MIF contributes to Trypanosoma brucei associated immunopathogenicity development

Abstract

African trypanosomiasis is a chronic debilitating disease affecting the health and economic well-being of many people in developing countries. The pathogenicity associated with this disease involves a persistent inflammatory response, whereby M1-type myeloid cells, including Ly6C(high) inflammatory monocytes, are centrally implicated. A comparative gene analysis between trypanosusceptible and trypanotolerant animals identified MIF (macrophage migrating inhibitory factor) as an important pathogenic candidate molecule. Using MIF-deficient mice and anti-MIF antibody treated mice, we show that MIF mediates the pathogenic inflammatory immune response and increases the recruitment of inflammatory monocytes and neutrophils to contribute to liver injury in Trypanosoma brucei infected mice. Moreover, neutrophil-derived MIF contributed more significantly than monocyte-derived MIF to increased pathogenic liver TNF production and liver injury during trypanosome infection. MIF deficient animals also featured limited anemia, coinciding with increased iron bio-availability, improved erythropoiesis and reduced RBC clearance during the chronic phase of infection. Our data suggest that MIF promotes the most prominent pathological features of experimental trypanosome infections (i.e. anemia and liver injury), and prompt considering MIF as a novel target for treatment of trypanosomiasis-associated immunopathogenicity.

Figure 1. MIF expression exhibits biphasic profiles during T. brucei infection.

Mif gene expression levels in the liver (A), spleen (B), bone marrow (BM) (C) and serum MIF protein levels (D) during infection in C57Bl/6 mice. The dashed line delineates the transition from the acute to the chronic phase of infection. Gene expression levels were normalized against s12 and expressed relative to expression levels in non-infected mice. Results are representative for 2–3 independent experiments and presented as mean of 2–3 individual mice ± SEM.

Figure 2. MIF deficiency confers protection and reduces inflammatory immune responses during T. brucei infection.

Parasitemia (A) and survival (B) during infection in C57Bl/6 (WT, black box; Mif ^−/−, white box) Mice. Results are representative of 2–5 independent experiments and expressed as mean of 5 individual mice ± SEM. (C) Serum cytokine levels of IFN-γ (upper left panel) (1 U = 100 pg), TNF (upper right panel), IL-6 (lower left panel) and IL-10 (lower right panel) in Mif ^−/− (white bars) and WT (black bars) mice. The dashed line delineates the transition from the acute to the chronic phase of infection. Results are representative of 3 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01, p-value: ***≤0.001).

Figure 3. MIF deficiency reduces liver damage and alters liver cell composition during T. brucei infection.

(A) Liver weight (left panel), serum ALT (middle panel) and AST (right panel) levels in Mif ^−/− (white bars) and WT (black bars) C57Bl/6 mice at day 25 p.i. (B) Representative liver immune cell gating strategy: Ly6c versus CD11b plot following gating on CD45+ and 7AAD- cells allows identifying CD11b⁺Ly6C⁺ cells (middle panel). This population was plotted in a Ly6C versus Ly6G plot to distinguish CD11b⁺Ly6C^highLy6G⁻ inflammatory monocytes and CD11b⁺Ly6C^intLy6G⁺ neutrophils (right panel). Percentage of CD11b⁺Ly6C⁺ cells within the liver CD45+ cells in WT (black bars) and Mif ^−/− (open bars) mice (left panel). (C) Number of CD11b⁺Ly6C^highLy6G⁻ and CD11b⁺Ly6C^intLy6G⁺ cells within the liver CD45+ cells (upper left panels). The dashed line represents values in non-infected mice. Total liver CCL2, KC (CXCL1) and LIX (CXCL5) gene expression levels in Mif ^−/− (white bars) and WT (black bars) mice at day 25 p.i. (lower panels). Gene expression levels are normalised using s12 and expressed relatively to expression levels of non-infected mice. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01).

Figure 4. Anti-MIF treatment reduces serum ALT/AST levels and affects liver cell composition during T. brucei infection.

(A) Serum ALT and AST levels of non-infected (dashed line), isotype control antibody treated (black box) and anti-MIF IgG treated (white box) WT C57Bl/6 mice at day 25 p.i. (B) Total numbers of liver CD11b⁺Ly6C^highLy6G⁻ and CD11b⁺Ly6C^intLy6G⁺ cells in the chronic (day 25 p.i.) phase of infection calculated using the gating strategy described in Fig. 3B. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01).

Figure 5. Neutrophil-derived MIF and monocyte-derived MIF contribute to different extent to TNF production and liver injury in T. brucei infected mice.

TNF and MIF levels from liver cell cultures (A, B), serum ALT (C) and AST (D) levels of infected (day 24 p.i.) Mif ^−/− mice (white box), of infected Mif ^−/− mice treated with neutrophils from WT (dark grey box) or Mif ^−/−(white hatched box) infected mice, of infected Mif ^−/− mice treated with monocytes from WT (light grey box) or Mif ^−/− (darker grey hatched box) infected mice and of infected WT mice (black box). Results are representative of 3–4 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01, ***: p-value ≤0.001).

Figure 6. MIF deficiency correlates with reduced anemia, restored hemoglobin/serum iron levels and restored iron homeostasis during T. brucei infection.

(A) Anemia development during infection in C57Bl/6 (WT, black box; Mif ^−/−, white box) mice. Results are representative of 2–5 independent experiments and expressed as mean of 3–5 individual mice ± SEM. (B) At day 18 p.i., (left panel) hemoglobin levels and (right panel) serum iron levels in Mif ^−/− (open bars) and WT (black bars) mice. (C) Expression levels of the iron-homeostasis associated genes Hmox1 (iron import), Dmt1 (iron transport), Fth1 (iron storage) and Fpn1 (iron export) were quantified by RT-QPCR in total livers from Mif ^−/− (white bars) and WT (black bars) mice at day 18 p.i. Gene-expression levels are normalised using s12 and expressed relatively to expression levels of non-infected mice. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05).

Figure 7. MIF deficiency correlates with restored/enhanced erythropoiesis during T. brucei infection.

(A) Representative profile for the gating of mature (Ter-119⁺ CD71⁻) and immature (Ter-119⁺ CD71⁺) RBCs in bone marrow of non-infected C57Bl/6 mice (left panel). The percentage of mature RBCs within the total Ter-119⁺ population in bone marrow, spleen and blood at day 18 p.i. (right panels). Dashed line represents percentage of mature RBCs in non-infected mice, which was similar in WT and Mif−/− mice. Results are representative of 3 independent experiments and shown as mean of 3 individual mice ± SEM. (B) Expression levels of genes involved in erythropoiesis in total bone marrow (upper panels) and spleen (lower panels) from Mif ^−/− (white bars) and WT (black bars) mice at day 18 p.i. Gene-expression levels are normalised using s12 and expressed relatively to expression levels of non-infected mice. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01).

Figure 8. MIF deficiency correlates with enhanced terminal RBC differentiation and reduced RBC clearance during T. brucei infection.

(A) Gating strategy used to discriminate different stages of erythroid development, starting from nucleated erythroblasts (P1 (pro), P2 (Basophilic + polychromatic) and P3 (orthochromatic)) till enucleated erythrocytes (P4 (reticulocyte) and P5 (erythrocyte)). 7AAD⁻CD45⁻Ter-119+ cells were selected and plotted in a CD44 versus FSC plot (upper panel). The percentage of the different erythroid populations at day 18 p.i. in WT (black box) and Mif ^−/− (white box) mice is shown for the spleen (B) and bone marrow (C). Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM. (D) RBC clearance in non-infected (WT, black bars; Mif ^−/−, white bars) and day 12 p.i. T. brucei infected (WT, dark grey bars; Mif ^−/−, light grey bars) C57Bl/6 mice following i.v injection with 200 µl GFP⁺ RBCs. At different time points after injection the presence of GFP⁺ RBCs following gating on Ter-119⁺ cells in the blood was evaluated. GFP⁺ RBC numbers were normalized, whereby GFP⁺ RBCs present after 1 day post infection are being referred as 100%. Results are representative of 2 independent experiments and presented as mean of 5 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01).