pHrodo erythrophagocytosis assay using naïve PECs.

<p>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.</p

Different myeloid sub-populations expressed as percentage within the CD45+ hematopoietic compartment or as absolute numbers within the organ.

<p>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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s002" target="_blank">S2 Fig</a>, respectively, from non-infected (white bars) or <i>T</i>. <i>brucei</i> 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 <i>T</i>. <i>brucei</i> 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.</p

Flow chart of the <i>in vitro</i> erythrophagocytosis assay protocol.

<p>Flow chart of the <i>in vitro</i> erythrophagocytosis assay protocol.</p

A. Chart representing the lipid composition of mature RBCs from naïve and <i>T</i>. <i>brucei</i> infected mice.

<p>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 <i>T</i>. <i>brucei</i> 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 <i>T</i>. <i>brucei</i> 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 <i>T</i>. <i>brucei</i> 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.</p

<i>In vivo</i> pHrodo erythrophagocytosis assay on liver and spleen cells during the acute stage of infection.

<p>A. Flow chart of the <i>in vivo</i> 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 <i>T</i>. <i>brucei</i> 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 <i>T</i>. <i>brucei</i> 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.</p

pHrodo erythrophagocytosis assay using naïve spleen and liver cells.

<p>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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s001" target="_blank">S1 Fig</a>. Lower panel (B): Different spleen cell populations (neutrophils, monocytes, CD11b⁺F4/80⁺ myeloid cells and Rest fraction) were identified as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003561#pntd.0003561.s002" target="_blank">S2 Fig</a>. 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.</p

Baseline characteristics at enrolment (during pregnancy) for the mothers of the children examined at age 5 years.

<p>Baseline characteristics at enrolment (during pregnancy) for the mothers of the children examined at age 5 years.</p

Effect of praziquantel treatment during pregnancy on <i>S. mansoni</i> infection status in children at age five years.

<p>Effect of praziquantel treatment during pregnancy on <i>S. mansoni</i> infection status in children at age five years.</p

Antibody levels to SWA or SEA among children at age five years.

<p>Shown are plasma levels of antibodies to SWA (A) or SEA (B) among uninfected children (n = 390) and infected children (n = 20) at age five years. ** Wilcoxon rank-sum test p<0.001.</p

Cytokine responses to SWA (A) or SEA (B) among children at age five years.

<p>Comparisons are shown between uninfected children (n = 390) and infected children (n = 20). Presented are Wilcoxon rank-sum test p-values.</p