Friedrich Frischknecht

Robert Ménard

Rogerio Amino

Sabine Thiberge

Spencer Shorte

CR Biologies

C. R. Biologies 329 (2006) 858–862http://france.elsevier.com/direct/CRASS3/Immunology / ImmunologieQuantitative imaging of Plasmodium sporozoitesin the mammalian hostRogerio Amino a, Sabine Thiberge a, Spencer Shorte b, Friedrich Frischknecht a,Robert Ménard a,∗a Unité de biologie et génétique du paludisme, Institut Pasteur, 25–28, rue du Docteur-Roux, 75724 Paris cedex 15, Franceb Plate-forme d’imagerie dynamique, Institut Pasteur, 25–28, rue du Docteur-Roux, 75724 Paris cedex 15, FranceReceived 9 March 2006; accepted after revision 12 April 2006Available online 17 August 2006Presented by Gérard Orth & Philippe SansonettiAbstractMalaria, the disease caused by Plasmodium, kills more than 1 million people annually. Little is known of the pre-erythrocyticphase of the parasite life cycle, i.e., after the sporozoite stage is inoculated in the dermis by a mosquito and before the erythrocyte-infecting stage is released from hepatocytes. We present here a quantitative, real-time analysis of the fate of parasites transmittedin a rodent system. We describe previously unrecognized steps in the parasite’s journey to the liver of the host, which are likely toplay an important role in the host immune response. To cite this article: R. Amino et al., C. R. Biologies 329 (2006).© 2006 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.RésuméImagerie quantitative des sporozoïtes de Plasmodium chez l’hôte mammifère. Le paludisme, la maladie causée par lesparasites du genre Plasmodium, tue plus d’un million de personnes par an dans le monde. La partie pré-érythrocytaire du cycle devie du parasite, depuis l’inoculation des sporozoïtes dans le derme par un moustique jusqu’à la libération dans le sang des stades quiinfectent les érythrocytes, reste peu connue. Nous présentons ici une analyse quantitative et en temps réel du devenir des sporozoïtesdans un modèle rongeur de l’infection. Nous décrivons des étapes préalablement inconnues dans le trajet du sporozoïte jusqu’aufoie de l’hôte, qui jouent vraisemblablement un rôle important dans la réponse immune de l’hôte. Pour citer cet article : R. Aminoet al., C. R. Biologies 329 (2006).© 2006 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.Keywords: Malaria; Sporozoite stage; In vivo imaging; Lymph nodeMots-clés : Paludisme ; Stade sporozoïte ; Imagerie in vivo ; Ganglion lymphatique* Corresponding author.E-mail address: rmenard@pasteur.fr (R. Ménard).1631-0691/$ – see front matter © 2006 Académie des sciences. Published bdoi:10.1016/j.crvi.2006.04.0031. IntroductionPlasmodium infection starts when an infected Ano-pheles mosquito, while probing the host in search of ablood source, ejects parasites into the dermis. At thisstage of its life cycle, the parasite is a sporozoite, any Elsevier Masson SAS. All rights reserved.R. Amino et al. / C. R. Biologies 329 (2006) 858–862 859elongated and actively motile cell whose final destina-tion is the liver. Inside hepatocytes, the parasite multi-plies and transforms into the erythrocyte-invasive form,which initiates the parasite replication cycles inside ery-throcytes and the clinical symptoms of the disease.The so-called pre-erythrocytic stages of Plasmod-ium, i.e., the sporozoite and the liver stages, have longbeen recognized as attractive targets for anti-malarialvaccines. As was first discovered more than 60 yearsago using an avian Plasmodium model of infection, andis now well established in rodent [1] and primate mam-malian models as well as in humans [2], injection ofseveral thousands of irradiated sporozoites induces a ro-bust and lasting immune response in the host that canprotect against subsequent injection of virulent sporo-zoites. Since irradiated sporozoites normally reach he-patocytes, but fail to develop into erythrocyte-infectingforms (they fail to undergo normal nuclear divisions),such a live, attenuated vaccine would aim at prevent-ing erythrocyte infection and the onset of the disease.However, such a vaccine remains experimental, becausethousands of mosquito bites are required to deliver theprotective dose of irradiated sporozoites, and the lat-ter, once dissected out from mosquito salivary glands,rapidly lose their infectivity and cannot be stored.In the last twenty years, the nature of the host im-mune responses initiated by irradiated sporozoites hasbeen under intense scrutiny [3]. Much of what we knowon these protective responses comes from studies usingthe P. berghei and P. yoelii species, which infect ro-dents. The primary effector cells of protection inducedby irradiated sporozoites are thought to be CD8+ Tcells [4], which recognize parasite peptides presented onthe surface of infected hepatocytes in association withMHC class-I molecules. The mechanisms underlyingthe CD8+-mediated effects are not completely under-stood, but IFN-γ, IL-12, and nitric oxide, an effectivekiller of the Plasmodium liver stage, play central roles.Recently, the demonstration that protection can also beinduced by genetically modified sporozoites unable tocomplete their development in the host liver [5,6] hasrenewed interest in developing live attenuated vaccines[7,8]. However, if such a live sporozoite vaccine is tobe pursued, it is particularly important to know the ex-act route that sporozoites take from the site of mosquitobite to hepatocytes, as well as the nature of the host–parasite interactions along the way.A quantitative and real-time view of sporozoite in-fection in vivo is now possible due to recent advances inimaging techniques and to the generation of transgenicparasites that express fluorescent tags. Using clones ofP. berghei that express the green fluorescent protein[9,10], we have investigated by epifluorescence time-lapse microscopy the fate of sporozoites naturally trans-mitted by Anopheles stephensi mosquitoes [11].We first imaged sporozoites in the dermis of the host.For this, a single mosquito was allowed to probe theear of an anesthetized hairless mouse for 1 min, andthe probed site was observed by wide-field or confo-cal microscopy. An average of ∼20 fluorescent sporo-zoites was detected in the imaged tissue volume. Ap-proximately 75% of them were gliding in the dermis(upon observation intervals of 10 s), and displayed arobust forward locomotion (at speeds of 1–2 µm s−1)that followed a tortuous and apparently random path(Fig. 1). The motility of dermis sporozoites thereforediffers from the motility of sporozoites in vitro (oncoverslips), where sporozoites glide at similarly highspeeds, but in an almost perfectly circular fashion, aswell as from the motility of sporozoites in the mosquitosalivary ducts, where only 10% (upon observation inter-vals of 100 s) glide at much slower speed (∼0.1 µm s−1)and in a typically back-and-forth fashion [12]. How-Fig. 1. Sporozoite motility in the dermis. The figure shows the dis-persion of sporozoites in the dermis, represented by maximum fluo-rescent intensity projections of parasites moving over 400 s at 5 min(red), 16 min (green) and 48 min (blue) after the mosquito bite. Themovement of most sporozoites is localized at the site of bite and de-creases over time.860 R. Amino et al. / C. R. Biologies 329 (2006) 858–862ever, despite their strongly induced motility, most der-mis sporozoites remained in the imaged volume overtime, due to both their sinuous gliding path and declin-ing average velocity (Fig. 1). This allowed a quantitativeassessment of the sporozoite fate in the dermis.We then addressed how sporozoites were leavingthe site of mosquito bite. Blood vessels were revealedby tracking the movement of erythrocytes with trans-mission light at high image acquisition rates prior toor after imaging fluorescent sporozoites, or the bloodflow was tracked by intravenous injection of fluores-cent bovine serum albumin (BSA). As already de-scribed [13], sporozoites invaded blood vessels. Sporo-zoites gliding in the dermis were seen interacting withblood vessel walls, frequently decreasing their speedwhen gliding along them (Fig. 2), before being suddenlycarried away at speeds similar to that of erythrocytes inthe dermal capillary bed, i.e., ∼40–50 µm s−1.We also observed, for the first time, sporozoites in-vading lymphatic vessels. Sporozoites gliding in thedermis anterior pole first were subsequently seen drift-ing in a sideward and low-velocity movement alonga path that did not coincide with the trajectory of ablood vessel. Confocal microscopy after intra-dermalinjection of sporozoites with red fluorescent dextranconfirmed the presence of sporozoites inside lymphat-ics. Based on data collected from 1-h-time-lapse analy-Fig. 2. Sporozoite invasion of a blood vessel in the dermis. The max-imum intensity projection shows a sporozoite gliding in the dermis(red; 20 confocal images over 120 s) and gliding along the blood ves-sel wall (green; 25 confocal images over 150 s) before invasion. Theblood vessel, tracked by fluorescent BSA, is depicted in blue.sis of dermis sporozoites delivered during 12 indepen-dent mosquito bite experiments, we estimate that in 1 h∼50% of the inoculated sporozoites leave the site of biteby invading either blood (∼70%) or lymphatic (∼30%)vessels. Unexpectedly, an important proportion of thesporozoites remained in the dermis, where they were de-tected up to 7 h post-injection.The traditional assumption is that sporozoites poten-tially drained by lymphatic vessels would eventuallyreach the blood and the liver [14,15], where they caninvade the only cells in which they are known to de-velop in vivo, i.e., hepatocytes. To analyze sporozoitesin the lymphatic system, 10 infected mosquitoes wereallowed to bite for 1 min the footpad of anesthetizedhairless mice, and at various times post-bite the proxi-mal (popliteal) and distal (iliac) draining lymph nodeswere dissected to determine the number of fluorescentsporozoites. An average of ∼50 parasites was found inthe popliteal node at 1 h, 2 h, and 4 h post-bite, rep-resenting ∼25% of the inoculated sporozoites. In theiliac node, however, sporozoites were not encounteredup to 4 h post-bite. Similar results, i.e., a very smallproportion of inoculated sporozoites found beyond thepopliteal lymph node, were also obtained using C57/Bl6mice and in Brown–Norway rats, which are both highlypermissive to P. berghei. We conclude that most of thesporozoites drained by lymphatics stop their journey inthe proximal lymph node. Importantly in the context ofa potential live sporozoite vaccine, we also found that a∼20-fold higher proportion of sporozoites reached thepopliteal node when sporozoites are injected by mos-quitoes than intra-dermally with a syringe.We next investigated the fate of sporozoites in thepopliteal lymph nodes. Early after injection (1–2 h),sporozoites were found mainly in the sub-capsular zoneof the lymph nodes close to the afferent lymph vesseland sub-capsular sinuses, but re-localized towards theefferent vessel over time. In the process, the proportionof parasites displaying a normal shape sharply declined.While at 1 h most parasites displayed the characteristicsporozoite shape, at 4 h most parasites appeared dam-aged, and at 8 h most fluorescence had disappeared.When extracted lymph nodes were stained with anti-bodies to CD11c, a marker specific of dendritic cells(DC), ∼20% and ∼50% of the lymph node parasiteswere closely associated with DC at 2 h and 4 h post-bite, respectively. Sporozoites were either extracellular,with the surface areas of the two cells apposed, or weredetected inside DC as normally shaped or partially de-graded parasites.Most surprisingly, examination of lymph nodes overtime revealed that lymph sporozoites could mature intoR. Amino et al. / C. R. Biologies 329 (2006) 858–862 861exo-erythrocytic forms (EEF). Normal development ofP. berghei inside rodent hepatocytes takes ∼52 h, andconsists in a remodelling process from the elongatedsporozoite to an enlarged and spherical EEF, in whichnuclei divide and from which daughter cells eventu-ally bud off. To investigate parasite development in thelymph nodes, the footpads of C57/Bl6 mice were sub-jected to 15 mosquito bites or to intra-dermal injec-tion of 105 salivary gland sporozoites. Following de-livery by mosquito or syringe, a small proportion offluorescent parasites was detected in the lymph node at15 h and 24 h. The location of these parasites insidelymph nodes was investigated by staining with antibod-ies to CD11c; CD11b, a macrophage/monocyte marker;CD45, a generic leukocyte antigen; and podoplanin,which is specifically expressed by lymphatic but notblood vascular endothelial cells. Parasites co-localizedonly with podoplanin-positive cells (Fig. 3). However,parasite development was not complete in lymph nodes.Indeed, although at 15 h lymph node parasites had asize, shape and fluorescence intensity that were simi-lar to those of EEF developing inside hepatocytes ofFig. 3. Lymph node EEF co-localize with endothelial cells.Twenty-four hours after parasite injection into the footpad of mice,popliteal lymph nodes were removed and stained with antibodies topodoplanin (red). Lymph node exo-erythrocytic forms (EEF) of theparasite (green) co-localize with labeled endothelial cells.the same animals, at 24 h the lymph node parasites hada smaller size than that of concomitant liver EEF, andat 52 h post-injection, fluorescence was not detectedin popliteal nodes, while large fluorescent EEF werepresent in the liver. Finally, RT-PCR experiments wereperformed to confirm that parasites were partially de-veloping inside lymph nodes. Expression of the TRAPgene, which normally peaks in sporozoites and de-creases in liver EEF, was not detected at both 15 h and24 h, while expression of the HEP17 gene, which isweak in sporozoites and sharply increases in liver EEF,was detected in lymph node EEF at both 15 and 24 h.This demonstrates the presence of developing parasitesinside lymph nodes.These observations indicate that Plasmodium sporo-zoites inoculated by Anopheles mosquitoes have variousfates in the mammalian host. Sporozoites can either re-main in the dermis, or invade blood or lymphatic ves-sels. Sporozoites drained by lymphatics are trapped inthe proximal lymph node, where most are degraded butsome can partially develop and deliver early EEF anti-gens. Many questions remain about the pre-erythrocyticphase of the parasite life cycle. What is the fate of thesporozoites left in the dermis? How do sporozoites leavethe liver sinusoids and reach the parenchyma? How dosporozoites resist phagocytosis by macrophages, bothin the dermis and in the liver sinusoids? It will also beimportant, in view of the renewed interest in live attenu-ated sporozoite vaccines, to investigate the contributionof dermal as well as lymph node parasites in mount-ing a protective immune response, or instead in inducingtolerance to parasite antigens. The increasingly power-ful imaging tools should help in dissecting these newhost–parasite interactions and the host responses theygenerate in a physiological context.References[1] R. Nussenzweig, J.-P. Vanderberg, H. Most, C. Orton, Specificityof protective immunity produced by X-irradiated Plasmodiumberghei sporozoites, Nature 222 (1969) 488–489.[2] S.L. Hoffman, L.M. Goh, T.C. Luke, I. Schneider, T.P. Le, D.L.Doolan, J. Sacci, P. de la Vega, M. Dowler, C. Paul, D.M. Gor-don, J.A. Stoute, L.W. Church, M. Sedegah, D.G. Heppner, W.R.Ballou, T.L. Richie, Protection of humans against malaria byimmunization with radiation-attenuated Plasmodium falciparumsporozoites, J. Infect. Dis. 185 (2002) 1155–1164.[3] A.V. Hill, Pre-erythrocytic malaria vaccines: towards greater ef-ficacy, Nat. Rev. Immunol. 6 (2006) 21–32.[4] P. Romero, J.L. Maryanski, G. Corradin, R.S. Nussenzweig,V. Nussenzweig, F. Zavala, Cloned cytotoxic T cells recognizean epitope in the circumsporozoite protein and protect againstmalaria, Nature 341 (1989) 323–326.862 R. Amino et al. / C. R. Biologies 329 (2006) 858–862[5] A.K. Mueller, M. Labaied, S.H. Kappe, K. Matuschewski, Ge-netically modified Plasmodium parasites as a protective experi-mental malaria vaccine, Nature 433 (2005) 164–167.[6] A.K. Mueller, N. Camargo, K. Kaiser, C. Andorfer, U. Fre-vert, K. Matuschewski, S.H. Kappe, Plasmodium liver stagedevelopmental arrest by depletion of a protein at the parasite-host interface, Proc. Natl Acad. Sci. USA 102 (2005) 3022–3027.[7] R. Ménard, Knockout malaria vaccine?, Nature 433 (2005) 113–114.[8] A.P. Waters, M.M. Mota, M.R. van Dijk, C.J. Janse, Malariavaccines: back to the future, Science 307 (2005) 528–530.[9] R. Natarajan, V. Thathy, M.M. Mota, J.C. Hafalla, R. Menard,K.D. Vernick, Fluorescent Plasmodium berghei sporozoites andpre-erythrocytic stages: a new tool to study mosquito and mam-malian host interactions with malaria parasites, Cell. Micro-biol. 3 (2001) 371–379.[10] B. Franke-Fayard, H. Trueman, J. Ramesar, J. Mendoza, M. vander Keur, R. van der Linden, R.E. Sinden, A.P. Waters, C.J.Janse, A Plasmodium berghei reference line that constitutivelyexpresses GFP at a high level throughout the complete life cycle,Mol. Biochem. Parasitol. 137 (2004) 23–33.[11] R. Amino, S. Thiberge, B. Martin, S. Celli, S. Shorte, F.Frischknecht, R. Menard, Quantitative imaging of Plasmodiumtransmission from mosquito to mammal, Nat. Med. 12 (2006)220–224.[12] F. Frischknecht, P. Baldacci, B. Martin, C. Zimmer, S. Thiberge,J.-C. Olivo-Marin, S.L. Shorte, R. Menard, Imaging movementof malaria parasites during transmission by Anopheles mos-quitoes, Cell. Microbiol. 6 (2004) 687–694.[13] J.-P. Vanderberg, U. Frevert, Intravital microscopy demonstrat-ing antibody-mediated immobilisation of Plasmodium bergheisporozoites injected into skin by mosquitoes, Int. J. Parasitol. 34(2004) 991–996.[14] J.A. Vaughan, L.F. Scheller, R.A. Wirtz, A.F. Azad, Infectivity ofPlasmodium berghei sporozoites delivered by intravenous inoc-ulation versus mosquito bite: implications for sporozoite vaccinetrials, Infect. Immun. 67 (1999) 4285–4289.[15] A.U. Krettli, L.A. Dantas, Which routes do Plasmodium sporo-zoites use for successful infections of vertebrates?, Infect. Im-mun. 68 (2000) 3064–3065.

Quantitative imaging of <i>Plasmodium</i> sporozoites in the mammalian host

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Quantitative imaging of <i>Plasmodium</i> sporozoites in the mammalian host

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