Fig 1.
Markers of oxidative stress and ageing in RBCs from HE-preconditioned mice infected with Plasmodium chabaudi adami.
BALB/c mice received intravenous injection of sulfo-NHS-LC-biotin (1 mg/100μl/mouse) to label RBCs in circulation, one day prior to a first HE treatment. Saline (control) or HE (10 mg/kg/day) were administered by the intraperitoneal (ip) route for 3 days and followed by ip injection of 5x105 P. c. adami iRBCs, on the 4th day. Tail-tip blood smears stained with Giemsa were performed daily to follow the parasitemia (A), determine the magnitude of peak parasitemia (B) and estimate cumulative parasitemia (calculated as the sum of daily parasitemia) (C). Three hours after the 3rd and last saline/HE injection, the mean volume of RBCs was estimated from FSC values (D), and gmeanFI relative to Annexin-V binding (E), DCFDA labeling (F) and CD47 expression (G) were estimated by flow cytometry on 40 000 RBCs positive for streptavidin. These parameters were also analysed 7 days after P. c. adami infection (H-K). Data in A-C are the means ± SEM of three independent experiments (n = 11), and data in D-K are the means ± SEM of one experiment (Ctrl; n = 4, HE; n = 3). An unpaired Student t test was performed to compare the control group to the HE-treated one, *p<0.05, **p<0.01, *** p<0.001.
Fig 2.
RBCs turnover in HE-treated infected mice.
The percentage of sulfo-NHS-LC-biotinylated RBCs was followed by flow cytometry, starting 24 hours after the biotin injection, preceding HE injection (A). During the course of infection, RBCs turnover was estimated as the concentrations of biotinylated (strep+) and non-biotinylated (strep-) RBCs per mL of blood (B) by combining FACS streptavidin staining analysis with a flow cytometry blood cell count. Hemoglobin levels (C) and reticulocytes (positive gating of CD71+ cells with FACS) (D) were measured in whole blood. Results in A and D represent the means ± SEM of three independent experiments (A; n = 11, D; n = 7–11) and data in B and C are the means ± SEM of two experiment (n = 4–8). All data for saline and HE-conditioned mice were compared with an unpaired Student t test, *p<0.05, **p<0.01.
Fig 3.
Plasmodium chabaudi adami schizogony and invasion stage.
Six days post-infection, thin blood smears of iRBCs were prepared at the time of schizogony and stained with DAPI (blue). DIC and fluorescence images were captured with Nikon A1 confocal microscope (objective plan Apo VC 60x, NA 1.4, λs oil immersion), and analysed with NIS-Elements Viewer 4.20 imaging software. The proportion of merozoites (nuclei) per mature schizont (containing ≥4 merozoites) was determined in >150 iRBCs per mouse (A). DAPI-stained blood smears were also analysed at the time of merozoite invasion, and the proportion of extra-erythrocytic merozoites was measured in ≥600 merozoites (number of extra-erythrocytic merozoites/total number of merozoites) (B). DIC, DAPI fluorescence and merged images at the time of merozoite invasion are shown for a control and HE-treated mouse (C). The results represent the mean ± SEM of one experiment (Ctrl; n = 4, HE; n = 3) and were compared with an unpaired Student t test, *p<0.05.
Fig 4.
Plasmodium chabaudi adami selectivity for red blood cells.
At day 6 and 7 post- P. c. adami infection, thin blood smears of parasite ring stages iRBCs were fixed and stained with DAPI (blue). DIC and fluorescence images were captured with Nikon A1 confocal microscope (plan Apo VC 60x, NA 1.4, λs oil immersion), and analysed with NIS-Elements Viewer 4.20 imaging software. Single and multiple-iRBCs were counted in >150 ring-iRBCs per mouse (A, C). Parasite SI was calculated for each day by dividing the observed value of multiple iRBCs by the value expected from a Poisson distribution (B, D). DIC, DAPI fluorescence, and merged channels are shown at day 6 post-infection during ring stage for a control and a HE-treated mouse (E). The results represent the means ± SEM of one experiment (Ctrl; n = 4, HE; n = 3) at day 6 post-infection and two experiments (Ctrl; n = 8, HE; n = 7) at day 7 post-infection. Data were compared with an unpaired Student t test, **p<0.01, ***p<0.001.
Fig 5.
Plasmodium chabaudi adami parasites preferentially infect RBCs that have not been conditioned by HE.
At peak parasitemia (day 7 post-infection), whole blood was fixed in 1% paraformaldehyde overnight, washed, stained and analyse with confocal microscopy. The parasites were labeled with DAPI (blue), and distinction between saline/HE-treated RBCs (strep+) and RBCs generated after treatments (strep-) was made by APC-conjugated streptavidin staining (red): reticulocytes were stained with FITC-labeled anti-CD71 antibody (green). DIC and fluorescence images were captured with Nikon A1 confocal microscope (plan Apo VC 60x, NA 1.4, λs oil immersion), and analysed with NIS-Elements Viewer 4.20 imaging software. Parasitemia within strep+ and strep- RBCs (A; infected strep+/- RBCs / total strep+/- RBCs) and proportions of strep+ and strep- RBCs charge with multiple ring (B; multiple infected strep+/- RBCs / infected strep+/- RBCs) were evaluated on >150 ring-iRBCs per mouse. DAPI, anti-CD71-FITC, streptavidin-APC fluorescence, DIC and merged channels are shown for a control and a HE-treated mouse (C). Data are the means ± SEM of one experiment (n = 4). All data from Ctrl and HE-treated mice were compared with an unpaired Student t test, **p<0.01, (Fig 5B; Ctrl strep- vs HE strep-, p = 0.0593).
Fig 6.
Plasmodium falciparum parasitemia and selectivity in HE-pretreated RBCs.
Human RBCs were treated with saline/HE for 18 h and added to a culture of P. falciparum Dd2 parasites. Parasitemia was estimated from Giemsa-stained smears analysis (A), as was the percentage of multiple-iRBCs (B, D) and the SI (C, E), 4 and 6 days post-infection (during ring stage). The results are the means ± SEM of three independent experiments (n = 3), and data from Ctrl and HE-pretreated RBCs were compared with an unpaired Student t test, *p<0.05, **p<0.01, *** p<0.001.