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

Authors:
Jordan R. Yaron at Arizona State University
Jordan R. Yaron
Liqiang Zhang at Arizona State University
Liqiang Zhang
Qiuyun Guo
Qiuyun Guo
Qiuyun Guo
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Enkidia Awo at Midwestern University
Enkidia Awo
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Abstract and Figures

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.
M-T7 accelerates full-thickness wound healing in mice. (A) Experimental design overview. Mice were wounded on day 0 (green arrow) and followed to day 15 post-wounding (red arrow), at which time mice were euthanized and tissue was collected. Mice were treated with saline or M-T7 on day 0 with a second bolus give on day 3 post-wounding (blue arrow). Mice were anesthetized and splints were removed on day 7 post-wounding (yellow arrow). Mice were assessed daily and images were collected for planimetric measurement of wound closure (gray arrows). N = 5 in each group. (B) Planimetric measurements of wound closure normalized to day 0 for each mouse. Mean and standard error are shown. Statistics were calculated by T-test per-day with correction for multiple comparisons by the Holm-Sidak method. For significance, a equates to p < 0.05, b equates to p < 0.01 and c equates to p < 0.001. (C) Representative wound images for saline and M-T7-treated mice on the day of wounding (day 0) and on days 2, 4, 7 and 15 post-wounding. The same mouse is shown in each image per condition. Scale bars are 5 mm.
M-T7 accelerates full-thickness wound healing in mice. (A) Experimental design overview. Mice were wounded on day 0 (green arrow) and followed to day 15 post-wounding (red arrow), at which time mice were euthanized and tissue was collected. Mice were treated with saline or M-T7 on day 0 with a second bolus give on day 3 post-wounding (blue arrow). Mice were anesthetized and splints were removed on day 7 post-wounding (yellow arrow). Mice were assessed daily and images were collected for planimetric measurement of wound closure (gray arrows). N = 5 in each group. (B) Planimetric measurements of wound closure normalized to day 0 for each mouse. Mean and standard error are shown. Statistics were calculated by T-test per-day with correction for multiple comparisons by the Holm-Sidak method. For significance, a equates to p < 0.05, b equates to p < 0.01 and c equates to p < 0.001. (C) Representative wound images for saline and M-T7-treated mice on the day of wounding (day 0) and on days 2, 4, 7 and 15 post-wounding. The same mouse is shown in each image per condition. Scale bars are 5 mm.
… 
Quantitative assessment of collagen maturation in wounds treated with M-T7. (A) Representative micrographs of Herovici's polychrome-stained normal skin and wounds at 15 days post-wounding. Top panels show brightfield data, while middle and bottom panels show colordeconvoluted fields for the pink and blue chromophores. (B) Quantification of collagen maturation in saline and M-T7 treated wounds by the Herovici Ratio, calculated by the densitometric ratio of the pink and blue chromophores in Herovici's polychrome. Mean and standard error are shown. Statistics were calculated by T-test. N = 4 saline, N = 5 M-T7.
Quantitative assessment of collagen maturation in wounds treated with M-T7. (A) Representative micrographs of Herovici's polychrome-stained normal skin and wounds at 15 days post-wounding. Top panels show brightfield data, while middle and bottom panels show colordeconvoluted fields for the pink and blue chromophores. (B) Quantification of collagen maturation in saline and M-T7 treated wounds by the Herovici Ratio, calculated by the densitometric ratio of the pink and blue chromophores in Herovici's polychrome. Mean and standard error are shown. Statistics were calculated by T-test. N = 4 saline, N = 5 M-T7.
… 
Assessment of peri-wound angiogenesis in wounds treated with M-T7. (A) ELISA quantification of TNFα and VEGF in wound tissues treated with saline or M-T7 collected on days 1, 4 and 7 post-wounding normalized to total protein. Bars are mean and standard error. Statistics were calculated by two-way ANOVA with Fisher's LSD post-hoc analysis. (B) Quantification of CD31+ cells and vessels per 20× field in the peri-wound area of wounds treated with saline or M-T7 collected on days 4 and 7 post-wounding. Bars are mean and standard error. Two non-overlapping fields were quantified per mouse and statistics were performed on the average per mouse with the N = 4 per group. Statistics were calculated by two-way ANOVA with Fisher's LSD post-hoc analysis. (C) Representative peri-wound CD31 IHC fields (10×) collected on days 4 and 7 post-wounding. Scale bars are 50 μm. Zoom areas indicated by boxes. N = 3-4 in each group and time point.
Assessment of peri-wound angiogenesis in wounds treated with M-T7. (A) ELISA quantification of TNFα and VEGF in wound tissues treated with saline or M-T7 collected on days 1, 4 and 7 post-wounding normalized to total protein. Bars are mean and standard error. Statistics were calculated by two-way ANOVA with Fisher's LSD post-hoc analysis. (B) Quantification of CD31+ cells and vessels per 20× field in the peri-wound area of wounds treated with saline or M-T7 collected on days 4 and 7 post-wounding. Bars are mean and standard error. Two non-overlapping fields were quantified per mouse and statistics were performed on the average per mouse with the N = 4 per group. Statistics were calculated by two-way ANOVA with Fisher's LSD post-hoc analysis. (C) Representative peri-wound CD31 IHC fields (10×) collected on days 4 and 7 post-wounding. Scale bars are 50 μm. Zoom areas indicated by boxes. N = 3-4 in each group and time point.
… 
M-T7 modulates the immune response in the healing wound. (A) ELISA quantification of CCL2 in wounds treated with saline or M-T7 at days 1, 4 and 7 post-wounding, normalized to total protein. (B) Quantification of Arginase-1+ cells per 20× field of wounds treated with saline or M-T7 at days 2, 4 and 7 post-wounding. (C) Representative Arginase-1 IHC fields at day 7. (D) Quantification of TGF-beta+ cells per 20× field on days 2, 4 and 7 post-wounding. (E,F) Quantification of CD3+ cells per 20× field of wounds treated with saline or M-T7 at days 2, 4 and 7 post-wounding, specifically in the (E) wound bed or (F) epithelial tongue. (G) Quantification of CD4+ cells per 20× field of wounds treated with saline or M-T7 at days 2, 4 and 7 post-wounding normalized to the numbers on day 2. (H) Representative CD4 IHC fields in the epithelial tongue at day 7. Full 20× field is given in Figure S2. All bars are mean and standard error. Statistics are calculated by two-way ANOVA with Fisher's LSD post-hoc analysis. N = 3-4 in each group and time point.
M-T7 modulates the immune response in the healing wound. (A) ELISA quantification of CCL2 in wounds treated with saline or M-T7 at days 1, 4 and 7 post-wounding, normalized to total protein. (B) Quantification of Arginase-1+ cells per 20× field of wounds treated with saline or M-T7 at days 2, 4 and 7 post-wounding. (C) Representative Arginase-1 IHC fields at day 7. (D) Quantification of TGF-beta+ cells per 20× field on days 2, 4 and 7 post-wounding. (E,F) Quantification of CD3+ cells per 20× field of wounds treated with saline or M-T7 at days 2, 4 and 7 post-wounding, specifically in the (E) wound bed or (F) epithelial tongue. (G) Quantification of CD4+ cells per 20× field of wounds treated with saline or M-T7 at days 2, 4 and 7 post-wounding normalized to the numbers on day 2. (H) Representative CD4 IHC fields in the epithelial tongue at day 7. Full 20× field is given in Figure S2. All bars are mean and standard error. Statistics are calculated by two-way ANOVA with Fisher's LSD post-hoc analysis. N = 3-4 in each group and time point.
… 
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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,thewoundsweretreatedbyapplicationofeither20μL
sterilenormal0.9%NaClsalinesolution(N=17)or20μLsterilenormalsaline(0.9%NaCl)containing
1μgrecombinantM‐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,and20μLsalineor20μLsalinecontaining1μgM‐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(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
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‐T7topicallyinadoseof1μgin20μL
saline,withasecondbolusof1μgin20μLsaline3dayspost‐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,TNFαiscriticalforthedownstreamproductionof
vascularendothelialgrowthfactor(VEGF),anessentialgrowthfactorinregulatingangiogenesis[46].
WeperformedELISAanalysesofthehealingwound’sbedtissueondays1,4and7post‐wounding
toquantitativelymeasurelevelsofTNFα andVEGF(Figure3A).RecombinantM‐T7induceda
significantincreaseinwoundbedlevelsofTNFαondays1and4(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‐T7stimulatesanearlyTNFαresponse,
whichstimulatesamorerobustVEGFresponse,ultimatelyleadingtoacceleratedangiogenesisinthe
peri‐woundareaassociatedwithacceleratedwoundclosure.
Pharmaceutics2020,12,10038of16
Figure3.Assessmentofperi‐woundangiogenesisinwoundstreatedwithM‐T7.(A)ELISA
quantificationofTNFαandVEGFinwoundtissuestreatedwithsalineorM‐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
barsare50μm.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
TNFαapplieddirectlytowoundsacceleratestheearlystagesofwoundhealing[59].Thetherapeutic
effectofTNFαintheearlystageofwoundhealingmayproceedviatheinductionofangiogenesis,as
TNFα isapowerfulinducerofVEGFandendothelialtipcellformation[45,46].Othertreatments
showntoacceleratewoundhealingalsoactviatheinductionofangiogenesis.Forexample,topical
simvastatinandasperosaponinVIbothacttoacceleratewoundhealingbyenlistingtheVEGF
signalingcascade,andVEGF‐Capplieddirectlytowoundsalsoaccelerateshealing[60–62].Here,we
showthatarecombinantM‐T7treatmentresultedinasignificantincreaseinlocalTNFαduringthe
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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