itgb8 expression mediated by adil1β is decreased by adcre. … · 2014-01-30 · ii in combination...

Kitamura,etal SupplementalData

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SupplementalFigure1:Increaseditgb8expressionmediatedbyAdIL1β isdecreasedbyAdCre.Thelevelsofitgb8mRNAinwholelunghomogenateswereassessed7dwithorwithoutintratracheal(IT)Ad‐IL‐1β. IT‐Ad‐IL‐1βtreateditgb8F/‐miceweretreatedwithincreasingdosesofAd‐CreorAd‐GFP.Shownaretheitgb8genecopynumbersrelativetogapdhandactb.NotethattheeffectsofIT‐Ad‐Crearerestrictedtofieldofbiodistribution,whichistheairwayandimmediatelysurroundingcellsandnotthedistallungparenchyma(seeSupplementalfigure2).Therefore,changesinexpressionofitgb8fromwholelunghomogenatesrepresentonlytheairwayanditgb8expressioninthedistallungisunaffected.


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SupplementalFigure2:IntratrachealAdCremediatedrecombinationinmultipleairwaycelltypes,andTamoxifendependentrecombinationonlyinairwayandlungfibroblasts.A)Theairwaycell‐typesexpressingAd‐CrefollowingIT‐Ad‐CreinjectionorbyCol1a2‐Cre‐ER(T)micewereassessedusingR26‐LacZreportermice.LacZexpressioninCre‐expressingcellsandtheirprogenyisactivatedbyCre‐mediatedexcisionofaloxP‐flankedpolyadenylationsequenceintheR26reportergene72.Col1a2‐Cre‐ER(T)miceexpress,onlyinfibroblasts,afusionproteinconsistingofthecatalyticdomainofCre‐recombinasewithamutatedligand‐bindingdomainoftheestrogenreceptorwhichonlybindstamoxifenor4‐hydroxyTamoxifen,butnotestradiol67.a)IT‐Ad‐GFPtreatmentofR26‐LacZmicedidnotrevealrecombinationincellsfromcytospinpreparationsofBALfluid,whileb)IT‐Ad‐Cretreatmentrevealedrecombinationinmacrophages(arrows).C,d)Col1a2‐Cre‐ER(T)micewerecrossedtoR26‐LacZreportermiceintheabsence(c)orpresence(d)oftamoxifen(Tamox).Cytospinpreparationswerestainedtodetectβ‐galactosidaseactivity.Bar=20µm.Nocellsinthebronchoalveolarlavage(BAL)fluidorairwayepithelialcellsshowedevidenceofrecombinationintamoxifentreatedmice.B)IT‐Ad‐Cre‐mediatesrecombinationinairwayepithelialcells,airwaymesenchymalcellsandmacrophages;Tamoxifen‐dependentrecombinationinCol‐Cre‐ER(T)miceisfibroblastspecific.a)ITAd‐LacZrevealsthefieldofadenoviralinfectioninairwayepithelialcells(largearrow),mesenchymalcells(smallarrows)andmacrophages(arrowhead).b)ITAd‐CremediatedrecombinationinR26‐LacZmicerevealsrecombinationinallcelltypeswithintheadenoviralfieldofinfection.Insetshowsclose‐upofmesenchymalcellssurroundingairways(smallarrows).c,d)AdultCol1a2‐Cre‐ER(T)micecrossedtoR26‐LacZreportermicerevealtamoxifen‐dependentrecombinationinspindlecellsresemblingfibroblastssurroundingtheairwaysandinparenchymalinterstitialfibroblasts(smallarrows).Recombinationisnotseenintheabsenceoftamoxifen(c).Insetshowshighmagnificationimageofmesenchymalcellstainingsurroundingairways(smallarrows).Bar=75µm,ThesedatademonstratethefibroblastspecificityandefficiencyofdeletionofadultmouselungairwayandparenchymalfibroblastsusingtransgenicCol1a2‐Cre‐ER(T)mice.


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SupplementalFigure3.Deletionofitgb8inallairwaycelltypesinhibitsAdIL1βinducedairwayremodeling.AD:Histologic(micrographsrepresent14daysposttreatment)andmorphometricanalysisofinflammation(C,H&E)orfibrosis(D,trichrome),7,14,21or42daysaftermicewithasinglefloxeditgb8allele,andasingleknock‐outitgb8allele(itgb8F/‐)weretreatedwithITAd‐IL1‐βwith(solidlines)orwithoutAd‐Cre(dottedlines).InB,Arrowspointtothickcollagenfibers.Shownarevaluesminusthenon‐IT‐Ad‐IL‐1βtreatedcontrols.


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SupplementalFigure4.Liganddependentrecombinationofitgb8infibroblastsdecreasesitgb8expressionandαvβ8mediatedTGFβ activationinculturedlungfibroblasts.Lungfibroblastsenrichedabove40/60%PercollinterfacefromwholelunghomogenatesofCol‐Cre‐ER(T);itgb8F/‐micetreatedwithoutorwithTamoxifen,werecultureduntilconfluentandtestedforitgb8expressionusingA:qPCRorB:TGF‐βactivationusingaTMLCTGF‐βbioassay.*p<0.05


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SupplementalFigure5:LungDCscanbeseparatedfromalveolarmacrophagesbasedonforwardandsidescatter,CD11c,MHCIIandLy6cstaining:FlowcytometricseparationoflungDCsfromalveolarmacrophagesusingCD11candMHCIIincombinationwitheitherLy6c(A)orF4/80(B).Forwardandsidescattergateswerebasedon“back‐gating”onCD11chi,autofluorescent,orCD11chigh,non‐autofluorescentcells.A:Highsidescatter,highforwardscatter,MHCIIintermediate,Ly6cintermediatecellsweremostlyautofluorescent(AF)alveolarmacrophages(AM).Lowsidescatter,lowforwardscatter,Gr1,TCRβ,NK1.1,CD19negative,MHCIIhigh,Ly6cintermediatecellswerenon‐AFlungDCs.B:Highsidescatter,highforwardscatter,MHCIIintermediate,F4/80negativecellsweremostlyautofluorescent(AF)alveolarmacrophages(AM).Lowsidescatter,lowforwardscatter,Gr1,TCRβ,NK1.1,CD19negative,MHCIIhigh,F4/80positivecellswerenon‐AFlungDCs.ShowninB,secondcolumnareoverlays(isotypecontrolinbluevs.F4/80inred).HistogramsintherightupperpanelofbothAandBshowautofluorescentseparationofAMvs.DCs,andinthelowerrightpanels,overlaysofAM(blue)andDC(red)usingAFvs.CD11cstaining.


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SupplementalFigure6:Lossofitgb8onfibroblastsdoesnotaffectlungorMLNDCmaturation.A,B:LungsorMLNwereharvestedfromCol‐Cre‐ER(T);itgb8F/‐micetreatedwithIT‐Ad‐LacZorIT‐Ad‐IL‐1β,withorwithoutTamoxifen.ShownisthemeanfluorescenceintensityinarbitraryunitsforMHCIIexpressedby103+or103‐DCsfromtheMLN(A)orlung(B).Datarepresentsaminimumof6miceforAd‐LacZcontrolsandaminimumof12miceforAd‐IL‐1βgroups.C:LungCD11c+DCsfromCol‐Cre‐ER(T);itgb8F/‐micetreatedwithIT‐Ad‐LacZorIT‐Ad‐IL‐1β,withorwithoutTamoxifenwerepurifiedusingCD11cmicrobeads(MiltenyiBiotech,Auburn,CA)andwereassessedusingqPCRforccr2,ccr6,andccr7(lefttoright).Shownarerelativegenecopynumbers.*p<0.05


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SupplementalFigure7:Cytokinearrayanalysisofitgb8dependentchangesincytokineexpressionfromIL1β treatedlungsandculturedlungfibroblasts.Cytokineantibodyarrayofpooledmouselunghomogenates(n=6)harvested14dafterIT‐Ad‐IL‐1β (A)orofpooledmousefibroblast(n=5)supernatants48hrsafterIL‐1βtreatment(B).Shownisthespotintensityrelativetointernalpositivecontrolforeachcytokinelistedfromhighesttolowestexpression,lefttoright.C,D:ChangeinexpressionofeachcytokinedetectedonthearrayaftertreatmentwithAd‐Crefromwholelunghomogenates(C)orprimaryculturesofitgb8F/Flungfibroblasts(D).ShownispercentchangeinexpressionofIL‐1βtreatedvs.IL‐1βtreated+Ad‐Cre.


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SupplementalFigure8:Ovalbuminchallengestimulatesinflammasomeactivation,IL1β secretion,andincreaseditgb8expression.ColcreER(T);itgb8

F/‐miceunderwentamock(PBS,openbars)oracuteovalbumin(Ova,filledbars)sensitizationandchallengeprotocol.EighthrsafterthefinalOvachallenge,themiceweresacrificed,BALharvested,andA)totalcellscounted.B)PelletedBALcellswerestainedwithafluorescentcleavedcaspase‐1detectionkitandnumbersoflabeledcellsdeterminedusingflowcytometry.C)LunghomogenatesweremadefromtherightlungofeachmouseandIL‐1βELISAwasperformedfromanequalvolumeoflunghomogenate.D)Genecopynumberforitgb8wasdeterminedfromtheleftlungofeachmouseusingqPCR.Resultswerenormalizedtoβ‐actinandgapdh.n=8.*p<0.05,***p<0.001.


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SupplementalFigure9:IL1β treatedhumannormalandCOPDfibroblastssecretealimitedrepertoireofcytokines.Normal(n=5)orCOPD(n=5)fibroblastsweretreatedwithrecombinantIL‐1βfor24hrinthepresenceorabsenceofisotype‐matchedcontroloranti‐β8(37E1B5).Pooledsupernatantswereappliedtocytokineantibodyarrays(RayBiotech,Inc.,NorcrossGA)anddetectedbychemiluminescence(Chemidoc,BioRad,Hercules,CA).ShownisdatanotrepresentedinFigure6D.A)Relativespotintensity(minusnegativecontrol)wasexpressedasaratiotothepositivecontrol.B)RelativespotintensityofeachdetectedcytokinefromIL‐1βtreatedCOPDfibroblastsupernatantsareplottedfromthemosthighlyexpressedtothelowest.Cytokinesthatwerenotdetectedareindicated.


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SupplementalTable1:SummaryofAirwayMorphometryMouse group Genotype n Time

(d) Airways

examined Inflammation Fibrosis

Ad-LacZ Control WT 2 14 29 3.1±0.4 4.4±0.4 Ad-Lac Z 3G9 WT 2 14 40 2.7±0.4 4.1±0.4 Ad-Lac Z 1D11 WT 2 14 42 2.9±0.3 2.8±0.2

Ad-IL-1β Control WT 3 14 57 33.3±2.4 10.0±0.8 Ad-IL1β 1D11 WT 3 14 48 17.0±1.8 6.9±0.7 Ad-IL1β 3G9 WT 3 14 55 28.9±3.1 9.7±0.9

Ad-LacZ no Tam Col-Cre ER(T); itgb8 F/- 3 14 46 2.9+/-0.3 7.6+/0.6 Ad-LacZ Tam Col-Cre ER(T); itgb8 F/- 3 14 67 3.6+/-0.3 9.9+/0.6

Ad-IL-1β no Tam Col-Cre ER(T); itgb8 F/- 4 14 95 36.5+/3.3 24.2+/-1.4 Ad-IL-1β Tam Col-Cre ER(T); itgb8 F/- 6 14 148 10.5+/1.0 13.1+/-0.7

Ad-LacZ no Tam Col-Cre ER(T); itgb8 F/- 3 21 62 4.4+/-0.5 9.3+/-0.8 Ad-LacZ Tam Col-Cre ER(T); itgb8 F/- 3 21 59 3.2+/-0.3 8.0+/-0.7

Ad-IL-1β no Tam Col-Cre ER(T); itgb8 F/- 5 21 104 17.3+/1.5 13.0+/0.9 Ad-IL-1β Tam Col-Cre ER(T); itgb8 F/- 5 21 122 11.4+/1.1 12.1+/0.9

Ad-LacZ no Tam Col-Cre ER(T); itgb8 F/- 1 42 15 2.7+/-0.3 4.4+/-0.4 Ad-LacZ Tam Col-Cre ER(T); itgb8 F/- 1 42 20 3.1+/-0.4 4.1+/-0.4

Ad-IL-1β no Tam Col-Cre ER(T); itgb8 F/- 2 42 35 5.9+/-0.8 7.3+/-0.7 Ad-IL-1β Tam Col-Cre ER(T); itgb8 F/- 2 42 39 3.1+/-0.3 4.2+/-0.5

Ad-LacZ no Tam K14 Cre-ER(T); itgb8 F/- 7 14 80 3.1+/-0.2 11.2+/-1.0 Ad-LacZ Tam K14-Cre ER(T); itgb8 F/- 6 14 72 3.2+/-0.2 8.7+/-0.8

Ad-IL-1β no Tam K14-Cre ER(T); itgb8 F/- 9 14 111 24.3+/-1.7 17.1+/-1.6 Ad-IL-1β Tam K14-Cre ER(T); itgb8 F/- 9 14 114 20.5+/-1.4 15.6+/-1.7

Ad-LacZ no Tam WT 2 14 41 3.2±0.3 ND Ad-LacZ Tam WT 2 14 43 3.1±0.2 ND

Ad-IL-1β no Tam WT 3 14 53 38.8±2.5 ND Ad-IL-1β Tam WT 3 14 58 43.3±3.1 ND PBS no Tam Col-Cre ER(T); itgb8 F/- 2 70 34 3.9+/-0.4 4.8+/-2.7

PBS Tam Col-Cre ER(T); itgb8 F/- 2 70 42 3.4+/-0.3 5.5+/- 3.3 Ova no Tam Col-Cre ER(T); itgb8 F/- 7 70 142 15.3+/1.2 14.0+/-1.0

Ova Tam Col-Cre ER(T); itgb8 F/- 6 70 110 6.1+/-0.7 7.6+/-0.7 Ad-LacZ/Ad-GFP Itgb8 F/- 6 7 76 5.4+/-0.6 12.6+/-1.0 Ad-LacZ/Ad-Cre Itgb8 F/- 6 7 77 6.9+/-0.9 7.1+/-0.6 Ad-IL-1β/Ad-GFP Itgb8 F/- 8 7 96 28.2+/-1.8 28.2+/-1.9 Ad-IL1β/Ad-Cre Itgb8 F/- 8 7 103 20.9+/-1.3 20.8+/-1.3

Ad-LacZ/Ad-GFP Itgb8 F/- 6 14 112 5.9+/-0.8 9.5+/-0.6 Ad-LacZ/Ad-Cre Itgb8 F/- 4 14 74 5.4+/-0.6 9.1+/-0.8 Ad-IL-1β/Ad-GFP Itgb8 F/- 8 14 145 25.1+/-1.8 14.8+/-0.8 Ad-IL1β/Ad-Cre Itgb8 F/- 8 14 135 12.3+/-1.1 12.8+/-0.7

Ad-LacZ/Ad-GFP Itgb8 F/- 8 21 176 13.1+/-1.0 9.3+/-0.5 Ad-LacZ/Ad-Cre Itgb8 F/- 8 21 165 20.6+/-1.5 12.6+/-0.6 Ad-IL-1β/Ad-GFP Itgb8 F/- 11 21 217 22.3+/-1.4 12.4+/-0.6 Ad-IL1β/Ad-Cre Itgb8 F/- 11 21 229 22.7+/-1.4 11.8+/-0.5

Ad-LacZ/Ad-GFP Itgb8 F/- 8 42 131 6.4+/-0.4 8.9+/-0.3 Ad-LacZ/Ad-Cre Itgb8 F/- 9 42 201 5.6+/-0.3 8.6+/-0.3 Ad-IL-1β/Ad-GFP Itgb8 F/- 14 42 245 9.8+/-0.5 12.4+/-0.6 Ad-IL-1β/Ad-Cre Itgb8 F/- 13 42 276 8.1+/-0.4 7.7+/-0.3 Ad-LacZ/Ad-GFP WT 1 14 12 7.2±1.6 ND1

Ad-LacZ/Ad-Cre WT 1 14 16 4.1±1.1 ND Ad-IL-1β/Ad-GFP WT 3 14 54 20.6±2.5 ND Ad-IL1β/Ad-Cre WT 3 14 43 21.2±2.5 ND

1. ND, Not Done


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SupplementalTable2:Primersgene name Species application 5' primer name Sequence (5’ to 3’) 3' primer name Sequence (5’ to 3’)

itgb8 Mouse genotyping mutant F AGAGGCCACTTGTGTAGCGGCCAAG mutant R GGAGGCATACAGTCTAAATTGT

itgb8 Mouse genotyping Flox F GAGATGCAAGAGTGTTTACC Flox R CACTTTAGTATGCTAATGATGG

cre Mouse genotyping colcre F ATCCGAAAAGAAAACGTTGA colcre R ATCCAGGTTACGGATATAGT

itgb8 Mouse recombination B8 intron 3 F GTGGTTAAGAGCACCGATTG exon 4-5 R CTAAATTGTAGTCACTGCACTGATTG

itgb8 Mouse SYBR Green exon 3-4 F CCAACTGCATCCAGGAGCTGAAGCT exon 4-5 R CTAAATTGTAGTCACTGCACTGATTG

itgb8 Mouse SYBR Green B8.2 F GGGAGTGTGAAGGTGGCAGA B8.2 R CTGAGGCTGATGGGCACTGA

serpine1 Mouse SYBR Green Pai1(2) F AGTGATGGAGCCTTGACAG Pai1(2) R AGGAGGAGTTGCCTTCTCTT

col1a2 Mouse SYBR Green coll1a F AAGGTGCTGATGGTTCTCC coll 1a R TCTTTCTCCTCTCTGACCG

tgfb1 Mouse SYBR Green tgfb F GCAACATGTGGAACTCTACCAGAA TGFB R GACGTCAAAAGACAGCCACTC

ifng Mouse SYBR Green infg F ATGAACGCTACACACTGCATC inf g R CGACTCCTTTTCCGCTTCCTG

il4 Mouse SYBR Green il-4(2) F AGGTCACAGGAGAAGGGACGCC il-4(2) R TGCGAAGCACCTTGGAAGCCC

il5 Mouse SYBR Green il-5 F CTCTGTTGACAAGCAATGAGACG il-5 R TCTTCAGTATGTCTAGCCCCTG

il6 Mouse SYBR Green il-6 F AGTTGCCTTCTTGGGACTGA il-6 R TCCACGATTTCCCAGAGAAC

il13 Mouse SYBR Green il-13(1) F CTTGCTTGCCTTGGTGGTCTCGC il-13(1) R GCAGTTTTGTTATAAAGTGGGCT

il17a Mouse SYBR Green il-17A(2) F ACCCTGGACTCTCCACCGCA il-17A(2) R CAGGTGCAGCCCACACCCAC

il17f Mouse SYBR Green il-17F(2) F TCGAGACCCCCACCGGTTCC il-17F(2) R AGCGTTGTCAGGCCGCTTGG

ccl2 Mouse SYBR Green ccl2(2) F GAGGAAGGCCAGCCCAGCAC ccl2(2) R TGGGGCGTTAACTGCATCTGGC

ccl20 Mouse SYBR Green ccl20(2) F GCAGCCAGGCAGAAGCAGCA ccl20(2) R CGCCCCCATGGATTGTGGGA

ITGB8 Human SYBR Green B8 JM F AGGATCTTCTACCCCTCTTGC B8 JM R ATCTGGACAGATGGCGGTAATG

ITGB8 Human SYBR Green ITGB8 ja F TGGTCGAGGAGTTTGTGTTTG ITGB8 ja R AGCCACTGAAGCATTGGCA

COL1A2 Human SYBR Green Type1coll F TCTGGATGGATTGAAGGGACA Type1coll R CCAACACGTCCTCTCTCACC

Kitamura,etal.SupplementalMethods

Cellsandreagents:CellculturemediaandantibioticswerepreparedbytheUniversityofCalifornia,SanFranciscoCellCultureFacilityusingdeionizedwaterandanalyticalgradereagents.FetalcalfserumwasobtainedfromInvitrogen(Carlsbad,CA),humanrecombinantIL‐1βandTGF‐β1wereobtainedfromR&DSystems(Minneapolis,MN).Hybridomaclone1D11(AmericanTissueTypeCollection(ATCC),Manassus,VA),isapan‐TGF‐βisoformmonoclonalantibodyproducerwhichgeneratesanantibodycrossreactivetoTGF‐β1,2,3ofhuman,minkandmouseorigin.Anti‐SV5(1)andanti‐humanMHCclassI(IgG2a,ATCC)hybridomasweregrownandpurifiedusingFPLC,aspreviouslydescribed(2).Antibodiesweretestedforendotoxintoconfirmendotoxinlevels<0.2EU/μgasdeterminedbyLALmethod,(Genscript,Piscataway,NJ).Anti‐mouseCCL2(MAB479)andCCL20(MAB760)werepurchased(R&Dsystems).ELISAkitshumanCCL2(Cat#DY279),humanCCL20(Cat#DY360),mouseCCL2(Cat#DY479),mouseCCL20(Cat#DY760)andmouseIL‐1β(Cat#MLB00B)werefromR&Dsystems.HumanCytokineantibodyarray‐6(cat#AAH‐CYT‐6)andmouseinflammatoryantibodyarray‐1(cat#AAM‐INF‐1)werefromRayBiotech,(Norcross,GA).TMLCTGF‐βreportercellsweremaintainedin10%FCSinDMEM(giftofJohnMunger,NewYorkUniversityMedicalCenter,NYC,NY).Antibodyengineeringandaffinitymaturationofclone37E1:Thevariableregionsofanti‐humanintegrinβ8(clone37E148)weresequenced.Mutationswereintroducedintothevariableregionsbyerror‐pronePCR,themutantrepertoiredisplayedasscFvantibodiesonthesurfaceofSaccharomycesCerevisiaeandhigheraffinityscFvselectedbyusingflowcytometry90.AnscFvwithincreasedaffinityovertheparentalscFvwasidentified(37E1B5)andclonedintoamouseIgG2abackboneDetailsof37E1B5antibodyengineeringandproductionaretobepublishedinaseparatemanuscript.Theparentalmouseantibody37E1wasaffinitymaturatedto37E1B5,usingantibodyyeastdisplaytechnology.Briefly,totalRNAwasextractedfrom5x107cellsof37E1(3),andtheheavyandlightchainvariabledomaingenes(VHandVL)wereamplifiedseparatelyusingusing5’‐RACEkit(InvitrogenCatalogNumber18374‐058)andmouseV‐genefamilyspecificprimers(4).TheVH&VLgenesweresubclonedintopYD2vectorsandexpressedassinglechainvariabledomain(scFv)onthesurfaceofyeaststrainEBY100asdescribed(1).Thebindingspecificityoftheparental37E1scFvwasconfirmedusingsolubletruncatedhumanintegrinανβ8inFACSanalysis(5).AscFvyeastdisplaylibraryatthesizeof2x107wascreatedviaerror‐pronePCR(GeneMorph®IIRandomMutagenesisKit,Strategene)amplificationofbothVHandVLgenesandwasdisplayedasscFvonthesurfaceofyeast(1).ThisyeastdisplayedscFvlibrarywassorted6timeswithdecreasedconcentrationofsolubletruncatedhumanintegrinανβ8,andthebindingofanyscFvwasdetectedwithanon‐overlappingmonoclonalantibodydirectedagainsttheαvintegrinsubunit(clone8B8(3))duringtheFACSsortingandanalysisprocess.Twenty‐fourrandomlypickedbestbindersafterthelastsortingwereaffinitycomparedandsequenced.Clone37E1B5waschosentoconvertintofulllengthimmunoglobulin.Thefull‐lengthimmunoglobulin37E1B5wasproducedfromstabletransformedCHOcelllinesaschimericIgG1andmouse


IgG2aaftersubcloningtheV‐genesintotwodifferentmammaliancellexpressionvectors(6)(U.S.PatentApplicationNo.61/305,749).Ovalbuminsensitizationandchallenge:Six‐to12wk‐oldsex‐matchedandlittermatecontrolCol‐Cre‐ER(T);itgb8F/‐miceweresensitizedwith50μgOvalbumin(Sigma)inatotalvolumeof200µlsalineintraperitoneally(IP)ondays0,4,and7.Startingonday10,micewereinjectedIPfor5consecutivedayswith1mgoftamoxifenemulsifiedincornoilorcontrolsinjectedwithcornoilalone.Intranasalchallengewithovalbumin(20µgin30µlofsaline)orsalinealonewasperformedonceaweekfor9weekswithtamoxifenorcornoilinjectedIPonthesameday.Bronchoalveolarlavage(BAL)wasperformedandlungharvested10wksaftersensitizationandusedforELISA.Todeterminetheroleofitgb8inDCmigration,6to12wk‐oldsex‐matchedandlittermatecontrolCol‐Cre‐ER(T);itgb8F/‐miceweresensitizedondays0,7,and14byIPinjectionof50µgovalbumin(Ova)(Sigma‐Aldrich)emulsifiedin1mgofaluminumpotassiumsulfateinatotalvolumeof200µlinPBS.ControlanimalsreceivedanequalvolumeofPBS/aluminumpotassiumsulfate.Onday16,micewereinjectedIPfor5consecutivedayswith1mgoftamoxifenemulsifiedincornoilorcontrolsinjectedwithcornoilalone.Intranasalchallengewithovalbumin(100µgOva/40µlofPBS)orwithPBSalonewasgivenondays21,22,and23tolightlyanesthetizedmice(isofluraneinhalation).Insomeexperiments,Ova‐FITC(Invitrogen,Carlsbad,CA)deliveredintranasally(30µlofa5mg/mlstock)wasco‐administeredwiththeday23challenge,24hpriortoorganharvestforDCanalysis.RecombinantAdenovirus:TherecombinantE1‐E3deletedtype5adenovirus,eitherempty(Ad‐C)orexpressinghumanactiveIL‐1β(Ad‐IL‐1β),hasbeendescribedindetailelsewhere(7).Thereplication‐deficientviruswascommerciallyamplifiedandpurifiedbycesiumchloridegradientcentrifugationandPD‐10Sephadexchromatography,plaquetiteredon293cellsandcheckedforwild‐typecontamination(ViraQuestInc.,NorthLiberty,IA).Recombinanttype5AdenoviralvectorsexpressingCre‐eGFPfusionprotein,eGFP,orLacZwereobtainedfromtheGeneTransferVectorCore(UniversityofIowa,IowaCity,IA)InductionofTamoxifendependentrecombination:4‐hydroxyTamoxifen(Sigma)wasdissolvedinethanol(100mg/ml)andsuspendedin4partscornoil(20mg/ml)andstoreda‐20°C.Forintraperitoneal(IP)injections,thetamoxifensolutionwasdilutedtwiceincornoil,sonicatedfor2min(2x1minbyusing3‐spulses).Sixto12weekoldmicereceivedIPinjectionof1mgoftamoxifenperdayorcornoilalonefor5consecutivedayspriortoIT‐Adtreatment.Intratrachealinjections:MicewereanesthetizedwithIPinjectionofAvertin(250mg/kg,IP).ThenAd‐hIL‐1βorAd‐LacZ(2.5x108pfuin75μlsterilePBS)wasinstilledintratracheallywithaneedle(Popper®24G‐1’Straight1.25mmball)usingthedirectvisualizedinstillation(DVI)technique(8).ForAd‐Credose‐responseexperiments,thefollowingdosesofAd‐CrewerepremixedwithAd‐IL‐1βpriorto


intratrachealinstillationintoitgb8F/‐mice:1.25,2.5or7.5x108pfu.Allotherexperimentswithcombinationsofviruseswereperformedwith2.5x108pfuofeach.PairsofvirusesandtheirappropriatecontrolswereAd‐IL‐1β/Ad‐LacZ,orAd‐IL‐1β/Ad‐CandAd‐Cre‐eGFP/Ad‐eGFP.Foranti‐β6oranti‐TGF‐βsystemictreatment,micewereinjectedIPwith3G9(neutralizinganti‐β6,giftofShielaViolette,Stromedix,Cambridge,MA),1D11antibody(4mg/kg/mouse)for3consecutivedayspriortoIT‐Ad‐IL‐1βtreatment(day0)andtheninjected3times/wk(4mg/kg/mouse)with1D11untiltheorganharvestonday14.FieldofrecombinationofAdCreandColCreER(T)andactivationoftheCreER(T)fusionprotein:ROSA26‐STOP‐LacZ(R26‐LacZ)reportermicehavetheLacZreporterwithanupstreamSTOPcassetteflankedbyLoxPsequences.InthepresenceoffunctionalCre‐recombinasethisSTOPcassetteisexcisedandtheLacZgeneisexpressed(9).ThusIT‐Ad‐Cre(2.5x108pfu)orIT‐Ad‐eGFP(negativecontrol)wasadministeredtoR26‐LacZmicetodeterminethefieldofrecombinationinthelungandcomparedtoWTmiceinfectedwithIT‐Ad‐LacZ(2.5x108pfu)todeterminethefieldofadenoviralinfectioninthelung.Rosa‐LacZ;Col‐Cre‐ER(T)micewereinjectedIPfor5consecutivedayswith1mgoftamoxifenorwithcornoilalone,asabove,priortoIT‐Ad‐IL‐1βorAd‐C.Thesemiceweresacrificedat7dafterinjection.StainingforβGalactosidase:7daysafterinjection,thelungswerefixedbyintratrachealperfusionwith1%glutaraldehydeinPBS(pH7.3)at4°Cfor1h.ThelungswererinsedtwicebyPBSandX‐Galstainingsolution(5mMK4Fe(CN)6,5mMK3Fe(CN)6,2mMmagnesiumchloride,1mg/mlX‐GalinPBS)wasinstilled.Aftertrachealligation,harvestedlungswereincubatedinX‐Galsolutionovernight,washedwithPBS,andthelungswerepost‐fixedin10%formalinandparaffinembedded.CytospinpreparationsofBALwereairdriedfor1h,andstoredat‐80°Cuntiluse.Theseslideswerefixedwith0.5%glutaraldehydefor10minatRT,washedtwiceinPBS,andstainedfor3hinX‐Galstainingsolution.Mouseorganharvestsandbronchoalveolarlavage(BAL):ForBAL,thetracheawascannulatedandthelungswerelavaged5timesusing0.8mlofsterilePBSwith5mMEDTA.Therecoveryofthetotallavageexceeded90%.Thefractionswerecentrifuged(600gfor10minutes),andthesupernatantfromthefirstfractioncollectedandkeptat–80°CforELISAassaysorTGF‐βbioassays,performedasdescribed(3);thecellpelletsfromallfractionswerepooledandresuspendedin1.0mlsterilePBSandthetotalcellcountwasdeterminedusingahemocytometer.Differentialcellcountswereperformedusingcytospinpreparations(Cytospin3;ThermoShandon),whichwerepreparedbycentrifugingat800rpmfor6min.Differentialcellcountsweremadebycounting200cellsusingstandardmorphologicalcriteria.Caspase‐1activationwasdeterminedinBALcellsusingaGreenFLICA™Caspase1AssayKit(ImmunoChemistryTechnologies,Bloomington,MN).Theheartwaspuncturedandthelungsperfusedwith10mlsterilePBScontaining


50Uofheparinperml(Sigma‐Aldrich).Therightlungwasisolatedanddividedintoitslobes,placedin20mlof10%formalinandshakenvigorouslyfor30stoinflate.Theleftlungwasdividedinto5portions,theleftupperandtherightlowerportionusedforDNAharvest,themiddlefrozenforproteinanalysisandtherightupperandtheleftlowerplacedinRNAlater(AppliedBiosystems/Ambion,Austin,TX).Airwaymorphometryandimmunohistochemistry:MeasurementsofairwayinflammationwereestimatedusingHematoxylinandEosin(H&E)stainedslidesandwallfibrosiswasassessedbythepresenceofthickcollagenbundlesstainedbythetrichromemethodessentiallyasdescribedbyHogg(10),whichexpresseswallthicknessasafunctionofareaoftheairwaywall/basementmembranelengthdeterminedusingimageanalysissoftware(ImageJ,v1.36b).MicrotomesectionsfromH&Eortrichromestainedsectionsofparaffinembeddedmouselungsweredigitallyimagedat200Xmagnification(QCapturev2.68.2,Surry,BC,Canada).Theslideswerecodedbyaninvestigator(H.K.)whileanotherinvestigator(S.L.N)whowasblindedtotheexperimentalgroupsacquired5digitalimagesrepresentingeachlunglobe(andtwoimagesfromthelargestlobe)andtheimagescodedandcatalogued.Airwayinflammationwasdefinedastheinflammatoryinfiltrateextendingfromtheairwaybasementmembranetowardsthelungparenchyma.Airwayfibrosiswasdefinedasthickcollagenbundles(stainedblueintrichromestains)belowtheairwaybasementmembrane.Digitalimageswereanalyzedbyathirdinvestigator(S.C)whowasblindedtotheexperimentalgroups.Alearningdatasetwasacquiredonatestgroupusingthismethodologyandbasedonanexperimentallydeterminedstandardizedwidthoftheconfidenceinterval(W/S),thenumberofairwaymeasurements/mouserequiredfor90,95and99%confidenceintervalswasdeterminedtobe9,12and21,respectively.Aminimumof12airwayswasexamined/mouse.

Microtomesectionsfrom22(15COPDand7normal)lungsamplesfromacohortofCOPDpatientswereimmunostainedwithamonoclonalrabbitanti‐humanCD11c(Epitomics)orwithgoatanti‐β8(G17,SantaCruzBiotechnology,SantaCruz,CA).Theslideswerecodedandblindedpriortopathologicassessmentanddigitalimaging,andβ8immunostainingassessedaspreviouslydescribed(2).DigitalimageswereassessedforCD11cimmunostainingbasedona0‐3scale,with0beingabsentstaining;grade1,1‐3positivecells/airway;grade2,4‐10positivecells/airway,grade3,>10positivecells/airway.Correlationbetweenβ8andCD11cwasperformedusingthePearsoncorrelationtest.Thedegreeofpulmonarydisability(stages0to4,with4beingtheworst)wasdeterminedusingtheGlobalInitiativeforChronicObstructiveLungDisease(GOLD)criteria(www.goldcopd.com).Thenumberofpatientsineachstagewere;Normaln=7;Stage1‐2,n=12Stage3,n=3;Stage4,n=0.BonemarrowdendriticcellcultureandCFSElabeling:Non‐adherentgranulocyticlineageswereremovedatday2‐7ofculture,atwhichpointnon‐adherentimmatureBMDCwereremovedandsubculturedforanadditional2‐3dinDCculturemedium(RPMI1640supplementedwith5%FCS,50µM2‐


mercaptoethanol[Sigma‐Aldrich,StLouis,MO],50µg/mlgentamicin[Invitrogen,Carlsbad,CA],and20ng/mlofmurineGM‐CSF(PeproTech,RockyHill,NJ)].Attheendofthesubcultureperiod,nearly95%ofthecellswereCD11chi,CD11bhi.BMDCwereCFSE‐labeledaccordingtomanufacturer’sinstructions(Invitrogen)andusedforadoptivetransfer.Briefly,cellswereresuspendedinprewarmedPBSwith0.1%BSAat1x106cells/ml.CFSEwasaddedtoafinalconcentrationof10μMandcellswereincubatedat37°Cfor10min.Labelwasquenchedbyadditionof5volumesofice‐cold10%FCS/RPMI,cellswerewashed3xandthenresuspendedinPBSataconcentrationof2x106/500μl.Cells(2x106/500μl/mouse)wereinjectedintothelateraltail‐vein.Thechronicovalbuminmodelhasbeenpreviouslydescribed(11).Preparationofcellsuspensions:Singlecellsuspensionswerepreparedfromthelungsbymincinganddigestingwith0.28U/mlLiberaseTM,(Roche,Indianapolis,IN),0.1%hyaluronidaseand30μg/mlDNAseI(SigmaAldrich,St.Louis,MO)inRPMI1640for90mininashaking37°Cwaterbath.Digestedlungsweremechanicallydisruptedbypassingthrougha70μMnyloncellstrainer(BD‐Biosciences,Bedford,MA).LivemononuclearleuokocyteswereenrichedfromlungsuspensionsbydensitygradientcentrifugationinPercoll(GEHeathcare,Piscataway,NJ)andharvestedatthe40/60%interface.CD11c+lungDCcellswerepurifiedusingCD11cMicroBeads(MiltenyiBiotech,Auburn,CA).SinglecellsuspensionswereobtainedfromMLNsandspleensbymashingthrough70μMnyloncellstrainers(BD‐Biosciences)withoutenzymaticdigestion.RBCswerelysedusingRedBloodCellLysisSolution(Sigma).Cellstainingandflowcytometry:Forcellsurfacestaining,Fcreceptorswereblockedwith10μg/ml(2.4G2,BDBiosciences,),followedbyfluorochrome‐labeledantibodiesfromBDBiosciences(SanJose,CA)[Ly6c(AL21),CD103(M290),F4/80(BM8),GR1(RB6‐8C5),CD62L(MEL‐14),CD11b(M1/70),TCRb(H57‐597),CD19(ID3),NK1.1(PK136)]eBiosciences(SanDiego,CA)[CD103(2E7),CD40(1C10),CD80(16‐10A1),CD86(GL1),MHCII(M5/114.14.2),CD11c(N418),CD4(GK1.5),B220(RA3‐6B2)],BioLegend(SanDiego,CA)[CCR6(29‐2L17)],andInvitrogen[CD8a(5H10)].Cellswerewashedtwiceaftera15minincubationat4°Candwashedonceafter15minat4°Cwithstreptavidin‐Qdot605(Invitrogen).Forintracellularstaining,cellsweretreatedwithPMA(SigmaAldrich)andIonomycin(SigmaAldrich)for1hrat37°C,priortoaddingGolgiplug(BDBiosciences)followedby4hrsat37°C.Fcreceptorswereblockedwith10μg/ml(2.4G2,BDBiosciences),followedbyfluorochrome‐labeledantibodiestoTCRβ(H57‐59,eBiosciences),NK1.1(PK136,BDBiosciences),CD4(GK1.5,eBiosciences),CD8α(Invitrogen).Cellswerewashedafter15minincubationat4°C.CellswerethenpermeabilizedwithCytofix/Cytoperm(BDBiosciences)at4°Cfor20minutesandFcreceptorsblockedagainwith10μg/ml2.4G2,followedbystaininginPermWash(BDBiosciences)withIL‐17‐PE(BDBiosciences,TC11‐18H10)andIFN‐γ‐APC(eBiosciences,XMG1.2).CellswerewashedtwicewithPermWashandoncewithstainingbuffer.FoxP3wasstainedusingacommercialkit(BDBiosciences).ForELISpotassays,cellswerecountedandimmediatelyplatedinananti‐cytokinemAb‐


coated96‐wellmicroplate(ELISpotmouseIFN‐γandIL‐17kits;BD).Eightserial2‐or3‐folddilutionsweredoneinduplicate,percondition.SpotswerecountedautomaticallybyusinganAIDELISpotReader.FlowcytometryacquisitionwasperformedonaLSRIIFlowCytometer(BD‐Biosciences)availablethroughtheLiverCenterFlowCytometryCoreFacilityattheUniversityofCalifornia,SanFranciscoandanalysiswasperformedwithFlowjosoftware(Treestar,Ashland,OR).Humanandmouselungfibroblastcellculture:Left‐overhumanexplantlungsamplesfrompulmonarypneumonectomiesperformedatMoffit‐LongHospital,UniversityofCaliforniaSanFrancisco,forsevereemphysema(GlobalInitiativeforChronicObstructiveLungDiseaseCriteria,stagesIIIorIV)weregatheredduringthestudyperiod(2006‐2010).Controllungtissueswereobtainedfromdonorlungsnotutilizedforlungtransplantation,usuallyforreasonsofsizemismatching.Adultlungparenchymawerecollectedfromresectionsperformedforprimarylungcancerorfromnormallungsnotusedfortransplantation.InformedconsentwasobtainedfromallsurgicalparticipantsaspartofanapprovedongoingresearchprotocolbytheUniversityofCaliforniaSanFranciscoCommitteeonHumanResearchinfullaccordancewiththedeclarationofHelsinkiprinciples.AirwayfibroblastswereculturedfromthelungparenchymabytheexplanttechniqueandusedP1toP4,aspreviouslydescribed(2).Adultmouselungfibroblastswereharvestedfrommouselung,usinganidenticalexplanttechniqueasusedforhumanlungfibroblasts,andusedatP1toP4.SomefibroblastsweretreatedwithhumanrecombinantIL‐1β(1ng/ml)fromR&DSystems.Sircolassayofcelllysateswasperformedtodeterminecollagenconcentrationaccordingtothemanufacturer’sinstructions(AcurateChemicals,Westbury,NY).GenomicDNAisolation,genotyping,RNAisolation,cDNAsynthesisandqPCR:Genotypingwasperformedusingtail‐tipDNA,asdescribed(3).GenomicDNAwasisolatedusingtheWizardGenomicpurificationkit(Promega,Madison,WI).PCRtodetectrecombinationofLoxPsiteswasperformedusingprimersspanningtheExon4junctions.RNAisolation,cDNAsynthesisandSYBRgreenPCR(qPCR)wasperformed,aspreviouslydescribed(2).DetailsonprimersusedforamplificationsarelistedinSupplementalTable2.MigrationAssays:Humanadultlungfibroblasts(n=4Normalandn=4COPD)ormouseitgb8F/Ffibroblastswereplatedon6‐wellplates(2.5x105/well)onday1.Onday2humanscellsweretreatedwithorwithoutIL‐1β(1ng/ml),anti‐TGF‐β(Clone1D11,40μg/ml)anti‐β8(37E1B5,20μg/ml)orcontrolMab(W6/32,40μg/ml)for24h.MousefibroblastsweretreatedasabovewiththeexceptionthattheyweretreatedwithAd‐CreorAd‐GFP(2.5x108pfu/ml)vectorsonday2,thenantibodiesandIL‐1βonday3.Ad‐Crewasusedsinceneutralizinganti‐mouseβ8antibodiesdonotexist.Onday3human/day4mouse,themediawasremoved,cellswashedinPBSandserum‐freeDMEMmediaadded.After24hthemediawascollected,pooled,sterilefiltered(0.2μm)and750μlofthepooledsamplewasadded


tothebottomchamberofatranswellplate,5.0μmporesize(Costar).BMDCsorlivehumandendriticcells[derivedfromCD34+cordbloodcellsobtainedfromMatTrekCorporation(Lot#1118DC)(Ashland,MA)]wereadded(1.5x105cells)totheupperchamberin200μlofRPMI1640andincubatedat37°CinahumidifiedCO2incubator.After4h,themediainthebottomchamberwascollected,spundownandthecellscountedusingahemocytometer.Supplementalreferences:1. Razai,A.,Garcia‐Rodriguez,C.,Lou,J.,Geren,I.N.,Forsyth,C.M.,Robles,Y.,

Tsai,R.,Smith,T.J.,Smith,L.A.,Siegel,R.W.,etal.2005.MolecularevolutionofantibodyaffinityforsensitivedetectionofbotulinumneurotoxintypeA.JMolBiol351:158‐169.

2. Araya,J.,Cambier,S.,Markovics,J.A.,Wolters,P.,Jablons,D.,Hill,A.,Finkbeiner,W.,Jones,K.,Broaddus,V.C.,Sheppard,D.,etal.2007.Squamousmetaplasiaamplifiespathologicepithelial‐mesenchymalinteractionsinCOPDpatients.JClinInvest117:3551‐3562.

3. Mu,D.,Cambier,S.,Fjellbirkeland,L.,Baron,J.L.,Munger,J.S.,Kawakatsu,H.,Sheppard,D.,Broaddus,V.C.,andNishimura,S.L.2002.Theintegrinαvβ8mediatesepithelialhomeostasisthroughMT1‐MMP‐dependentactivationofTGF‐β1.JCellBiol157:493‐507.

4. Amersdorfer,P.,Wong,C.,Chen,S.,Smith,T.,Deshpande,S.,Sheridan,R.,Finnern,R.,andMarks,J.D.1997.MolecularcharacterizationofmurinehumoralimmuneresponsetobotulinumneurotoxintypeAbindingdomainasassessedbyusingphageantibodylibraries.InfectImmun65:3743‐3752.

5. Nishimura,S.L.,Sheppard,D.,andPytela,R.1994.Integrinalphavbeta8.Interactionwithvitronectinandfunctionaldivergenceofthebeta8cytoplasmicdomain.JBiolChem269:28708‐28715.

6. Kehoe,J.W.,Velappan,N.,Walbolt,M.,Rasmussen,J.,King,D.,Lou,J.,Knopp,K.,Pavlik,P.,Marks,J.D.,Bertozzi,C.R.,etal.2006.Usingphagedisplaytoselectantibodiesrecognizingpost‐translationalmodificationsindependentlyofsequencecontext.MolCellProteomics5:2350‐2363.

7. Kolb,M.,Margetts,P.J.,Anthony,D.C.,Pitossi,F.,andGauldie,J.2001.TransientexpressionofIL‐1betainducesacutelunginjuryandchronicrepairleadingtopulmonaryfibrosis.JClinInvest107:1529‐1536.

8. Ganter,M.T.,Roux,J.,Miyazawa,B.,Howard,M.,Frank,J.A.,Su,G.,Sheppard,D.,Violette,S.M.,Weinreb,P.H.,Horan,G.S.,etal.2008.Interleukin‐1betacausesacutelunginjuryviaalphavbeta5andalphavbeta6integrin‐dependentmechanisms.CircRes102:804‐812.

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10. Hogg,J.C.,Chu,F.,Utokaparch,S.,Woods,R.,Elliott,W.M.,Buzatu,L.,Cherniack,R.M.,Rogers,R.M.,Sciurba,F.C.,Coxson,H.O.,etal.2004.Thenatureofsmall‐airwayobstructioninchronicobstructivepulmonarydisease.NEnglJMed350:2645‐2653.


11. Yu,M.,Tsai,M.,Tam,S.Y.,Jones,C.,Zehnder,J.,andGalli,S.J.2006.Mastcellscanpromotethedevelopmentofmultiplefeaturesofchronicasthmainmice.JClinInvest116:1633‐1641.

itgb8 expression mediated by adil1β is decreased by adcre. … · 2014-01-30 · ii in combination...

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