Evaluation of the cervicovaginal environment in asymptomatic Human T-cell lymphotropic virus type 1 infected women

w w w . e l s e v i e r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

Evaluation

of

the

cervicovaginal

environment

in

asymptomatic

Human

T-cell

lymphotropic

virus

type

1

infected

women

Alisson

de

Aquino

Firmino

_,

_Adenilda

_Lima

_Lopes

_Martins

_,

_Luana

_Leandro

_Gois

_,

Taiane

Silva

Paixão

_,

_Everton

_da

_Silva

_Batista

_,

_Bernardo

_{Galvão-Castro}

_,

Maria

Fernanda

Rios

Grassi

a_{EscolaBahianadeMedicinaeSaúdePública,Salvador,BA,Brazil} b_{Fundac¸ãoOswaldoCruz,InstitutoGonc¸aloMoniz,Salvador,BA,Brazil}

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Received14September2018 Accepted7February2019 Availableonline6March2019

Keywords: HTLV-1 Inflammatorycytokines Proviralload Cervicovaginalcytopathology

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Introduction:HumanT-celllymphotropicvirustype1(HTLV-1)issexuallytransmittedand causes persistent infection. This virusinduces activation of the immune system and productionof inflammatorycytokines.Thisstudy aimed toassess thecytokine profile andcytopathologicalfindingsinthecervicovaginalfluidofasymptomaticHTLV-1-infected women.

Methods:HTLV-1-infectedanduninfectedwomenwereselectedattheCentrode Atendi-mentoaoPortadordeHTLVinSalvador-Brazil.NoneoftheincludedHTLV-1-infectedwomen reportedanyHTLV-1-associateddiseases.Allvolunteersunderwentgynecological exam-ination tocollectcervicovaginalfluid.Cytokinequantificationwasperformedusingthe CytometricBeadArray(CBA)HumanTh1/Th2/Th17kit.Lightmicroscopywasusedto eval-uate cervicovaginalcytopathology.Inaddition, proviralloadin cervicovaginalfluidand peripheralbloodwasmeasuredbyreal-timequantitativepolymerasechainreaction. Results:112women(63HTLV-1-infectedand49uninfected)wereevaluated.Nodifferences werefoundwithrespecttocytopathologicalcervicovaginalfindingsbetweenthegroups. IL-2,TNF,IL-4,IL-10,andIL-17levelsweresignificantlyhigherincervicovaginalfluidofthe HTLV-1-infectedwomenthaninuninfectedwomen(p<0.05).Conversely,IFN-␥wasfound tobelowerintheHTLV-1-infectedwomen(p<0.001)comparedtouninfectedindividuals. Cervicovaginalproviralloadwasdetectablein53%oftheHTLV-1-infectedwomenandwas foundtobeconsistentlylowerthantheproviralloadinperipheralblood.

Conclusions: HTLV-1infectioninducesimmuneactivationincervicovaginalenvironment, characterizedbyelevatedconcentrationsofTh1,Th2,andIL17inthecervicovaginalfluid.

©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/ licenses/by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:fernanda.grassi@fiocruz.br(M.F.Grassi). https://doi.org/10.1016/j.bjid.2019.02.001

1413-8670/©2019SociedadeBrasileiradeInfectologia.PublishedbyElsevierEspa ˜na,S.L.U.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

HumanT-celllymphotropicvirustype1(HTLV-1)isaretrovirus distributedworldwide,withendemicareasfoundinAfrica, SouthandCentralAmerica,theCaribbean,Japan,Melanesia, andtheMiddle East.InBrazil,itisestimatedthat approxi-mately800,000peopleareinfected.1_{Thisvirusistransmitted} vertically from mother to child, mainly by breastfeeding, orhorizontally throughtransfusion ofblood,contaminated needles,orsexualintercourse.Arecentstudyconductedin Salvador,Brazil,underscored the importanceof thesexual routeinHTLV-1transmission.2_{Inthiscity,HTLV-1-infection} ismoreprevalentinwomen,reaching10%ofthoseoverthe ageof50.3

HTLV-1 is the etiological agent of adult T-cell leukemia/lymphoma (ATLL),4 _{HTLV-1-associated} myelopa-thy/tropical spastic paraparesis (HAM/TSP),5 _HTLV-1 associated uveitis,6 _{and infective dermatitis in children.}7 Inaddition, inflammatorydiseases,suchas keratoconjunc-tivitissicca(KCS),8_{bronchiectasis}9 _{and arthritis}10 _{are often} associatedwiththisviralinfection.

MemoryCD4+_{T-lymphocytesarethemaintargetcellsfor} HTLV-1,althoughCD8+_{T-lymphocytes,glialcells,and} circulat-ingdendriticcellsmayalsorepresenttargetsforinfection.11–13 Immune system activation is a hallmark of HTLV-1 infec-tion,resultinginspontaneouslymphoproliferation,increased expressionofactivationmarkers(HLA-DR,CD25),and exacer-batedproductionofproinflammatorycytokines(IFN-␥,TNF) andchemokines(IL-8,CXCL9andCXCL10).14,15_Studies indi-cate that elevatedproviral load (>5% ofinfectedcells) and immune activation are commonly found in the context of HTLV-1-associated diseases, as comparedto asymptomatic HTLV-1carriers.16–20_{However,asymptomaticindividualswith} low proviral load may also present pronounced immune activation.21

FewstudieshaveevaluatedtheeffectofHTLV-1infection inthevaginalmucosa.DetectionofHTLV-1DNAinthe cer-vicalfluidofinfectedwomenwaspreviouslyassociatedwith cervicitis.22_{Inaddition,presenceofanti-HTLV-1inthevaginal} fluidhasbeendescribed,including inasymptomatic HTLV-1infectedwomen.23_{Thisstudyevaluatesthecervicovaginal} environmentofHTLV-1infectedwomenbyassessingproviral load,cytopathologicalalterations,andcytokinelevels.

Methods

Recruitmentandstudydesign

The present cross-sectional observational study was con-ductedattheCentrodeAtendimentoao PortadordeHTLV oftheEscolaBahianadeMedicinaeSaúdePública,Salvador, Bahia, Brazil. Asymptomatic HTLV-1 infected women were sequentiallyincludedinthecourseofroutinemedical con-sultations from August2014to March2016.All individuals wereincludediftheirneurologicexaminationwasnormaland nocomplaintsforHTLV-1-associateddiseaseswerereported. Uninfectedwomen(controls)wereselectedfromcompanions or relativesof patients who attended consultations. These

individualswerepairedtoHTLV-1-infectedwomenmatched for age, presence ofcomorbidities, smoking, contraceptive methods,and presenceofother sexuallytransmitted infec-tions.ThesamplesizefortheHTLV-1asymptomatic group wascalculatedbasedona30%estimatedprevalenceofsexual dysfunctionforHTLV-1uninfectedwomen,withanestimated prevalenceratio(PR)of2.0amongboththeHTLV-1-infected and uninfectedwomen.Adoptinganalphaerrorof5%and power of 80%,the necessary sample size was determined to be49 womenineach group. Theinclusioncriteria con-sisted of age ranging from 20 to 50 years and report of sexualactivitywithinfourweeksprecedingtheconsultation. Women with HTLV-1-associated diseases, those who were menopausalordiagnosedwithdepression,aswellasthose takingmedicationknowntoaffectsexualdesire(beta block-ers,antidepressants,centralnervoussystemdepressantsor anticholinergics)wereexcluded.HTLV-1infection was diag-nosedbyEnzyme-LinkedImmunosorbentAssay(ELISA)with WesternBlotusedforconfirmation.Thisstudywasapproved bytheInstitutionalResearchBoardoftheEscolaBahianade Medicina eSaúdePública (registered underprotocol: CAAE 33098414.4.0000.5544)andallincludedwomensignedaterm ofinformedconsent.

Collectionandanalysisofsamples

Following the routine medical consultation, demographic, medical, sexual, and gynecological data were obtained through specific standardized data collection forms, and physical and gynecological examinations were performed. Papanicolaousmearswerecollectedfromtheectocervixand endocervixusinganAyresspatulaandcytobrush,respectively. Cottonswabswereusedtocollectvaginalfluidfromthe ecto-cervix, endocervixandvaginalwallsforproviral load(PVL) measurementandcytokinequantification.

Cell abnormalities detectedinthe Papanicolaou smears wereclassifiedinaccordancewiththeBethesdaSystem.24_To measurePVLinthecervicovaginalfluid,swabswereplacedin tubescontaining400␮Lofhydroxymethyl-ethylenediamine tetra acetic acid (Tris-EDTA) solution and stored at −20◦_C untiluse.Forcytokinequantification,swabswerepreserved in cryotubes containing 1mL ofsterilephosphate-buffered saline(PBS)storedat−70◦_{C.Cytokinelevelswereassessedby} flowcytometryusingtheCytometricBeadArray(CBA)Human Th1/Th2/Th17kit(Becton,DickinsonandCompany,New Jer-sey,USA).WholebloodsampleswerecollectedinEDTAtubes andperipheralbloodmononuclearcells(PBMC)wereobtained by density gradient centrifugation and cryopreserved until use.HTLV-1PVLincervicovaginalcellsandPBMCswas deter-minedbyreal-timeTaqManPCR,asdescribedelsewhere.25

Statisticalanalyses

Data were expressedasmediansand percentiles(25thand 75th)ormeansandstandarddeviation.Differencesbetween HTLV-1 infected and uninfected women concerning fam-ily income, schooling, length of relationship, number of partners, parity, proviral load, and cytokine levels were assessedbythenon-parametricMann–Whitney Utest, and differences in age were evaluated by the Student’s t-test.

Table1–SociodemographicprofileofHTLV-1-infectedanduninfectedwomen.

Variable HTLV-1-Infected(n=63) HTLV-1uninfected(n=49) p-value

Age(years)a _{34.68(7.18) 35.61(7.87) 0.52}

Familyincome(no.ofminimumwages)b _{1.2(1.0–2.0) 2.0(1.0–2.25) 0.01}

Educationallevel(years)b _{10.0(5.0–12.0) 12.0(8.0–12.0) 0.08}

Skincolorn(%)c _0.14 Black 33(52.4) 25(51.0) Mixed 21(33.3) 22(44.9) White 9(14.3) 2(4.1) Maritalstatusn(%)c _0.13 Married/Stableunion 50(79.4) 35(71.4) Single 13(20.6) 11(22.4) Divorced/Separated – 3(6.1)

Lengthofrelationship(years)b _{8.0(3.75–12.0) 3.0(1.15–7.5) 0.01}

Numberofpartnersb _{4.0(3.0–10.0) 4.0(2.0–7.0) 0.38}

Parityb _{2.0(1.0–3.0) 2.0(0.5–2.0) 0.40}

a _{Datapresentedasmean(standarddeviation)–p-value:Student’sttest.}

b _{Datapresentedasmedianandinterquartilerange(p25–p75)–p-value:Mann–WhitneyUtest.}

c _{Datapresentedasfrequency/proportion–p-value:Chi-squaretest.}

Differencesinqualitative variables (skincolor,marital sta-tusand cytopathological/vaginalmicrobiota) were assessed usingtheChi-squaretest.Spearman’srankcorrelation coeffi-cientwasusedtodetermineassociationsbetweencytokine levels and proviral load.p-values less than 0.05 were con-sideredstatisticallysignificant.Allanalyseswereperformed usingGraphPadsoftwareversion5.0andSPSSsoftware ver-sion17.0forWindows.

Results

Atotalof112women(63infectedwithHTLV-1and49 unin-fected) were evaluated (Table 1). No significant differences betweenthegroupswere seenregardingsociodemographic

profile,numberofpartners,orparity.HTLV-1-infectedwomen hadlongerrelationshipsthanuninfectedwomen(p=0.01)and themedianincomeofHTLV-1-infectedwomen(1.2minimum wage)was lowercomparedtothatofuninfectedwomen(2 minimumwages)(p=0.01).HTLV-1proviralloadwasdetected in94%ofthePBMCsamplesfromtheinfectedwomen,witha medianof28,665copies/106_{cells(IQR4868–69,408copies/10}6 cells).

Regarding cytopathological findings were similaramong HTLV-1infectedoruninfectedwomenwithnegativeresults for neoplasia (p=0.71) (Table 2). Atypical squamous cells of undetermined significance (ASC-US) was identified in 1.6% and 2% of infected and uninfected women, respec-tively (p=0.86). In addition, similar vaginal microbiota (Lactobacillus vaginalis, Gardnerella vaginalis/Mobiluncus spp.,

Table2–FrequencyofcervicovaginalcytopathologicfindingsandHTLV-1proviralloadincervicovaginalandPBMC

samples.

Variable HTLV-1-infected(n=63) HTLV-1uninfected(n=49) p-value Cervicovaginalcytopathologyn(%)a

Negativeforneoplasia 61(96.8) 48(98.0) 0.71

ASC-USb _{1(1.6) 1(2) 0.86} Unsatisfactory 1(1.6) 0 0.38 Vaginalmicrobiotan(%)a Lactobacillusvaginalis 11(17.5) 8(16.3) 0.87 Gardnerellavaginalis/Mobiluncusspp. 13(20.6) 15(30.6) 0.23 Coccus/Bacillus 23(36.5) 16(32.7) 0.67 Candidaspp. 16(25.4) 9(18.4) 0.37 Trichomonasvaginalis – 1(2) 0.25 HTLV-1PVLincervicovaginalfluidc,d _{62(0–2057) NA NA} HTLV-1PVLinPBMCc,e _{28,665(4868–69,408) NA NA}

a _{Datapresentedasfrequency/proportion–p-value:Chi-squaretest.}

b _{AtypicalSquamousCellsofUndeterminedSignificance.}

c _{Datapresentedasmedianandinterquartilerange(p25–p75)–p-value:Mann–WhitneyUtest.}

d _{Considering57women,numberofHTLV-1copies/10}6_cells.

Coccus/Bacillus, Candida spp., and Trichomonas vaginalis) were found in the two groups. HTLV-1 proviral load was detectablein53%ofthecervicovaginalsamplesfrom HTLV-1-infectedwomen.Themediancervicovaginalproviralload [62copies/106 cells (IQR0–2057copies/106cells)]was consis-tentlylowerthanthatinperipheralblood[28,665copies/106 cells(IQR4868–69,408)].

HTLV-1-infectedwomenhadsignificantlyhigher concen-trationsofIL-2,TNF,IL-10,IL-4,andIL-17inthecervicovaginal fluid than uninfected women (p<0.05). Conversely, these womenpresentedlowerconcentrationsofIFN-␥inthe vagi-nalfluidcomparedtothoseuninfectedbyHTLV-1(p<0.001) (Fig.1).TheIFN-␥/IL-10ratioinHTLV-1-infectedwomen(8.87, IQR 8.77–9.10) was significantly lower than in uninfected

2,5 2,5 2,5 2,0 1,5 1,0 0,5 0,0 21 20 19 18 2,4 2,4 2,3 2,2 2,1 2,0 1,9 2,2 2,0 1,8 1,6 1,4 25 20 20 15 15 10 2,5 2,0 1,5 1,0 0,5 0,0 3 2 1 0 2,0 1,5 1,0 0,5 0,0 HTLV-1 Infected p = 0,001

A

D

B

C

F

G

E

HTLV-1 Uninfected HTLV-1 Infected HTLV-1 Uninfected

HTLV-1 Infected HTLV-1 Uninfected

HTLV-1 Infected HTLV-1 Uninfected HTLV-1 Infected HTLV-1 Uninfected

HTLV-1 Infected HTLV-1 Uninfected HTLV-1 Infected HTLV-1 Uninfected

Fig.1–LevelsofIL-2(A),TNF(B),IFN-␥(C),IL-4(D),IL-6(E),IL-10(F)andIL-17(G)incervicovaginalfluidofHTLV-1-infected (n=63)anduninfectedwomen(n=49).

women(9.05,IQR8.88–9.23),(p<0.001).Cytokinelevelswere notfoundtobecorrelatedwithHTLV-1proviralloadineither thecervicovaginalfluidorinperipheralbloodmononuclear cells.

Discussion

Thepresentresultsindicatethat,inadditiontoelevated lev-elsofregulatoryIL-10,bothTh1(IL-2andTNF)andTh2(IL-4) cytokines,aswellasIL-17,wereallhigherinthe cervicovagi-nalfluidofHTLV-1-infectedwomenascomparedtouninfected individuals.Thisimmuneactivationofvaginalenvironment maybeconsequenttothe presenceofinfectedcellsinthe vaginalmucosa.Indeed,HTLV-1proviralloadwasfoundtobe detectableinthevaginalfluidofmorethan50%oftheinfected women.Corroboratingourresults,Belecetal.foundviralDNA inthreeoutof15HTLV-1-infectedwomenandsuggestedthat thevirusinducedalocalimmuneresponsethatresultedin increasedlevelsofHTLV-1antibodiesinthevaginalfluid.23 Inaddition,the presenceofHTLV DNAincervical samples obtainedfrom HTLV-1-infectedsexworkerswas associated withthediagnosisofcervicitis.22

Ithasbeen wellestablishedthatHTLV-1induces activa-tionoftheimmunesystem,whichisreflectedbyspontaneous proliferationofperipheralbloodmononuclearcellsand pro-ductionofcytokines.IndividualswithadiagnosisofHAM/TSP commonly present higher plasma levels of proinflamma-torycytokines(e.g.IFN-␥,TNF,IL-6)andchemokines(CXCL9, CXCL10)thanasymptomaticoruninfectedindividuals.14,16,26 Moreover,otherHTLV-1-associatedconditions,suchas neuro-genicbladder,27_{infectivedermatitis}17_{andsiccasyndrome,}18 havealsobeen reportedinassociation withhigherplasma levels of IFN-␥ and IL-6.16 _{In addition, high production of} IFN-␥isfoundinasymptomaticHTLV-1-infectedindividuals comparedtouninfectedcontrols.14_{Inthepresentstudy,low} levelsofIFN-␥andlowerIFN-␥/IL-10ratioswerefoundinthe cervicovaginalsamplesfromtheHTLV-1-infectedgroup.The regulatorycytokineIL-10hasbeenreportedtohavean antago-nisteffectontheproductionofIFN-␥.28_{Thepredominanceof} IL-10andTGF-␤iscommonlyfoundinthemucosaofhealthy individuals,whichcreatesaregulatorymilieuthatmaintains animmunologicaltoleranceagainstantigensfrommicrobiota andtheexternalenvironment.29,30_{Thesignificantlyhigher} IL-10levelsfoundinthecervicovaginalfluidofHTLV-1-infected womencomparedtouninfectedwomenseemstosuggestthat thepresenceofthevirusinducedincreasedimmune regula-tion.

Similarly, women infected withHIV, another retrovirus, alsopresentincreasedlevelsofTh1andTh2cytokinesinthe vaginalfluidascomparedtouninfectedcontrols.30,31 Regard-ingIL-17,astudyconductedinHIV-1-infectedwomenfound higherIL-17concentrationsinthevaginalfluidofwomenwith sexuallytransmittedbacterialinfectionsthaninthose with-outthesetypesofinfections,whereaswomenwithCandida spp.hadlowerIL-17concentrationscomparedtothosewithout candidalinfections.32

Ithas been demonstrated that localimmune activation induced byHIVin thevaginal environmentmay modulate

virus shedding incervicovaginal secretions.33 _Itispossible that HTLV-1 inducesa similar immune activation, thereby increasing cytokine production in situ. On the other hand, it is theoreticallypossiblethat elevated levels ofcytokines detectedinvaginalfluidmayalsobetheresultofanother phe-nomenon,suchasvaginaltransudation,anaturalprocessthat allowsvaginallubricationthroughvaso-dilatation.34

A positive correlation between inflammatory cytokine levelsandproviralloadinthebloodwasdescribedin HTLV-1-infected individuals diagnosed with Sicca syndrome.18 However,ourstudy foundnocorrelations betweenthe lev-els ofcytokinesin vaginalfluidand proviralload ineither cervicovaginal fluidor PBMCs.Ofnote, theHTLV-1-infected women evaluated herein were asymptomatic for diseases associatedwiththisvirusandhadverylowPVLinthe vagi-nalfluid,62copies/106_{cells,about0.006%ofinfectedcells,in} additiontointermediatelevelsofproviralloadintheblood (28,665copies/106 _{cells, about 2.9% ofinfected PBMCs).For} comparison,HTLV-1PVLinperipheralbloodisconsideredlow when<1%ofPBMCsareinfectedandintermediateonlywhen 1–5%ofPBMCsareinfected.20,35

Withrespecttothecytopathologicalfindingsandvaginal microbiota, nodifferences were observed between HTLV-1-infectedanduninfectedwomen.HigherlevelsofTh1andTh2 cytokines were foundin women with cervical intraepithe-lialneoplasia,withincreasinglevelsseeninaccordancewith lesionseverity.29,36_{Inourstudy,onewomanfromeachgroup} hadatypicalsquamouscellsofundeterminedsignificance,a cytopathologicalalterationthatrequiresperiodicmonitoring. Alimitationofthepresentstudywastheabsenceof HTLV-1-infectedwomendiagnosedwithHAM/TSP,whoconsistently presenthigherproviralloadsintheperipheralbloodas com-paredtoasymptomaticindividuals.However,HTLV-1proviral loadinthecervicovaginalfluidwasindeeddetectableinthe majority of HTLV-1-asymptomatic women herein. Another limitationwasthatascytokinelevelsintheplasmawerenot quantified,itwasimpossibletopairthesewiththoseinthe cervicalfluid.

Conclusions

The results presented herein show that HTLV-1 infection inducesimmuneactivationinthecervicovaginalenvironment ofasymptomaticwomen,characterizedbyelevatedlevelsof Th1,Th2,andIL17cytokinesinthecervicovaginalfluid. Fur-therstudiesshouldbeconductedinvolvingHTLV-1-infected womenwhopresenthighlevelsofproviralloadinthe periph-eralbloodtodeterminewhethercorrelationsexistregarding proviralloadincervicovaginalfluid.

Financial

support

This work was supported by the Fundac¸ão de Amparo à PesquisadoEstadodaBahia(BOL0575/2015).Thisstudywas financedinpartbytheCoordenac¸ãaodeAperfeic¸ãoamentode PessoaldeNívelSuperior(CAPES)-FinanceCode001

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

WethankMr.NoilsonLazaroandVivianaOlavarriafor tech-nicalassistance.Theauthors wouldliketothankAndrisK. WalterforEnglishrevision/editingservices.

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