Hydrogen sulfide inhalation ameliorates allergen induced airway hypereactivity by modulating mast cell activation

(1)

ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al ho me p a g e :ww w . e l s e v i e r . c o m / l o c a t e / y p h r s

Hydrogen

sulﬁde

inhalation

ameliorates

allergen

induced

airway

hypereactivity

by

modulating

mast

cell

activation

Fiorentina

Roviezzo

a,1

_,

_Antonio

_Bertolino

a,1

_,

_Rosalinda

_Sorrentino

b

_,

_Michela

_Terlizzi

b

_,

Maria

Matteis

d

_,

_Vincenzo

_Calderone

c

_,

_Valentina

_Mattera

a

_,

_Alma

_Martelli

c

_,

Giuseppe

Spaziano

d

_,

_Aldo

_Pinto

b

_,

_Bruno

_D’Agostino

d

_,

_Giuseppe

_Cirino

a,∗

a_Università_di_Napoli_Federico_II,_Italy

b_Dipartimento_di_Farmacia,_Università_di_Salerno,_Italy

c_Dipartimento_di_Farmacia_Università_di_Pisa,_Italy

d_Dipartimento_di_Medicina_{Sperimentale,}_Sezione_di_Farmacologia_L._Donatelli,_Seconda_Università_degli_Studi_di_Napoli,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received9June2015

Receivedinrevisedform30July2015

Accepted30July2015

Availableonline1August2015

Keywords: Hydrogensulﬁde Mastcell Fibroblast Pulmonaryinﬂammation Airwayhypereactivity

a

b

s

t

r

a

c

t

Compellingevidencesuggeststhathydrogensulfiderepresentsanimportantgaseoustransmitterinthe mammalianrespiratorysystem.Inthepresentstudy,wehaveevaluatedtheroleofmastcellsin hydro-gensulfide-inducedeffectsonairwaysinamousemodelofasthma.Miceweresensitizedtoovalbumin andreceivedaerosolofahydrogensulfidedonor(NaHS;100ppm)startingatday7afterovalbumin challenge.Exposuretohydrogensulfideabrogatedovalbumin-inducedbronchialhypereactivityaswell astheincreaseinlungresistance.Concomitantly,hydrogensulfidepreventedmastcellactivityaswell asFGF-2andIL-13upregulation.Conversely,pulmonaryinflammationandtheincreaseinplasmatic IgElevelswerenotaffectedbyhydrogensulfide.Alackofhydrogensulfideeffectsinmastcell defi-cientmiceoccurred.Primaryfibroblastsharvestedfromovalbumin-sensitizedmiceshowedanincreased proliferationratethatwasinhibitedbyhydrogensulfideaerosol.Furthermore,ovalbumin-induced trans-differentiationofpulmonaryfibroblastsintomyofibroblastswasreversed.Finally,hydrogensulfidedid abrogateinvitrothedegranulationofthemastcell-likeRBL-2H3cellline.Similarlytotheinvivo exper-imentstheinhibitoryeffectwaspresentonlywhenthecellswereactivatedbyantigenexposure.In conclusion,inhaledhydrogensulfideimproveslungfunctionandinhibitsbronchialhyper-reactivityby modulatingmastcellsandinturnfibroblastactivation.

1. Introduction

Compellingevidencesuggeststhathydrogensulfiderepresents animportantgaseoustransmitterinthemammalianrespiratory system[1].Hydrogensulfideisanendogenousgaseoustransmitter that isproduced in manytissues primarilybycystathionine ␤-synthase(CBS)andcystathionine␥-lyase(CSE),twoPLPdependent enzymes.Athirdenzymeinvolvedinhydrogensulfideproduction is3-mercaptopyruvatesulfurtransferase(MST)thatalsoproduces hydrogensulfidebutdoesnotrequirePLP[2,3].Theseenzymesare widelyexpressedinpulmonaryandairwaytissues,theexpression patternsandtheextentoftheseenzymesarevariabledepending

∗ Correspondingauthor.

E-mailaddress:[email protected](G.Cirino).

1 _These_authors_have_equally_contributed.

onthespeciesandcelltypes[4].Ithasbeendemonstratedthat administrationofthehydrogensulfide-donor,sodium hydrosul-fide(NaHS)directlyrelaxesairwaysmoothmuscle[5].Inaddition, intraperitonealadministrationofanhydrogensulfidedonororan inhibitorofhydrogensulfidebiosynthesisalleviatesoraggravates allergen-inducedairwayhypereactivity,respectively[6,7].Studies performedusingCSEKOmicehavefurtherconfirmedthatlower hydrogen sulfide levels worsen airway hyperesponsiveness [7]. Recently,Huangetal.[5]showedthattreatingmicewithan hydro-gensulfide donorbyaerosol, airway hyperesponsiveness(AHR) wassignificantlyrelieved.Thiseffectonairwayhypereactivityhas beenassociatedtoadirectrelaxanteffectofhydrogensulfideon airway smooth muscle.Moreover,Zhang etal. [7]demonstrate thatsystemicadministrationofhydrogensulfidedonor,in addi-tiontothedirectrelaxingeffect,modulateseosinophilinfiltration aswellasTh2cytokineproduction.Hydrogensulfidecanrelaxalso humanairwaysmoothmuscle[8]andapositivecorrelationamong http://dx.doi.org/10.1016/j.phrs.2015.07.032

(2)

thedeclineinlungfunction,thedecreasesinCSEexpressionand theendogenousplasmahydrogensulﬁdeconcentrationshasbeen foundinasthmaticpatients[9].

AHRisthemainasthmafeature.Severalcausativefactorshave beenproposedsuchaschronicinflammation,airwayremodeling, smoothmusclegrowthandepithelialdamage,butthemechanisms arenotasyetclear[10].ThisisprobablybecauseAHRis multifacto-rialandthereforedifferentindistinctasthmaphenotypes[11].An essentialroleofmastcellsinthedevelopmentofmouseairway hyperesponsivenesshasbeenwidelyproposed.It is well estab-lishedthatcross-linkingofIgEAbsonmastcellsbyantigentriggers thereleaseofchemicalmediators,whichinturncausetheearly allergicreactions.Activationofmastcellsalsoleadstothe synthe-sisofcytokines,whichinturncontributetochronicinflammation. Severalstudieshavedemonstratedelevatednumberofmastcells indifferentsitesinthelungoftheasthmaticpatientsandtheir correlationwithinflammationandimpairmentofpulmonary func-tion[12–14].Recentlyithasbeenalsodemonstratedthathydrogen sulfidepreventsheartfailuredevelopmentviainhibitionofrenin releasefrommastcellsinisoproterenol-treatedrats[15].

Inlightofthesefindings,weinvestigatedifthehydrogensulfide effectonairwaysinvolvesmastcells.Thestudyhasbeenperformed byusingawellknownmurinemodelofasthmainducedby oval-bumin.ThecontributionofmastcellsinthedevelopmentofAHR hasbeenalsoassessedbyusingmastcell-deficientmice.Hydrogen sulfidehasbeenadministeredbyaerosolandbothairwayreactivity andpulmonaryinflammationhavebeenevaluated.

2. Materialandmethods

2.1. RBL-2H3cellcultureandˇ-hexosaminidasemeasurement RBL-2H3(RatBasophilicLeukemiaMastCellLine;JapanHealth SciencesFoundation)[16] weresensitized withthemonoclonal anti-dinitrophenylantibodyanti-DNPIgE(0,50␮g/ml).TheH2 S-donorNaHS(10,100␮Mand1mM;5min)or thevehiclewere addedafter24h. RBL-2H3celldegranulationwasinducedby(i) theantigen dinitrophenyl-seralbumin10ng/ml(DNP);(ii) iono-mycin1␮Mand(iii)thapsigargin1␮M.Ttriton-X-100wasadded tocauseexhaustivereleaseof␤-hexosaminidase.Allreagentswere purchasedfromSigma–Aldrich,MilanItaly.

2.2. Mice

BALB/c, mast cell-deﬁcient KitW−sh/W−sh mice [17,18] and C57Bl6/Jmice(CharlesRiver)werehousedin acontrolled envi-ronmentandprovidedwithstandardrodentchowandwater.All mousestrains(20–25g)werehousedwitha12hlightdarkcycle andwereallowedfoodandwateradlibitum.Animalcarewasin compliancewithItalianregulationsonprotectionofanimalsused forexperimentalandotherscientiﬁcpurposes(D.M.116192)as wellaswiththeEECregulations(O.J.ofE.C.L358/112/18/1986).All studieswereperformedinaccordancewithEuropeanUnion reg-ulationsforthehandlinganduseoflaboratoryanimalsandwere approvedbythelocalcommittee.

2.2.1. Antigenexposureanddrugtreatmentofmice

Micereceivedsubcutaneousadministrationofovalbumin(OVA, 100␮g)adsorbedontoAl(OH)3 atday0and7.Inapreliminary screening,wedeterminedthat theoptimaldosetobegivenby aerosolofhydrogensulﬁdewas100ppmofNaHS.Rodentswere exposedtoaerosolizedNaHSorvehicleforupto5min,dailyfor2 weeks.NaHSwasadministeredtomiceusinganaerosolexposure device(InToxProducts,N.Mex.).Thesystemconsistedofacentral chamberhavingseparateaerosol supplyandexhaustpaths.The centralchamberhad24portsthatweredirectlyconnectedtothe

aerosolsupplysystem.Micewereplaced intoindividualaerosol exposuretubesandrestrainedwithanadjustablepushplateand end capassembly sotheycouldnot turnaroundor backaway fromtheendofthetube.Therestrainttubescontainingthemice wereloadedontoportsonthecentralchamberandtheairﬂow adjustedto1l/min.Theair(breathablequalityair)ﬂowtothePARI LCStarnebulizerwasheldconstantat6.9l/min.NaHSnebulizedat 0.2ml/minanddeliveredtothemiceviatheaerosolsupplypath. Additionalair(hereafterreferredtoasdilutionair)wasdelivered tonebulizer(where)tobalancetheoverpressureoftheairused todeliveraerosolizeddrugstothemice.Twentyfourhoursafter thelastadministrationmicewereanesthetizedandsubjectedto euthanasia(Fig.2A).

2.3. Airwayreactivity 2.3.1. Bronchialreactivity

Miceweresacriﬁcedand bronchiwererapidlydissectedand cleanedfromfatandconnectivetissue. Ringsof1–2mmlength werecutandplacedinorganbaths(2,5ml)ﬁlledwithoxygenated (95%O2–5%CO2)Krebssolutionat37◦Candmountedtoisometric forcetransducers(type7006,UgoBasile,Comerio,Italy)and con-nectedtoa Powerlab800(ADInstruments).Ringswereinitially stretcheduntilarestingtensionof0.5gwasreachedandallowedto equilibrateforatleast30minduringwhichtensionwasadjusted, whennecessary,toa0.5gandbathingsolutionwasperiodically changed.Inapreliminarystudy,arestingtensionof0.5gwasfound todevelop theoptimal tensiontostimulation withcontracting agents.Ineachexperiment,bronchialringswerepreviously chal-lengedwithacetylcholine(10−6mol/l)until theresponseswere reproducible.Subsequently,bronchiwerechallengedwith carba-chol.

2.3.2. Isolatedperfusedmouselungpreparation

Themouse lungswere perfusedin a non-recirculating fash-ionthroughthepulmonaryarteryataconstantflowof1ml/min resultinginapulmonaryarterypressureof2–3cmH2O.The perfu-sionmediumusedwasRPMI1640lackingphenolred(37◦C).The lungswereventilatedbynegativepressure(−3and−9cmH2O) with90breathmin−1 and a tidalvolumeofabout200␮l.Every 5minahyperinflation(−20cmH2O)wasperformed.Artificial tho-raxchamberpressurewasmeasuredwithadifferentialpressure transducer(ValidyneDP45-24)andairflowvelocitywith pneumo-tachographtubeconnected toa differentialpressuretransducer (ValidyneDP45-15).Thelungsrespiredhumidifiedair.The arte-rialpressurewascontinuouslymonitoredbymeansofapressure transducer(IsotecHealthdyne)whichwasconnectedwiththe can-nulaendinginthepulmonaryartery.Alldataweretransmittedto acomputerandanalysedwiththePulmodynsoftware(HugoSachs Elektronik,MarchHugstetten,Germany).Thedatawereanalyzed throughthefollowingformula:P=V×C−1+RL×dV×dt−1,where Pischamberpressure,Cpulmonarycompliance,Vtidalvolume,RL airwayresistance.Theairwayresistancevalueregisteredwas cor-rectedfortheresistanceofthepneumotacometerandthetracheal cannulaof0.6cmH2Osml−1.Lungswereperfusedandventilated for45minwithoutanytreatmentinordertoobtainabaselinestate. Subsequently,lungs werechallengedwithcarbachol. Repetitive doseresponsecurveofcarbacholwasadministeredas50␮lbolus, followedbyintervalsof15min,inwhichlungswereperfusedwith bufferonly.

2.4. FlowCytometryAnalysis

Lungs weredigestedwithcollagenase (Sigma–Aldrich,Italy). Pulmonaryinﬂammatorycellsweredeterminedbyﬂowcytometry

(3)

(BD FacsCalibur, Italy) using CD11c-APC, CD11b-PeCy5.5, cKit-PeCy5.5or-PE,IgE-FITC(Bioscience,SanDiego,CA,USA).

2.5. Immunohistochemistry

Left lung lobes were paraffin-embedded and 7␮m sections wereobtained.Thedegreeofinflammationwasscoredbyblinded observers by using hematoxylin and eosin (H&E) and Periodic acid/Alcianblue/Schiffstaining(PAS).PASStaining(Sigma–Aldrich, Milan Italy) was performed according to the manufacturer’s instructionstodetectglycoprotein.PAS+cryosectionsweregraded withscores0–4todescribelowtoseverelunginflammationas follows:0:<5%;1:5–25%;2:25–50%; 3:50–75%;4:<75% pos-itivestaining/total lungarea. FGF2detectionwasperformedby usinganti-FGF-2orIgGisotypecontrol(SantaCruz,USA).Mastcell recruitmentwasevaluatedbymeansoftoluidinebluestaining(1% solution).

2.6. MeasurementofserumIgElevels

PlasmaticserumIgElevelsweremeasuredbyELISAaccordingly tothemanufacturer’sinstructions(BDPharmingen,USA). 2.7. Fibroblastactivity

2.7.1. Fibroblastculture

Miceweresacriﬁcedandthethoraxwasopened, thenlungs wereremoved. Lungs were mincedand incubated withDMEM containing15%FCS.Cultureswerereplacedthreetimesweekly. Allcultures wereevaluatedbyimmunohistochemistrytoassess vimentinandallstainedpositivelywithvimentin.Selected cul-tureswereevaluated.Fibroblastswerepassagedwithtrypsin/EDTA (0.05% trypsin,0.53mM EDTA; GIBCO). Fibroblast betweenthe thirdandthesixthpassageswereusedforassays.

2.7.2. Fibroblastproliferationanddifferentiation

Pulmonaryfibroblastswereharvestedfrommicetreatedwith vehicle,OVAorOVAandexposedtohydrogensulfide.Fibroblast proliferationwasassessedbytheMTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl)tetrazoliumbromide,colorimetricassay. Fibro-blastdifferentiationwastested by incubatingcells withmouse monoclonalantibodyagainst␣-SMA(Sigma–Aldrich,Italy)or rab-bit polyclonal antibody against vimentin (Santa Cruz, CA). For detectionfluoresceinlabeledanti-mouseIgG (ABNOVA,Italy)or Texas-Redlabeledanti-rabbitIgG(ABNOVA,Italy)wereused.The levelofmyofibroblasticdifferentiationwasmeasuredbycounting atleast200cells.

2.8. Statisticalanalysis

Dataaremeans±SEMfromatleast6miceineachgroup.The levelofstatisticalsigniﬁcancewasdeterminedbyone-wayor two-wayanalysisofvariance(ANOVA)followedbyBonferroni’stestfor multiplecomparisonsbyusingtheGraphPadPrismsoftware(USA). 3. Results

3.1. Hydrogensulﬁdeinhibitsantigen-induceddegranulationin RBL-2H3cells

A direct effect of hydrogen sulﬁde on mast cell degranula-tion wasinvestigated in vitro by using mast cell-like RBL-2H3 cellline.TheadditionoftheantigenDNP-HSAtopre-sensitized RBL-2H3cellscausedasigniﬁcantdegranulationevaluatedas ␤-hexosaminidase release (Fig.1A).Analmostequivalentlevel of

Fig.1.HydrogensulﬁdereducesIgE-mediatedRBL-H3cellsdegranulation.(A)

RBL-2H3cell degranulationwaspharmacologically inducedby(i) theantigen

dinitrophenyl-seralbumin10ng/ml(DNP),(ii)ionomycin(ION)1␮Mor(iii)

thap-sigargin(TPS)1␮M.Inmatchedwells,triton-X-100wasadded,toelicitcelllysis

andexhaustivereleaseof␤-hexosaminidase.Theconcentrationofp-nitrophenate

releaseof␤-hexosaminidasewasmeasuredat405nm.(B)Cellsweresensitised

withDNPandafter24hincubatedfor5minwitheithertheH2S-donorNaHS(10,

100␮Mand1mM)orthevehicle.CelldegranulationwasinducedbyDNP.(C)Cells

wereincubatedeitherwithNaHS(1mM)orvehiclefor5minandcelldegranulation

inducedbyIONorTPS.(**p<0.01vsvehicle;p<0.001vsvehicle).

degranulationwasinducedbythenon-antigenicstimuli thapsigar-ginandionomycin(Fig.1A).Hydrogensulfidecausedaremarkable andconcentration-dependentinhibitionoftheRBL-2H3 degranu-lationinducedbyDNP-HAS(Fig.1B).Incontrast,hydrogensulfide didnotexhibitanysignificantinhibitoryactivityonthe degranu-lationinducedeitherbythapsigarginorionomycin(Fig.1C). 3.2. HydrogensulfideinhibitsOVA-inducedairwayhypereactivity

Sensitized micewereexposedtoNaHS inhalation(100ppm; 5min) daily from day7 to21 (Fig. 2A). This dosewas shown not to cause any effect “per se”. Bronchial reactivity (Fig. 2B) andlungresistance(Fig.2C)aftercarbacholchallengewere mea-sured. Bronchi harvested fromOVA-sensitized miceshowedan increasedreactivitytocarbacholincomparisontovehicletreated mice(Fig.2B).Similarly,increasedlungresistanceswaspresentas determinedbyusingtheisolatedandperfusedlungpreparation (Fig.2C).NaHSinhalationreversedbothbronchialhypereactivity (Fig.2B)andtheincreaseoflungresistance(Fig.2C)inOVA-treated mice.Conversely,hydrogensulfideinhalationdidnotalter OVA-inducedcellinfiltrationandinflammation(Fig.2D).PASpositive stainingwasstillvisibleinpulmonaryhistologyoflungsharvested frommicesensitizedandexposedtoNaHSinhalation(Fig.2E),as alsoobservedbytheH&Estaining(Fig.2D)showingastillaltered

(4)

Fig.2.HydrogensulﬁdeinhalationabrogatesOVA-inducedairwayhyper-reactivity.(A)Schematicrepresentationoftheprotocolused.Micereceivedsubcutaneous

admin-istrationofOVAadsorbedontoalumatday0and7.Aerosoladministrationofvehicleorhydrogensulﬁde(NaHS,100ppm)wasperformeddailyfromday7today21.Twenty

fourhoursafterthelastadministrationmiceweresacriﬁced.(B)Measurementofbronchialreactivitytocarbachol.(C)Measurementoflungresistancebyusingisolated

perfusedlungpreparation.***p<0.001vsvehicle.(D)Histologicalanalysisofpulmonarysections(Hematoxylin&Eosin).(E)Gobletcellhyperplasiaevaluationbyperiodic

acid/Alcianblue/Schiffstaining(**p<0.01vsvehicle).Lungsectionshavebeenphotographedunderlightmicroscopyat10×magniﬁcation,PASpositivecriosectionswere

gradedwithscores0–4todescribelowtoseverelunginﬂammation(0:<5%;1:5–25%;2:25–50%;3:50–75%;4:<75%positivestaining/totallungarea).Dataaremeans±SEM

from6miceineachgroup.

bronchialepitheliuminOVA+NaHStreatedmicecomparedto vehi-clemice.

3.3. Hydrogensulﬁdeaffectsmastcelldegranulationandinhibits ﬁbrogeniccytokinesproduction

FlowcytometryanalysisoflungsharvestedfromOVA-sensitised micedemonstratedasignificantincreaseofmastcellrecruitment. ThemastcellswereidentifiedasCD11c+c−Kit+Ige+positivecells (Fig. 3A). Treatment of mice with NaHS neither affected mast cellcount(Fig.3B)norplasmaticIgElevels(Fig.1B).Conversely NaHSinhalationsignificantlyaffectedmastcelldegranulationas

evidentbytoluidinepositivestaining(Fig.3C).Hydrogensulﬁde reversedalsoOVA-inducedup-regulationoftwoimportant ﬁbro-geniccytokinesIL-13(Fig.3D)andFGF-2(Fig.3E).

3.4. Mastcell-deﬁcientKitW−sh/W−sh_mice_fail_to_develop_airway hyperesponsiveness

Mast cell-deﬁcient KitW−sh/W−sh _mice _exposed _to _OVA sen-sitizationdidnot developbronchial hypereactivitytocarbachol (Fig.4A).ExposureofmicetoNaHSduringsensitizationdidnot furtheraffectbronchialtone(Fig.4A).Ontheotherhand,a sig-niﬁcantairwayhypereactivitywasobservedinthewildtypemice

(5)

Fig.3.Hydrogensulfideinhibitsmastcelldegranulationandcytokinesexpressioninthelung.(A)Quantificationoflungmastcellsbyflowcytometryidentifiedas

CD11c+c−Kit+Ige+positivecells(**p<0.01vsvehicle).(B)IgEseralevelsweredeterminedbyusingspeciﬁcELISA(*p<0.05vsvehicle).(C)Toluidinebluestainingof

parafﬁn-embeddedlungsections.(D)PulmonaryIL-13expressionhasbeendeterminedbyELISA.(E)ImmunohistochemistryofFGF-2expressioninlungs,(*p<0.05vs

vehicle,#_p_<_0.05_vs_OVA,##_p_<_0.01_vs_OVA)._Data_are_means_±_SEM_from₆_mice_in_each_group.

(C57Bl6/J,datanotshown).SimilarlytowhatobservedinBALB/c mice,NaHStreatmentofsensitizedC57Bl6/Jmicereversed OVA-inducedairwayhypereactivity(datanotshown).Sensitizationof mastcell-deficientKitW−sh/W−sh_mice_failed_to_induce_a_significant increaseofbothIL-13(Fig.4B)andFGF-2(Fig.4B)expressionin lung.Treatmentofmastcell-deficientKitW−sh/W−shmicewithNaHS didnotaffectallparametersevaluated(Fig.4A–D).Thesedataimply thatmastcellistheprimarytargetofhydrogensulfide.

Inordertofurtheraddresstheroleofmastcellswetreatedmice withOVAwithoutalumusingtheprotocolreportedin supplemen-talFig.1A.Thisisachronicallergicasthmamodelmainlymastcell dependent[19].Similarly,towhatwefoundintheothermodel theincreaseinmastcellrecruitmentdrivenbythesensitization procedurewasnotaffectedbyNaHS(SupplementalFig.1B),while mastcelldegranulationwasblunted(SupplementalFig.1C).Also inthisexperimentalsettingbronchialhypereactivitytocarbachol wasabrogatedbyhydrogensulﬁdeaerosol(SupplementalFig.1D). PASstainingevidencedaweakincreaseofgobletcellssuggesting amarginalroleofmastcellsintheregulationofmucusproduction (SupplementalFig.1E).Finallyhydrogensulﬁdetreatmentdidnot alterpulmonarymorphology.

3.5. FibroblastsharvestedfrommiceexposedtoNaHSdiplayeda reducedactivation

Primary pulmonary fibroblasts were harvested from mice exposed toOVA and theirproliferation rate and differentiation evaluatedin vitro. Our datashowthat primary fibroblasts har-vestedfromsensitizedmicedisplayedanincreasedproliferation rate(Fig.5A)aswellasincreasedexpressionof␣-SMA(Fig.5B and C) as compared to vehicle treated mice When fibroblasts wereharvestedfrommicetreatedwithhydrogensulphideaerosol in vivoboth theincreasedproliferation rateand differentiation were reverted(Fig. 5).Thus, miceexposed to hydrogensulfide inhalationdisplayedasignificantreductionoffibroblastactivation beyondreduction ofairwayhyper-reactivity.Inordertofurther demonstratethatthefibroblastscontributetomast-cell-dependent airwayhyper-reactivity,weevaluatedfibroblastactivationalsoin themodelofmastcell-dependenthyper-reactivityusingthe proto-coldescribedinSupplementalFig.1A.Ourdatashowthatprimary fibroblastsharvestedfromthesemicedisplayedanincreasedrate of proliferation (Supplemental Fig. 2A) aswell as an increased expressionof ␣-SMA(SupplementalFig.2Band C).Whenmice

(6)

Fig.4. Lackofeffectofhydrogensulfideinmastcell-deficientKitW−sh/W−sh_mice._Mast_{cell-deficient}_KitW−sh/W−sh_mice_received_subcutaneous_{administration}_of_OVA_adsorbed

ontoalumatday0and7.Miceweretreatedwitheithervehicleorhydrogensulﬁdeaerosoldailyfromday7to21.Twentyfourhoursafterthelastadministrationmice

weresacriﬁced.(A)Bronchialreactivitytocarbacholwasassessed.(B)Gobletcellhyperplasiaevaluationbyperiodicacid/Alcianblue/Schiffstaining.Lungsectionshave

beenphotographedunderlightmicroscopyat10×magniﬁcation,PASpositivecriosectionsweregradedwithscores0–4todescribelowtoseverelunginﬂammation(0:

<5%;1:5–25%;2:25–50%;3:50–75%;4:<75%positivestaining/totallungarea,**p<0.01vsvehicle).(C)PulmonaryIL-13expressionhasbeendeterminedbyELISA.(D)

ImmunohistochemistryofFGF-2pulmonaryexpression.Dataaremeans±SEMfrom6miceineachgroup.

weretreatedwithaerosolofhydrogensulﬁde,boththeincreased ﬁbroblastproliferationanddifferentiationmeasuredexvivowere prevented (Supplemental Fig. 2) as well as the airway hyper-reactivity.

4. Discussion

Thereisa growingbody of evidence suggestingthat hydro-gensulfidemightbeofclinicalbenefitinpulmonarydiseases.In particular,aroleforhydrogensulfideinmodulatingairway hypere-activityandremodelinghasbeensuggested.Airwayhypereactivity isanimportantpathophysiologicalcharacteristicofasthmaandit islinkedtobothairwayinflammationandremodeling.Although thepathogenicmechanisms arenot completely understood,an essentialroleisattributedtomastcells.Here wehaveassessed whetherthebeneficialeffectsofhydrogensulfideonlungfunction arerelatedtomastcellactivation.

Preliminarily,wehavedeterminedtheeffectofNaHSonmast cellsbyusingthemastcell-likeRBL-2H3cellline.Incubationof RBL-2H3withNaHSdidnothaveanyeffectbyitselfuptoadose of1mM.Conversely,hydrogensulfide causedaremarkableand concentrationdependentinhibitionoftheRBL-2H3degranulation whencellsweresensitizedandchallengedwithantigen. Hydro-gensulfidedidnotaffecteitherthapsigarginorionomycin-induced RBL-2H3degranulation.Theseresultsclearlysuggestthathydrogen sulfidecanexplicateitsactiononlyonsensitizedcells.

Inordertobetterunderstandthisphenomenonwetestedthe effectofhydrogensulﬁde invivobyusingOVA-sensitizedmice. Exposureofallergen-sensitizedmicetohydrogensulﬁdeaerosol

reversedbronchialhyperactivityasalreadydemonstratedby oth-ers [6]. In addition, we also show that the aerosol treatment abrogatedtheincreaseinlungresistance.Allergen-inducedAHR wascoupledto anincreased presenceof mast cells withinthe pulmonarytissue,asdeterminedbyFACSanalysis.Aerosol admin-istrationofhydrogensulfide neitherinfluenced therecruitment oflungmastcellsorplasmalevelsofIgEinducedbyOVA sensiti-zation.However,inanalogywithRBL-2H3cells,hydrogensulfide abrogatedinvivomastcelldegranulation.Thiseffectwascoupled toinhibitionofthepulmonaryexpressionofFGF-2aswellasIL-13. Ontheotherhand,hydrogensulfidedidnotaffectallergen-induced gobletcellmetaplasia,mucushypersecretionandpulmonarycell infiltration,allparametersindicativeoflunginflammation[20,21]. Therefore,thehydrogensulfidebeneficialeffectsonairway hyper-eactivitywellcorrelateswiththereversionofmastcellactivation. Indeed, the data obtained with the experiments in vitro with RBL cells, taken together with the in vivo functional data and the modulationof IL-13 and FGF2pulmonary expression were allindicativeforacentralroleformastcellinhydrogen sulfide-mediatedactions.Tofurtherunderstandthelinkamonghydrogen sulfide, mast cells and bronchial hypereactivity, we tested hydrogen sulfide effect in mast cell-deficient KitW−sh/W−sh mice.Bronchi,harvestedfromOVA-sensitizedmastcell-deficient KitW−sh/W−sh_mice,_did_not_develop_bronchial_{hypereactivity.}_The upregulation of IL-13 and FGF2 was absent, too. Conversely, theinflammatory features i.e. allergen-induced cellinfiltration, gobletcellmetaplasiaandmucushypersecretionwereallstill evi-dent.ExposureofOVA-sensitizedmastcell-deficientKitW−sh/W−sh micetohydrogensulfideaerosoldidnotaffecttheinflammatory

(7)

Fig.5.Hydrogensulfideinhibitsfibroblastactivationinthelung.(A)LungprimaryfibroblastswereharvestedfromOVA-sensitizedmiceexposedtovehicleorNaHSaerosol.

FortheinvivoprotocolseeFig.2A.TheMTTcolorimetricassaywasusedtoassessﬁbroblastproliferationinvitro(**p<0.01vsvehicle).(B)Percentageofmyoﬁbroblasts

(*p<0.05vsvehicle).(C)Fibroblastswerelabelledwithanti-vimentinantibody(red).NucleiwerestainedwithDAPI(blue).Myoﬁbroblastdifferentiationwasdeterminedby

detectionofexpressionofalphaSMA(green).Lowestpanelsrepresentthemergeofupperandcentralpanels.Theimageswereacquiredwithconfocalmicroscope(Leica).

Thepercentageofmyoﬁbroblastsareeasilydistinguishablesincetheydisplaywell-developed,thickandalphaSMA-positivestressﬁbers.(Forinterpretationofthereferences

tocolorinthisﬁgurelegend,thereaderisreferredtothewebversionofthisarticle.)

reaction.ThereforethelackofmastcellsinKitW−sh/W−shmice mim-icsthehydrogensulfideeffectsinOVA-sensitizedmice.Inaddition thesedataimplythattheupregulationofOVA-inducedIL-13and FGF-2expressioninthelungismastcelldependentandtherefore hydrogensulfideeffectisstrictlydependentfromantigen-activated mast cells. However,we still had to definehow this hydrogen sulfideeffectonmastcells translatesintoa beneficial effecton airwayhypereactivity.Mastcells synthesizeandsecretea large

number ofpro-inflammatorycytokineswhich regulateboth IgE synthesisandthedevelopmentofinflammationandseveral pro-fibrogeniccytokines.Someofthesemastcellproductse.g.,IL-13 areknowntoinducebronchialhypereactivityinthemouse inde-pendentoftheinflammatoryresponseandenhancedtheresponses in cultured humanairway smooth muscle[12,13].Accordingly, aerosolofhydrogensulfidepreventedFGF2andIL-13 upregula-tion,thatareknowntoactivatelungfibroblastsinhumansandin

(8)

experimentalanimals[22,23].Inadditionthereisincreasing evi-denceinhumansforastrongcorrelationbetweensub-epithelial fibrosisand airwayhyper-reactivity[24,25]assuggestedbythe increasednumbersoffibroblasts/myofibroblastsfoundinthe air-waysofasthmaticpatients[26,27].Inordertoaddressthisissue weharvestedfibroblastsfromOVA-sensitizedmice.These fibrob-lastsdisplayedanincreasedproliferationrateinvitroaswellasa significantincreaseinthepercentoffibroblastsconvertedin myofi-broblasts. When fibroblasts were harvested from mice treated invivowithhydrogensulfideaerosol,theincreasedfibroblast pro-liferationrateaswellastheirdifferentiationintomyofibroblasts weresignificantlyinhibited.Thesedatasuggestthatthehydrogen sulfide beneficial effects involvesalsomodulationof mast cell-mediatedfibroblastactivation.Thishypothesisissupportedbythe findingthatinmastcelldeficientmiceFGF-2aswellasIL-13 up-regulationarenotinducedbyOVA-sensitization.Thisisalsointune withthefindingthatIL-13+cells,presentwithintheairwaysmooth muscleofasthmaticpatients,arepredominantlymastcells[27]. IL-13hasbeenalsoshowntoelicitmanyofthefeaturesofhuman asthmaaswellastoregulateproductionofseveralpro-fibrogenic cytokinesincludingFGF-2[23,28].

Inconclusion,herewedemonstratethatexposureofsensitized micetohydrogensulfideinhalationreversesairwayhypereactivity. Thehydrogensulfideeffectstemsfromthemodulationoperatedby hydrogensulfideonmastcellasdemonstratedbythelackofeffect ofNaHSinKitW−sh/W−sh_mice._The_beneficial_effect_on_bronchial hypereactivityinvolvesthemastcell-inducedfibroblastactivation andinturninhibitionofIL-13andFGF-2expression.Thisis par-ticularinterestingontheclinicalside sincerecentlyit hasbeen demonstratedthatFGF-2levelsininduced sputumsignificantly correlatewithpulmonaryfunctionandhavebeenproposedtobe apossiblebiomarkerofasthmaticairwayremodeling[28].These dataalsosuggestthataerosol treatmentwithH2Sdonorscould beexploitedastherapeuticcomplementarytherapeuticapproach inlungdiseasessuchasthmaaswellasotherrespiratory fibrotic-relateddiseasessuchasidiopathicpulmonaryfibrosisandchronic obstructivepulmonarydisease.

Conﬂictofinterest

Iwishtoconfirmthattherearenoknownconflictsofinterest associatedwiththispublicationandtherehasbeennosignificant financialsupportforthisworkthatcouldhaveinfluencedits out-come.

Iconfirmthatthemanuscripthasbeenreadandapprovedbyall namedauthorsandthattherearenootherpersonswhosatisfied thecriteriaforauthorshipbutarenotlisted.Wefurtherconfirm thattheorderofauthorslistedinthemanuscripthasbeenapproved byallofus.

Acknowledgments

ThisworkhasbeenﬁnanciallysupportedbytheprojectPRIN 2012andCREME

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.phrs.2015.07.032 References

[1]Y.Chen,R.Wang,Themessageintheair:hydrogensulﬁdemetabolismin chronicrespiratorydiseases,Respir.Physiol.Neurobiol.184(2012)130–138.

[2]N.Nagahara,M.Nagano,T.Ito,K.Shimamura,T.Akimoto,H.Suzuki, Antioxidantenzyme,3-mercaptopyruvatesulfurtransferase-knockoutmice

exhibitincreasedanxiety-likebehaviors:amodelforhuman mercaptolactate-cysteinedisulﬁduria,Sci.Rep.3(2013)1986.

[3]J.E.Dominy,M.H.Stipanuk,Newrolesforcysteineandtranssulfuration enzymes:productionofH2S,aneuromodulatorandsmoothmusclerelaxant,

Nutr.Rev.62(2004)348–353.

[4]P.Wang,G.Zhang,T.Wondimu,B.Ross,R.Wang,Hydrogensulﬁdeand asthma,Exp.Physiol.96(2011)847–852.

[5]J.Huang,Y.L.Luo,Y.Hao,Y.L.Zhang,P.X.Chen,J.W.Xu,M.H.Chen,Y.F.Luo, N.S.Zhong,J.Xu,W.L.Zhou,Cellularmechanismunderlyinghydrogensulﬁde inducedmousetrachealsmoothmusclerelaxation:roleofBKCa,Eur.J. Pharmacol.741(2014)55–63.

[6]Y.H.Chen,R.Wu,B.Geng,Y.F.Qi,P.P.Wang,W.Z.Yao,C.S.Tang,Endogenous hydrogensulﬁdereducesairwayinﬂammationandremodelinginaratmodel ofasthma,Cytokine45(2009)117–123.

[7]G.Zhang,P.Wang,G.Yang,Q.Cao,R.Wang,Theinhibitoryroleofhydrogen sulﬁdeinairwayhyperresponsivenessandinﬂammationinamousemodelof asthma,Am.J.Pathol.182(2013)1188–1195.

[8]R.Fitzgerald,B.DeSantiago,D.Y.Lee,G.Yang,J.Y.Kim,D.B.Foster,Y.Chan-Li, M.R.Horton,R.A.Panettieri,R.Wang,S.S.An,H2Srelaxesisolatedhuman airwaysmoothmusclecellsviathesarcolemmalK(ATP)channel,Biochem. Biophys.Res.Commun.446(2014)393–398.

[9]M.Tian,Y.Wang,Y.Q.Lu,M.Yan,Y.H.Jiang,D.Y.Zhao,Correlationbetween serumH2Sandpulmonaryfunctioninchildrenwithbronchialasthma,Mol.

Med.Rep.6(2012)335–338.

[10]W.W.Busse,Therelationshipofairwayhyperresponsivenessandairway inﬂammation:airwayhyperresponsivenessinasthma:itsmeasurementand clinicalsigniﬁcance,Chest(2010)4S–10S.

[11]S.E.Wenzel,Asthmaphenotypes:theevolutionfromclinicaltomolecular approaches,Nat.Med.18(2012)716–725.

[12]P.Bradding,S.T.Holgate,Immunopathologyandhumanmastcellcytokines, Crit.Rev.Oncol.Hematol.31(1999)119–133.

[13]P.Bradding,A.F.Walls,S.T.Holgate,Theroleofthemastcellinthe pathophysiologyofasthma,J.AllergyClin.Immunol.117(2006)1277–1284.

[14]C.E.Brightling,F.A.Symon,S.T.Holgate,A.J.Wardlaw,I.D.Pavord,P.Bradding, Interleukin-4and-13expressionisco-localizedtomastcellswithinthe airwaysmoothmuscleinasthma,Clin.Exp.Allergy33(2003)1711–1716.

[15]Y.H.Liu,M.Lu,Z.Z.Xie,F.Hua,L.Xie,J.H.Gao,Y.H.Koh,J.S.Bian,Hydrogen sulﬁdepreventsheartfailuredevelopmentviainhibitionofreninrelease frommastcellsinisoproterenol-treatedrats,Antioxid.RedoxSignal.20 (2014)759–769.

[16]A.Rashid,E.Sadroddiny,H.T.Ye,A.Vratimos,S.Sabban,E.Carey,B.Helm, Diagnosticandtherapeuticapplicationsofratbasophilicleukemiacells,Mol. Immunol.52(2012)224–228.

[17]T.Tono,T.Tsujimura,U.Koshimizu,T.Kasugai,K.Isozaki,S.Nishikawa,c-kit Genewasnottranscribedinculturedmastcellsofmastcell-deﬁcient Wsh/Wshmicethathaveanormalnumberoferythrocytesandanormalc-kit codingregion,Blood80(1992)1448–1453.

[18]G.Berrozpe,I.Timokhina,S.Yukl,Y.Tajima,M.Ono,TheW(sh),W(57),and PhKitexpressionmutationsdeﬁnetissue-speciﬁccontrolelementslocated between−23and−154kbupstreamofKit,Blood94(1999)2658–2666.

[19]C.M.Williams,S.J.Galli,Mastcellscanamplifyairwayreactivityandfeatures ofchronicinﬂammationinanasthmamodelinmice,J.Exp.Med.192(2000) 455–462.

[20]O.Boucherat,J.Boczkowski,L.Jeannotte,C.Delacourt,Cellularandmolecular mechanismsofgobletcellmetaplasiaintherespiratoryairways,Exp.Lung Res.39(2013)207–216.

[21]D.R.Curran,L.Cohn,Advancesinmucouscellmetaplasia:aplugformucusas atherapeuticfocusinchronicairwaydisease,Am.J.Respir.Cell.Mol.Biol.42 (2010)268–275.

[22]M.S.Wilson,T.A.Wynn,Pulmonaryﬁbrosis:pathogenesis,etiologyand regulation,MucosalImmunol.2(2009)103–121.

[23]H.Kanazawa,J.Yoshikawa,Effectofbeclomethasonedipropionateonbasic ﬁbroblastgrowthfactorlevelsininducedsputumsamplesfromasthmatic patients,Ann.AllergyAsthmaImmunol.95(2005)546–550.

[24]L.P.Boulet,M.Laviolette,H.Turcotte,A.Cartier,M.Dugas,J.L.Malo,M.Boutet, Bronchialsubepithelialﬁbrosiscorrelateswithairwayresponsivenessto methacholine,Chest112(1997)45–52.

[25]K.Shiba,K.Kasahara,H.Nakajima,M.Adachi,Structuralchangesofthe airwaywallimpairrespiratoryfunction,eveninmildasthma,Chest122 (2002)1622–1626.

[26]W.R.Roche,R.Beasley,J.H.Williams,S.T.Holgate,Subepithelialﬁbrosisinthe bronchiofasthmatics,Lancet1(1989)520–524.

[27]M.Hoshino,Y.Nakamura,J.J.Sim,Expressionofgrowthfactorsand remodellingoftheairwaywallinbronchialasthma,Thorax53(1998)21–27.

[28]E.Y.Bissonnette,A.M.Madore,J.Chakir,M.Laviolette,L.P.Boulet,Q.Hamid,C. Bergeron,K.Maghni,C.Laprise,Fibroblastgrowthfactor-2isasputum remodelingbiomarkerofsevereasthma,J.Asthma51(2013)119–126.