Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells.

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by  Claessens, Antoine

JournalofVisualizedExperiments www.jove.com
VideoArticle
Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human
Brain Endothelial Cells
Antoine Claessens,J.Alexandra Rowe
CentreforImmunity,InfectionandEvolution,UniversityofEdinburgh
Correspondenceto:[email protected]
URL:http://www.jove.com/video/3122/
DOI:10.3791/3122
Keywords:Immunology,Issue59,Plasmodiumfalciparum,cerebralmalaria,cytoadherence,sequestration,endothelialcell,HBEC-5i,
DatePublished:1/3/2012
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense,whichpermits
non-commercialuse,distribution,andreproduction,providedtheoriginalworkisproperlycited.
Citation:Claessens, A.,Rowe, J.A. SelectionofPlasmodiumfalciparumParasitesforCytoadhesiontoHumanBrainEndothelialCells.J.Vis.Exp.
(59),e3122,DOI:10.3791/3122(2012).
Abstract
Mosthumanmalariadeathsarecausedbyblood-stagePlasmodiumfalciparumparasites.Cerebralmalaria,themostlife-threatening
complicationofthedisease,ischaracterisedbyanaccumulationofPlasmodiumfalciparuminfectedredbloodcells(iRBC)atpigmented
trophozoitestageinthemicrovasculatureofthebrain2-4.Thismicrovesselobstruction(sequestration)leadstoacidosis,hypoxiaandharmful
inflammatorycytokines(reviewedin5).Sequestrationisalsofoundinmostmicrovasculartissuesofthehumanbody2,3.Themechanismbywhich
iRBCattachtothebloodvesselwallsisstillpoorlyunderstood.
TheimmortalizedHumanBrainmicrovascularEndothelialCellline(HBEC-5i)hasbeenusedasaninvitromodeloftheblood-brainbarrier6.
However,PlasmodiumfalciparumiRBCattachonlypoorlytoHBEC-5iinvitro,unlikethedensesequestrationthatoccursincerebralmalaria
cases.Wethereforedevelopedapanningassaytoselect(enrich)variousP.falciparumstrainsforadhesiontoHBEC-5iinordertoobtain
populationsofhigh-bindingparasites,morerepresentativeofwhatoccursinvivo.
Asampleofaparasiteculture(mixtureofiRBCanduninfectedRBC)atthepigmentedtrophozoitestageiswashedandincubatedonalayerof
HBEC-5igrownonaPetridish.Afterincubation,thedishisgentlywashedfreefromuRBCandunboundiRBC.FreshuRBCareaddedtothefew
iRBCattachedtoHBEC-5iandincubatedovernight.Asschizontstageparasitesburst,merozoitesreinvadeRBCandtheseringstageparasites
areharvestedthefollowingday.Parasitesarecultureduntilenoughmaterialisobtained(typically2to4weeks)andanewroundofselectioncan
beperformed.DependingontheP.falciparumstrain,4to7roundsofselectionareneededinordertogetapopulationwheremostparasitesbind
toHBEC-5i.Thebindingphenotypeisprogressivelylostafterafewweeks,indicatingaswitchinvariantsurfaceantigengeneexpression,thus
regularselectiononHBEC-5iisrequiredtomaintainthephenotype.
Insummary,wedevelopedaselectionassayrenderingP.falciparumparasitesamore"cerebralmalariaadhesive"phenotype.Wewereableto
select3outof4P.falciparumstrainsonHBEC-5i.Thisassayhasalsosuccessfullybeenusedtoselectparasitesforbindingtohumandermal
andpulmonaryendothelialcells.Importantly,thismethodcanbeusedtoselecttissue-specificparasitepopulationsinordertoidentifycandidate
parasiteligandsforbindingtobrainendothelium.Moreover,thisassaycanbeusedtoscreenforputativeanti-sequestrationdrugs7.
VideoLink
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Protocol
Generalrecommendations
Humanbrainmicrovascularendothelialcell(HBEC-5i)culturehasbeenpreviouslydescribedin6,8.P.falciparumparasiteswereculturedasin9.
BothHBEC-5iandP.falciparumparasiteculturesshouldbekeptinsterileconditionsatalltimes.Allreagentsshouldbepre-warmedat37°C.We
recommendtoregularlycheckformycoplasmacontamination10byPCR(MineveraBiolabs,followingmanufacturer'sinstructions).Theprotocolis
summarizedinFigure1.
1. Endothelial cell routine culture
1. Preparethenecessaryreagents.
Mediumtoprepare Reagents Quantity
"DMEMincomplete" DMEM-F12Ham 500ml
  L-glutamine200mM 5ml
  Penicillin/streptomycin100X 5ml
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  NaOH1M 1.3ml(adjustpHto7.4)
"DMEMcomplete" "DMEMincomplete" 450ml
  FoetalBovineSerumheat-inactivated 50ml
  Endothelialcellgrowthsupplement 5ml

2. CultureHBEC-5iinavented25cm2flaskwith10mlDMEMcompletemediumina37°Cincubatorwith5%CO2.
3. Passagecellswhentheybecomeconfluent.RemovetheoldmediumbysuctionandwashtwiceusingDMEMincompletemediumortissue
culturegradePBS(Ca2+andMg2+free)pre-warmedto37°C.
4. Add1mlofpre-warmedTrypsin-EDTA(0.025%Trypsin,0.5mMEDTA),swirltocovertheentiretyoftheflaskandincubatefor˜2minat37°C.
5. Checkunderinvertedmicroscopethatatleast90%ofcellshavebeendetached.Ifnecessary,gentlyknockthebottomoftheflasktodislodge
adherentcells.Add10mlofDMEMcompletemediumtoblockthetrypsinandtransfercellsintoa15mlconicaltube.Centrifugefor4minat
300gatroomtemperature(RT).
6. Discardthesupernatantandresuspendthepelletwith10mlofDMEMcompletemedium.Pipettethesolutionupanddowntothoroughly
resuspendthecells.
7. Add1or2mlofthecellsuspensionintoanewcultureflaskandadd8mloffreshmediumtomaintaintheculture.Assessculturegrowth
everydayunderaninvertedmicroscopeandchangemediumevery2or3daysbeforeitturnsyellow.
2. Preparing endothelial cells for a selection
1. Twodayspriortothedayoftheselection,addfibronectindilutedinsterilePBS(2μg/cm2)inone(ormore)60mmPetridish.Incubatethe
dishfor5to20minat37°C,thenremovethefibronectinsolution,whichcanbestoredat4°Cforamonthandre-usedonce.
2. Passagecellsasdescribedinsections1.3.to1.5.Assuming100%confluentcellsweredetachedandresuspendedin10mlDMEMcomplete
medium(section1.6.,resuspendwithanequivalentvolumeofmediumiftheconfluencyislower,e.g.resuspendwith8mliftheconfluency
was80%),add1.5mlofthesuspendedcellstothefibronectincoatedPetridishandanother1.5mlofDMEMcompletemedium.
3. PlacetheseededPetridishintheincubator.Note:Ideally,theconfluencywillbearound90%atthetimeofselectiontwodayslater.
4. Optional.ToactivateHBEC-5i,addtheTNF(TumourNecrosisFactor)cytokineatafinalconcentrationof50μg/ml24hourspriortothe
selection.
3. Plasmodium falciparum routine culture
1. Preparethenecessaryreagents(seetablehereunder).Preparehumanredbloodcells(RBC)byseparatingwholeblood(groupO+)by
passagethroughaleukocytedepletionfilter(see"MethodsinMalariaResearch"publication11forgeneralmalariaparasitesculturing).Wash
RBCtwicebycentrifugingat400gfor5minandresuspendingthemwith10mlRPMIincomplete.KeepwashedRBCat4°Cinincomplete
mediumat50%haematocrit.
Mediumtoprepare Reagents Quantity
"RPMIincomplete" RPMI1640(withbicarbonate) 500ml
  Hepes1M 12.5ml
  Glucose20% 5ml
  L-glutamine200mM 5ml
  Gentamycin50mg/ml 250μl
  NaOH1M 0.7ml(adjustpHto7.2)
"RPMIcomplete" "RPMIincomplete" 450ml
  Pooledhuman(non-immune)serum 50ml

2. CultureP.falciparuminfectedRBCwithRPMIcompletemediumat2%haematocritandincubateat37°Cwith3%CO2,1%O2,and96%N2.
MakeGiemsasmear11dailytoassessthestageofdevelopmentofparasites(Figure2).
3. Regularly(approximatelyonceaweek)synchronizeculturebysorbitoltreatment12.Thedaypriortheselectionassay,aimforaring-stage
cultureat5%parasitaemiaormore(ideallyatleast10%).
4. Selection of P. falciparum for cytoadhesion to endothelial cells
1. Onthedayoftheassay,theparasitecultureshouldbeatpigmentedtrophozoitestage(Figure2)(ideally10%parasitaemia)whileHBEC-5i
cultureshouldbeat50to100%confluency(ideally90%).30μlpackedcellvolumeofparasitecultureisneededperHBEC-5iPetridish.
2. Washparasitestwicebycentrifuging(500gfor5min)1.5mlofparasiteculture.Discardsupernatantandresuspendwith10mloffreshly
made,warmed,DMEMincompletemedium.Repeatthewashasecondtime.Resuspendthe30μlpackedcellvolumewith1.5mlofDMEM
incompletewith1%BSA.
3. WashtheHBEC-5icoatedPetridishtwiceaspiratingmediumandadding3mlofincompleteDMEM.
4. AddthesolutionofparasitestotheHBEC-5idishandincubateat37°Cfor75min.Resuspendparasitestwice(after30and60min)duringthe
incubationbygentlyrockingthedishinthefourdirections,aswellasclockwiseandanti-clockwise.
5. Afterincubation,washthedish5timesbyaspiratingmedium,usingaplasticPasteurpipettetoadd3mlofwarmDMEMincompletemedium,
andgentlerocking.Ifmanydishesarebeingused,keepthemonawarmsurface,suchasalargeflaskfilledwithwaterat37°C.
6. Checkthedishunderaninvertedmicroscope.IfmanyuninfectedRBCarestillvisible,domorewashesasdescribedabove.HandlethePetri
dishverycarefullytoavoidanyriskofcontamination.
7. Removemediumfromthedishandadd3mlofwarmRPMIcompletemediumwith40μlpackedcellvolumeoffreshuRBC.
8. Placethedishinanairtightincubatingchamber,gasitfor3minandplacethechamberinanincubatorat37°Covernight.
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9. Thedayafter,harvesttheparasitesbywashingwithRPMIincompletemedium,inasimilarwayasdescribedinsection4.4.butmore
vigorously.Keepallmediumused(containingresuspendedRBC)ina15mlconicaltube.CheckunderinvertedmicroscopethatallRBChave
beenremovedfromthedish.
10. Pellettheparasites,discardthesupernatant,resuspendin5mlRPMIcompletemediumandplacethemixtureinaflaskfornormalculturing.
5. Representative Results
UnselectedparasitesshowalowlevelofbindingtoHBEC-5i(Figure3A).Thus,afterthefirstroundofselection,veryfewparasiteswillbe
harvestedanditmaytakeuptoamonthofculturingtoreachaparasitaemiasufficientforthesecondroundofselection.Aftereachroundof
selection,moreandmoreparasitesbindtotheendothelialcellsandtheselectionscanberepeatedatshortertimeintervals.After4-5roundsof
selection,high-bindingparasitepopulationsareobtained(Figure3).
Inourhands,thebindingofHB3-HBECtoHBEC-5iorTNFactivatedHBEC-5iwasofsimilarlevel(datanotshown).
Theprotocoldescribedherewastestedwith4P.falciparumstrains:HB3,IT/FCR3,3D7andDd2(Table1).Onlythelatterprovednotabletobind
toHBEC-5i,evenafter5roundsofselection.
HB3parasiteswerealsoselectedforcytoadherencetoHumanDermalandPulmonaryMicrovascularEndothelialCells(HDMECandHPMEC).
After4roundsofselection,usingthemethoddescribedhere,ahigh-bindingpopulationwasobtainedonbothHDMECandHPMEC(Figure4).

Figure1.Overviewoftheselectionprocess.

Figure2.DevelopmentstagesofP.falciparuminfectedredbloodcells.Giemsasmearvisualizedundermicroscopeat1000Xmagnification.
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Figure3.TypicalexampleofparasitesbindingtoHBEC-5i.(A)ThebluelayerisHBEC-5ifixedwithglutaraldehydeandstainedwithGiemsa,
visualizedundermicroscopeat1000Xmagnification.PicturesweretakenafteruRBCandunboundiRBCwerewashedaway.Theleftpanel
showsasingleP.falciparumHB3(unselected)parasiteboundtoHBEC-5i.IntherightpaneltheHB3-HBECparasiteshadbeenselectedfor5
rounds.(B)Datarepresentstheaverageoftwoindependentexperiments,eachperformedinduplicate.Thenumberofparasitesboundper
endothelialcellwascounted.

Table1.SummaryofPlasmodiumfalciparumstrainsthatweresuccessfullyselectedforbindingtoHBEC-5i.NotethatHB3wasalso
selectedonTNF-activatedHBEC-5i.After5roundsofselection,theDd2strainshowednoincreaseinbindingtoHBEC-5icomparedto
unselected-Dd2.
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Figure4.BindingofP.falciparumHB3parasitestodermal(HDMEC)andpulmonary(HPMEC)endothelialcells,beforeandafter4
roundsofselection.AlthoughtheculturemediumforHDMECandHPMECslightlydiffers(seesupplier'sinstructions),theprotocolusedforthe
selectionwasidenticalaswithHBEC-5i.Picturestakenat400Xmagnification.
Nameofthereagent Company Catalogue Comments
number
DMEM-F12Ham Sigma D6421 ForDMEMcompletemedium
L-glutamine200mM GIBCO 25030 ForDMEMandRPMIcompletemedium
Penicillin/streptomycin100X(10000units/mland10mg/ml) ScienCell 0503 ForDMEMcompletemedium
FoetalBovineSerumheat-inactivated ScienCell 0025 ForDMEMcompletemedium
Trypsin-EDTA(0.025%Trypsin, 0.5mMEDTA) ScienCell 0103  
Endothelialcellgrowthsupplement ScienCell 1052 ForDMEMcompletemedium
Tissueculturetreated60mmX15mmPetridishes BD 353002  
HumanFibronectin Millipore FC010 Useat2μg/cm2
TNF R&D 210-TA optional,useat50μg/ml
Systems
RPMI1640 Lonza BE12-167F ForRPMIcompletemedium
Gentamycin50mg/ml Lonza 17-518Z ForRPMIcompletemedium
Hepes1M Lonza BE17-737E ForRPMIcompletemedium
HDMEC ScienCell 2000 Primarycellline
HPMEC ScienCell 3000 Primarycellline
HBEC-5i     ObtainedfromFrancisco
Candal ([email protected])
Table2.Materials
Discussion
ThehallmarkofcerebralmalariaissequestrationofP.falciparumiRBCwithinthebrainmicrovasculature2,3.However,invitroculturesofP.
falciparumonlypoorlycytoadheretoHBEC-5i,amodelforhumanbrainmicrovascularendothelium.Herewedevelopedastraightforwardassay
toenrichapopulationforbindingtoHBEC-5i,amore"in-vivolike"phenotype.Threeoutof4P.falciparumstrainsweresuccessfullyselected
usingthismethod.Furthermore,HB3wasalsoselectedonHDMECandHPMEC,indicatingthatthisprotocolcanbeusedforvariousparasite
andendothelialcelltypes.
DifferenthypothesesmayexplainthelackofbindingwiththeDd2strain.Themostlikelybeingthefactthatthisparasitelineisknobless,which
deeplyimpedesthecytoadherence13,14.
Werecommendculturingunselectedparasitesalongsidetheselectionprocesstoprovideacontrolforcomparison.Thiswillallow,forexample,
comparingthetranscriptomeofbindingandnon-bindingparasites,withthehopeofdiscoveringparasiteligandcandidates.
TNFisacytokinefoundathighlevelincerebralmalariapatientsandhasbeenshowtoinducetheexpressionofmanysurfaceproteinsof
HBEC-5i(Claessensetal,inpreparationand8,15,16).Inthiscase,theamountofboundiRBCwassimilarinnormalHBEC-5icomparedto
activatedHBEC-5i.
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This"sequestrationmodel"canalsobeusedtostudythemolecularinteractionbetweentheiRBCandtheendothelialcell,aswellastheeffectof
putativeanti-cytoadherencedrugs.Inthiscase,werecommendplatingHBEC-5iinsmallerwells,suchas"8-wellchamberslides"(BD354628)or
"CultureWell"(SigmaC7735-20EA).
Disclosures
Wehavenothingtodisclose.
Acknowledgements
WethankFranciscoCandal,CDCTechnologyTransferOffice,AtlantaGeorgiaforHBEC-5icells.ThisworkwasfundedbytheWellcomeTrust(4
yearPhDstudentshiptoACandSeniorFellowshipinBasicBiomedicalSciencetoJAR,grantnumber084226).
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