MIF contributes to Trypanosoma brucei associated immunopathogenicity development

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

MIF-mediated hemodilution promotes pathogenic anemia in experimental African trypanosomosis

Animal African trypanosomosis is a major threat to the economic development and human health in sub-Saharan Africa. Trypanosoma congolense infections represent the major constraint in livestock production, with anemia as the major pathogenic lethal feature. The mechanisms underlying anemia development are ill defined, which hampers the development of an effective therapy. Here, the contribution of the erythropoietic and erythrophagocytic potential as well as of hemodilution to the development of T. congolense-induced anemia were addressed in a mouse model of low virulence relevant for bovine trypanosomosis. We show that in infected mice, splenic extramedullary erythropoiesis could compensate for the chronic low-grade type I inflammation-induced phagocytosis of senescent red blood cells ( RBCs) in spleen and liver myeloid cells, as well as for the impaired maturation of RBCs occurring in the bone marrow and spleen. Rather, anemia resulted from hemodilution. Our data also suggest that the heme catabolism subsequent to sustained erythrophagocytosis resulted in iron accumulation in tissue and hyperbilirubinemia. Moreover, hypoalbuminemia, potentially resulting from hemodilution and liver injury in infected mice, impaired the elimination of toxic circulating molecules like bilirubin. Hemodilutional thrombocytopenia also coincided with impaired coagulation. Combined, these effects could elicit multiple organ failure and uncontrolled bleeding thus reduce the survival of infected mice. MIF ( macrophage migrating inhibitory factor), a potential pathogenic molecule in African trypanosomosis, was found herein to promote erythrophagocytosis, to block extramedullary erythropoiesis and RBC maturation, and to trigger hemodilution. Hence, these data prompt considering MIF as a potential target for treatment of natural bovine trypanosomosis

MIF deficiency reduces liver damage and alters liver cell composition during <i>T. brucei</i> infection.

<p>(A) Liver weight (left panel), serum ALT (middle panel) and AST (right panel) levels in <i>Mif</i>^−/− (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 <i>Mif</i>^−/− (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 <i>Mif</i>^−/− (white bars) and WT (black bars) mice at day 25 p.i. (lower panels). Gene expression levels are normalised using <i>s12</i> 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).</p

MIF deficiency correlates with restored/enhanced erythropoiesis during <i>T. brucei</i> infection.

<p>(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 <i>Mif−/−</i> 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 <i>Mif</i>^−/− (white bars) and WT (black bars) mice at day 18 p.i. Gene-expression levels are normalised using <i>s12</i> 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).</p

MIF deficiency correlates with enhanced terminal RBC differentiation and reduced RBC clearance during <i>T. brucei</i> infection.

<p>(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 <i>Mif</i>^−/− (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; <i>Mif</i>^−/−, white bars) and day 12 p.i. <i>T. brucei</i> infected (WT, dark grey bars; <i>Mif</i>^−/−, 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).</p

Neutrophil-derived MIF and monocyte-derived MIF contribute to different extent to TNF production and liver injury in <i>T. brucei</i> infected mice.

<p>TNF and MIF levels from liver cell cultures (A, B), serum ALT (C) and AST (D) levels of infected (day 24 p.i.) <i>Mif</i>^−/− mice (white box), of infected <i>Mif</i>^−/− mice treated with neutrophils from WT (dark grey box) or <i>Mif</i>^−/−(white hatched box) infected mice, of infected <i>Mif</i>^−/− mice treated with monocytes from WT (light grey box) or <i>Mif</i>^−/− (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).</p

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

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004414#ppat-1004414-g003" target="_blank">Fig. 3B</a>. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM (*: p-values ≤0.05, **: p-values ≤0.01).</p

MIF expression exhibits biphasic profiles during <i>T. brucei</i> infection.

<p><i>Mif</i> 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 <i>s12</i> 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.</p

MIF contributes to heme catabolism and tissue iron accumulation during <i>T</i>. <i>congolense</i> infection.

<p>At 3 months p.i., <b>(A)</b> iron deposition was quantified on spleen and liver sections of WT (black bar) and <i>Mif</i>^<i>-/-</i> (open bar) mice and expressed as % of stained area analyzed in a region of interest. Non-infected (dashed line) mice. <b>(B)</b> Representative Perl’s Prussian staining on spleen sections. <b>(C, D)</b> At 1.5, 3 and 4 months p.i., serum <b>(C)</b> total bilirubin and <b>(D)</b> albumin levels in WT and <i>Mif</i>^<i>-/-</i> mice. Values represent mean ± SEM of 5 mice per group. One representative of 2 independent experiments is shown. **: p<0.01; ***: p<0.005.</p

MIF contributes to hemodilution during <i>T</i>. <i>congolense</i> infection.

<p>At 1.5, 3 and 4 months p.i., 10 μg APC-conjugated hydroxyethyl starch (HES) were injected i.v. in WT (black bar) or <i>Mif</i>^-/- (open bar) mice. Mice were exsanguinated 5–10 minutes later via cardiac puncture and tested for <b>(A)</b> HES concentration, <b>(B)</b> the total blood volume collected and <b>(C)</b> Pack cell volume (PCV). <b>(D)</b> Total plasma (white bar) and PCV (black bar) volumes calculated based on the total blood (B) and the % PCV (C). <b>(E)</b> Concentration of CD41⁺ platelets (gated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.s008" target="_blank">S7B Fig</a>) was determined at 3 months p.i. <b>(F)</b> Total number of platelets in the total blood volume. <b>(G)</b> Bleeding time of non-infected (N) and 3 months infected (I) mice. For ethical reasons the bleeding of WT mice was stopped by sealing the wound after 15 minutes. Results are representative of 2 independent experiments and presented as mean of 7 individual mice ± SEM. **: p<0.01; ***: p<0.005.</p