LETTERRESEARCH
ExtendedDataFigure4|NeuroglialdamageinMPN-affectedBMstroma.a–d,ExpressionofneuroglialgenesetsinMPNBMNes-GFP1cells.a–c,Selectedtranscriptexpressioninfragmentsperkilobaseofexonpermillionfragmentsmapped(FPKM)ofmesenchymalgenes(a),HSC-nicherelatedgenes(b)andneural-relatedgenes(c).RNA-Seqin
CD452CD312Ter1192GFP1cellsisolatedfromcompoundMPNandcontrolmice6weeksafterpIpCtreatment(samplespooledfrom3mice).d,Genesetenrichmentanalyses(GSEA)ofRNA-Seqdata.e,f,Enrichmentplotofcoordinatedchangesinneuroactiveligand-receptorinteractions(e)andthetranscriptomeofneurons,astrocytesandoligodendrocytes(f).g,h,BMNes-GFP1cellsaredifferentfromsympatheticnervefibresandmatureSchwanncells.Immunostainingoftyrosinehydroxylase(TH)tovisualizesympatheticnervefibres(g)andglialfibrillaryacidicprotein(GFAP)formatureSchwanncells(h)inNes-gfpBM.NotethecloseassociationofNes-GFP1(green)cellstodistinctiveTH1orGFAP1(red)cells.i,BMbiopsiesfrom(leftandmiddle)controlor(right)MPNpatientsimmunostainedwith(left)secondaryAbasnegativecontrolor(middleandright)anti-THantibodies.Nucleiwere
counterstainedwithDAPI(blue).Scalebars,75mm(g),50mm(h),100mm(i).
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ExtendedDataFigure5|ContributionofIL-1btoearlyMPNpathogenesis.a,IncreasedBMIL-1blevelsearlyinMPN.MultiplexELISAanalysisofpro-inflammatory(IL-1b,TNF-a,IFN-c,IL-6),regulatory(IL-17)andanti-inflammatory(IL-10)mediatorsinplasma(control,n516;MPN,n513)andBMextracellularfluidsamples(control,n511;MPN,n56;onlyIL-1bandTNF-aweredetectable)fromcontrolandMx1-cre;JAK2(V617F)mice
4–8weeksafterpIpCtreatment.b–f,Nes-gfpmiceweretransplantedwithBMcellsfromMPN(n56)andcontrol(n53)miceandanalysed2weekslater.b–d,mRNAexpressionofIL-1b(b)anditsactivatingenzymecaspase-1(Casp1,c),andBMfrequenciesoflin2sca-11c-kit1haematopoieticprogenitorsandCD11b1Ly-6G(1A8)2monocytes(d).e,f,mRNA
expressionofIL-1receptor(IL1r)(e)anditsantagonist(IL1ra)(f)inBMCD452CD312Ter1192Nes-GFP1/2cells.g,NumberofcirculatingplateletsinWTmicetransplantedwithMPNandcontrolBMcellsandtreatedover18weekswithIL1ra,starting2weeksaftertransplant.h,FrequencyofBMCD452CD312Ter1192CD901cellsinmiceing.i,j,qPCRanalysesofIL-1b(i)andCasp1(j)mRNAexpressioninhaematopoieticprogenitorsand
monocytesisolatedfromtheBMofmiceing.Gapdhwasusedashousekeepinggene(n55).Mean6s.e.m.*P,0.05,**P,0.01,***P,0.001(unpairedtwo-tailedttest).
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ExtendedDataFigure6|Compensatorytreatmentwithb3-adrenergicagonistBRL37344blocksMPNprogression.a,Expressionofb2-andb3-adrenergicreceptorsinimmunomagnetically-enrichedCD451andCD452BMcellsfromcontrolandpIpC-inducedMx1-cre;JAK2(V617F)mice(n53).b-c,BloodcountsofWTmice(b)4weeksaftertransplantationwithMPNorcontrolBMcells,before(control,n511;MPN,n513)and(c)4-12weeksafterchronicBRL37344(2mgkg21)orvehicletreatment(i.p.,twiceaday;n55).CD11b1monocytesandgranulocytes,B2201BcellsandCD31Tcellsweredeterminedbyflowcytometry.d,BloodcountsofWTmicetransplantedwithcontrolBMcellsandtreatedwithBRL37344(n56)orvehicle(n54)asdescribedabove.Mean6s.e.m.*P,0.05,**P,0.01,***P,0.001(unpairedtwo-tailedttest).
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ExtendedDataFigure7|b3-adrenergicagonistsorneuroprotectivedrugspreventneuroglialgeneinductioninnestin1cellsandindirectlyaffectmutanthaematopoieticcells.a,InhibitionofmutantHSCexpansionbyb3-adrenergicagonistsorneuroprotectivedrugsisHSC-nichedependent.Immunomagnetically-enrichedCD451haematopoieticcellswereobtainedfromtheBMofNes-gfp;Mx1-cre;JAK2(V617F)miceandcontrollittermates16weeksafterpIpCtreatment(n53).BRL37344(BRL)and4-methylcatechol(4MC)wereaddedinvitroattheindicatedconcentrationsandthefrequencyofcolony-formingunitsinculture(CFU-C)wasscoredafteroneweekinculture.b,Compensatorytreatmentwiththehumanb3-adrenergicagonistMirabegrondelaysMPNinmice.BloodcountsofWTmice8weeksaftertransplantation
withMPNorcontrol(n55)BMcells.Mirabegron(2mgkg21,n58)orvehicle(n57)treatment(i.p.,twiceaday)wasadministeredthelasttwoweeks.c,BMNes-GFP1cellsfromMPNmicetreatedwithBRL37344donotexpressneuroglialgenes;mRNAexpressionoftheSchwanncellmarkersmyelinbasicprotein(Mbp),myelin-associatedoligodendrocytebasicprotein(Mobp)andpleckstrinhomologydomaincontaining,familyB(evectins)member1
(Pleckhb1)inBMCD452CD312Ter1192GFP1cellssortedfromNes-gfpmicetreatedover8weekswithBRL37344orvehicle,starting4weeksaftertransplantationwithMPNorcontrolBMcells(n54).Mean6s.e.m.*P,0.05,**P,0.01,***P,0.001(unpairedtwo-tailedttest).
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ExtendedDataFigure8|IncipientMPNsignsareimprovedbyBRL37344or4-methylcatechol.a,b,HaematologicalparametersofWTmice8weeksaftertransplantationwithBMcellsfrompIpC-treatedcontroland
Mx1-cre;JAK2(V617F)mice.Thelatterreceivedtheneuroprotectivedrug4-methylcatechol(10mgkg21,oncedaily),BRL37344(2mgkg21)orvehicleinjections(twiceaday)forthelast4weeks(i.p.;n55).MPNprogressionwasmonitoredinperipheralbloodandmicewerekilledwhentheyshowedonly
incipientsymptoms.a,Bloodcounts,spleensize,weightandnucleatedcellnumber,BMnucleatedcells(limbsandsternum),CD11b1myeloid,B2201B-lymphoidandCD31Tcells.b,BMlin2sca-11c-kit1(LSK)haematopoieticprogenitors,Ter1191CD712matureerythroblastsandCD411/CD421megakaryocyteprogenitors.Mean6s.e.m.*P,0.05,**P,0.01,***P,0.001(unpairedtwo-tailedttest).
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