Original Article

59

Introduction

Toll-likereceptors(TLRs)havebeendemonstratedtobeakeycomponentoftheinnateimmunesystem.Atleast11TLRshavebeenidentifiedinmammals,andeachonerecognizesuniquemolecularpatternsassociatedwithdifferentclassesofpathogens.TLR-9recognizeshypomethylatedCpG-richsequencesofDNApresentinmanybacteriaandviralspecies[1,2].Althoughitis

CpG ODN ligation of TLR-9 attenuates TNF-α production in untreated active SLE patients Jeng-Ting Tsao1,2, Chia-Li Yu2, Song-Chou Hsieh2, Tsui-Wen She3, Chia-Chen Kuo1,

Shih-Chang Lin1,4

From the Division of Allergy and Immunology, Department of Internal Medicine, Cathay General Hospital, Taipei, Taiwan1Division of Allergy and Immunology, Department of Internal Medicine, Cathay General Hospital, Taipei, Taiwan2Division of Rheumatology and Immunology, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan3Nursing department, Cathay General Hospital, Taipei, Taiwan4The Laboratory of Allergy and Immunology, Cathay Medical Research Institute, and School of Medicine, Department of Medicine, Fu Jen Catholic University, Taipei, Taiwan

Correspondingauthor:Shih-ChangLinM.D.,Ph.D.DivisionofAllergyandImmunology,DepartmentofInternalMedicine,CathayGeneralHospital.No.280,Sec.4,Ren-aiRd.,Da-anDistrict,TaipeiCity106,Taiwan.Tel:+886-2-27082121ext3210;Fax:+886-2-27095346E-mail:p95421019@ntu.edu.twReceived: June 30, 2009Revised: July 18, 2009Accepted: August 14, 2009

Objective:Inthepresentstudy,wedeterminedtheimmunomodulatoryeffectoftoll-likereceptor-9(TLR-9)ligationwithhypomethylatedCpGoligodeoxynucleotides(CpGODNs)onTNF-αreleaseinuntreatedsystemiclupuserythematosus(SLE)patientsandinacohortofsex-andage-matchedhealthyindividuals.Methods:Peripheralbloodmononuclearcells(PBMCs)from13SLEpatientsand8healthyindividualswerestimulatedwithCpG-ODNsinthepresenceofphorbolmyristateacetateandionomycin.RT-PCRandELISAassayswereusedforanalysisofTNF-αmRNAexpressionandproteinsecretion.Results:CpGODNsincreasedTLR9-mediatedTNF-αproteinreleaseinsupernatantofPBMCcultureinSLEandhealthyindividualsalthoughtherewerenosignificantdifferencesofTNF-αmRNAexpressionbetweenthesetwogroups.ThelevelofTNF-αproteinproductionwassignificantlylowerinSLEpatientsthaninnormalcontrols(592.5±202.8ng/mLvs.1369.8±277.3ng/mL,p=0.033).Conclusion:ThelowerTNF-αproductionbyPBMCsafterTLR-9ligationinuntreatedSLEpatientsreflectedthedysregulationof this importantproinflammatorycytokine.ThisdysregulationmaybeinvolvedinthepathogenesisofattenuatedinnateimmunitywhenpatientsareexposedtobacteriaorviruseswithCpG-richDNA.

Key words:Systemiclupuserythematosus,tumornecrosisfactor-α,tolllikereceptor-9,CpG-ODN

Formosan Journal of Rheumatology 2009;23:59-65

60

CpG ligation of TLR-9 and TNF-α in active SLE

knownthatTLR-9playsakeyroleintheproductionofpathogenicautoantibodiesandinthedevelopmentofclinical featuresofsystemic lupuserythematosus(SLE), themechanismbywhich it recognizesself-antigensandproducesautoantibodiesisunclear.Tworecentstudiesprovidedcontradictoryfindings.Inonestudy,anti-dsDNAantibodyproductionwasimpairedinTLR-9gene-knockoutlupus-pronemice[3],whereasintheotherstudytherewasnodifferenceinproductionbetweenTLR-9gene-knockoutmiceandmicewithanintactTLR-9gene[4].InananimalstudyontheeffectivenessofTLR9,Marcetal. foundthatTLR-9-deficientautoreactiveBcellsdonotundergoclassswitchingtothepathogenicimmunoglobulinisotypesIgG2aandIgG2b[5].Somestudieshaveassessedtheefficacyofusing inhibitoryoligodeoxynucleotides(ODNs) toblock theactivationofTLR-9 in lupusmice.Notably,administrationofODNswasshowntosignificantlyreducetheseverityofclinicalfeaturesofSLEinonestudyandwasassociatedwithdelayedonsetofrenaldiseaseandprolongedsurvivalinanotherstudy[6,7].ItisknownthatCpGODNsareendocytosedbyBcells,plasmadendriticcells(pDCs),andmonocytes,andaresubsequently recognizedbyTLR-9.TLR-9thenrecruitstheadaptorproteinmyeloiddifferentiationfactor88(MyD88).ThisrecruitmentofMyD88initiatesasignalingpathwayandfinallyinducestheexpressionofvariousoncogenesandproinflammatorycytokines,suchasIL-1,IL-6,andTNF-α.ThelinkbetweenTLR-9,CpGODNs,andTNF-αhasbeenfurtherestablishedbyinvitrostudies.DNA-containingimmunecomplexesstimulatemousebone-marrowderivedDCstoproducelargeamountsofTNF-α,andthisresponsewasfoundtobeblockedbyinhibitorsofTLR9andtobemarkedlyreducedincellsfromTLR9-deficientmice[8].OneinvitrostudyshowedthatDNA-containingimmunecomplexesfromtheseraofSLEpatientscanstimulatetheproductionofTNF-αmRNAinhumanembryonicdendritic cells [9]. Moreover, CpG-stimulatedmacrophages can also secreteTNF-αandvariouscytokinesinvolvedinimmunereactions[10,11].

TNF-αhasbeenfoundtobeapotentimmunoregulatorycytokineandhasbeen implicated inautoimmunityofSLE.The relationshipbetweenTNF-αandSLEis controversial. Elevated levels ofTNF-α havebeenobserved inSLEpatients ina limitednumberof studies,while inother studies the resultswereinconsistent[12-15].ItismoreobviousnowthatTNFinhibitoronatheoreticallevelappliedinhumanswithautoimmunediseasemayleadtothepresenceofanti-dsDNAantibody,aserologicalmarkerofSLE,and

evenclinicalmanifestationsofSLE[16].Ithasbeenshownthatanti-TNF-αantibodiesdecreaseproductionof immunoglobulinsby culturedperipheral bloodmononuclearcells(PBMCs)inpatientswithSLE[17].TheresultsfromsomeanimalstudiessuggestalocalpathogenicroleofTNF-αinlupusglomerulonephritisrather than systemic dysregulation [18]. DetailsregardingtheeffectsofTLR-9ligationonTNF-αarestillnotclear.ToidentifytherelationshipbetweenTNF-αandTLR-9 inSLEpatients, thisstudyexplored thechangeinTNF-αexpressionatthemRNAandproteinlevelsinPBMCssubjectedtoTLR-9ligationwithCpGODNsinuntreatedSLEpatients.

Materials and Methods

Patients and healthy controlsWeenrolled13activeuntreatedSLEpatientsfrom

theCathayGeneralHospital andNationalTaiwanUniversityHospital,Taipei,TaiwanduringtheperiodMarch2007toJune2008.Allstudysubjectsprovidedinformedconsent.ThediagnosisofSLEwasbasedontheclassificationcriteriaoftheAmericanCollegeofRheumatology[19].Sex-andage-matchedhealthyvolunteerswere recruited as thenormal controls.DemographiccharacteristicsofblooddonorsareshowninTable1.DiseaseactivityofSLEineachgroupwasdeterminedusingtheSystemicLupusErythematosusDiseaseActivityIndex(SLEDAI)scoringmethod[20].

Preparation of blood samples and cell cultureApproximately20mLofheparinizedwholeblood

wasobtainedfromeachstudy individualandbloodsamplesweresubjectedtoanalysiswithin4hoursaftercollection.Plasmawasisolatedbycentrifugationandstoredat–80ºC.Peripheralbloodmononuclearcells(PBMCs)were isolatedbyFicoll-Hypaquedensitygradientcentrifugation.PBMCswereplatedatadensityof1×106cells/mLin12-wellcellcultureplateswithRPMI1640supplementedwith10%heat-inactivatedfetalcalfserum(FCS)and100µg/mLstreptomycin.CellswerestimulatedwithCpGODN(1µM)inthepresenceofPMA(100ng/mL)and ionomycin(500ng/mL)topromotecell-cellinteraction.Cultureplateswerethenincubatedina5%CO2incubatorat37ºCfor24hours.Afterculture,totalRNAwasextractedfromPBMCs.Supernatantsofcellcultureswerecollectedandstoredat–80ºC.

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Tsao et al

Reagents for cell culture and final concentrations

The CpG-ODNs sequences used in this studywereselectedaccordingtopublishedreportsandwerecommerciallysynthesized.Thefinalconcentrationwas1µM.Phorbolmyristateacetate(PMA)waspurchasedfromSigma-Aldrich,St.Louis,MO,USA.Thefinalconcentrationwas100ng/mL.IonomycinwaspurchasedfromSigma-Aldrich,St.Louis,MO,USA.Thefinalconcentrationwas500ng/mL.

CpG-ODNs sequenceThesequenceofCpG-ODNswasTCGTCGTTTTG

TCGTTTTGTCGTT.

TNF-α primer pairsThesequencesofprimersspecificforTNF-αgene

were5’-TGAGCACTGAAAGCATGATCC-3’ and5’-GGTTTGCTACAACATGGGCTA-3’.Theexpectedproductsizewas335basepairs.

Total RNA isolation and Reverse transcription-polymerase chain reaction (RT-PCR)

TotalmRNAinPBMCs fromSLEpatientsandhealthy individuals was extracted using an RNAextractionkit (SuperScript™IIRnaseH– reversetranscriptase,Invitrogen)accordingtothemanufacturer’sinstructions.ThetargetgenewasTNF-α.Forreversetranscription,1μgoftotalRNAwasusedtosynthesizecDNA.Then,1μLofRTproductwasusedfor thesubsequentPCRexperiments. TheamplificationofGAPDmRNAwasusedasaninternalcontrol. PCRwasprogrammedforoptimalcycleswithdenaturationfor10secondsat95ºC,annealingfor50secondsat59ºC, andextension for45 secondsat72ºC. TheDNAfragmentswereseparatedon2%agarosegelsandvisualizedbyethidiumbromidestaining. ThequantityofDNAfragmentswasdeterminedusingtheChemiGenus2imagingsystemandGeneToolsimageanalysiscomputersoftware(Syngene,Frederick,MD).AstandardDNAsamplewithaknownconcentrationwasusedtocalculatetheabsoluteconcentrationofDNAfragmentsinPCRproducts.ThenumberoftranscriptswasnormalizedusingGAPDtranscript levelsasaninternalcontrol,andwascomputedusingthefollowingformula:

Enzyme-linked immunosorbent assay (ELISA)

Cytokinesinplasmaandcellculturesupernatantswerequantifiedusing a ‘‘sandwich-type’’ELISAtechnique.Thematchedantibodypairs,consistingofunlabelledcaptureantibodyandbiotinylateddetectionantibody,andrecombinantproteinstandardsforTNF-αwerepurchasedfromBenderMedSystems(Vienna,Austria).ThedetectablerangeofTNF-αis310-20,000ng/mL.

Statistical analysisNon-parametricMann-Whitneyrank-sumtestwas

usedtocomparedifferencesinTNF-αatthetranscriptandproteinlevelsbetweenSLEpatientsandnormalcontrols.Alltestsweretwo-tailed.Ap-value<0.05wasconsideredsignificant.

Results

Demographic characteristics of study populations

TherewasnosignificantdifferenceinmaletofemaleratiobetweentheSLEgroupandcontrolgroup.TheSLEDAIscoreforSLEpatientsrangedfrom6to17.Oneof theSLEpatientshadcentralnervoussysteminvolvementand11SLEpatientshadlupusnephritis.All13SLEpatientshadpositiveantinuclearandanti-dsDNAantibodies.DemographiccharacteristicsofactiveSLEpatientsandnormalindividualswereshowninTable1.

mRNA of cytokine expression in PBMCsTo investigate theeffectofmicrobialagentson

TNF-αreleasefromPBMCs,PBMCsobtainedfromactive lupuspatients andnormal individualswerestimulatedwith theTLR-9ligandCpGODNfor24

Table 1. Demographic characteristics of active SLE patients and normal individuals.

Normal individuals Active SLE patientsCase numbers 8 �3Female:Male (% female) 6:2 (75%) ��:2 (85%)

Age (years, mean ± standard error) 31.4 ± 3.3 28.5 ± 2.2

Disease status - Disease active or flare lupus patients

SLEDAI score - 9.6 ± 0.9a

a Mean ± standard error

Levelofexpression= Intensityofecahgene ×100%IntensityofGAPDgene

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CpG ligation of TLR-9 and TNF-α in active SLE

hours.The resultsofTNF-αmRNAexpressionofuntreatedPBMCswere35.6±11.1ng/mLinSLEand24.0±6.6ng/mLinnormalindividuals(p=0.56).AsforCpGODN-treatedPBMCs,TNF-αRNAexpressionlevelswere26.6±4.4ng/mLinlupuspatientsand22.0±7.3ng/mLinnormalindividuals(p=0.56).Therewerenosignificantdifferences inTNF-αtranscript levelsbetweenthesetwogroups(Table2).

Dysregulation of cytokine production in PBMCs from SLE patients

TNF-αproteinconcentrations in thesupernatantofPBMCcultureafter ligationofTLR-9withCpGODNsandinplasmaofactiveSLEpatientsandnormalindividualsweredeterminedbyELISA.TheTNF-αprotein levels inplasmadidnotdiffersignificantlybetween the twogroups (SLE:13.8±6.6ng/mL;normal:241.9±211.2ng/mL;p=0.14);however,intheinvitroexperimentsofPBMCculturedinthepresenceofCpGODNsforTLR-9activation,theTNF-αproteinlevelsintheculturesupernatantsofSLEPBMCsweresignificantlylowerthanthoseintheculturesupernatantsofnormalPBMCs(SLE:592.5±202.8ng/mL;normal:1369.8±277.3ng/mL;p=0.033) (Table3).ThesedatasuggestedthatTNF-αproductioninPBMCsafterligationofTLRwasdysregulatedinSLEpatients.

Discussion

TLR-9hasbeenshowntoplayanimportantroleinthepathogenesisofSLE.PreviousstudieshaveshownthatactiveSLEpatientshave increasedproportionsofmatureBcells,plasmacells,andmonocytes that

expressTLR-9, and that theexpansionofTLR-9-expressingBcellscorrelateswithanti-dsDNAantibodyproduction[19].TherelevantpointisthatTLR-9exertspowerfulsynergisticeffectindeterminingclass-switchrecombinationandmaturationofBcells tobecomeautoantibody-secretingcells,alongwithBcellreceptorligationandTcellhelp.OurstudyinvestigatedtheeffectofTLR-9ligationwithCpGODNonproinflammatorycytokineproductionfromPBMCs.Ofmanycytokinesinvolved inpathogenesisofSLE,TNF-αhasbeenchosen,becausethelevelofTNF-αmayreflectthelevelof inflammationinpatientswithSLEfrompreviousstudies,andthatlupuspatientsarecharacterizedbyahigherlevelofTNF-αcomparedtoahealthypopulation[20].InahumanSLEstudy,TNF-αconcentrationishigherintheglomerulartissueofSLEpatients,whilesystemicTNF-αconcentrationisnot increased[21].Our findings,however, showed that therewerenosignificantdifferences inTNF-αmRNAexpressionbetweenSLEpatientsandnormalindividualsthoughtheTNF-αproteinexpressionwaslesspotentiatedinSLEgroupsafterTLR-9ligationwithCpGODNonPBMCs(Table2andTable3).ThesefindingssuggestedalocalpathogeniceffectofTNF-αratherthansystemicdysregulationof thiscytokine inSLEpatients frombaselineconditions.Furthermore, theup-regulationofTNF-αwasindeedinsufficientwhenSLEpatientsencounteredinvasionofpathogencontainingCpGODN.

ThereasonwhyPBMCsinsteadofasubpopulationofBcellsorpDCswereused in this studywas toinvestigate theeffectofTLR-9 ligationwithCpGODNoncell-cellcommunication.There isgrowingevidencethatTLR-9expressionand/orfunctioncanbeupregulatedinBcells[22]andothercell types,suchasmacrophages/monocytes,cDCs,granulocytesandevenepithelialcells.Theseobservationsunderscorethatthosevariouscelltypes,otherthanBcellsandpDCs,potentiallyplayTLR9-mediatedprotectiveorpathogenicrolesininfectionandotherimmunologicaldisorders.AcombinationofPMA(aphorbolester/PKCactivator)andionomycin(acalciumionophore)wereusedincellculture tostimulateTcellactivationandsubsequentinteractiontoothercellpopulations.

Ourresultsshowedthat theproductionofTNF-αprotein by PBMCswas increased in both SLEpatients andnormal controls afterTLR-9 ligationwithCpGODN.However,therangeofTNF-αlevelswassignificantlylowerintheSLEgroup.In2006,avitrostudypublishedbyLiuetal.demonstratedthatpretreatmentwith low-doseCpGODNsuppressesTNF-αreleaseinresponsetoasubsequentchallenge

Table 2. Cytokine mRNA levels of PBMCs activated via toll-like receptor (TLR) ligation

cytokine receptor ligand

Responders (ng/mL)p

Active SLE Normal

TNF-αTLR-9 (CpG ODN) 26.6 ± 4.4a 22.1 ± 7.3 0.56

Un-treated 35.6 ± 11.1 24.0 ± 6.6 0.56aData are represented as mean ± standard error

Table 3. Cytokine protein levels in cell culture supernatant after PBMC activated via toll-like receptor (TLR) ligation

cytokine receptor (ligand)

Responders (ng/mL)p

active SLE Normal

TNF-αTLR-9(CpG ODN) 592.5 ± 202.8a 1369.8 ± 277.3 0.033

Un-treated 13.8 ± 6.6 241.9 ± 211.2 0.14aData are represented as mean ± standard error.

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withhigh-doseCpGODN[23].Thesedataprovideapossibleexplanationforourhypothesis that,withdefectiveclearanceofapoptoticcells,SLEpatientswithprecedingexposuretoendogenouslyderivedCpGODNmaydemonstratesuppressedTNF-αreleaseinresponsetosubsequentchallengeofCpGODN.Theresulting“tolerantstate”ofTLR-9ligationwithCpGODNinSLEmaybeassociatedwithTLRsignal tolerance,whichhasbeenprovedinahyporesponsivestateofmacrophagesasamarkeddecreaseinproinflammatorycytokineproductionfollowingsecondarychallengewithmicrobialligands,suchaslipopolysaccharide,bacteriallipoprotein,andCpGODN[24-26].OurfindingthatTLR-9 ligation lesseffective inpotentiatingTNF-αproductionbyPBMCsinSLEpatientsmayalsoprovideamechanismtoexplaintheimpairedimmuneresponseagainstmicro-organismsinpatientswithSLE.WithregardtotheinconsistentresultsbetweenTNF-αproteinandmRNAexpressioninourstudy,webelievethatthedilutedeffectofpooledPBMCsincellcultureinvitromayhavebeenacontributingfactor.Inotherwords,theincreasedlevelofTNF-αtranscriptinmonocytescouldhavebeenmaskedbyothercellpopulations.TherehasbeenanotherpossibilitythatTNF-αisapro-inflammatorycytokinewhoseproduction isusuallyreflectedveryquicklytoinfectiouscircumstances.TheincreaseofTNF-αmRNAexpressioncouldhappenearlierthan24hours.ItneedsfurtherstudytoinvestigatewhetherTNF-αmRNAexpressionwillsynchronizedincreaseasproteinproductionwithin two toeighthoursafterTLR-9ofPBMCsligatingwithCpGODN.AlthoughtherehasbeendoubtthatPMAorionomycincan stimulateTcells toproducemanycytokines,includingTNF-α, the influenceon results canbeoverlookedsincethesamestimulantswereaddedincellculturesofbothstudiedgroups.

Conclusion

TLR-9ligationwithCpGODNwaslesseffectiveinpotentiatingTNF-αproductionbyPBMCsinSLEpatientswithactivediseasethaninhealthyindividuals.ThissuppressedTNF-αproductionfromPBMCsinSLEmaybeassociatedwithTLR-9toleranceandmightpartlyexplainthepathogenesisofdiseaseflareandthedevelopmentoffurther immuno-compromisedstatuswhenSLEpatientsencounterCpGODN-containingpathogens.ItremainstobeseenwhetherTLR-9ligationsuppressesTNF-αexpression indifferent immunecellsubpopulations inorder to identify thespecific

dysregulatedimmunecellsinSLEpatients.

Acknowledgement

SponsorbygrantsfromCathayMedicalResearchInstitute,Hsichih,TaipeiShien,Taiwan.

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在未經治療活動性全身性紅斑性狼瘡病患以CpG去氧核糖寡

核甘酸連結類鐸蛋白受體-9會減弱腫瘤壞死因子-α的產生

曹正婷�,2 余家利2 謝松洲2　徐翠文3　郭家禎�　林世昌�

�國泰綜合醫院 過敏免疫科

2台灣大學醫學院 附設醫院 風濕免疫科

3國泰綜合醫院 護理部

目的：本研究探討在未經過治療且處於疾病活動期的全身性紅斑性狼瘡患者，與性別、年齡對應的

健康受試者以CpG去氧核糖寡核甘酸連結類鐸蛋白受體9對於腫瘤壞死因子-α（TNF-α）的產生，是

否有差異。材料與方法：13位全身性紅斑性狼瘡患者與8位健康受試者的周邊血液單核球，以CpG

去氧核糖寡核甘酸和phorbolmyristateacetate及 ionomycin刺激，分別以反轉錄聚合酶反應和酵素

免疫法分析TNF-α的mRNA表達量和蛋白的產量。結果：全身性紅斑性狼瘡患者和健康受試者，以

CpG去氧核糖寡核甘酸連結類鐸蛋白受體-9，都會增加週邊血液單核球產生TNF-α，但是在全身性

紅斑性狼瘡患者產生的TNF-α的量比健康受試者低，並且達統計上顯著差異，對週邊血液單核球

TNF-α核糖核酸的表達量在這兩組則未達顯著差異。結論：未經過治療且處於疾病活動期的全身

性紅斑性狼瘡患者，其周邊血液單核球在類鐸蛋白受體-9被CpG去氧核糖寡核甘酸連結後減弱了

TNF-α的產生，此一結果反應出這個重要的前驅發炎激素在全身性紅斑性狼瘡患者失去正常調節的

現象，而這個現象可能和全身性紅斑性狼瘡患者感染到帶有CpG序列的細菌或病毒時其內生性免疫

力會減弱有關。

關鍵詞：全身性紅斑性狼瘡，腫瘤壞死因子，類鐸蛋白受體-9，CpG去氧核糖寡核甘酸