Benznidazole treatment following acute Trypanosoma cruzi infection triggers CD8+ T-cell expansion and promotes resistance to reinfection

Abstract

Many studies have shed light on the mechanisms underlying both immunoprotection and immune dysregulation arising after Trypanosoma cruzi infection. However, little is known about the impact of benznidazole (N-benzyl-2-nitroimidazole acetamide), the drug available for clinical treatment of the infection, on the immune system in the infected host. In the present study we investigated the effect of benznidazole therapy on the lymphoid compartment during the course of experimental T. cruzi infection. Although amelioration of a variety of clinical and parasitological signs was observed in treated mice, amelioration of splenocyte expansion was not detected. Interestingly, this sustained splenomegaly observed in benznidazole-treated mice showed a preferential expansion of CD8(+) T lymphocytes. Moreover, although benznidazole treatment blocked the expansion of recently activated CD4(+) and CD8(+) T cells seen in infected hosts, benznidazole treatment led to a selective expansion of effector and memory CD8(+) T lymphocytes in association with a lower rate of apoptosis. In addition, the surviving treated animals were protected from reinfection. Together, these data suggest that, in addition to its well-known direct role in blocking parasite replication in vivo, benznidazole appears to directly affect immune regulation in T. cruzi-infected hosts.

FIG. 1.

Benznidazole improves the resistance of mice to T. cruzi infection. Each point consists of data for 16 to 22 mice in the noninfected (open squares), infected, nontreated (filled squares), and infected, benznidazole-treated (filled triangles) groups from three independent experiments. (a) Quantitation of parasitemia, with vertical bars representing the means + standard errors for each time point; (b) survival rates; (c) body weight evolution of the experimental groups.

FIG. 2.

Sustained absolute spleen mass and cellularity in benznidazole-treated mice following T. cruzi infection. Data are for noninfected (open squares), infected, nontreated (filled squares), infected, benznidazole-treated (filled triangles), and noninfected, benznidazole-treated (open triangles) mice. Absolute spleen mass values (a and b) and spleen cell numbers (c and d) on days 9 (a and c) and 14 (b and d) after infection. Horizontal bars represent the median values of three independent experiments for the analysis at day 9 and nine experiments for the analysis at day 14.

FIG. 3.

Prominent CD8⁺ expansion in the spleens of T. cruzi-infected, benznidazole-treated mice. Phenotypically defined CD4⁺, CD8⁺, and B lymphocytes in noninfected (N; open squares), infected, nontreated (I; filled squares), infected, benznidazole-treated (IBz; filled triangles), and noninfected, benznidazole-treated (NBz; open triangles) mice were analyzed after 14 days of infection in three to four different experiments. The numbers of CD4⁺, CD8⁺, and B lymphocytes in the spleens of individual mice are depicted (a, b, and c, respectively), with the horizontal bars indicating the median value for each experimental group.

FIG. 4.

Diminished CD69 expression in CD4⁺ and CD8⁺ lymphocytes together with downregulation of CD62L expression on CD8⁺ lymphocytes after infection and benznidazole treatment. Cytofluorometric studies were performed with noninfected (N), infected, nontreated (I), and infected, benznidazole-treated (IBz) mice. (a) A representative dot plot of CD69 expression versus CD4 or CD8 expression is shown, with the percentages of CD4⁺CD69⁺ or CD8⁺CD69⁺ cells indicated inside the inner square boxes; data are from four experiments with three to four mice per group. Positive staining was calculated by defining a positivity marker from the end of the unrelated immunoglobulin isotype control fluorescence. (b) Representative histograms of CD62L fluorescence intensity in CD4⁺ and CD8⁺ lymphocytes from noninfected (N; dotted lines), infected, nontreated (I; solid lines), and infected, benznidazole-treated (IBz; shaded curves) mice in a representative experiment. The solid horizontal lines depict positive staining defined by an immunoglobulin isotype control. (c) Individual absolute CD4⁺CD62L⁻ and CD8⁺CD62L⁻ numbers are shown, with solid horizontal lines representing median values. CD62L phenotyping was done in three experiments, with similar results, by using three mice from each group.

FIG. 5.

Benznidazole treatment confers immune protection against a secondary challenge with T. cruzi. The survival rate depicts data for 40 infected, benznidazole-treated, reinfected mice (filled circles) and 21 primary infected control mice of the same age (filled squares) in two independent experiments.