An Animal Model of Acute and Chronic Chagas Disease With the Reticulotropic Y Strain of Trypanosoma cruzi That Depicts the Multifunctionality and Dysfunctionality of T Cells

Abstract

Chagas disease (ChD), a complex and persistent parasitosis caused by Trypanosoma cruzi, represents a natural model of chronic infection, in which some people exhibit cardiac or digestive complications that can result in death 20-40 years after the initial infection. Nonetheless, due to unknown mechanisms, some T. cruzi-infected individuals remain asymptomatic throughout their lives. Actually, no vaccine is available to prevent ChD, and treatments for chronic ChD patients are controversial. Chronically T. cruzi-infected individuals exhibit a deterioration of T cell function, an exhaustion state characterized by poor cytokine production and increased inhibitory receptor co-expression, suggesting that these changes are potentially related to ChD progression. Moreover, an effective anti-parasitic treatment appears to reverse this state and improve the T cell response. Taking into account these findings, the functionality state of T cells might provide a potential correlate of protection to detect individuals who will or will not develop the severe forms of ChD. Consequently, we investigated the T cell response, analyzed by flow cytometry with two multicolor immunofluorescence panels, to assess cytokines/cytotoxic molecules and the expression of inhibitory receptors, in a murine model of acute (10 and 30 days) and chronic (100 and 260 days) ChD, characterized by parasite persistence for up to 260 days post-infection and moderate inflammation of the colon and liver of T. cruzi-infected mice. Acute ChD induced a high antigen-specific multifunctional T cell response by producing IFN-γ, TNF-α, IL-2, granzyme B, and perforin; and a high frequency of T cells co-expressed 2B4, CD160, CTLA-4, and PD-1. In contrast, chronically infected mice with moderate inflammatory infiltrate in liver tissue exhibited monofunctional antigen-specific cells, high cytotoxic activity (granzyme B and perforin), and elevated levels of inhibitory receptors (predominantly CTLA-4 and PD-1) co-expressed on T cells. Taken together, these data support our previous results showing that similar to humans, the T. cruzi persistence in mice promotes the dysfunctionality of T cells, and these changes might correlate with ChD progression. Thus, these results constitute a model that will facilitate an in-depth search for immune markers and correlates of protection, as well as long-term studies of new immunotherapy strategies for ChD.

Keywords: Chagas disease; T cell response; clonal exhaustion; immune activation; inhibitory receptors; multifunctionality.

Figure 1

Analysis of the parasite load and inflammatory cell infiltration in mice with acute and chronic experimental ChD. (A) Parasite loads in the colon, heart, liver, skeletal muscle, and blood samples from mice with acute (left panel) and chronic (right panel) T. cruzi infections. The bar graphs show the median parasite equivalent per 50 ng of DNA (LOG₁₀) in each tissue from each mouse. The dotted line represents the cut-off for the limit of detectable quantification (LOQ) based on serially diluted T. cruzi-spiked tissue DNA as described in the Materials and Methods (0.1 parasite equivalents per 50 ng of DNA). (B) Images of representative histopathological staining of cross-sections of tissues from mice with acute and chronic experimental ChD. (C) Inflammatory infiltrate scores in the colon, heart, liver, and skeletal muscle tissues from mice with acute (left panel) and chronic (right panel) T. cruzi infections. The bar graphs show the average inflammatory infiltrate scores in the tissues. The inflammatory infiltrate score was obtained as described in the Materials and Methods. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, Mann-Whitney U-test.

Figure 2

Functional activity profiles of T. cruzi–specific CD4⁺ Th1 cells from mice with acute and chronic experimental ChD. (A) Representative dot plot (left panel) and percentages (right panel) of CD4⁺ T cells producing IFN-γ, TNF-α, or IL-2 in mice with acute and chronic experimental ChD. The gates applied for the identification of cytokine production on the total population of CD4⁺ T cells were defined according to the cells cultured with Mock for each mouse. The number on the upper right side corresponds to the frequency detected in spleen cells cultured with Mock or TcSAs. The dotted line represents the cut-off for the assessment of a positive cytokine response, as described in the Materials and Methods. (B) Proportions of the functional profiles of CD4⁺ T cells with one, two, or three functions after stimulation with TcSA. The boxes (25–75th percentiles) and whiskers (minimum to maximum) show the median percentages (A) or proportions (B) of Ag-specific CD4⁺ T cells. The pie chart depicts the median proportion of Ag-specific CD4⁺ T cells, and the color depicts the T cells with one, two, or three functions. *p < 0.05 and **p < 0.01, Mann-Whitney U-test (boxes and whiskers) or permutation test (pie charts).

Figure 3

Functional activity profiles of T. cruzi–specific CD8⁺ Tc1 cells from mice with acute and chronic experimental ChD. (A) Representative dot plot (left panel) and percentages (right panel) of CD8⁺ T cells producing IFN-γ, TNF-α, IL-2, granzyme B, or perforin in mice with acute and chronic experimental ChD. The gates applied for the identification of cytokine, granzyme B or perforin production on the total population of CD8⁺ T cells were defined according to the cells cultured with Mock for each mouse. The number on the upper right side corresponds to the frequency of molecules detected in spleen cells cultured with Mock or TcSAs. The dotted line represents the cut-off for the assessment of a positive response, as described in the Materials and Methods. (B) Proportions of the functional profiles of CD8⁺ T cells with one, two, or three functions after stimulation with TcSA. (C) Proportions of the cytotoxic profiles of CD8⁺ T cells producing granzyme B or perforin from mice with acute and chronic experimental ChD stimulated with TcSA. The boxes (25–75th percentiles) and whiskers (minimum to maximum) show the median percentages (A) or proportions (B,C) of Ag-specific CD8⁺ T cells. The pie chart depicts the median proportion of Ag-specific CD8⁺ T cells, and the color depicts the T cells with one, two, or three functions (B) or the production of granzyme B, perforin, or both (C). *p < 0.05 and **p < 0.01, Mann-Whitney U-test (boxes and whiskers) or permutation test (pie charts). GzmB, granzyme B; UD, undetected.

Figure 4

Inhibitory receptor expression and co-expression on CD4⁺ T cells from mice with acute and chronic experimental ChD. (A) Representative dot plot of the gating strategy for CD4⁺ T cell expressing 2B4, CD160, CTLA-4, or PD-1. The number on the upper right side corresponds to the frequency of molecules detected in CD4⁺ T cells. (B) Percentages (top panel) and median proportions (bottom panel) of CD4⁺ T cells expressing 2B4, CD160, CTLA-4, or PD-1 in mice with experimental ChD. The boxes (25–75th percentiles) and whiskers (minimum to maximum) show the median percentages and range of expression of inhibitory receptors on CD4⁺ T cells. The pie chart depicts the median proportion of inhibitory receptors. *p < 0.05 and **p < 0.01, Mann-Whitney U-test (boxes and whiskers) or permutation test (pie charts).

Figure 5

Inhibitory receptor expression and co-expression on CD8⁺ T cells from mice with acute and chronic experimental ChD. (A) Representative dot plot of the gating strategy for CD8⁺ T cell expressing 2B4, CD160, CTLA-4, or PD-1. The number on the upper right side corresponds to the frequency of molecules detected in CD8⁺ T cells. (B) histopathological Percentages (top panel) and median proportions (bottom panel) of CD8⁺ T cells expressing 2B4, CD160, CTLA-4, or PD-1 in mice with experimental ChD. The boxes (25–75th percentiles) and whiskers (minimum to maximum) show the median percentages and range of expression of inhibitory receptors on CD8⁺ T cells. The pie chart depicts the median proportion of inhibitory receptors. *p < 0.05 and **p < 0.01, Mann-Whitney U-test (boxes and whiskers) or permutation test (pie charts).

Figure 6

Effector function of T cells and inhibitory receptor co-expression on T cells according to the inflammatory infiltrate score in the liver tissue in chronically T. cruzi-infected mice. Comparison of the effector function of T cells and the inhibitory receptor co-expression on T cells between chronically T. cruzi-infected mice with moderate and low inflammatory infiltrate in the liver tissue. The comparative analysis with CD4⁺ and CD8⁺ T cells is shown in (A,B), respectively. The bar graphs show the median percentages of total cytokine-producing T. cruzi-specific T cells, percentages of multifunctional T cells endowed with two and three functions, or percentages of T cells co-expressing three and four inhibitory receptors on T cells. *p < 0.05, Mann-Whitney U-test. FC, fold change.

Figure 7

Schematic depicting the multifunctionality and dysfunctionality of T cells in an animal model of acute and chronic Chagas disease. In the present study, whether experimental acute (10 and 30 days) and chronic (100 and 260 days) ChD alters the CD4⁺ Th1 and CD8⁺ Tc1 cell multifunctional capacities and inhibitory receptor co-expression on T cells was analyzed in a murine model. Representative results of CD8⁺ T cells across the experimental infection conditions with T. cruzi are shown. The black, blue, and red lines show the median proportions of functional and cytotoxic activity and the co-expression of inhibitory receptors, respectively. The colors in the pie charts depict the number of T cell functions, as well as the inhibitory receptors co-expressed on CD8⁺ T cells in the acute and chronic phases of experimental ChD. Based on our results, an acute T. cruzi infection induces a multifunctional T cell response and high inhibitory receptor co-expression on T cells, whereas chronic T. cruzi infection is characterized by a monofunctional T cell response, high cytotoxic activity, and high levels of inhibitory receptor co-expression. GzmB, granzyme B; Perf, perforin.