Fig 1.
MIF deficiency confers partial protection and reduces hepatosplenomegaly and white blood cell accumulation during T. congolense infection.
(A) Parasitemia, (B) survival, serum (C) AST and (D) ALT levels in infected mice. During the course of infection, (E) liver and spleen weight as well as (F) 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); Mif-/- (white symbol) mice. Results are representative of 2 (E) or 3 independent experiments and presented as mean (A, C, D, E, F) or median (B) of 3–5 individual mice ± SEM. *: p≤0.05, **: p≤0.01, ***: p≤0.001.
Fig 2.
MIF contributes to pathogenic chemokine and cytokine production during T. congolense infection.
At 3 months p.i., ex-vivo protein levels of MIF, CXCL1, CCL2, TNF, IL-6, IFN-γ and IL-10 in (A-C) supernatant from the liver, spleen, and bone marrow cell cultures, and (D) in the blood. IL-12p70 levels in blood are shown for WT (black bar) and Mif-/- (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.
Fig 3.
MIF deficiency preserves parasite-specific IgG responses, maintains germinal center formation and prevents B-cell apoptosis during T. congolense infection.
(A) Parasite-specific blood IgG level (1/500 dilution) in WT (black bar) and Mif-/- (open bar) mice at 1.5, 3 and 4 months p.i.. At 3 months p.i. (B) absolute numbers of B220+MHC-II+ B-cells and (C) of germinal GL-7+Fas+ B-cells and (D) percentage of Annexin-V+ B220+ cells in the spleen of WT (black bar) and Mif-/- (open bar) mice were determined using the gating strategy described in S5A and S5B Fig. Results are representative of 2 independent experiments and presented as mean of 3 individual mice ± SEM. *: p≤0.05, **: p≤0.01, ***: p≤0.001.
Fig 4.
MIF deficiency partially reduces anemia during T. congolense infection.
(A) Anemia development in infected WT (closed symbol) and Mif-/- (open symbol) mice. Number of RBCs in non-infected mice was set as 100%. At 1.5, 3 and 4 months p.i., (B) serum hemoglobin levels, (C) serum iron levels, and (D) 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.
Fig 5.
MIF contributes to dyserythropoiesis during T. congolense infection.
At 3 months p.i., (A-C) numbers of mature RBCs (erythrocytes, Ery.) and immature RBCs (reticulocytes, Ret.) defined as described in S6A Fig in (A) blood, (B) bone marrow and (C) spleen of WT and Mif-/- mice. WT (black bar), Mif-/- (open bar) and non-infected (grey bar) mice. Results are representative of 3 independent experiments and shown as mean of 3 individual mice ± SEM. (D, E) Numbers of the different erythroid populations (defined as described in S6B Fig) in (D) bone marrow and (E) spleen. (F) Gene expression levels of Vcam1 and Maea in total spleen from WT (black bar) and Mif−/− (white bar) mice at 3 months p.i. Gene expression levels were normalized using S12 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.
Fig 6.
MIF contributes to erythrophagocytosis during T. congolense infection.
(A) Representative Annexin-V gating strategy and percentage of Annexin-V+ RBCs in blood of WT (black bar) and Mif-/- (open bar) mice. (B, C left panel) At 3 months p.i., 109 pHrodo labelled RBCs isolated from non-infected WT mice were injected i.v. in WT (black bar) or Mif-/- (open bar) mice. 18 h later, mice were sacrificed, liver (B) and spleen (C) myeloid phagocytic cells (MPC), namely CD11b+Ly6CintLy6G+ PMNs, CD11b+Ly6ChighLy6G- monocytes and CD11b+Ly6C-Ly6G-F4/80+ macrophages (identified as described in S7A Fig) 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, C right panel) Numbers of MPC in the (B) liver and (C) spleen of WT (black bar) or Mif-/- (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.
Fig 7.
MIF contributes to heme catabolism and tissue iron accumulation during T. congolense infection.
At 3 months p.i., (A) iron deposition was quantified on spleen and liver sections of WT (black bar) and Mif-/- (open bar) mice and expressed as % of stained area analyzed in a region of interest. Non-infected (dashed line) mice. (B) Representative Perl’s Prussian staining on spleen sections. (C, D) At 1.5, 3 and 4 months p.i., serum (C) total bilirubin and (D) albumin levels in WT and Mif-/- mice. Values represent mean ± SEM of 5 mice per group. One representative of 2 independent experiments is shown. **: p<0.01; ***: p<0.005.
Fig 8.
MIF contributes to hemodilution during T. congolense infection.
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 Mif-/- (open bar) mice. Mice were exsanguinated 5–10 minutes later via cardiac puncture and tested for (A) HES concentration, (B) the total blood volume collected and (C) Pack cell volume (PCV). (D) Total plasma (white bar) and PCV (black bar) volumes calculated based on the total blood (B) and the % PCV (C). (E) Concentration of CD41+ platelets (gated as described in S7B Fig) was determined at 3 months p.i. (F) Total number of platelets in the total blood volume. (G) 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.
Fig 9.
rMIF treatment in T. congolense-infected MIF-/- mice recapitulates anemia and hemodilution development.
At 3 months p.i., Mif-/- mice received 200 ng rMIF every second day for 1 week. (A) Percentage of RBCs in blood, whereby the number of RBCs in non-infected mice was set as 100%; (B) Numbers of blood mature (erythrocytes, Ery.) and immature (reticulocytes, Ret.) RBCs defined as described in S6A Fig; (C) Percentage of the different erythroid populations defined as described in S6B Fig in the spleen; (D) Percentage of blood Annexin-V+ RBCs gated as in Fig 6A; (E) Total plasma (white bar) and PCV (black bar) volumes calculated based on the total blood volume and the % PCV; (F) Spleen and liver weights; (G, H) Concentration and number of CD41+ platelets gated as described in S7B Fig in total blood volume were determined in rMIF-treated Mif-/- mice (grey bar) as well as in Mif-/- (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.
Fig 10.
Proposed model for the contribution of MIF to pathogenic anemia during T. congolense infection in trypanotolerant C57Bl/6 mice.
In the chronic phase of T. congolense 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 T. congolense 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.