Toxoplasma gondii Upregulates Interleukin-12 To Prevent Plasmodiumberghei-Induced Experimental Cerebral Malaria
Erik W. Settles,a Lindsey A. Moser,a Tajie H. Harris,b Laura J. Knolla
Department of Medical Microbiology and Immunology, University of Wisconsin Medical School, Madison, Wisconsin, USAa; Department of Neuroscience, School ofMedicine, University of Virginia, Charlottesville, Virginia, USAb
A chronic infection with the parasite Toxoplasma gondii has previously been shown to protect mice against subsequent viral,bacterial, or protozoal infections. Here we have shown that a chronic T. gondii infection can prevent Plasmodium bergheiANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. Treatment with soluble T. gondii antigens (STAg) re-duced parasite sequestration and T cell infiltration in the brains of P. berghei-infected mice. Administration of STAg also pre-served blood-brain barrier function, reduced ECM symptoms, and significantly decreased mortality. STAg treatment 24 hpost-P. berghei infection led to a rapid increase in serum levels of interleukin 12 (IL-12) and gamma interferon (IFN-�). By 5days after P. berghei infection, STAg-treated mice had reduced IFN-� levels compared to those of mock-treated mice, suggestingthat reductions in IFN-� at the time of ECM onset protected against lethality. Using IL-10- and IL-12�R-deficient mice, wefound that STAg-induced protection from ECM is IL-10 independent but IL-12 dependent. Treatment of P. berghei-infectedmice with recombinant IL-12 significantly decreased parasitemia and mortality. These data suggest that IL-12, either induced bySTAg or injected as a recombinant protein, mediates protection from ECM-associated pathology potentially through early in-duction of IFN-� and reduction in parasitemia. These results highlight the importance of early IL-12 induction in protectionagainst ECM.
Cerebral malaria (CM) is a fatal neurological complication thatcan arise during Plasmodium falciparum infection (1). Hall-
marks of P. falciparum-induced CM that occur in the central ner-vous system include sequestration of parasitized erythrocytes, leu-kocytes, and platelets at the blood-brain barrier (BBB) (2–5). Evenwith current antimalaria drug treatment, progression to fatal CMremains high (2–7). Patients that survive CM infections can de-velop subsequent neurological complications (8, 9), highlightingthe need for additional treatments. Because the exact contribu-tions of immune and parasitic events to CM cannot be studied inhumans (1, 10), relevant animal models are key for developingnovel therapies.
The murine experimental cerebral malaria (ECM) model in-duced by Plasmodium berghei ANKA has many similarities to hu-man CM, which include infected red blood cell (RBC) (iRBC)sequestration at the BBB, vascular leakage, and neurologicalsymptoms (10–12). In addition, the ECM model has allowed therole of the immune response in ECM to be examined and dis-sected. Removal of immune cells, such as T or NK cells, or thecytokines gamma interferon (IFN-�) and lymphotoxin alpha(LT�) prevents P. berghei ANKA-induced ECM (13–21). Under-standing the immune response in ECM has allowed the investiga-tion of potential treatments via regulation of innate immunity(22–27). Additionally, investigating the immune response duringcoinfection, either with other Plasmodium spp. (28, 29), hel-minths (30–33), or LP-BM5, the murine leukemia virus that in-duces murine AIDS (30, 34), has allowed the identification ofimmune components necessary for protection from ECM.
Toxoplasma gondii is an obligate intracellular apicomplexanparasite that infects any warm-blooded animal. The asexual lifecycle consists of an acute systemic phase of disease with fast-rep-licating tachyzoites that transitions into a life-long chronic infec-tion of bradyzoite cysts located primarily in striated muscle andbrain tissue (35). Immune suppression of the host leads to brady-
zoite cyst activation and reversion back to the tachyzoite stage ofreplication (36). T. gondii induces a robust Th1 immune responseand stimulates innate Toll-like receptors, which leads to the pro-duction of interleukin 12 (IL-12), gamma interferon (IFN-�), anda cytotoxic T cell response (37, 38). The induction of a Th1 im-mune response does not require live parasites (39–41). T. gondiiinfection also induces the proinflammatory cytokines IL-8 andIL-12, which play a major role in clearing the tachyzoite stage andmaintaining the chronic stage (42–44). The anti-inflammatorycytokine IL-10 plays a critical role in limiting inflammation dur-ing T. gondii infection. Deletion of IL-10 results in enhancedmouse morbidity compared to that for control mice, and IL-10-dependent morbidity is reduced by IFN-� and T cell depletion(45–47).
Animals with a chronic infection of the parasite Toxoplasmagondii can survive subsequent lethal challenges with bacteria, pro-tozoa, or viruses (48–52). In addition, T. gondii chronic infectioncan reduce the number of Plasmodium yoelii blood-stage parasites(53) and prolong the survival of mice infected with P. bergheiANKA (54), suggesting a potential protective role during Plasmo-dium-induced disease. Furthermore, immunization with frozenand thawed T. gondii antigen or lysates induced an adaptive im-
Received 4 October 2013 Returned for modification 24 October 2013Accepted 31 December 2013
mune response that was protective against future P. berghei ANKAinfections (55, 56). For this study, we examined how the immuneresponse elicited by either chronic T. gondii infection or treatmentwith soluble T. gondii antigen (STAg) could inhibit P. bergheiANKA-induced ECM. We determined that IL-12 signaling plays acentral role in the reduction of P. berghei parasitemia and theprevention of P. berghei-induced ECM.
MATERIALS AND METHODST. gondii cell culture and STAg preparation. T. gondii parasites wereserially passaged on human foreskin fibroblasts (HFF) in Dulbecco’smodified Eagle medium (DMEM) supplemented with 10% fetal bovineserum, 1% penicillin-streptomycin, and 2 mM L-glutamine. STAg wasgenerated as previously described (57), using the Pru�HPT strain (58)with the following alterations. Parasites were egressed from HFF cells byreplacing the DMEM with Hanks balanced salt solution (Thermo Scien-tific) containing 1 �M calcium ionophore (Sigma) for 6 min at 37°C.Freshly egressed parasites were collected by centrifugation at 550 � g for10 min and washed twice with Dulbecco’s phosphate-buffered saline(DPBS) (128 mM NaCl, 2.7 mM KCl, 8 mM NaH2PO4, and 1 mMKH2PO4). Parasites were suspended at a concentration of 4 � 108 para-sites/ml in DPBS and sonicated. The protein mix was centrifuged at100,000 � g for 45 min, and the soluble fraction was collected, aliquoted,and stored at �80°C.
ECM model and treatments. P. berghei ANKA-induced ECM and theimmune components involved in the induction of ECM are well docu-mented. Thus, P. berghei ANKA was the preferred Plasmodium species/strain for our ECM studies. P. berghei ANKA was recovered from bloodglycerol stocks in BALB/c mice, which do not develop ECM. C57BL/6Jmice are highly susceptible to P. berghei ANKA-induced ECM, and nu-merous immune deletion mouse lines are available in this background. Toexamine ECM, experimental infections were initiated in C57BL/6J miceusing blood from P. berghei ANKA-infected BALB/c mice. BALB/c andC57BL/6J mice were purchased from NCI (Frederick, MD). IL-12�2R�/�
(B6.129S1-Il12b2tm1Jm/J) and IL-10�/� (B6.129P2-Il10tm1Cgn/J) micewere purchased from Jackson Laboratory (Bar Harbor, ME) and bred atthe University of Wisconsin—Madison. All deletion strains were in aC57BL/6J background and backcrossed at least 10 generations. All ani-mals use was approved by and in accordance with the policies of theInstitutional Animal Care and Use Committee at the University of Wis-consin—Madison.
Percent parasitemia was determined by counting 500 to 1,000 RBCfrom Giemsa-stained thin blood smears and defined as the ratio of iRBCto total RBC. The universally lethal P. berghei ANKA clone 4 (59) was usedin all experiments and was maintained as cryopreserved stabilates ofmouse blood at 10% parasitemia. Infections were initiated by intraper-itoneal (i.p.) injection with 50 �l of stabilate (106 iRBC) into BALB/cmice. At 10% parasitemia, blood was collected, and the ECM model wasinitiated by infecting age-matched C57BL/6J, IL-12�2R�/�, or IL-10�/�
mice i.p. with 1 � 106 iRBC. Doses lower than 1 � 106 iRBC producedinconsistent ECM results. Experiments with the IL-12�2R�/� mice used6-week-old mice, while all other experiments used 8-week-old mice.For STAg treatment, equivalent volumes of PBS or STAg were intrave-nously (i.v.) injected at the indicated times. Recombinant murine IL-12(p70) derived from CHO cells had endotoxin levels of 1 endotoxin unit(EU)/�g or 0.1 ng/�g of protein (PeproTech, Rocky Hill, NJ) and wasadministered i.v. at the indicated amount 1 day after P. berghei ANKAinfection. Mice were scored for ECM symptoms using a rapid behavioralscale from 1 to 18 and euthanized at scores of �5 (60). Lower scores weregiven for a loss of coordination (gait and balance) exploration (motorperformance), strength and tone (body position and limb strength), re-flexes/self-preservation (touch escape, pinna reflex, and toe pinch), andhygiene (grooming).
Assessment of BBB permeability. Mice were administered 0.1 ml of10 mg/ml Evans blue dye (MP Biomedicals) dissolved in DPBS 7 days after
P. berghei ANKA infection or when the percent parasitemia was �8%.After 1 h, the mice were sacrificed and perfused with 0.9% NaCl saline,and tissues were removed and weighed. Evans blue was extracted by im-mersing the tissue in formamide and quantified by measuring absorbanceof the formamide at 610 nm (61).
qPCR. To measure the amount of P. berghei ANKA DNA associatedwith the brain, mice were sacrificed 7 days after P. berghei infection andperfused with 0.9% saline to remove P. berghei genomic DNA present inthe circulatory system. Brain tissues were collected and minced, andgenomic DNA was purified using the High Pure PCR template purifica-tion kit (Roche) from a portion of the minced brain. Quantitative PCR(qPCR) was performed using primer-probe sets specific to mouse albu-min and P. berghei ANKA 18S rRNA and genomic DNA sequences aspreviously described (62). The previously published P. falciparum primerswere modified to detect P. berghei 18S rRNA. The primer-probe sets usedwere as follows: P. berghei ANKA 18S primers, 5=-TCAACTACGAGCGTTTTAACTGCAAC-3= and 5=-TTGGAATGATGGGAACTTAAAATCTTCCC-3=; probe, 5=-6-carboxyfluorescein (FAM) TGCCAGCAG ZENCCGCGGTAATTC Zen Iowa BlackFQ (IBKFQ). Murine albumin prim-ers were as follows: 5=-CAATCCTGAACCGTGTGTGTCT-3= and 5=-TTCATCAACTGTCAGAGCAGAGAAG-3=; probe, 5=-FAM CCAAGTGCTZEN GTAGTGGATCCCTGGTGG IBKFQ. Probes were labeled with the6-FAM fluorophore and IBKFQ double quencher (Integrated DNA Tech-nologies). Target genes were amplified using Absolute Blue QPCR mix(Thermo Scientific) using iCycler real-time PCR (Bio-Rad). Thresholdcycle (CT) values were determined automatically by the Bio-Rad software.The relative levels of P. berghei genomic 18S rRNA (PbA18SCT) were nor-malized to mouse albumin levels (murine albuminCT) using the followingequation: (Emurine albumin
CT)/(EPbA18SCT), where E is efficiency, m is the
slope of the dilution series/standard curve, and E � 10(�1/m) (63, 64). Theefficiency of amplification was calculated to be 100% for both primer sets.The P. berghei ANKA 18S primers were not able to detect murine genomicDNA (gDNA). In addition, the amplification efficiency of the P. bergheiANKA 18S region was not altered when murine gDNA was included inefficiency curves. The limit of detection was approximately 20 pg of P.berghei purified gDNA in water or murine gDNA-spiked samples.
Cell purification and flow cytometry. Brain mononuclear cells(BMNC) were isolated as previously described (47). Mice were perfusedwith 0.9% saline, and brains were collected, cut into small pieces, andfurther disrupted by passing through an 18-gauge needle. Brain tissueswere digested with 25 �g/ml collagenase/dispase (Roche) and 750 �g/mlDNase I (Roche) for 45 min at 37°C. Cells were washed and passedthrough a 70-�m cell strainer (Falcon). BMNC were purified over a 30%to 60% discontinuous Percoll gradient centrifuged at 1,000 � g for 25 minat room temperature. Cells were collected from the interface, washed, andenumerated by trypan blue stain for viability. For flow cytometry, cellswere washed with fluorescence-activated cell sorting (FACS) buffer (PBS[pH 7.4], 0.2% bovine serum albumin [BSA], and 1 mM EDTA) andincubated for 15 min with Fc block (0.1 �g/ml CD16/32; ebiosciences)prior to incubation with conjugated antibody. After antibody incubation,cells were fixed with 2% formaldehyde for 10 min. Cells were stainedwith CD3-eFluor 450, CD4-Alexa Fluor 700, and CD8 allophycocyanin(APC)-eFluor 780 (ebioscience) to monitor T cell accumulation at thebrain. Events were collected on a BD LSR II flow cytometer (BD). Com-pensation and analyses were performed using the FlowJo software pro-gram (TreeStar).
Serum cytokine quantification. Serum samples were obtained by tailbleed at the given time points. Cytokines were quantified from 12.5 �l ofserum using a mouse inflammation cytokine bead array (CBA) kit (BDBiosciences), which measures IL-12p70, IFN-�, tumor necrosis factor al-pha (TNF-�), monocyte chemoattractant protein 1 (MCP-1), IL-6, andIL-10 in the same serum sample. Events were collected and gated using theBD LSR II flow cytometer and FACSDiva software program (BD Biosci-ences).
Statistical analysis. A 2-way analysis of variance (ANOVA) analysisused to compare independent experimental repeat data sets to determineif significant variation between experiments existed and if the data couldbe merged. The dependent variables were parasitemia or time of death,and the independent variable were treatment, infection, and experiment.Kaplan-Meier survival curves were statistically compared using a Mantel-Cox log rank analysis. Log rank analysis P values was used to comparePBS-treated or untreated P. berghei ANKA-infected animals to coinfectedanimals or results with protein treatment. For single comparisons, signif-icance was determined by the Mann-Whitney test. For multiple compar-isons, data were compared by a Kruskal-Wallis test followed by Dunn’spairwise comparison. A P value of �0.05 was considered significant. Allstatistical analysis was performed in the Prism software program (Graph-Pad Software, Inc.).
RESULTSChronic T. gondii infection decreases P. berghei ANKA-in-duced morbidity and P. berghei parasitemia. To determine ifchronic T. gondii infection prevents P. berghei ANKA-induceddisease, 7- to 8-week-old C57BL/6 mice were infected with 1,000T. gondii parasites and allowed to established chronic infection for28 days. T. gondii-infected or age-matched uninfected mice werethen challenged with a lethal dose of P. berghei ANKA and moni-tored daily for ECM symptoms to determine if they needed to beeuthanized. Ninety percent of the T. gondii-infected mice survivedsubsequent infection with P. berghei, whereas P. berghei challengewas uniformly lethal in mice not infected with T. gondii (Fig. 1A)(P 0.01). Coinfection with T. gondii significantly reduced P.berghei parasitemia (Fig. 1B) (P 0.05). Without T. gondiiinfection, P. berghei parasitemia reached 20% by the time themice needed to be euthanized for ECM symptoms, whereas withchronic T. gondii infection, 95% of mice did not display ECMsymptoms despite the fact that P. berghei parasitemia increaseduntil it plateaued at 40%. These data suggest that T. gondii coin-
fection can reduce P. berghei ANKA parasitemia and prevent theonset of ECM.
To begin to elucidate the mechanism of T. gondii protection,we tested whether treatment with soluble T. gondii antigens, calledSTAg, could protect against P. berghei ANKA-induced ECM. Weinfected mice with P. berghei ANKA and administered STAg i.v. atvarious times post- P. berghei infection. Treatment of P. berghei-infected mice with STAg 1 day (P 0.001 compared to resultswith PBS treatment) or 2 days (P 0.01 compared to results withPBS treatment) after P. berghei infection resulted in �90% sur-vival, but ECM was not prevented when mice were treated withSTAg at 3 or 4 days post- P. berghei infection (Fig. 1C). STAgtreatment 1 day after P. berghei infection significantly reducedparasitemia when it was measured 6 days after P. berghei infection(P 0.01); however, parasitemia was not reduced when STAgtreatment occurred 2 days after infection even though the micewere protected from ECM-induced disease (Fig. 1D). In addition,the STAg treatments on day 1 and to a lesser extent on day 2reduced the level of parasitemia until 8 days after P. berghei infec-tion (Fig. 1E), a time frame similar to that seen for P. bergheiANKA infection of mice with a chronic T. gondii infection (Fig.1B). Treatment with a lysate of the host cells from which STAg wasprepared did not prevent ECM or reduce parasitemia (data notshown). These data show that post- P. berghei infection, treatmentwith STAg can mimic T. gondii chronic infection and prevent P.berghei-induced ECM with or without reduction of P. berghei par-asitemia.
STAg reduces P. berghei ANKA-induced vascular leakageand parasite sequestration in the brain. Breakdown of the BBBand vascular leakage have been associated with CM in humans andECM in mice (31, 65–67). To determine if STAg treatment couldprevent this hallmark of P. berghei ANKA-induced ECM, we used
FIG 1 Chronic T. gondii infection or STAg prevents P. berghei ANKA (PbA)-induced ECM and lowers parasitemia. (A and B) Seven- or eight-week-old C57BL/6mice inoculated with T. gondii (Tg PbA) (n � 6) or medium (PbA) (n � 7) were challenged 28 days later with 1 � 106 P. berghei ANKA iRBC. Symptoms of ECMwere monitored, and mice with scores � 5 were euthanized (A). Parasitemia was determined from Giemsa-stained thin blood smears at various timespostinfection (B). Data shown are from a representative experiment repeated twice. (C to E) Approximately 8-week-old C57BL/6 mice were inoculated with 1 �106 iRBC and treated with PBS or STAg at days 1 (D1), 2 (D2), 3 (D3), or 4 (D4) post-P. berghei ANKA infection. The average data from two to five experimentsare shown. (C) Symptoms of ECM were monitored, and mice with scores � 5 were euthanized up to 17 days after P. berghei infection. (D and E) Thin bloodsmears were prepared at 6 days (D) or at the indicated days (E) after P. berghei infection. The average percentage of iRBC was determined after treatment withGiemsa stain. Survival significance was determined by Mantel-Cox log rank analysis comparing PBS or P. berghei ANKA alone to coinfection or STAg treatments.iRBC significance was determined with a Kruskal-Wallis test by comparing results for P. berghei ANKA alone or P. berghei ANKA plus PBS controls (PbA PBS)to those for coinfection or STAg treatments (PbA/STAg). �, P 0.05, ��, P 0.01; ���, P 0.001.
T. gondii-Induced IL-12 Averts Murine Cerebral Malaria
Evans blue dye as an indicator of vascular leakage and BBB integ-rity (68). Mice were infected with P. berghei ANKA and treatedwith STAg 2 days postinfection to allow for similar parasitemia ininfected mice. At 7 days post- P. berghei infection, PBS-treatedmice showed vascular leakage of Evans blue into the brain tissue,whereas STAg-treated mice had reduced infiltration of dye intothe brain, similar to results for naive mice (Fig. 2A). The reducedinfiltration of dye was confirmed by quantifying Evans blue accu-mulation in brain tissue (Fig. 2B) (P 0.05). As a control, we alsomeasured vascular leakage into the mouse brain once their para-sitemia was greater than 8% (Fig. 2C). Yet again, the accumulationof dye was significantly decreased by STAg treatment (P 0.01compared to results with PBS treatment). Histological examina-tion of mouse brains detected vascular hemorrhage in both STAg-and PBS-treated mice, with a reduced hemorrhage size and fre-quency in brains of STAg-treated mice (Fig. 2D). The hemorrhagefrequency and size were further reduced if mice were treated withSTAg 1 day after infection (data not shown). These results suggestthat STAg treatment of P. berghei ANKA-infected mice can pre-serve BBB integrity.
Sequestration of iRBC and T cells in the brains of P. bergheiANKA-infected mice is required to induce ECM (15, 18, 20, 31,65, 69). To determine if STAg treatment affected the ability ofparasites to sequester in the brains of infected mice, the presence
of P. berghei ANKA genomes in brain tissue was quantified byqPCR after intracardiac perfusion. Analysis of the brains fromnaive mice showed no P. berghei ANKA genomic DNA (data notshown). P. berghei genomic DNA was 4-fold less after STAgtreatment than was seen for PBS-treated mice (Fig. 2E) (P 0.01),even though the parasitemia was similar when the tissues werecollected (data not shown). STAg treatment did not completelyeliminate the sequestration of parasite genomes compared to thatfor naive animals, which suggests that STAg treatment reduces butdoes not eliminate the sequestration of iRBC in the brains of P.berghei ANKA-infected animals.
STAg reduces P. berghei ANKA-induced brain T cell localiza-tion and late levels of IFN-�. Sequestration of parasites in thebrains of P. berghei ANKA-infected mice is enhanced by the pres-ence of CD8 and CD4 T cells in the brain (31, 65, 70). Due tothe reduction of parasites in the brains of STAg-treated mice, wehypothesized that STAg treatment would also reduce P. berghei-induced T cell accumulation in the brain. To this end, we moni-tored the numbers of CD4 and CD8 T cells in the brains byFACS analysis in naive mice, P. berghei-infected mice treated withSTAg or PBS, or uninfected mice treated with STAg (Fig. 3A).Quantification of this FACS analysis showed that P. berghei ANKAinfection increased the number of CD4 and CD8 T cells local-ized to the brain compared to results for naive animals or with
FIG 2 STAg treatment of P. berghei ANKA (PbA)-infected mice maintains BBB integrity and reduces parasite sequestration. Approximately 8-week-old C57BL/6mice were inoculated with 1 � 106 iRBC or PBS and then treated with PBS or STAg 2 days after infection. The percentage of iRBC was monitored after Giemsastain treatment of thin blood smears. At 7 days postinfection (A, B, D, and E) or once parasitemia exceeded 8% (C), mice were injected with Evans blue dye (Ato C and E). The mice were euthanized and perfused with saline, and the brains were collected, weighed, photographed, or processed for histology. (B and C)Evans blue was extracted from the tissue using formamide. The concentration of Evans blue per gram of brain tissue (�g of EB/g of brain) was determined byabsorbance. Each symbol represents an individual mouse. (E) Brains from P. berghei ANKA-infected mice were collected, and genomic DNA was prepared. Thenumbers of P. berghei 18S and mouse albumin genomic DNA copies were determined by qPCR. The number of P. berghei copies was normalized to mousealbumin. Each symbol represents an individual mouse. Significance was determined by a Mann-Whitney test. �, P 0.05. Shown are results from a representativeexperiment repeated two times.
STAg treatment alone, and STAg treatment of P. berghei-infectedmice reduced brain-localized CD4 and CD8 T cells comparedto results for PBS treatment (Fig. 3B and C). Although STAg treat-ment did not result in significant reductions in brain-localized Tcells, the general reduction suggests that STAg treatment during P.berghei infection reduces T cell localization during P. berghei in-fection, which may in turn reduce damage to the brain vascula-ture.
The cytokine IFN-� has been implicated in T cell-inducedECM pathology (19, 71, 72). Because mice deleted for the IFN-�receptor chain (IFN-��/�) do not develop ECM (19, 71, 72), wemeasured serum cytokine levels of IFN-� at 7 days post-P. bergheiANKA infection (5 days posttreatment), which is just prior to theonset of ECM symptoms. STAg-treated mice had lower levels ofIFN-� than PBS-treated animals (Fig. 3D). In combination with Tcell reductions, these data suggest that STAg treatment may lowerT cell localization by reducing late IFN-� production, similar toresults seen in IFN-��/� mice.
Analysis of early cytokine response. STAg is known to inducea strong Th1 response that includes initial induction of interleukin12 (IL-12), which in turn stimulates IFN-� production (73, 74).To confirm that STAg treatment increased Th1 cytokine produc-tion and to understand how STAg treatment was affecting theearly stages of P. berghei ANKA infection, we measured serumlevels of IFN-�, IL-12p70, IL-10, monocyte chemoattractant pro-tein 1 (MCP-1), and IL-6 by CBA. Mice were infected with P.berghei ANKA and treated with STAg or PBS 1 day post-P. bergheiinfection, and serum cytokines were measured at 2 h, 14 h, and 4days after treatment. Two hours after STAg treatment, P. berghei-
infected mice had increases in IL-12p70, MCP-1, and IL-6 com-pared to levels for P. berghei-infected mice treated with PBS (Fig.4). By 14 h after STAg treatment, IFN-� had also increased. Incontrast, STAg increased serum levels of IL-10 and TNF-� by 14 hafter infection, but this elevation was not seen in STAg-treated P.berghei-infected animals. By 4 days after treatment, STAg treat-ment reduced the level of IFN-� compared that for to PBS-treatedanimals, similar to the results day 7 after infection. However, thelevels of IL-12, IL-10, IL-6, MCP-1, or TNF-� were not different.These results suggest that STAg treatment of P. berghei-infectedmice stimulates a rapid Th1 response that can reduce parasitemiaand subsequent ECM.
STAg-induced protection requires IL-12 but not IL-10. IL-10suppresses a Th1 response and has been shown in multiple studiesto play a major role in preventing ECM (28, 33, 34, 75). To inves-tigate whether STAg-induced protection against ECM requiresIL-10, we infected IL-10�/� mice with P. berghei ANKA andtreated mice with STAg after 24 h. STAg treatment was just aseffective in wild-type (wt) and IL-10�/� mice, as evidenced bysimilar percent survivals (Fig. 5A) (P 0.001 when results for PBSwere compared with those for STAg treatment within each geno-type) and parasitemias (Fig. 5B) (P 0.01), suggesting that IL-10is not required for STAg-induced protection.
Analysis of the STAg-induced cytokine response showed a sub-stantial induction of IL-12 (Fig. 4). To understand the contribu-tion of IL-12 to STAg-induced protection from ECM, we infectedIL-12�R�/� mice with P. berghei ANKA and monitored mice forECM symptoms after STAg or PBS treatment. In contrast to re-sults for the IL-10�/� mice, STAg treatment did not prevent P.
FIG 3 STAg reduces brain-localized T cells and late IFN-� production. Approximately 8-week-old C57BL/6 mice were uninfected and treated with PBS (naive)or STAg (STAg) or inoculated with 1 � 106 P. berghei ANKA iRBC and treated with PBS (PbA/PBS) or STAg (PbA/STAg) 2 days post-P. berghei infection. At 6days post-P. berghei infection, mice were euthanized and perfused, and brain tissue was collected. Brain mononuclear cells were isolated, and fluorescence-conjugated antibodies were used to detect CD3 CD8 or CD3 CD4 cells. (A) Representative CD4 and CD8 gates are shown from two experiments. Thetotal number of brain CD8 (B) or CD4 (C) cells was determined. Each symbol represents an individual mouse. (D) Whole blood was collected 7 days afterinfection, and serum IFN-� levels were quantitated by CBA (n � 6 to 7 for P. berghei-infected mice, and n � 4 for noninfected mice). Significance was determinedby a Kruskal-Wallis test, but no comparisons were statistically significant.
T. gondii-Induced IL-12 Averts Murine Cerebral Malaria
berghei-induced ECM (Fig. 5C) or reduce and delay parasitemia(Fig. 5D; see also Fig. S1A in the supplemental material) in IL-12�2R�/� mice. Thus, STAg-induced protection from ECM re-quires IL-12. To test whether IL-12 alone was sufficient to preventP. berghei ANKA-induced ECM, we treated with increasingamounts of recombinant murine IL-12p70 24 h after P. berghei
infection and monitored for ECM symptoms. Murine IL-12p70 isa 75-kDa heterodimeric glycoprotein consisting of disulfide-linked 35-kDa and 40-kDa subunits. While mice that received lessthan 0.1 �g of recombinant IL-12p70 succumbed to infection atrates similar to those for PBS-treated animals, 50% of mice thatreceived 0.1 �g and 100% of mice that received 1 �g (P 0.01
FIG 4 STAg treatment increases serum cytokine levels. Approximately 8-week-old C57BL/6 mice were uninfected or inoculated with 1 � 106 P. berghei ANKAiRBC and treated with PBS or STAg 1 day post-P. berghei infection. Blood was collected at 2 or 14 h after treatment, and serum cytokine levels of IL-12p70, IFN-�,MCP-1, IL-6, TNF-�, and IL-10 were quantitated by CBA (n � 3 per time point). A representative of two experiments is shown. Significance was determined bya Kruskal-Wallis test, but no comparisons were statistically significant.
FIG 5 IL-12 but not IL-10 contributes to STAg-induced protection. Approximately 8-week-old C57BL/6 wt or IL-10�/� (A and B) or 6-week-old wt orIL-12�R�/� (C and D) mice were inoculated with 1 � 106 iRBCs and treated with PBS or STAg 24 h later. (E and F) wt C57BL/6 mice were inoculated with 1 �106 iRBCs and treated with increasing amounts of recombinant purified IL-12p70 at 24 h postinfection. Symptoms of ECM were monitored, and mice with scores� 5 were euthanized (A, C, and E). Thin blood smears were prepared 6 days after P. berghei ANKA infection, and the percentage of iRBC was determined aftertreatment with Giemsa stain (B, D, and F). Survival significance was determined by Mantel-Cox log rank analysis comparing PBS or P. berghei alone tocoinfection or STAg treatments. iRBC significance was determined by a Kruskal-Wallis test. �, P 0.05; ��, P 0.01; ���, P 0.001. Two independentexperiments are shown in panels A to C, and a representative experiment is shown for panels D to F.
compared to results for PBS treatment) of recombinant IL-12p70survived P. berghei infection (Fig. 5E). Treatments with 1 �g and0.1 �g of IL-12 showed significant reductions in parasitemia at 6days post-P. berghei ANKA infection compared to that for PBS-treated mice (Fig. 5F). In addition, these two doses of IL-12 de-layed the accumulation of parasitemia, similar to results seen dur-ing stag treatment (see Fig. S1B in the supplemental material).Contaminating endotoxins, including lipopolysaccharide (LPS),were below the limit of detection of 0.1 ng/�g of protein, or0.01 ng endotoxin per treatment as reported by the manufac-turer. These results demonstrate that exogenous IL-12p70 is suf-ficient to prevent P. berghei ANKA-induced ECM.
To further investigate the signaling cascade induced by IL-12treatment of a P. berghei ANKA infection and how it compares toresults with STAg treatment, we treated P. berghei ANKA-infectedmice 1 day after infection with STAg or 1 �g IL-12 and comparedthe levels of serum cytokines at 2 h, 14 h, and 5 days after treatment(Fig. 6). Importantly, STAg induced concentrations of IL-12 inserum similar to those with IL-12 treatment alone, which suggeststhat the 1-�g dose was biologically relevant. STAg and IL-12 treat-ments both induced IFN-� and MCP-1, which suggests that theinduction of IL-12 by STAg induces the downstream productionof IFN-� and MCP-1 and that these cytokines can contribute toSTAg-induced protection.
DISCUSSION
These studies have shown that T. gondii chronic infection and thesubset of soluble T. gondii antigens called STAg can lessen thedisease induced by Plasmodium species in rodents. Specifically, asingle STAg treatment protected mice against P. berghei ANKA-induced ECM symptoms by reducing parasite sequestration and Tcell accumulation in the brains. Early induction of Th1 cytokineslikely lead to decreases in overall parasite levels in the blood, aswell as reductions in IFN-� levels at the time ECM symptoms arenormally induced. Previous studies have shown that STAg is apotent inducer of IL-12 responses in vivo (74). In this study, we
showed that STAg treatment is not effective at preventing ECMsymptoms in IL-12�2R�/� mice and that treatment with recom-binant IL-12 alone was sufficient to reduce parasitemia and pre-vent ECM. These results stress the role of IL-12 in the develop-ment of ECM.
To determine how STAg prevented P. berghei ANKA-inducedECM, we evaluated the role of STAg in BBB permeability anddiscovered that the permeability induced by P. berghei infectionwas reduced by STAg treatment. Consistent with this result, wefound that STAg treatment reduced the level of P. berghei genomesin the brain after perfusion, suggesting that STAg treatment re-duced parasite sequestration. This reduction in parasite sequestra-tion is especially relevant because mice treated with STAg on day 2postinfection do not show a reduction in parasitemia compared tocontrols (Fig. 1D). It is possible that STAg directly influences par-asite cytoadherence. Binding of P. berghei iRBCs to brain vascularendothelial cells is dependent on vascular cell adhesion protein 1(VCAM-1) (76). Our future studies will examine the effect ofSTAg on VCAM-1 levels in brain vascular endothelial cells and ifSTAg blocks cytoadherence of P. berghei ANKA.
While STAg treatment did not significantly reduce T cell local-ization in the brain, there is a trend toward reduction in brain-localized T cells. Perforin and granzyme B produced by CD8 Tcells are major contributors to the disruption of BBB integrityduring P. berghei ANKA infection (18, 77). The reduced permea-bility observed upon STAg treatment may result from the limitedpresence of these enzymes due to the STAg-dependent decrease inthe numbers of CD8 T cells in the brain. The reduction of local-ized T cells also correlated with reduced serum IFN-� levels.IFN-� contributes to the localization of T cells to the brain duringP. berghei ANKA infection (71, 78, 79). Thus, reductions in IFN-�levels following STAg treatment may contribute to the preventionof ECM symptoms by reducing the number of T cells localized tothe brain during P. berghei infection.
Knowing that STAg is a strong inducer of IL-12 (74), we inves-tigated the role of IL-12 in ECM. STAg treatment of P. berghei
FIG 6 STAg treatment increases serum cytokine levels. Approximately 8-week-old C57BL/6 mice were uninfected or inoculated with 1 � 106 P. berghei ANKAiRBC and treated with PBS or STAg 1 day post-P. berghei infection. Blood was collected at 2 h, 14 h, or 5 days after treatment, and serum cytokine levels ofIL-12p70, IFN-�, MCP-1, IL-6, TNF-�, and IL-10 were quantitated by CBA (n � 2 to 4 per time point). Significance was determined by a Kruskal-Wallis testfollowed by a Dunn’s pairwise comparison. �, P 0.05; ��, P 0.01.
T. gondii-Induced IL-12 Averts Murine Cerebral Malaria
ANKA-infected IL-12�R�/� mice did not prevent P. bergheiANKA-induced ECM. While the IL-12 receptor has been reportedto be required for the induction of ECM (13), we found that IL-12�2R�/� mice are susceptible to P. berghei ANKA-induced ECMat 6 weeks of age. When infected with P. berghei after 7 weeks ofage, IL-12�R�/� mice were resistant to ECM induction. Why theIL-12�R�/� mice become resistant after 7 weeks of age is unclear,because other IL-12 components and receptors can be deletedwith an ECM response still being produced (13). Using 6-week-old IL-12�R�/� mice, we showed that STAg-induced protectionfrom ECM is dependent on IL-12 signaling. Treatment of P. ber-ghei ANKA-infected mice with recombinant IL-12 preventedECM symptoms, lethality, and reduced parasitemia. STAg andIL-12 treatment alone induced similar concentrations of IL-12 inserum, suggesting that the 1-�g dose determined to be 100% ef-fective (Fig. 5E) was appropriate. Along with IFN-�, both STAgand IL-12 treatments induced early MCP-1 production, suggest-ing that these cytokines likely contribute to STAg-induced protec-tion. Other cytokines, such as TNF-�, are highly induced only inresponse to IL-12 treatments and thus are unlikely to be essentialfor STAg-induced prevention of ECM symptoms and reduction ofparasitemia.
While the importance of IL-12 for the prevention of P. bergheiANKA-induced ECM is clear, the precise mechanism of action isnot fully understood. IL-12 induces downstream cytokines thatmediate the immune response during Plasmodium infection.IL-12 induces IL-10 production (80), and IL-10 plays a major rolein preventing ECM (28, 33, 34, 76). Due to the immunosuppres-sive nature of IL-10, the induction of IL-10 could have preventedthe localization of T cells to the brain. However, STAg treatmentdid not change serum levels of IL-10 at any of the time points weexamined, and STAg treatment was still protective in IL-10�/�
mice. Thus, IL-10 was not a major contributor to STAg-inducedprotection.
Early induction of IL-12 stimulates IFN-� production (73, 74),and IFN-� was observed in response to STAg treatment (Fig. 4 and6). IL-12-induced IFN-� plays a major role in protecting againstblood-stage parasite replication during Plasmodium chabaudi in-fection as well as that with human malaria parasites (81–83). Wetried to prevent ECM with a wide dose range of exogenous IFN-�treatments (0.001 to 1 �g/mouse) without success (data notshown). IFN-� levels were higher in STAg- than in PBS-treatedmice 14 h after treatment; however, this trend reverses by 4 daysposttreatment, when IFN-� levels were higher in PBS- than inSTAg-treated mice (Fig. 3D and 4). Furthermore, we see that theTh1 cytokine response remains low in P. berghei ANKA-infectedPBS-treated mice for at least the first 38 h postinfection (Fig. 4).The correct source and/or tissue concentration of IFN-� may becritical in order to reduce parasitemia and prevent ECM, and thismay explain why i.v. inoculation of IFN-� was not successful. Incontrast, bloodstream inoculation of IL-12 is able to recapitulatethe systemic response induced by STAg. IL-12 may induce anadditional factor, such as the chemokine MCP-1, which may be nec-essary either alone or in combination with IFN-� to protect againstECM by decreasing parasitemia or directing immune cell localiza-tion. This factor may not be induced by exogenous IFN-� and thusmay account for the lack of protection with IFN-� treatment.
STAg has been shown to induce paralysis of IL-12 productionfrom dendritic cells (DCs), which protects animals from lethalparasite-induced immunopathology (84). In isolated spleen cells,
IL-12 production in response to STAg is transient, with peak pro-duction 6 to 12 h poststimulation and a return to baseline levels by24 h (74). We see similar results for IL-12 stimulation in mice afterSTAg treatment with or without P. berghei ANKA infection (Fig. 4and 6). STAg also induces systemic paralysis of IL-12 productionfrom DCs in vivo, since stimulation with a second dose of STAg 24h after the first does not induce IL-12 production from DCs (84).DC paralysis upon STAg reinjection has been connected to thefunctional downregulation of the chemokine receptor CCR5 byendogenously produced lipoxin A4 (85, 86). Because mice defi-cient in the enzyme responsible for lipoxin A4 synthesis (5-LO�/�) are unable to induce DC paralysis, future studies of theIL-12 signaling cascades during P. berghei ANKA infection willexamine STAg protection in 5-LO�/� mice. Likewise, becauseSTAg contains cyclophilin 18, a chemokine mimic that signalsthrough CCR5 (87), future studies will examine the ability of pu-rified cyclophilin 18 to protect against P. berghei-induced ECM.Together, these studies will further elucidate the role of IL-12 inSTAg-mediated protection from ECM.
ACKNOWLEDGMENTS
We sincerely thank Bill Weidanz for the P. berghei ANKA strain used inthese studies and Chris Hunter and Kasturi Haldar for training in brain-specific flow cytometry and the ECM rapid behavioral screen, respec-tively.
This research was supported by the American Heart Association,0840059N (to L.J.K.) and 5 T32 HL007899 (to E.W.S.), and a BurroughsWellcome travel grant (to E.W.S.).
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T. gondii-Induced IL-12 Averts Murine Cerebral Malaria
