Exhaled nitric oxide in interstitial lung diseases

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ContentslistsavailableatScienceDirect

Respiratory

Physiology

&

Neurobiology

j o u r n a l ho me p ag e :w w w . e l s e v i e r . c o m / l o c a t e / r e s p h y s i o l

Exhaled

nitric

oxide

in

interstitial

lung

diseases

P. Cameli

∗

,

E. Bargagli,

R.M.

Reﬁni,

M.G.

Pieroni,

D. Bennett,

P. Rottoli

DepartmentofMedicalandSurgicalSciencesandNeurosciences,RespiratoryDiseaseandLungTransplantationSection,LeScotteHospital,Siena,Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Accepted21March2014 Availableonline2April2014 Keywords:

Nitricoxide

Idiopathicpulmonaryﬁbrosis Non-speciﬁcinterstitialpneumonia biomarker

Oxidativestress Nitrosativestress

a

b

s

t

r

a

c

t

Nitricoxide(NO)isabiomarkerofnitrosativestress,whichisinvolvedinthepathogenesisofidiopathic interstitialpneumonias(IIP).ThisstudyevaluatesexhaledNOlevelsinIIPpatientsandrelates alveo-larconcentrationsofNO(CalvNO)topulmonaryfunctiontest(PFT)and6-minutewalkingtest(6MWT)

parameters.Wemeasuredfractionalexhalednitricoxide(FeNO),CalvNOandmaximumconducting

air-waywallﬂux(J’awNO)in30healthysubjectsand30patientswithIIP(22idiopathicpulmonaryﬁbrosis

and8idiopathicnon-specificinterstitialpneumonias).IIPpatientshadhigherFeNOatflowratesof 50–100–150ml/sandhigherCalvNOlevelsthanhealthycontrols(p<0.0001).CalvNOwassignificantly

correlatedwith6-minutewalkingdistance(p<0.0001),recoverytime(p<0.0005),TLC(p<0.001),FVC (p=0.01)andTLCO(p<0.01).IIPpatientsshowedabnormalnitricoxideproduction,probablyduetolung ﬁbrosisandoxidative-mediatedlunginjury.CalvNOwascorrelatedwithPFTand6MWTparametersand

isproposedasapotentialbiomarkeroflungﬁbrosisandexercisetolerance.

1. Introduction

Nitricoxide(NO)isamoleculeproducedbythehumanlungs

andispresentinexhaledbreath.Itplaysahomeostaticroleina

widerangeofphysiologicalprocesses.Sincetheearly1990s,

sys-temsformeasuringNOin exhaledbreathhave beendeveloped

andusedtomeasurethefractionofexhalednitricoxide(FeNO)

inmanylungdiseases,includingasthma,chronicobstructive

pul-monarydisease(COPD),cysticﬁbrosis,primaryciliarydyskinesia

andpulmonaryarterialhypertension(AmericanThoracicSociety

EuropeanRespiratorySociety,2010).TheanatomicaloriginofNO

productioncanbeseparatedusingatwo-compartmentmodelof

the lung (airway and alveolar compartments) and partitioning

NOintoalveolarconcentrationofexhaledNO(CalvNO)and

max-imumconductingairwaywallﬂux(J’awNO)(TsoukiasandGeorge,

1998).Atpulmonarylevel,NOactsasavasodilator,bronchodilator,

Abbreviations: NO,nitricoxide;eNO,exhalednitricoxide;FeNO,fractionof exhaledNO;VeNO,exhaledNOoutput;CalvNO,alveolarconcentrationofNO;J’awNO,

maximumconductingairwaywallflux;IPF,idiopathicpulmonaryfibrosis;BAL, bronchoalveolarlavage;NSIP,non-specificinterstitialpneumonia;PFT,pulmonary functiontests;6MWT,6-minutewalkingtest;TLC,totallungcapacity;FVC,forced vitalcapacity;TLCO,transferfactorofthelungforcarbonmonoxide;COPD,chronic obstructivepulmonarydisease;IIP,idiopathicinterstitialpneumonia;2CM, two-compartmentmodel;TMAD,trumpetmodelofaxialdiffusion.

∗ Correspondingauthor.Tel.:+390577586710/+393297856833; fax:+390577280744.

E-mailaddress:paolocameli88@gmail.com(P.Cameli).

neurotransmitterandinﬂammatorymediator.Itisinvolvedin

sev-eralpathologicalconditions,includingﬁbrogenesisandoxidative

stress.NOistheprincipalreactivenitrogenspeciesencountered

invivo,andathighconcentrationscausestoxicitybycombining

withsuperoxide anion to produce peroxynitrite.This molecule

maycause nitration(additionof −NO2)ofmost classesof

bio-logicalmolecules,determiningproteindysfunction,inhibitionof

mitochondrialrespirationandDNAdamage.Experimentalstudies

supporttheroleofoxidativeandnitrosativestressinthe

patho-genesisofinterstitiallung diseases(ILD),thoughfewstudieson

exhaledNOanalysisareavailableforidiopathicinterstitial

pneu-monia (IIP) and particularlyidiopathicpulmonary ﬁbrosis (IPF)

patients(Chapmanetal.,2010;Chowetal.,2012;Hua-Huyetal.,

2010;MoodleyandLalloo,2001).Inparticular,Pullamssettietal. (2011)notedapotentialroleofNOinIPF.IPFandidiopathic

non-speciﬁcinterstitialpneumonia(NSIP)withﬁbrosingpatternarethe

mostcommonidiopathicinterstitialpneumonias:theyare

charac-terizedbyapoorprognosis(3–5yearssurvivalforIPF),duetothe

chronicprogressiverestrictivefunctionalimpairmentofthelung,

causedbyﬁbroticdegenerationofthelungparenchyma.Inaddition

tothat,IPFandNSIPpatientsearlydevelopasevereimpairmentof

physicaltolerance,duetoprogressivehypoxemia,whichseriously

deterioratestheirperformancestatusandqualityoflife.Inorder

toanalyzethisperspectiveinthesepatients,theuseof6-minute

walkingtest(6MWT)hasbeenalreadyvalidatedinIPF,becauseof

itssafetyanditspracticality:therefore,unlikemaximal

cardiopul-monaryexercisetesting,6MWTcanbeperformedbyalmostall

patientsand,becauseitisself-paced,isbettertoleratedandmore

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reﬂectiveofdailyactivitiesthanotherfunctionaltests:Muraetal. (2012)hasdemonstratedthat6minwalkdistanceatthetimeof

diagnosisisasigniﬁcantpredictorofsurvivalinIPFpatients.The

progressofthesediseasesisalsounpredictableatthetimeof

diag-nosis,sotheneedfor newseveritybiomarkersthatcanpredict

thenaturalhistoryandthephysicaltoleranceofthesepatientsis

intenselyfelt

Regardingotherdiffuselungdiseases,Tievetal.(2012)reported

increasedproductionofalveolarnitricoxideassociatedwithlung

ﬁbroblastproliferationinpatientswithpulmonaryﬁbrosis

associ-atedwithsystemicsclerosisandproposedCalvNOasanon-invasive

biologicalmarkerofalveolitispredictiveofdeteriorationoflung

function.Evaluation ofNO in exhaledbreath condensatesfrom

patientswithsarcoidosis(amultisystemgranulomatousdiseaseof

unknownetiology,inwhichinﬂammationcanplayafundamental

physiopathologicalrole,onlyevolvingtodiffusepulmonaryﬁbrosis

inaminorityofpatients)hasproducedconﬂictingresults.Moodley

etal.(1999)reportedhigherconcentrationsofFeNOinexhaled

breathsamplesfrom12patientswithsarcoidosisthanincontrols,

whileWilsheretal.(2005)describedcontraryﬁndingsinacohort

of52patients.Choietal.(2009)failedtoﬁndanysigniﬁcant

dif-ferencesinFeNOconcentrationsbetweenpatientswithsarcoidosis

andhealthynon-smokercontrols,howevertheyreported

signiﬁ-cantnegativecorrelationsbetweenCalvNOandpercentagevaluesof

forcedvitalcapacity(FVC)andtransferfactorofthelungforcarbon

monoxide(TLCO).Theauthorsconcludedbynotrecommending

exhaledFeNOdetectioninroutineclinicalpractice,duetothelarge

inter-subjectvariabilitytheyfound.HigherCalvNOlevelshavealso

beenreportedin pneumoconiosispatientsthanincontrols, and

exhaledNOhasbeenproposedasapotentialclinicalbiomarkerof

silica-andasbestos-exposedsubjectssusceptibletosilicosis(Fubini

andHubbard,2003;Saunietal.,2012)andasbestosis(Lehtimäki etal.,2010).

AslimitedliteratureisavailableonexhaledNOlevelsinpatients

withidiopathic interstitialpneumonias (Furukawaet al., 2011;

Schildge,2011;Zhaoetal.,2012),wedesignedthepresentstudyto

compareexhaledFeNOconcentrationsofpatientswithidiopathic

pulmonaryﬁbrosisandnon-speciﬁcinterstitialpneumonia with

thoseofapopulationofhealthysex-andage-matchedcontrols,

inordertocontributetothestudyofFeNOinIIP.Instead,toour

knowledge,noliteratureisavailableontheeffectofmildphysical

exerciseinexhaledNOlevelsinIPFandNSIPpatients.Forthis

rea-son,tobettercharacterizethepatternofNOinﬁbroticpatients,we

alsoexaminedeNOparametersatrestandafterthe6-minute

walk-ingtest(6MWT)inrelationtoreducedphysicaltolerance(which

inﬂuencesqualityoflifeandthereproducibilityoflungfunction

tests).

2. Materialsandmethods

2.1. Studypopulationandstudydesign

ThirtyILDpatientswereenrolledinthestudythatwas

per-formed at Siena Regional Referral Centre for Sarcoidosis and

InterstitialLungDiseasesbetweenSeptember2012andMay2013.

Twenty-twopatientshadidiopathicpulmonaryﬁbrosis(17males,

meanage64±10years),andeightpatientshadidiopathic

non-speciﬁc interstitialpneumonia withﬁbrosing pattern (2males,

mean age63±9 years). Diagnosis wasperformed accordingto

international guidelines (Raghu et al., 2011) and all patients

underwentpulmonaryfunctiontests(PFT),includingsingle-breath

diffusingcapacity forcarbon monoxideatdiagnosisandalmost

every six months thereafter. An accurate medical history was

obtainedfromallpatientsinordertoevaluateprofessional

expo-sure,smokingand medicationhistory. Chestx-raysweretaken

inposterior-anteriorandlateralprojections.Highresolution

com-putedtomographyofthechestwasperformedinallpatientsfor

diagnosticpurposes.Allpatientsandcontrolswereaskedto

per-formacomplete6-minutewalkingtest(6MWT).NineIPFpatients

were non-smokers and 13 were ex-smokers (mean packs/year

10.27±12.23).SixNSIPpatientswerenon-smokersandtwoNSIP

patients were ex-smokers (mean packs/year 5.62±10.5). The

patientshadnohistoryofatopy,concomitantasthma,canceror

autoimmunedisorders,andNSIPsecondarytosystemicsclerosis

orrheumatoidarthritiswasspeciﬁcallyexcluded.Atthemoment

ofNOdetection,patientswithIPFandNSIPwereinstablephaseand

hadbeenfreeofrespiratoryinfectionsand/oracuteexacerbations

foratleast4weeks.

The control group included 30 healthy volunteers (16

male, mean age 62±4.73 years), 15 non-smokers and 15

ex-smokers (mean packs/year 5.25±7.2). Patients treated with

phosphodiesterase-ﬁve inhibitors and ICS were excluded. All

healthyvolunteershadnormallungfunctionandhadsufferedno

respiratorysymptomsorinfectionsinthelast4weeks.Allpatients

and controlsgave theirwritteninformed consenttothe study,

whichwasapprovedbythelocalethicscommittee,andall

por-tionsofthestudywereperformedaccordingtotheDeclarationof

Helsinki.

2.2. Studyprotocol

Participantswereaskedtoavoidalcoholicdrinks, coffeeand

otherfoodorbeveragescontainingcaffeine(cola,tea,cocoa)for

atleast3hbeforetheexamination.Theyalsohadtoabstainfrom

foodscontainingnitrates(lettuce,spinach,cabbage,sausages)and

high-fatfoodsfor atleast12hbeforetheexamination.Patients

onbronchodilatorshadtosuspendtherapy 12hbeforethetest.

All participants had a mouthwash with water just before the

test.Baselinedeterminationofsystolicpressure,diastolicpressure,

heart rate,pulsed arterialoxygensaturationand exhaled nitric

oxidemeasurementswere takenafter10minofrestin a quiet

andcomfortableenvironment.Allsubjectsthenperformeda

com-plete6MWT,afterwhichexhalednitricoxidemeasurementswere

repeatedwithin5min.IPFandNSIPpatientsalsounderwentPFT

onthesameday.

2.3. Pulmonaryfunctiontests

The following lung function measurements were recorded

accordingtoATS/ERSstandards(Milleretal.,2005),usingaJaeger

BodyPlethysmographwithcorrectionsfortemperatureand

baro-metric pressure: forced expiratory volume in the ﬁrst second

(FEV1), forcedvitalcapacity(FVC),FEV1/FVC,totallungcapacity

(TLC),residualvolume(RV),carbonmonoxidelungtransferfactor

(TLCO)andcapacitycarbonmonoxidelungtransferfactor/alveolar

volume(TLCO/VA).Allparameterswereexpressedaspercentages

ofpredictedreferencevalues.TLCOmeasurementcouldnotbe

per-formedinfourpatientswhowereonoxygentherapy.PFTswere

performedatleast2hafterexhaledNOmeasurements.

2.4. Six-minutewalkingtest

IPFandNSIPpatientsandcontrolsperformedthesix-minute

walkingtestaccordingtothestandardsoftheATS/ERSguidelines

(ATSStatement,2002).

2.5. Exhalednitricoxidemeasurements

Nitricoxidewasmeasuredusingachemiluminescenceanalyzer

(modelHypairFeNOmedisoftCardiolineExp’air,2010)according

toATS-ERSrecommendationsforonlinemeasurementofFeNOin

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Table1

Demographicﬁndings,clinicalfeatures,6MWTparameters,lungfunctiontestparametersincludingTLCOinpatientswithIPFandNSIPpatientscomparedthancontrols.

Parameters IPF NSIP Controls p-Value

No. 22 8 30

Age(aa) 64.81±10.45 63.25±9.48 62±4.73 ns

Male(n) 17(59%) 2(25%) 16(51%) ns

BMI(kg/m2₎ _26.25_±_3.49 _27.65_±_2.51 _25.9_±_2.9 _ns

Tobaccouse(pack/year) 10.27±11.8 5.62±10.5 5.2±7.2 ns

SystolicPA(mmHg) 125±12.23 123.75±10.26 121.3±11.9 ns DiastolicPA(mmHg) 73.18±10.86 75±9.25 72.6±9.8 ns Heartrate(bpm) 78.95±11.63 76.5±5.2 73±9 ns SpO2% 95.4±1.14 95.7±2.3 97.6±0.9 <0.001† 6MWT baselineSpO2% 95.2±0.94 95.87±1.88 97.4±0.6 <0.0001*

endtestSpO2% 89.63±2.92 90.5±2.32 96.5±1.8 <0.0001*

baselineHR(bpm) 77.5±8.4 78.75±6.4 71.9±8.4 <0.0001*

posttestHR(bpm) 100.36±17.49 100.87±12.11 85±10.9 <0.0001*

Walkdistance(m) 297.72±55.02 282.5±73.63 366.8±39 <0.0001*

SpO2recoverytime(s) 110.9±59.19 88.75±65.56 7.6±11 <0.0001*

Borgindexbaseline 0.68±1.24 0.75±1.48 0 <0.0001*

Borgindexposttest 4±2.37 5.25±2.96 0.4±0.8 <0.0001*

PFT(%referencevalues) FVC% 69.52±18.15 77.71±25.51 ns‡ FEV1% 69.95±18.33 76.81±25.82 ns‡ FEV1/FVC% 78.22±9.44 78.96±8.64 ns‡ ITGV% 88±16.72 91.31±26.52 ns‡ RV% 92.51±18.06 93.03±25.74 ns‡ TLC% 77.45±14.95 76.47±16.36 ns‡ RV/TLC 48.29±8.19 48.21±8.75 ns‡ TLCO% 43.33±16.65 41.06±21.25 ns‡ TLCO/VA% 68.51±19.22 59.81±11.07 ns‡

Lungfunctiontestareexpressedaspercentagesofpredictedvalues:FVC,ForcedVitalCapacity;FEV1,ForcedExpiratoryVolumein1s;ITGV,IntrathoracicGasVolume;RV,

ResidualVolume;TLC,TotalLungCapacity;TLCO,TransferFactoroftheLungforCarbonMonoxide;VA,AlveolarVolume.

* _p-value_between_IIP_(IPF_and_NSIP)_patients_and_controls. † _p-value_between_IPF_and_controls.

‡ _p-value_between_IPF_and_NSIP_patients.

2010).TheanalyzerwassensitivetoNOfrom1to500ppbwitha

resolutionof1ppb.Allmeasurementswereundertakenat

ambi-entNOlevelsof<10ppb.Subjectswerestudiedinsittingposition.

Exhaled NO was measured during slow exhalation from total

lungcapacityagainstapositivepressurekeptconstantlybetween

5–20cm H2O togenerate exhalationﬂow rates of 50, 100 and

150ml/s.Theexhalationﬂowratewaskeptasconstantas

pos-sibleusing a biofeedback visualdisplay. For each ﬂow rate, at

leasttwo technicallyadequate measurements wereperformed.

In orderto evaluateNO concentrationproﬁleversus time

dur-ingexhalation,wealsomeasuredexhaledNOoutput(VeNO)in

patients and controls. A third measurement was performed if

thedifferencebetweenﬁrst twomeasurementswasmore than

10%.Thefourpatientsonoxygentherapyweretakenoffit

dur-ingexhalednitricoxidemeasurements.Theﬂow-independentNO

parameters,CalvNOandJ’awNO,werecalculatedusingtheTsoukias

two-compartment model of NO exchange (2CM; Tsoukias and

George,1998).Anotheranalyticalmethodtorepresentourresults

wasthetrumpetmodelofaxialdiffusion(TMAD)ofCondorellietal.

(2007).Alinearrelationshipbetweenthethreepoints(50,100and

150ml/s)oftheNOﬂuxagainsttheﬂowwasevaluatedforeach

subjectbyalinearitytest.

2.6. Datacollectionandreproducibility

Eachmeasurement was consideredacceptable witha

conﬁ-dence rate >95% and a ﬂow stability >90%. All measurements

weremadebyasingleinvestigator,guaranteeinginter-and

intra-observeragreement.

2.7. Statisticalanalysis

Datawasexpressedasmean±standarddeviation(SD).

Compar-isonsbetweenIPF,NSIPpatientsandcontrolswereperformedby

Mann–Whitneytest.ChangesinclinicalandexhaledNO

param-eters observedafter the6MWTwerecompared using two-way

ANOVA,withBonferroni’sposttest.CorrelationsbetweenCalvNO,

PFTvaluesand6MWTparametersweremadebySpearman’stest.

Ap-value<0.05wasconsideredstatisticallysigniﬁcant.Statistical

analyseswereperformedusingGraphPadPrism5.

3. Results

3.1. Clinicalandfunctionalcharacteristics

Clinicaland demographicdataof patientswithIPFand NSIP

andcontrolsare reportedinTable1,togetherwithPFT results,

including TLCO percentages, and 6MWT parameters. Globally,

IIP patients showed mild to moderate restrictive impairment

and 6MWT parameters (baseline and end test SpO2, heart

rate,Borgindex,6-minutewalkingdistanceandrecoverytime)

were signiﬁcantly different from those measured in controls

(p<0.0001).

3.2. eNOvaluesinIPFandNSIPandhealthycontrols

FeNO values at ﬂow rates of 50, 100 and 150ml/s were

signiﬁcantlyhigherinIPFandNSIPpatientsthaninhealthy

con-trols(p<0.0001)(Fig.1,Table2).Atbaseline,ﬂow-independent

parameters included CalvNO, that was signiﬁcantly higher in

IPF and NSIP patients than controls (p<0.0001), and J’awNO,

which did not differ between the two groups (p>0.05)

(Table 2). These results were obtained applying the 2CM

and TMAD. In Table 2, exhaled VeNO was also reported to

evaluate NO concentration proﬁle versus time during

exhala-tion. VeNO values at ﬂow rates of 50, 100 and 150ml/s were

signiﬁcantlyhigherinIPFandNSIPpatientsthanhealthycontrols

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Fig.1. ComparisonofexhalednitricoxideparametersbetweenIPF+NSIPpatientsandhealthycontrols,beforeandafter6MWT.FeNO,fractionofexhalednitricoxide; CalvNO,alveolarconcentrationofNO;ppb,parsperbillion;IPF,idiopathicinterstitialpneumonia;NSIP,non-speciﬁcinterstitialpneumonia;***p<0.0001.**_p_<_0.001.*

p<0.05.

VeNO150(156±24.5vs 144.9±27.6nl/min; p>0.05) werenot

signiﬁcantlydifferenttoIPFandNSIPpatientswhilewere

signif-icantlyhigherthanhealthycontrols(17.7±8.2vs10.8±2.9ppb;

156_±24.5vs95_±10nl/min;p<0.01andp<0.001,respectively).

Therewerenosigniﬁcantdifferencesbetweennon-smokersand

ex-smokersinanypopulation

3.3. eNOvaluesand6MWT.

Afterthe6MWT,FeNO50values(p<0.001),VeNO50 values

(p<0.0001)andJ’awNOlevels(p<0.001)weresigniﬁcantlyhigher,

andFeNO100values(p<0.05),FeNO150values(p<0.05),VeNO

100(p<0.05),VeNO150levels(p<0.05)andCalvNO(p<0.001)

signiﬁcantlylower,inIPFandNSIPpatientswithrespecttobefore

6MWT(Figs.1and2,Table2).IPFandNSIPpatientsdidn’t

sig-niﬁcantly differ in any baseline or post6MWT parameters. No

signiﬁcantchangeswereobservedinhealthycontrolsafter6MWT.

Inthepatientsgroup,baselineCalvNOwassigniﬁcantlycorrelated

withsix-minutewalkingdistance(r=−0.73,p<0.0001),recovery

time(r=0.63;p<0.0005)andSpO2posttest(r=−0.54;p<0.01).

CalvNOwasalsoinverselyrelatedtoFVC(r=−0.47;p<0.01),TLC

(r=−0.62; p<0.001) and TLCO percentages (r=−0.60; p<0.01)

(Fig.3).NosigniﬁcantcorrelationswerefoundbetweenFeNO50,

Fig.2.ComparisonofJ’awNOatbaselineandafter6MWTbetweenpatientsand

healthycontrols.J’awNOdidnotsigniﬁcantlychangeinpatientswithrespectto controlsatbaseline,whereasitwassigniﬁcantlyhigherinIIPpatientsthan con-trolsafter6MWT.J’awNOdidnotchangeinhealthysubjectsafter6MWT.J’awNO,

maximumconductingairwaywallflux;6MWT,6-minutewalkingtest;IPF, idio-pathicpulmonaryfibrosis;NSIP,non-specificinterstitialpneumonia;***_p_<_0.001_in

patients;ns:nonsigniﬁcant.

Table2

eNOparametersinIPFandNSIPpatientsandhealthycontrols.

eNoparameters IPF NSIP Controls p-Value

Baseline Post6MWT Baseline Post6MWT Baseline Post6MWT

FeNO50ml/s(ppb) 22.3±8.4 26.1±10.5† 21±8 27.3±11.2‡ 15.8±4.1 16.1±4.1 <0.0001* FeNO100ml/s(ppb) 18.2±6.8 16.1±6.8† 17.8±5.3 16.6±6.4 13.1±2.8 12.2±3.5 <0.0001* FeNO150ml/s(ppb) 15.2±7.7 13.2±5.5† _15.1_±_5.1 _13.6_±_4.6 _10.8_±_2.9 _9.7_±_2.4 _<0.0001* J’awNO(nl/min)a 42.6±24.6 69.5±37.1† 40.3±30.9 83.2±71.3‡ 45.3±22.3 48.7±20.5 ns CalvNO(ppb)a 11.5±6.6 7±3.8† 12.6±6.8 6.7±3.2‡ 4.7±2.3 3.9±1.6 <0.0001* J’awNO(nl/min)b 71.4±44 124±81† 67.6±49.2 135.6±114‡ 77.1±38 82.8±35 Ns CalvNO(ppb)b 10.7±5.9 6.2±3.3† 11.2±5.7 6.1±2.7‡ 4.3±2.08 3.6±1.4 <0.0001* VeNO50ml/s(nl/min) 81.8±18.4 94.6±22.5† 78.3±24 100.5±32‡ 47.7±10.4 51.5±8.4 <0.0001* VeNO100ml/s(nl/min) 115±25 105±21† 104±30 118±45 82.5±12 84.2±9 <0.0001* VeNO150ml/s(nl/min) 149±27 132±18† ₁₄₄_±₃₂ ₁₃₇_±₃₄ ₉₅_±₁₀ ₉₉_±₇ _<0.0001*

FeNO,fractionalofexhalednitricoxide;CalvNO,alveolarconcentrationofnitricoxide;J’awNO,maximumconductingairwaywallﬂux. *_p-value_between_IIP_(IPF_and_NSIP)_patients_and_controls.

†_p_<_0.05_with_baseline_IPF. ‡_p_<_0.05_with_baseline_NSIP.

a_{Two-compartment}_model_by_Tsoukias_and_George.

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100,150,J’awNOandsix-minutewalkingdistance,recoverytime,

FVC,TLCandTLCO.

4. Discussion

Thepresentstudyevaluatedexhalednitricoxideparametersin

IPFandNSIPpatients,comparedwithagroupofhealthysubjects,

demonstratinganincreaseatrestinFeNOatﬂowratesof50,100

and150ml/sandanincreaseinCalvNOinIPFandNSIPpatients

comparedwithhealthycontrols.

VerylittledataisavailableonFeNOconcentrationsinexhaled

breath of patients with IPF and NSIP (Furukawa et al., 2011;

Schildge,2011;Zhaoetal.,2012),whichpromptedustoexplore

NOpatterninthesediseases,consideringthepotentialimplications

ofNOasamediatorinthepathogenesisofpulmonaryﬁbrosis.We

alsocomparedeNOvaluesatrestandafterexerciseinour

popu-lationasacontributiontotheanalysisofexercise-inducedeffects

oneNOconcentrationsinIIPpatients,asnoliteratureisavailable

onthistopicandthesepatientssoondeveloplowphysical

toler-ance(whichimpairsqualityoflifeandthereproducibilityofPFT).

PhysicalexerciseisacrucialtriggerofdesaturationinIPFandNSIP

patientsandwedecidedtoestablishitseffectonNOkinetics.

4.1. NOandpulmonaryﬁbrosis

The increase in eNO parameters in our IIP patients can be

explainednot onlyby a reducedgas diffusionthrougha

thick-enedalveolar-capillarymembrane,butalsobyoverexpressionof

induciblenitric oxidesynthase(iNOS)and consequentlyhigher

productionofNOinthelungparenchyma(Pullamsettietal.,2011).

TheobservationthatNOisimplicatedinpulmonaryﬁbrogenesis

isdemonstrated by studiesreportingabnormal NOand

deriva-tiveconcentrations inBAL of murineﬁbrotic lungs (Janget al.,

2004;Wellsetal.,2009)andBALﬂuidofIPFpatientsPullamsetti

et al., 2011) as a result of overexpression of dimethylarginine

dimethylaminohydrolases(DDAH)by type II pneumocytes.Our

studystronglysupportstheseﬁndingsindicatingpotential

involve-mentofNOandderivatesinthepathogenesisofinterstitiallung

diseases.

4.2. NOandoxidative/nitrosativestress

AnotherpotentialpathogenicroleofNOininterstitiallung

dis-easepathogenesisisNOinvolvementinoxidation-mediatedlung

damage(afunctionreportedsincetheearly2000s).FeNOhasbeen

proposedasabiomarkerofoxidativestressinotherﬁbroticdiffuse

lung diseases, includingbleomycin-induced pulmonary ﬁbrosis,

systemicsclerosisandpneumoconiosis(FubiniandHubbard,2003;

Inghillerietal.,2006;Saunietal.,2012;Tievetal.,2012),while

therehavebeenfewstudiesontheroleofFeNOin

oxidative-stress-mediatedlung ﬁbrotic injuryin IPFand NSIP. Asmanyauthors

considernitricoxideanessentialmoleculeinthemodulationof

oxidant-antioxidantbalance(Allenetal.,2009;Lanzettietal.,2012;

Rahmanetal.,2006),theincreasewefoundinourIIPpatientsisof interest.

LiteraturesustainingapossibleroleofNOintheoxidativeand

nitrosativestressthatoccurs indiffuselung diseases formsthe

backgroundofthepresentstudy.Wedemonstratedhigher

alve-olarconcentrationsofNOinIIPpatientsthanincontrolsandan

inversecorrelationbetweenCalvNOandlungfunctiontest

param-eters,suggestingthatrespiratoryimpairmentwasassociatedwith

higherNOconcentrations.

ComparingIPFandNSIPpatientswithanasthmaticpopulation,

whereofmoreisknownabouteNOlevelsintheliterature,itcan

bereportedthatFeNO50–100andJ’awNOlevelsofourpatientsare

ingenerallowerthaninasthmaticpatients.TheincreaseofFeNO

andofthemaximumﬂuxofNOintheairwaysregardingasthma

hasbeenalreadyexploredinmanymanuscripts(Chládkováetal.,

2012;Lehtimäkietal.,2001;Spearsetal.,2011):thedifference

betweenasthmaticandIIPpatientscouldbeexplainedby

distinc-tivepathogeneticpathways:inparticular,elevatedFeNOvaluesin

asthmaisjustiﬁedbythepresenceofeosinophilicinﬂammation

intheairways,whichisnotcommonlyobservedinIPFandNSIP

patients.

4.3. NOandlungfunctiontests

InourstudyweanalyzedexhaledNOconcentrationsinpatients

andcontrolsaccordingtotwodifferentanalyticmodels.The2CM

is a simple and attractive way tocalculate CalvNO and J’awNO.

Since itprobably doesnotrepresent theheterogeneityof

alve-olarcompartmentsofIPFandNSIP,wealsoappliedCondorelli’s

trumpetmodelofaxialdiffusion(TMAD)thatconsidersincreasing

airwaycross-sectionwithdistanceintotheairwaytreeand

gas-phaseaxialdiffusionofNO,bothcriticalaspectsofNOexchange.

ThesigniﬁcantdifferenceinCalvNObetweenpatientsandcontrols

wasconﬁrmedbybothmodels,suggestingtheimportanceofthis

parameter,recentlyproposedasanon-invasivebiomarkerof

alve-olitis,predictiveofdeteriorationoflungfunction(Tievetal.,2012).

Atthesametime, reducedgasdiffusionmaypartiallyinﬂuence

theincrease in CalvNO by reducing theremoval of alveolarNO

acrossthethickenedalveolar-capillarymembrane.Interestingly,

ina recentmanuscript, inwhich NOmeasurementswere

com-paredindifferentILDpopulations,Schildgeaddedairwaydiffusing

capacityofNO(DAWNO)toevaluatereducedNOdiffusionfrom

alveolitothebloodduetothickeningofalveolarmembranes.In

ourspeciﬁcpopulation,TLCOwasmildlytoseverelyreduced:of

theﬁvepatientsreportingsimilarCalvNOlevelstohealthysubjects,

onlytwoshowedmildTLCOimpairment(Fig.3),suggestingthat

increasedCalvNOmaynotonlybeduetoalowereliminationrate

butalsotohigherlocalproduction(Pullamssettietal.,2011).Tiev

etal.(2013)obtainedsimilarresultstooursinaveryrecentstudy

ofapopulationofpatientswithpulmonaryﬁbrosisassociatedwith

systemicsclerosis,inwhomincreasedCalvNOinexhaledbreathwas

associatedwithseverereductioninTLCandTLCO.AlthoughFeNO

at50ml/sisrecognizedasacentralairwaysparameter,itselevation

inIPFpatientshasalsobeenreportedbyGuilleminaultetal.(2013),

withvaluescomparabletoours,suggestingthatelevatedalveolar

concentrationsofNOcoulddrivetheincreasedlevelsmeasuredat

allﬂowrates.

Since earlier airway closure is a possibility in IPF and NSIP

patients,analysisofVeNOwasaddedtoobtainNOconcentration

proﬁlesduringexhalation. VeNOvalues wereclosely correlated

withFeNOvaluesforallﬂowratesinpatientsandcontrols,

indicat-ingtheabsenceofairwayclosure,whichthereforecouldnotalter

theinterpretationoftheresultsinpatientswithdiffuselung

dis-eases.Onelimitationofthisstudyisthelackoftheevaluationof

parallelinhomogeneityoflungparenchymainIPFandNSIP:

unfor-tunately,therearenoexperimentalmodelsintheliteraturethat

couldhelpusstandardizeeNOvaluesfordifferentlungareas.

Fur-therresearchcouldbeusefultocreateanewmodelforevaluation

ofeNOvaluesinpatientswithinterstitiallungdiseases.

OurresultsthereforeindicateCalvNOasapotentialbiomarker

ofdiseaseseverity,alsoonthebasis oftheinversecorrelations

observedwithTLCO percentages,6-minutewalkingdistanceand

endtest SpO2,aswellasevidenceofa directrelationship with

recoverytime.CalvNOprovedtobeaninterestingpotential

pro-gnosticparameterworthyoffurtherstudy,assuggestedbyTiev

inpulmonaryﬁbrosisassociatedwithsystemicsclerosis,

correlat-ingwithwell-knownclinicalprognosticbioindicators,suchasTLCO,

FVCand6-minutewalkingdistance.Thisparametercouldbe

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Fig.3.CorrelationsbetweenCalvNOatbaselineand6MWTandPFTparametersinpatients:(A)6-minwalkingdistance(r=−0.7317,p<0.0001);(B)recoverytime(r=0.6332,

p<0.0005);(C)endtestSpO2(r=−0.5419,p<0.01);(D)TLC%(r=−0.6267,p<0.001);(E)FVC%(r=−0.4745,p<0.01);(F)TLCO%(r=−0.6009,p<0.01).6MWT,6-minute

walkingtest;PFT,pulmonaryfunctiontest;CalvNO,alveolarconcentrationofNO;ppb,parsperbillion;TLC,totallungcapacity;FVC,forcedvitalcapacity;TLCO,carbon

monoxidelungtransferfactor.

therapy(Macintyreetal.,2005).Moreover,theprocedureisless

hardtoperformthanTLCOdetection.

4.4. NOand6-minutewalkingtest

Inourstudy,exercise-inducedvariationsinairwayand

alve-olar exhaled NO parameters were examined for the ﬁrst time,

revealingsigniﬁcantlydifferentpatterns.Afterthe6MWT,FeNO

50values(p<0.001)andJ’awNOlevels(p<0.001)weresigniﬁcantly

higher,andFeNO100values(p<0.05),FeNO150values(p<0.05)

andCalvNO(p<0.001)signiﬁcantlylowerinIPFandNSIPpatients

thanbefore6MWT.Physicalexertionpresumablyaffectedthese

parametersbecauseitcauseschangesinventilationinIIPpatients,

favouringclearanceof NOfromthealveoles andincreasingthe

airwayﬂuxofNO.Hypoxemiacausedbyphysicalexertionmay

alsoinducelungchangesthatincreaseoxidant-antioxidant

imbal-ance,affectingNOconcentrations.Verylittledataisavailableon

exercise-inducedNOconcentrations, particularlyin IIPpatients.

Mostrelatestostudiesin asthmapatientsandhealthy controls

(Barretoetal.,2013;Therminariasetal.,1998).However,ourstudy

doesnot explain thephysiopathogenetic causesof these

mod-iﬁcations which are worthyof furtherinvestigations fora new

interestingscenario.

Inconclusion, ourdatashows higherlevelsofexhalednitric

oxideinpatientswithidiopathicinterstitialpneumonia,probably

duetooxidative/nitrosativestressrelatedtopulmonaryﬁbrosis.

Alveolarconcentrationsof NOmaybeproposedasamarkerof

severityin pulmonaryﬁbrosis sincetheywere mainlyelevated

inIPFandNSIPpatientswithsevererestrictivedeﬁcitandaltered

6MWT.Thepresentresultsalsohighlightthepotentialinterestof

thisnon-invasiveandreproduciblemethodasanadditional

param-eterforlungfunctionalassessmentindiffuselungdiseaseslikeIPF

andNSIP.

Thestudywasconductedat theDepartmentofMedical and

SurgicalSciencesandNeurosciences,RespiratoryDiseaseandLung

TransplantationSection;nofundingsponsorstodeclare.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in

theonlineversion,athttp://dx.doi.org/10.1016/j.resp.2014.03.011.

References

Allen,B.W.,Demchenko,I.T.,Piantadosi,C.A.,2009.Twofacesofnitricoxide: impli-cationsforcellularmechanismsofoxygentoxicity.J.Appl.Physiol.106(2), 662–667.

AmericanThoracicSociety;EuropeanRespiratorySociety,2010.ATSERS recom-mendationsforstandardizedproceduresfortheonlineandofﬂinemeasurement ofexhaledlowerrespiratorynitricoxideandnasalnitricoxide,2005.Am.J. Respir.Crit.CareMed.171,912–930.

(7)

ATSstatement,2002.Guidelinesforthesix-minutewalktest.ATSCommitteeon Pro-ﬁciencyStandardsforClinicalPulmonaryFunctionLaboratories.Am.J.Respir. Crit.CareMed.166(1),111–117.

Barreto,M.,Zambardi,R.,Villa,M.P.,2013.Exhalednitricoxideandotherexhaled biomarkersinbronchialchallengewithexerciseinasthmaticchildren:current knowledge.Paediatr.Respir.Rev.,pii:S1526-0542(13)00148-6.

Chapman,E.A.,Thomas,P.S.,Yates,D.H.,2010.Breathanalysisinasbestos-related disorders:areviewoftheliteratureandpotentialfutureapplications.J.Breath Res.4(3),034001.

Chládková,J.,Senkerík,M.,Havlínová,Z.,Krcmová,I.,Chládek,J.,2012Nov.Alveolar concentrationandbronchialﬂuxofnitricoxide:twolinearmodelingmethods evaluatedinchildrenandadolescentswithallergicrhinitisandatopicasthma. Pediatr.Pulmonol.47(11),1070–1079.

Choi,J.,Hoffman,L.A.,Sethi,J.M.,Zullo,T.G.,Gibson,K.F.,2009.Multipleﬂowrate measurementofexhalednitricoxideinpatientswithsarcoidosis:apilot feasi-bilitystudy.SarcoidosisVasc.DiffuseLungDis.26(2),98–109.

Chow,S.,Thomas,P.S.,Malouf,M.,Yates,D.H.,2012.Exhaledbreathcondensate (EBC)biomarkersinpulmonaryﬁbrosis.J.BreathRes.6(1),016004. Condorelli,P.,Shin,H.W.,Aledia,A.S.,Silkoff,P.E.,George,S.C.,2007.Asimple

techniquetocharacterizeproximalandperipheralnitricoxideexchangeusing constantﬂowexhalationsandanaxialdiffusionmodel.J.Appl.Physiol.102(1), 417–425,Epub2006Aug3.

Fubini,B.,Hubbard,A.,2003.Reactiveoxygenspecies(ROS)andreactivenitrogen species(RNS)generationbysilicaininﬂammationandﬁbrosis.FreeRadic.Biol. Med.34(12),1507–1516.

Furukawa,K.,Sugiura,H.,Matsunaga,K.,Ichikawa,T.,Koarai,A.,Hirano,T., Yana-gisawa,S.,Minakata,Y.,Akamatsu,K.,Kanda,M.,Nishigai,M.,Ichinose,M.,2011. Increaseofnitrosativestressinpatientswitheosinophilicpneumonia.Respir. Res.12,81.

Guilleminault,L.,Saint-Hilaire,A.,Favelle,O.,Caille,A.,Boissinot,E.,Henriet,A.C., Diot,P.,Marchand-Adam,S.,2013.Canexhalednitricoxidedifferentiatecauses ofpulmonaryﬁbrosis?Respir.Med.,pii:S0954-6111(13)00260-6.

Hua-Huy,T.,Tiev,K.P.,Chéreau,C.,Duong-Quy,S.,Cabane,J.,Dinh-Xuan,A.T.,2010. Increasedalveolarconcentrationofnitricoxideisrelatedtoserum-inducedlung ﬁbroblastproliferationinpatientswithsystemicsclerosis.J.Rheumatol.37(8), 1680–1687.

Inghilleri,S.,Morbini,P.,Oggionni,T.,Barni,S.,Fenoglio,C.,2006.Insituassessment ofoxidantandnitrogenicstressinbleomycinpulmonaryﬁbrosis.Histochem. CellBiol.125(6),661–669.

Jang,A.S.,Lee,J.U.,Choi,I.S.,Park,K.O.,Lee,J.H.,Park,S.W.,Park,C.S.,2004.Expression ofnitricoxidesynthase,aquaporin1andaquaporin5inratafterbleomycin inhalation.IntensiveCareMed.30,489–495.

Lanzetti,M.,daCosta,C.A.,Nesi,R.T.,Barroso,M.V.,Martins,V.,Victoni,T.,Lagente, V.,Pires,K.M.,eSilva,P.M.,Resende,A.C.,Porto,L.C.,Benjamim,C.F.,Valenc¸a,S.S., 2012.Oxidativestressandnitrosativestressareinvolvedindifferentstagesof proteolyticpulmonaryemphysema.FreeRadic.Biol.Med.53(11),1993–2001. Lehtimäki,L.,Kankaanranta,H.,Saarelainen,S.,Hahtola,P.,Järvenpää,R.,Koivula, T.,Turjanmaa,V.,Moilanen,E.,2001Jun.ExtendedexhaledNOmeasurement differentiatesbetweenalveolarandbronchialinﬂammation.Am.J.Respir.Crit. CareMed.163(7),1557–1561.

Lehtimäki,L.,Oksa,P., Järvenpää,R.,Vierikko,T., Nieminen,R.,Kankaanranta, H.,Uitti,J.,Moilanen,E., 2010.Inﬂammation inasbestos-exposedsubjects withborderlineparenchymalchangesonHRCT.Respir.Med.104(7),1042– 1049.

Macintyre,N.,Crapo,R.O.,Viegi,G.,Johnson,D.C.,vanderGrinten,C.P.,Brusasco,V., Burgos,F.,Casaburi,R.,Coates,A.,Enright,P.,Gustafsson,P.,Hankinson,J.,Jensen, R.,McKay,R.,Miller,M.R.,Navajas,D.,Pedersen,O.F.,Pellegrino,R.,Wanger,J., 2005Oct.Standardisationofthesingle-breathdeterminationofcarbon monox-ideuptakeinthelung.Eur.Respir.J.26(4),720–735.

Miller,M.R.,Crapo,R.,Hankinson,J.,Brusasco,V.,Burgos,F.,Casaburi,R.,Coates,A., Enright,P.,vanderGrinten,C.P.,Gustafsson,P.,Jensen,R.,Johnson,D.C., MacIn-tyre,N.,McKay,R.,Navajas,D.,Pedersen,O.F.,Pellegrino,R.,Viegi,G.,Wanger, J.,ATS/ERSTaskForce.,2005.Generalconsiderationsforlungfunctiontesting. Eur.Respir.J.26(1),153–161.

Moodley,Y.P.,Chetty,R.,Lalloo,U.G.,1999.Nitricoxidelevelsinexhaledairand induciblenitricoxidesynthaseimmunolocalizationinpulmonarysarcoidosis. Eur.Respir.J.14(4),822–827.

Moodley,Y.P.,Lalloo,U.G.,2001.Exhalednitricoxideiselevatedinpatientswith progressivesystemicsclerosiswithoutinterstitiallungdisease.Chest119(5), 1449–1454.

Mura,M.,Porretta,M.A.,Bargagli,E.,Sergiacomi,G.,Zompatori,M.,Sverzellati,N., Taglieri,A.,Mezzasalma,F.,Rottoli,P.,Saltini,C.,Rogliani,P.,2012Jul.Predicting survivalinnewlydiagnosedidiopathicpulmonaryﬁbrosis:a3-yearprospective study.Eur.Respir.J.40(1),101–109.

Pullamsetti,S.S.,Savai,R.,Dumitrascu,R.,Dahal,B.K.,Wilhelm,J.,Konigshoff,M., Zakrzewicz,D.,Ghofrani,H.A.,Weissmann,N.,Eickelberg,O.,Guenther,A., Leiper,J.,Seeger,W.,Grimminger,F.,Schermuly,R.T.,2011.Theroleof dimethy-largininedimethylaminohydrolaseinidiopathicpulmonaryﬁbrosis.Sci.Transl. Med.3(87),87ra53.

Raghu,G.,Collard,H.R.,Egan,J.J.,Martinez,F.J.,Behr,J.,Brown,K.K.,Colby,T.V., Cordier,J.F.,Flaherty,K.R.,Lasky,J.A.,Lynch,D.A.,Ryu,J.H.,Swigris,J.J.,Wells, A.U.,Ancochea,J.,Bouros,D.,Carvalho,C.,Costabel,U.,Ebina,M.,Hansell,D.M., Johkoh,T.,Kim,D.S.,KingJr.,T.E.,Kondoh,Y.,Myers,J.,Müller,N.L.,Nicholson, A.G.,Richeldi,L.,Selman,M.,Dudden,R.F.,Griss,B.S.,Protzko,S.L., Schüne-mann,H.J.,ATS/ERS/JRS/ALATCommitteeonIdiopathicPulmonaryFibrosis., 2011.Idiopathicpulmonaryﬁbrosis:evidence-basedguidelinesfordiagnosis andmanagement.Am.J.Respir.Crit.CareMed.183(6),788–824.

Rahman,I.,Biswas,S.K.,Kode,A.,2006.Oxidantandantioxidantbalanceinthe airwaysandairwaydiseases.Eur.J.Pharmacol.533(1–3),222–239.

Sauni,R.,Oksa,P.,Lehtimäki,L.,Toivio,P.,Palmroos,P.,Nieminen,R.,Moilanen,E., Uitti,J.,2012.Increasedalveolarnitricoxideandsystemicinﬂammationmarkers insilica-exposedworkers.Occup.Environ.Med.69(4),256–260.

Schildge,J.,2011Mar.StickstoffmonoxydinderAtemluftvonPatientenmit inter-stitiellenLungenkrankheiten(Nitricoxideinexhaledbreathofpatientswith interstitiallungdiseases).Pneumologie65(3),143–148.

Spears,M.,Weir,C.J.,Smith,A.D.,McSharry,C.,Chaudhuri,R.,Johnson,M.,Cameron, E.,Thomson,N.C.,2011Dec.Bronchialnitricoxideﬂux(J’aw)issensitivetooral corticosteroidsinsmokerswithasthma.Respir.Med.105(12),1823–1830. Therminarias,A.,Oddou,M.F.,Favre-Juvin,A.,Flore,P.,Delaire,M.,1998.Bronchial

obstructionandexhalednitricoxideresponseduringexerciseincoldair.Eur. Respir.J.12(5),1040–1045.

Tiev,K.P.,Hua-Huy,T.,Kettaneh,A.,Allanore,Y.,Le-Dong,N.N.,Duong-Quy,S., Cabane,J.,Dinh-Xuan,A.T.,2012.Alveolarconcentrationofnitricoxidepredicts pulmonaryfunctiondeteriorationinscleroderma.Thorax67(2),157–163. Tiev, K.P., Hua-Huy, T., Rivière, S., Le-Dong, N.N., Febvre, M., Cabane, J.,

Dinh-Xuan, A.T.,2013 Jan 15.High alveolarconcentrationof nitricoxide is associated with alveolitis in scleroderma. Nitric Oxide 28, 65–70, http://dx.doi.org/10.1016/j.niox.2012.10.005.Epub2012Oct23.

Tsoukias,N.M.,George,S.C.,1998.Atwo-compartmentmodelofpulmonarynitric oxideexchangedynamics.J.Appl.Physiol.85,653–666.

Wells,S.M.,Buford,M.C.,Migliaccio,C.T.,Holian,A.,2009.Elevatedasymmetric dimethylargininealterslungfunctionandinducescollagendepositioninmice. Am.J.Respir.Cell.Mol.Biol.40,179–188.

Wilsher,M.,Fergusson,W.,Milne,D.,Wells,A.U.,2005.Exhalednitricoxidein sarcoidosis.Thorax60(11),967–970.

Zhao,Y.,Cui,A.,Wang,F.,Wang,X.J.,Chen,X.,Jin,M.L.,Huang,K.W.,2012. Char-acteristicsofpulmonaryinﬂammationincombinedpulmonaryﬁbrosisand emphysema.Chin.Med.J.(Engl)125(17),3015–3021.