Reticulocyte-prone malaria parasites predominantly invade CD71hi immature cells: implications for the development of an in vitro culture for Plasmodium vivax

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Gating strategy used for the flow cytometry analysis. Blood cells from non-infected mice were labelled with CD71 and TER119 specific antibodies and analysed for GFP transgenic parasites in a BD LSRFortessa Flow Cytometer. (A) The gate was set to exclude debris and include non-nucleated erythroid cells. (B) Numbers in the upper-right insets show percentages of TER119⁺CD71⁺ stained reticulocytes. TER119⁺CD71⁺ cells were gated into two populations according to differences in CD71 intensity. (C) TER119⁺ cells from infected mice were analysed according CD71 and GFP (parasite) expression. Data are represented as mean ± SEM of three mice per group.

Figure 2

Flow cytometric analysis of cells from peripheral blood on different days post-infection. BALB/c mice were infected ip with 10⁵P. yoelii 17X-GFP parasites and peripheral blood was analysed at different days post-infection. Cells were labelled with CD71 and TER119 specific antibodies and analysed for GFP transgenic parasites in a BD LSRFortessa Flow Cytometer. The gate was set to exclude debris and include erythroid cells. TER119⁺CD71⁺ cells were gated into two populations according to differences in CD71 intensity. (A) Percentages of CD71^hi, CD71^lo and CD71^- as a function of erythroid cells from blood are expressed as the mean ± SEM of three mice. (B) Percentages of infected cells (CD71^hiGFP⁺, CD71^loGFP⁺ and CD71^-GFP⁺) as a function of erythroid cells from blood are expressed as the mean ± SEM of three mice. (C) Parasitaemias and (D) percentages of infected cells (CD71^hiGFP⁺, CD71^loGFP⁺ and CD71^-GFP⁺) as a function of CD71^hi, CD71^lo and CD71^- respectively are expressed as the mean ± SEM of three mice. Data were evaluated by analysis of variance for each cell type in the different time points post-infection and versus non-infected (*P <0.05, **P <0.01 and ***P <0.001) (Dunnet post hoc test versus NI). Non-infected animals (NI); animals infected with the P. yoelii 17X nonlethal-GFP strain (NL).

Figure 3

Flow cytometric analysis of cells from erythopoietic organs on different days post-infection. BALB/c mice were infected ip with 10⁵P. yoelii 17X-GFP parasites and cells from bone marrow and spleen were analysed at different days post-infection. Cells were labelled with CD71 and TER119 specific antibodies and analysed for GFP transgenic parasites in a BD LSRFortessa Flow Cytometer. The gate was set to exclude debris and include non-nucleated erythroid cells. TER119⁺CD71⁺ cells were gated into two populations according to differences in CD71 intensity. Analyses of bone marrow (A, B) and spleen (C, D) erythroid cells. (A, C) Percentages of CD71^hi, CD71^lo and CD71^- as a function of erythroid cells are expressed as the mean ± SEM of three mice (B, D). Percentages of infected cells (CD71^hiGFP⁺, CD71^loGFP⁺ and CD71^-GFP⁺) as a function of erythroid cells are expressed as the mean ± SEM of three mice. Data were evaluated by analysis of variance for each cell type in the different time points post-infection and versus non-infected (*P <0.05, **P <0.01 and ***P <0.001) (Dunnet post hoc test versus NI). Non-infected animals (NI); animals infected with the P. yoelii 17X nonlethal-GFP strain (NL).

Figure 4

Analysis of erythroid cells at day 15 pi. Blood, spleen and bone marrow cells from mice infected with the GFP transgenic parasites were stained with specific antibodies. (A) The mean fluorescence intensity (MFI) of CD71^hi cells was measured in the erythroid population at day 15 pi. Results represent the mean ± SEM of two independent experiments with three mice each. Data were evaluated by analysis of variance for each cell type separately and versus blood (*P <0.05 and **P <0.01) (Dunnet post hoc test). Electron microscopy of non-infected (B) and infected (C) blood CD71^hi cells obtained after sorting showed early reticulocytes containing different organelles. Mitochondria (m), vesicles (v) and parasite (p).

Figure 5

CD71^hi immature cells as a target for the development of the Plasmodium yoelii 17X-mCherry strain in vitro culture. (A) CD71 expression of blood cells from wild type mouse (black line) and a PKD mouse (grey line). Results were representative of three independent experiments. Percentages of CD71⁺ cells were 47,82% for PKD mice and 6,89% in control mice. CD71^hi cells represent 24,40% for PKD mice and 4,92% for control mice. (B) Percentages of parasitaemia during in vitro culture of P. yoelii using PKD reticulocytes. Results were representative of four independent experiments. (C) Giemsa staining images of blood smears after 72, 96 and 120 hours of in vitro culture. (D) Representative dot plot of CFSE-PKD/mCherry-parasite signal for erythroid cells after 72 hours of in vitro culture. (E) Representative dot plot of CD71-PE/mCherry-parasite signal for erythroid cells after 72 hours of in vitro culture.