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. 2019 Nov 8;15(11):e1008145.
doi: 10.1371/journal.ppat.1008145. eCollection 2019 Nov.

Trypanosoma Brucei Infection Protects Mice Against Malaria

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Free PMC article

Trypanosoma Brucei Infection Protects Mice Against Malaria

Margarida Sanches-Vaz et al. PLoS Pathog. .
Free PMC article

Abstract

Sleeping sickness and malaria are parasitic diseases with overlapping geographical distributions in sub-Saharan Africa. We hypothesized that the immune response elicited by an infection with Trypanosoma brucei, the etiological agent of sleeping sickness, would inhibit a subsequent infection by Plasmodium, the malaria parasite, decreasing the severity of its associated pathology. To investigate this, we established a new co-infection model in which mice were initially infected with T. brucei, followed by administration of P. berghei sporozoites. We observed that a primary infection by T. brucei significantly attenuates a subsequent infection by the malaria parasite, protecting mice from experimental cerebral malaria and prolonging host survival. We further observed that an ongoing T. brucei infection leads to an accumulation of lymphocyte-derived IFN-γ in the liver, limiting the establishment of a subsequent hepatic infection by P. berghei sporozoites. Thus, we identified a novel host-mediated interaction between two parasitic infections, which may be epidemiologically relevant in regions of Trypanosoma/Plasmodium co-endemicity.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. T. brucei infection protects mice from malaria.
(A) Assessment of P. berghei prepatency period following inoculation of 500 P. berghei sporozoites into naïve BALB/cByJ mice (Pb—blue line) or BALB/cByJ mice infected 5 days earlier with T. brucei (Tb/Pb—green line). Percentage of mice displaying P. berghei parasitemia, as measured by flow cytometry. The pooled data of 10 mice from in two independent experiments is shown. (B) Assessment of P. berghei prepatency period following inoculation of 500 P. berghei sporozoites into naïve C57BL/6J mice (Pb—blue line) or C57BL/6J mice infected 5 days earlier with T. brucei (Tb/Pb—green line). Percentage of mice displaying P. berghei parasitemia, as measured by flow cytometry. The pooled data of 10 mice from two independent experiments is shown. (C) Mouse survival following inoculation of 500 P. berghei sporozoites into naïve C57BL/6J mice (Pb—blue line) or C57BL/6J mice infected 5 days earlier with T. brucei (Tb/Pb—green line). Percentage of live mice from a pool of 10 mice employed in two independent experiments. (D) Assessment of P. berghei parasitemia after inoculation of 1 x 106 iRBCs into naïve C57BL/6J mice (Pb—blue line) or C57BL/6J mice infected 5 days earlier with T. brucei (Tb/Pb—green line). The mean bioluminescence and SEM of the pooled data of 10 mice from two independent experiments is shown. (E) Mouse survival following inoculation of 1 x 106 iRBCs into naïve C57BL/6J mice (Pb—blue line) or C57BL/6J mice infected 5 days earlier with T. brucei (Tb/Pb—green line). Percentage of live mice from a pool of 10 mice from two independent experiments. For A and B, the Mantel-Cox (log rank) test was employed to compare the onset of P. berghei parasitemia, indicating statistically significant differences for the Tb/Pb group compared to the Pb control group. For C and E, the Mantel-Cox (log rank) test was employed to compare survival curves, indicating statistically significant differences for Tb/Pb compared to the Pb control, and the time window for ECM development is depicted by the grey-shaded area.
Fig 2
Fig 2. T. brucei attenuates hepatic infection by P. berghei.
(A) Schematic illustration of the co-infection experimental design. The arrows indicate the times of T. brucei and/or P. berghei inoculation, and of liver dissection for quantification of P. berghei liver load. (B) P. berghei liver infection load (bars–primary YY axis) determined by qRT-PCR 46 h after sporozoite injection into naïve mice (blue bar) or mice previously infected by T. brucei (green bars), and T. brucei parasitemia (dots–secondary YY axis) determined daily by microscopy. All P. berghei infections were performed on the same day, with prior T. brucei infections staggered to give a consistent day for subsequent P. berghei infection. The time points indicated on the XX-axis correspond to the number of days that elapsed between T. brucei inoculation and sporozoite injection. Bars represent the mean values of two independent experiments and error bars indicate the SEM. The one-way ANOVA with post-test Dunnett was employed to assess the statistical significance of differences between the experimental groups. ns, not significant, * P < 0.05 and **** P < 0.0001. (C) Representative bioluminescence images of mouse livers 46 h after inoculation of 3 x 104 P. berghei sporozoites into either naïve mice (Pb—top) or mice infected 5 days earlier with T. brucei (Tb/Pb—bottom). (D) Quantification of the P. berghei liver infection load measured by bioluminescence 46 h after sporozoite injection into naïve mice (blue bar) or mice previously infected by T. brucei (green bar). Bars represent the mean values of three independent experiments and error bars indicate the SEM. The Mann-Whitney test was employed to assess the statistical significance of differences between experimental groups (**** P < 0.0001). (E) Assessment of P. berghei prepatency period following inoculation of 3 x 104 sporozoites into naïve mice (Pb—blue line) or mice infected 5 days beforehand with T. brucei (Tb/Pb—green line). Percentage of mice displaying P. berghei parasitemia, as measured by flow cytometry. The pooled data from 10 mice employed in two independent experiments is shown The Mantel-Cox (log rank) test was employed to compare the onset of P. berghei parasitemia curves, indicating statistically significant differences for Tb/Pb compared to the Pb control. (F) Mouse survival following inoculation of 3 x 104 P. berghei sporozoites into naïve C57BL/6J mice (Pb—blue line) or C57BL/6J mice infected 5 days earlier with T. brucei (Tb/Pb—green line). Percentage of live mice from a pool of 10 mice employed in two independent experiments. The Mantel-Cox (log rank) test was employed to compare survival curves, indicating statistically significant differences for Tb/Pb compared to the Pb control, and the time window for ECM development is depicted by the grey-shaded area.
Fig 3
Fig 3. T. brucei impairs early liver stage of P. berghei infection.
(A) P. berghei liver infection load quantified by qRT-PCR 30 min, 2 h, 6 h, 12 h, 24 h and 46 h after injection of 3 x 104 P. berghei sporozoites into naïve mice (Pb–blue bars) or mice infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of three to five independent experiments and error bars indicate the SEM. The two-way ANOVA with post-test Bonferroni was employed to assess the statistical significance of differences between experimental groups. *** P < 0.001 and **** P < 0.0001. (B) Number of P. berghei-infected hepatocytes per square millimeter of liver section quantified by immunofluorescence microscopy 6 h, 12 h, 24 h and 46 h after injection of 3 x 104 P. berghei sporozoites into naïve mice (Pb–blue bars) or mice infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of one representative experiment out of two independent experiments and error bars indicate the SEM. The two-way ANOVA with post-test Bonferroni was employed to assess the statistical significance of differences between experimental groups. *** P < 0.001 and **** P < 0.0001. (C) Representative confocal microscopy images of EEFs at 12 h, 24 h and 46 h after injection of 3 x 104 P. berghei sporozoites into naïve mice or mice infected 5 days earlier with T. brucei. White: Hoechst—nuclear staining; green: P. berghei GFP labeling showing the parasite heat shock protein 70; purple: P. berghei UIS4 labeling showing the parasitophorous vacuole membrane. Scale bars, 10 μm. (D) EEF area at 12 h, 24 h and 46 h after injection of 3 x 104 P. berghei sporozoites into naïve mice (Pb—blue dots) or mice infected 5 days earlier with T. brucei (Tb/Pb—green dots), assessed by immunofluorescence microscopy. Results are expressed as the mean values of one representative experiment out of two independent experiments and error bars indicate the SEM.
Fig 4
Fig 4. T. brucei-activated lymphocytes are required to inhibit P. berghei liver infection.
(A) Representative microphotographs of liver from non-infected and T. brucei-infected mice (3–4 mice per time-point); depicted are the inflammatory cell infiltrates (black arrow) and hepatocellular damage/apoptosis seen at different time-points (XX axis) of T. brucei infection. Hematoxylin and Eosin. Original magnification 10x (upper panel; scale bar, 200 μm) and 40x (lower panel; scale bar, 50 μm). (B) Graphic representation of the severity of inflammatory cell infiltration (bars) and hepatocellular damage (dots), both scored through histopathology using a 5-tier system with 0–4 grading scale (0, absent; 1, minimal; 2, mild; 3, moderate; 4, marked). Time points indicated on the XX-axis correspond to the days of T. brucei infection. (C) P. berghei liver infection load quantified by qRT-PCR 6 h after injection of 3 x 104 P. berghei sporozoites into naïve mice (Pb–blue bars) or mice infected 5 days earlier with T. brucei (Tb/Pb–green bars), non- or clodronate-treated 48 h prior to P. berghei infection. Bars represent the mean values of four independent experiments and error bars indicate the SEM. (D) P. berghei liver infection load quantification by qRT-PCR 6 h after injection of 3 x 104 P. berghei sporozoites into wild-type and RAG2-/- mice, either naïve (Pb–blue bars) or infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of three independent experiments and error bars indicate the SEM. (E) P. berghei liver infection load quantification by qRT-PCR 6 h after injection of 3 x 104 P. berghei sporozoites into wild-type, TCRβ-/-, TCRδ-/-, JHT-/- and NKT depleted mice, either naïve (Pb–blue bars) or infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of two to three independent experiments with error bars indicating the SEM. For C to E, the Mann-Whitney test was employed to assess the statistical significance of differences between the experimental groups. ns, not significant, * P < 0.05, *** P < 0.001 and **** P < 0.0001.
Fig 5
Fig 5. T. brucei elicits a strong immune response systemically and in the liver.
(A) IFN-γ, IL-1B, GM-CSF, IL-2, IL-4, IL-6, IL-10, IL-12p70, MCP-1 and TNF-α quantification in serum by immunoassay from mice non-infected or infected only with T. brucei. The time points indicated on the XX-axis correspond to the days of T. brucei infection. Dots represent the mean values of three to four mice from one independent experiment with error bars indicating the SEM. (B) Quantification of IFN-γ by immunoassay in the lung (light grey bar) and liver (dark grey bar) of both non-infected mice and mice infected for two and five days with T. brucei. The time points indicated on the XX-axis correspond to the days of T. brucei infection. Bars represent the mean values of three to four mice from one independent experiment and error bars indicate the SEM. (C-D) Multi-parameter flow cytometry-based quantification of IFN-γ-producing TCRαβ, TCRγδ and NK1.1+ lymphocytes in the spleens (C) and livers (D) of mice infected only with P. berghei (Pb—blue dots), infected with P. berghei on the fifth day of T. brucei infection (Tb/Pb–green dots) and infected for 5 days only with T. brucei (Tb—yellow dots). The time points indicated on the XX-axis correspond to the time after injection of 3 x 104 P. berghei sporozoites, with 0 h corresponding to day 5 of T. brucei infection. Dots represent the mean values of four mice from one independent experiment with error bars indicating SD.
Fig 6
Fig 6. IFN-γ mediates the inhibitory effect of T. brucei on P. berghei liver infection.
(A) P. berghei liver infection load quantification by qRT-PCR 6 h after injection of 3 x 104 P. berghei sporozoites into naïve mice (blue bar), IFN-γ-treated mice (blue bar), or mice infected 5 days earlier with T. brucei (green bar). Bars represent the mean values of four independent experiments and error bars indicate the SEM. The one-way ANOVA with post-test Dunnett was employed to assess the statistical significance of differences between experimental groups. *** P < 0.001 and **** P < 0.0001. (B) P. berghei liver infection load quantification by qRT-PCR 6 h after injection of 3 x 104 P. berghei sporozoites into wild-type and IFN-γ-/- mice, either naïve (Pb–blue bars) or infected 5 days earlier with T. brucei (Tb/Pb–green bars). Bars represent the mean values of three independent experiments and error bars indicate the SEM. The Mann-Whitney test was employed to assess the statistical significance of differences between experimental groups. ns, not significant and **** P < 0.0001.

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