Red Blood Cells Preconditioned with Hemin Are Less Permissive to <i>Plasmodium</i> Invasion <i>In Vivo</i> and <i>In Vitro</i>

<div><p>Malaria is a parasitic disease that causes severe hemolytic anemia in <i>Plasmodium</i>-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of <i>Plasmodium</i> immunity <i>in vitro</i> and <i>in vivo</i>, mice preconditioned with hemin develop lower parasitemia when challenged with <i>Plasmodium chabaudi adami</i> blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and <i>P</i>. <i>c</i>. <i>adami</i> infection. This strategy allowed discriminating hemin-treated from d<i>e novo</i> generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased <i>P</i>. <i>c</i>. <i>adami</i> red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in <i>in vitro P</i>. <i>falciparum</i> cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during <i>in vivo</i> infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.</p></div

Markers of oxidative stress and ageing in RBCs from HE-preconditioned mice infected with <i>Plasmodium chabaudi adami</i>.

<p>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<i>P</i>. <i>c</i>. adami iRBCs, on the 4^th 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 3^rd 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 <i>P</i>. <i>c</i>. <i>adami</i> 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 <i>t</i> test was performed to compare the control group to the HE-treated one, *p<0.05, **p<0.01, *** p<0.001.</p

<i>Plasmodium falciparum</i> parasitemia and selectivity in HE-pretreated RBCs.

<p>Human RBCs were treated with saline/HE for 18 h and added to a culture of <i>P</i>. <i>falciparum</i> 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 <i>t</i> test, *p<0.05, **p<0.01, *** p<0.001.</p

RBCs turnover in HE-treated infected mice.

<p>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 <i>t</i> test, *p<0.05, **p<0.01.</p

<i>Plasmodium chabaudi adami</i> parasites preferentially infect RBCs that have not been conditioned by HE.

<p>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 <i>t</i> test, **p<0.01, (Fig 5B; Ctrl strep^- vs HE strep^-, p = 0.0593).</p

<i>Plasmodium chabaudi adami</i> schizogony and invasion stage.

<p>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 <i>t</i> test, *p<0.05.</p

<i>Plasmodium chabaudi adami</i> selectivity for red blood cells.

<p>At day 6 and 7 post- <i>P</i>. <i>c</i>. <i>adami</i> 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 <i>t</i> test, **p<0.01, ***p<0.001.</p

Preconditioning with Hemin Decreases <em>Plasmodium chabaudi adami</em> Parasitemia and Inhibits Erythropoiesis in BALB/c Mice

<div><p>Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during <em>Plasmodium chabaudi adami</em> infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and T_h1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following <em>P.c.adami</em> infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology.</p> </div

Bone marrow and spleen erythroid parameters.

<p>Control (Ctrl) and hemin (HE)-treated mice were euthanized 24 h after the last injection with PBS or HE. Populations of erythroid cells were analyzed in the bone marrow (A) and in the spleen (B) by staining cells with anti-CD71-FITC and anti-Ter119-PE antibodies. The percentage of reticulocytes (CD71⁺ cells) (C) and hemoglobin levels were measured in the blood (D). Macrophages (F4-80⁺ cells) were quantified in femoral bone marrow cell suspensions (E) by staining with anti-F4/80-PE antibody. Erythropoietin (EPO) was measured in the plasma by ELISA (F). Data are mean ± SEM from two independent experiments (n = 4–11 mice per group) and values were compared using a non-parametric Student <i>t</i> test. <i>*p<</i>0.05; **<i>p</i><0.01; ***P<0.001.</p

Impact of heme on the clearance of <i>Plasmodium</i> infection.

<p><i>P. chabaudi adami</i> DK (10⁵ parasitized RBCs) were inoculated by the intravenous route in PBS (Ctrl)- and hemin (HE)-preconditioned mice (5 mg/kg, for 3 consecutive days) 24 h after the last injection. Parasitemia was followed daily from tail-tip blood smears for estimation of the kinetics of infection (A) and cumulative (B) and peak parasitemia (C). Values represent the mean ± SEM from two independent experiments (n = 8) and were compared using a non-parametric Student <i>t</i> test. **<i>p</i><0.01; ***<i>p</i><0.001.</p