Neuropathy of haematopoietic stem cell niche is essential fo

2019-06-17 12:52

LETTER

doi:10.1038/nature13383

Neuropathyofhaematopoieticstemcellnicheisessentialformyeloproliferativeneoplasms

′nchez-Aguilera1,DanielMart?′n-Pe′rez1,JoanIsern1,XavierLanga1,AlexandarTzankov2,LorenaArranz1,AbelSa

′n3,Yi-ShiuanTzeng4,Dar-MingLai4,Ju¨rgSchwaller2,RadekC.Skoda2PontusLundberg2,SandraMuntio

′nMe′ndez-Ferrer1&Simo

Myeloproliferativeneoplasms(MPNs)arediseasescausedbymuta-tionsinthehaematopoieticstemcell(HSC)compartment.MostMPNpatientshaveacommonacquiredmutationofJanuskinase2(JAK2)geneinHSCs1–4thatrendersthiskinaseconstitutivelyactive,leadingtouncontrolledcellexpansion.Thebonemarrowmicroenvironmentmightcontributetotheclinicaloutcomesofthiscommonevent.Wepreviouslyshowedthatbonemarrownestin1mesenchymalstemcells(MSCs)innervatedbysympatheticnervefibresregulatenormalHSCs5,6.HerewedemonstratethatabrogationofthisregulatorycircuitisessentialforMPNpathogenesis.Sympatheticnervefibres,supportingSchwanncellsandnestin1MSCsareconsistentlyreducedinthebonemarrowofMPNpatientsandmiceexpressingthehumanJAK2(V617F)mutationinHSCs.Unexpectedly,MSCreductionisnotduetodifferentiationbutiscausedbybonemarrowneuraldamageandSchwanncelldeathtrig-geredbyinterleukin-1bproducedbymutantHSCs.Inturn,invivodepletionofnestin1cellsortheirproductionofCXCL12expandedmutantHSCnumberandacceleratedMPNprogression.Incontrast,

ControlhumanControl human

MPN

10 mRNA (ratio)86420

NES

(human)

20 mRNA (ratio)151050

Nes(mouse)

CJAK2(V617F)

CD34C MPNCMPNNes-GFP

administrationofneuroprotectiveorsympathomimeticdrugspre-ventedmutantHSCexpansion.Treatmentwithb3-adrenergicagonists

thatrestoredthesympatheticregulationofnestin1MSCs5,6preventedthelossofthesecellsandblockedMPNprogressionbyindirectlyreducingthenumberofleukaemicstemcells.Ourresultsdemon-stratethatmutant-HSC-drivennichedamagecriticallycontributestodiseasemanifestationinMPNandidentifyniche-formingMSCsandtheirneuralregulationaspromisingtherapeutictargets.

Thestemcellnichehasrecentlyemergedasanoncogenicunitandanimportantelementinregulatingcancerstemcells,includingHSCs7–11.MostMPNpatientswhodonotcarrytheBCR-ABLfusionhaveanacquiredmutationinJanuskinase2(JAK2(V617F))inHSCsthatresultsincon-stitutivekinaseactivity,leadingtouncontrolledexpansionofHSCsanderythroid,megakaryocyticandmyeloidprogenitors1–4.Somaticmutationsinthrombopoietinreceptor12,13orcalreticulin14,15genesarefoundinsomeMPNpatientsandadditionalHSCmutationsalsoaffectdiseaseprogres-sion16,17.ChangesintheHSCmicroenvironmentmightalsocontributetoMPNdevelopment,andexpansionofbonemarrow(BM)fibroblastsandbone-formingcellssuggeststheparticipationofMSCs.

WepreviouslyreportedthatmouseBMnestin1MSCsarerequiredtomaintainHSCs5andthathumanBMnestin1cellscanexpandHSCs18.Herewefoundthat,despiteelevatedBMblood-vesseldensityinMPNpatients,nestin1cellnumberandnestinmessengerRNAexpressionweremarkedlyreduced(Fig.1a,b).ThiswasreproducedinMx1-cre;JAK2(V617F)

Figure1|ApoptosisofBMnestin1HSCnichecellscontributestoMPNprogression.a,BMsectionsofcontrols(left)andMPNpatients(right)immunostainedwithnestin(brown,allpanels)andCD34(red,lowerpanels;magnification3200).Numbersofnestin1nichespermm2(mean6s.d.)were1.1560.3(control)and0.1760.18(MPN;n540;P51026,Mann–WhitneyUtest).b,c,NestinmRNAexpressioninBMcellsfrom

controls(n52),MPNpatients(n511)andmice(n54).d,Nes-GFP1cellsinskullBMofcontrol(left)andMPNmice(right;n510).e–g,CD452CD312Ter1192Nes-GFP1cells(e),clonalself-renewing

mesenchymalsphere(mesensphere)-formingcells(f)andfibroblasticcolony-formingunits(CFU-F,g)inBMcellsfromwild-typemice30weeksaftertransplantationwithcontrol(n57)orMPNBMcells(n512).h,i,Lineage-tracingstudiesofnestin1cells.Femoralsectionsoftamoxifen-treated

Nes-creERT2;RCE:loxPmice28weeksaftertransplantationwithcontrol(h)orMPNBMcells(i),showingfluorescentsignalsfromGFPandnuclei

counterstainedwithDAPI(n54).j,Fractionoflive,earlyandlateapoptoticBMstromalNes-GFP1cellsfromcontrol(n57)orMPNmice(n55).k–m,Bloodcounts(k),femoraltrichromic(l)andspleenhaematoxylinandeosinstainings(m)ofNes-creERT2;iDTAandcontrolmice20weeksaftertransplantationofMPNBMcells(n53).n,Frequencyoflin2sca-11c-kit1(LSK)haematopoieticprogenitorsinBMnucleatedcells(BMNC)of

Nes-creERT2;Cxcl12fl/fl(n55)andcontrollittermates(n57)30weeksaftertransplantationwithMPNBMcellsand24weeksaftertamoxifen

treatment.c–e,j,6-8weeksafterpIpCtreatment.Scalebars(d,h),200mm.Magnification(l,m)3100.Mean6s.e.m.*P,0.05;**P,0.01(unpairedtwo-tailedttest).BM,bonemarrow.C,control(disease-free)mice.

NestinNestinBM mesenssphere-forming cells e

BM Nes-GFP+ cells2,5002,0001,5001,0005000

5,000

500

3,0002,0001,000

ControlJAK2(V617F)hNes-creERT2;RCE

i**

BM CFU-F4,0004003002001000

CJAK2(V617F)

ControlJAK2(V617F)ERT2ET2;iDTA Nes-creERiDTAT

C JAK2

(V617F)

Control

CJAK2(V617F)

BM Nes-GFP+ cells (%)6040200

MxCre;JAK2(V617F)

Control iDTAT

Alive

iDTAT6420

Late apop. Early apop.Late apop.

ERT2ET2;iDTANes-creERT

Neutrophils per ml of blood (×106) m

Control iDTATERT2ET2;iDTA Nes-creERiDTAT

1.51.00.50.0Haematocrit (%) **

6040200

LSK cells (% BMNC)Cxcl12?/?

Nes-creERT2;Cxcl12 *1StemCellNichePathophysiologyGroup,CentroNacionaldeInvestigacionesCardiovasculares(CNIC),28029Madrid,Spain.2UniversityHospitalBasel,CH-4031Basel,Switzerland.3DepartmentofHaematology,IBSAL-HospitalUniversitariodeSalamanca,37007Salamanca,Spain.4NationalTaiwanUniversity,Taipei10002,Taiwan.00MONTH2014|VOL000|NATURE|1

?2014Macmillan Publishers Limited. All rights reservedRESEARCHLETTER

302010Principal component 2 0–10–20–30

–80

–60

–40P1 BM

Nes-GFP–Pdgfrα– E12.5

Schwann cellprecursors

Pdgfrα+ Sca1+

P1 BM Nes-GFP– Pdgfrα+

ControlNes-GFP+ P1 BM

Nes-GFP+ Pdgfrα+P0 Schwann cells

0mRNA (fold)–200–4000–500–1,000–1,500–2,0000–100,000–200,000–300,0000–1,000–2,000–3,000–4,000–5,000108642Lifrc

Nes-GFP

Sympatheticfibres

Merge

Schwann cells

JAK2(V617F)Nes-GFP+

Pdgfrα– Sca1–

–600–2,500–400,000Cxcl12Angpt1Kitl100mRNA (fold)80604020040302010Mbp0Mobp806040202520151052015105ControlControlP1 BM Nes-GFP+ Pdgfrα–

–20

Principal component 10000Plekhb1S100bPlp1Pkp4JAK2(V617F)JAK2(V617F)Nes GFP+ BM stromal cells Apoptotic (%)Sympathetic nerve fibresBM area (% control) e

0.25Th+ area (% BM)0.200.150.100.050.00f

0.6mRNA (ratio)0.40.20.0GFAP1.00.80.60.40.2C MPNGfapg

Schwann cellsBM area (% control) 150100500P = 0.06248CD105+ cells(% BMNC) 3020100241.51.00.5TUNEL+ cells (fold)200*C MPN**35030025020015010050040h

2.0****i

32***ControlLSK*8JAK2(V617F)124****Time (weeks)

0.0CJAK2(V617F)

Time (weeks)Time (weeks)

0.0JAK2(V617F)–

IL1ra–

–++–

0+JAK2(V617F)–IL1ra–+

+–––+–++

IL1raJAK2(V617F)DAPI TUNEL

MSCSchwann

Figure2|BMSchwanncelldeathtriggeredbyHSPC-derivedinterleukin-1bprecedesnestin1MSCapoptosisinMPN.a,PrincipalcomponentanalysisofcontrolandMPNBMCD452CD312Ter1192Nes-GFP1cellscomparedwithmesenchymalstromal(adultBMPdgfra1Sca11andneonatalBMPdgfra1Nes-GFP1/2cells;purpleshadedarea)andSchwanncells(embryonicday12.5(E12.5)SchwanncellprecursorsandneonatalSchwanncells;greenshadedarea;seeMethods).b,qPCRvalidation(n53).c,Nes-gfpskullBM5weeksaftertransplantationwithcontrolorMPNBMcells;fluorescentsignalsofGFP(green)andsympatheticnervefibresdetectedwithanti-tyrosinehydroxylase(Th)antibodies(red).d,Immunostainingofglialfibrillaryacidicprotein(Gfap)tovisualizeSchwanncellsincontrolandMPNBM.Scalebar,200mm(c),100mm(d).e,QuantificationofTh1fibresinBMfromcontrols(n52)andMPN

Neutrophilsper ml of blood (x106)patients(n516).f,GFAPmRNAexpressioninBMcellsofcontrols(n52),MPNpatients(n511)andmice(n54).g,Time-courseanalysesofSchwanncells(Gfap),sympatheticfibres(Th)andNes-GFP1cellapoptosisinNes-gfp;Mx1-cre;JAK2(V617F)1andcontrolmice2,4(n55)and8(n57)weeksafterpIpCtreatment.h,FrequencyofBMCD452CD312Ter1192CD1051cellsafter18-weekinterleukin-1receptorantagonist(IL1ra)treatment(n55).

i,ApoptoticrateofMSCsandSchwanncellsinvitroderivedfromneonatalBMNes-GFP1cellsandco-culturedfor24hwithcontrolorMPNadultBMlin2sca-11c-kit1(LSK)cells(6200ngml21IL1ra)(n53experiments).TUNELstaining(pink)ofSchwanncells;nucleiwerecounterstainedwithDAPI(blue).Scalebar,100mm.Mean6s.e.m.*P,0.05;**P,0.01;***P,0.001

(unpairedtwo-tailedttest).HSPC,haematopoieticstemandprogenitorcells.

a Schwann cells

Control Vehicle

4-methylcatechol b

2.01.51.00.5****Erythrocytesper ml of blood (×109)Neutrophilsper ml of blood (×106)Plateletsper ml of blood (×109)c

2520151050806040200P = 0.096420P = 0.063210*d WT0.0JAK2(V617F)–+–4MC–

BRL37344––

β3-AR KO

++–+–+

micethatdevelopedMPNuponpIpCtreatment19,20(Fig.1c).MPNmicewereintercrossedwithaNes-gfplinetolabelMSCs5withgreenfluorescentprotein(GFP).BothcompoundmutantmiceandNes-gfpanimalstrans-plantedwithmutantBMcellsdevelopedMPNandhadfewerBMMSCsdefinedbyGFP,surfacemarkerexpressionandfunctionalassays(Fig.1d–gandExtendedDataFig.1a–f).BecauseMSClosswascon-comitantwithincipientBMfibrosis,weconductedlong-terminvivolineage-tracingstudiestodeterminewhethernestin1MSCsdifferenti-ateintofibroblastsorosteoblastsinMPN,therebycontributingtothestromalchangesinthesemice19,20(ExtendedDataFig.1g–j).Unexpect-edly,thevascularpatternsofGFP1cellsweresimilartothoseinunaffected

Figure3|Treatmentwithb3-adrenergicagonistorneuroprotectivedrugblocksMPNprogression.a,b,NeurotrophicrescueofBMSchwanncellsblocksMPNprogression.Wild-typemicetransplantedwithMPNBMcellsweretreatedoveramonthwithBRL37344,theneuroprotectivedrug4-methylcatechol(4MC)orvehicle.a,SkullBMimmunostainingofglialfibrillaryacidicprotein(red)tovisualizeSchwanncells(n55);scalebar,100mm.b,Circulatingneutrophils(control,JAK2(V617F)4MC,JAK2(V617F)BRL37344,n55;JAK2(V617F)vehicle,n59).c,Circulatingerythrocytes,neutrophilsandplatelets16weeksaftertransplantationofMPNBMcellsintob3-adrenergicreceptor-deficient(KO,n58)andwild-type(WT)mice(n57).d,VanGiesonstainingsoffemoralBMofmiceinc.e–g,CompensationofBMsympatheticdamagebyselectivesympathomimeticdrugsblocksMPNprogressionandpreventsfibrosis.e,BloodcountsofWTmicetransplantedwithMPNBMcellsandchronicallytreatedwithselectiveb3-adrenergicagonist(BRL37344)orvehicle(n55).f,ReticulinandVanGiesonstainingsoffemoralBMofmiceine(magnification,3200).g,Invivodepletionofnestin1cellsreducesthe

therapeuticeffectofBRL37344.BloodcountsofiDTAcontrol(n55)andNes-creERT2;iDTAmicetreatedwithvehicle(n54)orBRL37344(n55)for6weeks.Drugtreatmentsina,b,e,fstarted4weeksaftertransplantation;treatmentingstarted2weeksaftertransplantationcombinedwithtamoxifen(4weeks).Mean6s.e.m.*P,0.05,**P,0.01,***P,0.001(unpairedtwo-tailedttest).

Plateletsper ml of blood (×109)e

Neutrophilsper ml of blood (×106)WTKOWTKO6,0004,0002,0000WTKOf

JAK2(V617F) vehicleReticulin fibresVan Gieson staining

***********Haemoglobin per dl of blood (g)20100**Haematocrit (%)481216Time (weeks)JAK2(V617F) Vehicle30481216Time (weeks)

JAK2(V617F) BRL37344 80*6040200481216Time (weeks)

Leukocytesper ml of blood (x106)2015105Neutrophils per ml of blood (x106)g

**43210+–

*0Nestin+ cells+BRL37344–

–––+–––+

2|NATURE|VOL000|00MONTH2014

JAK2(V617F) BRL37344 481216Time (weeks)

Nes-gfpmice(Fig.1h,i).Thus,aswasrecentlyreportedinBCR-ABL1MPN21,Nes-GFP2cellsmightgenerateexcessivefibroblastsandoste-oblasts.Nestin1MSCreductionwasinsteadexplainedbyathreefoldincreasedapoptoticrateinmutantmice(Fig.1j),andwasnotpreventedbytheJAKinhibitorruxolitinib(ExtendedDataFig.2).

Todeterminewhethernestin1MSCdeathcouldinturnstimulateMPNprogression,weselectivelydepletednestin1cellsinvivo.ThisdepletiondidnotaffectmatureBMSchwanncells,reportedtoexpressnestin22,butreducedMSCs,associatedwithincreasedwhiteandredbloodcells(Fig.1kandExtendedDataFig.3a–e).BMsectionsrevealedexcessivefibroblastsandboneformation,notyetdetectableincontrolmice(Fig.1l),consistentwithnestin1cellsnotgeneratingfibroblastsorbonecellsinMPN.Diseaseaccelerationfollowingnestin1celldepletionalsomanifestedasseverespleeninfiltration,stillabsentincontrolmice(Fig.1m).Atanearlydiseasestage,mostprimitiveHSCsshowedhighestexpan-sion,leadingtoincreasedhaematopoieticprogenitorsinBM,peripheralbloodandspleen.ThechemokineCxcl12regulatesHSCmigrationandquiescence23,24andishighlyexpressedbynestin1MSCs5.EarlyHSCmobilisationcorrelatedwithdecreasedBMCxcl12,consistentwithMSCreduction.Inaddition,Cxcl12expressiondropped70-foldinMPNBMNes-GFP1cells(ExtendedDataFig.3f–k).DeletionofCxcl12(ref.24)innestin1cellsinvivoincreasedBMhaematopoieticprogenitorsandcir-culatingplatelets(Fig.1nandExtendedDataFig.3l).MSC-derivedCxcl12canthusnegativelyregulateJAK2(V617F)1HSCexpansion.TobetterunderstandBMnestin1cellalterations,genome-wideexpress-ionwasprofiledbynext-generationsequencing.ExpressionofMSCandHSC-relatedgeneswaslowerinMPNNes-GFP1cells,whichinsteadshowedenrichmentinSchwanncellgenesandneural-relatedfunctionalcategor-ies(ExtendedDataFigs4a–fand10).PrincipalcomponentanalysesofindependentbiologicalsamplescomparedwithpubliclyavailabledatashowedthatcontrolNes-GFP1cellswereclosesttomesenchymalpro-genitors,whereasMPNNes-GFP1clusteredawayfromthemandclosetoSchwanncells(Fig.2a).Thesechanges,confirmedbyquantitativePCR(Fig.2b),suggestedanalteredHSCniche’sneuralcomponentinMPN.SympatheticnervefibresandensheathingSchwanncells,adjacenttodistinctiveNes-GFP1cells,andGFAPmRNAexpressionweremarkedlyreducedinBMofMPNpatientsandmice(Fig.2c–fandExtendedDataFig.4g–i).Time-courseanalysisshowedthatBMneuraldamageprecedesNes-GFP1cellapoptosis(Fig.2g),indicatingthatsympatheticneuro-pathycouldsensitisenestin1cellstocelldeathtriggeredbymutantcells.MultiplexELISAdetectedearlyincreasedinterleukin-1binMPNBM(ExtendedDataFig.5a).Thiscytokineanditsactivatingenzymecaspase-1wereexpressedbymonocytes,aspreviouslyreported25,butalsobyhaematopoieticprogenitors(ExtendedDataFig.5b–d).ComparedwithBMNes-GFP2stromalcells,mRNAlevelsofinterleukin-1receptoranditsantagonistwere10-and1,000-foldhigherinNes-GFP1cells,respectively,andspecificallyupregulatedinNes-GFP1cellsinMPN(ExtendedDataFig.5e,f).Thereforewechronicallytreatedmicewithanantagonistofinterleukin-1receptor.ThistreatmentreducedplateletcountsandincreasedBMMSCfrequency,associatedwithreducedcaspase-1mRNAexpressioninhaematopoieticprogenitors(Fig.2handExtendedDataFig.5g–j).WestudiedwhetherJAK2(V617F)1HSCsmightdirectlycauseBMSchwanncelldeath.UnlikeMSCs,BM-derivedSchwanncellsco-culturedwithJAK2(V617F)1haematopoieticprogenitorsshowedthreefoldhigherapoptoticrate,whichwasblockedbyinterleukin-1receptorantagonist(Fig.2i).Together,thesedatasuggestthatHSC-derivedinterleukin-1bcontributestoneuroglialdamage,whichcompromisesMSCsurvival.WethereforeinvestigatedwhethersympatheticneuropathymightunderlieHSCnichealterationsandthusrepresentatherapeutictargetinMPN.WetreatedsymptomaticMPNmicewiththeneuroprotectiveagent4-methylcatechol,whichcanprotectBMsympatheticnervefibresdur-ingchemotherapy26.Schwanncellswerepreservedin4-methylcatechol-treatedmice,associatedwithpreventedneutrophilia(Fig.3a,b).SympatheticnervefibresregulateBMHSCtrafficviab3-adrenergicreceptoractivationinnestin1MSCs5,6.ThisreceptorisnotexpressedinnormalorJAK2(V617F)1haematopoieticcells(ExtendedDataFig.6a).Diseasedevelopmentwas

Neutrophils per ml of blood (×106) Platelets per ml of blood (×106) ?2015105011IL-1β (pg per mg of protein) ?Erythrocytes per ml of blood (×109) a

12108642025???8,0006,0004,0002,0000?????b

4003002001000*******?*11????????**????1315Time (weeks)1315Time (weeks)

JAK2(V617F), veh

111315Time (weeks)JAK2(V617F), BRL37344

*Control, veh Control, BRL37344

c d

Gfap (average size) 806040200e

IL-1β (pg per mg of protein) 806040200*****JAK2(V617F), vehicle, vehicle

CXCL12 (ng per 2 femurs)JAK2(V617F), BRL37344

CFU-C per ml of blood f h g j i

BM Nes-GFP cells 2,0001,5001,0005000Control, veh CFU-C (% BMNC) *CFU-C (% BMNC) 2,500*0.30.20.10.0***LSK cells (% BMNC) 1.51.00.50.020,00015,00010,0005,00001.20.80.40.00.80.60.40.20.0****Control, BRL3734476543210JAK2(V617F), veh

2.0ST-HSCs (×106) JAK2(V617F), 4-methylcatechol JAK2(V617F), BRL37344

Negative mice (%) k

LSK cells (×106) 2.52.01.51.00.50.0***LT-HSCs (×105) ****l ****1001 in 192,641 10**1.51.00.50.0*BRL37344veh1 in 42,167 **1010203040Bone marrow nucleated cells (×104)

mBone marrowSchwann cellHealthNA SNS β3-AR Normal HSC CXCL12 Nestin+MSC CXCL12 SNS compensation (β3 adrenergic agonists) Cytokine stormNestin+ MSC apoptosis β3-AR agonistsβ3-AR No fibrosis or osteosclerosisSNS rescue/protection (4-methylcatechol) Glial and neural damageFibrosis &osteosclerosisMPN IL-1βMutant HSC CXCL12 CXCL12 Figure4|CompensationofBMneuropathyrescuesMSCsandpreventsmutantHSCexpansioninMPN.a–d,EfficacyofBRL37344treatmentinadvancedMPN;WTmicetransplantedwithcontrolorMPNBMcellsweretreatedwithBRL37344orvehicle(veh)uponthrombocytosis(8696233106and1,96862643106plateletspermlofblood,respectively;n55).a,Bloodcounts;{P,0.05,{{P,0.01,{{{P,0.001vsvehicle-treatedcontrolmice.b,IL-1bcontentinBMsupernatant.c,VanGiesonstainingoffemoralBMsections(magnification,3100).d,QuantificationofBMglialfibrillaryacidicprotein(Gfap)immunostaining.e–l,SympathomimeticdrugrestoresBMCxcl12levelsandpreventsHSCexpansionandmobilisation.WTmicetransplantedwithcontrolorMPNBMcellsweretreated4weekswithBRL37344,4-methylcatecholorvehicleover4(e,h),8(f)or16weeks(g,i,j).e,IL-1bcontentinBMsupernatant(controlvehicle,n510;

JAK2(V617F)vehicle,n511;JAK2(V617F)4-methylcatechol,JAK2(V617F)BRL37344,n55).f,BMCD452CD312Ter1192Nes-GFP1cellsinvehicle(n58)andBRL-treated(n510)MPNmice.g,Cxcl12contentinBM

supernatant(n55).h–j,Frequencyofcolony-formingunits(CFU-C)inBMnucleatedcells(BMNC)(h,n55;j,n53)andblood(i),andBMfractionoflin2sca-11c-kit1(LSK)cells(n53)(j).k,LSKcells,long-term(LT-)andshort-term(ST-)HSCs(n55)inBMfrommiceina–d.l,ReductionofleukaemicstemcellsinBRL37344-treatedmice.CD45.2WTmiceweretransplantedwithBMcellsfromCD45.1micetogetherwithBMcellsfromMPNmicetreatedwithvehicleorBRL37344(n55).Frequencyofmicewith,50%donorLSKcellchimaerism16weeksaftertransplantationisplottedagainsttestedcellnumber.MPN-initiatingcellfrequencyisindicated.*P,0.05,Pearsonchi-squaredttest.m,ModelillustratingHSCnichealterationsandrescueinMPN.MPN,myeloproliferativeneoplasm;HSC,haematopoieticstemcell;SNS,sympatheticnervoussystem;MSC,

mesenchymalstemcell;NA,noradrenaline;AR,adrenergicreceptor;C,control(disease-freemice).a–k,Mean6s.e.m.*P,0.05,**P,0.01,***P,0.001(unpairedtwo-tailedttest).

00MONTH2014|VOL000|NATURE|3

acceleratedinmicelackingtheb3-adrenergicreceptor(Fig.3c,d),unco-veringaprotectiveroleofthisreceptorinMPN.Symptomaticmicewerechronicallytreatedwithaselectiveb3-adrenergicagonist(BRL37344)tocompensatefordeficientsympatheticstimulationofnestin1MSCs.Notably,BRL37344treatmentpreventedMPN-associatedneutrophiliaandthrombocytosis,anddelayedredbloodcellreduction,butdidnotaffectbloodcountsinwild-typemice(Fig.3b,eandExtendedDataFig.6b–d).Contrastingwiththeseverefibrosisinvehicle-injectedmice,theBMofBRL37344-treatedanimalswasvirtuallydevoidofexcess-iveboneandfibroblastictissue(Fig.3f).TheseeffectswereHSCniche-dependent,becauseneither4-methylcatecholnorBRL37344affectedthegrowthofculturedhaematopoieticprogenitorsandleukocytosiswasnotrescuedbyBRL37344inmicewithnestin1celldepletion(Fig.3gandExtendedDataFig.7a).Similarly,severalMPNmarkerswereimprovedbytreatmentwiththeclinically-approvedb3-adrenergicagonistmir-abegron(ExtendedDataFig.7b),albeittoalowerextent,probablyowingtoitspoorsolubilityandrelativelylowaffinityforthemurinereceptor.Toinvestigatethepotentialtherapeuticbenefitwhenadmi-nisteredatmoreadvancedstages,thrombocytoticandcontrolmiceweretreatedwithBRL37344.Thistreatmentreducedneutrophilia,erythrocytosis,thrombocytosis,BMinterleukin-1b,fibrosisandosteo-sclerosis,rescuedBMSchwanncells(Fig.4a–d)andblockedSchwanncellprogramactivationinBMnestin1cells(ExtendedDataFig.7c).MPNprogressioncanthusbeblockedbyprotectionorrescueofBMneurogliaandbycompensationofdeficientsympatheticstimulationofnestin1MSCsbyb3-adrenergicagonists.

WenextaskedwhetherMPNblockadecouldbemediatedbypre-servationofMSCsandtheirHSCregulatoryfunction.BRL37344reducedIL-1b,restoredNes-GFP1cellnumberandincreasedCxcl12levelsinBM(Fig.4e–g).EarlyBRL37344or4-methylcatecholtreatmentpreventedmutanthaematopoieticprogenitorexpansion(Fig.4handExtendedDataFig.8).Long-termBRL37344treatmentdidnotcompromisenor-malHSCsbutefficientlydecreasedmutanthaematopoieticprogenitors(Fig.4i,jandExtendedDataFig.9),evenwhenadministeredatthrom-bocytosisstage(Fig.4k).Moreover,BRL37344-treatedMPNmiceshowed4.5-foldreductioninleukaemicstemcells(Fig.4l).

OurfindingspointtomutantHSCsasthecauseofBMneuroglialdamagethatcompromisesMSCsurvivalandfunction,criticallycon-tributingtoMPNpathogenesis(Fig.4m).ThestudyshowsthatthenichedamagetriggeredbythemutantHSCisessentialforthedevelopmentofahaematopoieticmalignancypreviouslyconsideredtobecausedbytheHSCalone.TargetingHSCniche-formingMSCsandtheirneuralregu-lationmaypavethewaytomoreefficienttherapeuticstrategiesinMPN.

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Mx1-cre;JAK2(V617F)19,Nes-gfp27,Nes-creERT2(REF28),RCE-loxP29,iDTA30,Cxcl12-floxed24,Adrb3tm1Lowl,CD45.1andCD45.2C57BL/6Jmice(JacksonLaboratories)werehousedinspecific-pathogen-freefacilities.ProtocolswereapprovedbytheAni-malWelfareEthicalCommittee.Invivotreatments,cellextraction,culture,FACS,histologicalanalyses,ELISA,qPCR,genomicandstatisticalanalysesaredetailedintheMethods.

OnlineContentMethods,alongwithanyadditionalExtendedDatadisplayitemsandSourceData,areavailableintheonlineversionofthepaper;referencesuniquetothesesectionsappearonlyintheonlinepaper.Received7July2013;accepted15April2014.Publishedonline22June2014.1.2.3.4.5.James,C.etal.AuniqueclonalJAK2mutationleadingtoconstitutivesignallingcausespolycythaemiavera.Nature434,1144–1148(2005).Kralovics,R.etal.Again-of-functionmutationofJAK2inmyeloproliferativedisorders.N.Engl.J.Med.352,1779–1790(2005).Levine,R.L.etal.ActivatingmutationinthetyrosinekinaseJAK2inpolycythemiavera,essentialthrombocythemia,andmyeloidmetaplasiawithmyelofibrosis.CancerCell7,387–397(2005).Baxter,E.J.etal.AcquiredmutationofthetyrosinekinaseJAK2inhumanmyeloproliferativedisorders.Lancet365,1054–1061(2005).′ndez-Ferrer,S.etal.MesenchymalandhaematopoieticstemcellsformaMeuniquebonemarrowniche.Nature466,829–834(2010).′n-Salamanca,A.M.Mart?′n,A.B.Ricote,AcknowledgementsWethankS.Mart?J.M.Ligos,S.BartlettandtheCNICComparativeMedicine,Genomicsand′n?ezgroupsandBioinformaticsUnitsforassistance;membersofS.M.-F.andB.Iba′ndezfordatadiscussion;G.Enikolopov,G.FishellandD.Riethmacher′a-FernaM.Garc?′nCNIC,SpanishMinistryofforprovidingmice.ThisworkwassupportedbyFundacioEconomyandCompetitiveness(TerCel,SpanishCellTherapyNetwork;PlanNacionalSAF-2011-30308)andConSEPOC-ComunidaddeMadridS2010/BMD-2542grants′nyCajalProgramgrantsRYC-2009-04703/2011-09726andMarietoS.M.-F.;RamoCuriegrantsFP7-PEOPLE-2011-RG-294262/294096toA.S.-A.andS.M.-F.;SwissNationalScienceFoundation310000-120724/1,32003BB_135712/1andSwissCancerLeagueKLS-02398-02-2009grantstoR.C.S.;A.S.-A.receivedaResearchFellowshipfromtheEuropeanHematologyAssociation.S.M.-F.issupportedinpartbyanInternationalEarlyCareerScientistgrantoftheHowardHughesMedicalInstitute.AuthorContributionsL.A.designedandperformedmostexperimentsandanalyses,preparedfiguresandwrotethemanuscript.A.S.-A.performedinvivotransplantationassays.D.M.-P.performedqPCR,genome-wideexpressionanalysesandtogetherwithX.L.providedtechnicalassistance.J.I.performedneonatalcellisolationandculture.X.L.andA.T.performedhistologicalanalyses.S.M.providedhumansamples.Y.-S.T.andD.-M.L.providedCxcl12-floxedmice.P.L.,J.S.andR.C.S.providedsamples,designedexperimentsandhumanstudies.S.M.-F.designedtheoverallstudy,supervisedtheexperimentsandwrotethemanuscript.AuthorInformationThegeneexpressiondatahavebeendepositedintheGeneExpressionOmnibus(GEO)databank(http://www.ncbi.nlm.nih.gov/geo)undertheaccessionnumberGSE55802.Reprintsandpermissionsinformationisavailableatwww.nature.com/reprints.Theauthorsdeclarenocompetingfinancialinterests.Readersarewelcometocommentontheonlineversionofthepaper.CorrespondenceandrequestsformaterialsshouldbeaddressedtoS.M.-F.(smendez@cnic.es).4|NATURE|VOL000|00MONTH2014

METHODS

Humanstudy.Thestudywasapprovedbyinstitutionalreviewboards.WritteninformedconsentwasobtainedfromallpatientsinaccordancewiththeDeclarationofHelsinki.ThediagnosisofMPNwasestablishedaccordingtotherevisedcriteriaoftheWorldHealthOrganization.

Invivopharmacologicaltreatments.Theminimalrequirednumberofanimalswasused.Micewererandomizedfortreatments.ProtocolswereapprovedbytheAnimalWelfareEthicalCommittee.InMx1-cre;JAK2(V617F)19double-transgenicmice,expressionofthehumanJAK2(V617F)mutationisdrivenbytheendogenousJak2promoterandcanbeconditionallyexpressedinhaematopoieticcellsuponMyxovirusresistance-1(Mx1)-drivenCrerecombinaseactivationbypolyinosine-polycytosine(pIpC).pIpC-inducedtransgenicmiceandwild-typemicetransplantedwithBMcellsfromthesemicedevelopprogressivesymptomsofPV19,20.

Age-matched,femalewild-typeC57BL/6JorNes-gfp27micewereusedasrecipi-entsinbonemarrow(BM)transplantationassaysandforinvivopharmacologicaltreatments.Lethallyirradiated(12Gy)recipientmiceweretransplantedwith23106BMcellsfromMx-Cre;JAK2(V617F)miceinducedwithpIpC8weeksbefore,orfromCre-negativemiceasdisease-freecontrols.Theselectiveb3-adrenergicagonistBRL37344(Sigma,St.Louis,MO)wasadministeredat2mgkg21throughintraper-itoneal(i.p.)injectiontwiceperday(every10–12h).Vehicle(salinesolution)dailyinjectionswereperformedinthesameway.Unlessindicated,treatmentwasinitiated4weekspost-transplant,whenanimalsevidencedsignsofMPNinperipheralblood.Micewererandomlydistributedbeforetreatmentinitiation,anddiseasedevelop-mentwasmonitoredovertimeinperipheralbloodsamples,usinganautomatedbloodcounter.Asimilartreatmentprotocolwasperformedwiththeselectiveb3-adrenergicagonistmirabegron(2mgkg21,i.p.,twoinjectionsperdayseparated10–12h).Theneuroprotectivedrug4-methylcatechol(10mgkg21,i.p.)wasinjectedoncedaily.TheJAKinhibitorINCB018424(S-ruxolitinib,AbmoleBioscience)wasadministeredbyoralgavage(30mgkg21,twiceperdayseparated10–12h)in0.5%hydroxypropylmethylcellulose(Sigma)aftersolubilisationinDMSO.TheIL-1recep-torantagonist(Kineret,Sobi,Stockholm,Sweden)wasadministeredbysubcutaneousosmoticpumps(Alzet)infusingacontinuousdosingof40mgkg21perday.Micewereeuthanizedatdifferenttimepointsandsubjectedtocompletenecropsy;BMandspleenwereanalysedblindlybyhistologyandflowcytometry;femoraandskullwereusedforimmunostainings,andhaematopoieticandstromalcellswereaddi-tionallyusedforfunctionalassaysexvivo.

ForquantificationofHSCs,BMcellsfromtreatedmicewereusedforcompet-itiverepopulationassaysusinglimitingcelldilutions.Briefly,CD45.11competitorBMcells(23106)weretransplantedintolethallyirradiatedCD45.21recipientmice,aftermixwith43105,43104and43103donorBMcells.Peripheralbloodchimaerismwasassessedbyflowcytometryevery2–4weeks.Micewerekilled16weeksafterthetransplantandtheBMLSKcellchimaerismwasassessedbyflowcytometry.Aminimumof5í45.21chimaerismintheLSKcellcompartmentwasconsideredaspositiveinrecipientsofcontrolBMcells.InrecipientsofmutantBM,micewereconsideredaspositive(‘leukaemic’)above50í45.21chimaer-ism.ThesoftwareL-Calc(StemCellTechnologies)wasusedforquantificationofHSCsandMPN-initiatingcellsincontrolandmutantdonorBM,respectively.Nes-creERT2(ref.28)micewereinducedwithtamoxifen(Sigma)fortheindicatedtimeperiods.Controlandexperimentalmicewereinjectedi.p.with140mgkg21oftamoxifen(14mgml21solutionincornoil),3timesonalternatedays,andweresimultaneouslyfedwithphytoestrogen-freedietforoneweek,followedbytamox-ifen-containingdietTM400(Harlan).Nes-creERT2;RCE:loxP29micewereusedforinvivolineage-tracingstudies.Toselectivelydepletenestin1cells,Nes-creERT2micewerecrossedwithaCrerecombinase-induciblediphtheriatoxinmouseline(iDTA30).Nes-creERT2micewereadditionallycrossedwithconditionalCxcl12-deficientmice24toselectivelydeleteCxcl12innestin1cellsupontamoxifeninduction.

BMcellextraction,flowcytometryandfluorescence-activatedcellsorting.Forhaematopoieticcellrecovery,boneswerecrushedinamortar,filteredthrougha40-mmmeshtoobtainsinglecellsuspensions,anddepletedofredbloodcellsbylysisin0.15MNH4Clfor10minat4uC.Spleensampleswerehomogenizedandfilteredbeforelysis;bloodsamplesweredirectlylysed.Cells(13106–23106cellspersample)wereincubatedwiththeappropriatedilution(2–5mgml21)offluorescentanti-bodyconjugatesand49,6-diamidino-2-phenylindole(DAPI)fordeadcellexclusion,andanalysedonLSRFortessaflowcytometer(BDBiosciences,FranklinLakes,NJ)equippedwithFACSDivaSoftware(BDBiosciences).Thefollowingantibodieswereused:fluorescentanti-CD45.1(A20),anti-CD45.2(104),anti-B220(RA3-6B2),anti-CD11b(M1/70),anti-CD3e(145-2C11),anti-Ly-6G(1A8),anti-Sca1(E13-161.7),anti-CD34(RAM34),anti-CD135/Flt3(A2F10.1),andbiotinylatedlineageantibod-ies(anti-CD11b,anti-Gr-1,anti-Ter119,anti-B220,anti-CD3e),allfromBDBio-sciences;anti-c-kit(2B8)fromeBioscience(SanDiego,CA).Biotinylatedantibodiesweredetectedwithfluorochrome-conjugatedstreptavidin(BDBiosciences).PhenotypicpopulationsofHSCweredefinedaslong-termhaematopoieticstemcells(HSCs)

(lin2Sca11c-kit1(LSK)CD342Flt32),short-termHSCs(LSKCD341Flt32)andmultipotentprogenitors(MPP)(LSKCD341Flt31).

Forisolationofnestin1cells,boneswerecleanedfromsurroundingtissue,crushedinamortarwithapestle,andcollagenase-digested(cataloguenumber07902,StemCellTechnologies)inashakingwaterbathat37uCfor45min.Cellswerefilteredthrougha40-mmmeshanderythrocyteswerelysedaspreviouslydescribed.Theresultingbonemarrow-enrichedcellsuspensionswerecentrifuged,washedandsuspendedinPBSbuffercontaining2ttalcalfserum(FCS)forfurtheranalyses.Forcellsorting,cellswereenrichedbyimmunomagneticdepletionusingbiotinylatedanti-CD45(104),anti-CD31(MEC13.3),andanti-Ter119antibodiesfollowedbyadditionofstreptavidinmagneticbeads(BDBiosciences),accordingtothemanufacturer’srecommendations.BMstromalCD452CD312Ter1192cellswerefurtherpurifiedaccordingtoGFPfluorescenceusingaFACSAriacellsorter(BDBioscience).Forthedeterminationofapoptoticcells,sampleswerewashedwithPBSaftersurfaceantibodystainingandsubsequentlystainedwithAnnexinV-PacificBlueandSYTOXAADvanced(Invitrogen,LifeTechnologies,Paisley,UK).Forfunctionalassays,cellswereenrichedbyimmunomagneticdepletionusingbio-tinylatedCD45andTer119.Forimmunophenotypiccharacterization,totalBMsampleswerestudiedbyflowcytometryusingthefollowingadditionalantibodies:anti-CD63(NVG-2),anti-CD105(MJ7/18),anti-CD140a(APA5),anti-Vcam,anti-CD51(RMV-7),allfromBiolegend;andanti-CD90.2fromBDBiosciences.Cellculture.Colony-formingunitsinculture(CFU-C)assaywasperformedaspreviouslydescribed31.BRL37344and4-methylcathecolwereaddedtothemethyl-celluloseatthespecifiedconcentrations.Coloniesofmorethan50cellswerescoredafter7daysofincubationat37uC,5%CO2,20%O2inawater-jacketedincubator.ForCFU-Fassays,BMCD452Ter1192cellswereplatedinto6-welldishesandculturedinmaintenancemedium(a-MEM,15üSwithantibiotics).After10–12daysinculture,adherentcellswerefixedwith100%methanolandstainedwithGiemsastain(Sigma)torevealfibroblasticclusters.Colonieswithmorethan50cellswerescoredasCFU-F.

Forclonalself-renewingmesenchymalsphereformation,cellswereplatedinultra-lowadherence35-mmdishes(StemCellTechnologies).Thegrowthmediumcontained15%chickenembryoextract,preparedasdescribed32,33;0.1mMb-mercaptoethanol;1%non-essentialaminoacids(Sigma);1%N2and227supplements(Invitrogen);recombinanthumanfibroblastgrowthfactor(FGF)-basic,recombinanthumanepidermalgrowthfactor(EGF),recombinanthumanplatelet-derivedgrowthfactor(PDGF-AB),recombinanthumanoncostatinM(227aminoacidsOSM)(20ngml21)andrecombinanthumaninsulin-likegrowthfactor-1(IGF-1;40ngml21)(Peprotech)inDMEM/F12(1:1)/humanendothelial(1:2)serum-freemedium(Invitrogen).Thecultureswerekeptat37uCwith5%CO2,20%O2inawater-jacketedincubator.One-halfmediumchangeswereperformedweekly.Mesensphereswerescoredatday10.

Forco-cultureofBM-derivedSchwanncellsandMSCswithBMLSKcellsfromcontrolandmutantmice,neonatalBMCD452CD312Ter1192Nes-GFP1Pdgfra2/1cellscontainingSchwanncellprecursorsandMSCs,respectively(Isern,J.etal.,sub-mitted),weresortedandculturedunderSchwanncelldifferentiationconditions,aspreviouslydescribed34,ormesenchymalcultureconditions,inMEMasupplementedwith10ngml21PDGF-AB,15?S.SchwanncellsandMSCswereco-culturedfor24hwithprimaryBMLSKcellsisolatedbyFACSfromcontrolormutantmice.SchwanncellsderivedfromsortedGFP1Pdgfra2BMprecursorswereadditionallyincubatedwithIL1ra(200ngml21)fortheco-cultureperiod.After24hofco-culture,haematopoieticcellswerewashedawayandtheremainingadherentSchwanncellswerefixedandblindlyanalysedforapoptosisbyTUNEL.

Histologicalanalyses,immunohistochemistryandimmunofluorescence.Haema-toxylinandeosinconventionalstainingwasperformedindeparaffinisedsectionsfollowedbyre-hydration.HarrishaematoxylinsolutionwasusedforstainingandeosinYsolutionwasusedforcounterstaining.Afterbrieflyrinsingtheslidesindistilledwater,dehydrationwasquicklyperformedin70%,95%andabsoluteethylalcohol.SectionswereclearedwithxyleneandmountedinDPX(Sigma).

ForMasson’strichromestainingofcollagen,sectionswerefixedagaininBouin’ssolutionfor1hat56uCandstainedwithWeigert’sironhaematoxylinworkingsolutionfor10min,followedbyBiebrichscarlet-acidfuchsinsolutionfor10–15min.Phosphomolybdic-phosphotungsticacidsolutionwasaddedfor10–15minoruntilcollagenlosttheredstaining.Sectionsweretransferreddirectlytofastgreensolutionandstainedfor1min,brieflyrinsedandincubatedwith1?eticacidsolutionfor2-5min.Afterabriefrinseindistilledwater,dehydrationwasquicklyperformedin70%,95%andabsoluteethylalcohol;sectionswereclearedwithxyleneandmountedwithDPX.

GordonandSweet’sstainingprotocolwasusedtovisualizereticulinfibres.Briefly,deparaffinisedsectionswereoxidisedin1?idifiedpotassiumpermanganatefor5min,followedby1%oxalicacidtodecolourise,andmordantin2.5%ironalumfor15min.Sectionswereimpregnatedinammoniacalsilversolutionfor2minandreducedwith10%aqueousformalinfor2min.Afterwardsthesectionswereincubated

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