- Open Access
Reticulocyte-prone malaria parasites predominantly invade CD71hi immature cells: implications for the development of an in vitro culture for Plasmodium vivax
© Martín-Jaular et al.; licensee BioMed Central Ltd. 2013
- Received: 3 September 2013
- Accepted: 22 November 2013
- Published: 1 December 2013
The lack of a continuous in vitro culture system for blood stages of malarial parasites with a unique tropism for reticulocytes, such as Plasmodium vivax and the Plasmodium yoelii 17X reticulocyte-prone strain, hinders research in these organisms. The maturation of reticulocytes into erythrocytes is a complex process involving the selective removal of membrane proteins such as the transferrin receptor, CD71. In order to advance in the characterization of infected cells during experimental infections of BALB/c mice with P. yoelii 17X, CD71 expression in erythroid cells (TER119+) was assessed and in vitro culture of P. yoelii 17X was attempted by adding reticulocytes highly expressing CD71.
BALB/c mice were infected with P. yoelii 17X-GFP transgenic parasites and erythroid cells (TER119+) were analysed in blood, spleen and bone marrow cells. TER119, CD71 and GFP expression was assessed at different points post-infection by flow cytometry. Moreover, in vitro culture of P. yoelli 17X was attempted by adding red blood cells (RBCs) from mice with a pyruvate kinase deficiency, which contain high percentages of CD71hi cells in peripheral blood as compared to healthy animals.
A predominance of erythroid cells lacking expression of CD71 (CD71-) was observed in peripheral blood and spleen in normal and infected animals up to ten days post-infection (pi). At ten days pi, however, a dramatic temporal switch to erythroid cells highly expressing CD71 (CD71hi) was observed in the spleen and at day 15 pi in peripheral blood of the infected cells. A distribution of erythroid cells expressing differently CD71 was noticed in the bone marrow. Yet, similar to peripheral blood and spleen, a predominance of CD71hi cells was observed at 15 days pi. Remarkably, CD71hi cells were the cells predominantly infected in these organs as well as in peripheral blood. Attempts were thus made to culture in vitro the P. yoelli 17X strain by adding RBCs from pyruvate kinase-deficient mice containing high percentages of CD71hi cells in peripheral blood.
The parasite preference for immature cells that are rare in normal peripheral blood could have important implications for the development of an in vitro culture system for P. vivax.
- Plasmodium yoelii
- Plasmodium vivax
- In vitro culture
- CD71 cells
Reticulocytes represent the invasion target for a number of malarial parasites, including Plasmodium vivax and the rodent malaria Plasmodium yoelii 17X strain [1, 2]. Arguably, it is thought that this tropism has hindered the development of a continuous in vitro culture system for blood stages of these species. Yet, several attempts to establish the in vitro culture system for blood stages of P. vivax by adding different sources of reticulocytes  have been unsuccessfully tried over the past 100 years.
Enucleation of erythroblasts in the bone marrow is the source point of reticulocytes that after a brief period of time in the erythropoietic tissue are released into circulation where they mature into erythrocytes. Interestingly, it has been long established that reticulocytes are a heterogeneous cell population composed of cells in different stages of differentiation [4–6]. The transformative process of maturation of reticulocytes includes loss of organelles through apoptosis and autophagy , and the remodelling of the plasma membrane through the selective removal of proteins, notoriously the transferrin receptor CD71 in nanovesicles termed exosomes .
Here, to better characterize the cells infected during P. yoelii 17X-BALB/c experimental infections, CD71 expression in parasitized erythroid cells (TER119+) from peripheral blood and erythropoietic organs have been assessed. TER119 is a marker for erythroid cells from the early pro-erythroblast to mature erythrocyte stages of development  and CD71 is the transferrin receptor known to be released in exosomes during erythrocyte maturation , The results suggest that the more immature reticulocytes highly expressing CD71 are the predominant target cell for invasions. As a proof of concept, the in vitro culture of the P. yoelii 17X strain was tried by adding red blood cells (RBCs) from mice with a pyruvate kinase deficiency (PKD) containing higher percentages of CD71hi cells in peripheral blood than healthy animals .
All the animal studies were performed at the animal facilities of Hospital Clinic in Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (No 87/12). Female BALB/c mice of seven to nine weeks old were obtained from Charles River Laboratories. AcB55 (C57Bl/6 × A/J) mice (also recorded as PKD mice) carrying a point mutation at nucleotide 269 (T → A) of the pklr gene  were obtained from Emerillon Therapeutics (Montreal, Quebec, Canada) and bred at the animal house of CIEMAT (registration number 28079-21A). When needed, eight to ten-week old PKD mice were shipped to Barcelona and after an eight-day housing period, mice were used as reticulocyte donors for in vitro culture experiments.
Plasmodium yoelii-GFP transgenic lines were generated as described before . To generate P. yoelii 17X parasites transgenic for the red fluorescent protein mCherry, schizonts of P. yoelii 17X were transfected with the pL0017-mCherry plasmid (kindly donated by V T Heussler, University of Bern, Switzerland) following standard methodologies .
Flow cytometry analysis
Blood, spleens and bone marrow were obtained from control and infected mice on days 1, 3, 5, 10, 15 and 30 post-infection (pi) and single-cell suspensions were prepared. Blood was obtained by intracardiac puncture. Spleens were homogenized and passed through a nylon mesh of 70 μm (352350, BD) to create a single-cell suspension. Bone marrow cells were collected by flushing the femurs and tibias with RPMI medium. The cells were counted by an automatic counter (Countess™, Invitrogen™) and 5 × 106 cells were used for the staining. Before addition of specific fluorescently labelled antibodies, the cells were pre-incubated with Seroblock anti-Fc receptor antibody (BUF041A, AbDSerotec) for 10 min. Cells were stained with PerCP anti-TER119 (116225, Biolegend), PE conjugated anti-CD71 (ab25622, Abcam) at 4°C for 30 min. Samples were acquired on a BD LSRFortessa Flow Cytometer and analysed with FlowJo software. Non-nucleated erythroid cells were defined according SSC/FSC profile and TER119 expression. TER119+ cells were analysed for CD71 and GFP expression. The percentage of different populations of cells was calculated as a function of erythroid cells. Data are representative of 200,000 events collected. Results are expressed as the mean ± SEM and were analysed using GraphPad Prism software.
Electron microscopy analysis
Blood cells from P. yoelii 17X infected BALB/c mice at day 15 pi were stained with CD71 antibody (ab25622, Abcam) as described above. CD71hi cells were sorted using a BD Aria II. For ultrastructural analysis, cells were fixed in 2% paraformaldehyde and treated with Karnovsky’s fixative. Small pieces of 1 sq mm were fixed in the same fixative at 4°C for at least 24 hours, post-fixed in 1% osmium tetroxide and dehydrated in acetones before embedding in Spurr resin. Ultrathin (70–90 nm) sections of the spleen were obtained on an ultramicrotom Ultracut E (Reichert-Jung) equipped with a diamond knife (Diatome). Ultrathin sections were stained with 2% uranyl acetate and lead citrate  to allow high-contrast imaging. Image acquisition was performed using a JEOL 1010 TEM operating at 80 kV with a Bioscan 792 camera (Gatan MultiScan cameras).
In vitro culture
Blood from PKD and infected mice was obtained by intracardiac puncture and passed through a hand-made CF11 cellulose filter to remove the leucocyte population . Non-synchronized infected blood was mixed with blood from PKD mice in order to obtain parasitaemia between 0.25 and 4% and resuspended at a density of 1.5×107 cells/ml in McCoy medium containing 10% FBS and penicillin/streptomycin. Two-hundred μl of cells prepared as described above were cultured in 96-well plates at 37°C and with 5% C02, 5% 02 and 90% nitrogen. Cells from PKD mice were maintained at 4°C with no major variations in the percentages of reticulocytes (around 45%). 0.5×105 cells from PDK mice maintained at 4°C were added to each well every day. For examination of cultures, the entire content of each well was drawn off and cells counts were monitored by Giemsa staining and flow cytometry. In some experiments, blood from PKD mice was stained with the viable dye CFSE  and used in experiments with mCherry transgenic parasites in the same conditions as described above. Briefly, CFSE Vybrant CFDA-SE Cell Tracer Kit (C34554, Invitrogen) was used to label cells according to the manufacturer’s instructions. Cells were suspended in 5 μM CFSE solution in PBS for six minutes at room temperature. The reaction was stopped by dilution and cells were then washed in PBS. Parasitaemia was assessed on a BD LSRFortessa Flow Cytometer and analysed with FlowJo software.
These data strongly indicate that immature reticulocytes highly expressing CD71 (CD71hi), rarely observed in peripheral blood circulation in normal BALB/c mice, are the cells predominantly invaded by the reticulocyte-prone P. yoelii 17X strain. In humans, these cells probably represent R1 reticulocytes formed immediately after enucleation in the marrow as opposed to mature R2 reticulocytes released into the peripheral blood circulation . Blood R2 reticulocytes could be in a late maturation process with advanced autophagic events that impairs parasite survival . The use of immature CD71hi reticulocytes obtained from the bone marrow and/or from patients with different haemolytic anaemias containing high percentages of immature CD71hi reticulocytes is guaranteed to try advancing the development of a continuous in vitro culture system of blood stages for P. vivax.
We are grateful to Juan A Bueren and Oscar Quintana-Bustamante for support and scientific discussions, to Volker Heussler for the gift of the plasmid containing m-Cherry, to Isabel Crespo at the Citomics unit of the IDIBAPS for the technical help, to Nuria Cortadellas for helping with EM facilities. Work in the laboratory of HAP is funded by the European Community’s Seventh Framework Programme (grant agreement N° 242095) and by the Ministerio Español de Economia y Competitividad (SAF2011-30526-C02-01and SAF2012-35133), Spain.
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