J. Infect. Dis. (2015) 132–140] myocarditis”[Braz. effects of IL-28A in coxsackievirusB3-induced Retraction notice to “Antiviral and myocyteprotective INFECTIOUS DISEASES

www .e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Retraction

notice

Retraction

notice

to

“Antiviral

and

myocyte

protective

effects

of

IL-28A

in

coxsackievirus

B3-induced

myocarditis”

[Braz.

J.

Infect.

Dis.

(2015)

132–140]

Shihong

Wang

_,

_Xingyuan

_Huang

_,

_Jing

_Zhang

_,

_Congxin

_Huang

a_{DepartmentofPediatrics,RenminHospitalofWuhanUniversity,WuhanUniversity,Hubei,PRChina} b_{DepartmentofCardiology,RenminHospitalofWuhanUniversity,WuhanUniversity,Hubei,PRChina}

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/ withdrawalpolicy).

ThisarticlehasbeenretractedattherequestoftheEditorandthePublisher.Afterathoroughinvestigation,thePublisher hasconcludedthatthepeer-reviewprocesshasbeencompromisedandthescientificintegrityofthepapercannotbe guaran-teed.Reviewerreportsweresubmittedfromemailaccountswhichwereprovidedtothejournalassuggestedreviewersbythe correspondingauthorduringthesubmissionofthepaper.Howevernoconfirmationhasbeenreceivedfromthoseaccounts uponfurtherrequestfromthePublishertoendorsethattheywereindeedthepersonswhocompletedthereviewsandthatthe non-institutionalemailaddresseswereofthem.

Thismanipulationofthepeer-reviewprocessrepresentsaclearviolationofthefundamentalsofpeerreview,ourpublishing policies,andpublishingethicsstandards.Apologiesareofferedtothereadersofthejournalthatthisdeceptionwasnotdetected duringthesubmissionprocess.

DOIoforiginalarticle:http://dx.doi.org/10.1016/j.bjid.2014.10.007.

http://dx.doi.org/10.1016/j.bjid.2015.09.001

www .e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Antiviral

and

myocyte

protective

effects

of

IL-28A

in

coxsackievirus

B3-induced

myocarditis

Shihong

Wang

_,

_Xingyuan

_Huang

_,

_Jing

_Zhang

_,

_Congxin

_Huang

a_{DepartmentofPediatrics,RenminHospitalofWuhanUniversity,WuhanUniversity,Hubei,PRChina} b_{DepartmentofCardiology,RenminHospitalofWuhanUniversity,WuhanUniversity,Hubei,PRChina}

a

r

t

i

c

l

e

i

n

f

o

Received22May2014 Accepted6October2014

Availableonline17December2014

Keywords: CoxsackievirusB3 IL-28A Apoptosis Myocarditis

a

b

s

t

r

a

c

t

Objective:Thisstudyaimedtoinvestigatewhetherinterleukin-28A(IL-28A)playsarolein murinemyocarditisinducedbycoxsackievirusB3(CVB3),andtoexploreitspossible mech-anisminvolved.

Methods:MaleBALB/cmicebothinfectedandnotinfectedbyCVB3wererandomlydivided intofourgroups (n=40), untreatedortreatedwith differentdoses ofIL-28A for4 days, andthensacrificedondays4and7post-infection.Theheartsampleswerecollectedfor histopathologicexamination.Cardiacviralloadwasdeterminedbyaplaqueassay. Addi-tionally,immunoblotanalysis,TUNELassay,andimmunohistochemistrywereperformed toexaminetheexpressionofsignaltransducer,activatoroftranscription1and2(STAT1 andSTAT2),CVB3-inducedapoptosisandtheexpressionofBcl-2,BAXandCaspase-3.

Results:Comparedtouninfectedmice,theCVB3infectedmiceexhibitedhighermortality rate(p<0.001),apparentinflammationandmyocardial lesion(p<0.01),and higher car-diacviral load(p<0.01). After CVB3infection, IL-28A treated micepresented nodeath (p<0.001),reduced inflammationand myocardial lesion (p<0.01), and lower viral load (p<0.01)comparedtountreatedmice.Besides,treatmentwithIL-28Amarkedlyincreased theexpressionsofSTAT1andSTAT2,andinhibitedCVB3-inducedapoptosisinmyocardial cellswithincreasedratioofBcl-2/BAX.

Conclusion:The antiviral and myocyte protective effects of IL-28A in CVB3-induced myocarditisareregulatedbySTAT1andSTAT2.

©2014PublishedbyElsevierEditoraLtda.

Introduction

Myocarditisisacardiacdiseaseassociatedwithinflammation andinjuryofthemyocardium.Severaltypesofviruseshave

∗ _{Correspondingauthorat:DepartmentofCardiology,RenminHospitalofWuhanUniversity,WuhanUniversity,238JiefangRoad,Wuchang}

District,Wuhan,Hubei,430060,PRChina.

E-mailaddress:huangfornew@163.com(C.Huang).

beenimplicatedinthedevelopmentofhumanmyocarditis.1 However, coxsackievirus B3 (CVB3)is considered the most common pathogen.CVB3-inducedmyocarditisisfrequently complicatedbycardiomyopathy,leftventriculardysfunction, andchronicheartfailure.2_{ThemurinemodelofCVB3-induced}

http://dx.doi.org/10.1016/j.bjid.2014.10.007

1413-8670/©2014PublishedbyElsevierEditoraLtda.

myocarditis shares many features with human disease.3 Despite decades of extensive efforts, the pathogenesis of CVB3-inducedmyocarditisisstillnotwelldefined.Although CVB3causescellulardysfunctioneitherthroughinducedcell apoptosis,shut downofcell RNAand proteinsynthesisor viralproteasecleavageofcontractileproteins,4–7 _thestrong hostTh1immuneresponsesmaybemoreresponsibleforthe courseofmyocytedamage,verifiedbytheimprovementof heartinjuryandfunctionbyimmunemodulatingand inhibi-tingagents.8–10_{Clinicalstudieshavealsofoundcytokinessuch} asIL-1␤,IL-6andTNF-␣thatwereinvolvedinthecourseof cardiacdysfunction.11 _{Besides,thepoorprognosisformore} thanhalfofpatientspresentingwithclinicalsymptoms,since currentlynoeffectivetherapyisavailableforthisdisease,12,13 muchworkonthedevelopmentofvaccinesandtherapeutic agentsagainstCVB3hasbeendone.

HumanIL-28A,IL-28B,andIL-29,alsoknownasinterferon (IFN)-␭2,-␭3 and -␭1,respectively,belongingto thetypeIII IFNs.14 _{Like other typical IFNs, these cytokines have been} alsodemonstratedtoplayakeyroleinthenegative regula-tionofcytopathicvirusessuchasencephalomyocarditisvirus (EMCV),15_{vesicularstomatitisvirus(VSV),}15_{cytomegalovirus} (CMV),16_{influenzaAvirus(IAV),}17_{respiratorysyncytialvirus} (RSV),hepatitisBvirus(HBV),18_{hepatitisCvirus(HCV),}18_and herpessimplexvirus-2(HSV-2).19_{Agrowingnumberof} pre-viousreportshavesuggestedthattypeIIIIFNsexertantiviral activitybytheinducedupregulationofIFNeffectorproteins suchasSTATproteins.14,20_{However,arecentstudyhasshown} that certain types of viruses can develop mechanisms to inhibittheantiviralactivityoftypeIIIIFNs.21

ThisstudyaimedtoinvestigatewhetherIL-28Aplaysarole inmurinemyocarditisinducedbyCVB3,andtoexploreits pos-siblemechanisminvolvedbydeterminingtheexpressionsof STAT1andSTAT2,Bcl-2,BAX,andCaspase-3inCVB3-infected hearttissues.Itwillprovideareferenceforthedevelopment ofvaccinesandtherapeuticagentsagainstCVB3inhuman.

Materials

and

methods

Mice,CVB3infection,andIL-28Atreatment

The research protocol of this study was approved by the ethicalCommitteeofWuhanUniversitySchoolofMedicine. Animal handling was carried out according to the Wuhan Directivefor AnimalResearch. Atotal of240healthy male BALB/cmice(4-weekold),weighing10–12g,werepurchased from the AnimalCenter ofHubeiProvince (Wuhan, Hubei, China) and had free access to food and water during the experiments.Amongthem,160randomlyselectedmicewere inoculated intraperitoneally with CVB3 (Nancy strain) at a doseof105_{plaque-formingunits(pfu)inavolumeof100␮L}

usingphosphatebufferedsaline(PBS)asavehicle,anddivided evenlyinto4groups(n=40),includinginfectedbutuntreated group, low-dose, mild-dose and high-dose IL-28A treated groups. Starting 1h after infection, these CVB3 inoculated micereceivedintraperitoneal injectionwith PBS,10,20,or 40␮g/kgofrecombinantmurineIL-28A(Peprotech,RockyHill, NJ), respectively, followed by daily subcutaneous injection for4days.The80uninfectedmice,dividedevenlyintotwo

groupsthatwereeitherintraperitoneallyinoculatedwithPBS (n=40,controlgroup)or40␮g/kgILrecombinantmurine IL-28A (n=40,IL-28Agroup). Tenmice from each groupwere euthanizedondays4and7post-infection,respectively.The heartswereexposed,perfusedwith5mLPBSthroughtheleft ventricle,andthenisolated.Approximately10mgoftissues fromcardiacapexwereexcisedandweighedforplaqueassay, whereastheremainderoftheheartwasfixedin10%neutral formalinforhistopathologicexaminationasdescribedbelow. The remaining20 miceof each group were monitoredfor healthstatusandsurvivalanalysis.

Histopathology

Theheartswerefixedin10%neutralformalinfor24h, embed-dedinparaffin,andthencutinto4␮m-thicksections(about foursectionsperheart).Thenthesectionswerestainedwith hematoxylinandeosin(H&E)andusedtoassess inflamma-toryinfiltration,myocardiallesionornecrosis.Theextentof inflammationandgeneralizedmyocardialfibernecrosiswere blindlyscoredbyapathologistbasedonthefollowingscale: 0,absent;0.5,rare;1.0,10%ofmyocardialareaaffected;2.0, 25% areaaffected;3.0,25–50%areaaffected;4.0,>50% area affected.Thetwoscoresreflectingtheextentofinflammation and myocardiallesionwerethen averagedtoyieldan inte-gratedpathology(IP)scoreasanindicatorofoverallinjury.22

Viralplaqueassays

Theviralplaqueassaywasperformedaspreviouslydescribed to determine cardiac viral load.22 _{One pieceof heart apex} (10mg) was removed aseptically, weighed, and stored at −80◦_{C. Then the frozen heart tissue was homogenized in}

200␮LPBSandcentrifugedat12,000rpmfor5min.Viraltiter, expressedaspfu/mL,wasdeterminedfromthesupernatant ofthehearthomogenatebyaplaqueassayonHeLacell mono-layersaccordingtopreviousstudy.23

Westernblotanalysis

Western blot analysis was performed as described in the literature to evaluate the expressions of STAT1 and STAT2.24 _{The ventricles of mice were homogenized in a} lysis buffer consisting of 10mM HEPES, pH 7.4, 150mM NaCl, 5mM EDTA, 1mM sodium-orthovanadate, 20␮M 4-(2-aminoethyl)-benzenesulfonylfluoride,0.2%Nonidet P-40, 5␮g/mLleupeptin,andproteaseinhibitorcocktail(Sigma,St. Louis,MO).Thehomogenateswerecentrifugedat12,000rpm for10minat4◦C,andsupernatantswerecollectedto deter-minetheproteinconcentrationsusingtheDCproteinassay (Bio-Rad,Hercules,CA).Theequalamountsofprotein(40␮g) from each samplewere separated by sodiumdodecyl–poly acrylamiegelelectrophoresis(SDS-PAGE)using4–12%NuPAGE NovexprecastBis-Trisgradientgels(Invitrogen,Carlsbad,CA) andtransferredontopolyvinylidenedifluoride(PVDF) mem-branes(Bio-Rad,Hercules,CA).Themembraneswereblocked in 5%BSA for40minatroom temperature and then incu-batedindividuallyat4◦Covernightwithprimaryantibodies, includingrabbitpolyclonalantibodiesagainstSTAT1(1:1000) andactin(1:2000),andmousemonoclonalantibodiesagainst

STAT2(1:200)(CellSignalingTechnology,Inc.,Danvers,MA). AfterthreewashingswithTBST,membraneswereincubated atroomtemperaturefor40minwiththesecondaryantibodies includinghorseradishperoxidase(HRP)-conjugatedgoat anti-rabbitIgG(1:10,000)andgoatanti-mouseIgG(1:5000)(Santa Cruz Biotechnology, Inc., Dallas,TX). Thesignals from the boundantibodiesweredetectedusingtheSuperSignalWest PicoChemiluminescentSubstrateKit(Pierce,Rockford,IL).

Immunohistochemistry

Immunohistochemistrywasperformedtoassessthe expres-sionsofBcl-2,BAXandCaspase-3accordingtotheinstructions ofthemanufacturer.Briefly,paraffin-embeddedmyocardial sectionswere deparaffinized inxyleneandrehydrated ina seriesofgradedconcentrationsofethanol.Protein antigenic-itywasenhancedbyboilingthe sectionsinacitratebuffer withmicrowavefor15min.Endogenousperoxidaseactivity wasblockedwith3%hydrogenperoxideinPBSfor20minat 37◦C.Thenthesectionswerewashedwithdistilledwater,and incubated withthe primaryantibodies againstBAX (1:100), Bcl-2(1:100), and caspase-3(1:100) (Santa Cruz Biotechnol-ogy,Inc.,Dallas,TX)at4◦Covernight,followedbystaining withastreptavidin–biotin–peroxidasekit(BeijingZhongshan BiotechnologyCo.,Ltd,Beijing,China).Theimmunoreactions were then visualized witha 3,3-diaminobenzidinesolution (Beijing Zhongshan Biotechnology Co., Ltd, Beijing, China), counterstainedwith hematoxylin, and mounted.For nega-tivecontrols,anequivalentvolumeofPBSwasusedinplace ofthespecificprimaryantibodies.Theimmunostaining tis-suesampleswerevisualizedunderalightmicroscope(Eclipse E800, Nikon,Japan)and fivephotomicrographs were taken fromhighpowerfields(400×magnifications)randomly.The averageopticaldensity(AOD)fromthephotomicrographswas thenquantitatedusingthehighresolutionpathologicalimage analysissystem(HPIAS-1000;QianPingCorporation,Wuhan, China)tomeasureproteinexpressionlevelsofBcl-2,BAXand Caspase-3.TheratioofBAXtoBcl-2wasdetermined individ-uallyfromeachphotographaccordingly.

TUNELassay

TUNEL (terminal transferase-mediated 20-deoxyuridine 50-triphosphate-biotinnick endlabeling) stainingon paraffin-embeddedsectionswasusedtoevaluatetheeffectofIL-28A onCVB3-inducedapoptosisaccordingtotheinstructions pro-videdbythemanufacturer.Briefly,4␮m-thicksectionswere dewaxed,rehydrated,andincubatedwithterminal deoxynu-cleotidyltransferaseenzymeinahumectouschamberat37◦C for1h. Thereaction wasdetectedbyincubationwith anti-digoxigenin-peroxidasefor30mininahumidified chamber atroomtemperature and visualized ina buffer containing diaminobenzidine. The specimens were counterstained by immersioninhematoxylin.Thestainedtissuesampleswere observedusingafluorescencemicroscope (OlympusBX40, Japan)andfivephotomicrographswere obtainedfromhigh powerfields (400× magnifications).Thecells withapparent nuclearlabelingwereconsideredTUNELpositive.The percent-ageofTUNEL-positivecellsinthetotalcellpopulationwas thenquantitatedtoassesstheapoptosisindex.25

1.0 0.8 0.6 0.4 0 2 4 6 8 10 12 14 Postinfective day Survival function Infected IL-28A treated Groups Cum sur viv al

Fig.1–IL-28ApreventedmicewithCVB3-induced

myocarditisfromdying.Thesurvivalratewasanalyzedby

theKaplan–Meierestimate.TheIL-28Atreatedmicehad

highersurvivalratesthantheuntreatedinfectedgroup

followingCVB3infection(p<0.001).

Statisticalanalysis

Datawerepresentedasmeans±SD.One-wayanalysisof vari-ance (ANOVA) was used todetermine differences between individualmiceforIPscoresandmyocardialviraltiters.The survivalratewasanalyzedbytheKaplan–Meierestimate.All statisticalanalyseswereperformedusingSPSS13.0(SPSSInc., Chicago,IL).Anyp-values<0.05wereconsideredstatistically significant.

Results

IL-28AtreatmentprotectsmicefromCVB3 infection-associateddeath

Uninfected BALB/c mice appeared healthy whereas CVB3-inoculatedmicedisplayedsignsofseriousillness,including ruffled and dirty fur, soft feces, lethargy, and huddling behavior.Twelvemiceintheinfectedgroupdiedafter infec-tion, with six, five and one mice dying on days 6, 9 and 12 post-infection,respectively. However,treatmentwith IL-28A improved mices’ general appearance, and completely preventedCVB3infection-associateddeathupto14days post-infection,evenwiththelowerdoseofIL-28A(10␮g/kg).The Kaplan–Meieranalysisclearlyshowedmuchhighersurvival rate in IL-28A treated mice than untreated mice(infected group)followingCVB3infection(Fig.1)(p<0.001).

IL-28AtreatmentinhibitsCVB3infection-induced inflammationandmyocardiallesion

Histological examinationofthe H&E-stainedslidesshowed evidenceoflymphocyteinfiltrationandmyocardialnecrosis onday7afterCVB3infection(Fig.2A)comparedtocontrols.

Control IL-28A

B

A

Uninfected IL-28A (μg/kg) treated

Infected N.D N.D 4 3 2 1 0

Control IL-28A treated (40 μg/kg)

50 μm 50 μm 50 μm Infected 10 20 40 P athology score

Fig.2–IL-28AdecreasedtheIPscoreofCVB3-infectedmice.(A)UninfectedandCVB3-infectedmiceweretreatedwithor

without40␮g/kgIL-28Afor4days.Hearttissuewasremovedonday7post-infectionandsubjectedtoH&Estaining(200×

magnification)toexaminemyocardialinflammationandinjury.Thearrowindicatedasiteoflymphocyteinfiltrationinthe

hearttissuefollowingCVB3infection.(B)UninfectedBALB/cmiceweretreatedwithorwithout40␮g/kgIL-28A.

CVB3-infectedmicewereexposedtodifferentdosesofIL-28Afor4days.Hearttissuewasthenremovedonday7

post-infectionandsubjectedtoH&EstainingtoquantitateIPscores.TreatmentwithIL-28AsignificantlyreducedtheIP

scoresintheheartsofIL-28Atreatedmicecomparedwithinfectedmice(**p<0.01).

However,theseverityofcellularinfiltrationwassignificantly reducedinIL-28Atreatedgroup(40␮g/kg).AsshowninFig.2B, CVB3infectionincreasedtheIPscoreto2.81±0.22,whilein uninfectedcontrolandIL-28Agroupsthebasalscorewas0, withinflammationor generalizedmyocardialfiber necrosis undetected(N.D.).Despitenoeffectonuninfectedmice, IL-28AtreatmentreducedtheIPscoreofCVB3-infectedmicein adose-dependentmanner(p<0.01).Adoseof40␮g/kgalmost completelyeliminatedcardiacinflammatoryinfiltrationand protectedcardiacmyocytesfromdamage,withtheaverage IP scoreon day7 post-infection being reduced to 0.4±0.1 (p<0.01)comparedwiththeinfectedgroup.Treatmentwith alow-doseIL-28A(10␮g/kg)alsostatisticallyreducedtheIP scorecomparedwiththeuntreatedinfectedgroup,butata levelinferiortothatwith20␮g/kgIL-28A.

IL-28Areducescardiacviralload

InoculationofCVB3inBALB/cmiceresultedinconsiderably highlevelsofcardiacviralload(6432±291pfu/mg)detected bytheplaqueassayonday4post-infection(Fig.3A),which thenspontaneouslydeclinedto550±50pfu/mghearttissue

onpost-infectiveday7(Fig.3B).Asexpected,treatmentwith IL-28Adose-dependentlyreducedcardiacviralloadfourdays afterCVB3infections(p<0.01),evenatthelowdose(10␮g/kg). Onpost-infectiveday7,therewasafurtherreductionin car-diacviralloadinmicetreatedwithIL-28Acomparedwiththat ofuntreatedinfectedgroup(p<0.01,Fig.3B).

IL-28AupregulatestheexpressionofSTAT1andSTAT2

Treatment with 40␮g/kg IL-28A dramaticallyincreased the proteinlevelsofSTAT1andSTAT2inthehearttissuesofmice4 daysafterCVB3infection(Fig.4).Nosignificantdifferencewas observedinthelevelsofSTAT1andSTAT2betweenuntreated infectedandcontrolgroups.

IL-28AinhibitsCVB3-inducedapoptosisofthemyocardial cellsinmice

FollowingCVB3infection,apoptosiswastriggeredwitha sig-nificantincreaseinapoptosisindexintheinfectedmice(0.7%) comparedtocontrolgroup(0.1%)(p<0.01,Fig.5).However,a significantdecreaseinapoptoticindexwasobservedinIL-28A

Control IL-28A

Uninfected IL-28A (μg/kg) treated Infected N.D N.D 0 200 400 600 800 1000

B

A

Uninfected IL-28A (μg/kg) treated Infected N.D N.D 0 2000 4000 6000 8000 10000 10 20 40 PFU/mg hear t tissue

Fig.3–IL-28Adecreasedviralloadofhearttissuein

CVB3-infectedmice.UninfectedandCVB3-infectedmice

weretreatedwithorwithoutIL-28Afor4days.Hearttissue

ofinfectedmicewereremovedonday4(A)andday7(B)

post-infectionandsubjectedtoplaqueassays.Treatment

withIL-28Asignificantlyreducedmyocardialviralload

comparedwithinfectedmice(**p<0.01).

(0.39%)treatedmicecomparedtotheuntreatedinfectedgroup (p<0.01,Fig.5),suggestingthattreatmentwithIL-28Acould inhibitCVB3-inducedapoptosisinmyocardialcells.

AsshowninFig.6A,infectionwiththeCVB3substantially decreasedtheexpressionlevelsofBcl-2andupregulatedthe expressionsofBAXandCaspase-3comparedtocontrols,while IL-28Atreatmenthadareverseeffectontheexpressionlevels oftheseapoptosis-relatedproteins,withincreasedexpression levelsofBcl-2anddecreasedexpressionofBAXand Caspase-3.ThequantitativedatafurthershowedthatCVB3infection induceda2-folddown-regulationofBcl-2expression,5-fold upregulationofBAXexpression,aswellasa10-folddecrease intheratioofBcl-2toBAXcomparedwithuninfectedcontrol mice(Fig.6BandC).TreatmentwithIL-28Aupregulatedthe expressionofBcl-2by5-fold,down-regulatedtheexpression ofBAXby2.5-fold,andconsequentlyincreasedtheratioof Bcl-2toBAXby10-foldcomparedtotheuntreatedgroupfollowing CVB3infection(Fig.6BandC).

Discussion

It was demonstrated that CVB3 infection in humans frequently progresses to cardiomyopathy, left ventricular

Control STAT2

STAT1

Actin

Treated Infected

Fig.4–IL-28AincreasedtheexpressionofSTAT1and

STAT2inhearttissue.UninfectedandCVB3-infectedmice

weretreatedwithorwithoutIL-28A(40␮g/kg)for4days,

thenthehearttissuesofinfectedmicewereremovedon

day4post-infectionandsubjectedtoWesternblotanalysis.

Actinwasusedasaloadingcontrol.TheamountofSTAT2,

STAT1andActinwasquantifiedusingNIHImageJandthe

ratioofSTAT2orSTAT1toActinwasgraphedinthelower

panels.IL-28Atreatmentresultedinasignificantincrease

intheexpressionofSTAT1andSTAT2comparedtoinfected

group(**p<0.01).

dysfunction,and subsequentchronicheartfailure.2 _Despite thegrowingknowledgeaboutviralmyocarditis,itremained challengingtomanagepatientswithviralmyocarditisdueto lackofeffectiveantiviraltherapeuticregimens.26_Inthisstudy, the resultsshowed that IL-28Aprotectedmicefrom CVB3-induced death, inhibited viral replication in heart tissues followingCVB3infection,andreducedtheinduced inflamma-tionandmyocardiallesion.

Fromtheresults,noinflammationorgeneralized myocar-dialfibernecrosiswasdetected(N.D.)inuninfectedcontrol andIL-28Agroups(40␮g/kgIL),suggestingthatthiscytokine does not cause apparent side effects in mice. A relatively lowdose(10␮g/kg)ofIL-28Awassufficienttoreduce CVB3-induced myocarditis in mice as evidenced by significantly decreasingIPscoresandcardiacviralload,letalonetreatment with40␮g/kgIL-28A,whichalmostcompletelyinhibited car-diacinflammatoryinfiltrationandprotectedmyocytesfrom injurywithIPscoredecreasedto0.4±0.1.Itseemedthat IL-28Awasahighlyeffectiveandtolerableantiviralagentagainst CVB3-inducedmyocarditis,althoughfurtherclinicaltrialswill beconductedtoensureitseffectivenessandsafetyinhuman patients.

Consistentwithpreviousreports,27_{thisstudyalso} demon-strated that viral replication was a dominant pathological processintheearlystagesofCVB3infection,asevidencedby averyhighviralloadwithrelativelylowlevelsof inflamma-toryinfiltrationandtissueinjuryintheheartonday4.This wasfollowedbyaspontaneousreductionofviralloadbuta furtherdevelopmentofinflammationand injuryincardiac tissueonday7,whichwoulddeteriorate generalhealth,as evidencedbythepresenceofdeathonday6to12.The spon-taneousdecayofviralloadmightbeattributedtoanactivation

Control Infected Treated

Control Infected Treated

0 0.5 1.0 2.0 1.5

A

B

C

Fig.5–EffectofIL-28Aonapoptosisincardiacmyocytes.IL-28Adecreasedtheapoptoticindexinhearttissuecomparedto

theinfectedgroup(**p<0.01).UninfectedandCVB3-infectedmiceweretreatedwithorwithoutIL-28A(40␮g/kg)for4days,

thenhearttissuesofmicewereremovedonday4post-infectionandsubjectedtoTUNELassay.Thestainedtissuefrom

control(A),CVB3-infected(B),andIL-28A-treatedmice(C)werephotographedunderafluorescencemicroscope(400×

magnification).ThecellswithapparentnuclearlabelingwereconsideredTUNELpositive.ThepercentageofTUNEL-positive

cellsinthetotalcellpopulationwasthenquantitatedtoassesstheapoptosisindex(D).

oftheinnatecellulardefensemechanismsincludingtheIFN system.28–30 _{AstypeIIIIFNsincludingIL-28A,IL-28Band} IL-29arefrequentlyupregulatedinresponsetocertaintypesof virus infectionsand exerted antiviralactivity in vitro.15,18,31 ThisstudyfirstdocumentedtheefficacyofIL-28Aininhibiting CVB3replicationinhearttissuesofinfectedmice.UnliketypeI

IFNsusingIFNAR(composedofsubunitsIFNAR1andIFNAR2c) asareceptor,typeIIIIFNssignaledthroughaheterodimeric receptorcomposedofIL-28R␣(IFN-␭R1orCRF2-12)specificto IFN-␭,andIL-10R␤(IL-10R2orCRF2-4)sharedwithallother IL-10-related cytokines.15,31,32 _{However, upon binding their} specificcellsurfacereceptors,typeIandtypeIIIIFNstrigger thesameJak-STATsignaltransductionpathway,33,34_leadingto theupregulationofmanygenes,calledIFN-stimulatedgenes (ISGs).35

OurfindingsdemonstratedthatIL-28Amarkedlyincreased theexpressionsofSTAT1andSTAT2,indicatingthatthesetwo proteinswere thekey antiviral regulatorsin CVB3-infected hearttissues ofmice.Itwas welldocumented that activa-tionofSTAT1andSTAT2wasessentialforantiviraleffectof IFNs.Followingvirusnucleicacidsrecognizedby transmem-branetoll-likereceptors(TLRs),cytoplasmicDNAsensorsand RNAhelicases,kinaseswereactivated.Thesekinaseswould promotetheactivationoftranscriptionfactorsandtheir sub-sequenttranslocationtothenucleuswheretheystimulated

IFN genetranscription.Afterbindingtothe cognate recep-tors, type I and type III IFNs induced the same Jak/STAT pathwaytoactivateSTAT1and STAT2transcriptionfactors. ThenphosphorylatedformsofSTAT1andSTAT2werefurther associatedwiththeDNA-bindingproteinIFNregulatory fac-tor9(IRF-9)toformanIFN-stimulatedgenefactor3(ISGF3) complex,16,35_{whichwasalsoacrucialmediatorinthetype1} interferonreceptor(IFNAR)signalingpathway.36_ThenISGF3 was translocated to the nucleus, bound to IFN-stimulated responseelements(ISREs)withinthepromotersof interferon-stimulated genes (ISGs) and activated the transcription of ISGs.16,35_{Althoughthecompletesignalingcascadeinitiatedby} thebindingofIFNtoitsreceptorhadnotyetbeenfully eluci-dated,currentevidenceindicatesthatthesignalingpathways activatedbyIFN-␣/␤andIFN-␭maybesimilaroroverlap.14,37 Thus,thefindingsabouttheexpressionsofSTAT1andSTAT2 expressioninducedbyIL-28Aprovidedareasonable mecha-nismresponsibleforIL-28A-mediatedantiviraleffect.

Asapoptosiswasimplicatedinvirus-inducedmyocardial injury,5,6_{theeffectofIL-28AonCVB3-inducedapoptosiswas} alsodetermined.Theresultsshowedthattreatmentwith IL-28AcouldinhibitCVB3-inducedapoptosisinmyocardialcells asevidencedbyasignificantdecreaseinapoptoticindexin IL-28A(0.39%)treatedmicecomparedtotheuntreatedinfected group (p<0.01). This could be attributed to the regulating

Control Infected Treated

Control Infected Treated

Control T reated Inf ected Caspase 3 Bcl-2 Bcl-2 BAX BAX 0 0 2 4 6 8 10 12 0.5 1 1.5 2 2.5

A

B

C

age optical density (%)

Fig.6–ImmunohistochemistrystainingofBcl-2,BAXandcaspase-3.IL-28AenhancedtheexpressionofBcl-2(p<0.01)and

decreasedtheexpressionofBAX(p<0.01)andcaspase-3(p<0.01)inhearttissue.UninfectedandCVB3-infectedmicetreated

withorwithoutIL-28A(40␮g/kg)for4days,thenhearttissueofmicewereremovedonday4post-infectionandsubjected

toimmunohistochemistrystaining.Theimmunostainedtissuesampleswerevisualizedandphotographedunderalight

microscope(A).Atotaloffivephotomicrographsweretakenfromfivehighpowerfields(400×magnification)randomly.AOD

fromthephotomicrographswasthenquantitatedtomeasuretheproteinexpressionlevelsofBcl-2andBAX(B)asdescribed

intheSectionofMaterialsandMethods.TheratioofBAXtoBcl-2wasdeterminedindividuallyfromeachphotograph(C).

effectofIL-28Aontheexpressionofcaspase-3,BAXandBcl-2, whichwerekeymoleculesregulatingapoptosis.38–40_TheBcl-2 couldinhibitapoptosisbybindingtoBAXwhileBAXpromoted apoptoticcelldeathbybindingtoitself.41_{Specifically,incells} with BAX overexpression, BAX/BAX homodimers predomi-nated,andturningthecellssusceptibletoinducedapoptosis. Conversely,incellswithBcl-2overexpression,BAX/Bcl-2 het-erodimerspredominated and the cells became resistant to apoptosis.41_{SinceBAX,aproapoptoticprotein,antagonized} thefunctionofBcl-2,it hadbeengenerallyconsideredthat theBcl-2/BAXratiocoulddeterminethesusceptibilityofacell toapoptotic stimulus.42,43 _{Besides, caspase-3,amember of} thecysteine-asparticacidprotease(caspase)family,actedas theexecutantofcellularapoptosis.44,45_{Accordingtotheabove} reports,theresultalsoshowedthatIL-28Areducedthe expres-sionsofBAXandcaspase-3butupregulatedBcl-2expression, therebyresultinginanincreaseintheBcl-2/BAXratioand sub-sequentinhibitionofCVB3-inducedmyocardialcellapoptosis inmice.Interestingly,previousstudiesdemonstratedthatthe activationofSTAT1notonlyservedtoincreasethe expres-sionofBcl-246_{butalsonegativelyregulatedBAXexpression.}47 Therefore,it seemed that, inadditiontoits antiviraleffect as discussed above, STAT1 activated byIL-28A might bind to the promoters of Bcl-2 or BAX, contributingto positive andnegativeregulationofBcl-2andBAXexpression, respec-tively.Hence,IL-28AmightincreasetheratioofBcl-2/BAXand consequentlyinhibitCVB3infection-inducedapoptosisofthe myocardiuminmicebytheabovemechanism.

Inconclusion,thisstudydemonstratedthatIL-28Aplayed animportantrolein thehostinnatedefenseagainstCVB3 infectionandcouldinhibitCVB3-inducedmyocarditisinmice. ThestudyalsosuggestedthatSTAT1andSTAT2arethekey antiviral regulatorsin heart tissuesofCVB3-infected mice, withtheformerIL-28AcouldincreasetheratioofBcl-2/BAX andconsequentlyinhibit CVB3infection-inducedapoptosis ofthemyocardium.Thusitcould beconcludedthatIL-28A hadagreatpotentialtobecomeatherapeuticinterventionfor CVB3infectioninhumans;however,theprecisemechanisms bywhichIL-28Aexertedtheantiviraldefensestillneedtobe exploredinthefuture.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WethankDr.ZhanqiuYangforgenerouslyprovidinguswith theCVB3virus.

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