Trypanosoma Infection Favors Brucella Elimination via IL-12/IFNγ-Dependent Pathways

Abstract

This study develops an original co-infection model in mice using Brucella melitensis, the most frequent cause of human brucellosis, and Trypanosoma brucei, the agent of African trypanosomiasis. Although the immunosuppressive effects of T. brucei in natural hosts and mice models are well established, we observed that the injection of T. brucei in mice chronically infected with B. melitensis induces a drastic reduction in the number of B. melitensis in the spleen, the main reservoir of the infection. Similar results are obtained with Brucella abortus- and Brucella suis-infected mice and B. melitensis-infected mice co-infected with Trypanosoma cruzi, demonstrating that this phenomenon is not due to antigenic cross-reactivity. Comparison of co-infected wild-type and genetically deficient mice showed that Brucella elimination required functional IL-12p35/IFNγ signaling pathways and the presence of CD4⁺ T cells. However, the impact of wild type and an attenuated mutant of T. brucei on B. melitensis were similar, suggesting that a chronic intense inflammatory reaction is not required to eliminate B. melitensis. Finally, we also tested the impact of T. brucei infection on the course of Mycobacterium tuberculosis infection. Although T. brucei strongly increases the frequency of IFNγ⁺CD4⁺ T cells, it does not ameliorate the control of M. tuberculosis infection, suggesting that it is not controlled by the same effector mechanisms as Brucella. Thus, whereas T. brucei infections are commonly viewed as immunosuppressive and pathogenic, our data suggest that these parasites can specifically affect the immune control of Brucella infection, with benefits for the host.

Keywords: Brucella abortus; Brucella melitensis; Mycobacterium tuberculosis; Trypanosoma brucei brucei; Trypanosoma cruzi; brucellosis; infection control.

Figure 1

Brucella persisted in CD11c⁺ reservoir cells in the spleen of chronically infected wild-type C57BL/6 mice. Wild-type and DTR-CD11c C57BL/6 mice received i.n. 2 × 10⁴ CFU of mCherry-Brucella melitensis in PBS or PBS alone (control group). 26 or 48 days later, the mice received 500 ng of diphtheria toxin by i.p. route in 500 µl of PBS or PBS alone. Two days later (28 or 50 days post Brucella infection), the mice were sacrificed, and the spleens were harvested. (A) Flow cytometry analysis of CD11c and MHCII expression on spleen cells. The data show a representative dot plot for individual mice. (B) The data represent the number of colony-forming units per gram of spleen for each group of mice at the indicated time of infection. Gray bars represent the median. n denotes the number of mice used for each group (*p < 0.05, **p < 0.01). These results are representative of at least two independent experiments.

Figure 2

Brucella reservoir cells were resistant to the protective memory response. Wild-type C57BL/6 mice were infected i.n. with 2 × 10⁴ CFU of wild-type Brucella melitensis and challenged i.p. or i.n. with 2 × 10⁴ CFU of mCherry-B. melitensis. The mice were euthanized at the selected time, and the spleen was harvested, as described in panel (A). The data in panels (B,C) represent the number of colony-forming units per gram of spleen for each group of mice. Gray bars represent the median. n denotes the number of mice used for each group (***p < 0.001). These results are representative of at least two independent experiments.

Figure 3

Trypanosoma brucei co-infection strongly reduced the CFU level of Brucella melitensis in the spleen. Wild-type C57BL/6 mice were infected i.n. with PBS or 2 × 10⁴ CFU of mCherry-B. melitensis and, at 7 or 45 days postinfection, received an i.p. injection of PBS or 5,000 T. brucei or PBS alone, as described in panel (A). The data in panel (B) represent the number of T. brucei per milliliters of blood. The data in panels (C,D) represent the number of colony-forming units per gram of spleen. Note that, to avoid a possible effect of repeated blood sampling, mice that were bled to measure the number of parasites in the blood were not used to measure the number of Brucella CFUs in the spleen. Gray bars represent the median. n denotes the number of mice used for each group (**p < 0.01, ***p < 0.001). These results are representative of at least two independent experiments.

Figure 4

Impact of Trypanosoma brucei co-infection on Brucella suis and Brucella abortus infection and impact of Trypanosoma cruzi on Brucella melitensis infection. Wild-type C57BL/6 mice were infected i.n. with 2 × 10⁴ CFU of B. abortus (A) or B. suis (B) and, at 7 days postinfection, received an i.p. injection of 5,000 T. brucei in 200 µl of PBS or PBS alone. (C) Wild-type C57BL/6 mice were infected i.n. with 2 × 10⁴ CFU of mCherry-B. melitensis and, at 7 days postinfection, received an i.p. injection of 1,000 T. cruzi in 200 µl of PBS or PBS alone. The mice were euthanized at 28 days post Brucella infection, and the spleen was harvested. The data represent the number of colony-forming units per gram of spleen. Gray bars represent the median. n denotes the number of mice used for each group. These results are representative of at least two independent experiments (***p < 0.001).

Figure 5

Elimination of Brucella during Trypanosoma brucei infection required IL-12 and CD4⁺ T cells. (A) Wild-type and various genetically deficient C57BL/6 mice were infected i.n. with 2 × 10⁴ CFU of mCherry-Brucella melitensis and, at 7 days postinfection, received an i.p. injection of 5,000 T. brucei in 200 µl of PBS (co-infection group) or PBS alone (B. melitensis group). The mice were euthanized at 28 days post Brucella infection, and the spleen was harvested (A). The data represent the number of colony-forming units per gram of spleen. Gray bars represent the median. n denotes the number of mice used for each group. (B,C) The data represent the frequency of IFNγ- and iNOS-producing cells in the spleen as determined by flow cytometry analysis. The control (cont) group consisted of naive mice receiving PBS only (**p < 0.01, ***p < 0.001). These results are representative of at least two independent experiments.

Figure 6

Impact of Trypanosoma brucei co-infection on the course of Mycobacterium tuberculosis infection in mice. Wild-type C57BL/6 mice were infected by aerosol with 5 × 10³ CFU of M. tuberculosis and, at 15 or 80 days postinfection, received an i.p. injection of 5,000 T. brucei in 200 µl of PBS (M. tuberculosis/T. brucei group) or PBS alone (M. tuberculosis group). The mice were euthanatized at the selected time post M. tuberculosis infection, and the spleen was harvested, as described in panels (A,B). The data represent the frequency of IFNγ-producing cells in the spleen as determined by flow cytometry analysis. (C,D) The data represent the number of mRLU/g of spleen. Gray bars represent the median. n denotes the number of mice used for each group (***p < 0.001). These results are representative of at least two independent experiments.

Figure 7

Attenuated dominant-negative adenylate cyclase (DNac) mutant of Trypanosoma brucei induced the elimination of Brucella. Wild-type C57BL/6 mice were infected i.n. with 2 × 10⁴ CFU of mCherry-Brucella melitensis and, at 7 days postinfection, received an i.p. injection of 5,000 wild-type T. brucei or attenuated DNac mutant of T. brucei in 200 µl of PBS or PBS alone. The mice were euthanized at 12, 19, and 28 days post Brucella infection and the spleen was harvested, as described in panel (A,B). The data represent the number of colony-forming units per gram of spleen. Gray bars represent the median. n denotes the number of mice used for each group. (C,D) The data represent the mean of the frequency of IFNγ-producing cells (C) and CD3⁺CD4⁺ IFNγ-producing cells (D) in the spleen from five individual spleens as determined by flow cytometry analysis for each group. n denotes the number of mice used for each group (*p < 0.05, **p < 0.01, ***p < 0.001). These results are representative of at least two independent experiments.

Figure 8

Trypanosoma brucei co-infection did not affect the protective memory against Brucella melitensis. As described in panel (A), wild-type C57BL/6 mice were infected i.p. or i.n. with 2 × 10⁴ CFU of wild-type B. melitensis and treated with antibiotic at 28 days postinfection for 15 days. At 65 days, the mice received an i.p. injection of 5,000 T. brucei in 200 µl of PBS (Sec Br/T. brucei group) or PBS alone (Sec Br cont group) were treated with Berenil at 86 days and were challenged at 102 days with i.p. or i.n. injection of 2 × 10⁴ CFU of mCherry-B. melitensis. The pri Br cont group received only PBS, antibiotic, and Berenil until the Brucella challenge. (B,C) i.p. model: the data represent the number of colony-forming units per milliliter of blood or colony-forming units per gram of spleen at the selected time post challenge, as indicated. (D) i.n. model: the data represent the number of colony-forming units per gram of spleen. Gray bars represent the median. n denotes the number of mice used for each group (**p < 0.01, ***p < 0.001). These results are representative of at least two independent experiments.