Neutrophils and neutrophil serine proteases are increased in the spleens of estrogen-treated

21 Pages·2017·1.81 MB·English

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Open Access Publications
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Rujuan Dai
Virginia Tech
Catharine Cowan
Virginia Tech
Bettina Heid
Virginia Tech
Deena Khan
Virginia Tech
Zhihong Liang
Virginia Tech
See next page for additional authors
Follow this and additional works at: https://digitalcommons.wustl.edu/open_access_pubs
RReeccoommmmeennddeedd CCiittaattiioonn
Dai, Rujuan; Cowan, Catharine; Heid, Bettina; Khan, Deena; Liang, Zhihong; Pham, Christine T. N.; and
Ahmed, S. Ansar, ,"Neutrophils and neutrophil serine proteases are increased in the spleens of estrogen-
treated C57BL/6 mice and several strains of spontaneous lupus-prone mice." PLoS One. 12,2. e0172105.
(2017).
https://digitalcommons.wustl.edu/open_access_pubs/5762
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Rujuan Dai, Catharine Cowan, Bettina Heid, Deena Khan, Zhihong Liang, Christine T. N. Pham, and S. Ansar
Ahmed
This open access publication is available at Digital Commons@Becker: https://digitalcommons.wustl.edu/
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RESEARCHARTICLE
Neutrophils and neutrophil serine proteases
are increased in the spleens of estrogen-
treated C57BL/6 mice and several strains of
spontaneous lupus-prone mice
RujuanDai1,CatharineCowan1,BettinaHeid1,DeenaKhan1,ZhihongLiang1¤,
ChristineT.N.Pham2,S.AnsarAhmed1*
a1111111111 1 InfectiousDiseaseResearchFacility(IDRF),DepartmentofBiomedicalSciencesandPathobiology,
Virginia-MarylandCollegeofVeterinaryMedicine(VMCVM),VirginiaTech,Blacksburg,Virginia,United
a1111111111
StatesofAmerica,2 DepartmentofMedicine,DivisionofRheumatology,WashingtonUniversitySchoolof
a1111111111
Medicine,St.Louis,Missouri,UnitedStatesofAmerica
a1111111111
a1111111111 ¤ Currentaddress:LaboratoryofFoodSafetyandMolecularBiology,CollegeofFoodScienceand
NutritionalEngineering,ChinaAgriculturalUniversity,Beijing,PRChina.
*[email protected]
Abstract
OPENACCESS
Citation:DaiR,CowanC,HeidB,KhanD,LiangZ,
Estrogen,anaturalimmunomodulator,regulatesthedevelopmentandfunctionofdiverse
PhamCTN,etal.(2017)Neutrophilsandneutrophil
serineproteasesareincreasedinthespleensof immunecelltypes.Thereisnowrenewedattentiononneutrophilsandneutrophilserinepro-
estrogen-treatedC57BL/6miceandseveralstrains teases(NSPs)suchasneutrophilelastase(NE),proteinase3(PR3),andcathepsinG(CG)
ofspontaneouslupus-pronemice.PLoSONE
ininflammationandautoimmunity.Inthisstudy,wefoundthatalthoughestrogentreatment
12(2):e0172105.doi:10.1371/journal.
significantlyreducedtotalsplenocytesnumber,itmarkedlyincreasedthesplenicneutrophil
pone.0172105
absolutenumbersinestrogen-treatedC57BL/6(B6)micewhencomparedtoplacebocon-
Editor:CharafBenarafa,UniversitatBern,
trols.Concomitantly,thelevelsofNSPsandmyeloperoxidase(MPO)werehighlyupregu-
SWITZERLAND
latedinthesplenocytesfromestrogen-treatedmice.DespitethecriticalroleofNSPsinthe
Received:October3,2016
regulationofnon-infectiousinflammation,byemployingNE-/-/PR3-/-/CG-/-tripleknockout
Accepted:January31,2017
mice,wedemonstratedthattheabsenceofNSPsaffectedneitherestrogen’sabilityto
Published:February13,2017 increasesplenicneutrophilsnortheinductionofinflammatorymediators(IFNγ,IL-1β,IL-6,
Copyright:©2017Daietal.Thisisanopenaccess TNFα,MCP-1,andNO)fromexvivoactivatedsplenocytes.Depletionofneutrophilsinvitro
articledistributedunderthetermsoftheCreative insplenocyteswithanti-Ly6GantibodyalsohadnoobviouseffectonNSPexpressionor
CommonsAttributionLicense,whichpermits
LPS-inducedIFNγandMCP-1.ThesedatasuggestthatestrogenaugmentsNSPs,which
unrestricteduse,distribution,andreproductionin
appearstobeindependentofenhancingexvivoinflammatoryresponses.Sinceestrogen
anymedium,providedtheoriginalauthorand
sourcearecredited. hasbeenimplicatedinregulatingseveralexperimentalautoimmunediseases,weextended
ourobservationsinestrogen-treatedB6micetospontaneousautoimmune-pronefemale
DataAvailabilityStatement:Allrelevantdataare
withinthepaperanditsSupportingInformation MRL-lpr,B6-lprandNZB/W mice.Therewasaremarkablecommonalitywithregardsto
F1
files. theincreaseofneutrophilsandconcomitantincreaseofNSPsandMPOinthespleniccells
Funding:Thisworkwassupportedbythegrants ofdifferentstrainsofautoimmune-pronemiceandestrogen-treatedB6mice.Collectively,
fromNationalInstitutesofHealth(NIH,5RO1 sinceNSPsandneutrophilsareinvolvedindiversepro-inflammatoryactivities,thesedata
AI051880)andInterdepartmentalFundtoSAA.The
suggestapotentialpathologicimplicationofincreasedneutrophilsandNSPsthatmeritsfur-
generationofNSPtripleknockoutmousemodel
wassupportbythegrantsfromNIH(AI049261 therinvestigation.
andAI051436)toCP.Thepublicationfeeforthis
articleissupportedbyVirginiaTech’sOpenAccess
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 1/19
Estrogenregulationofneutrophilserineproteases
SubventionFund.Thefundershadnoroleinstudy Introduction
design,datacollectionandanalysis,decisionto
Estrogenhasbeenshowntoregulatetheimmunesystemofbothnormalandautoimmune
publish,orpreparationofthemanuscript.
individualseitherviaactivationofestrogenreceptorα(ERα)and/orERβorthroughER-inde-
Competinginterests:Theauthorshavedeclared
pendentmechanisms[1–5].Ithasbeenreportedthatinvivoestrogenexposurepromotesthe
thatnocompetinginterestsexist.
productionofinflammatorycytokinessuchasinterferon-gamma(IFNγ),Interleukin(IL)-6,
IL-1β,chemokinessuchasmonocytechemoattractantprotein(MCP)-1andMCP-5),and
inflammatorymoleculessuchasnitricoxide(NO)inConcanavalinA(ConA)orlipopolysac-
charide(LPS)-activatedmousespleniclymphoidcellsand/orperitonealmacrophages[6–9].
Further,estrogeniscapableofpromotingBcellsurvivalandactivationorbreakdownofBcell
tolerancetoinducelupus-relatedserologyandpathologyinnon-autoimmunemice[10–13].
Together,thesedatademonstrateapivotalroleofestrogenintheregulationofTandBlym-
phocyte-mediatedinflammationandautoimmuneresponses.Whiletheregulatoryroleof
estrogenonTandBlymphocytesiswelldocumented,itseffectsonneutrophilsremainslargely
unknown.
Neutrophils,amajorleucocytesubsetofinnateimmunecells,arethefirstlineofcellular
defenseagainstinvadingpathogens.Neutrophilscounterinvadingpathogensviaavarietyof
mechanismsthatincludephagocytosis,respiratoryburst,andrecentlyidentifiedNETosis[14,
15].Neutrophilderivedserineproteases(NSPs)includingneutrophilelastase(NE),proteinase
3(PR3),andcathepsinG(CG)areessentialforneutrophilsscavengingofinfectiousagents.In
addition,NSPsplayimportantrolesintheregulationofnon-infectiousinflammatory
responsesviaproteolyticprocessingofcytokines,chemokines,andsignalingmoleculessuchas
NF-κBandp21[14,16,17].Furthermore,NSPscanregulateinflammationviaactivationof
cellsurfacereceptorssuchasintegrins,protease-activatedreceptors(PARs),andtoll-like
receptors(TLRs)[14,16–18].Inadditiontotheirprimaryroleininnateimmuneresponses
andinflammation,neutrophilsarealsocriticallyinvolvedintheadaptiveimmuneresponseby
attractingTcellstositesofinflammationand/orbyprimingandengagingTcellactivation
[19,20].
Sexdifferencesinneutrophilcountshavelongbeenobservedinmenandwomen.Women
usuallyhavehigherneutrophilnumbersthanmen[21],whichcould,inpart,contributeto
strongerimmuneresponsesinwomencomparedtomen.Thepotentialeffectofestrogenon
neutrophilsissuggestedbytheobservationthattheneutrophilcountsinwomenfluctuatedur-
ingthemenstrualcycleandthatincreasedneutrophilpercentagesareassociatedwithhigher
serumestradiol[22–24].Todate,thereislimiteddatawithregardtoestrogeneffectsonneu-
trophilsandneutrophils-mediatedonsiteinflammatoryresponses.Previousstudiesin1980s’
havedocumentedthatestrogenimpairedhematopoiesiswithdecreasedbonemarrowcellular-
ity,causinglymphopeniaandneutropeniainestrogen-treatedmice[25,26].Alaterstudy
howeverhasshownthatestrogenanditsmetaboliteswereabletostimulategranulocyticdiffer-
entiationinmyoblastsandinducedneutrophiliainmice[27].Estrogentreatmentwasableto
reducethevascularinjuryresponseviainhibitinginflammatorymediatorproductionand
thenattenuatingneutrophilinfiltrationtoinjuredarteries[28].However,inadifferentmurine
influenzainfectionmodel,estrogentreatmentenhancedpulmonaryrecruitmentofneutro-
philstopotentiatevirus-specificCD8+Tcellsresponseforvirusclearance[29].Thissuggests
thattheeffectofestrogenonneutrophilsdependsontheexperimentalcontextandtissuetype.
Inthisstudy,weinvestigatedtheestrogeneffectonneutrophilsinbonemarrow,bloodand
spleniclymphoidtissuestoenableabetterunderstandingofthepotentialbroadtissueeffects
ofestrogenonneutrophils.Ourstudyclearlyshowsthatestrogenupregulatesneutrophilsin
theabovelymphoidorgansandpromotesNSPexpressioninwholesplenocytes.Abnormal
expressionandfunctionofNSPshasbeenimplicatedinthepathogenesisofmanychronic
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 2/19
Estrogenregulationofneutrophilserineproteases
autoimmuneinflammatorydiseasesincludingSLE[14,30].Nevertheless,depletionofNSPsin
vivoinmiceanddepletionofneutrophilsinvitroinsplenocyteshadnoobviouseffectonLPS
inducedinflammatoryresponsesinsplenocytesofestrogen-treatedmice.Thissuggeststhat
increasedneutrophilsandNSPsarenotdirectlyinvolvedinestrogen-mediatedpromotionof
inflammatoryresponses.Sinceestrogenhasbeenreportedtoinducelupus-relatedautoim-
muneinflammatoryparameters[10–13],wethereforeinvestigatedwhethertherearealso
changesofneutrophilsandNSPsinthespleenofthreedifferentgeneticallylupus-prone
murinemodels(MRL-lpr,B6-lpr,andNZB/W ),whichmanifestvariedformsoflupusand
F1
othersystemicautoimmunediseases.Thefindingofasimilaraugmentationofneutrophils
andNSPsinthespleensofdifferentspontaneousmurinelupusmodelsandestrogen-treated
wild-typeB6micesuggestsapotentialsignificanceofestrogenupregulationofneutrophilsand
NSPsinautoinflammation.
Materialsandmethods
Ethicsstatementandmice
AllanimalexperimentalproceduresandhousinghavebeenapprovedbytheInstitutionalAni-
malCareandUseCommittee(IACUC)ofVirginiaTech(ProtocolID#12-131-CVM).The
experimentalmicewereeuthanizedbycervicaldislocationinstrictaccordancewithapproved
IACUCprotocolandregulations.Tominimizesufferingandtoensureasuccessfuleuthanasia
ofmicewithinseconds,cervicaldislocationwascarriedoutonlybywell-trainedandapproved
researchstaff.AllmicewerehousedinourAAALACaccreditedanimalfacilityattheVirginia-
MarylandCollegeofVeterinaryMedicine(VMCVM),VirginiaTech.Micewerefedwitha
commercial7013NIH-31Modified6%Mouse/RatSterilizableDiet(HarlanLaboratory,
Madison,WI,USA)andgivenwateradlibitum.
Threetofourweek-oldmaleC57BL/6(B6)micewerepurchasedfromtheCharlesRiver
Laboratories,USA.TheNE-/-/PR3-/-/CG-/-tripleknockout(NECGPR3deficient[31],referred
asNSP-/-inthetext)breedersonB6backgroundwerekindlyprovidedbyDr.ChristineT.N.
PhamfromtheWashingtonUniversityMedicalSchoolandbredinouranimalfacility.Geneti-
callyautoimmune-pronefemaleMRL/MpJ-Faslpr/J(MRL-lpr,stockNo:006825),B6.
MRL-Faslpr/J(B6-lpr,stockNo:000482),NZBWF1/J(NZB/W ,stockNo:100008),andtheir
F1
respectivecontrolfemaleMRL/MpJ(MRL,stockNo:000486),B6,andNZW/LacJ(NZW,
stockNo:001058)micewerepurchasedfromTheJacksonLaboratory,ME,USA.Thesethree
strainsofmicespontaneouslydevelopdifferentmanifestationsoflupusandotherformsofsys-
temicautoimmunediseases.ThefemaleMRL-lpr,B6-lpr,andtheircontrolfemaleMRLand
B6micewereeuthanizedat3–4monthsold;thefemaleNZB/W anditscontrolfemaleNZW
F1
micewereeuthanizedat7–9monthsoldfortheexperimentalanalysis.
Forestrogenimplanttreatment,the4–5week-oldmaleB6andNSP-/-micewereorchidec-
tomizedandimplantedwith17-βestradiol(Sigma-Aldrich,SaintLouis,MO,USA)orempty
(placebocontrol)silasticimplantsfollowingourstandardizedlabprotocol[8,32–34].After
seventoeightweeksoftreatment,themicewereeuthanizedtoisolatesplenocytesand/orbone
marrowcellsforexperimentalanalysis.Theserumestrogenlevelsinestrogen-implantedmice
generatedbythisstandardlabpracticehavebeenreportedpreviously(rangedfrom140to270
pg/mlat7wksoftreatment)[8,33].
Splenocytepreparationandculture
Mousesplenocyteswereisolatedusingwell-establishedlabproceduresthathavebeen
describedindetailbefore[8,32–34].Thesplenocyteswereresuspendedat5×106cells/mlin
phenolredfreeRPMI-1640medium(MediatechInc,Manassas,VA,USA)supplementedwith
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 3/19
Estrogenregulationofneutrophilserineproteases
10%charcoal-strippedfetalbovineserum(AtlantaBiologicals,FloweryBranch,GA,USA),2
mML-glutamine(HyCloneLabsInc,Logan,UT,USA),100IU/mlpenicillinand100μg/ml
streptomycin(HyClone),and1%non-essentialaminoacids(HyClone).Theinadvertent
potentialestrogeniceffectofculturemediawascarefullyavoidedbytheuseofphenolredfree
RPMIandcharcoal-strippedfetalbovineserum.
Toactivatesplenocytes,2.5x106cellswereseededin24-wellplatesandstimulatedwith500
pg/mlofLPS(Sigma-Aldrich)asdesignatedintheensuingfiguresandfigurelegends.Thecell
pelletswerecollectedforWesternblotanalysisandtheculturesupernatantswerecollectedfor
analysisofcytokineandchemokinelevelsbytheELISAassay.
Westernblotting
ThewholecellextractswerepreparedbylysingthecellpelletwithCelLyticMCellLysis
Reagent(Sigma-Aldrich).Westernblottingwasperformedperourpreviouslypublishedpro-
tocoltoanalyzetheproteinexpressioninthewholecellextracts[33,35].Theanti-NE(N18,
sc-9518),anti-PR3(p20,sc-19748),andanti-iNOS(M19,sc-650)antibodieswerepurchased
fromSantaCruzBiotechnologyInc.,PasoRobles,CA,USA.Theanti-CC/DPPI(AF1034-SP)
andanti-MPO(AF3667-SP)antibodieswerepurchasedfromNovusBiologicals,Littleton,CO,
USA.Theproteinloadingcontrolantibody,anti-β-actinantibody(ab8227),wasobtained
fromAbcamInc.,Cambridge,MA,USA.
Proteaseandelastaseactivityassay
TheEnZCheckproteaseAssay(E6638)kitandEnZCheckelastaseassay(E12056)kit(Invi-
trogen,GrandIsland,NY,USA)wereusedtomeasuretheproteaseandelastaseactivities,
respectivelyinwholesplenocytes.Briefly,thefreshlyisolatedsplenocyteswerelysedwith
CelLytic™MCellLysisReagent(Sigma-Aldrich).Allsampleswereadjustedwiththelysis
buffertothesameconcentration,andequalamountsofproteinfromeachsamplewereincu-
batedwithBODIPY1FLdyeconjugatedsubstrates(caseinforproteaseassayandDQ™elas-
tinforelastaseassay)in1Xdigestionbufferfor24hrs.Thefluorescencesignalthatreflects
proteaseactivitywasmeasuredbyusingaSpectraMaxGeminiXPSmicroplatereader
(MolecularDevice,Sunnyvale,CA,USA)withexcitation485nm/emission538nm.Since
DQ™elastincanalsobedigestedbyproteasesotherthanelastase,aselectiveinhibitorofelas-
taseN-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone(25μM-100μM)wasadded
totheseparateparallelassayreactionfollowingmanufacturer’sinstructiontodeterminethe
specificityofelastaseactivity.
Assayofserineproteaseactivityinlivingsplenocytes
TheFluorescent-LabeledInhibitorsofSerineProtease(FLISP)™detectionkits(Immuno-
ChemistryTechnologiesLLC,Bloomington,MN,USA)wereutilizedtomeasureintracellular
serineproteaseactivityinthelivingsplenocytes.TheCarboxyfluorescein(FAM)-Spacer-Phe-
nylalanine(Phe)-ChloromethylKetone(CMK)FLISPassaykit(FSFCK)andFAM-Spacer-
Leucine(Leu)-CMKFLISPassaykit(FSLCK)candetectchymotrypsin-likeserineproteases
thatfavortargetingaminoacidphenylalanineandleucine,respectively.Briefly,200μlofsple-
nocytesat5x106/mlwereplatedinUbottom96wellcultureplatesandincubatedwith7.5μM
ofFSFCKorFSLCKreagentat37˚C,5%CO incubatorfor1.5to2hrs.Then,thecellswere
2
washedthreetimeswithwashbuffer,resuspendedwithPBSbuffercontaining0.5%BSA,and
analyzedbyFlowCytometertodeterminetheFAMsignalintensity,whichreflectstheserine
proteaseactivity.
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 4/19
Estrogenregulationofneutrophilserineproteases
Depletionofneutrophilsinvitrofromsplenocytes
TheEasySep™MouseStreptavidinRapidSpheres™IsolationKit(STEMCELLTechnologies
Inc.,Vancouver,BC,Canada)wasusedtodepletesplenicneutrophilsinvitro.Briefly,thesple-
nocyteswerecollectedandsuspendedinMACSbuffer(PBScontaining0.5%BSAand2mM
EDTA)at1x108/ml.NormalRatserum(50μl/ml)wasaddedtothecellsbeforetheadditionof
specificantibody.Todepleteneutrophils,biotinconjugatedanti-mouseLy6Goranti-mouse
Gr-1antibody(Biolegend,SanDiego,CA,USA)wasaddedintoa5mlpolystyreneround-bot-
tomtubecontaining1.5x107splenocytes(150μl)ataconcentrationof2μg/mlandthenincu-
batedatRTfor10minutes,andthenincubationwithstreptavidinbeads(25μl/ml)atRTfor
2.5minutes.Afteradding1.5mlMACSbuffer,thetube(withoutcap)wasplacedintomagnet
immediately,incubatedatRTfor2.5minutes.Subsequently,theneutrophil-depletedspleno-
cyteswerecollectedinanewcollectiontube,pelleted,andresuspendedincompleteRPMI
mediumat5x106/mlforcellculture.Aliquotofsplenocytesthatwasincubatedwithstreptavi-
dinbeadsonlywasservedascontrol(No-Abcontrol).
Real-timeRT-PCR
TotalRNAwasisolatedfromfreshlypreparedsplenocytesandpurifiedspleniccellssubsets
usingRNAeasyminikit(Qiagen,Valencia,CA,USA)ormirVanamiRNAisolationkits
(Ambion).AnycontaminatedresidualDNAwasremovedeitherbyperformingon-column
DNasedigestionduringRNAisolation(RNAeasyminikit)orbyusingRQ1RNase-freeDNA-
ase(Promega,Madison,WI,USA)afterRNAisextracted(mirVanamiRNAisolationkit)per
themanufacturer’sinstruction.TheiScriptone-stepRT-PCRwithSYBRgreenkit(Bio-Rad,
Hercules,CA,USA)wasusedforquantifyinggeneexpression.Quantitect10×PCRprimer
mixesformouseNE,PR3,CG,myeloperoxidase(MPO),andβ-actinwerepurchasedfrom
Qiagen.TherelativeNSPmRNAexpressionlevelswerenormalizedtoβ-actinlevelsandcalcu-
latedusingthe2−ΔΔCt(Livak)method.
Detectionofinflammatorymoleculesinculturesupernatant
Asreportedpreviously[33–35],theleveloftheNOindicator,nitriteincellculturesupernatant
wasmeasuredbytheGriessassay.ThelevelsofTh1cytokinesIFNγ,IL-1β,IL-6,TNFα,and
Th2cytokineIL-10weremeasuredbyCiraplexmultiplexChemiluminescentAssaykit
(AushonBiosystemInc.,Billerica,MA,USA).ThechemokineMCP-1levelwasmeasured
withmouseMCP-1ELISAMAX™Deluxekit(BiolegendInc.,SanDiego,CA,USA).
Statisticalanalysis
Allvalueswerereportedasmean±SEM.Twotailed,unpairedttestswereperformedtoassess
statisticalsignificanceofmRNAexpressionlevels,proteaseactivitiesbetweentwobiological
groups(placebovsestrogen;MRLvsMRL-lpr;B6vsB6-lpr;NZWvsNZB/W ).Pairedttests
F1
wereperformedtoassessthestatisticalsignificancebetweencontrolandspecifictreatedsam-
ples(withoutinhibitorvswithinhibitor;No-Abcontrolvsanti-Ly6Goranti-Gr-1).
Results
Increasedproteaseandelastaseactivityinsplenocytesfromestrogen-
treatedB6mice
WehavepreviouslyreportedaserineproteasemediatedtruncationofSTAT-1andNF-κBp65
proteinsinthenuclearextractsofsplenocytesfromestrogen-treatedB6mice[35],which
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 5/19
Estrogenregulationofneutrophilserineproteases
Fig1.Theproteaseandelastaseactivitiesareincreasedinsplenocytesfromestrogen-treatedmice
whencomparedtoplacebocontrols.(A)Thetotalproteaseactivityinfreshlyisolatedsplenocytesfrom
placebo-andestrogen-treatedB6micewasdeterminedbyEnzChekProteaseAssaykit.Thegraphshowsthe
mean±SEM(n(cid:21)7).(B)Thechymotrypsin-likeserineproteaseactivityinwholelivingsplenocyteswasdetected
withFLISP™serineproteasedetectionkit.FreshlyisolatedsplenocyteswerestainedwithFSFCK(upperleft
panel)orFSLCK(upperrightpanel)FLISPreagent,whichdetectedchymotrypsin-likeserineproteasesfavoring
phenylalanineandleucine,respectively.TheFAMsignalintensityinlivingsplenocytes(gatedonpropidium
iodide(PI)negativecells)wasanalyzedbyflowcytometry.Therepresentativeflowscatterplotsareshown.The
tableinthelowerpanelshowstheFAMvalue(mean±SEM)summarizedfromtheFlowdata(n(cid:21)6).(C)The
elastaseactivityinfreshlyisolatedsplenocytesfromplacebo-andestrogen-treatedB6micewasdeterminedby
EnZCheckelastaseassaykitwithorwithouttheadditionofinhibitorinthereaction.Meanactivity±SEM(n(cid:21)4)
isshown.(D)Thegraphdemonstratesthattheactivityofpurifiedporcinepancreaticelastasewascompletely
blockedbyaselectiveinhibitor,N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketoneat100μM
concentration.Eitherunpairedstudentttest(placebovsestrogen)orpairedstudentttest(withoutinhibitorvs
withinhibitor)wereperformed;*,p<0.05,**p<0.01,and***,p<0.001.
doi:10.1371/journal.pone.0172105.g001
suggestedapotentialregulatoryeffectofestrogenonserineproteases.Wethereforemeasured
thetotalproteaseactivityinwholecelllysatesoffreshlyisolatedsplenocytesfromplacebo-and
estrogen-treatedmice.Asexpected,theproteaseactivitywassignificantlyincreasedinspleno-
cytelysatesfromestrogen-treatedB6micecomparedtoplacebocontrols(Fig1A).Byusing
FLISPSerineProteaseDetectionAssay,wefurtherdemonstratedthattherewasasignificantly
higherchymotrypsin-likeserineproteaseactivityinlivingsplenocytesfromestrogen-treated
micewhencomparedtoplacebocontrols(Fig1B).Wethenmeasuredthespecificelastase
activityinsplenocytesusingacommerciallyavailableEnZCheckelastaseassaykit.Asshown
inFig1C,theproteaseactivitywassignificantlyincreasedinsplenocytesfromestrogen-treated
mice(withoutinhibitor:placebovsestrogen).Theadditionofaselectiveelastaseinhibitor,N-
methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone(100μM)significantlyreducedthe
proteaseactivity(estrogen:withoutinhibitorvswithinhibitor),suggestingamajor
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 6/19
Estrogenregulationofneutrophilserineproteases
contributionofelastasetotheincreasedproteaseactivityobservedinsplenocytesfromestro-
gen-treatedmice(Fig1C).Whiletheselectiveelastaseinhibitorat100μMcompletelyblocked
theactivityofthepositivecontrol(purifiedporcinepancreaticelastase,Fig1D),itonlypar-
tiallyinhibitedtheproteaseactivityinsplenocytesfromestrogen-treatedmice(Fig1C).This
suggeststhatinadditiontoelastase,estrogenalsoupregulatedothertypesofproteases/serine
proteases.
Enhancedneutrophilserineproteaseexpressioninsplenocytesfrom
estrogen-treatedB6mice
Whilethereareavarietyofserineproteasesproducedindifferentimmunecelltypes,NSPsare
ofparticularinterestbecauseoftheirroleintheregulationofnon-infectiousinflammation
[17].Withthefindingofincreasedelastaseactivityinestrogen-treatedsplenocytes,wenext
examinedwhetherestrogentreatmentaffectstheexpressionofNEorotherNSPsinspleno-
cytes.Real-timeRT-PCRanalysisindicatedthatmRNAexpressionlevelsofallthreeNSPs
(NE,PR3,andCG)weresubstantiallyincreasedinthefreshlyisolatedsplenocytesfromestro-
gen-treatedmicewhencomparedtoplacebocontrols(Fig2A).Estrogentreatmentledtoan
over10-foldincreaseinNEmRNAlevelandanover80-foldincreaseinPR3andCGmRNA
levelsinsplenocytes.InaccordancewithincreasedmRNAlevels,theproteinexpressionof
NSPssuchasNEandPR3werealsosignificantlyenhancedinsplenocytesfromestrogen-
treatedB6micewhencomparedtoplacebocontrols(Fig2B).WewereunabletoanalyzeCG
proteinlevelinthesesamplesbecausetheCGantibodydoesnotworkwellwithourwestern
blottingsystem.CathepsinC(CC,alsocalledDipeptidylpeptidaseI(DPPI))isrequiredforthe
fullactivationofneutrophilderivedNE,PR3,andCG[36].Westernblottingrevealedsimilar
expressionlevelsofCC/DPPIinthesplenocytesofplaceboandestrogen-treatedmice(Fig2C).
Invivoestrogenexposureincreasesthepercentageofneutrophilsin
spleen,blood,andbonemarrow
Wenextdeterminedwhetherestrogenaltersthepercentageofneutrophilsinvariouslym-
phoidtissues.Theflowcytometricanalysisrevealedthatthepercentageofmatureneutrophils
(Gr-1highCD11b+)wassignificantlyincreasedinsplenocytes(Fig3A),peripheralblood(Fig
3B),andbonemarrow(Fig3C)cellsfromestrogen-treatedmicewhencomparedtoplacebo-
treatedmice.Consistentwithourlong-termobservationthatestrogendecreasesspleencellu-
larity,therewasasignificantdecreaseoftotalsplenocytescounts(Fig3D).However,therewas
stillasignificantincreaseinabsoluteneutrophilnumberinthespleen(Fig3E).Myeloperoxi-
dase(MPO),amajorcomponentofazurophilicgranules,ismostabundantlyexpressedinneu-
trophilgranulocytes.Accompanyingtheincreasedneutrophilcountsinthespleen,therewas
alsoasubstantialupregulationofMPOmRNA(Fig3F)andprotein(Fig3G)levelsinthesple-
nocytesfromestrogen-treatedB6mice.
Estrogen-mediatedpromotionofinflammatorymoleculesand
neutrophilsoccursdespitethedepletionofNSPs
NSPshavebeenshowntoplayanimportantroleintheregulationofinflammatoryresponses,
especiallyatthesiteofinflammation[14,16,36].TounderstandwhetherincreasedNSPscon-
tributetoestrogen-mediatedpromotionofinflammationinsplenocytes,weutilizedtripleNSP
knockout(NSP-/-)miceandsubjectedthesemicetoestrogentreatment.AsindicatedinFig
4A,NEandPR3proteinswerenotdetectableinthesplenocytesofNSP-/-mice(inbothpla-
ceboandestrogentreatedmice).SimilartothatobservedinwildtypeB6mice,MPOwasalso
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 7/19
Estrogenregulationofneutrophilserineproteases
Fig2.NSPexpressionlevelsareupregulatedinthesplenocytesfromestrogen-treatedB6micewhen
comparedtoplacebocontrols.(A)Real-timeRT-PCRanalysisoftherelativemRNAexpressionlevelsof
NE,PR3,andCGinthesplenocytesfromplacebo-andestrogen-treatedB6mice.Thegraphrepresentsthe
means±SEMs(n(cid:21)4).Studentttests(placebovsestrogen)werepreformed;*,p<0.05;**,p<0.01;and
***,p<0.001.(BandC)WesternblotanalysisofNEandPR3(B),CC/DPPI(C)proteinexpressionlevelsin
thewholesplenocyteextractsfromplacebo-andestrogen-treatedmice.β-actinwasprobedasaprotein
loadingcontrol.
doi:10.1371/journal.pone.0172105.g002
increasedinsplenocytesfromestrogen-treatedNSP-/-whencomparedtoplacebo-treated
NSP-/-mice.CC/DPPIexpressionlevelinwildtypemicewassimilartothatinNSP-/-mice,
eitherwithorwithoutestrogentreatment(Fig4A).ByusingtheEnZCheckelastaseassaykit,
wedemonstratedthatdepletionofNSPsabolishedtheincreaseofproteaseactivityinestro-
gen-treatedB6mice(Fig4B).ThisdatafurthersuggeststhattheupregulationofNSPsdirectly
contributedtotheobservedincreaseinproteaseactivityinsplenocytesfromestrogen-treated
B6mice.
WeinitiallyhypothesizedthatdepletionofNSPswouldimpedetheeffectofestrogenon
inflammatorymediatorsinexvivo-activatedsplenocytes.Nevertheless,similartoestrogen-
treatedwildtypeB6mice,inLPS-activatedsplenocytesfromestrogen-treatedNSP-/-triple
knockoutmicetherewasenhancedproductionofinflammatorymediatorsthatincludeIFNγ
PLOSONE|DOI:10.1371/journal.pone.0172105 February13,2017 8/19