Folia Biologica (Praha) 62, 26-33 (2016)

Original Article

Impact of Induced Th1/Th2 Shift on Trichobilharzia regenti Infection in Mice(birdschistosomes/schistosomulamigration/Th1/Th2polarization/immunomodulation/CNS/neurotropicparasite)

M. CHANOVÁ, J. HRDÝ

Institute of Immunology and Microbiology of the First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic

Abstract. Bird schistosomes parasitize mammals as non-specific hosts. Neurotropic Trichobilharzia re-genti migrates extravasally via nervous tissue in ex-perimentally infected mice. The majority of success-fully penetrated larvae remain in the skin; the rest migrate through peripheral nerves to the spinal cord and brain. The potential of schistosomula to leave the skin and enter the central nervous system vary, and may be associated with Th1/Th2 polarization of the host cell immune response. The aim of the pre-sent study was to evaluate the impact of induced shift in polarization of cell immune response on the mi-gration of T. regenti larvae in mammals. For this purpose, non-specifically immunomodulated mice were infected. The localization and abundance of schistosomula and associated histopathological chan-ges were followed using routine histological tech-niques. Markers characterizing Th1 and Th2 systemic immune responses were followed using flow cytome-try. The study revealed that the shift towards Th1 re-sponse at the time of infection correlates with the speed and intensity of schistosomula migration to-wards the brain and with the severity of accompany-ing pathologies. This indicates increased health risks associated with T. regenti infection for mammals (po-tentially including human) with previously modu-lated cell immune response that may occur under natural conditions, e.g. due to the exposure to another infectious agent.

Received:February20,2015.AcceptedJuly25,2015.

Funding sources: Czech Science Foundation (P302/12/P548);Charles University in Prague (PRVOUK P25/LF1/2; UNCE204017).

Correspondingauthor: Marta Chanová, Institute of Immunology and Microbiology of the First Faculty of Medicine, Charles Uni-versity in Prague and General University Hospital in Prague, Studničkova7,12800Prague,CzechRepublic.E-mail:marta.chanova@lf1.cuni.cz

Abbreviations: CFA – Complete Freund’s Adjuvant, CNS – cen-tral nervous system, p.i. – post infection.

Introduction

Birdschistosomesundergodixeniclifecycle,usuallywith fresh water pulmonate snails as intermediates and waterfowlasdefinitivehosts.Theycovereightgenerawithapproximately58species,thevastmajorityofthemwiththelifehistorytypicalofschistosomes,reflectingthat of well-known human species. However, a unique strategy was described for Trichobilharziaregenti, the nasal bird schistosome with intravertebrate stages show-ing neurotropism and obligatory migration via peri-pheral and central nervous tissue (Horák et al., 1999;HrádkováandHorák,2002).

Besides birds, cercariae of bird schistosomes also at-tack the skin of mammals including humans. Cercarial dermatitis, the defensive skin immune reaction that eliminates invading cercariae directly in the skin, has long been recognized as the only risk associated with humanexposuretobirdschistosomes.However,recentfindingsshowthatcercarialdermatitisdevelopsastypeI immediate hypersensitivity reaction concurrent with sensitization, and thus that the entrapment and destruc-tion of penetrating cercariae usually occurs only after repeatedcontactwiththeselarvae(HorákandKolářová,2001;Kouřilováetal.,2004a,b).Experimentalstudiesof parasite interaction with unsensitized mammalian host not only showed weaker to none skin reaction, but alsoconfirmedsuccessfulcercarialtransformationintoschistosomula, their further development, migration throughseveral tissues/organsand feedingofhost tis-sues, although resulting in death of immature worms andparasitelife-cyclefailure(e.g.Olivier,1953;Bachaetal.,1982;HaasandPietsch,1991;HorákandKolá-řová,2000;HrádkováandHorák,2002;Chanováetal.,2007;Lichtenbergováetal.,2011).Basedonthesestudiesperformed with various bird schistosomes in several mammalian host species (including primates; Olivier,1953),asimilarsituationisproposed,althoughnotcon-firmed, inprimarily infectedhumans. (e.g.,Bayssade-Dufouretal.,2001,HorákandKolářová,2001).

Various studies showed the transient presence of bird schistosomula in mammalian tissues being associated

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with more or less severe pathologies; the pathologycaused by T. regenti is the most serious. The details were describedinmice:first,aninflammatoryreactionwithoedema, vasodilatation and tissue infiltrates occurs inthe skin. Subsequently, migrating schistosomula cause neuronal inflammation in deeper dermis and subcutis(Kouřilováetal.,2004b).Furthermigrationthroughpe-ripheralnerves,spinalcordandbrainleadstoaninflam-matory cell reaction with granuloma formation at the sites of worms’ location, along with increased prolifera-tion and activation of astrocytes, as well as pathologic changes of adjacent neurons (Kolářová et al., 2001;Lichtenbergováetal.,2011).Clinicalsymptomsduetothe central nervous system (CNS) invasion vary fromminortoseriousbalancedisordersand/orlegparalysisand death (Horák et al., 1999; personal observation).The development of neurological symptoms correlates with the number of schistosomula localized in nervous tissue (Lichtenbergová et al., 2011). The number ofschistosomula that reach the nervous tissue depends, ob-viously, on the infection dose, but also on the host im-mune status. Studies of the schistosomula migratory pattern revealed the majority of successfully penetrating larvae remaining in the skin without further migration. Only a minor proportion of skin schistosomula undergo invasionofCNS;however,thisnumberissignificantlyhigher in immunosuppressed(SCID) than in immuno-competent (SKH1 hr/hr)mice infectedwith the samedoseofcercariae(Kouřilováetal.,2004a,b).Efficiententrapmentofpenetratingwormsintheskin

of a sensitized immunocompetent host with cercarial dermatitis is attributed to Th2 polarization of the im-mune response (CD3-deficient SCIDmice do not de-velopthisskinreaction;Kouřilováetal.,2004a,b).Theresponse during the initial phase of primary infection, when schistosomula are able to leave the skin, was char-acterized by the same authors as Th1/Th2mixed.Thisledus to the hypothesis that the change of the host immuno-logical status (particularly the shift towards the Th1 cell immuneresponse)beforeorduringexposuretomigrat-ingschistosomulawillalsoinfluencethecourseof in-fection in the immunocompetent host.

Thus, the aim of the present study was to evaluate the impact of induced shift in the polarization of systemic cell response on the course of Trichobilharzia infection in mammals. For this purpose, the migratory pattern and development of schistosomula-associated pathologies werestudiedinBALB/candC57BL/6miceexposedto

T. regenticercariaeafterpreviousnon-specificTh1 cell immune response inducer application. Higher speed and intensityofmigrationtoCNSwasconfirmed.

Material and Methods

Experimental models

Bird schistosomes of Trichobilharziaregenti are kept in complete laboratory cycle using snails Radix lagotis and ducks Anas platyrhynchos f. domestica as interme-diateanddefinitivehosts, respectively.All theexperi-mentswere performedwith adult females ofBALB/cand C57BL/6mice (Charles River Laboratories, Inc.,Wilmington,MA)and freshly shedcercariaeofT. re-gentiinthefollowingpattern(overviewedinTable1):Group I – Immunostimulated infected mice: mice

were subcutaneously injected with 100 µl of Complete Freund’s Adjuvant (CFA, #F5881 Sigma, St. Louis,MO) and subsequently, 7 days post application, theywereroutinelyexposedtocercariae(40minutesoffeetand tail immersion into a beaker containing approxi-mately2000cercariaein50mloftapwater).Groups II–IV – Experimental conditions followed

thoseforGroupI,exceptforphysiologicalsalineappli-cation instead of CFA (Group II – Non-stimulated in-fectedmice);cercariae-freetapwaterexposureinsteadof water containing cercariae (Group III – Immuno-stimulated non-infectedmice) and both, physiologicalsalineapplicationandcercariae-freetapwaterexposureinstead of CFA and cercariae, respectively (Group IV – Controlnon-stimulatedandnon-infectedmice).Miceweresacrificedbyoverdosedanaesthesia(keta-

mine/xylazine)3–14dayspost infection (p.i.).All theexperimentswere consistentwith current animalwel-fare laws of the Czech Republic and EU, and were ap-proved by the Animal Experiment Board at CharlesUniversity in Prague.

Tissue processingHeart, lungs, liver and kidney were extracted and

tested for the parasite presence on fresh squashed slides using routine light microscopy. Spinal cord and brain wereprocessedforhistology(fixedinBouin’sfixative,paraffinembedded,cutinto4µmsectionsandhaema-toxylin/eosin or Luxol fast blue/neutral red stained).Whole heparinized blood of a single mouse per particu-

Table1. Overview of experimental model groups

Group numberDescription

Group IImmunostimulated and

infected mice

Group IINon-stimulated infected

mice

Group IIIImmunostimulated non-

infected mice

Group IVControl non-stimulated and non-infected mice

Mouse strain BALB/c C57BL/6 BALB/c C57BL/6 BALB/c C57BL/6 BALB/c C57BL/6CFA injection + + - - + + - -T. regenti infection + + + + - - - -Number of mice 15 15 15 15 3 3 3 3

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lar experimental group and period was processed forflowcytometry.

Flowcytometry(FACS)Heparinized whole blood was incubated with the fol-

lowingfluorescentantibodies:Allophycocyanin/Cyanine7-conjugated anti-mouse CD4, #100414; phycoeryth-rin-conjugatedanti-mouseCCR5,#107006;Peridinin-chlorophyll-protein/Cyanine5.5-conjugated anti-mouseCXCR3,#126514–allsuppliedbyBioLegendInc.,SanDiego, CA and fluorescein isothiocyanate-conjugatedanti-CCR8,#ABIN732870Antibodies-onlineInc.,Atlan-ta, GE. Subsequently, red blood cells were lysed (BD FACS™ Lysing Solution, #349202, BD Biosciences,SanJose,CA),thesampleswerewashed3xwithphos-phate-buffered saline and immediately acquired using BDFACSCantoII(BDBiosciences).Atleast100,000events per sample were recorded. Data were evaluated usingFlowJo(TreeStarInc.,Ashland,OR).Firstly,thelymphocytepopulationwasgatedandthenCD4+ T cells were selected for further detailed analyses for the pres-enceofCXCR3,CCR5andCCR8chemokinereceptors(seegatingstrategyinFig.1).Relativenumbersofcellswith particular surface marker were acquired (reported aspercentofCD4+cellsinthebloodsample).Through-

out the text,CXCR3+ and CCR5+ cells are considered Th1,andCCR8

+ cells are considered Th2,basedontypi-cal expression of chemokine receptors on these cells(e.g.Saxenaetal.,2012).

Results

Th1 / Th2balance

Thecountsofcells labelledwithanti-CXCR3,anti-CCR5 and anti-CCR8 antibodies differed betweenGroupIIIandGroupIVmiceonthedayofexposuretoT. regenti larvae(day0;7dayspostCFA/salineinjec-tion; Fig. 2). Particularly, the percentage of CXCR3+ cellswashigher,andthepercentageofCCR8+ cells was lower in CFA-injected mice of both strains than in the corresponding controls. The percentage of CCR5+ cells inCFA-injectedmicewashigheronlyinBALB/candequal in C57BL/6 compared to controls. The highestdifference between CFA-injected and control mice was observedforCXCR3+cells(approximately4%forbothmouse strains).Among allmouse groups, the highestnumber of Th1 cells on the day of infection was meas-uredinCFA-injectedBALB/cmice.Thehighestnum-

Fig. 1.Typicalflowcytometrydataforthepresenceofchemokinereceptorswithgatingstrategy;GroupIBALB/cmouse7daysp.i.

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ber of Th2andthelowestnumberofTh1 cells were seen incontrolBALB/cmiceatthesametime.ShiftsintheratioofparticularCD4+cellsinthenext

14 days after infection differed between the mousegroupsandstrains:In infected BALB/c mice previously injected with

CFA(GroupI), theinitialsituationwithmoreTh1 and less Th2cellscomparedtonon-injected(GroupII)miceremained in existence also after the infection. Parti-cularly,therelativenumberofCCR8+ cells was lower in Group I than in Group II mice and the relative number ofboth,CXCR3+ and CCR5+ cells, was higher in Group I than in Group II mice at corresponding time intervals during the entire period under study. Nevertheless, the number of Th1 cells was continually decreasing in both groups.InC57BL/6mice,theinitialsituationswitchedsoon

aftertheinfection.TherelativenumberofCXCR3+ cells inGroupIImiceexceededthatofGroupIandtherela-tivenumberofCCR8+ cells in Group II mice declined belowthecountinGroupImice;almostnodifferencein

CCR5+ cell counts was measured between both groups aftertheinfection.InbothC57BL/6mousegroups,thenumber of Th1cellsexceededtheinitialmaximalnum-bers soon after infection.

Schistosomula localization and invasion of CNSNo worms were detected in visceral organs of any

mousetested.Thefirstschistosomulainthespinalcordweredetected3daysp.i.inallgroupsofinfectedmice.In most mice, schistosomula were also observed in me-dullaoblongata.Afewworms(usually1–3permouse)were found in Group II BALB/cmice at this period,contrary toothergroups(GroupIBALB/cwith5–10,andboth,GroupIandIIC57BL/6,withmorethan10schistosomulapermouse). Initially, schistosomula ap-pearedinwhitematter(Fig.3a),andsubsequentlyalsoingreymatter(Fig.3b);earliestspreadtothegreymat-teroccurredinGroupIIBALB/cmice.Intactschistoso-mula were present in the spinal cords of all mice for the rest of the period under study, with highest numbers re-corded inGroup IBALB/cmice5daysp.i.Although

Fig. 2. Presence of chemokine receptors typical of Th1(CXCR3,CCR5)andTh2(CCR8)cellsinCFA-immunostimulatedandnon-stimulatedmiceofBALB/candC57BL/6strainsindifferentperiodspostinfectionwithT. regenti. Flow cyto-metry analysis

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the number of larvae decreased with time, the differ-ences in the intensity of spinal cord invasion in different mouse groups remained clear until the end of the study. Thefirstwormsinthebrainweredetected7daysp.i.inGroupIBALB/cmice,9daysp.i.inGroupIC57BL/6mice and as late as 11 days p.i. in Group II mice of both strains. The number of worms found in the brain did not exceed three per mouse. Schistosomula were foundmostly in the pons, cerebellum and pia mater; ratherrandom schistosomula distribution in the brains of all mouse groups was observed. For a review of the speed

and intensity of CNS invasion in different mouse groups, seeTable2.

The presence of schistosomula was associated with infiltrationofimmunecells(mainlymacrophages,lym-phocytes and eosinophils) in BALB/c, but rarely inC57BL/6mice.InBALB/cmice,theextentofinfiltra-tionseemed to reflect theschistosomulanumber: sub-stantial granulomas were formed around the accumu-lated worms as well as in their migratory route traces in Group IBALB/cmice from day 5 p.i. (Fig. 3c); lessextensive infiltrations surrounding isolated larvae in

Fig. 3. Invasion of murine CNS by schistosomula of Trichobilharziaregentiwithorwithoutpreviousnon-specificim-munomodulationwithCFA.Haematoxylin/eosinstaininga–schistosomuluminthewhitematterofspinalcord;GroupIIC57BL/6mouse3daysp.i;b – schistosomulum in the greymatterofspinalcord;GroupIIBALB/cmouse7daysp.i;c – massiveinfiltratescomposedmainlyofmacrophages,lymphocytesandeosinophilssurroundingaccumulatedschistosomulainthespinalcord;GroupIBALB/cmouse5daysp.i;d–infiltratessurroundingindividualschistosomuluminthespinalcord;GroupIIBALB/cmouse8daysp.i;e – schi-stosomuluminthespinalcord–notetheabsenceofinflammatorycells;GroupIC57BL/6mouse9daysp.i;f – schisto-somuluminthebrainhemisphere;GroupIBALB/cmouse8daysp.i.;arrows – schistosomula.

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GroupIIBALB/cmiceappearedthreedayslater(Fig.3d).Contrarytothisfinding,onlyaweakinfluxofin-flammatorycellswasseen inGroupIIC57BL/6micefromday9p.i.andalmostnocellularreactionwasre-cordedinGroupIC57BL/6mice(Fig.3e).Nocellularreaction was observed in the brains throughout the in-vestigatedmousegroups(Fig.3f).Luxolfastbluestain-ing for myelin did not reveal any demyelination in any specimen.

Discussion Trichobilharziaregentiwasdescribedin1998(Horák

etal.,1998),andestablishmentofthelaboratorylifecy-cle followed. Since that time, host-parasite interactions of T. regenti with immunocompetent murine hosts has been studied using several mouse strains (BALB/c,C57BL/6andSKH1hr/hr),withdetailson themigra-tionandpathogenesisdescribedforBALB/cmiceonly.MiceoftheC57BL/6strainwereusedinthestudiesofthe skin phase focused on cercarial dermatitis, and no details on the migration to CNS were provided. In the present study, the impact of cell immune response po-larization on the infection cruise was in focus. Therefore, mice of Th1 dominant C57BL/6 and Th2 dominantBALB/c strains (Watanabe et al., 2004)were used inorder to compare schistosomula migration in mice with differently polarized response, either due to previous immunomodulation or genetic predisposition. To confirm the differences in T-helper cell counts

among mouse groups before and during the infection, flowcytometryanalysiswasperformed.UseofCCR5,CXCR3 and CCR8 chemokine receptors as surfacemarkers for T-helper cell subsets was based on their typical expression on Th1 and Th2 cells, respectively.Although the employment of chemokine receptor pres-ence for phenotyping T-helper cells has been debated (e.g.Chiuetal.,2002),theauthorsbelievethattheiruseinthepresentstudyfulfilsitsobjective.

T. regenti is kept in the laboratory cycle running for several years. Migration in mice was described in detail in the last decade and authors are familiar with its ob-ligatory pattern. However, variability in the penetration success, survival in mammalian tissues, speed and in-tensity of CNS invasion, and severity of associated pa-

thologies have been reported since the species descrip-tionin1998(Horáketal.,1998).Theabilitytoparasitizea host depends on the particular mouse strain used, but alsoseemstobeinfluencedbylong-termpassageinthelaboratory lifecycle (personalobservation).The lattershould be taken in consideration when comparing the above-mentionedvariables(especially theexactwormnumbersdetectedinselectedlocationorinfectionphase)with published data. Therefore, here reported results re-ferring schistosomula quantity were compared with controls from the present study only. The route and tim-ing of migration are discussed with all the data available.

CFA is a strong Th1inducer(Jensenetal.,1998).Itsapplication in the present study showed a desired effect – change in Th1cells’relativecounts7dayspostinjec-tioninmiceofbothstrains(moresignificantinBALB/cthanC57BL/6mice).

The migratory route of schistosomula reported here did not show any dissimilarities either among the mouse groups tested in the present study, or in comparison with thepublisheddata (HrádkováandHorák,2002;Lich-tenbergováetal.,2011).Thetimeframeofthemigra-tion and number of schistosomula detected in various locations/periods,however,differedsignificantlyamongthe tested mouse groups.Theperiodoffirstschistosomuladetectioninthespi-

nal cord and medullaoblongata reported in the present study(3daysp.i.forallmousegroups)isinagreementwithboththepublisheddataandthelong-termexperi-enceofthefirstauthor(theearliestfindinginspinalcord2daysp.i.was reported,e.g.byHrádkováandHorák(2002)).Theperiodoffirstschistosomulaoccurrenceinthe brain (minus the above-mentioned m. oblongata)differed among the mouse groups in the present study (7to 11 days p.i. in particular groups).According topublisheddata, thefirst schistosomula reach thebrainaroundday11p.i.; the same timingwasobserved forGroupIIBALB/cmiceinthepresentstudy.Aregularpresence of worms in the brain as soon as in Group I BALB/cmicedescribedherehasnotbeenreportedyet.

The speed and intensity of migration seems to corre-late with the relative numbers of investigated CD4+ cells. Comparing all mouse groups, the highest number of Th1cellswasdetected inCFA-injectedBALB/conthedayofinfection,andwasexceededinC57BL/6mice

Table2.LocalizationofT.regentischistosomulainCNSofnon-stimulated(GroupII)andCFA-injected(GroupI)BALB/candC57BL/6mice3–11dayspostinfection(dpi;3miceforeachgroupandperiodwereinvestigated)

dpi

Group II BALB/c Group I BALB/c Group II C57BL/6 Group I C57BL/6spinalcord&

medullaoblongatabrain spinalcord&

medullaoblongatabrain spinalcord&

medullaoblongatabrain spinalcord&

medullaoblongatabrain

3 dpi +(wm) - ++(wm) - +++(wm) - +++(wm) -5 dpi +(wm) - +++(wm) - ++(wm) - N/A N/A

7/8 dpi +(wm) - +++(wm,gm) + ++(wm) - ++(wm) -9 dpi +(wm,gm) - ++(wm,gm) + ++(wm,gm) - ++(wm) +

11 dpi +(wm) + ++(wm,gm) + +(wm,gm) + ++(wm) ++ 1–5schistosomula;++ 5–10schistosomula;+++>10schistosomula;N/A –datanotavailable;wm–whitematter;gm–greymatter.

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of both groups soon after the infection. The number of schistosomula that had reached CNS in these mice (GroupIBALB/c,GroupsIandIIC57BL/6)wassig-nificantly higher than in non-stimulated (Group II)BALB/cmice.

The differences in speed of further schistosomula mi-gration were also obvious. The difference between Group I and Group II BALB/cmice was substantial,with faster establishment in the brain of CFA-injected BALB/c mice. In contrast, differences in the speedamongtwogroupsofC57BL/6micewereminorandinboth groups, schistosomula appeared in the brain sooner than in Group II BALB/c mice.We believe that thehigher intensity and speed of migration from the skin to CNS was caused by the initial rise in Th1 cell numbers.

The number of Th1 cells was continually decreasing inBALB/cmice,butincreasinginC57BL/6miceafterinfection, probably due to their different predisposition to Th1/Th2responsedevelopment.AstrongTh1 response inC57BL/6micemayhavepreventedgranuloma for-mation in spite of numerous schistosomula present in thespinalcord.Incontrast,thereactioninBALB/cmicewas obvious and its intensity correlated with the schisto-somula number, as previously reported by Lichten ber-gováetal.(2011).Thus,infectionwithT. regenti resulted in most significant pathologies in immunomodulated(CFA-injected)BALB/cmice.Thisfindingsupportsourhypothesis that the shift in polarization of the cell im-mune response increases the risks associated with T. re-genti infection in mammals.

Bird schistosomes of the genus Trichobilharzia with morethan40speciesoccurworldwideandstillspreadoverformerlyunaffectedareas.Humanexposuretobirdschistosomes manifested with cercarial dermatitis is fre-quently reported from many regions, including those in temperate and cold zones, and has been recently de-scribedasare-emergingdisease(deGentileetal.,1996;HorákandKolářová,2011).Whenspeculatingaboutthepossible schistosomula survival in human, CNS in-volvement is unlikely, mainly due to the host body size with distances invincible for these worms. However, en-tering the peripheral nerves and induction of local in-flammatoryresponseclosetothepenetrationsiteseemsrealistic.Althoughnoexactevidenceonthemigrationin human has been provided (possibly due to the lack of ausefuldiagnostictoolaccordingtoHoráketal.,2008),several studies reviewed by these authors pointed to the necessity to also consider schistosomula-associated in-juries. The present study even enhances this suggestion.

In conclusion, our results show that the rise in Th1 cell counts in initial phase of T. regenti infection is associ-ated with faster and more intensive migration towards CNS. We therefore suppose that natural conditions in-fluencingtheTh1/Th2balance,e.g.previousorsimulta-neous contact with other infectious agents, may also act andsignificantly increase therisksconnectedwith theexposuretobirdschistosomes.Thisshouldalsobekeptin mind in human cases with history of contact with these parasites.

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