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Genetic variation in IL-10 influences the progression of

hepatitis B infection.

Magda Rybicka, Anna Woziwodzka, Alicja Sznarkowska, Tomasz

Romanowski, Piotr Stalke, Marcin Dręczewski, Eloi Verrier, Thomas F.

Baumert, Krzysztof Piotr Bielawski

To cite this version:

Magda Rybicka, Anna Woziwodzka, Alicja Sznarkowska, Tomasz Romanowski, Piotr Stalke, et al..

Genetic variation in IL-10 influences the progression of hepatitis B infection.. International Journal

of Infectious Diseases, Elsevier, 2020, 96, pp.260 - 265. �10.1016/j.ijid.2020.04.079�. �hal-02917279�

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Genetic

variation

in

IL-10

in

fluences

the

progression

of

hepatitis

B

infection

Magda

Rybicka

a,

*

,

Anna

Woziwodzka

a

,

Alicja

Sznarkowska

a,b

,

Tomasz

Romanowski

a

,

Piotr

Stalke

c

,

Marcin

Dre

˛czewski

c

,

Eloi

R.

Verrier

d

,

Thomas

F.

Baumert

d,e

,

Krzysztof

Piotr

Bielawski

a,

*

a

DepartmentofMolecularDiagnostics,IntercollegiateFacultyofBiotechnology,UniversityofGdanskandMedicalUniversityofGdansk,Abrahama58, 80-307Gdansk,Poland

bInternationalCentreforCancerVaccineScience,UniversityofGdansk,ul.WitaStwosza63,80-308Gdansk,Poland cDepartmentofInfectiousDiseases,MedicalUniversityofGdansk,ul.PowstaniaStyczniowego9b,81-519Gdynia,Poland d

UniversitédeStrasbourg,Inserm,InstitutdeRecherchesurlesMaladiesViralesetHépatiquesUMR_S1110,F-67000Strasbourg,France

e

PôleHépato-Digestif,InstitutHospitalo-Universitaire,HôpitauxUniversitairesdeStrasbourg,Strasbourg,France

ARTICLE INFO Articlehistory:

Received17February2020

Receivedinrevisedform27April2020 Accepted28April2020 Keywords: HBV Cirrhosis Fibrosis IL10 Single-nucleotidepolymorphism ABSTRACT

Objectives: The outcomes of hepatitis B virus (HBV) infection vary substantially among affected individuals,providingevidenceoftheroleofhost geneticbackgroundinthesusceptibilitytoHBV persistenceandthedynamicsofliverinjuryprogressiontocirrhosisandhepatocellularcarcinoma(HCC). Methods:Sixsingle-nucleotidepolymorphismswithintheinterleukin10gene(IL10)weregenotypedby MALDI-TOFmassspectrometryin857patientswithchronicHBV infection(CHB),48patientswith resolvedHBVinfection,and100healthyvolunteers.Associationsoftheselectedpolymorphismswith susceptibilitytochronicHBVinfection,liverinjuryprogression,andoutcomeswereinvestigated. Results:IL10819T(rs1800871),592A(rs1800872),and+504T(rs3024490)alleleswereassociated with treatment-inducedhepatitisB surfaceantigen(HBsAg) seroclearance.Additionally, IL10 ATAC haplotypeincreasedthechanceofHBsAglossandwassignificantlymorefrequentinpatientswithless liver injury. Moreover rs1800871TT, rs1518110TT, rs1800872AA,and rs3024490TTgenotypes were identifiedaspredictorsofalowerFIB-4score(<0.5).

Conclusions:Thisstudyindicatesthatpolymorphismswithinthepromoterregionandintronicsequences ofIL10areassociatedwithchronicityofhepatitisBandwithHBV-inducedliverdamage.

©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense( http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Despiteprogressinimplementingvaccinationprogrammesand in thedevelopment of newtreatment perspectives, hepatitis B virus(HBV)infectionsremainamajorhealthproblemworldwide, contributing considerably to cirrhosis- and hepatocellular carcinoma(HCC)-relatedmortalityof0.5–1millionperyear.As muchas one-thirdofthegeneral populationcarries serological markersofHBVinfection(HuandRen,2017).

OutcomesofHBVinfectionvarysubstantiallyamongaffected individuals, providing evidence of the role of host genetic background in the susceptibility to HBV persistence and the

dynamics of liver injury progression tocirrhosis and HCC. The responseoftheimmunesystemtoHBVinfectioniscomplex,anda broadrangeofcytokinessuchasinterferon(IFN)-α/β,IFN-

g

,and tumournecrosisfactor(TNF)-αareinvolvedintheearlyphaseof infection.Aneffectiveantiviralresponse,mainlymediatedbyCD4+ andCD8+T-cells,naturalkillercells,andmonocytes,mayresultin immunecontrolledHBVreplication(functionalcure).Bycontrast, inchildrenandadultswithacompromisedimmunesystem,active viralreplicationcanbecomepersistent.InchronicHBVinfection, both the number of regulatory T-cells and levels of inhibitory interleukin10(IL-10)andtransforminggrowthfactorbeta(TGF-β) increase, leading to HBV-specific CD8+ T-cell exhaustion and rendering viral eradication from the liver impossible (Peeridogahehetal.,2018).

IL-10isrecognizedasakeycytokineregulatingtheimmune responsetoHBVinfection.Recently,asubsetofIL-10-producing B-cells,knownasregulatoryB-cells(Bregs),havebeenshownto

*Correspondingauthors.

E-mailaddresses:magda.rybicka@biotech.ug.edu.pl(M.Rybicka),

krzysztof.bielawski@biotech.ug.edu.pl(K.P.Bielawski).

https://doi.org/10.1016/j.ijid.2020.04.079

1201-9712/©2020TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

International

Journal

of

Infectious

Diseases

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regulateHBV-specificCD8+T-cellimmunity(Dasetal.,2012;Liu et al., 2016). Moreover, down-regulation of IL-10 restores the functionofexhaustedHBV-specificCD8+T-cells(Dasetal.,2012). InpatientswithchronichepatitisB(CHB),boththelevelsofIL-10 andnumberofregulatoryB-cellsincrease(Dasetal.,2012).Serum IL-10levelsreflectthedynamicsofviralloadandliverin flamma-tion,andarecorrelatedwithspontaneousflaresofliver disease (Dasetal.,2012).Moreover,IL-10mightsubstantiallyaffectthe antiviral immune response, as it inhibits the production of proinflammatorycytokinessuchasIFN-

g

,TNF-α,IL-1β,andIL-6 (Walter,2014).

The capacity for IL-10 production is regulated on the transcriptionallevelandisaffectedbysingle-nucleotide polymor-phisms(SNPs)locatedinthe50-flankingpromoterregionof the IL10 gene: 1082A/G (rs1800896), 819C/T (rs1800871), and 592C/A (rs1800872) (Edwards-Smith et al.,1999; Reuss etal., 2002; Höhler et al., 2005). Numerous studies have aimed to elucidatetheroleoftheseSNPsintheanti-HBVresponse,anda recent meta-analysis conducted by Shu et al. confirmed their clinical significance in the course of HBV infection (Shuet al., 2015). Additionally, the 1353G/A (rs1800893) promoter and +954G/T (rs1518110) intronic IL10 SNPs have been shown to influence the outcome of HBV infection in African Americans (Truelove et al., 2008). Another IL10 intronic variant +504G/T (rs3024490) has been related to susceptibility to CHB in the ChineseHanpopulation(Zhangetal.,2014).

InCHB,activationoftheimmuneresponse,requiredforvirus eradicationfromhepatocytes,isalsoassociatedwithinflammation thatleadstoliverdamage.ThehostIL10geneticbackgroundisa riskfactorforliverinjuryandthedevelopmentofcirrhosisinCHB (Ghaleh Baghi et al., 2015; Guo et al., 2015; Yao et al., 2015). Interestingly,theroleofIL-10inliverdiseaseisnotlimitedto HBV-infected patients, and the interactions of the IL10 promoter genotypeandliveroutcomehavealsobeenobservedinhepatitisC virus (HCV) infection (Guo et al., 2015) and alcohol-related cirrhosis(Yangetal.,2014).Indeed,thereisevidencesuggesting thatIL-10,apartfromitswell-documentedroleinregulatingthe

immuneresponsetoHBVinfection,alsoexertsamodulatoryeffect inliverfibrogenesis(Louisetal.,1998;Thompsonetal.,1998).

ThisstudywasperformedtoinvestigatetheimpactoffourSNPs located in the promotorregion of IL10 (rs1800896, rs1800871, rs1800872,rs1800893)andtwoIL10intronicvariants(rs1518110, rs3024490)onthecourseofCHBinaEuropeanpopulation. Materialsandmethods

Patients

The studygroupconsistedof 857patientswithCHB and48 untreated patientsin the hepatitis Bsurface antigen (HBsAg)-negative phase of disease (HBsAg-negative, hepatitis B core antibody(anti-HBc)reactive).Thebloodsamplesof648patients werecollectedfromtheANRSCO22HEPATHERcohort (Clinical-Trials.govregistrynumberNCT01953458).Theremaining257CHB patients were recruited from the Department of Infectious Diseases, Medical University of Gdansk, and the Hepatology OutpatientsClinicofthePomeranianCentreforInfectiousDiseases and Tuberculosis in Gdansk in 2014–2016. The control group comprised100blooddonorswithconfirmedabsenceofHIV,HBV, and HCV infections from theGdansk Regional Centre of Blood DonationsandHaemotherapy(Figure1).Patientswithahistoryof schistosomiasisinfection,heavyalcoholabuse,non-alcoholicfatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or inheritedliverdiseaseswereexcludedtopreventinterferencewith theassociationwithHBVinfection.

Bloodtests,includinghepatitisBserology(HBsAg,hepatitisBe antigen (HBeAg), hepatitis B e antibody (anti-HBe)) and the quantification of HBV DNA, were performed on all recruited patients. Liver biopsies were collected from 132 patients. The specimens wereassessedforinflammationactivityandstageof fibrosisaccordingtoScheuerscores.For725patients,liverfibrosis orcirrhosiswasassessedaccordingtotheMetavirscoringsystem, asdescribedbyCarratetal.(2019).Significantfibrosiswasdefined asF2fortheMetavirandScheuerstagingsystemsandcirrhosis

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wasdefinedasF4.TheFibrosis-4score(FIB-4)(Sterlingetal., 2006)andaspartateaminotransferase(AST)-to-plateletratioindex (APRI) (Wai 2003) were also used to estimate the amount of scarringintheliver.

Ofthe675patientswhoreceivedtreatmentaccordingtoPolish orFrenchNationalHealth Servicerecommendations,99 (14.5%) weretreated with pegylated interferon alpha(PEG-IFN-α), 502 (74.5%)withnucleoside/nucleotideanalogues(NAs),and74(11%) with combination NA therapy. The treatment response was monitoredbythemeasurementofHBVDNAand HBsAglossat week24 after treatment discontinuation for PEG-IFN-α-treated individuals,andatweek72oftreatmentforNA-treatedpatients.At the 48-week follow-up, an analysis was conducted to further monitor HBsAg loss. Sustained virological response (SVR) was defined as an undetectable HBV DNA level at 24 weeks after treatmentdiscontinuation.

The study was performed with the approval of the local independent bioethics committee at the Medical University of GdanskincompliancewiththeDeclarationofHelsinki,andwritten consentwasobtainedfromeachpatient.

SNPgenotyping

GenomicDNAwasisolatedfromthewholebloodsamplesusing the MagNA Pure Compact Nucleic Acid Isolation Kit I (Roche, Germany)accordingtothemanufacturer’sprotocol.The polymor-phisms of the IL10 gene (592C/A, rs1800872; 819C/T, rs1800871; 1082G/A, rs1800896; 1353C/T, rs1800893; +504G/T, rs3024490; +954G/T, rs1518110) were analysed by MassARRAYplatformwithiPLEXProchemistry(AgenaBioscience, USA) according to the standard protocol. Amplification and extension primers (Table 1) were designed with Agena Assay DesignSuitev2.DuringtheinitialamplificationPCR,sixdifferent products of 100 bp containing the SNPs of interest were amplified.Next,unincorporateddNTPswereremovedwithshrimp alkaline phosphatase. Finally, after single-nucleotide extension reaction,allele-specificproductsofdistinctmasseswereobtained. Thepurifiedextensionreactionproductswithananion-exchange resin,werespottedontoSpectroCHIPusingaMassARRAYRS1000 Nanodispenser.Mass spectra wereacquiredwitha MassARRAY Analyzer4massspectrometerandwereanalysedwithMassARRAY Typer4.0software.

Statisticalanalysis

The statistical analysis was conducted using Statistica 13.3 (StatSoft,USA). MIDASsoftwarewas usedtoassess thelinkage disequilibrium (LD) and deviations from Hardy–Weinberg equilibrium (HWE) of the analysed SNPs. The Chi-square test andFisher’sexacttestwereappliedtoanalysethedistributionof nominal variables. Haplotype blocks were constructed using HaploView 4.2 program. Quantitative variables were expressed as the median values (unless stated otherwise) and compared withtheMann–WhitneyU-test or Kruskal–Wallis test. Logistic

regressionwasconductedtodeterminetheassociationsbetween analysedvariablesadjustedforpossibleconfounders.Allstatistical testsweretwo-tailed.p-Valuesoflessthan0.008wereconsidered significantaftertheBonferronicorrectionwasappliedtoaccount formultipletesting.

Results

Studygroupcharacteristics

AllenrolledindividualswereunrelatedCaucasianadults;857 werepatientswithCHB,48wereindividualswhohadrecovered spontaneously from HBV infection (HBsAg-negative phase), constituting the functional cure control group, and 100 were volunteers,constitutingthehealthycontrolgroup.Theirmeanage was 52.0 years, 56.9 years, and 27.3 years, respectively. The baselinecharacteristicsofthestudyandcontrolgroupsareshown inTable2.

ThegenotypesofsixSNPswithinIL10wereobtainedforall1005 subjectsincludedinthestudy,withasuccessrateof100%.AllSNPs werevariableinthestudygroup.Genotypicandallelicfrequencies oftheanalysedSNPsfortheCHBandcontrolgroupsareshownin SupplementaryMaterialTableS1andTableS2.Forthecontrol groups,thedistributionofgenotypesfollowedHWE(p>0.05), exceptforrs1800871,rs1800872,andrs1518110inindividualswith functional cure. For CHB patients, the distributions were not consistent with HWE (p < 0.05). Significant differences in genotypic distribution of rs1800871, rs1800872, and rs151811 werefoundbetweenhealthyblooddonorsandindividualswith HBsAg loss. Moreover, the genotypic distribution of rs1800893 differedbetweentheCHBpatientsandthehealthycontrolgroup (Supplementary MaterialTable S1). No statistically significant differences in allelic distribution of IL10 polymorphisms were observed between the CHB and the control groups (SupplementaryMaterialTableS2).

Thehaplotypeanalysisshowedlinkagedisequilibriumbetween rs1800871and rs1800872(r2 =0.88),rs1518110(r2=0.74),and rs3024490(r2=0.78);betweenrs1800893and1800896(r2=0.89);

between rs1518110 and rs1800872 (r2 = 0.68); and between

rs1518110andrs3024490(r2=0.72).AllremainingpairsofSNPs wereindependent(r2<0.2).

Inthisstudy,rs1800896CCandrs1800893CCgenotypeswere morecommon inpatientswithlowerbaseline HBVDNAlevels (<2000IU/ml)(sex-adjustedGG,GAvs.AA:oddsratio(OR)1.72, 95%confidenceinterval(CI)1.16–2.57,p=0.007;sex-adjustedTT, CTvs.CC:OR1.82,95%CI1.22–2.70,p=0.003).

IL10polymorphismsandtreatmentresponse

FortheCHBpatientswhounderwentantiviraltreatment, an analysiswasperformedtodeterminewhethertheIL10 polymor-phisms affected the treatment response expressed as HBsAg clearance.ThreeoftheanalysedpolymorphismswithinIL10were significantlyassociatedwithtreatment-inducedHBsAglossat24

Table1

PrimersusedforMALDI-TOFMassARRAYsingle-nucleotidepolymorphismgenotyping. NCBIID PCRforwardprimera

PCRreverseprimera

Extensionprimer

rs1800871 ATGCTAGTCAGGTAGTGCTC GGTGTACCCTTGTACAGGTG CCCCCTTGTACAGGTGATGTAA rs1800872 AAAGGAGCCTGGAACACATC TCCTCAAAGTTCCCAAGCAG CAAGAGACTGGCTTCCTACAG rs1800893 CTGACTATAGAGTGGCAGG CCTGCCATTCCAGTTTAGAC TGTAACTGGGAGGAACA rs1800896 GACAACACTACTAAGGCTTC ATTCCATGGAGGCTGGATAG TTACCTATCCCTACTTCCCC rs1518110 ATGCGGTCTTTTTGATGCCC TGATTTTTTTGGGCCAGAGC GGGCCAGAGCCAATTT rs3024490 GGGAGCATCTTCACCTCGAA CTTATAAGATCCTGCTGGCG GGCTAGGAGAAGTAAAGAAA MALDI-TOF,matrix-assistedlaserdesorption/ionizationtime-of-flight.

a

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weeks after discontinuation of PEG-IFN-α. Significantly higher minor allele frequencies at 819T (rs1800871), 592A (rs1800872), and +504T (rs3024490) wereobservedin patients withHBsAg loss(Table 3).In thegroup of patientspreviously treated with NAs, rs1800896 was associated with a higher predispositiontotreatment-inducedlossofHBsAg(ageand sex-adjusted: OR 0.037, 95% CI 0.002–0.66, p = 0.01); however significance was lost after Bonferroni correction. Additionally, the IL10 ATAC haplotype (1082A/819T/592A/1353C) increasedthechanceof achievingtheHBsAg-negativephase in thePEG-IFN-α-treated(sexandage-adjusted:OR9.85,95%CI1.13– 85,p=0.0034)andNA-treated(sexandage-adjusted:OR20.33, 95%CI3.25–127,p=0.0012)groups.

TheincidenceofHBsAglosswasassessedin344patientsfrom theoriginalcohortof675CHBpatientswhounderwentantiviral treatment, and the clearance rate was found to be 6.98%. Comparison of the genotype and allele frequencies between individuals withand without treatment-inducedloss of HBsAg revealedsignificantdifferences inallsix polymorphismswithin IL10(Table4).

IL10polymorphismsandliverinjury

Toexaminepossibleriskfactorsforliverinjuryassessedby FIB-4 in the study population, logistic regression analyses were conductedonvariablesthatwereIL10genotypes,sex,andage.In themultivariateanalysisrs1800871TT,rs1518110TT,rs1800872AA, and rs3024490TT genotypes were identified as predictors of a lowerFIB-4score(p<0.5)(Table5).Similarobservationswere foundfortheAPRIindexresults,whichweresignificantlylowerin patientswithahomozygosityoftheminorallelewithinthesefour SNPsintheIL10gene(p=0.003).Additionally,theIL10geneATAC haplotype (1082A/819T/592A/1353C) was significantly more frequentin patientswith less liver injury (OR 8.94, 95% CI 1.78–44, p = 0.007). Moreover, the IL10 GCCT haplotype (1082G/819C/592C/1353T)increasedtheriskofdeveloping cirrhosis(OR2.61,95%CI1.58–4.30,p=0.0003).

Discussion

Initskeyrole,IL-10actsasanimmunosuppressivecytokineby suppressingT-cellproliferationandantigen-presentingcell(APC) functions,andbymodulatingcytokineandchemokinesynthesis (Saraiva and O’Garra, 2010).IL10 expressionis elevated during severalchronicviralinfections,whichservesasaviralstrategyto downregulatethehostimmuneresponseandallowviral persis-tenceinthehost(Hyodoetal.,2004;Ohgaetal.,2004;Brooks etal.,2006;Kaplanetal.,2008;Brockmanetal.,2009).Overrecent yearsithasbeenestablishedthatpolymorphismwithintheIL10 promoterregioninfluencesexpressionandserumlevelsofIL-10. ThreefunctionalSNPsintheIL10promoterhavebeeninvestigated intensively:1082A/G,819T/C,and592A/C.TheseSNPspresent threemajorhaplotypes:ATA,ACC,andGCC,whichareassociated withlow,medium,andhighlevelsofIL10expression,respectively (Turneretal.,1997;Eskdaleetal.,1998).Thesethree polymor-phismshavebeendemonstratedtobeessentialmodulatorsofthe immuneresponseagainsthepatitisviralantigens,suggestingtheir roleintheaetiologyofHBVinfection(Höhleretal.,2005).

Inthisstudy,sixcommonpolymorphismsintheIL10gene(four from the promoter region and two intronic variants) were genotyped in a cohort of HBV chronically infected patients, a groupofindividualsintheHBsAg-negativephase,andinhealthy blood donors. Differences in genotype distribution in one SNP (rs1800893)betweenCHBpatientsandhealthyblooddonors,and inthreeSNPs(rs1800871,rs1800872,rs1518110)betweenhealthy blooddonorsandindividualsintheHBsAg-negativephaseofHBV infection, were observed. Strikingly, all of the analysed SNPs

Table3

IL10polymorphismsandchanceofPEG-IFN-α-inducedHBsAgloss.a

IL10genotype MAF Oddsratio 95%CI p-Value rs1800896[A/G] AAvs.GA G=0.27 0.43 0.15–1.27 0.12 AAvs.GG 0.19 0.02–1.61 0.10 rs1800871[C/T] CCvs.CT T=0.43 4.43 1.33–14.76 0.016a CCvs.TT 19.59 1.76–217 0.0068b* rs1800872[C/A] CCvs.CA A=0.39 3.60 1.31–9.89 0.013a CCvs.AA 12.97 1.72–97 0.007b * rs1800893[G/A] CCvs.CT T=0.28 0.42 0.15–1.26 0.12 CCvs.TT 0.19 0.022–1.62 0.10 rs3024490[G/T] GGvs.GT T=0.43 3.67 1.35–10 0.012a GGvs.TT 13.46 1.81–100 0.009b * rs1518110[G/T] GGvs.GT T=0.42 1.54 0.66–3.63 0.31 GGvs.TT 2.39 0.43–13.18 0.31 CI,confidenceinterval;HBsAg,hepatitisBsurfaceantigen;IL10,interleukin10gene; PEG-IFN-α,pegylatedinterferonalpha;MAF,minorallelefrequency.

a

Multivariatelogisticregressionanalysisadjustedforage;HBsAglossdefinedas HBsAgnegativityatweek24aftertreatmentdiscontinuation.

b Significant

p-values(p<0.05);thosemarkedwithanasterisk(*)remained significantafterBonferronicorrection.

Table2

Baselinedemographicandclinicalcharacteristicsofthestudyandcontrolgroups. CHBpatients

(n=857)

Individualswithfunctionalcure (n=48) Healthycontrols (n=100) Age,years 511 55.71.4 27.30.9 Sex,%female 37% 30% 22% Origin,%Caucasian 100% 100% 100% ALT,IU/l 9753 29.501.91 – AST,IU/l 21991 30.252.83 – HBVDNA,kIU/ml 69571728 – – HBsAg,%positive 100% 0% – HBeAg,%positive 13% – – Anti-HBsAg,%positive 0% 100% – Anti-HBcAg,%positive 100% 100% – Anti-HBe,%positive 82% – –

Liverinflammationgradea

1.5(0–2) – –

Liverfibrosisstagea 1(0–2) 0.62(0–4)

ALT,alanineaminotransferase;AST,aspartateaminotransferase;CHB,chronichepatitisB;HBeAg,hepatitisBeantigen;HBsAg,hepatitisBsurfaceantigen;HBV,hepatitisB virus.Dataarereportedasthemeanstandarderrorofthemean.

a

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showedsignificantlydifferentgenotypicandallelicdistributions between chronically infected patients and the spontaneously recoveredgroup.Hyodoetal.(2004)showedthatIL-10production iselevatedinresponsetotheHBVcoreantigen(HBcAg)inpatients withchronicinfection,suggesting that itsexcessive production maycontributetoHBVpersistencyinthesepatients(Hyodoetal., 2004).KnowingthatalloftheanalysedSNPsareassociatedwith the IL10 expression level, it is justified to conclude that IL10 polymorphismsplay an essential role in the immune response againstHBVinfection.

As well as determining the possible association of IL10 polymorphismswiththechronicityofHBVinfection,thisstudy alsoinvestigatedwhetherthesecouldhaveapredictivevaluein assessingtheresponseofCHBpatientstoantiviraltherapy(IFN-α andanalogues)measuredbyserumHBsAgloss.Inthisstudy,three of the IL10 treatment response to PEG-IFN-α, and it was determined that the ATAC haplotype increased the chance of treatment-inducedHBsAg loss. The ATA haplotypehas already beencorrelatedwithalowlevelofIL10expression.ATAindividuals secrete on average two or three times less IL-10 than GCC individuals(Turneretal.,1997).Heterogeneityin thepromoter regionoftheIL10geneaffectstheinitialresponsetoIFN-αtherapy

in patients with hepatitis C. Patients who are genetically predisposedtohighIL-10productionhaveshownapoorresponse toIFN-α.Abetterresponsetotreatmenthasalsobeenassociated withlowerliverfibrosis.Similarlyinthepresentstudy,allelesthat wererelatedtotreatment-inducedHBVlosswereassociatedwith adecreasedliverfibrosisscoredbyFIB-4andAPRIindexes.This indicatesthatIL10polymorphismsmighthaveaprognosticvalue in assessingtheliverconditionin chronicallyinfected patients. Moreover, the ATAC haplotype, which was associated with a decreasedIL10expressionandanincreasedchanceofeliminating viralinfection,wasmoreoftenfoundinpatientswithlessintense liverinjury.Incontrast,theGCCThaplotype,associatedwithhigh IL10expression,hasbeenshowntoincreasetheriskofprogression tocirrhosis(Shin,2003).ThisprovesthatIL10polymorphismsmay not only impact the antiviral immune response, but also HBV infection-inducedliverfibrosis.Instudiesonalcohol-inducedliver cirrhosis,itwasthelowexpressionoftheATAhaplotypethatwas associatedwithincreasedfibrosisanda higherriskofalcoholic cirrhosisinTaiwanesepatients(Yangetal.,2014).IL-10hasbeen shown to protect against inflammation-induced liver damage, fibrosis, and cirrhosis due to its immunosuppressive action (Thompsonet al.,1998).Interestingly,in the presentstudy the ATAChaplotypewas found tobeassociated witha betterliver conditionand less fibrosisin chronicallyinfected patients. This leadstotheconclusionthatlowerlevelsofIL-10allowthevirusto beeradicatedmorequicklyandmoreeffectively,andinthisway protecttheliverfromthevirus-inducedfibrosis.

Whatemergesfromthisstudyisthatpolymorphismswithin thepromoterregionandintronicsequencesofIL10arestrongly associated with chronicity of hepatitis B and with the virus-inducedliverdamage.

Thisstudyhassomelimitations.Theinfluenceoftheparticular polymorphismsonIL10expressionwasnotanalysed.However,in termsoftheSNPslocalizedinthepromoterregion,therearemany studiespointingtotheirimpactonIL10expression(throughthe modulationofthetranscriptionfactorbindingsites),althoughwe donotknowhowtheintronicvariantsaffecttheIL-10leveland/or activity. It can only be hypothesized that intronic SNPs affect splicingandthereforemodulateIL10mRNAlevelsorstability,but thishypothesisawaitsverification.

Table4

GenotypicandallelicdistributionofanalysedIL10polymorphismsbetweenCHBpatients(HBsAg-positive)andindividualswhoachievedHBsAglossinducedbyantiviral treatment(HBsAg-negative).

SNPID Genotypicdistribution(%) Allelicdistribution(%)

Genotype HBsAg(+)(n=320) HBsAg()(n=24) p-Value Allele HBsAg(+)(n=640) HBsAg()(n=48) p-Value 592C/A(rs1800872) CC 185(58) 6(25) C 471(74) 22(46) CA 101(31) 10(42) 0.0007a A 169(26) 26(54) 0.000039a AA 34(11) 8(33) 819C/T(rs1800871) CC 182(57) 6(25) C 470(73.5) 22(46) CT 106(33) 10(42) 0.0005a T 170(26.5) 26(54) 0.000044a TT 32(10) 8(33) 1082G/A(rs1800896) GG 61(19) 1(4) G 263(41) 10(21) GA 141(44) 8(33) 0.03a A 377(59) 38(79) 0.005653a AA 118(37) 15(63) 1353C/T(rs1800893) CC 122(38) 14(58) C 363(57) 37(77) CT 119(37) 9(38) 0.041a T 277(43) 11(23) 0.005809a TT 79(25) 1(4) +504G/T(rs3024490) GG 173(54) 7(29) G 462(72) 24(50) GT 116(36.5) 10(42) 0.005a T 178(28) 24(50) 0.001132a TT 31(9.5) 7(29) +954G/T(rs1518110) GG 188(59) 12(50) G 476(74.5) 28(58) GT 100(31) 4(17) 0.0021a T 164(25.5) 20(42) 0.015446a TT 32(10) 8(33)

CHB,chronichepatitisB;HBsAg,hepatitisBsurfaceantigen;HBsAg(+),HBsAg-positivepatients;HBsAg(),HBsAg-negativepatients;IL10,interleukin10gene;SNP,single nucleotidepolymorphism.

a

Significantp-values(p<0.05).

Table5

RelationshipbetweenIL10polymorphismsandseverityofliverdiseasein HBV-infectedpatients.a

IL10genotype MAF Oddsratio 95%CI p-Value rs1800871[C/T] CC,CTvs.TT T=0.43 8.94 1.78–44.80 0.007b * rs1800872[C/A] CC,CAvs.AA A=0.39 9.15 1.82–45.89 0.006b * rs3024490[G/T] GG,GTvs.TT T=0.43 9.02 1.79–45 0.007b* rs1518110[G/T] GG,GTvs.TT T=0.42 8.84 1.76–44.29 0.008b*

CI,confidenceinterval;HBV,hepatitisBvirus;IL10,interleukin10gene;MAF,minor allelefrequency.

a

LiverfibrosiswasassessedbytheFIB-4index.

b Significant

p-values(p<0.05);thosemarkedwithanasterisk(*)remained significantafterBonferronicorrection.

(7)

Funding

ThisworkwassupportedbytheNationalCentreforResearch and Development (NCBR, grant number INFECT-ERA/01/2014) andbytheFrenchNationalResearchAgency(ANR,grantnumber ANR-13-IFEC-0006-02)intheframeoftheEuropeanconsortium Infect-ERA hepBccc and ST79 from the Medical University of Gdansk,and the MOBI4Health project, which is funded by the EuropeanUnion’s Seventh Framework Programme under grant agreementnumber316094.

Conflictofinterest Nonetodeclare. Acknowledgements

Wethanktheparticipantsand participatingcliniciansof the ANRS CO22 HEPATHER cohort at each study site. We thank F. Carrat,S.Pol,andH.Fontaine,thecoordinatorsofthecohort,C. Dorival,projectmanager,andtheANRSCO22HEPATHERscientific committee.TheANRSCO22HEPATHERcohort issponsoredand fundedbyInserm-ANRS(FranceRechercheNord&SudSida-vih Hepatites), ANR (Agence Nationale de la Recherche), DGS (Direction Ge’ne’rale de la Sante’), and MSD,Janssen,Gilead, AbbVie,BMSandRoche.

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Figure

Figure 1. Diagram presenting study group.

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