INFECTION AND IMMUNITY,0019-9567/99/$04.00!0

Sept. 1999, p. 4939–4944 Vol. 67, No. 9

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Coinfection with Toxoplasma gondii Inhibits Antigen-Specific Th2Immune Responses, Tissue Inflammation, and Parasitism

in BALB/c Mice Infected with Leishmania majorH. C. SANTIAGO,1 M. A. P. OLIVEIRA,1 E. A. BAMBIRRA,2 A. M. C. FARIA,1

L. C. C. AFONSO,3 L. Q. VIEIRA,1 AND R. T. GAZZINELLI1,4*Department of Biochemistry and Immunology—ICB1 and Department of Pathologic Anatomy—Medical School,2

Federal University of Minas Gerais, 31270-910 Belo Horizonte, Centro de Pesquisas Rene Rachou,FIOCRUZ, 30190-002 Belo Horizonte,4 and Department of Biological Sciences—NUPEB,

Federal University of Ouro Preto, 35400-000 Ouro Preto,3 Minas Gerais, Brazil

Received 15 March 1999/Returned for modification 16 April 1999/Accepted 9 June 1999

Lesion size, cellular infiltration, and tissue parasitism in the footpads of BALB/c mice infected withLeishmania major were all dramatically inhibited during acute but not chronic infection with Toxoplasma gondii.Similarly, acute but not chronic toxoplasmosis at the time of infection with L. major had a strong inhibitoryeffect on development of acquired immune responses mediated by Th2 lymphocytes. In contrast, no majorchanges in Leishmania-specific Th1-mediated responses were observed in mice coinfected with T. gondii.

Infection with Leishmania major, an obligatory intracellularparasite in mammals that multiplies in macrophages (21), leadsto highly polarized Th1 and Th2 immune responses in resistant(C57BL/6) and susceptible (BALB/c) mice, respectively (26).The resistance is determined by the host’s ability to producehigh levels of gamma interferon (IFN-") and low levels ofinterleukin 4 (IL-4), culminating in the release of reactivenitrogen intermediates that are highly effective in controllingLeishmania replication (10, 12, 28, 30, 32). In contrast, T cellsfrom susceptible mice fail to produce high levels of IFN-" andreactive nitrogen intermediate release by macrophages, allow-ing the spread of the parasite and leading to visceralization anddeath. This difference in the immune response and diseaseoutcome has been attributed to the fact that C57BL/6 andBALB/c mice bias their immune responses to Th1 and Th2,respectively (15).

In contrast, infection with the intracellular protozoan Toxo-plasma gondii, which can infect and replicate in any nucleatedcell from the vertebrate host, induces a highly polarized Th1response, independent of the host genetic background (e.g.,BALB/c and C57BL/6) (1). The ability of T. gondii to trigger aTh1 response, instead of the expected Th2 response, in theBALB/c mouse can be attributed to the ability of this parasiteto trigger the synthesis of overwhelming levels of IL-12 andIFN-" during early stages of infection (7, 8). Thus, for the earlycontainment of parasite replication, the IFN-" synthesis (ini-tially by NK cells and later by Th1 lymphocytes) leading tomacrophage activation is crucial (11).

In the present study, we show that susceptible BALB/c micepreviously infected with T. gondii become protected againstlesion development in the footpad from infection with L. ma-jor. The basis of this T. gondii-induced protection was evalu-ated. As shown in Fig. 1A, animals infected with L. major (106

promastigotes) alone developed intense footpad swelling, andall had to be sacrificed at 7 weeks of infection, due to the sizeof the footpad lesion. Interestingly, infection with T. gondii 5

days prior to challenge with L. major resulted in protectionagainst lesion development in BALB/c mice. These resultswere similar to the ones observed in C57BL/6-resistant mice(Fig. 1A, insert). Even at 16 weeks postinfection, the duallyinfected animals showed no major lesions in the footpads in-fected with L. major. In contrast, we found normal develop-ment of lesions when BALB/c mice were coinfected with L.major during chronic toxoplasmosis (Fig. 1B). Furthermore,when T. gondii cysts were given 2 weeks after infection with L.major, we observed only a small delay in footpad lesion devel-opment induced by L. major infection (Fig. 1C).

The results presented in Fig. 2 illustrate the tissue pathologyobserved in mice infected with L. major alone (Fig. 2A and C)or infected with L. major 5 days postinfection with the ME-49strain of T. gondii (Fig. 2B and D). BALB/c mice infected withL. major alone showed an intense diffuse inflammatory infil-trate, tissue necrosis, ulceration areas, abscess formation (Fig.2A) and intense tissue parasitism (Fig. 2C) in their footpads. Incontrast, as observed in C57BL/6 mice infected with L. majoralone (data not shown), dually infected mice showed a smallinflammatory infiltrate with little tissue destruction, well-de-limited nodules (Fig. 2B) with lymphocytes, plasma cells, fewpolymorphonuclear cells, many noninfected macrophages, andfew infected ones (Fig. 2D). No necrotic areas, ulceration, orabscess formation was present in the latter group.

In order to measure the tissue parasitism, we used as atemplate in PCR, DNA extracted from the footpads of animalsinfected with L. major. The PCR was performed in a volume of25 #l containing GP63 (22) primers 5$ GTGCGCACGTGAACTGG 3$ (sense, nucleotides 499 to 517) and 5$ CACCCGCAGTAGTTGTAG 3$ (antisense, nucleotides 917 to 934) anddifferent concentrations of footpad DNA, as indicated in theright panel of Fig. 3. The PCR was performed with a 30-cycleprogram, and the product was electrophoresed in a 6% poly-acrylamide gel developed by silver staining (23). PCR specificfor L. major GP-63 DNA (Fig. 3) showed a small difference oftissue parasitism in footpads from mice infected only with L.major and mice chronically infected with T. gondii and coin-fected with L. major. Our PCR yielded 100 to 1,000 times lessGP63-specific product, when DNA extracted from footpad tis-sue of BALB/c mice infected with L. major 5 days after infec-

* Corresponding author. Mailing address: Department of Biochem-istry and Immunology, Federal University of Minas Gerais, 31270-901Belo Horizonte, MG, Brazil. Phone: 031 295 3566. Fax: 031 295 3115.E-mail: ritoga@mono.icb.ufmg.br.

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tion with T. gondii was used as a template, compared with thatfrom BALB/c mice infected with L. major alone.

In order to compare the immune responses in mice infectedwith L. major alone versus those in dually infected animals,lymph node and spleen cells from mice were collected at 7weeks postinfection with L. major, and IL-4 and IFN-" re-sponses were evaluated after lymphocyte stimulation with sol-uble tachyzoite antigens (STAg) (5), Leishmania antigens(LAg) (31), or mitogen (concanavalin A [ConA]). Our ex vivoexperiments show that lymph node but not spleen cells fromanimals infected with L. major alone produced high levels ofIL-4 after stimulation with LAg (Fig. 4). Interestingly, STAgtriggered the synthesis of IL-4 by lymph node cells from miceinfected only with L. major. Denkers et al. (2) have shown asuperantigen activity in STAg. Thus, we assume that STAg isactivating, in a nonspecific manner, T cells from BALB/c micethat have differentiated into Th2 lymphocytes after infectionwith L. major. Infection with T. gondii 5 days prior to infectionwith L. major had a major modulatory activity on IL-4 synthesisinduced by parasite antigens (Fig. 4B) or mitogen (Fig. 4B andD). Intriguingly, coinfection with T. gondii did not result in acorresponding enhancement of IFN-" synthesis by lymph nodeor spleen cells from mice infected with L. major (Fig. 4A andC).

Experiments were also performed to analyze the in vivoeffects of T. gondii infection on L. major-elicited humoral im-mune responses. STAg- and LAg-specific total immunoglobu-lin G (IgG) or IgG1 and IgG2a isotypes in sera of animalsinfected with L. major and/or T. gondii were measured aspreviously described (3). Our results support the conclusion ofour previous experiments by showing that coinfection with T.gondii had a major inhibitory effect on the in vivo synthesis ofLeishmania-specific IgG (Fig. 5A). More importantly, the re-sults presented in Fig. 5B indicate that most of the inhibitoryactivity on Leishmania-specific IgG is due to inhibition of Igfrom the IgG1 isotype, which is stimulated by IL-4. Again, noenhancement of Leishmania-specific IgG2a, an IgG isotypedriven by Th1 lymphocytes, was observed.

Different studies have demonstrated that infection with T.gondii results in protection against different nonrelated patho-gens, such as parasites (19, 20), bacteria (27), and viruses (6),as well as development of certain types of tumor cells (14). In

most of these studies, it is suggested that activation of cellsfrom an innate immune system, such as macrophages, ratherthan induction of cross-reactive immunity is responsible for theprotective activity elicited by T. gondii infection.

In addition to leading to microbiostatic or microbicidal ac-tivity of macrophages, different studies have suggested that thisearly activation of the innate system has an important role indirecting the differentiation of Th precursor cells to the Th1phenotype (4, 16). However, this question has been difficult toevaluate during infection with T. gondii, because the two cyto-kines IL-12 and IFN-", which are crucial for driving T-celldifferentiation to the Th1 phenotype, are also essential ele-ments in resistance to T. gondii. Thus, in the absence of en-dogenous IL-12 or IFN-", animals infected with T. gondii suc-cumb to infection in approximately 9 days (8), before theprocess of T-cell differentiation is completed.

In contrast, infection with L. major is an extremely interest-ing model with which to study T-cell differentiation (12, 16, 17,30). First, L. major is a less virulent parasite and will take amuch longer time to cause pathology and lethality, in theabsence of endogenous IL-12 and IFN-" (13). Second, thesynthesis of IL-12 and/or IFN-" is not high enough (or fastenough) to change the tendency of T cells from BALB/c miceto differentiate into Th2 cells. Interestingly, our results showthat infection with T. gondii 5 days prior L. major infectionmakes BALB/c mice highly resistant to the latter parasite. Amajor question raised by these results is related to the mech-anism of protection against L. major observed in mice coin-fected with T. gondii. A major argument against cross-reactiveprotection is the fact that no protection was observed whenBALB/c mice chronically infected with T. gondii were chal-lenged with L. major. Furthermore, little or no cross-reactivitywas observed when parasite antigens were used to stimulate Tcells as well as to measure the levels of antiparasite antibodyresponses in serum.

In our previous studies (8, 9), we have shown that the peakof IL-12 and IFN-" synthesis during acute infection with T.gondii occurs at approximately 5 to 8 days postinfection. There-fore, it is tempting to speculate that the protective effect of T.gondii infection is probably related to the overwhelming levelsof IL-12 and IFN-" synthesis during acute toxoplasmosis. It isnoteworthy that the protective effect of T. gondii infection

FIG. 1. Course of lesion development in BALB/c mice infected with L. major (circles) or with both L. major and T. gondii (squares). The animals were infectedintraperitoneally with 20 cysts of the ME-49 strain derived from the brains of C57BL/6 mice chronically infected with T. gondii and challenged in the hind footpads with106 stationary forms of the WHO MHOM/80/Friedlin strain of L. major during acute (A) or chronic (B) toxoplasmosis. (C) Mice were previously infected with L. majorand, after 14 days, were challenged with T. gondii. The animals in the different experiments were monitored for 7 weeks, except for animals infected with T. gondii 5days prior to infection with L. major, which were monitored for 16 weeks (A). The insert in panel A shows a curve of footpad swelling of resistant C57BL/6 mice infectedwith 106 stationary forms of L. major. Each point represents the mean (% standard deviation) lesion size for 10 mice. Asterisks indicate that differences are statisticallysignificant (P & 0.05), as evaluated by Students’ t test. Results from one representative experiment of two separately performed are shown.

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against L. major is equivalent to treatment with recombinantIL-12, if not more efficient. Protection persisted even up to 16weeks of infection, when the animals were sacrificed. However,our results also show that after establishment of L. majorinfection, the challenge with T. gondii induced only a discreetdelay in footpad swelling, similar to that observed after admin-istration of IL-12 in late stages of infection with L. major (13,24, 33).

A recent study illustrated the ability of acute and chronicinfection with T. gondii to enhance a Th1 response duringvaccination with a nonparasite-related antigen (25). However,our data show that the change in lesion size, cytokine, and IgGisotype response to Leishmania antigens was only observed inmice acutely, but not chronically, infected with T. gondii. Fur-thermore, unexpectedly we found an insignificant enhance-ment of Leishmania-specific Th1 lymphocyte activity. In fact

FIG. 2. Histopathology of BALB/c mouse footpad infected with L. major alone (A and C) or infected with T. gondii 5 days earlier to infection with L. major (B andD). Groups of animals were sacrificed 7 weeks after infection with L. major, and the infected footpads were fixed, included in paraffin, cut, and stained with hematoxylinand eosin. Footpads from BALB/c mice infected with L. major alone (A and C) showed an intense and diffuse inflammatory infiltrate, tissue necrosis (T), ulcerationareas (U), and abscess formation (A). (A) Hematoxylin and eosin; original magnification, '52.8; 1.05 cm ( 200 #M). An intense tissue parasitism (C) is observed inthe footpads of mice infected with L. major alone. (C) Hematoxylin and eosin; original magnification, '528; 1.05 cm ( 20 #M). Arrows indicate replicating amastigotesinside macrophages. In contrast, dually infected mice at 7 weeks postinfection with L. major showed a small inflammatory infiltrate with little tissue destruction andwell-delimited inflammatory foci (I). No necrotic areas, ulceration, or abscess formation was present in the footpads of dually infected animals (B) Hematoxylin andeosin; original magnification, '132; 1.32 cm ( 100 #M). An inflammatory focus, at higher magnitude, shows the presence of lymphocytes, plasma cells, fewpolymorphonuclear cells, many noninfected macrophages, and few infected macrophages (arrows).

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FIG. 3. PCR analysis of gp63 expression in footpads from mice infected with L. major alone, during the acute (T. gondii Ac-L. major) or chronic (T. gondii Chr-L. major) phase of T. gondii infection. Mice were infected intraperitoneally or not with 20 cysts of T. gondii and challenged 5 days (acute) or 30 days (chronic) laterwith 106 stationary forms of L. major. At the 7th week of L. major infection, mice were sacrificed, and the footpads were used as a DNA source employed in aGP63-specific PCR analysis. The left panel shows the PCR products of a reaction using 100 ng of footpad DNA and primers specific for L. major GP63. Each curverepresents the average (% standard deviation) of two animals, indicating the intensity of the GP63-specific PCR product employing different concentrations of footpadDNA, as measured by densitometric analysis. No PCR products were found in samples obtained from uninfected animals or animals infected with T. gondii alone.Similar results were found in two different experiments.

FIG. 4. IFN-" and IL-4 production by spleen and draining lymph node cells from BALB/c mice infected with L. major alone or coinfected with T. gondii. Mice wereintraperitoneally infected or not with 20 cysts of T. gondii and challenged 5 days later with 106 stationary forms of L. major. Seven weeks later, the mice were sacrificed.Lymph node and spleen cells were harvested and cultured for 72 h. The culture was done in medium alone (white bars) or in the presence of LAg (black bars), STAg(crosshatched bars), or ConA (gray bars). IFN-" and IL-4 were assayed in the culture supernatants by capture enzyme-linked immunosorbent assay. Bars representmeans (% standard deviations) of four mice per group. Different letters indicate that differences are statistically significant (P & 0.05) by nonparametric Kruskal-Wallis’ test,when comparing cytokine responses from animals from different groups by lymph node or spleen cells stimulated with a single stimulus (i.e., medium, LAG, STAg, or ConA).

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our major finding in terms of the cytokine synthesis of thedually infected animals was the complete suppression of IL-4synthesis compared to that in animals infected with L. majoralone. These findings were also confirmed by measurement ofLeishmania-specific IgG isotypes. We found that the synthesisof Leishmania-specific IgG1, but not IgG2a, was suppressed inthe dually infected mice.

Therefore, our study suggests that, at least at the level of Thcell differentiation, the major mechanism of action operatingduring acute toxoplasmosis, and possibly responsible for pro-tection against immunopathology elicited by L. major, is theinhibition of Th precursor cells from developing into the Th2phenotype. Consistent with this interpretation are the findingsthat if given 2 weeks postinfection with L. major, T. gondiibecomes unable to protect against lesion development in thefootpad. In fact, earlier reports indicate that although IL-4does not appear to be sufficient to make C57BL/6 mice sus-ceptible to infection with L. major (29), neutralizing monoclo-nal antibodies against IL-4 protect BALB/c mice against le-sions caused by L. major, only if given in the first week ofinfection (18, 28).

Finally, our earlier studies demonstrate that infection withT. gondii in mice results in induction of persistent T-cell-me-diated immunity characterized by production of high levels ofIFN-" and IL-2 as well as low levels of IL-4 and IL-5, whenstimulated with tachyzoite antigens (5, 8). This induction ofTh1 lymphocytes by T. gondii occurs even in BALB/c mice (5)that present a genetic propensity for the development of anti-gen-specific Th2 lymphocytes (15). Together, the results pre-sented here suggest that inhibition of differentiation of Thprecursor cell into Th2 lymphocytes during acute infectionwith T. gondii may be one important component for the devel-opment of parasite-specific, highly polarized Th1 immune re-sponses that persist during chronic toxoplasmosis.

We thank Wagner Taffuri, Denise C. Cara, and Luiz Antonio R.Freitas for the analysis of histopathology data and helpful discussions.We also gratefully acknowledge Elaine Speziali and Marileia C. An-drade for technical support in IgG isotype measurement. H.C.S. isgrateful to Gilton Santiago for support and encouragement during thiswork.

This work was supported in part by FAPEMIG and CNPq (522.056/95-4). R.T.G. and L.Q.V. received a research fellowship from CNPq.

H.C.S. is a medical student supported by FAPEMIG. M.A.P.O. is agraduate student and received a scholarship from CAPES.

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FIG. 5. Total IgG antibodies (A) specific for L. major (white bars) or T. gondii (black bars) antigens and (B) specific IgG1 (white bars) or IgG2a (black bars) isotypesagainst L. major in mice infected or not with T. gondii and challenged with L. major. Mice were infected with 20 cysts of T. gondii and 5 days later were challenged with106 stationary forms of L. major in the hind footpads. Mice were sacrificed 7 weeks later, and sera were obtained for total IgG and IgG isotype assays by enzyme-linkedimmunosorbent assay. Bars represents means (% standard deviations) of eight mice per group. These data are pooled from two separate experiments.

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Editor: J. M. Mansfield

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