CD47 regulates the phagocytic clearance and replication of the Plasmodium yoelii malaria parasite

Abstract

Several Plasmodium species exhibit a strong age-based preference for the red blood cells (RBC) they infect, which in turn is a major determinant of disease severity and pathogenesis. The molecular basis underlying this age constraint on the use of RBC and its influence on parasite burden is poorly understood. CD47 is a marker of self on most cells, including RBC, which, in conjunction with signal regulatory protein alpha (expressed on macrophages), prevents the clearance of cells by the immune system. In this report, we have investigated the role of CD47 on the growth and survival of nonlethal Plasmodium yoelii 17XNL (PyNL) malaria in C57BL/6 mice. By using a quantitative biotin-labeling procedure and a GFP-expressing parasite, we demonstrate that PyNL parasites preferentially infect high levels of CD47 (CD47(hi))-expressing young RBC. Importantly, C57BL/6 CD47(-/-) mice were highly resistant to PyNL infection and developed a 9.3-fold lower peak parasitemia than their wild-type (WT) counterparts. The enhanced resistance to malaria observed in CD47(-/-) mice was associated with a higher percentage of splenic F4/80(+) cells, and these cells had a higher percentage of phagocytized parasitized RBC than infected WT mice during the acute phase of infection, when parasitemia was rapidly rising. Furthermore, injection of CD47-neutralizing antibody caused a significant reduction in parasite burden in WT C57BL/6 mice. Together, these results strongly suggest that CD47(hi) young RBC may provide a shield to the malaria parasite from clearance by the phagocytic cells, which may be an immune escape mechanism used by Plasmodium parasites that preferentially infect young RBC.

Keywords: CD47; F4/80; Plasmodium yoelii; RBC; malaria.

Fig. 1.

In vivo biotinylation separated young and aged populations of RBC that allowed determining the association between CD47 expression level and parasitemia. Three dosages of biotin were administered in WT C57BL/6 mice (n = 5) on consecutive days, and on the following day, mice were infected with GFP-PyNL pRBC. On the day of parasite challenge, 100% of RBC in all mice were biotin-coated, as determined by in vitro staining with streptavidin-APC (Fig. S1). A gradual accumulation of young RBC was observed as the population of the aged RBC decreased during a 13-d observation period. (A) A representative diagram showing the percentage GFP-PyNL parasitemia in young and aged populations of RBC in biotin-injected mice on day 9 p.i. (B) The parasite burden during the course of infection was determined in the young and aged populations of RBC and plotted as the mean ± SEM. (C) CD47 intensity in the young and aged populations of RBC in biotin-injected mice was assayed on day 9 after GFP-PyNL infection. (D) The CD47 levels on young and aged populations of RBC in biotin-injected mice were measured throughout the course of infection and expressed as MFI values (mean ± SEM, n = 5). Bonferroni comparison test was applied after two-way ANOVA.

Fig. 2.

GFP-PyNL parasites prefer to infect CD47^hi RBC. (A) The parasitized RBC (pRBC) and nonparasitized RBC (npRBC) were differentiated on the basis of GFP expression by the GFP-PyNL, and the CD47 intensity was measured in the pRBC and npRBC groups assayed on day 9 post-GFP-PyNL infection. (B) The CD47 MFI of pRBC and npRBC from the same infected mouse (n = 10) were plotted throughout the course of infection. Statistically significant differences in the CD47 MFI values were noted between the two groups (P < 0.0001). (C) A representative diagram shows the GFP-PyNL burden in CD47^hi and CD47^lo RBC populations. (D) The percentage of parasitemia in the CD47^lo and CD47^hi group was plotted as mean ± SEM throughout the course of infection. Data were analyzed using the two-way ANOVA followed by Bonferroni test.

Fig. 3.

Absence of CD47 confers resistance against PyNL malaria. Course of parasitemias in the CD47^−/− (n = 5) and the WT C57BL/6 mice (n = 5) after infection with GFP-PyNL parasite and percentage parasitemia were expressed as mean ± SEM. Data were compared using two-way ANOVA, followed by Bonferroni test.

Fig. 4.

Modulation of CD47 level affects the severity of GFP-PyNL infection. (A) Two dosages of PHZ were given on consecutive days to WT C57BL/6 mice (n = 5), and on the following day the PHZ-treated and PHZ-nontreated mice were infected with GFP-PyNL. Parasitemia was measured on day 3; results are shown as mean ± SEM values. (B) WT C57BL/6 mice (n = 5) were injected with either the neutralizing anti-CD47 mAb (miap301) or an isotype control antibody and were challenged with GFP-PyNL the same day. The parasite burden was measured on day 3 p.i.; results are shown as mean ± SEM. The percentage parasitemias between the neutralizing anti-CD47 antibody and isotype control mice were compared using the Student’s t test (B), whereas comparisons between the PHZ-treated and PHZ-nontreated mice were made using Mann–Whitney U test (A).

Fig. 5.

Immunologic analysis of resistance to GFP-PyNL infection in CD47^−/− mice. The WT and CD47^−/− C57BL/6 mice were infected with GFP-PyNL, and spleen cells from the day 7 p.i. mice were stained with anti-F4/80-Percp antibody; results were analyzed by flow cytometry. (A) Percentage of F4/80⁺ cell population in GFP PyNL-infected WT and CD47^−/− mice are expressed as mean ± SEM. (B) Absolute number of F4/80⁺ cells in the WT and CD47^−/− mice on day 7 p.i. was calculated and presented as mean ± SEM. (C) Serum IL-10 levels in the WT and CD47^−/− mice on day 7 p.i. were measured by the bioplex assay and presented as picograms per milliliter. (D) Percentage of Il-10 cytokine producing F4/80⁺ cells was determined from the WT and CD47^−/− mice on day 7 p.i. (E) Percentage of F4/80⁺ cells that internalized the GFP-PyNL parasitized RBC was measured by flow cytometry. The statistical methods used were Student’s t test (A, B, D, and E) and repeated measure ANOVA (C).