Induction of Antiviral Immune Response through Recognition of the Repeating Subunit Pattern of

17 Pages·2017·1.42 MB·English

RESEARCH ARTICLE
crossm
Induction of Antiviral Immune Response
through Recognition of the Repeating
Subunit Pattern of Viral Capsids Is Toll-
Like Receptor 2 Dependent
D
KellyM.Shepardson,aBenjaminSchwarz,b*KyleLarson,aRachelleV.Morton,a o
JohnAvera,bKimberlyMcCoy,bAlaynaCaffrey,aAnnHarmsen,aTrevorDouglas,b w
n
AgnieszkaRynda-Applea lo
DepartmentofMicrobiologyandImmunology,MontanaStateUniversity,Bozeman,Montana,USAa; a
d
DepartmentofChemistry,IndianaUniversity,Bloomington,Indiana,USAb e
d

ABSTRACT Although viruses and viral capsids induce rapid immune responses, little fr
o
is known about viral pathogen-associated molecular patterns (PAMPs) that are ex- Received1August2017 Accepted10 m
hibited on their surface. Here, we demonstrate that the repeating protein subunit October2017 Published14November2017 h
CitationShepardsonKM,SchwarzB,LarsonK, t
pattern common to most virus capsids is a molecular pattern that induces a Toll- MortonRV,AveraJ,McCoyK,CaffreyA, tp
like-receptor-2 (TLR2)-dependent antiviral immune response. This early antiviral im- HarmsenA,DouglasT,Rynda-AppleA.2017. ://
m
mune response regulates the clearance of subsequent bacterial superinfections, Inductionofantiviralimmuneresponse
throughrecognitionoftherepeatingsubunit b
which are a primary cause of morbidities associated with influenza virus infections. patternofviralcapsidsisToll-likereceptor2 io
Utilizing this altered susceptibility to subsequent bacterial challenge as an outcome, dependent.mBio8:e01356-17.https://doi.org/ .a
s
we determined that multiple unrelated, empty, and replication-deficient capsids initi- 10.1128/mBio.01356-17. m
ated early TLR2-dependent immune responses, similar to intact influenza virus or InvitedEditorDennisW.Metzger,Albany .o
MedicalCollege r
murine pneumovirus. These TLR2-mediated responses driven by the capsid were not g
EditorMaryK.Estes,BaylorCollegeof /
dependent upon the capsid’s shape, size, origin, or amino acid sequence. However, Medicine o
n
they were dependent upon the multisubunit arrangement of the capsid proteins, Copyright©2017Shepardsonetal.Thisisan
A
because unlike intact capsids, individual capsid subunits did not enhance bacterial open-accessarticledistributedundertheterms p
clearance. Further, we demonstrated that even a linear microfilament protein built oInfttehrneaCtrioenatailvleicCeonmsem. onsAttribution4.0 ril
from repeating protein subunits (F-actin), but not its monomer (G-actin), induced 4
AddresscorrespondencetoAgnieszkaRynda- ,

similar kinetics of subsequent bacterial clearance as did virus capsid. However, al- Apple,[email protected]. 2
0
though capsids and F-actin induced similar bacterial clearance, in macrophages they *Presentaddress:BenjaminSchwarz,Rocky 1
MountainLaboratories,Hamilton,Montana, 9
required distinct TLR2 heterodimers for this response (TLR2/6 or TLR2/1, respectively)
USA. b
and different phagocyte populations were involved in the execution of these re- y
K.M.S.andB.S.contributedequallytothiswork.
sponses in vivo. Our results demonstrate that TLR2 responds to invading viral parti- g
u
cles that are composed of repeating protein subunits, indicating that this common e
s
architectureofviruscapsidsisapreviouslyunrecognizedmolecularpattern. t
IMPORTANCE Rapid and precise pathogen identification is critical for the initiation
of pathogen-specific immune responses and pathogen clearance. Bacteria and fungi
express common molecular patterns on their exteriors that are recognized by cell
surface-expressed host pattern recognition receptors (PRRs) prior to infection. In
contrast, viral molecular patterns are primarily nucleic acids, which are recognized
after virus internalization. We found that an initial antiviral immune response is in-
duced by the repeating subunit pattern of virus exteriors (capsids), and thus, induc-
tion of this response is independent of viral infection. This early response to viral
capsids required the cell surface-expressed PRR TLR2 and allowed for improved
clearance of subsequent bacterial infection that commonly complicates respiratory
viral infections. Since the repeating protein subunit pattern is conserved across viral
capsids, this suggests that it is not easy for a virus to change without altering fit-
ness. Targeting this vulnerability could lead to development of a universal antiviral
vaccine.
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Shepardsonetal.
KEYWORDS patternrecognition,bacterialsuperinfection,innateimmunity,Toll-like
receptors,virology,virus-hostinteractions
Rapidrecognitionofpathogensbytheimmunesystemisessentialfortheinitiation
ofanefficientimmuneresponse.Naturehasevolvedadelicatesystemofimmunity
that allows for the recognition of harmful and/or damaging entities based on con-
served elements that mark them as dangerous (1, 2). This early immune response
depends on a pathogen recognition event mediated by the host pattern recognition
receptors(PRRs)thatcaneitherbesecreted,expressedonthecellsurface,orexpressed
intracellularly(1).Regardlessoftheircellularlocation,PRRsidentifyspecificmolecules
commonamongforeignorganisms,knownaspathogen-associatedmolecularpatterns
(PAMPs)(3).Formanybacteriaandfungi,therearewell-knowncellsurface-expressed
PRRs,suchasToll-likereceptors(TLRs),thatareabletoalerttheimmunesystemtoa D
o
threat before infection can be established. In contrast, viruses are typically identified w
after internalization, based on their nucleic acid recognition by intracellular PRRs (4). n
lo
While early reports implicated cell surface-expressed TLRs in immune responses to a
viruses, including respiratory syncytial virus (RSV) or Epstein-Barr virus (EBV) (5, 6), d
e
classicalexternalPAMPsarenotknowntobepresentoneithertheviralcapsidorthe d

f
viral envelope, the most external structures of nonenveloped and enveloped viruses, r
o
respectively(4).Consequently,littleisknownaboutcommonexternalviralPAMPsand m

howtheycouldbeinvolvedinearlyantiviralimmuneresponses. h
t
To address this gap in our understanding of virus recognition, we utilized empty tp
:
capsids, the protein shells of viruses, as a probe to determine whether and how the //
m
immunesystemrespondstotheinitialrecognitionoftheviruspriortoestablishment
b
of infection. Capsids are composed of repeating copies of either a single protein io
.
subunit or a small number of protein subunits (7). Importantly for this study, capsids a
s
retainthebasicexternalarchitectureofavirus,butaredevoidofcommonviralPAMPs m
.
suchasnucleicacids(4).Forthesereasons,capsidsprovideausefultoolforexamining o
r
howthehostmayinitiallysenseexternalviralstructure. g
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Previously,weobservedsimilaritiesbetweentheimmuneresponsesinducedbyan o
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infectious enveloped virus and by empty protein capsids in the lungs of mice (8–10).
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We showed that antigen-nonspecific priming of the murine lung by a capsid derived p
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fromtheSalmonellabacteriophageP22resultedininnateimmuneimprintingsimilarto il
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influenza A virus (IAV) infection, and that this imprinting accelerated the immune ,

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responsetolethalIAVchallenge(8,11,12).Wealsoshowedthatevenanon-pathogen-
0
associatedcapsid,smallheatshockprotein(sHSP)fromMethanocaldococcusjannaschii, 1
9
induced immune responses similar to IAV and protected mice from a range of respi- b
ratorypathogens,includingIAV,severeacuterespiratorysyndrome(SARS)coronavirus, y

g
Coxiellaburnetii,andStaphylococcusaureus(9–14).Immuneimprintingbyvirusescan u
e
eitherreduceorenhancehostsusceptibilitytosubsequentbacterialinfections(15,16). s
t
Indeed,ourprecedinginvestigationrevealedthatprotectionfromS.aureuschallenge
is time-dependent after IAV, with enhanced bacterial clearance 2 to 3 days post-IAV
infectionandreducedclearance7dayspost-IAVinfection(11,14).ConsideringthatIAV
andthecapsidsinducedsimilarinitialimmuneresponses(9),wehypothesizedthatthe
time-dependentchangeinhostsusceptibilitytosubsequentS.aureuschallenge,which
wereportedtooccurafterIAVinfection,isnotlimitedtoIAVinfection.Therefore,we
speculatedthatthisresponseisinducedbyrecognitionofcertaincommonpatternson
thevirusexteriorbyhostPRRsindependentlyofinfection.
Here,wereportinductionofasimilartime-dependentchangeinhostsusceptibility
topost-IAVS.aureusinfection(14)afterinoculationofmicewithvariousemptycapsids
unrelatedtoIAV.Thisprovidedustheopportunitytousethischangeinhostsuscep-
tibilitytoasubsequentS.aureusinfectionasareadoutforthecapsid-inducedimmune
responses (11, 14). We found that recognition of the multisubunit protein assembly
architecture of virus capsids by the cell surface-expressed PRR TLR2 was required for
inductionoftheseresponses.Ourinvestigationalsorevealedthatthistime-dependent
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EarlyAntiviralImmuneResponseRequiresTLR2
changeinhostsusceptibilitytoasubsequentS.aureusinfectionwasinducedregardless
ofthesize,shape,origin,oraminoacidsequenceoftheviruscapsid,butthatinduction
ofthisresponserequiredtherepeatingproteinsubunitarrangementcommontothe
majorityof,ifnotall,viruscapsids.Importantly,becausetherepeatingarrangementof
protein subunits extends beyond just capsid structure, we also found that linear
multisubunit protein assemblies not derived from viruses, such as actin filaments
(F-actin),alsoregulatedhostsusceptibilitytoasubsequentS.aureuschallenge.While
wedeterminedthatTLR2wasrequiredforinitiationoftheseresponsestobothcapsular
andlinearrepeatingproteinassemblies,differentTLR2heterodimers(TLR2/6orTLR2/1)
and different effector cells (neutrophils and macrophages) were involved in the exe-
cutionoftheseresponseswheninducedbyeitherviruscapsidsorF-actin.
Taken together, these data support a novel role for TLR2 as a receptor that
recognizesrepeatingproteinsubunitpatternsregardlessoftheproteinsinvolved.The
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significance of TLR2-mediated recognition of multivalent protein subunit architecture
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expandsourcurrentunderstandingofimmunitytovirusesanditsimpactonimmunity w
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tobacteria,andprovidesanewlayerofmotif-directedrecognition.Importantly,these lo
datademonstratethattherecognitionofviralcapsidsisindependentofinfectionand a
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establishesTLR2’sroleintheinitialrecognitionofviruses. e
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RESULTS o
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Exposure to empty virus capsid from P22 bacteriophage induces time-
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dependent kinetics of S. aureus bacterial clearance, similar to IAV. Building on t
t
previousresultsdemonstratingeithersHSPcapsid-orIAV-inducedclearanceofsubse- p
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quent S. aureus challenge, we examined another capsid, derived from the bacterio- /m
phageP22,andanaturalmousevirustoensurethatthisphenomenonwasnotcapsid b
orvirusspecific.AswithsHSPcapsid,IAVandP22capsidssharenoproteinsimilarity, io
.
a
althoughtheydoshareasimilarsphericalshapewithapolyvalentproteinsurface(17) s
m
(Fig. 1A). The ability of the host to clear secondary post-IAV S. aureus pneumonia
.
changes over time (11, 14), and we hypothesized that this time dependence and the o
r
g
associatedimmuneresponsesmaybeutilizedtocharacterizesimilaritiesintherecog- /

nition of the virus and the capsid. To test this initial hypothesis, wild-type (WT) mice o
n
wereinoculatedwitheitherIAVorP22capsidandthenchallengedwithS.aureus3,7, A
or14dayslater(Fig.1B).Ascapsidsarereplicationdeficient,weinoculatedmicewith p
r
3consecutivedailydoses,mimickingthereplicationofaninfectiousvirus. il
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Compared to mock-inoculated mice, mice inoculated with P22 capsid displayed a ,

2
similar increase in clearance of S. aureus as did IAV-inoculated mice at day 3 post- 0
1
inoculation (p.i.) (Fig. 1C). By day 7 p.i., both P22 capsid- and IAV-inoculated mice 9
showed at least a 10-fold decrease in bacterial clearance (Fig. 1C). At day 14 p.i., b
y
IAV-inoculatedmice,whichbythispointwouldstillhaveresultingimmuneeffectsfrom
g
a productive viral infection (18, 19), exhibited improved bacterial clearance, whereas u
e
bacterial clearance in P22 capsid-inoculated mice was similar to that of mock- s
t
inoculated mice (Fig. 1C). Inoculation of mice with pneumonia virus of mice (PVM), a
causeofnaturalmouseviralinfectionthatresultsin/inducespathologysimilartothat
causedbyhumanrespiratorysyncytialvirus(20),resultedinasimilartime-dependent
clearance of S. aureus challenge as found with P22 capsids or IAV (see Fig. S1 in the
supplementalmaterial).
P22capsidinoculationinducescytokineandcellularimmuneresponses,similarto
IAV, both before and after S. aureus challenge. To determine whether the similar
patterns of bacterial clearance induced by IAV and by P22 capsid inoculation are also
associatedwithacommoncytokineresponse,wecomparedlevelsofinterleukin-13(IL-13),
whichcontributestoclearanceofS.aureusinfectioninthelung(11,14),andgamma
interferon(IFN-(cid:2)),whichbothweandothershavefoundtobediseaseenhancing(11,
14,21).Comparedtomock-inoculatedWTmice,bothP22capsid-andIAV-inoculated
miceexhibitedincreasedIL-13productionwhenchallengedwithS.aureusatday3p.i.
(Fig.1D).However,whenchallengedatday7p.i.withS.aureus,bothP22capsid-and
IAV-inoculatedmiceshoweddiminishedIL-13production.ForIFN-(cid:2)production,com-
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Shepardsonetal.
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FIG 1 Inoculation of mice with P22 capsid or with IAV induces similar immune responses to subsequent S. aureus 9

challenge and similar patterns of time-dependent alteration of bacterial clearance. (A) Structural representation of b
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influenza A virus (IAV) (courtesy of CDC) and P22 capsid (PDB accession no. 2XYY). (B) Schematic of inoculation and
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S.aureuschallengetimeline.(C)WTmicewereeitherinoculatedwithIAV(blacktriangles)onday0orinoculatedwithP22
u
capsid(graysquares)orPBS(mock;whitecircles)ondays0,1,and2.Differentgroupsofmicewerechallengedwith e
S.aureusonday3,7,or14andweresacrificed24hafterchallengetodetermineS.aureuslungbacterialload.(D)Fold s
t
change of cytokine concentration determined by ELISA on the BALFs of mice from day 3 and day 7 p.i. in panel C
normalizedtocytokineconcentrationinmiceinoculatedwithPBS(mockcontrol;statisticalanalysescompareindividual
treatmentstomocktreatment).(EandF)WTmicewereinoculatedasdescribedforpanelC,CD11c(cid:2)(E)orLy6G(cid:2)(F)cells
werepurifiedfromthelungsatday3or7,andtheirabilitytokillS.aureusfollowinga1.5-h(E)or3-h(F)incubationex
vivowasdetermined.TheexperimentinpanelChadaminimumof5animalspergroup,theexvivoexperimentinpanel
Dhad5biologicaland3technicalreplicates,andtheexvivoexperimentinpanelsEandFhad3combinedbiologicalwith
5technicalreplicates.Eachwasrepeatedthreetimes.Dataarerepresentedasmean(cid:3)SD.*,P(cid:4)0.05;**,P(cid:4)0.01;***,
P(cid:4)0.001.
paredtomock-inoculatedWTmice,bothP22capsid-andIAV-inoculatedmiceshowed
diminishedproductionofthiscytokinewhenchallengedwithS.aureusatday3p.i.,but
when challenged at day 7 p.i., both P22 capsid- and IAV-inoculated mice showed
increasedIFN-(cid:2)production(Fig.1D).
IFN-(cid:2)can impair bacterial clearance by macrophages (21), whereas IL-13 can be
involved in neutrophil recruitment (22). We previously reported that both CD11c(cid:2)
(alveolarmacrophages)andLy6G(cid:2)cells(neutrophils)isolatedfromIAV-inoculatedmice
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EarlyAntiviralImmuneResponseRequiresTLR2
onday3p.i.haveanincreasedabilitytokillS.aureusexvivocomparedtothosefrom
mock-inoculatedmice,whereasneutrophilsisolatedfromthelungsofIAV-inoculated
miceonday7p.i.showedanimpairedabilitytokillS.aureus(14).SimilartoIAVonday
3p.i.,bothalveolarmacrophagesandneutrophilsharvestedfromlungsofWTmiceat
day3post-P22capsidinoculationkilledbetween6and12timesmoreS.aureusthan
cells from mock-inoculated mice (Fig. 1E and F). Additionally, similar to neutrophils
harvested from mice at day 7 post-IAV inoculation, neutrophils harvested at day 7
post-P22capsidinoculationexhibitedimpairedbactericidalactivitycomparedtothose
frommock-inoculatedmice(Fig.1F).AlveolarmacrophagesisolatedfromP22capsid-,
IAV-, or mock-inoculated mice at day 7 showed similar bactericidal activities toward
S.aureusexvivo(Fig.1E).
Host responses involved in dictating susceptibility to S. aureus challenge during
earlyviralinfectionareinducedpriortobacterialchallenge.Specifically,productionof
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IFN-(cid:3)over the course of early viral infection (day 2 to 3) contributes to increased
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bacterial clearance of S. aureus during subsequent challenge. However, decreased w
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production of IFN-(cid:3)and increased production of IFN-(cid:4)later in viral infection reduce lo
bacterialclearance(11,14).Therefore,weinvestigatedwhetherthecytokineresponses a
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inducedbyP22capsidinoculationpriortoS.aureuschallengewerealsosimilartothose e
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inducedbyIAV.ThetwoconditionsinducedsimilarearlyproductionofIFN-(cid:3)(day3)
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andlateproductionofIFN-(cid:4)(day7)inWTmicecomparedtomock-inoculatedWTmice ro
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(Fig. S2). This suggests that the induction of a protective immune response was a

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consequence of the initial viral or capsid recognition events and not a response to t
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activechallengewithS.aureus.Activationofadaptiveimmuneresponsesatlatertimes p
:
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followingactiveviralinfectionincreasesboththemagnitudeandthecomplexityofthe /m
immuneresponse.Thus,fortheremainderofthisstudy,improvedbacterialclearance b
on day 3 p.i. was used as a metric for better understanding the mechanisms of early io
.
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recognitionofviruscapsid. s
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TLR2-dependentsignalingisrequiredforIAV-andP22capsid-mediatedclear-
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anceofS.aureusonday3p.i.Ourresultsthusfarsuggestthatactivevirusinfection o
r
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isnotrequiredforinductionoftheimmunologicresponsesthatregulatehostsuscep- /

tibility to subsequent bacterial infection. Thus, we hypothesized that a common o
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structuralpatternofviralcapsidswasrecognizedbyaPRRwithbroadspecificity,such A
as the TLRs. To reduce the number of possible candidate PRRs involved, the MyD88 p
r
adapter molecule, which is used by nearly all members of the TLR family to induce il
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inflammatorysignalinguponrecognitionofPAMPs,wasexamined(23).myd88(cid:5)/(cid:5)mice ,

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inoculatedwitheitherIAVorP22capsidshowedimpairedclearanceofS.aureusfrom 0
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thelungatday3p.i.comparedtosimilarlyinoculatedWTmice(Fig.2A).Inadditionto 9
acting as a mediator for TLRs, MyD88 also complexes and transmits signals from the b
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inflammation-mediating receptor IL-1R (24, 25). However, il-1r(cid:5)/(cid:5) mice, either IAV or
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P22inoculated,demonstratedasimilarpatternofclearanceofS.aureusuponchallenge u
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asthatseeninWTmice(Fig.S3),implicatingTLRsandnotIL-1Rasalikelycandidate s
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intherecognitionofandsubsequentimmuneactivationbyIAVandP22capsid.
To determine which TLRs were induced in response to IAV or P22 capsid, we
quantified transcript levels for each TLR, in either whole-lung samples or purified
alveolarmacrophages,6hfollowingeitherIAVorP22capsidinoculation,aknowntime
frameforTLRtranscriptinduction(26,27).The6-htimepointwaschosentoidentify
TLRs involved in early recognition rather than in downstream immune activation
events, which could be complicated by recognition of other viral structures, such as
nucleic acids. Both IAV and P22 capsid inoculation induced TLR2 and TLR6 mRNA
expression. While TLR2 was upregulated in both whole-lung samples and purified
alveolar macrophages, TLR6 was upregulated only by alveolar macrophages (Fig. 2B).
Based on the consistent upregulation of TLR2, we assessed the requirement for this
receptor in the immune response induced by the capsid. In contrast to WT mice,
inoculationoftlr2(cid:5)/(cid:5)miceonday3p.i.witheitherIAV(Fig.2C)orP22capsid(Fig.2D)
didnotresultinanincreaseofS.aureusclearancecomparedtomock-inoculatedmice.
This indicates that TLR2 signaling was required for the initiation of protective re-
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FIG 2 TLR2-dependent signaling is required for IAV- and P22 capsid-mediated bacterial clearance of S. aureus during subsequent bacterial h
infection.(A)WTandmyd88(cid:5)/(cid:5)micewereinoculatedwithIAV(day0;blackbars),P22capsid(days0,1,and2;graybars),orPBS(days0,1,and t
t
2;mock;whitebars)andchallengedwithS.aureusonday3,andlungbacterialburdenwasmeasured24hlater.(B)Geneexpressionoftlr2and p
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tlr6wasanalyzedbyqRT-PCRonRNAisolatedfromwholeWTlungorCD11c(cid:2)lungalveolarmacrophages6haftertreatmentwitheitherIAV, //
P22capsid,orPBS(black,gray,orwhitebars,respectively).(CandD)WTandtlr2(cid:5)/(cid:5)micewereinoculatedwithIAV(C)(day0;blackbars)orP22 m
capsid(D)(days0,1,and2;graybars)orPBS(day0ordays0,1,and2;mock;whitebars)andchallengedwithS.aureusonday3,andlung b
bacterial burden was measured 24 h later. (E) IL-13 and IFN-(cid:3)cytokine concentrations in cell-free BALF from mice in panels C and D were io
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determinedbyELISA(dashedredlineisPBS).(F)WTmicewereinoculatedwithPAM2(hatchedgraybar)orPBS(mock;whitebar)(day0)and a
challengedwithS.aureusonday3,andlungbacterialburdenwasmeasured24hlater.(G)LevelsofIL-13andIFN-(cid:3)weremeasuredintheBALFs sm
frommiceinpanelFbyELISA(dashedredlineisPBS,andstatisticalanalysiscomparestreatmenttoPBS).Mouseexperiments(A,C,D,andF)
.
hadaminimumof5animalspergroup,theexvivoexperimentinpanelBhad3biologicaland3technicalreplicates,andtheexvivoexperiment o
inpanelsEandGhad5biologicaland3technicalreplicates.Eachwasrepeatedthreetimes.Dataarerepresentedasmean(cid:3)SD.*,P(cid:4)0.05; rg
**,P(cid:4)0.01;***,P(cid:4)0.001;****,P(cid:4)0.0001. /
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sponses.tlr2(cid:5)/(cid:5)micedidnotpresentthecharacteristiccytokinesequelaeoftypeIIFNs p
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andIL-13atday3p.i.Specifically,tlr2(cid:5)/(cid:5)miceinoculatedwithIAVorP22capsidhad il
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noincreaseinproductionofIL-13orIFN-(cid:3)comparedtomock-inoculatedtlr2(cid:5)/(cid:5)mice, ,

2
whileWTmiceinoculatedwithIAVorP22capsidhada2-foldincreaseinproduction 0
of IL-13 and IFN-(cid:3)compared to mock-inoculated WT mice (Fig. 2E; Table S1). This 19
indicatedthatTLR2signalingcontributestotheupregulationofIFN-(cid:3)andIL-13after b
y
eitherIAVorP22capsidrecognitiononday4p.i.(Fig.2E).Furthermore,wefoundthat
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killingofS.aureusexvivobyneutrophilsandmacrophagesisolatedfromtlr2(cid:5)/(cid:5)mice u
e
on day 3 after inoculation with either IAV or P22 capsid did not induce increased s
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bactericidalactivitycomparedtothesecellsfromWTmice(Fig.S4).
ToexaminewhetherTLR2activationalonewassufficienttoelicitprotectionagainst
subsequent bacterial challenge, the known TLR2 agonist PAM2CSK5 (PAM2) was ad-
ministeredinplaceofIAVorP22capsid.PAM2treatmentresultedinincreasedbacterial
clearancecomparedtomock-inoculatedmiceanddisplayedasimilarpatternofboth
IL-13 and IFN-(cid:3)production at day 3 p.i. (Fig. 2F and G) as found with IAV and P22
capsid. These results support a role for TLR2 signaling in immune responses to
secondarypost-viralandpost-capsidS.aureuschallenge.
Capsid-mediated immune events are independent of the capsid size and
shape, but are dependent on TLR2 and recognition of intact capsid. TLR2’s
involvement in the recognition of both IAV and P22 capsid suggested that TLR2 was
respondingtosimilarstructuralpatternsonthesurfaceofthetwoparticles.Thisraised
thepossibilityofTLR2actingasagenericreceptorforcommonstructuralelementsof
viral capsids. Despite having no sequence similarity and having distinct host specific-
ities, IAV and P22 capsid have similar sizes (~80 nm for IAV [28] and 60 nm for P22
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FIG 3 Repeating protein subunit structure, but not spherical shape or size, is required for time-dependent
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alterationofS.aureusclearance.(A)To-scalestructuralrepresentationsofTMVcapsid,P22capsid,andferritin //
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capsid(assembledusingPDBaccessionnumbers4UDV,2XYY,and3AJO,respectively).(B)Micewereinoculated
b
withP22capsid(graysymbols),TMVcapsid(greensymbols),murine(openbluesymbols)orP.furiosus(solidblue
io
symbols)ferritincapsid,orPBS(mock;whitesymbols)(days0,1,and2)andchallengedwithS.aureusoneither
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day3orday7.(C)tlr2(cid:5)/(cid:5)micewereinoculatedwithTMVcapsid(greensymbols),murine(openbluesymbols)or a
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P.furiosus(solidbluesymbols)ferritincapsid,orPBS(mock;whitesymbols)(days0,1,and2)andchallengedwith m
S.aureusonday3.(D)StructuralrepresentationofP22capsidandP22dimer.Sizesarenottoscale(PDBaccession .
o
no. 2XYY). (E) WT mice were inoculated with P22 capsid (gray symbols), P22 dimer (white symbols with gray
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outline),orPBS(mock;whitesymbolswithblackoutline)(days0,1,and2)andwerechallengedwithS.aureuson g
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eitherday3orday7.(B,C,andE)S.aureuslungbacterialburdenwasdetermined24hafterchallenge.Experiments o
hadaminimumof5animalspergroupandwererepeatedtwo(C)orthree(BandE)times.Dataarerepresented n
asmean(cid:3)SD.*,P(cid:4)0.05;***,P(cid:4)0.001. A
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capsid[29])andsphericalshapes.Toconsiderthepossibilitythatsizeand/orshapemay ,

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play a role in TLR2-mediated recognition of these particles, we tested a library of 0
1
capsids with various sizes and shapes for their ability to induce a similar pattern of 9
susceptibilitytoS.aureuschallengeasfoundwithP22capsidandIAV.Toexaminethe b
y
impact of shape, mice were inoculated with a large (~300-nm) rod-shaped capsid
g
derived from a tobacco mosaic virus (TMV) coat protein (CP) mutant (which forms a u
e
capsid in the absence of the nucleic acid cargo [30]). To examine the impact of size, s
t
micewereinoculatedwithsmall(~12nmindiameter[31])ferritincapsidsofeithera
self(murine)orforeign(Pyrococcusfuriosus)origin(Fig.3A).Similartomiceinoculated
withP22capsid,miceinoculatedwithTMVcapsidorwitheitherpyrococcal(nonself)or
murine (self) ferritin capsids showed improved bacterial clearance when challenged
with S. aureus at day 3 p.i., but not at day 7 p.i. (Fig. 3B). Additionally, tlr2(cid:5)/(cid:5) mice
inoculated with TMV or with either ferritin capsid did not clear subsequent S. aureus
challenge better than mock-inoculated mice on day 3 p.i. (Fig. 3C). These results
demonstrated that the capsid recognition resulting in time-dependent alteration of
hostsusceptibilitytoS.aureuspneumoniarequiredTLR2,andwasnotdeterminedby
the capsid’s size or shape, and suggested that the capsid recognition preceded the
recognitionofselfversusnonself.
Viral capsids have long been known to induce more-efficient humoral responses
thantheirisolatedsubunitsastheirrepeatedsubunitstructurebettercross-linksB-cell
receptors (32, 33). While IAV is an enveloped virus, proteins (including primarily
hemagglutinin, but also neuraminidase and matrix 2 protein) on the IAV surface are
November/December2017 Volume8 Issue6 e01356-17 mbio.asm.org 7
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Shepardsonetal.
tightlydistributed,withanaveragespacingbetweenhemagglutininsubunitsof~9nm,
leadingtoasimilarlyspacedpolyvalentproteinsurfaceasthecoatproteinoftheP22
capsid(averagespacingof~6nmbetweensubunits)(28,34).Toconsiderwhetherthe
repeatingproteinsubunitarrangementwassimilarlyrequiredfortheTLR2-dependent
recognition, we inoculated WT mice with the disassembled P22 coat protein subunit,
whichcanbestablymaintainedinsolutionasadimer(29,35)F(ig.3D).Incontrastto
miceinoculatedwithanintactP22capsid,inoculationofmicewithP22dimerneither
showed improved bacterial clearance at day 3 p.i. nor decreased the clearance of
S.aureusuponchallengeonday7p.i.(Fig.3E).Infact,miceinoculatedwithP22dimer
showedasimilarpatternofsusceptibilitytoS.aureuschallengeasdidmock-inoculated
mice (Fig. 3E). This finding further supports the hypothesis that antiviral immune
responsesmediatetime-dependentchangesinhostsusceptibilitytosubsequentS.au-
reus challenge via sequence-independent recognition of the structural pattern of the
D
viral capsid. These results strongly suggest/imply a pivotal role for repeating protein o
subunit structures in the induction of antiviral immune responses by a mechanism w
n
involvingacommonrecognitionpathwayforthispattern. lo
TLR2 recognizes repeating protein structures and does not require the sub- a
d
units to be spherically organized. The induction of common antiviral immune e
d
responses and the requirement for TLR2 by all of the intact capsids examined sug-
f
r
gested that TLR2 might be involved in recognizing the repeating subunit pattern of o
m
viral capsids. In nature, repeating protein subunit patterns are not limited to capsids,

h
i.e., multisubunit protein assemblies surrounding a hollow cargo volume. Thus, we t
t
p
rationalized that if TLR2 recognition is initiated solely by multisubunit protein assem-
:
/
blies, then it might extend to noncapsid structures. Therefore, we examined filamen- /m
tous (F) actin as a model noncapsid repeating protein subunit assembly. Unlike the b
io
capsidstestedthusfar,F-actinisanextendedhelicalmicrofiber,butitisstillcomposed
.
a
ofarepeatingproteinsubunit(Fig.4A). s
m
InoculationofmicewithF-actinallowedfora10-foldincreaseinbacterialclearance
.
o
atday3p.i.,butrenderedmicelessabletoclearS.aureusfollowingchallengeatday
r
g
7p.i.thanmock-inoculatedmice(Fig.4B).Thus,F-actininoculationresultedinasimilar /

o
time-dependentpatternofhostsusceptibilitytobacterialchallengeasdidinoculation
n
witheitherthecapsidsorviruses(Fig.1C,3B,andS1).Additionally,aswasfoundwith A
P22 dimer (Fig. 4C), the globular F-actin monomers (G-actin) did not induce the p
r
time-dependentalterationofsusceptibilitytoS.aureuschallengecomparedtoF-actin- il
4
inoculated mice, further supporting our hypothesis that repeating protein assemblies ,
2
composedofrepeatingproteinsubunitsareinducingthisresponse.F-actininoculation 0
1
also did not increase clearance of S. aureus in tlr2(cid:5)/(cid:5) mice at day 3 p.i. compared to 9

F-actin-inoculated WT mice, indicating a requirement for TLR2 in bacterial clearance b
y
after F-actin inoculation (Fig. 4D), similar to IAV and P22 capsid (Fig. 2C and D).
g
However, in contrast to IAV and P22 capsid (Fig. 2C), there was not a significant u
e
difference in the production of IFN-(cid:3)or IL-13 in the F-actin-inoculated tlr2(cid:5)/(cid:5) mice s
t
comparedtothatfoundinF-actin-inoculatedWTmice(Fig.4E),suggestingthatF-actin
inducedadifferentTLR2-mediatedmechanismthanP22capsid.AsF-actinisknownto
be recognized by the c-type lectin receptor Clec9a (DNGR1) (36, 37), we determined
whetherClec9awasinvolvedintheantiviralimmuneresponseregulatinghostsuscep-
tibilitytoasubsequentbacterialchallenge.Inoculationofclec9a(cid:5)/(cid:5)micewithF-actin
resultedinincreasedclearanceofS.aureuscomparedtomock-treatedclec9a(cid:5)/(cid:5)mice
(Fig. S5), indicating that Clec9a is not required for the F-actin-mediated immune
responses that regulate host susceptibility to a subsequent S. aureus pneumonia.
Together, these results support a general role for TLR2 recognition of proteins with
repeating subunit structure regardless of their origin, but also suggest that TLR2
recognition can be promiscuous and, as such, can result in different downstream
cytokineresponses.
The repeating protein subunit assemblies of P22 capsid and F-actin mediate
inductionofprotectiveimmuneresponsesthroughTLR6andTLR1,respectively.
TheinductionofdifferentcytokineresponseswithasharedTLR2dependencebyP22
November/December2017 Volume8 Issue6 e01356-17 mbio.asm.org 8
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EarlyAntiviralImmuneResponseRequiresTLR2
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FIG4 F-actininoculationinducessimilartime-dependentalterationofbacterialclearanceafterS.aureus A
challenge,butdifferentcytokinescomparedtoIAVorP22capsidinoculation.(A)Astructuralrepresen- p
tmaticioenwoefrFe-iancoticnualantdedGw-aicthtinF-gaecntienra(oteradnfgroemsyPmDbBoalsc)coerssPiBoSn(nmuomcbke;wrsh3iJte8Ksyamndbo1lJs6)Z(d,raeyssp0e,c1ti,vaenlyd.(2B))aWndT ril 4
werechallengedwithS.aureusoneitherday3orday7,andlungbacterialburdenwasevaluated24h ,

afterchallenge.(C)WTmicewereinoculatedwith50(cid:5)gofeitherF-actin(orangesymbols),G-actin(dark 2
0
redsymbols),orPBS(mock;whitesymbols)(days0,1,and2)andwerechallengedwithS.aureusonday 1
3,andlungbacterialburdenwasevaluated24hafterchallenge.(D)WTortlr2(cid:5)/(cid:5)micewereinoculated 9
with F-actin (orange symbols) or PBS (mock; white symbols) (days 0, 1, and 2) and challenged with b
S. aureus on day 3, and lung bacterial burden was evaluated 24 h after challenge. (E) Cytokine y

concentrationdeterminedbyELISAontheBALFsofmicefrompanelC.ExperimentsshowninpanelsB, g
u
C,andDhadaminimumof5animalspergroup,andtheexvivoexperimentinpanelEhad5biological e
and3technicalreplicates.Eachwasrepeatedthreetimes.Dataarerepresentedasmean(cid:3)SD.*,P(cid:4) s
0.05;**,P(cid:4)0.01;***,P(cid:4)0.001;N.S.,notsignificant. t
capsid and F-actin suggested that there might be differences in cellular recruitment
and/oractivation.Cellularanalysisofthebronchoalveolarlavagefluid(BALF)revealed
thatP22capsidinoculationinducedrecruitmentofneutrophils(CD11b(cid:2)Ly6G(cid:2);similar
to previous results from IAV [14]) while F-actin did not (Fig. 5A and S6A). In fact, the
cellular composition of BALF from mice inoculated with F-actin resembled that of
mock-inoculated mice and consisted of mostly resident alveolar macrophages (~93%
CD11c(cid:2)SiglecF(cid:2)[Fig.5AandS6A]).Todeterminewhetherthesedifferencesincellular
recruitmentintothelungtranslatedintodifferencesinthefunctionofthesecells,we
tested their bactericidal capacity against S. aureus in the ex vivo assay. P22 capsid
increased the bactericidal capacity of both neutrophils (Ly6G(cid:2)) and macrophages
(CD11c(cid:2))isolatedfromWTmiceonday3p.i.,whileF-actinincreasedthebactericidal
capacity of only macrophages (Fig. 5B). These results indicated that while both P22
November/December2017 Volume8 Issue6 e01356-17 mbio.asm.org 9
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Shepardsonetal.
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A
FIG5 P22capsidandF-actinrequiredifferentTLR2heterodimersfortheirabilitytoinduceS.aureuskillingbymacrophages.(A)WTmicewereinoculatedwith p
r
P10202(cid:5)camp)s.id(B,)FW-aTctmin,icoerwPBerSe(dinaoycsu0la,t1e,danwdit2h),eaitnhderdPif2fe2rceanptisaildc(oguranytssoymfwbhoiltse),bFl-oaoctdince(ollrsafnrgoemsBymALbFowls)e,roerdPoBnSe(monocdka;yw3hpit.ei.s(yrempbreosles)n(tdaatiyvse0i,m1a,gaensds2h)o;wCDn1;b1ca(cid:2)r, il 4
orLy6G(cid:2)cellswerepurifiedfromthelungsatday3;andtheirabilitytokillS.aureusfollowinga1.5-h(CD11c(cid:2))or3-h(Ly6G(cid:2))incubationexvivowas ,
2
determined.(C)PercentinitialbodyweightsofmiceinoculatedwithP22capsid(graysymbols),F-actin(orangesymbols),orPBS(mock;whitesymbols)(days 0
0,1,and2)andchallengedwithS.aureusonday3.(D)PercentinitialbodyweightsofmiceinoculatedwithPAM2,PAM3,orPBS(mock)(day0)andchallenged 1
withS.aureusonday3.(EandF)DifferentialcountsofwhitebloodcellsfromBALFofmicetreatedwithPAM2,P22capsid,orPBS(mock)(E)orPAM3,F-actin, 9

orPBS(mock)(F)andchallengedwithS.aureusonday3.(GandH)ImmortalizedBMmacrophagesfromWT,tlr2(cid:5)/(cid:5),tlr1(cid:5)/(cid:5),andtlr6(cid:5)/(cid:5)micewerestimulated b
y
withP22capsid(graysymbols)(G)orF-actin(orangesymbols)(H)orPBS(mock;whitesymbols)for12h.Cellswerewashedandincubatedata10:1ratioof
g
S.aureustocellsfor5h.CFUweredeterminedafterovernightincubationofsamplesonTSAplatesfollowingcelllysis.ExperimentsshowninpanelsAand
u
CtoFhadaminimumof5animalspergroup,theexvivoexperimentinpanelBhad3combinedbiologicalwith5technicalreplicates,andtheexperiment e
showninpanelsGandHhad5technicalreplicatespercellline.Eachwasrepeatedthreetimes.StatisticsforpanelsCandDareforthechangeinweightloss s
t
fromday3today4(mock,F-actin,andPAM3)orfromday0today3(P22capsidandPAM2).Dataarerepresentedasmean(cid:3)SD.**,P(cid:4)0.01;***,P(cid:4)0.001.
capsidandF-actinutilizeTLR2tomodulatehostsusceptibilitytosubsequentS.aureus
challenge, the P22 capsid-induced responses are more inflammatory than the re-
sponsesinducedbyF-actin.Differencesincellularrecruitmentandinflammationoften
leadtodifferencesinbiologicaleffects;thus,wetestedwhetherP22capsidandF-actin
haddistinctbiologicaleffects.P22capsidinoculationresultedinsignificantweightloss
fromday0today3inWTmice(Fig.5C),butthemicewerethenprotectedfromweight
lossuponS.aureuschallenge(day3to4,P(cid:6)0.315)(Fig.5C).F-actininoculationhad
noimpactonweightlossinitially(day0to3,P(cid:6)0.501)(Fig.5C),butfollowingS.aureus
challenge, the mice lost a significant amount of weight, similar to mock-inoculated
mice(day3to4,Fig.5C).
The observed differences between mice inoculated with P22 capsid and those
inoculatedwithF-actinintermsofcellularrecruitmentandactivation,aswellasbody
November/December2017 Volume8 Issue6 e01356-17 mbio.asm.org 10