Journal of Reproduction and Development, Vol. 63, No 4 ...
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—Original Article— Advanced glycation end products regulate interleukin-1 β production in human placenta Kotomi SENO 1) , Saoko SASE 1) , Ayae OZEKI 1) , Hironori TAKAHASHI 2) , Akihide OHKUCHI 2) , Hirotada SUZUKI 2) , Shigeki MATSUBARA 2) , Hisataka IWATA 1) , Takehito KUWAYAMA 1) and Koumei SHIRASUNA 1) 1) Laboratory of Animal Reproduction, Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan 2) Department of Obstetrics and Gynecology, Jichi Medical University, Tochigi 329-0498, Japan Abstract. Maternal obesity is a major risk factor for pregnancy complications, causing inflammatory cytokine release in the placenta, including interleukin-1β (IL-1β), IL-6, and IL-8. Pregnant women with obesity develop accelerated systemic and placental inflammation with elevated circulating advanced glycation end products (AGEs). IL-1β is a pivotal inflammatory cytokine associated with obesity and pregnancy complications, and its production is regulated by NLR family pyrin domain- containing 3 (NLRP3) inflammasomes. Here, we investigated whether AGEs are involved in the activation of NLRP3 inflammasomes using human placental tissues and placental cell line. In human placental tissue cultures, AGEs significantly increased IL-1β secretion, as well as IL-1β and NLRP3 mRNA expression. In human placental cell culture, although AGE treatment did not stimulate IL-1β secretion, AGEs significantly increased IL-1β mRNA expression and intracellular IL-1β production. After pre-incubation with AGEs, nano-silica treatment (well known as an inflammasome activator) increased IL-1β secretion in placental cells. However, after pre-incubation with lipopolysaccharide to produce pro-IL-1β, AGE treatment did not affect IL-1β secretion in placental cells. These findings suggest that AGEs stimulate pro-IL-1β production within placental cells, but do not activate inflammasomes to stimulate IL-1β secretion. Furthermore, using pharmacological inhibitors, we demonstrated that AGE-induced inflammatory cytokines are dependent on MAPK/NF-κB/AP-1 signaling and reactive oxygen species production in placental cells. In conclusion, AGEs regulate pro-IL-1β production and inflammatory responses, resulting in the activation of NLRP3 inflammasomes in human placenta. These results suggest that AGEs, as an endogenous and sterile danger signal, may contribute to chronic placental cytokine production. Key words: Advanced glycation end products, Inflammation, NLRP3 inflammasome, Placenta (J. Reprod. Dev. 63: 401–408, 2017) M aternal obesity has recently been increasing in developed countries [1]. Obesity is a major risk factor in pregnancy complications, such as gestational diabetes, spontaneous miscarriage, intrauterine growth restriction, and preeclampsia [1, 2]. Obesity represents a low-grade chronic systemic inflammation [3], and maternal obesity increases the risk of the offspring developing obesity and insulin resistance in the later stages of life [4–8]. The placenta is a vital organ for pregnancy and produces cyto- kines to regulate placental function [9]. Levels of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor-α (TNFA), are increased in the serum and placental tissue of pregnant women with obesity when compared with those in pregnant women of normal weight [4, 8, 10, 11]. In addition, pregnant women with obesity also develop accelerated systemic and placental inflam- mation with elevated circulating advanced glycation end products (AGEs) [10, 12]. AGEs consist of heterogeneous, reactive, and irreversibly crosslinked molecules formed from the non-enzymatic glycation of proteins, lipids, and nucleic acids [13, 14]. In general, AGEs interact with the receptor for advanced glycation end product (RAGE) and/or toll-like receptor 4 (TLR4) to induce inflammatory responses (the production of cytokines) [15, 16]. However, the mechanism underlying these inflammatory responses via AGEs during pregnancy remains unclear. IL-1β is one of the pivotal inflammatory cytokines associated with obesity and pregnancy complications, and its production is regulated by nucleotide-binding oligomerization domain-like recep- tor pyrin domain-containing 3 (NLRP3) inflammasomes [17–20]. NLRP3 inflammasomes comprise three different proteins—NLRP3, an apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1 (CASP1, known as IL- 1β-converting enzyme). In most cells, optimal IL-1β production is dependent on two separate signals [17]. Signal 1 is a stimulus involving lipopolysaccharide (LPS) and is needed to produce the IL-1β precursor (pro-IL-1β) in cells before inflammasome activation. Signal 2 consists of IL-1β secretion and is regulated by CASP1 activation, which is induced by NLRP3 inflammasome activators, such asATP, Received: March 12, 2017 Accepted: April 27, 2017 Published online in J-STAGE: May 18, 2017 ©2017 by the Society for Reproduction and Development Correspondence: K Shirasuna (e-mail: [email protected]) This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/) Journal of Reproduction and Development, Vol. 63, No 4, 2017
Transcript of Journal of Reproduction and Development, Vol. 63, No 4 ...
—Original Article—
Advanced glycation end products regulate interleukin-1β production in human placentaKotomi SENO1), Saoko SASE1), Ayae OZEKI1), Hironori TAKAHASHI2), Akihide OHKUCHI2), Hirotada SUZUKI2), Shigeki MATSUBARA2), Hisataka IWATA1), Takehito KUWAYAMA1) and Koumei SHIRASUNA1)
1)Laboratory of Animal Reproduction, Department of Animal Science, Tokyo University of Agriculture, Kanagawa 243-0034, Japan
2)Department of Obstetrics and Gynecology, Jichi Medical University, Tochigi 329-0498, Japan
Abstract. Maternalobesityisamajorriskfactorforpregnancycomplications,causinginflammatorycytokinereleaseintheplacenta,includinginterleukin-1β(IL-1β),IL-6,andIL-8.Pregnantwomenwithobesitydevelopacceleratedsystemicandplacentalinflammationwithelevatedcirculatingadvancedglycationendproducts(AGEs).IL-1βisapivotalinflammatorycytokineassociatedwithobesityandpregnancycomplications,anditsproductionisregulatedbyNLRfamilypyrindomain-containing 3 (NLRP3) inflammasomes. Here, we investigated whetherAGEs are involved in the activation of NLRP3inflammasomesusinghumanplacentaltissuesandplacentalcellline.Inhumanplacentaltissuecultures,AGEssignificantlyincreasedIL-1βsecretion,aswellasIL-1β and NLRP3mRNAexpression.Inhumanplacentalcellculture,althoughAGEtreatmentdidnotstimulateIL-1βsecretion,AGEssignificantlyincreasedIL-1βmRNAexpressionandintracellularIL-1βproduction.Afterpre-incubationwithAGEs,nano-silica treatment (wellknownas an inflammasomeactivator) increasedIL-1β secretion in placental cells. However, after pre-incubation with lipopolysaccharide to produce pro-IL-1β, AGEtreatmentdidnotaffectIL-1βsecretioninplacentalcells.ThesefindingssuggestthatAGEsstimulatepro-IL-1βproductionwithinplacentalcells,butdonotactivateinflammasomestostimulateIL-1βsecretion.Furthermore,usingpharmacologicalinhibitors,wedemonstratedthatAGE-inducedinflammatorycytokinesaredependentonMAPK/NF-κB/AP-1signalingandreactiveoxygenspeciesproductioninplacentalcells.Inconclusion,AGEsregulatepro-IL-1βproductionandinflammatoryresponses,resultingintheactivationofNLRP3inflammasomesinhumanplacenta.TheseresultssuggestthatAGEs,asanendogenousandsteriledangersignal,maycontributetochronicplacentalcytokineproduction.Key words: Advancedglycationendproducts,Inflammation,NLRP3inflammasome,Placenta
(J. Reprod. Dev. 63: 401–408, 2017)
Maternalobesityhasrecentlybeenincreasingindevelopedcountries[1].Obesityisamajorriskfactorinpregnancy
complications,suchasgestationaldiabetes,spontaneousmiscarriage,intrauterinegrowthrestriction,andpreeclampsia[1,2].Obesityrepresentsalow-gradechronicsystemicinflammation[3],andmaternalobesityincreasestheriskoftheoffspringdevelopingobesityandinsulinresistanceinthelaterstagesoflife[4–8].Theplacentaisavitalorganforpregnancyandproducescyto-
kinestoregulateplacentalfunction[9].Levelsofpro-inflammatorycytokines,suchasinterleukin(IL)-1β,IL-6,IL-8andtumornecrosisfactor-α(TNFA),areincreasedintheserumandplacentaltissueofpregnantwomenwithobesitywhencomparedwiththoseinpregnantwomenofnormalweight[4,8,10,11].Inaddition,pregnantwomenwithobesityalsodevelopacceleratedsystemicandplacentalinflam-
mationwithelevatedcirculatingadvancedglycationendproducts(AGEs)[10,12].AGEsconsistofheterogeneous,reactive,andirreversiblycrosslinkedmoleculesformedfromthenon-enzymaticglycationofproteins,lipids,andnucleicacids[13,14].Ingeneral,AGEsinteractwiththereceptorforadvancedglycationendproduct(RAGE)and/ortoll-likereceptor4(TLR4)toinduceinflammatoryresponses(theproductionofcytokines)[15,16].However,themechanismunderlyingtheseinflammatoryresponsesviaAGEsduringpregnancyremainsunclear.IL-1βisoneofthepivotalinflammatorycytokinesassociated
withobesityandpregnancycomplications,anditsproductionisregulatedbynucleotide-bindingoligomerizationdomain-likerecep-torpyrindomain-containing3(NLRP3)inflammasomes[17–20].NLRP3inflammasomescomprisethreedifferentproteins—NLRP3,an apoptosis-associated speck-like protein containing a caspase recruitmentdomain(ASC),andcaspase-1(CASP1,knownasIL-1β-convertingenzyme).Inmostcells,optimalIL-1βproductionisdependentontwoseparatesignals[17].Signal1isastimulusinvolvinglipopolysaccharide(LPS)andisneededtoproducetheIL-1βprecursor(pro-IL-1β)incellsbeforeinflammasomeactivation.Signal2consistsofIL-1βsecretionandisregulatedbyCASP1activation,whichisinducedbyNLRP3inflammasomeactivators,suchasATP,
Received:March12,2017Accepted:April27,2017PublishedonlineinJ-STAGE:May18,2017©2017bytheSocietyforReproductionandDevelopmentCorrespondence:KShirasuna(e-mail:[email protected])This is anopen-access articledistributedunder the termsof theCreativeCommonsAttributionNon-CommercialNoDerivatives(by-nc-nd)License.(CC-BY-NC-ND4.0:https://creativecommons.org/licenses/by-nc-nd/4.0/)
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nano-silica,nigericin,orcholesterolcrystal[17–20].NLRP3inflam-masomesareinvolvedinthepathogenesisofinflammatorydiseases,includingmetabolicsyndrome,type2diabetes,cardiovasculardiseases,gout,andsilicosis[17–20].Ithasbeenreportedthaturicacidandnano-silica,aknownNLRP3activator,stimulatesIL-1βsecretionviatheNLRP3inflammasomeinhumantrophoblastcells[21].Inaddition,weandothergroupshaverecentlyreportedthatpregnancycomplicationsareimprovedinNLRP3-deficientmicebecauseplacentalinflammationissuppressed[22–24].However,theroleofNLRP3inflammasomesinplacentalfunctionispoorlyunderstood.Inthepresentstudy,wehypothesizedthatobesity-relatedAGEs
stimulateIL-1βproductionandsecretionviatheNLRP3inflamma-some.Totestthishypothesis,weinvestigatedtheeffectofAGEsonmRNAexpressionofNLRP3inflammasomeconstitutedmoleculesandIL-1βsecretionusinghumanplacentaltissuesandhumantrophoblastcell line in vitro.
Materials and Methods
Tissue collectionHumanplacentaewereobtainedfromatotalofthreewomen,
whodeliveredhealthy,singletoninfantsatterm(patientswhogaveinformedconsentwereobtainedusingprotocolsapprovedbytheJichiMedicalUniversityEthicsCommittee).Tissueswereobtainedwithin10minofdeliveryanddissectedfragmentswereplacedinice-coldphosphatebufferedsaline(PBS).Placentaltissuewasbluntdissectedtoremovevisibleconnectivetissueandcutintosmallpieces(40–60mgwetweight).Theseplacentaltissuepieceswereplacedonacellcultureinsertmembrane(0.4µmmembrane,24-wellplate,ThermoFisherScientific,Waltham,MA,USA)with1mlDulbecco’smodifiedEagle’smedium/F-12(DMEM/F-12;LifeTechnologies,Carlsbad,CA,USA)supplementedwithantibioticsincludingamphotericinBandgentamicin(Sigma-Aldrich,St.Louis,MO,USA),and5%fetalcalfserum(FCS,ICNPharmaceuticals,CostaMesa,CA,USA).
Cell culture of human trophoblast cell lineHumanfirst-trimestertrophoblastcells(Sw.71,trophoblastcell
line)werekindlyprovidedbyProfessorGilMor[25].TheSw.71cellsproducedvarioustypesofcytokines,includingIL-1β,IL-6,IL-8,andTNFA,wereusedasin vitromodelofuterineimplantation[26].IthasalsobeenreportedthatSw.71cellsexpresskeyinflammasomecomponents,includingNLRP3andASC[27].CellswereculturedinDMEM/F-12supplementedwithantibiotics,sodiumpyruvate(WakoPureChemicalIndustries,Osaka,Japan),non-essentialaminoacids(WakoPureChemicalIndustries),and5%FCS.Afterreachingconfluence,thecellswereharvestedandplatedataconcentrationof1×105cells/wellina48-wellcultureplate(ThermoFisherScientific).
Experimental conditionsPlacentaltissuecultureexperiment;humanplacentaltissueswere
incubatedwithglycoaldehyde-AGEs-BSA(200μg/ml;BioVision,Milpitas,CA,USA),BSAasacontrolforAGEs(BioVision),orLPS(100ng/ml;Sigma-Aldrich)for6hat37°C.Supernatantandtissueswerecollectedforenzyme-linkedimmunosorbentassay
(ELISA),westernblotting,andreal-timeRT-PCR,andstoredat–20°Cor–80°Cbeforeuse.Placentalcellcultureexperiment;Sw.71placentalcellswere
washedtwiceinPBSandtreatedwithAGEs-BSA(200μg/ml),BSA,orLPS(100ng/ml)for24hat37°C.AfterserumstarvationsandprimingwithAGEsorLPSfor24h,cellsweretreatedwithnano-silica(100μg/ml,MicromodPartikeltechnologieGmbH,Rostock,Germany)for24hat37°C,toinvestigatetheroleofAGEsoninflammasomeactivation.TofurtherexploretherelationshipsbetweenAGEsandinflammatorycytokineproduction,variouspharmacologicalinhibitors,includingSB203580[amitogen-activatedproteinkinase(MAPK)p38inhibitor,20μM,MerckMillipore,Belize,MA,USA],SP600125[aJunN-terminalkinase(JNK)inhibitor,20μM,MerckMillipore],Bay11-7082(aNF-κBactivationinhibitor,2μM,MerckMillipore),SR11302(anAP-1inhibitor,10μM,MerckMillipore),N-acetyl-L-cysteine(NAC,anantioxidant,5mM,WakoPureChemicalIndustries),ordiphenyleneiodonium(DPI,aNADPHinhibitor,2μM,Sigma-Aldrich)werepre-incubatedinthemediumfor1h.SupernatantandcelllysatewerecollectedforELISA,real-timeRT-PCR,orwesternblotanalysis,andstoredat–20°Cor–80°Cbeforeuse.
Determination of cytokinesLevelsofIL-1β,IL-6,orIL-8weredeterminedusingahuman
ELISAkit(R&DSystems,Minneapolis,MN)accordingtothemanufacturer’sinstructions.
Real-time RT-PCRTotalRNAwaspreparedusingISOGEN(NipponGene,Toyama,
Japan)accordingtothemanufacturer’sinstructions.RNAextrac-tionandcDNAproductionwereperformedusingacommercialkit(ReverTraAce;Toyobo,Osaka,Japan).Real-timeRT-PCRwasperformedusingtheCFXConnectTMRealTimePCR(Bio-Rad,Hercules,CA,USA)andacommercialkit(ThunderbirdSYBRqPCRMix;Toyobo) todetectmRNAexpressionsof IL-1β, NLRP3, ASC, CASP1, IL-6, or glyceraldehyde 3-phosphate dehy-drogenase (GAPDH).Thefollowingantisenseandsenseprimerswereused:IL-1β(5′-TGATGGCTTATTACAGTGGCAATG-3′and5′-GTAGTGGTGGTGGGAGATTCG-3′,NM000576),NLRP3 (5 ′ -GAGAGACCTTTATGAGAAAGCA-3 ′ and5 ′ -GCATATCACAGTGGGATTCGAA-3 ′ , BC143363) ,A S C ( 5 ′ - AACCCAAGCAAGATGCGGAAG - 3 ′ a n d5 ′ - TTAGGGCCTGGAGGAGCAAG-3 ′ , BC013569 ) ,C A S P 1 ( 5 ′ - GAAGCTCAAAGGATATGGAAACAAA-3′ and 5′-AAGACGTGTGCGGCTTGACT-3′, X65019),I L - 6 ( 5 ′ - AAATTCGGTACATCCTCGACGG -3 ′ and5 ′ - GGAAGGTTCAGGTTGTTTTCTGC-3 ′ , M54894)and GAPDH (5′-AAATGAGCCCCAGCCTTCT-3′ and5′-AGGATGTCAGCGGGAGCCGG-3′,M33197).RT-qPCRwasperformedinduplicatewithafinalreactionvolumeof20µlcontaining10µlSYBRGreen,7.8µldistilledwater,0.1µl100µMforwardandreverseprimers,and2µlofcDNAtemplate.Theamplificationprogramconsistedofa5mindenaturationat95°Cfollowedby40cyclesofamplification(95°Cfor15sec,60°Cfor30sec,and72°Cfor20sec).ExpressionlevelsofeachtargetgenewerenormalizedtocorrespondingGAPDHthresholdcycle(CT)valuesusingtheΔΔ
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CTcomparativemethod[28].
Western blot analysisLysatesfromthecellculturewerepreparedusingRIPAbuffer
(WakoPureChemicalIndustries).CellswerewashedwithcoldPBSandincubatedwithRIPAbufferfor15minonice.Celllysatesweresubsequentlytransferredinto1.5mltubesandcentrifugedat12,000×gfor20minat4°C.Supernatantsweretransferredtoafreshtubeandstoredat–80°Cbeforeanalysis.Atotalof10µgproteinwasloadedperlaneandseparatedby10%SDS-PAGE.Theexpressionofpro-IL-1βandβ-actin(ACTB)wasanalyzedbywesternblot.Aftertransferontopolyvinylidenefluoridemembranes,nonspecificantibodybindingwasblockedfor1hatroomtemperatureusingImmunoblock(DSPharmaBiomedical,Osaka,Japan).Then,membraneswereincubatedfor24hat4°Cwithanti-IL-1βantibody(1:1000,SantaCruzBiotechnology,Dallas,TX,USA)andanti-ACTBantibody(1:10000,Sigma-Aldrich),followedbyanincubationfor1hwithsecondaryantibodyconjugatedhorseradishperoxidase(HRP;1:1000,GEHealthcare,UK,Buckinghamshire,UK).ImmunoreactivebandswerevisualizedbyWesternBLoTQuantHRPSubstrate(GEHealthcare)usingImageQuantLAS4000(GEHealthcare).Theresultsrepresentatleast3independentexperiments.QuantitativeanalysisofbandswasperformedusingImageJ(NationalInstitutesofHealth,Bethesda,MD,USA).
Statistical analysisDataareexpressedasmean±standarderrorofthemean(SEM).
Differencesbetweentreatmentgroupswereidentifiedusingunpairedt-tests.Multiplecomparisonsweremadeusingone-wayanalysisofvariance(ANOVA),followedbyBonferroni’smultiplecomparisontest.AP-valueof<0.05wasconsideredstatisticallysignificant.
Results
Effects of AGEs on inflammatory cytokine production in human placental tissuesFirst,wetestedwhetherAGEscouldinduceinflammatoryresponses
usinghealthyhumanplacentaltissuecultures.LPSwasusedasapositivecontrolforinductionofinflammatoryresponses.BothAGEandLPStreatmentssignificantlyincreasedIL-1β and NLRP3 mRNAexpression(Fig.1AandB),butdidnotaffectASC and CASP1mRNAexpressioninhumanplacentaltissues(Fig.1CandD).Ontheotherhand,bothAGEsandLPSsignificantlyincreasedpro-IL-1βproduction(Fig.1E)andIL-1βsecretion(Fig.1F)inhumanplacentaltissues.Inthepresentstudy,proinflammatorycytokines,includingIL-6andIL-8,wereusedaspositivemarkersforinflammatoryresponses,becausewepreviouslyreportedthatAGEsandLPSstimulatedIL-6andIL-8secretionfromhumanplacentalcells[16].Asexpected,bothAGEsandLPSsignificantlyincreasedIL-6,andIL-8secretionfromhumanplacentaltissues(Fig.1GandH).ThesefindingssuggestthatAGEsup-regulateinflammatorycytokineproductionandsecretioninhumanplacenta.
Effects of AGEs on IL-1β production in the Sw.71 human placental cell lineNext,weinvestigatedtheeffectsofAGEsandLPSonIL-1β
productionsystemsinSw.71cells(ahumanplacentalcellline).BothAGEsandLPSsignificantlyincreasedIL-1βmRNAexpression(Fig.2A)andpro-IL-1βproteinproduction(Fig.2B)inSw.71cells.Contrarytothehumanplacentaltissuedata,AGEandLPStreatmentdidnotaffectNLRP3, ASC and CASP1mRNAexpression(Fig.2C–E).Previously,wereportedthatAGEsandLPSstimulateinflammatorycytokinesecretion,includingIL-6andIL-8,fromhumanSw.71placentalcells[16].Tosupportthepreviousresults,bothAGEsandLPSsignificantlyincreasedIL-6mRNAexpressioninSw.71cells(Fig.2F).
Effects of AGEs on IL-1β secretion in the Sw.71 human placental cell lineTwosignalsarerequiredformatureIL-1βsecretionfromthe
NLRP3inflammasome[17].Thefirstsignalisthetranscriptionandtranslationofpro-IL-1βbyprimingstimuli,includingLPS.Thesecondsignalactivatesautocatalyticcleavageofpro-CASP1toCASP1,tosecreteIL-1βfrompro-IL-1β.Weusednano-silicaforthesecondsignaltoactivatetheNLRP3inflammasome.AsshowninFig.3A (leftside),bothAGEsandLPSalone(withoutnano-silica)didnotaffectIL-1βsecretioninSw.71placentalcells.However,afterAGEorLPSprimingfor24htoinducepro-IL-1βproductionwithinthecells,nano-silicatreatmentclearlystimulatedIL-1βsecretioninSw.71placentalcells(Fig.3A,rightside),indicatingtheroleofAGEsandLPSas“Signal1”forpro-IL-1βproduction.WecheckedthepotentialroleofAGEsas“Signal2”toactivatetheinflammasomesysteminSw.71placentalcells.AfterLPSpriming,AGEtreatmentdidnotaffectIL-1βsecretion,whereasnano-silicatreatmentsignificantlystimulatedIL-1βsecretioninSw.71cells(Fig.3B).ThesefindingssuggestthatAGEsstimulatepro-IL-1βproductionwithinthecells,butdonotactivatetheinflammasometostimulateIL-1βsecretion.
Intracellular AGE signaling pathways on inflammatory cytokine production in the Sw.71 human placental cell lineItiswellknownthatMAPKandtranscriptionfactors,including
NF-κBandAP-1areimportantinAGEandLPSsignaltransduction[29].Toelucidatethesignalingmechanisms,weaimedtoclarifywhichofthesepathwaysisinvolvedinAGE-andLPS-amplifiedinflammatorycytokineproductionusingspecificpharmacologicalinhibitorstoblockeachindividualsignalingpathway.Blockageofeachindividualpathway(SB203580asap38MAPKinhibitor,SP600125asaJNKinhibitor,Bay11-7082asaNF-κBinhibitor,andSR11302asanAP-1inhibitor)significantlyattenuatedAGEs-inducedintracellularIL-1βproduction(Fig.4A),extracellularIL-1βsecretionstimulatedbynano-silica(Fig.4B),andextracellularIL-6secretion(Fig.4C)inhumanSw.71placentalcells.ThesefindingsindicatethattheactivationofallofthesefourpathwaysisnecessaryforeffectiveinductionofcytokinesbyAGEsinhumanSw.71cells.
Role of ROS for AGE-induced cytokine production in the Sw.71 human placental cell lineTheincreaseininflammatorycytokinesecretionafterAGE
treatmentledustohypothesizethatreactiveoxygenspecies(ROS)productioninvolvesAGE-inducedinflammationinSw.71cells.Indeed,wealreadyreportedthatAGEsstimulatedROSproductionandthatAGE-inducedROSproductionisimportantforstimulating
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cytokineproduction,includingIL-6andIL-8[16].ToinvestigatetheimportanceofROSforAGE-inducedIL-1βproduction,wetestedtheeffectoftheantioxidantsNACandDPIonSw.71cells.TreatmentwithNACorDPIsignificantlydecreasedtheextracellularIL-1βsecretioninducedbyAGEprimingwithnano-silica(Fig.5A).Inaddition,weshowedthatpre-treatmentofNACattenuatedAGE-inducedIL-1βmRNAexpression(Fig.5B).Thus,ourfindingsindicatedthatROSproductionisanupstreamfactorinAGE-inducedinflammatorycytokineproduction,includingIL-1βandIL-6,inhumanplacentalcells.
Discussion
IncreasingevidenceindicatesthatAGEsandIL-1βareassociatedwithpreeclampsiaandobesityinpregnantwomen[1,2,30–32].
Inthepresentstudy,weinvestigatedwhetherAGEsareinvolvedinIL-1βproductionandsecretionviaNLRP3inflammasomesinhumanplacenta.Ournovelresultsdemonstratedthat(1)inhumanplacentaltissues,AGEsdirectlyincreaseboththetranscriptionandsecretionofIL-1β;(2)Inhumanplacentalcellline,AGEsstimulatepro-IL-1βproduction,resultingintheaccelerationofmatureIL-1βsecretionbyNLRP3inflammasomeactivation;(3)AGE-inducedinflammatorycytokinesaredependentonMAPK/NF-κB/AP-1signalingandROSproduction.Inhumanplacentaltissues,AGEtreatmentdirectlystimulated
matureIL-1βsecretion,togetherwithanincreaseinIL-1β and NLRP3 mRNAtranscription.Similartoourpresentresults,ithasbeenreportedthatAGEsincreasedthein vitroreleaseofIL-1βfromhumanplacentainatissueculturesystem[12].However,weshowedthatAGEsmarkedlyinducedpro-IL-1βproduction,regulatingtranscriptionof
Fig. 1. EffectsofAGEsoninflammatorycytokineproductioninhumanplacentaltissues.Humanplacentaltissueswereincubatedfor6hwithBSA,AGEs(200μg/ml),orLPS(100ng/ml).(A–D)Afterincubation,theIL-1β, NLRP3, ASC and CASP1mRNAlevelsweremeasuredusingqRT-PCR.Pro-IL-1βandACTBproteinlevelsweresubsequentlyquantifiedinthetissuelysatesbywesternblot(E).IL-1β(F),IL-6(G),andIL-8(H)levelsinthesupernatantweredeterminedusingELISA.Dataareexpressedasmeans±SEM(n=4).SignificantdifferenceswereidentifiedusingANOVAfollowedbyBonferroni’smultiplecomparisontest;*P<0.05and**P<0.01.
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IL-1βmRNA,whereasAGEsdidnotstimulateIL-1βsecretioninhumanplacentalcells.WebelievedthatthesedifferencesregardingIL-1βsecretionbyAGEswereduetotheconditionaldifferencesbetweenplacentalcells(celllines)andplacentaltissues(composedofmanytypesofcells,includingplacentalcells,endothelialcells,and
immunecells).Indeed,numerousimmuneandinflammatorycells,includingmacrophages,NKcells,lymphocytesandneutrophils,resideintheplacentaanddecidua[9].Inaddition,activatedmacrophagesarethemajorsecretorsofIL-1βviaNLRP3inflammasomes.Infact,AGEs,aswellasLPS,independentlyincreasedIL-1βsecretion
Fig. 2. EffectsofAGEsonIL-1βproductionintheSw.71humanplacentalcellline.(AandB)Sw.71cellswereincubatedfor24hwithBSAorAGEs(200μg/ml),andLPS(100ng/ml);TheIL-1βmRNAlevelsweremeasuredusingqRT-PCR.Pro-IL-1βandACTBproteinlevelsweresubsequentlyquantifiedinthecelllysatesbywesternblot.(C–F)Afterincubation,theNLRP3, ASC and CASP1mRNAlevelsweremeasuredusingqRT-PCR.SignificantdifferenceswereidentifiedusingANOVA,followedbyBonferroni’smultiplecomparisontest;*P<0.05and**P<0.01.
Fig. 3. EffectsofAGEsoninflammasomeactivationintheSw.71humanplacentalcellline.(A)Sw.71cellswerepre-incubatedfor24hwithBSA,AGEs(200μg/ml),orLPS(100ng/ml).Afterpriming,cellsweretreatedwithnano-silica(100μg/ml)for24htoactivateNLRP3inflammasomes.(B)Sw.71cellswerepre-incubatedfor24hwithLPS(100ng/ml).Afterpriming,cellsweretreatedwithBSA,AGE(200μg/ml)ornano-silica(100μg/ml)for24h.IL-1βlevelsinthesupernatantweredeterminedusingELISA.Dataareexpressedasmean±SEM(n=4).SignificantdifferenceswereidentifiedusingANOVA,followedbyBonferroni’smultiplecomparisontest;*P<0.05and**P<0.01.
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byhumanperitonealmacrophagesinadose-dependentmanner[33].Inaddition,inflammatoryimmunecellsaccumulateintheadiposetissuesofobesepatients,andAGEsdirectlyincreasedIL-1βsecretionwithoutLPSprimingintheadiposetissueofwomenwithgestationaldiabetes[12].Thesefindingsledustohypothesizethat(1)immunecellswithinhumanplacentaltissuesrespondtoAGEtreatmenttoinduceIL-1βsecretion,and(2)cell-to-cellinteractions(communicationbetweenplacentacellsandimmunecells)withintheplacentaareimportantforAGEstodirectlyinducematureIL-1βsecretion.FurtherinvestigationisneededtoclarifythemechanismsofAGE-inducedIL-1βsecretion,itsrelatedsignalingpathways,andthekeycelltypesinvolvedinIL-1βproductionandsecretioninhumanplacentaltissues.Next,weinvestigatedthepossibleroleofAGEsonIL-1βproduction
andsecretionusingthehumanSw.71placentalcellline.WeshowedthatAGEsdidnotregulatematureIL-1βsecretion,butregulatedpro-IL-1βproduction,andnano-silicatreatmentstimulatedIL-1βsecretionafterAGE-priminginplacentalcells.Therefore,theseresultsindicatethatAGEsactas“Signal1”toproducepro-IL-1β,notas“Signal2”toactivateNLRP3inflammasomeswithinhumanplacentalcells,suggestingthatAGEshaveakeyroleinpro-IL-1βproductionfollowingNLRP3inflammasomeactivation.Contrarytoourpresentstudy,Songet al.[34]recentlydemonstratedthatusinghumanprimarynucleuspulposuscells,AGEsalonecanactivatenotonlypro-IL-1βproductionas“Signal1”withinthecells,butalsotheNLRP3inflammasomeassemblyonmitochondriaas“Signal2”,resultinginelicitationofmatureIL-1βsecretion.Weconsideredcelltypeorcellcondition(primarycellsisolatedfromtissuesorestablished
Fig. 4. IntracellularAGE signaling pathways on inflammatory cytokine production in the Sw.71 human placental cell line. Sw.71 cells were pre-incubatedfor1hwithspecificpharmacologicalinhibitors(SB203580asap38MAPKinhibitor,SP600125asaJNKinhibitor,Bay11-7082asaNF-κBinhibitor,andSR11302asanAP-1inhibitor).(A)Afterinhibitortreatments,cellswereincubatedfor24hwithAGEs(200μg/ml).(B)Afterinhibitortreatments,cellswereincubatedfor24hwithAGEs(200μg/ml),followedbynano-silica(100μg/ml)treatmentfor24h.(C)Afterinhibitortreatments,cellswereincubatedfor24hwithAGEs(200μg/ml).IntracellularIL-1β,andIL-1βandIL-6levelsinthesupernatantweremeasuredusingELISA.Dataareexpressedasmean±SEM(n=4).SignificantdifferenceswereidentifiedusingANOVA,followedbyBonferroni’smultiplecomparisontest;*P<0.05.
Fig. 5. ROSpathwaysofAGEsand theireffecton inflammatorycytokineproduction in theSw.71humanplacentalcell line.Sw.71cellswerepre-incubatedfor1hwithspecificpharmacologicalinhibitors(NACasanantioxidant,DPIasaNADPHinhibitor).(A)Afterinhibitortreatments,cellswereincubatedfor24hwithAGEs(200μg/ml),andthentreatedwithnano-silica(100μg/ml)for24h.IL-1βlevelsinthesupernatantweredeterminedusingELISA.(B)AfterNACtreatments,cellswereincubatedfor24hwithAGEs(200μg/ml).TheIL-1βmRNAlevelsweremeasured using qRT-PCR.Data are expressed asmean±SEM (n= 4). Significant differenceswere identified usingANOVA, followed byBonferroni’smultiplecomparisontest;*P<0.05.
AGE-INDUCED INTERLEUKIN-1βPRODUCTION 407
celllines)aspossiblereasonsfordifferencesinexperimentaloutcome.Wefurtherinvestigatedthepossiblesignalingpathwaysof
AGE-inducedinflammatorycytokineproductioninhumanSw.71placentalcells.Recently,wereportedthatexposuretoAGEsresultedintheactivationofinflammatoryresponses,inducingIL-6andIL-8secretion,andAGE-inducedIL-6secretionwasdependentonNF-κBactivationthroughRAGEand/orTLR4inhumanSw.71placentalcells[16].AccumulatingevidencehasindicatedthatactivationofbothMAPK(p38,JNK,extracellularsignal-regulatedkinase1/2)andtranscriptionfactors,includingNF-κBandAP-1,arerequiredforAGE-amplifiedinflammatorycytokineproduction[29,35,36].Inthepresentstudy,weconfirmedthatbothMAPK(p38andJNK)andNF-κB/AP-1areessentialforAGE-inducedIL-1βproductionandsecretion,aswellasIL-6secretion,inhumanplacentalcells.ROSareharmfulmediatorsofinflammation,andROSinhibition
suppressesNLRP3inflammasomeactivationandinflammatoryresponses[22].WepreviouslyreportedthatAGEssignificantlystimulatedROSproduction,ROSinhibitorexperimentsconfirmedthatROSareessentialforIL-6secretionbyAGEsinhumanSw.71cells[16].Inthepresentstudy,weusedtheROSinhibitorsNAC(anantioxidant)andDPI(anNADPHoxidaseinhibitor)toexaminetheimportanceofROS,andfoundthatthesesignificantlyblockedAGE-inducedIL-1βproduction,duetotheinhibitionofIL-1βmRNAtranscription,resultinginthesuppressionofIL-1βsecretionviaNLRP3inflammasomeactivation.Indeed,ROSgenerationises-sentialasanupstreamfactorforNLRP3inflammasome-dependentIL-1βsecretion[37].Therefore,itappearsthatROSproductionisimportantforbothinflammatorycytokineproductionandinflam-masomeactivationinplacentalcells.Inconclusion,wedemonstratedthatexposuretoAGEsresulted
intheactivationofNLRP3inflammasomeviapro-IL-1βproductionandinflammatoryresponses,inducingIL-6andIL-8secretioninhumanplacenta.MAPK/NF-κB/AP-1signalingandROSproductionplaypivotalrolesinAGE-inducedinflammatoryresponses.TheseresultssuggestthatAGEs,asanendogenousandsteriledangersignal,maycontributetochronicplacentalcytokineproduction,resultinginpregnancyrelatedcomplicationssuchaspreeclampsia.Furtherinsightintotherelationshipsbetweenplacentalinflammation,obesity,andpregnancycomplicationswillbeusefulfordevelopingtherapeuticstrategiestotreatthesecomplications.
Conflict of interest:Theauthorsdeclarenoconflictofinterest.
Acknowledgments
ThisstudyreceivedgrantsfromtheJapanSocietyforthePro-motionofScience(JSPS)throughtheScientificResearch(C)(KSandHS),andStrategicResearchProjectfromTokyoUniversityofAgriculture(KS).
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