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Coinfection with Toxoplasma gondii Inhibits Antigen-Specific Th2 Immune Responses, Tissue Inflammation, and Parasitism in BALB/c Mice Infected with Leishmania major

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Coinfection with Toxoplasma gondii Inhibits Antigen-Specific Th2 Immune Responses, Tissue Inflammation, and Parasitism in BALB/c Mice Infected with Leishmania major
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  I NFECTION AND  I MMUNITY ,0019-9567/99/$04.00  0Sept. 1999, p. 4939–4944 Vol. 67, No. 9Copyright © 1999, American Society for Microbiology. All Rights Reserved. Coinfection with  Toxoplasma gondii  Inhibits Antigen-Specific Th2Immune Responses, Tissue Inflammation, and Parasitismin BALB/c Mice Infected with  Leishmania major  H. 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. GAZZINELLI 1,4 *  Department of Biochemistry and Immunology—ICB 1  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 with  Leishmania 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 of interleukin 4 (IL-4), culminating in the release of reactivenitrogen intermediates that are highly effective in controlling  Leishmania  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   (10 6 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  5days prior to challenge with  L. major   resulted in protectionagainst lesion development in BALB/c mice. These results were 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 with  L. 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. major  alone (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 of 25   l containing GP63 (22) primers 5   GTGCGCACGTGAA CTGG 3   (sense, nucleotides 499 to 517) and 5   CACCCGC AGTAGTTGTAG 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 of tissue 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.4939  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 infected with  L. major   alone versus those in dually infected animals,lymph node and spleen cells from mice were collected at 7 weeks 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 of IL-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 infection with  L. major  . Infection with  T. gondii  5 days prior to infection with  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 of our previous experiments by showing that coinfection with  T. gondii  had a major inhibitory effect on the in vivo synthesis of   Leishmania -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). Inmost 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. A major 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-reactivity was 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 with10 6 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. major  and, 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 infected with 10 6 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. 4940 NOTES I NFECT . I MMUN .  against  L. major   is equivalent to treatment with recombinantIL-12, if not more efficient. Protection persisted even up to 16 weeks of infection, when the animals were sacrificed. However,our results also show that after establishment of   L. major  infection, 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 during vaccination 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; srcinal 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; srcinal 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 and well-delimited inflammatory foci (I). No necrotic areas, ulceration, or abscess formation was present in the footpads of dually infected animals (B) Hematoxylin andeosin; srcinal 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). V OL  . 67, 1999 NOTES 4941  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) later with 10 6 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 10 6 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). 4942 NOTES I NFECT . I MMUN .  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. major  alone. These findings were also confirmed by measurement of   Leishmania -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. gondii becomes 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 of infection (18, 28).Finally, our earlier studies demonstrate that infection with T. gondii  in mice results in induction of persistent T-cell-me-diated immunity characterized by production of high levels of IFN-   and IL-2 as well as low levels of IL-4 and IL-5, whenstimulated with tachyzoite antigens (5, 8). This induction of Th1 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 infection with  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 Antoˆnio R.Freitas for the analysis of histopathology data and helpful discussions.We also gratefully acknowledge Elaine Speziali and Marile´ia C. An-drade for technical support in IgG isotype measurement. H.C.S. isgrateful to Gilton Santiago for support and encouragement during this work.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. REFERENCES 1.  Denkers, E. Y., and R. T. Gazzinelli.  1998. 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W. Hartley, T. N. Fredrickson, S. K. Chattopadhyay, A.Sher, and H. C. Morse III.  1992. Opportunistic infections and retrovirus-induced immunodeficiency: studies of acute and chronic infections with Toxoplasma gondii  in mice infected by LP-BM5 murine leukemia viruses.Infect. Immun.  60: 4394–4401.7.  Gazzinelli, R. T., S. Hieny, T. Wynn, S. Wolf, and A. Sher.  1993. IL-12 isrequired for the T-cell independent induction of IFN-   by an intracellularparasite and induces resistance in T-deficient hosts. Proc. Natl. Acad. Sci.USA   90: 6115–6119.8.  Gazzinelli, R. T., M. Wysocka, S. Hayashi, E. Y. Denkers, S. Hieny, P.Caspar, G. Trinchieri, and A. Sher.  1994. Parasite-induced IL-12 stimulatesearly IFN-gamma synthesis and resistance during acute infection with  Toxo- plasma gondii . J. Immunol.  153: 2533–2543.9.  Gazzinelli, R. T., M. Wysocka, S. Hieny, T. Scharton-Kersten, A. Cheever, R.Ku¨hn, W. Mu¨ller, G. Trinchieri, and A. Sher.  1996. In absence of endoge-nous IL-10 mice acutely infected with  Toxoplasma gondii  succumb to a lethalimmune response dependent on CD4   T cells and accompanied by over-production of IL-12, IFN-  , TNF-  . J. Immunol.  157: 798–805.10.  Green, S. J., R. M. Crawford, J. T. Hockmeyer, M. S. Meltzer, and C. A.Nacy.  1990.  Leishmania major   amastigotes initiate the L-arginine-dependentkilling mechanism in IFN-   stimulated macrophages by induction of tumornecrosis factor-  . J. Immunol.  145: 4290–4297.11.  Hayashi, S., C. C. Chan, R. T. Gazzinelli, and F. G. Roberge.  1996. Contri-bution of nitric oxide to host parasite equilibrium in toxoplasmosis. J. Im-munol.  156: 1476–1481.12.  Heinzel, F. P., M. D. Sadick, B. J. Holaday, and R. M. Locksley.  1989.Reciprocal expression of IFN-  or IL-12 during the resolution or progressionof murine leishmaniasis. Evidence for expansion of distinct helper T cell4subsets. J. Exp. Med.  169: 59–72.13.  Heinzel, F. P., R. M. Rerko, F. Ahmed, and E. Pearlman.  1995. EndogenousIL-12 is required for control of Th2 cytokine responses capable of exacer-bating leishmaniasis in normally resistant mice. J. Immunol.  155: 730–739.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 with10 6 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. V OL  . 67, 1999 NOTES 4943
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