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

Proposed model for the contribution of MIF to pathogenic anemia during <i>T</i>. <i>congolense</i> infection in trypanotolerant C57Bl/6 mice.

<p>In the chronic phase of <i>T</i>. <i>congolense</i> infection, anemia did not result from the impaired production of mature RBCs, the total amount of RBCs being similar in infected and non-infected mice. The infection- and MIF- induced spleen extramedullary erythropoiesis (1) could thus compensate for the impaired differentiation of erythroblasts in the bone marrow (2) and for the enhanced clearance of RBCs by phagocytic cells colonizing the liver and the spleen (3). Anemia induced by <i>T</i>. <i>congolense</i> mainly occurred through hemodilution (4), that was partially mediated by MIF. Hemodilution could also account for thrombocytopenia and for impaired coagulation in infected mice (5). The heme catabolism following the chronically-induced erythrophagocytosis (3) led to iron accumulation in myeloid cells (6) and to hyperbilirubinemia (7). The combination of liver injury and hemodilution resulted in declined serum albumin levels (8) in infected mice, thereby preventing efficient removal of toxic molecules from the circulation, including bilirubin. This could cause multiple organ failure (MOF, 9) that culminates in reduced survival of the infected host.</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

MIF deficiency partially reduces anemia during <i>T</i>. <i>congolense</i> infection.

<p><b>(A)</b> Anemia development in infected WT (closed symbol) and <i>Mif</i>^<i>-/-</i> (open symbol) mice. Number of RBCs in non-infected mice was set as 100%. At 1.5, 3 and 4 months p.i., <b>(B)</b> serum hemoglobin levels, <b>(C)</b> serum iron levels, and <b>(D)</b> numbers of splenic white blood cells (WBC, black bar) and RBCs (white bar) were determined. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM. **: p≤0.01, ***: p≤0.001.</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 pathogenic chemokine and cytokine production during <i>T</i>. <i>congolense</i> infection.

<p>At 3 months p.i., <i>ex-vivo</i> protein levels of MIF, CXCL1, CCL2, TNF, IL-6, IFN-γ and IL-10 in <b>(A-C)</b> supernatant from the liver, spleen, and bone marrow cell cultures, and <b>(D)</b> in the blood. IL-12p70 levels in blood are shown for WT (black bar) and <i>Mif</i>^-/- (open bar) mice. Protein levels in non-infected mice (dashed line) were similar in both mouse strains. (N.D.: not detectable). Results are representative of 3 independent experiments and presented as mean of 3 individual mice ± SEM. *: p≤0.05, **: p≤0.01, ***: p≤0.001.</p

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

<p>At 3 months p.i., <b>(A-C)</b> numbers of mature RBCs (erythrocytes, Ery.) and immature RBCs (reticulocytes, Ret.) defined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.s007" target="_blank">S6A Fig</a> in <b>(A)</b> blood, <b>(B)</b> bone marrow and <b>(C)</b> spleen of WT and <i>Mif</i>^<i>-/-</i> mice. WT (black bar), <i>Mif</i>^-/- (open bar) and non-infected (grey bar) mice. Results are representative of 3 independent experiments and shown as mean of 3 individual mice ± SEM. <b>(D, E)</b> Numbers of the different erythroid populations (defined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.s007" target="_blank">S6B Fig</a>) in <b>(D)</b> bone marrow and <b>(E)</b> spleen. <b>(F)</b> Gene expression levels of <i>Vcam1</i> and <i>Maea</i> in total spleen from WT (black bar) and <i>Mif</i>^−/− (white bar) mice at 3 months p.i. Gene expression levels were normalized using <i>S12</i> and expressed relatively to expression levels in non-infected mice. Results are representative of 2 independent experiments and presented as mean of 3–5 individual mice ± SEM. *: p≤0.05, **: p≤0.01, ***: p≤0.001.</p

rMIF treatment in <i>T</i>. <i>congolense</i>-infected MIF^-/- mice recapitulates anemia and hemodilution development.

<p>At 3 months p.i., <i>Mif</i>^-/- mice received 200 ng rMIF every second day for 1 week. <b>(A)</b> Percentage of RBCs in blood, whereby the number of RBCs in non-infected mice was set as 100%; <b>(B)</b> Numbers of blood mature (erythrocytes, Ery.) and immature (reticulocytes, Ret.) RBCs defined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.s007" target="_blank">S6A Fig</a>; <b>(C)</b> Percentage of the different erythroid populations defined as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.s007" target="_blank">S6B Fig</a> in the spleen; <b>(D)</b> Percentage of blood Annexin-V⁺ RBCs gated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.g006" target="_blank">Fig 6A</a>; (E) Total plasma (white bar) and PCV (black bar) volumes calculated based on the total blood volume and the % PCV; <b>(F)</b> Spleen and liver weights; <b>(G, H)</b> Concentration and number 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> in total blood volume were determined in rMIF-treated <i>Mif</i>^-/- mice (grey bar) as well as in <i>Mif</i>^-/- (open bar) and WT (black bar) mice. Results are representative of 2 independent experiments and presented as mean of 3–5 individual mice ± SEM. *: p≤0.05, **: p≤0.01, ***: p≤0.001.</p

MIF deficiency confers partial protection and reduces hepatosplenomegaly and white blood cell accumulation during <i>T</i>. <i>congolense</i> infection.

<p><b>(A)</b> Parasitemia, <b>(B)</b> survival, serum <b>(C)</b> AST and <b>(D)</b> ALT levels in infected mice. During the course of infection, <b>(E)</b> liver and spleen weight as well as <b>(F)</b> liver, spleen and bone marrow white blood cell (WBC) numbers. For bone marrow WBCs, only data from 3 months p.i. are shown. Wild type (WT, black symbol); <i>Mif</i>^-/- (white symbol) mice. Results are representative of 2 <b>(E)</b> or 3 independent experiments and presented as mean <b>(A, C, D, E, F)</b> or median <b>(B)</b> of 3–5 individual mice ± SEM. *: p≤0.05, **: p≤0.01, ***: p≤0.001.</p

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

<p><b>(A)</b> Representative Annexin-V gating strategy and percentage of Annexin-V⁺ RBCs in blood of WT (black bar) and <i>Mif</i>^-/- (open bar) mice. <b>(B, C left panel)</b> At 3 months p.i., 10⁹ pHrodo labelled RBCs isolated from non-infected WT mice were injected i.v. in WT (black bar) or <i>Mif</i>^-/- (open bar) mice. 18 h later, mice were sacrificed, liver <b>(B)</b> and spleen <b>(C)</b> myeloid phagocytic cells (MPC), namely CD11b⁺Ly6C^intLy6G⁺ PMNs, CD11b⁺Ly6C^highLy6G^- monocytes and CD11b⁺Ly6C^-Ly6G^-F4/80⁺ macrophages (identified as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005862#ppat.1005862.s008" target="_blank">S7A Fig</a>) were tested for delta median fluorescent intensity (MFI) of the intracellular pHrodo signal determined by subtracting the PE signal of cells from mice receiving unlabeled RBCs from the PE signal of cells from mice receiving pHrodo-labeled RBCs. <b>(B, C right panel)</b> Numbers of MPC in the <b>(B)</b> liver and <b>(C)</b> spleen of WT (black bar) or <i>Mif</i>^-/- (open bar) mice. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM. *: p<0.05; **: p<0.01.</p