INTRODUCTION

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Chagas' disease, caused by the protozoan Trypanosoma cruzi, afflicts about 18 million people on the American continent (29). Although the disease has been extensively studied, its pathogenesis is far from completely elucidated. Results obtained by using mice as an experimental model indicate that the outcome of infection is determined by many different factors. These include the genetic backgrounds of both the host (19, 26, 30) and the parasite (7, 18) and the experimental conditions under which infections are carried out (4). The role of concurrent infections in influencing the pathogenesis of T. cruzi has not been defined, although it is well known that such infections may seriously compromise host resistance. For example, endogenous viral infections alter host responsiveness through their multiple immunomodulatory effects (3, 6, 8, 9).

Recently, we have shown that specific-pathogen-free CBA/J mice infected with the Y strain of T. cruzi, which is maintained by serial mouse passage of Y_UEC stock, had higher levels of parasitemia and shorter survival times than animals infected with a stock of parasites (Y_CT) which was maintained exclusively in cell culture. Tests for virus-specific antibody using filtered plasma from Y_UEC-infected animals indicated the presence in these animals of murine hepatitis virus type 3 (MHV-3) (20). The cultures in which Y_CT is maintained are virus free, and thus, mice infected with this strain did not acquire the virus. Both MHV-3- and Y_UEC-infected animals show a marked lymphopenia in peripheral blood as early as 48 h postinfection (p.i.), whereas this does not occur in Y_CT-infected animals (27).

These data suggest that the greater pathology shown by Y_UEC-infected animals results from combination of viral and protozoal infection and is correlated with the observed lymphopenia. To investigate this point, changes in the cellularity and structure of the thymus were monitored in four groupsmice infected with MHV-3 alone, mice infected with Y_UEC, mice infected with Y_CT, and mice double infected with MHV-3 and Y_CTand these were compared with uninfected controls. In each group, total thymus cell numbers; percentages of Thy1.2, CD4⁺, and CD8⁺ cells; thymus weight; and histological changes were quantified during the course of infection. In addition, in order to clarify points related to the biological significance of the observed lymphopenia, we investigated whether cortical thymocytes in infected mice undergo DNA fragmentation (apoptosis). A demonstration of apoptosis should provide clues for understanding how concurrent infection might be associated with reduced host resistance.

	MATERIALS AND METHODS

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Animals. CBA/J (8- to 10-week-old) mice of both sexes were obtained from specific-pathogen-free colonies bred in isolators at Centro Multi-Institucional de Bioterismo, Unicamp. These colonies have been periodically screened since 1989 with consistently negative results for the following microorganisms: MHV-3, Sendai virus, lymphocytic choriomeningitis virus, rotavirus, pneumonia virus of mice, reovirus 3, Theiler's GD-VII virus, mouse minute virus, K virus, ectromelia virus, mouse adenovirus, mouse cytomegalovirus, and lactate dehydrogenase-elevating virus; Mycoplasma pulmonis; pathogenic bacteria; ectoparasites; and endoparasites. The animals were maintained in plastic isolators under aseptic conditions throughout the study.

Parasites. Two different stocks of the Y strain of T. cruzi were used. The stock originally received from Z. Brener in 1972 was labelled Y_UEC and is maintained in CBA/J mice by infecting animals, every week, with 10⁵ blood parasites intraperitoneally. The stock Y_CT was obtained by culturing Y_UEC parasites in monolayers of LLC-MK₂ cells as reported earlier (5). This parasite stock produces an active infection when injected in mice, as indicated by the presence of parasitemia that peaked on the 7th day p.i., but was unable to kill CBA/J mice when 10⁵ parasites were injected subcutaneously.

Virus. MHV-3 isolated in our laboratory was used throughout the experiments. MHV-3 was cultured in L-929 cells and stored in liquid nitrogen (14). The 50% lethal dose (LD₅₀) of the virus preparations was determined by the method of Reed and Muench (22).

Experimental design. Four experimental groups of 25 CBA/J mice (total, 100 mice) were inoculated subcutaneously in the left hind limb with (i) 10⁵ trypomastigotes of the Y_CT stock of T. cruzi, (ii) 10⁵ trypomastigotes of the Y_UEC stock of T. cruzi, (iii) 0.1 LD₅₀ of coronavirus (MHV-3), and (iv) 10⁵ trypomastigotes of the Y_CT stock plus 0.1 LD₅₀ of MHV-3. Five mice in each group were sacrificed 1, 2, 3, 5, and 7 days post-inoculation, after which the thymuses were removed and weighed. Single-cell suspensions were obtained from these organs by gently teasing the tissue in RPMI 1640 medium. The cells thus obtained were washed twice and resuspended in the same medium, their viability was determined by the trypan blue exclusion test, and they were counted in a hemacytometer. Twenty-five mice were used as uninfected control group (normal group).

Histopathological study. At sacrifice, thymuses were collected and immediately fixed in buffered 10% formaldehyde fixative, and then they were routinely processed and embedded in paraffin. Sections (4 µm) for histological examination were stained with hematoxylin-eosin and examined by light microscopy.

Lymphocyte subpopulation measurement. Briefly, thymuses were individually collected after sacrifice, and cells were obtained by gently teasing with tweezers in RPMI 1640 medium. Cell suspensions were adjusted to give desired cell concentrations (2 × 10⁵/ml), laid in slides by centrifugation, air dried, fixed in cold acetone, and subjected to indirect immunoperoxidase assays. Endogenous peroxidase activity was abolished by immersing the slides for 20 min in methanol containing 0.3% H₂O₂. Different lymphocyte populations were identified by using a three-step staining procedure. First, the cells were incubated with monoclonal antibody (MAb) to T helper cells (GK 1.5 clone), MAb to T suppressor/cytotoxic cells (YTS 169 clone), or MAb to total T cells (Thy 1.2 antigen), followed by incubation with biotinylated goat anti-rat immunoglobulin G (Dako A/S) and finally with avidin-peroxidase complex (Vector Laboratories). The stain was developed by adding 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) solution containing 0.03% hydrogen peroxide and incubating for 20 min in darkness. The slides were weakly counterstained with Mayer's hematoxylin (Merck) and examined by light microscopy. Cells showing positive staining were identified by the peripheral rim of brown color outlining the cell membrane. The percentage of positive cells was scored quantitatively by counting 100 cells in five microscopic fields and by two independent investigators.

In situ identification of nuclear DNA fragmentation. Tissue sections were analyzed by in situ end labelling of fragmented DNA as previously described (28). Briefly, after a 10% formaldehyde fixation, thymuses from infected and control mice were paraffin embedded. Sections were treated with proteinase K solution and washed with phosphate-buffered saline, and residues of digoxigenin-nucleotide were catalytically added to the DNA by terminal deoxynucleotidyl transferase. Antidigoxygenin antibodies labelled with peroxidase (ApopTag-Oncor) were used to detect the reaction. Staining was developed in diaminobenzidine-H₂O₂, and sections were counterstained with Harris hematoxylin.

Statistical analysis. Student's t test was used as described by Zar (31).

	RESULTS

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Thymus weight and cellularity were strikingly diminished in mice infected intraperitoneally with MHV-3, with the Y_UEC stock of T. cruzi, or with Y_CT plus MHV-3.

Figure 1A shows that the only group able to maintain thymic weight was that infected with the avirulent stock of parasites (Y_CT). All other groups that were infected with MHV-3, alone or associated with parasites, showed a marked decrease in thymic weight. The atrophy started 24 to 48 h p.i. and on day 7 p.i. represented approximately 50% of thymic weight in normal uninfected animals or the Y_CT-infected group.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Thymic alterations during MHV-3, YCT, YUEC, and YCT-MHV-3 infection of animals. Weight (A) and cellularity (B) are dramatically diminished in groups infected with MHV-3, the YUEC stock of parasites, and YCT plus MHV-3 but remain stable in animals infected only with the YCT stock of parasites. The values for each day are means with standard deviations for experiments involving five mice analyzed individually. Normal levels in uninfected animals are delineated by horizontal lines.

As shown in Figure 1B, numbers of thymic cells decreased in MHV-3-infected animals as early as 24 h p.i. until day 3 p.i., when numbers showed some recovery. Moreover, these animals were able to survive for more than 30 days after infection. Y_UEC- and Y_CT-MHV-3-infected mice showed thymic cell depletion around 48 and 72 h p.i., respectively, and numbers continued to fall until the animals died. No significant differences were observed in the numbers of thymic cells in Y_CT-infected animals. Virus infection, alone or in association with parasites, provoked significant decreases in the percentages of Thy1.2⁺, CD4⁺, and CD8⁺ cells in the thymus. This reduction in thymocyte subpopulations started at 24 h p.i. and reached maximum values on day 7 p.i. (Table 1).

Comparative histopathological studies of thymuses from stocks of T. cruzi- and MHV-3-infected mice showed differences in morphology and in the cortical/medullary content ratio. Thymus tissue from Y_CT-infected mice (Fig. 2A) was similar to that from control mice (not shown). In contrast, animals infected with Y_CT plus MHV-3 (Fig. 2B), MHV-3 alone (Fig. 2C), or Y_UEC stock (Fig. 2D) showed significant thinning and cellular depletion in the thymic cortex and a decreased mitotic index (data not shown). Besides, these animals showed a "starry sky" pattern, caused probably by interspersed histiocytes.

View larger version (162K): [in this window] [in a new window]   FIG. 2.   Histology of thymuses from YCT-infected CBA/J mice (A), YCT-plus MHV-3-infected CBA/J mice (B), MHV-3-infected CBA/J mice (C), and YUEC-infected CBA/J mice (D). No alterations were observed in YCT-infected animals compared with uninfected controls (data not shown). The other groups showed dramatic depletion in the cortical zone. Hematoxylin-eosin staining was used. Magnification, ×140.

In order to verify the origin of thymic atrophy and the fate of subpopulations of thymocytes, we investigated the degree of programmed cell death in tissue sections by in situ end labelling of fragmented DNA. Y_CT-infected mice showed a few cells with fragmented DNA (Fig. 3A). In contrast, the number of apoptotic cells in the thymuses of mice infected with Y_CT plus MHV-3 (Fig. 3B) was dramatically increased. Thymuses of MHV-3-infected animals (Fig. 3C) and those infected with the Y_UEC stock of parasites (Fig. 3D) showed significant numbers of apoptotic cells, but these numbers were lower than those in Y_CT- plus MHV-3-infected animals and only slightly higher than those in Y_CT-infected animals. In addition, different patterns of DNA fragmentation were observed in different groups. While animals infected with the Y_CT or Y_UEC stock of parasites (Fig. 3A and D) showed a round nuclear pattern, animals infected with Y_CT plus MHV or MHV alone (Fig. 3B and C) showed large clusters of irregular masses scattered in a disorganized cortico-medullary region.

View larger version (164K): [in this window] [in a new window]   FIG. 3.   Apoptosis in a thymus section from a YCT-infected animal (A), a YCT- plus MHV-3-infected animal (B), a MHV-3-infected animal (C), and a YUEC-infected animal (D). YCT-infected animals showed rare apoptosis granules, whereas the animals infected with the virus alone or in association with the parasite showed increased numbers of apoptotic cells. Hematoxylin counterstaining was used. Magnification, ×420.

	DISCUSSION

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Conventionally housed laboratory mice may sustain infections with one or more murine virus (10). The method of breeding mice under barrier-sustained conditions only recently became available to some laboratories, and so, earlier isolates of T. cruzi were maintained in mice that were coinfected by other pathogens.

Here we have observed a marked thymic cell depletion when animals were infected with MHV-3, alone or associated with one stock of the parasite. The diminished cellularity in thymus correlated well with decreased numbers of Thy1.2, CD4⁺, and CD8⁺ thymocyte subpopulations. These findings, showing that MHV-3 is able to exacerbate the parasite infection, together with the observed effects upon numbers of circulating lymphocytes (unpublished data) and thymic cells, suggest that the enhanced pathology associated with Y_UEC infection reflects underlying alterations in the immune system. There are examples in the literature which show aggravation of both T. cruzi and murine leukemia virus by concomitant infections (25). It is known that MHV induces lymphoid organ atrophy (15) and shows a tropism to T and B lymphocytes (13). However, the mechanisms responsible for immunodeficiency associated with MHV-3 infection remain unknown.

The present results suggest that virus-induced programmed cell death could account for the loss of T lymphocytes from the thymuses observed after either Y_CT plus MHV-3 or Y_UEC infection. Together with the inhibition of thymocyte mitotic index in these animals, cellular death by apoptosis would be responsible for the thinning or atrophy of the thymus cortex. Apoptosis of T lymphocytes in animals infected with T. cruzi has already been demonstrated in spleen CD4⁺ cells (16). Similarly, several viruses, including some strains of MHV, are able to induce apoptosis (11, 21, 24). The unchanged numbers of both peripheral lymphocytes (unpublished data) and thymic cells, as well as the maintenance of a normal cortical/medullary content ratio in Y_CT-infected mice, are consistent with the low level of programmed cell death in this group. On the other hand, the more extensive apoptosis observed in the thymus in T. cruzi- plus MHV-3-infected mice suggests a severe immunosuppression which is probably triggered by the virus replication.

T-cell-dependent immune responses are crucial in the control of T. cruzi infection (2, 17, 23). Cellular immune responses mediated by helper and cytotoxic T lymphocytes are also involved in the elimination of viral infection (13). Therefore, control of both infections is dependent upon the capacity of the thymus to generate and maintain normal T lymphopoiesis. In the present work we have observed a marked thymic involution in animals that were infected with the MHV-3 alone or with MHV-3 associated with T. cruzi, and this may well explain the greater pathology seen in these mice.

Several mechanisms including secretion of different mediators (1) could be responsible for the apoptosis seen during the course of the infections reported here. Further work is necessary to determine whether this apoptosis occurs as a result of direct lymphocyte or stromal cell infection or is caused by stress-induced glucocorticoid release and/or secretion of other soluble factors from intra- and extrathymic sites (12).