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Pharmaceutics2020,12,1003;doi:10.3390/pharmaceutics12111003www.mdpi.com/journal/pharmaceutics
Article
RecombinantMyxomaVirus‐DerivedImmune
ModulatorM‐T7AcceleratesCutaneousWound
HealingandImprovesTissueRemodeling
JordanR.Yaron
1,†
,LiqiangZhang
1,
*
,†
,QiuyunGuo
1
,EnkidiaA.Awo
1
,MichelleBurgin
1
,
LaurenN.Schutz
1
,NathanZhang
1
,JacquelynKilbourne
2
,JulianeDaggett‐Vondras
2
,
KennethM.Lowe
2
andAlexandraR.Lucas
1,
*
1
CenterforPersonalizedDiagnosticsandCenterforImmunotherapy,VaccinesandVirotherapy,
TheBiodesignInstitute,ArizonaStateUniversity,Tempe,AZ85287,USA;jyaron@asu.edu(J.R.Y.);
qguo27@asu.edu(Q.G.);eawo1@asu.edu(E.A.A.);mburgin@asu.edu(M.B.);lschutz2@asu.edu(L.N.S.);
nzhang55@asu.edu(N.Z.)
2
DepartmentofAnimalCareandTechnologies,ArizonaStateUniversity,Tempe,AZ85287,USA;
jacki.kilbourne@asu.edu(J.K.);juliane.daggett@asu.edu(J.D.‐V.);kenneth.m.lowe@asu.edu(K.M.L.)
*Correspondence:liqiang.zhang@asu.edu(L.Z.);arlucas5@asu.edu(A.R.L.);Tel.:+1‐301‐803‐8881(L.Z.);
Tel.:+1‐352‐672‐2301(A.R.L.)
†Theseauthorscontributedequallytothiswork.
Received:21August2020;Accepted:21October2020;Published:22October2020
Abstract:Complexdermalwoundsrepresentmajormedicalandfinancialburdens,especiallyinthe
contextofcomorbiditiessuchasdiabetes,infectionandadvancedage.Newapproachestoaccelerate
andimprove,or“finetune”thehealingprocess,soastoimprovethequalityofcutaneouswound
healingandmanagement,arethefocusofintenseinvestigation.Here,weinvestigatethetopical
applicationofarecombinantimmunemodulatingproteinwhichinhibitstheinteractionsof
chemokineswithglycosaminoglycans,reducingdamagingorexcessinflammationresponsesina
splintedfull‐thicknessexcisionalwoundmodelinmice.M‐T7isa37kDa‐secreted,virus‐derived
glycoproteinthathasdemonstratedtherapeuticefficacyinnumerousanimalmodelsof
inflammatoryimmunopathology.TopicaltreatmentwithrecombinantM‐T7significantly
acceleratedwoundhealingwhencomparedtosalinetreatmentalone.Healedwoundsexhibited
propertiesofimprovedtissueremodeling,asdeterminedbycollagenmaturation.M‐T7treatment
acceleratedtherateofperi‐woundangiogenesisinthehealingwoundswithincreasedlevelsofTNF,
VEGFandCD31.TheimmunecellresponseafterM‐T7treatmentwasassociatedwitharetention
ofCCL2levels,andincreasedabundancesofarginase‐1‐expressingM2macrophagesandCD4T
cells.Thus,topicaltreatmentwithrecombinantM‐T7promotesapro‐resolutionenvironmentin
healingwounds,andhaspotentialasanoveltreatmentapproachforcutaneoustissuerepair.
Keywords:recombinantproteintherapeutic;woundhealing;chemokine;tissueremodeling;
immunemodulator
1.Introduction
Dermalwounds,especiallythosecomplicatedbyfactorssuchasdiabetes,infectionandage,
representamajormedicalburden,estimatedtoaccountforanannualexpenditureofmorethanUSD
20billionby2024[1].Newapproachestomanageandimprovethehealingofcutaneouswoundsare
thefocusofintenseinvestigation.Asthefirstbarrierprotectingthehostfromexternalinsults,the
skincontainsanintricateimmunesystemthatrapidlyrespondstolimitinfection,toremovedebris
Pharmaceutics2020,12,10032of16
andtorepairdamage.Cutaneouswoundhealingisacomplexmulti‐stageprocessthatreliesupon
numerouscelltypesandmediatorsactinginacoordinatedtemporalsequence.Thewoundhealing
processiscommonlydescribedinfourphases:(i)hemostasis(ii)inflammation,(iii)tissuegeneration
(proliferation)and(iv)remodeling[2].Dysregulationatanystageofwoundhealingprolongsthe
resolutionprocess,worsensscarringandleadstotissuedisruptionandtheriskofinfection.The
immunesystemplaysacriticalroleinsuccessfulwoundhealing.Inadditiontocontributingtohost
defensesagainstinfection,immunecellsarecriticalregulatorsofwoundhealingthroughthe
secretionofcytokines,chemokinesandgrowthfactorsthatorchestratelocalinflammatorycell
invasionandresponses,cellulardifferentiation,andtissueregeneration[3,4].Theimmunecells
involvedinwoundhealingincludeallclassesofneutrophils,macrophages,andTandBlymphocytes.
Animbalanceofimmunecellfunctionordiscordanceincellorchestrationatanystagecanresultin
impairedwoundhealing.Toolsto“finetune”theimmuneresponsemyleadtobettertreatment
managementandimprovewoundhealing.
Chemokinesarekeyplayersinthecellularorchestrationthatregulateswoundhealing.While
theyareinvolvedinallstagesofwoundhealing,chemokinesaremostabundantandvariedduring
theinflammationandproliferationstages[5].Chemokinesrecruitleukocytes,stimulatetheactivity
ofneutrophils,drivemacrophageactivityandpolarization,andregulatetheproliferationof
fibroblastsandkeratinocytes,whichmediatecollagendeposition[6].Chemokinesarealsoclosely
involvedinangiogenesis,newvesselgrowthandextension[6].Thereareapproximately20
chemokinesthatareknowntobeinvolvedinwoundhealing,fromacrossallfourchemokine
subfamilies(C,CC,CXC,andCX3C).Chemokinesreleasedfrominjuredtissuesbindto
glycosaminoglycans(GAGs)onthesurfacesofcells,inthevascularlumenglycocalyxorwithin
extracellularmatrices,wheretheymaysignaldirectlytocells,stimulatinginter‐andintra‐cellular
cascades,ordrivechemotacticmigrationtowardsthesiteofinjury[7].Basedontheunderstanding
ofthestructureandfunctionofthesechemokines,researchinteresthasintensifiedaroundthe
developmentofnewtherapeuticmethodstoacceleratewoundhealing,ortorepairwoundswith
impairedhealingbychemokinemodulation[8,9].Thesemethodsincludechemokinedepletionwith
biomaterialscontainingGAGs[10],immunotherapywithmonoclonalantibodiesagainstindividual
chemokinesandchemokinereceptors[9],orthetopicalapplicationofrecombinantchemokines[11].
Whilethebasisofsomeoftheseapproachesremainsincompletelyunderstood,eachhas
demonstratedsuccessinpreclinicalexperiments.
Ourgroupandothershaveinvestigatedtheapplicationofimmunemodulatorsfromvirusesas
anapproachtowardderivingnovelproteintherapeutics[12].Theco‐evolutionofviruseswiththeir
naturalhostsinvokesanadaptationarmsrace,wherebyasuccessfulstrategyforthevirusrelieson
immuneevasion,oftentargetingkeypathwaysthatdriveimmuneactivation[13].LargeDNA
viruses,suchaspoxvirusesandherpesviruses,areadeptatevadingtheinnateimmunesystemviaa
suiteofvirulencefactors[14,15].Translationally,thesefactorsconstitutearichtoolboxfordeveloping
immunemodulatorsfortreatingdisease[12].
Myxomavirus(MYXV)isaleporipoxviruswithwell‐knownstrictspecies‐specificityandhost‐
tropismtotheEuropeanrabbit(Oryctolaguscuniculus)[16].Wehavedemonstratedthesafetyand
immunotherapeuticefficacyofseveralMYXVimmunemodulatorsinawidearrayofpreclinical
models[17–27].M‐T7isanMYXV‐derivedimmunemodulatorwithbroadchemokine‐binding
activityandproventherapeuticpotentialininflammation‐relateddiseases.M‐T7isexpressedearly
inMYXVinfection,andisthemostabundantlysecretedimmunemodulator[28].InMYXVinfection,
M‐T7blockslymphocyteinfiltrationintoinfectedlesionsbypreventingchemokinegradient
formation[29].M‐T7isasolubleglycoproteinwiththeabilitytodirectlybindallclassesof
chemokines(C,CC,CXC)testedinvitro,anddecoupletheirinteractionswithGAG[30].M‐T7
treatment,givenwithorwithoutconcomitantcyclosporine,reducedacuterenaltransplantrejection,
vasculopathyandscarringinrats[31].M‐T7alsomarkedlysuppressedinflammatorycellinvasion,
andreducedacuteandchronicaorticandrenaltransplantrejection,inmiceinamannerdependent
onheparansulfation[23,32].Thus,M‐T7hasthepotentialtopromotearesolutionphenotypeanda
mechanismoftissuehealingwhereintheregulationofinflammationiscritical.Here,weinvestigate
Pharmaceutics2020,12,10033of16
thepotentialforrecombinantM‐T7tomodulatehealingandlocalinflammatorycellresponsesatsites
offull‐thicknesscutaneouswoundsinamousemodel.
2.MaterialsandMethods
2.1.RecombinantM‐T7Production
Purified,recombinantM‐T7protein(m007L;NCBIGeneID#932081)wasproducedand
providedbyVironTherapeutics(London,ON,Canada)andexpressedandpurifiedaspreviously
described[33].Briefly,theM‐T7codingsequenceisinsertedintopFastBacDualwithaC‐terminal
His‐tagandtransformedintoDH10Baccellstogeneratebacmidsasbaculovirusshuttlevectors.
PurifiedbacmidsaretransfectedintoSf21cellsusingCellfectinIIreagent.Supernatantscontaining
baculovirusareusedtotransduceHighFivecells.SecretedM‐T7ispurifiedbyaffinitytag
purificationoveraNi‐NTAcolumnwithfurtherpurificationbysizeexclusionchromatographyvia
FPLCwithaHiLoad16/60Superdex75column.
2.2.Animals
AllanimalproceduresinthisstudywereapprovedbytheInstitutionalAnimalCareandUse
CommitteeofArizonaStateUniversityunderprotocol#17‐1549R.Maleandfemalewildtype
C57BL6/Jmicewerebredon‐siteatArizonaStateUniversity.Miceaged8–12weekswereselectedby
simplerandomization[34]andusedinthisstudy.Micewerekeptonastandard12hlight–12hdark
cycleinaspecificpathogen‐freeenvironmentandgivenfoodandwateradlibitum.Miceweresingle‐
housedafterthewoundingproceduretopreventinterferencewithwoundhealing,aspreviously
described[35].
2.3.WoundingSurgeryandMeasurement
Weperformedasplinted,full‐thicknesswoundhealingmodelaspreviouslydescribed[35].In
thismodel,asiliconesplintisusedtoprohibitthewoundcontractionofmouseskinaroundasingle,
intrascapularfull‐thicknessbiopsypunchwoundduringthefirstsevendays,effectivelyforcing
second‐intentionhealingasoccursinhumanskin(whereasmouseskinprimaryhealsbycontraction).
Briefly,micewereanesthetizedbyintraperitonealinjectionof0.1mLper25gbodyweightofa
cocktailof120mg/kgketamineand6mg/kgxylazine.Oncereachingtheanestheticsurgicalplane(as
determinedbytoepinch),micewerepreppedbyshavinga1x1inchareaspanningfrombetweenthe
earstotheapexofthespineandcenteredbetweeneachshoulder.Theshavedareawassterilizedby
twosuccessivewashesof2%chlorhexidinegluconatesolution(Dyna‐Hex2®,XttriumLaboratories,
Prospect,IL,USA)followedby70%ethanolwithsterilecottonswabs.Asmallamountofveterinary
ocularointmentwasappliedtoeacheyetopreventcornealdrying.Micewerekeptonamonitored
heatingpadforthedurationoftheprocedure.
Afull‐thicknessexcisionalwoundwascreatedwitha3.5mmbiopsypunchtoolcenteredinthe
shavedarea.Carefulattentionwaspaidtopreventdamagetothepanniculuscarnosusbeneaththe
skin.Immediatelyaftercreatingthepunch,thewoundsweretreatedbyapplicationofeither20μL
sterilenormal0.9%NaClsalinesolution(N=17)or20μLsterilenormalsaline(0.9%NaCl)containing
1μgrecombinantM‐T7(N=16)applieddirectlytothewoundbedwithamicropipette.Adonut‐
shapedsiliconsplint(O.D.15mm;I.D.5.0mm;Culture‐Well™,GraceBiolabs,Bend,OR,USA)with
Tegaderm™(3MCompany,SaintPaul,MN,USA)affixedtoonesidewascoatedwithcyanoacrylate
glue(KrazyGlue®)ontheoppositesideandcarefullyplacedonthebackofthemousewhilekeeping
thewoundcenteredwithintheinnerdiameterofthesplint.Sixinterruptedsutures(4‐0blackEthilon
monofilamentwithanFS‐2reversecuttingneedle;Ethicon,Inc.,Somerville,NJ,USA)wereplaced
aroundtheoutercircumferenceofthesplint(approximately2mminsetfromtheedge)tocomplete
theprocedure.Miceweremonitoredonheatingpadsuntilawakeandmotilepriortoreturningto
single‐housedcagesfortheremainderoftheexperiment.Onday3post‐wounding,themicewere
anesthetizedwith1–3%isoflurane,toeffect,and20μLsalineor20μLsalinecontaining1μgM‐T7
Pharmaceutics2020,12,10034of16
wascarefullyappliedtopicallytothewounds(drop‐wiseabovethewound)byinsertinganinsulin
syringethroughthesiliconsplint,withcarenottodisruptthehealingwoundbedduringapplication.
Topreventself‐inducedsecondaryskindamagefromscratching,micewereagainanesthetizedwith
1–3%isoflurane,toeffect,onday7post‐woundingandthesplintswerecarefullyremovedwith
sterilesurgicalscissorsbeforebeingreturnedtosingle‐housedcagesaspreviouslydescribed[35].
TheexperimentaldesignisoutlinedinFigure1A.
Figure1.M‐T7acceleratesfull‐thicknesswoundhealinginmice.(A)Experimentaldesignoverview.
Micewerewoundedonday0(greenarrow)andfollowedtoday15post‐wounding(redarrow),at
whichtimemicewereeuthanizedandtissuewascollected.MiceweretreatedwithsalineorM‐T7on
day0withasecondbolusgiveonday3post‐wounding(bluearrow).Micewereanesthetizedand
splintswereremovedonday7post‐wounding(yellowarrow).Micewereassesseddailyandimages
werecollectedforplanimetricmeasurementofwoundclosure(grayarrows).N=5ineachgroup.(B)
Planimetricmeasurementsofwoundclosurenormalizedtoday0foreachmouse.Meanandstandard
errorareshown.StatisticswerecalculatedbyT‐testper‐daywithcorrectionformultiplecomparisons
bytheHolm–Sidakmethod.Forsignificance,aequatestop<0.05,bequatestop<0.01andcequates
top<0.001.(C)RepresentativewoundimagesforsalineandM‐T7‐treatedmiceonthedayof
wounding(day0)andondays2,4,7and15post‐wounding.Thesamemouseisshownineachimage
percondition.Scalebarsare5mm.
2.4.WoundPlanimetry
Micewereassessedwhileawakeonthedayoftheprocedure(day0)andoneverysubsequent
dayoffollow‐upforatotalof15days.Digitalimageswerecollectedalongwithaknownsizemarker.
PlanimetricmeasurementsofthewoundhealingprogresswereperformedinImageJ/FIJIand
calibratedagainsttheknownsizemarker[36].
2.5.ImmunohistochemistryandHerocivi’sPolychromeStaining
Micewereeuthanizedinindividualcohortsondays2,4,7and15post‐wounding,andtissues
werecollectedandfixedin10%neutral‐bufferedformalinfor1weekbeforeprocessing.Fixedtissues
wereprocessedandperfusedwithparaffinwithaLeicaTP1050processorthroughgradedalcohols
andxylene,thenembeddedintoparaffincassettesonaLeicaEG1160embeddingstation.Blockswere
sectionedusingaLeicaRM2165microtome(5μmsections)andstainedwithhematoxylinandeosin
(H&E)accordingtostandardprocedures.Slideswithsectionsthatreachedthewoundsiteas
Pharmaceutics2020,12,10035of16
determinedbyH&Escreeningwerefurtherstainedbyimmunohistochemistryandcollagenspecial
staining.
Immunohistochemistry(IHC)wasperformedaspreviouslydescribed.Briefly,theslideswere
rehydratedthroughgradedxyleneandgradedalcohols.Rehydratedslidesweresubmergedin
sodiumcitratebuffer,sandwichedwithacleanglassslidetopreventtissuelossandincubatedat60
°Ctoretrieveepitopes.Endogenousperoxidaseswerequenchedwith3%hydrogenperoxideinPBS
andnon‐specificproteinbindingwasblockedwith5%bovineserumalbumininTBS/0.1%Tween20.
Sectionswereprobedovernightat4°CwithrabbitpolyclonalantibodiesagainstArginase‐1(Cell
Signaling,#93668,1:200),CD31(Abcam,ab28364,1:200),CD3(Abcam,ab5690,1:200)andCD4
(Abcam,ab183685,1:1000),rabbitmonoclonalantibodiesagainstHSP47(Abcam,ab109117,1:300)or
TGF‐beta1(Abcam,ab215715,1:500),ormousemonoclonalantibodyagainstLy6G(Invitrogen,#14‐
5931‐82,1:200).HRP‐conjugatedsecondaryantibodiesagainstrabbitandmouseIgG(Jackson
ImmunoResearch,WestGrove,PA,USA)wereappliedatadilutionof1:500for1to2hatroom
temperature.AntigenswererevealedwithImmPACTDAB(VectorLabs,Burlingame,CA,USA),
counterstainedwithGil’sformula#3HematoxylinandmountedwithCytosealXYL.
Herovici’sPolychromecollagenstainkitwaspurchasedfromAmericanMasterTech(Lodi,CA,
USA).Theslideswereprocessedaccordingtomanufacturer’sprocedureandmountedwithCytoseal
XYL.
2.6.HistopathologyImagingandAnalysis
TheslideswereassessedonanOlympusBX51uprightmicroscopeequippedwithanOlympus
DP74CMOShigh‐resolutioncameraoperatedbycellSensDimensionsv1.16.Objective‐calibrated
TIFFswereanalyzedandprocessedinImageJ/FIJI.Positivelystainedcellswerequantifiedperhigh
powerfieldusingtheCellCounterplugindevelopedbyKurtDeVosandpackagedwithFIJIunder
aGPLv3license.Herovici’sPolychromestainswerequantifiedtoproducea“HeroviciRatio”ofpink
stain(TypeIcollagen)versusbluestain(TypeIIIcollagen),whereahigherratioindicatesmore
maturecollagenandthelessactivedepositionofimmaturecollagen.Briefly,imageswere
deconvolutedwiththepluginColourDeconvolution1.7usingthemethodsdescribedbyRuifrokand
Johnson[37].Aregionofinterestwasdrawninthedermisofthewoundareaandreplicatedtoboth
redandbluechannels.Theintegratedintensity(densitometry)oftheregionofinterestwasmeasured
foreachchannelandthevalueswereusedtoproducetheHeroviciRatio.
2.7.ELISAs
Enzyme‐linkedimmunosorbentassays(ELISA)wereperformedusingDuo‐SetkitsforTNFα
(DY410),VEGF(DY493)andCCL2(DY479),allfromR&DSystems(Minneapolis,MN,USA).ELISAs
wereperformedusingtissuescollectedfromindividualcohortsofmiceeuthanizedondays1,4and
7.A1cmtissuesamplecenteredonthewoundwascollectedforeachmouse,snapfrozenand
homogenizedinRIPAlysisbufferaccordingtomanufacturer’sprocedures.Resultswerenormalized
tomgtotalproteinasdeterminedbyBCAproteinassay(Pierce,ThermoFisherScientific,Carlbad,
CA,USA).
2.8.Statistics
AnalysisofstatisticalsignificancewasperformedbyTwo‐WayAnalysisofVariance(ANOVA)
andStudent’sunpairedT‐testusingGraphPadPrismv8.2.1.Allanalysespassednormalitytests
accordingtoAnderson–Darling(A2*),D’Agostino–Pearsonomnibus(K2),Shapiro–Wilk(W)and
Kolmogorov–Smirnov(distance)withanalpha=0.05.P‐valueswereconsideredsignificantat*p<
0.05,**p<0.01,***p<0.001and****p<0.0001,exceptinFigure1Bwhereaequatestop<0.05,b
equatestop<0.01andcequatestop<0.001forthepurposesofclarityindatapresentation.
Pharmaceutics2020,12,10036of16
3.Results
3.1.RecombinantM‐T7PromotesFull‐ThicknessWoundHealing
WeanalyzedtheeffectsofrecombinantM‐T7onthetreatmentoffull‐thicknesswoundsina
splintedwoundhealingmodelinwildtypeC57BL6/Jmice[35,38–40].Basedoneffectivedosesand
ourpriorworkwithasecondunrelatedMYXV‐derivedimmunemodulatingserineproteinase
inhibitor,Serp‐1,inthesamemodel,wegaverecombinantM‐T7topicallyinadoseof1μgin20μL
saline,withasecondbolusof1μgin20μLsaline3dayspost‐wounding(Figure1A)[35].Thecontrol,
saline‐treatedmiceweresimilarlytreatedwithabolusofsaline3dayspost‐wounding.Daily
planimetricmeasurementsofwoundhealingprogressdemonstratethatM‐T7significantly
acceleratesfull‐thicknesswoundhealingwhengivenasarecombinantproteininatopicalsaline
solution(Figure1B,C).Noevidenceofwoundsiteinfection(pus,discharge,discoloration)was
observedineitherthesalineorM‐T7‐treatedgroups.Wenotedthatduringthefirst8daysofhealing
(day0andupto7dayspost‐wounding),healingwaslimitedtoonlysecond‐intentionmechanisms
duetothesiliconsplintpreventingwoundcontraction(Figure1C).Thisisamajorbenefitofthe
siliconsplintmodelbecause,leftonitsown,mouseskincontractswithin1–2days,whichlimitsthe
interpretationofthehealingprocess.Byday7,salinecontrol‐treatedwoundshadonlyachieveda
meanof23%closure,whileM‐T7significantlyacceleratedthehealingprocessanda78%mean
woundclosurewasachieved(p=0.0007).Evenafterremovingthesiliconsplints,thewoundsofM‐
T7‐treatedmicecontinuedtoclosemorequicklythanmicetreatedwithsalinealone,andachieved
fullclosure3–4daysbeforesaline‐treatedmice.Thus,recombinantM‐T7givenduringtheearlystage
ofhealinghasasustainedeffectonacceleratingwoundclosure.
3.2.RecombinantM‐T7PromotesCollagenMaturationinWounds
Collagendepositionandmaturationarekeycomponentsofthewoundhealingprocess.The
inappropriatedepositionorimpairedmaturationofcollagenareassociatedwithscarringandlimited
angiogenesis[41].Theimprovedremodelingofcollageninthehealingwoundcanthusimproveboth
scarringandhealedtissuehealthviathepromotionofimprovedangiogenesis.Herovici’s
polychromeisahistologicspecialstainwhichdifferentiatesbetweenimmatureTypeIIIcollagen
(stainedblue)andmatureTypeIcollagen(stainedpink)[42].WeusedHerovici’spolychrometo
evaluatethecollagenmaturationofhealedwoundsafterM‐T7treatment.Quantitativeimage
analysisoftheamountofpinkTypeIcollagenstainingversustheamountofblueTypeIIIcollagen
showedwhatwerefertoastheHeroviciRatioforthetissue.AhigherHeroviciRatio(morepink,less
blue)indicatesmoreadvancedcollagenmaturation,whereasalowerHeroviciRatio(lesspink,more
blue)indicateslesscollagenmaturationandmoreactivedepositionofimmaturecollagen.Atday15
post‐wounding,thehealedwoundsofmicetreatedwithM‐T7hadsignificantlyhigherHerovici
Ratios,indicatingmorematureTypeIcollagenthansaline‐treatedmice(Figure2).Interestingly,this
wasnotassociatedwithanincreaseinfibroblastmarkerHSP47(FigureS1).Thesedataindicatethat,
inadditiontoacceleratedclosure,woundstreatedwithM‐T7hadahigherfidelity,healingwitha
moreproperlyorganizedcollagenarchitecture.
Pharmaceutics2020,12,10037of16
Figure2.QuantitativeassessmentofcollagenmaturationinwoundstreatedwithM‐T7.(A)
RepresentativemicrographsofHerovici’spolychrome‐stainednormalskinandwoundsat15days
post‐wounding.Toppanelsshowbrightfielddata,whilemiddleandbottompanelsshowcolor‐
deconvolutedfieldsforthepinkandbluechromophores.(B)Quantificationofcollagenmaturation
insalineandM‐T7treatedwoundsbytheHeroviciRatio,calculatedbythedensitometricratioofthe
pinkandbluechromophoresinHerovici’spolychrome.Meanandstandarderrorareshown.Statistics
werecalculatedbyT‐test.N=4saline,N=5M‐T7.
3.3.M‐T7StimulatesPeri‐WoundAngiogenesis
Angiogenesisisanessentialcomponentoftheproliferativestageofcutaneouswoundhealing,
characterizedbyanearlyandabundantburstofimmaturevesselswhicheventuallyregressintoa
maturevascularnetworkviatheactivityofanti‐angiogenicfactors,suchasSprouty2andPEDF[43].
Therapeuticstrategiesarenowactivelysoughttoenhanceangiogenesisduringwoundhealing[44].
Angiogenesisintheearlystagesofwoundhealingisdrivenbytheprimingofendothelialcellswith
pro‐inflammatorycytokines,suchastumornecrosisfactoralpha(TNFα),therebyinducingatipcell
phenotype[45].Inthecontextoftissueinjury,TNFαiscriticalforthedownstreamproductionof
vascularendothelialgrowthfactor(VEGF),anessentialgrowthfactorinregulatingangiogenesis[46].
WeperformedELISAanalysesofthehealingwound’sbedtissueondays1,4and7post‐wounding
toquantitativelymeasurelevelsofTNFα andVEGF(Figure3A).RecombinantM‐T7induceda
significantincreaseinwoundbedlevelsofTNFαondays1and4(p<0.05),andofVEGFbyday7(p
<0.05),versussalinetreatmentalone.Weperformedimmunohistochemistryofwoundtissueson
days4and7post‐woundingtodeterminethedegreeofangiogenesisbystainingforCD31(alsocalled
PECAM‐1),acanonicalmarkerforendothelialcellsinthevasculature(Figure3B,C).Aquantification
ofthenumberofCD31+cellsandvesselsper20×fieldintheperi‐woundareaindicatedasignificant
increaseonday4post‐wounding(p<0.05)versussalinetreatmentalone.Weobservednosignificant
differenceonday7post‐wounding.Qualitatively,wenotedthattheCD31+cellsformedmorerobust
vesselsinthewoundstreatedwithM‐T7,withincreasedlengthandthicknessversussalinetreatment
alone(Figure3C).Takentogether,theseresultssuggestthatM‐T7stimulatesanearlyTNFαresponse,
whichstimulatesamorerobustVEGFresponse,ultimatelyleadingtoacceleratedangiogenesisinthe
peri‐woundareaassociatedwithacceleratedwoundclosure.
Pharmaceutics2020,12,10038of16
Figure3.Assessmentofperi‐woundangiogenesisinwoundstreatedwithM‐T7.(A)ELISA
quantificationofTNFαandVEGFinwoundtissuestreatedwithsalineorM‐T7collectedondays1,
4and7post‐woundingnormalizedtototalprotein.Barsaremeanandstandarderror.Statisticswere
calculatedbytwo‐wayANOVAwithFisher’sLSDpost‐hocanalysis.(B)QuantificationofCD31+
cellsandvesselsper20×fieldintheperi‐woundareaofwoundstreatedwithsalineorM‐T7collected
ondays4and7post‐wounding.Barsaremeanandstandarderror.Twonon‐overlappingfieldswere
quantifiedpermouseandstatisticswereperformedontheaveragepermousewiththeN=4per
group.Statisticswerecalculatedbytwo‐wayANOVAwithFisher’sLSDpost‐hocanalysis.(C)
Representativeperi‐woundCD31IHCfields(10×)collectedondays4and7post‐wounding.Scale
barsare50μm.Zoomareasindicatedbyboxes.N=3–4ineachgroupandtimepoint.
3.4.M‐T7ModulatesImmuneResponsesintheHealingWound
M‐T7bindstoallclassesofchemokines(C,CCandCXC)andinhibitstheirinteractionwith
glycosaminoglycans,therebypreventingchemokinegradientformation[23,30,47].CCL2,alsocalled
monocytechemoattractantprotein‐1(MCP‐1),isaCC‐classchemokinepreviouslyshowntohavea
criticalroleintheregulationofwoundhealing[48].WeperformedtheELISAanalysisofCCL2on
woundtissuestreatedwithsalineorM‐T7,collectedondays1,4and7post‐wounding(Figure4A).
WoundstreatedwithM‐T7hadanelevatedlevelofCCL2onday4post‐wounding,which
approachedsignificance(p=0.0763),whilelevelsofCCL2werenotdifferentbetweensalineandM‐
T7treatmentondays1and7post‐wounding.Independentofitschemotacticfunction,thesignaling
ofCCL2withitsreceptor,CCR2,waspreviouslyshowntopromotethepolarizationofmacrophages
towardsapro‐resolution(i.e.,M2)phenotype[49].Weperformedimmunohistochemistryofwounds
treatedwithsalineorM‐T7ondays2,4and7post‐wounding,stainingforArginase‐1,acanonical
markerofM2macrophagepolarization.ThequantificationofArginase‐1+cellsrevealedatrend
Pharmaceutics2020,12,10039of16
towardselevatedM2macrophagesondays2and4post‐wounding,whichachievedsignificance(p<
0.05)byday7post‐wounding(Figure4B,C).Accordingly,thenumberofTGF‐beta+cellsperfield
trendedtowardssignificanceonday4(p=0.0836)andreachedsignificancebyday7post‐wounding
(p<0.05)(Figure4D).WefurtherinvestigatedtheeffectsofM‐T7treatmentonTcellinfiltrationin
thehealingwound.WefoundthatM‐T7treatmentsignificantlyinhibitedtheinfiltrationofCD3+T
cells,ageneralTcellmarker,intothebedofthehealingwoundondays4and7post‐wounding
(Figure4E),withoutinhibitingtheaccumulationofCD3+cellsintheepithelialtongueofthewounds
(Figure4F).RegulatoryTcells,aCD4Tcellsubtype,arecrucialforthenormalandaccelerated
healingofcutaneouswounds[50].WefoundthatM‐T7treatmentsignificantlyincreasedthe
accumulationofCD4+cellsintheepithelialtongueofhealingwoundsversussalinetreatmentalone
(Figure4G,H,FigureS2).Wedidnotobserveaneffectonneutrophilinfiltration(FigureS3).Thus,
decouplingthechemokine‐glycosaminoglycangradientwithM‐T7modulatestheimmuneresponse
inthewoundenvironmenttoacceleratehealing.
Figure4.M‐T7modulatestheimmuneresponseinthehealingwound.(A)ELISAquantificationof
CCL2inwoundstreatedwithsalineorM‐T7atdays1,4and7post‐wounding,normalizedtototal
protein.(B)QuantificationofArginase‐1+cellsper20×fieldofwoundstreatedwithsalineorM‐T7at
days2,4and7post‐wounding.(C)RepresentativeArginase‐1IHCfieldsatday7.(D)Quantification
ofTGF‐beta+cellsper20×fieldondays2,4and7post‐wounding.(E,F)QuantificationofCD3+cells
per20×fieldofwoundstreatedwithsalineorM‐T7atdays2,4and7post‐wounding,specificallyin
the(E)woundbedor(F)epithelialtongue.(G)QuantificationofCD4+cellsper20×fieldofwounds
treatedwithsalineorM‐T7atdays2,4and7post‐woundingnormalizedtothenumbersonday2.
(H)RepresentativeCD4IHCfieldsintheepithelialtongueatday7.Full20×fieldisgiveninFigure
S2.Allbarsaremeanandstandarderror.Statisticsarecalculatedbytwo‐wayANOVAwithFisher’s
LSDpost‐hocanalysis.N=3–4ineachgroupandtimepoint.
4.Discussion
Largecutaneouswounds,particularlywithassociatedpoorhealing(e.g.,diabeticoraged),
scarringandsuper‐imposedinfections,areacomplexandcostlymedicalburden,withanannual
incidenceofmorethan6millioncutaneouswoundcasesandacollectiveyearlycostofmorethan
Pharmaceutics2020,12,100310of16
USD20billion,notinclusiveofthemorethan170,000scarrevisionsurgeriesannuallyintheUnited
States[51].Comorbiditiessuchasadvancedageanddiabetes,orcomplicationssuchasinfection,
burnsandbattlefieldconditions,furtherincreasethedifficultyofwoundmanagementandtherisk
ofadverseoutcomes[52].Investigationhasthusintensifiedtoaddressanunmetneedfornovel
treatmentstoacceleratewoundhealing.
Inthisstudy,wetestedthetherapeuticefficacyofrecombinantMyxomavirus‐derivedimmune
modulatingproteinM‐T7inamousemodeloffull‐thicknesswoundhealing.Weadministeredtwo
dosesofrecombinantM‐T7ondays0and3post‐wounding,mirroringthedosingregimenthatwe
foundtobeoptimalforanotherMyxomavirus‐derivedimmunemodulator,Serp‐1,inaprevious
study[35].Planimetricanalysisrevealedasignificantaccelerationofwoundclosurebytreating
woundstopicallywithM‐T7.Accelerationoccurredduringtheearlieststagesofhealingandwas
independentofcontraction,asthesiliconesplintswerenotremoveduntilday7post‐wounding
(Figure1B).Thisfindingispromising,asthefirstphaseofhealingisknowntobeacrucialperiodfor
protectionagainstinfectionandthepreventionofadditionaltraumaasgranulationtissueisformed
[53].
Onepotentialriskofacceleratedwoundhealingisthedepositionofdisorganizedconnective
tissueleadingtoscarring,particularlyinfull‐thicknessskinwoundswithoutcontraction[54].
Drueckeandcolleaguesinvestigatedtheuseofdifferentdermalregenerationtemplatesonfull‐
thicknesswoundsinaporcinemodel[55].TheyfoundthatwhiletheIntegramaterial,acomposite
scaffoldofbovinehidecollagenandsharkchondroitin‐6‐sulfate,improvedcollagenmaturation,
therewasslowertissueingrowth,andtissueintegritywaslost[55].Incontrast,theauthorsfound
thatabovinehidecollagenspongescaffold,producedviachemicalcrosslinkingwith1‐ethyl‐3‐(3‐
dimethylaminopropyl)carbodiimide(EDC),allowedmorerapidingrowthandoveralltissue
integrity,withnobenefittocollagenmaturation.Here,wefoundthatinadditiontoaccelerating
woundhealing,recombinantM‐T7alsoresultedinearlier,improvedcollagenmaturation,as
determinedbyquantificationwithHerovici’spolychromeinametricwetermthe“HeroviciRatio”
(Figure2).AsimilarquantificationofTypeIII:IcollagenwasobservedbyO’Rourkeandcolleagues
intheirinvestigationofacceleratedwoundhealingbysurfactantpolymerdressingscontaining
siRNAtoFidgetin‐Like2,withtheauthorsnotingincreasedredTypeIcollagen,illustratinghigher‐
fidelityhealing[56].
Angiogenesisisacriticalcomponentofwoundhealing,andseveralcomorbiditiesassociated
withimpairedwoundhealing,suchasdiabetes,alsoexhibitreducedangiogenesis[57,58].
Angiogenesisplaysacriticalroleinprovidingthesufficientdeliveryofbloodandnutrients,and
accesstoreparativeimmunecells,duringthecourseofhealing.Accordingly,numerousgroupshave
investigatedapproachestoacceleratehealingbypromotingangiogenesis.Forexample,recombinant
TNFαapplieddirectlytowoundsacceleratestheearlystagesofwoundhealing[59].Thetherapeutic
effectofTNFαintheearlystageofwoundhealingmayproceedviatheinductionofangiogenesis,as
TNFα isapowerfulinducerofVEGFandendothelialtipcellformation[45,46].Othertreatments
showntoacceleratewoundhealingalsoactviatheinductionofangiogenesis.Forexample,topical
simvastatinandasperosaponinVIbothacttoacceleratewoundhealingbyenlistingtheVEGF
signalingcascade,andVEGF‐Capplieddirectlytowoundsalsoaccelerateshealing[60–62].Here,we
showthatarecombinantM‐T7treatmentresultedinasignificantincreaseinlocalTNFαduringthe
earlieststagesofwoundhealing,whichtemporallytransitionsintoasignificantincreaseinVEGFin
thehealingbed(Figure3A).Thiscoincideswiththecanonical,earlyinflammatoryphaseofhealing,
andthetransitiontotheproliferationphaseofhealing[63].Accordingly,increasedangiogenesiswas
directlyobservedbyimmunohistochemistryforCD31intheperi‐woundarea(Figure3B,C).Thus,
thedatasuggestthattopicalM‐T7modulatesthechemokineenvironment,resultinginthe
augmentationofpro‐healingmoleculesinthewoundenvironment,engagingthepro‐angiogenesis
signalingcascadeatthelevelofbothcytokinesandgrowthfactors,andresultinginasignificant
inductionofangiogenesisattheboundariesofhealingwoundsassociatedwithincreasedwound
closure.
Pharmaceutics2020,12,100311of16
WesoughttodeterminetheeffectofrecombinantM‐T7onlocalimmuneresponsesinthe
healingwounds.Somevirus‐derivedimmunemodulators,suchastheherpesvirusM3chemokine
decoyreceptorandMyxomavirusMT1,inhibittheabilityofchemokinestosignaltotheirreceptor.
M3alsouniquelyblockschemokinebinding,bothtoGAGsandalsotoreceptors[64].Incontrast,M‐
T7actsprimarilyatthelevelofchemokine‐glycosaminoglycaninteractions[30,47,65,66].M‐T7isan
interferongammareceptorhomologuewithspecificityforrabbitinterferongamma[28].
Interestingly,theM‐T7inhibitionofchemokinetoGAGbindingisfoundforallmammalstestedto
date,e.g.,rabbits,rats,miceandhumancells[30].Thus,M‐T7treatmentisexpectedtoinhibit
chemokinegradientformation[47].Indeed,wepreviouslyfoundthatM‐T7losttherapeuticefficacy
intheabsenceofnormalactiveheparansulfationinmicewithconditionalendothelialdeficiencyof
theheparansulfotransferaseenzymeNdst1,withpresumedconsequencesinmodifyingthe
formationofchemokinegradients[23].CCL2/MCP‐1signalingisknowntobecriticalinregulating
physiologicwoundhealing.Lowandcolleaguesreportedthatwoundsmadeinmicedeficientof
CCL2exhibitdelayedre‐epithelialization,reducedcapillarydensityandimpairedcollagen
remodeling[48].Incontrast,multiplegroupshaveshownthatrecombinantCCL2treatmentreverses
impairedwoundhealingindiabeticmicebyrestoringmacrophageresponses[67,68].Receptor
engagementbychemokinesinducestheinternalizationofboththereceptoranditsligand,resulting
inintracellulardegradationandrecycling[69].WhileCCL2existsindynamicequilibriumasbotha
monomeranddimer,onlythemonomericformiscapableofreceptorengagement,andobligate
dimericmutantsofCCL2areincapableofsignaling[70].Importantly,thedimerizationofCCL2
requiresglycosaminoglycaninteractions[71].WefoundincreasedlevelsofCCL2inhealingwounds
whentreatedwithrecombinantM‐T7(Figure4A),consistentwiththeroleofCCL2inimproved
healing.WehypothesizethatM‐T7treatmentinhibitedtheoligomerizationofCCL2,slowing
receptorengagementanddelayingitssubsequentdegradation.Investigatingfurther,wefoundM‐
T7‐dependenteffectsintwocellpopulationsknowntobeaffectedbyCCL2andotherchemokine
signaling:macrophagesandTcells.Specifically,wefoundanincreaseinM2‐polarized,pro‐
resolutionmacrophages(Figure4B).ThisfindingagreeswithpriorworkshowingthatCCL2
signalingresultsinM2polarization[49].WealsofoundincreasedCD4+Tcellsintheepithelial
tonguesofhealingwoundstreatedwithM‐T7(Figure4F,G),inagreementwiththeabilityforCCL2
topromoteCD4recruitment,andintheCD4‐lineagecellsdrivingacceleratedwoundhealing[50,72].
CCL2actsdirectlyonTcellsviatheactionofCCR2andCCR4[73],butcanalsoinducethe
recruitmentofCD4cellsintotissuesinapromiscuousmanner,usingotherreceptors[72].Further,
M‐T7interactswithmanychemokines,andmayinduceabroadmilieuchangeinarangeof
chemokines.ItwillbeimportanttoprobethespecifictargetedmechanismsofCD4recruitmentusing
geneticknockoutsorneutralizingantibodytreatmentsinfuturestudies.Wespeculatethatthe
continuedexpressionofCCL2bylocalcells,combinedwithanoff‐rateofCCL2:M‐T7interactions,
mayhavecontributedtosustainedCCL2signaling.However,wecannotexcludetheroleofother
chemokinesorcytokinesinthewoundhealingmilieuinthisstudy,asM‐T7interfereswithGAG
bindingforC,CCandCXCchemokinesinvitro.Thus,thedecouplingofglycosaminoglycan
interactionswithchemokinesbyM‐T7hasaneffectonCCL2signaling,anddownstreameffectson
macrophageandTcellpopulations,leadingtoacceleratedwoundhealing.Furtherworkisnecessary
todeterminethespecificchemokineandGAGpathwaysmodulatedbyM‐T7intheorchestrationof
localandinfiltratingimmunecellsinthehealingwoundbed.
M‐T7isnowthesecondMyxomavirus‐derivedimmunemodulatortoexhibitefficacyin
promotingwoundhealing.Serp‐1,aserineproteaseinhibitor(serpin),isaglycosylated,secreted
proteinwhichtargetsserineproteasesinboththethrombotic(FXa,thrombin)andthrombolytic(uPA,
tPA,plasmin)cascades[20].WerecentlyreportedthatrecombinantSerp‐1acceleratedfull‐thickness
woundhealinginmice,withnumerousobservedsimilaritiestotheM‐T7treatmentasperformedin
thisstudy[35].Inadditiontotheaccelerationofwoundclosure,bothSerp‐1andM‐T7resultedinan
increaseinVEGFandperi‐woundangiogenesis,aswellasanincreaseinpro‐resolutionM2‐polarized
macrophages.Thesesimilaritiesunderscorethebenefitsassociatedwithdevelopingtherapeutics
fromvirus‐expressedimmunemodulators.Thesestudiesalsoagainemphasizethesafetyofthese
Pharmaceutics2020,12,100312of16
immunemodulatorswhenusedastherapeutics.First,thelimitedgenomicspaceinavirus
necessitatestheevolutionofmultipotency(i.e.,multipletargetsofinhibition),providinghighly
potentandeffectiveimmunemodulatingmolecules[74].WhileSerp‐1targetsnumerousproteinsin
theclottingcascade,M‐T7targetsthepanoplyofchemokines.Second,virus‐derivedimmune
modulatorsoftenexhibitpotencyatextremelylowconcentrations.BothSerp‐1andM‐T7functionat
dosesofonly100nanogrampergrambodyweight,theequivalentofmicrogramperkilogramdosing
inhumans;thatis,thelowestendrangefortherapeuticbiologics[75].Indeed,Serp‐1hasbeenshown
toexhibittherapeuticefficacydowntothepicogrampergramrange[76].Third,virus‐derived
immunemodulatorshaveundergoneextensive“researchanddevelopment”intheevolutionary
armsracebetweenthevirusanditshost.InthecaseofMyxomavirus,anestimated10millionyears
ofevolutionhavegoneintodevelopingexpertmodulatorsofthehostimmuneresponse[16].Thus,
thesuiteofimmunemodulatingproteinsinMyxomavirusareavaluable,highlyoptimized
“medicinecabinet”fortargetingimmune‐drivenpathologies,andforharnessingimmunefunction
toenhancetissuerepair[12].Furtherworkwillinvestigatethepotentialforcombiningtheseproteins
incocktails,towardsfurtherenhancingtherapeuticresponsesinwoundhealingandothermodels.
5.Conclusions
WereportherethattreatmentwithrecombinantM‐T7,aMyxomavirus‐derivedchemokine
signalingmodulator,acceleratestherateofhealinginfull‐thicknesswoundsinwildtypemice.M‐T7
treatmentimprovedconnectivetissueremodeling,andincreasedangiogenesisandpro‐resolution
immunecellphenotypes.Thechemokinemilieuofthehealingwoundbedishighlycomplex,andM‐
T7caninteractwithallclassesoftheC,CCandCXCchemokines.WeobservedaneffectonCCL2in
thepresentstudy,whichwasassociatedwitheffectsonmacrophages,Tcellsandendothelialcells.
FurtherworkisneededtodelineatetheprecisemechanismsofM‐T7’stherapeuticeffectsonwound
healing.Further,itwillbeimportanttoinvestigatetheeffectsofrecombinantM‐T7incomplex
comorbiditiessuchasdiabetes,infectionandburns,todevelopnext‐generationversionsofM‐T7with
enhancedfunction,andtolearnmoreaboutthefundamentalroleofchemokinesincutaneouswound
healing.Thus,M‐T7representsapromisingvirus‐derivedtherapeutic,anewclassofprotein
biologicswiththepotentialtoaddressthesignificantmedicalburdencreatedbydermalwounds.
SupplementaryMaterials:Thefollowingareavailableonlineatwww.mdpi.com/xxx/s1,FigureS1:
QuantificationofIHCstainingforHSP47+cellsper20×fieldontissuesofmiceondays4and7post‐wounding
andtreatedwithsalineorM‐T7.Statisticsanalyzedbytwo‐wayANOVAwithFisher’sLSDpost‐hocanalysis.
Allbarsaremeanandstandarderror.N=3–4micepertreatmentpertimepoint,FigureS2:Full‐frame20×fields
ofCD4IHConDay7post‐woundingformicetreatedwithsalineandM‐T7.CorrespondstoFigure4Hinthe
mainmanuscript.Imagesarerepresentativeof3–4micepertreatment,FigureS3:QuantificationofIHCstaining
forLy6G+cellsper20×fieldontissuesofmiceondays4and7post‐woundingandtreatedwithsalineorM‐T7.
Statisticsanalyzedbytwo‐wayANOVAwithFisher’sLSDpost‐hocanalysis.
AuthorContributions:J.R.Y.,L.Z.,andA.R.L.designedthestudy.J.R.Y.andL.Z.developedmaterials.J.R.Y.,
L.Z.,J.K.,J.D.‐V.andK.M.L.performedthewoundingstudies.J.R.Y.,L.Z.,Q.G.,E.A.A.,M.B.,L.N.S.andN.Z.
performedhistology.J.R.Y.andL.Z.performedtheELISAs.J.R.Y.performedquantitativeimageanalysis.J.R.Y.,
L.Z.andA.R.L.analyzedthedata.J.R.Y.,L.Z.andA.R.L.wrotethemanuscript.Allauthorsreviewedthe
manuscript.Allauthorshavereadandagreedtothepublishedversionofthemanuscript.
Funding:ThisstudywasfinanciallysupportedbygrantsfromtheNIH(1R01AI10Supple0987‐01A1and
1RC1HL100202),AmericanHeartAssociation(17GRNT33460327),UniversityofFloridaGatoradeFund
(00115070)andstart‐upfundsfromtheBiodesignInstituteatArizonaStateUniversityalltoARL.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
Pharmaceutics2020,12,100313of16
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