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Infection and Immunity, August 2005, p. 4777-4786, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.4777-4786.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Species-Specific Inhibition of Cerebral Malaria in Mice Coinfected with Plasmodium spp.

Tatiana Voza,1 Ana M. Vigário,2 Elodie Belnoue,2 Anne Charlotte Grüner,2 Jean-Christophe Deschemin,2 Michèle Kayibanda,2 Florian Delmas,1 Chris J. Janse,3 Blandine Franke-Fayard,3 Andrew P. Waters,3 Irène Landau,1,{dagger} Georges Snounou,1,4,{dagger}* and Laurent Rénia2,{dagger}

Equipe Parasitologie Comparée et Modèles Expérimentaux USM 307, CNRS IFR 101, Laboratoire de Protozoologie et Parasitologie Comparée, EPHE, Muséum National d'Histoire Naturelle, CP52, 61 Rue Buffon, 75231 Paris Cedex 05, France,1 Département d'Immunologie, INSERM U567, CNRS UMR 8104, Université René Descartes, Hôpital Cochin, 75014 Paris, France,2 Department of Parasitology, Malaria Group, Leiden University Medical Center, Leiden, The Netherlands,3 Unité de Parasitologie Biomédicale, CNRS URA 2581, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France4

Received 4 November 2004/ Returned for modification 8 January 2005/ Accepted 10 March 2005


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ABSTRACT
 
Recent epidemiological observations suggest that clinical evolution of Plasmodium falciparum infections might be influenced by the concurrent presence of another Plasmodium species, and such mixed-species infections are now known to occur frequently in residents of most areas of endemicity. We used mice infected with P. berghei ANKA (PbA), a model for cerebral malaria (CM), to investigate the influence of experimental mixed-species infections on the expression of this pathology. Remarkably, the development of CM was completely inhibited by the simultaneous presence of P. yoelii yoelii but not that of P. vinckei or another line of P. berghei. In the protected coinfected mice, the accumulation of CD8+ T cells in the brain vasculature, a pivotal step in CM pathogenesis, was found to be abolished. Protection from CM was further found to be associated with species-specific suppression of PbA multiplication. These observations establish the concept of mixed Plasmodium species infections as potential modulators of pathology and open novel avenues to investigate mechanisms implicated in the pathogenesis of malaria.


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INTRODUCTION
 
Recent observations in Africa (6), Asia (14), and Oceania (50) have suggested that associations between different malaria parasite species might actually have a beneficial influence on the clinical activity of Plasmodium falciparum infections, the major cause of malaria mortality in humans. This notion has been reinforced through detailed longitudinal observations in Thailand (44), where the simultaneous presence of P. vivax in patients presenting with P. falciparum was observed to reduce the risk of developing severe disease or complication (25), treatment failure (33), and anemia during follow-up (34).

The epidemiological and clinical relevance of interactions between parasite species found in the same host, derives from the emerging realization that mixed-species infections are a common feature of malaria in humans. Three of the parasite species—P. falciparum, P. vivax, and P. malariae-to which humans are susceptible are globally distributed, although P. vivax is rare in West Africa. A fourth species, P. ovale, is predominantly found in West Africa but has not been recorded in the Americas. Residents in most areas of endemicity for malaria are subjected to infection by two, and often three, Plasmodium species. Scrutiny of the early epidemiological records revealed that mixed-species infections were generally found significantly less frequently than expected (21), a pattern common to past and present surveys where parasite detection is achieved through microscopic examination. The development (43) and subsequent use of sensitive PCR-based species detection methods has clearly established that mixed Plasmodium species infections are actually very common (28, 42), even in areas of relatively low endemicity (32, 39).

Given that in many areas of endemicity mixed-species infections are likely to be the rule rather than the exception, it is important to investigate whether interactions between Plasmodium species have any consequences on the pathological evolution of the infections. Detailed investigations of the influence of mixed infections on pathology and elucidation of underlying mechanisms are best undertaken through carefully controlled experimental infections that can only be envisaged in animal models. Cost, ethical considerations, and the dearth of immunological reagents preclude primates as initial experimental models for such investigations. Laboratory rodents, though imperfect models for the human infection, offer the most practical alternative, in particular through the availability of cloned lines from different Plasmodium species to which they are susceptible.

Laboratory mice infected with the P. berghei ANKA cloned line (PbA) provide a suitable experimental model for the investigations of the pathogenesis of cerebral malaria (CM) (23). PbA-infected susceptible mice develop overt clinical signs between day 6 (D6) and D9 and die within 48 h of that onset. The development of CM in mice was recently shown to be associated with the migration of pathogenic CD8+ T cells to the brain, a mechanism thought to be initiated by PbA parasite sequestration in the brains of the CM-susceptible mice (5, 15).

We report here the first detailed study of the influence of mixed-species infections on the expression of PbA-induced CM. Infection by PbA was combined with one of five different parasite lines, from the same or different species, chosen for their inability to induce CM in the mice used.


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MATERIALS AND METHODS
 
Mice and parasites. All studies were carried out with 6- to 10-week-old 129,B6 mice from a mixed 129/Ola x C57BL/6J genetic background (5). In these mice, CM has been established to occur reproducibly (3-5). The mice were bred in a specific-pathogen-free animal facility. Mice were matched for age and sex for each experiment. All experiments and procedures conformed to the French Ministry of Agriculture Regulations For Animal Experimentation (published in 1987). The following parasite lines were used: P. berghei ANKA clone BdS (PbA), P. berghei NK 65 (PbNK65), P. yoelii yoelii 17X clone 1.1 (Pyy 1.1), P. yoelii yoelii 17X clone YM (Pyy YM), P. vinckei vinckei strain 67 (Pvv), and P. vinckei petteri strain 106HW (Pvp). Infections were initiated by injection of thawed stabilates (107 infected red blood cells [IRBC]/ml in Alsever's solution) stored in liquid nitrogen, except for Pvp, where the injection was initiated by using fresh blood from infected donor mice. The P. berghei ANKA clone cl15cy (12), hereafter referred to as PbA-GFP, constitutively expresses an integrated green fluorescent protein (GFP) gene. Mice were infected intraperitoneally (i.p.) with 5 x 105 IRBC of a given parasite line; thus, in mixed infections a total of 106 IRBC (5 x 105 of each parasite line) were inoculated. All control and experimental groups consisted of five mice, except for the brain perfusion experiments where a larger number (indicated in the legend) was used. The results presented derive from experiments that were duplicated at least once, and no significant differences were observed between repeated experiments.

In the 129,B6 mice nearly all the PbA-infected or PbA-GFP-infected animals develop CM early in the course of the infection (between days 6 and 9, as in other CM-susceptible mouse strains), and death generally intervenes within 48 h after the onset of symptoms. In the minority of infected mice where CM does not develop by D10, the parasitemia continues to increase and reaches high levels resulting in anemia and death 7 to 10 days later. In preliminary experiments, the incidence of CM was not observed to vary with an inoculum size of 105 to 107 PbA or PBA-GFP parasites per mouse. A diagnosis of CM was established only when the mice, which were examined daily from D4 postinoculation (p.i.), displayed the following neurological symptoms: paralysis, deviation of the head, ataxia, convulsions, and coma.

Parasitemia. Parasitemia was enumerated by microscopic examination of methanol-fixed tail blood smears stained for 45 min with 10% Giemsa diluted in phosphate buffer at pH 7.2. The number of parasites in 500 erythrocytes was obtained when parasitemias exceeded 1%, whereas 5,000 erythrocytes were examined for lower parasitemias. Mice were monitored as described above every 2 days up to D30 p.i., and every 4 days thereafter. Enumeration of the PbA-GFP line parasites in both control (single infection) and experimental groups (mixed infection) was obtained by fluorescence-activated cell sorting (FACS) analysis with tail-vein blood obtained every 2 days. Briefly, 1 µl of blood was diluted in 500 µl of FACS buffer (phosphate-buffered saline [PBS] containing fetal calf serum and NaN3 at final concentrations of 1 and 0.01%, respectively), and the numbers of fluorescent IRBC were evaluated by acquisition in a FACSCalibur instrument and analysis by the CellQuest Software (Becton Dickinson, Le Pont de Claix, France).

Hematological parameters. Anemia was estimated by measuring hemoglobin (Hb) and enumerating erythrocytes as previously described (49). Hb concentrations were determined every 2 days. Briefly, 2 µl of tail vein blood were diluted in 500 µl of Drabkin's solution (Sigma), and Hb was assayed in 96-well microtiter plates (Costar, Cambridge, MA) in a volume of 100 µl by measuring the absorption at 405 nm with a microplate reader (Victor 1420; Wallac, Turku, Finland). Values were converted to milligrams per milliliter by means of a standard curve of human Hb (Sigma) dissolved in Drabkin's solution. RBC were counted in a Malassez chamber from 2 µl of tail blood diluted in 1 ml of PBS. Reticulocytemia was enumerated by microscopic examination of methanol-fixed tail blood smears that were stained for 45 min with 10% Giemsa diluted in phosphate buffer at pH 7.2. Mice were monitored as described above every 2 days up to D30 p.i. and every 4 days thereafter.

Purification of whole-brain-sequestered leukocytes (BSL). Sacrificed mice were perfused intracardially with PBS to remove both circulating and nonadherent RBC and leukocytes from the brain. The brain was then removed, and adherent leukocytes were isolated as previously described (5). Brains were removed and crushed in RPMI medium (Life Technologies, Paisley, United Kingdom). The tissue extract was then centrifuged at 400 x g for 5 min. The pellet was resuspended with 10 ml of an HEPES buffer containing 100 mM NaCl, 2 mM KCl, 0.3 mM Na2HPO4 · 12H2O, and 0.01 M HEPES (Sigma-Aldrich, St. Quentin l'Arbresles, France), supplemented with 100 IU of penicillin-streptomycin (Life Technologies)/ml, 0.05% collagenase (Boehringer Mannheim, Meylan, France), and 2 U of DNase (Sigma-Aldrich)/ml. The mixture was stirred at room temperature for 30 min. The tissue extract was passed through a sterile gauze and centrifuged at 80 x g for 30 s to remove debris. The supernatant was deposited on a 30% Percoll gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) and centrifuged at 1,400 x g for 10 min. The pellet was collected, and residual RBC were removed by hypotonic shock using an ammonium-chloride-potassium lysis buffer. BSL were resuspended in FACS buffer (PBS containing 1% fetal calf serum and 0.01% NaN3) and counted.

Immunolabeling and flow cytometry analysis of BSL. BSL were identified by their size (forward light scatter) and granulosity (side light scatter) as previously described (19). Macrophages were identified as F4/80+ (biotinylated rat immunoglobulin G2b [IgG2b] monoclonal antibody [MAb] anti-mouse F4/80, clone C1:A3-1; Tebu, Le Perray-en-Yvelines, France). Neutrophils were identified as F4/80– and Gr-1+ (rat IgG2b MAb anti-mouse Gr-1 conjugated to fluorescein isothiocyanate [FITC], clone RB6-8C5; BD Pharmingen, San Diego, CA). Lymphocytes were identified by their small size and with the following antibodies: hamster IgG MAb anti-mouse CD3 conjugated to phycoerythrin (PE; clone 17A2; BD Pharmingen), rat IgG2a MAb anti-mouse CD8{alpha} conjugated to FITC (clone 53-6.7; BD Pharmingen), and rat IgG2a MAb anti-mouse CD4 conjugated to QR (clone H129-19; Sigma-Aldrich), diluted at the appropriate concentration in FACS buffer. Ultravidin-PE-conjugated (Leinco Technologies, St. Louis, MO) and goat anti-rat IgG conjugated to FITC (Polysciences, Warrington, PA) were used as secondary reagents. For each sample, 10,000 cells were analyzed. The data were collected by using a FACSCalibur flow cytometer and analyzed by using CellQuest software (BD Biosciences, le Pont de Claix, France).

In vivo depletions. CD8+-T-leukocyte subpopulation depletion was performed by i.p. injection of a total of 1 mg of purified rat anti-mouse CD8 MAb (clone 2.43; TIB 210; American Type Culture Collection, Manassas, VA) at D6 after parasite injection before the onset of CM. More than 98% of blood CD8+ T cells were depleted by this procedure, as verified by cytofluorometry (FACSScan; BD Biosciences, Mountain View, CA) with an anti-mouse CD8 MAb (clone 53-6.7; BD Pharmingen, San Diego, CA) that recognized a different epitope from the one recognized by the depleting MAb.

Macrophages were depleted at days 0 and 4 after parasite injection by intravenous injection of 0.2 ml of PBS containing ca. 1 mg of dichloromethylenediphosphonate (Cl2-MDP) encapsulated in liposomes (5) (kindly provided by Nico van Rooijen, Faculty of Medicine, Amsterdam University, Amsterdam, The Netherlands). More than 90% of blood F4/80+ cells were depleted, as verified by FACS analysis 2 days later.

Natural killer (NK) cell depletion was performed by treatment of mice with 1 mg of anti-interleukin-2Rß (IL-2Rß) MAb TMß1 (48) (kindly provided by Didier Fradelizi, Institut Cochin, Paris, France) on the day of parasite injection. Depletion of DX5+ CD3– NK cells was >80% as verified by FACS analysis on spleen cells obtained from the MAb-treated mice using the DX5 and anti-CD3 MAbs (clone 17A2), respectively, coupled to FITC and phycoerythrin (BD Pharmingen, San Diego, CA).

Neutrophils were depleted by i.p. injection at D6 of 1 mg of purified rat IgG anti-mouse CR3 MAb, 5C6 (35), after PbA infection. Depletions of blood neutrophils was >80% as verified by FACS analysis with a rat anti-mouse GR1 MAb (clone RB6-8C5; BD Pharmingen).

Cytolytic-T-lymphocyte-associated protein 4 (CTLA4)-bearing T cells were depleted by i.p. administration of 250 µg of hamster IgG anti-mouse CTLA-4 MAb (18) (clone 91OH, kindly provided by James P. Allison, UC Berkeley, Berkeley, CA) 1 day before and on days 2, 5, and 7 after parasite inoculation. This treatment resulted in the depletion of >90% of splenic CTLA4+ T cells, as verified by FACS analysis.

CD25+-T-cell depletion was carried out by i.p. administration of 250 µg of rat IgG1 anti-mouse CD25 MAb (38) (PC61, kindly provided by Ana Cumano, Institut Pasteur, Paris, France) 1 day before and on days 2, 5, and 7 after parasite inoculation. The efficacy of CD25 depletion was confirmed by FACS analysis, with FITC-labeled anti-mouse CD25 MAb (7D4; BD Pharmingen) and anti-mouse CD4 MAb conjugated to Quantum Red (clone 53-6.7; Sigma Aldrich, Saint Quentin L'Arbresle, France). More than 80% of blood CD4+ CD25+ T cells were depleted by D2 after treatment.

Double CTLA4+ and CD4+ CD25+ T-cell depletion was obtained by the administration of 500 µg of each of the MAbs above on days 3 and 1 before and on D5 after infection of the mice.

In vivo neutralization. IL-12, IL-6, and IL-4 neutralization were performed by i.p. injection of 1 mg of purified rat IgG2a anti-mouse IL-12 MAb (51) (clone C17.8.20 [cell line provided by Georgio Trinchieri, Wistar Institute, Philadelphia, PA]), rat IgG1 anti-IL-6 MAb (1) (clone MP5-20F3 [cell line provided by Paola Minoprio, Institut Pasteur, France]), and rat IgG1 anti-IL-4 MAb (30) (clone 11B11 [kindly provided by Mireille Viguier, Institut Cochin, Paris, France]), respectively, on days 0 and 4 after parasite inoculation.

IL-10 and transcription growth factor ß (TGF-ß) neutralizations were performed by i.p. injection of 250 µg of rat IgG1 anti-mouse IL-10 (1) (clone JE5A10 [cell line provided by Paola Minoprio, Institut Pasteur, France]) or mouse anti-TGF-ß (24) (clone 2G7 [kindly provided by Didier Fradelizi, Institut Cochin, Paris, France]) 1 day before and on days 2, 5, and 7 after parasite inoculation, respectively.

Double neutralization of IL-10 and TGF-ß was achieved by simultaneous i.p. injection of 100 µg of each of the above MAbs 1 day before and on days 0, 2, and 5 after parasite inoculation.

Nitric oxide (NO) was neutralized by daily i.p. administration of 100 µg of S-methyl-thio-urea (SMT; Sigma) (45) from D1 before infection until the death of the animals.

Statistical analysis. At a given time point, preliminary analysis established that parasitemia values for the mice within a defined group are normally distributed; thus, differences between mean parasitemia values were analyzed for statistical significance with the unpaired t test when only two groups were compared. The log-rank test was used to analyze the statistical difference in survival among different groups. the Fisher exact test was used to compare the statistical differences in the proportions of animals dying of CM among the different groups. Differences between means of brain sequestered leukocyte values in the different groups of mice on days 7 and 8 p.i. (when PbA-infected animals displayed neurological signs) were analyzed for statistical differences with GraphPad Prism software (version 3.0; San Diego, CA) by using the one-factor analysis of variance, followed by the post hoc Tukey test. In all cases, a P value of <0.05 was used as the level of significance.


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RESULTS
 
Course of infections. The course of infection by each of the species used is presented in Fig. 1. Mice infected with 5 x 105 PbA IRBC, generally died of cerebral malaria between D7 and D11 p.i. when the parasitemia was on average 26.5% (± 7.5 standard deviation [SD]). Survival past this period was observed occasionally (<5% of the PbA-infected mice), and in these mice death due to severe anemia and high parasite loads intervened 1 to 2 weeks later. In mice infected with the nonlethal parasite lines, Plasmodium vinckei petteri (Pvp) or P. yoelii yoelii clone 1.1, (Pyy 1.1) peak parasitemias not exceeding 30% were observed by D10 p.i., and the infection resolved spontaneously by D20 p.i. Whereas in mice infected with the lethal parasite lines, P. berghei strain NK65 (PbNK65), P. vinckei vinckei (Pvv), or P. yoelii yoelii clone YM (Pyy YM), peak parasitemias reached high levels which were maintained until the death of the 129,B6 mice through hyperparasitemia (Fig. 1) and anemia (Fig. 2).



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  		FIG. 1. Course of parasitemia in single and mixed infections. Tail blood smears were collected from each mouse in the group (five mice per group) every 2 days p.i., and parasitemias were expressed as the mean of the log10 of the number of parasitized erythrocytes for 105 erythrocytes. The SD is indicated for each point; only the upper half of the bar is shown for the sake of clarity. The curves are presented for one experiment, and similar data were obtained in the duplicate experiments. The course of the mixed-infection group is represented by open triangles linked by a thick line, whereas the courses of single-infection groups are represented by thin lines joining open circles for PbA or open squares for the second species used in the mixed infection. The identity of this second species is indicated in each of the panels.



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  		FIG. 2. Hematological parameters during the course of single and mixed infections. Mean reticulocytemia levels are expressed as the percentage of reticulocytes enumerated in peripheral blood. Anemia is expressed as the number of RBC (108) per ml of blood (means ± the SD [three to five mice per group]). Abbreviations: PbA, P. berghei ANKA clone BdS; Pyy 1.1, P. yoelii yoelii clone 1.1; Pyy YM, P. yoelii yoelii clone YM; Pvp, P. vinckei petteri strain 106 HW; Pvv, P. vinckei vinckei strain 67; PbNK65, P. berghei strain NK65. The course of the mixed-infection group is represented by open triangles linked by a thick line, whereas the courses of single-infection groups are represented by thin lines joining open circles for PbA or open squares for the second species used in the mixed infection. The identity of this second species is indicated in each of the panels. D p.i., indicated days postinoculation.

Groups of mice were simultaneously inoculated with PbA and one of the following parasite lines: PbNK65, Pvp, Pvv, Pyy 1.1, or Pyy YM. Where sufficient numbers of mice survived in the three groups (two single-infection groups and the mixed-infection group), the overall courses of the parasitemia did not generally appear to differ significantly. However, for the mice in the Pyy 1.1 mixed-infection group, the mice survived beyond D10 and displayed a parasitemia pattern akin to that of PbA-infected mice that do not develop CM, an exacerbation of the parasite load and death 2 to 3 weeks later.

Species-specific protection from CM. Mortality and CM incidence for single species infections are given in Fig. 3A. The combination of the lethal PbA parasite with each of the five other parasite lines did not reduce the overall mortality, since in all cases the mice eventually succumbed to the infection. Since PbA parasites were always present, it was expected that mortality would occur between D8 and D12 and would be due to the cerebral manifestations. This was indeed the case when the otherwise-lethal PbNK65 or Pvv or the nonlethal Pvp were coinfected with PbA (Fig. 3B). However, although death was observed for all mice coinfected with PbA and the otherwise-nonlethal Pyy 1.1 or the lethal Pyy YM, mortality from CM was nearly completely prevented (Fig. 3B). Evident total protection from CM was provided by the simultaneous presence of Pyy 1.1 since in three groups of five mice coinfected with Pyy 1.1, only one mouse died of CM, whereas in the other mice death mainly occurred 3 or more weeks postinfection, as a result of hyperparasitemia and anemia (Fig. 2). Given that mortality observed in the mice singly or coinfected with Pyy YM occurred by D8 and that mortality due to CM intervenes between D7 and D10 after PbA infection (Fig. 1), inhibition of mortality from CM by Pyy YM could not be conclusively derived from these simultaneous infections. However, the observation of CM mortality in groups of mice where P. yoelii yoelii YM infections were initiated 2 or 4 days after PbA inoculation (data not shown) suggested that this parasite combination was not protective.



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  		FIG. 3. Mortality and CM incidence in single and mixed infections. (A) Cumulative mortality in mouse groups (n = 5 for each experiment) inoculated with a single species of parasite as indicated. Open bars indicate overall mortality, and solid bars indicate the proportion of animals succumbing to CM. (B) Cumulative CM incidence in the infection groups in which a different species or line, as indicated, was simultaneously inoculated with PbA. Overall, mortality in all of theses groups was total and was not indicated. {cjs3669}{cjs3669}{cjs3669}, P < 0.0001 (Fisher exact test [versus PbA single infections] on D12 p.i.). Values on the x axes represent the overall percentages of the total number of infected animals.

Inoculations with Pyy 1.1 parasites on different days before injection of PbA were also performed. In one group of mice Pyy 1.1 was inoculated 7 days before superinfection with PbA, whereas in three other groups the Pyy 1.1 parasites were inoculated 2, 4, or 7 days after the injection of PbA parasites. The degree of protection from CM mortality proved to vary with the timing of the coinfections (Fig. 4). As for simultaneous mixed infections, full protection from CM was observed when PbA parasites were introduced in mice injected with Pyy 1.1 parasites 7 days previously, at a time when Pyy 1.1 parasitemia had reached 5 to 10%. Full protection from CM was also observed when the Pyy 1.1 parasites were introduced 2 days after PbA injection, and partial protection was still observed when they were introduced 4 days after infection by PbA. However, the Pyy 1.1 infection did not alter CM mortality when it was initiated 7 days after PbA inoculation.



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  		FIG. 4. Mortality and CM incidence in PbA infections superinfected with P. yoelii yoelii clone 1.1. Solid and open bars indicate the percentages of animal dying from CM and anemia/hyperparasitemia, respectively. The left panel represents single infections with PbA or P. yoelii yoelii clone 1.1 (Pyy 1.1). In the right panel, Pyy 1.1 was inoculated before, at the same time as, or after inoculation of PbA on the days indicated. {cjs3669}{cjs3669}{cjs3669}, P < 0.0001; {cjs3669}{cjs3669}, P < 0.001 (Fisher exact test [versus PbA single infections] on D12 p.i.).

Effects on pathogenic CD8+ T cells. The pathological alterations in the brain found in PbA-infected mice at the time of CM, e.g., hemorrhages and sequestration of parasite and leukocytes, were not observed in the brains of mice infected with Pyy 1.1 or in those simultaneously coinfected with both parasite species (data not shown).

A central role in the pathogenesis of CM in PbA-infected mice has been recently demonstrated for a minor subset of CD8+ T cells that specifically migrate and sequester in the brains of infected mice (5). Depletion of CD8+ T cells inhibited mortality from CM in the mice coinfected with PbA and Pvp (results not shown), as it does in PbA-infected mice (5). Migration of leukocytes, including CD8+ T cells, to the brain was measured in the groups of mice infected singly or concurrently with PbA and Pyy 1.1 (Table 1). Compared to naive mice, the overall level of BSL was significantly increased in the mice infected with PbA alone but not in those infected with Pyy 1.1 alone or in the coinfected animals. The brain-sequestered CD8+ T cells levels in the coinfected group were similar to those observed in the Pyy 1.1-infected mice and significantly lower than in mice infected with PbA (Table 1). A similar pattern was observed for the other leukocytes subsets, namely, macrophages, neutrophils, and CD4+ as well as double-negative T cells (Table 1).


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  		TABLE 1. Numbers of whole-brain-sequestered leukocyte subsets in singly infected, coinfected, and naive micea

Protection from CM is not mediated by immune factors known to modify pathologies induced by the infection or parasite development. A number of cytokines, soluble mediators, and immune cells have been linked to the suppression (IL-10, TGF-ß, IL-4, IL-6, NO, CD4 T cells, macrophages, and neutrophils) or exacerbation (IL-6, IL-12, NO, CD4 T cells, macrophages, and neutrophils) of immunopathological responses (7, 17, 20, 36, 37, 47). We have therefore used a panel of neutralizing antibodies individually against IL-4, IL-6, IL-10, IL-12, or TGF-ß or in combination against IL-10 and TGF-ß, as well as depleting antibodies individually against CD25+-bearing CD4+ T cells, CTLA4+-bearing CD4+ T cells, or neutrophils. Macrophages were pharmacologically depleted, and NO synthesis was similarly inhibited.

In none of these experiments was the protection conferred by Pyy 1.1 against CM mortality reversed by the neutralizing or the depleting treatments. Macrophage and NK cell depletion exacerbated the total parasitemia in coinfected mice, as detected by microscopy (results not shown), but did not alter protection from CM.

Species-specific suppression of parasitemia. We hypothesized that suppression of the PbA parasitemia in the coinfection groups could account for the species-specific protection against mortality from CM and the reduced migration of leukocytes to the brain. Supportive indications for this hypothesis could be derived from comparison of the patterns of anemia and reticulocytemia in the various mouse groups (Fig. 2). For the first 10 days of the infection, these patterns in the mixed-infection groups where no protection from CM mortality occurred were similar to those measured in the control mice infected with PbA, whereas in the mixed-infection groups where protection from CM mortality was observed, these patterns were akin to those measured in the control mice infected with the non-PbA parasite line. In the coinfected mice that survived beyond D10, by which time all mortality from CM is observed, anemia persisted and increased until the death of the animals 2 weeks later, as is observed in the PbA-infected mouse strains resistant to CM.

Evidence for species-specific suppression of parasitemia was observed in previous rodent parasite coinfection experiments (41). However, the molecular technique used to distinguish between the microscopically similar malaria parasites of rodents is neither sufficiently sensitive nor adequately quantitative (40) to allow for an accurate differential enumeration of the two lines present in the coinfected animals. Consequently, selected experimental infections were repeated with PbA-GFP, a line where GFP is expressed constitutively (12). Accurate comparison of the evolution of PbA parasitemia in single- and mixed-infection groups could thus be obtained. Since patterns of CM mortality can differ between independent PbA clones (2), the behavior of the PbA-GFP line was tested in single and mixed infections (Fig. 5). Although there was a tendency for the mice to present with clinical CM signs early (D6 p.i.), the behavior of the PbA-GFP line proved to be comparable to that of the PbA BdS line. Two notable exceptions were, however, noted in sequential mixed infections with P. yoelii yoelii parasites. Protection from CM was not obtained when Pyy 1.1 parasites were introduced on Day 4 of the PbA-GFP infection (compare Fig. 4 with Fig. 5B). Unexpectedly, inhibition of CM mortality was observed in 50% of the animals in the group, where the Pyy YM clone was introduced on D2 of the PbA-GFP infection (Fig. 6D).



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  		FIG. 5. Influence of simultaneous and sequential mixed infections on PbA-GFP CM incidence and mortality. (A and B) CM incidence and survival curves in the simultaneous coinfection groups (A) and mortality rates and mortality and survival curves in groups of sequential coinfections with P. yoelii yoelii clone 1.1 (Pyy 1.1) (B). Solid and open bars indicate the percentages of animals dying from CM and anemia/hyperparasitemia, respectively. {cjs3669}{cjs3669}{cjs3669}, P < 0.0001; {cjs3669}{cjs3669}, P < 0.01 (Fisher exact test on D12 p.i. [versus PbA-GFP]). Comparisons between the PbA survival curve and another group survival curve were performed by using the log-rank test.



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  		FIG. 6. PbA-GFP specific parasitemias in various mixed-infection groups. (A to C) Simultaneous infections of PbA-GFP with P. yoelii yoelii clone YM (Pyy YM) (A), P. vinckei vinckei strain 67 (Pvv) (B), and P. berghei strain NK65 (PbNK65) (C). It is interesting that in the PbA-GFP/PbNK65 mixed-infection group, a single mouse was pro-tected from CM and survived beyond D10. In this mouse the level of PbA-GFP dropped sharply after D6 and was still four times less numerous than PbNK65 parasites on D20 when this mouse died of severe anemia. (D and E) Group in which Pyy YM parasites were introduced 2 days after PbA-GFP inoculation. Mean parasitemia levels are expressed as in Fig. 1, except that PbA-GFP parasitemias in single- and mixed-infection groups were enumerated by flow cytometric analysis of GFP fluorescence. The parasitemia course of the mixed-infection group is represented by open triangles linked by a thick line, whereas the courses of the infection in animals infected with PbA-GFP alone are represented by open circles joined by a thin line, and the specific PbA-GFP parasitemia within the mixed-infection groups is represented by solid circles joined by a thin line. Statistical comparison between the parasitemia values recorded for mice infected with PbA-GFP alone and for the PbA-GFP present in the mixed-infection groups was done by using unpaired Student t test (PbA-GFP versus PBA-GFP in coinfected animals): {cjs3669}{cjs3669}{cjs3669}, P < 0.0005; {cjs3669}{cjs3669}, P < 0.001; and {cjs3669}, P < 0.01. This analysis was only conducted and reported when sufficient numbers of surviving mice (n = 3) were present in both groups. In panel E, PbA-GFP parasitemia data are presented separately for two subgroups in the PbA-GFP+Pyy YM (D+2) group, the first consisting of three mice that developed CM (open squares) and the second consisting of three mice in which CM did not develop (solid squares); the open circles represent the control PbA-GFP infection.

In coinfections of PbA-GFP with Pvv or PbNK65, where mice die of CM, the evolution of PbA-GFP did not differ significantly in the control and mixed-infection groups (Fig. 6B and C). In contrast, significant suppression of the multiplication of P. berghei was observed during the first 2 weeks p.i. in PbA-GFP-infected mice coinfected with Pyy 1.1 (Fig. 7A) or clone YM (Fig. 6A), where mortality from CM was not observed. In the mice where the timing of the coinfection was varied, suppression of P. berghei was only observed in the animals where protection from CM was obtained, i.e., in the five mice in which PbA-GFP parasites were inoculated 7 days after the Pyy 1.1 infection was initiated (Fig. 7B) and in the three mice that survived in the group of six mice in which Pyy YM parasites were inoculated 2 days after infection with PbA-GFP (Fig. 6E). It was interesting that suppression of PbA-GFP numbers by P. yoelii was initiated only 5 days after these parasites were introduced to form a mixed infection (Fig. 6A, D, and E and Fig. 7A and B).



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  		FIG. 7. PbA-GFP specific parasitemias in mixed infections of PbA-GFP and P. yoelii yoelii clone 1.1. (A) simultaneous infection; (B) PbA-GFP inoculated 7 days after Pyy 1.1; (C and D) Pyy 1.1 injected 4 days (C) and 7 days (D) after PbA-GFP. Mean parasitemias are expressed as in Fig. 1, except that PbA-GFP parasitemias in single- and mixed-infection groups were enumerated by flow cytometric analysis of GFP fluorescence. The parasitemia course of the mixed-infection group is represented by open triangles linked by a thick line, whereas the courses of the infection in animals infected with PbA-GFP alone are represented by open circles joined by a thin line, and the specific PbA-GFP parasitemia within the mixed-infection groups is represented by solid circles joined by a thin line. {cjs3669}{cjs3669}{cjs3669}, P < 0.0005; {cjs3669}{cjs3669}, P < 0.001; {cjs3669}, P < 0.05 (as determined by unpaired Student t test [PbA-GFP alone versus PbA-GFP present in the mixed-infection groups]). This analysis was only conducted and reported when sufficient numbers of surviving mice (n = 3) were present in both groups.


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DISCUSSION
 
In the present work, we sought to ascertain whether mixed-species infections have an influence on pathogenesis in malaria infections. We focused on CM, for which the PbA model has served to illuminate key aspects of CM pathogenesis of potential relevance to the human host (5, 10, 13, 29).

The first key finding was that mortality due to CM in PbA-infected mice can be inhibited as a consequence of mixed infection. Remarkably, this protection was only conferred by the simultaneous presence of P. yoelii yoelii but not by that of P. vinckei (2 lines) or another line of P. berghei. A strong indication that protection from CM was further linked to a specific combination of parasite lines from each of the species was provided by the different patterns of protection observed in simultaneous and sequential infections using the genetically different P. yoelii yoelii lines with the PbA or the PbA-GFP parasites. Defined parasite combinations were selected to investigate the underlying mechanisms, in particular the PbA/Pyy 1.1 mixed infection in which mortality from CM was nearly totally inhibited.

Sequestration of leukocytes and CD8+ T cells in the brain was observed for PbA-infected mice during CM but not for Pyy 1.1-infected mice in which CM does not occur. In the mice simultaneously coinfected with these parasite lines, the levels of brain-sequestered cells were found to be akin to those observed in Pyy 1.1-infected mice. This observation strongly suggested that the basis of CM protection through mixed infection lay in an inhibition of the induction and/or migration of pathogenic CD8+ T cells to the brain vasculature. However, depletion and neutralization experiments excluded a role for immune factors known to regulate CD8+-T-cell activation and function, including CD25+ CD4+ T cells, IL-10, and TGF-ß (22, 31). The reduced numbers of pathogenic CD8+ T cells in the brain of coinfected mice could simply be due to an insufficient antigenic stimulation by PbA parasites, since this will prevent T-cell expansion and thus migration.

A previous study of mixed infection in rodents had indicated that P. yoelii yoelii suppresses P. berghei early in the infection (41). Since the development of CM only occurs early in the infection, it was hypothesized that this suppression could be linked to protection from CM. GFP-expressing PbA parasites provided conclusive evidence of species-specific suppression of P. berghei parasitemia in mixed-infection groups, the second key finding. Indeed, suppression of PbA was observed only when P. yoelii yoelii parasites, but not Pvv or PbNK65, were inoculated simultaneously. Strong evidence for a causal link between PbA suppression and the inhibition of CM was provided by the group in which Pyy YM parasites were inoculated 2 days after the initiation of the infection by the GFP-expressing PbA parasite. In these six mice, suppression of PbA was only observed in the three animals that did not succumb of CM but not in the other three that did (Fig. 6E).

The observations presented here do not allow us to identify an underlying mechanism for species-specific suppression of parasitemia. Some factors can, however, be discounted. The suppression is unlikely to be accounted for by the red blood cell tropism of the parasite lines used. Thus, Pvv, a predominant mature red blood cell invader, would not be expected to compete for the low numbers of reticulocytes, rarely exceeding 3% of total red blood cells in naive mice, that PbA invades predominantly early in the infection. Furthermore, both Pyy 1.1 and PbNK65 parasites share the preference of PbA for reticulocytes, and yet suppression of PbA is observed for the first but not the second parasite in mixed infections. Moreover, strong suppression of PbA is also observed when this parasite was introduced 7 days after Pyy 1.1 inoculation, when reticulocytes have reached high levels (ca. 10%). The fact that the delayed introduction of P. yoelii yoelii parasites still leads to the suppression of an ongoing PbA infection (Fig. 7), further excludes differences in multiplication rate as a mechanism.

A role for cross-reactive acquired immune responses could not be excluded since evidence of suppression was consistently observed starting from D5 of the mixed infection, a time when some acquired responses become effective (46). However, the lack of inhibition of PbA in the presence of PbNK65 suggests that such cross-reactive responses do not play a central role. Innate immune mechanisms might also be involved, although the largely nonspecific nature of the effectors contrasts with the species specificity of the suppression. The effectiveness of P. yoelii yoelii, but not Pvv or PbNK65, in checking PbA could be explained if one postulates that different parasites lines do not activate the same set of effectors and/or that these distinct lines are differentially susceptible to a given effector. It is possible that species-specific suppression might actually derive from a particular combination of innate and acquired immune responses. Finally, it could be speculated that direct interactions between the different parasite species leads to the suppression observed. It is clear that elucidation of the mechanisms underlying the phenomenon of species-specific suppression merits further investigations.

In conclusion, the observations presented establish that a defined lethal pathology specific to an infection by one parasite species can be alleviated by the presence of another species. In the rodent model, this is effected by a mitigation of the parasitemia. It has long been considered that an antagonism exists between the different Plasmodium species that infect humans. The general observation that episodes of relatively high parasitemia due to the different species tend to occur in succession (11, 16, 21) contributed to explain the oft-observed deficit in mixed infections and suggest such an antagonism. This notion was supported by a small number of human experimental mixed infections with P. falciparum and P. vivax infections (8, 9), as well as with P. malariae and P. vivax (26). Recent observations from Thailand revealed that cryptic P. falciparum infections are present in a significant proportion of patients presenting with patent P. vivax infections (27, 44). The possibility that the less virulent parasite species (P. vivax, P. ovale, and P. malariae) might dampen the growth of the virulent P. falciparum and thereby contribute to diminish severe pathology and mortality has important implications for malaria control strategies. An understanding of the mechanism of species-specific suppression may lead to a novel approach to control the parasite and reduce morbidity.

It would now be quite important to determine whether modulation of pathology, as seen in mixed infections in the rodent model, is also observed in malaria infections of humans, and if so to assess its magnitude. The possibility exists that measures aimed at reducing the prevalence of one parasite species alone might lead to alteration of the morbidity resulting from infection by the other species present in that area. This phenomenon could have a significant impact on the interpretation of past and future clinical observations, as well as of data from drug and vaccine trials conducted in areas of endemicity. It might be judicious to include an assessment of mixed-species infections in future epidemiological surveys and field interventions.


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ACKNOWLEDGMENTS
 
T.V. and E.B. were supported by grants from the Ministère National de la Recherche et de la Technologie, France, and T.V. was further supported by the Conseil Régional de la Martinique, France. A.M.V. held a fellowship from the Junta Nacional de Investigação Cientifica Tecnologia of Portugal. A.C.G. was in part supported by the Carlsberg Foundation (Denmark).


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FOOTNOTES
 
* Corresponding author. Mailing address: Muséum National d'Histoire Naturelle, Equipe Parasitologie Comparée et Modèles Expérimentaux USM 307, CNRS IFR 101, CP52, 61 Rue Buffon, 75231 Paris Cedex 05, France. Phone: 33-1-40-79-34-98. Fax: 33-1-40-79-34-99. E-mail: snounou{at}mnhn.fr. Back

Editor: J. F. Urban, Jr.

{dagger} I.L., G.S., and L.R. contributed equally to this study. Back


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Infection and Immunity, August 2005, p. 4777-4786, Vol. 73, No. 8
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.8.4777-4786.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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