Proc. Natl. Acad. Sci. USAVol. 91, pp. 8097-8101, August 1994Medical Sciences

Murine AIDS protects mice against experimental cerebral malaria:Down-regulation by interleukin 10 of a T-helper type 1 CD41cell-mediated pathologyMICHEL ECKWALANGA*, MYRIAM MARUSSIG*, MARISA DIAS TAVARES*t, JEAN CLAUDE BOUANGA*,ELISABETH HULIER*, JANA HENRIETTE PAVLOVITCHt, PAOLA MINOPRIO§, DENIS PORTNOi¶,LAURENT RtNIA*, AND DOMINIQUE MAZIER*II*Institut National de la Sant6 et de la Recherche Mddicale, Unite 313, D6partement de Parasitologie, 91 Boulevard de l'H6pital, 75013, Paris, France; tCentreNational de la Recherche Scientifique, 583, H6pital Necker, Paris, France; and Unite de Recherche Associde §Unitd d'Immunoparasitologie,and ILaboratory of Immunobiology, Institut Pasteur, Paris, France

Communicated by William Trager, May 5, 1994 (received for review November 5, 1993)

ABSTRACT The retrovirus LP-BM5 murine leukemia vi-rus induces murine AIDS in C57BL/6 mice that has manysimilarities with human AIDS; Plasmodium berghei ANKAcauses experimental cerebral malaria in the same strain ofmice. The outcome of malaria infection was studied in miceconcurrently infected with the two pathogens. The retrovirussignificantly reduced the gravity of the neurological manifes-tations associated with Plasmodium berghei ANKA infection.The protection against experimental cerebral malaria inducedby murine AIDS increased with duration of viral infection and,hence, with the severity of the immunodeficiency. Interleukin10, principally from splenic T cells, was shown to play a crucialrole in this protection.

Infection by human immunodeficiency virus (HIV) and en-demic malaria represent major public health problems at thepresent time in many developing countries. Since the immu-nosuppression induced by HIV is in part due to depletion ofCD4+ T lymphocytes [the cell population shown experimen-tally to be necessary for establishment of an anti-malarialimmunity (1, 2)], there was concern that it could aggrave themalaria situation. However, apart from reports of a fewisolated cases of severe malaria apparently associated withHIV-1 (3), most cross-sectional (4-9) and longitudinal (10)studies have shown that malaria is not more common inpatients with HIV-1 infection, in contrast to diseases causedby other organisms that are phylogenetically related to Plas-modium (11, 12).

Besides their role in the establishment of anti-malarialimmunity, CD41 T cells have also been described as beinginvolved in the induction of experimental cerebral malaria(ECM). Grau et al. (13), using Plasmodium berghei ANKA(PBA) in CBA/Ca mice, have proposed a model wherebyinterferon y (IFN-y) produced by T-helper type 1 (TH1)CD41 lymphocytes acts on macrophages, stimulating thetumor necrosis factor a (TNF-a) production responsible forthe pathology. Thus immunosuppression resulting from HIVinfection could interfere with the severity of neurologicalmanifestations associated with malaria. Detailed studies onconcurrent HIV and Plasmodium infections are difficult tocarry out; consequently, we used murine AIDS (MAIDS) (14,15), as a model ofhuman AIDS, and determined the outcomeof Plasmodium infection in mice immunosuppressed to var-ious degrees by the virus.The MAIDS model differs from HIV infection in a number

of ways but, like HIV, the MAIDS LP-BM5 murine leukemiavirus (MuLV) induces abnormal activation and proliferation

of B cells (16, 17), dysfunction of T and B cells (18, 19),aberrant secretion of cytokines (20, 21), and abnormal re-sponses to mitogens and antigens (22, 23), making it avaluable model of the human disease.The interactions were studied in C57BL/6 mice, a strain

that is susceptible to LP-BM5 MuLV and develops cerebralpathology when infected with PBA (24).

MATERIALS AND METHODSReagents. Rat IgG1 anti-murine interleukin (IL) 10 (JES

2A5) and isotype control rat IgG1 anti-,B-galactosidase(GL113) monoclonal antibodies (25) were prepared in theUnitd d'Immunoparasitologie. They were used at a dose of 2mg injected i.p. 1 h before experimental infection. Murinerecombinant IL-10 (Genzyme and Bachem) was injected i.p.at a dose of 1 gg at the time of experimental infection.

Mice. Female C57BL/6 mice (4-6 weeks old) were ob-tained from Charles River Breeding Laboratories.

Virus Preparation. The cell-free mixture ofLP-BM5 MuLVcontains the disease-causing defective retrovirus BM5d, anecotropic helper virus, and a mink cell focus-forming virusthat are constitutively produced by chronically infected SC-1cells. These reagents (26) were obtained through the AIDSResearch and Reference Reagent Program, Division ofAIDS, National Institute of Allergy and Infectious Diseases,National Institutes of Health (Bethesda, MD) from HerbertMorse and Janet Hartley. Freshly thawed cell-free prepara-tions of LP-BM5 MuLV (0.2 ml) containing 3.4 x 103plaque-forming units/ml on Fg 10S+L- cells assayed asdescribed (27, 28) were inoculated i.p., giving disease to100% mice; from 8 to 12 weeks after infection, mice showprogressive axillary and cervical lymph node enlargementand death occurs 14-22 weeks after infection (29).

Parasite Preparations. Blood stages of PBA were injectedinto C57BL/6 female mice and blood was taken when micepresented neurological manifestations of cerebral malaria.Blood samples were diluted in Alseveer's solution, separatedinto 1.0-ml samples containing 107 parasitized erythrocytesper ml, and preserved in liquid nitrogen. Infection wasobtained by injecting each mouse i.p. with 106 parasitizederythrocytes.

Abbreviations: MAIDS, murine acquired immunodeficiency syn-drome; MuLV, murine leukemia virus; PBA, Plasmodium bergheiANKA; HIV, human immunodeficiency virus; ECM, experimentalcerebral malaria; IFN-y, interferon--y; IL, interleukin; TNF-a, tumornecrosis factor alpha; EBV, Epstein-Barr virus; TH, T helper.tOn leave from: Hospital Universitario CFF-Universidade Federaldo Rio de Janeiro, Brazil."To whom reprint requests should be addressed.

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8098 Medical Sciences: Eckwalanga et al.

In Vivo Experiments. In the first series of experiments, 40mice were infected on day 0 with the virus preparation. PIBAwas given to groups of 10 mice on days 0, 15, 30, or 60 aftervirus infection. Controls consisted ofmice injected with PBAalone or with supernatants from noninfected cell cultures. Inthe second series of five experiments, a total of 62 mice werecoinfected at day 0 with virus and PBA preparations, and 45mice were injected with PBA alone. In a third series, (i) 20mice were infected with PBA and, from this group, 10 micewere treated with IL-10; (ii) 30 mice were coinfected withvirus and parasite, and from this group, 10 mice were treatedwith anti-IL-10 antibody and 10 received control IgG1 anti-body. This last series of experiments was repeated.

Parasitemia. Parasitemia was monitored daily by countingthe number of infected erythrocytes per 2000 erythrocytes inGiemsa-stained slides.

Detection of the Defective LP-BM5 MuLV Genome by PCR.Template DNAs were prepared by phenol/chloroform ex-traction from spleens of mice 6 days after virus inoculation(30). The PCR primers were 5'-CCTCTTCCTTTATCGA-CACT-3' and 5'-ATTAGGGGGGGAATAGCTCG-3'. Theprimers correspond to the sequences located in defectiveLP-BM5 MuLV (BM5d) gag gene, nt 1282-1301, and werecomplementary to nt 1499-1518 of the published sequences(31). Template DNAs were added to a solution containing 10mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01%gelatin, 1% Triton X-100, all four dNTPs (each at 200 AM),200 nM of each primer, and 0.5 unit of Taq polymerase(ATGC Biotechnologie) in a total volume of 50 ,l and weresubjected to 40 cycles of amplification. In each cycle ofPCR,the mixture was denatured at 95°C for 30 sec (7 min for thefirst cycle), annealed at- 55°C for 1.30 min, and extended at72°C for 1 min on a program temperature control system(Omnigene temperature cycler, Hybaid, Middlesex, U.K.).The reaction product was visualized after electrophoresis of10 ,l4 of the reaction products at 120 V for 60 min in 2%agarose in Tea buffer (40 mM Tris/20 mM sodium acetate/2mM EDTA, with the pH adjusted to 7.7 with glacial aceticacid) containing ethidium bromide (0.5 ,ug/ml). EcoRI- andAva II-digested pBR322 DNA (Bio-Rad; 0.2 ,ug) was elec-trophoresed in parallel as size markers providing bands at1746, 1434, 800, 634, 303, 279, 249, 222, 88, and 42 bp. Theprimer pair from the defective virus sequence produced aproduct of the predicted size (237 bp).

Lymphoproliferative Assay. Spleens (four per group) wereexcised aseptically on days 5, 15, 60, and 90 after viralinfection and from age-matched controls. Each spleen was

A100

80 +CUECE 60

CU

20J

0

teased apart in RPMI 1640 medium (GIBCO) supplementedwith 10% (vol/vol) heat-inactivated fetal calf serum (FCS;GIBCO), 50 ,uM 2-mercaptoethanol (Merck), penicillin (Eu-robio, Paris; 100 units/ml), streptomycin (Eurobio; 100 pg/ml), 10 ,uM L-glutamine (Eurobio), and 10 ,jM sodium pyru-vate (Eurobio). Spleen cells were filtered through a cellstrainer (Falcon, Becton Dickinson) and washed three timeswith RPMI 1640 medium (GIBCO). The pooled cells werecultured in 0.2 ml of complete RPMI medium in a 96-wellflat-bottom tissue culture plate (Costar), with or withoutconcanavaline A (Con A) (Sigma) at 2.5 pug/ml. Each test wasperformed in triplicate. After 54 h of culture (37°C in 5%C02/95% air), the cells were pulse-labeled for 18 h with[3H]thymidine (aqueous solution; 185 GBq/mmol or 5 Ci/mmol; Amersham) at 1 ,Ci per well and then processed forscintillation counting.

Characterization of IL-1O-Producing Cell Populations. One-hundred mice were infected with PBA and 50 of these micealso received the virus preparation simultaneously. Twentynaive mice were used as controls. Spleens were removed andperitoneal cells were collected on days 4 (n = 20 mice) and7 (n = 13 mice) after infection. After erythrocyte lysis, poolsof splenic or peritoneal cells (3 x 107 cells per ml) wereseeded overnight (37°C in 5% C02/95% air) on 100-mm Petridishes. Nonadherent cells were collected and processed toseparate B and T cells. B cells were obtained by "panning"on dishes coated with goat anti-mouse immunoglobulins(Nordic) at 50 pg/ml in PBS (pH 7.2). T cells were enrichedon nylon wool columns (Polysciences), washed twice, andsuspended in complete RPMI medium. Macrophages wererecovered by scraping, washed, and suspended in completeRPMI medium. Macrophage- and B-cell-enriched popula-tions (3.8 x 106 cells per ml) were stimulated with lipopoly-saccharide (Escherichia coli 0111: B4, Difco), at 10 Pg/ml for67 h (37°C in 5% C02/95% air) in 24-well flat-bottom tissueculture plates. T-cell-enriched populations (3.8 x 106 cellsper ml) were stimulated with Con A at 5 pg/ml for 67 h.Supernatants were collected and stored at -200C before theIL-10 level was determined by ELISA. Plates (Nunc) werecoated with anti-IL-10 monoclonal antibody (JES2A5). Serialdilutions of the culture supernatants were added and thebound cytokine was detected with a biotinylated rat 1gManti-IL-10 (SCX1) and peroxidase-labeled streptavidin(Kirkegaard & Perry Laboratories). Cytokine concentrationswere determined by reference to a standard curve using asreference IL-10 produced by a hybridoma transfected withmouse IL-10 gene (J558-10).

B100

0 10 20 30 0 10 20Days after infection Days after infection

30

FIG. 1. Survival ofand parasitemia in mice infected with PBA alone (A) or simultaneously with LP-BM5 MuLV (B). Nine of 10 mice infectedwith PBA alone died with parasitemias <15% before day 11 as a result of ECM. The mouse that did not present ECM manifestations died onday 28 after infection from severe anemia. Eight of 10 mice coinfected with PBA and LP-BM5 MuLV developed ECM but in four of these mice,neurological manifestations disappeared and mice died much later (of severe anemia). +, Death of one mouse.

Proc. Natl. Acad Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 8099

Table 1. Effect of LP-BM5 MuLV infection on the outcome of ECM in micePBA + LP-BM5 MuLV

ExperimentPBA 1 2 3 4 5 Total

Mice, no. 45 7 8 8 11 28 62No. with clinical signs of ECM 39 5 8 6 8 28 55No. deaths from ECM 39 (100) 2 3 2 2 16 25 (46)No. reversions 0 (0) 3 5 4 6 12 30 (55)Mice were injected with LP-BM5 MuLV and PBA malaria on the same day. All mice became infected

with PBA and details are given only of those that developed ECM. Reversion refers to the loss of allclinical signs that characterize ECM. The viral infection was verified by PCR in additional mice infectedwith the virus alone or with both the virus alone or with both the virus and PBA. The total number ofexperiments for PBA-infected mice was five; the total number of experiments for PBA- plus LP-BM5MuLV-infected mice was five. Numbers in parentheses are the percent of total of the mice with clinicalsigns of ECM.

RESULTS

Effect of Virus Infection on the Outcome of PlasmodiumInfection. When C57BL/6 mice are infected with PBA,80-100% of the mice die between days 6 and 11 afterinfection. Fig. 1A shows a typical experiment where micedied with parasitemias <15% as a result of ECM. ECM isdefined by a variety of neurological symptoms (hemiplegia,paraplegia, ataxia, convulsions, and coma) leading to deathwithin 24 h of onset. When mice were coinfected withLP-BM5 MuLV and PBA (Fig. 1B), eight developed ECM,but in four infected mice, neurological manifestations pro-gressively disappeared and were not detectable 24 h later.However, these four mice died of severe anemia and hyper-parasitemia after 20 days of infection. These results were

confirmed in five further experiments (Table 1). No effectwas obtained after injection of supernatants from noninfectedSC-1 cells (data not shown), ruling out a nonspecific antigeniccompetition.MAIDS progression is characterized by abnormal func-

tioning of CD4+ T cells, including loss of proliferative re-

sponse to mitogens and antigens (14, 22, 32). We firstconfirmed that viral infection diminished the proliferativeresponses of spleen cells to Con A since indices were

proliferative, respectively, 1.2, 0.58, 0.39, and 0.02 on days5, 15, 60, and 90 after infection.Experiments were then performed to evaluate the effect of

duration of the viral infection on the subsequent PBA infec-tion. As seen in Fig. 2, as the viral infection developed, fewermice died of ECM. Moreover, when PBA was injected into

Day of infection

LP-BM5 PBAMuLV

n

0

0

0

0

0

15

30

60

0 20 40 60 80 100% mortality from ECM

FIG. 2. Effect of the duration of the LP-BM5 MuLV infection onthe outcome of a subsequent malaria infection. Groups of 10 miceeach were infected on day 0 with the MAIDS virus LP-BM5 MuLVand challenged 0, 15, 30, or 60 days later with PBA.

mice infected with the virus for >1 month, none presentedany clinical symptoms of ECM.

Effect of IL-10 in Vivo. LP-BM5 MuLV infects B cells (33,34), macrophages (15, 20), and T cells (15, 35). These threepopulations are known to produce IL-10 (36-38), a lympho-kine that inhibits secretion ofIFN-y (37) and TNF-a (39), twocytokines implicated in development of ECM. We thusinvestigated the potential role of this cytokine in the effectsinduced by viral infection (Fig. 3). Two approaches weretaken. (i) One hour before coinfection, mice received 2 mg ofanti-IL-10 antibodies. Seventy percent of mice died fromECM. In contrast, in coinfected mice treated with IgG1control antibodies, progressive development ofthe syndromewas reversed in the same way as in nontreated coinfectedmice. (ii) Mice infected with PBA received, at the same time,1 ,ug of murine recombinant IL-10. Significantly fewer micedeveloped and died from the neurological manifestations.Treatment with IL-10 or anti-IL-10 antibody had no effect onparasitemia (data not shown).

Characterization ofIL-10-Producing Cell Populations. Sple-nic T cells from mice coinfected 4 days previously producedmore IL-10 than similar cells from PBA-infected or normalmice (Fig. 4). There were however no differences 7 days afterinfection (Fig. 4) or between peritoneal T-cell populations(data not shown). No significant differences were seen whenenriched macrophage and B-cell populations from eitherspleen or peritoneum were compared (data not shown).

DISCUSSIONFrom our results, we conclude that the retrovirus LP-BM5MuLV significantly alters the gravity of the neurologicalmanifestations associated with the PBA malaria infection.The "protection" against ECM induced by MAIDS increaseswith the severity of the immunodeficiency and IL-10 plays acrucial role in this protection. These changes are not aconsequence of an effect of the viral infection on multipli-cation of the malaria parasite.

In a recent study, Gazzinelli et al. (40) evaluated cytokineexpression oflymphocytes from C57BL/6 mice infected withLP-BM5 MuLV. During the first week of viral infection, theyfound that spleen cells from infected mice were producinghigh levels of cytokines characteristic of THO cells-i.e.,IL-2, IFN-y, IL-4, IL-5, and IL-10. After 2-4 weeks, aug-mented synthesis of IL-4, IL-5, IL-6, and IL-10 (TH2 cyto-kines) was observed, while levels of IFN-y and IL-2, theproducts of TH1 cells, were diminished. This result canexplain our observation that the capability of PBA-infectedmice to develop ECM decreases with the severity of theimmunodeficiency due to viral infection.LP-BM5 MuLV infects B cells (33, 34), macrophages (15,

20), and T cells (15, 35). These three populations are knownto produce IL-10 (36-38), a lymphokine that inhibits secre-

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8100 Medical Sciences: Eckwalanga et al.

PBA

PBA + LP-BM5 MuLV

PBA + IL-10

PBA + LP-BM5 MuLV + anti-IL-10

PBA + LP-BM5 MuLV + IgGl

A*

]**

-1l,T~ I

0 20 40 60% mortality from ECM

80 100

FIG. 3. LP-BM5 MuLV-mediated protection against ECM is dependent on IL-10. The results from two experiments were similar and areshown together with standard deviations. *, Significantly different at P = 0.0377 (unpaired t test); **, significantly different at P = 0.0395(unpaired t test).

tion of IFN-y (37) and TNF-a (39), two cytokines implicatedin the development of ECM. Consequently, we decided toinvestigate the role of IL-10 in the establishment of ECM.The experiments with murine recombinant IL-10 and mono-clonal antibody against IL-10 confirmed our prediction thatIL-10 could prevent ECM pathology. The cells implicated inIL-10 production seem to be the splenic T cells; IL-10production by the other cell types was at a much lower leveland was not enhanced in mice with the concurrent infections.However, we cannot completely exclude participation of Bcells and macrophages in IL-10 secretion in vivo.There is a well-established association between Burkitt

lymphomas, caused by Epstein-Barr virus (EBV), and ma-laria; Plasmodium infection increases the number of circulat-ing B cells carrying EBV (41). Recently, Watier et al. (42),proposed that EBV could in turn protect infected individualsfrom the pathological effects of malaria by a mechanismanalogous to the mechanism we have shown. EBV secretes

30 -

20 -

*......

-

10-

0Nive PBA

in1-.

BCRF-1, a peptide related to IL-10 that has many propertiesof IL-10 (43). In addition, lymphoblastoid cells infected withEBV secrete large quantities ofIL-10 itself. Thus natural IL-10and BCRF-1 could down-regulate the secretion of inflamma-tory cytokines, specifically, IFN-y from TH1 cells and TNF-afrom monocytes, and thus, give protection against the severeforms of Plasmodium infection, including cerebral malaria.

Pathogens like Cryptosporidium parvum (44) and Toxo-plasma gondii (45) become opportunistic microorganisms inmice concurrently infected with LP-BM5 MuLV. For thoseparasites, the TH1 subset of CD41 cells protects against theprogression of disease, while the TH2 cytokines induced by thevirus infection enhance host susceptibility (46). In contrast,cytokines produced by the THi cells are responsible for patho-genicity of PBA malaria and down-regulation of these cells asa result of the viral infection explains why the malaria infectionis not exacerbated. Though PBA malaria is always fatal in mice,the MAIDS protects against early death due to ECM.

- Con A

+ Con A

PBAday 7

PBA %lAIDS >BA '>I.. AAID Sdae A,

FIG. 4. IL-10 production by splenic T cells from mice infected 4 or 7 days previously with PBA either alone or simultaneously with the MAIDSvirus. Cells were unstimulated (solid bars) or exposed to Con A at 5 jtg/ml (hatched bars).

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 8101

Results from rodent model infections should be extrapo-lated to corresponding human diseases only with caution.However, the production of IFN-y and TNF-a in the largeamounts seen in rodent malarias also characterizes cerebralmalaria in humans (47) and secretion of IL-10 is described inHIV-infected people (48). Moreover, the opportunistic in-fections associated with HIV-1 in humans include C. parvumand T. gondii. Based on our experimental results, futurestudies on HIV and malaria should perhaps concentrate moreon modulation of malaria pathogenesis by the virus infectionrather than looking for increases in severity or prevalence ofmalaria.

We thank Pr. G. A. T. Targett and Dr. C. Leclerc for criticalreview ofthe manuscript and M. Bauza for her invaluable assistance.M.D.T. received a fellowship from Coordenqao de Aperfeigoamentode Pessoal de Nival Superior Ministerio da Educasao-Brazil. Thisresearch was supported in part by Agence Nationale pour la Re-cherche sur le Sida.

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