Fig 1.
Flow chart of the in vitro erythrophagocytosis assay protocol.
Fig 2.
pHrodo erythrophagocytosis assay using naïve PECs.
A (left panel): CD11b versus F4/80 profile following gating on CD45+ cells allows identifying two distinct populations; (i) CD11b+F4/80+ cells which represent macrophages within the PEC population and (ii) cells which are negative or low for CD11b and F4/80 expression referred by as the Rest fraction. A (right panel): histogram showing the PE signal obtained following gating on CD11b+ F4/80+ cells when PECs are incubated alone (black line), with unlabeled RBCs (blue line), with pHrodo-labeled RBCs (orange line) or stimulated with LPS and incubated with pHrodo-labeled RBCs (green line). B: Histogram showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. PECS were unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Results are presented +/- SEM and are representative of 4 independent experiments (for each point triplicates were used). (*: p-values ≤ 0.05) C: Microscopic pictures from PECs incubated with pHrodo-labeled RBCs.
Fig 3.
pHrodo erythrophagocytosis assay using naïve spleen and liver cells.
Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells incubated with unlabeled RBCs from cells incubated with pHrodo-labeled RBCs. Cells were either unstimulated (white bars) or stimulated with 1μg/ml LPS (black bars). Upper panel (A): Different liver cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified as described in S1 Fig. Lower panel (B): Different spleen cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified as described in S2 Fig. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.
Fig 4.
Anemia development during T. brucei infection and pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.
Upper panel (A): Anemia profile during the course of T. brucei infection, whereby the boxed area indicates the time point of interest. Lower panels: Histograms showing delta medium fluorescence intensity (ΔMFI) obtained by subtracting the PE signal for cells in presence of unlabeled RBCs from cells in presence of pHrodo-labeled RBCs following in vitro incubation (B and C). Different cell populations (neutrophils, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) were identified for liver and spleen as described in S2 Fig and S3 Fig, respectively, from naïve (white bars) or T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.
Fig 5.
Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.
Left panels: Percentage of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) within the CD45+ hematopoietic compartment obtained following gating of liver (A) and spleen (B) as described in S1 Fig and S2 Fig, respectively, from non-infected (white bars) or T. brucei infected (day 6 p.i.) animals. Right panels: Absolute numbers of the different cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ myeloid cells and Rest fraction) in total liver and spleen of non-infected (white bars) and T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01; ***: p-values ≤ 0.005 and if nothing is mentioned the differences were not significant.
Fig 6.
A. Chart representing the lipid composition of mature RBCs from naïve and T. brucei infected mice.
This result is a representative of three independent experiments, including three mice per time point. B. Osmotic fragility profile of RBCs following incubation of naïve (black line) or T. brucei infected (day 6 p.i.) (white line) with decreasing concentrations of NaCl, resulting in hemolysis of RBCs. The percentage of hemolysis was plotted against the concentration of NaCl in the medium and the NaCl concentrations corresponding with 50% hemolysis were determined. As positive control RBCs were exposed to 100% distilled H2O and as negative control RBCs were exposed to 100% HBSS-solution. Results are representative of 3 independent experiments and expressed +/- SD. C. pHrodo erythrophagocytosis assay using RBCs from naïve and T. brucei infected mice co-cultured with PECs from non-infected (left panel) or infected (left panel) animals. Erythrophagocytosis by non-infected PECs of non-infected RBCs (white bars) or T. brucei infected (day 6 p.i.) (black bars). Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05; **: p-values ≤ 0.01.
Fig 7.
In vivo pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.
A. Flow chart of the in vivo erythrophagocytosis assay protocol. To asses erythrophagocytosis in infected mice, donor mice should be sacrificed at day 5 post infection. Labeled blood is then transferred to 5 days-infected acceptor mice. At day 6 post infection the acceptor mice are sacrificed for organ isolation and flow cytometry analysis. As a control the same procedure is performed with naïve donor and acceptor mice. B. Erythrophagocytic potential of liver cell populations (neutrophils/PMN, monocytes, monocyte-derived macrophages, resident macrophages and rest fraction) from naïve (white bars) or T. brucei infected (day 6 p.i.). C. Erythrophagocytic potential of spleen cell populations (neutrophils/PMN, monocytes, CD11b+F4/80+ macrophages and rest fraction) from naïve (white bars) or T. brucei infected (day 6 p.i.) mice. Results are presented +/- SEM and are representative of 3 independent experiments (for each point triplicates were used). Of note, *: p-values ≤ 0.05 and if nothing is mentioned the differences were not significant.