B)thatwereuntreated((cid:1))orstimulatedwithC.albicanshyphae(MOI0.3),LPS(500ng/ml),orPam3CSK4(50ng/ml)for16hrwereanalyzedbyELISA.
(C)IL-1binthesupernatantsofWTandVav1/2/3(cid:1)/(cid:1)BMDCsleftuntreated((cid:1))orstimulatedwithC.albicanshyphae(MOI1)for6hr.
(D)Quantitativereal-timePCRanalysisofIL-1btranscriptsinWTandVav1/2/3(cid:1)/(cid:1)BMDCsleftuntreatedorstimulatedwithC.albicanshyphae(MOI1)(left)orLPS
(100ng/ml)(right)fortheindicatedtimes;resultsarerelativetothoseofb-actinmRNA.
(E)ImmunoblotanalysisofmatureCaspase-1(p10)incellculturesupernatantsofWTandVav1/2/3(cid:1)/(cid:1)((cid:1)/(cid:1))BMDCsleftuntreated((cid:1)),stimulatedwithC.albicans
hyphae(MOI1)for4hr(C.a.),orpre-stimulatedwithLPS(50ng/ml)for6hrpriortotheadditionofATP(5mM)for45min(A+L).QuantificationofmatureCaspase-1
p10bydensitometryisshowntotherightofthewesternblot.
(legendcontinuedonnextpage)
CellReports17,2572–2583,December6,2016 2575
target organ of the fungus (Brieland et al., 2001). Although and S1B), this response is almost completely abolished in the
wild-type mice cleared the fungus readily, the kidneys of all absenceofVavproteins(Figure4C;FigureS1A)orPKCd(Fig-
investigated Vav1/2/3(cid:1)/(cid:1) mice were enlarged and infiltrated ureS1B;Strasseretal.,2012).Consistentwiththesefindings,
withmacroscopicallyvisiblefungalcolonies(Figure3B).Quanti- C.albicans-inducednucleartranslocationofthetranscriptional
tatively, we detected more than 100-fold higher titers of activeNF-kBsubunitsp65,c-Rel,andRelBwerealsoseverely
C. albicans in the kidneys of Vav1/2/3 triple-deficient animals defectiveinVav1/2/3(cid:1)/(cid:1)BMDCs,astheyareinCard9-deficient
compared with the wild-type (Figure 3C), and histopathology cells(Figures4Dand4E;FiguresS1CandS1D),demonstratinga
confirmed intravital fungal growth (Figure 3D). The fungal bur- criticalroleforVavproteinsinCLR-dependentNF-kBcontrol.
dens in Vav3 single knockout and Vav2/3 double knockout As indicatedabove, the activation of the Card9/Bcl10/Malt1
mice were increased by trend compared with the wild-type, complex not only regulates IKK activation but also induces
butaselectivelossofVav1orVav2didnotresultinhigherfungal Malt1 proteolytic activity (Jaworski et al., 2014). Consistently,
titerscomparedwithwild-typemice(Figure3C).Finally,westud- stimulationofwild-typeBMDCswithCLRligandsresultsinthe
iedtheroleofVavsignalinginthesurvivalofmiceafterfungal rapidproteolyticprocessingoftheMalt1substrateRelB(Figures
infection. In line with the incapacity to control fungal invasion 4Fand4G;FiguresS1EandS1F;Gewiesetal.,2014;Hailfinger
oftheorgans,Vav1/2/3-deficientmicediedrapidlyafterinjection etal.,2011;Jaworskietal.,2014).Curdlan-inducedRelBcleav-
of13105CFUofC.albicans,whereasthewild-typecontrolan- agewascompletelydefectiveinBMDCsfromMalt1(cid:1)/(cid:1)miceand
imals survived this challenge (Figure 3E). These experiments frommicewithgene-targetedinactivationoftheMalt1paracas-
reveal that Vavproteins play essential roles in fungus-induced pase function (Malt1PM/(cid:1)) (Gewies et al., 2014; Figure 4F; Fig-
cytokineproductionandantifungalimmunityinvivo,withVav3 ure S1E) and, therefore, can be used as a marker for CBM
beingthesinglemostimportantVavfamilymemberforprotec- complex activation. Likewise, Malt1-mediated RelB cleavage
tionagainstC.albicansinfection. uponC.albicansstimulationwasalsodefectiveinVav1/2/3triple
knockout cells (Figure 4G; Figure S1F), which, together with
VavProteinsAreRegulatorsofCLR-InducedNF-kB the defective IKK activation in Vav1/2/3(cid:1)/(cid:1) BMDCs, indicates
Activation that Vav proteins operate upstream of the Card9/Bcl10/Malt1
The susceptibility of Vav1/2/3 triple-deficient animals and the signalosome.
failuretoinduceCLR-mediatedcytokineresponsesarereminis- Finally, we studied the role of Vav proteins in CLR-induced
centoftheseverephenotypeobservedinCard9-deficientmice, geneexpressioninaglobalmanner.Tothisend,weperformed
suggestingthatVavproteinsandCard9mayoperateinacom- high-throughput cDNA sequencing (RNA sequencing [RNA-
mon signaling cascade. Because of the partially overlapping seq]) followed bygene setenrichment analysis (GSEA) of cur-
functions of the three Vav isoforms, we focused subsequent dlan-stimulated and untreated BMDCs. In Dectin-1-stimulated
biochemical studies on Vav1/2/3 triple knockout BMDCs. wild-typeBMDCs,wedetectedasignificantenrichmentofupre-
UponC.albicansstimulation,phosphorylationofthetyrosineki- gulated NF-kB-dependent transcripts compared with Vav1/
naseSykandoftheDectin-1signaltransducerPLCg2(Xuetal., 2/3(cid:1)/(cid:1) cells (Figure 4H), which isconsistent with the defective
2009)didnotdifferbetweenwild-typeandVav1/2/3(cid:1)/(cid:1)BMDCs IKK activation described above. These results further validate
(Figure 4A), indicating that Vav proteins are not required for Vav proteins as essential regulators of CLR-mediated NF-kB
the immediate receptor-proximal events. Moreover, because control.
Erk1/2, p38, and Jun N-terminal kinase (JNK) were similarly
phosphorylated in C. albicans-stimulated wild-type and Vav1/ AHumanVav3PolymorphismIsAssociatedwith
2/3(cid:1)/(cid:1)BMDCs(Figure4B),weconcludethatVavsarenotessen- SusceptibilitytoCandidemia
tialfortheregulationofmitogen-activatedproteinkinase(MAPK) AfteridentifyingVavproteinsasintegralregulatorsoftheDec-
signalingafterfungalinfection.However,activationofproteinki- tin-1/Card9/NF-kB signaling cascade in murine cells, we were
nase C d (PKCd), which controls Card9 engagement (Strasser interestedinthepotentialrolesforVavmoleculesinhumananti-
etal.,2012),waspartiallyimpairedinBMDCslackingallthree fungalimmunity.Therefore,weinvestigatedwhethergeneticvar-
Vavisoforms(Figure4C;FigureS1A).Likewise,PKCddeficiency iationslinkedtoanyoftheVAVgenescorrelatewithsusceptibility
partially compromised Vav1 phosphorylation in response to to candidemia in patients by studying a previously described
C. albicans or curdlan stimulation (Figure S1B). Moreover, cohortofcandidemiapatientsandanappropriatecontrolgroup
althoughC.albicansorcurdlanstimulationofwild-typeBMDCs (Jaegeretal.,2015).Interestingly,theanalysisofSNPsassoci-
inducestherapidactivationoftheIKKcomplex,asdetermined atedwithVAV3revealedasignificantassociationwithcandide-
by measuring IKKa/bphosphorylation (Figure 4C;Figures S1A mia, with the significant SNPs distributed across the linkage
(FandG)BMDCsfromWTandVav1/2/3-/-mice(F),orVavsingle-ordoublemutantmiceoftheindicatedgenotypes(G)wereuntreated((cid:1)),stimulatedwiththe
Dectin-1ligandcurdlan(10mg/ml),plate-boundanti-Dectin-2(12.5mg/ml)orisotypecontrolantibodies(Isotype),ortheMincleligandTDB(100mg/ml).TNFlevels
inthecellculturesupernatantswerequantifiedbyELISA.
(H)WTandVav1/2/3(cid:1)/(cid:1)micewereinjectedintravenouslywithTDMcontainingoil-in-wateremulsionsorwiththevehiclecontrol((cid:1)).24hrpost-injection,TNF,
IL-1b,andIL-10levelsinseraweredeterminedbyCBA.
(A,B,F,andG)DataareexpressedaspercentofWT±SEMofthreeindependentexperiments.
(C–EandH)Dataofatleastthreeindependentexperimentsarepresentedasmean±SEM.
*p<0.05,**p<0.01,and***p<0.001,Student’sttest(A,C,F,andH),andone-wayANOVAandposthocTukey-Kramertest(BandG).
2576 CellReports17,2572–2583,December6,2016
A B C
D E
Figure3. VavProteinsAreRequiredforSystemicAntifungalHostDefense
(A–E)Miceoftheindicatedgenotypeswereintravenouslyinfectedwith13105CFUsofC.albicans.
(A)SerumIL-6andTNFlevelsinWTandVav1/2/3(cid:1)/(cid:1)miceweredeterminedbyCBA6hrafterinfection.
(BandC)After4days,kidneysweremacroscopicallyexamined(B;scalebar,10mm),andC.albicanstitersweredeterminedinthekidneys(C).
(D)KidneysectionsfromC.albicans-infectedWTandVav1/2/3(cid:1)/(cid:1)micewerestainedwithH&Eorperiodicacid-Schiff(PAS;scalebars,100mm).
(E)WT(n=5)andVav1/2/3(cid:1)/(cid:1)(n=4)miceweremonitoreddailyforhealthandsurvival.Statisticalsurvivalanalyseswereperformedusingthelog-ranktest
(p<0.005).
(AandC)Eachsymbolrepresentsanindividualmouse;smallhorizontallinesindicatethemean;errorbarsindicatetheSEM.Dataofoneexperimentineachcase
arepresented.*p<0.05,Student’sttest(AandC,left)andone-wayANOVA(C,right,p=0.06).
disequilibrium(LD)regionofthegene.Theassociationdoesnot dependent NF-kB activation similar to Card9 deficiency, with
extend beyond the LD region, and the strongest association Vav3beingthemostimportantfamilymemberinthispathway
waswiththeSNPrs4914950(Figure5A).Theriskgenotypeof inmiceand,presumably,inhumans.
theSNPrs4914950TTisincreasedfrom10%(controlpopulation) When engaged by fungal particles, Syk-coupled CLRs acti-
to18%(candidemiapatients)(Figure5B). vate various intracellular signaling pathways that regulate
phagocytosis, microbicidal activities, and gene transcription.
DISCUSSION Using C. albicans as a clinically relevant model organism that
issensedbymultipleCLRsandinducesCLRsignaling(Robin-
Using a complementary approach of immunology, molecular son et al., 2009; Taylor et al., 2007; Wells et al., 2008), we
biology,andgeneticstudies,theresultspresentedinthismanu- observedVavactivationdownstreamofSyk.Inaddition,using
scriptdefineanessentialroleforVavproteinsinCLR-mediated defined agonists of Dectin-1, Dectin-2, and Mincle, we estab-
inflammatory responses and establish these molecules as lishedageneralroleforVavproteinsinCLRsignalinginmyeloid
essential signaling platforms that relay proximal events from antigen-presenting cells. However, our results that CLR-medi-
Syk-coupled CLRs to the Card9 signaling complex for NF-kB ated MAPK activation and inflammasome signaling are not
control and antifungal defense (Dambuza and Brown, 2015; impairedinVav1/2/3triple-deficientBMDCsrevealthatVavpro-
Osorio and Reis e Sousa, 2011; Roth and Ruland, 2013). teins only mediate specific CLR effector mechanisms. Recent
Although individual Vav isoforms can compensate for each studies have indicated the involvement of Vav1 and Vav3 in
other, their combined absence results in a blockade of CLR- Dectin-1-mediatedzymosanphagocytosisandreactiveoxygen
CellReports17,2572–2583,December6,2016 2577
A B
C
D
E
H
F
G
Figure4. VavProteinsControlCLR-TriggeredNF-kBActivation
(A–E)BMDCsfromtheindicatedgenotypeswerestimulatedwithC.albicanshyphae(MOI0.5)forvarioustimesorwithLPS(100ng/ml)for45min.
(A)SykandPLCg2phosphorylationwasdeterminedincelllysatesbyimmunoblotwithanti-phospho-Sykandanti-phospho-PLCg2antibodies.
(B)ActivationoftheMAPkinasesErk1/2,p38,andJNKwasanalyzedwithanti-phospho-Erk1/2,anti-phospho-p38,andanti-phospho-JNKantibodies.
(C)Celllysateswereanalyzedbyimmunoblotwithanti-phospho-PKCdandanti-phospho-IKKa/bantibodies.b-actinservedasaloadingcontrol.
(DandE)NuclearextractsfromWTandVav1/2/3-/-(D),orCard9-/-BMDCs(E)wereanalyzedbyimmunoblotwithantibodiesagainsttheNF-kBsubunitsp65,
c-Rel,andRelB;anti-laminBantibodiesindicateequalproteinloading.
(legendcontinuedonnextpage)
2578 CellReports17,2572–2583,December6,2016
species(ROS)productioninneutrophilsandtherecruitmentof dependence of these mechanisms should be defined in the
Vav1tophagocyticcupsforC.albicansengulfmentbymacro- future.
phages (Li et al., 2011; Strijbis et al., 2013). Although these Inaccordancewithourmolecularmodel,similartoCard9defi-
effector mechanisms are important during innate immune re- ciency, a complete deficiency in all Vav isoforms results in a
sponses,theyarenotregulatedbytheCard9pathway(Drewniak massivesusceptibilitytoC.albicansinfection.Likewise,acondi-
etal.,2013;Goodridgeetal.,2009;Grossetal.,2006).However, tional deficiency of Syk only in dendritic cells also abolishes
theCard9signalingpathwayappearstobethecentralmamma- innateresistancetoacutesystemicC.albicansinfection(Whit-
lianhostdefensemechanismagainstfungi,giventhatmultiple ney et al., 2014), and Whitney et al. (2014) elegantly demon-
geneticstudiesinhumanshaverecentlyidentifiedthatseveral strated that the early innate cytokine response is required to
Card9loss-of-functiondefectsarecausativeforseveralfungal orchestrate innate anti-fungal functions, including neutrophil
diseases, including various types of mucocutaneous candidi- anti-fungal activity. Nevertheless, in our infection models pre-
asis;superficial,extensive,anddeepdermatophytosiswithTri- sentedhere,itmustbenotedthattheVavmutantmiceanalyzed
chophyton spp.; subcutaneous phaeohyphomycosis; invasive were germline Vav-deficient animals and thatVav proteins are
Candidainfectionsofthedigestivetractandcentralnervoussys- not selectively expressed in Syk and Card9-containing innate
tem;Candidaendophthalmitisandosteomyelitis;anddissemi- immunecells,includingdendriticcells,macrophages,andneu-
natedExophialadiseaseoftheliver,brain,andlung(Grumach trophils, but are also expressed in lymphocytes, which signal
etal.,2015;Pe´rezdeDiegoetal.,2015). through Card9-independent mechanisms (Gross et al., 2006;
WeassessedenzymaticMalt1paracaspaseactivityasadirect Hara et al.,2007). Still, the rapid deathof Vav1/2/3 triple-defi-
functional marker for Card9 complex activity in Vav-deficient cientmiceuponfungalinfectionindicatesthataninnateimmune
mice.GiventhatthisactivitywasseverelyimpairedinVav1/2/3 defectisindeed responsible forthe observedphenotype. This
triple-deficientBMDCs,ourresultsestablishVavmoleculesas hypothesisisalsoinlinewiththeestablishedrolesofVav1and
regulators of the Card9/Bcl10/Malt1 complex. Consistent with Vav3inmediatingphagocytosisandROSproductioninmacro-
theessentialfunctionofaVav/Card9-mediatedmechanismfor phagesandneutrophilsuponfungalsensing(Lietal.,2011;Strij-
CLR-inducedNF-kBactivation,theNF-kBresponseisblocked bisetal.,2013).FuturestudieswithconditionalVavmutantmice
in Vav1/2/3-deficient mice in response to C. albicans infection will further dissect the individual roles of Vav signaling in den-
or selective CLR triggering, and Vav1/2/3-deficient animals driticcells,macrophages,andneutrophilsaswellasinadaptive
exhibitdramaticallyimpairedproinflammatorycytokineproduc- immune subsets during complex fungal infection scenarios
tion in response to C. albicans infection and CLR stimulation in vivo. In addition, in vivo results in individual Vav1-, 2-,
invitroandinvivo.Therefore,weproposeamechanisticmodel and 3-deficient animals also revealed that, although there is
inwhichSykisengagedbyCLRsuponfungalsensing,thereby functionalredundancyamongthethreeVavfamilymembersin
triggering the phosphorylation and activation of Vav proteins, infection control, Vav3 is the single most important Vav family
whichsubsequentlyactivatetheCard9complexforNF-kBcon- memberforantifungaldefenseinmice.Thisfindingisconsistent
trolandantifungalgenetranscription. withourresultsinpatientswholinkapolymorphismintheVAV3
HowVavproteinsaremechanisticallycoupledtoCard9con- genetosusceptibilitytoCandidainfection,suggestingthatthe
trol remains to be determined. We previously demonstrated functionofVavmoleculesissimilartoCard9functionconserved
that Card9 is phosphorylated by PKCd at Thr231 to induce betweenmiceandmen.
Card9 effector function. However, the activation of PKCd is Inadditiontofungalcomponents,CLRsalsorecognizestruc-
only partially impaired in Vav-deficient BMDCs, whereas IKK tures present on mycobacteria (Ishikawa et al., 2009; Lobato-
activation is almost completely defective. Similarly, in PKCd- Pascualetal.,2013;Miyakeetal.,2013;Schoenenetal.,2010),
deficientcells,IKKphosphorylationisabolished,whereasphos- virusessuchasdenguevirus(Chenetal.,2008),andparasites
phorylation of Vav1 is only slightly compromised, suggesting, such as Schistosoma mansoni (Ritter et al., 2010), as well as
together,thatVavandPKCdoperateatthesamelevelupstream endogenousligandsundersterileinflammatoryconditions(Roth
ofCard9.BecauseVavproteinscancontrolcytoskeletalreorga- andRuland,2013).Wethereforebelievethatourfindingshave
nization and serve as scaffolding platforms in other signaling implicationsbeyondantifungalimmunity.Consistentwiththishy-
systems (Acton et al., 2012; Bustelo, 2014; Spurrell et al., pothesis,Vav1/2/3(cid:1)/(cid:1)micewerealmostcompletelyimpairedin
2009;Tybulewicz,2005),wespeculatethatVavfamilymembers systemicinflammatoryresponsesuponinnateimmunestimula-
could bring Card9 into the vicinity of key upstream regulators tionwiththemycobacterialpathogen-associatedmolecularpat-
such as PKCd and potential, still unknown factors to allow terns (PAMP) analog TDM, implying that, like Card9 (Dorhoi
Card9activation.Moreover,becauseVav1/2/3donotonlyregu- etal., 2010), Vavproteinsmight alsobe criticalforinnateanti-
lateCLR-inducedCard9-NF-kBsignalingbutalsocontrolother mycobacterialimmuneresponses.Inaddition,accumulatingevi-
cellularresponses,suchasphagocytosisandROSproduction denceindicatesimportantrolesforCLR/Card9signalingininflam-
(Lietal.,2011;Strijbisetal.,2013),theprecisemolecularinter- matory disorders. Several independent studies have shown an
(FandG)BMDCsfrommiceoftheindicatedgenotypeswerepretreatedwiththeproteasomeinhibitorMG132for30minandthenstimulatedwithcurdlan
(0.5mg/ml)(F)orC.albicanshyphae(MOI1)(G)for2or4hr.CelllysateswereanalyzedbyimmunoblotusingantibodiesagainstRelBandb-actin.
(H)WTandVav1/2/3(cid:1)/(cid:1)BMDCswerestimulatedwithcurdlan(0.2mg/ml)for3hr.ShownistheGSEAenrichmentscoreofNF-kBtargetgenesdifferentially
expressedincurdlan-stimulatedwild-typecellscomparedwithVav1/2/3-deficientBMDCs.
Dataarerepresentativeofatleastthreeindependentexperiments.SeealsoFigureS1.
CellReports17,2572–2583,December6,2016 2579
A B
Figure5. AVAV3PolymorphismIsAssociatedwithIncreasedSusceptibilitytoCandidemia
(A)Polymorphismsassociatedwithcandidemiawereinvestigatedinagroupof227patientswithCandida-positivebloodculturesandcomparedwith176
patientsfromthesameclinicalwardswithoutinfection.SNPsassociatedwiththeVAV3locusrevealedasignificantassociationwithcandidemia,withthe
significantSNPsdistributedacrosstheLDregionofthegene.TheVAV3generegiononchromosome1isshown.UsingtheSNAPserver,theSNPofinterest
(rs4914950)(biggestorangediamond)wasplottedwithallannotatedpolymorphismsinthisgene(otherdiamonds).R2andrecombinationrateareindicatedon
theleftandrightyaxis,respectively.DiamondsonthesamehorizontallineastheSNPofinterest(biggestorangediamond)areincompletelinkagedisequilibrium
(R2=1)withthisSNPandhavethesamediseaseassociationvalues.DiamondsbelowthislinehavealowerLDvaluewiththeSNPofinterestand,therefore,differ
intheassociationwiththedisease.Thetablebelowshowsthetop15polymorphismsthatarelinkedtors4914950.
(B)Genotypefrequenciesforthers4914950SNPthatshowedthestrongestassociationwithcandidemia.Thetablebelowshowsthetotalnumbersandper-
centages(inparentheses)ofgenotypesinCandida-infected(candidemiapatients)andnon-infected(controls)individuals.Statisticalcomparisonsofgenotype
frequenciesbetweenthetwogroupsweremadeusingc2test.
associationbetweenCard9polymorphismsandulcerativecolitis nephropathy(Kiryluketal.,2014).Inconjunctionwiththeresults
and Crohn’s disease (Roth and Ruland, 2013), which are sup- presented here, these data suggest that the described Vav/
portedbyarecentstudyinmicedemonstratingaprotectiverole Card9-dependentsignalingmechanismcouldalsoplayarolein
forCard9inintestinalinflammation(Sokoletal.,2013).Deepre- sterileinflammatorydiseases.
sequencingofinflammatoryboweldiseasegenome-wideassoci-
ationstudy(GWAS)lociconfirmedanassociationbetweenCard9
EXPERIMENTALPROCEDURES
andulcerativecolitisandCrohn’sdisease(Beaudoinetal.,2013;
Hongetal.,2016).Moreover,additionalgenome-wideassociation
Mice
studies in humans have identified significant associations be- Vav1(cid:1)/(cid:1)(Turneretal.,1997),Vav2(cid:1)/(cid:1)(Doodyetal.,2001),Vav3(cid:1)/(cid:1)(Sauzeau
tweenCARD9andVAV3genelociandimmunoglobulinA(IgA) etal.,2006),Card9(cid:1)/(cid:1)(Grossetal.,2006),Prkcd(cid:1)/(cid:1)(Leitgesetal.,2001),
2580 CellReports17,2572–2583,December6,2016