Case
Report
Immune
dissociation
during
acute
hepatitis
E
infection
Jose
D.
Debes
a,b,
Zwier
M.A.
Groothuismink
b,
Michail
Doukas
c,
Robert
A.
de
Man
b,
Andre
Boonstra
b,*
a
DepartmentofMedicine,UniversityofMinnesota,Minneapolis,MN,USA
b
DepartmentofGastroenterologyandHepatology,ErasmusMC,UniversityMedicalCenter,Rotterdam,TheNetherlands
c
DepartmentofPathology,ErasmusMC,UniversityMedicalCenter,Rotterdam,TheNetherlands
ARTICLE INFO
Articlehistory: Received2May2019
Receivedinrevisedform2August2019 Accepted7August2019
CorrespondingEditor:EskildPetersen, Aar-hus,Denmark Keywords: HEV Immune-regulation Cholestatichepatitis ABSTRACT
Objective:WereporttheimmuneresponseduringacaseofacuteHEVresponseinapatientwithanacute
hepatitisEvirus(HEV)genotype3infectionintheNetherlands.
Methods:Cytokineevaluationwasperformedviamultiplexcytokinearrayfor65immunemarkersin
plasmaduringthedifferentphasesofhepatitis.
Results:Thepatientinitiallypresentedwithfeaturestypicalofacuteviralhepatitis,withdetectableHEV
RNAinblood.Thisevolvedintoacholestaticdiseasefollowingperipheralclearanceofthevirus,leading
tothedemiseofthepatient.RealtimePCRrevealedthepresenceofHEVinlivertissue,suggestiveof
activeintrahepaticinfectiondespiteclearanceinblood.DuringthephaseofdetectableHEVRNAin
serum,therewasasurgeinT-cell-relatedimmunemediators,aswellasinterferonalphaandinterferon
gamma-inducedprotein10(IP-10),characteristicofaviralinfection.Afterclearanceofthevirusinthe
bloodanddevelopmentofcholestatichepatitis,severalinflammatorymarkerssubsided,followedbyan
increaseinimmune factorsrelatedtoanti-inflammatoryactivity,aswellas
monocyte/macrophage-relatedmarkers,likelyduetotheintrahepaticpresenceofthevirus.
Conclusions:Thisreportdescribesthedissociationofintra-andextra-hepaticimmuneresponsesduring
acuteHEVinfection.Assheddingofthevirusbecamesolelyintrahepatic,animmuneprofilereflectiveof
theactivityofhepaticresidentcellswasobserved.
©2019TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.
ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(
http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Hepatitis E virus (HEV), specifically genotype 3, has been recognizedasacauseofchronichepatitisinimmunosuppressed individuals, particularly in solid organ transplant recipients in high-resourcecountries(Debesetal.,2016;Kamaretal.,2008).In recentyears,somelimitedinsightintotheimmunemodulation triggeredbychronicHEVinfectionhasbeenobtained. However, acutesymptomaticHEVgenotype3infectionisrarein resource-rich countries (Brost et al., 2010; Saint-Jacques et al., 2016). Consequently,thereisapaucityofknowledgeabouttheimmune processesinvolvedduringacuteHEVinfectionwithgenotype3. This case report describes a patient with fatal autochthonous cholestatic HEV in the Netherlands, with an analysis of the patient’speripheralimmuneresponses.
Casereport
A73-year-oldmalewithahistoryofdiabetesmellitus,presented witha2-weekhistoryoffatigueanddarkurine.Hisvitalsignswere withinnormallimitsandhisphysicalexaminationwasremarkable forjaundice.
Laboratoryinvestigationsshowedanaspartateaminotransferase (AST)levelof4700IU/ml,alanineaminotransferase(ALT)of5600IU/ ml,totalbilirubinof15mg/dl,alkalinephosphataseof149IU/ml, internationalnormalizedratio(INR)of1.2,andferritinlevelof19 000ng/ml. A complete blood count and chemistry panels were normal.Thepatientdeniedtakinganynewmedications, over-the-counterproducts,oringestingmushrooms.Hedeniedtheuseof alcohol,cigarettes,orillicitdrugs.Hehadnohistoryofrecenttravel andnoclosecontactsreportedanyrecentillnesses.
Aninfectiouswork-uprevealednegativeserologyforcommon viralhepatitisincludinghepatitisA,B,andCviruses,Epstein–Barr virus(EBV),herpeszostervirus(HSV),andcytomegalovirus(CMV). SerologiesforHEVIgGandIgMwerepositive,andPCRforHEVwas positiveat8.3104IU/ml.FurtherHEVampli
ficationdetermined thepresence of HEVgenotype3. Computedtomographyof the
* Correspondingauthorat:Department ofGastroenterologyandHepatology, ErasmusMC,UniversityMedicalCenterRotterdam,Wytemaweg80,RoomNa-1011, 3015CERotterdam,TheNetherlands.
E-mailaddress:p.a.boonstra@erasmusmc.nl(A.Boonstra).
https://doi.org/10.1016/j.ijid.2019.08.006
1201-9712/©2019TheAuthor(s).PublishedbyElsevierLtdonbehalfofInternationalSocietyforInfectiousDiseases.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
InternationalJournalofInfectiousDiseases87(2019)39–42
ContentslistsavailableatScienceDirect
International
Journal
of
Infectious
Diseases
abdomen revealed a possible nodular liver contour, but no evidenceofportalhypertension.Thepatientwasdiagnosedwith acuteautochthonousHEVinfection.Duetothepossiblenodular contour,therewasconcernforlivercirrhosisinthispatient,buta fibroscanstudydidnotindicateadvancedfibrosis.
Followingadmission,thepatient’sliverenzymelevelsshoweda downwardtrend.Heshowed signsof clinicalrecoveryand was discharged home with no treatment and a diagnosis of acute hepatitisEinarecoveringphase.
Atthepatient’sfollow-upvisit2weekslaterintheoutpatient clinic,thepatientcontinuedtoexpressfatigue.HisALTandAST levelswere372IU/ml and 470IU/ml, respectively, but his total bilirubin level had increased to 33mg/dl. The patient was re-admittedtohospital(forachronologyofevents,seeFigure1A). Repeat PCR for HEV RNAin blood was negative,indicating no peripheralviralactivity(atthetimenofecesweresentforHEV assessment).Itwasdecidedtoobtainaliverbiopsytoaidinthe diagnosis,which revealedmild lobularandportal inflammatory infiltrates with obvious bilirubinostasis and slight periportal fibrosis, with no evidence of cirrhosis (Figure 1B). The biopsy showednoevidenceofmedication-ortoxin-inducedliverdisease. Adiagnosisofcholestatichepatitiswasmade.Overthefollowing week, the patient developed an upper gastrointestinal bleed secondarytoduodenalulcers,sufferedfurtherhepaticaswellas renaldecompensation,andpassedaway.
Results
Toobtaingreaterinsightintotheimmuneprocessesactiveduring the acute HEV infection in this patient, a microsphere-based multiplexproteinassaywasperformed.Itwasobservedthatduring
theacutephaseoftheHEVinfection,expressedbypositiveHEVRNA inserumandhighlevelsoftransaminases,dramaticallyincreased levels of all immune analytes were present in the patient as compared totwohealthy controlsofsimilar ageand sex(Figure 2,left panel). Theseincluded elevated T-cell-related cytokines such as interleukin(IL)-2 andinterferongamma(IFN-
g
), and monocyte-derivedcytokinessuchastumornecrosis factor(TNF),IL-12p70, IL-18,andmacrophagecolony-stimulatingfactor(M-CSF),aswellas markerscharacteristicofviralinfectionssuchasinterferonalpha (IFN-α)andIP-10.Thesemediatorshaveallbeenshowntoincrease duringacuteviralhepatitisfromdifferentviruses,suggestingno majordifferencesininitialimmuneresponseforHEVcomparedto otherviralinfections(Staceyetal.,2009).FollowingnegativizationofserumHEVRNAandthedecreasein livertransaminases,severalmarkers,includingsolubleIL-2receptor (sIL-2R),IP-10,andeotaxin3normalized,aswouldbeexpectedwith resolutionoftheviralhepatitis(Figure2,rightpanel).However,after peripheralHEVclearance,afurtherincreaseintheT-cell-derived cytokinesIL-2,IFN-
g
,andIL-10wasobserved,likelyreflectingstrong inductionofproinflammatoryTh1responsesthatarecounteracted bytheactivityoftheanti-inflammatorycytokineIL-10.Also,the levels of IFN-αas well as important proinflammatorymediators,such asIL-15,TNF,andIL-1βproducedbyKupffercells,monocytes,and hepatocytes,remainedelevatedorwereevenfurtheraugmented. DespitetheabsenceofdetectableHEVRNAinserum,significantly elevatedlevelsofbilirubin wereobserved.Surprisingly,examination oftheliverbiopsybyHEVRNAPCRdemonstratedapositiveresult (5.7105arbitraryunits(AU)).Forcomparison,weaddedanegative biopsyfromachronicHCVpatient(<200copies/g)andapositive sample from apatientwith acuteHEV(6.1108arbitrary units (AU)).StainingofthebiopsywiththeJ2antibodyfordouble-strandedRNA
Figure1.(A)Graphicdescribingthechronologicalorderofpresentation,laboratoryvalues,andclinicalcourse.(B)Liverbiopsy(hematoxylin–eosin200):mildportal(blue arrow)and mild lobular(greenarrow) inflammation; bilirubinostasis (yellow arrows) and Mallory hyalin in the cytoplasm of hepatocytes (yellow circle).(C) Immunohistochemistryofliverbiopsy(J240):anti-ds-RNAantibodystaining(brown)incytoplasmicandperinuclearareas.
(ds-RNA) was positive, whereas uninfected control liver was negative(Figure 1C). Thesefindingsindicate that although HEV RNAwasclearedfromblood,theviruswasstillpresentandactivein theliver.
Discussion
Thiscaseemphasizesthepossibilityofacutecholestaticviral hepatitis E due tothe presence of intrahepatic HEVRNA with negative peripheral RNA, which has not been reported before. Interestingly,duringthecholestaticphase andperipheral clear-anceofHEVinblood,therewasaninflammatorycascaderelatedto monocyte/Kupffer cell activation. This clinical manifestation possibly suggestsa response of the intrahepaticcomponent of theimmunesystemthatcouldhaveledtothecholestaticpattern observed.Therefore,onecouldspeculatethatwhiletheperipheral inflammatory pattern normalized, an augmented intrahepatic immuneresponsewasobservedduetothepresenceofthevirusin thisenvironment,arguingforadissociationofintra-and extra-hepaticimmuneresponses.
Thiscasealsohighlightspotentialexplanationsforthelackof correlationbetweenHEVRNAdetectionandHEVIgMlevelswhen diagnosing acute HEV. The authors of several studies have reporteddifficultiesindiagnosingacuteHEVduetoindividuals presentingpositivityineitheroneortheothertest,butnotboth. ThishasgenerallybeenattributedtothelackofstandardizedPCR techniquesorpoorsensitivitiesofIgMELISAtests(Debesetal., 2016;Cattoiret al.,2017).However,asinourpatient, alackof
peripheral blood HEV RNA concomitant with intrahepatic replication could explainthis dissociation andshould be kept inmindintheappropriateclinicalsetting.Moreover,asHEVcan replicateintheliverwithoutreplicationinblood(suchasinour patient), in situations where liver biopsy is not possible, consideration should be given to stool HEV PCR assessment (whichrepresentssheddingfrombile)orrepetitionofHEVIgM. These tests, if properly applied, could increase the odds of diagnosisincasesofhighsuspicion.
Methods
Assessmentoflevelsofcytokines/chemokines
Plasmawascollectedandmulti-analyteprofilingofcytokines, chemokines, growthfactors,and otherproteins was performed using the Procarta Plex Human Immune Monitoring Panel (Affymetrix; eBioscience, Vienna,Austria). The panelmeasured 65 proteins simultaneously (Spaan et al., 2016).The assaywas conductedaccordingtothemanufacturer’sinstructions,similarly to our previous studies (Spaan et al., 2016).All samples were analyzed in one run. Data were analyzed using Procarta Plex Analyst1.0.
LivertissueHEVPCR
RNA was isolated from formalin-fixed paraffin-embedded (FFPE)coreneedleliverbiopsiesusingtheRNAisolationRNAeasy
Figure2.DifferentiallevelsofimmunemarkersinplasmafollowingchangesinHEVRNAlevelsinblood.Arrowspointingupdescribeanincreaseandarrowspointingdown describeadecrease.Theleftpaneldescribesthefoldchangecomparedtocontrols;thepanelontherightdescribesthefoldchangeafternegativizationofHEVRNAinblood. J.D.Debesetal./InternationalJournalofInfectiousDiseases87(2019)39–42 41
FFPE Kit (Qiagen, Hilden, Germany). Positive (acute HEV) and negative(chronic HCV)biopsies were included as controls. All samples were screened for the presence of HEV RNA using an ISO15189:2012-validated, internally controlled quantitative real-timeRT-PCR,asdescribedpreviously(Pasetal.,2012). Immunohistochemicalstainingfordouble-strandedRNA
Immunohistochemistry was performed with an automated, validated,and accredited staining system (Ventana Benchmark ULTRA; Ventana Medical Systems, Tucson, AZ, USA) using the Optiview Universal DAB Detection Kit (#760-700). In brief, following deparaffinization and heat-induced antigen retrieval, the tissue samples were incubated with mouse monoclonal antibodyanti-ds-RNA(J2,Englishand ScientificConsulting Kft.) for30min.Incubationwas followedbyhematoxylinII counter-staining for 12min and then blue coloring reagent for 8min, accordingtothemanufacturer’sinstructions.
Funding
ThisworkwassupportedbytheVIRGOconsortium,theRobert WoodJohnsonFoundation(AMFP),American Collegeof Gastro-enterology,andNIH-NCIgrantnumberR21CA215883-01A1.
Roleofthefundingsource
Noneofthefundingsourcesstatedhadanyinfluenceonthe studydesign,interpretationofdata,orwritingofthemanuscript.
Ethicalapproval
ThisstudywasapprovedbytheEthicsCommitteeofErasmus MC,Rotterdam,theNetherlands.
Conflictofinterest
Noconflictofinterestdeclaredbyanyoftheauthors. References
BrostS,WenzelJJ,GantenTM,FilserM,FlechtenmacherC,BoehmS,etal.Sporadic casesofacuteautochthonoushepatitisEvirusinfectioninSouthwestGermany. JClinVirol2010;47:89–92.
CattoirL,VanHoeckeF,VanMaerkenT,NysE,RyckaertI,DeBoulleM,etal. HepatitisEvirusserologyandPCR:doesthemethodologymatter?.ArchVirol 2017;38:1062–9.
DebesJD,PisanoMB,LottoM,ReV.HepatitisEvirusinfectionintheHIV-positive patient.JClinVirol2016;80:102–6.
KamarN,SelvesJ,MansuyJM,OuezzaniL,PéronJM,GuitardJ,etal.HepatitisEvirus and chronic hepatitis in organ-transplant recipients. N Engl J Med 2008;358:811–7.
PasSD,deManRA,MuldersC,BalkAH,vanHalPT,WeimarW,etal.HepatitisEvirus infectionamongsolidorgan transplantrecipients,theNetherlands.Emerg InfectDis2012;18:869–72.
Saint-JacquesP,Tissot-DupontH,ColsonP.AutochthonousinfectionwithhepatitisE virusrelatedtosubtype3a,France:acasereport.AnnHepatol2016;15:438–41. SpaanM,vanOordG,KreefftK,HouJ,HansenBE,JanssenHL,etal.Immunological analysisduringinterferon-freetherapyforchronichepatitisCvirusinfection reveals modulation of the natural killer cell compartment. J Infect Dis 2016;213:216–23.
StaceyAR,NorrisPJ,QinL,HaygreenEA,TaylorE,HeitmanJ,etal.Inductionofa striking systemiccytokine cascadepriorto peakviremia inacutehuman immunodeficiency virustype1infection,incontrasttomoremodest and delayed responses in acute hepatitis B and C virus infections. J Virol 2009;83:3719–33.
