Trypanosoma cruzi - Digitum

Histol Histopathol (2002) 17: 837-844

Histology and Histopathology

http://www.hh.um.es

Cellular and Molecular Biology

Trypanosoma cruzi: infection patterns in intact and athymic mice of susceptible and resistant genotypes S.C. Gonçalves da Costa1, K.S. Calabrese1, T. Zaverucha do Valle1 and P.H. Lagrange2 1Immunomodulation 2Central

Laboratory, Protozoology Department, Oswaldo Cruz Institut, FIOCRUZ, Rio de Janeiro, RJ, Brasil and

Laboratory of Microbiology, Hôpital Saint-Louis, Paris Cedex 10, France

Summary. Inbred strains of mice inoculated with the T.

cruzi Y strain behaved as susceptible (A/J, C3H/HeN), intermediate (BALB/c) or relatively resistant (C57BL/6) with respect to the magnitude of parasitaemia and mortality rate. C57BL/10 mice were susceptible in relation to parasitaemia but resistant when mortality was analyzed. Infection with T. cruzi CL strain presented the same results, except for C57BL/6 which behaved as susceptible mice. Athymic mice of various backgrounds revealed no differences in susceptibility, presenting the same dramatic parasitaemia, tissue colonization pattern and no inflammatory reaction in any of the tissues studied. Infection of euthymic and athymic BALB/c mice elicited the production of parasite-specific antibodies, which reached similar levels on the first 9 days but differed after day 13. Serum transfer experiments in BALB/c mice did not show great differences in parasitaemia but altered T. cruzi polymorphism reducing the slender forms in athymic mice. Histopathology of athymic BALB/c mice showed the same tissue tropism when infected either with T. cruzi Y or CL strain. Key words: Trypanosoma cruzi, Athymic mice,

Susceptibility, Resistance, Inbred mice Introduction

Mouse strains vary markedly in natural resistance to Trypanosoma cruzi infection. It has been established that C3H/HeN is susceptible, BALB/c is intermediate and B10A is resistant to the T. cruzi Brazil strain (Trischmann et al., 1978). It was also shown that the quantity of hydrogen peroxide released by macrophages from susceptible C3H/HeN mice was significantly higher than that released by the other two inbred mouse Offprint requests to: Sylvio Celso Gonçalves da Costa, Laboratório de Imunomodulação, Departamento de Protozoologia, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brazil. Fax: 55 21 2598-4323. e-mail: sycosta@ ioc.fiocruz.br

strains and by athymic outbred mice (Russo et al., 1989). Otherwise, it was shown that outbred OF1 and inbred BALB/c athymic mice presented exceptionally high parasitaemia when infected either with reticulotropic or myotropic T. cruzi strains (Gonçalves da Costa et al., 1984); in contrast with BALB/c nude mice infected with T. rhodesiense, which is slightly more resistant than intact mice (Campbell et al., 1978). Because of the striking mouse strain variations in correlation with resistance to parasitic infection, the interest in nude mice is increasing, since athymic mice of various genotypes are available, despite their high cost for research (Hansen, 1978). On the other hand, it has been shown that T-cells were not responsible for the natural resistance to T. musculi. In this model it was demonstrated that T-cell-deprived C3H/HeN mice were more susceptible than T-cell deprived C57BL/6 (Magluilo et al., 1983). Similar observations were made by Gros et al. (1983) studying the time course of BCG infection in congenitally athymic mice caring the nude mutation on bcg r (AKR/J) or bcg s (BALB/c) background. These authors showed that the functional absence of T lymphocytes did not influence the expression of the bcg gene. In the present report we investigate the possibility that some parameters of the natural and specific immunity will influence the course of T. cruzi infection, using athymic and intact mice of susceptible and resistant genotypes. Materials and methods Animals

The A/J, C3H/HeN, BALB/c, C57BL/6 and C57BL/10 inbred mouse strains were maintained in our own animal facilities. Congenitally athymic homozygous mice (nude, Nu/Nu) and their littermates, heterozygous thymusbearing mice (Nu/+) on BALB/c, C57BL/6 and C3H/HeN backgrounds were used. These animals were purchased from Gl. Bomholtgard Ltd., Denmark.

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T. cruzi infection in susceptible and resistant mice

OF1-specific pathogen-free outbred female mice, purchased from IFFA-CREDO (Domaine des Oncins, St.Germain sur l'Arbresle, France), were used for routine serial passages of parasites. The experimental groups consisted of 15 female mice, 6 to 8 weeks old, in each group, for every strain. Parasites

The Y and CL strains of T. cruzi were used. The Y strain was originally isolated in São Paulo (Brazil) from a patient in the acute phase of the disease (Pereira da Silva and Nussenzweig, 1953), while the CL was isolated from naturally infected Triatoma infestans bugs collected in Rio Grande do Sul, Brazil (Brener and Chiari, 1963). The parasites were routinely maintained by serial passages in mice. For challenge, blood was drawn by cardiac punction and diluted in PBS (phosphate buffer saline solution) to adjust the concentration of parasites, which were intraperitoneally (ip) inoculated in OF1 mice. Cloned Y - A1.2 TM (Trypomastigotes) and CL-1 TM were maintained in tissue culture by weekly infection of J 774 tumor cell monolayers as described elsewhere (Ralph et al., 1975). The Y- A1-2 TM suspensions used in the present experiments contained 8 to 9 TM for 1 amastigote (Plata et al., 1984). Experimental schedule

Two groups of experiments were developed: 1) groups of different euthymic inbred mice were infected (i.p.) with 104 blood forms of T. cruzi Y strain; 2) groups of athymic nude mice (Nu/Nu) as well as their heterozygous littermates (Nu/+) were infected (i.p.) with 104 blood forms of T. cruzi either Y or CL strains. The evaluation of parasitaemia was made by a standard microscopic procedure developed by Pizzi and Prager (1952) with slight modifications. Briefly, 5 µl of tail blood vein were placed under a 22x22mm cover slip. Parasites were counted in 50 fields and the number was converted to the total number of parasites per ml of blood. In the 5th, 9th and 13th day after the experimental infection, three animals from each group were sacrificed. Serum was collected for determination of antibody levels. The rest of the animals, six mice of each group, were observed to evaluate mortality rate until 4 weeks when we considered the study to be completed. Experiments were made in triplicate and arithmetic means as well as standard error of the mean (SEM) were calculated. Evaluation of animals was made until 4 weeks when the experiments were considered finished. Histopathology

Fragments of the organs (heart, pancreas, liver, spleen and brain) were removed and immediately fixed in Millonig's fluid (Carson et al., 1973). This material was processed and embedded in paraffin. Several 5 µm

sections were cut and stained with haematoxylin and eosin. Stimulation of the mononuclear phagocytic system (MPS)

Stimulation of the MPS was measured through the spleen index. It was calculated after individual evaluation of the relative spleen index (SI) (spleen weight/mouse weight). The arithmetic mean was then calculated. Serum transfer

Serum samples were obtained from 50 BALB/c mice infected with 2x106 trypomastigotes of T cruzi Y strain. Mice were bled 13 days after infection and the serum was filtered twice (0.45 µm and 0.22 µm) in a Schleicher and Scull disposable filter holder. After this, the pool of sera was stored at -20 °C. Before transfer, experimental haemagglutination titre was determined as described elsewhere (Camargo et al., 1971) and heat-inactivated serum presenting high titre was injected intravenously (iv) into groups of normal recipients in a volume of 0.2ml daily (the first dose was given 1 hour before challenge) for 12 days. Serum-treated and control groups of mice received an infective dose of 2 X 10 6 trypomastigotes 1 hour after the first serum dose and levels of parasitaemia, mortality and morphology of T. cruzi blood forms were followed. Results Susceptibility of inbred mice using T. cruzi Y strain

The behavior of T. cruzi Y strain in different inbred mice is presented in Fig. 1. C57BL/10 mice had the highest peak of parasitaemia, followed by A/J and C3H/HeN mice. The parasitaemia of C57BL/6 mice was significantly lower than other strains, presenting a peak on the day 8 after infection. BALB/c mice appeared as an intermediate group. Death occurred early in A/J mice, reaching 50% of the total population 17 days after infection and eliminating more than 60% of animals on day 26. The same occurred with C3H/HeN mice, while the C57BL/6 and C57BL/10 mice remained alive until the end of the study (week 4). Course of infection in Nu/Nu and Nu/+ mice of susceptible, intermediate and resistant genotypes

Groups of 6 congenitally athymic nude mice with BALB/c (intermediate), C3H/HeN (susceptible) and C57BL/6 (relatively resistant) backgrounds and their heterozygous littermates were infected with 10 4 trypomastigotes of the Y strain and the course of parasitaemia was followed. When the parasitaemia of nude mice and their littermates was compared, at the moment of the peak, an

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enhancement of the parasitaemia of all athymic mice was observed, regardless of their genetic background. The greatest difference was observed in BALB/c mice,

whose parasite level was up to 10 times higher than that observed in their littermates (Fig. 2a-c). On day 17 after infection, all athymic mice were dead, no matter which genetic background of inbred strain was being used (Fig. 2d). Humoral immune response of nude mice and their littermates to T.cruzi infection

The ability of BALB/c nude mice to produce immunoglobulins in response to T. cruzi infection was compared with the antibody levels observed in their littermates, using the Y strain. The greatest difference in antibody titre was observed on day 13 after infection as shown in Table 1. The cross reactivity was tested with CL strain. Serum transfer experiments in BALB/c mice Fig. 1. Time course infection in different inbred mouse strains infected with 104 trypomastigotes of T. cruzi Y strains. Mean of six mice SEM (in triplicate experiments).

Immune serum transfer was not able to reduce the parasitaemia significantly (Fig. 3) and presented little

Fig. 2. Comparison of parasitaemia in C57BL/6 (a), C3H/HeN (b) and BALB/c (c) nude mice (Nu/Nu) and their littermates (Nu/+) infected with 104 bloodstream trypomastigotes of T. cruzi Y strain. Fig. d shows the cumulative mortality (%) of these mice. Experiments were made in triplicate and arithmetic means, of six mice, as well as SEM were calculated.

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influence on mortality rate of infected nude mice. However, when T. cruzi polymorphism was analyzed, a reduction of slender form percentage was found (data

Fig. 3 - Serum transfer experiments in which athymic BALB/c mice infected with Y strain received a daily dose of immune sera from late acute phase. Difference with control group was not significant (mean ± SEM).

not shown). Histopathology of Nu/Nu and Nu/+ mice with C3H/HeN, C57BL/6 and BALB/c backgrounds

Athymic nude mice of different genetic backgrounds, infected with Y strain, did not show any inflammatory reaction in the different tissues studied (Fig. 4A-C). In contrast, littermates of all inbred strains studied presented an inflammatory infiltrate in different organs and tissues (Fig. 4D). The density of inflammatory cells appeared in exceptionally high levels, sometimes without correlation to parasite load with both strains. In athymic mice parasites, Y and CL strains appeared in almost all tissues and organs, with the exception of the brain, showing an extraordinary level of amastigote nests. BALB/c nude mice infected either with the reticulotropic Y strain or the myotropic CL strain presented the same pattern of tissue colonization, in the absence of inflammatory reaction (Fig. 5A,B), while

Fig. 4. Histopathology of heart and pancreas from athymic susceptible and resistant mice 13 days after infection with the T. cruzi Y (A, B and C) or CL (D) strain. A. shows nests of amastigotes in acine of pancreas (arrows) from C57BL/6 athymic mice. x 1000. B. The colonization of heart from C3H/HeN athymic mice by amastigotes (arrow) is observed. x 160. C. Shows heart of BALB/c nude mice with several nests of T. cruzi amastigotes (arrows), nests are shown in detail (window, zoom). In all of the above cases no inflammatory reaction was observed. x 400. D. shows myocarditis of BALB/c infected with the CL strain associated with a large parasite nest (arrow). x 160. Haematoxylin and eosin stain.

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their littermates presented many inflammatory cells (Fig. 5C). An unexpected case was observed in C57BL/6 nude mice in which no amastigote nests were observed in the myocardium. Parasites were found below the pericardium in a region colonized by macrophages and adipocytes (Fig. 6A). Adipocytes were dramatically infected in all athymic mice (Fig. 6B). In nude mice, parasites frequently appeared near nervous terminations without any sign of inflammatory reaction or lesions (data not shown). Euthymic BALB/c mice infected with CL strain did, in fact, behave differently from those infected with Y strain. A minor degree of stimulation of the MPS was observed using the CL strain, as measured by the spleen index (SI). Mice infected with the Y strain presented a greater SI than the ones infected with CL strain. No difference was observed in SI of athymic BALB/c mice infected with any of these strains (data not shown).

Discussion

In the early seventies the manipulation of Tdependent response was introduced in experimental Chagas' disease in view of a better understanding of host defense mechanism against T. cruzi infection. Depletion of T-cells by treatment with anti-thymocyte sera (Roberson et al., 1973) or surgical neonatal thymectomy (Schmunis et al., 1971) markedly exacerbates the time course infection. In addition, another fact demonstrated that cell-mediated immunity plays a central role in the resistance against T. cruzi infection in mice: athymic mice are dramatically susceptible to T. cruzi infection (Gonçalves da Costa et al., 1984) in contrast with mice infection by T. rhodesiense (Campbell et al., 1978). Still another piece of evidence that favors the acceptance of this view is that a T-cell transfer – from infected littermates to nude mice at the beginning of the acute

Table 1. Serum antibody of T. cruzi Y strain in infected mice. PHENOTYPE

Nu/+ Nu/Nu Nu/+ Nu/Nu Nu/+ Nu/Nu

DAY AFTER INFECTION 5 5 9 9 13 13

HAEMAGGLUTINATION THITRE UPON Y-A1.2 1/10 1/10 1/2.430 1/2.430 1/21.800 1/810

HAEMAGGLUTINATION TITRE UPON CL-1 1/10 1/10 1/270 1/270 1/270 1/30

Fig. 5. Histopathology of liver from BALB/c mice infected with the CL or Y strain of T. cruzi on day 13 after infection. A. hows nest of amastigotes (arrow) in complete absence of inflammatory reaction when athymic mice were infected with Y strain. x 400. B. shows the same athymic mice infected with the CL strain. Arrow shows a nest of amastigotes. x 400. C. shows mononuclear inflammatory cells infiltration (arrow) which was always observed in euthymic mice infected with T. cruzi Y strain. x 160. Haematoxylin and eosin stain.

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phase – induces focal inflammatory reaction in the myocardium, which can be correlated with the lysis of infected myocyte. Littermates bearing an intact thymic structure presented the common pattern of T. cruzi Y strain infection in mice, showing a parasitaemia peak on day 8-9 of infection and death with a low level of parasitaemia. The same occurred when thymic reconstitution of homozygous BALB/c nude mice was attempted (Ribeiro dos Santos and Rossi, 1985). Furthermore, altered electrocardiograms were observed in euthymic but not in athymic mice infected with the Tulahuén T. cruzi strain (Subias et al., 1982). The course of the experimental infection acute phase in euthymic mice, when considering either parasitaemia levels or tissue invasion, depends upon the genetic background of the parasite strain (Brener and Chiari, 1963; Postan et al., 1983; Arruda, 2000), as well as the host’s background (Pizzi et al., 1949; Trischman et al., 1978; Arruda, 2000). The present results show that, in absence of

functional T lymphocytes, no factor can influence the expression of host resistance against T. cruzi. This occurs when two strains, considered in the literature as polar strains (CL and Y strains) (Melo and Brener, 1978), are used to infect athymic mice. The present data is quite different from what has been previously observed in the T. musculi model, where the expression of natural immunity seems to be important in the course of infection (Magluilo et al., 1983). Transfer experiments of T-cells obtained from lymph nodes or spleen of chronic chagasic euthymic BALB/c to athymic mice – eight days after infection – or to naive syngeneic recipients – 24 hours before infection – promoted an enhancement of myocarditis with mild tissue parasitism, as well as a reduction of both parasitaemia and mortality (Gonçalves da Costa et al., 1984; Rottenberg et al., 1992). Selective CD4+ T-cell depletion in C3H/HeN mice by injection of monoclonal anti-CD4 antibody during the course of T. cruzi CL strain led to higher parasitaemias (Russo et al., 1988).

Fig. 6. Aspects of athymic C57BL/6 mice infected by T. cruzi Y strain on day 13 after infection. A shows an unexpected high level of macrophages and adipocytes colonized by amastigotes (arrow) on the sub-pericardium layer. In contrast, no parasites or inflammatory reaction were found in myocardium. x 160. B shows adipocytes heavily infected (arrow) x 400. Haematoxylin and Eosin stain.

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The same was observed in athymic mice from the relatively resistant C57BL/6 mice (Minoprio et al., 1989). In addition, syngenic CD4 + T-cell-transferred athymic mice were able to control parasitaemia better than the euthymic control group. Nevertheless, the role of CD4 + T cells is more general and has been implicated in the pathology of experimental Chagas' disease by mediating parasitespecific delayed type hypersensitivity (HontebeyrieJoskowicz et al., 1987). The murine model of T. cruzi infection has proven to be useful for studies of human Chagas' disease (Gonçalves da Costa, 1999). Neonates infected with 104 trypomastigotes develop a hard acute phase with systemic parasite invasion, showing intense myocardite and encephalitis (Gonçalves da Costa et al., 1986) which appear closely related with human Chagas' disease, as described by Carlos Chagas in children at the beginning of the 20th century (Chagas et al., 1929). Different euthymic adult mice infected with the same trypomastigote dose developed the two characteristic phases of the disease, with a parasitaemia peak and tissue tropism which is dependent of both T. cruzi and mouse strains. T. cruzi strain variations, which have been correlated with differences in severity in the human acute phase, may explain the regional prevalence of a specific pathology such as myocarditis or mega syndromes (Melo and Brener, 1978). Tissue invasion and pathology induced by T. cruzi results also from host immunity. Association of T. cruzi infection with the acquired immunodeficience syndrome (AIDS) led to a high incidence of amastigote proliferation in the central nervous system (CNS) (Jesus et al., 1995). The present report shows that, in adult athymic mice, the CNS is preserved. Therefore, we believe that nude mice may represent a good model to investigate the interaction between this parasite and the CNS. In the late acute and chronic phases, focal and diffuse inflammatory reactions composed by a mononuclear infiltrate, consisting basically of macrophages and lymphocytes, have been related to a progressive destruction of the myocardium and skeletal muscles, muscles of the digestive tract, and also the liver and nervous system (Gonçalves da Costa et al., 1984; Calabrese et al., 1994; Lenzi et al., 1996). These findings can be correlated with evidence presented in humans such as the abundance and systemic diffusion of inflammatory infiltrate associated with the low level of parasites in tissues (Torres, 1941) – the so called "allergic" approach to explain the mechanism by which T. cruzi causes cardiomyopathy. The other concept developed was the neurogenic theory of Köberle (1968), proposing that the cardiac enlargement was due to the loss of autonomic heart control, thus leading to a progressive destruction of the cardiac vagal neurons. Based on the fact that cardiac denervation may be of nonspecific nature, and that in non-chagasic heart diseases the blockage of autonomic heart rate control is proportional to the degree of cardiac dysfunction, a new

pathological pathway was proposed by which T. cruzi inducement of focal myocardial fibrosis and necrosis lead to a progressive ventricular dilatation. This results in the impairment of cardiac receptor parasympathetic abnormalities, a sympathetic predominance, which is followed by a cardiac failure and subsequent death (Dávila et al., 1989). The present data is based on morphological studies and gives support to Magarinos Torres view (Torres, 1929, 1941) that was recently complemented by Dávila et al. (1989). It has been proposed, however, that CD4+ T cells are not the only kind of cells implicated in these mechanisms. More recently, Tarleton argued that CD8+ T cells are also implicated in the pathogenesis of experimental Chagas' disease by following the time course infection with Brazil T. cruzi strain of the relatively resistant C57BL/6 mice. After treatment with monoclonal anti-CD8 antibody these mice showed higher parasitaemias and died earlier than the untreated control group (Tarleton, 1990; Sun and Tarleton, 1993). These findings are correlated with the partial control of parasitaemia observed in athymic C57BL/6 mice receiving a CD8+ T cell transfer (Minoprio et al., 1989) and other evidence that has been exhaustively discussed by Kierszenbaum (1995). He emphasized that the susceptible C3H/HeSn mice receiving anti-CD8 antibody one week before infection did not show levels of increased parasitaemia as statistically significant as was expected. The present data show that C3H/HeN and C57BL/6 athymic/euthymic mice are useful models to investigate immunopathology in T. cruzi experimental infection. Death of athymic C57BL/6 mice, relatively resistant to the T. cruzi Y and CL strains, may occur without myocarditis or any sign of parasite colonization; brains of both athymic mouse strains were consistently negative for parasite, which may be an important model for investigations about immunopathology of CNS induced by T. cruzi infection. Acknowledgements. The authors wish to thank Mrs. Luciana F. Pereira for secretarial work. This paper was supported by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Proc. 305170/88-0); CNPq-INSERM (Proc. 91.0320/92.6) and Instituto Oswaldo Cruz (FIOCRUZ).

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