Short telomere length in IPF lung associates with fibrotic lesions and predicts survival

15 Pages·2017·7.34 MB·English

RESEARCHARTICLE
Short telomere length in IPF lung associates
with fibrotic lesions and predicts survival
ReinierSnetselaar1☯,AernoudA.vanBatenburg1☯,MatthijsF.M.vanOosterhout2,Karin
M.Kazemier1,3,SuzanM.Roothaan4,TonPeeters4,JoanneJ.vanderVis1,5,
RoelGoldschmeding4,JanC.Grutters1,3,ColineH.M.vanMoorsel1,3*
1 DepartmentofPulmonology,StAntoniusILDCenterofExcellence,StAntoniusHospital,Nieuwegein,the
Netherlands,2 DepartmentofPathology,StAntoniusILDCenterofExcellence,StAntoniusHospital,
Nieuwegein,theNetherlands,3 DivisionofHeartandLungs,UniversityMedicalCenterUtrecht,Utrecht,the
Netherlands,4 DepartmentofPathology,UniversityMedicalCenterUtrecht,Utrecht,theNetherlands,
5 DepartmentofClinicalChemistry,StAntoniusILDCenterofExcellence,StAntoniusHospital,Nieuwegein,
theNetherlands
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☯Theseauthorscontributedequallytothiswork.
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*[email protected]
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Abstract
TelomeremaintenancedysfunctionhasbeenimplicatedinthepathogenesisofIdiopathic
PulmonaryFibrosis(IPF).However,themechanismofhowtelomerelengthisrelatedto
OPENACCESS
fibrosisinthelungsisunknown.SurgicallungbiopsiesofIPFpatientstypicallyshowahet-
Citation:SnetselaarR,vanBatenburgAA,van
erogeneouspatternofnon-fibroticandfibroticareas.Therefore,telomerelength(TL)inboth
OosterhoutMFM,KazemierKM,RoothaanSM,
PeetersT,etal.(2017)ShorttelomerelengthinIPF lungareasofpatientswithIPFandfamilialinterstitialpneumoniawascompared,specifically
lungassociateswithfibroticlesionsandpredicts inalveolartype2(AT2)cells.
survival.PLoSONE12(12):e0189467.https://doi.
FluorescentinsituhybridizationwasusedtodetermineTLinnon-fibroticandfibrotic
org/10.1371/journal.pone.0189467
areasof35subjects.Monochromemultiplexquantitativepolymerasechainreaction
Editor:GernotZissel,Universitatsklinikum
(MMqPCR)wasusedfor51wholelungbiopsiesandbloodTLmeasurements.
Freiburg,GERMANY
ForsporadicIPFsubjects,AT2cellTLinnon-fibroticareaswas56%longerthanin
Received:January13,2017
fibroticareas.Nosuchdifferencewasobservedinthesurroundinglungcells.Insubjects
Accepted:November28,2017 carryingatelomerasereversetranscriptase(TERT)mutation,AT2cellTLwassignificantly
Published:December27,2017 shorterthaninsporadicsubjects.However,nodifferenceinsurroundingcellTLwas
observedbetweenthesesubjectgroups.Finally,usingbiopsyMMqPCRTLmeasurements,
Copyright:©2017Snetselaaretal.Thisisanopen
accessarticledistributedunderthetermsofthe itwasdeterminedthatIPFsubjectswithshortestlungTLhadasignificantlyworsesurvival
CreativeCommonsAttributionLicense,which thanpatientswithlongTL.
permitsunrestricteduse,distribution,and
ThisstudyshowsthatshorteningoftelomerescriticallyaffectsAT2cellsinfibroticareas,
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. implyingTLasacauseoffibrogenesis.Furthermore,shortlungtelomerelengthisassoci-
atedwithdecreasedsurvival.
DataAvailabilityStatement:Datarestricted
becausetheycontainpotentiallyidentifyingor
sensitivepatientinformation.Restrictionsare
imposedbytheMedicalResearchEthics
CommitteesUnited(MEC-U).Allanonymizeddata
areavailablefrom:ILDCenterofExcellence Introduction
Biobank,M.Struik,MSc,Koekoekslaan1,3435
Idiopathicpulmonaryfibrosis(IPF)isararelungdiseasecharacterizedbyprogressivefibrosis
CM,Nieuwegein,TheNetherlands,m.
oflungparenchyma[1].Patientswiththediseasehaveamedianpost-diagnosticsurvivalof
[email protected]
wasuploadedinaseparateexcelfile(S1Dataset). 2–5years[2].IPFcanbebothasporadicandafamilialdisease.Thefamilialformcanbe
PLOSONE|https://doi.org/10.1371/journal.pone.0189467 December27,2017 1/15
TelomerelengthinIPFlung
Funding:ThisresearchwasenabledbyZonMW- causedbymutationsinsurfactantrelatedgenes,orgenesthatinfluencetelomeremaintenance
TopZorgStAntoniusScienceCornergrant(grant [3–10].AnalysisoffamilialIPFpatientswithmutationsintelomerasereversetranscriptase
number842002003;www.zonmw.nl;JCGCHMvM
(TERT)ortelomeraseRNAcomponent(TERC)showedadiminishedtelomeraseactivityand
JJvdV)and’Pendersfonds’ofthelungfibrosis
prematurelyshortenedtelomerelength(TL)inbloodleukocytes.Similarresultswerefoundin
patientassociation(CHMvM).
sporadicpatientsnotcarryingtelomerasemutations,whencomparedtohealthycontrols[11–
Competinginterests:Theauthorshavedeclared
13].ItwasalsoshownthatTLofthelungalveolartype2(AT2)cellsofIPFpatientswasshorter
thatnocompetinginterestsexist.
comparedtocontrols[11].Together,thesefindingsindicatethattelomererelatedpathology
playsaroleinbothfamilialandsporadicIPF.However,itremainsunknownwhetherthe
shortTLinAT2cellsisrelatedtofibrosis.
AcontemporaryviewonthepathogenesisofIPFfocusesontheroleofAT2cellduringdis-
easedevelopment[9,14–16].Evidenceforthiscanbefoundinpatientsdiagnosedwithasur-
factant-relatedfamilialinterstitialpneumonia(FIP).SinceAT2cellsaretheexclusive
producersofsurfactantprotein-C,thesecellsareconsideredtobetheprecursorcellsleading
topulmonaryfibrosis[17].Conversely,alinkbetweenmutationsintelomeraserelatedgenes
andtheAT2cellisnotclear.Inhealthylungtissue,theAT2cellprovidestheregenerative
capacityofthelungalveoli[18].Faultytelomeremaintenancecouldunderlieanimpairedpro-
liferativecapacityoftheAT2cells[19].Recentlyithasbeendemonstratedthatmicewithtelo-
mererepeatbindingfactor1(TRF1)-deletedAT2cellsdeveloplungfibrosisandshowedshort
telomeresinAT2cells[20,21].ThismightexplainthehumanAT2cellTLshorteninginIPF,
whichcouldresultinasimilarresponsecharacterizedbyprogressivefibrosis[22].Iftelomere
shorteningplaysaroleinIPFdiseasedevelopment,itwouldbeexpectedtooccurprimarilyin
AT2cells.
IPFlungsshowapatchydistributionofaffectedfibroticandrelativelypreserved,non-
fibrotictissue[23,24].ThisheterogeneousdistributionallowsforacomparisonofTLbetween
non-fibroticandfibrotictissueinasinglesurgicalbiopsy.Inthisstudyweinvestigatedhow
thedistributionoftelomereshorteninginlungtissuebiopsiesofpatientsisrelatedtofibrotic
remodelingofthetissue.WeshowthatinsporadicIPF,AT2TLwassignificantlylongerin
non-fibroticareasthaninfibroticregions,therebyimplicatingtelomereshorteningasacause
offibroticremodelingoflungtissueinIPF.Inaddition,familialpatientswithaTERTmuta-
tionshowsignificantshortertelomeresthaninsporadicIPF.Furthermore,shortwholebiopsy
telomerelengthinsporadicIPFpatientsisassociatedwithworsesurvival.
Materialandmethods
Humansubjects
Inthisstudy,63patientsdiagnosedwithIPFattheSt.AntoniusILDCenterofExcellence
Nieuwegeinwereincludedretrospectively(Table1).Inthesepatients,TLwasmeasuredin
Table1. Patientgroupcharacteristics.
IPF FIP-TERT FIP-nonTERT
Totaln(%male) 39(90%) 10(80%) 14(57%)
Mean(SD)
Ageatdiagnosisinyears 61(10) 64(7) 54(12)
DLCO%predicted 47(18) 47(10) 43(15)
FVC%predicted 69(22) 85(8) 63(22)
IPF:idiopathicpulmonaryfibrosis,FIP:familialinterstitialpneumonia,SD:standarddeviation,FVC:forced
vitalcapacity,DLCO:diffusingcapacityofthelungsforcarbonmonoxide
https://doi.org/10.1371/journal.pone.0189467.t001
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TelomerelengthinIPFlung
AT2cells,wholelungbiopsiesandwhitebloodcells.Patientswereclassifiedaseithersporadic
IPF(n=39)orfamilialinterstitialpneumonia(FIP)(n=24).Diagnoseswerebasedonthe
ATS/ERS/JRS/ALATguidelinesaftermultidisciplinarydiscussion[1,25].Thediseasewasdes-
ignatedasfamilialiftwoormorefirst-degreefamilymemberssufferedfromidiopathicinter-
stitialpneumonia.FIPpatientswerescreenedonmutationsinTERT,TERC,surfactantprotein
C(SFTPC),surfactantproteinA2(SFTPA2)exon6andTRF1-InteractingNuclearFactor
2(TINF2)exon6.Basedontheseresults,theFIPgroupwassubdividedintwosubgroups:
patientsthatcarriedamutationinTERT:FIP-TERT(n=10,S1Table)andpatientsthatdid
notcarryaknownmutationintelomererelatedgenes:FIP-nonTERT(n=14).Thelattersub-
groupincluded3patientswithaSFTPCorSFTPA2mutation.Oftheremaining11patients,no
knownpathogenicmutationswerefound.Toassesslungfunctionparameters,diffusingcapac-
ityofthelungsforcarbonmonoxide(DLCO)andforcedvitalcapacity(FVC)datawerecol-
lectedwithina3-monthwindowbeforeorafterdiagnosis(n=39).Tocrossreferenceresults,
acontrolgroupwasformedusingnormallungtissueobtainedduringpost-mortemexamina-
tionoffivesubjectsnotsufferingfromlungrelatedpathology.Patientcharacteristicswere
retrievedfrommedicalreports.ThestudywasapprovedbytheMedicalresearchEthics
CommitteesUnited(MEC-U)oftheStAntoniusHospital(approvalnumberW14.056and
R05.08A).Allpatientdatawereanonymized.
Lungtissue
Residuallungtissuewasobtainedfrombiopsiescarriedoutfordiagnosticpurposesandwas
fixedinformalinandembeddedinparaffin(FFPE).Serialsectionsof4μmwerecut.Non-
fibroticandfibroticareaswereidentifiedonhematoxylin&eosin(H&E)stainedsections(Fig
1Aand1B).Allidentificationsweredonebypathologists(MvOandSR),whoarehighlyexpe-
riencedinthefieldofinterstitiallungdiseases.
Fluorescentinsituhybridization
Afteridentificationoffibroticandnon-fibroticareas,thesequentialsectionofthebiopsywas
usedforafluorescenceinsituhybridization(FISH).Tissueslidesweredeparaffinizedusinga
xyleneseries.Next,theywereplacedinH O blockbuffer(1.5%),washedinPhosphate-buff-
2 2
eredsaline(PBS)andtreatedwithBorax(1mg/mL).Forantigenretrieval,specimenswere
boiledinacitratesolutionfor20min(2.94g/L,pH6).Telomereswerelabeledwithatelo-
mere-Cy3PNAprobe(Panagene,Daejeon,South-Korea)andpro-SPC(AB3786,1/500,
MerckMillipore,Darmstadt,Germany)wasfluorescentlylabeledtoidentifyAT2cells(sec-
ondaryantibody;A-11008,1/300,ThermoFisherScientific,Waltham,MA,USA)(Fig1Cand
1D).Pro-SPCnegativesurroundingcellswereusedasreference.Surroundingcellswere
locatedwithin2cellsofAT2cells.Finally,DNAofsampleswasstainedwith4’,6-diamidino-
2-phenylindole(DAPI,25μg/mL)andfinishedwithVectashieldantifademountingmedium
(Vectorlaboratories,Burlingame,CA,USA).Slideswerestoredat4˚Cuntilanalysis.
Imagingandsignalquantification
FISH-TLwasmeasuredusingamethodadoptedfromMeekeretal.[11,26,27].Imageswere
capturedusingaFluorescencemicroscope(LeicaDM5500B)athighmagnification(100x).
Perbiopsy,upto15imagesweremadeperarea.Z-stackingof9focalplaneswith0.5μminter-
valswasusedtomaximizethecoverageofcellnuclei.Totaltelomere(cy3)fluorescentsignal
wasquantifiedpernucleususingtheTelometerimageanalysisplugin(availableathttp://
demarzolab.pathology.jhmi.edu/telometer/index.html)ofImageJ(http://rsb.info.nih.gov/ij/).
Toaccountforsub-optimalcapturingofthenucleicausedbythecuttingplanes,totaltelomere
PLOSONE|https://doi.org/10.1371/journal.pone.0189467 December27,2017 3/15
TelomerelengthinIPFlung
Fig1.Imagesshowingrepresentativeregionsfornon-fibroticandfibroticareaswithinonelung
biopsy.(A)LowmagnificationimageofH&EstainedIPFlungtissueshowingnon-fibrotic(NF)andfibrotic(F)
areas,andcharacteristichoneycombing(HC).(B)EnlargedboxedareaofimageArepresentingafibrotic
areainIPFlungbiopsy.Characteristicfibroblastfocus(FF)andalveolartype2(AT2)cellhyperplasia(black
arrows)arehighlighted.(C,D)CombinedfluorescentimagesofAT2andsurroundingcellsinnon-fibrotic
areas(C)andfibroticareas(D)inIPFlungtissue.DNAinnucleusisdisplayedinblue(DAPI),pro-SPCin
greenandtelomeresinred.NotethelowersignalofAT2cell(whitearrows)telomeresignal(reddots)in
fibroticareas(D)vsnon-fibrotic(C)tissue,reflectingshortertelomeresinAT2cellsinfibroticareas.Examples
ofpro-SPCnegativesurroundingcellsaremarkedwithanasterisk(*).
https://doi.org/10.1371/journal.pone.0189467.g001
signalwasdividedbythetotalDNA(DAPI)signal.Allimagesweretakenatfixedtimepoints
between1to3daysafterstainingtocircumventdatavariabilitybyDAPIfluorescencefading.
MMqPCRfortelomerelengthinFFPEtissue
DNAwasisolatedfromFFPEtissuesectionsusinganAllPrepDNA/RNAFFPEKit(Qiagen,
Hilden,Germany)accordingtomanufacturerinstructions.Slideswerecutfromsequential
sectionsusedforFISH.Theparaffinwasremovedusingparaffindissolver(Macherey-Nagel,
Du¨ren,Germany).DNAwasquantifiedusingaNanodrop(ThermoFisherScientific,Wal-
tham,MA,USA)usinganabsorbanceratioof260and280nm.Sampleswithinaratioof1.8–
2.0wereincluded.TomeasurewholelungbiopsyandwhitebloodTL,monochromemultiplex
qPCR(MMqPCR)wasperformedasdescribedearlier[13,28].Becauseamplificationof
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TelomerelengthinIPFlung
telomereandβ-globininFFPEDNAisdelayedcomparedtobloodderivedDNAweadjusted
cyclecountsforallFFPEsampleswith-5and-7respectively.TherelativeTLforeachsample
wasestimatedfromtheratiotelomererepeatcopynumber(T)toasinglehumanβ-globin
genecopynumber(S)(T/Sratio),usingstandardcurvesfromaserialdilutionofagenomic
DNA-pool[28].QuadruplicatereactionswereperformedonaMyiQ™Single-ColorReal-Time
PCRDetectionSystem(Bio-Rad,Hercules,CA,USA)usingiQSYBRGreenSupermix(Bio-
Rad,Hercules,CA,USA).MMqPCRisproventobeasensitivemethodtodiscriminate
betweenpatientswithhighandlowTL[13].
Statistics
Ratioswerecalculatedfornon-fibrotic/fibroticandAT2cell/surroundingcellcomparisons.
Valuesbelow1indicateshorterFISH-TLinnon-fibroticareasandAT2cellrespectively.
Allanalyseswereperformedusingnon-parametricstatisticaltests.Mann-Whitneyand
WilcoxonsignedrankedtestswereusedtocompareTL.P-valuesfortwo-sidedt-testsare
shown.CorrelationsweredeterminedusingSpearman’srankcoefficienttest.Survivalanalysis
wasdoneusingKaplan-Meierestimation.ForstatisticalanalysisIBMSPSSStatistics22.(IBM
Corp.,Armonk,NY,USA)andGraphPadPrism5and6(GraphPadSoftware,SanDiego,CA,
USA)wereused.
Results
DAPIisavalidmeasuretocorrectfortotalDNApercell
FortheFISH-TLmeasurements,DAPIwasusedtoaccountforthetotalamountofDNAper
cell.ToverifywhetherDAPIstainingwasvalidmeasure,wecomparedDAPIwithacentro-
mereFISH[29].Similarresultswerefoundbetweenbothassays(n=4,datanotshown).
ThereforeweconcludethatusingDAPIasacounterstainisavalidmethod,aswasfoundby
MeekerandcoworkersandKropskiandcoworkers[27,30].
Telomeresinnon-fibroticareasofsporadicIPFsubjectsarelongerthan
infibroticareas
InordertoinvestigatewhetherAT2telomereshorteningisrelatedtofibrosis,weperformeda
FISHstainingonFFPEmaterialinagroupof16sporadicIPFsubjects.MedianAT2cellTL
wassignificantlylonger(p<0.001)innon-fibroticareascomparedtofibroticareas(Fig2),
resultingin2.24timesdifference(FISH-TLratioinTable2).TogetanideaofthegeneralTL
innon-fibroticandfibroticareas,wemeasuredFISH-TLinpro-SPCnegativesurrounding
cells.Here,nosignificantdifference(p=0.30,datanotshown)wasfoundbetweennon-
fibroticandfibroticareas(FISH-TLratio:1.15,Table2).
Fig2showsthatFISH-TLisvariablebetweensubjects.ToassesshowFISH-TLdiversity
withinsubjectsisdistributed,thecorrelationbetweennon-fibroticandfibroticFISH-TLwas
analyzed(datanotshown).ThisresultedinasignificantcorrelationforbothAT2(r=0.855,
p=2(cid:1)10−5)andsurroundingcells(r=0.689,p=0.003),indicatingthatFISH-TLvariability
amongsubjectsishigh,butcorrelatespositivelybetweennon-fibroticandfibroticareaswithin
asubject.
Non-fibroticandfibroticAT2celltelomerelengthinsporadicIPFsubjects
isshorterthaninsurroundingcells
Next,toelucidatefurtherontelomereshorteninginAT2cellsspecifically,wecomparedtelo-
merelengthofAT2cellswithsurroundingcells.Innon-fibroticareas,thetelomeresinAT2
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TelomerelengthinIPFlung
Fig2.TelomerelengthinsporadicIPFlungs.(A)Representativeexampleoftelomerelengthperalveolartype2(AT2)cellforonesporadicIPFsubject.
Eachdotrepresentsonecell.Non-fibroticAT2FISH-TLissignificantlyhighercomparedtoFISH-TLinfibroticareas(p<0.0001).Barsrepresentmediansand
p-valueiscalculatedusingaMann-Whitneytest.(B,C,D)MediansurroundingandAT2cellFISH-TLinnon-fibroticandfibroticareasfor16sporadicIPF
subjects.Eachlineconnects,persubject,theFISH-TLof(B)AT2cellsinthenon-fibroticandfibroticareaormedianFISH-TLdifferencesbetweensurrounding
andAT2cellsin(C)non-fibroticareasor(D)fibroticareas.AT2FISH-TLdifferencesbetweennon-fibroticandfibroticareasandbetweensurroundingandAT2
cellsweresignificant(2-tailedp<0.0006andp<0.0001respectively),whichwereindicatedbyasterisks(***=p<0.001,****=p<0.0001).
https://doi.org/10.1371/journal.pone.0189467.g002
cellswere4timesshorterthaninsurroundingcells(p<0.0001,Fig2CandFISH-TLratio:
0.26,Table3).Thedifferencewasevenlargerinfibroticareas:telomeresinAT2cellswere8
timesshorterthaninsurroundingcells(p<0.0001,Fig2DandFISH-TLratio:0.13,Table3).
Toplacethisinperspective,wedeterminedtheFISH-TLratiobetweenAT2cellsandsur-
roundingcellsincontrolsubjects(n=5).Incontrolsnosignificantdifferencewasfound
betweenAT2andsurroundingcells,indicatingthatundernon-pathologicalconditions,AT2
cellsdonothaveshortenedtelomeres(FISH-TLratio:0.90,Table3).
LungtelomerelengthinFamilialInterstitialPneumonias:TERT
ToinvestigateTLdifferencesbetweensporadicIPFsubjectsandsubjectswithanestablishedtelo-
meresyndrome,FISH-TLwasdeterminedinTERTmutationcarriers(FIP-TERT).FIP-TERT
Table2. Mediantelomerelengthfornon-fibroticversusfibroticareaspercohort.
AT2FISH-TL SurroundingcellFISH-TL
Subgroup n Non-Fibrotic(nf) Fibrotic(f) Ratio(nf/f) Non-Fibrotic(nf) Fibrotic(f) Ratio(nf/f)
Controls 5 23.47 N/A N/A 26.01 N/A N/A
SporIPF 16 3.22 1.44* 2.24 12.30 10.73 1.15
FIP-nonTERT 10 3.74 2.15* 1.74 12.60 7.46 1.68
FIP-TERT 9 1.00# 1.00 1.00 9.21 10.20 0.90
FISH:fluorescenceinsituhybridization,TL:telomerelength,AT2:alveolartype2cell,IPF:idiopathicpulmonaryfibrosis,FIP:familialinterstitialpneumonia.
Numbersindicatemediantelomeresignal,ofwhichratioswerecalculated.Ratio(nf/f)=Non-fibrotic/Fibrotic,i.e.ifratio=2telomeresinnon-fibroticareas
aretwotimeslongerthaninfibroticareas.
*=Innon-fibroticareas,AT2FISH-TLissignificantlylongerthaninfibroticregions(sporIPF:p=0.0006,FIP-nonTERT:p=0.02).
#=AT2FISH-TLinFIP-TERTissignificantlyshorterthaninsporadicIPF(p=0.02).
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TelomerelengthinIPFlung
Table3. Mediantelomerelengthforalveolartype2(AT2)cellsversussurroundingcellspercohort.
Non-fibroticareaFISH-TL FibroticareaFISH-TL
Subgroup n AT2 Surr. Ratio(AT2/Surr.) AT2 Surr. Ratio(AT2/Surr.)
Controls 5 23.47 26.01 0.90 N/A N/A N/A
IPF 16 3.22 12.30* 0.26 1.44 10.73* 0.13
FIP-nonTERT 10 3.74 12.60* 0.29 2.15 7.46* 0.29
FIP-TERT 9 1.00# 9.21* 0.11 1.00 10.20* 0.10
FISH:fluorescenceinsituhybridization,TL:telomerelength,AT2:alveolartype2cell,IPF:idiopathicpulmonaryfibrosis,FIP:familialinterstitialpneumonia,
surr.:pro-SPCnegativesurroundingcells.Numbersindicatemediantelomeresignal,ofwhichratioswerecalculated.Ratio(AT2/Surr.)=AT2cell/
surroundingcell,i.e.ifratio=2telomeresinAT2cellsaretwotimeslongerthaninsurroundingcells.
#=AT2FISH-TLinFIP-TERTissignificantlyshorterthaninsporadicIPF(p=0.02).
*=Inbothnon-fibroticandfibroticareas,AT2FISH-TLissignificantlyshorterthaninsurroundingcells(sporIPF:p<0.0001,FIP-nonTERTandFIP-TERT:
p<0.01).
https://doi.org/10.1371/journal.pone.0189467.t003
showednodifferenceinAT2FISH-TLbetweennon-fibroticandfibroticareas(p=0.36,Fig3A).
However,AT2FISH-TLwassubstantiallyshorterinnon-fibroticareascomparedtosporadicIPF
(median1.00vs3.22,p=0.02,Table2).InsurroundingcellsFISH-TLwasconcordantbetween
FIP-TERTandsporadicIPFinbothareas.FISH-TLinAT2cellswassignificantlyshorterthanin
surroundingcellsinbothnon-fibroticandfibroticareas(p<0.01,Fig3Band3C).Thesedata
showthatAT2TLdistributionbetweennon-fibroticandfibrotictissueinFIP-TERTdiffersfrom
sporadicIPFsubjects,underliningtheeffectofadefectivetelomeraseenzyme.
LungtelomerelengthinFamilialInterstitialPneumonias:NonTERT
Next,weanalyzedthefamilialsubjects,whodidnotcarryaknowntelomererelatedmutation
(FIP-nonTERT).Forthesesubjects,FISH-TLpatternswerethesameasinthesporadicIPF
group;non-fibroticAT2cellFISH-TLwas1.74timeslongerthanfibroticAT2cells(p=0.02,
Fig3D,Table2).Furthermore,inbothnon-fibroticandfibroticareastheAT2cellFISH-TL
was3.5timesshorterthansurroundingcells(p<0.01,Fig3Eand3F,FISH-TLratio:0.29,
Table3).ComparedtoFIP-TERT,FIP-nonTERTsubjectshadsimilarAT2FISH-TLinnon-
fibroticareas(p=0.081).
Inordertogetanoverview,wecomparedthesporadicIPF,FIP-TERTandFIP-nonTERT
subjectgroups.AlldataissummarizedinTables2and3.Nostatisticalagedifferenceswere
foundbetweengroups.Lookingspecificallyatfibroticareas,AT2FISH-TLwasnotsignificantly
different(p=0.16)betweensubjectgroups.Innon-fibroticareasasignificantdifferenceinAT2
cellFISH-TLwasobservedbetweenFIP-TERTandsporadicIPFsubjects(p=0.02)andatrend
betweenFIP-TERTandFIP-nonTERTsubjects(p=0.081).Thesedataindicatethatshortest
AT2telomeresinnon-fibroticareaswerefoundinFIP-TERTsubjects.Inaddition,nosignifi-
cantdifferenceinsurroundingcellsFISH-TLwasfoundbetweenthethreesubgroupsineither
area(p=0.67),suggestingthatinallsubjectgroupsTLshorteningismostevidentinAT2cells.
TelomerelengthbyMMqPCR:Lung
TotestwhetherwholelungbiopsyTLasmeasuredbyMMqPCR(biopsyT/S)correlateswith
FISH-TLofAT2cells,weextractedDNAfrombiopsysectionsandperformedMMqPCRas
describedbyCawthonetal.(IPFn=15,FIP-TERTn=9,FIP-nonTERTn=10)[28].Inspo-
radicIPFsubjects,asignificantpositivecorrelationwasfoundbetweenbiopsyT/SandAT2
FISH-TLinbothnon-fibrotic(r2=0.53,p=0.002)andfibrotic(r2=0.73,p<0.0001)areas
(Fig4).NocorrelationswerefoundintheFIP-TERTandFIP-nonTERT(datanotshown).
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TelomerelengthinIPFlung
Fig3.FISHtelomerelengthinlungsofFIP-TERTandFIP-nonTERTsubjects.(A)MedianFISH-TLinalveolar
type2(AT2)cellsinnon-fibroticandfibroticareasof9FIP-TERTsubjects.Telomeresaregenerallyshortandno
significantdifferencebetweenareaswasobserved(2-tailed,p=0.36).(B,C)MedianFISH-TLofsamesubjectsas
infigureA,showingdifferencesbetweensurroundingandAT2cellsin(B)non-fibroticand(C)fibroticareas.(D)
MedianFISH-TLinalveolartype2(AT2)cellsinnon-fibroticandfibroticareasof10FIP-nonTERTsubjects.Non-
fibroticAT2FISH-TLissignificantlyhighercomparedtoFISH-TLinfibroticareas(2-tailed,p=0.02).(E,F)Median
FISH-TLofsamesubjectsasinfigureD,showingdifferencesbetweensurroundingandAT2cellsin(E)non-fibrotic
and(F)fibroticareas.AsterisksindicatesignificantdifferencescalculatedbyWilcoxonmatched-pairssignedrank
analysis(*=p<0.05,**=p<0.01).
https://doi.org/10.1371/journal.pone.0189467.g003
TelomerelengthbyMMqPCR:Blood
WetestedforacorrelationbetweenperipheralwhitebloodcellTLmeasuredwithMMqPCR
(bloodT/S)andFISH-TLoflungtissue.WefoundnosignificantcorrelationbetweenAT2
FISH-TLinfibroticareasandbloodT/S,exceptinFIP-TERTsubjects.(r2=0.67,p=0.007)
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TelomerelengthinIPFlung
Fig4.CorrelationbetweenFISH-TLandbiopsyT/SinIPFsubjects.Correlation(Spearman)between
FISH-TLmeasuredinalveolartype2(AT2)cellsandbiopsyT/SmeasuredbyMMqPCR(n=15)innon-
fibroticareas(blackdots)andfibroticareas(opensquares).Significantcorrelationswereestablishedbetween
biopsyT/SandAT2cellFISH-TLinnon-fibrotic(r2=0.53,p=0.002)andfibrotic(r2=0.73,p<0.0001)areas.
https://doi.org/10.1371/journal.pone.0189467.g004
(Fig5).AlsonosignificantcorrelationwasfoundbetweenMMqPCRmeasurementsbiopsyT/
SandbloodT/S(sporadicIPF;n=26,FIP-nonTERT;n=12andFIP-TERT;n=10,Fig6).
Shorttelomeresareassociatedwithworsesurvival
Intheliterature,shortperipheralleukocytetelomerelengthhasbeenassociatedwithworsesur-
vivaltimeinIPF[31,32].Hereweinvestigatedwhetherashortersurvivaltimeissimilarlyasso-
ciatedwithFISH-TL.Weshowedthatinnon-fibroticregionsAT2FISH-TLvariabilitybetween
IPFsubjectswassubstantial(Figs2and3).Totestwhetherthisvariabilityisassociatedwithsur-
vivalwedividedthisgroup(n=15)atthemedianAT2cellTL.Survivalwascalculatedfrom
dateofbiopsyuntildeath(n=9)orcensoringofthepatient(lungtransplantationn=3,still
aliven=3).KaplanMeiersurvivalanalysisshowedthatpatientswithshortestAT2cellTLhada
lowermediansurvivalratethanpatientswithlongestAT2cellTL(26monthsvs60months,
p=0.353,Fig7A).Becauselackofsignificancecouldbecausedbyunderpoweredanalysisand
becauseasignificantpositivecorrelationbetweenbiopsyT/SandAT2FISH-TLwasestablished
above,wealsoperformedasurvivalanalysisusingbiopsyT/S(n=34).Dividingthepatient
groupatthemedianT/S,asignificantdifferenceinsurvivalrate(p=0.003)wasfound.Patients
withalowT/Shaddecreasedmediansurvivalof22monthsandlived41monthsshorterthan
patientswithhighT/S(Fig7B).Therewerenosignificantdifferencesinmeanageatdateof
biopsybetweenthegroupwithTLabovemedianandthegroupwithTLbelowmedianineither
AT2FISH-TLandbiopsyT/Sanalyses.
Discussion
Inthisstudy,wefoundthattelomereshorteningispredominantlyobservedinAT2cellsand
associateswithfibroticlesionsinIPFlungbiopsies(Figs2and3).Furthermore,patientswith
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TelomerelengthinIPFlung
Fig5.CorrelationbetweenFISH-TLandMMqPCRleukocyteTL(bloodT/S)inFIP-TERTfibroticareas.
PositiveFIP-TERTcorrelation(Spearman)betweenalveolartype2(AT2)cellFISH-TLinfibroticareasand
MMqPCRbloodT/S(n=9,r2=0.67,p=0.007).
https://doi.org/10.1371/journal.pone.0189467.g005
shortlungtelomereshadsignificantlyworsesurvivalthanpatientswithlongertelomeres
(Fig7).
TelomereshorteninginAT2cellsisinaccordancewithtwoexperimentalobservations
wheremicewithtelomererepeatbindingfactor1(TRF1)-deletedAT2cellsdeveloplungfibro-
sisandpresentshorttelomeresinAT2cells[20,21].
Todate,nostudyinvestigatedtheassociationbetweenAT2TLandthecharacteristicnon-
fibroticandfibroticregionsinhumanIPFlungswith(FIP-TERT)orwithoutamutationin
Fig6.CorrelationsbetweenMMqPCRmeasurementsbiopsyT/SandbloodT/SinIPF.Nocorrelation
betweenbiopsyT/SandbloodT/SwasobservedinsporadicIPF(n=26),FIP-nonTERT(n=12)and
FIP-TERT(n=10)subjects.
https://doi.org/10.1371/journal.pone.0189467.g006
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