The liver plays a major role in clearance and destruction of blood trypomastigotes in Trypanosoma cruzi chronically infected mice

Abstract

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5x10(6) trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1(+), CD8(+) and CD4(+) cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4(+) and CD8(+) cells were activated, increased frequencies of CD69(+)CD8(+), CD69(+)CD4(+) and CD25(+)CD122(+)CD4(+) cells were observed at 24 and 48 h after challenge, and of CD25(-)CD122(+)CD4(+) cells at 48 h. The major role of CD4(+) cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-gamma-producing CD4(+) cells 24 h after challenge. In contrast, liver CD8(+) cells produced little IFN-gamma, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge.

Figure 1. Tissue parasitism in chronic mice challenged with T. cruzi trypomastigotes.

Chronic mice were analyzed for tissue parasitism 1 h and 48 h after i.v. challenge with 5×10⁶ trypomastigotes. Unchallenged chronic mice were used as controls. (A) Comparison of parasite loads in different tissues using real-time PCR for T. cruzi DNA. Average values and standard error for each experimental group (n = 3) are represented. Differences between groups were evaluated by unpaired T test (* p<0.05, ** p<0.0001, compared to 1 h). (B) Presence of live T. cruzi parasites in the liver of challenged chronic mice. Parasites in liver tissue fragments of 1.6 and 0.4 mg weight were amplified by LIT culture. Results represent the frequency of T. cruzi-positive cultures in each group of mice (quadruplicate cultures per liver; n = 3). The results are representative of two experiments.

Figure 2. Presence of T. cruzi parasites at the liver parenchyma of chronic mice challenged with trypomastigotes.

T. cruzi-infected chronic mice and non-infected controls were injected i.v. with 5×10⁶ culture trypomastigotes and 1 h later, the animals were sacrificed and the liver tissue screened for the presence of parasite antigen by immunohistochemistry as described in material and methods. Unchallenged chronic mice were included as controls. Pictures are representative of three mice in each group. Challenged control mice (A); unchallenged chronic mice (B); challenged chronic mice (C). Bar: 20 µm.

Figure 3. Histopathological analysis of liver in unchallenged and challenged chronic mice.

C57BL/6 mice were infected with T. cruzi and after 8 months challenged i.v. with 5×10⁶ homologous culture trypomastigotes. Chronic mice (A) and chronic mice, 24 h (B) or 48 h (C) after challenge. Lesions are representative of at least 6–10 animals in each group. Bar: 100 µm.

Figure 4. Phenotypic characterization of cellular infiltrates in the liver of unchallenged and challenged chronic mice.

C57BL/6 mice infected for 7–10 months with T. cruzi parasites were challenged i.v. with 5×10⁶ homologous trypomastigotes and, after 24 and 48 h, liver leukocytes analyzed by flow cytometry. Control and chronic mice were also included. (A) Mean±SD (n = 3) of total liver numbers of B (B220⁺), Mac1⁺ (Mac1⁺CD4⁻CD8⁻B220⁻), NK (NK1.1⁺CD4⁻CD8⁻), CD4⁺ and CD8⁺ cells; Differences between groups were evaluated by ANOVA and Tukey's multiple comparison tests (* p<0.05 and ** p<0.005, compared to chronic mice). A representative experiment out of three is shown. (B) CD69 expression by gated CD4⁺ and CD8⁺ cells. Numbers indicate the mean±SD (n = 3) of the percentage of CD69⁺ cells. Representative histograms of one experiment out of two are shown.

Figure 5. Expression of CD25 and CD122 by CD4⁺ cells in the liver of chronic mice challenged with T. cruzi trypomastigotes.

C57BL/6 mice infected for 7 months with T. cruzi were challenged i.v. with 5×10⁶ trypomastigotes and, after 24 and 48 h, the liver leukocytes analyzed by flow cytometry. (A) Expression of CD25 and CD122 by CD4⁺ cells. Frequency of large cells (B) and total liver cell numbers (C) of CD4⁺ subpopulations defined by expression of CD25 and CD122 molecules. Numbers in dot blots indicate the mean frequencies of cells in quadrants. Numbers in histograms indicate the mean frequencies of large cells among cell subsets defined by quadrants in A. Data in C correspond to the mean±SD of total liver cell numbers of the indicated subpopulations. A representative experiment (n = 3) of two is shown. Differences between groups were evaluated by ANOVA and Tukey's multiple comparison tests (* p<0.01; ** p<0.001, compared to chronic mice).

Figure 6. IFN-γ production by CD4⁺ and CD8⁺ liver cells after challenge of chronic mice with T. cruzi tripomastigotes.

Frequency (A) and total numbers (B) of IFNγ-producing cells in gated CD4⁺ and CD8⁺ cells of unchallenged and T. cruzi-challenged chronic mice. The spontaneous and anti-CD3/CD28-stimulated production of IFNγ was evaluated 24 and 48 h after challenge. Numbers inside dot-blots represent the mean±SD (n = 3) of IFNγ-producing cell frequencies. Bars in (B) represent the mean±SD (n = 3) of IFNγ-producing cell numbers per liver of mice in (A); * p<0.05; ** p<0.01; *** p<0.001 (compared to chronic mice by ANOVA and Tukey's multiple comparison tests). # p<0.05; ## p<0.0001 (compared to spontaneous production by unpaired T test). A representative experiment out of two is shown.