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NATURE | LETTER

Thelungisasiteofplateletbiogenesisandareservoirfor
haematopoieticprogenitors
EmmaLefranais,GuadalupeOrtizMuoz,AxelleCaudrillier,BeatMallavia,FengchunLiu,DavidM.Sayah,EmilyE.
Thornton,MarkB.Headley,TovoDavid,ShaunR.Coughlin,MatthewF.Krummel,AndrewD.Leavitt,EmmanuellePassegu&
MarkR.Looney

Nature (2017) doi:10.1038/nature21706

Received 24April2016 Accepted 14February2017 Publishedonline 22March2017 Print

Plateletsarecriticalforhaemostasis,thrombosis,andinflammatoryresponses1,2,buttheeventsthatleadtomatureplateletproduction
remainincompletelyunderstood3.Thebonemarrowhasbeenproposedtobeamajorsiteofplateletproduction,althoughthereis
indirectevidencethatthelungsmightalsocontributetoplateletbiogenesis4,5,6,7.Here,bydirectlyimagingthelungmicrocirculationin
mice8,weshowthatalargenumberofmegakaryocytescirculatethroughthelungs,wheretheydynamicallyreleaseplatelets.
Megakaryocytesthatreleaseplateletsinthelungsoriginatefromextrapulmonarysitessuchasthebonemarrowweobservedlarge
megakaryocytesmigratingoutofthebonemarrowspace.Thecontributionofthelungstoplateletbiogenesisissubstantial,accounting
forapproximately50%oftotalplateletproductionor10millionplateletsperhour.Furthermore,weidentifiedpopulationsofmatureand
immaturemegakaryocytesalongwithhaematopoieticprogenitorsintheextravascularspacesofthelungs.Underconditionsof
thrombocytopeniaandrelativestemcelldeficiencyinthebonemarrow9,theseprogenitorscanmigrateoutofthelungs,repopulatethe
bonemarrow,completelyreconstitutebloodplateletcounts,andcontributetomultiplehaematopoieticlineages.Theseresultsidentifythe
lungsasaprimarysiteofterminalplateletproductionandanorganwithconsiderablehaematopoieticpotential.

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Authorinformation
Theseauthorscontributedequallytothiswork.
EmmaLefranais&GuadalupeOrtizMuoz

Affiliations
DepartmentofMedicine,UniversityofCalifornia,SanFrancisco(UCSF),SanFrancisco,California94143,USA
EmmaLefranais,GuadalupeOrtizMuoz,AxelleCaudrillier,BeatMallavia,FengchunLiu,AndrewD.Leavitt,EmmanuellePassegu
&MarkR.Looney

DepartmentofMedicine,UniversityofCalifornia,LosAngeles(UCLA),LosAngeles,California90095,USA
DavidM.Sayah

DepartmentofPathology,UniversityofCalifornia,SanFrancisco(UCSF),SanFrancisco,California94143,USA
EmilyE.Thornton,MarkB.Headley&MatthewF.Krummel

CardiovascularResearchInstitute,UniversityofCalifornia,SanFrancisco(UCSF),SanFrancisco,California94143,USA
TovoDavid&ShaunR.Coughlin

DepartmentofLaboratoryMedicine,UniversityofCalifornia,SanFrancisco(UCSF),SanFrancisco,California94143,USA
MarkR.Looney

Contributions
E.L.designedandconductedmostoftheexperiments,analysedthedata,andwrotethemanuscript.G.O.M.designedandconducted
experimentsandanalysedthedata.A.C.andB.M.conductedexperimentsandanalyseddata.F.L.performedthelungtransplantation
experiments.D.M.S.,E.E.T.,M.B.H.andT.D.assistedindesigningandconductingexperiments.S.R.C,M.F.K.andA.D.L.assistedin
designingexperimentsandprovidededitorialsupportonthemanuscript.E.P.assistedindesigningexperiments,providedtechnical
expertisewithhaematopoieticprogenitoranalyses,andprovidededitorialsupportonthemanuscript.M.R.L.designedtheexperiments,
conductedexperiments,analyseddata,andwrotethemanuscript.

Competingfinancialinterests
Theauthorsdeclarenocompetingfinancialinterests.

Correspondingauthor
Correspondenceto:MarkR.Looney

ReviewerInformationNaturethanksF.Ginhoux,S.Morrison,G.Zimmermanandtheotheranonymousreviewer(s)fortheir
contributiontothepeerreviewofthiswork.

Publisher'snote:SpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.

Extendeddatafiguresandtables

ExtendedDataFigures
1.ExtendedDataFigure1:Megakaryocytesandproplateletsobservedinlungcirculationarefromanextrapulmonarysource.
(1,087KB)
a,Lung2PIVMofaPF4nTnGmouse(nuclearGFP).ThepresenceofthemobileGFP+nucleatedcells(circled)indicatesthe
presenceofanucleusincirculatingmegakaryocytes.b,Plateletcountsinthebloodbeforeandafterimaging.n.s.,not
 significant(n=3).c,ExperimentalschemaoftransplantationoflungsfrommTmGmouse(perfuseddonorlung)toPF4mTmG
(recipient)mouseandviceversafollowedby2PIVM.d,2PIVMofamTmGmouselungshowingnoGFPsignal.e,2PIVMof
anmTmGmouselungtransplantedintoaPF4mTmGrecipientmouseshowingGFP+cellsfromrecipientandplatelet
productioninthelung.f,Bonemarrow2PIVMapparatus.g,Representativeimageofproplateletreleaseinbonemarrow(BM)
sinusoids(arrows).h,2PIVMofPF4mTmGmouseliver.Smallplatelets(GFP,green)wereseeninthecirculationbutneither
residentnorcirculatingmegakaryocytesorproplateletswereobserved.i,2PIVMofPF4mTmGmousespleen.Sequential
imagesshowresidentmegakaryocytes(GFP,green)releasingproplatelets(arrows)inthespleenvasculature(inred).

2.ExtendedDataFigure2:Residentmegakaryocytesinthelungsandotherorgans.(1,177KB)
a,SurveyofPF4tomatomouselungvisualizedby2PIVM.PF4tomatoexpressingcells(red)arefoundinhighnumbersinthe
lungs.LungvasculatureislabelledbyintravascularinjectionofFITCdextran(green).Atotalareaof2.49mm2(1.6mm1.6
mm)wasimaged.b,Resident(static)GFP+cellsarefoundinaPF4mTmGlungtransplantedintoanmTmGmouse.c,d,
2PIVMimagesofbonemarrow(c)andspleen(d)fromPF4mTmGmice.Manylargemegakaryocytes(GFP,green)arefound
inthebonemarrowandspleen.e,Sizecharacterizationofresident(static)GFP+cellsbyimageanalysisofPF4mTmGlungs
(n=16),bonemarrow(n=12),andspleen(n=16).Minimumtomaximumboxplotsareshown:thelineinthemiddleofthebox
isplottedatthemedian,theboxextendsfromthe25thto75thpercentilesandthewhiskersrangefromthesmallesttothe
largestvalues.The+indicatesthemean.

3.ExtendedDataFigure3:Surfaceexpressionoflungmegakaryocytescomparedtobonemarrowmegakaryocytes.(353KB)
a,FlowcytometricanalysisofnGFP+cellsfromPF4nTnGlungs.b,CD41expressiondefinestwopopulationsof
megakaryocytes:CD41+(red)andCD41(green).c,PositivesurfaceexpressionofGPVI,cMplandCD45wasdetectedin
bothpopulations.Unstainedcellsareplottedinblue.d,SurfaceexpressionofF4/80,CD34,CD11b,Sca1andcKitwasnot
detected.ei,TheCD41+populationhasahigherpercentageofCD61+cells(e),CD42b+cells(f)andlargercells(g)andhad
higherDNAcontent(h),assummarizedini(n=3).j,FlowcytometricanalysisofnGFP+cellsfromPF4nTnGbonemarrow.
km,Comparedtothelungs,thebonemarrownGFP+populationhasahigherpercentageofCD41+cells(n=2123)(k),
CD61+cells(n=3)(l),andCD42b+cells(n=3)(m).Dataarerepresentativeofthreeormorereplicates.Means.d.are
presented.Unpairedttest:*P<0.05,**P<0.01,***P<0.001.

4.ExtendedDataFigure4:Geneexpressionanalysisoflungversusbonemarrowmegakaryocytesandbacterialpneumonia
experimentrevealanimmatureprofileandapotentialroleinimmunityforlungmegakaryocytes.(449KB)
Megakaryocytes(nGFP+CD41+)weresortedfromPF4nTnGlungsandbonemarrowfollowedbymRNAisolationand
sequencing.a,RelativemRNAexpressionisshownonascalefromlow(green)tohigh(red).Threeindependentexperiments
(fourmiceeach)wereusedforstatisticalanalysis.Heatmapofallsignificantlydifferentiallyexpressedgenes(FDR<0.05).b,
Readcountsformegakaryocyterelatedgenes.Thesegenesarefoundinbothlungandbonemarrowmegakaryocytes,but
someareunderrepresentedinlungmegakaryocytes.c,d,Analysisofgeneontologybiologicalprocessesrelatedtogenesthat
aredownregulated(c)orupregulated(d)inlungmegakaryocytes.Thetop20biologicalprocessesareshown.Theverticalaxis
representsgeneontologycategories,andthehorizontalaxisindicatesthenumberofgenesineachontologycategory.e,f,
ReadcountsforTLRgenepathways(e)andchemokines(f)thatwereoverexpressedinlungmegakaryocytes.FPKM,
fragmentsperkilobaseofexonpermillionfragmentsmapped.gk,Lungmegakaryocytesandprogenitorpopulationsare
alteredduringinfection.FlowcytometricanalysisofnGFP+cellsfromPF4nTnGlungs24hafterintratrachealadministrationof
S.aureus(MRSA,510 7colonyformingunits(c.f.u.)).CD41APCwasinjectedintravenouslybeforelungdigestionand
stainingwithCD41FITC.ThenumberofcellsinnormalorinfectedlungsareshownforallnGFP+cells(g),lessmaturecells
(nGFP+CD41h),andmaturecells(nGFP+CD41+i).j,k,Percentageofintravascularmegakaryocytes(j)andextravascular
megakaryocytes(k)inthematurepopulation(nGFP+CD41+).n=46micepergroup.

5.ExtendedDataFigure5:PlateletreconstitutionafterlungtransplantationwithorwithoutTPOinjection.(378KB)
Bloodwascollectedfromthemandibularveineveryweekfollowinglungtransplantation.Aftertransplantation,agroupofmice
(redandorange)receivedTPOinjection(250mgkg1,days3and40).Theothergroupwasleftuntreated(blueandpurple).
Somemiceineachgroupshowedsustainedplateletproductionformorethan3months(blueandred).Intheothermice
(purpleandorange),theplateletproductionwaslowerandtransient(lessthan3months).Thepercentagesofmiceineach
groupareindicated.Datafromindividualmice(a,c,e,g)orgroupaverages(b,d,f,h)areplotted.ad,i,j,Percentageof
donorderivedplatelets.PercentagewasanalysedbyFACS,countingthetomato+platelets(CD41+FSCsmallgate).eh,k,
Overallplateletcountsintheperipheralblood.j,k,Plotsfrommicewith10monthsofsustainedplateletproduction.i,
Percentageoftomato+plateletsincontrollungtransplants(n=46).l,Colourcodeforthedifferentlungtransplantgroups
accordingtolungorigin(donor),recipientmouse,treatmentreceived(withorwithoutTPO)andobservedresponse(sustained
ortransient).
 6.ExtendedDataFigure6:Characterizationofplateletsproducedafterlungtransplantation.(294KB)
a,b,Flowcytometricanalysisoftomato+plateletsobservedinthebloodandstainedwithantibodiesagainstCD41,CD42d,
GPVIandcMpl.BloodfromPF4tomatomice(a)orcmpl /micethathadreceivedPF4tomatolungtransplants(b).c,
Experimentalschemaforplateletactivation.d,Flowcytometricanalysisoftomato+plateletsafterstimulationwiththrombin(10
nM)stainedwithantibodyagainstCD62P.e,PercentageofCD62P+plateletsbeforeandafterthrombinactivation.Ctrl,PF4
tomatoLungtx,PF4tomatolungstransplantedintocmpl /mice.Means.d.arepresented(n=23micepergroup).
Unpairedttest:**P<0.01,***P<0.001

7.ExtendedDataFigure7:Lungandbonemarrowanalysisoftransplantedmice.(504KB)
Micewithsustainedproductionoflungderivedplateletswerekilledatleast3monthsafterlungtransplantation.a,
Representative2PIVMimageofaPF4tomatolungaftertransplantationintoacmpl /mouse.b,Representative2PIVM
imageofflushedbonemarrowcellsfromPF4tomatoandcmpl /mice,andwildtypeandcmpl /micetransplantedwith
PF4tomatolungs.c,ExperimentalschemafortransplantationofmTmGlungsintocmpl /mice.Bloodwascollectedfromthe
mandibularveinevery2weekstotestfordonorderivedplatelets(tomato+platelets)andmeasureoverallbloodplateletcounts.
d,PercentageofdonorderivedplateletsanalysedbyFACScountingoftomato+eventsintheCD41+FSCsmallgate.e,Platelet
countsinperipheralblooddeterminedbyCBC.d,e,Means.e.m.arepresented.fl,BonemarrowcellsfrommTmGmice
(D),cmpl /mice(R),orcmpl /micetransplantedwithmTmGlungs(Tx)thatshowedsustaineddonorderivedplatelet
productionfor3monthswereanalysed.PopulationfrequencieswithintheCD45+compartmentweremeasuredformyeloid
progenitors(f,MP:LinSca1cKit+),MkPs(g),LSKcells(h,LinSca1+cKit+),LTHSCs(i),STHSCs(j),MPP2s(k)and
MPP3/4s(l).m,PopulationfrequencieswithintheLSKcompartmentfromindicatedgroups.np,Totalcellpopulations(grey),
donororigintomato+cells(red),andpercenttomato+cells(abovebars)inbonemarrow(n),recipientnativelung(rightlung
o)andspleen(p).Means.d.arepresented(n=23micepergroup).Unpairedttest:*P<0.05,**P<0.01,***P<0.001.

8.ExtendedDataFigure8:Lunghaematopoieticprogenitorsareextravascularandhavemultilineagecapabilities.(454KB)
a,RepresentativespleenFACSplotsofhaematopoieticprogenitorswithintheLSKcompartmentandthemyeloidprogenitor
compartment(LinSca1cKit+).b,Cellcountsofhaematopoieticprogenitorpopulationsinthespleen(n=6).ce,Lungs
wereperfusedbeforedigestion.c,RepresentativeFACSplotofLSKandmyeloidprogenitorcompartmentswithanterograde
perfusionandwithorwithoutretrogradeperfusion.d,e,Frequencies(d)andcellcounts(e)oflunghaematopoieticprogenitor
populations(n=3).fh,CD45APCmAbwasinjectedintravenouslyviathetailvein5minbeforelungdigestionandstaining
withCD45FITCmAb.g,HaematopoieticprogenitorpopulationswereexaminedforlabellingwithinjectedCD45mAbbyflow
cytometry.h,PercentageofCD45FITC+cellspositiveornegative(extravascularcells)fortheintravenousCD45APCmAb(n
=36).ik,Peripheralbloodwasanalysed23monthsaftertransplantationofmTmGlungsintocmpl /mice.i,
RepresentativeFACSplotofbloodcellanalysis.j,Percentageoflungderivedcells(Tom+CD41)inthebloodafter
transplantation.k,PercentageofBcells(CD19+),Tcells(CD3+)orneutrophils(CD11b+Ly6G+)inthelungderivedcells(Tom+
CD41).Means.e.m.arepresented(n=5micepergroup).

9.ExtendedDataFigure9:Proposedschemaoflunginvolvementinplateletbiogenesis.(268KB)
Theroleofthelungsinplateletbiogenesisistwofoldandoccursintwodifferentcompartments.a,Plateletproductioninthe
lungvasculature.Afterbeingreleasedfromthebonemarroworthespleen,proplatelets(a1)andmegakaryocytes(a2)are
retainedinthelungvasculature,thefirstcapillarybedencounteredbyanycellleavingthebonemarrow,whereproplatelet
formationandextensionandfinalplateletreleaseareobserved.b,Matureandimmaturemegakaryocytesalongwith
haematopoieticprogenitorsarefoundinthelunginterstitium.Inthrombocytopenicenvironments,haematopoieticprogenitors
fromthelungmigrateandrestorebonemarrowhaematopoieticdeficiencies.

ExtendedDataTables
1.ExtendedDataTable1:Variablesusedtocalculatethenumberandpercentageofplateletsproducedbythelungs(85KB)

Supplementaryinformation

Video
1.Video1:Dynamicreleaseofplateletsinthelungvasculature(8.15MB,Download)
Lung2PIVMinPF4mTmGmicewherePF4CredrivesmembraneGFPexpressioninMKsandplateletswhileallothercells
arelabelledwithmembranetomato.SeveralexamplesareshownoflargeGFP+MKsthatenterthelungcirculationand
undergoproplateletformationandextension.Scalebarsandacquisitiontimeareindicated.
 2.Video2:MKswithnucleicirculatinginthelungvasculature(11.27MB,Download)
Lung2PIVMinPF4mTmGmicewherePF4CredrivesmembraneGFPexpressioninMKsandplateletswhileallothercells
arelabelledwithmembranetomato.SeveralexamplesareshownoflargeGFP+MKsthatenterthelungcirculationand
undergoproplateletformationandextension.TheevidenceofanucleusintheGFP+cellsissupportedbythepresenceofa
darkcentrethatappearsinsidetheGFP+cells.Attheendoftheprocess,whenallthecytoplasmhasbeenreleased,anaked
nucleusisobserved.Scalebarsandacquisitiontimeareindicated.

3.Video3:MKnucleicirculatinginthelungvasculature(2.89MB,Download)
Lung2PIVMinPF4nTnGmicewherePF4CredrivesnuclearGFPexpressioninMKswhileallothercellsarelabelledwith
nucleartomato.DepictedareseveralexamplesofmobileGFP+nucleicirculatinginthelung.Scalebarsandacquisitiontime
areindicated.

4.Video4:SurfaceanalysisofMKsandplateletsinthelung(10.83MB,Download)
Surfacerendered3DreconstructionofGFP+MKsandplateletsrecordedduringlung2PIVMinaPF4mTmGmouse.Notein
example2thatthenuclearvolumeisnotincludedintherenderedGFP+surfacevolume.

5.Video5:QuantificationofMKsreleasingplatelets(12.97MB,Download)
Lung2PIVMinaPF4mTmGmousethatincludes0.7m3oflungvolume.ThenumberofMKsreleasingplateletsduringthis2
hourvideowerecounted(whitecircles).

6.Video6:MKsreleasingplateletsinthelungareofextrapulmonaryorigin(15.03MB,Download)
a)Lung2PIVMofmTmGlung(noCreexpression)transplantedintoaPF4mTmGrecipient.IntravascularGFP+MKsfromthe
PF4mTmGrecipient(extrapulmonary)wereobservedreleasingproplatelets.b)Reversetransplant(PF4mTmGlung
transplantedintomTmGrecipient)showsextravascularMKs(GFP+),butnointravascularMKsorproplateletformation.Scale
barsandacquisitiontimeareindicated.

7.Video7:ProplateletreleasebyMKsintheBM(28.23MB,Download)
CalvariumBM2PIVMinPF4mTmGmice.ThevideosshowextravascularMKs(GFP+)releasingproplateletsintheBM
sinusoids(arrows).

8.Video8:MKmigrationintheBMsinusoids(13.07MB,Download)
CalvariumBM2PIVMinPF4mTmGmice.ThevideosshowlargeMKs(circled)enteringtheBMsinusoidsandexitingthe
imagedBMspace.

9.Video9:MKsreleasingproplateletsinthespleen(19.84MB,Download)
Spleen2PIVMinPF4mTmGmice.ThevideosshowlargeextravascularMKsinthespleen(greencircles=inactiveMKswhite
circles=MKsreleasingproplatelets).ThehigherpowerviewsshowexamplesofextravascularMKsreleasingproplateletsinto
thesplenicsinusoids.

10.Video10:SessileMKsareobservedinthelunginterstitium(23.96MB,Download)
Lung2PIVMinPF4mTmGmice(examples1and2)orPF4tomatomice(examples3and4)wherePF4Credrives
membraneGFPorcytoplasmictomatoexpressioninMKsandplatelets.ShownareexamplesoflargeMKsobservedinthe
lunginterstitiumthatremainsessileduringseveralhoursofimaging(upto4hours).

Excelfiles
1.SupplementaryTable1(6.7MB)
ThisfilecontainsSupplementaryTables1ag.

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