Effect of dihydroartemisinin (DHA) on human erythroid cell differentiation : implications for malaria treatment in pregnancy

BACKGROUND: Severe malaria in pregnancy causes maternal anemia, low birth weight increased mortality of both mother and infants. WHO recommends few antimalarials due to safety problems. Artemisinin combination therapy is the first line treatment, however artemisinin derivatives showed animal embryotoxicity with a reduction of embryonic erythrocytes when treatment is performed on certain days of gestation. AIMS: To investigate the effect of Dihydroartemisinin (DHA), the metabolite of artemisinins, on an in vitro model reproducing human erythropoiesis and to characterize the erythroid target stage, in order to predict the window of susceptibility to DHA in human pregnancy. METHODS: The mononuclear cells derived from pheripheral blood of healthy volunteers were enriched for CD34+ cells by positive selection using anti-CD34-tagged magnetic beads. CD34+ cells were cultured for 14 days with a specific medium containing erythropoietin to induce erythroid differentiation. DHA at 0,5 or 2 µM, according to the dosages of previous animal experiments, was added for the first time at day 0 (on isolated stem cell), at day 2 (on early erythroid progenitors), at day 4 (in presence of both early progenitors and pro-erythroblasts), at day 7 (on basophilic erythroblasts) or at day 11 (polychromatic erythroblasts) then continuously every 3 days up to 14 days, because of its short half life. Cells growth and viability were evaluated by trypan blue exclusion; erythroid differentiation was investigated by cytofluorimetric analysis of Glycophorin A (GPA) expression, by morphological analysis on benzidine-May-Grunwald-Giemsa stained smears and by erythroid specific gene expression analysis with real-time PCR. RESULTS: DHA was added on stem cells or early erythroid progenitors caused a transient inhibition of both cell growth and differentiation up to day 7, but then the treated cells started growing and completed their erythroid differentiation at day 14 of culture. When DHA was added on basophilic erythroblasts, a significant and long lasting effect decrease in proliferation as well as a delay in erythroid differentiation was observed. Up to day 14. DHA added on mature stages i.e. polychromatic erythroblasts, only a small reduction of cell growth has been observed without any consequence for the erythroid cell differentiation. CONCLUSIONS: These data suggest that DHAâ s specific target is the basophilic erythroblast, since DHA added at this stage causes a significant inhibition of erythroid differentiation. Based on these in vitro results, we hypothesize that DHA could affect human primitive erythropoiesis, which occurs during the late phase of human secondary yolk sac erythropoiesis (weeks 4-8 of gestation), when foetal blood is formed of only primitive erythroblasts. This means that if the treatment with DHA or artemisinin derivatives is performed during the first trimester of human pregnancy, toxic effects on embryo could be expected.