Hemoglobinopathic Erythrocytes Affect the Intraerythrocytic Multiplication of Plasmodium falciparum in Vitro

The hemoglobinopathies comprise a group of heritable disorders that can be clinically benign or malignant. Haldane's “malaria hypothesis,” that β-thalassemia trait protects against severe Plasmodium falciparum malaria [1], has been extended to hemoglobin (Hb) S heterozygosity (HbAS), α-thalassemia trait, and other hemoglobinopathies that are prevalent in malaria endemic areas. Epidemiological data indicate that HbAS and α-thalassemia trait protect against life-threatening P. falciparum malaria syndromes (reviewed in [2]). Cellular and molecular mechanisms of this protection have not been fully elucidated (reviewed in [3, 4]) but likely include impaired cytoadherence of parasitized erythrocytes to microvascular endothelial cells and uninfected erythrocytes, two interactions associated with the development of severe disease [5–7]. Weakened host-pathogen interactions may minimize microvascular inflammation and associated pathologies (reviewed in [8]). Additional mechanisms of protection may limit parasite densities in individuals with sickle cell trait (HbAS) and disease (HbSS): restricted parasite development under low O2 tension [9], enhanced removal of parasitized erythrocytes in the spleen [10], impaired parasite egress from dehydrated erythrocytes [11], and inhibition of parasite replication by human erythrocyte miRNAs [12]. While only some of these specific mechanisms have been documented for α- or β-thalassemia traits, restriction of parasite growth in thalassemia trait erythrocytes has been observed (reviewed in [4]). The association of severe P. falciparum malaria with high parasite densities [13] led us to hypothesize that hemoglobinopathic erythrocytes may protect against severe malaria by producing fewer merozoites per schizont than normal erythrocytes, thereby slowing parasite replication. While this mechanism has previously been considered, few studies have addressed the multiplication of P. falciparum within individual erythrocytes in vitro [14–17]. Previous approaches in these studies involved counting merozoites in 2-dimensional images of schizonts, which has the potential to underestimate merozoite number. We developed a novel in vitro method to accurately count the number of merozoites released from an individual schizont, which we term the “intraerythrocytic multiplication factor” (IMF). Here we investigate the effect of HbAS, HbSS, and α- and β-thalassemia trait erythrocytes on the IMF of a laboratory-adapted line and 2 clinical isolates of P. falciparum in vitro. We show that all 3 parasite strains produce a significantly lower number of merozoites per cycle in thalassemia trait erythrocytes than in control erythrocytes, and that the IMF correlates significantly with mean corpuscular Hb concentration (MCHC), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) in all cell types. To explore how this reduction in IMF, in the presence of only the innate immune system, might affect the development of severe malaria, we used a mathematical model of host-parasite interaction. The model was used to predict increases in parasite densities and anemia over 14 days of parasite multiplication in vivo, after which the adaptive immune response presumably comes into play.