Morgan M. Goheen

Host iron status and iron supplementation mediate susceptibility to erythrocytic stage plasmodium falciparum

Iron deficiency and malaria have similar global distributions, and frequently co-exist in pregnant women and young children. Where both conditions are prevalent, iron supplementation is complicated by observations that iron deficiency anaemia protects against falciparum malaria, and that iron supplements increase susceptibility to clinically significant malaria, but the mechanisms remain obscure. Here, using an in vitro parasite culture system with erythrocytes from iron-deficient and replete human donors, we demonstrate that Plasmodium falciparum infects iron-deficient erythrocytes less efficiently. In addition, owing to merozoite preference for young erythrocytes, iron supplementation of iron-deficient individuals reverses the protective effects of iron deficiency. Our results provide experimental validation of field observations reporting protective effects of iron deficiency and harmful effects of iron administration on human malaria susceptibility. Because recovery from anaemia requires transient reticulocytosis, our findings imply that in malarious regions iron supplementation should be accompanied by effective measures to prevent falciparum malaria.

Influence of host iron status on Plasmodium falciparum infection

Iron deficiency affects one quarter of the worlds population and causes significant morbidity, including detrimental effects on immune function and cognitive development. Accordingly, the World Health Organization (WHO) recommends routine iron supplementation in children and adults in areas with a high prevalence of iron deficiency. However, a large body of clinical and epidemiological evidence has accumulated which clearly demonstrates that host iron deficiency is protective against falciparum malaria and that host iron supplementation may increase the risk of malaria. Although many effective antimalarial treatments and preventive measures are available, malaria remains a significant public health problem, in part because the mechanisms of malaria pathogenesis remain obscured by the complexity of the relationships that exist between parasite virulence factors, host susceptibility traits, and the immune responses that modulate disease. Here we review (i) the clinical and epidemiological data that describes the relationship between host iron status and malaria infection and (ii) the current understanding of the biological basis for these clinical and epidemiological observations.

Anemia Offers Stronger Protection Than Sickle Cell Trait Against the Erythrocytic Stage of Falciparum Malaria and This Protection Is Reversed by Iron Supplementation

Background Iron deficiency causes long-term adverse consequences for children and is the most common nutritional deficiency worldwide. Observational studies suggest that iron deficiency anemia protects against Plasmodium falciparum malaria and several intervention trials have indicated that iron supplementation increases malaria risk through unknown mechanism(s). This poses a major challenge for health policy. We investigated how anemia inhibits blood stage malaria infection and how iron supplementation abrogates this protection. Methods This observational cohort study occurred in a malaria-endemic region where sickle-cell trait is also common. We studied fresh RBCs from anemic children (135 children; age 6–24 months; hemoglobin < 11 g/dl) participating in an iron supplementation trial (ISRCTN registry, number ISRCTN07210906) in which they received iron (12 mg/day) as part of a micronutrient powder for 84 days. Children donated RBCs at baseline, Day 49, and Day 84 for use in flow cytometry-based in vitro growth and invasion assays with P. falciparum laboratory and field strains. In vitro parasite growth in subject RBCs was the primary endpoint. Findings Anemia substantially reduced the invasion and growth of both laboratory and field strains of P. falciparum in vitro (~ 10% growth reduction per standard deviation shift in hemoglobin). The population level impact against erythrocytic stage malaria was 15.9% from anemia compared to 3.5% for sickle-cell trait. Parasite growth was 2.4 fold higher after 49 days of iron supplementation relative to baseline (p < 0.001), paralleling increases in erythropoiesis. Interpretation These results confirm and quantify a plausible mechanism by which anemia protects African children against falciparum malaria, an effect that is substantially greater than the protection offered by sickle-cell trait. Iron supplementation completely reversed the observed protection and hence should be accompanied by malaria prophylaxis. Lower hemoglobin levels typically seen in populations of African descent may reflect past genetic selection by malaria. Funding National Institute of Child Health and Development, Bill and Melinda Gates Foundation, UK Medical Research Council (MRC) and Department for International Development (DFID) under the MRC/DFID Concordat.

Independent lineages of highly sulfadoxine-resistant Plasmodium falciparum haplotypes, eastern Africa.

Parasites with increased resistance to sulfadoxine might undermine malaria control measures.

RBC Barcoding Allows for the Study of Erythrocyte Population Dynamics and P. falciparum Merozoite Invasion

Plasmodium falciparum invasion of host erythrocytes is essential for the propagation of the blood stage of malaria infection. Additionally, the brief extracellular merozoite stage of P. falciparum represents one of the rare windows during which the parasite is directly exposed to the host immune response. Therefore, efficient invasion of the host erythrocyte is necessary not only for productive host erythrocyte infection, but also for evasion of the immune response. Host traits, such as hemoglobinopathies and differential expression of erythrocyte invasion ligands, can protect individuals from malaria by impeding parasite erythrocyte invasion. Here we combine RBC barcoding with flow cytometry to study P. falciparum invasion. This novel high-throughput method allows for the (i) direct comparison of P. falciparum invasion into different erythrocyte populations and (ii) assessment of the impact of changing erythrocyte population dynamics on P. falciparum invasion.

The role of the red blood cell in host defence against falciparum malaria: an expanding repertoire of evolutionary alterations

The malaria parasite has co‐evolved with its human host as each organism struggles for resources and survival. The scars of this war are carried in the human genome in the form of polymorphisms that confer innate resistance to malaria. Clinical, epidemiological and genome‐wide association studies have identified multiple polymorphisms in red blood cell (RBC) proteins that attenuate malaria pathogenesis. These include well‐known polymorphisms in haemoglobin, intracellular enzymes, RBC channels, RBC surface markers, and proteins impacting the RBC cytoskeleton and RBC morphology. A better understanding of how changes in RBC physiology impact malaria pathogenesis may uncover new strategies to combat the disease.

Biopreservation of RBCs for in vitro Plasmodium falciparum culture

Falciparum malaria remains a devastating infectious disease, killing nearly 700 000 people annually. Further understanding of malaria pathogenesis will help identify molecular and cellular targets of next-generation therapeutics. The morbidity and mortality of malaria infection occur during the erythrocytic stage. Study of erythrocytic stage malaria is critical not only for new anti-malarial development, but also for increasing our understanding of the host pathogen relationship. For example, many host genetic polymorphisms known to impact red blood cell (RBC) physiology also alter malaria susceptibility. Methods for culturing erythrocytic stage Plasmodium falciparum were developed more than 30 years ago (Jensen & Trager, 1977). However, optimization of in vitro P. falciparum culture is still under investigation. The aspects of intraerythrocytic development that are impacted by RBC storage, as well as the effects of RBC biopreservation on the intraerythrocytic life cycle, remain unknown. Studies on RBC storage for human clinical use reveal a relationship between RBC storage and transfusion complications (Aubron et al, 2013). Current blood banking standards involve RBC storage in SAGM (a solution containing saline, adenine, mannitol and glucose) or closely related solutions for up to 42 d at 4°C (Sparrow, 2012). Many RBC storage lesions have been documented in these acidic medias, including decreased intracellular ATP, 2,3-diphosphoglycerate (2,3-DPG) and potassium; increased intracellular NaCl; oxidative damage; lipid peroxidation; membrane phospholipid changes and vesiculation; decreased deformability; reduced glycolytic capacity; decreased vasodilatory capacity; and increased cytoadhesion (Bennett-Guerrero et al, 2007; Aubron et al, 2013). Overall, these storage lesions are similar to physiological changes occurring with normal RBC ageing in the bloodstream (Franco et al, 2013) and could also impact parasite growth, as P. falciparum preferentially infects younger RBCs in circulation (Lim et al, 2013; Clark et al, 2014a). The use of biopreserved RBCs for human transfusion has been validated (Fabricant et al, 2013) and cryopreserved umbilical cord blood cells can propagate Plasmodium vivax (Borlon et al, 2012). Here we examine the impact of RBC storage and biopreservation on P. falciparum growth and development in vitro. Prolonged RBC shelf-life and biopreservation could enhance malaria research by: (i) enabling standardization of the RBC source for multiple experiments, and (ii) increasing access to RBCs from individuals with unusual blood types, nutritional deficiencies, or from remote locations. Given lingering discrepancies in standard P. falciparum culture protocols, we sought to definitively assess RBC shelflife. To begin, fresh RBCs were collected into acid citrate dextrose (ACD) and stored in aliquots as packed RBCs at 4°C for up to 6 weeks. At 2-week intervals, fresh RBCs were obtained and identically stored to allow for simultaneous comparisons of parasite growth in vitro using blood stored for 0, 2, 4 and 6 weeks. Growth in RBCs stored for 2 weeks showed no decrease in standard 96-h growth assays (Clark et al, 2014a). After 4 weeks of storage, growth rates diminished significantly (42% decline for Dd2, 65% for FCR3FMG). In RBCs stored for 6 weeks, there was very little growth (over 90% decline for both strains Dd2 and FCR3-FMG) (Fig 1A). We next compared parasite growth in RBCs stored for 0, 2 and 4 weeks at 4°C in four different storage buffers. Buffers tested were: (i) ACD; (ii) citrate-phosphate-dextrose-adenine (CPDA), commonly used for malaria culture; (iii) Alsever’s Solution, a balanced salt solution routinely used for RBC washing prior to parasite culture; and (iv) “RBC buffer” an alternative balanced salt solution. Growth rates decreased proportionally to storage length in each of the buffers, with a significant decrease after 4 weeks of storage (62% for ACD, 56% for CPDA, 54% for Alsever’s and 51% for “RBC buffer”) (Fig 1B). This confirms RBCs destined for parasite culture must be used within 2 weeks of collection and that differential storage media does not prolong their shelf-life for P. falciparum culture. We next sought to determine whether parasite replication and/or invasion were decreased in stored RBCs. To assess replication, we measured the parasite erythrocyte multiplication rate (PEMR), which reflects the number of infectious merozoites produced per schizont (Clark et al, 2014a). We found no statistically significant differences in replication between RBCs stored for 0–4 weeks (Fig 1C). Invasion rates were assayed using a RBC barcoding assay (Clark et al, 2014b) in which differentially labelled RBCs (with CellTrace membrane dyes; Life Technologies Corp., Grand Island, NY, USA) were combined in the same wells and seeded with unlabeled trophozoite stage parasitized RBCs. This assay allows direct comparison of parasite invasion into two different RBC populations. Invasion rates decreased as RBC storage time increased (Fig 1D). We hypothesize that this Correspondence