Nitrated Fatty Acids Reverse Cigarette Smoke-Induced Alveolar Macrophage Activation and Inhibit

16 Pages·2016·7.7 MB·English

RESEARCHARTICLE
Nitrated Fatty Acids Reverse Cigarette
Smoke-Induced Alveolar Macrophage
Activation and Inhibit Protease Activity via
Electrophilic S-Alkylation
AravindT.Reddy1,2,SowmyaP.Lakshmi1,2,RamamohanR.Muchumarri1,2,Raju
C.Reddy1,2*
1 DepartmentofMedicine,DivisionofPulmonary,AllergyandCriticalCareMedicine,UniversityofPittsburgh
SchoolofMedicine,Pittsburgh,PA,15213,UnitedStatesofAmerica,2 VeteransAffairsPittsburgh
a11111
HealthcareSystem,Pittsburgh,PA,15240,UnitedStatesofAmerica
*[email protected]
Abstract
OPENACCESS Nitratedfattyacids(NFAs),endogenousproductsofnonenzymaticreactionsofNO-derived
Citation:ReddyAT,LakshmiSP,MuchumarriRR, reactivenitrogenspecieswithunsaturatedfattyacids,exhibitsubstantialanti-inflammatory
ReddyRC(2016)NitratedFattyAcidsReverse activities.Theyarebothreversibleelectrophilesandperoxisomeproliferator-activatedrecep-
CigaretteSmoke-InducedAlveolarMacrophage torγ(PPARγ)agonists,butthephysiologicalimplicationsoftheirelectrophilicactivityare
ActivationandInhibitProteaseActivityvia
poorlyunderstood.Wetestedtheireffectsoninflammatoryandemphysema-relatedbiomark-
ElectrophilicS-Alkylation.PLoSONE11(4):
e0153336.doi:10.1371/journal.pone.0153336 ersinalveolarmacrophages(AMs)ofsmoke-exposedmice.NFA(10-nitro-oleicacidor12-
nitrolinoleicacid)treatmentdownregulatedexpressionandactivityoftheinflammatorytran-
Editor:YunchaoSu,AugustaUniversity,UNITED
STATES scriptionfactorNF-κBwhileupregulatingthoseofPPARγ.Italsodownregulatedproductionof
inflammatorycytokinesandchemokinesandoftheproteasecathepsinS(CatS),akeymedia-
Received:December24,2015
torofemphysematousseptaldestruction.CatSdownregulationwasaccompaniedby
Accepted:March28,2016
decreasedAMelastolyticactivity,amajormechanismofseptaldestruction.NFAsdownregu-
Published:April27,2016
latedbothCatSexpressionandactivityinAMsofwild-typemice,butonlyinhibiteditsactivity
Copyright:Thisisanopenaccessarticle,freeofall inAMsofPPARγknockoutmice,pointingtoaPPARγ-independentmechanismofenzyme
copyright,andmaybefreelyreproduced,distributed,
inhibition.WehypothesizedthatthismechanismwaselectrophilicS-alkylationoftargetCatS
transmitted,modified,builtupon,orotherwiseused
cysteines,andfoundthatNFAsbinddirectlytoCatSfollowingtreatmentofintactAMsand,as
byanyoneforanylawfulpurpose.Theworkismade
availableundertheCreativeCommonsCC0public suggestedbyinsilicomodelingandcalculationofrelevantparameters,elicitS-alkylationof
domaindedication. Cys25whenincubatedwithpurifiedCatS.TheseresultsdemonstratethatNFAs’electrophilic
DataAvailabilityStatement:Allrelevantdataare activity,inadditiontotheirroleasPPARγagonists,underliestheirprotectiveeffectsinchronic
withinthepaperanditsSupportingInformationfiles. obstructivepulmonarydisease(COPD)andsupporttheirtherapeuticpotentialinthisdisease.
Funding:ThisworkwassupportedbyaMerit
ReviewawardfromtheU.S.DepartmentofVeterans
AffairsandNationalInstitutesofHealthgrant
HL093196toRCR.Thefunderhadnoroleinstudy
design,datacollectionandanalysis,decisionto
Introduction
publish,orpreparationofthemanuscript.
Chronicobstructivepulmonarydisease(COPD)isthethirdleadingcauseofdeathintheUS
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. [1],characterizedbychronicinflammationofsmallairwaysanddestructionofalveolarsepta
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 1/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
withconsequentemphysema[2].Suchseptaldestructionreducesthesurfaceareaavailablefor
pulmonaryO andCO exchange,andcombineswithairwayinflammationandmucuspro-
2 2
ductiontoimpairrespiratorymechanics.BecauseCOPDisprogressiveandoftenfatal,andno
effectivetherapiesareavailabletoimpedeitscourse,identificationofnewpotentialtherapeutic
targetsisneeded.
AmongthemanycelltypesthatcontributetoCOPDpathophysiology,alveolarmacro-
phages(AMs)areespeciallycritical[3].Theyaredirectlyexposedtocigarettesmoke,the
majorriskfactorforCOPD,andothernoxiousairborneagents.SuchagentsactivateAMsto
producepro-inflammatorycytokinesthatactivateothercells,andchemokinesthatattractneu-
trophilsandTlymphocytes,prominentinCOPD-associatedinflammation.Suchactivatedand
recruitedcellslikewisesecretemediatorsthatfurtheractivateAMsinaviciouspositive-feed-
backcycle.YetAMsalsoexertaninflammation-dampeninginfluencebyphagocytosingapo-
ptoticneutrophilsandepithelialcellsthat,ifallowedtobecomenecrotic,wouldfurtherfeed
theinflammatorycycle.SuchphagocytoticactivityofAMsisreducedinCOPD,therebyexac-
erbatingpathogenesis[4].
AMsarealsotheprimarysourcesofelastin-degradingproteasesthatlargelydriveseptal
destruction[5].MostrecentattentionhasfocusedontheZn-containingmatrixmetalloprotei-
nases(MMPs),butcathepsins,includingcathepsinsK,L,andS,arealsoimportantmediators
ofseptaldegradation[6].Productionofinflammation–associatedoxidantsbyAMscanalso
contributetoseptaldestruction.
Inflammation-associatedoxidantsincludeNOandderivedreactivenitrogenspeciesthat
reactnonenzymaticallywithunsaturatedfattyacidstoproducenitratedfattyacids(NFAs),
including10-nitro-oleicacid(OA-NO )and12-nitrolinoleicacid(LNO )[7],themostpreva-
2 2
lentNOreactionproductsinthehumanbloodstream[8].NFAsarepotentiallyimportant
endogenousmodulatorsofinflammatoryprocesses[9–11],actingatleastinpartasagonistsof
thenuclearhormonereceptorperoxisomeproliferatoractivatedreceptor-γ(PPARγ)[12].
Theyarealsoreversibleelectrophiles[13].Assuch,theycanreactwiththiolgroupsincluding
thoseofcysteineresidueswithinproteinstoproduceanS-alkylbondthatiscleavable(thus
reversible)byreactionwithfreeintracellularthiolssuchasglutathione(GSH).Ithaslongbeen
observedthatspecificcysteinesarepreferentiallyalkylated,butthefactorsthatrendersuchspe-
cificcysteinesmorereadilyorstablyalkylatedareunknown.KnowncysteinetargetsofNFAS-
alkylationincludeonewithintheligand-bindingsiteofPPARγthatcontributessignificantlyto
agonistpotency[14].
ReversibleCysS-alkylationprovidesauniqueregulatorymechanismforbiologicallyactive
proteins.Forexample,reversibleelectrophilesregulateoxidativestressbyreversiblyS-alkylat-
ingtheinhibitoryproteinKeap-1,allowingactivationoftheantioxidanttranscriptionfactor
Nrf2[15].NFAsmayalsoalleviateinflammationbyS-alkylatingthepro-inflammatorytran-
scriptionfactorNF-κB[10].NosuchinfluenceofreversibleelectrophilesingeneralorNFAsin
particularhasbeenrecognizedinthepathophysiologyofCOPD.Wehypothesizedthatanti-
inflammatoryandrelatedactionsofNFAsmaymitigateCOPDpathogenesis,includingsecre-
tionoftheproteasesthatmediateseptaldestruction,andtestedthisideaandthemechanisms
involvedinAMsfromsmoke-exposedmice.
MaterialsandMethods
Animals
C57BL/6andTie2Cre-PPARγflox/floxmice[16]wereexpandedfrombreedingpairs.Micewere
housedinmicroisolatorcagesunderspecificpathogen-freeconditionsandfedautoclaved
food.Malemiceaged6–8weeks(20–25g)wereusedinallexperiments.Allstudieswere
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 2/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
performedaccordingtoprotocolsreviewedandapprovedbytheVAPittsburghHealthcare
SystemInstitutionalAnimalCareandUseCommittee.
SmokeExposure
Micewereexposedtocage-aircigarettesmoke(CS)ortofilteredairfor14days.CSwasgener-
atedbyburningfive3RF4researchcigarettes(TobaccoResearchInstitute,UniversityofKen-
tucky,Lexington,KY)accordingtotheFederalTradeCommission(FTC)protocol,eachpuff
beingof2-seconddurationand35-mlvolume,inanautomatedTE-10smokingmachine(Tea-
gueEnterprises,Davis,CA).Themachinewasadjustedtoproduce89%sidestreamand11%
mainstreamsmoke.ThechamberatmospherewasmonitoredtomaintainTPMat250mg/m3.
Twenty-fourhoursfollowingthelastexposure,micewereeuthanizedandBALfluidandlungs
werecollectedforfurtheranalysis.
BALFluidCollection,CellCountandAMIsolationfromMice
BALfluidwascollectedbyflushing3×1mlofPBScontaining0.1mMEDTAintothelungvia
atrachealcannula.ThepooledBALfluidwascentrifugedat500×gat4°Cfor5min.Pelleted
cellswerethenresuspendedin1mlofPBS.Totalcellnumberwascountedbyhemocytometer
anddifferentialcellcountwasperformedusingcytospinpreparationsstainedwithDiff-Quik
(Siemens,Newark,DE).AMswereisolatedasdescribedpreviously[17].
MeasurementofCytokine,ChemokineandCathepsinSLevels
Cellculturemediumwascollectedandstoredat–80°C.LevelsofTNF-αandKCweremea-
suredusingELISAkits(R&DSystems,Minneapolis,MN)accordingtothemanufacturer’s
instructions.CatSlevelsinmediasamplesweremeasuredusinganELISAkit(MyBioSource,
Inc,SanDiego,CA)accordingtothemanufacturer’sinstructions.
WesternBlotting
TotalproteinextractswerepreparedandWesternblottingwasperformedasdescribedprevi-
ously[18].PrimaryantibodiesagainstNF-κBp65(372),PPARγ(7196),laminB1(20682),bio-
tin(57636),CatS(6505),andβ-actin(1616)werefromSantaCruzBiotechnology(SantaCruz,
CA).Thesecondaryantibodiesdonkeyanti-mouseIR-680RD(926–68072),donkeyanti-rabbit
IR-680RD(925–68073),donkeyanti-goatIR-680RD(926–68074),goatanti-rabbitIR-800CW
(925–32211),andgoatanti-mouseIR-800CW(926–32210),werefromLI-COR(Lincoln,NE).
TheinfraredsignalwasdetectedusinganOdysseyInfraredImager(LI-COR).
Immunoprecipitation
TotalproteinextractswereimmunoprecipitatedusingtheDynabeadsProteinGImmunopre-
cipitationkit(Invitrogen,Carlsbad,CA).AntibodieswereboundtoDynabeadsProteinG,and
theDynabeads-Abcomplexwasusedtoprecipitatetargetproteinsfromthetotalprotein
extracts.Precipitateswerewashedtoremoveunboundproteinsandcomplexeswereeluted.All
sampleswereseparatedbyelectrophoresisonSDS-polyacrylamidegels,transferredtomem-
branes,andWesternblottingwasperformed.
EMSA
Nuclearproteins(5μg)wereincubatedwith2.5nMinfrareddye(IRDye)700end-labeleddou-
blestrandedconsensusoligonucleotidesforNF-κBorPPARγ(Table1)in10×bindingbuffer
(100mMTris,500mMKCl,10mMDTT;pH7.5),polydIdC(1μg/μlin10mMTris,1mM
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 3/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
Table1. Consensusoligonucleotidesemployed.
Bindingsite Prime Sequence Endlabelling
κBSite F 50-AGTTGAGGGGACTTTCCCAGGC-30 IRDye700
R 30-TCAACTCCCCTGAAAGGGTCCG-50 IRDye700
PPRESite F 50-AGACAAGTCAGAGGCCACGGT-30 IRDye700
R 30-TCTGTTCAGTCTCCGGTGCCA-50 IRDye700
NonspecificControl F 50-AGACTGGGGCTGGAGTGCGGTT-30 IRDye700
R 30-TCTGACCCCGACCTCACGCCAA-50 IRDye700
doi:10.1371/journal.pone.0153336.t001
EDTA),25mMDTTand2.5%Tween20.Sampleswerethenseparatedon5%non-denaturing
polyacrylamidegelsin1×Tris-BorateEDTAbuffer(130mMTris,pH8.3,45mMboricacid,
2.5mMEDTA).TheinfraredsignalwasdetectedusinganOdysseyInfraredImager(LI-COR).
MonocyteChemotaxisAssay
MousebloodmonocyteswereisolatedusinganEasySepmousemonocyteenrichmentkit(Stem-
CellTechnologiesInc,Vancouver,BC,Canada).Briefly,leukocytepoolsfrombloodwereincu-
batedwithanantibodymixtureagainstnon-monocytesfollowedbyabiotinselectionmixture
andmagneticparticles.LabeledcellswereremovedusingDynaMag-2magnet(Invitrogen),
resultinginhighlysignificantenrichmentofmonocytesintheremainingcellpreparation.
Blind-wellchemotaxischambersseparatedwith8-mmporepolycarbonatemembrane(Neuro
ProbeInc.,Gaithersburg,MD)wereusedtoassessmigrationofmonocytestowardcellculture
media.Theupperwellscontainedmonocytesisolatedasdescribedabove;thelowerwellswere
loadedwithculturemediasupernatantfrommacrophagesisolatedfrommiceexposedtociga-
rettesmokefor14daysandthentreatedeitherwithvehicleorwithOA-NO for6h.Thecham-
2
berswereincubatedat37°Cfor1h,afterwhichthemembraneswerefixedandstainedwithDiff-
Quikstains.Themembraneswereretrievedandmountedonglassslides,coveredwithacover-
slip,andexaminedunderamicroscope.Thenumberofcellsthatmigratedthroughthemem-
braneintothelowerchamberandcellsthatwereattachedonthelowersideofthemembrane
werecountedandtransmigrationwasexpressedas%ofmonocytesaddedinupperchamber.
ElastolyticActivityAssay
Intracellularandextracellularelastindegradationstudieswereperformedusingsolubleand
insolublefluorescein(FITC)labeledelastinrespectively(ElastinProductsCompany,Inc,
Owensville,MO).TodetermineextracellularelastolyticactivityinsolubleFITC-elastinwas
addedtoculturemediaorBALfluidandtreatedasindicated.Afterincubation,insolubleelas-
tinwasremovedbycentrifugationandfluorescenceinsupernatantswasdeterminedusing
excitationat490nmandemissionat520nm.Intracellularelastolyticactivitywasdetermined
byincubatingAMstreatedasindicatedabovewithsolubleFITC-elastin.Afterincubationcells
werewashedandmountedwithVectashieldmountingmediumwithDAPI(VectorLaborato-
ries,Burlingame,CA).TheslideswereviewedbyanOlympusFluoviewFV1000confocal
microscope(Olympus,CenterValley,PA)usinga60×objectivelensalongwithFluoviewcon-
focalsoftware(FV10-ASWv1.7,Olympus).
ChIPAssay
TheChIPassaywasperformedusingSimpleChIPEnzymaticChromatinImmunoprecipitation
kitwithmagneticbeads(CellSignalingTechnology,Beverly,MA,USA).Briefly,cellular
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 4/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
Table2. Oligonucleotideprimersemployed.
Gene PrimerSequence Tm(°C) AmpliconSize(bp)
CD36Promoter F 50-AATGCTTTATTCCTCCTTGTTTCC-30 62 96
R 50-TGCTAGAAAGGAAGTAGCTTCAG-30 62
CatSPromoter F 50-GGCTCTTCTTGATGGCTTACT-30 62 77
R 50-AGCTAGTACAGTCACCTCTAGTC-30 62
α-satellite Cat#4486,CellSignalingTechnology
doi:10.1371/journal.pone.0153336.t002
chromatinwascrosslinkedwith1%formaldehydefor10minatroomtemperature,thecross-
linkingwasstoppedwith0.125Mglycine,andcellswerewashedtwicewithicecoldPBS.
Nucleiwerepelletedanddigestedbymicrococcalnuclease.Followingsonicationandcentrifu-
gation,equalamountsofshearedchromatinwereincubatedovernightat4°Cwithantibodies,
IgGasnegativecontrol,orRNApolymeraseIIaspositivecontrol.ProteinGmagneticbeads
werethenaddedandthechromatinwasincubatedwithrotationfor2hat4°C.Analiquotof
chromatinthatwasnotincubatedwithanyantibodywasusedastheinputcontrolsample.
Antibody-boundprotein/DNAcomplexeswereelutedandsubjectedtoreal-timePCRas
describedpreviously[18]withspecificprimersforCtss(CatS),CD36,andα-satellite
(Table2).
MolecularModelingandComputerSimulationsofBindingofOA-NO
2
withCatS
Molecularmodelingandcovalentdockinganalysiswascarriedoutasdescribedpreviously
[19].TheX-raystructureofhumanCatS{PDBID1NPZ;[20]}anditsC25Smutant{PDBID
1GLO;[21]}wereobtainedfromtheRCSBProteinDataBank.Covalentdockingwasper-
formedwithOA-NO (ligand)usingDiscoveryStudio2.5(AccelrysInc.,SanDiego,CA).
2
CatSEnzymeInhibitionAssay
CatSenzymeinhibitionassaywasperformedusingtheCathepsinSDrugDiscoveryKit
(BML-AK431-0001;EnzoLifeSciences,Farmingdale,NY)accordingtothemanufacturer’s
instructions.
CatSEnzymeActivityAssay
CatSenzymeactivityincellculturemediasampleswasmeasuredbyincubatingthesesamples
withCatS-specificfluorescentsubstrate(Ac-KQKLR-AMC;600742,CaymanChemical)
accordingtothemanufacturer’sinstructions.
CatSCysMutants
AplasmidcontainingthehumanCatSgenewasobtainedfromAddgene(#11251)andsub-
clonedintoapGEX6P1plasmid.Cysteineresiduesatpositions12,25and110orallthree(12,
25and110)weremutatedtoserineusingsitedirectedmutagenesis(customcloningcorefacil-
ityatEmoryUniversity,Atlanta,GA).RecombinantproteinswereexpressedinBL21-DE3
cells(Sigma-Aldrich,St.Louis,MO),purifiedusingGSTSpinTrapcolumns(GEHealthcare
LifeSciences),andtheGSTtagwasremovedwithPreScissionProtease(GEHealthcareLife
Sciences)accordingtothemanufacturer’sinstructions.
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 5/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
CatSS-alkylation
TodemonstrateS-alkylationofCatS,humanrecombinantCatS(EMDMilliporeBillerica,
MA)andvariants−CatSWT,C12;25;110Smutant,C12Smutant,C25Smutant,andC110S
mutant−wereincubatedwithtestcompoundsfortheindicatedtimeperiodsandS-alkylation
wasanalyzedbyrunningthesesamplesinaSDS-PAGEgelundernon-reducingconditions
andperformingWesternblotsusingantibodytobiotin(SantaCruzBiotechnology).
ElectrosprayIonization-LiquidChromatography-MassSpectrometry/
MassSpectrometry(ESI-LC-MS/MS)
ESI-LC-MS/MSanalyseswereperformedonaQExactiveHybridQuadrupole-Orbitrapmass
spectrometer(ThermoScientific,Pittsburgh,PA).Theelectrospraysystememployeda3.5kV
sprayvoltageandacapillarytemperatureof260°C.
StatisticalAnalysis
Dataarepresentedasmean±SD.Differencesbetweengroupswereanalyzedusinganunpaired
t-testoranalysisofvariance,followedbyBonferronimultiplecomparisoncorrection.Analyses
wereperformedusingGraphPadPrism5.03(GraphPadSoftware,LaJolla,CA).Differences
withP<0.05wereconsideredsignificant.
Results
OA-NO reducescigarettesmoke-inducedAMactivationinmice
2
ConsideringthepivotalroleofAMsintheresponsetosmokeinhalationandCOPDpathogen-
esis,wetestedtheeffectsofNFAsonAMactivitiesrelevanttoinflammationandseptal
destructioninamousemodelofCOPD.WeexposedmicetoCSdeliveredviacageairfor14
days.SmokeexposuregreatlyincreasedthenumbersofmacrophagesinBALfluid,withlesser
increasesinthenumberofneutrophils(Fig1Aand1B).BecauseAMsrepresentedthemajority
ofinflammatorycellsfoundinBALfluidofsmoke-exposedmice,weassessedthemfurther.
InvitroOA-NO treatment(notoxicitywasobserved,S1Fig)ofAMsisolatedfromBALfluid
2
ofsmoke-exposedmicereducedbothnuclearexpressionandDNA-bindingactivityofthepro-
inflammatorytranscriptionfactorNF-κB(Fig1Cand1D),butincreasedthoseoftheanti-
inflammatorynuclearhormonereceptorPPARγ(Fig1Cand1E).Basedonthesechangesin
activityofpro-andanti-inflammatorytranscriptionalregulators,wepredictedthatexpressionof
pro-inflammatorygeneswouldbereduced,andindeedfoundthatsecretionofTNF-α(Fig1F)
andKC(Fig1G)byAMswassuppressed.Secretionofchemokineswasalsoreduced,asseen
usingamonocytetransmigrationassay(Fig1Hand1I).Assessingemphysema-causingprotease
secretionandelastindegradation,wefoundthatOA-NO treatmentmarkedlyreducedsecreted
2
CatSlevelsandactivityinculturemedium(Fig1Jand1K).Degradationofelastinbyculture
mediumofOA-NO -treatedAMs(Fig1L)andintracellularelastindegradationbywholeAMs
2
(Fig1M)weresimilarlyreduced.Thus,OA-NO suppressedboththeinflammatoryandproteo-
2
lyticactivitiesinducedbysmokeexposureinAMs.AsthesecellsplayaprominentroleinCOPD
pathophysiology[3],theseeffectscarrysignificantimplicationsformodulationofthisdisease.
OA-NO EffectsonCathepsinSExpression,butnotActivity,are
2
PPARγ-dependent
AsNFAsareknowntoactbothasreversibleelectrophilesandviaPPARγactivation,wetested
whichofthesemechanismswasresponsiblefortheobservedreductionsinCatSexpression
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 6/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
Fig1.NFAsmitigatecigarettesmoke-inducedalveolarmacrophageactivation.(A,B)C57BL/6micewereexposedtocigarettesmoke(CS)orto
filtered-airfor14days.Twenty-fourhafterthefinalexposure,BALsampleswerecollected.(A)Photomicrographs(40×objectivelens)ofDiff-Quik-stained
cellsand(B)total(Tot),neutrophil(PMN),andmacrophage(MAC)countsinBALfluidfromtheindicatedtreatmentgroups.(C-M)Alveolarmacrophages
(AMs)wereisolatedfromBALfluidofmiceexposedtoCSasdescribedaboveandwerethentreatedwith1μMOA-NO orvehicle(Veh)for6h.(C)Western
2
blotsforPPARγandp65innuclearextracts.(D,E)DNA-bindingactivityof(D)NF-κB,and(E)PPARγ,inOA-NO orVehtreatedAMextractsmeasuredby
2
EMSAusingIRDyeoligonucleotides.(F,G)Releaseof(F)TNFα.and(G)KC.(H,I)Mousemonocyteswereisolatedandtransmigrationwasassayedusing
achemotaxischamberinwhichtheywereseparatedbyan8-mmporepolycarbonatemembranefromchemoattractantculturemediafromtheabove
treatmentgroups.(H)Percentagetransmigration,and(I)photomicrographs(40×objectivelens)ofthelowersideofthepolycarbonatemembrane.(J)CatS
expressionand(K)activityinculturemediafromabovetreatmentgroups.(L,M)AMswerecollectedandtreatedasindicatedabove,thenincubatedwith
FITC-elastinforfurther1htodetermine(L)extracellularand(M)intracellularelastolyticactivity(60×objectivelens).Dataarerepresentativeoftwotothree
independentexperimentswithcellsobtainedfromn=14–18mice/group(cellsfrom3micepooledtogether).**P<0.01,***P<0.001.
doi:10.1371/journal.pone.0153336.g001
andactivityusingAMsfromwild-type(WT)andPPARγknockout(KO)micefollowing
14-dayinvivosmokeexposureand6-hinvitroexposuretoOA-NO orvehicle.Absenceof
2
PPARγinAMsfromKOmicewasconfirmedbyWesternblotting,whichalsorevealedmodest
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 7/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
Fig2.NFAs’inhibitionofCatSexpressionbutnotactivity,isPPARγ-dependent.(A)WesternblotsforPPARγandCatSinwholecellextractsfrom
AMsisolatedfromBALfluidofC57BL/6(WT)andTie2Cre-PPARγflox/flox(KO)mice.(B,C)Micewereexposedtocigarettesmoke(CS)for14days,AMs
wereisolatedandthentreatedwith0.5and1μMOA-NO orVehfor6h.(B)CatSexpressionand(C)activityinculturemedia.(D)FollowingAMisolation
2
andOA-NO (1μM)treatmentasabove,chromatinwascrosslinkedandimmunoprecipitatedwithPPARγantibody;theantibody-boundDNA-protein
2
complexeswerethensubjectedtoreal-timePCRwithprimersspecificforPPREsitesinCatSandCD36promoterregions,α-satellitewasusedascontrol.
(E)Asabove,AMswerecollectedfromCS-exposedmiceandtreatedwith5μMbiotin-labeledOA-NO orVehfor6h.Totalproteinextractswereprepared,
2
CatSwasimmunoprecipitatedandWesternblottingwasperformedwithanti-biotinand-CatSantibodies.Dataarerepresentativeoftwotothree
independentexperimentswithcellsobtainedfromn=14–18mice/group(cellsfrom3micepooledtogether).*P<0.05,**P<0.01,***P<0.001,n.s.=non-
significant.
doi:10.1371/journal.pone.0153336.g002
increasesinCatS(Fig2A).OA-NO treatmentreducedCatSexpressioninAMsfromWTbut
2
notPPARγKOmice(Fig2B),indicatingthatNFA-inducedsuppressionofCatSexpression
requiresPPARγactivation.Incontrast,NFAsreducedCatSactivityinculturemediaofAMs
toapproximatelythesameextentirrespectiveofthepresenceorabsenceofPPARγ(Fig2C).
Therefore,inhibitionofCatSactivitydoesnotdependonPPARγ,andwethushypothesizedit
reflectsS-alkylationbyNFAs.Supportingthishypothesis,wefoundthatNFAbounddirectly
toCatSfollowingincubationofCatSwithbiotin-labeledOA-NO ,immunoprecipitation,and
2
Westernblottingusinganti-biotinantibody(Fig2E).Conversely,supportingaPPARγ-depen-
dentmechanismofNFAeffectsonCatSexpression,wefoundbychromatinimmunoprecipi-
tation(ChIP)thatOA-NO treatmentincreasedPPARγbindingtoboththeCd36promoter,
2
whichisknowntobeupregulatedbyPPARγactivation,andalsotheCtss(CatS)promoter(Fig
2D).Thiswasconsistentwithpriorfindingsthatundercertaincircumstancesbindingof
PPARγtoDNAinhibitsratherthanstimulatestranscription[22].Thus,theobservedNFA
effectsonCatSexpressionandactivityweremediatedbydistinct,complementarymechanisms
inwhichelectrophilicadditionplaysaprominentrole.
OA-NO InhibitsCathepsinSbyS-alkylatingCys25
2
WenextsoughttospecificallyidentifythesiteofNFA-CatSbindingandthetypeofbond
involved.Moleculardockingstudiesrevealedcovalentbondformationbetweentheelectro-
philiccarbonofOA-NO andsulfuratomCys25ofCatS(Fig3A).Nocovalentbond
2
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 8/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
Fig3.NFAsinhibitCatSenzymaticactivitybyS-alkylation.(A-C)BindingofOA-NO toCatSwasmodeledinsilicoandevaluatedinvitro.(A)Schematic
2
representationshowingcovalentinteractionofOA-NO withCatSCys25residue.(B)Schematicrepresentationshowingabsenceofanycovalentinteraction
2
ofOA-NO withCatSCys25Sermutation.(Leftpanels)CatSisshownasaflatribboncoloredaccordingtosecondarystructure;OA-NO (yellow)isshown
2 2
asastickmodelandinteractingaminoacidsareshowninscaledball-and-stickrepresentation.(Rightpanels)CatSisshownwithinteractingaminoacid
residuesasascaledball-and-stickmodelwithlabelsandOA-NO (yellow)asastickmodel.Inbothfiguresthecovalentbondisindicatedbywhitearrowand
2
hydrogenbondsareindicatedbygreendottedlines.(C)HumanrecombinantCatSinactiveform(200ng)wasincubatedfor30minwiththeindicated
compounds.Followingincubation,S-alkylationofCatSbybiotin-labeledOA-NO intestsampleswasassessedbyWesternblottingundernon-reducing
2
conditions.(D)HumanrecombinantCatSinactiveform(6μU/μl)wasincubatedfor1hwiththeindicatedcompoundsandenzymeactivitydeterminedby
cleavageoffluorescentsubstratepeptide.Dataarerepresentativeofthreeindependentexperimentswithn=4/group.*P<0.5,***P<0.001.
doi:10.1371/journal.pone.0153336.g003
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 9/16
NFAsInhibitEmphysema-relatedCathepsinviaS-Alkylation
formationwasseenwhenCatSwithCys25toSermutatedwasused,astheoxygeninserineis
notsufficientlynucleophilictobondwiththeNFA’selectrophiliccarbon(Fig3B).
WeconfirmedthatNFAbindstoCatSproteinbyincubatingrecombinanthumanCatSin
vitrowithbiotin-labeledOA-NO followedbyWesternblotting(Fig3C).Incubationwith
2
eitherOA-NO orLNO dose-dependentlyinhibitedhumanCatSactivity,andatthehighest
2 2
dosetestedexhibitedinhibitionsimilartothatseenwiththeclassicCatSinhibitorE-64(Fig
3D).Thisinhibitionwasblockedbythethiolexchangeinhibitorbacitracin,thusconfirmingS-
alkylationasthemechanismofinhibition.
WenextsoughttospecificallyidentifythesiteofNFA-CatSbindingandthetypeofbond
involved.WemodeledCatSinsilico(Fig4)to1)determinethepresenceorabsenceofdisul-
fidebondingforeachCysresidueandtheSatom’spartialpositivecharge(Fig4A);and2)cal-
culatesolventaccessibility(Fig4B),H-bondingpotential(Fig4C),andpKa(Fig4D)foreach
ofitscysteineresidues.TheresultssuggestedthatCys25isthemostnucleophilicandthusthe
mostlikelysiteofS-alkylation.Thisconclusionwassupportedbyinsilicomodelingof
OA-NO bondedtoCys25,indicatingappropriatesolventaccessibilityandthepresenceofsta-
2
bilizingH-bonds(Fig4B–4D).
TotestwhetherCys25wasthespecificCatSresiduetowhichNFAsbondcovalently,we
incubatedbiotin-labeledOA-NO withWTCatSorCatSwithCysresiduesmutatedtoSer
2
[eitherindividuallyatCys12(C12S),Cys25(C25S),orCys110(C110S),oratallthreepositions,
(C12;25;110S)].OA-NO bindingwasthendeterminedbyWesternblotting(Fig5).OA-NO
2 2
wasboundtoCatSbyS-alkylationonlyinCatSWT,C12S,andC110Smutants;theinterac-
tionwasabsentwhenCys25wasmutatedtoSer(C12;25;110SandC25S).Theseresultsindicate
thattheS-alkylationofCatSbyOA-NO occursspecificallyatCys25.
2
TofurtherconfirmthecovalentinteractionweincubatedOA-NO (orvehicle)withasyn-
2
theticCatSpeptide(CatS23-29)containingeitherthenativeCys25(sequenceGACWAFS)or
aCys25Sersubstitution(GASWAFS),andanalyzedtheproductsbymassspectrometry,which
indicatedthatNFAwasS-alkylatedtotheCys25-containingfragment(Fig6Bandtable).As
predicted,Cys25SermutationabolishedNFAS-alkylation(Fig6Dandtable)andnoaddition
wasseenintheabsenceofNFA(Fig6Aand6Candtable).Ourinitialprediction,basedonthe
theoreticalcalculationsshowninFig4,thatCys25wouldbethealkylatedresiduewasthus
confirmed.
Discussion
WefindthatNFAtreatmentofactivatedAMsfromCS-exposedmicedownregulatesexpres-
sionandactivityofCatS,aproteaseheavilyinvolvedintheseptaldestructionthatleadsto
emphysema.BecauseNFAsareknownPPARγagonists[23],wetestedwhetherPPARγmedi-
atestheirsuppressiveeffectsonCatS,findingthatNFAsdownregulatebothCatSgeneexpres-
sionanditsenzymaticactivity.Theseactionsaremechanisticallydistinct,however,asPPARγ
deletioneliminatedNFA-induceddownregulationofCatSexpressionbutnotNFA-induced
inhibitionofCatSenzymaticactivity.FurtherstudiesdemonstratedthatNFAsinhibitCatS
activityviaelectrophilicS-alkylation.Asourdatashow,thisreversibleadditionofanNFAto
theCatSproteinisnotrandom,butisdirectedtowardaspecificcatalyticcysteine(Cys25)that
exhibitsappropriatenucleophilicity,solventaccessibility,andpotentialofsurroundingamino
acidsforformationofstabilizingH-bonds.Thus,wefoundthatNFAsactthroughdistinct
PPARγ-dependentand-independentmechanismsthatnonethelessactinadual,functionally
concertedmannertoreduceCatSlevels,leadingtotheobserveddecreaseinAMs’extracellular
andintracellularelastolyticactivity.Suchareductioninvivowouldbeexpectedtoalleviate
COPDseverityandprogressiontowardemphysema.
PLOSONE|DOI:10.1371/journal.pone.0153336 April27,2016 10/16