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Recombinant Myxoma Virus-Derived Immune Modulator M-T7 Accelerates Cutaneous Wound Healing and Improves Tissue Remodeling

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Complex dermal wounds represent major medical and financial burdens, especially in the context of comorbidities such as diabetes, infection and advanced age. New approaches to accelerate and improve, or "fine tune" the healing process, so as to improve the quality of cutaneous wound healing and management, are the focus of intense investigation. Here, we investigate the topical application of a recombinant immune modulating protein which inhibits the interactions of chemokines with glycosaminoglycans, reducing damaging or excess inflammation responses in a splinted full-thickness excisional wound model in mice. M-T7 is a 37 kDa-secreted, virus-derived glycoprotein that has demonstrated therapeutic efficacy in numerous animal models of inflammatory immunopathology. Topical treatment with recombinant M-T7 significantly accelerated wound healing when compared to saline treatment alone. Healed wounds exhibited properties of improved tissue remodeling, as determined by collagen maturation. M-T7 treatment accelerated the rate of peri-wound angiogenesis in the healing wounds with increased levels of TNF, VEGF and CD31. The immune cell response after M-T7 treatment was associated with a retention of CCL2 levels, and increased abundances of arginase-1-expressing M2 macrophages and CD4 T cells. Thus, topical treatment with recombinant M-T7 promotes a pro-resolution environment in healing wounds, and has potential as a novel treatment approach for cutaneous tissue repair.
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Pharmaceutics2020,12,1003;doi:10.3390/pharmaceutics12111003www.mdpi.com/journal/pharmaceutics
Article
RecombinantMyxomaVirusDerivedImmune
ModulatorMT7AcceleratesCutaneousWound
HealingandImprovesTissueRemodeling
JordanR.Yaron
1,†
,LiqiangZhang
1,
*
,†
,QiuyunGuo
1
,EnkidiaA.Awo
1
,MichelleBurgin
1
,
LaurenN.Schutz
1
,NathanZhang
1
,JacquelynKilbourne
2
,JulianeDaggettVondras
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.:+13018038881(L.Z.);
Tel.:+13526722301(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
splintedfullthicknessexcisionalwoundmodelinmice.MT7isa37kDasecreted,virusderived
glycoproteinthathasdemonstratedtherapeuticefficacyinnumerousanimalmodelsof
inflammatoryimmunopathology.TopicaltreatmentwithrecombinantMT7significantly
acceleratedwoundhealingwhencomparedtosalinetreatmentalone.Healedwoundsexhibited
propertiesofimprovedtissueremodeling,asdeterminedbycollagenmaturation.MT7treatment
acceleratedtherateofperiwoundangiogenesisinthehealingwoundswithincreasedlevelsofTNF,
VEGFandCD31.TheimmunecellresponseafterMT7treatmentwasassociatedwitharetention
ofCCL2levels,andincreasedabundancesofarginase1expressingM2macrophagesandCD4T
cells.Thus,topicaltreatmentwithrecombinantMT7promotesaproresolutionenvironmentin
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.Cutaneouswoundhealingisacomplexmultistageprocessthatreliesupon
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‐andintracellular
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].Thecoevolutionofviruseswiththeir
naturalhostsinvokesanadaptationarmsrace,wherebyasuccessfulstrategyforthevirusrelieson
immuneevasion,oftentargetingkeypathwaysthatdriveimmuneactivation[13].LargeDNA
viruses,suchaspoxvirusesandherpesviruses,areadeptatevadingtheinnateimmunesystemviaa
suiteofvirulencefactors[14,15].Translationally,thesefactorsconstitutearichtoolboxfordeveloping
immunemodulatorsfortreatingdisease[12].
Myxomavirus(MYXV)isaleporipoxviruswithwellknownstrictspeciesspecificityandhost
tropismtotheEuropeanrabbit(Oryctolaguscuniculus)[16].Wehavedemonstratedthesafetyand
immunotherapeuticefficacyofseveralMYXVimmunemodulatorsinawidearrayofpreclinical
models[17–27].MT7isanMYXVderivedimmunemodulatorwithbroadchemokinebinding
activityandproventherapeuticpotentialininflammationrelateddiseases.MT7isexpressedearly
inMYXVinfection,andisthemostabundantlysecretedimmunemodulator[28].InMYXVinfection,
MT7blockslymphocyteinfiltrationintoinfectedlesionsbypreventingchemokinegradient
formation[29].MT7isasolubleglycoproteinwiththeabilitytodirectlybindallclassesof
chemokines(C,CC,CXC)testedinvitro,anddecoupletheirinteractionswithGAG[30].MT7
treatment,givenwithorwithoutconcomitantcyclosporine,reducedacuterenaltransplantrejection,
vasculopathyandscarringinrats[31].MT7alsomarkedlysuppressedinflammatorycellinvasion,
andreducedacuteandchronicaorticandrenaltransplantrejection,inmiceinamannerdependent
onheparansulfation[23,32].Thus,MT7hasthepotentialtopromotearesolutionphenotypeanda
mechanismoftissuehealingwhereintheregulationofinflammationiscritical.Here,weinvestigate
Pharmaceutics2020,12,10033of16
thepotentialforrecombinantMT7tomodulatehealingandlocalinflammatorycellresponsesatsites
offullthicknesscutaneouswoundsinamousemodel.
2.MaterialsandMethods
2.1.RecombinantMT7Production
Purified,recombinantMT7protein(m007L;NCBIGeneID#932081)wasproducedand
providedbyVironTherapeutics(London,ON,Canada)andexpressedandpurifiedaspreviously
described[33].Briefly,theMT7codingsequenceisinsertedintopFastBacDualwithaCterminal
HistagandtransformedintoDH10Baccellstogeneratebacmidsasbaculovirusshuttlevectors.
PurifiedbacmidsaretransfectedintoSf21cellsusingCellfectinIIreagent.Supernatantscontaining
baculovirusareusedtotransduceHighFivecells.SecretedMT7ispurifiedbyaffinitytag
purificationoveraNiNTAcolumnwithfurtherpurificationbysizeexclusionchromatographyvia
FPLCwithaHiLoad16/60Superdex75column.
2.2.Animals
AllanimalproceduresinthisstudywereapprovedbytheInstitutionalAnimalCareandUse
CommitteeofArizonaStateUniversityunderprotocol#171549R.Maleandfemalewildtype
C57BL6/JmicewerebredonsiteatArizonaStateUniversity.Miceaged8–12weekswereselectedby
simplerandomization[34]andusedinthisstudy.Micewerekeptonastandard12hlight–12hdark
cycleinaspecificpathogenfreeenvironmentandgivenfoodandwateradlibitum.Miceweresingle
housedafterthewoundingproceduretopreventinterferencewithwoundhealing,aspreviously
described[35].
2.3.WoundingSurgeryandMeasurement
Weperformedasplinted,fullthicknesswoundhealingmodelaspreviouslydescribed[35].In
thismodel,asiliconesplintisusedtoprohibitthewoundcontractionofmouseskinaroundasingle,
intrascapularfullthicknessbiopsypunchwoundduringthefirstsevendays,effectivelyforcing
secondintentionhealingasoccursinhumanskin(whereasmouseskinprimaryhealsbycontraction).
Briefly,micewereanesthetizedbyintraperitonealinjectionof0.1mLper25gbodyweightofa
cocktailof120mg/kgketamineand6mg/kgxylazine.Oncereachingtheanestheticsurgicalplane(as
determinedbytoepinch),micewerepreppedbyshavinga1x1inchareaspanningfrombetweenthe
earstotheapexofthespineandcenteredbetweeneachshoulder.Theshavedareawassterilizedby
twosuccessivewashesof2%chlorhexidinegluconatesolution(DynaHex2®,XttriumLaboratories,
Prospect,IL,USA)followedby70%ethanolwithsterilecottonswabs.Asmallamountofveterinary
ocularointmentwasappliedtoeacheyetopreventcornealdrying.Micewerekeptonamonitored
heatingpadforthedurationoftheprocedure.
Afullthicknessexcisionalwoundwascreatedwitha3.5mmbiopsypunchtoolcenteredinthe
shavedarea.Carefulattentionwaspaidtopreventdamagetothepanniculuscarnosusbeneaththe
skin.Immediatelyaftercreatingthepunch,thewoundsweretreatedbyapplicationofeither20μL
sterilenormal0.9%NaClsalinesolution(N=17)or20μLsterilenormalsaline(0.9%NaCl)containing
1μgrecombinantMT7(N=16)applieddirectlytothewoundbedwithamicropipette.Adonut
shapedsiliconsplint(O.D.15mm;I.D.5.0mm;CultureWell™,GraceBiolabs,Bend,OR,USA)with
Tegaderm™(3MCompany,SaintPaul,MN,USA)affixedtoonesidewascoatedwithcyanoacrylate
glue(KrazyGlue®)ontheoppositesideandcarefullyplacedonthebackofthemousewhilekeeping
thewoundcenteredwithintheinnerdiameterofthesplint.Sixinterruptedsutures(40blackEthilon
monofilamentwithanFS2reversecuttingneedle;Ethicon,Inc.,Somerville,NJ,USA)wereplaced
aroundtheoutercircumferenceofthesplint(approximately2mminsetfromtheedge)tocomplete
theprocedure.Miceweremonitoredonheatingpadsuntilawakeandmotilepriortoreturningto
singlehousedcagesfortheremainderoftheexperiment.Onday3postwounding,themicewere
anesthetizedwith1–3%isoflurane,toeffect,and20μLsalineor20μLsalinecontaining1μgMT7
Pharmaceutics2020,12,10034of16
wascarefullyappliedtopicallytothewounds(dropwiseabovethewound)byinsertinganinsulin
syringethroughthesiliconsplint,withcarenottodisruptthehealingwoundbedduringapplication.
Topreventselfinducedsecondaryskindamagefromscratching,micewereagainanesthetizedwith
1–3%isoflurane,toeffect,onday7postwoundingandthesplintswerecarefullyremovedwith
sterilesurgicalscissorsbeforebeingreturnedtosinglehousedcagesaspreviouslydescribed[35].
TheexperimentaldesignisoutlinedinFigure1A.
Figure1.MT7acceleratesfullthicknesswoundhealinginmice.(A)Experimentaldesignoverview.
Micewerewoundedonday0(greenarrow)andfollowedtoday15postwounding(redarrow),at
whichtimemicewereeuthanizedandtissuewascollected.MiceweretreatedwithsalineorMT7on
day0withasecondbolusgiveonday3postwounding(bluearrow).Micewereanesthetizedand
splintswereremovedonday7postwounding(yellowarrow).Micewereassesseddailyandimages
werecollectedforplanimetricmeasurementofwoundclosure(grayarrows).N=5ineachgroup.(B)
Planimetricmeasurementsofwoundclosurenormalizedtoday0foreachmouse.Meanandstandard
errorareshown.StatisticswerecalculatedbyTtestperdaywithcorrectionformultiplecomparisons
bytheHolm–Sidakmethod.Forsignificance,aequatestop<0.05,bequatestop<0.01andcequates
top<0.001.(C)RepresentativewoundimagesforsalineandMT7treatedmiceonthedayof
wounding(day0)andondays2,4,7and15postwounding.Thesamemouseisshownineachimage
percondition.Scalebarsare5mm.
2.4.WoundPlanimetry
Micewereassessedwhileawakeonthedayoftheprocedure(day0)andoneverysubsequent
dayoffollowupforatotalof15days.Digitalimageswerecollectedalongwithaknownsizemarker.
PlanimetricmeasurementsofthewoundhealingprogresswereperformedinImageJ/FIJIand
calibratedagainsttheknownsizemarker[36].
2.5.ImmunohistochemistryandHerocivi’sPolychromeStaining
Micewereeuthanizedinindividualcohortsondays2,4,7and15postwounding,andtissues
werecollectedandfixedin10%neutralbufferedformalinfor1weekbeforeprocessing.Fixedtissues
wereprocessedandperfusedwithparaffinwithaLeicaTP1050processorthroughgradedalcohols
andxylene,thenembeddedintoparaffincassettesonaLeicaEG1160embeddingstation.Blockswere
sectionedusingaLeicaRM2165microtome(5μmsections)andstainedwithhematoxylinandeosin
(H&E)accordingtostandardprocedures.Slideswithsectionsthatreachedthewoundsiteas
Pharmaceutics2020,12,10035of16
determinedbyH&Escreeningwerefurtherstainedbyimmunohistochemistryandcollagenspecial
staining.
Immunohistochemistry(IHC)wasperformedaspreviouslydescribed.Briefly,theslideswere
rehydratedthroughgradedxyleneandgradedalcohols.Rehydratedslidesweresubmergedin
sodiumcitratebuffer,sandwichedwithacleanglassslidetopreventtissuelossandincubatedat60
°Ctoretrieveepitopes.Endogenousperoxidaseswerequenchedwith3%hydrogenperoxideinPBS
andnonspecificproteinbindingwasblockedwith5%bovineserumalbumininTBS/0.1%Tween20.
Sectionswereprobedovernightat4°CwithrabbitpolyclonalantibodiesagainstArginase1(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
TGFbeta1(Abcam,ab215715,1:500),ormousemonoclonalantibodyagainstLy6G(Invitrogen,#14
593182,1:200).HRPconjugatedsecondaryantibodiesagainstrabbitandmouseIgG(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
DP74CMOShighresolutioncameraoperatedbycellSensDimensionsv1.16.Objectivecalibrated
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
Enzymelinkedimmunosorbentassays(ELISA)wereperformedusingDuoSetkitsforTNFα
(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
AnalysisofstatisticalsignificancewasperformedbyTwoWayAnalysisofVariance(ANOVA)
andStudent’sunpairedTtestusingGraphPadPrismv8.2.1.Allanalysespassednormalitytests
accordingtoAnderson–Darling(A2*),D’Agostino–Pearsonomnibus(K2),Shapiro–Wilk(W)and
Kolmogorov–Smirnov(distance)withanalpha=0.05.Pvalueswereconsideredsignificantat*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.RecombinantMT7PromotesFullThicknessWoundHealing
WeanalyzedtheeffectsofrecombinantMT7onthetreatmentoffullthicknesswoundsina
splintedwoundhealingmodelinwildtypeC57BL6/Jmice[35,38–40].Basedoneffectivedosesand
ourpriorworkwithasecondunrelatedMYXVderivedimmunemodulatingserineproteinase
inhibitor,Serp1,inthesamemodel,wegaverecombinantMT7topicallyinadoseof1μgin20μL
saline,withasecondbolusof1μgin20μLsaline3dayspostwounding(Figure1A)[35].Thecontrol,
salinetreatedmiceweresimilarlytreatedwithabolusofsaline3dayspostwounding.Daily
planimetricmeasurementsofwoundhealingprogressdemonstratethatMT7significantly
acceleratesfullthicknesswoundhealingwhengivenasarecombinantproteininatopicalsaline
solution(Figure1B,C).Noevidenceofwoundsiteinfection(pus,discharge,discoloration)was
observedineitherthesalineorMT7treatedgroups.Wenotedthatduringthefirst8daysofhealing
(day0andupto7dayspostwounding),healingwaslimitedtoonlysecondintentionmechanisms
duetothesiliconsplintpreventingwoundcontraction(Figure1C).Thisisamajorbenefitofthe
siliconsplintmodelbecause,leftonitsown,mouseskincontractswithin1–2days,whichlimitsthe
interpretationofthehealingprocess.Byday7,salinecontroltreatedwoundshadonlyachieveda
meanof23%closure,whileMT7significantlyacceleratedthehealingprocessanda78%mean
woundclosurewasachieved(p=0.0007).Evenafterremovingthesiliconsplints,thewoundsofM
T7treatedmicecontinuedtoclosemorequicklythanmicetreatedwithsalinealone,andachieved
fullclosure3–4daysbeforesalinetreatedmice.Thus,recombinantMT7givenduringtheearlystage
ofhealinghasasustainedeffectonacceleratingwoundclosure.
3.2.RecombinantMT7PromotesCollagenMaturationinWounds
Collagendepositionandmaturationarekeycomponentsofthewoundhealingprocess.The
inappropriatedepositionorimpairedmaturationofcollagenareassociatedwithscarringandlimited
angiogenesis[41].Theimprovedremodelingofcollageninthehealingwoundcanthusimproveboth
scarringandhealedtissuehealthviathepromotionofimprovedangiogenesis.Herovici’s
polychromeisahistologicspecialstainwhichdifferentiatesbetweenimmatureTypeIIIcollagen
(stainedblue)andmatureTypeIcollagen(stainedpink)[42].WeusedHerovici’spolychrometo
evaluatethecollagenmaturationofhealedwoundsafterMT7treatment.Quantitativeimage
analysisoftheamountofpinkTypeIcollagenstainingversustheamountofblueTypeIIIcollagen
showedwhatwerefertoastheHeroviciRatioforthetissue.AhigherHeroviciRatio(morepink,less
blue)indicatesmoreadvancedcollagenmaturation,whereasalowerHeroviciRatio(lesspink,more
blue)indicateslesscollagenmaturationandmoreactivedepositionofimmaturecollagen.Atday15
postwounding,thehealedwoundsofmicetreatedwithMT7hadsignificantlyhigherHerovici
Ratios,indicatingmorematureTypeIcollagenthansalinetreatedmice(Figure2).Interestingly,this
wasnotassociatedwithanincreaseinfibroblastmarkerHSP47(FigureS1).Thesedataindicatethat,
inadditiontoacceleratedclosure,woundstreatedwithMT7hadahigherfidelity,healingwitha
moreproperlyorganizedcollagenarchitecture.
Pharmaceutics2020,12,10037of16
Figure2.QuantitativeassessmentofcollagenmaturationinwoundstreatedwithMT7.(A)
RepresentativemicrographsofHerovici’spolychromestainednormalskinandwoundsat15days
postwounding.Toppanelsshowbrightfielddata,whilemiddleandbottompanelsshowcolor
deconvolutedfieldsforthepinkandbluechromophores.(B)Quantificationofcollagenmaturation
insalineandMT7treatedwoundsbytheHeroviciRatio,calculatedbythedensitometricratioofthe
pinkandbluechromophoresinHerovici’spolychrome.Meanandstandarderrorareshown.Statistics
werecalculatedbyTtest.N=4saline,N=5MT7.
3.3.MT7StimulatesPeriWoundAngiogenesis
Angiogenesisisanessentialcomponentoftheproliferativestageofcutaneouswoundhealing,
characterizedbyanearlyandabundantburstofimmaturevesselswhicheventuallyregressintoa
maturevascularnetworkviatheactivityofantiangiogenicfactors,suchasSprouty2andPEDF[43].
Therapeuticstrategiesarenowactivelysoughttoenhanceangiogenesisduringwoundhealing[44].
Angiogenesisintheearlystagesofwoundhealingisdrivenbytheprimingofendothelialcellswith
proinflammatorycytokines,suchastumornecrosisfactoralpha(TNFα),therebyinducingatipcell
phenotype[45].Inthecontextoftissueinjury,TNFαiscriticalforthedownstreamproductionof
vascularendothelialgrowthfactor(VEGF),anessentialgrowthfactorinregulatingangiogenesis[46].
WeperformedELISAanalysesofthehealingwound’sbedtissueondays1,4and7postwounding
toquantitativelymeasurelevelsofTNFα andVEGF(Figure3A).RecombinantMT7induceda
significantincreaseinwoundbedlevelsofTNFαondays1and4(p<0.05),andofVEGFbyday7(p
<0.05),versussalinetreatmentalone.Weperformedimmunohistochemistryofwoundtissueson
days4and7postwoundingtodeterminethedegreeofangiogenesisbystainingforCD31(alsocalled
PECAM1),acanonicalmarkerforendothelialcellsinthevasculature(Figure3B,C).Aquantification
ofthenumberofCD31+cellsandvesselsper20×fieldintheperiwoundareaindicatedasignificant
increaseonday4postwounding(p<0.05)versussalinetreatmentalone.Weobservednosignificant
differenceonday7postwounding.Qualitatively,wenotedthattheCD31+cellsformedmorerobust
vesselsinthewoundstreatedwithMT7,withincreasedlengthandthicknessversussalinetreatment
alone(Figure3C).Takentogether,theseresultssuggestthatMT7stimulatesanearlyTNFαresponse,
whichstimulatesamorerobustVEGFresponse,ultimatelyleadingtoacceleratedangiogenesisinthe
periwoundareaassociatedwithacceleratedwoundclosure.
Pharmaceutics2020,12,10038of16
Figure3.AssessmentofperiwoundangiogenesisinwoundstreatedwithMT7.(A)ELISA
quantificationofTNFαandVEGFinwoundtissuestreatedwithsalineorMT7collectedondays1,
4and7postwoundingnormalizedtototalprotein.Barsaremeanandstandarderror.Statisticswere
calculatedbytwowayANOVAwithFisher’sLSDposthocanalysis.(B)QuantificationofCD31+
cellsandvesselsper20×fieldintheperiwoundareaofwoundstreatedwithsalineorMT7collected
ondays4and7postwounding.Barsaremeanandstandarderror.Twononoverlappingfieldswere
quantifiedpermouseandstatisticswereperformedontheaveragepermousewiththeN=4per
group.StatisticswerecalculatedbytwowayANOVAwithFisher’sLSDposthocanalysis.(C)
RepresentativeperiwoundCD31IHCfields(10×)collectedondays4and7postwounding.Scale
barsare50μm.Zoomareasindicatedbyboxes.N=3–4ineachgroupandtimepoint.
3.4.MT7ModulatesImmuneResponsesintheHealingWound
MT7bindstoallclassesofchemokines(C,CCandCXC)andinhibitstheirinteractionwith
glycosaminoglycans,therebypreventingchemokinegradientformation[23,30,47].CCL2,alsocalled
monocytechemoattractantprotein1(MCP1),isaCCclasschemokinepreviouslyshowntohavea
criticalroleintheregulationofwoundhealing[48].WeperformedtheELISAanalysisofCCL2on
woundtissuestreatedwithsalineorMT7,collectedondays1,4and7postwounding(Figure4A).
WoundstreatedwithMT7hadanelevatedlevelofCCL2onday4postwounding,which
approachedsignificance(p=0.0763),whilelevelsofCCL2werenotdifferentbetweensalineandM
T7treatmentondays1and7postwounding.Independentofitschemotacticfunction,thesignaling
ofCCL2withitsreceptor,CCR2,waspreviouslyshowntopromotethepolarizationofmacrophages
towardsaproresolution(i.e.,M2)phenotype[49].Weperformedimmunohistochemistryofwounds
treatedwithsalineorMT7ondays2,4and7postwounding,stainingforArginase1,acanonical
markerofM2macrophagepolarization.ThequantificationofArginase1+cellsrevealedatrend
Pharmaceutics2020,12,10039of16
towardselevatedM2macrophagesondays2and4postwounding,whichachievedsignificance(p<
0.05)byday7postwounding(Figure4B,C).Accordingly,thenumberofTGFbeta+cellsperfield
trendedtowardssignificanceonday4(p=0.0836)andreachedsignificancebyday7postwounding
(p<0.05)(Figure4D).WefurtherinvestigatedtheeffectsofMT7treatmentonTcellinfiltrationin
thehealingwound.WefoundthatMT7treatmentsignificantlyinhibitedtheinfiltrationofCD3+T
cells,ageneralTcellmarker,intothebedofthehealingwoundondays4and7postwounding
(Figure4E),withoutinhibitingtheaccumulationofCD3+cellsintheepithelialtongueofthewounds
(Figure4F).RegulatoryTcells,aCD4Tcellsubtype,arecrucialforthenormalandaccelerated
healingofcutaneouswounds[50].WefoundthatMT7treatmentsignificantlyincreasedthe
accumulationofCD4+cellsintheepithelialtongueofhealingwoundsversussalinetreatmentalone
(Figure4G,H,FigureS2).Wedidnotobserveaneffectonneutrophilinfiltration(FigureS3).Thus,
decouplingthechemokineglycosaminoglycangradientwithMT7modulatestheimmuneresponse
inthewoundenvironmenttoacceleratehealing.
Figure4.MT7modulatestheimmuneresponseinthehealingwound.(A)ELISAquantificationof
CCL2inwoundstreatedwithsalineorMT7atdays1,4and7postwounding,normalizedtototal
protein.(B)QuantificationofArginase1+cellsper20×fieldofwoundstreatedwithsalineorMT7at
days2,4and7postwounding.(C)RepresentativeArginase1IHCfieldsatday7.(D)Quantification
ofTGFbeta+cellsper20×fieldondays2,4and7postwounding.(E,F)QuantificationofCD3+cells
per20×fieldofwoundstreatedwithsalineorMT7atdays2,4and7postwounding,specificallyin
the(E)woundbedor(F)epithelialtongue.(G)QuantificationofCD4+cellsper20×fieldofwounds
treatedwithsalineorMT7atdays2,4and7postwoundingnormalizedtothenumbersonday2.
(H)RepresentativeCD4IHCfieldsintheepithelialtongueatday7.Full20×fieldisgiveninFigure
S2.Allbarsaremeanandstandarderror.StatisticsarecalculatedbytwowayANOVAwithFisher’s
LSDposthocanalysis.N=3–4ineachgroupandtimepoint.
4.Discussion
Largecutaneouswounds,particularlywithassociatedpoorhealing(e.g.,diabeticoraged),
scarringandsuperimposedinfections,areacomplexandcostlymedicalburden,withanannual
incidenceofmorethan6millioncutaneouswoundcasesandacollectiveyearlycostofmorethan
Pharmaceutics2020,12,100310of16
USD20billion,notinclusiveofthemorethan170,000scarrevisionsurgeriesannuallyintheUnited
States[51].Comorbiditiessuchasadvancedageanddiabetes,orcomplicationssuchasinfection,
burnsandbattlefieldconditions,furtherincreasethedifficultyofwoundmanagementandtherisk
ofadverseoutcomes[52].Investigationhasthusintensifiedtoaddressanunmetneedfornovel
treatmentstoacceleratewoundhealing.
Inthisstudy,wetestedthetherapeuticefficacyofrecombinantMyxomavirusderivedimmune
modulatingproteinMT7inamousemodeloffullthicknesswoundhealing.Weadministeredtwo
dosesofrecombinantMT7ondays0and3postwounding,mirroringthedosingregimenthatwe
foundtobeoptimalforanotherMyxomavirusderivedimmunemodulator,Serp1,inaprevious
study[35].Planimetricanalysisrevealedasignificantaccelerationofwoundclosurebytreating
woundstopicallywithMT7.Accelerationoccurredduringtheearlieststagesofhealingandwas
independentofcontraction,asthesiliconesplintswerenotremoveduntilday7postwounding
(Figure1B).Thisfindingispromising,asthefirstphaseofhealingisknowntobeacrucialperiodfor
protectionagainstinfectionandthepreventionofadditionaltraumaasgranulationtissueisformed
[53].
Onepotentialriskofacceleratedwoundhealingisthedepositionofdisorganizedconnective
tissueleadingtoscarring,particularlyinfullthicknessskinwoundswithoutcontraction[54].
Drueckeandcolleaguesinvestigatedtheuseofdifferentdermalregenerationtemplatesonfull
thicknesswoundsinaporcinemodel[55].TheyfoundthatwhiletheIntegramaterial,acomposite
scaffoldofbovinehidecollagenandsharkchondroitin6sulfate,improvedcollagenmaturation,
therewasslowertissueingrowth,andtissueintegritywaslost[55].Incontrast,theauthorsfound
thatabovinehidecollagenspongescaffold,producedviachemicalcrosslinkingwith1ethyl3(3
dimethylaminopropyl)carbodiimide(EDC),allowedmorerapidingrowthandoveralltissue
integrity,withnobenefittocollagenmaturation.Here,wefoundthatinadditiontoaccelerating
woundhealing,recombinantMT7alsoresultedinearlier,improvedcollagenmaturation,as
determinedbyquantificationwithHerovici’spolychromeinametricwetermthe“HeroviciRatio”
(Figure2).AsimilarquantificationofTypeIII:IcollagenwasobservedbyO’Rourkeandcolleagues
intheirinvestigationofacceleratedwoundhealingbysurfactantpolymerdressingscontaining
siRNAtoFidgetinLike2,withtheauthorsnotingincreasedredTypeIcollagen,illustratinghigher
fidelityhealing[56].
Angiogenesisisacriticalcomponentofwoundhealing,andseveralcomorbiditiesassociated
withimpairedwoundhealing,suchasdiabetes,alsoexhibitreducedangiogenesis[57,58].
Angiogenesisplaysacriticalroleinprovidingthesufficientdeliveryofbloodandnutrients,and
accesstoreparativeimmunecells,duringthecourseofhealing.Accordingly,numerousgroupshave
investigatedapproachestoacceleratehealingbypromotingangiogenesis.Forexample,recombinant
TNFαapplieddirectlytowoundsacceleratestheearlystagesofwoundhealing[59].Thetherapeutic
effectofTNFαintheearlystageofwoundhealingmayproceedviatheinductionofangiogenesis,as
TNFα isapowerfulinducerofVEGFandendothelialtipcellformation[45,46].Othertreatments
showntoacceleratewoundhealingalsoactviatheinductionofangiogenesis.Forexample,topical
simvastatinandasperosaponinVIbothacttoacceleratewoundhealingbyenlistingtheVEGF
signalingcascade,andVEGFCapplieddirectlytowoundsalsoaccelerateshealing[60–62].Here,we
showthatarecombinantMT7treatmentresultedinasignificantincreaseinlocalTNFαduringthe
earlieststagesofwoundhealing,whichtemporallytransitionsintoasignificantincreaseinVEGFin
thehealingbed(Figure3A).Thiscoincideswiththecanonical,earlyinflammatoryphaseofhealing,
andthetransitiontotheproliferationphaseofhealing[63].Accordingly,increasedangiogenesiswas
directlyobservedbyimmunohistochemistryforCD31intheperiwoundarea(Figure3B,C).Thus,
thedatasuggestthattopicalMT7modulatesthechemokineenvironment,resultinginthe
augmentationofprohealingmoleculesinthewoundenvironment,engagingtheproangiogenesis
signalingcascadeatthelevelofbothcytokinesandgrowthfactors,andresultinginasignificant
inductionofangiogenesisattheboundariesofhealingwoundsassociatedwithincreasedwound
closure.
Pharmaceutics2020,12,100311of16
WesoughttodeterminetheeffectofrecombinantMT7onlocalimmuneresponsesinthe
healingwounds.Somevirusderivedimmunemodulators,suchastheherpesvirusM3chemokine
decoyreceptorandMyxomavirusMT1,inhibittheabilityofchemokinestosignaltotheirreceptor.
M3alsouniquelyblockschemokinebinding,bothtoGAGsandalsotoreceptors[64].Incontrast,M
T7actsprimarilyatthelevelofchemokineglycosaminoglycaninteractions[30,47,65,66].MT7isan
interferongammareceptorhomologuewithspecificityforrabbitinterferongamma[28].
Interestingly,theMT7inhibitionofchemokinetoGAGbindingisfoundforallmammalstestedto
date,e.g.,rabbits,rats,miceandhumancells[30].Thus,MT7treatmentisexpectedtoinhibit
chemokinegradientformation[47].Indeed,wepreviouslyfoundthatMT7losttherapeuticefficacy
intheabsenceofnormalactiveheparansulfationinmicewithconditionalendothelialdeficiencyof
theheparansulfotransferaseenzymeNdst1,withpresumedconsequencesinmodifyingthe
formationofchemokinegradients[23].CCL2/MCP1signalingisknowntobecriticalinregulating
physiologicwoundhealing.Lowandcolleaguesreportedthatwoundsmadeinmicedeficientof
CCL2exhibitdelayedreepithelialization,reducedcapillarydensityandimpairedcollagen
remodeling[48].Incontrast,multiplegroupshaveshownthatrecombinantCCL2treatmentreverses
impairedwoundhealingindiabeticmicebyrestoringmacrophageresponses[67,68].Receptor
engagementbychemokinesinducestheinternalizationofboththereceptoranditsligand,resulting
inintracellulardegradationandrecycling[69].WhileCCL2existsindynamicequilibriumasbotha
monomeranddimer,onlythemonomericformiscapableofreceptorengagement,andobligate
dimericmutantsofCCL2areincapableofsignaling[70].Importantly,thedimerizationofCCL2
requiresglycosaminoglycaninteractions[71].WefoundincreasedlevelsofCCL2inhealingwounds
whentreatedwithrecombinantMT7(Figure4A),consistentwiththeroleofCCL2inimproved
healing.WehypothesizethatMT7treatmentinhibitedtheoligomerizationofCCL2,slowing
receptorengagementanddelayingitssubsequentdegradation.Investigatingfurther,wefoundM
T7dependenteffectsintwocellpopulationsknowntobeaffectedbyCCL2andotherchemokine
signaling:macrophagesandTcells.Specifically,wefoundanincreaseinM2polarized,pro
resolutionmacrophages(Figure4B).ThisfindingagreeswithpriorworkshowingthatCCL2
signalingresultsinM2polarization[49].WealsofoundincreasedCD4+Tcellsintheepithelial
tonguesofhealingwoundstreatedwithMT7(Figure4F,G),inagreementwiththeabilityforCCL2
topromoteCD4recruitment,andintheCD4lineagecellsdrivingacceleratedwoundhealing[50,72].
CCL2actsdirectlyonTcellsviatheactionofCCR2andCCR4[73],butcanalsoinducethe
recruitmentofCD4cellsintotissuesinapromiscuousmanner,usingotherreceptors[72].Further,
MT7interactswithmanychemokines,andmayinduceabroadmilieuchangeinarangeof
chemokines.ItwillbeimportanttoprobethespecifictargetedmechanismsofCD4recruitmentusing
geneticknockoutsorneutralizingantibodytreatmentsinfuturestudies.Wespeculatethatthe
continuedexpressionofCCL2bylocalcells,combinedwithanoffrateofCCL2:MT7interactions,
mayhavecontributedtosustainedCCL2signaling.However,wecannotexcludetheroleofother
chemokinesorcytokinesinthewoundhealingmilieuinthisstudy,asMT7interfereswithGAG
bindingforC,CCandCXCchemokinesinvitro.Thus,thedecouplingofglycosaminoglycan
interactionswithchemokinesbyMT7hasaneffectonCCL2signaling,anddownstreameffectson
macrophageandTcellpopulations,leadingtoacceleratedwoundhealing.Furtherworkisnecessary
todeterminethespecificchemokineandGAGpathwaysmodulatedbyMT7intheorchestrationof
localandinfiltratingimmunecellsinthehealingwoundbed.
MT7isnowthesecondMyxomavirusderivedimmunemodulatortoexhibitefficacyin
promotingwoundhealing.Serp1,aserineproteaseinhibitor(serpin),isaglycosylated,secreted
proteinwhichtargetsserineproteasesinboththethrombotic(FXa,thrombin)andthrombolytic(uPA,
tPA,plasmin)cascades[20].WerecentlyreportedthatrecombinantSerp1acceleratedfullthickness
woundhealinginmice,withnumerousobservedsimilaritiestotheMT7treatmentasperformedin
thisstudy[35].Inadditiontotheaccelerationofwoundclosure,bothSerp1andMT7resultedinan
increaseinVEGFandperiwoundangiogenesis,aswellasanincreaseinproresolutionM2polarized
macrophages.Thesesimilaritiesunderscorethebenefitsassociatedwithdevelopingtherapeutics
fromvirusexpressedimmunemodulators.Thesestudiesalsoagainemphasizethesafetyofthese
Pharmaceutics2020,12,100312of16
immunemodulatorswhenusedastherapeutics.First,thelimitedgenomicspaceinavirus
necessitatestheevolutionofmultipotency(i.e.,multipletargetsofinhibition),providinghighly
potentandeffectiveimmunemodulatingmolecules[74].WhileSerp1targetsnumerousproteinsin
theclottingcascade,MT7targetsthepanoplyofchemokines.Second,virusderivedimmune
modulatorsoftenexhibitpotencyatextremelylowconcentrations.BothSerp1andMT7functionat
dosesofonly100nanogrampergrambodyweight,theequivalentofmicrogramperkilogramdosing
inhumans;thatis,thelowestendrangefortherapeuticbiologics[75].Indeed,Serp1hasbeenshown
toexhibittherapeuticefficacydowntothepicogrampergramrange[76].Third,virusderived
immunemodulatorshaveundergoneextensive“researchanddevelopment”intheevolutionary
armsracebetweenthevirusanditshost.InthecaseofMyxomavirus,anestimated10millionyears
ofevolutionhavegoneintodevelopingexpertmodulatorsofthehostimmuneresponse[16].Thus,
thesuiteofimmunemodulatingproteinsinMyxomavirusareavaluable,highlyoptimized
“medicinecabinet”fortargetingimmunedrivenpathologies,andforharnessingimmunefunction
toenhancetissuerepair[12].Furtherworkwillinvestigatethepotentialforcombiningtheseproteins
incocktails,towardsfurtherenhancingtherapeuticresponsesinwoundhealingandothermodels.
5.Conclusions
WereportherethattreatmentwithrecombinantMT7,aMyxomavirusderivedchemokine
signalingmodulator,acceleratestherateofhealinginfullthicknesswoundsinwildtypemice.MT7
treatmentimprovedconnectivetissueremodeling,andincreasedangiogenesisandproresolution
immunecellphenotypes.Thechemokinemilieuofthehealingwoundbedishighlycomplex,andM
T7caninteractwithallclassesoftheC,CCandCXCchemokines.WeobservedaneffectonCCL2in
thepresentstudy,whichwasassociatedwitheffectsonmacrophages,Tcellsandendothelialcells.
FurtherworkisneededtodelineatetheprecisemechanismsofMT7’stherapeuticeffectsonwound
healing.Further,itwillbeimportanttoinvestigatetheeffectsofrecombinantMT7incomplex
comorbiditiessuchasdiabetes,infectionandburns,todevelopnextgenerationversionsofMT7with
enhancedfunction,andtolearnmoreaboutthefundamentalroleofchemokinesincutaneouswound
healing.Thus,MT7representsapromisingvirusderivedtherapeutic,anewclassofprotein
biologicswiththepotentialtoaddressthesignificantmedicalburdencreatedbydermalwounds.
SupplementaryMaterials:Thefollowingareavailableonlineatwww.mdpi.com/xxx/s1,FigureS1:
QuantificationofIHCstainingforHSP47+cellsper20×fieldontissuesofmiceondays4and7postwounding
andtreatedwithsalineorMT7.StatisticsanalyzedbytwowayANOVAwithFisher’sLSDposthocanalysis.
Allbarsaremeanandstandarderror.N=3–4micepertreatmentpertimepoint,FigureS2:Fullframe20×fields
ofCD4IHConDay7postwoundingformicetreatedwithsalineandMT7.CorrespondstoFigure4Hinthe
mainmanuscript.Imagesarerepresentativeof3–4micepertreatment,FigureS3:QuantificationofIHCstaining
forLy6G+cellsper20×fieldontissuesofmiceondays4and7postwoundingandtreatedwithsalineorMT7.
StatisticsanalyzedbytwowayANOVAwithFisher’sLSDposthocanalysis.
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(1R01AI10Supple098701A1and
1RC1HL100202),AmericanHeartAssociation(17GRNT33460327),UniversityofFloridaGatoradeFund
(00115070)andstartupfundsfromtheBiodesignInstituteatArizonaStateUniversityalltoARL.
ConflictsofInterest:Theauthorsdeclarenoconflictofinterest.
Pharmaceutics2020,12,100313of16
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